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