Comparative studies of rapid and conventional methods for malaria diagnosis

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1 Journal of Scientific Research and Studies Vol. 3(9), pp , September, 2016 ISSN Copyright 2016 Author(s) retain the copyright of this article MRP Full Length Research Paper Comparative studies of rapid and conventional methods for malaria diagnosis Musa D. 1 *, Uba A. 2 and Yahaya O. 3 1 Department of Human Trypanosomiasis Research, Nigerian Institute for Trypanosomiasis Research, Kaduna, Nigeria. 2 Department of Microbiology, Abubakar Tafawa Balewa University, Bauchi, Nigeria. 3 Department of Science Lab. Tech, Federal Polytechnic, Idah, Kogi State, Nigeria. *Corresponding author. musakaus78@gmail.com, Tel: Accepted 19 September, 2016 Malaria in Nigeria is a serious life threatening infection to the all more especially pregnant mother, the foetus and the less privileged. A study was conducted to test sensitivity of rapid and conventional methods for malaria diagnosis amongst patients presenting at the General Outpatient of Kaduna State University Teaching Hospital, Kaduna. Blood samples of 1,000 were tested for malaria parasite by Giemsa stained smear and Malaria HPR2 (Pf) Rapid Diagnostic Test (RDT). 76.8% of the samples were positive by thin film microscopy, 79.6% thick film while on the average 8.3% were positive by the RDT when compared. The prevalence of malaria was slightly higher with thick than thin film. The sensitivity of the Malaria HPR2 (Pf) test was between and 10.7%, while the specificity was between 99.1 and 100%. The positive predictive value was between 97.6 and 100% while the negative predictive value was between and 25.1%. For rapid clinical evaluation of patients and the General Outpatient Departments, the Malaria HPR2 (Pf) test should be employed as a viable alternative to ensure a reliable parasite based diagnosis of malaria for prompt and accurate treatment. Key words: Malaria, Plasmodium falciparum, rapid diagnostic tests, Malaria HPR2 (Pf), Kaduna State. INTRODUCTION Malaria is a mosquito-borne infectious disease of humans and other animals caused by protozoan parasites of the genus Plasmodium (Bartoloni and Zammarchi, 2012; WHO Fact Sheet, 2014). It begins with a bite from an infected female Anopheles mosquito, which introduces the parasites through saliva into the circulatory system. The disease is widespread in tropical and subtropical regions in a broad band around the equator, including much of Sub-Saharan Africa, Asia, and the Americas (Bartoloni and Zammarchi, 2012). Five species of Plasmodium can infect and be transmitted by humans. The vast majority of deaths are caused by Plasmodium falciparum and Plasmodium vivax, while Plasmodium ovale, and Plasmodium malariae cause a generally milder form of malaria that is rarely fatal (Bartoloni and Zammarchi, 2012). The zoonotic species Plasmodium knowlesi, prevalent in Southeast Asia, causes malaria in macaques but can also cause severe infections in humans (Olasehinde et al., 2010). Malaria is prevalent in tropical and subtropical regions because rainfall, warm temperatures, and stagnant waters provide habitats ideal for mosquito larvae. Disease transmission can be reduced by preventing mosquito bites by distribution of mosquito nets and insect repellents, or with mosquito-control measures such as spraying insecticides and draining standing water (WHO Fact Sheet, 2014). Malaria Rapid Diagnostic tests (RDTs) assist in the diagnosis of malaria by providing evidence of the presence of malaria parasites in human blood (Reyburn et al., 2007). RDTs are an alternative to diagnosis based on clinical grounds or microscopy, particularly where good quality microscopy services cannot be readily provided (Olasehinde et al., 2010). Variations occur between products, such as targets and formats, though the principles of the tests are similar. Malaria RDTs detect specific antigens (proteins)

2 175 J. Sci. Res. Stud. produced by malaria parasites in the blood of infected individuals. Some RDTs can detect only one species (P. falciparum) while others detect multiple species (P. vivax, P. malariae and P. ovale). Blood for the test is commonly obtained from a finger-prick (WHO, 2006, Reyburn et al., 2007). The aim of this study is to make comparative analysis of the rapid and conventional methods in the diagnosis of malaria at the Kaduna State University Teaching Hospital. MATERIALS AND METHODS Study area The study was carried out in the Kaduna State University Teaching Hospital. The state is located in the North-West geo political zone of Nigeria with coordinates N, 7 45 E. It has a total population of 6,827,966 (2006 Nigerian census) inhabitants distributed in 23 Local Government Areas (LGAs) and 225 political wards. It shares borders with Zamfara, Katsina, and Kano states to the North; Plateau and Bauchi states to the East, Niger state to the West, Nasarawa state and the FCT Abuja to the South. The predominant religion in the population in the northern part of the state is Islam while in the south is Christianity. Farming and trading are the major occupation of the people. There are over 15 ethnic groups in the state. Kaduna state is located within guinea savannah vegetation belt where malaria transmission is meso-endemic and parasitaemia is season dependent. The peak rainy season is between June and September. The temperature range is C and the annual rainfall is mm. Sampling technique Febrile patients: the patients were stratified into those presenting with a febrile illness and those without. The patients (n=400) in the eligible group were selected consecutively and enrolled in the study in line with the eligibility criteria. Each patient was grouped into males and females. Each group was further stratified into patients who have ever had fever and a study group of seven each was then selected purposively. Study design The study design was a combination of cross-sectional, laboratory study (accuracy), consent approval, biographic data and questionnaire administration. Study population All individuals presenting with fever at General Out Patient Department of Kaduna State University Teaching Hospital during the study period and their parents/guardians, and members of the environ and beyond. Eligibility criteria (a) Inclusion criteria: All patients above 5 years, presenting with fever and temperature 37.5 C (axillary body temperature) or history of febrile illness in the last 24 h, whose parents have consented to participate in the study, were enrolled. Clinical fever was ascertained using a clinical infrared non - contact thermometer; otherwise history of fever in the last 24 h was to be used. (b) Exclusion criteria: Any patient < 5years or any patient without history of febrile illness and or with fever with temperature < 37.5 C and any parent who declines to participate in the study. Patients with fever and rash or any form of bleeding disorder, patients with history of major psychiatric disorders, patient currently on antimalaria, patients currently on treatment for mental health, the very sick that need immediate attention will also be excluded. Sample size The sample size is determined by using the Fisher s statistical formula for estimating minimum sample size in health studies = 2 Where n = desired sample size (when population is >10,000). Z = standard normal deviation, usually set at 1.96 which corresponds to 95% confidence interval. P = 0.357, at a prevalence value of malaria in patients attending General Hospital Makarfi, Kaduna State, North Western Nigeria (Millicent and Gabriel, 2013). q = 1 p d = degree of accuracy desired usually set at 0.05 Therefore, = ( ) 0.05 n = ~ 400 Consent approval Permission was obtained from the ethical and research committee of the Department of Microbiology Kaduna State University, Nigerian Institute for Trypanosomiasis Research, Kaduna State Ministry of Health and the

3 Musa et al. 176 Table 1. Statistical values of malaria PF test. SENS (%) SPEC (%) PPV (%) NPV (%) FPR (%) FNR (%) LR+ LR- F-MEASURE (%) SENS = Sensitivity, SPEC = Specificity, PPV = Positive predictive value, NPV = Negative predictive value, FPR = False positive rate, FNR = False negative rate, LR+ = Likelihood ratio for positive test, LR- = Likelihood ratio for negative test, F-Measure = Harmonic mean of Precision (positive predictive value) and Recall (Sensitivity). management of the Kaduna State University Teaching Hospital (KSUTH). All participants were recruited voluntarily as they come in. They were given copies of patient s information leaflets and consent form to read and sign. Participants who decline consent to participate were still consulted and managed without compromising quality of care they received. Oral Informed consent was taken from the caregivers and patients with fever. Confidentiality of information given was assured and maintained. Rapid diagnostic tests Malaria HPR2 (Pf) MFD NOV. 2013, LOT MO13L01, expiration date APR. 2017, a two-band Malaria RDT that detects Histidine-Rich Protein II antigen of Plasmodium falciparum was used. It is one of the RDTs currently deployed by the NMCP and selected from the list of WHO Prequalified RDT kits. Blood samples of eligible patients were taken by finger prick under aseptic conditions and screened with the Malaria HPR2 (Pf) RDT kits using microscopy as the gold standard. On the Malaria HPR2 (Pf) RDT cassette, positive result will show one red line in window C (Control) and one red line in window T (Test) meaning that the patient have falciparum malaria. For a negative result: one red line in window C and no line in window T meant the patient do not have falciparum malaria. Absence of a line in window C meant the test was damaged and result was invalid. The results of the RDT were read within 15 min. Thick and thin film Microscopic examination of the Giemsa-stained blood smear was carried out. Thin and thick blood films were made to identify the different species of Plasmodium and assessed parasitaemia from the samples respectively. The results of the blood film was processed daily or every other day depending on workload. The two trained laboratory scientists from the Department of Microbiology (each of whom will be responsible for handling each test separately and non-staff were blinded to the results of RDT and microscopy. Flow chart was used for diagnostic accuracy component of the study. Parasite density in the thick blood smear would be assessed by counting the number of asexual parasites against 200 leucocytes in a thick film, and converting the value to parasites/µl using the standard of 8,000 leucocytes/µl. Hand tally-counters were used to count a field to the end. The parasitaemia was calculated according to the formula below: Parasite density (number of parasites/µl) = Parasite count x 8,000 / No of WBC counted. A blood slide was deemed negative when the examination of 100 thick film fields did not show the presence of asexual forms of P. falciparum. In addition, 100 fields of the thick film were examined for the exclusion of mixed infections. Quality assurance The malaria rapid test kits were stored at room temperature throughout the period of the study. The tests were carried out by trained and experienced medical laboratory scientists. Quality assurance for the identified microscopists: two microscopists were engaged to ensure accuracy of test results and blinded rechecking was also carried out. The microscopists were blinded to the results of RDTs. RESULTS A total of 1,000 patients children and adults were tested. The results were statistically analysed by SPSS software version 22 which showed that the prevalence of malaria was between 76.8 and 79.6% (Table 1). About 29.2% were aged between 0 19 years (Figure 1), which falls within the children and adolescence, and 62.3% were between the ages years (working class). On the average, 782 patients were positive by microscopy while 84 (8.4%) were positive by the Malaria HPR2 (Pf) test. About 10.74% of those positive by microscopy were negative by Malaria HPR2 (Pf). Compared with microscopy, the sensitivity (Altman and Bland, 1994a) of the Malaria HPR2 (Pf) was between and 10.7% and the specificity was between 99.1 and 100%. The positive predictive value (Altman and Bland, 1994b) was between 79.6 and 100% and negative predictive value was between and 25.1% (Table 1). The age range 0 19 years (children and adolescence)

4 177 J. Sci. Res. Stud. Figure 1. Age distribution of patients presenting with fever at the Kaduna State Teaching Hospital. Table 2. Distribution of slide positive rate of malaria in febrile patients presenting at Kaduna State University Teaching Hospital. RDT Thin microscopic examination RTD, thin film and thick film slide RTD % of total Thick microscopic examination Slide positive rate (thin film) (%) (N = 1,000) Slide positive rate (thick film) (%) N =1,000 RTD positivity rate (%) (N = 1,000) were more infected than the older age group (Table 2). In addition, the age group years were more infected than other age groups (Figure 1). DISCUSSION This study revealed a malaria prevalence of between 76.8 and 79.6% by microscopy and amongst patients attending General out-patient Clinic of Kaduna State University Teaching Hospital. Children and adolescence were more infected (29.2%) than the rest. In addition, the age group (20 39 years) were more infected (21.7%) than the other age groups. This is similar to the findings of Tay et al. (2013). It has also revealed that the Malaria Pf Rapid Diagnostic Test has a sensitivity of to 10.7%, specificity of 99.1 to 100%, and a positive predictive value of 97.6 to 100% and negative predictive value of to 25.1% when compared with the gold standard that is microscopy for the diagnosis of malaria. This sensitivity is lower than the World Health Organization prescribed sensitivity of over 90% for an acceptable rapid diagnostic test for malaria (WHO, 2006). The false negative rate of 10.6% is worrisome. It means that up to 10.6% of the patients who have malaria could be left untreated if patients are treated solely on the basis of this test. It is interesting to point out that majority of the false negative specimens in this study had low parasitaemia. This agrees with similar studies with other rapid diagnostics tests where false negative results were reported in patients with parasitaemia of less than 50 parasites/µl of blood (WHO, 2006; Enuwuru et al., 2011). In contrast, Tagbor et al. (2008) and Murray and Benneth (2009) have at different times suggested the possibility of more sensitive rapid diagnostic tests than microscopy at low parasite densities. Furthermore, some studies have reported that false negative results with rapid diagnostic tests could be as a result of mutant parasites that escape the antigenic determinant of the RDT (Lee et al., 2006). The false positive rate in this study is 0.92%. This could possibly be explained by the fact that patients who were possibly already on treatment and whose parasitaemia has cleared could still have tested positive by the rapid diagnostic test picking up remnant antigens. Studies have shown antigens to be present several days after clearance of parasitaemia from the blood stream, making

5 Musa et al. 178 the RDT unsuitable for monitoring treatment and level of parasitaemia. The sequestration of parasitized erythrocytes in tissue capillaries and placental malaria in the absence of peripheral blood parasitaemia may have also yielded positive RDT and negative microscopy for malaria parasites (Murray and Bennett, 2009). There is also a reported cross reactivity between RDTs that detect histidine-rich protein 2 and rheumatoid factor, which gives a false positive result for malaria parasites (Murray and Benneth, 2009; Adekunle et al., 2014). Certain challenge exists in providing the facilities for accurate microscopic diagnosis of malaria at the rural communities. This is the setting where the malaria menace threatens the life of the pregnant woman, the unborn baby and the less privileged. There is risk involved in exposing every pregnant woman to random choice of antimalarial drugs. It can strongly be asserted that the findings of this study were good to suggest the adoption of the Malaria Pf Rapid Diagnostic Test in the parasitological confirmation of malaria diagnosis (Enwuru et al., 2011). As stated by WHO (2006), this will, with some degree of certainty, help to rule out malaria in patients (Murray and Benneth, 2009). It is further suggested that RDT will curb malaria miss-diagnosis and help identify true antimalarial drug resistance (which seems to have been exaggerated), in the face of counterfeit antimalarial drugs. Some of these fake antimalarial drugs also receive false reports of efficacy when taken during the course of self-limiting febrile conditions. In addition, money will be saved by only treating those who are infected. Conclusion and Recommendation The few shortcomings of the RDT were observed in the study of Sarah et al. (2013); not withstanding, the Malaria HPR2 (Pf) Rapid Diagnostic Test is a veritable tool for parasite based diagnosis of malaria in patient. This is so especially in the rural communities where facilities for microscopic diagnosis of malaria are not readily available and where experienced laboratory personnel may also not be available. REFERENCES Adekunle NO, Sam-Wobo SO, Dedeke GA, Ojo DA, Abimbola WA, Adleke MA, Sukurat OA (2014). Evaluation of rapid methods in malaria diagnosis from persons attending primary health facilities, Ogun State Nigeria. Nig. J. Parasitol. ISSN (1-2): Altman DG, Bland JM (1994a). Diagnostic Tests 1: Sensitivity and Specificity. BMJ 308(6943):1552. Altman DG, Bland JM (1994b). Diagnostic Tests 2: Predictive Values. BMJ 309(6947):102. Bartoloni A, Zammarchi L (2012). Clinical aspects of uncomplicated and severe malaria. Med. J. Hematol. Infect. Dis. 4(1):e doi: /MJHID PMC PMID Enwuru CP, Umeh SI, Agbasi UM, Egbuobi RC (2011). Laboratory diagnosis of malaria in children under 5 years in a rural community: microscopy versus malaria pf test. Afr. J. Clin. Exp. Microbiol. 12(2): Lee N, Baker J, Andrews KT, Gatton ML, Bell D, Cheng Q, McCarthy J (2006). Effect of sequence variation in Plasmodium falciparum Histidine-Rich Protein 2 on binding of specific monoclonal antibodies: Implications for Rapid Diagnostic Tests for malaria. J. Clin. Microbiol. 44(8): Millicent LU, Gabriel NU (2013). Prevalence of malaria in patients attending the General hospital Makarfi, Kaduna State, Northwestern Nigeria. Am. J. Infect. Dis. Microbiol. (3):1-5. Murray CK, Benneth JW (2009). Rapid Diagnosis of Malaria. Interdiscip. Persp. Infect. Dis. doi: /2009/ Olasehinde GI, Ajayi AA, Taiwo SO, Adekeye BT, Adeyeba OA (2010). Prevalence and Management of falciparum Malaria among Infants and Children in Ota, Ogun State, Southwestern Nigeria. Afr. J. Clin, Exp. Microbiol. 11(3):160. Reyburn H, Mbakilwa H, Mwangi R, Mwerinde O, Olomi R, Drakeley C, Whitty CJM (2007). Rapid diagnostic tests compared with malaria microscopy for guiding outpatient treatment of febrile illness in Tanzania: randomised trial. Br. Med. J. doi: /bmj AE Sarah IU, Chika PE, Richard CE (2013). Diagnosis of malaria in pregnancy: A comparison of microscopy with rapid diagnostic tests. Microbiol. Res. Int. 1(3): ISSN: Tagbor H, Bruce J, Browne E, Greenwood B, Chandramohan D (2008). Performance of the OptMal dipstick in the diagnosis of malaria infection in pregnancy. Ther. Clin. Risk Manag. 4(3): Tay SCK, Agboli E, Abruquah HH, Walana W (2013). Malaria and anaemia in pregnant and non-pregnant women of child-bearing age at the University Hospital, Kumasi, Ghana. Open J. Med. Microbiol. 3: WHO (2006). The role of Laboratory diagnosis to support malaria disease management: Focus on the use of Rapid Diagnostic Tests in areas of high transmission. Report of a WHO technical consultation. WHO (2014). Fact sheet on the World Malaria Report. World Health Organization. Retrieved 2 February Acknowledgement Special thanks to my wife, Maryam Musa Danjuma; daughter, Yaasmeen Musa Danjuma; Mallam Mohammad Ahmad and Mr Meshack Anthony Kugama for helping me go through this work.

HUMASIS MALARIA ANTIGEN TEST HIGH SENSITIVE DIFFERENTIAL DIAGNOSIS OF MALARIA INFECTION

HUMASIS MALARIA ANTIGEN TEST HIGH SENSITIVE DIFFERENTIAL DIAGNOSIS OF MALARIA INFECTION References 1) World Malaria Report 2010, WHO 2) Rapid diagnostic tests for malaria parasites, Clin. Microbiol. Rev.