Evaluation of the Effects of Artesunate on the Liver of Plasmodium Berghei Infected Mice

J E. Toryila

Department of Human Physiology Faculty of Basic Medical Sciences,

College of Health Sciences, Bingham University, Karu.

G. P Oderinde; W.O hamman, E.U Umana

Department of Human Anatomy, Faculty of Basic Medical Sciences,

College of Health Sciences, Ahmadu Bello University Zaria

All correspondence to: J E. Toryila Department of Human Physiology Faculty of Basic Medical Sciences,

College of Health Sciences, Bingham University, Karu.

ABSTRACT

Background: Antimalaria drugs play important role in treating malaria but some medicinal agents when taken in overdoses or even within therapeutic ranges maybe toxic. Artesunate is one of the successfully used over-the-counter antimalaria drug in treating and preventing malaria but many studies have shown validation of toxicity on several organs. The aim of this study was to investigate the effect of Artesunate on the liver of Plasmodium berhei. infected mice. Methods: 25 mice were divided into 5 groups of 5 animals each, Group 1 received 0.5 ml/kg distilled water, Group 2 received 4 mg/kg/day Artesunate only, Group 3 was inoculated with Plasmodium berghei only, Group 4 and 5 were inoculated with P. berghei and treated with 4 and 8 mg/kg/day Artesunate respectively 72 hours after inoculation. The treatment lasted for a period of 5 days after which the mice were humanly sacrificed. Blood samples were collected by cardiac puncture and stored in 5 ml plain and EDTA bottles for biochemical analysis. The liver tissues were removed and placed in neutral buffered saline. RESULTS: There was no significant difference in the liver function biomarkers (ALT, AST and ALP) examined when compare among the groups. There was also significant increase in SOD activities in Group 5 when compared with Group 1, 2, 3 and 4, also there was a significant decrease in CAT and GSH activities in Group 1 when compared with Group 3, 4 and 5, the MDA activities in Group 5 showed a significant increase when compared with the other Group. There was increase in the liver weight and liver somatic index of Group 3 when compared to Group 1 Histological changes were observed in Group 2, 3, 4 and 5 when compared with Group 1. This research shows that Artesunate and malaria has adverse effect on the liver There is need for caution during the administration of Artesunate in the treatment of malaria. CONCLUSION: The effects of Artesunate on the liver of Plasmodium berghei infected mice were evaluated in this study. The results obtained show that Artesunate induces oxidative stress in mice infected with Plasmodium berghei and causes cytoplasmic vacuolation, traces of periportal mononuclear cells infiltration, and severe and diffused hepatic necrosis with karyolysis in the liver of mice infected with Plasmodium berghei.

INTRODUCTION

Malaria is a preventable and treatable disease. The primary objective of treatments is to ensure complete cure that is the rapid and full elimination of the Plasmodium parasite from the patient’s blood, in order to prevent progression of uncomplicated malaria to severe disease or death and to prevent chronic infection that leads to malaria-related anaemia (WHO 2018).

Artemisinin is currently the most widely used antimalarial drug against drug-resistant malaria. It is a Chinese herb that has been used over thousands of years for the treatment of fever. It is extracted from the leaves of Artemisia annua (sweet wormwood). Artemisinin and its derivatives (artesunate, artemether, arteether and dihydroartemisinin) are highly potent, rapidly eliminated antimalarial drugs

with a broad stage specificity of action. They clear parasitemia more rapidly than all other currently available antimalarial agents. Artesunate is a hemisuccinate derivative of the active metabolite dihydroartemisin. Currently it is the most frequently used of all the artemisinin-type drugs. Its only effect is mediated through a reduction in the gametocyte transmission. It is used in combination therapy and is effective in cases of uncomplicated P. falciparum (Izunya et al., 2010; Ashley et al., 2014).

The liver is a vital organ only found in vertebrates (Abdel-Misih and Bloomston, 2010). The liver is a reddish-brown wedge-shaped organ with four lobes of unequal size and shape. A human liver normally weighs 1.44 – 1.66 kg (3.2 – 3.7 Ib.), and has a width of about 15 cm, it is both the heaviest internal organ and the largest gland in the human body. Located in the right upper quadrant of the abdominal cavity, it rests just below the diaphragm, to the right of the stomach and overlies the gallbladder. The numerous functions of the liver are carried out by the liver cells. The liver is thought to be responsible for up to 500 separate functions usually in combination with other systems and organs. It plays a role in metabolism, regulation of glycogen storage, decomposition of red blood cells and hormone production. It also helps in detoxification of various metabolites, protein synthesis, and the production of biochemical necessary for digestion (Tortora and Derrickson, 2008).

Artesunate is one of the successfully used over-the-counter antimalarial therapies in treating and preventing malaria, M any studies on toxicity of Artesunate have shown validation of toxicity on the stomach, brain stem, and the liver. Administration of Artesunate for the treatment of malaria maybe an additional burden on the liver which is already undergoing insult from malaria parasite invasion of the hepatocytes, this therapy even in normal doses may result in severe hepatic injury to an already damaged organ. Some studies have suggested evidence of toxicity on the brainstem, superior colliculus, stomach, testis and liver in artesunate treated rats (Maude et al., 2009). Moreover, artesunate has been reported to destroy cancer cells and also reduces proliferation, interferes in DNA replication and cell cycle and enhance apoptosis through the intrinsic death pathway by ROS generation. It has been reported that artesunate is toxic at nanomolar concentration to malaria parasites, while micromolar concentration produced toxicity in mammalian cells (Turschner and Efferth, 2009). Evidences showed the neurotoxicity of artesunate at high doses (50 – 100 mg/kg per day oral and IM) in laboratory animals (Brewer et al., 1994) including the cytotoxicity of artesunate on tumor cell lines have been reported (Maudeet al., 2009). Therefore, this research was carried out further help in understanding the adverse effect of Artesunate and malaria on the liver and also provide the need for caution during the administration of Artesunate in the treatment of malaria.

MATERIALS AND METHODS:

Regeants: Randox test kit, Microscope, glass slides and cover slips, Formalin and bottles for fixing tissues, Dissecting set, Beakers and syringes, Anaesthesia (Ketamin), Normal saline, Haematoxylin and eosin. Experimental Desig: The animals were grouped into five groups of five (5) male mice per group. One control group and four treated groups; group III to V were inoculated with malaria on the first (1st) day of the experiment. Group I served as the control group and received 1 ml/kg normal saline intraperitoneally during the first day of the experiment and were given distilled water orally subsequently for a period of 5 days, Group II served as the Artesunate group and received 1 ml/kg normal saline intraperitoneally during the first day of the experiment and was latter administered with 4 mg/kg body weight of Artesunate starting from the 4th (fourth) day of the experiment, Group III served as the Untreated malaria infection groups while Group IV, and V were treated malaria infection group in different doses; the animals were infected with malaria and treated with Artesunate at 4 mg/kg body weight, and 8 mg/kg body weight respectively. Malaria infection was established on the 3rd day before commencement of treatment. The mice were treated with Artesunate for five (5) days before sacrifice. Artesunate was dissolved in distilled water and administered orally with the aid of a stainless metallic feeding cannula. The animals were then humanely sacrificed under anesthesia using Ketamine, through a midline incision on the thoracic wall, the heart was accessed and blood samples were collected by cardiac puncture. The blood samples were stored in 5 ml plain bottle for biochemical evaluation. The liver was quickly excised, rinsed in normal saline, gently blotted between folds of a filter paper and then weighed using an electronic compact digital scale. The liver tissue was cut into two pieces; the first piece was immediately fixed in Neutral Buffered Formalin for subsequent histological and histochemical analysis. The second piece was weighed and homogenized in Phosphate Buffer (pH 7.4) (at a ratio of 1 g to 5 ml respectively) for estimation of Oxidative stress parameters. The liver Enzymes and Oxidative Biomarkers were determined by ELISA technique (Kavishe et al., 2017). The tissues were Processed with Tissue Processing Machine and stained using haematoxylin and eosin (H and technique for general liver histology, Statistical Analysis: Data obtained were expressed as mean ± SEM (Standard Error of Mean). One-way analysis of variance (ANOVA) was used to compare the mean differences followed by LSD post-hoc test, P-value less than 0.05 was considered to be statistically significant. All the results were analyzed using the Statistical Package (SPSS version 20)

RESULTS AND DISCUSSION:

This study revealed that there was no significant difference in the liver weight and liver somatic index when compared between the Groups, although there was increase in the liver weight and liver somatic index of Group 3 when compared to Group 1 (Table 1.0). Group 4 also showed a slight increase in the liver weight and liver somatic index when compared to Group 1, Group 5 showed increase in the liver weight and liver somatic index when compared to Group 1 but it was not significant stastistically (Table 4.2). Rungruang et al (2013) reported that the liver of Plasmodium infected mice showed no significant difference in weight when compared with the control,hepatomegaly was observed on day 5 and 6 post-infection of Swiss Albino mice infected with Plasmodium yoelli nigeriensis (Ahmad and Srivastava, 2007). The reason for increase in liver weight and liver somatic index could be that the liver of infected mice is under very severe condition; part of systemic sequestration and anemia reduce the chance of circulating cells passing into the organs (Thuma et al., 1998).

There were no statistical differences (p<0.05) in all the liver injury biomarkers: Alanine transaminase (ALT), Aspartate aminotransferase (AST), Alanine phosphate (ALP) investigated between the experimental groups and the control groups. However, there was a slight increase in the ALT of Group 3 (128.00±20.06) when compared to other groups, there was also a slight decrease in ALT of Group 2 (106.75±15.34) when compared to the other groups (Table 1.0).

There was also no statistically significant difference (p<0.05) in the AST of the all the experimental groups when compared to the control groups (Table 1.0). There was slight increase in AST of Group 4 when compared to other groups. The AST activities of Group 2 were lower when also compared with the other groups

This investigation also revealed no statistically significant difference (p<0.05) in the ALP of all the groups when compared. Group 5 (155.40±25.52) showed the highest level of ALP when compared with the other Groups. Group 4 (148.40±49.36) showed a slight increase in ALP level when compared with Group 1 (137.25±32.42) and Group 2 (142.00±52.38).

Liver function tests are groups of blood tests that give information about the state of the liver (Lee, 2009). Injury to the liver may affect the integrity of hepatocytes leading to the release of liver enzymes such as alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphate (ALP) since these enzymes are confined to hepatocytes and released into the blood following liver injury. Hence, these enzymes are commonly used as markers of hepatic injuries (Ozer et al., 2008). The liver aspartate transaminase (AST) and alanine transaminase (ALT) are useful biomarkers of liver injury with some degree of intact liver function (Mengel and Schwiebert, 2005). These tests can be used to detect the presence of liver disease, distinguish among different types of liver disorders, gauge the extent of known liver damage and follow the response to treatment. In the present study, there was no significant difference statistically in serum level of AST, ALT and ALP as compared between the groups.

The level of liver dysfunction may be determined by the level of parasitemia in the body (Onyesom and Onyemakonor, 2011). Hamman et al (2011) also reported no statistical significant differences in ALP, AST and ALT levels in Wistar rats that were administered Artesunate at various doses.

The result revealed statistically significant (p<0.05) increase in Superoxide Dismutase (SOD) activities in all the groups; Group 4 (21.24±1.35); Group 5 (24.80±1.64) when compared to Group 1 (17.22±0.84) and Group 2 (16.58±0.45). Group 5 (24.80±1.64) also showed a significant statistical (p<0.05) increased in the SOD activities when compared to Group 1 (17.22±0.84), Group 2 (16.58±0.45), Group 3 (19.52±0.96) and Group 4 (21.24±1.35) There was significant statistical increase in Catalase (CAT) activities in Group 5 (10.14±0.50); Group 4 (10.24±0.39); and Group 3 (9.92±0.36) when compared with Group 1 (7.96±0.43) and Group 2 (8.00±0.33). There was also slight decrease in CAT activities in Group 1 (7.96±0.36) when compared to Group 2 (8.00±0.33) however it was not statistically significant (Table 2.0)

There was significant statistical increased in Glutathione (GSH) activities in Group 5 (38.16±2.97); Group 4 (43.88±1.81); and Group 3 (39.40±2.54) when compared with Group 1 (25.06±2.18) and Group 2 (31.04±1.43). There was also slight decrease in GSH activities in Group 1 when compared to Group 2 but was statistically insignificant (Table 2.0).

There was significant statistical increase in the Malondialdehyde (MDA) activities in Group 5 (37.68±3.15) when compared with other groups. There was also significant statistical increase in the MDA activities in Group 2 (34.44±2.75) when compared to Group 3 (25.58±1.94). This result also showed slight decrease in MDA activities in Group 3 (25.58±1.94) when compared to Group 1 (27.68±1.29) and Group 4 (29.06±2.08), however it was statistically insignificant (p<0.05) (Table 2.0).

The role of oxidative stress during malaria infection is still unclear. Some researchers argued a protective role, whereas other claims a relation to the pathophysiology of the disease (Sohail et al., 2007). However, other studies suggested that the generation of reactive oxygen and nitrogen species (ROS and RNS) associated with oxidative stress, plays a critical role in the development of systemic complications caused by malaria. Malaria infection induces the generation of hydroxyl radicals (OH-) in the liver, which most probably is the main reason for the induction of oxidative stress and apoptosis (Guha et al., 2006). The iron-rich environment within the parasite enables rapid production of specific redox active drug molecules and perhaps ROS that can lead to a rapid reaction and destruction of several specific systems within the parasite such as components of cellular membranes, the redox systems of the parasites, and the mitochondrial electron transport chain, and this may explain the rapid elimination of parasites by Artesunate (Kavishe et al., 2017).

In the present study there was significant increase in the GSH activities in Group 3, Group 4 and Group 5 when compared to Group 1 and Group 2 (Table 4.4). In Plasmodium parasites, GSH is also involved in the degradation of the toxic ferriprotoporphyrin IX (FP IX), which escapes from hemozoin formation. Additionally, GSH functions as an electron donor for the enzyme ribonucleotide reductase (RNR), and also crucial for DNA synthesis and cellular proliferation (Buchholz et al., 2010). Artesunate may induce DNA double-strand breaks in Plasmodium infection in a physiologically relevant dose and time dependent manner which is accompanied by an increase in the ROS level in the parasites (Gopalakrishnan and Kumar, 2015). Mannitol, a ROS scavenger, reversed the cytotoxic effect of Artesunate and reduced DNA damage and modulation of GSH activities impacted ROS and DNA damage induced by Artesunate (Gopalakrishnan and Kumar, 2015). The present study also showed no statistically significant difference between the group that was inoculated with malaria but not treated and the groups that were treated with Artesunate after malaria inoculation, it was also discovered that at increased dose of Artesunate there was reduce GSH activities when compared with the malaria treated group. This could occur because Plasmodium possesses two redox systems which are the thioredoxin and the glutathione system, glutathione is the most abundant low molecular weight redox active thiol in the parasites existing primarily in its reduced form representing an excellent thiol redox buffer. This allows for an efficient maintenance of the intracellur reducing environment of the parasite cytoplasm and its organelles. During development, malaria parasites are exposed to environmental and metabolic stresses.

H&E stain result of the liver sections from Group 1 showed normal cytoarchitecture of the liver parenchyma; the characteristic appearance of the hepatocytes radiating from the central vein. Fine vascular spaces separate the thin plate of hepatocytes, the sinusoids and Kupffer cells found within the sinusoid lining (Plate I). Liver section from Group 2 showed some abnormal morphological characteristics showing Cytoplasmic Vacuolation (CyV) and Sinusoidal Congestion (SC) (Plate II). Liver section from Group 3 showed Cytoplasmic Vacuolation (CyV) with Kupffer cells Hyperplasia (KH), Central vein congestion (CCV) and Hemozoin (H) (Plate III). Liver section from Group 4 showed Cytoplasmic Vacuolation (CyV), Sinusoidal congestion (SD) and traces of Periportal Mononuclear cells infiltration (PM) (Plate IV). Liver section from Group 5 showed distortion in the cytoarchitecture with severe and difused Hepatic Necrosis with karyolysis (HN), Cytoplasmic vacuolation (CyV), traces of Periportal Mononuclear cells infiltration (PM) and Sinusoidal Congestion (SC) (Plate V).

Table 1 Liver functions biomarkers

Groups Treatments ALT (IU/L) AST (IU/L) ALP (IU/L)
Mean±SEM Mean±SEM Mean±SEM
1 Distilled water 110.25±10.05 77.00±17.31 137.25±32.42
2 Artesunate 4 mg/kg/day only 106.75±15.34 65.00±11.79 142.00±52.38
3 Malaria Induced (Untreated) 128.00±20.06 83.75±24.33 153.00±33.52
4 Malaria Induced and treated 125.40±23.98 83.40±13.78 148.40±49.36
4 mg/kg/day Artesunate
5 Malaria Induced and treated 126.40±22.44 80.80±22.04 155.40±45.52
8 mg/kg/day Artesunate
F 0.091 0.284 0.223
p 0.984 0.884 0.920

Table 2. Oxidative stress markers

Groups Treatments SOD(U/ml) CAT(U/mg) GSH(ug/ml) MDA(nMols/mg)
Mean±SEM Mean±SEM Mean±SEM Mean±SEM
1 Distilled water 17.22±0.84ae 7.96±0.43ace 25.06±2.18ace 27.68±1.29a
2 Artesunate 4mg/kg/day only 16.58±0.45bf 8.00±0.33bdf 31.04±1.43bdf 34.44±2.75bd
3 Malaria Induced (Untreated) 19.52±0.96c 9.92±0.36ab 39.40±2.54ab 25.58±1.94d
4 Malaria Induced and treated 21.24±1.35def 10.24±0.39cd 43.88±1.81cd 29.06±2.08c
4mg/kg/day Artesunate
5 Malaria Induced and treated 24.80±1.64abcd 10.14±0.50ef 38.16±2.97ef 37.68±3.15abc
8mg/kg/day Artesunate
f 8.693 8.183 10.930 4.614
p <0.001 <0.001 <0.001 0.008

Oneway ANOVA test followed by Tukey poct hoc test; Results expressed as mean±SEM, cells carrying same superscripts on each column are significantly different (p<0.05). SOD: Superoxide Dismutase; CAT: Catalase; GSH: Glutathione; MDA: Malondialdehyde.

H K
CV S CV CyV
K SC
Plate I: Transverse section of the Liver from Group 1 Plate II: Transverse section of the Liver from Group 2
showing normal cytoarchitecture; Hepatocytes (H) Central Vein showing the Central Vein (CV), Cytoplasmic Vacuolation
(CV), Sinusoid (S) and Kupffer Cells (K). (H&E x250). (CyV) and Sinusoidal Congestion (SC) (H&E x250)

 

CCV CyV

H

KH

Plate III: Transverse section of the Liver from

Group 3 showing Cytoplasmic Vacuolation (CyV) with Kupffer cells Hyperplasia (KH), Central vein Congestion (CCV), Hemozoin (H) (H&E x250)

CyV

SC

CV

HN

PLATE V: Transverse section from the Liver from Group 5 showing distortion in the cytoarchitecture with severe and diffused Hepatic Necrosis with karyolysis (HN), and Sinusoidal Congestion (SC), Cytoplasmic vacuolation (CyV) (H&E x250)

CONCLUSION: This research shows that Artesunate and malaria has adverse effect on the liver There is need for caution during the administration of Artesunate in the treatment of malaria.

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A Histological, Histochemical and Ultrastructural Characterization of Uterine Vessels at Early Stages of Pregnancy

Abd-Elhafez EA

Department of Anatomy and Histology, Faculty of Veterinary Medicine, Assiut University, 71526, Egypt

All Correspondences to: Fatma, El-Zahraa AM, Department of Anatomy and Histology, Faculty of Veterinary Medicine,

Assiut University, 71526, Egypt e-mail: f.histology@aun.edu.eg

The integrity of uterine circulation is essential for normal pregnancy outcome. The present study describes for the first time the histology, histochemistry, and ultrastructure of the different types of vessels in the uterus at critical early stages of pregnancy. Arteries were congested and showed PAS-positive granules. veins characterized by its wide lumen and thick adventitia rich in collagen and elastic fibers and showed PAS-positive reaction in their wall. Lymphatics showed a positive reaction with PAS-AB combination. Many arteriols observed in the uterine wall and showed PAS-positive granules and venules appeared congested in the endometrium. Spiral artery and arterial sinuses also demonstrated. Anastomosis between arterioles and venules was recorded. Blood vessels of a special structure were demonstrated presenting a vascular wall of variable thickness, unevenly distributed smooth muscle in the wall, smooth muscle fibers arranged in a different direction. Spirally oriented arterioles and venules were also demonstrated. Sclerotic changes observed in the wall of some vessels. Glomus cells observed in the wall of many arteries, arterioles, spirally oriented arterioles, and arteriovenous anastomosis. filopodia-like protrusions and caveolae were the most characteristic feature of the endothelium.

Key Words: Blood vessels; Lymphatics; Glomus cells; Telocyte; Filopodia; Early pregnancy.

The integrity of uterine circulation is essential for normal pregnancy outcome. The clinical relevance of maternal uterine vascular adaptation during pregnancy is underscored by the fact that its aberrance is associated with several common gestational pathologies, including intrauterine growth restriction, gestational diabetes, and preeclampsia [1]. Preeclampsia is a major cause of maternal and neonatal mortality and morbidity

It is known that the vascularization of the uterus is of primary importance in the pregnancy success [3], and vascular disorders may play a role in pathological pregnancies [4]. Furthermore, normal uterine vascular development may have critical effects on the growth and development of the fetus and insufficient uterine vascular establishment is associated with increased risk for cardiovascular morbidity in adult life [5]. The lymphatic circulation plays an important role in regulating content of interstitial fluid and adaptive immunity [6] and lymphatic vessels could also play a role in maternal and fetal immunity [7]. The uterine lymphatics during pregnancy have been scantily studied. Rabbit has a duplex uterus, with the main uteroovarian arteries and veins running parallel to, but well outside of, the uterine wall within the mesometrium. The vessels of the mesometrium are perfused by arterial blood coming from either the uterine or the ovarian end. Secondary vessels analogous to the arcuate arteries in humans may form redundant loops with the main artery, and tertiary radial arteries connect the arcuate loops with the uterine wall. These radial arteries divided into premyometrial arteries.

The premyometrial arteries enter the uterine wall supplies the myometrium [1]. A better understanding of the basic morphological structure of the blood vessels of the uterus of the rabbit may guide the clinicians to the design of strategies to improve their reproductive efficiency and help in solve pregnancy problems which related to the blood vessels. Yet, the available literature lacks any detailed

information concerning the uterine vessels during early stages of pregnancy. The aim of the present investigation was to study the different types of vessels in the uterus at critical early stages of pregnancy. The current study focused on arteries, veins, lymphatics, arterioles, venules, blood vessels of special structure.

MATERIAL AND METHODS Sample collection

The study was approved by the Ethics Committee of Assiut University, Egypt. The material included in this work was originated from the female genitalia of 6 healthy New Zealand rabbits at 0 and 7days of pregnancy. The uterus was taken immediately after slaughtering.

Histological preparation

The collected materials were dissected as soon as possible and immediately fixed in Bouin• fs fluid for 22 hours. The fixed materials were dehydrated in an ascending series of ethanol, cleared in methyl benzoate and then embedded in paraffin wax. Transverse paraffin sections at 1-7 ƒÊm in thickness were cut and stained with Harris haematoxylin and eosin for general histological examination, Crossmon• fs Trichrome for identification of collagenous and muscle fibers and Wigert• fs resorcin fuchsine for identification of elastic fibers. For carbohydrate histochemistery, sections were stained with Periodic Acid Schiff (PAS) technique. For demonstration of neutral mucopolysaccharides, combined alcian blue-PAS technique were used [8].

Semithin sections and transmission electron microscopic preparations

Other small specimens of the uterus were fixed in a mixture of 2.5% paraformaldehyde and 2.5% glutaraldehyde in 0.1M Na-cacodylate buffer, pH 7.3 for 4 hours at 4 Co. They were washed in the same buffer used and then post-

37

 

fixed in 1% osmic acid in 0.1M Na-cacodylate buffer for further 2 hours at room temperature. The samples were then dehydrated in ethanol and embedded in Araldite-Epon mixture. Semithin sections (1ƒÊm in thickness) were cut and stained with Toluidine blue and examined under a light microscope. Thin sections, obtained by a Reichert ultramicrotome, were stained with uranyl acetate and lead citrate [9] and examined with a Philips EM 400.

Myometrial artery (A) showed tunica media (TM) and vascular tunica adventitia (TA) (Harris haematoxylin and eosin). (G) Longitudinal section in myometrial artery (A) demonstrate PAS-positive granules (blue arrow) (PAS). (H) Myometrial artery (A) showed well developed internal elastic membrane (black arrow) (Wigert’s resorcin fuchsine).

RESULTS

O day of pregnancy

Mesometrial arteries were congested and demonstrated in varies directions cross and longitudinally oriented arteries (Figure 1A and 1B). The wall of these arteries was formed mainly of 3 – 5 layers of smooth muscle fibers tunica media, intermingled by elastic fibers. The tunica intima consisted of a thin layer of flat endothelial cells separated from the coating muscular media by a well-developed highly tortuous internal elastic membrane. The adventitia contained abundant elastic fibers and rich in collagenous fibers (Figure 1A-1D). In addition, there were arteries recorded in the mesometrium with spirally oriented smooth muscle fibers in the tunica media (Figure 1E). Myometrial arteries possessed a wide lumen and surrounded by a 2-4 layer of smooth muscle fiber. In addition, the adventitia showed several blood vessels. These arteries demonstrate PAS-positive granules in their wall and well developed internal elastic membrane (Figure 1F-1H). Mesometrial veins were numerous wide thin-walled vessels of different size. The tunica intima was surrounded by a thin tunica media composing of a 1-2 layer of smooth muscle fibers encircled by a relatively thicker adventitia rich in collagen and elastic fibers (Figure 2A and 2B). At the myometrial region, venae showed PAS-positive reaction in their wall (Figure 2C). Several thin-

Figure 1) Arteries at 0 day pregnancy. (A) Cross section in mesometrial artery (A) showed tunica media (TM) and internal elastic membrane (black arrow) (Combined Alcian blue-PAS). (B) Longitudinal section in the congested mesometrial artery (A) and tunica media smooth muscle fibers (TM) (Harris haematoxylin and eosin). (C) Cross section in mesometrial artery (A) showed tunica media (TM), internal elastic membrane (black arrow), tunica adventitia (TA) and elastic fibers (red arrow) (Wigert’s resorcin fuchsine). (D) Showed tunica media (TM) and tunica adventitia rich in collagenous fibers (TA) (Crossmon’s Trichrome).

  1. Showed tunica media formed from spirally oriented smooth muscle fibers (TM) and internal elastic membrane (black arrow) (PAS). (F)

Figure 2) Veins and lymphatics at 0 day pregnancy. (A) Mesometrial vein appeared as wide thin-walled vessels (V) and lymph vessel (L) showed valve (red arrow) (Harris haematoxylin and eosin). (B) Mesometrial vein

  1. showed thick tunica adventitia (TA) rich in elastic fibers (blue arrows) (Wigert’s resorcin fuchsine). (C) PAS-positive reaction in the wall of myometrial vein (V) (PAS). (D) Myometrial lymph vessel (L) with a thin valve (black arrow) (Harris haematoxylin and eosin). (E) Myometrial lymph vessel (L) with a valve (black arrow) and showed a positive reaction with PASAB combination (red arrows) (Combined Alcian blue-PAS). (F) Endometrial lymph vessel with a wide irregular lumen (L) (Crossmon’s Trichrome).

Figure 3) Arterioles and venules at 0 day pregnancy. (A-C) Mesometrial arterioles (red arrows), venules (black arrows), tunica media (TM), internal elastic membrane (blue arrow) and tunica adventitia which composed mainly of collagen fibers (TA) (figure A stained with Harris haematoxylin and eosin, figure B stained with Wigert’s resorcin fuchsine and figure C stained with Crossmon’s Trichrome). (D and E) Myometrial arterioles (red arrows), tunica media (TM) and internal elastic membrane (blue arrow) (Figure D stained with Harris haematoxylin and eosin and figure E stained with Wigert’s resorcin fuchsine). (F-I) Mesometrial arterioles (red arrows), venule (black arrow), smooth muscle (black arrow head), interrupted internal elastic membrane (green aarrow) and PAS-positive granules (blue arrows) (Figure F stained with Harris haematoxylin and eosin, figure G stained with Crossmon’s Trichrome figure H stained with Wigert’s resorcin fuchsine and figure I stained with PAS).

walled wide-lumened lymphatics were recorded within the mesometrium. Their wall is composed of an intimal tunic surrounded by a thick fibrous layer consisting of collagenous, elastic fibers and connective tissue cells. Some of these vessels demonstrate valves (Figure 2A). Myometrial lymphatics were composed of an intimal tunic surrounded by a fibrous layer of collagenous and elastic fibers. The valves of these vessels were mainly thin and consisted of a single cellular layer. The wall of myometrial lymph vessels showed a positive reaction with PAS-AB combination (Figure 2D and 2E). The lymphatic vessel in the endometrium appeared as wide irregular thin walled vessels (Figure 2F). Several arterioles were demonstrated within the mesometrium. These blood vessels were composed of 1-2 smooth muscle fiber-thick tunica media encircling a thin tunica intima with interrupted internal elastic membrane and surrounded by tunica adventitia which composed mainly of collagen fibers (Figure 3A-3C). In addition, arterioles of the myometrium were composed of 1-2 smooth muscle fiber-thick tunica media surrounded a thin intimal tunic with interrupted internal elastic membrane and was surrounded by fibrous adventitial tunic which mainly consisted of collagen fibers (Figure 3D and 3E). Many arterioles were observed within the endometrium with 1-2 smooth muscle fiber-thick tunica media encircling a thin tunica intima with interrupted internal elastic membrane and surrounded by tunica adventitia which contained mainly elastic and collagenous fibers. In addition, few PASpositive granules observed on the wall (Figure 3F-3I). Venules wall consists of an intimal tunic surrounded by one cell-thick tunica media of circularly arranged smooth muscle fibers coated by thick adventitial tunic consisting of collagenous fibers and connective tissue cellular elements.

However, venules of endometrium appeared as dilated thin wall vessels engorged with blood (Figure 3A and 3G). Spiral artery also demonstrated and characterized by the wide lumen and spiral course. Its wall consists of tunica intima of endothelial cells surrounded by a thin layer of smooth muscle fibers and thin adventitia (Figure 4A). In addition, arterial sinuses demonstrated and characterized by wide irregular lumen, one layer of smooth muscle tunica

media and unevenly disreputed elastic fibers (Figure 4B-4D). Anastomosis between arterioles and venules was recorded in mesometrium and showed an abrupt change from the arteriole to a venule. Another arteriovenous anastomosis in the mesentery with several glomus cells and its lumen

is closed (Figure 4E and 4F). Anastomosis between arterioles and venules was also recorded in the myometrium and (Figure 4G). Blood vessels of a special structure were demonstrated within the mesometrium presenting a vascular wall of variable thickness. The thicker portions of the wall showed a tunica media of 2-3 layers of smooth muscle fibers. However, the thin portions of the wall showed tunica media consisting of an interrupted layer of smooth muscle fibers (Figure 5A). In addition, another vessel showed 2-3 layers of longitudinally-arranged smooth muscle fibers at one side only (Figure 5B). Various special structures arteries observed within the mesometrium.

Characterized by the wide lumen and 2-3 layer of glomus cells (Figure 5C). Another special type of artery characterized by smooth muscle fibers arranged at various directions and fibrinoid deposition (Figure 5D). Various special structures veins observed within the mesometrium and characterized by a wide irregular lumen. The flattened endothelial cell lining was surrounded by 2-3 cell thick muscular media composing of circularly arranged smooth muscle fibers. These muscle cells were oriented into several protrusions, within the consecutively irregular lumen, bearing as muscular pads. The relatively thick

Figure 4) Spiral artery, arterial sinuses, and Anastomosis between arterioles and venules at 0 day pregnancy. (A) Spiral artery (SA) with the wide lumen and spiral course (Harris haematoxylin and eosin). (B-D) arterial sinuses (AS), arteriole (red arrow) and elastic fibers (black arrows) (figure B stained with Harris haematoxylin and eosin, figure C stained with Crossmon’s Trichrome and figure D stained with Wigert’s resorcin fuchsine). (E and F) Anastomosis between arterioles and venules in the mesometrium and (G) Anastomosis between arterioles and venules in the myometrium, arterioles (blue arrows), venules (red arrows) and glomus cells (black arrows) (figure E, F and G stained with Harris haematoxylin and eosin).

Figure 5) Blood vessels of a special structure in the mesometrium at 0 day pregnancy. (A) Blood vessel showed vascular wall of variable thickness (blue arrows) (Harris haematoxylin and eosin). (B) Special vessel showed longitudinally-arranged smooth 3 muscle fibers at one side only (black arrow) (Harris haematoxylin and eosin). (C) Special structures arteries characterized by the wide lumen and glomus cells (red arrows) (PAS). (D) Special type of artery characterized by smooth muscle fibers arranged at various directions (black arrows) and fibrinoid deposition (black arrowhead) (PAS). (E and F) Special structure vein characterized by smooth muscle cells oriented into several protrusions within the consecutively irregular lumen an showed PAS-positive granules (black arrows) (E stained with Harris haematoxylin and eosin and F stained with PAS). (G) Small arterioles of a special structure with few glomus cells (red arrows) (Harris haematoxylin and eosin). (H) Arteriole with an occlusive lumen and one layer of glomus cells (red arrows) (Harris haematoxylin and eosin).

39

 

Figure 6) Blood vessels of the special structure in the myometrium at 0 day pregnancy. (A) Artery with triangular shape lumen and surrounded by tunica media of spirally oriented smooth muscle fibers (black arrow) (Harris haematoxylin and eosin). (B) Artery with patches of longitudinally smooth muscle (blue arrow) in between circularly arranged smooth muscle (black arrow) (Harris haematoxylin and eosin).

  1. Spirally oriented artery (A) with a clear internal elastic membrane (green arrow) (Wigert’s resorcin fuchsine). (D) Spirally oriented arterioles with glomus cells in the tunica media (red arrows) (Wigert’s resorcin fuchsine). (E) Artery of a special structure with the narrow lumen (red arrow) and unevenly distributed elastic fibers in the wall (green arrows) (Wigert’s resorcin fuchsine).

Figure 7) Blood vessels of the special structure in the myometrium at 0 day pregnancy. (A) Arteries characterized by the wide irregular lumen (A) and unevenly distributed elastic fibers in the wall (green arrows) (Wigert’s resorcin fuchsine). (B) Veins of special structure (V) with irregular lumen and thick adventitia (TA) (Harris 4 haematoxylin and eosin). (C) Vein of special structure (V) with an irregular lumen and thick adventitia rich in elastic fibers (green arrows) (Wigert’s resorcin fuchsine). (D) Small arterioles surrounded by glomus cells (red arrows) (Harris haematoxylin and eosin).

adventitia mainly consists of collagenous and elastic elements. In addition, several islets of longitudinally-arranged smooth muscle fibers surrounded these vessels and characterized by PAS-positive granules (Figure 5E and 5F).

Small arterioles of a special structure were also observed and lined with 2-3 endothelial cells and surrounded by smooth muscle fibers of tunica media with few glomus

Figure 8) Digital colored TEM micrographs of uterine blood vessels at 0 day pregnancy. (A) Endometrial arteriole endothelial cells (EC), red blood cell (green arrow), smooth muscle cells (black arrows). cytoplasmic processes between endothelial cells and smooth muscle cells (red arrows) and filopodia-like protrusions of the endothelial cell toward the lumen (blue arrows). (B) Capillary with many caveolae (blue arrows), vesicles (red arrows), free ribosomes (black arrowhead), mitochondria (black arrow) and myaline figure (green arrow). (C) Spirally oriented capillary

(C). (D) Endothelial cells contained abundant ribosomes (black arrows) and small caveolae (red arrows) and large caveolae (green arrow).

cells (Figure 5G). Another arteriole with an occlusive lumen and one layer of glomus cells also demonstrated (Figure 5H). Blood vessels of the special structure were demonstrated also within the myometrium. Our observation revealed that there were arteries with triangular shape lumen and surrounded by tunica media of spirally oriented smooth muscle fibers (Figure 6A). In addition, some arteries demonstrate 2-4 layer of circularly arranged smooth muscle fibers and patches of longitudinally smooth muscle fibers in between (Figure 6B). Spirally oriented arteries were present within the myometrium with a wide lumen, clear internal elastic membrane and elastic fibers distributed at a various layer of the vessels wall (Figure 6C). Another spirally oriented arterioles observed and characterized by clear glomus cells in the tunica media (Figure 6D). In addition, there was another special type of arteries which characterized by the narrow lumen, thick wall and unevenly distributed elastic fibers in the wall (Figure 6E). Although, there was another type of arteries characterized by the wide irregular lumen and unevenly distributed elastic fibers in the wall (Figure 7A). Veins of special structure could also be observed within myometrium bearing an irregular lumen and relatively thick fibrillar adventitia mainly consists of collagenous and elastic elements (Figure 7B and 7C). Small arterioles of a special structure were lined with 2-3 endothelial cells and surrounded by glomus cells (Figure 7D).

Transmission electron microscopic observations of uterine blood vessels at 0day pregnancy showed that there were endometrial arterioles with endothelial cells surrounded by two smooth muscle layers. Endothelial cells and the inner smooth muscle cells gave cytoplasmic processes to each other and numerous filopodia-like protrusions of the endothelial cell toward the lumen (Figure 8A). Capillary with overlapping endothelial cell ends also observed and characterized by many caveolae of different size distributed singly or in small groups, numerous small vesicles, many free ribosomes, some mitochondria, and myaline figure (Figure 8B). In addition, spirally oriented capillary demonstrated and characterized by abundant ribosomes and rich in caveolae which small in size and arranged in rows or large in size and distributed singly (Figure 8C and 8D)

7 days of pregnancy

The most prominent characteristic features of this stage of pregnancy were the observation of numerous vessels of special morphological character. Some arteries in the myometrium contained batches of longitudinal smooth muscle in between the circular smooth muscle of the tunica media (Figure 9A). In some arteria, smooth muscle of the tunica media appeared in different directions (Figure9B). Other arterial tunica media showed different thickening of the smooth muscle from 1 to 3 layer (Figure 9C). In addition, there was arteria showed inner circular and outer longitudinal epitheloid cells in tunica media and thick adventitia (Figure 9D).

Our observation also revealed that Internal elastic lamina of some arteries appeared less developed but the external elastic lamina clearly well developed (Figure 9E) and in other arteries the elastic fibers distributed in one side of the wall only (Figure 9F). PAS-positive granules and PAS-positive material demonstrated in the wall of the blood vessels in the large amount (Figure 10A and 10B). Moreover, our observation also revealed that some arterioles surrounded by numerous venules (Figure 10C). Other arterioles of different size characterized by a very narrow lumen and some of them with dumbbelllike appearance (Figure 10D and 10E).

At this stage of pregnancy Arteriovenous anastomosis also observed with thick fibrous adventitia. The arterial side of the anastomosis may show narrow lumen and thick media (Figure 10F). However, another artery tunica media formed from epitheloid cells (Figure 10G).

Endometrial arteries showing special character one of them showed extensive venous or arterial profiles at the tunica adventitia of many arteries (Figure 11A). Spirally oriented arterioles and venules also observed at the endometrium (Figure 11B and 11C). In addition, arteriovenous anastomosis observe at the endometrium (Figure 11D). Extensive capillary network observed in lamina propria and take strong PAS positive reaction (Figure 11E).

At this stage of pregnancy transmission electron microscopic observations of uterine blood vessels showed that endothelial cells of the arteriole appeared swell to a plump with abundant mitochondria, numerous cytoplasmic filopodic extensions toward the lumen and few microvacuoles. The nuclei of these endothelial cells change to large nuclei (Figure 12A). However, the endothelium of the venule characterized by the presence of abundant free ribosomes, many mitochondria, some caveolae, few rough endoplasmic reticulum, scant microvacuoles and several filopodic extensions toward the lumen (Figure 12B-12D). Capillary also observed and encircled by telocyte (Figure 13A). Endothelium of the capillary contained caveolae and

Figure 9) Arteries at 7 days pregnancy. (A) Myometrial artery showed batches of longitudinal smooth muscle (blue arrows) in between the circular smooth muscle of the tunica media (TM) (Harris haematoxylin and eosin). (B) Myometrial artery with smooth muscle of the tunica media appeared in different directions (blue arrows) (Harris haematoxylin and eosin). (C) Myometrial artery tunica media showed different thickening of the smooth muscle (blue arrows) (Harris haematoxylin and eosin). (D) Myometrial artery showed inner circular epitheloid cells (blue arrow) and outer longitudinal epitheloid cells (red arrow) in tunica media and thick adventitia (TA) (Harris haematoxylin and eosin). (E) Myometrial artery showed less developed internal elastic lamina (red arrow) and well developed external elastic lamina (green arrows) (Wigert’s resorcin fuchsine). (F) Myometrial artery with elastic fibers distributed in one side of the wall only (green arrows) (Wigert’s resorcin fuchsine).

Figure 10) Myometrial vessels. (A and B) PAS-positive granules (black arrow) and a large amount of PAS-positive material demonstrated in the wall of the blood vessels (blue arrows) (figure A and B stained with PAS).

  1. Arteriole (blue arrow) surrounded by numerous venules (black arrows) (Harris haematoxylin and eosin). (D and E) Semithin sections of arterioles of different size characterized by the very narrow lumen (black arrows) and some of them with dumbbell-like appearance (red arrows). (F and G) Arteriovenous anastomosis showed the arterial side of the anastomosis (blue arrow), venous side of the anastomosis (black arrow) and epitheloid cells (red arrows) (figure F and G stained with Harris haematoxylin and eosin).

 

Figure 11) Endometrial vessels. (A) Artery with extensive venous and arterial profiles at the tunica adventitia (black arrows) (Harris haematoxylin and eosin). (B and C) Semithin sections showed spirally oriented arterioles (black arrows) and venules (red arrows). (D) Arteriovenous anastomosis showed the arterial side of the anastomosis (blue arrow), venous side of the anastomosis (black arrow). (E) Extensive capillary network observed in lamina propria showed strong PAS positive reaction (black arrows) (PAS).

Figure 12) Digital colored TEM micrographs of uterine blood vessels at 7 days pregnancy. (A) Arteriole endothelium (E), nucleus (N), mitochondria (blue arrows), cytoplasmic filopodic extensions (black arrows) and microvacuoles (red arrow). (B) Venule endothelium (E), nucleus (N) and filopodic extensions (black arrows). (C and D) Venule endothelium (E), nucleus (N) and filopodic extensions (black arrows), free ribosomes (black arrowheads), mitochondria (red arrows), caveolae (green arrows), rough endoplasmic reticulum (blue arrows), scant microvacuoles (red arrowhead).

Figure 13) Digital colored TEM micrographs of uterine blood vessels at 7 days pregnancy. (A) Capillary (C), telocyte (T), telopodes (black arrows) and microvacuoles 6 (red arrow). (B) Capillary (C), telocyte (T), nucleus (N), caveolae (green arrows) and free ribosomes (blue arrow), microvacuoles (red arrows) and rough endoplasmic reticulum (black arrows).

free ribosomes and large microvacuoles resemble the microvacuoles which present in the nearby telocyte (Figure 13B).

DISCUSSION

Rabbit is considered one of the widely used laboratory animals. Rabbits have been used extensively for basic research in drug and bacteria, toxicology, healing, tissue and organ culture, mycology, skin sensitivity, immunology, ophthalmology, oncology and reproductive biology [10]. The rabbit is believed to be a helpful model for comparative biology in humans, concerning sperm capacitation and the general reactivity of the female genital tract during the reproductive cycle [11].

In this study, we established for the first time a detailed investigation of the arterial, venous and lymphatic mapping of the uterus at early stages of pregnancy in the rabbit. The viability of this vessels were very important for the female reproduction and fetus maintenance. It was recognized in the present study that arteries were congested and demonstrated in varies directions in the mesometrial region.

Rabbit has uteroovarian arteries running within the mesometrium and giving secondary and tertiary vessels (radial arteries). These radial arteries give the premyometrial radial arteries enter the uterine wall supplies the myometrium. The uterus is drained by a venous system that parallels the arterial tree, with closely apposed arteries and veins [1]. The present study demonstrated that arteries of mesometrial and myometrial regions were characterized by well-developed elastic lamina which helps the vessel to bear changes in blood pressure during pregnancy.

Large increases in uteroplacental blood flow during gestation are essential for normal foetal growth and survival and occur in every mammalian species studied, including human beings [12]. During pregnancy, the passive (fully dilated) diameter of the human uterine artery is approximately doubled [13], with similar changes reported in rodents, sheep, pigs and guinea pigs [14,15]. The myometrial arterial supply that course between the outer and middle thirds of the myometrium and this zone is referred to as the ‘vascular zone’.

Because of their semicircular course, these arteries are referred to as ‘arcuate’ arteries [16]. Our observation revealed that myometrial arteries characterized by its wide lumen, thin muscular coat, and vascularized adventitia. Arteries and veins are arranged in close apposition in a number of species [17]. Our observation revealed that mesometrial and myometrial veins characterized by its thin wall encircled by a thick adventitia rich in collagen and elastic fibers. The uterus is an immunologically unique organ in that it must be able to protect itself effectively from invading pathogens, but at the same time is faced with periodic exposures to foreign cells and tissues, namely allogeneic spermatozoa and the fetal-placental unit. Even though the uterus seems to be an effective site of immunization for some antigens [18]. In rats, mice, and similar small mammals, there is only one main lymphatic plexus lying between the circular and longitudinal muscles. In addition, Endometrium possessed few or no lymphatics [19].

Our observation revealed that several thin- walled wide-lumened lymphatics were recorded within the mesometrium. Their wall is composed of an intimal tunic surrounded by a thick fibrous layer consisting of collagenous, elastic fibers and connective tissue cells. Some of these vessels demonstrate valves. The rat showed similar observation [20].

Myometrial lymphatics were composed of an intimal tunic surrounded by a fibrous layer of collagenous and elastic fibers. The valves of these vessels were mainly thin and consisted of a single cellular layer. The wall of myometrial

lymph vessels showed a positive reaction with PAS-AB combination. The lymphatic vessel in the endometrium appeared as wide irregular thin walled vessels similar observation obtained in the rat [21]. Rabbit arterial sinuses as arterial device affecting the pulse pressure and linear velocity of maternal blood flow and regulate optimal condition for nutritional exchange [22]. The present study revealed that Several arterioles, venules observed at the different layer of the uterus. in addition, spiral artery also observed with a wide lumen and spiral course. As well as, arterial sinuses characterized by wide irregular lumen, a single layer of smooth muscle and unevenly distributed elastic fibers.

Arteriovenous anastomosis demonstrated at a various layer of the uterus. Some of this vessels showed several glomus cells. Arteriovenous anastomoses are one of the key adaptations for thermoregulation [23]. The glomus cell observed at this study in the wall of arteries, arterioles, arteriovenous anastomosis, and spirally oriented arterioles. the glomus cells can be considered as a highly reactive special type of cell, which cause a reduction in the diameter of the lumen by their ability to swell or even complete obliteration of the vessel lumen [24-26].

The histological and histochemical investigation of the uterine vasculature in the rabbit revealed several blood vessels of special regulatory devices which indicate a unique function. These vessels were observed at different uterine parts.

Our observation revealed that there was fibrinoid deposition at some arteries similar observation reported in women [27,28]. In addition, Fibrinoid deposition and elastic fibers contents of vessels will increase at 7 days pregnancy of pregnancy. gestation sclerosis discused as a type of sclerosis developed due to circulatory demands increased [29]. However, gestational sclerosis considered as physiological processes as they are not uniform in nature, intensity and distribution [26].

The present study demonstrated some spirally oriented arterioles with extreme thickening of their wall by glomus cells in addition to arterioles with a narrow lumen. This constriction of arterioles increases

peripheral resistance to blood flow giving an important role in the regulation of systemic blood pressure. Similar observations were recorded in camel ovaries [26] and camel skin [30].

In our study, Various vessels of special structure detected at different parts of the uterus. The vessels of the special structure are supposed to possess an important regulatory function for the peripheral circulation. They exert an active function on the blood flow and pressure regulation. This function is attained either through the contraction of the smooth muscle fibers or by the presence of glomus cells which cause a reduction in the diameter of the lumen by their ability to swell [25]. Ultrastructure study revealed that numerous filopodia-like protrusions at the arteriole endothelium toward the lumen. The formation of filopodial structures described under inflammatory conditions and in response to the inflammatory mediators [31]. Mild but significant inflammatory activity is involved in the development of normal pregnancy, which might have important physiological roles [32,33]. The maternal inflammatory response is supposed to be modulated to allow the establishment and maintenance of a viable pregnancy.

Abundant caveolae of different sizes and different arrangement were observed in the endothelial cells of the capillary. The permeability of endothelial cells is usually described in electron microscope studies as related to the presence of caveolae [34]. With the advancement of pregnancy, endothelial cells of some vessels appeared swell to a plump and Similar observation obtained at early stages of human pregnancy [35].

Our data revealed that telocyte encircled the capillary with similar vacuoles in both. The telocyte discussed as play a role in juxta and paracrine signaling [36]. In addition, telocyte expressed estrogen and progesterone receptors and

act as a hormonal sensor [37,38]. However, it always needs to be noted the exceptional cases out of such rules, and cutaneous horns at the gross level should prompt consideration of biopsy for the definite diagnosis. Our case

#10 also seems to support such speculation.

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Evaluation of Rapid Diagnostic Tests for Typhoid Fever

,1 Jim Pruckler,1 William Bibb,1 Nguyen Thi My Thanh,2 Tran My Trinh,2

Nguyen Thi Minh,3 Sumathi Sivapalasingam,1 Amita Gupta,1 Phan Thu Phuong,4 Nguyen Tran Chinh,5 Nguyen Vinh Chau,5 Phung Dac Cam,4 and Eric D. Mintz1*

Foodborne and Diarrheal Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia,1 and Pasteur Institute2 and Hospital for Tropical Diseases,5 Ho Chi Minh City, Cai Lay Medical Center, Cai Lay,3 and National Institute of Hygiene and Epidemiology, Hanoi,4 Vietnam

Corresponding author. Sonja J. Olsen Mailing address: Foodborne and DiarrhealDisease Branch, Centers for Disease Control and Prevention.

ABSTRACT

Laboratory diagnosis of typhoid fever requires isolation and identification of Salmonella enterica serotype Typhi. In many areas where this disease is endemic, laboratory capability is limited. Recent advances in molecular immunology have led to the identification of sensitive and specific markers for typhoid fever and technology to manufacture practical and inexpensive kits for their rapid detection. We evaluated three commercial kits for serologic diagnosis of typhoid fever. Patients presenting with > 4 days of fever were enrolled at two hospitals in Southern Vietnam. Cases were patients with serotype Typhi isolated from blood samples, and controls were patients with other laboratory-confirmed illnesses. Serotype Typhi isolates were confirmed and tested for antimicrobial susceptibility at the Pasteur Institute in Ho Chi Minh City. The Widal test was run at the hospitals and the Pasteur Institute. Sera were shipped frozen to the Centers for Disease Control and Prevention and tested by using Multi-Test Dip-S-Ticks, TyphiDot, and TUBEX to detect immunoglobulin G (IgG), IgG and IgM, and IgM, respectively. Package insert protocol instructions were followed. We enrolled 59 patients and 21 controls. The sensitivity and specificity findings were as follows: 89 and 53% for Multi-Test Dip-S-Ticks, 79 and 89% for TyphiDot, 78 and 89% for TUBEX, and 64 and 76% for Widal testing in hospitals and 61% and 100% for Widal testing at the Pasteur Institute. For all assays, the sensitivity was highest in the second week of illness. The Widal test was insensitive and displayed interoperator variability. Two rapid kits, TyphiDot and TUBEX, demonstrated promising results.

Typhoid fever, caused by Salmonella enterica serotype Typhi, is a major cause of morbidity and mortality worldwide, causing an estimated 16.6 million new infections and 600,000 deaths each year (14). In Vietnam, typhoid fever is highly endemic, with the southern provinces most heavily affected. In a study conducted in Dong Thap Province in 1995 and 1996, the incidence of confirmed serotype Typhi infection was 198 per 100,000 for all ages (11).Isolation of serotype Typhi from blood, urine, or stool is the most reliable means of confirming an infection. However, this requires laboratory equipment and technical training that are beyond the means of most primary health care facilities in the developing world. Most serotype Typhi infections are diagnosed purely on clinical grounds and treated presumptively. As a result, the diagnosis may be delayed or missed while other febrile illnesses are considered, and patients without typhoid fever may receive unnecessary and inappropriate antimicrobial therapy. Emerging drug resistance among circulating serotype Typhi strains in Vietnam (6, 15) and elsewhere (16) has complicated the treatment of typhoid fever and heightened the need for rapid accurate diagnosis and the appropriate and selective use of antimicrobial agents to which the organism has thus far remained susceptible.

Serodiagnosis of typhoid fever has been attempted since the late 19th century when Widal and Sicard showed that the serum of patients with typhoid fever agglutinated typhoid bacilli (20). Unfortunately, neither the Widal test, which remains in widespread use in the developing world, nor any of the serodiagnostic tests that have since been developed has proven sufficiently sensitive, specific, and practical to be of value in areas where this disease is endemic (9). Recent advances in molecular immunology have led to the identification of potentially more sensitive and specific markers in the blood and urine of patients with typhoid fever and have enabled the manufacture of practical and inexpensive kits for their detection.

Here we report the results of an evaluation of three commercial serodiagnostic assays for diagnosis of acute serotype Typhi infection with specimens collected in southern Vietnam.

MATERIALS AND METHODS

Specimen collection. Specimens were collected from patients at two hospitals in Southern Vietnam: Cai Lay District Hospital (180 beds) in Tien Giang Province and the Hospital for Tropical Diseases (Cho Quan Hospital) (500 beds) in Ho Chi Minh City. Patients 3 years old who presented with 4 days of fever between October 2000 and April 2002 were eligible for enrollment. Patients who met the criteria were asked to give informed consent and answer a brief questionnaire about clinical signs and symptoms, antimicrobial treatment, and history of typhoid fever and vaccination. Participants gave 5 ml of blood (3 ml from children 3 to 5 years old) upon routine venipuncture for blood culture. Only patients with a laboratory-confirmed etiology of their fever were included in the analysis.

Blood samples were centrifuged, and the serum was divided into aliquots and stored at 20°C. In order to minimize the degradation of the antibodies in the serum, the specimens were frozen immediately and remained frozen until the time of testing. At routine intervals, personnel from the Pasteur Institute retrieved the isolates and serum specimens from the hospitals; serum was stored at 70°C. All isolates were confirmed at the Pasteur Institute, and serum was reevaluated by using the Widal test. Serum specimens from all patients with a laboratory-confirmed illness were batched and shipped on ice every

few months to the Centers for Disease Control and Prevention (CDC) in Atlanta, Ga., for further testing with the commercial assays. Patients with serotype Typhi isolated from blood were compared to patients with other laboratory-diagnosed pathogens by three commercial kits for rapid diagnosis of acute typhoid fever.

Laboratory analysis. (i) Blood culture. At Cai Lay Hospital, 5 ml of patient blood was added to blood culture medium (biphasic tryptic soy agar and brain heart infusion broth with SPS [0.6 mg/ml]) supplied by the Pasteur Institute. The blood culture bottle was then incubated at 37°C for 24 h before being tilted so that the liquid flowed over the solid medium. The broth was subcultured on blood agar after 1, 2, 3, and 7 days, and the solid medium was subcultured any time there was a colony visible on the slant. Isolates were Gram stained and identified by standard biochemical methods. Serotyping was performed by using agglutination with Salmonella O, H, and Vi antisera. If there was no growth after 10 days, the culture was considered negative. The Hospital for Tropical Diseases used the BACTEC system and surveyed the results after 5 days. If there was any growth, colonies were subcultured to blood agar and identified as described above.

  1. Confirmation and antimicrobial susceptibility testing of isolates at the Pasteur Institute. The identification of suspect serotype Typhi isolates was confirmed at the Pasteur Institute by standard biochemical tests and Salmonella serotyping. Antimicrobial susceptibility testing was done by using the Kirby-Bauer disk diffusion method. The following antimicrobial agents (zone size for resistance) were used: ampicillin ( 17 mm), tetracycline (19 mm), chloramphenicol ( 18 mm), ceftriaxone ( 21 mm), ciprofloxacin ( 21mm), ofloxacin (16 mm), norfloxacin ( 17 mm), nalidixic acid ( 19 mm), and gentamicin (15 mm).
  2. Laboratory confirmation of other pathogens. Confirmation of other pathogens was done as follows: blood smear for malaria, acid-fast bacilli (AFB) sputum smear for tuberculosis, blood or urine cultures for other bacterial pathogens, or serum immunoglobulin M (IgM) detection by antibody-capture enzyme immunosorbent assay (MAC EIA) for dengue. AFB smears and blood and urine cultures were done in the hospitals; sera were sent from Cai Lay Hospital to the Center for Preventive Medicine in Tien Giang province for dengue testing by using a MAC EIA kit produced by the Pasteur Institute (validated by comparison to an Omega, UK, commercial kit). The Hospital for Tropical Diseases did not test or refer specimens for dengue serology.
  3. Widal test. Widal testing was done by using the Sanofi qualitative agglutination test kits (Bio-Rad) by two different methods. In both methods, serum was serially diluted, starting at 1/10, in physiological saline and then further diluted 1/10 in suspensions containing serotype Typhi O and H antigens, separately. Cai Lay Hospital used the rapid centrifugation technique in which the tubes were centrifuged at 3,000 rpm for 5 min. The precipitate was resuspended by tapping the bottom of the tube; if agglutination was visible, the results were considered positive. The Hospital for Tropical Diseases and the Pasteur Institute used the classical technique with incubation in which the tubes were incubated in a 37°C water bath for 2 h for H suspensions and at room temperature overnight for O suspensions; if agglutination was visible, the results were considered positive.
  4. Rapid tests. Serum was evaluated by using the following three commercially available rapid diagnostic kits: Multi-Test Dip-S-Ticks (PANBIO INDX, Inc., Baltimore, Md.), TUBEX (IDL Biotech, Sollentuna, Sweden), and Typhi-Dot (Malaysian Biodiagnostic Research SDN BHD, Singapore, Malaysia).

Briefly, the Multi-Test Dip-S-Ticks tests for five pathogens, including Salmonella serotype Typhi. The test is in a dipstick format that detects anti-O, anti-H, anti-Vi, IgM, or IgG antibodies in patient serum, plasma, or heparinized whole blood. We evaluated the IgG kit only. The TyphiDot is a DOT enzyme immunoassay that detects either IgM or IgG antibodies against a specific antigen on the outer membrane protein of serotype Typhi. For specimens that are indeterminate (IgM negative and IgG positive), a confirmatory test, TyphiDot-M, is recommended by the manufacturer. Due to manufacturing problems with the TyphiDot-M, only the TyphiDot was used in this evaluation. These first two tests, the Multi-Test Dip-S-Ticks and the TyphiDot, are qualitative. The third test was the TUBEX, a semiquantitative test that uses polystyrene particle agglutination to detect IgM antibodies to the O9 antigen. Specimens were run according to the protocol listed on the packet inserts.

Ethical review. The study protocol was approved by the institutional review boards of the CDC and the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam.

Statistical analysis. Analyses were performed by using SPSS version 11.0.1 (SPSS, Inc., Chicago, Ill.). Medians were compared by using the median test for nonparametric data that calculates a chi-square statistic. For each assay, we calculated the sensitivity, specificity, and positive and negative predictive values.

Fleiss quadratic 95% confidence intervals were calculated by using Epi Info 6 (CDC, Atlanta, Ga.). The patient age was calculated by using a mid-year birth date and date of interview.

RESULTS

We enrolled 59 serotype Typhi cases and 20 controls with other laboratory-confirmed febrile illnesses. The control diagnoses were as follows: 7 subjects with dengue fever, 4 subjects with Escherichia coli cultured from blood, 1 subject with E. coli cultured from urine, 2 subjects with malaria (Plasmodium falciparum), 2 subjects with tuberculosis, 2 subjects with Klebsiella pneumoniae cultured from blood, and 2 subjects with S. enterica serotype Paratyphi A cultured from blood. Serum was missing from one case and one control (serotype Paratyphi A).

The demographic characteristics of the serotype Typhi patients and controls are listed in Table 1. In the Hospital for Tropical Diseases there was a slightly longer, but nonsignificant, time between fever onset and enrollment compared to Cai Lay Hospital (median number of days, 11 versus 8 [P 0.07]). Twenty-five patients recalled taking antibiotics after fever onset (10 of 15 cases versus 6 of 10 controls), however, 74 of 75 (99%) reported taking any medicine in the same week.

Most persons (54 of 79 [68%]) did not know if the medicine they took was an antibiotic or not. The dates of fever onset in patients with serotype Typhi ranged from January 2001 to March 2002, peaking in April through October (Fig. 1). None of the participants reported vaccination for typhoid fever; one patient and two controls reported having a history of typhoid fever.

A comparison of the three assays in presented in Table 2. A refrigerator is needed for storage of all of the kits but very little additional equipment is needed. The Multi-Test Dip-S-Ticks method requires a water bath, and the TyphiDot requires a calibrated pipette. At approximately $10 per test, the Multi-Test Dip-S-Ticks is the most expensive, followed by TUBEX at approximately $4 per test and the TyphiDot at approximately $1 per test. The sensitivity, specificity, and predictive values are shown in Table 3. Although the sensitivity of the Multi-Test Dip-S-Ticks was quite high (89%), it had low specificity (50%). The Typhi-Dot and the TUBEX both had high sensitivities (79 and 78%, respectively) and specificities (89 and 94%, respectively). The Widal test was the least sensitive of the assays, and the results varied by place performed (64% sensitive and 76% specific in the hospitals and 61% sensitive and 100% specific at the Pasteur Institute).

We examined the sensitivity of the assays by week after fever onset (Fig. 2). These were not tests performed on specimens from the same patient at weeks 1, 2, and 3 but rather the merged results of single samples collected at a single time point from each patient. The sensitivity of all tests was highest for serum specimens obtained during the second week of illness. The numbers were too small to do a meaningful evaluation of the specificity of the assays by the week after fever onset (data not shown).

There were no discernible differences between the 10 controls with a false-positive result on one of the three commercial assays and the 9 other controls, although the numbers were small. The controls with a false-positive result were slightly younger (mean age, 31 versus 37 years [P 0.6]) and less likely to be women (50% versus 55%, P 1.0); these differences are not statistically significant. Nine of the ten controls were positive on the Multi-Test Dip-S-Ticks, and four of these had dengue fever. One control with

FIG. 1. Time of fever onset in patients with Salmonella Typhi by hospital. a false-positive result reported having a history of typhoid fever.

Antibiotic susceptibility. A total of 58 of the 59 serotype Typhi isolates were available for testing. Of the 58 isolates tested, 14 (24%) were pansensitive. All of the remaining 44 isolates were resistant to nalidixic acid; 33 were also resistant to chloramphenicol and tetracycline, and 29 of these were also resistant to ampicillin. Only two isolates were also resistant to cefotaxime, one of which was also resistant to norfloxacin. Among the 57 cases with serologic results, there was no statistically significant difference in the typhoid assay results by sensitivity as defined by pansensitivity or resistance to at least one antimicrobial agent.

DISCUSSION

We evaluated three commercial rapid diagnostic kits for serotype Typhi with sera collected from patients with acute febrile illness of 4 days’ duration at two hospitals in Vietnam. Overall, the TyphiDot and TUBEX, both of which detect IgM antibodies, demonstrated the most promising results. However, the performance of the TyphiDot assay may not have been optimized since we were unable to run our 15 indeterminate specimens (seven cases and eight controls) on the TyphiDot-M assay for confirmation. The Multi-Test Dip-S-Ticks, which only detects IgG antibodies, had poor specificity. The Multi-Test Dip-S-Ticks to detect IgM was not evaluated. The Widal test had low sensitivity and was highly operator dependent. Since the other three assays were performed at only one laboratory, we could not assess their operator variability.

The hospitals participating in this evaluation were quite different and had the potential to enroll patients at different stages of illness. Cai Lay is a small, rural hospital with minimal laboratory capability, whereas the Hospital for Tropical Diseases is a large, urban referral hospital with good laboratory capability. Despite this, there were few differences in the patients enrolled. Patients at the Hospital for Tropical Diseases were slightly older, more likely to be

women, and were seen a median of 2 days later in their illness.

In our evaluation, the sensitivity of the TyphiDot was high beginning in the first week of illness onset. Presumably, this is because the TyphiDot relies more heavily on IgM results that occur earlier in the course of the illness, whereas IgG rises later; however, we did not see this effect with the TUBEX, which also detects IgM antibodies. In the Widal test, the O and H agglutinins usually appear around day 8 and days 10 to 12, respectively. The Multi-Test Dip-S-Ticks was the most costly assay, presumably because the dipstick measures antibodies to five different pathogens. Although all three assays were relatively easy to use, the TUBEX was the simplest. A limitation of the TUBEX test, which uses a colorimetric reaction, is the potential for difficulty in interpreting the results of hemolyzed samples. Another concern is that the TUBEX may produce a false positive in persons with recent S. enterica serotype Enteritidis infection and result in inappropriate antibiotic treatment (13). Ideally, the Widal test should be run on both acute- and convalescent-phase sera to detect an increase in the agglutination titer. However, to inform treatment decisions before convalescent samples can be obtained, it is common for a single acute-phase serum sample to be run. The results from a single sample are difficult to interpret because high background rates of circulating antibodies to serotype Typhi or other Salmonella serotypes may produce a false-positive result. In Vietnam, an area of high endemicity, a single Widal test can lead to many false-positive and false-negative results (17). Operator variability also contributes to unreliable results, as evidenced in the present study. Each of these three commercial kits has been previously evaluated but, to our knowledge, this is the first time they have all been included together in the same evaluation. In a recent evaluation of the Multi-Test Dip-S-Ticks in Singapore, the sensitivity varied greatly depending upon the case definition (5). Among clinical and blood culture-positive cases it was 51%, and among blood culture-positive patients only it was 78%. The specificity among control patients who had a clinical diagnosis of typhoid but were culture negative, had other laboratory diagnoses, or had pyrexia of unknown origin was 81%. The TyphiDot was evaluated in Pakistan and Singapore by using a variety of case definitions (2, 5). The sensitivity ranged from 59 to 93% for the TyphiDot and 73 to 84% with the addition of the TyphiDot-M. Specificity was consistently higher when TyphiDot-M was used; 89% compared to 77% or lower with only the Typhi-Dot. An evaluation of the TyphiDot in India was 100% sensitive and 80% specific compared to a blood culture “gold standard” (8). In an early evaluation, the TUBEX demonstrated 100% sensitivity and specificity (10). However, this was before the kit was commercially available. In Vietnam, it was 87% sensitive among blood culture positive patients and 76% sensitive among hospitalized patients with fever (7).

One limitation in the previous and current evaluations is that blood culture-confirmed cases were used as the gold standard. Since blood culture is less sensitive than bone marrow culture for diagnosing typhoid fever (4), the results should be interpreted with caution. It is possible that the rapid diagnostic tests are more sensitive than blood culture. If so, a result that appears to be a false-positive test compared to a blood culture may in fact be a true positive. This hypothesis requires further evaluation. Alternatively, a false-positive may be the result of past infection with serotype Typhi or another nontyphoidal Salmonella serotype that shares common antigens. Researchers continue to search for the ideal rapid test to diagnose acute typhoid fever. Several urine assays have been developed (1, 3, 12, 18), although none has proved optimal. In the field, there is a definite advantage to collecting urine instead of blood; urine collection is simple, less invasive than venipuncture, and requires less training and equipment. In addition, some antigens may be excreted in higher concentration in the urine. With the recent sequencing of the entire serotype Typhi genome, it may now be possible to identify other antigens, such as fimbrial antigens, that may produce an antibody response specific to serotype Typhi (19). More sophisticated molecular techniques for diagnosis, such as PCR, are also being explored. However, their use in developing countries will most likely be limited.

FIG. 2. Sensitivity by week after fever onset.

ACKNOWLEDGMENTS

We thank Tran Thi My Trinh and Bui Thu Hien of the Pasteur Institute in Ho Chi Minh for their assistance with laboratory testing.

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Early Diagnosis of Acute Myocardial Infarction (Heart Attack): Immunoassay for Serum Troponin Marker

Tunji Akande

Department Of Medical Laboratory Sciences Bingham University Karu, Nigeri a

All Correspondences to:Tunji Akande Department Of Medical Laboratory Sciences Bingham University Karu, Nigeri a

ABSTRACT

Background: Heart disease is a common and delibilitating condition that affects millions of patients globally. Measurement of troponin assays has been a tremendous boon to clinical diagnosis. Objective: To evaluate the clinical utility and the biochemical characteristics of serum troponin as an early and accurate cardiac marker for diagnosis of acute myocardial infarction. Method: a review of serum troponin as choice biomarker for diagnosis of Acute Myocardial Infarction (AMI). Result: Troponin T allows for early and late diagnosis of AMI. Troponin is only found in the myocardium in adults making it extremely specific for cardiac disease. Troponin I or T is replacing total CK and CK-MB detection as the marker of choice for cardiac dysfunction as seen in AMI. Conclusion: There is value in detection myocardial damage early after its onset. Clinical laboratories should move rapidly to implement the new cardiac troponin standard for the early and accurate diagnosis of AMI.

Key Words: AMI, Troponin, Cardiac Marker, Early diagnosis.

INTRODUCTION

Cardiovascular diseases are the leading cause of death globally. Cardiovascular disease includes coronary artery disease (CAD) such as angina and myocardial infarction commonly known as a heart attack The World Health Organization has established three criteria for the diagnosis of acute myocardial infarction (AMI) including a history of chest pain, evolutionary changes on the ECG, and elevations of serial cardiac markers [2] however it was rare for a diagnosis of AMI to be made in the absence of biochemical evidence of myocardial injury. [3] .Numerous biomarkers have been monitored to assess myocardial injury. Most are myocardial proteins and differ in their location within the myocyte, release kinetics after damage and clearance from the circulation [4] . The European Society of Cardiology/American College of Cardiology (ESC/ACC) has published consensus guidelines for the diagnosis of AMI [5,6 7]. A Corner stone of the consensus document is predicated on cardiac biomarker especially cTnI or CTnT [8,9]. Because of its dire consequence, great efforts have been made to determine the best tools for the early and accurate diagnosis of AMI. Therefore this article aimed at assessing some cardiac markers that could provide an early and accurate diagnosis for acute myocardial infarction

Myocardial infarction (MI)

Acute myocardial infarction is one of the largest killers globally. Its diagnosis is usually made on the clinical presentation and electrocardiagraphic (ECG) findings and confirmed by the characteristic changes in plasma enzyme activities or troponin levels Symptoms and ECG abnormalities however may be absent or non specific. Thus, the diagnosis of an acute MI has increasingly depended upon evaluation of Cardiac biomarkers particularly troponins [10,11]. Acute myocardial infarction is defined as an imbalance between myocardial oxygen supply and demand resulting in injury and the eventual death of myocyte. When the blood supply to the heart is interrupted, gross necrosis of myocardium results. Such extensive necrosis is most often associated with a thrombotic occlusion superimposed on coronary atherosclerosis. Myocardial infarction is seen as a spectrum of disease ranging from angina pectoris through to acute myocardial infarction; this stratification is based upon cardiac markers reflecting ischemic damage. [12,13]. Myocardial ischemia and infarction are usually segmental diseases. The American College of Cardiology and European Society of cardiology have defined acute myocardial infarction as a typical rise and fall of biochemical marker, for example plasma creatine kinase isoenzyme MB (CK-MB) or troponin, with at least one of the following: Ischemic symptoms, new pathological Q waves, on electro cardiogram (ECG), ischemic ECG changes (ST.depression or elevation) and coronary artery intervention [14]

The patients previously classified as having unstable angina or minor myocardial injury are now reclassified as having non-ST segment elevation myocardial infarction (NSTEMI), therefore myocardial infarction is now regarded as a spectrum of disease ranging from angina pectoris through to acute myocardial infarction. Acute myocardial infarction and NSTEMI have a common pathophysiological pathway (15)

Cardiac markers

Cardiac markers are biomarkers measured to evaluate heart function. They are clinical laboratory tests useful for detecting AMI or minor myocardial injury. They are most useful when patients have non-diagnostic ECG tracings.

Most efforts to date have been placed on the development of an ideal cardiac marker for the early and accurate diagnosis of AMI. Many factors must be considered in the selection of the most clinically diagnostic effective, cost effective and cost efficient laboratory tests for patients with chest pain which include

  1. the time that has elapsed after onset of chest pain;
  2. Any concomitant diseases;
  3. The possibility of skeletal muscle injury
  4. The ease of measurement and turn around time for results.
  5. Assay specificity, sensitivity and interferences. (17, 18)

Serum enzymes such as aspartate aminotransferase (AST), Creatine Kinase (CK), Lactate dehydrogenase (LD) and their iso enzymes have all been used as biomarkers of AMI. Decades ago they were thought to be sensitive indicators of myocardial necrosis and could be used to correlate with other signs and symptoms such as abnormalities in ECG pattern [19,20] All three of these enzymes, however are found in other tissues as well, making them less specific to myocardial damage. AST for example is also found in skeletal muscle, liver parenchyma cells, and erythrocytes, while CK is found in skeletal muscles, brain tissue and embryonic and malignant tissue. LD is the least specific of these three enzymes in that it is found in virtually all tissues and is associated with damage to liver, skeletal muscle, cardiac muscle, erythrocytes renal cells and many other tissues as well as ovarian and testicular tumors. [21] Historically myocardial infarction was detected by looking for the CK Iso enzyme CK-MB. This marker is released into circulation from necrotic heart muscle. As the heart muscle becomes damaged this CK Isoenzyme is released into the blood stream and may be detected.

Cardiac proteins

Several proteins may be monitored in suspected cases of AMI to give significant diagnostic information myoglobin an oxygen-binding protein is rapidly released from striated muscles (both skeletal and cardiac muscle0 when damaged. However because of its small size, myoglobin is rapidly cleared by the kidney, making it an unreliable long-term marker of cardiac damage. [22] Myoglobin is significantly more sensitive them CK and CK-MB activities during the first hours after chest pain onset. It starts to rise within 1-4hrs and is detectable in essentially all AMI patients between 6 and 9 hours from chest pain onset returning to baseline levels within 18-24 hours. If myoglobin concentration remains within the reference range 8 hours after onset of chest pain, AMI can essentially be ruled out [23] Myoglobin is released into circulation with any damage to muscle tissue, including myocardial necrosis. Since skeletal muscle contains myoglobin, this measurement is quite non-specific for myocardial infarctions. The benefit lays in the fact that a detectable increase is seen only 30 minutes after injury occurs, unlike

troponin and creatine Kinase which can take 3-4 hours [24,25,26] Troponin I and troponin T are normal proteins important in the contractile apparatus of the cardiac myocyte. They are released into the circulation about 3-4 hours after myocardial infarction and are still detectable for 10 days afterwards. The long half-life allows for the late diagnosis of myocardial infarction [27,28]. All of the initial cardiac markers were enzymes, so the earliest techniques measured the catalytic activity of the marker. Immunoassay techniques measure the mass of a marker and they are the predominant methodology used in clinical laboratory practice today. Analytically, they offer lower limits of detection, improved precision, and faster assay time on both highly automated central laboratory Platforms and Point of Care Testing (POCT). [29,30] Over the past 15 years numerous manufacturers have described the development of monoclonal anthody –based diagnostic immunoassays for the measurement of troponins (CTnI and CTnT) in serum. Troponin assays has been a tremendous boon to clinical diagnosis. Troponins released from heart muscle remains in the blood stream for up to 10-14 days after onset of AMI, making them preferred marker for detection of an AMI [31]. Troponins as cardiac markers appear to have many advantages primarily due to their quick release following heart muscle damage.

Recommendations

The National Academy of clinical Biochemistry recommends that two biochemical markers be used for routine diagnosis of AMI: an early marker that is reliably increased within 6 hours after onset of symptoms and a definitive marker that remains increased after 6-9 hours but has high sensitivity and specificity for myocardial injury and remains abnormal for several days. [33]. To assist in differentiating patients with AMI from non – AMI, the European Society of cardiology/American College of cardiology (ESC/ACC) has published consensus guidelines for redefinition of AMI [34]. A cornerstone of this is predicated on cardiac biomarkers, especially CTnI or CTnT. The cardiology recommendations imply that for clinical laboratories that cannot move as rapidly as others to implement the new cardiac troponin standard, CK-MB (Preferable mass should be used. Although it is suggested that CK-MB be used together with cardiac troponin for assisting in timing the onset of myocardial injury, infarct sizing or determination of reinfarction at present there is no strong evidence to support dual testing for cTn and CK-MB.

Conclusion

The diagnosis of acute myocardial infarction can be difficult, but it is important to make prompt diagnosis as thrombolysis needs to be given early. The diagnosis of an acute myocardial infarction has increasingly depended upon evaluation of cardiac biomarker particularly troponins. Troponins are more specific cardiac marker than the CK. Also, plasma troponin stays elevated for longer than CK after an infarct. Troponins are therefore useful for the late diagnosis of myocardial infarction. Clinical Laboratories should move rapidly to implement the new cardiac troponin standard for the early and accurate diagnosis of myocardial infarction.

Table I. The time sequence of changes in plasma cardiac markers after myocardial infarction

Cardiac marker Starts to rise Time after Infarction Duration of
(hours) for peak rise (hours) rise (days)
CK (total) 4-6 24-48 3-5
AST 6-8 24-48 4-6
LDH/HBD 12-24 48-72 7-12
MYOGLOBIN 2-4 12-24 2-4
TROPONIN 4-6 12-24 7-10

CK= Creatine kinase; AST= aspartate transaminase

LDH= lactate dehydrogenase; HBD= Hydroxybutyrate dehydrogenase

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Local Blend of Plant Improves Blood Parameters In Anaemia-induced Albino Wistar Rats.

Kevin E. Aghatise

Department of Medical Laboratory Science, Igbinedion University Okada

Ivie Osula and Anslem O. Ajugwo

Department of Medical Laboratory Science, Madonna University Nigeria

All Correspondences to: Kevin E. Aghatise, Department of Medical Laboratory Science, Igbinedion University Okada

ABSTRACT

Anaemia constitutes a serious health problem in many tropical countries including Nigeria because of the prevalence of malaria and other parasitic infections which possibly leads to decrease of hemoglobin. Telfairia occidentalis (fluted pumpkin), Vernonia amygdalina (bitter leaf) and Gongrone malatifolium (utazi leaf) have been reported to be very good in building the constituents of the blood and also replacing them. This study was designed to investigate the effect of fluted pumpkin, bitter leaf and utazi leaf extracts on haematolgical parameters of anaemia induced albino wistar rats. Fifteen Wistar albino rats with weight range from (122-190g) obtained from the animal house of Madonna University, Elele Nigeria were separated into three groups of five rats each for the study. Rats in group B and C were injected 400mg/kg 2,4 dinitrophenylhydrazine for five days to induce anaemia, while rats in group A were fed with grower’s mash and water throughout the experiment. All analysis were done using the standard methods. The result of this study show that, there was a significant (p<0.05) difference when the PCV was compared among the Groups, the control groups had 41.0+2.65% anemia induced group had 25.3+6.8% and the treatment group recorded 33.6+4.9%. The anemia induce group recorded a reduced PCV but was raised in the treatment group and this was statistically significant (p<0.05). There was also a significant difference when the Haemoglobin concentration was compared among the groups, the control group had 14.1+0.7g/dl, anaemia-induced group has 8.3+1.9g/dl and the treatment group had 10.+2.15g/dl. There was no significant difference (p>0.05) in all other parameters estimated. The result of this research indicate that aqueous leaf extract of fluted pumpkin, bitter leaf and utazi leaf could elevate the packed cell volume, red blood cells and hemoglobin concentration in anaemia-induced rats. It can be concluded that aqueous leaf extract of the combination of fluted pumpkin, bitter leaf and utazi leaf are a potential blood booster.

Keywords: anaemia-induced, blood booster, haematological, dinitrophenylhydrazine.

INTRODUCTION

The use of herbal products for medicinal benefits has an important role in nearly every culture on earth. Herbal medicine was practiced by ancient people of Africa, Asia, Europe and the Americas (Abosi and Raseroka, 2003). Over 50% of all modern clinical drugs are of natural product origin and natural products play an important role in drug development programs of the pharmaceutical industry. The consumption of a variety of local herbs and vegetables by man is believed to contribute significantly to the improvement of human health, in terms of prevention, or cure of disease because plants have long served as a useful and rational source of therapeutic agents (babalolaet al., 2003). Regular consumption of plant foods are associated with numerous health benefits rooted in their various physiological effects as a result of their physiochemical and nutritional constituent (Hunter and Fletcher, 2002).

Green leafy vegetables are particularly important in

promoting health because of their rich sources of nutrients (Gupta and Prakash, 2009). Telfairia occidentalis (fluted pumpkin) is a common tropical green leafy vegetables native to many African countries especially Eastern Nigeria (Burkett, 1968). It thrives in humid climate and well drained soils and is usually cultivated in garden and family farms around homes. Telfairia occidentalis is traditionally used by an estimated 30 to 35 million people indigenous people in Nigeria, including the Efik, Ibibio, and Urhobo (Akoroda, 1990). However, it is predominantly used by the Igbo tribe, who continue to cultivate it for food sources and traditional medicines (Okoli and Mgbeogu, 1983). A reoccurring subject in the Igbo’s folklore, the fluted gourd is noted to have healing properties and was used as a blood tonic, to be administered to the weak or ill (Akoroda, 1990). Bitter leaf is a medicinal plant of the family Asterceae. It is a small perennial shrub that grows in tropical Africa. Vernonia amygdalina is commonly called bitter leaf because of its bitter taste and it can be propagated either by cutting or seedling. Today, the plant is widely known throughout the continent and nearly 85% of Nigerians cultivates the plant due to its nutritional and medicinal values. It is locally called ‘ewuro; in Yoruba land (Abosi and Raseroka, 2003). The leaves of the plant may be consumed either as a vegetable (macerated leaves in soup) or aqueous extract as tonic for the treatment of various illnesses (Abosi and Reseroka, 2003).

Gongrone malatifolium also called utazi belongs to class of medicinal plants that are beneficial in preventing and treating certain disease and ailments that are detrimental to human health. Gongrone malatifolium leaf, which can be chewed infused or used for cooking is mainly used in the western part of America for nutritional and medicinal reason. Pharmacological studies suggest that utazi has both analgesic, antimicrobial, antibacterial, anti ulcer and antioxidant properties (Eguyoni et al., 2009). Utazi plant is highly medicinal in nature, which suggest why its health benefits cannot be overemphasized. Researchers agree that the fundamental ingredients used for medical purposes are stored in the various parts of the utazi plants such as leaves, fruits, roots etc (Eguyoni et al., 2009). The vital medicinal ingredients stored in the various parts of the utazi leaf can be extracted either through blending the fresh leaves, chewing the utazi seed, leaves or fruits, infusing either the dry or fresh utazi leaves and decoction. However these various methods of preparing and using utazi leaves for medical purpose mainly depends on the part of utazi plants where the active medicinal ingredient are present (Eguyoni et al., 2009).

Anaemia is decrease in the amount of red blood cells (RBCs) or hemoglobin in the blood. It can also be defined as a lowered ability of the blood to carry oxygen (Rodak and Bernadtte, 2007). When anaemia sets in slowly, the symptoms are often vague and may include: feeling tired, weakness, shortness of breath or a poor ability to exercise. Anaemia that comes on quickly often has greater symptoms, which may include; confusion, feeling like one is going to pass out, loss of consciousness, or increased thirst. Anaemia must be significant before a person becomes noticeably pale (Rodak and Bernadette, 2007).Use of plants has long been known to be used as local remedy for treatment of anaemia especially pumpkin leaf which is used in villages to treat anaemia especially when quality drugs cannot be accessed. This work was carried out to determine the effects of combination of herbal plants in anaemia-induced condition using haematological parameters.

Materials and Methods

Animal Model/Experimental design

Fifteen (15) Wistar strain albino rats with weight range of

(122 – 190g) obtained from the animal house, Madonna University Elele, Nigeria were used for the study. The rats were housed in wire meshed cage under standard conditions (temperature 25 – 29°C, 12 hours light and 12 hours darkness cycles) and fed with standard rat pelleted diet and water. They were housed in 3 meshed cages containing five

  1. rats in each cage. The rats were made to acclimatize for 1 week before the experiment began. They were allowed to feed on standard feed and water freely throughout the period the experiment lasted.

Ethical Approval

The research was approved by the ethical committee of the institution. The standard, rules and regulations of use of animal for research purposes was strictly adhered to as approved by the committee.

Order of Placement

Group1: The rats in this group served as control and were

administered with feed and water only. Group2: The rats in this group served as test and were administered with feed, water and 1 ml of phenyl hydrazine for five days. Group3: The rats in this group served as treatment group and were administered with feed, water, 1 ml of phenyl hydrazine for five days and subsequently treated with 0.5 ml of a blend of the local plants for another five days. Preparation and Administration of the plant blend

Large quantities of fresh leaves of Telfairia occidentalis (fluted pumpkin), Vernonia amygdalina (bitter leaf) and Gongrone malatifolium (utazi leaf) were purchased from the Central Market Elele. Identification and authentication of the plants was done by Mr Eze of the Faculty of Science. The leaves were washed and dried air-dried for eight days until there was no sign of moisture and grinded into powder. The powder was dissolved in 100 ml of deionized water and administered to the treatment group 0.5 ml in the morning and evening for 5 days.

Sample Collection

2ml of blood sample was collected by ocular puncture from each of the animal model using capillary tube and was dispensed into commercially prepared concentrations of ethylene diamine tetra acetic acid containers.

Sample Analysis

Blood samples collected were analyzed within six hours of collection for Hb, PCV, RBC count, Total WBC and differential analysis using standard manual methods (Cheesebrough, 2000)

RESULTS

Mean±S.D of hematological parameters of the control group (A), test group (B) and treatment group ©

PARAMETERS GROUP A GROUP B GROUP C P-VALUE
PCV (%) 41.0+2.65 25.3+6.8 33.6+4.9 P<0.05
HB (g/dl) 14.1+0.7 8.3+1.9 10.5+2.15 P<0.05
RBC (X1012/l) 6.0+0.56 4.4+0.62 5.2+1.0 p>0.05
TWBC X109/1) 9.3+1.9 13.7+5.4 11.7+2.11 p>0.05
Platelet (x109/1) 272+24.0 261+39.5 302+39.2 p>0.05
NEUTROPHIL (%) 56.7+3.51 63.7+6.11 55.7+5.86 p>0.05
LYMPHOCYTE (%) 34+4.0 29.7+7.23 33.7+11.06 p>0.05
EOSINOPHIL (%) 6.0+1.0 4.0+1.0 4.3+1.53 p>0.05
MONOCYTE (%) 3.3+1.15 3.3+1.52 3.3+1.15 p>0.05

20

 

DISCUSSION

The consumption of a variety of local herbs and vegetables by man is believed to contribute significantly to the improvement of human health, in terms of prevention, and or cure of diseases because plants have long served as a useful and rational sources of therapeutic agents (Babalola et al., 2003). Regular consumption of plant foods are associated with numerous health benefits rooted in their various physiological effects as a result of their physiological and nutritional constituents (Hunter and Fletcher, 2002).

From the research work there was a significant (p<0.05) difference when the PCV was compared among the groups, the control group has 41.0±2.65%, anaemia induced group has 25.3±6.8% and the treatment group recorded 33.6±4.9%. The anaemia induced group recorded a reduced PCV but was raised in the treatment group and this was statistically significant (p<0.05). This definitely is attributable to the local blend of plants. Previous work have shown that pumpkin is capable of improving haematological parameters (Alada,2000; Adias et al., 2013).

There was also a significant difference when the Hb was compared among the group, the control group had 14.1±0.7g/dl, anaemic induced group had 8.3±1.9g/dl and the treatment group had 10.5±2.15g/dl, the anaemic group recorded a reduction 8.3+1.9/dl but was elevated in the group given the extract. The reason for the significant increase in these haematological parameters could be related to the chemical composition of the leaves used in this research work. The chemical composition of Telfairia occidentals includes protein, fat, carbohydrates, calcium, iron, vitamin A, thiamine and riboflavin(Tindal, 1968; Adisa et al., 2014). Most of these constituents have positive impact on blood production (Akube, 1980). For instance, iron is a well established haemopoetic factor and deficiency of if produces anaemia (Ganong, 1997). There was no significant (p>0.05) difference in the RBC, the control group had 6.0±0.56×1012/1, the anaemic group had 4.4±062×1012/1, while the treatment group had 5.2±1.0×1012/l. The extract increased the RBC count of the anaemia-induced group from 4.4+062×1012/1 to 5.2+1.0×1012/1 but this was not significant. For TWBC there was no significant (p>0.05) difference when the test groups B (13.7±5.4×109/1) was compared to the control group A (9.3±1×109/1) although the TWBC was increase in the anaemic group (13.7±5.4109/1) and reduced in the treatment group (11.7±2.11×109/1).

There was also no significant (p>0.05) difference in platelets count when the groups B (261±39.5×109/1) and C (302±39.2×109/1) was compared with control group

(272±24.0×109/1) although the platelets count decreased in the anaemic group and increased in the treatment group but this was not significant (p>0.05). No significant difference was seen in all the differential count parameters. In conclusion, the combination of the extract of Telfairia occidentalis (fluted pumpkin), Vernonia amygdalina (bitter leaf) and Gongrone malatifolium (utazi leaf) have been shown to improve blood parameters (PCV and haemoglobin concentration). This extract can be used in management of anaemia especially when the conventional blood builders are not readily available.

Conflict of interest

The Authors declare that we have no conflict of interest.

REFERENCES

  • Abosi, A. O. and Raserok, B. H. ( 2003) “In vivo antimalarial activity of Vernonia amygdalina” British Journal of Biomedical Science. 8 (60) 89 – 91.
  • Adias, T. C., Ajugwo, A. O., Erhabor, T. and Nyenke, C. U. (2013). “Effect of Pumpkin Extract (Telfairia occidentalis) on Routine Haematological Parameters in Acetone-Induced Oxidative Stress Albino Rats.” American Journal of Food Science and Technology 1 (4): 67 – 69. doi: 10.12691/ajfst-1-4-1.
  • Adisa, W. A., Okhiai, O., Bankole, J. K., Iyamu, O. A. and Aigbe, O. (2014). Testicular damage in Telfairia occidentalis extract treated Wistar rats” American Journal of Medical and Biological Research. 2 (2): 37 – 45.
  • Akoroda, M. O. (1990) “Ethnobotany of Telfairia occidentalis among Igbos of Nigeria” Economic Botany. 1 (230): 29 – 39.
  • Akube, A. (1980). Chemical composition of Telfairia occidentalis. Plantamedia 38 33 – 43.
  • Alada, A. R. (2000). The haematological effect of t diet preparation” African Journal of Biomedical Research. 3 (3): 185 – 186.
  • Babalola, O. O., Anetor, J. I., and Adeniyi, F. A. (2001) “Amelioration of carbon tetrachloride induced hepatoxicity by terpenoid extract from leaves of Vernonia amygdalina” African Journal Medicine Science. 2 (30): 91 – 93.
  • Burkett, H. M. (1968). The results of plants of West Africa. Vol 1. Royal Botanic Garden Kews, England. 603 – 604.
  • Cheesebrough, M. (2000). Haematological tests. In: District laboratory practice in tropical countries. Part 2 Cambridge University Press U.K. 297.
  • Eguyoni, A., Moody, J. O. and Eletu, O. M. (2009). “Anti-sickling activities of two ethnomedicinal plant recipes used for the management of sickle cell anaemia in Ibadan, Nigeria” African Journal of Biotechnology. 8 (1): 20 – 25.
  • Ganong, W. F. (1997). A review of medical physiology. Appleton and Large. 496
  • Gupta, S. and Prakash, J. (2009).”Studies on Indian green leafy vegetables for their antioxidant activities” Plant Foods Human Nutrition. 64 (1): 39 – 45.
  • Hunter, K. J. and Fletcher, J. M. (2002) “The antioxidant activity and composition of fresh, jarred, frozen and canned vegetables” Innovative Food Science and Emergency Technology. 3 (90): 34 – 37.
  • Okoli, B. E. and Mgbeogu, C. M. (1983).”Fluted pumpkin: West African Vegetable crop” School of Biological sciences, University of Port Harcourt. 37 (2): 145 – 149.
  • Rodak, A.and Bernadette, F. (2007). “Hematology: Clinical Principles and applications (3rd edition). Philadelphia
  • Tindal, H. D. (1968). Commercial vegetable growing. Oxford Press, London. 69

 




Assessment of Antibiotic Susceptibility Pattern on Staphylococcus aureus isolated from Suya (Roasted Meat) and Nunu (Milk) Sold in Jos Metropolis

Assessment of Antibiotic Susceptibility Pattern on Staphylococcus aureus isolated from Suya (Roasted Meat) and Nunu (Milk) Sold in Jos Metropolis

Ejinaka, OR; ; Lote-Nwaru IE; Agbalaka P.I; Ajik, H.

Federal School of Medical Laboratory Science, Jos-Nigeria

Jwanse, R.I

Health and Development Support Programme (Hands), Jos, Nigeria

All correspondence to: Obeta M.U. E-mail: uchejesoobeta@gmail.com;

ABSTRACT

The assessment of antibiotic susceptibility pattern on Staphylococcus aureus isolated from Suya (Roasted Meat) and Nunu (Milk) sold in Jos was carried out in Jos Metropolis. This study was aimed at the occurrence of staphylococcus aureus in suya and nunu and its sensitivity to antibiotics. A total of 15 Suya and 15 Nunu sample were collected, the sample were cultured and identified by routine bacteriological method. Prevalence of S.aureus in Nunu was 40% and Suya was 33.3%. The susceptibility profile of staphylococcus aureus isolates from Suya (roasted meat) on an antimicrobial agents revealed a high susceptibility to Gentamycin (80%), Levofloxacin (80%), Rifampin (80%) Norfloxacin (80%) and had high resistance to Chloramphericol (80%), Ciprofloxacin (80%), Amoxil (80%), Ampiclox (80%) and Erythromycin (60%). The susceptibility profile of staphylococcus aureus isolates from Nunu (milk) on an antimicrobial agents revealed a high susceptibility to Gentamycin (80%), Levofloxacin (83.3%), Norfloxacin (83.3%), Erythromycin and Ciprofloxacin (66.7%) respectively, Rifampin and Levofloxacin 50% respectively and had high resistance to Chloramphericol, Amoxil and Ampiclox (66.7%) respectively; Rifampin and Levofloxacin 50% respectively. The S. aureus was present in Suya (Roasted Meat) and Nunu

(Milk) sold in Jos and resistant to some commonly used antibiotics in Jos.

Keywords: Staphylococcus aureus, Antibiotic Susceptibility, Suya, Nunu, Jos.

INTRODUCTION
Staphylococcus aureus is one of the major food borne abdominal cramping, with or without diarrhea. The disease is usually self-limiting and typically resolves within 24 – 48h after onset. Occasionally it can be severe enough to pathogens, frequently causing disease globally as a warrant hospitalization, particularly when infants, elderly result of food ingestion contaminated with or debilitated people are concerned (Argud´ın et al., 2012;
staphylococcal endotoxin (Mendoza, et al., 2010). S.
Mendoza et al, 2013).
S. aureus can cause wide spectrum of infections, ranging from superficial skin infections to severe, and potentially fatal, Jhalka et al., (2014) suggests the contributing factors for invasive disease due to combination of toxin-mediated the low incidence of SFD to include misdiagnosis, virulence, invasiveness, and antibiotic resistance. An improper sample collection in laboratory during important impediment to the control of the Staphylococcus examination, lack of seeking medical attention by the aureus infection is the tendency to gain resistance to almost affected persons, and lack of routine surveillance of all classes of antimicrobial agent (Jhalka et al., 2014).
clinical for S. aureus or its enterotoxins and unavailability
Staphylococcus aureus does not compete well with
of implicated foods for confirmation of laboratory testing
indigenous microbial in raw foods, contamination is
at the time of outbreak.
mainly associated with improper handling of cooked or
processed foods. However, S. aureus is also present in food,
Food handlers such as Suya meat and Nunu vendors are
animals, and dairy cattle, sheep and goats (Larkin et al.,
carrying enterotoxin-producing S. aureus in their noses or
2009). The S. aureus bacteria were then termed methicillin-
their hands which are regarded as the main source of food
resistant S. aureus (MRSA) usually show resistant to many
contamination, via manual contact or through respiratory
antibiotic (Ray et al., 2013). Staphylococcal food
secretions thereby contaminating raw foods, as a result of
poisoning (SFP) is an intoxication that results from the
improper handling of cooked or processed foods, followed
consumption of foods containing sufficient amounts of one
by storage under condition which allows growth of S.
(or more) preformed enterotoxin symptoms of SFP have a
aureus in food and animals. Food that have been frequently
rapid onset (2-8h), and include nausea, violet vomiting,
incriminated in staphylococcus intoxication include meat

and meat products, poultry and egg products, milk and dairy products, salads, bakery products, particularly cream-filled pastries and cakes, and sandwich filling. Salted food products (SFP) such as ham, have also been implicated according to the capacity of S. aureus to grow at relatively low water activity.

S. aureus have been found to survive in materials, clothes, hand nails, ATMs, mobile phones aside skins and nasal areas of the body (Oviasogie et al., 2014; Ekrakene & Igeleke, 2007; Adetona & Olabisio, 2010). It is noteworthy that Suya and Nunu hawkers come in contact with clothes, phones, ATMs and other materials while doing the merchandise without any consideration of washing before handling the food.

SFP is common disease whose real incidence is probably underestimated for a number of reasons, which include misdiagnosis, unreported minor outbreaks, improper sample collection and improper laboratory examination. (Argud´ın et al., 2010)

Nowhere has this issue been of great concern than with the gram-positive bacteria pneumococci, enterococci and staphylococci where multidrug resistance has become a norm among these pathogens. S. aureus is perhaps the pathogen of great concern because of its intrinsic virulence its ability to cause a diverse array of life-threatening infection, and its capacity to adapt to different environment conditions (Argud´ın et al., 2012, Hennekinne et al., 2012). The mortality of S. aureus bacteremia remains approximately 20-40% despite the availability of effective antimicrobial agents (Hassen et al., 2006). S. aureus is now the leading overall cause of nosocomial infections and, as more patients are treated outside the hospital setting is an increasing concern in the community as it is popular practice in Jos where drug vendors and Chemists are patronized without diagnosis or medical advice and such could also lead to increasing resistance to a great number of antimicrobial agents. Inevitably this has left fewer effective bactericidal antibiotics to treat these often life-threatening infections. As rapidly as new antibiotics are introduced, staphylococci have developed efficient mechanism to neutralize them.

In Jos metropolis, most infection are been treated with available drugs in the clinic or just by visiting drug vendors otherwise called Chemists where they are given antibiotics without recourse to medical laboratory diagnosis to identify the disease causing organism and possible susceptible drugs for the disease. Such common antibiotics have been found to be resistant to S. aureus as reported by Wang et al., (2017).

This study is aimed to determine the presence and antibiotics susceptibility profile of pathogenic staphylococcus aureus in Nunu and and Suya meat sold in Jos metropolis.

Materials and Methods

Study Area

The study was carried out in Jos Metropolis, Jos North Local Government Area in locations: Hamaz, Gangare, Railway, Yan Taya and Plateau riders Park. Sample Size / Collection

A total of 15 roasted meats (Suya) and 15 raw milk (Nunu) were collected from the select areas in and around Jos metropolis which includes: Hamaz, Gangare, Railway, Yan Taya and Plateau Riders Park. The Suya (roasted meat) and Nunu samples were obtained 3 each from every point of sales in Hamaz, Gangare, Railway, Yan Taya and Plateau riders Park into a sterilized universal bottle, packed into a polythene bags, and transported into the medical laboratory for analysis. While the Nunu sample were also collected in sterile polythene bags and transported in an icebox and transported to the Federal School Research Laboratory, Jos for analysis within 3 hours.

Isolation of Staphylococcus Aureus in Nunu and Suya 1mL of each sample was transferred to flask containing 9ml of peptone water for enrichment. According to ISO 6888, the plates were incubated under aerobic conditions at 370C for 24hr and those that yielded growth were subjected to another 24hr incubation for susceptibility assessment.

The total counts were carried out using nutrient agar. In serial dilution preparation, 1.0g of sample was aseptically, transferred into 9.0ml of diluted water and homogenized by vortex. Subsequent serial dilutions up to 10-5 were made in line with Kalalou et al. (2004). The enumeration of microorganisms in the samples was by the pour plate technique. At the end of the incubation, resultant microbial colonies (staphylococcus aureus) were counted.

Identification of Staphylococcus Aureus from Nunu and Suya

Pure isolates of microorganism grown on the different chocolate agar plates were further identified biochemically using Cowan and Steel (1974) technique using oxidase, coagulase, maltose, glucose and gram’s staining. The staphilococcus aureus were the only organisms isolated from suya meat and nunu samples obtained from the food vendors in Jos metropolis as identified from the samples.

Antibiotic Susceptibility Test of the Isolates of Staphilococcus Aureus

All S. aureus isolates were screened for multi-drug-resistance using disc diffusion in line with Bauer et al., (1966). This was performed on nutrient agar plates. Furthermore, the antibiotic susceptibility pattern of S. aureus strains was determined by disc diffusion method for Ciprofloxacin (10mcg), Gentamycin (10mcg), Norfloxacin (10mcg), Erythromycin (30mcg), Streptomycin (30mcg), Levofloxacin (20mcg), Chloramphenicol (30mcg), Amoxil, Ampiclox and Rifampin. Chloramphenical, Erythromycin and Norfloxacin susceptibility was determined by the e-test according to the manufacturers’ guidelines (Biometriux, France).

The diameter of the zone of inhibition produced each antibiotic disc was measured, recorded and the isolates were classified as “resistant”, “intermediate” and “sensitive” based on the standard interpretative chart updated according to the CLSI (2015). Multi-resistance was defined by resistance of the strain to at least three antibiotic agents.

15

 

RESULTS

Prevalence of Staphylococcus aureus from suya (roasted meat) sample

S/N Meat sample S. aureus x(y) Cell count Percentage
prevalence (%)
1. A 2(3) 7 x 1010 66.7
8 x 1010
2. B 1(3) 4 x 1010 33.3
3. C 1(2) 9 x 1010 50.0
4. D 0(2) 0.0
5. E 1(5) 5 x 1010 40.0
Total 5(15) 33.3

Key: X = Number Positive, Y = Number of sample examined, A = Hamas, B = Gangare, C = Rail Way, D = Yantaya, E = Plateau riders

Prevalence of Staphylococcus aureus from Nunu in Jos Metropolis

S/N Meat sample S. aureus x(y) Cell count Prevalence (%)
1. A 2(3) 6x 1010 66.7
8 x 1010
2. B 1(2) 4 x 1010 50.0
3. C 0(5) 0.0
4. D 2(3) 4 x 1010 66.7
7 x 1010
5. E 1(2) 3 x 1010 50.0
Total 6(15) 40.0

Key: X = Number Positive, Y = Number of sample examined, A = Hamas, B = Gangare, C = Rail Way, D = Yantaya, E = Plateau riders

Antibiotic Susceptibility Profile for Suya (Roasted Meat)

Antibiotics Disc t Samples Percentage Percentage
Conten A2 B1 C1 D3 E1 Resistance Sensitive
(ng) (%) (%)
Norfloxacin (NB) 10 R S S S S 20 80
Chloramphenicol (CH) 30 R S R R R 80 20
Ciprofloxacin (CPF) 10 R R S R R 80 20
Erythromycin (E) 30 S R R R S 60 40
Levofloxacin (LV) 20 R S S S S 20 80
Ampiclox (AML) 20 R R S R R 80 20
Amoxil (AML) 20 R R S R R 80 20
Gentamycin (CN) 10 R S S S S 20 80
Rifampin (RD) 5 S S R S S 20 80

Key: S = Sensitive, R = Resistance

16

Assessment of Antibiotic Susceptibility Pattern on Staphylococcus…

Antibiotic Susceptibility Profile for Suya (Roasted Meat)

Antibiotics Disc t Samples Percentage Percentage
Conten A2 B1 D2 E1 B3 E2 Resistance Sensitive
(ng) (%) (%)
Norfloxacin (NB) 10 S S R S S S 16.67 83.34
Chloramphenicol (CH) 30 R R S S R R 66.67 33.34
Ciprofloxacin (CPF) 10 S R S S S R 33.34 66.67
Erythromycin (E) 30 S R S S S R 33.34 66.667
Levofloxacin (LV) 20 S R R S R S 50.00 50.00
Ampiclox (AML) 20 R S R R S R 66.67 33.34
Amoxil (AML) 20 R R R R S S 66.67 33.34
Gentamycin (CN) 10 S S R S S S 16.67 83.34
Rifampin (RD) 5 S S R R R S 50.00 50.00

Key: S = Sensitive, R = Resistance

Discussion

Staphylococcus aureus is an important health care and community acquired infection in every region of the world (Wang et al., 2017). This studies shows that Staphylococcus aureus is prevalent in Suya (33.3%) and Nunu (40%) and could possess a potential health hazard (Sofos, 2008) to consumers in Jos Metropolis. Studies on isolation and antibiotic susceptibility frequency of isolation of S. aureus isolates from meat and milk may be due to the possible susceptibility of S. aureus isolates from Suya and Nunu to Levofloxacin, Erythromycin, Norfloxacin, Gentamycin and Rifampin as observed in this study. However, this report was not different from other studies reported by Waters et al. (2011). The high susceptibility of S. aureus to these antibiotics may be due to high costly or injectable forms and the possibility of abuse of such antibiotics may be low. The low susceptibility of chloramphenicol, amoxil, ampiclox observed in this study was not different from the other studies reported by Le et al., (2003). The low susceptibility of S. aureus to antibiotics mentioned may be due to inappropriate use of antibiotics without doctor’s prescription and inclusion of antibiotics in animal feeds as growth promoter (Le et al., 2003). From this study, it was however observed that the frequency of isolation of S. aureus observed in this study was high and isolates were more susceptible to flouroquinolones and aminoglycosides. In view of this, such drugs may be useful for treatment of staphylococcal infections, but there is a need to conduct appropriate medical laboratory diagnosis to find out the causative organism S. aureus for example before treatment especially in Jos Metropolis. The indiscriminate use of antibiotics/antimicrobials agents for prophylactic as well as other therapeutic purpose could be the reasons for increased antimicrobial resistance of S. aureus in animals and humans.

Conclusion

Staphylococcal food poisoning (SFP) is a major concern in public health programs worldwide. S. aureus may be present in cow milk as a result of milk collection from the animal suffering from disease condition and excreting S. aureus in milk or due to unhygienic conditions during production, processing, storage and handling of milk products, which are the main causes of food borne

diseases. The result of this study clearly indicated that some Nunu and Suya available in the Jos metropolis were contaminated with S. aureus, which could pose a risk of food poisoning to Jos metropolis. Thus, more hygienic preventive measures are required to reduce the bacterial contamination, so as to increase the wholesomeness of these Nunu and Suya. The study also revealed common antibiotics used in Jos which are resistant to S. aureus. This study highlights the need for continuous surveillance of antibiotic sensitivity pattern of staphylococcus aureus with a view to selecting appropriate therapy. This study also recommends that:

  1. All Suya meat and Nunu vendors should imbibe adequate hygiene measures while handling the food products
  2. The Suya and Nunu vendors should use nose masks while processing handling their products
  3. The Suya and Nunu vendors in Jos metropolis should avoid using dirty rags to clean their hands while processing and dispensing their products to the public

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Influence of antibiotic treatment on the detection of S. aureus in whole blood following pathogen enrichment

Matthias Pilecky, Anita Schildberger, Viktoria Weber

Center for Biomedical Technology, Department for Health Sciences and Biomedicine, Danube University Krems,

Dr.-Karl-Dorrek-Strasse 30, 3500 Krems, Austria.

Viktoria Weber

Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Health Sciences and Biomedicine, Danube University Krems, Dr.-Karl-Dorrek-Strasse 30, 3500 Krems, Austria.

Ludwig Knabl and Dorothea Orth-Höller

Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Schöpfstraße 41, A-6020 Innsbruck, Austria.

Viktoria Weber

Department for Biomedical Research, Danube University Krems, Dr.-Karl-Dorrek-Strasse 30, 3500 Krems, Austria.

All Correspondences to: Viktoria Weber E-mail: viktoria.weber@donau-uni.ac.at

ABSTRACT

Background: Early pathogen detection and identification are crucial for an effective and targeted antibiotic therapy in patients suffering from blood stream infection. Moleculardiagnostic methods can accelerate pathogen identification as compared to blood culture, but frequently suffer from the inhibition of polymerase chain reation (PCR) by sample matrix components, such as host DNA, anticoagulants, or plasma proteins. To overcome this limitation, molecular diagnostic methods commonly rely on pathogen enrichment by selective lysis of blood cells and pelleting of intact pathogens prior to analysis. Results: Here, we investigated the impact of antibiotic treatment on the recovery of pathogen DNA using an established pathogen enrichment protocol. Based on the hypothesis that induction of bacterial cell wall disintegration following antibiotic administration leads to incomplete pelleting of pathogen DNA, S. aureus was grown in human whole blood with or without addition of cell wall active (vancomycin, piperacillin) or non cell wall active (ciprofloxacin, clindamycin) antibiotics at clinically relevant concentrations. Pathogen detection remained unaffected by non cell wall active antibiotics or even increased in the presence of cell wall active antibiotics, indicating improved accessibility of pathogen DNA. Likewise, mechanical lysis of S. aureus prior to pathogen enrichment resulted in increased recovery of pathogen DNA. Quantification of pathogen and human DNA after selective lysis of blood cells and pathogen enrichment confirmed partial depletion of human DNA, leading to a net enrichment of pathogen DNA over human DNA. Conclusion: Concurrent antibiotic administration does not reduce the recovery of pathogen DNA during pathogen enrichment by selective lysis and centrifugation. Leads to a 10-fold human DNA depletion as compared to pathogen DNA. Moreover, we confirm that the recovery of pathogen DNA after pathogen enrichment is not negatively

influenced by concurrent antibiotic administration.

Keywords: Molecular diagnostics, Pathogen detection,Blood stream infection, DNA extraction, Selective lysis, Antibiotics.

BACKGROUND management of sepsis [10] recommend immediate
. aureus is an opportunistic pathogen with the antibiotic therapy as well as pathogen identification, since
S early and targeted antibiotic treatment can significantly
potential to cause community-associated and
nosocomial infections [1]. It is the most common improve the survival of sepsis patients [11–14]. Blood
gram-positive pathogen associated with sepsis, with a culture is the current reference method for the
prevalence of up to 20% of all blood culture positive sepsis identification of pathogens and for the characterization of
cases [2–4]. This high prevalence results from its ability to their antibiotic susceptibility. It may, however, fail to detect
adapt to its environment by modulating the host immune slow-growing or intracellular pathogens and yield
response [5], to switch from a highly proliferative inconclusive results due to concurrent antibiotic treatment.
disseminative state into a slow growing, biofilm producing Moreover, definitive results of culture are usually not
state [6], to grow intracellularly [7, 8], and to acquire available before 48 h [11, 13, 15]. Molecular diagnostic
antibiotic resistance [9]. Clinical guidelines for the methods provide a time-to-result of 4–7 h, compatibility

Nigerian Biomedical Science Journal Vol. 16 No 3 2019 57

Influence of antibiotic treatment on the…

with antibiotic treatment, and do not depend on pre-selecting culture steps [13, 16, 17]. Still, the direct detection of pathogen DNA in blood samples (e.g. SeptiFast, Roche, Basel, Switzerland) is prone to interference of matrix components, such as host DNA [18,

19], heparin [20, 21], or plasma proteins [22–24]. To avoid

inhibition of PCR and to increase the sensitivity of pathogen detection, virtually all current molecular diagnostic systems, including FAST ID BSI (QVella, Richmond Hill, Canada), Hybcell Pathogen Array (CubeDX, St. Valentin, Austria), T2 Bacteria (T2 Biosystems, Lexington, MA), SepsiTest (Molzym, Bremen, Germany), as well as Magicplex Sepsis Test (Seegene, Seoul, Korea), rely on pre-analytical pathogen enrichment [25]. Common to all pathogen enrichment protocols is the selective lysis of blood cells by addition of a hypotonic detergent solution. Pathogens withstand the osmotic pressure and are enriched by subsequent centrifugation prior to DNA quantification. We hypothesized that antibiotic treatment, by affecting bacterial cell wall integrity, might lead to incomplete pelleting of pathogens after selective lysis of the blood cells and centrifugation, and, consequently, result in a loss of pathogen DNA prior to analysis. Therefore, we investigated the impact of both, cell wall active and non cell wall active antibiotics on the pre-analytical enrichment of DNA of different S. aureus strains from human whole blood.

Methods

Bacteria and reagents

Antibiotics (Table 1), 4 – (2 – hydroxyethyl) – 1 – piperazineethanesulfonic acid buffer (HEPES, pH 7.0, cell culture grade) and adenine were purchased from Sigma-Aldrich (St. Louis, MO). D-Glucose was obtained from Merck

(Darmstadt, Germany). S. aureus culture strains (ATCC 12600, 29213, and 29737) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Wild-type strains (WT32217, 32237, and 32248) were isolated from patient material at the Division of Hygiene and Medical Microbiology, Medical University

of Innsbruck, Austria. Strains were cultivated on lysogeny broth (LB, Lennox formulation) agar plates (Carl Roth, Karlsruhe, Germany) at 37 °C. Overnight cultures were obtained by inoculating single colonies into LB medium. To determine the minimal inhibitory concentration for individual antibiotics used in this study, overnight cultures were diluted 1:5,000 in LB medium in 96-well polystyrene microwell plates (CELLSTAR®, Greiner Bio-One GmbH, Frickenhausen, Germany), serial dilutions of antibiotics were added, and incubation was performed for 24 h at 37 °C.

Human whole blood

Venous human whole blood was collected from healthy

adult volunteers into tubes (Vacuette, Greiner Bio-One,

Kremsmuenster, Austria) containing sodium heparin or

EDTA. Blood collection was approved by the Ethical

Review Board of Danube University Krems, and written

informed consent was obtained from all donors.

Cultivation of S. aureus in human whole blood Freshly

drawn human whole blood anticoagulated with heparin

was buffered with 1/50 volume of 1M HEPES and

supplemented with 2 mg/L glucose and 48 μg/L adenine

per hour. Overnight cultures of each strain were diluted

1:1000 in LB medium, spiked into supplemented whole

blood (typically 4 mL) at a ratio of 1:2560 (ATCC 12600),

1:205 (ATCC 29213), 1:1700 (ATCC 29737), 1: 730

(WT32217), 1:1700 (WT32237), and 1:128

(Wt32248), and incubated at 37 °C with gentle shaking.

These spiking ratios were chosen based on growth curves

for the individual strains to obtain pathogen concentrations

of approximately 5000 colony forming units (CFU) per mL

after 4 h. For each strain, growth was assessed in whole

blood from six donors (n = 3 per donor) for up to 8 h.

Antibiotic pretreatment of S. aureus

To investigate whether weakening or disruption of bacterial cell walls by antibiotic pretreatment would influence subsequent pathogen enrichment and, consequently, PCR-based quantification of pathogen DNA, S. aureus was spiked into whole blood and incubated for 4 h as described above to ensure logarithmic growth. Subsequently, final concentrations of 15 μg/mL

Table 1 Antibiotics used in this study and their mechanism of action

Antibiotic Cell Wall Active Mechanism of Action
Vancomycin (VAN) + interferes with cell wall synthesis by preventing formation and crosslinking of peptidoglycan strands [26–28]
Piperacillin (PIP) + Interferes with cell wall synthesis by binding to enzymes required for the extracytoplasmatic stage of
cell wall formation [28, 29]
Ciprofloxacin (CIP) prevents replication of bacterial DNA by inhibiting DNA gyrase [30, 31]
Clindamycin (CLI) inhibits bacterial protein synthesis by binding to 50S ribosomal subunits [32]

vancomycin (VAN), 20 μg/mL piperacillin (PIP), 1 μg/mL ciprofloxacin (CIP), or 2 μg/mL clindamycin (CLI), respectively (Table 1), were added, and incubation was continued for another 90 min at 37 °C with gentle agitation. Subsequent pathogen enrichment and DNA extraction were performed as described below. Spiked blood without antibiotic treatment served as control. Strain ATCC 29213 was additionally grown in the presence of CIP and VAN for up to 72 h to investigate potential differences between blood culture based and qPCR based pathogen detection.

Mechanical lysis of S. aureus

To achieve complete pathogen disintegration, fresh S. aureus overnight cultures (ATCC 29213) were diluted 1:5000 in LB medium, incubated for 2 h at 37 °C, mixed with an equal volume of 0.1mm zirconium beads (Biozym, Hessisch Oldendorf, Germany) in a 0.3 mL PCR tube (Bio-Rad, Hercules, CA), and vortexed using a regular benchtop vortex (VortexGenie2, Carl Roth) at maximum speed for up to 120 min. Samples drawn after 30 s, 5 min, and 120 min were characterized by scanning electron microscopy,

58 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

 

and bacterial lysis was confirmed by quantification of viable bacteria (CFU) as described below. Suspensions of lysed bacteria obtained at the indicated time points were spiked into freshly drawn whole blood anticoagulated with EDTA at a ratio of 1:100, and pathogen enrichment as well as isolation and quantification of pathogen DNA were performed as described below.

Quantification of viable bacteria

To quantify viable bacteria, spiked blood samples were diluted 1:5 in 0.9% NaCl (Fresenius Kabi, Bad Homburg, Germany), and 100 μL of the dilutions were plated onto LB agar (Carl Roth, n = 3). Plates were incubated overnight at 37 °C, and colonies were counted manually on the next day. Pathogen enrichment and extraction of pathogen DNA Pathogen DNA was isolated from spiked blood samples using a commercial pathogen enrichment system (GINA Pathogen Enrichment, CubeDX, St. Valentin, Austria) according to the instructions of the manufacturer. The enrichment protocol relies on the selective lysis of blood cells by detergent buffer, followed by centrifugation to pellet intact pathogens, lysis of pelleted pathogens by heating in alkaline buffer, and extraction of pathogen DNA using MiniSpin columns. For comparison, total DNA was extracted without previous pathogen enrichment using a total DNA extraction kit (MagMAX DNA Multi-Sample Kit, Thermo Fisher Scientific, Waltham, MA) according to the protocol of the manufacturer.

Quantification of DNA

qPCR was performed using Staphylococcus spp. specific 16S rRNA gene primers and primers specific for the beta-actin gene as control for human DNA. All primers are specified in Additional file 1: Table S1. Samples were mixed with an equal volume of SsoAdvanced SYBR Green Master Mix (Bio-Rad, Hercules, CA) containing 0.5 μM of each primer to yield a total reaction volume of 20 μL. qPCR was performed using a Roche LightCycler 96 (Basel, Switzerland) and comprised a 2 min preheating step at 94 °C, followed by 45 cycles of denaturation at 94 °C for 5 s, annealing at 53 °C for 10 s, and extension at 72 °C for 15 s. Correct amplification was verified by melting curve analysis. cT values were calculated using the Roche LightCycler96 Software version 1.1.0.1320.

Scanning electron microscopy

Sample preparation for scanning electron microscopy included filtration of bacterial suspensions through 0.22 μm polycarbonate isopore membranes (Merck Millipore, Darmstadt, Germany), followed by fixation with 2.5% glutaraldehyde (Carl-Roth) in 0.9% NaCl for 2 h at room temperature and dehydration in a graded alcohol series (30–100%). Samples were sputtered with gold using a Q150R (Quorum Technologies Ltd., Laughton, UK) at 30 kV in DC mode for 60 s, and images were acquired using a FlexSEM 1000 scanning electron microscope (Hitachi, Mannheim, Germany) at an accelerated voltage of 20 kV.

Statistical analysis

Statistical analysis was performed using SigmaPlot 13.0 (Systat Software, Erkrath, Germany) and graphs were plotted using GraphPad Prism version 7.02 (La Jolla, CA). At least three replicates were performed for each

Viktoria Weber

experiment. Data are presented as mean ± standard deviation or as mean and 95% confidence intervals (CI). The significance of differences was calculated using paired t-test for comparison of a sample to a reference value, or, in case of multiple time points, by Friedman repeated measures analysis of variance on ranks, followed by multiple comparisons versus reference using Dunnett’s Method. P-values < 0.05 were considered as statistically significant.

RESULTS

Growth of S. aureus in human whole blood and antibiotic susceptibility The experimental setup of this study is outlined in Fig. 1. All S. aureus strains exhibited logarithmic growth in human whole blood supplemented with glucose and adenine after a lag phase of 2–4 h (Fig. 2). All strains were susceptible to VAN, PIP, CIP and CLI. Minimal inhibitory concentrations for individual

Fig. 1 Background and outline of the study. a Blood-borne pathogens can be detected using blood culture (detection of viable pathogens) or by molecular diagnostic methods, such as PCR (detection of pathogen DNA). PCR is either performed after extraction of total (host and pathogen) DNA or following pathogen enrichment to deplete human DNA as well as blood-borne inhibitors of PCR. We hypothesized that antibiotic treatment might

induce disintegration and, consequently, incomplete pelleting of pathogens during sample processing with established pathogen enrichment protocols, resulting in partial loss of pathogen DNA. b Commonly used pathogen enrichment protocols comprise the selective lysis of blood cells by detergent treatment, pelleting of intact pathogens, and extraction of pathogen DNA

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Influence of antibiotic treatment on the…

using spin columns. c To assess the influence of antibiotic treatment on PCRbased pathogen detection following pathogen enrichment, S. aureus was spiked into human whole blood and grown for 4 h. Thereafter, spiked samples were incubated without further treatment (control) or received antibiotic treatment as described in Materials and Methods. For comparison, S. aureus was mechanically lysed using zirconium beads to achieve complete disintegration as described in Materials and Methods, spiked into whole blood, and processed in parallel to untreated and antibiotic-treated samples. CFU counts (viable pathogens) were determined in all samples, and pathogen DNA was quantified after pathogen enrichment as shown in panel b

antibiotics are listed in Additional file 1: Table S2. Influence of antibiotic treatment on the recovery of pathogen DNA

To assess whether the presence of antibiotics would influence the recovery of S. aureus during pathogen enrichment, spiked whole blood was treated with either cell wall active (VAN, PIP) or non cell wall active (CIP, CLI) antibiotics (Table 1). cT values remained constant for CIP and CLI (ΔcT -0.5 ± 0.9 and 0.4 ± 0.9 as compared to the reference taken prior to the addition of antibiotics), or decreased significantly for the cell wall active antibiotics PIP and VAN (ΔcT -2.9 ± 1.4 and − 2.4 ± 1.3) (Fig. 3a), suggesting enhanced accessibility of pathogen DNA in the presence of cell wall active antibiotics. VAN, PIP, and CIP, which exhibit bactericidal activity [33–35], induced a reduction of CFU counts as compared to the reference, while CLI exerted bacteriostatic effects with stable CFU counts (Fig. 3b). No significant differences were detected between individual S.aureus strains (data not shown). Upon incubation with VAN and CIP for up to 72 h, pathogen DNA remained constant in the presence of VAN or even increased in the presence of CIP, providing further

evidence that antibiotic treatment does not induce a loss of pathogen DNA during pathogen enrichment (Fig. 3c). CFU counts decreased continuosly over time, and no viable bacteria were detected beyond 48 h (Fig. 3d). Scanning electron microscopy did not provide evidence for cell wall degradation or reduced density of culture (Fig. 3e). I

nfluence of mechanical lysis on the recovery of

pathogen DNA

Next, we performed mechanical lysis of S.aureus and spiked the lysates into whole blood to assess the impact of pathogen disintegration on the recovery of pathogen DNA during subsequent pathogen enrichment. Culture as well as scanning electron microscopy confirmed the efficient lysis of S. aureus (Fig. 4a). The recovery of pathogen DNA increased with progressing pathogen disintegration, confirming the efficient enrichment of pathogen DNA despite the absence of intact bacteria (Fig. 4b).

Selectivity of DNA isolation: pathogen versus host DNA

Based on the enhanced recovery of pathogen DNA following pathogen disintegration by mechanical lysis, we went on to assess the ability of the pathogen enrichment protocol to selectively isolate S. aureus DNA, i.e. to deplete human DNA. S.aureus DNA and human DNA were quantified following DNA isolation from spiked whole blood using (i) pathogen enrichment (protocol A) or (ii) total DNA extraction without previous pathogen enrichment (protocol B). qPCR (Table 2)

Fig. 2 Growth of S. aureus in supplemented human whole blood. a S. aureus strains obtained from ATCC and b wild-type strains isolated from patient material reached exponential growth (log phase) after 2–4 h of incubation in whole blood supplemented with glucose and adenine as described in Materials and Methods. CFU counts are indicated relative to the initial spiking concentration of 103–104 CFU/mL (n = 6)

revealed the presence of residual human DNA in samples isolated with the pathogen enrichment protocol, however, samples obtained by total DNA extraction contained significantly higher amounts of human DNA (ΔcT 6.1 ± 0.3). Pathogen DNA was nearly equally well enriched with both protocols (ΔcT 2.1 ± 0.4), resulting in an approximately 16-fold enrichment of pathogen DNA over human DNA using the pathogen enrichment protocol.

DISCUSSION

Early adequate antimicrobial therapy is crucial in the management of blood stream infection, where broadspectrum antibiotics are administered upon the first suspicion of infection [10], followed by targeted antimicrobial treatment after identification of the causative pathogens by blood culture or molecular diagnostic methods [36, 37]. While there is ample evidence that antimicrobial treatment can interfere with blood culture

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[38, 39], the impact of antibiotic therapy on molecular diagnostic pathogen detection remains incompletely defined. The only prospective study published to date in this context revealed a significantly higher rate of positive results with SeptiFast multiplex PCR as compared to blood culture in children under concurrent antimicrobial therapy

  1. Since PCR-based pathogen detection in whole blood is, however, susceptible to inhibition by blood-borne factors and host DNA, pathogens are commonly enriched from whole blood prior to analysis. Established enrichment

protocols rely on the selective lysis of blood cells in hypotonic detergent and subsequent pelleting of intact pathogens, followed by extraction of pathogen DNA and PCR. We hypothesized that antimicrobial therapy, in particular administration of cell wall active antibiotics, might result in a release of DNA from damaged bacteria and, consequently, in decreased DNA recovery during pathogen enrichment. To test this hypothesis, we chose S. aureus as model pathogen, since it is a leading cause of blood stream infection worldwide.

After an adaptation phase of several hours, both, S.aureus culture and wild-type strains reached stable growth in supplemented heparinized whole blood under our experimental conditions, while earlier studies with MRSA had observed rapid uptake of spiked S. aureus by neutrophils [7], with constant or decreasing CFU counts

Viktoria Weber

over time. The use of a methicillin-susceptible strain in our study could explain these divergent findings, since carriage of antibiotic resistance genes can reduce bacterial growth rates [41, 42]. Furthermore, we performed experiments for up to 72 h, after 4 h of adaptation of S.aureus to its environment. This required buffering as well as supplementation of whole blood with glucose and adenine, as commonly used for the storage of blood products [43]. Since wild type strains required a lag phase of 4 h for adaptation, we chose to incubate all strains for 4 h in supplemented whole blood prior to antibiotic administration, and we adjusted initial spiking concentrations to achieve a density of about 5000 CFU/mL after this adaptation phase. While this bacterial load was clearly higher than in septic patients, where pathogen loads of typically 1–10 CFU/mL have been reported [13], it allowed for the quantification of S. aureus using both, microbiological cultivation and qPCR and enabled the reliable determination of changes in qPCR signal intensity, which would not have been feasible at lower CFU counts.

To assess the influence of concurrent antibiotic treatment on pathogen enrichment, we used PIP and VAN according to clinical guidelines for the treatment of S.aureus infections [44, 45]. For comparison, we chose CIP, which is widely prescribed by general practitioners for the treatment of pneumonia, suspected gastrointestinal infections, or infections of the genitourinary tract [46, 47],

Fig. 3: Influence of antibiotic treatment on the recovery of pathogen DNA following pathogen enrichment. a-b S. aureus was grown in human whole blood, followed by a 90 min incubation in the presence of antibiotics, and CFU counts as well as pathogen DNA were quantified as described in Materials and Methods. Spiked whole blood without adsorbent treatment served as control (ctrl.). ΔcT values and CFU counts are given relative to the reference (sample taken prior to the addition of antibiotics). Recovery of pathogen DNA was not influenced by CIP and CLI, while DNA recovery was increased after treatment with the cell wall active antibiotics VAN and PIP. CFU counts were significantly reduced for PIP, VAN, and CIP (bactericidal), or remained constant for CLI (bacteriostatic), confirming efficacy of antibiotic treatment. Data are presented as mean ± standard deviation (n = 18; paired t-test). c-d Upon incubation of spiked blood samples with VAN and CIP over 72 h, the recovery of pathogen DNA remained stable (VAN) or even increased (CIP), while no viable bacteria were detectable beyond 48 h (n = 4; Friedman repeated measures and Dunnett’s Method). e Scanning electron micrographs of S. aureus incubated for 24 h in the presence of VAN and CIP failed to provide evidence for cell wall degradation or reduced culture density, as compared to the untreated control. Scale bar, 5 μm. Data are given as mean ± standard deviation.

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as well as CLI, which is commonly used for the treatment of skin and soft tissue infections in outpatient care [48]. All antibiotics were applied to achieve concentrations equivalent to mean plasma levels reported in the literature [26, 27, 29–32]. We used an established manual pathogen enrichment kit, since it allows for intervention at multiple steps of the pre-analytical protocol,

while protocols provided by other suppliers are fully integrated systems, which process blood samples automatically and do not allow for pathogen quantification at individual stages of the pre-analytical protocol. Still, our findings are likely to be valid for most other current pathogen enrichment protocols, as these systems differ

only insignificantly regarding the composition of lysis reagents.

Regardless of the antibiotic used, S. aureus DNA was not lost during the pre-analytical pathogen enrichment and was still detectable after 3 days of incubation in supplemented whole blood. Scanning electron microscopy revealed that S. aureus stayed largely intact over the course of the experiment despite antibiotic treatment, suggesting that pathogen DNA remained encapsulated by the bacterial cell wall and was protected from bloodborne degradation factors such as DNase [49]. Notably, the administration of cell wall active antibiotics resulted in a significant increase in the qPCR signal. It is highly probable that this effect was

Fig. 4 Influence of mechanical lysis on the viability and recovery of S. aureus DNA after pathogen enrichment. a S. aureus was mechanically lysed using zirconium beads and spiked into human whole blood as described in Materials and Methods. Culture confirmed the absence of viable bacteria after 5 min of lysis. Fragmentation of bacteria was confirmed by scanning electron microscopy. b Lysed S. aureus was spiked into whole blood, and DNA was quantified after pathogen enrichment as described in Materials and Methods. DNA recovery increased in parallel with pathogen disintegration. Data are given as mean ± standard deviation (n = 3; Friedman repeated measures and Dunnett’s Method). Scale bar, 5 μm

not related to the process of pathogen enrichment as such, but was rather due to incomplete lysis of gram-positive bacteria in alkaline solution after the actual pathogen enrichment step, and we assume that cell-wall active antibiotics support the release of pathogen DNA by reducing the thickness of the peptidoglycan layer of gram-positive bacteria [50]. Even mechanical disintegration of S. aureus prior to pathogen enrichment resulted in enhanced detection of pathogen DNA. This further confirms previous reports on the incomplete solubilization of bacterial DNA, which was particularly reported for

Table 2 Selective depletion of human DNA by pathogen enrichment

qPCR after cT value
Human DNA S. aureus DNA
spiked whole blood protocol A 23.15 ± 0.38 25.98 ± 0.75
protocol B 17.06 ± 0.08 23.85 ± 0.64

Human whole blood was spiked with S. aureus and processed in parallel with an estalished pathogen enrichment protocol (protocol A) and by total (host and pathogen) DNA extraction with magnetic beads (protocol B) as described in Materials and Methods. qPCR revealed the presence of residual human DNA (positive PCR for beta-actin) in samples isolated with the pathogen enrichment protocol, but samples obtained by total DNA extraction contained significantly higher amounts of human DNA. Pathogen DNA was nearly equally well enriched with both protocols, resulting in a net enrichment of pathogen DNA over host DNA using the pathogen enrichment protocol. Data are presented as mean ± standard error of the mean (n = 4)

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gram-positive bacteria [51], most likely due to its association with the cell walls [45].

While a comparison of pathogen DNA recovery from whole blood, either after selective lysis and pathogen enrichment or after extraction of total DNA, revealed that pre-analytical pathogen enrichment was not associated with enhanced detection of pathogen DNA, human DNA was depleted by 4–5 cT values, which might be relevant for the pre-analytical depletion of blood-borne PCR inhibitors. Likewise, the reduction of human DNA background may be crucial for emerging analytical approaches including next-generation sequencing [52].

Conclusion

In conclusion, our study provides evidence that concurrent antibiotic administration is not associated with decreased recovery of pathogen DNA after pathogen enrichment by selective lysis and centrifugation. On the contrary, cell wall active antibiotics seem to increase the yield of pathogen DNA, presumably by supporting preanalytical DNA solubilization. Moreover, we confirmed a depletion of human DNA as compared to pathogen DNA by at least a factor of 10 during pathogen enrichment.

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Reference intervals for serum cystatin C and serum creatinine in an adult sub-Saharan African population

Bertille Elodie Edinga-Melenge Suzanne Belinga, Eric Minkala, Prisca Armel Noudjeu, Michel Ondhoua, Samuel Walter Kokola, and Catherine Bilong

Department of Biochemistry, Centre Pasteur of Cameroon, Yaoundé, Cameroon.

Bertille Elodie Edinga-Melenge and Vicky Joceline Ama Moor

Department of Physiological Sciences and Biochemistry, Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, Yaoundé, Cameroon.

Adrienne Tchapmi Yakam

Ebebda District Hospital, Centre Regional Delegation, Ministry of Public Health, Ebebda, Cameroon.

Jobert Richie Nansseu

Department for the Control of Disease, Epidemics and Pandemics, Ministry of Public Health, Yaoundé, Cameroon.

Jobert Richie Nansseu

Department of Public Health, Faculty of Medicine and Biomedical Sciences of the University of Yaoundé I, PO Box 1364, Yaoundé, Cameroon.

Vicky Joceline Ama Moor

Laboratory of Biochemistry, Yaoundé University Teaching Hospital, Yaoundé, Cameroon.

Gloria Ashuntantang

Cardiology and Nephrology Unit, Yaoundé General Hospital, Yaoundé, Cameroon.

Gloria Ashuntantang

Department of Internal Medicine and Specialties, Faculty of Medicine and Biomedical Sciences of the University of Yaoundé I, Yaoundé, Cameroon.

All Correspondences to: Jobert Richie Nansseu E-mail: jobertrichie_nansseu@yahoo.fr

ABSTRACT

Background: Serum cystatin C (SCysC) and serum creatinine (SCr) are two biomarkers used in common practice to estimate the glomerular filtration rate (GFR). For SCysC and SCr to be used in a given population, normal values need to be determined to better assess patients. This study aimed to determine SCysC and SCr reference intervals (RIs) in a Cameroonian adult population and factors susceptible of influencing them. Methods: We carried-out a cross-sectional study from November 2016 to May 2017 in Yaoundé, Cameroon. Participants were Cameroonians aged 18 years and above, residing inside the country and found in good health at study inclusion. SCysC and SCr were determined by particle-enhanced turbidimetric immunoassay standardized against the ERM-DA471/IFCC reference material and by the IDMS reference modified Jaffe kinetic method, respectively. RIs were determined using the 2.5th and 97.5th percentiles and their respective 90% confidence intervals (CIs). The quantile regression served to identify potential factors likely influencing SCysC and SCr values. Results: We included 381 subjects comprising 49.1% females.. RIs for SCysC varied between 0.57 (90%CI: 0.50–0.60) and 1.03 mg/L (90%CI: 1.00–1.10) for females, and from 0.70 (90%CI: 0.60–0.70) to 1.10 mg/L (90%CI: 1.10–1.20) for males. Concerning SCr, its RIs ranged from 0.58 (90%CI: 0.54–0.61) to 1.08 mg/dL (90%CI: 1.02–1.21) for females, and from 0.74 (90%CI: 0.70–0.80) to 1.36 mg/dL (90%CI: 1.30–1.45) for males. Men had significantly higher SCysC and SCr values than women (p < 0.001). Likewise, subjects aged 50 years and above had higher SCysC values in comparison to younger age groups (p < 0.001), which was not the case for SCr values (p = 0.491). Moreover, there was a positive and significant correlation between SCysC and SCr in women (ρ = 0.55, p < 0.001), in men (ρ = 0.39, p < 0.001) and globally (ρ = 0.58; p < 0.001). Furthermore, the sex influenced both biomarkers’ values across all quantile regression models while age and body surface area (BSA) influenced them inconsistently. Conclusion: This study has determined serum cystatin C and serum creatinine reference intervals in an adult Cameroonian population, whose interpretations might take into account the patient’s sex

and to a certain extent, his/her age and/or BSA.

Keywords: Reference interval, Cystatin C, Creatinine, Yaoundé, Cameroon.

BACKGROUND

Glomerularfiltrationrate(GFR)iswidelyacceptedas the mostusefuloverallindexofkidneyfunctionin healthand disease(1).It is best evaluated by clearance measurement ofexogenousmarkerssuchasinuline,but thecomplex proceduresofthesemeasureslimittheir routineuse(2,3). GFR is therefore commonly estimated fromserumlevelof endogenousfiltrationmarkers.The mostwidelyusedand recommendedendogenousmarker forinitialassessment ofGFRisserumcreatinine(4).

Despitethecheapestcostandthesimpleuseof creatinine-basedmeasurementsofGFR,estimationof thelevelof

renalfunctionobtainedisquiteimprecise.

Indeed, the steady-state serum creatinine level is determined byfactorsthatincludeleantissuemass; hence, it mayvarywithsex,age,weightandheight(3,5,6).

Asaresultoftheselimitations,alternativeendogenous markersforGFRsuchasserumcystatinChavebeen proposed.CystatinCisabiomarkerformedataconstant rate by all nucleated cells of the body which do not correlate with lean tissue mass (5). Evidence has demonstrated improved accuracy and sensitivity of cystatinC comparedtocreatinine(7).

For an accurate interpretation of biomarkers levels,

Nigerian Biomedical Science Journal Vol. 16 No 3 2019 49

Reference intervals for serum cystatin C…

referenceintervalsspecifictoapopulationneedtobe established.Intriguinglyandalthoughserumcreatinine is widely used in Cameroon, no previous study had yet focused at determining its reference intervals, interpretations relying on western countries’ data. Moreover consideringthegrowingimportanceofcystatin Casa prospectivemarkertoassesstherenalfunction,itis obvious thatthismarkerwouldbeintroducedinroutine clinicalpracticeinCameroonverysoon.Therefore,we conductedthepresentstudytodeterminethereference valuesofserumcreatinineandcystatinCinahealthy adult Cameroonianpopulationlivinginsidethecountry. Besides, weaimedtoidentifypotentialfactorslikely influencing thesereferenceintervals.

Methods

Studydesignandsetting

This was a population based cross-sectional study conducted betweenNovember2016andMay2017in Yaounde’thecapitalcityofCameroon.Participantswere recruitedfromthe4mostpopulatedhealthdistrictsout of the6thatconstitutesthecity,namely:Yaounde’1,2,4 and 7

(8). Biological analyses were performed at the Centre PasteurofCameroun.

Descriptionofthestudypopulation

ParticipantswereadultCamerooniansresidinginsidethe country,aged18yearsandabove,foundingoodhealthat studyinclusion-afterageneralexaminationincludinga briefmedicalinterview,urinalysisandmeasurementof bloodpressureandglycaemia-withnoevidenceofany acuteorchronicillnesssusceptibleofaffectingcreatinine orcystatinClevels.Weexcludedknownorsuspected hypertensives,thosewithanimpairedglucosemetabolism

(pre-diabetes or diabetes mellitus) or an abnormal dipstick urinetest.Pregnantandbreastfeedingwomenwerealso excluded, as well as drug users. No special dietary recommendations were required. Participants were consecutively recruited during the study period and a minimumof 120 participantswasrequiredforeachsex group,inline withtheInternationalFederationofClinical Chemistry’s (IFCC) recommendations (9).

Datacollection

Participants were mostly recruited in churches, sc hools/universities/collegesandmosques.Onthedaysof recruitment,eachpotentialparticipantwasrequiredto sign aconsentformasthetestimonyofhis/hervolunteering participation. Subsequently, he/she underwent a brief interview using a preconceived, standardized and pre-testedquestionnaire(Additionalfile 1);then a summary physicalexaminationwasconducted,duringwhich blood pressurewasmeasured.Weusedthesimplified calculation procedurefromMostellerRDtoderiveeach participant’s bodysurfacearea(BSA)(10).In addition, a urinesample wascollectedfordipstickurineanalysis andacapillary glycaemiawasperformedusinga OneTouch*analyzer.

Biochemicalassays

Ten milliliters of venous blood were collected by

venipuncture in 2 dry tubes of 5ml each. Serum was separatedbycentrifugationat3000rpmwithin10min. Biochemical assays were conducted using the autoanalyzer CobasC501/6000,RocheDiagnostics,USA. Serum cystatinCwasmeasuredinincrementsof0.1mg/L by particle-enhanced turbidimetric immunoassay using Tina-quant*CystatinCreagentkits(RocheDiagnostics, USA).Themethodappliedwasstandardizedagainstthe ERM-DA471/IFCCreferencematerial.Meanwhile, serum creatininewasdeterminedbytheIsotopeDilution Mass Spectrometry (IDMS) reference modified Jaffe kinetic methodusingCreatinineJaffeCobas*reagentkits (Roche

Diagnostics, USA).

Statisticalanalysis

DatawerecodedandenteredusingtheCensusand Survey ProcessingSystemversion7.1.Statisticalanalysis was performed using the Statistical Package for Social Sciencesversion23.0(IBMSPSSInc.,Chicago,Illinois, USA) and STATA version 12.0(STATACORP, Texas, USA).Categoricalvariablesarepresentedusingfrequency (percentage) while continuous variables are summarized withtheirmedian(interquartilerange,[IQR].

Edinga-Melengeetal.BMCClinicalPathology (2019) 19:4Page2 of 9

TheKolmogorovSmirnovtestwasusedtoassessthe normality of continuous variables’ distributions. Reference intervals(RIs) were determined by the nonparametric methodasdescribedintheIFCCguidelines (11).

This method was used to determine the 2.5 and 97.5 percentilesandtherespective90%confidenceintervals (CI) around these estimates. The Mann–WhitneyU-test and the Kruskal-Wallis H-test were used for bivariate analyses, to compare the distributions of continuous variables, consideringthatthesevariablesdidnotfollowa

Gaussianshape.Forthesamereason,itistheSpearman correlationtest(withitsrho(ρ)coefficient) that was used

to investigate existence of any correlation between continuousvariablesincludingserumcystatinC,serum creatinineandage.Furthermore,weuseda25th,50th and 75thpercentilequantileregressionanalysistoidentify any factor likely influencing serum cystatin C or serum creatininereferenceintervalsinamodelincluding theage, sex,andBSA.Statisticalsignificancewasset atap-value

lower than 0.05.

Results

Atotalof 485healthysubjectswerescreenedofwhom 104 wereexcludedbecauseofunderlyingdiabetesmellitus, pre-diabetes,hypertensionorabnormaldipstick urinetest. Thereferencepopulationcomprised 381 healthyadults (including49.1% females)agedbetween 18and71years oldwithamedianageof28years[IQR 23-40].Therewere nodifferencesinthedistributionof agebetweenmaleand femaleparticipants(p=0.290).By contrast,maleshad significantlyhigherBSAvaluesthan females:p=0.002 (Table 1).

Thenon-parametricreferenceintervalsforserum cystatin Cwere0.57-1.03mg/Lforwomenand0.70-1.10mg/Lfor

50 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

Jobert Richie Nansseu

men; the reference intervals for the whole studypopulation were0.60 -1.10mg/L(Table 1).For serumcreatinine,these intervalswere0.58-1.08mg/dL forwomen,0.74-1.36 mg/dLformen,and0.61-1.30 mg/dLforallsubjects (Table1).As compared to women,menhadsignificantly highertitersofserum cystatinC(median0.90vs.0.80 mg/L; p<0.001;Table 1) thanwomen,exceptforthose aged50yearsandabove (p=0.125;Table 2).Similarly, men had significantly higher serum creatinine values (median1.06vs.0.79mg/dL; p<0.001;Table 1)than women, this tendency being thesameinallagegroups (Table3).

Additionally, serum cystatin C levels were higher in persons aged 50 years and above compared to their counterparts agedlessthan50yearsold(p<0.001;Table 2);on thecontrary,thisdifferencewasnotobservedwith serum creatininevalues(p=0.491;Table 3).Moreover, we found apositiveandsignificantcorrelationbetweenserum cystatinCandserumcreatininebothinfemales(p = 0.55; p

<0.001),in males(p =0.39 < 0.001) and in the total study population(p = 0.58;p<0.001).

Furthermore,thecorrelationbetweenserumcystatin C logarithmically-transformedvaluesandagewasweak and non-significantinmales(p=- 0.006,p=0.930;Fig. 1 a),but becamesignificantinfemales(p= 0.265, p< 0.001;Fig. 1b. Contrariwise,thecorrelationbetween serumcreatinine logarithmically-transformed values and age was significantinmales(p = 0.162, p=0.023;Fig. 2a),but insignificantinfemales(p = 0.127, p=0.082; Fig.2b).On theotherhand,resultsofthequantile regressionwhichare presentedinTable4showed thatacrossthevariousmodels, thesexremainedthe onlyfactorlikelyinfluencingboth serumcystatinC andserumcreatininevalues.Theage seemedtocontribute inexplainingserumcystatinCvalues inthe 75thpercentilequantileregressionmodel,which was identicalforserumcreatininevalues.TheBSAwas contributiveinexplainingserumcreatininevalues onlyin the50thpercentilequantileregressionmodel (Table 4).

Table 1 Reference intervals for serum cystatin C and serum creatinine according to sex

Parameter All (n = 381) Males (n = 194) Females (n = 187) p*
Age (years) 28 [23–40] 28 [24–40] 26 [22–43] 0.290
BSA (m2) 1.68 [1.55–1.79] 1.70 [1.81] 1.65 [1.52–1.76] 0.002*
Serum cystatin C (mg/L)
Median [IQR] 0.80 [0.70–0.90] 0.90 [0.80–1.00] 0.80 [0.70–0.90] < 0.001
2.5th percentile (90%CI) 0.60 (0.60–0.61) 0.70 (0.60–0.70) 0.57 (0.50–0.60)
97.5th percentile (90%CI) 1.10 (1.10–1.11) 1.10 (1.10–1.20) 1.03 (1.00–1.10)
Serum creatinine (mg/dL)
Median [IQR] 0.92 [0.77–1.06] 1.06 [0.96–1.14] 0.79 [0.71–0.88] < 0.001
2.5th percentile (90% CI) 0.61 (0.59–0.64) 0.74 (0.70–0.80) 0.58 (0.54–0.61)
97.5th percentile (90% CI) 1.30 (1.28–1.35) 1.36 (1.30–1.45) 1.08 (1.02–1.21)

BSA body surface area, CI confidence interval, IQR interquartile range, SCysC serum cystatin C, SCr serum creatinine; †The Mann-Whitney U-test was used for variable comparisons; *p < 0.05

Table 2 Reference intervals for serum cystatin C by age and sex

Age N=381 Serum cystatin C (mg/L)
(years) Median (IQR) 2.5th percentile (90%CI) 97.5th percentile (90%CI) p„©
< 20 Male: 9 0.9 (0.85.1.05) 0.7 (0.70.0.90) 1.1 0.003
Female: 14 0.75 (0.70.0.80) 0.6 (0.60.0.70) 0.9
All: 23 0.8 (0.70.0.90) 0.6 (0.60.0.70) 1.1
[20.30] Male: 95 0.9 (0.80.1.0) 0.6 (0.60.0.70) 1.1 (1.10.1.10) < 0.001
Female: 89 0.7 (0.70.0.80) 0.6 (0.50.0.60) 1.0 (0.90.1.00)
All: 184 0.8 (0.70.0.90) 0.6 (0.60.0.60) 1.1 (1.10.1.10)
[30.40] Male: 40 0.9 (0.80.0.90) 0.7 (0.70.0.80) 1.0 (1.00.1.00) 0.001
Female: 31 0.8 (0.60.0.80) 0.5 (0.50.0.50) 1.0
All: 71 0.8 (0.80.0.90) 0.5 (0.50.0.58) 1.0 (1.00.1.00)
[40.50] Male: 32 0.9 (0.80.0.98) 0.7 (0.70.0.70) 1.1 0.002
Female: 21 0.8 (0.70.0.90) 0.6 (0.60.0.70) 1.0
All: 53 0.8 (0.80.0.90) 0.6 (0.60.0.70) 1.0 (1.00.1.00)
50 Male: 18 1.0 (0.88.1.0) 0.7 (0.70.0.80) 1.2 0.125
Female: 32 0.9 (0.80.0.90) 0.6 (0.60.0.80) 1.1
All: 50 0.9 (0.80.1.00) 0.7 (0.60.0.73) 1.2 (1.10.1.20)

CI confidence interval, IQR interquartile range. Some 90% confidence intervals are not presented due to the small number of participants in corresponding age groups. The Mann-Whitney U-test was used to compare the distribution of serum cystatin C values between males and females. The difference between agegroups was significant when using the Kruskal-Wallis H-test (p < 0.001)

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Reference intervals for serum cystatin C…

Discussion

In agreement with IFCC recommendations [11], the reference intervals for serum cystatin C and serum creatinine were determined in the present study among a healthy Cameroonian adult population. Our results revealed that the reference intervals for serum cystatin C

varied between 0.6 and 1.1 mg/L, with men having higher values than women (p < 0.001), except in the 50+ years age group. Concerning serum creatinine, the reference intervals ranged from 0.6 to 1.3 mg/dL; similarly, men had significantly higher levels than women (p < 0.001) across all age groups. Participants

Table 3 Reference intervals for serum creatinine by age and sex

Age N=381 Serum creatinine (mg/dL)
(years) Median (IQR) 2.5th percentile (90%CI) 97.5th percentile (90%CI) p„©
< 20 Male: 9 1.09 (1.00.1.15) 0.80 (0.80.1.04) 1.18 < 0.00
Female: 14 0.76 (0.72.0.79) 0.66 (0.66.0.70) 0.88
All: 23 0.79 (0.75.1.08) 0.66 (0.66.0.70) 1.18
[20.30] Male: 95 1.03 (0.94.1.12) 0.80 (0.70.0.85) 1.30 (1.29.1.35) < 0.001
Female: 89 0.79 (0.71.0.87) 0.60 (0.54.0.61) 1.07 (1.01.1.28)
All: 184 0.91 (0.78.1.05) 0.61 (0.59.0.65) 1.30 (1.26.1.33)
[30.40] Male: 40 1.06 (0.95.1.13) 0.75 (0.69.0.81) 1.29 < 0.001
Female: 31 0.73 (0.65.0.83) 0.57 (0.57.0.60) 1.26
All: 71 0.94 (0.74.1.08) 0.58 (0.57.0.62) 1.27 (1.22.1.32)
[40.50] Male: 32 1.08 (0.99.1.17) 0.68 (0.68.0.74) 1.45 < 0.001
Female: 21 0.74 (0.69.0.90) 0.51 (0.51.0.66) 1.02
All: 53 0.99 (0.74.1.10) 0.61 (0.51.0.66) 1.38 (1.24.1.45)
≥50 Male: 18 1.17 (0.99.1.30) 0.79 (0.79.0.96) 1.52 < 0.001
Female: 32 0.85 (0.79.0.93) 0.60 (0.60.0.65) 1.05
All: 50 0.93 (0.81.1.06) 0.64 (0.60.0.66) 1.43 (1.33.1.52)

CI confidence interval, IQR interquartile range. Some 90% confidence intervals are not presented due to the small number of participants in corresponding age groups. ┼ The Mann-Whitney U-test was used to compare males and females; the difference in the distribution of serum creatinine values between age-groups was not significant with the Kruskal-Wallis H-test (p = 0.491)

Fig. 1 a Relationship between serum cystatin C (log) values and age in males [(n = 194); y = 0.0009x – 0.0114, ρ = 0.162 (p = 0.024)]. b Relationship between serum cystatin C (log) values and age in females [(n = 187); y = 0.0016x – 0.164, ρ = 0.265 (p < 0.001)]

52 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

 

aged 50 years and above had higher serum cystatin C values than those aged less than 50 years (p < 0.001), which was not the case for serum creatinine values (p = 0.491). Moreover, the correlation between serum cystatin C and serum creatinine was positive and significant (ρ = 0.58; p < 0.001) and the quantile regression pointed mostly the sex, and to a certain extent the age and BSA as independent factors susceptible of influencing serum cystatin C and/or serum creatinine values. Reference intervals for serum cystatin C obtained in this study (0.60–1.10 mg/L) are in compliance with those from previous studies which have also used turbidimetric assay. For instance, Köttgen et al. recorded in a US population a reference interval varying between 0.61– 1.04 mg/L; Okonkwo et al. in a Nigerian population recorded a reference interval ranging between 0.64–1.12mg/L and Li et al. in a Chinese population recorded a reference interval varying from 0.60 to 1.08 mg/L [12–14]. By contrast, the reference intervals for serum creatinine obtained in this study (0.61–1.3 mg/dL)

Jobert Richie Nansseu

seem to differ from that of Caucasians. Indeed, Pottel et al. found reference intervals around 0.48–0.93 mg/dL in women and 0.63–1.16 mg/dL in men within a healthy adult Caucasian population [15]. These intervals concur with those of Ceriotti et al. obtained in a multicenter analysis of three studies based on Caucasian adults. In this study indeed, the reference intervals for serum creatinine varied between 0.45–0.92mg/dL in women and 0.59–1.05mg/dL in men [16]. These differences could be explained by the fact that the measurement of serum creatinine used enzymatic methods in the two studies just cited, which could give slightly lower values than colorimetric assays that were used in our study. Additionally, evidence has accumulated that black people have a more important lean tissue mass and a lower GFR compared to Caucasians [3, 17]. However, our results corroborate those from other African authors such as Sakande et al. in Burkina Faso and Dosoo et al. in Ghana. Indeed, Sakande et al. reported reference intervals ranging between 0.63–1.41 mg/dL in

Fig. 2 a Relationship between serum creatinine (log) values and age in males [(n = 194); y = 0.0009x – 0.0114, ρ = 0.162 (p = 0.024)]. b Relationship between serum creatinine (log) values and age in females [(n = 187); y = 0.0007x – 0.129, ρ = 0.127 (p = 0.082)]

men and 0.45–1.24 mg/dL in women; reference intervals obtained by Dosoo et al. were 0.63–1.35 mg/dL in men and 0.60–1.20mg/dL in women [18, 19]. Furthermore, Lim et al. conducted a study among afro-Americans and found similar results with men having serum creatinine reference intervals around 0.73–1.45 mg/dL and women, around 0.52–1.15 mg/dL [20]. The sex-related differences in the non-parametric reference intervals for serum creatinine are in line with previous studies and reinforced by results of our quantile regression analysis indicating that the sex influenced serum creatinine values across all models, while adjusting for age and BSA. Indeed, muscular mass is

higher in men compared to women [3, 5, 6, 21]. Concurring with previous findings, our results indicate that serum cystatin C levels seem to be slightly affected by factors such as sex and age [22–24]. Pottel et al. showed for instance that cystatin C increases with age, after the age of 70 years old [24]. The influence of sex on serum cystatin C levels is still unclear. In fact, some studies have reported that serum cystatin C levels are independent of sex unlike other studies have claimed that sex influences significantly serum cystatin C values [13, 23, 25–28]. In our study for instance, we found that the sex constituted one independent explanatory factor for serum cystatin C values, whatever

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Reference intervals for serum cystatin C…

Table 4 Regression coefficients and p-values for the 25th, 50th and 75th percentiles quantile regression models

Serum cystatin C Serum creatinine
25th 50th 75th 25th 50th 75th
Sex -0.1 (< 0.001)* – 0.1 (< 0.001)* -0.09 (< 0.001)* -0.24 (< 0.001)* -0.28 (< 0.001)* -0.27 (< 0.001)*
Age 7.67e-19 (1.000) – 3.36e- 18 (1.000) 0.003 (0.001)* 0.0007 (0.171) 0.001 (0.162) 0.002 (0.031)*
BSA 9.39e-17 (1.000) 7.35e-16 (1.000) 0.038 (0.475) 0.08 (0.072) 0.15 (0.001)* 0.127 (0.073)
*p < 0.05
the quantile regression model considered; additionally, perhaps because they used the Pearson correlation test and
serum cystatin C levels were 11% higher in men than in rescaled their biomarkers. The inconsistent influence of
women (0.90 mg/L vs 0.80 mg/L; p < 0.001). These results age on both serum cystatin C and serum creatinine values
corroborate those from Köttgen et al. in the US who was observed after applying the quantile regression
reported a difference of 8% between males and females analysis. Indeed, we found that age influenced
[12]. However, Al Wakeel et al. in a Saudi adult population significantly both serum cystatin C and serum creatinine
reported lower serum cystatin C levels in men compared to values only at the 75th percentile quantile regression
women (0.72 mg/L vs 0.77 mg/L; p < 0.001) as well as Li et model, the estimator being insignificant at the 25th and
al. in China (0.84 mg/L vs 0.85 mg/L; p < 0.05) [14, 29]. In 50th percentile models. We need further well-designed
the Saudi study, women had higher body mass index that studies to better investigate the influence of age (and BSA)
men and the positive correlation between serum cystatin C on serum cystatin C and serum creatinine values in our
and body mass index could have explained the higher context. However, our findings need to be interpreted in the
serum cystatin C levels in women [13, 29, 30]. In Li et al.’s context of some limitations, mainly occurring from the
study, the sex difference was observed only between 30 non-random sampling method used and single
and 60 years [14]. Likewise, we found in our study that measurement of serum cystatin C and serum creatinine. In
from 50 years old and beyond, differences of serum fact, the representativeness of our study population and
cystatin C levels between men and women became non- generalization of our results to the entire Cameroonian
significant (median 1.00 vs 0.90 mg/L; p = 0.125) while the population would have been better obtained with
difference persisted for serum creatinine levels (median randomization. Nevertheless, we selected the most
1.17 vs 0.85 mg/dL; p < 0.001). Actually, the influence of populated health districts among the 6 that compose
sex on serum cystatin C levels seems non-significant with Yaoundé, the cosmopolitan capital city of Cameroon. On
increasing age, suggesting a physiological or pathological the other hand, participants were selected on the basis of
condition which should be more investigated in elderly. their normal renal function which could be attested only by
Further studies are warranted in this respect. On the other measurement of GFR by the gold standard (inuline).
hand, subjects aged 50 years and over had 11% higher Nonetheless, the absence of risk factors for kidney disease
serum cystatin C levels compared to lower age groups and the normal clinical and biological tests performed
(0.90 vs. 0.80; p < 0.001). Concurring with these results, among our participants could be some indirect indicators
several other studies have demonstrated an increase in of normal kidney function. Furthermore, we used rigorous
cystatin C values above a threshold age varying from 40 to statistical procedures and applied the IFCC guidelines to
70 years [12, 14, 24, 29–32]. The higher levels of serum depict our estimates. Notwithstanding and to the very best
cystatin C in older subjects could be due to the of our knowledge, this is the first
physiological decrease in GFR which starts from 40 years study providing the reference values for serum cystatin C
[33]. Serum creatinine levels are also expected to increase and serum creatinine in Cameroon, which could be
around the same age (≥50 years); however, we observed translatable to similar sub Saharan African populations.
that the distributions of serum creatinine values were Conclusion
similar across the various age groups (p = 0.491).
Likewise, Pottel et al. using a Caucasian population This study depicted serum cystatin C and serum creatinine
noticed that between 20 and 70 years old, the mean serum reference intervals in a healthy adult Cameroonian
creatinine level was stable [24]. This could be explained by population. Men had significantly higher levels of both
the drop in creatinine rate production due to reduction in biomarkers compared to women. Subjects aged 50 years
the muscle mass which appears concomitantly with the old and above had significantly higher serum cystatin C
decrease in GFR [33]. The physiological increase in values than those aged less than 50 years old. Therefore,
creatinine levels will be therefore lately observed around the interpretation of both biomarkers should probably take
65–70 years [3, 15]. We found a positive and significant into account the patient’s sex and to a certain extent, his/her
correlation between serum cystatin C and serum age (and/or body surface area) for an appropriate diagnosis
creatinine, both in males (ρ = 0.39, p < 0.001), in females (ρ of a renal disease. Moreover, it is hoped that our data
= 0.55, p < 0.001) and in the total population (ρ = 0.58; p < stimulate further research on a larger population that will
0.001). These findings mirror those from Pottel et al. who be more representative of the whole country’s diversity.
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  10. Solberg HE. Approved recommendation on the theory of reference values. Part 5. Statistical treatment of collected reference values. Determination of reference limits. Clin Chim Acta. 1987;170:S13–32.
  11. Köttgen A, Selvin E, Stevens LA, Levey AS, Van Lente F, Coresh J. Serumcystatin C in the United States: the third National Health and nutrition examination survey (NHANES III). Am J Kidney Dis. 2008;51:385–94.
  12. Ijoma C, Ijoma U, Okonkwo I, Ogbu I, Ulasi I. Reference intervals for serumcystatin C and creatinine of an indigenous adult Nigerian population. Niger J Clin Pract. 2015;18:173.
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Jobert Richie Nansseu

et al. Reference intervals and factors contributing to serum cystatin C levels in a Chinese population: reference intervals of cystatin C. J Clin Lab Anal. 2012;26:49–54.

  1. Pottel H, Vrydags N, Mahieu B, Vandewynckele E, Croes K, Martens F. Establishing age/sex related serum creatinine reference intervals from hospital laboratory data based on different statistical methods. Clin Chim Acta. 2008;396:49–55.
  2. Ceriotti F, Boyd JC, Klein G, Henny J, Queraltó J, Kairisto V, et al. Reference intervals for serum creatinine concentrations: assessment of available data for global application . Clin Chem . 2008;54:559–66.
  3. Wang X, Xu G, Li H, Liu Y, Wang F. Reference intervals for serum creatinine with enzymatic assay and evaluation of four equations to estimate glomerular filtration rate in a healthy Chinese adult population. Clin Chim Acta Int J Clin Chem. 2011;412:1793–7.
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  5. Dosoo DK, Kayan K, Adu-Gyasi D, Kwara E, Ocran J, Osei-Kwakye K, et al. Haematological and biochemical reference values for healthy adults in the Middle Belt of Ghana. PLoS One. 2012;7:1–9.
  6. Lim E, Miyamura J, Chen JJ. Racial/ethnic-specific reference intervals for common laboratory tests: a comparison among Asians, blacks, Hispanics, and white. Hawaii J Med Public Health. 2015;74:302–10.
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  9. Delanaye P, Chapelle J-P, Gielen J, Krzesinski J-M, Rorive GL. intérêt de la cystatine C dans l’évaluation de la fonction rénale. Néphrologie. 2003;24:457–68. Edinga-Melenge et al. BMC Clinical Pathology (2019) 19:4 Page 8 of 9
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  2. Uhlmann EJ, Hock KG, Issitt C, Sneeringer MR, Cervelli DR, Gorman RT, et al. Reference intervals for plasma cystatin C in healthy volunteers and renal patients, as measured by the Dade Behring BN II system, and correlation with creatinine. Clin Chem. 2001;47:2031–3.
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An Enhanced Computer Vision Platform for Clinical Diagnosis of Malaria

Arnon Houri-Yafin, Yochay Eshel, Natalie Lezmy, Sarah Levy-Schreier,

Caitlin Lee Cohen, Joseph Joel Pollak1 and Seth J. Salpeter

Sight Diagnostics Ltd., Israel

Benedicta Larbi and Emma Wypkema

Department of Clinical Hematology Lancet Laboratories, Lancet Corner, South Africa

Veena Dayan

Department of Parisitology, City Hospital Mangalore, India

All Correspondences to: Seth Salpeter, Sight Diagnostics Ltd., Jerusalem Technology Park, Jerusalem 96951, Israel, E-mail: seth@sightdx.com

ABSTRACT

Accurate malaria diagnosis is necessary to prevent unnecessary deaths and curb malaria drug resistance related to unnecessary treatment. While numerous diagnostic assays exist, the need for a low-cost, rapid and highly accurate malaria test remains. Here we evaluate the diagnostic performance of a computer vision platform, the Sight Diagnostic P2 device for malaria diagnosis, speciation and parasite quantification. The trial was conducted at two centers on Plasmodium falciparum and Plasmodium vivax samples, using different testing protocols: 374 samples were collected at City Hospital Mangalore India and 167 samples were collected at Lancet Laboratories Johannesburg South Africa. At City Hospital, the device diagnoses were compared to RT-PCR results while at Lancet Laboratories the device diagnoses were compared to a panel of tests provided by the clinic. For identification of malaria, the device demonstrated a sensitivity of 97% and a specificity of 99.5% at City Hospital India, and a sensitivity of 97.8% and a specificity of 97.5% at Lancet Laboratories Johannesburg. For speciation, the device correctly identified 87.5% for Plasmodium Vivax and 93.5% for Plasmodium Falciparum at City Hospital India. Lastly, comparing the device parasite count with that of trained microscopes, produced an average pearsons correlation of 0.87.

Keywords: Malaria; Diagnostic; Computer vision; Machine Learninga

Introduction

ccurate diagnosis of malaria is imperative to Areduce morbidity, prevent resistance to anti-malarials, and limit the number of adverse treatment effects from unnecessary use [1]. Furthermore, many studies show that infectious malaria carriers maintain a very low parasitemia, making sensitive detection technologies imperative for treatment targeted at epidemiologic control [2,3]. Many governmental health organizations now require patients to undergo malaria testing before receiving any anti-malarial medicine. As a result, the WHO forecasts an increase in global demand for malaria tests from 500 million in 2012 to nearly 1 billion

tests by 2020 [1,4].

Due to increased demand for malaria tests, reliable, simple and highly accurate malaria diagnostic is needed. Globally, only 77% of suspected cases in the public sector are tested, while in Africa only 47% of cases are assayed [5]. A recent report showed that in some regions 81% of people taking ACT therapy are not infected, while only 31% of the positive cases received the treatment [6]. This glaring disparity not only leaves the needy untreated but encourages the further development of drug resistant malaria strains.

While new diagnostic modalities for malaria have emerged in recent years, none have the ideal set of test characteristics. According to the World Health Organization, an ideal test would be inexpensive,

consistent, highly-sensitive, adequately specific, quantitative, and species-differentiating. Microscopy remains the gold standard malaria test worldwide [7,8], as it supports direct parasite identification and also provides monitoring of systemic inflammation and its response to therapy [9]. However, microscopy can be very inaccurate, needs extensive analysis, and requires highly trained staff [10,11]. Notably, malaria is associated with systemic spiraling of innate inflammation and additional blood abnormalities, further complicating microscopy examination [12]. Numerous reports have also shown inconsistent sensitivity of microscopist due to the high volume of tests and varied level of skill among malaria technicians [13,14]. Rapid diagnostics tests (RDTs) continue to increase in popularity and market share as they have significantly improved the diagnosis of malaria in remote, inaccessible areas [15]. However, RDTs have significant limitations that make many practitioners wary of their use, including decreased sensitivity at low parasitemia, inability to quantitate parasite burden, and inconsistencies between brands in their ability to detect and differentiate different malaria species [16,17]. Recent improvements to malaria diagnostic technologies include Polymerase Chain Reaction (PCR) and loop-mediated isothermal amplification (LAMP), which offer superior sensitivity, speciation and parasitemia but are impractical for the vast majority malaria-endemic areas [18,19].

Malaria diagnosis using computer vision offers a potential solution to the shortcomings of other technologies. An

40 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

An Enhanced Computer Vision Platform…

automated microscopist maintains the advantages of a microscopist with significant improvements in speed, cost, and consistency. Previous attempts at a creating a computer microscopist have not surpassed the development stage [20,21]. A recent report from our group, described a clinically available automated microscopist which was tested with the National Institute of Malaria Research India

  1. The device, the SightDx P1 malaria platform, was tested on 431 patients and demonstrated a sensitivity of 97.05%, and a specificity of 96.33% when compared with PCR. Furthermore, the device was able to accurately speciate 73.3% of the PCR Plasmodium falciparum and 91.4% of the PCR Plasmodium vivax samples, and showed a parasitemia correlation with microscopists of 0.89.

Here, we present an enhanced version of the Sight Diagnostic malaria device, the SightDx P2 platform for malaria detection. The device is intended for laboratories performing high volumes of malaria tests as it is capable of scanning a sample in 4 minutes and can hold up to 30 tests. The system has dimensions of 45 x 50 x 58 cm (DxWxH) and can easily fit onto a standard laboratory bench top with minimal installation requirements.

In the following report, we describe the results of clinical studies performed in Mangalore India, and Johannesburg South Africa to evaluate the sensitivity, specificity, speciation and parasite count calculation as compared to standard diagnostic procedures.

Methods

Study design

The study was a double center, prospective, non-randomized, non blinded study conducted at City Hospital, Mangalore India with 374 blood samples from clinically-suspected malaria patients, and at Lancet Laboratories Johannesburg South Africa on 167 clinically suspected malaria patients.

Study Procedures

City Hospital, Mangalore India: Determination of eligibility for malaria treatment was solely based on the clinic’s standard diagnosis protocol and the patients course of treatment and was not altered due to the study or the SightDx diagnostic device. In most cases blood was scanned by the device within 24 hours of sampling. Samples more than 48 hours old were not included in the study. In addition, 100 μL of blood was collected on GE Healthcare FTA Whatman filter paper spots for RT-PCR evaluation. RT-PCR results were considered the standard of comparison for determining the sensitivity, specificity and speciation of the various methods. Lancet Laboratories, Johannesburg South Africa: Samples were provided from malaria tests performed at Lancet Laboratories Johannesburg and at surrounding Lancet Laboratory clinics in South Africa. Samples were tested on the Sight Diagnostic device within 1 week of drawing. RDT and microscopy were performed on all samples. Discrepancies between these tests were evaluated by PCR. Positive samples which were not Plasmodium falciparum or had a parasitemia under 1000 parasites/μL also underwent PCR. All negative samples were reviewed with QBC.

Laboratory Methods

Sight Diagnostic Device Analysis

In all locations digital imaging scanning was carried out onsite. To begin sample diagnosis, 5 μL of patient blood was mixed with a fluorescent dye solution that stained intracellular DNA and RNA. The sample was then loaded into a plastic cartridge and incubated for 5 minutes, during which time the cells formed a monolayer. The stained cells were then excited using 3 different LED light sources (370 nm, 475 nm and 530 nm) after which the imaging system recorded 600 images analyzing ~1.8 million cells. The total scan time per sample was 4 minutes and the device held up to 30 samples which can be loaded in batch. Samples which registered an error on the device due to incorrect user preparation were repeated. Computer vision and statistical models were used to detect the malaria parasites. Using statistical models, the device determined infection status, parasitemia levels, and species.

Parasitemia

Parasitemia counts were performed on 24 positive samples at Lancet laboratories Johannesburg. An expert microscopist analyzed 10 fields at 100X with approximately 100 RBCs counted per field. Parasitemia was calculated as a ratio of infected RBCs to total RBCs.

Real Time PCR Analysis

For PCR experiments performed on samples from City Hospital India, a whole punch was removed from the blood spot on the GE FTA Whatman paper and eluted as previously reported [23]. Real time PCR was performed with Fast Syber Green Master Mix at a volume of 10 μL (Applied Biosystems) using previously published primer sequences [24] for identifying falciparum, vivax and for general Plasmodium (Plu). All reactions were performed in 384 well qPCR plates (Bio-Rad) on a CFX384 real time PCR machine from Bio-rad.

Results

The Sight Diagnostic P2 malaria scanning device is a desktop system for computerized malaria diagnostics (Figures 1A and 1B). The stained blood is loaded into a cartridge which holds five patients samples. To evaluate device performance in a clinical setting, 374 samples were collected and scanned at City Hospital Mangalore and 167 samples were collected and scanned at Lancet Laboratories Johannesburg.

Figure 1: The SightDx Malaria Platform. (A) The P2 malaria scanning device. (B) The loading cartridge holds 5 patient samples.

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Sensitivity and specificity

Sensitivity and specificity were analyzed for all trials (Table 1). For samples scanned at City Hospital India device results were compared to qPCR while for samples scanned at Lancet Johannesburg device results were compared to a final diagnosis based on a combination of several malaria diagnostic assays (Table 1). At City Hospital Mangalore sensitivity was calculated as 97%

Seth J. Salpeter

(167/172) and specificity was calculated as 99.5%

(201/202). For samples scanned at Lancet Laboratories Johannesburg sensitivity was 97.8% (46/47) while specificity was 97.5% (117/120). Positive predictive values (PPV) were 99.4% at City Hospital and 93.8% at Lancet Laboratories, and negative predictive values (NPV) were 97.5% at City Hospital and 99.1% at Lancet Laboratories.

Sensitivity Specificity
Percent Ratio 95% CI Percent Ratio 95% CI
City Hospital India 97% 167/172 0.934-0.988 99.50% 201/202 0.972-0.999
Lancet Labs South Africa 97.80% 46/47 0.843-0.994 97.50% 117/120 0.929-0.991

Speciation

Speciation studies were conducted on samples provided at City Hospital Mangalore (Table 2). At City Hospital, the device distinguished between P.v (Plasmodium vivax ) and P.f (Plasmodium falciparum ) and results were compared to qPCR analysis. A total of 167 samples were identified as positive by the device and were analyzed for species type. The device correctly identified samples with Plasmodium vivax at 87.5% sensitivity (119/136) and Plasmodium falciparum at 93.5% sensitivity (29/31).

City Hospital (India)
Percent Ratio 95% CI
Plasmodium Vivax 87.50% 119/136 0.809-0.92
Plasmodium Falciparum 93.50% 29/31 0.793-0.982

Table 2: Speciation accuracy divided according to treatment groups. Speciation percentages of the trials from City Hospital India are presented in the table, as well as the specific number of patients and confidence Index.

Parasitemia

For cases diagnosed at Lancet Laboratories with thin smear microscopy, parasitemia was provided and compared to values from the device (Figure 2).

Figure 2: Micrscopist compared to the device. At Lancet Laboratories a trained microscopist analyzed 24 slides and the results were compared to the parasitemia reported by the device. The correlation between the two produced a Pearsons correlation coefficient of 0.87.

A comparison of the percentage of infected RBC determined by the microscopist and the device yields a Pearsons correlation coefficient of 0.87. The microscopist calculated parasitemia by analyzing the number of infected red blood cells out of the total number of blood cells.

Discussion

This study evaluated the SightDx P2 malaria detection platform, an enhanced computer vision platform for rapid and automated malaria diagnostics. Previous attempts to develop vision based malaria detection devices have had varying levels of success [21,25-28]. While a specific report showed high sensitivity and specificity [29], others demonstrated relatively low performance numbers. Notably, these papers describe development stage technologies showing initial device construction or preliminary algorithm designs for malaria detection. Previous studies showed problems in cartridge design and focus mechanisms, yielding slow scanning times and poor results. While these reports used complicated microfluidics systems, our study presents an easy to use plastic cartridge which fills quickly upon loading using capillary forces activated by mixing the blood with our stain solution. Moreover, we have solved image focus difficulties, by implementing unique algorithms which allow the scanning system to quickly autofocus on each new field allowing for high quality images of all cells scanned. In a previous study [22] we presented the first clinically available computer vision based reader for malaria diagnostics. The P1 device showed a sensitivity of 97.05%, specificity of 96.33% and speciation of 73.3% Plasmodium falciparum and 91.4% for Plasmodium vivax

  • The P2 device features many functional and performance based improvements over the earlier system. While the P1 device holds only 5 patient samples, the P2 machine holds 30 samples and is capable of asynchronous batch loading. Moreover, the P1 device requires 8 minutes to scan a sample while the P2 device requires only 4 minutes, allowing for the rapid scanning of large volumes of specimens. On a performance level, the device showed a similar sensitivity at an average of 97.4% but a significantly improved specificity at an average of 98.5%. Speciation of Plasmodium vivax was comparable to the previous study at 87.5% while speciation of Plasmodium falciparum was significantly improved at 93.5%.

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Importantly, in contrast to the study on P1 which was performed only in India, the current trial was conducted on samples from both continental Africa and India. Numerous studies have shown the ability of strains of malaria to develop mutations causing significant difficulties in diagnosis [1,30,31]. In particular, it has been shown that RDTs which identify HRP-2 from Plasmodium falciparum can yield false negatives due to specific antigen mutations

  1. Specific regions are known to develop unique genetic variants of even the most standard species of Plasmodium. Our results confirm the devices ability to detect strains of malaria in variety of geographical regions. In the current study, the device maintains a limit of detection of 50 parasites/ μL. While the system is capable of identifying as few as 5 parasites/ μL the current algorithm only identifies a positive sample if it detects more than 50 objects identified as malaria parasites. This limitation was evident from the six cases of false negatives where the parasitemia was found to be under 50 parasites/ μL, explaining the misdiagnoses. Decreasing the current limit of detection causes an increase of false positives reported by the system. False positives have been found to be caused by particles that have flourescence morphology similar to the stained malaria within the RBC. The four false positives found in our study were determined to result from Howell Jolly bodies which are malaria-like DNA/RNA fragments found in RBCs. By collecting larger libraries of samples, as well as samples with Howell Jolly bodies, we will be able to apply machine learning to improve the accuracy of the algorithm classification and overall diagnosis. Additional data collection and algorithm design work will be necessary to further improve the differentiation between malaria and these objects to lower the limit of detection. Several device improvements are currently under development to strengthen diagnostic performance and provide additional clinical information to assist in patient treatment. Speciation for P.v was calculated at 87.5% and P.f at 93.5%, leaving room for increased accuracy. As the device speciation is based on a machine learning algorithm which improves with an increased database, the collection of additional scanned samples of both P.v and P.f should significantly improve speciation results. Moreover, to expand speciation capabilities to Plasmodium ovale , Plasmodium malarie and mixed infection a large library of P.o and P.m and mixed infection samples will need to be collected and analyzed. Additional studies will also be necessary which feature completely blinded data collection as well as PCR ground truth for all samples.

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Quantification bias in blood alcohol determination by headspace gas chromatography

Quantification bias in blood alcohol determination by headspace gas chromatography

Elia Mattarucchi, Carlo Peruzzo, Ramona Consuelo Maio, Ursula Andreotta, Marco Mario Ferrario

Ospedale di Circolo e Fondazione Macchi, Laboratory of Toxicology, Varese, Italy – 21100

Marco Mario Ferrario

Università dell’Insubria, Research Centre in Epidemiology and Preventive Medicine (EPIMED), Varese, Italy

All Correspondences to: Elia Mattarucchi Ospedale di Circolo e Fondazione Macchi, Laboratory of Toxicology, Varese, Italy – 21100

ABSTRACT

The quantification of blood alcohol concentration is routinely performed in many forensic laboratories by gas chromatography. In this short report, the influence of the matrix miss-match between samples and calibrators (e.g. the use of aqueous standards to quantify blood samples) and the actual efficiencies of different internal standards used to normalise the ethanol signal were considered. Test samples in blood and water were prepared from a certified reference standard of ethanol. The samples were analysed by gas chromatography using n-propanol and t-butanol as internal standards. The collected data provided evidences that a sub-optimal setting of the considered variables can be responsible for a quantification bias up to 15%. These issues are of practical relevance in the medico-legal sector and should be considered during the process of

method development.

KEY WORDS: blood alcohol concentration, calibrator, internal standard, gas chromatography, bias.

INTRODUCTION:

uman blood samples are the primary type of Hknown2, 3. Method development should start from evidence received for medico-legal testing of tuning of the most important parameters; in this alcohol

concentration by gas chromatography. context we feel that further attention should be paid Whole blood is the sample of choice, since plasma typically entails an overestimation1. For the purpose of forensic testing, different thresholds of alcohol concentration are set before a person is charged with a fine or crime. The consequent need to produce medico-legal reports underlines the importance of the careful development and validation of the analytical methods applied. The literature on this argument is vast and many possible influencing factors are to the choice of the internal standard and calibrators. The quantification of unknown samples is achieved by interpolation of a calibration curve. For this reason, reference materials at known concentrations of ethanol are widely available on the marketplace4. The majority of these materials are aqueous solutions produced according to ISO Guide 34 and satisfy the regulatory needs posed by ISO 17025. However, the opportunity to use aqueous calibrators to quantify blood samples should be carefully evaluated, even if an internal standard is used to correct for the differences among samples and calibrators5, 6. The actual efficacy of the internal standard needs to be experimentally verified. A “good” internal standard is c h e m i c a l l y s i m i l a r t o e t h a n o l w i t h o u t a n y chromatographic overlap with the target substance or other volatiles that could be present in the samples (e.g., acetone, methanol or acetaldehyde). n-propanol is commonly considered the best suited substance for this purpose, even if t-butanol is also used5,6. The samples can also be diluted

with water up to practically correct for the matrix mismatch (usually a 1:10 dilution is sufficient)7. However, this procedure inevitably increases the limit of quantification with evident constraints in dealing with the “zero tolerance” threshold posed for young drivers or commercial vehicle operators.

MATERIALS AND METHODS:

In this report we present some data retrieved from the development of the analytical procedure adopted at our laboratory. The method was arranged starting from the protocol suggested by the producer of the available equipment (i.e. a PerkinElmer Clarus 500 gas chromatographer equipped with a Turbomatrix40 headspace autosampler). The original parameters were: sample equilibration: 30 min 70 °C, vial pressurisation: 15 psi for 3 min, transfer line temperature: 110 °C, injection: 200 °C, oven: 40 °C isothermal, column: Elite-BAC1 (PerkinElmer Cod. N9316579), carrier gas: N2.n-propanol and t-butanol were both tested as internal standard. Samples were mixed with ammonium sulphate in order to increase the partition coefficient of alcohol in the gas phase and amplify the analytical response8. A fixed volume of sample (1 ml) was added to 0.9 ml of 1 M ammonium sulphate and to 0.1 ml of 8 g/L t-butanol or n-propanol (internal standard).

Two type of test samples were prepared: an intermediate series of sample at 0.30, 0.75, 1.50, 3.75, 7.50 g/L of ethanol in water were produced independently from a mother solution 15 g/L. All the samples (including the mother solution) were further diluted 1:5 in water and blood (previously checked for the absence of ethanol). At the end of the process, 2 independent series at 0.06, 0.15, 0.30, 0.75, 1.50 and 3 g/L in blood and water were obtained.

32 Nigerian Biomedical Science Journal Vol. 16 No 3 2019

Quantification bias in blood alcohol…

This dilution scheme ensured that each concentration was independent, and the matrix composition uniform within each series. The second type of samples consists of 4 solutions at the nominal concentrations of 0.5 and 2 g/L in water and blood. These samples were prepared by adding 100 and 400 µl of a 0.5 g/L ethanol standard to a final volume of 1 ml of water or blood.

RESULTS:

The first type of test samples described in the “materials and method” session (i.e. 2 independent series at 0.06, 0.15, 0.30, 0.75, 1.50 and 3 g/L of ethanol in blood and water) were prepared and analysed independently 10 times. For each repetition, blood and water samples were analysed within the same analytical session. For each concentration, it was expected that the average analytical response (i.e. the ethanol/internal standard area ratio) of the blood and water samples would be similar due to normalisation with the internal standard. However, a progressive response difference was observed (Table 1). The disparity becomes clearly significant among samples normalised by t-butanol, while it is not supported by a statistical evidence when n-propanol is used. In order to quantify the possible bias, test samples at the nominal concentrations of 0.5 and 2 g/L in water and blood were used (i.e. the second type of test samples described in the “Materials and methods” section). The 2 couples of samples were produced and analysed independently 10 times using a quantitative method calibrated by commercial certified reference materials based on water solutions. As reported in Table 2, the average error ranges from 2% at 0.5 g/L with n-propanol up to 16% at 2 g/L with t-butanol. The error is indeed negligible, if the analytical samples are matrix matched with the calibrators (i.e. water samples quantified against water calibrators).

DISCUSSION:

The presented data show how a matrix difference between samples and calibrators and/or the actual efficacy of the internal standard can bias the outcome of the alcohol test by gas-chromatography. These factors should be carefully considered during method development. Our data point out the superiority of npropanol for BAC determination. However, a few additional aspects should be considered, especially if t-butanol is selected as internal standard. As the error seems to increase with the amount of ethanol, the use of a fixed correction factor becomes difficult. On the other hand, the practice of making up “in-house” standards by spiking known concentrations of ethanol into “blank” blood samples can lead to regulatory problems associated with the medico-legal sector. The whole process should be carefully controlled and documented, even though it would be unrealistic for a medical laboratory to have enough resources to produce certified reference materials. A possible solution could be the use of commercial blood standards that are industrially manufactured by spiking ethanol into a blood matrix. However, the availability of such materials is still limited and the assigned concentration is usually an average value derived from a number of independent certified laboratories (usually these standards don’t comply with the ISO guide 34 requirements).

CONCLUSIONS:

In conclusion, this report offers some helpful thoughts on the calibration of the analytical methods used to assess blood alcohol concentration for medico-legal purposes. These considerations may seem trivial, but a recent comment by Schug9 shows that these analytical issues are still highly relevant. In our practice, the guidelines of the Italian Forensic Toxicologist Group don’t report any specific indication about the internal standard and the calibrators used for alcohol tests10 and the only way to control the risk of possible bias is to asses experimentally the real efficacy of such measures adopted to minimize the matrix mismatch and to participate periodically to suitable proficiency tests on blood samples.

Table 1. Analytical response of water and blood samples

t-BUTANOL

Blood samples Water samples

0.06 0.0096 0.0009 0.0130 0.0016 -0.0034 3.E-02
0.15 0.0301 0.0031 0.0323 0.0036 -0.0021 5.E-01
0.30 0.0746 0.0020 0.0712 0.0038 0.0034 2.E-01
0.75 0.2281 0.0022 0.1994 0.0034 0.0286 3.E-04
1.50 0.4736 0.0121 0.4073 0.0065 0.0663 1.E-03
3.00 0.9754 0.0407 0.8224 0.0148 0.1530 4.E-03
t-PROPANOL
Blood samples Water samples

0.06 0.0197 0.0012 0.0288 0.0006 -0.0091 1.E-02
0.15 0.0613 0.0034 0.0718 0.0017 -0.0105 6.E-02
0.30 0.1557 0.0136 0.1669 0.0159 -0.0112 5.E-01
0.75 0.4880 0.0287 0.4788 0.0245 0.0093 8.E-01
1.50 0.9824 0.0599 0.9699 0.0424 0.0124 8.E-01
3.00 2.0218 0.0969 1.9658 0.0551 0.0560 6.E-01

Average analytical response (ethanol/internal standard area ratio) of blood and water samples at different concentrations (0.06-3.00 g/L of ethanol) analysed using n-propanol or t-butanol as internal standards (n=10).

Nigerian Biomedical Science Journal Vol. 16 No 3 2019 33

 

Table 2. Ethanol quantification in water and blood samples

Internal Standard t-butanol

Cerilliant® calibrators (water)

Nominal Av. Quant. Av. Quant. Error on blood
Conc. g/L Water Blood samples samples %
samples
0.50 0.50 0.56 12%
2.00 2.01 2.32 16%

Internal Standard t-butanol

Cerilliant® calibrators (water)

Nominal Av. Quant. Av. Quant. Error on blood
Conc. g/L Water Blood samples samples %
samples
0.50 0.50 0.51 2%
2.00 2.01 2.11 5%

Average alcohol quantification of 0.5 and 2 g/L ethanol spikes in blood and water analysed using n-propanol or tbutanol as internal standard and Cerilliant® reference materials (water solutions) as calibrators (n = 10).

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