Expression of Vitamin D Receptor (VDR) in Triple Positive Breast Cancer Tissues

FASOGBON, Samuel Ayobami
Public Health In-vitro Diagnostic Control Laboratory, Medical Laboratory Science Council of Nigeria, Yaba-Lagos, Nigeria.
ABDULLAHI, Kabiru
Department of Histopathology, Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria.
OKORIE, Nnaemeka
N.K.S.T. Len Gabrielse’ School of Medical Laboratory Science Mkar, Benue State, Nigeria.
OGUNJIMI, Tolulope Samuel
Population Council, Nigeria, Yaba, Lagos State.
All Correspondences to: Fasogbon, Samuel Ayobami Public Health In-vitro Diagnostic Control Laboratory,
Medical Laboratory Science Council of Nigeria, 8 Harvey Road, Yaba, Lagos State, Nigeria. E-mail: samfash4best@gmail.com

ABSTRACT
Background: The study was to immunohistochemicallydetermine the Correlation of the Expression of Vitamin D Receptor (VDR) and Triple Positive Invasive Ductal Carcinoma (IDC) of Breast tissues. Materials and Method: Fifty six (56) archived female breast Invasive Ductal Carcinoma tissue blocks were experimented Immunohistochemically with ER, PR, HER-2 and VDR.Seven (7) confirmed cases of those that were positive to ER, PR, and HER-2 (Triple Positive) were correlated with VDR expressionand the results were compared. Result: The results show that there was no significant difference (P?0.05) found comparing the immunohistochemical expression of VDR with Triple positive IDC tissues. Conclusion: This study shows that no significant difference was found in the expression of VDR and triple positive IDC tissues; it can therefore be said that VDR can be of therapeutic target and of additional antibody in immunohistochemical diagnosis of breast cancer.

Keywords: Vitamin D receptor (VDR), Triple Positive, Breast cancer, Invasive ductal carcinoma (IDC).

INTRODUCTION

Breast cancer is the predominant malignancy where oncologists use predictive markers clinically to select treatment options, with steroid receptors having been used for many years. Immunohistochemistry has taken over as the major assay method used for assessing markers [1]. The surfacing of molecular technology has brought about new biomarkers along with immunohistochemical and serum biomarkers. Immunohistochemical markers [Estrogen receptor (ER), Progesterone receptor (PR), and Human epidermal growth factor receptor 2 (HER-2)] are often included inguiding treatment decisions, to classify breast cancer into subtypes that are biologically distinct and behave differently, and both as prognostic and predictive factors[1]. Invasive ductal carcinoma (IDC), also known as infiltrating ductal carcinoma, is cancer that began growing in the duct and has invaded the fatty tissue of the breast outside of the duct. The commonest type of breast cancer is IDC, which represent 80 percent of all breast cancer diagnoses [2]. Breast cancer is the most frequent cancer among women, being a heterogeneous disease, with distinct morphologies, metastatic behaviour and therapeutic response [3]. Approximately, 90% of breast cancer deaths are caused by local invasion and distant metastasis of tumor cells [4].According to [5], different types of this neoplasm exhibit variable histopathological and biological features, different clinical outcome and different response to systemic interventions. In fact, global gene-expression analyses have provided an appealing molecular classification for breast carcinomas, which is highly associated with patients’ prognosis [6].In the last decade; a major effort has been made to better inform the choice of the systemic treatment for breast cancer patients.
Vitamin D receptor (VDR) can be called Calcitriol receptor, and is also known as NR1I1 (nuclear receptor subfamily 1, group I, member 1), is a member of the nuclear receptor family of transcription factors [7]. Upon activation by vitamin D, the VDR forms a heterodimer with the retinoid-X receptor and binds to hormone response elements on DNA resulting in expression or transrepression of specific gene products. In addition to VDR regulation of transcriptional responses, it is also involved in microRNA-directed post transcriptional mechanisms [8]. In humans, the vitamin D receptor is encoded by the VDR gene [9].Glucocorticoids are known to decrease expression of VDR, which is expressed in most tissues of the body and regulate intestinal transport of calcium, Iron and other minerals [10].Also, it has recently been identified that VDR as an additional bile acid receptor alongside FXR and may function to protect gut against the toxic and carcinogenic effects some endobiotics[11]. Different researches have shown the evidence of linkage between vitamin D and breast cancer. Women who have breast cancer tend to have low levels of vitamin D in their body. Researchers have found how vitamin D might have a role in breast cancer. On the surface of a cell are also found Vitamin D receptors where they receive chemical signals. These chemical signals direct a cell to do something by attaching themselves to a receptor, for example to act in a certain way, or to divide or die. Vitamin D can bind to vitamin D receptors which are in breast tissue. These can oncogenes to die or stop growing, and can stop the cancer cells from spreading to other parts of the body. It is then said that vitamin D may help in preventing breast cancer, by making cells in the breast smarter. However, the relationship between breast cancer and vitamin D is complex, not fully understood, and is still being studied [12][13][14].A study carried out by [19] discovered VDR expression in patient of large population as inversely related with more aggressive breast cancer, but not with breast cancer survival outcomes. This research therefore, has set out to correlate the immunohistochemical expression of VDR with Triple Positive Breast IDC tissues.

MATERIALS AND METHODS
Area of Study
This study was carried out at Department of Histopathology, National Hospital Abuja, FCT, Nigeria. The Hospital serves most of the states of Nigeria and therefore serving a significant population of the region.

Ethical Consideration
Approval for this research work was obtained and given from the Health Research Ethics Committee (HREC) with assigned number NHA/EC/062/2015 of National Hospital Abuja, FCT, Nigeria.

Sample Size
Fifty six (56) archived female breast Invasive Ductal Carcinoma tissue blocks were experimented. The Seven (7) cases that wereconfirmed triple positive Invasive ductal carcinoma tissues were then obtained for correlation with VDR expression.

Sample Collection/Histopathological Procedures
Paraffin tissue blocks diagnosed of invasive ductal carcinoma of the female breast were experimented and those positive to ER, PR, and HER-2 (Triple positive) of the female breast were selected.
The tissue blocks were sectioned at not more than 2µm each. Seven (7) sections were obtained from each block from which one (1) section was used for Haematoxylin and Eosin staining technique while four(4) sectionswere treated with VDR, ER, PR and HER-2antibodies, while the other two (2) sections were used as negative and positive control.

Haematoxylin and Eosin Staining Technique
The sections were taken to water, stained using Harris Haematoxylin for 5minutes, washed in tap water then differentiated in 1% acid alcohol for few seconds. They were washed in tap water then blued in tap water for 10minutes. The sections were then counterstained in 1%
Eosin for 1minutes. They were then washed in tap water, dehydrated, cleared and mounted using DPX [15].

Immunohistochemical Technique
The method used is the Avidin Biotin Complex (ABC) method and the antibodiesthat are used were Novocastramanufactured. The antibody dilution factor used was 1:100 dilutions for all the antibody markers.
The processed tissues were sectioned at 2µm on the rotary microtome and placed on the hot plate at 700C for at least 1hour. Sections were brought down to water by passing them in 2 changes of Xylene, then 3 changes of descending grades of alcohol and finally to water. Antigen retrieval was performed on the sections by heating them on a Citric Acid solution of pH 6.0 using the Microwave at 1000C for 15minutes. The sections were equilibrated gradually with cool water to displace the hot Citric Acid for at least 5min. Peroxidase blocking was done on the sections by covering them with 3% hydrogen peroxide (H2O2) for 15min. Sections were washed with PBS and protein blocking was performed using avidin for 15min. Sections were washed with PBS and endogenous biotin in tissue was blocked using biotin for 15min. After washing with PBS, sections were then incubated with the respective diluted primary antibody, diluted 1:100 for 60 min. Excess antibodies were washed off with PBS and a secondary antibody (link) was applied on section for 15min. Sections were washed and the (label, in this case which is the Horseradish Peroxidase HRP) was applied on the sections for 15min. A working DAB solution is made up by mixing 1 drop (20µl) of the DAB chromogen to 1ml of the DAB substrate. This working solution was applied on sections after washing off the HRP with PBS for at least 5min. The brown reaction began to appear at this moment especially for a positive target. Excess DAB solution and precipitate were washed with water. Sections were counterstained with Haematoxylin solution for at least 2min and blued briefly. Sections were dehydrated in alcohol, cleared in Xylene and mounted in DPX [16].

Immunohistochemical Analysis
Cells with specific brown colours in the cytoplasm, cell membrane or nuclei depending on the antigenic sites were considered to be positive. The Haematoxylin stained cells without any form of brown colours were scored negative. Non specific binding/brown artifacts on cells and connective tissue were disregarded [16].

Statistical Analysis
Photomicrograph was basically used for correlating the expression and where necessary, Paired T-test statistics method was used to analyze the data generated.

RESULTS
Seven confirmed cases of triple positives tissue blocks already diagnosed as invasive ductal carcinoma of the female breast (Age mean=46.4) were used in the study. The results were presented in tables and figures below:

TABLE 1: CORRELATION OF IMMUNOHIS-TOCHEMICAL EXPRESSION OF VDR WITH TRIPLE POSITIVE (ER, PR AND HER2) IDC

 

DISCUSSION
There is no significant difference found; comparing VDR expression with Triple positives Breast IDC tissues which indicate that VDR can be used as an additional antibody in the immunohistochemical diagnosis of Breast IDC. This result is supported by earlier related study done; VDR expression was analyzed immunohistochemically in breast cancer patients in whichstrong VDR immunoreactivity was observed in breast cancer specimens, supporting the body of evidence that breast cancer may be a target for therapeutically applied vitamin D analogues[17].
This also support a study carried out that said; there are vitamin D receptors in breast tissue, and vitamin D can bind to these receptors. This can cause oncogenes to die or stop growing, and can stop the cancer cells from spreading to other parts of the body. Therefore, it is thought that vitamin D may help in protecting against breast cancer [12][18].This can also be related to the findings by [19] that VDR expression in patient of large population is inversely related with more aggressive breast cancer.

CONCLUSION
On the basis of this study and relevant literatures review, VDR has statistically significant correlations when compared with Triple positives Breast IDC tissues; it can therefore be said that VDR can be of therapeutic target and of additional antibody in immunohistochemical diagnosis of breast cancer. Further research is recommended to fully ascertain this fact.

REFERENCES

1. Walker R. A. (2007): Immunohistochemical markers as predictive tools for breast cancer Department of Cancer Studies & Molecular Medicine, University of Leicester, Robert Kilpatrick Building, Leicester Royal Infirmary, Journal of Clinical Pathology; 61:10.1136.

2. Johns Hopkins (2016): Inasive Ductal Carcinoma
(IDC); http://www.hopkinsmedic-ine.org/breast_center/breast_cancers_other_conditions/invasive_ductal_carcinoma.html . Sited November, 2016.

3. Ricardo, S., Vieira.,A.F.,Gerhard, R.,Leitão, D.,Pinto, R., Cameselle-Teijeiro, J, F., Milanezi, F., Schmitt, F., Paredes, J (2011): Breast cancer stem cell markers CD44, CD24 & ALDH: expression distinguishing intrinsic molecular subtype: Journal of clinical pathology; 64 (11): 937- 946.

4. Yifau, W., and Binhua, P.Z (2011): Epithelial-mesenchymal transition in breast cancer progression and metastasis: Chinical journal of cancer; 30 (9): 603-611.

5. Viale, G (2012): The current state of breast cancer classification: Annual Oncology; 23(10):207-210.

6. Sotiriou, C., Neo, S.Y., McShane, L.M (2003): Breast cancer classification and prognosis based on gene expression profiles from a population-based study. Proclaimed National Academic Science; 100:10393–10398.

7. Hosoi, T. (2002): Polymorphisms of vitamin D receptor gene. .Nippon Rinsho. 60Suppl 3: 106–110.

8. Uitterlinden AG, Fang Y, Van Meurs JB, Pols HA, Van Leeuwen JP (2004): “Genetics and biology of vitamin D receptor polymorphisms”. Gene 338 (2): 143–156.

9. Norman A.W. (2007).”Minireview: vitamin D receptor: new assignments for an already busy receptor”.Endocrinology147 (12): 5542–5548.

10. Bollag W.B. (2007): Differentiation of human keratinocytes requires the vitamin d receptor and its coactivators. Journal of Investigative Dermatology127(4): 748–750.

11. Salashor, S., and Woodgett, J.R. (2002): Links between AXIN and carcinogenesis; 58:225-236

12. Rose A.A.N, Elser C, Ennis M. (2013): Blood levels of vitamin D and early stage breast cancer prognosis: a systematic review and meta-analysis. Breast Cancer Resilience Treatment; 141:331-339.

13. Wang, D., Velez de-la-Paz OI, and Zhai JX, (2013): Serum 25-hydroxyvitamin D and breast cancer risk: a meta-analysis of prospective studies. Tumor Biology; 1-9.

14. Welsh J. (2012): Cellular and molecular effects of vitamin D on carcinogenesis. ActaBichemistry and Biophysics; 523b:107-114

15. Avwioro, O. G. (2014): Histochemistry and Tissue Pathology in; Principle and Technique Third edition, page 133-168

16. Marc (2009): Standard Immunohistochemistry
Staining Method; Avidin Biotin Complex (ABC) Method. IHC world life science products and sevices. http://www.ihcworld.com /_protocols/general_IHC/standard_abc_method.htm[cited: 20th October, 2016]

17. Friedrich M, Villena-Heinsen C, Tilgen W, Schmidt W, Reichrat J, Axt-Fliedner R. (2002): Vitamin D receptor (VDR) expression is not a prognostic factor in breast cancer. Anticancer Research May-Jun; 22(3):1919-1924.

18. Fasogbon S.A.; Okechi O.O.; Adisa J. O.; and Madukwe J. U. Immunohistochemical Correlation between the Expression of Vitamin D Receptor (VDR) and Triple Negative Invasive Ductal Carcinoma (IDC) tissues; Am. J. Biomed. Sci. 2017; 9(4), 237-243 doi:10.5099/aj170400237

19. Al-Azhri J, Zhang Y, Bshara W, Zirpoli G, McCann SE, Khoury T, Morrison CD, Edge SB, Ambrosone CB, Yao S(2017): Tumor Expression of Vitamin D Receptor and Breast Cancer Histopathological Characteristics and Prognosis. Clin Cancer Res.23(1):97-103.

 

 

 

 

 




Evaluation of Antioxidant Enzyme Levels in HIV Subjects In Port Harcourt, Nigeria

K.N. Elechi-Amadi
Department of Medical Laboratory Science, Rivers State University, Port Harcourt
O.N. Briggs
Department of Medical Laboratory Science, Rivers State University, Port Harcourt
G.A. Obediah
Department of Biochemistry, Rivers State University, Port Harcourt.
All Correspondences to: kemzi89@gmail.com

ABSTRACT

HIV infection is one of the most prevalent infections in Nigeria. The high rate of transmission of HIV in Nigeria is of an immense public health concern. This study evaluated the levels of the antioxidant enzymes, superoxide dismutase and glutathione peroxidase in HIV positive subjects. A total of 173 subjects of age 20 to 70 years were involved in this study; 60 HIV positive patients on HAART (Highly Active Antiretroviral Therapy), 57 HIV patients who are HAART-naïve, and 56 apparently healthy control subjects. Patients who had severe malaria, tuberculosis or diabetes, were excluded from this study. Pregnant female subjects were also excluded. Five millilitres of venous blood was obtained from each subject by standard procedure. CD4 count was done using Fluorescent Activated Cell Sorter (FACSCount) automation, while the antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were determined using enzyme-linked immunosorbent assay (ELISA) technique. It was observed that HIV patients had significantly reduced levels of the SOD than control subjects, with HIV positive subjects on HAART even having lower levels than those who were not on HAART. However, there were no significant differences in the levels of GPx. The results indicate impairment of antioxidant function in HIV positive subjects. Inclusion of antioxidant therapies in the treatment and management of HIV infection may prove a useful treatment modality in ameliorating the negative health implications of a compromised antioxidant system in the subjects.

Keywords: HIV, Superoxide dismutase, Glutathione peroxidase, Antioxidants, HAART, Port Harcourt

INTRODUCTION

The human immunodeficiency virus (HIV) is a retrovirus that affects the human immune system. Retroviruses constitute a large and diverse family of enveloped RNA viruses that use as a replication strategy for the transcription of virion RNA into linear double-stranded DNA with subsequent integration into the host genome (Chatterjea & Shinde, 2008).
Chronic administration of Highly Active Antiretroviral Therapy (HAART) has been reported to induce metabolic disorders including insulin resistance. HIV drugs have also been reported to cause abnormalities in carbohydrate metabolism (Florescu & Kotler, 2007). For example, Protease inhibitors have been reported to cause glucose intolerance and hyperglycaemia (Mulligan et al., 2000, Savès et al., 2002).
The human body possesses a system of antioxidants that co-operate to protect the cells of the body against the dangerous effects of oxidants. Antioxidant system consists of a variety of components, endogenous and exogenous in origin, which functions to neutralize free radicals (Krishnamurthy & Wadhwani, 2012) in the body. The human antioxidant system consists of both enzymes and non-enzymatic systems (Panda, 2012). Essentially, antioxidants are compounds that dispose, scavenge and suppress the formation free radicals or oppose their actions (Oguntibeju et al., 2009). They prevent or delay the oxidation of other molecules by preventing oxidative chain reactions.
Antioxidant enzymes; superoxide dismutase, glutathione peroxide and catalase, are complex proteins that have minerals incorporated into their molecular structure (Kiefer, 2006). Antioxidant enzymes are often the first line antioxidant defence in the human body; they terminate the chain reaction of free-radicals by donating hydrogen or electrons to the free-radicals, thus, converting them to more stable products (Panda, 2012). All the cells in the body possess powerful antioxidant enzymes (Kabel, 2014).
Superoxide dismutase (SOD) belongs to the group of oxido-reductases (Vaskova et al., 2012). It catalyses the dismutation of superoxide (O2-) to oxygen and hydrogen peroxide. The mechanism of dismutation of superoxide by SOD involves successive oxidation and reduction of the particular transition metal in the molecule of the SOD (Krishnamurthy & Wadhwani, 2012). Superoxide dismutase has been reported to be the most powerful natural antioxidant enzyme (Kiefer, 2006).
Glutathione peroxidase (GPx) is responsible for protecting cells from damage due to hydrogen peroxide and lipid peroxides (Krishnamurthy & Wadhwani, 2012). It is an 80kDa enzyme that catalyses the oxidation of peroxide using glutathione substrate. This antioxidant enzyme contains a single seleno-cysteine (Sec) residue in each of the four identical subunits. L-selenocysteine is the most important amino acid at the active site of GPx, and is also responsible for the reduction of hydroperoxides, utilizing a tripeptide substance known as glutathione in the process (Alberto et al., 2010). This study evaluated the levels of superoxide dismutase and glutathione peroxidase in HIV positive subjects.

METHODOLOGY
This work was carried out in one hundred and seventy three subjects. One hundred and seventeen (117) were HIV subjects, sixty (60) of whom were receiving antiretroviral therapy while fifty-seven (57) were not on antiretroviral therapy. Fifty-six (56) HIV negative individuals were used as control subjects. The bio-data and medical history of the subjects were obtained using questionnaire. The subjects that participated in this research gave their informed consent, did not have tuberculosis, diabetes or severe malaria; those with any of these conditions were excluded from the research. Pregnant female subjects were also excluded from the study. Ethical consent was also obtained for this research study.
Five millilitres of blood was collected from each subject; 2ml of this were put in a sample bottle containing ethylene diamine tetraacetic acid ( EDTA), for the assay of CD4 count, while 3ml were put in plain bottles for the assay of superoxide dismutase and glutathione peroxidase. The blood samples were centrifuged at 3,000rpm for 10 minutes and the plasma separated and put in another plain bottle. The plasma samples were then preserved at -20oC in the refrigerator prior to analysis. Analysis of the samples for superoxide dismutase and glutathione peroxidase was done using ELISA technique while Fluorescent Activated Cell Sorter (FACSCount) automation was used for CD4 count. The data generated was analysed using SPSS version 22.

RESULTS

 

DISCUSSION

The results of this work indicate differences in the levels of antioxidant enzymes in HIV patients compared to the control subjects. SOD levels were significantly reduced in HIV patients than in control subjects. During HIV infection, there is enhanced oxidative stress due to activation of the immune system, which increases the generation of free radicals which results in the depletion in the levels of these antioxidant enzymes. The depletion is probably because they are consumed in the process of providing antioxidant defence in the affected subjects (Suresh et al., 2009). Therefore, HIV infection can cause a significant decrease in SOD levels in HIV-positive subjects.
The levels of the antioxidant enzymes were lower in HIV patients on HAART than in HAART-naive HIV subjects. A similar results have been reported by other researchers (Kostyushov et al.,2009; Brown & Elechi-Amadi, 2016). HAART increases chemically reactive species in humans (Awodele et al., 2012), and has also been associated with increased oxidative stress in human HIV positive subjects (Ngondi et al., 2006). Thus, antiretroviral therapy exerts a negative effect on the activities of antioxidant enzymes.

It was observed that the levels of GPx in HIV subjects did not significantly reduce compared to the control subjects. This finding agrees with the work of Kim et al., 2015. This is may be due to the synthesis of a selenoprotein that has significant homology to mammalian GPx by HIV (Zhao et al., 2000).
Alterations in the levels of antioxidant enzymes have been reported in different disease conditions (Gil et al., 2003). HIV patients suffer constant oxidative stress, and this can lead to malfunctioning of the antioxidant system in these individuals.

CONCLUSION
This study has shown that HIV patients suffer significant oxidative stress. There is impairment of the antioxidant defence mechanism in HIV patients, and this is further affected by drug therapy used in the management of the condition. It may prove useful for treatment of HIV infection to include antioxidant therapy.

REFERENCES

1. Chatterjea, M.N & Shinde, R.(2008). Textbook of Medical Biochemistry. New Delhi. Jaypee Brothers.

2. Mulligan, K., Grungfeld, C., Tai, V.W., Algren, H., Pang, M., Chernoff, D.N., Lo, J.C. & Sehambelam, M. (2000). Hyperlipidaemia as insulin resistance are induced by protease inhibitors independents with HIV infection. Journal of Acquired immune Deficiency Syndrome, 23, 35-43.

3. Savès, M., Raffi, F., Capeau, J., Rozenbaum, W., Ragnaud, J.M., Perronne, C., Basdevant, A., Leport, C. & Chene, G. (2002). Factors related to lipodystrophy and metabolic alterations in patients with HIV infection receiving HAART. Clinical and Infectious Diseases, 34, 1396 ? 1405.

4. Florescu, D. & Kotler, D.P. (2007). Insulin resistance, glucose intolerance and diabetic mellitus in HIV-infected patients. Antiviral Therapy (London), 12, 149-162.

5. Krishnamurhthy, P. & Wadhwani,A. ( 2012). Antioxidant enzymes and human health. In El-Missiry, M.A( Ed.). Antioxidant Enzymes. Croatia. InTech.

6. Oguntibeju, O.O., Esterhuyse, A.J.& Truter, E.J. ( 2009). Possible benefits of micronutrient supplementation in the treatment and management of HIV infection and AIDS. African Journal of Pharmacy and Pharmacology, 3(9), 404-412

7. Panda, S.K. (2012). Assay-guided comparism for enzymatic and non-enzymatic antioxidant activities with special reference to medicinal plants. in El-Missiry, M.A.(Ed.). Antioxidant Enzyme. Croatia. InTech.

8. Kiefer, D. ( 2006). Superoxide dismutase: boosting the body?s primary antioxidant defence. Life Extension Magazine,6, 73-78.

9. Kabel, A.M. (2014). Free Radicals and antioxidants: role of enzymes and nutrition. World Journal of Nutrition and Health, 2(3), 35-38.

10. Vaskova, J., Vasko, L., & Kron,I. ( 2012). Oxidative processes and antioxidative metaloenzymes. In El-Missiry, M.A(Ed.). Antioxidant Enzymes. Croatia.InTech.

11. Gil,L., Martinez,G., Gonzalez,I., Tarinas,A., Alvarez,A., Giuliani,A., Molina,R., Tapanes,R., Perez,J. & Leon,O.S. (2003). Contribution to characterization of oxidative stress in HIV/AIDS patients. Pharmacological Research, 47, 217-224

12. Suresh, D.R., Annam, V., Pratibha,K. & Prasad, B.V.M. ( 2009). Total antioxidant capacity-a novel early biochemical marker of oxidative stress in HIV infected individuals. Journal of Biomedical Science, 16(61), 186-196

13. Kostyushov, V.V., Bokal, I.I. & Petrov, S.A. (2011). The study of activity of blood antioxidant enzymes in HIV infection. Biochemistry (Moscow). Supplement series B: Biomedical chemistry, 5(2), 193-196.

14. Awodele, O., Olayemi, S.O., Nwite, J.A. & Adeyemo, T.A. (2012). Investigation of the levels of oxidative stress parameters in HIV and HIV-TB co-infected patients. Journal of Infection in Developing Countries, 6(1), 79-85

15. Ngondi, J.L., Oben, J., Forkah, D.M., Etame, L.H. & Mbanya, D. (2006). The effect of different combination therapies on oxidative stress markers in HIV infected patients in Cameroon. AIDS Research and Therapy, 3, 19.

16. Mgbekem, M.A., John, M.C., Umoh,I.B., Eyong, E.U., Ukam,N. & Omotola, B.D. (2011). Plasma antioxidant micronutrients and oxidative stress in people living with HIV. Pakistan Journal of Nutrition, 10(3), 214-219

17. Kim, S., Smith, A.J., Tan, J., Shytle,R.D. & Giunta,B. (2015). MSM ameliorates HIV-1 tat induced neuronal oxidative stress via rebalance of the glutathione cycle. American Journal of Translational Research, 7(2), 328-338

18. Zhao,L., Cox,A.G., Ruzicka,J.A., Zhang,W. & Taylor,E.W. (2000). Molecular modelling and invitro activity of an HIV-1 encoded glutathione peroxidase. Procedures of the National Academy of Science of USA, 97, 6356-6361

19. Brown, H. & Elechi-Amadi, K.N. (2016). Assessment of Antioxidant Enzymes and Cortisol Levels among HIV Patients on HAART. International Journal of Science and Research, 5(4), 633-636




Antimicrobial Resistance and Beta-lactamase Detection in Staphylococcus aureus isolates from Human Sources in Port Harcourt, Nigeria.

Easter Godwin Nwokah, Samuel Douglas Abbey and Confidence Kinikanwo Wachukwu.
Department of Medical Laboratory Science, Rivers State University, Port Harcourt, Nigeria
All Correspondences to: easternwokah@yahoo.com

ABSTRACT

The problem of Antimicrobial resistance has since become a global health challenge with renewed calls for global action, including surveillance, to contain the menace. Staphylococcus aureus strains are implicated in a wide range of diseases in humans and other animals. The aim of this study was to investigate the antimicrobial susceptibility pattern of Staphylococcus aureus isolates in Port Harcourt, Nigeria. Two hundred and five (205) isolates of Staphylococcus aureus from human sources were randomly collected from three health facilities- University of Port Harcourt Teaching Hospital, Braithwaite Memorial Specialist Hospital and De-Integrated Laboratories- all located in Port Harcourt. Isolates were grouped as Hospital-acquired (n=76) and Community-acquired (n=129) Staphylococcus aureus based on established criteria. The isolates collected were reconfirmed using standard laboratory protocols and thereafter, stored at +4°C. Using the disk diffusion method, the following antimicrobial agents (OXOID, UK) were tested- Cefoxitin(30µg) Vancomycin (30µg), Erythromycin (15µg), Fusidic acid (10µg), Penicillin G (10 Units), Tetracycline, Mupirocin (5 µg), Levofloxacin (30µg), Gentamicin (10 µg), Ceftazidime (30 µg), Cefuroxime (30µg), Clindamycin (2 µg), Amoxicillin/clavulanic acid (30µg), Tigecycline (15µg) and Linezolid (10µg), Quinupristine/dalfopristine (30µg), Ticarcillin/ clavulanic acid (85µg), Sulphamethoxazole/Trimethoprim (25µg), Piperacillin/Toxobactam (110µg). Organisms showed high levels of resistance to Cotrimoxazole (65.4%) and amoxicillin-clavulanic acid (44.9%) while only one isolate was resistant to Tegecycline. All isolates were susceptible to Vancomycin, Linezolid and Quinupristine/dalfopristine. MRSA detection was 12.2% and this, including the PCR methods for mecA status has been previously reported. Study further established the presence of multi-drug resistant (MDR) strains of Staphylococccus aureus (65.4%). Beta-lactamase production was detected in 94.1% of the isolates. There is need for sustained surveillance of antimicrobial resistance of S. aureus in this region to enhance guidance for treatment and also for infection control policies.

KEYWORDS: Antimicrobial Resistance, Staphylococcus aureus, MRSA,

INTRODUCTION

The problem of Antimicrobial resistance has since become a global health challenge with renewed calls for global action, including surveillance, to contain the menace. Staphylococcus aureus strains are implicated in a wide range of diseases in humans and other animals such as boils, deep tissue abscesses, enterocolitis, bacteriuria, osteomyelitis, pneumonia, carditis, meningitis, septicemia and arthritis, with associated morbidities and mortalities and medical costs. Following the introduction of the ß-lactam antibiotic, penicillin in the early 1940s, which improved outcomes, penicillin-resistant strains of S. aureus was reported, and by 1946 it was estimated that 60% of hospital isolates in the UK were resistant to this antibiotic. [1] Since then, successive introduction of new antibiotics- streptomycin, tetracycline, chloramphenicol and erythromycin has, in each case, been rapidly accompanied by the emergence of resistant organisms. [2,3]
It is interesting that many strains acquiring resistance to the latest antimicrobials also usually expressed a ß-lactamase (penicillinase), providing resistance to penicillin while some are resistant to all of the other antibiotics. Introduction of the semisynthetic ß-lactamase-resistant penicillins, such as methicillin and oxacillin, during the early 1960s, led to a general decline in the prevalence of multiple-resistant S. aureus. [3]. However, by the late 1960s to early 1970s, strains resistant to the ß-lactamase-resistant penicillins were isolated with increasing frequency. [4] Till date, there has been an increasing incidence of hospital-associated (nosocomial) and also community-acquired infections caused by multi-drug resistant strains of S. aureus, especially the methicillin-resistant S. aureus (MRSA), which term includes not only resistance to methicillin, but also to many other different antimicrobial compounds, including various biocides. Several genes have been found in strains of MRSA which confer on them high virulence and resistance to several antibiotic classes; these include mecA (that codes for penicillin resistance), lukS-lukF (responsible for widespread skin and soft tissue infections) and tetM (that codes for tetracycline resistance), erm (for macrolide resistance) among others.[5] The acquisition of genes such as mecA that codes for penicillin binding protein (PBP2A) by the strains, confers almost complete resistance to all beta-lactam antibiotics, including the semi-synthetic penicillin. [6-8]
A number of studies have indicated that multi-drug resistant S. aureus is among the most frequently encountered microorganisms in microbiology laboratories in Nigeria.[9-15] It is common knowledge that there is indiscriminate use of antibiotics and poor hygienic practices in our locality, as well as poor infection prevention and control practices in our health care facilities. These are some of the reasons for the success of this pathogen, accounting for is its great variability, occurrence at different periods and places with diverse clonal types and antibiotic resistance patterns within regions and countries.
The ability to characterize S. aureus and monitor antimicrobial susceptibility patterns is important for clinicians selecting empirical antimicrobial therapy, rational formulation of public health care polices, and providing useful information on the global surveillance of this pathogen. However, data on the antimicrobial susceptibility patterns of this pathogen in Rivers State are inadequate, and in most cases, isolates are screened against commonly available first line antibiotics only, most of which have lately become unhelpful. This study was aimed to investigate the antimicrobial susceptibility pattern and Beta-lactamase production in Staphylococcus aureus isolates in Port Harcourt, Nigeria.

MATERIALS AND METHODS
Study Area
Port Harcourt is a cosmopolitan city, located in Rivers State, one of the 36 States of Nigeria. There are two major tertiary healthcare facilities as well as other public and private health facilities. Clinical isolates were collected from the two tertiary health facilities- University of Port Harcourt Teaching Hospital and Braithwaite Memorial Specialist Hospital and De-Integrated Laboratories. Isolates were also grouped as Hospital in-patient (Nosocomial) or Out-patient isolates (Community-acquired) according to the criteria as prescribed by the US Centers for Disease Control and Prevention. [16]

Microbiological/ Identification Tests
Two hundred and five (205) non-duplicate human isolates of Staphylococcus aureus, were used for this study. Reconfirmation of isolates was done following standard microbiological protocols. [17] All confirmed isolates were stored at +4°C and later sub-cultured to carry out phenotypic characterizations.

Antimicrobial Susceptibility Testing
Antimicrobial Sensitivity Discs
The antibiotic sensitivity discs (Oxoid Ltd., Basingstoke, England) and their disc strengths are as follows: Oxacillin(1µg) Cefoxitin(30µg) Vancomycin(30µg), Erythromycin (15µg), Fusidic acid (10µg), Penicillin G (10 Units), Tetracycline (30µg), Levofloxacin (5µg),
Ciprofloxacin (5µg), Gentamicin (10µg), Ceftazidime (30µg), Cefuroxime (30µg), Clindamycin (2µg), Amoxicillin/clavulanic acid (Augmentin) (30µg), Tigecycline (15µg), Linezolid (30µg), Quinupristine/dalfopristine(30µg), Ticarcillin/clavulanic acid(85µg), Sulphamethoxazole/Trimethoprim (Bactrim) (25µg), Piperacillin/Toxobactam (110µg),

Disk Diffusion method
All isolates of Staphylococcus aureus were subjected to in-vitro antimicrobial susceptibility testing on Muller-Hinton agar (MH) as per the method described by Kirby and Bauer (1966) [18]. Briefly, inocula of bacteria were prepared to 0.5 McFarland standards and tested against all the aforementioned antibiotics disks. Following incubation at 37°C for 24 hrs, the zones of inhibition around the discs were measured with ruler and interpreted using the interpretation chart as prescribed by CLSI (2009) [19]. Multi-resistance was defined as resistance to at least three classes of antibiotics.

Test for Decreased Vancomycin Susceptibility
Isolates were screened further for vancomycin resistance using the vancomycin agar screening test whereby isolates were spot-inoculated onto Mueller Hinton agar supplemented with 6 µg/ml of vancomycin from a 0.5 McFarland standard suspension. The plates were incubated at 35°C for 24 h as recommended; and growth of two or more colonies on this agar would be considered as positive. [20]
Detection of Beta-lactamases production:
ß –lactamase test was also carried out on the S. aureus isolates to detect whether the organism would be able to produce the enzyme ß-lactamase- an enzyme that inactivates ß- lactam antibiotics. ß -lactamase production was detected by two different methods: Test tube iodometric technique and filter paper technique using 24 hour old culture and 10,000 units/ml of crystalline penicillin as per the method described by Sykes and Mathew (1979). [21]
Test tube method:
A loopful of heavy inoculum of 24 hours old culture from MH agar was mixed well with 1.0 ml Penicillin solution containing 10000 U per ml. The tubes were left for 60 minutes at room temperature with mixing at 15 minutes interval. Then 2 drops of 1% soluble starch solution was added, followed by one drop of Iodine solution. The tubes were mixed well and the results were recorded as follows- Instant discoloration: ++++ (Strong positive); Discoloration in 1-5 min: +++ (Average positive); Discoloration in 6 to 10 min: ++ (Moderately positive); Discoloration in 10-15 min: + (Weak positive) and No discoloration – (Negative). All test tubes showing discoloration within 10 minutes after adding iodine solution were taken as positive for beta-lactamase production.
Agar Plate method:
Isolates were inoculated on MH agar containing 1% soluble starch and incubated at 37°C for 48 hours. Then the plate was flooded with Penicillin solution containing 10000 U per ml and left at room temperature for 30 minutes. Then the penicillin solution was decanted completely and flooded with 1:5 dilution of iodine follows- More than 10 mm diameter discoloration around the culture: ++++ (Strong positive); 5-10 mm discoloration around culture: +++ (Average positive); 2-4 mm discoloration around culture: ++ (Moderately positive); 1 mm or discoloration below culture: + (Weakly positive) and No discoloration around or below culture: – (Negative).

RESULTS
Two hundred and five (205) non-duplicate isolates of Staphylococcus aureus collected from different clinical specimens were used in this study. Antimicrobial susceptibility pattern revealed varying degree of resistance to various types of antimicrobial agents tested (Table 1). Highest degree of antimicrobial resistance was recorded in cotrimoxazole- 134 (65.4%) out of 205 isolates of S. aureus and followed by amoxicillin-clavulanic acid (44.9%) while only one isolate was resistant to Tegecycline. All isolates were susceptible to vancomycin, teicoplanin, quinupristin/dalfopristin and linezolid. The oxacillin disc susceptibility testing showed that 25 (12.2%) out of 205 isolates of S. aureus were resistant to oxacillin (Table 1).
Table2 shows comparison of antimicrobial
susceptibility between in-patient S.aureus isolates and out-patient S.aureus isolates. Resistance was significantly higher in in-patient S.aureus isolates (p<0.05).
Multi-drug Resistance (resistance to three or more classes of antimicrobial agents) was significantly high (p<0.05) as detected in 134 (65.4%) of the isolates

(Table 3).
MRSA detection was significantly higher in in-patient isolates ((23.7% of 76) than out-patient (5.4% of 129) S. aureus (p = 0.000318) (Table 4).

Table 5 shows distribution of ß-lactamase producing S. aureus isolates. One hundred and ninety-three (94.1%) of the 205 isolates were positive. There was no significant difference (p > 0.05) in ß-lactamase production between MRSA and MSSA. There was also no significant difference (p > 0.05) in ß-lactamase production between in-patient and out-patient isolates.

 

 

DISCUSSION
Multi-Drug Resistance among pathogens is a significant challenge in both hospital and community settings that adds to the cost of medical care and the morbidity and mortality of patients. This becomes worrisome in the light of our local economic realities. Continual surveillance is essential to guide therapy and for the establishment of adequate infection control programmes.
This study revealed a multi-drug resistance rate of 65.4% (inclusive of all MRSA isolates) (Tables 3). MRSA detection was 12.2% and this, including the PCR methods for mecA status has been previously reported.[8] The study further confirmed that MRSA are more resistant to various groups of antibiotics compared to MSSA (Table 3), which also agrees with other authors. [22-23] Furthermore, selective pressure on the organisms, occasioned by misuse of antibiotics, as well as the ease of transferability of genetic elements is instructive. Study confirmed that Hospital Acquired-MRSA (nosocomial) are more resistant to antimicrobial agents than the Community-Acquired-MRSA (out-patients) among S. aureus isolates from clinical sources in Port Harcourt.
None of the isolates in this study showed resistance to the glycoprotein- vancomycin (Table 1). This is in consonance with some other investigations in Nigeria. [24-26] However, certain reports have revealed the presence of VRSA in Nigeria. Akambi and Mbe, (2013),[27] reported vancomycin resistance in 4 isolates out of 213 isolates of S. aureus in the University of Abuja Teaching Hospital, although only the disk diffusion method was employed. A VRSA prevalence rate of 57.7% has also been reported in Zaria, Northern Nigeria,[28] 6.3% among MRSA.[29] In another study, in non-clinical isolates, a prevalence rate of 89% was reported.[30] The difference in rates of vancomycin resistance probably reflects differences in levels of over-prescription and abuse in different parts of the country. Vancomycin-resistant strains are a source of concern because until recently, vancomycin was the only uniformly effective treatment for staphylococcal infections, particularly MRSA. Resistance to vancomycin severely limits therapeutic options. It is therefore cheering that there was zero resistance for vancomycin in this study. Also, none of the isolates was resistant to teicoplanin, another glycoprotein.

Nasal mupirocin plays an important role in the eradication of MRSA carriage. [31] Overall, 13 isolates were resistant to mupirocin (Table 1). The 6.3% prevalence of mupirocin resistance in this study is comparable to the 7% reported by Shittu and Lin, (2006) [32] but higher than a study (2%) in South Africa[33] and 1.5% in India.[34] This trend suggests that mupirocin resistance in S. aureus is an emerging feature in this locality, and therefore, underscores the need for routine testing for early detection of resistant isolates and prompt institution of infection control measures.
Five of the isolates in this study showed resistance to fusidic acid (Table 1). Some other investigators have reported full susceptibility of S. aureus to fusidic acid,[32, 35] indicating that fusidic acid is a good and effective agent for the treatment of S. aureus infections. Monotherapy with fusidic acid has been associated with the emergence of resistance and therefore, in order to minimize the emergence of fusidic-acid resistant strains, Howden and Grayson, (2006) [36] advised that monotherapy with fusidic acid should be discouraged and a combination with another anti-staphylococcal agent (ß-lactams, rifampicin or glycopeptides) be recommended.
A number of reports have indicated an increase in the resistance of staphylococci to trimethoprim/ sulphametoxazole (cotrimoxazole) in Nigeria. In one study, resistance rate among MRSA was 92.1%. In the present study, 65.4% of S. aureus isolates was resistant to cotrimoxazole, accounting for the highest level of resistance observed. Twenty-one (84%) of MRSA were also resistance to cotrimoxazole. This antimicrobial has wide clinical application, inexpensive, orally administered and available over-the-counter in Nigeria, where they are sold with or without prescription. This could possibly explain the high level of staphylococcal resistance observed in this study.
Erythromycin is one of the commonest and affordable antimicrobial agents available in Nigeria. An erythromycin-resistance rate of 21.5% in this study is therefore a cause for concern. More worrisome is that 23 out of the 38 erythromycin-resistant S. aureus were inducibly resistant to clindamycin and this has been previously reported.
This study also detected resistance to the fluoroquinolones. 22.9% (30 MSSA and 17 MRSA) of the 205 isolates were resistant to levofloxacin while 21.0% (28 MSSA and 15 MRSA) were also resistant to ciprofloxacin. Fayomi et al., (2011), had reported 12.2% and 9.6% ciproxin resistance among MRSA and MSSA respectively in Ido-Ekiti, Nigeria. These rates, although lower than the findings from one study in Okigwe, Nigeria where resistance to various brands of quinolones ranged from 44% to 88%, constitute a growing concern for a therapeutic option in our setting, especially where the preferred vancomycin are largely unavailable. The reasons for the disparity in rates of quinolone resistance between MSSA and MRSA strains are uncertain but could include antibiotic selective pressure, especially in the hospital setting, resulting in the spread of the more antibiotic-resistant MRSA strains.
In this study, the enzyme, ß–lactamase was detected in 193 (94.1%) of the 205 isolates (Table 5), a rate higher than 64% earlier reported by Terry-Alli et al., (2011) but lower than the 100% previously reported by Olowe et al. (2007). Twenty-five (13.0%) of the 193 ß-lactamase-producing isolates in this study were MRSA. However, there was no significant difference (p>0.05) in ß-lactamase-production between MRSA and MSSA. A previous study reported majority of ß-lactamase producers as methicillin-resistant. The indiscriminate use of antibiotics and the over-the-counter availability of antibiotics without prescription have, to a large extent, contributed to the emergence of resistant strains. ß-lactamase production by S. aureus had been identified to be a risk factor for the prevalence of MRSA in South Western, Nigeria. The finding in this study is instructive for urgent surveillance, control and other intervention programmes.
In Nigeria, systematic reporting of infectious diseases is still rudimentary and most health institutions lack proper infection control programmes. Because there is no national policy or guidelines for screening, reporting and control of MRSA outbreaks, the tendency is either under-reporting or over-reporting of the true prevalence of MRSA and therefore infections/ outbreaks are largely undetected and their contributions to mortality, morbidity and cost of care, associated with hospital-acquired infections are unknown. This problem is expected to be overcome with the recent establishment of a reference laboratory in Nigeria.

CONCLUSION
The degree of Multidrug resistance among S. aureus and especially among MRSA as detected in this study in Port Harcourt, Nigeria is high enough to warrant the need for continuous antimicrobial resistance surveillance as well as molecular epidemiological typing. This will enhance guidance for treatment and also for infection control policies.

ACKNOWLEDGEMENTS
We are grateful for the technical support of all staff of the Medical Microbiology departments in UPTH, BMSH and De-Integrated Laboratories in Port Harcourt, Nigeria.

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Comparison of Staining Reaction of Periodic Acid Schiff with Unripe Orange Extract-Schiff on Skin and Intestinal Tissues

Okorie Nnaemeka
N.K.S.T. Len Gebrielse’ School of Medical Laboratory Science Mkar, Benue State, Nigeria.
Fasogbon Samuel Ayobami
Public Health In-vitro Diagnostic Control Laboratory, Medical Laboratory Science Council of Nigeria, Lagos State, Nigeria
Adeluwoye Adekunle Oluwatosin
Department of Medical Laboratory Science, Lead City University, Ibadan, Oyo State.
Ajileye Ayodeji Blessing
Department of Medical Laboratory Science, College of Medicine and Health Sciences, AfeBabalola University, Ado Ekiti, Ekiti State.

ABSTRACT
The aim of this study is to compare thehistochemicalreaction of unripe orange extract juice-Schiff with that of Periodic acid Schiff reagent on skin and intestinal tissues.Four (4) tissue sections each obtained from a processed skin and intestine of a wistar rat were prepared and used for this study. The unripe orange juice was extracted and the various pH was measured with different timing. The control tissue sections were stained with periodic acid Schiff technique while the test tissue sections were stained with the unripe orange juice extraction obtained replacing the periodic acid solution in the PAS technique.Unripe orange juice (UOJ) which has similar pH with periodic acid Schiff react with the glycogen and mucosubstances on skin and intestinal tissue of the wistar rat used in this study, by staining them purple magenta similar to what is obtainable with periodic acid Schiff (PAS) staining protocol. In conclusion, unripe Orange JuiceSchiff technique has stained the skin and intestine of the Wister rat in similar manner to periodic acid Schiff technique.

Keywords: Histochemical, Periodic acid Schiff, Intestinal tissue, Skin tissue, Mucosubstances, Glycogen.

INTRODUCTION

Periodic Acid Schiff (PAS) is a staining method used to detect polysaccharide such as glycogen, and mucosubstances such as glycoproteins, glycolipid and mucins in tissues. The reaction of periodic acid oxidizes the vicinal diols in these sugars. Usually breaking up the bond between two adjacent carbons not involved in the glycosidic linkage or ring closure in the ring of the monosaccharide unit that are parts of long polysaccharides, and creating a pair of aldehydes at the two free tips of each broken monosaccrharide ring1. The oxidation condition has to be sufficiently regulated so as not tooxidize the aldehydes further. These aldehydes then react with the Schiff reagent to give a purple magenta color. A suitable basic stain is often used as a counter stain2.
The PAS stain is a histochemical reaction in that the periodic acid oxidizes the carbon to carbon bond forming aldehydes which react to the fuchsin-sulfurous acid of the Schiff solution forming the magenta color3.PAS staining is mainly used for staining structures containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans), typically found in e.g. connective tissues, mucus, the glycocalyx, and basal laminae.PAS diastase stain (PAS-D) is PAS stain used in
combination with diastase, an enzyme that breaks down glycogen. Alcian blue/periodic acid–Schiff (AB/PAS or AB-PAS) uses alcian blue before the PAS step4. Presence of glycogen can be confirmed on a section of tissue by using diastase to digest the glycogen from a section, then comparing a diastase digested PAS section with a normal PAS section. The diastase negative slide will show a magenta staining where glycogen is present within a section of tissue. The slide that has been treated with diastase will lack any positive PAS staining in those locations on the slide.
Among several immense use that has been found for Periodic acid Schiff staining in the diagnosis of medical conditions are glycogen storage disease, adenocarcinomas (which often secrete neutral mucins), paget disease of the breast, Alveolar soft part sarcoma, staining macrophages in Whipple’s disease, Pulmonary alveolar proteinosis, and erythroleukemia (leukemia of immature red blood cells). It can be used to diagnose a1-antitrypsin deficiency if periportal liver hepatocytes stain positive, as well as pulmonary interstitial glycogenosis (PIG), a condition where glycogen is identified in lung biopsy specimens of infants.
Orange is one of the largest citrus grown fruit in Nigeria5 and is one of the main sources of vitamin C (ascorbic acid) in Nigerian diets. Other vitamins that are of dietary significance are found in orange and include folic acid and thiamine.Orange juice also contains niacin, riboflavin and pantothenic acid vitamins although in minute quantities of about 2 to 4%6. Orange as one of the citrus fruits is non-climacteric, hence, they do not ripen or improve in quality after harvest. Various methods which can be used for processing of orange juice include the traditional thermal processing method (pasteurization) which kills bacterial and extend the shelf-life of juices, but affects the taste of the product. The pressurized carbon dioxide process, according to7 showed that this process is as effective as heat pasteurization, but does not change the taste and preserves more of the vitamins found in fresh squeezed juice. The periodic acid Schiff reaction is used to basement membrane, glycogen and neutral acidic mucosubstances will appear purple due to positive reaction with both alcianlue and periodic acid Schiff. According to8 periodic acid stain is useful for many things. It stain glycogen, mucin, mucoprotein as well as glycoprotein and fungi. A pre-digestion step with amylase will remove staining for glycogen periodic acid Schiff is useful for outlining tissue structure basement membranes capsules, blood vessels, etc8.

Orange juice have been found to be of medicinal importance in folkloric medicine. Among uses found for orange juice includes the reduction of the presence and effects of “bad” cholesterol, why increasing the amount of “good” cholesterol in the body, boosting of immune system, as well as dissolution of kidney stone.Orange juice neutralizes the pro inflammatory effect of a high-fat, high-carbohydrate meal and prevents endotoxin induced toxicity. Similarly, in cancer prevention, since one of the most important functions of antioxidants is to prevent cancer, orange juice is very rich in vitamin C which works as an antioxidant. Antioxidants keep the DNA of healthy cells from mutating into cancerous cells,hence antioxidants like vitamin C are the first line of defense for cancer and other serious diseases9. Having been observed for it acidity level, this study therefore compare the unripe orange juice with periodic acid as a component solution in the Periodic acid Schiff (PAS) staining technique, which is a staining protocol carried out at an acidic pH of 3.9.This is particularly important as naturally occurring dyes from plants are being viewed as an alternative to synthetic dyes for many developing countries that can no longer afford the ever increasing cost of synthetic dyes.

MATERIALS AND METHOD
Ethical Clearance
Ethical approval for this research study was obtained from the research ethics committee of the N.K.S.T. Len Gabrielse School of Medical Laboratory Science, the experiment was carried out in strict accordance with the guidelines for the care and use of animals for research.

Experimental Animal
Wister rat was used for this scientific research. The animal was handled humanely in conformity with the established ethical guideline for the care and use of laboratory animals.

Extraction of orange juice
For thisstudy, unripe orange fruit where obtained freshly from the orange tree stem at Mkar, Gboko Local Government area of Benue State, Nigeria.The unripe orange fruit were peel using knife and were then sliced into smaller pieces. The sliced unripe orange were parked inside the juice extractor machine, in order to extract the juice separating it from the chaff.The juice was filtered using four layers of gauze, and labeled with date.

Procedure for testing pH of unripe orange juice (UOJ)
The pH meter was inserted in the extracted unripe orange juice (UOJ) to measure the pH of the orange juice solution. The result of freshly prepared orange juice was 3.6. After 24 hours, the pH remains 3.6, only measuring 3.4 after 48hours, an indication of reduction due to fermentation.

Study Design
A total number of eight (8) tissue sections, four (4) each from skin and intestine of a wistar rat were prepared and used for this study. Skin and Intestine tissues were selected for this study as they have been histologically demonstrated to be rich tissue source of localized mucosubstances and glycogen.

Preparation of Slide
Four sections were obtained from a processed intestine tissue of the wistar rat and labeled as follow:
IC – Intestine control at pH 3.9
IT1 – Intestine test 1 at pH 3.6(pH of freshly prepared UOJ)
IT2 – Intestine test 2 at pH 3.6(pH after 24 hours of UOJ)
IT3 – Intestine test 3 at pH 3.4(pH after 48 hours of UOJ)
Also, another four tissue sections were obtained from a processed skin tissue of the wistar rat and labeled as follow:
SC – Skin control at pH 3.9
ST1 – Skin test 1 at pH 3.6(pH of freshly prepared UOJ)
ST2 – Skin test 2 at pH 3.6(pH after 24 hours of UOJ)
ST3 – Skin test 3 at pH 3.4(pH after 48 hours of UOJ)

Staining Procedure Using PAS (Control Slides)
i. Control slide IC and SC were dewaxed and hydrated using descending grades of alcohol (100%, 90%, 70%)
ii. They were taken to water
iii. They were oxidized using 1% Periodic acid at pH 3.9 for 10 minutes
iv. Both slides were rinsed with tapwater
v. They were then stained with Schiff reagent for 20 minutes
vi. Slides IC and SC were again rinsed thoroughly in tapwater
vii. They were counterstained with Cole’s haematoxylin for 10 minutes
viii. They were rinsed with tapwater
ix. Differentiated with 1% acid alcohol
x. Rinsed with water
xi. They were blued in tap water for 10 minutes grades of alcohol (70%, 90%, and 100%).
xiii. Cleared using xylene and mounted using Diphthalenexylene (DPX)

Staining Procedure Using Orange Juice Extract (Test Slides)
i. Sections marked IT1, IT2, IT3 and ST1, ST2, ST3 were dewaxed and hydrated in descending grades of alcohol (100%, 90%, 70%)
ii. Sections were taken to water
iii. Slide IT1, IT2, and IT3 were oxidized with unripe orange juice at pH 3.6 (fresh), 3.6(24hrs), and 3.4 respectively for 10 minutes
iv. Slide ST1, ST2, ST3 were oxidized with unripe orange juice at pH 3.6 (fresh), 3.6 (24hrs), and 3.4 respectively for 10 minutes
v. All the sections were rinsed thoroughly in tap water
vi. They were stained with Schiff solution for 20 minutes
vii. Rinsed thoroughly in tap water
viii. They were counterstained with Cole’s haematoxylin for 10 minutes
ix. They were again rinsed in tap water
x. Differentiated briefly with 1% acid alcohol
xi. They were blued in tap water for 10 minutes
xii. Dehydrated using ascending grades of alcohol (70%, 90%, 100%)
xiii. Cleared using xylene and mounted using Diphthalenexylene (DPX)
All slides were examined under the binocular microscope using 10x, 40x and 100x objectives respectively.

RESULTS
Histology of Intestinal Tissue Sections
Histology of Intestinal Control (IC) pH 3.9 micrograph

 

Discussion
Cell structures take up specific stains to varying degrees based on their biochemical characteristics. Many stains which are suited for particular purposes and allowing cell structures to be differentiated have been developed by histologists. The unripe orange juice which have the similar pH with that of periodic acid solution in the Periodic Acid Schiff (PAS) stain, stained clearly, the glycogen components of the intestinal tissue at pH3.6 (both fresh and 24hrs), as well as at pH 3.4. However for the intestinal tissue, mucosubstances (glycogen) were clearlydemonstrated at pH 3.4. Goblet cells of the intestine were also stained by the unripe orange juice due to its positive reaction with Schiff solution. For the skin tissue sections, similar to the PAS technique which serves as control,the unripe orange juice stained the glycogen, stratum adiposum, and hair follicles on skin tissues of wistar rat as indicated in figures 6-8 above. It is of note too that neoplastic cells on the skin test sections, (ST1, ST2, and ST3) were also demonstrated (indicated by yellow arrow).
Conclusion
Based on the findings of this study, it is observed that, unripe orange juice which has similar pH with periodic acid Schiff react with the glycogen and mucosubstances on skin and intestinal tissue of the wistar rat, by staining them purple magenta similar to what is obtainable with periodic acid Schiff (PAS) staining protocol.

REFERENCES

1. Thomas J, Lawton G. Breast. Cambridge University Press. 2009 16 November Pp. 55. ISBN 978-0-521-88159-3.

2 Carson, FL, Hladik C, Histotechnology: A Self-Instructional Text (3rdedition.). Hong Kong: American Society for Clinical Pathology Press.2009 Pp. 137–139. ISBN 978-0-89189-581-7.

3. Crookham J, Dapson R. Hazardous Chemicals in the Histopathology Laboratory, 2nd Edition, Anatech, 1991Pp. 28 – 37.

4. Hauser (2005): https://www.google.com.ng /search?q=Hausar+2005,+glycogen+PAS&dcr

5. Osbeck L. Citrus X sinensis (L) Osbeck (Pro sp) (Maxima x reticulate) sweet orange” plants. USDA. Gov Archived from the original on may 12, 2011. https://www.google.com.ng/search?q=Osbeck+2011,+orange&dcr=0&tbm=isch&t

6. Smoot JM, Nagy S.Effects of storage temperature and duration on total vitamin C content of canned single-strength grapefruit juice. J. Agric. Food Chem., 1980, 28 (2), Pp 417–421.

7. Nicolosi E, Deng ZN, Gentile A, La-Malfa S, Continella G, Tributlato E. citrus phylogeny and genetic origin of important species as investigated by molecular markers” TAG theoretical and Aplied Genetics. Theor Appl Genet 2000 100:1155-1166.

8. Manjunath M. Shenoy S, Teerthanath V K, Karnaker B S, Girisha, Krishna P, Jerome P.Periodic Acid Schiff’s staining. Indian J Dermatol Venereol Leprol 2009 75 (1): 73-74.

9. Lutsenko EA, Cárcamo JM,Golde DW. Vitamin C prevents DNA mutation induced by oxidative stress. J Biol Chem.2002: 277(30):27576-27578.




Prevalence of Haemoparasites (Plasmodium and Microfilaria) in Blood Donors Attending University Of Maiduguri Teaching Hospital (UMTH)

Bukar Alhaji, Mary Ann Amarachi Umeh, Obi Simon Osita, Waziri Gimba, Medugu Jessy Thomus
Department of Haematology, University of Maiduguri Teaching Hospital, Maiduguri,
Anthony Nwobi, Osakue Eguagie Osareniro
Department of Chemical Pathology, Igbinedion University Teaching Hospital, Okada
Olaniyan Matthew Folaranmi
Department of Medical Laboratory Science, Achievers University, Owo
Jeremiah Zaccheaus Awortu.
Department of Medical Laboratory Science, Niger Delta University
All correspondence to: alhajibukar@gmail.com.

ABSTRACT
Haemoparasite,such as Plasmodium and Microfilaria are animal parasite living in the blood of a vertebrate host. The study was aimed to determine the prevalence of Haemoparasite (Plasmodium and Microfilaria) in blood donors attending University of Maiduguri Teaching Hospital. A total of 230 blood donors were recruited for this study using simple random sampling. A semi-structured questionnaire was used to collect data regarding demographic and social profile of the subjects. Giemsa stained thick blood film was used for the detection of malaria parasite while wet preparation was used for the detection of microfilaria. A total of 78 blood donors had malaria parasites while no filarial parasite was recorded showing a prevalence of 33.9% and 0% respectively. The prevalence of malaria parasites in the blood donors was not significantly associated with usage of insecticide and/or insecticide treated net. The prevalence of malaria parasite was however significantly associated with treatment with antimalarial drugs. It is therefore necessary for the government to improve the sanitary condition of Maiduguri which will in turn reduce the availability of breeding sites for mosquitos.

Keywords: Malaria, microfilaria, blood donors.

INTRODUCTION
Blood transfusion is potentially a lifesaving therapeutic procedure and a common form of tissue transplantation which is aimed to provide patients with blood components which they are deficient.1. Although,blood transfusion is generally believed to save human lives, blood can nonetheless be a route for the transmission of infections generally referred to as transfusion transmissible infections (TTIs). TTI occurs when a patient is infected by the same parasite that was present in the donor’s blood. TTIs are broadly classified into viral, bacterial, amoebal or parasitic.Haemoparasite is an animal parasite such as a haemoflagellate or filarial worm living in the blood of a vertebrate host2. These parasites reside eitherin the blood cells or in the plasma. Malaria parasite and Babesiaare haemoparasites that resides in the red blood cells, while leishmania and filarial wormsresides in the white blood cells and the plasma respectively3.
In Nigeria, malaria and filariasis are more prevalent and over the years varying prevalence has been recorded among Nigerian blood donors4. Haemoparasites constitute a serious threat to human race as they can result in increased morbidity and mortality5. Malaria is sporozoan parasite of the genus Plasmodium, its infection is transmitted naturally through the bite of infected female Anopheles mosquitoes6. In endemic areas, malaria transmission is so intense that a large proportion of the population is infected but not made ill by these parasites7. These carriers harbour low levels of the parasitesand shows no clinical signs of infection as they are immune to parasitic illness but not to the infection and for this reason, blood from such donors contains malaria parasite which can easily be transmitted to recipients by blood transfusion.
A bite from an infected mosquito may cause malaria by introducing as few as 15 parasites while a single parasite identified on a thick film (4ul) is equivalent to approximately 10,000 parasites in 450ml unit thereby causing malaria in transfused patients8. Transfusion-transmitted malaria can however have serious consequences, as infection with P. falciparum may prove rapidly fatal when such blood is transfused especially into children under 5 years, pregnant women, trauma victims with acute blood loss and immuno-suppressed patients9. Malaria destroys red blood cells and converts it to methaemoglobin leading to methemoglobinemia causing illness especially in immune compromised individuals 7, 10.

Filariasis on the other hand is a parasitic disease that is caused by thread-like nematodes (roundworms) belonging to the superfamily Filarioidea. These parasites are transmitted from host to host by blood feeding arthropods, mainly black flies and mosquitoes.
As adults, the worms can survive and reproduce for up to 7 years within which the worms gradually build-up in the vessels of their host. This interfereswith the lymphatic system’s ability to fight infection and causes lymph fluid to accumulate in the arms, legs, breasts and male genitals leading to welling and disfigurement3, 11.
In all species, sexually mature female worms release microfilariae, which are their pre-larval stages into the bloodstream of their infected host.If, the blood from microfilaraemic individuals is transfused into a patient, the transfused microfilariae may persist in the recipient’s circulation for up to 3 years12. Recipient of these blood component usually develop post transfusion allergic reactions due to dying microfilariae13.
In Nigeria, screening forparasitic infections is not routinely done in blood banks, nor stipulated in the current National Blood Guidelines. This is because transmission of parasitic infections such as malaria through blood transfusion is generally not regarded as a serious problem in adult and adolescent whose level of immunity is thought to be sufficiently effective in combating post transfusion malaria in an endemic area like Nigeria6. These parasites are prevalent in Nigeria but the extent to which it currently affects blood donors attending UMTH is unknown, we therefore, considered it necessary to contribute some information on this subject.

MATERIALS AND METHOD
This study was conducted at the University of Maiduguri Teaching hospital (UMTH) from February 2017 to May 2017. A total of 230blood donors which are negative to HIV 1/2, HBsAg, Syphilis and HCVwere recruited for the study. These donors were recruited using simple random sampling. Two millitres of the donor’s venous blood was collected into an EDTA container.ABO and RhD blood groups of the donors were determined using tile method. Malaria parasite was qualitatively determined by making thick blood films in duplicates for each blood samples on a clean grease free glass slide. these was allowed to air-dry after which it was stained with Giemsa stain. Stained films were examined under x100 objective lens of microscope with Immersion oil for any stage of malaria parasite. A slide is defined as negative if no asexual stage of the parasite is found after counting 100 microscopic fields.For, microfilaria parasite, a drop of anticoagulated blood was dispensed on a cleaned grease-free slide and covered with cover slip. It was examined microscopically using x10 and x 40 magnification for motile microfilaria. Result were analysed using, percentage and SPSS 20.0 statistical package. Chi-square was used to determine if prevalence was dependent on certain factors. A P-value of less than or equal to 0.05 (p=0.05) was considered as statistically significant.

RESULTS
A total of two hundred and thirty subjects (230) were recruited for the study. The subjects were within the age group of 18-55 years with the age group 20-29 having the
highest mode (47.6%, 110/230) and age group 50-59 years with the least (3%, 7/230).
Table I shows the prevalence of malaria parasite and filarial worms in the blood donors attending UMTH.Out of the 230 blood donors studied, 78 donors had malaria parasite giving a prevalence rate of 33.9% while none had filarial worm giving a prevalence rate of 0%.Table II shows the prevalence of malaria parasite in blood donors attending UMTH in relation to blood group and donation history. Malaria parasite in respect to ABO blood group, group B donors had the highest prevalence rate of 38.1% (16/42) while blood group AB donors had no malaria parasite in their blood. This difference was not statistical significant (x2 = 1.513, df= 3, p-value = 0.679). Malaria parasite with relation to Rh D blood group, Rh D- donors had a higher prevalence rate of 37.5% (6/16) while Rh D+ donors had a lower prevalence of 33.6% (72/214). This difference was also not statistically significant (x2 =0.99, df =1, p-value = 0.753). Family replacement donors had a higher prevalence of malaria infection 34.2% (77/225) when compared to voluntary donors who had a prevalence rate of 20% (1/4), this difference was also not statistically significant (x2 =0.441, df= 1, p=0.506). There was no commercial blood donor in this study. Repeat donors had a higher prevalence rate of malarial infection 36.1% (49/119) while first-time donors had a lower prevalence rate 35.1% (35/111). This prevalence is not statistically significant (x2 =0.543, df= 1, p=0.461).
Table III shows the prevalence of malaria parasite in relation to some social factors.Female donors had higher prevalence rate of malaria parasite (36.4%, 4/11) compared to male donors who had a prevalence rate of 33.8% (74/219). This difference is not statistically significant (x2=0.31, df= 1, p=0.860). Donors below 20 years had the highest prevalence of malarial infection 40% (4/10) while those within the age range of 40-49 had the least prevalence.It is not statistically significant (x2=3.994, df= 4, p=0.479).The prevalence is higher among the singles, 40.7% (50/123) while no infection was detected among the divorced donor. It is not statistically significant (x2=5.66, df= 2, p=0.059).
Table IV shows the prevalence of malaria parasite in relation to usage of insecticides and/ or insecticide treated net. Blood donors who neither used insecticides nor insecticide treated net had the highest prevalence rate of 50% (18/18) while those who used both insecticides and insecticide treated net had the least prevalence rate of 23.5% (4/17). This difference is not statistically significant.
Table V shows the prevalence of malaria parasite in relation to treatment with antimalarial drugs. Donors who 

said to have never been treated with antimalarial drugs had the highest prevalence of 56.7% (17/30) while donors who self-administered antimalarial drug within the last six months had the least prevalence 14.5% (11/76). This difference is statistically significant. No Filarial parasite is found in any of the donors.
DISCUSSION
Results obtained from this study showed that 78 blood donors had malaria while none had filarial worm showing a prevalence of 33.9% and 0% respectively. The prevalence of malaria parasitaemia in this study was lower than that reported by Abioye et al.15 who recorded a prevalence rate of 56% (140/250) in Abuja and was higher than the report of Garba et al.16 who reported a prevalence of 7.5% (27/360) in Kaduna. These differences in regional prevalence could be attributed to variation in predisposing factors such as present of Anopheles species, environmental conditions, climatic conditions, period of study, the study population and diagnostic test method used. The high prevalence rate may be attributed to current security challenges in Borno which forced people from other villages within other towns of the state to relocate to Maiduguri which in turn increases the population and decreases sanitary condition of the city. The decreased sanitary condition has resulted in increased chocked drainage channels which provide a suitable breeding ground for Anopheles mosquito.However, in relation to filarial worms the result from this study was inconsistent with the report of Bolaji et al.3 who reported a prevalence of 2% with Loa loa, Brugria Malayi and Wuchereria Bancrofti in the following proportion; 4(1.33%), 1(0.33%) and 1(0.33%) respectively.This difference may be attributed to difference in the number of subjects and geographical locations. Although Ochocerca Volvulus is prevalent in some areas in Bornu such as Hawul18,it is not prevalent in Maiduguri possibly due to lacks fast flowing water which is a suitable breeding site for its biological vector (Backfly).

This study further revealed that malaria parasitsitaemia is higher among blood group B donors while no malaria parasite was recorded in AB blood donors. This result does not tally with the report of Agboola et al.5 who reported a higher prevalence among blood group O donors. This difference may be as a result of chance. The difference in malaria among ABO blood groups in this study was however not statistically significant, indicating that susceptibility to malaria parasite is independent of a person’s ABO blood group. Also, a higher prevalence of parasitaemia among Rh D negative blood donors was reported in this study compared to the Rh D+ blood donors. This finding was not similar to a report by Bankole et al.19 who reported a higher prevalence among Rh D+ blood donors. The difference between Rh D blood groups in this study was not statistically significant, indicating that susceptibility to malaria parasite is independent of a person’s Rh D blood group. The study clearly suggests that family replacement donors were the major source of blood for transfusion in UMTH. This is consistent with findings from other researchers19, 20, indicating that family replacement donors were the major source of blood for transfusion in most states in Nigeria.There is higher prevalence of malaria parasitaemia in family replacement donors when compared to voluntary blood donors, this result is in line with the report of Olawumi et al.20, however not statistically significant. The lower prevalence recorded in this study indicates that there is reduced risk of transmission ofmalaria when blood productsare derived from voluntary donors. Result from this study shows a higher prevalence of parasitaemia in repeat donors when compared to first-time donors. This result is consistent with the report of Garba et al.16. This could be as a result of the fact that first-time donors are apprehensive and those having mild symptoms of malaria such as headache are usually excluded to donate blood.

The prevalence of malaria parasite in this study shows high rate among female donors when compared to male donors. The difference in prevalence between the genders may not be conclusive owing to the relatively small number of female donors who participated in the study. Higher prevalence of parasitaemia was found in donors whose age where below twenty and the least prevalence was seen in donors within the age range of 40-49 years of age. This result does not tally with the report of Ekwunife et al.(2011)6 who reported the highest prevalence among donors within the range of 25-29 and the least prevalence among donors within the age range of 50-54 years of age. The difference may be due to chance.There is higher prevalence rate in single (unmarried) blood donors while malaria parasite was not detected in the blood of the divorced donor. The result does not tally with the report of Alli et al.,4 who reported a higher prevalence among married donors which might be probably by chance.
Overall, there is no significant relationship between the prevalence of malaria infection and the usage of personal protection against mosquitos. This indicatesthat the current prevalence of malaria parasite among the blood donors is not dependent on the use of insecticide and/or insecticide treated nets. The results also indicated that there is statistically significant relationship between the prevalence of malaria infection and treatment with anti-malaria drugs. This indicates that treatment with antimalarial drugs significantly reduces the prevalence of malaria among blood donors. This coincides with the report of UNICEF 21, which states that the two major ways to reduce the spread of malaria are the use of insecticide treated mosquito nets and early diagnosis and prompt treatment with antimalarial medications.

CONCLUSION
In conclusion the result from this study shows a progressive increase in the prevalence of malaria parasite among blood of donors attending UMTH when compared with previous results. This increase is alarming as these donors are apparently healthy subjects indicating an increased risk of transmission of malaria through transfusion in Maiduguri. No filarial worm was recorded in this study. No statistically significant relationship was established betweenmalaria infection and the usage of Insecticide and/ or insecticide treated net. However, statistically significant relationship between the prevalence of malaria parasite and treatment with antimalarial drugs is noted.

Recommendations Haemoparasites can be transmitted through transfusion of infected blood derived from asymptomatic donors. This may negatively affect patient’s health and increase the duration of their illness. State government should improve the sanitary condition of Maiduguri and environs which will in turn reduce mosquito breeding sites. Screening donors for parasitic infections should be included in the current nation’s transfusion guidelines. Enlighten donors on better ways of preventing infections with haemoparasites.Encourage prompt and effective treatment of infected prospective donors.In additions incentives such as insecticide treated mosquito net, insect repellent and refreshments should be given to donors as this may encourage voluntary donation and as well reduce the prevalence of haemoparasites in the blood of donors.

REFERENCES

1. Schmaier, A. H., & Petruzzelli, L.M. (2003). Haematology for the medical student. Lippincott Williams & Wikins.

2. Medical Dictionary for the Health Professions and Nursing (MDHPN). (2012). Retrieved May 23rd 2017 from http://medical-dictionary.thefreedic tionary.com/haemoparasite

3. Bolaji, O.S., Uthman-izobo, S.O., Ojurongbe, O., Opaleye, O.O & Adeyeba, O.A. (2014). Filariasis among asymptomatic blood donors in general hospital, Odan Marina-Lagos, Nigeria. International Journal of Research in Applied, Natural and Social Sciences, 2(6), 177-182.

4. Alli, J.A., Okonko, O.I., Abraham, O.A., Kolade,
A.F., Ogunjobi, P.N., Salak, A.O., Ojezele, M.O & Nwanze, J.C. (2010). A serosurvey of blood parasites (plasmodium, microfilaria, HIV, HBsAg, HCV antibodies) in prospective Nigerian blood donors. Research Journal of Medical Science, 4(4), 255-275.

5. Agboola, T.F., Ajayi, M.B., Adeleke, M.A. & Gyang, P.V. (2010). Prevalence of malaria parasite among blood donor in Lagos University teaching hospital, Lagos Nigeria. Scholars Research Library Annals of Biological Research, 1(3), 72-75.

6. Ekwunife, C.A., Ozumba, N.A., Eneanya, C. I. & Nwaorgu, O.C. (2011). Malaria infection among blood donors in Onitsha urban, Southeast Nigeria. Sierra Leone Journal of Biomedical Research 3(1), 21-26.

7. Anthony, C.N., Yee-Ling, L., Jia-Siang, S., Mun-Yik, F., Hany, A., Wai-Linn, Z., Indra, J. & Rohela, M. (2013). Malaysian child infected with plasmodium vivax via blood transfusion: a case report. Malaria Journal 12:308. Retrived on January 10th 2017 from http://www.malariajournal.com/content/12/1/308.

8. Alex. K.O., Christopher, P. & Imelda, B. (2010). Transfusion transmittable malaria in countries where malaria is endemic: A review of the literature from sub- Saharan African. Journal of Clinical Infectious Diseases, 1192-1193.

9. Kitchen, A.D., and Chiodini, P.L. (2006). Malaria and blood transfusion. Vox Sanguiis, 90, 77-84

10. Okeke, C.O., Agbasiere, F.N., Amilo, I.G. & Ifeanyichukwu, O.M. (2017). Methemoglobin levels among malaria parasite-infected blood donors in Nnewi, Southeastern, Nigeria. Tropical Journal of Medical Research, 20(1), 80-83.

11. Ojo-bola, T., Omisakin, C.T., Esan, A.J. & Owoseni, M.F. (2014). Filaria worm among prospective blood donors attending a tertiary health institution in south-west Nigeria. Journal of Dental and Medical Sciences, 13(1), 84-87.

12. Nagwa, M.E. (2015). Recent updates in transfusion transmitted parasitic diseases. Journal of Bacteriology, Virology and Parasitology, 2(1) 1-11

13. Bregant, E.T., Balzarinl, L., Ghiringhelli, C. & Tarsta, P. (2003). Transfusional Mansonella pertans microfiariasis. Parassitologia 45, 71-72

14. Chessbrough, M. (2000). District laboratory practice in tropical countries. Part 1, (2nd ed). Cambridge University Press:UK.

15. Abioye, J.O.K., Abdullahi, D.K., Alalade, O.M., & Olokun, A.L. (2015). Incidence of malaria parasite in blood donors at Kwali General Hospital, FCT Abuja. Journal of Emerging Trends in Engineering and Applied Sciences, 6(3), 212-216.

16. Garba, D.D., Ameh, B.J., Whong, C.M.Z. & Mukhtar, M.A. (2016). Prevalence of malaria parasites among blood donors in Kaduna, Nigeria. International Journal of Research in Medical Sciences, 4(6), 2112-2119.

17. Okoye, I.C., Dakul, D.A. & Wakawa, A.I. (2011). Perception of onchocerciasis by rural Hausa women in northeast Nigeria and the implications for onchocerciasis control. Animal Research International, 8(1), 1309 – 1314.

18. Bankole, H.O., Richard, O., Eguagie, O.O., & Tola,
O.O. (2014) Asymptomatic malaria among blood donors in Benin city Nigeria. Iranian Journal of Parasitology, 9(3), 415–422.

19. Olawumi, H.O., Fadeyi, A., Babatunde, S.K., Akanbi, A.A., Babatunde, A.S., Sani, M.A., & Aderibigbe, A.S. (2015). Malaria parasitaemia among blood donors in Ilorin, Nigeria. African Journal of Infectious Disease, 9(1), 10–13.

20. UNICEF. (2000). The Prescriber: Promoting rational use of drugs and correct case management in basic health services. Retrieved on 5th May 2017, from https://www.unicef.org/ prescriber/eng_p18.pdf




Development of a real-time PCR assay and comparison to CHROMagarTM STEC to screen for Shiga toxin-producing Escherichia coli in stool, Cape Town, South Africa

John B. Kalule, Karen H. Keddy, Anthony Smith, Mark P. Nicol,1 and Lourens Robberts

 

ABSTRACT

Introduction: Shiga toxin-producing Escherichia coli (STEC) is an emerging infectious pathogen which could lead to haemolytic uremic syndrome. Even though previous studies have compared the performance of CHROMagarTMSTEC to real-time polymerase chain reaction (PCR) in Europe, no study has been done to assess its performance on African isolates. Objectives: This project aimed to validate and test an in-house-developed duplex real-time PCR and use it as a reference standard to determine the performance of CHROMagarTMSTEC on African isolates from diarrhoeic stool samples. Methods: This study evaluated STEC diagnostic technology on African isolates. An in-house-developed duplex real-time PCR assay for detection of stx1 and stx2 was validated and tested on diarrhoeic stool samples and then used as a reference standard to assess the performance of CHROMagarTMSTEC. Real-time PCR was used to screen for stx in tryptic soy broth and the suspected STEC isolates, while conventional PCR was used to detect the other virulence genes possessed by the isolates. Results: The real-time PCR limit of detection was 5.3 target copies/?L of broth. The mean melting temperature on melt-curve analysis for detection of stx1 was 58.2 °C and for stx2 was 65.3 °C. Of 226 specimens screened, real-time PCR detected stx in 14 specimens (6.2%, 95% confidence interval = 3.43% – 10.18%). The sensitivity, specificity, negative predictive value and positive predictive value of the CHROMagarTMSTEC were 33.3%, 77.4%, 95.3% and 11.3%.
Conclusions: The in-house developed real-time PCR assay is a sensitive and specific option for laboratory detection of STEC as compared to CHROMagarTMSTEC in this setting.

INTRODUCTION

Globally, food- and water-borne outbreaks of both O157 and non-O157 Shiga toxin-producing Escherichia coli (STEC) have been successfully detected due to the availability of good baseline data and effective active laboratory-based surveillance systems.1,2,3,4 Early detection of outbreaks is important to minimise morbidity, mortality and associated economic losses.5 There is a lack of good baseline data on STEC in Africa, which can be attributed to a lack of laboratory resources and the surveillance strategy employed. STEC has been implicated in outbreaks of bloody diarrhoea in sub-Saharan countries;6,7,8 however, these have been difficult to track and manage due to laboratory weakness.9,10 Furthermore, typical haemolytic uremic syndrome, which is overwhelmingly caused by STEC, was reported as the leading contributor to acute renal failure in paediatric patients at a South African academic hospital.11 Even though several studies have evaluated the performance of CHROMagarTMSTEC by comparison to molecular and antigen detection methods in developed countries,12,13 no study has so far evaluated its performance in Africa. This is necessary, especially given that there are geographical differences in characteristics of STEC that are dependent on the index of suspicion for the different STEC serotypes and on the availability of suitable laboratory methods to detect them.14
In many South African (and African) laboratories, stool specimens are not routinely tested for STEC, although physicians may request tests specific for E. coli serotype O157:H7, if it is clinically suspected. Testing is based on the non-sorbitol fermenting property using sorbitol MacConkey and only on request by a physician. This practice is of concern, is misleading and underestimates the real magnitude of STEC, since not all serotype O157 strains are non-sorbitol fermenting (O157: NM), and over 470 non-O157 serotypes have been attributed to clinical disease.15 Laboratory capacity for molecular detection is increasingly available in African countries and may, in some cases, be simpler than culture-based detection. This project, therefore, aimed to validate and test diarrhoeic stool samples by using an in-house developed duplex real-time polymerase chain reaction (PCR) and use it as a reference standard to determine the performance of CHROMagarTMSTEC on African isolates. The duplex assay was used to screen tryptic soy broth (TSB) for stx following overnight stool enrichment, and conventional PCR was used to screen for the other diarrhoeagenic E. coli virulence genes. Diarrhoeagenic E. coli were serotyped, and stx-positive isolates were tested for Shiga toxin production using immunochromatography.

Methods
Ethical approval
This study was approved by the ethics and research committee of the Faculty of Health Sciences at the University of Cape Town (HREC REF: 014/2015).

Study design
This study validated an in-house-developed duplex real-time PCR assay for detection of stx1 and stx2. The assay was then tested on diarrhoeic stool samples at a tertiary referral hospital and was used as a reference standard to assess the performance of a commercial chromogenic medium (CHROMagarTMSTEC) for STEC screening (Figure 1).

Summary of methods used in this study.
Target plasmid preparation
The real-time PCR previously described by Grys et al.16 was used to amplify stx1 and stx2 gene targets from a STEC O157:H7 NCTC control strain (C4193-1) with both stx1 (subtype 1a) and stx2 (subtype 2a). PCR amplicon size was confirmed visually by agarose gel detection (~208 bp for stx1 and ~204 bp for stx2) before confirmation by sequencing using the Big Dye® Terminator v3.1 Cycle Sequencing Kit (Life Technologies Corporation, Carlsbad, California, United States). We used primers 1a and 2a (Table 1) for unidirectional Sanger sequencing of the amplicons. Resultant sequences were then trimmed and submitted for BLAST analysis against the NCBI database and confirming stx1 or stx2 target sequences in comparison to O157:H7 EDL933 (NCBI Reference: NC_002655.2).17
Purified amplicons (Mini Elute Gel extraction kit, Qiagen, Madrid, Spain) were cloned using CloneJet PCR cloning kit (Thermofisher Scientific, Austin, Texas, United States) into a pJet 1.2/blunt vector using the sticky end cloning protocol and transfected into the JM109 competent cells by calcium chloride transformation. Plasmids containing stx1 and stx2 were separately extracted using a Genopure plasmid Maxi kit (Roche Life Sciences, Rotkreuz, Switzerland) and quantified by spectrophotometry. To verify successful preparation purified plasmids were subjected to PCR amplification using primers 1a and 1b for stx1 and 2a and 2b for stx2 with amplicon size visually confirmed by agarose gel detection and subsequent sequence analysis. Plasmid quantification was determined spectrophotometrically employing the BioDrop-µLite (Isogen Life Science, B.V, Veldzigt, Netherlands). The A260 was used to calculate the plasmid concentration expressed as the number of molecules/µL.

To assess the potential for PCR cross-reactivity and assess the analytical specificity of the hybridisation probe-based real-time PCR described by Grys et al.,16 the primer and probe sequences were subjected to BLAST analysis on the NCBI database. The PCR reaction was optimised for use on

the LightCycler®480 Instrument II (Roche Life Sciences, Rotkreuz, Switzerland) employing the LightCycler® 480 Probes Master mastermix (Roche Life Sciences, Rotkreuz, Switzerland) with modification to the thermal cycling conditions for amplification consisting of denaturation at 95 °C for 10 min followed by 45 cycles of 95 °C for 5 s, 56 °C for 5 s and 72 °C for 15 s. A positive amplification signal was defined as an increase in fluorescence signal that crossed the threshold before 30 cycles. Amplicon identity was determined using the melt-curve analysis program of 95 °C for 30 s, 40 °C for 60 s and 85 °C for 5 s with continuous fluorescence acquisition. The Multi-color HybProbe detection format was used for analysis, combining the Red 610, Red 640 and FAM filter pairs (LightCycler®480 Instrument II Manual, Roche Life Sciences, Rotkreuz, Switzerland). The resulting amplicon size was visualised using agarose gel electrophoresis and subjected to DNA sequencing and BLAST alignment to reference stx1a and stx2a sequences (NC_002655.2). To mimic the sample matrix for sensitivity determination, TSB was inoculated with a pea-size amount of stool (from a single donor) shown to be stx-negative by PCR. To this inoculated broth 1 mL of plasmid stock (5.3*106 copies/µL) containing both stx1 and stx2 was added and serially diluted eight times in 9 mL of TSB, to a lowest dilution of 1:108 (53 plasmid copies/ml). Nucleic acid extraction was performed on 200 µL broth employing the MagNApure LC instrument (Roche Diagnostics, Rotkreuz, Switzerland) to yield 100 µL of extract. Initially, real-time PCR was performed in triplicate using a template from each of the eight dilutions to estimate a limit of detection (LOD). Subsequently, real-time PCR was performed in eight replicates on the dilution with the estimated LOD, as well as one dilution above and one dilution below the estimate. The LOD was defined as the lowest plasmid concentration spiked into TSB, before nucleic acid extraction, yielding a positive signal, as described above in all eight replicates. Nucleic acid extractions from STEC subtypes 1d (Reference strain MH1813, GenBank accession No. AY170851), 2b (Reference strain EH250, GenBank accession No. AF043627), 2c (Reference strain 031, GenBank accession No. L11079), 2d (Reference strain C165-02, GenBank accession No. DQ059012), 2e (Reference strain S1191, GenBank accession No. M21534), 2f (Reference strain T4/97, GenBank accession No. AJ010730) and 2g (Reference strain 7V, GenBank accession No. AY286000) were also subjected to PCR amplification to assess impact of strain variation on detection. The reproducibility of melting temperature assessment for stx1 and stx2 differentiation was determined by testing 24 replicates of TSB spiked with cloned stx1 and stx2 plasmids. To further assess the reproducibility of melting temperature across the subtypes, three stx1 subtypes and seven stx2 subtypes were tested similarly.

Clinical specimen testing
Between September 2014 and May 2015, we collected same day residual stool after routine testing from 226 consecutive stool specimens (the stool samples were transported in a temperature regulated box and processed within 12 h of collection) from the National Health
Laboratory Services located at the Groote Schuur Hospital in Cape Town, South Africa, a tertiary care academic teaching laboratory affiliated with the University of Cape Town. This tertiary academic hospital serves the greater Cape Town area.

A pea-sized stool sample was inoculated in 90 mL of TSB and vortexed before incubation at 37 °C for 18 h. Two hundred microlitres of broth were subsequently extracted employing the MagNA Pure LC Total Nucleic Acid isolation kit (Roche Diagnostics, Rotkreuz, Switzerland) using the total variable elution volume protocol and following the manufacturer’s manual (version 14) to yield 100 µL of nucleic acid extract.

In addition, CHROMagarTMSTEC (CHROMagar Microbiology, Paris, France) was inoculated with a loop full of overnight inoculated broth and incubated at 37 °C for 18 h. Bright mauve colonies (up to five mauve colonies were picked per sample, depending on the number of mauve colonies formed) were sub-cultured onto MacConkey agar with crystal violet, sorbitol MacConkey agar, and 2% blood agar (Green point Media, National Health Laboratory Service, Albertynshof, South Africa). E. coli was presumptively identified as lactose-positive, oxidase-negative, spot indole-positive and pyrrolidonyl arylamidase (PYR)-negative with confirmatory identification using VITEK 2 (bioMerieux, Inc., Durham, North Carolina, United States).

Isolate characterisation
Isolates yielding mauve colonies on CHROMagarTMSTEC and presumptively identified as E. coli were subjected to stx characterisation employing the real-time PCR assay characterised herein. Other diarrhoeic E. coli virulence genes, including the fimbrial adhesion gene for diffusely adherent E. coli, the anti-aggregation protein transporter gene for enteroaggregative E. coli, heat-stable and heat-labile enterotoxin genes of enterotoxigenic E. coli, the intimin coding gene eae for enteropathogenic E. coli (EPEC) and the bundle-forming pili gene for the typical EPEC, were determined using standard gel-based PCR as previously described using primers as shown in Table 2.18

ST, heat-stable; LT, heat-labile.
To confirm Shiga toxin production among stx-positive isolates, the Immunocard STAT!® EHEC (Meridian Biosciences, Inc., Cincinnati, Ohio, United States) was used to detect Shiga toxin 1 and 2 (by employing immunochromatography with toxin-directed monoclonal antibodies labelled with red-coloured gold particles). All mauve isolates found to carry virulence genes were serotyped at the Centre for Enteric Diseases, National Institute of Communicable Disease, Johannesburg, by employing antisera (Statens Serum Institut, Copenhagen, Denmark) and the detection of somatic O-antigens as previously described.19,20 H-antigen serotyping was not undertaken.
Statistical analysis
Data on the possession of virulence genes, cultural characteristics on different media, serotypes and Shiga toxin production was entered in Microsoft Office Excel 2010 (Microsoft Corp.,Redmond, Washington, United for Disease Control and Prevention, Atlanta, Georgia, United States) for analysis. Using the LightCycler 480 II software, efficiency of the in-house real-time PCR assay was determined. The amplification curves and the melting peaks were used to differentiate between stx1 and stx2.

Results
Real-time polymerase chain reaction validation
The BLAST analysis of the primers and probe sequence specificity yielded no significant homology to non-stx targets (data not shown). Real-time PCR amplicons generated were confirmed as 208 bp for stx1 and 204 bp for stx2 (Figure 2). Sequencing and BLAST analysis confirmed the identity of both stx1 and stx2 amplicons. The serially diluted plasmid-stool-TSB was successfully amplified in

8/8 replicates in the sixth dilution, whereas the seventh dilution yielded an amplification signal in 3/8 replicates, yielding a LOD of 5.3 target copies/µL of broth. All other stx subtypes investigated (stx1a, stx1b, stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g) were successfully amplified by this assay (data not shown). stx1 and stx2 were successfully distinguished by a melting temperature of 58.2 °C (SD = 0.033) and 65.3 °C (SD = 0.037) (Figures 3–6). The Tm for stx2 subtypes 2a, 2b, 2c, 2d, 2e, 2f and 2g were the same at 65.3 °C (SD = 0.037, 0.041, 0.035, 0.039, 0.034, 0.033 and 0.032, respectively), whereas that of 1d was 44.7 °C (SD = 0.042). The efficiency of the assay was 1.99 as calculated from the amplification curves generated using the Light Cycler® 480 software. The duplex assay detected both targets in the same run, and these could be differentiated by the melt curve with two distinct peaks at 58.2 °C for stx1 and 65.3 °C for stx2 (Figure 7).

Performance of CHROMagarTMSTEC
Of the 53 mauve isolates, 48 were negative for stx genes using the validated real-time PCR assay. Of the 14 broths that were positive on PCR, nine did not yield any mauve colonies on CHROMagarTMSTEC culture.

Isolate characteristics
Forty-four (83%) of the 53 mauve colonies fermented lactose on MacConkey agar with crystal violet. Eleven (25%) of the 44 lactose fermenters were non-sorbitol-fermenting. Real-time PCR on the 44 E. coli confirmed the presence of stx genes in five (11%), whereas 39 were negative for the stx gene. Real-time PCR was not done on the nine non-lactose fermenting isolates as these were found not to be E. coli on biochemical testing. Four of the five stx positive E. coli colonies were also positive in the real-time PCR broth assay. Of the 39 stx-negative E. coli, only four (12.5%) carried eae genes, whereas four possessed aat genes. Of the four eae positive isolates, two also had the bfp genes and were typical EPEC. The other two eae positive isolates did not possess the bfp genes and were classified as atypical EPEC. The four enteroaggregative stx-negative isolates all belonged to E 

coli serotype O104. All the typical EPEC belonged to serotype O55, whereas one of the two atypical EPEC belonged to serotype O101. The atypical EPEC serotype O101 was from stx2 positive broth. The other atypical EPEC isolate was untypeable. No diffusely adherent E. coli, enteroinvasive E. coli or enterotoxigenic E. coli were detected. None of the 53 E. coli isolates that were screened by immunochromatography was positive for Shiga toxins. For the CHROMagarTMSTEC, sensitivity was 33.3%, specificity was 77.4%, negative predictive value was 95% and positive predictive value was 9.4% (Table 3).

DISCUSSION
We validated the use of a previously described duplex real-time PCR assay with modification able to detect and differentiate stx1 (melting temperature = 58.2 °C) and stx2 (melting temperature = 65.3 °C) from overnight broth enrichment with a LOD of 5.3 target copies/µL broth. This assay was able to detect both stx1 and stx2 in the same run, thus potentially reducing process turn-around time in a busy laboratory setting. Timely reporting of STEC infections is important, because use of certain antibiotics is contraindicated in STEC infections.
Compared to the validated duplex real-time PCR, CHROMagarTMSTEC showed a sensitivity of 33%, specificity of 77.4%, negative predictive value of 9.4% and positive predictive value of 95% for detection of STEC in stool following TSB enrichment. Of the 53 mauve isolates, 48 were negative for stx genes on use of the validated real-time PCR, whereas nine of the 14 PCR-positive broths did not yield any mauve colonies when cultured on CHROMagarTMSTEC. Reasons for the poor performance of this medium in relation to the in-house developed duplex real-time PCR include the following: (1) delays in reporting of diarrhoea cases to a tertiary hospital (where samples were collected) may have led to loss of stx genes; STEC numbers are sharply reduced in stool after one week of illness, and the Shiga toxin genes might be lost by the bacteria.21 (2) CHROMagarTMSTEC selects for tellurite resistant strains but misses the tellurite susceptible STEC whose prevalence in this setting is not known.
For a chromogenic medium to be considered for routine screening purposes, it must have high specificity so as not to waste scarce laboratory resources on false positives. The false positivity rate in this setting (48/53 [90.6%]) is higher than has previously been reported in Europe (16.3% reported by Gouali et al., 2013 and 18.3% by Wylie et al., 2013).12,22
Similar studies to evaluate this medium were done in Canada, Finland, and Germany, all of which reported high sensitivities for STEC serotypes O26, O111, O121, O145, O118, and O157.13,22,23 The specificity of 77.4% noted in this study was low compared to values (between 95.8% and 98.9%) reported in similar studies done in Europe. The difference in sensitivity and specificity could be explained by the differences in the patient characteristics (whether they present with haemolytic uremic syndrome and or bloody diarrhoea or not). Unlike the studies in Europe, this study did not focus on only patients with haemolytic uremic syndrome or bloody diarrhoea. Additionally, the distribution of tellurite resistant STEC (which is targeted by CHROMagarTMSTEC) in this setting is not known. The prevalence of stx genes in stool samples (6.2%) was lower than the 9% previously reported by Kullin et al., 2015. Among the 53 isolates that formed mauve colonies on CHROMagarTMSTEC, five were STEC (two serotype O26 and others non-typeable), four were enteroaggregative E. coli (serotype O104), two were atypical EPEC (serotype O101 and non-typeable) and two were typical EPEC (serotype O55). Serotypes O26 and O104 are among the top six STEC serotypes globally.25,26 CHROMagarTMSTEC is intended for STEC culture; however, we detected other diarrhoeic E. coli pathotypes using this medium. The other pathotypes detected might have been hybrid strains that lost the stx genes or hybrid strains whose stx genes could not be detected using the primers employed in this study. Notably, bacteriophages carrying the stx genes are very quickly lost both in vivo and in vitro,27 and not all stx primers can detect all the stx gene variants.28

Limitations
Not all STEC are tellurite resistant and may have been missed on CHROMagarTMSTEC culture. This study only focused on the strains that formed mauve colonies. The stool samples in this study were collected between September 2014 and May 2015, and therefore the results of this study as regards prevalence of STEC may not reflect the entire year or areas of South Africa other than Cape Town.

Conclusions
The in-house developed real-time PCR assay is a sensitive and specific alternative to the currently used diagnostic strategy. Due to the high false positivity rate, CHROMagarTMSTEC can only be used as an adjunt to a more sensitive and specific assay such as real-time PCR.

Reliability
We confirm that the experiments conducted in this study will yield the same results during repeated trials using the same reagents and detection platforms.

Validity
To the best of our knowledge, the findings of this study, as obtained using the methods we employed, are valid for the study area and season. The in-house developed real-time PCR may, however, be adopted in other laboratories in developing countries.

Acknowledgements
We acknowledge the Center for Enteric Disease Research Unit, National Institute of Communicable Disease (Sandringham, Johannesburg, South Africa) for serotyping of the E. coli isolates. We also acknowledge the National Research Foundation, South Africa, the ACP-RISE scholarship fund and the ADB-HEST fund.

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Cellular and Molecular determinants of Embryo implantation: A Review.

Osazee Kehinde
Department of Obstetrics and Gynaecology, School of Medicine, College of Medical Sciences, University of Benin, Nigeria.
Orhue A.A. E
St. Augustine Medical Center, Ugbowo, Benin City, Edo State, Nigeria
All correspondence to: Osazee Kehinde Email; kehindeosazee@gmail.com

 

ABSTRACT
In the last two decades, advances have emerged in reproductive technologies. Only limited number of cases have resulted in a successful outcome, mainly due to implantation failure. The course of embryo implantation is dynamic, and it involves the interplay of cellular and molecular events between the endometrium and the nascent blastocyst in a well-coordinated manner, within a time frame called window of implantation. Although animal studies have tried to elucidate the various molecular and cellular pathways involved in embryo implantation, there is a paucity of knowledge about the humans. The direct study of the implantation events in human may be impossible for ethical and technical reasons. Hence, a good understanding of the factors involved may trigger research aim at modulating the cellular and molecular attributes for therapeutic purposes.

Keywords: Implantation, Cytokines, immune cells, microRNA

Introduction: The implantation of the embryo in the endometrium is crucial for the development of the pregnancy andis essential for the reproduction of organisms including humans. The act of accepting a blastocyst, a supposed foreign tissue because of the paternal components, by the maternal endometrium, involves the interplay of many factors. These factors,produced by the blastocyst as well as the maternal endometrium, are the cytokines, chemokines, adhesion molecules, certain class of major histocompatibility complex(MHC) and recently the microRNAs. For successful implantation, the interactive process of the blastocyst and endometrium must create controlled regulatory settings with these factors as theexcess of its pro-inflammatory or anti-inflammatory process will compromise the implantation process. So, these factors must play their regulatory role accordingly. The chemokinescreate good orientation of the blastocyst and the adhesion molecules establishing good interactions while the uterine Natural Killer cells (uNK) and the regulatory T cells play a pivotal role in the maternal immunological response to the blastocyst.
The concept of implantation involves the invasion of the maternal endometrium by the blastocystaim to establish a nutrient pathway essential for the developing embryo. Considering the allogeneic nature of the blastocyst and the immunological activity involved in the acceptance by the maternalendometrium, make the implantation a complex process. Before implantation, the endometrium undergoes structural and functional changes under the influence of the Ovarian steroid hormones in a process term implantation window. The endometrium, haven been primed by the oestrogen in the proliferative phase of the menstrual cycle, starts secreting various substances between 19-24 days from the stromal glands under the influence of the progesterone in the secretory phase of the cycle. This process forms the decidualization and it varies with species such that oestrogen and progesterone are required in humans and Rats while Pigs, Hamster and Rabbit only require oestrogen.

Cytokines
This structural modification induced by oestrogen and progesterone facilitate the apposition, attachment and invasion through theproduction of cytokines, growth factors, chemokines, adhesion molecules and immune cells. The blastocyst, on the other hand, is endow with receptors, adhesion molecules and production of cytokines and other mediators in the region proposed for the implantation process.
Implantation occurs 6-7 days post fertilisation and become fully embedded after ten days. The outcome depends on the successful establishment of the mother for the semi allogeneicembryo. In turn depends on the complex signalling network of the pro-inflammatory and the anti-inflammatory activities of the cytokines and the adhesion molecules. As the blastocyst approaches the endometrium, it develops polarity that influences its orientation and the trophectoderm over the inner cell mass move towards the endometrial surface for implantation in humans. Though the mechanism is not known, some authorities have proposed due to chemokine gradient in the uterus. These chemokines are CX3CL1, CCL7, CCL14, and CCL4. While the receptors on the blastocyst are CCR1, CCR3, CX3CR1.

The blastocyst in the uterus triggers the activities of the cytokines and interleukin-6(IL-6) family plays a very important role in this regard. These cytokines are Leukemia inhibitory factor(LIF), IL-11, Neurotrophin factor and cardiotrophin 1. They activate STAT3 through their intracellular signalling gp130. LIF is a pro-inflammatory cytokine and in combination with LIF-BR are produced during the secretory phase of the menstrual cycle to regulate aggregation of immune cells, migration of macrophages and restriction of NK cells in the endometrium during implantation. Its activity influenced by IL-1, Tumour Necrosis Factor(TNF-alpha), leptin, insulin-like growth factor(IGF) and Tissue growth factor(TGF-beta). Its role on the blastocyst is not known. The fact implantation is maternofetalinterplay; the blastocyst LIF may exert some control on the endometrial LIF to facilitate its receptivity towards achieving a successful implantation.It has been shown that LIFhas a role in the improvement of fertility rate in animal studies, but there is a conflictopinion abouthumans. However, it implantation potential has been established in a dose- dependent manner. IL-6is regulated by ovarian steroid hormones especially the oestrogen and Its production rises but drops during the late secretory phase of the menstrual cycle. While TGF and hCG inhibit its production. IL-11 is an anti-inflammatory, and its production is highest during decidualization with no cyclical variation because of it regulatory role to the pro-inflammatory cytokines especially TNF-alpha.

Growth Factors
IGF-1 and IGF-2 regulate the production of oestrogen and progesterone respectively. IGF-2 promotes invasion of the trophoblastand regulated by insulin-like growth factor binding protein (IGFBP) in an inhibitory manner to preventexcessive invasion of the trophoblast that could be detrimental.L-Selectin in the trophoblast enhances apposition byattraction to the carbohydrate ligand in the endometrium. Despite the perceived function of the L-selectin, its role in humans issubject to debate in the literature. Instead, the integrinalpha5beta3 is found to play a prominent role in the penetration phase. AndComplimented by the down -regulation of MuC-1 production especially in human and mice.

Immune cells
The immune cells of relevance in implantation are the uNK cells, macrophages, dendritic cells (DCs), and T cells. The uNK cells are more produced in the late secretory phase with an indirect regulation by ovarian steroid hormones and prolactin. It shares a common CD56+ receptor with the peripheral NK cells, but its larger proportion have CD56bright receptors. uNK cells enhance decasualization and regulate maternal immune response through the regulation of the cytokines production and the vascularization of the endometrium by the production of vascular endothelial growth factors (VEGF) which promotes angiogenesis.
The concept of T cells through TH1/TH2 balance has generated aconflict of opinions in the literature. The earlier belief that pregnancy has a bias for TH2 mediated events and TH1 cytokines such as interferon-y (IFN-y), and TNF-alpha can lead to infertility and abortion has become
thesubject of debate. Studies have shown in both human and mice that TH1 is essential during implantation. The cytokinesstimulate the production of LIF and enhance the process of angiogenesis. Furthermore, the TH1 triggers the production of TH2 cytokines. While, Treg with its receptor CD4+CD25+ plays a central role in peripheral tolerance and suppression of autoimmuneT cells. Though the mechanism is not known, some authors have proposed through the trigger of dendritic cells (DCs) to produce indoleamine 2, threedioxygenases (IDO) or through the inhibition of CD4+ and CD8+ T cells.While macrophages aid trophoblast invasion and as well as clearing of apoptotic materials to prevent trigger of theimmune response. In a bid to enhance tolerance from the maternal endometrium, the trophoblast through its MCP and CD59 factors inhibits the compliments C3, C5 and C5aR. However, it expresses various types of Major Histocom-patibility Complex molecules; HLA-C, HLA-G, HLA-E. While the HLA-C is polymorphic which could be a source of threat to the embryo, HLA-G has restricted polymorphism and does not involve any antigenic activity. It enhances tolerance through the regulation of cytokines from the uNK cells and the interaction of HLA-E with CD96/NKG2 receptors on the uNK cells. Also, it promotes the combination of HLA-C with the maternal killer immunoglobin-like receptor (KIRs) A and B expressed by uNK cells.

MicroRNAs
MicroRNAs arenon- coding RNAs expressed in many cell types where they regulate the expression of RNAs by modulating the stability and translocation of mRNAs. They regulate close to 50% of all protein- coding genes in mammals.Through their potential in regulating several genes expression, they are said to bebio regulator of several cellular processes that include differentiation, proliferation and apoptosis.
MicroRNAs plays a role in implantation process under the influence of ovarian steroid hormones. The oestrogen indirectly regulates microRNA by infringing on its biosynthesis through the alteration of enzymes. While Progesterone synergistically with oestrogen enhance microRNA activity in the uterus. Alteration in the microRNAs expression can constitute an adverse effect on the implantation process, for example, HLA-G expression down- regulation by microRNA-148 and microRNA-152 will compromise the immune tolerance and cytolysis from uNK cells. However, microRNA 29b is involved in the development of placental by promoting invasion, apoptosis and angiogenesis through its target on Nodal an important gene involved in early embryogenesis and cell differentiation. Several studies have shown the crucial role of microRNAs in regulating the fundamental pathway associated with pregnancy outcome. However, there is need for better understanding of the interactive process of microRNAs and mRNA to attain the needed pregnancy outcome.

Infertility affects 8-12% of couples worldwide, and a significant number of these couples require Assisted Reproduction Technology as a mainstay treatment modality. Though the advent of modern technology has enhanced the availability of relatively good quality embryo, only about 20-30% of such embryos result in a live birth. Epidemiological studies have shown that most of the pregnancy losses are due to implantation failure. Therefore, a good understanding of the complementary interaction of the endometrium and the nascent blastocyst may engender research aim at modulating the cellular and the molecular factors for therapeutic purposes.

Conclusion
Implantation constitute an inflammatory activity involving both the pro-inflammatory and anti-inflammatory factors with the background immunological complex aim at creating access for the blastocyst into the maternal endometrium. The embryo tends to exhibit an aggression through its pro-inflammatory cytokines resulting in a penetrating effect on the endometrium. While the endometrium, on the other hand,creates a receptive environment through the implantation window. The interplay must be in a regulatory environment that is ensured by the maternal endometrium through the influence of ovarian steroid hormones and production of anti-inflammatory cytokines. While these various factors play a key role in the implantation process, their dose- dependent manner must bebear in mind as theexcessive level could compromise pregnancy outcome.

REFERENCES:

1. Fu G, Brkic J, Hayder H, Peng C. MicroRNAs in Human Placental Development and Pregnancy Complications. Int J Mol Sci. 2013;14(3):5519-44.

2. Castro-Rendon WA, Castro-Alvarez JF, Guzman-Martinez C, Bueno-Sanchez JC. Blastocyst-endometrium interaction: intertwining a cytokine network. Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas. 2006;39(11):1373-85.

3. Singh M, Chaudhry P, Asselin E. Bridging endometrial receptivity and implantation: network of hormones, cytokines, and growth factors. Journal of Endocrinology. 2011;210(1):5-14.

4. Guzeloglu-Kayisli O, Kayisli UA, Taylor HS, editors. The role of growth factors and cytokines during implantation: endocrine and paracrine interactions. Seminars in reproductive medicine; 2009: NIH Public Access.
5. Dimitriadis E, White C, Jones R, Salamonsen L. Cytokines, chemokines and growth factors in endometrium related to implantation. Human reproduction update. 2005;11(6):613-30.

6. Guerin LR, Prins JR, Robertson SA. Regulatory T-cells and immune tolerance in pregnancy: a new target for infertility treatment? Human reproduction update. 2009;15(5):517-35.

7. Gnainsky Y, Granot I, Aldo PB, Barash A, Or Y, Schechtman E, et al. Local injury of the endometrium induces an inflammatory response that promotes successful implantation. Fertility and sterility. 2010;94(6):2030-6.

8. Chen D-b, Wang W. Human placental microRNAs and preeclampsia. Biology of reproduction. 2013;88(5):130, 1-11.

9. King AE, Critchley HO. Oestrogen and progesterone regulation of inflammatory processes in the human endometrium. The Journal of steroid biochemistry and molecular biology. 2010;120(2-3):116-26.

10. Cork B, Tuckerman E, Li T, Laird S. Expression of interleukin (IL)-11 receptor by the human endometrium in vivo and effects of IL-11, IL-6 and LIF on the production of MMP and cytokines by human endometrial cells in vitro. Molecular human reproduction. 2002;8(9):841-8.

11. Mourik MS, Macklon NS, Heijnen CJ. Embryonic implantation: cytokines, adhesion molecules, and immune cells in establishing an implantation environment. Journal of leukocyte biology. 2009;85(1):4-19.

12. Hosseini MK, Gunel T, Gumusoglu E, Benian A, Aydinli K. MicroRNA expression profiling in placenta and maternal plasma in early pregnancy loss. Molecular medicine reports. 2018;17(4):4941-52.

13. Farrokhnia F, Aplin JD, Westwood M, Forbes K. MicroRNA regulation of mitogenic signaling networks in the human placenta. The Journal of biological chemistry. 2014;289(44):30404-16.

14. Schofield G, Kimber SJ. Leukocyte subpopulations in the uteri of leukemia inhibitory factor knockout mice during early pregnancy. Biology of reproduction. 2005;72(4):872-8.

15. Dekel N, Gnainsky Y, Granot I, Mor G. Inflammation and implantation. American Journal of Reproductive Immunology. 2010;63(1):17-21.

16. Kimber SJ. Leukaemia inhibitory factor in implantation and uterine biology. Reproduction (Cambridge, England). 2005;130(2):131-45.

17. Doridot L, Miralles F, Barbaux S, Vaiman D. Trophoblasts, invasion, and microRNA. Frontiers in genetics. 2013;4:248.

18. Zhang S, Lin H, Kong S, Wang S, Wang H, Wang H, et al. Physiological and molecular determinants of embryo implantation. Molecular aspects of medicine. 2013;34(5):939-80.

19. Hayder H, O’Brien J, Nadeem U, Peng C. MicroRNAs: crucial regulators of placental development. Reproduction (Cambridge, England). 2018:REP-17-0603.

20. Fu G, Ye G, Nadeem L, Ji L, Manchanda T, Wang Y, et al. MicroRNA-376c impairs transforming growth factor-? and nodal signaling to promote trophoblast cell proliferation and invasion. Hypertension. 2013:HYPERTENSIONAHA. 111.203489.

21. Bidarimath M, Khalaj K, Wessels JM, Tayade C. MicroRNAs, immune cells and pregnancy. Cellular & molecular immunology. 2014;11(6):538.

22. Dominguez F, Yanez-Mo M, Sanchez-Madrid F, Simon C. Embryonic implantation and leukocyte transendothelial migration: different processes with similar players? The FASEB journal. 2005;19(9):1056-60.

23. Cuman C, Van Sinderen M, Gantier MP, Rainczuk K, Sorby K, Rombauts L, et al. Human blastocyst secreted microRNA regulate endometrial epithelial cell adhesion. EBioMedicine. 2015;2(10):1528-35.

24. Su L, Liu R, Cheng W, Zhu M, Li X, Zhao S, et al. Expression patterns of microRNAs in porcine endometrium and their potential roles in embryo implantation and placentation. PLoS One. 2014;9(2):e87867.

25. Dior UP, Kogan L, Chill HH, Eizenberg N, Simon A, Revel A, editors. Emerging Roles of microRNA in the embryo-endometrium cross talk. Seminars in reproductive medicine; 2014: Thieme Medical Publishers.

26. Liu W, Niu Z, Li Q, Pang RT, Chiu PC, Yeung WSB. MicroRNA and embryo implantation. American
Journal of Reproductive Immunology. 2016;75(3):263-71.

27. Xia HF, Jin XH, Cao ZF, Hu Y, Ma X. MicroRNA expression and regulation in the uterus during embryo implantation in rat. The FEBS journal. 2014;281(7):1872-91.

28. Geng Y, He J, Ding Y, Chen X, Zhou Y, Liu S, et al. The differential expression of microRNAs between implantation sites and interimplantation sites in early pregnancy in mice and their potential functions. Reproductive Sciences. 2014;21(10):1296-306.

29. Cheong AW, Pang RT, Liu W-M, Kottawatta KSA, Lee K-F, Yeung WS. MicroRNA Let-7a and dicer are important in the activation and implantation of delayed implanting mouse embryos. Human Reproduction. 2014;29(4):750-62.

30. Rosenbluth EM, Shelton DN, Wells LM, Sparks AE, Van Voorhis BJ. Human embryos secrete microRNAs into culture media-a potential biomarker for implantation. Fertility and sterility. 2014;101(5):1493-500.

31. Inhorn MC, Patrizio P. Infertility around the globe: new thinking on gender, reproductive technologies and global movements in the 21st century. Human reproduction update. 2015;21(4):411-26.

32. Singh H, Nardo L, Kimber SJ, Aplin JD. Early stages of implantation as revealed by an in vitro model. Reproduction (Cambridge, England). 2010;139(5):905-14.




Management of Ectopic Pregnancy: A 5-year Review at a Tertiary Hospital in Benin City.

*Osazee K and Oyagha F E
Department of Obstetrics and Gynaecology, School of Medicine, College of Medical Sciences, University of Benin, Nigeria.
All correspondence to:
College of Medical Sciences, University of Benin, Nigeria. Email; kehindeosazee@gmail.com
Osazee K Department of Obstetrics and Gynaecology, School of Medicine,

ABSTRACT
Background: Ectopic pregnancyis a common life-threatening emergency in pregnancy and leading cause of maternal mortality in the first trimester.Also, the morbidity associated with ectopic pregnancy could pose a severe challenge to reproductive potential.

Objective: To determine the trend, incidence, pattern of presentation, diagnostic and treatment modalities of ectopic pregnancy in University Benin Teaching Hospital, Benin City. Method: A retrospective review of ectopic pregnancy cases managed in our hospital between 1st January 2010 and 31stDecember 2014.
Results: The incidence of ectopic pregnancy was 0.87%. The mean age of the patient was 28.3 ± 6.9years, the most frequent presenting symptom was abdominal pain in 108(90.7%) patients, and it was commonest among the primiparous group (26.9%). Tubal ectopic pregnancy was the most typical (96.6%), followed by ovarian ectopic pregnancy (1.6%), then cornual ectopic (0.8%). Right tubal ectopic pregnancy (65.5%) was commoner than left tubal ectopic pregnancy (34.5%). Ampullary implantation was the most common site of ectopic tubal pregnancy (57.1%), and the least was interstitial implantation (5.9%). Ruptured ectopic pregnancy occurred in 60.5% of the cases while 28.6% were slow leaking, 2.5% were tubal abortions, and 8.4% were unruptured. Conclusion: Ruptured ectopic pregnancy is the most frequent variant necessitating laparotomy with salpingectomy as the mainstay treatment modality. However, some of the women were hemodynamically stable at presentation, and operative laparoscopy could have been a viable option.
Keywords: Ectopic Pregnancy, Salpingectomy, Laparotomy, Laparoscopy

INTRODUCTION:

Ectopic pregnancy is the pregnancy in which thefertilised ovum implants in any location other than the endometrial lining of the uterus [1]. It is a common life-threatening emergency in pregnancy and a leading cause of maternal mortality in the first trimester [2,3] which results in significant morbidity for the mother and inevitable loss of pregnancy [4].It constitutes a significant cause of maternal morbidity and mortality especially in developing countries where the majority of the patients often present late with ruptured
variant and hemodynamic compromise [5]. Ectopic pregnancy was first recognised by Busiere in 1693
on examination of an executed prisoner in Paris [6]. The incidence varies from country to country and within the same geographical region depending on the risk factors in the population concerned [5]. In Nigeria, the incidence ranges between 1.2-2.7% of deliveries [4,7-11] and 1.68% in a previous study in our centre[12].
Pregnancies in the fallopian tube account for 97% of ectopic pregnancies; 55% in the ampulla; 25% in the isthmus; 17% in the fimbria and 3% in the abdominal cavity, ovary and cervix [13]. Damage to the fallopian tubes from pelvic inflammatory disease, previous tubal surgery or previous ectopic pregnancy poses strongly associated with an increased risk of ectopic pregnancy [11,13]. Other risk factors include a history of cigarette smoking [13], age over 35years [11,12]and multiple lifetime partners [13], the use of ART [12] and progesterone contraceptive pill [13,14]. However, half of all women who have ectopic pregnancy do not have any known risk factor [14]. Ectopic gestation may present with typical features of acute abdomen and cardiovascular collapse [14,15]. The typical symptoms associated with ectopic pregnancy are amenorrhea, lower abdominal pain, vaginal bleeding and syncope [15].While the signs can be diffused or localised abdominal tenderness, adnexal mass, vagina bleeding which is of uterine origin caused by endometrial involution and decidual sloughing [16]. The association of cardiovascular collapse is suggestive of ruptured ectopic pregnancy [14]it is the most prevalent variant in the developing countries [16] necessitating radical surgeries [15,16].The unruptured variant tends to predominate in the developed countries.With early diagnosed in the course of early pregnancy evaluation [17]. Thus, allows for conservative surgeries such as linear salpingotomy, salpingostomy and milking of the tube [15-17]. The diagnostic approach of ectopic gestation depends on the clinical presentation [18]. The diagnosis of the unruptured variant, as evidenced by the stability of the cardiovascular parameters, relied on the high index of suspicion as well as the -subunit of human chorionic gonadotropin (-hCG) assessment and transvaginal ultrasonography (TVS) [17,18]. Where the facility is available, the use of laparoscopy remains the gold standard [19]. Typically, the serum level of hCG  approximately doubles every 48 hours within the first forty days of conception [18,19]. The trend lowers in extra-uterine pregnancy [18]. However, it is subject to debate as about 17% of extrauterine pregnancy exhibit doubling of hCG within 48 hours,and 15% of a healthy pregnancy associated with lower hCG level [18-20].Furthermore, the concept of discriminating zone is another diagnostic tool that determines the level of serum hCG at which TVS can demonstrate the intra-uterine gestational sac [17,18]. Despite the benefits of these diagnostic tools, the scenario in the developing countries tends to be the ruptured variant due to the paucity of resources and diagnostic facilities [18- 20]. Treatment options include expectant, medical and surgical managements [20]. The surgical approach could be laparoscopy or laparotomy [19]. Also, depending on the state of the tube involved, it could be radical or conservative surgery [18,19].The choice of treatment modality is informed by the state of the fallopian tubes, size of the gestational sac, serum -hCG level and the cardiovascular parameters of the patient [16-21]. Expectant management is considered if the tube involved is unruptured, hCG 1500iu and the gestational sac less than 4cm in diameter without cardiac activity and background haemodynamic stability [19].With rising hCG level and the onset of cardiac activity, the medical option is indicated [20].The treatment involves the use of methotrexate and mifepristone [17]. Where the facilities are available, laparoscopy could be used [18, 19]. Especially in a situation with diagnostic dilemma [19-21]as well as its advantages of less morbidity and shorter hospital stay [18]. In developing countries, laparotomy remains the main surgical approach [20,21]due to the scarcity of operative laparoscopy. However, it remains the most expedient route in the event of ruptured ectopic pregnancy with massive hemoperitoneum and haemodynamic instability [16-20].The conservative approach is indicated by an munruptured tube with compromised contralateral tube and desire to sustain future fertility potential [20].

MATERIALS AND METHOD
A retrospective analysis of all the cases of ectopic pregnancy admitted and managed at the University of Benin Teaching Hospital Benin city(UBTH) a major referral for Edo, Delta, Ondo and Kogi States during a five years period from 1st Jan 2010 and 31st December 2014. The study population consisted of all the patients admitted with ectopic pregnancy. There was a total of 119 patients admitted with ectopic pregnancy over the period under review. Case records of patients were obtained from the records department, wards and theatre operation register. Sociodemographic data, the pattern of presentation, treatment options, a cadre of the surgeon and the type of transfusion were obtained from case note. Other information include previous ectopic pregnancy, previous abdominal surgery, previous treatment of PID and contraceptive use. Also, data on the total number of deliveries, gynaecological admissions and surgery extracted from the hospital database. The data were analysed with simple descriptive statistics and presented in frequency charts and tables

 

RESULTS
The total number of delivery in the period under review was 13655,and a total of 119 cases of ectopic pregnancy were recorded giving the incidence as 0.87% (table 1). The commonest presentations were abdominal pain, 108
(90.7%), amenorrhoea 97(81.5%) and bleeding per vaginam 55(46.2%) Table 2. The occurrence of ectopic pregnancy was highest in 25-29 years age group, 47(39.5%) and was more in the primigravida 32(26.9%) Table 3. Tubal gestation was the most frequent type in 116(96.6%) of patients. The right side accounted for 75(65.5%) and the left side in 41(34.5%) Table 4.Ampullary tubal ectopic pregnancy constituted 68(57.1%), followed by isthmic ectopic with 32 (26.9%), fimbrial12(10.1%).Theleast being interstitial ectopic pregnancy constituting 7 (5.9%)as shown in table 5. 72(60.5%) of the patients had ruptured at presentation, and 10(8.4%) were unruptured.34(28.6%) were slowly leaking, while 3(2.5 %) were tubal abortion. Table 6. 108(90.8%) had total salpingectomy,and 9(7.6%) had partial salpingectomy. 2(1.6%) of the patients had a unilateral oophorectomy. Table 7. 81(68.1%) had a homologous transfusion. While 12(10.1%) had autotransfusion and 26(21.8%) were not transfused. Table 8

DISCUSSION
Ectopic pregnancy constitutes asignificant cause of morbidity and mortality in early pregnancy and constitutes a significantgynaecological emergency. The incidence of 0.87% in this study shows a downward trend in the incidence of ectopic in our centre when compared with 1.68% in a previous study by Gharoro et al [12]; this could be attributed to the increase in total deliveries in the period reviewed. However, other series in the country reported a relatively higher incidence [ 7, 8, 13,14].

The peak age incidence was amongst women age group of 25-29 years which corroborate with findings of Udigwe et al [4]. and Etuknwa et al [10], the relatively high frequency of ectopic gestation in the age group 16-35years was not surprising since this corresponds to the age of reproductive and peak sexual activity [7-13]. It occurred mainly among the primiparous group and was similar to that of other workers [2,10,11]

Most of the patient in this review were treated mainly by laparotomy and salpingectomy. This constituted 15.2% of all gynaecological operations and 72.5% ofall gynaecological emergencies. These findings are similar to reports of other series in developing countries [7,12,20]. The explanation could be due to late presentation of most patient to the hospital as well as the paucity of diagnostic tools at the various levels of health facilities. Thus, depriving the patients the benefit of early diagnostic and possibility of unruptured variant [7,10,16].Often, the diagnosismade by clinical presentation, transabdominal ultrasonography and positive serum or urine pregnancy test, may not elicit early diagnosis before it ruptures necessitating laparotomy and salpingectomy [6,21].

The occurrence of the right-sided tubal ectopic was observed in this study to be commoner than the left-sidedectopic tubal pregnancy. This finding isconsistent with studies byGharoro et. Al[12] insame centre as our study and Musa et al [11] in Jos.This occurrence may be explained by the infective process especially caused by sub-acute appendicitis because of its close proximity [14,15,19,20]. Also, other infections such as PID post-abortion and puerperal sepsis could cause tubal damage. These are not peculiar to either of the tubes [15-21]. The ampulla (57.1%) was themost common site in the fallopian tube followed by this isthmic portion (26.9%), fimbrial (10.1%) and the least being the interstitial portion (5.9%), comparable to other studies in Nigeria [22]and some parts in the developed countries [7,20]. The ampulla-isthmic junction is where fertilisation of ovum takes place,and any delay in transporting the embryo to the uterus may lead to implantation in the segment of the tube. Thus, explains why ectopic pregnancy occurs most in these two segments of the tubes [15].

In this review, total salpingectomy was preferred over partial salpingectomy and this preference was similar to other studies done in Markurdi, Jos [11]where 88.5 % had total salpingectomy. Also corroborated by the reports of Nayama et. Al [23]. Thus, may be due to the risk of recurrence and the advent of in-vitro fertilisationmay have rendered the need for partial salpingectomyunnecessary [17-22].

12(10.1%) of the patients had autologous transfusion with blood drainedfrom the peritoneal cavity. Hence, provided a quick means of correcting the post haemorrhagic anaemia, volume replacement and augmentation of oxygen-carrying capacity [13,17,23]. Furthermore, save the stress and time consuming associated with grouping and cross-matching of blood [24].

CONCLUSION
Ectopic pregnancy still has remained a criticalgynaecologicalcondition in our Centre. Although several risk factors for ectopic are known, the cause of a significant proportion of ectopic pregnancy remains unknown. The compliments of high index of suspicion and use of modern diagnostic techniques such as TVS and laparoscopy will assist in early diagnosis obviating the mobility and need for radical treatment. Furthermore, creating awareness for early presentation as well as prevention of pelvic inflammatory disease (PID) and adoption of safe abortion programme may help to reduce the scourge of ectopic pregnancy in our environment.

RECOMMENDATION
There is the need to create and adopt a protocol for the diagnosis and management of ectopic pregnancy, develop laboratory capacity for rapid serum hCG assay and make diagnostic laparoscopy a routine tool in evaluating any acute pain in the hospital. There is also the need to research intothe acceptability of vaginal ultrasound with the view to utilising it in assessing early pregnancy complications.

REFERENCES

of obstetric and gynecologic emergencies. 3rd Ed. The U.S.A. Lippincott Williams and Wilkins; 2005. P67-79

2. Arup KM, niloptalSk, Praclip KB, ectopic pregnancy an analysis of 180 cases, J India Med association 2007;105: 308-14

3. Shaw Jt, Dry SK, Critchley HO, Horne, Aw. Current knowledge of the aetiology of human tubal Ectopic pregnancy. Human Reprodupdate2010;16:32-44

4. Udigwe Go, Umeonihu OS Mbuchi II ectopic pregnancy: a five-year review of cases at NnamdiAzikiwe University Teaching Hospital (NAUTH) Nnewi, Niger Med J 2010;51:160-3

5. Stabile I, Grudzinski JG. Ectopic pregnancy, what's new? In: Studd J, editor. Progress in obstetrics and gynaecologyvol 11 Edinburgh: Churchill Livingstone: 1997 p. 281-309
6. DavorJurkovic: ectopic pregnancy. In: Dewhurst; (ed) Textbook of obstetrics &gynecology for postgraduate. 7th edition Blackwell publishing 2007.106-115

7. Aboyeji AP, Fawole AA Ijaija MA. Trends in ectopic pregnancy in Ilorin, Nigeria. The Nigeria J surge Res 2002;4:6-11.

8. Airede LR, EkeleBA Ectopic pregnancy in Sokoto, northern Nigeria Malawi Med J 2005:17: 14-6.

9. Anorlu RI, Oluwole, A, Abudu OO, Adebayo, S. risk factor for ectopic pregnancy in Lagos Nigeria. ActaObstetGynecolScand 2005; 84:184-8.

10. Etunkwa BT, Azu OO, Adebayo S, Risk Factor of Ectopic pregnancy in Lagos Nigeria, ActaObstetGynecolScand 2005;84:184-8.

11. Musa J. Daru PH Mutiihir JJ, Ujar IA ectopic pregnancy in Jos Northern Nigeria: prevalence and impact on subsequent fertility: Niger J Med 2009: 18: 35-8.

12. Gharoro EP, Igbafe AA. Ectopic pregnancy revisited in Benin City. Nigeria; analysis of 152 cases. ActaObstet Gynecology Scand 2002;81(12) 1139-1143

13. Della-Giustina D, Denny M. Ectopicpregnancy. Emerg Med Clin North Am 2003;21:565-84.

14. Jurkovic D. Ectopic Pregnancy in Dewhurst's Textbook of Obstetrics and gynaecology by Keith Edmonds. 8th Ed:Wiley-Blackwell;2012:76-87.

15. Peter SU, GarmelSH, Early pregnancy Risk. In: Dechermey AH, Nathan L, Goodwin TM, Laufer N(eds). Current Diagnosis & treatment, Obstetrics & Gynaecology. 10th edition. Washington, US McGraw Hill CompaniesInc. in 2007. PP 259-272

16. Farquhar CM(2005). Ectopic Pregnancy. The Lancet.366;583-591.

17. Ibekwe P. Ruptured advanced tubal ectopic pregnancy simulating uterine rupture: a case report. Nigerian journal of medicine: journal of National Association of Resident Doctors of Nigeria. 2004;13(2):196.

18. Sotubu O, Aboyeji A, Ectopic pregnancy in Ilorin, Nigeria; A-5 year review. Nig Med Pract,1994;27(314):25-27.

19. Eugene Okpere. Ectopic gestation. In: Clinical Gynaecology (ed), first edition. Uniben press, Benin. 2005. P 74-75.
20. Monga A (ed) .disorder of early pregnancy. In; gynaecology by Ten Teachers 18th edition London UK. Hodder Arnold. 2006; P89-102.

21. Igberase G.O Ebeigbe P.N igekoyi O.F ectopic pregnancy: an 11-year review in a tertiary centre in the Niger Delta in Uk. 2000-2002: London RCOG press.

22. Swede TZ Jogo AA; ruptured tubal pregnancy in Ilorin. Nigerian Journal of Surgical Research 2008;4(1): 75-77.

23. Nayamaet al., Management of ectopic pregnancy in developing countries: Nigerian reference maternity, GynecolObstetferti 2006,34(1): 14-18

24. Editorial. Autotransfusion. Brit Med. J 1967; 2: 393.

 

 




Hypertonic Xylose Agar Medium: A Novel Medium for Differentiation of Candida dubliniensis from Candida Albicans

Abiroo Jan, Gulnaz Bashir, Bashir Ahmad Fomda, Dekyong Angmo Urgain Khangsar,
Munazah Manzoor, Amrish Kohli, Sulmaz Reshi, Mohd Suhail, Saba Choudhary, Akeela Fatima
Department of Microbiology, Sher-i-Kashmir Institute of Medical Sciences, Srinagar, Jammu and Kashmir, India
All correspondence to: Dr. Gulnaz Bashir Department of Microbiology, Sher-i-Kashmir Institute of Medical Sciences, Srinagar – 190 011, Jammu and Kashmir India

 

ABSTRACT
Background: Candida dubliniensis is a pathogenic Candida species which shares many phenotypic features with Candida albicans. These similarities have caused significant problems in the identification of C. dubliniensis in an average clinical mycology laboratory. Several phenotypic-based tests have been developed to distinguish C. albicans from C. dubliniensis but none has been demonstrated being sufficient alone for accurate differentiation of the two species. Aim: To facilitate the differentiation of these species, we evaluated the utility of a novel medium ‘Hypertonic Xylose Agar Medium’ (HXAM). Materials and Methods: A total of 200 Candida spp. were tested in this study which included 186 stock strains of C. albicans and 14 strains of C. dubliniensis. Identification of all these strains was confirmed by polymerase chain reaction-restriction fragment length polymorphism using Bln I (Avr II) enzyme. All isolates were inoculated on HXAM, incubated at 28°C and examined for visible growth every day up to 7 days. Results: On this medium at 28°C, all 186 C. albicans isolates showed visible growth at 48 h of incubation whereas none of the 14 C. dubliniensis isolates did so even on extending the incubation period up to 7 days. Conclusion: Hence, we propose HXAM as a sole phenotypic method for identifying C. dubliniensis from germ-tube-positive isolates or from stock collections of known C. albicans.

Keywords: Candida albicans, Candida dubliniensis, hypertonic xylose agar medium, phenotypic identification, xylose assimilation.

INTRODUCTION

Rapid growth in clinical microbiological technologies over the past two decades has prompted laboratories to seek more efficient and cost-effective methods of identification of microorganisms in contrast to the previous labour-intensive methods requiring several days. With the emergence of new pathogens, especially the drug-resistant ones, it has also become necessary that the available diagnostic tools be constantly modified to keep abreast with the ever-changing spectrum of pathogens.

Candida dubliniensis is a fungal pathogen whose clinical significance seems to be its association with immunocompromised individuals and its ability to rapidly develop a stable fluconazole-resistant phenotype on exposure to this antifungal in vitro. C. dubliniensis have been overlooked and misidentified for a long time. This is because C. dubliniensis and its close relative Candida albicans share many features in common such as microscopic morphology and ability to form germ tubes in serum, production of blastoconidia with pseudohyphae, true hyphae and chlamydospores used routinely by average clinical mycology laboratory to identify C. albicans.

Several phenotypic-based tests have been developed to distinguish C. albicans from C. dubliniensis: the absence of growth at 42°C and 45°C, characteristic rough-looking colonies and chlamydospores on STAIBagar, initial dark green colonies on CHROMagar Candida, non-fluorescent colonies on methyl-blue Sabouraud agar, turquoise smooth colonies on Candida ID agar, colony morphology on other differential media such as niger seed agar, simplified sunflower seed agar or tobacco agar, reduction of tetrazolium salts, absence of beta glucosidase activity, antigenic differences with C. albicans by indirect immunofluorescence, physiological assimilation tests and coaggregation with Fusobacterium nucleatum. In multiple publications, variable success using each of these methods has been reported and all have been demonstrated insufficient alone for accurate differentiation of the two species.

To circumvent these difficulties, various molecular methods have been developed, polymerase chain reaction (PCR) being the ‘gold standard. However, these tests are not readily applicable for the identification of this species in most average mycology laboratories. Nevertheless, the concept of a reliable routine isolation medium that could be used instead of PCR in differentiation of the two species remains attractive. In the present study, the authors evaluated a new medium ‘hypertonic xylose agar medium’ (HXAM) for isolation and identification of C. dubliniensis and differentiation of this yeast from most other Candida spp. on the basis of growth on this medium.

MATERIALS AND METHODS
This study was conducted in the Mycology Division of Department of Microbiology of a tertiary care hospital in Kashmir, India. The study was approved by the Institute’s Ethics Committee.

TEST STRAINS
A total of 200 Candida spp. were tested in this study. These included 186 stock strains of C. albicans tentatively identified by phenotypic methods such as germ tube formation, colony colour on HiCrome Candida differential agar (HiMedia) and characteristic morphology on corn meal agar. These were isolated from cancer patients with oral candidiasis/colonization and held in stock collection of Mycology Laboratory, Department of Microbiology, SKIMS, Srinagar. Remaining 14 isolates were strains of C. dubliniensis which were kindly provided by Dr. Ziauddin Khan (Professor and Chairman Department of Microbiology, Kuwait University). Identification of all these strains was confirmed by PCR-restriction fragment length polymorphism (PCR-RFLP) using Bln I (Avr II) enzyme which produced two strong bands of 200 bp and 340 bp in C. dubliniensis and only one band of 540 bp in C. albicans.

REFERENCE STRAINS
C. albicans 90028 obtained from National Culture Collection of Pathogenic Fungi, Department of Medical Microbiology, PGIMER, Chandigarh and C. dubliniensis (type strain CD36) and C. dubliniensis (CBS 7987) which were kindly provided by Dr. Ziauddin Khan (Professor and Chairman Department of Microbiology, Kuwait University) were included in the study.

Preparation of hypertonic xylose agar medium
HXAM was prepared by suspending 10 g of xylose powder in 900 ml distilled water. To this 20 g of agar and 65 g of NaCl was added and the solution was heated to dissolve the medium completely. The medium was sterilized by autoclaving at 15 lbs pressure (121°C) for 15 min. After cooling to 50°C, 100 ml of yeast nitrogen base solution was added and mixed thoroughly. The medium was poured in sterile Petri dish More Detailses (20 ml in each 90 mm) and allowed to dry. Final pH of 6.3 ± 0.2 was maintained at 25°C.

Inoculation of hypertonic xylose agar medium
Colonies from a 24 h culture on Sabouraud agar plates were suspended in sterile distilled water adjusting to 0.5 McFarland standard. One loop full of each suspension was inoculated on HXAM which was then incubated at 28°C and examined for visible growth every day up to 7 days.

RESULTS
On HXAM medium at 28°C, the reference strain ‘C. albicans 90028’ and all 186 test strains of C. albicans of showed visible growth at 48 h of incubation. In contrast,
none of the 14 C. dubliniensis test isolates and the reference strains ‘C. dubliniensis (type strain CD36) and C. dubliniensis (CBS 7987)’ showed any growth on this medium at 28°C even on extending the incubation period up to 7 days [Figure 1] and [Figure 2]. These observations were reproducible when experiments were repeated on three different occasions.

DISCUSSION
C. albicans assimilates xylose while C. dubliniensis does not. Gales et al. and Pincus et al. in 1999, Gutiérrez et al. in 2002 and Loreto et al. in 2010 used this property to differentiate C. albicans from C. dubliniensis. Various carbohydrate assimilation systems (Vitek 2, API 20C AUX, ID 32 C, Micronault-Candida) are available for the identification of these two species but different investigators have reported variable results for each of them.

 

 

 

 

 

 

 

The usefulness of xylose assimilation in discriminating C. dubliniensis from C. albicans has also been evaluated by conventional assimilation method using xylose disc as well as by incorporating this carbon source into a carbon-free solid medium. The latter was done by Khan et al. in 2012, who proposed that this test should be performed on germ tube-positive Candida isolates only as Candida glabrata also does not grow on this medium. About 5% of C. dubliniensis isolates assimilate xylose and could be falsely identified as C. albicans as reported by Pincus et al. Hence, if used alone there is the possibility that a small number of C. dubliniensis isolates will be missed. Furthermore, some isolates of C. albicans do not assimilate xylose and could be falsely labelled as C. dubliniensis.

Differences in the regulation of specific stress genes have been observed between C. albicans and C. dubliniensis. In particular, ENA21 gene, encoding a sodium ion transporter, is strongly induced in C. albicans but not in C. dubliniensis. This difference of salt tolerance has been used by Alves et al. in 2002, Chowdhary et al. and Ells et al. in 2011 to discriminate between the two species.

Salt tolerance of the strains was first measured using hypertonic Sabouraud broth by Alves et al. and salt in YEPD agar by Cassone et al., but these methods did not reveal clear-cut and easily interpretable results. Furthermore, high percentage of false-positive results (11.4%) using hypertonic Sabouraud broth were reported by Silveira-Gomes et al. in 2011. Akgül and Cerikçio?lu in 2009 used modified agar-based medium which showed good results.

Keeping in view the results of salt tolerance test and xylose assimilation, we devised a novel medium ‘HXAM’ by incorporating xylose into a carbon-free hypertonic solid medium. In our study, we found that all C. albicans showed visible growth on this medium at 48 h of incubation while none of the C. dubliniensis was able to grow on this medium. By this method, we can test both salt tolerance and xylose assimilation on a single medium increasing accuracy, decreasing cost, saving time and manpower. C. glabrata being non-osmotolerant and not able to assimilate xylose [8] may be mistakenly identified as C. dubliniensis on this medium. This can be overcome by performing the test on germ-tube-positive isolates only.

We observed that, as compared to other methods, HXAM is a simple in-house, fast and easy to perform test saving time of technical staff. It is also less expensive (1.5 INR per isolate) since as many as eight isolates can be tested on a single plate and the extent of growth is assessed by the naked eye. Hence, we propose HXAM as a sole phenotypic method for identifying C. dubliniensis from germ-tube-positive isolates or from stock collections of known C. albicans.

CONCLUSION
As overall prevalence of C. dubliniensis is very low compared to other species of Candida in clinical laboratories, adoption of expensive, time consuming and labour intensive methods such as PCR-RFLP for its routine identification method that is 100% accurate, simple and easy to perform without consuming much time of the technical staff in addition to being cost effective.

ACKNOWLEDGMENT
The authors thank Sher-i-Kashmir Institute of Medical Sciences, Soura, Srinagar, for funding the project. Authors thank National Culture Collection of Pathogenic Fungi (NCCPF), Department of Medical Microbiology, PGIMER, Chandigarh and Dr. Ziauddin Khan, Professor and Chairman Department of Microbiology, Kuwait University, for providing control strains for the project.

REFERENCES

1. Kantarcioglu AS, Yücel A. The presence of fluconazole-resistant Candida dubliniensis strains among Candida albicans isolates from immunocompromised or otherwise debilitated HIV-negative Turkish patients. Rev Iberoam Micol 2002;19:44-8.

2. Ellepola AN, Khan ZU. Rapid differentiation of Candida dubliniensis from Candida albicans by early D-xylose assimilation. Med Princ Pract 2012;21:375-8.

3. Gutiérrez J, Morales P, González MA, Quindós G. Candida dubliniensis, a new fungal pathogen. J Basic Microbiol 2002;42:207-27.

4. Khan ZU, Ahmad S, Mokaddas E, Chandy R. Tobacco agar, a new medium for differentiating Candida dubliniensis from Candida albicans. J Clin Microbiol 2004;42:4796-8.

5. Akgül O, Cerikçio?lu N. Hypertonic Sabouraud dextrose agar as a substrate for differentiation of Candida dubliniensis. Mycopathologia 2009;167:357-9.
6. Jan A, Bashir G, Fomda BA, Fatima A, Lone MA, Roohi S. Molecular Identification of Candida dubliniensis among Candida albicans isolated from oral cavity of cancer patients using PCR-RFLP, in a tertiary care hospital in Kashmir, India. Br Microbiol Res J 2016;14:1-7.

7. Mirhendi H, Makimura K, Zomorodian K, Maeda N, Ohshima T, Yamaguchi H, et al. Differentiation of Candida albicans and Candida dubliniensis using a single-enzyme PCR-RFLP method. Jpn J Infect Dis 2005;58:235-7.

8. Khan Z, Ahmad S, Chandy R, Joseph L. A simple xylose-based agar medium for the differentiation of Candida dubliniensis and Candida albicans. Diagn Microbiol Infect Dis 2012;72:285-7.

9. Gales AC, Pfaller MA, Houston AK, Joly S, Sullivan DJ, Coleman DC, et al. Identification of Candida dubliniensis based on temperature and utilization of xylose and alpha-methyl-D-glucoside as determined with the API 20C AUX and vitek YBC systems. J Clin Microbiol 1999;37:3804-8.

10. Pincus DH, Coleman DC, Pruitt WR, Padhye AA, Salkin IF, Geimer M, et al. Rapid identification of Candida dubliniensis with commercial yeast identification systems. J Clin Microbiol 1999;37:3533-9.

11. Loreto ES, Scheid LA, Nogueira CW, Zeni G, Santurio JM, Alves SH, et al. Candida dubliniensis: Epidemiology and phenotypic methods for identification. Mycopathologia 2010;169:431-43.

12. Betty AF, Sahm DF, Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology. 11th ed. Philadelphia: Mosby Publications; 2004. p. 927-38.

13. Enjalbert B, Moran GP, Vaughan C, Yeomans T, Maccallum DM, Quinn J, et al. Genome-wide gene expression profiling and a forward genetic screen show that differential expression of the sodium ion transporter Ena21 contributes to the differential tolerance of Candida albicans and Candida dubliniensis to osmotic stress. Mol Microbiol 2009;72:216-28.

14. Alves SH, Milan EP, de Laet Sant’Ana P, Oliveira LO, Santurio JM, Colombo AL, et al. Hypertonic Sabouraud broth as a simple and powerful test for Candida dubliniensis screening. Diagn Microbiol Infect Dis 2002;43:85-6.

15. Chowdhary A, Randhawa HS, Kowshik T, Kathuria S, Roy P, Brandt ME, et al. Application of hypertonic Sabouraud glucose agar for differentiation of Candida dubliniensis from Candida albicans. Diagn Microbiol Infect Dis 2011;69:440-2.

16. Ells R, Kock JL, Pohl CH. Candida albicans or Candida dubliniensis? Mycoses 2011;54:1-6.

17. Silveira-Gomes F, Sarmento DN, Espírito-Santo EP, Souza Nde O, Pinto TM, Marques-da-Silva SH, et al. Differentiation between Candida albicans and Candida dubliniensis using hypertonic Sabouraud broth and tobacco agar. Rev Soc Bras Med Trop 2011;44:457-60.

 

 

 




Leukocyte Esterase as Predictor of Urine Culture Result

Willis John Rieker
Department of Pathology and Medical Laboratory, Beloit Memorial Hospital, Wisconsin, USA
Eileen Reilly
Department of Health Sciences, SSM Health Janesville, Wisconsin, USA
Balamurugan Pandiyan
Department of Mathematics, University of Wisconsin, Wisconsin, USA
Samantha Lom
Department of Medical Microbiology, Beloit Memorial Hospital, Wisconsin, USA
Andrew Merry
Department of Academic Affairs, International University Health Science, Wisconsin, USA
Correspondences to: Eileen Reilly, Department of Health Sciences, SSM Health Janesville, Wisconsin, USA. Email: etreilly2004@yahoo.com

 

ABSTRACT
Objective: Assess varying levels of leukocyte esterase presence on urine dipstick as a risk factor for positive urine culture. Materials and methods: Retrospective evaluation of outpatient laboratory data from Beloit Memorial Hospital obtained randomly from samples in outpatient settings in the year 2016. 2000 urine results obtained from automated urine dipstick and microscopy analysis. From the 2000 samples, 1123 patients randomly selected and grouped into controls and case participants based on positive urine cultures. Information gathered included age and gender. Conclusion: Leukocyte esterase on dipstick analysis at “large” or “moderate” levels are both independent positive predictors of positive urine culture. “Small” level of leukocyte esterase has no predictive value for positive urine culture and “trace” leukocyte level has a negative predictive value for a positive urine culture.

Keywords: Leukocyte esterase; Microscopy analysis; Positive urine culture.

INTRODUCTION

Urinary Tract infection (UTI) is a frequent outpatient diagnoses. It is common practice to obtain urine for dipstick and microscopy analysis for gastroenterology or genitourinary complaints. The challenge comes with interpretation since gold standard continues to be the delayed urine culture. Leukocyte esterase is an indicator on the urine dipstick analysis. Leukocyte esterase is an enzyme in neutrophils and macrophages that is released when those cells are injured. Based on Pappas article from 1991 “Leukocyte esterase may be used to detect >10 leukocytes per high power field (sensitivity of 75% to 96%; specificity of 94% to 98%” .
How well does varying levels of leukocyte esterase equate to a positive urine culture which is the gold standard?

Another 2004 article by Deville’ et al. reviewing the sensitivity and specificity of “BOTH nitrites and leukocyte esterase tests are positive then sensitivity (for bacterial UTI) 68% to 88%”. This article combined the data rather than assessing leukocyte esterase separately. Once again, literature review reviewed sensitivity and specificity data of leukocyte esterase but in combination with presence of blood, not as a separate entity. “Nitrites OR leukocyte esterase AND blood had sensitivity and specificity 75 and 66%” .

As stated by a meta-analysis of nitrites “overall the sensitivity of urine dipstick test for nitrites was low (45% to 60% in most situations) with high levels of specificity (85% to 98%). Nitrite positive dipstick allows provider to more confidently make the diagnosis of UTI. Unfortunately, nitrites generally only present if patient infected with Enterobacteriaceae organism that generates this by product by metabolism of nitrates. In order to detect nitrites in urine the patient cannot void for approximately 4 h. This is a problem when your patient is voiding less than every 4 h which is common scenario in patient with suspected UTI.

If a medical provider reviews Medical textbook regarding the diagnostic testing in urinary tract infections (UTI) the following information is available. Harrison’s Guide to Internal Medicine states “The leukocyte esterase dipstick method is less sensitive than microscopy in identifying pyuria but is useful alternative when microscopy is not feasible”. Rosen’s Emergency Medicine mentions “urine dipstick testing for leukocyte esterase has shown sensitivity 75% to 96% in detecting pyuria associated with urinary tract infection”. Merck Manual states “the leukocyte esterase is very specific for presence of >10 WBC’s/microliter and is fairly sensitive. “Epocrates”: A urinary tract infection is indicated by urinalysis test result of “positive for leukocyte esterase, nitrites, and Hgb.”

Previous literature suggests that treatment in afebrile uncomplicated adult patient with no vaginal discharge with classic genitourinary symptoms can be treated without urinalysis. “In women who present with 1 or more symptoms of UTI the probability of infection is approximately 50%. Specific combinations of symptoms (dysuria AND polyuria) raise the probability of UTI to more than 90%.

The problem is that patients will visit an outpatient setting for treatment with only one genitourinary complaint (“dysuria”), normal vitals and possibly another symptom such as mild suprapubic pressure. Often the initial assessment involves a urinalysis. A complete blood count may not be justifiable or immediately obtainable. Even more difficult is the urban or rural urgent care setting where urine microscopy may not be available. Often the urinalysis dipstick will indicate “small” or “trace” leukocytes and 5-10 WBC (if microscopy available). Previous literature has not looked at the varying levels of leukocyte esterase on the dipstick and the correlation of those levels with a positive urine culture. In the age of antibiotic resistance and thus the judicious use of antibiotics, medical providers have to decide based on a marginal urinalysis result with “trace” or “mild” leukocyte esterase to treat the patient with antibiotics or wait for culture and then utilize more resources later as you contact the patient if the urine culture is positive. Just on the 1123 samples in this study, 442 sample were “mild” or “trace” which is 40% of the data.

MATERIALS AND METHODS
Data base “Path Net” was accessed by Microbiology staff for patient urine laboratory information at Beloit Memorial Hospital. 2000 outpatient samples were randomly selected from each month of the year 2016. Urinalysis was initially completed by Siemens Atlas analyser. This laboratory equipment utilizes colorometric change for detection of leukocyte esterase. The degree of color change quantifies the classification of leukocyte esterase level. At the time this study was completed all urine samples at Beloit Memorial hospital are plated for growth. Leukocyte esterase levels are resulted as “large”, “moderate”, “small” and “trace”. Other hospital and clinic labs may result leukocyte esterase in numerical values. According to the CLINITEX Novus Operations Guide.
Large=3+; Moderate=2+; Small=1+; and Trace=Trace
492 of the samples were positive for urine culture growth indicative of true UTI bacturia based on laboratory standards from Cumitech 2C Laboratory Diagnosis of Urinary Tract Infections.
Utilizing accession numbers from laboratory data I was able to access patient medical record number to determine age and gender of the studied patients population (Figures 1 and 2)

Figure 2: Case Patients: Age and gender frequency.
Results
Odds ratio (OR) was utilized to determine association between Leukocyte esterase and positive urine culture given case control study. The data for the 2 × 2 frequency table was obtained from Figure 3. Formula ad/bc for odds ratio [11]. 2 × 2 data and OR values then applied to mathematical formula for Confidence interval:
With 5% level of significance, the p-value has been determined utilizing Fisher exact test (Tables 1 and 2).

“Large” (3+) LE 2 × 2 table
141 39
347 596
OR=6.21
Confidence Interval (CI) 4.25-9.07
p-value: 2.5 × 10-5<0.05
H0: OR>1, so “Large” LE has strong association with Positive Urine culture (+UC)

Table 1: Large leukocyte esterase.
“Moderate” (2+) LE 2 × 2 table
115 103
373 532
OR=1.59
Confidence Interval (CI) 1.18-2.14
p-value: 0.0014<0.05
H0: OR>1, so “Moderate” LE has strong association with Positive Urine culture (+UC)

Table 2: Moderate leukocyte esterase.
For all categories, “Large”, “Moderate”, “Small” and “Trace” Leukocyte Esterase, we define the null hypothesis (H0 : OR=1), that is to say, Leukocyte Esterase is not associated with the positive urine culture (Tables 3 and 4).

“Small” (1+) LE 2 × 2 table
140 170
348 465
OR=1.10
Confidence Interval (CI) 0.84-1.43
p-value: 0.2592>0.05
H0: OR=1.1, so “Small” LE has no association with Positive Urine culture (+UC)

Table 3: Small leukocyte esterase.

“Trace” LE 2 × 2 table
35 97
453 538
OR=0.43
Confidence Interval (CI) 0.29-0.65
p-value: 0.000015627<0.05
H0: OR<1, so “Trace” LE has strong negative association with Positive Urine culture (+UC)

Table 4: Trace leukocyte esterase.

Discussion
Leukocyte esterase has a strong predictive value of positive urine culture at “moderate” or “large” levels based on statistical hypothesis testing of odds ratio. Leukocyte esterase at “small” levels has no predictive value for association with positive urine culture thus accepting null hypothesis. “Trace” leukocyte esterase has NEGATIVE predictive value for association with positive urine culture therefore rejecting null hypothesis.
Out of 1123 urine samples only 398 were positive for “large” or “moderate” LE and 442 sample were “small” or “trace”. Determining association for all levels of LE is pertinent given that the majority of urine samples in this data set are not “large” or “moderate” and more closely resembles the distribution in the typical outpatient setting. Applying statistical analysis to the LE levels gives the provider more confidence in opting for medicinal intervention or not.

CONCLUSION
Specifically, the results can be applied in outpatient clinics where microscopy is not readily available, and symptoms are vague. Possibly the information may help to curb unnecessary antibiotic prescriptions. The data can also support the reduction of additional processing of select urine samples sent to laboratories for further evaluation. This reduction of higher level of testing will ultimately decrease health care costs for the patient and the clinic.

REFERENCES
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2. Deville WL, Yzermans JC, Van Duijn NP, Bezemer PD, Van Der Windt DAWM, et al. (2004) The urine dipstick test useful to rule out infections. A meta-analysis of the accuracy. BMC Urol 4: 4.

3. Little P, Turner S, Rumsby K, Jones R, Warner G, et al. (2010) Validating the prediction of lower urinary tract infection in primary care: Sensitivity and specificity of urinary dipsticks and clinical scores in women. Br J Gen Pract 60: 495.

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11. Szumilas M (2010) Explaining odds ratio. J Can Acad Child Adolesc Psychiatry 19: 227-229.