Malaria Rapid Diagnostic Test: A Study of Accuracy among Adults in North Central Nigeria

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1 Journal of Medicine in the Tropics (2011) 14:1: 7-11 Journal of Medicine in the Tropics Original Article Malaria Rapid Diagnostic Test: A Study of Accuracy among Adults in North Central Nigeria *Isa S. Ejiji, Comfort A. Daniyam, Okechukwu O. Ezekwesili, Celestine A. Ameh, Moses P. Chingle 1 2 Department of Medicine, Jos University Teaching Hospital, Jos, Plateau state, Nigeria. Federal Staff Hospital, Garki II, Abuja 3 Department of Community Medicine, Jos University Teaching Hospital, Jos, Plateau state, Nigeria. Abstract Background: There is increasing need for parasitological diagnosis of malaria in order to direct rational therapy and reduce the consequences of presumptive treatment. Because microscopy, the gold standard test, is laborious and technically difficult to perform, we evaluated the accuracy of a Rapid Diagnostic Test (RDT) that could be widely deployed for testing. Methodology: A hospital based cross-sectional study in which out-patients older than 15 years with presumptive (clinical) diagnosis of malaria from February to May 2010 were consecutively tested. Capillary blood from each consenting patient was tested for Plasmodium falciparum infection by both conventional Giemsa stain microscopy and Histidine Rich Protein-2 (HRP- 2) rapid diagnostic testing. The statistical test applied were mean ± SD for continuous variables and proportions for categorical variables as well as the estimation of accuracy of RDT. A p-value < 0.05 was considered statistically significant, and agreement (kappa statistics) between RDT and microscopy provided estimation of reliability of the RDT; > 80% was considered good reliability. Results: A total of 161 patients were studied. The mean age was 30.0 ± 11.4 years with 116 (72%) females and 45 (28%) males; p<0.05. Nine (5.6%) and 10 (6.2%) patients had parasitaemia on microscopy and Key Words RDT, Microscopy, Malaria, Accuracy, Adults. RDT testing respectively. The microscopy results were in agreement with the RDT results in 160(%) patients. The sensitivity and specificity of the RDT were 100% and.3% respectively while the positive and negative predictive values were 0% and 100% respectively. Conclusion: The RDT was highly sensitive for the detection of parasitaemia and could be useful in the accurate diagnosis of Plasmodium falciparum malaria. Introduction Malaria is a major cause of morbidity and mortality and according to the 2010 World Health Organisation (WHO) report, an estimated 225 million cases occurred resulting in about 781,000 deaths [1]. Approximately 1% deaths were recorded in Africa with 85% occurring in children under 5 years of age [2]. In Nigeria, malaria remains a major public health and developmental challenge. There are nearly 110 million clinically diagnosed cases of malaria per annum resulting in 60% of outpatient visits and 30% hospitalisations [3]. Furthermore, an estimated 300,000 children die of malaria each year, and up to 11% of maternal mortality is due to malaria. In addition to the direct health impact of malaria, there is also a severe social and economic burden on the communities and the country as a whole with about N132 billion lost *Corresponding author: ejijisa@yahoo.com to malaria yearly in form of treatment cost, prevention, and loss of man hours [3]. The dominant species of malaria parasites is the Plasmodium falciparum (>5%) with p. ovale and p. malariae playing a minor role; the latter being quite common as a double infection in children [3]. The National Malaria Control Programme (NMCP) plans to carry out diagnostic testing for presumptive malaria for at least 80% of patients older than 5 years with a fever by 2013 and subsequently scale up testing to include everyone, irrespective of their age, in line with the current WHO recommendation [1]. However, up till now, clinical diagnosis is the most widely used approach despite evidence that symptoms of malaria are nonspecific and overlap those of other febrile illnesses [4]. Confirmation of presumptive malaria diagnosis can lead to rational use of the now recommended artemisinin combination therapy (ACT) for treatment of uncomplicated malaria and therefore delay the development of resistance. Moreover, it may

2 Isa Samson E. Isa et al 8 reduce the risk of adverse drug reactions due to unnecessary use of ACTs. This, in turn, will increase the patient and caretaker's confidence in health services by providing accurate information for a proper diagnosis [5,6]. The gold standard diagnostic method remains the stained blood film microscopy which is time consuming, often requires trained technicians, good quality reagents and a well maintained microscope. As an adjunct to microscopy, the NMCP now promotes the use of RDTs for malaria diagnosis. According to the World Health Organisation, the RDT must be capable of reliably detecting at least 100 parasites/µl (0.002% parasitaemia) with 100% sensitivity and of giving rapid results (15-20min) [4]. A more recent pivotal clinical trial evaluating the performance of the RDTs in areas of malaria endemicity was presented at the American Society of Tropical Medicine and Hygiene annual conference in which the RDT sensitivity was found to be 5% [7]. The most commonly used RDT target is a glycoprotein called Histidine-rich protein-2 (HRP-2), an antigen specific for P. falciparum which have also been shown to be accurate in detecting P. falciparum infections [8]. HRP-2 is water-soluble, localized in the parasite cytoplasm and on the surface membrane of infected erythrocyte. It is present on protrusions, known as knobs, thought to account for sequestration of the trophozoites and schizonts in postcapillary venules. There is increasing concentration of HRP-2 as the parasite develops from ring stage to trophozoite, and it then readily diffuses into the plasma [,10]. HRP- 2 is predominately found in the asexual stages, but it is also found in young P. falciparum gametocytes. This allows detection of HRP-2 at lower parasitemias and an average of 28 days after clinical presentation; well after resolution of symptoms and apparent parasites clearance by microscopy following treatment [11,12]. Therefore, this antigen is not valuable in monitoring response to therapy. In addition, field conditions require that RDT be stable under extremes of temperatures and humidity during use and storage. The primary objective of this study was to evaluate the accuracy of SD Bio Line Malaria Test Kit (Standard Diagnostics Incoporation, USA), a HRP-2 based test, in adults with presumptive diagnosis of malaria. Methodology This cross-sectional study was carried out at the Federal Staff Hospital Abuja; Nigeria's Federal Capital between February to May 2010 (during dry season to the beginning of rains). Consecutive patients older than 15 years presenting to out patient clinic with presumptive diagnosis of malaria (axillary temperature 0 >37.5 C or a history of fever and/or other complaints indicating a possible malaria infection) were enrolled after obtaining consent. We excluded patients who volunteered history of auto-immune diseases and those who were treated for microscopically confirmed malaria within the past 4 weeks as at the time of enrolment. Basic demographic and clinical information were entered into a proforma, and followed by the collection of 50 µl capillary blood from each consenting patient. Thick and thin blood films were stained with 10% Giemsa stain for 10 min and examined by an experienced microscopist who had no knowledge of patient disease to avoid any bias in blood film readings. A minimum of 100 consecutive high power fields were viewed before classifying a slide as negative. The study was approved by the hospital ethics committe (HEC) of Federal Staff Hospital, Garki. The rapid test kits (SD Bio Line Malaria Test Kit ) were opened only after the patient had been selected and interviewed by the clinical staff and the tests performed according to the manufacturer's instructions. The RDTs were of traceable quality and had a guaranteed history of proper storage and transport conditions. The microscopists and RDT readers were all blinded to each other's diagnoses as additional attempt to eliminate bias. In addition, 73 randomly selected slides were read by a different, but experienced, microscopist for documentation of inter-observer variation. A third, external microscopist, unaware of the first two results, resolved any discordant results. Patients whose results were positive for malaria on microscopy or RDT were treated according to the National guidelines [13]. Principle of Malaria RDT: Briefly, malaria RDT employs lateral flow immunochromatographic technology. In these assays, the clinical sample migrates as a liquid across the surface of a nitrocellulose membrane by capillary action. For a given targeted parasite antigen, two sets of monoclonal or polyclonal antibodies were used, a capture antibody and a detection antibody. Monoclonal antibodies, like in HRP-2, in contrast to polyclonal antibodies can be very specific but less sensitive [14]. Data were collected and analyzed using the EPI- Info version statistical program. The statistical test applied were mean ± SD for continuous variables, chi-square for categorical variables, and the sensitivity

3 Malaria rapid diagnostic test: A study of accuracy among adults in North Central Nigeria was calculated as the proportion of positive test results obtained among samples containing malaria parasites by microscopy; the specificity was calculated as the proportion of negative test results obtained among samples whose thick blood films were negative. Positive and negative predictive values were calculated as the proportion of true-positive results among all positively reacting samples and as the proportion of true-negative results among all negatively reacting samples, respectively. A probability value < 0.05 was considered statistically significant, and agreement (kappa statistics) between RDT and microscopy provided an estimation of the reliability of the RDT; > 80% was considered as a measure of good reliability. Results A total of 161 eligible and consenting patients were tested. The mean age was 30.0 ± 11.4 years with 116 (72%) females and 45 (28%) males. The most frequent symptom was fever or a feeling that the body is hot in143 (88.8%) patients. A body temperature > 37.5 C was recorded in 70 patients (43.5%) at the time of the consultation. Headache and weakness were reported in 3 (57.7%) and 7 (4.0%) of patients respectively. Among the 161 patients with presumptive Table 1. Comparison of Parasitaemia by microscopy with RDT antigenaemia Microscopy POSITIVE NEGATIVE TOTAL Positive Negative Total 1 10 PPV=/10 x 100 = 0% PPV = Positive predictive value NPV = Negative predictive value Sensitivity = / x 100 = 100% diagnosis of malaria, only (5.6%) had microscopically confirmed p. falciparum malaria and 10(6.2%) had p. falciparum antiginaemia by rapid test. The microscopy results were in agreement with the RDT results (i.e congruent microscopy and RDT results) in 160(%) patients (kappa statistics 0.63, p<0.001). Therefore, RDT NPV=151/151 x 100 = 100% Specificity = 151/152 x 1 =.3% the sensitivity and specificity of the RDT were 100% and.3% respectively while the positive and negative predictive values were 0% and 100% respectively (Table I). Furthermore, body temperature >37.5 C was recorded at the time of consultation in four (44.4%) of the patients with positive microscopic malaria. In one (11%) patient with positive microscopic malaria, no fever or history of fever was recorded; the main symptom was body weakness. Discussion The study evaluated the performance of SD Bio Line Malaria Test Kit relative to conventional microscopy in a hospital setting and the main findings were RDT sensitivity 100%, specificity.3%, PPV 0%, NPV 100%, and kappa agreement of %. There were significantly more females 116 (72%) than males 45 (28%) p<0.05. This is not surprising as Okonkwo and co-workers in Ibadan [15] and Kamga and co-workers in Limbe [16] had reported higher malaria prevalence and parasite densities in females than males. On the symptomatology of malaria, this study has further demonstrated the inaccuracies of presumptive malaria diagnosis. Only (5.6%) patients had microscopically confirmed p. falciparum parasitaemia and 10(6.2%) patients had p. falciparum antiginaemia by rapid test. Also, body temperature of >37.5 C was recorded at the time of consultation in only four (44.4%) of the patients with positive microscopic malaria. While in general more than half of those with malaria presumptive diagnosis do not have parasitaemia on microscopy [17], our finding of 152(4.4%) is comparable with results from previous studies in which 7%- 3.6% [18-20] of patients with presumptive malaria were negative on microscopy. This study has reported especially low rates of confirmed parasitaemia possibly because of availability and abuse of antimalaria drugs in urban areas, the study been carried out during off transmission season and semi-immunity in adults which is associated with aparasitaemia or low parasitaemia. These are well established factors responsible for reduced malaria prevalence and parasite densities [15, 21, 22]. The result of this study also demonstrates that SD

4 Isa Samson E. Isa et al 10 Bio Line Malaria Test Kit is an accurate and reliable method for diagnosing P. falciparum malaria. Our measures of accuracy revealed sensitivity 100%, specificity.3%, PPV 0%, NPV 100%, and kappa agreement of %. These results are consistent with other published studies showing that the sensitivity and specificity of HRP-2-based tests usually are > 0% for P. falciparum [20,23-25]. The single false positive result in this study could have been due to persistence of antigenaemia which can be seen up to 35 days after successful treatment [11], presence of early gametocytes [26] or parasitaemia lower than the threshold for microscopic detection [27]. This study was not without limitations. Insufficient numbers of samples with parasites have been tested to draw definitive conclusions and thus, the reported RDT accuracy might not be generalizable. Furthermore, samples with negative results were not tested by the more sensitive polymerase chain reaction (PCR). Conceivably, some additional low-level malaria infections may have been missed by either microscopy or RDT, which would decrease the overall sensitivity of either or both tests. Notwithstanding, our study adds information to the scanty knowledge of accuracy of RDTs in adults with presumptive malaria diagnosis. In conclusion, malaria is over-diagnosed with the result that other common treatable conditions might be missed or wrongly treated with antimalaria drugs. This may worsen personal sufferings, institutional strain in a manner that is not sustainable and constitute a huge economic loss to the nation. RDTs have the potential to change the way we diagnose malaria and, expectantly, influence our antimalarial prescription habits. Acknowledgement We sincerely appreciate the National Malaria Control Programme (NMCP) for their support by providing the rapid test kits used in this study. We are also grateful to Dr. Igwilo C.I who facilitated the study at Federal Staff Hospital Garki II and provided linkage with the NMCP. Declaration of Conflict of interest We wish to state that there was no conflict of interest in this study. References 1. Maru A, Richard C, Yousuke K, etal. World Health Organisation global malaria programme. World Malaria Report. WHO press Geneva, Switzerland; 2010, Pp xi - xii. 2. Maru A, Richard C, Mac O, etal. World Health Organisation global malaria programme. World Malaria Report. WHO press Geneva, Switzerland; Pp viii. 3. Federal Ministry of Health. National Malaria Control Programme strategic plan ; A road map for malaria control in Nigeria. Abuja, Nigeria. 2008, p World Health Organization. New perspectives: malaria diagnosis. Report of a joint W.H.O./USAID informal consultation. W. H. O./MAL/ World Health Organization, Geneva, Switzerland; 1. Pp 4, D'Acremont V, Lengler C, Mshinda H, et al: Time to move from presumptive malaria treatment to laboratory confirmed diagnosis and treatment in African children with fever. PLoS med. 200, 6: e Bisoffi Z, Gobbi F, Angheben A, et al: The role of rapid diagnostic test in managing malaria. PLoS med 200, 6: e Gasser RA Jr, Magill AJ, Ruebush T, et al. Malaria diagnosis: performance of NOW ICT Malaria in a large scale field trial [abstract 2338]. In: Program and abstracts of the 54th Annual Meeting of the American Society of Tropical Medicine and Hygiene (Washington, DC). American Society of Tropical Medicine and Hygiene; Gatti S, Gramegna M, Bisoffi Z, et al. A comparison of three diagnostic techniques for malaria: a rapid diagnostic test (NOW Malaria), PCR and microscopy. Ann Trop Med Parasitol 2007, 101: Howard R.J, Uni S, Aikawa M, et al. Secretion of a malarial histidine-rich protein (Pf HRP II) from Plasmodium falciparum-infected erythrocytes. Int J cell Biol. 186, 4: Rock E.P, Marsh K, Saul A.J, et al. Comparative analysis of the Plasmodium falciparum histidine-rich proteins HRP-I, HRP-II and HRP-III in malaria parasites of diverse origin. Parasitology. 187, 5: Swarthout TD, Counihan H, Senga RKK, et al. Paracheck- Pf accuracy and recently treated Plasmodium falciparum infections: is there a risk of over-diagnosis? Malar J. 2007, 6: Richter J, Göbels K, Müller-Stöyer I, et al. Co-reactivity of plasmodial histidine-rich protein 2 and aldolase on a combined immuno-chromographic-malaria dipstick (ICT) as a potential semi-quantitative marker of high Plasmodium falciparum parasitaemia. Parasitol Res. 2004, 5: Federal Republic of Nigeria National antimalarial treatment guidelines, Federal Ministry of Health National malaria and vector control division. Abuja- Nigeria March Clinton KM, Jason WB. Rapid Diagnosis of Malaria. Interdiscip Perspect Infect Dis. 200, Article ID 41553, doi: /200/ Okonko IO, Donbraye-Emmanuel OOB, Donbraye E et al. Malaria parasitaemia among patients in Ibadan, Southwestern Nigeria. J. Appl. Biosci. 2010, 2:

5 Malaria rapid diagnostic test: A study of accuracy among adults in North Central Nigeria Kamga FHL, Akuro S A, Njunda AL. Relationships between blood cell counts and the density of malaria parasites among patients at the regional hospital, Limbe, Cameroon. Afr. J. Cln. Exper. Microbiol. 2010, 11: Benjamin SC, Eric NO, Obinna EO, et al. Cost-effectiveness analysis of rapid diagnostic test, microscopy and syndromic approach in the diagnosis of malaria in Nigeria: implications for scaling-up deployment of ACT. Malar J. 200, 8: Paul M, Waziri S, Raymond U, et al. Over-diagnosis of malaria is not a lost cause. Malar J. 2006; 5: Reyburn H, Mbakilwa H, Mwangi R et al. Rapid diagnostic tests compared with malaria microscopy for guiding outpatient treatment of febrile illness in Tanzania: randomised trial. Br Med J. 2007, 34: Alioune BL, Adama T, Robert P, et al. Use of HRP-2-based rapid diagnostic test for Plasmodium falciparum malaria: assessing accuracy and cost-effectiveness in the villages of Dielmo and Ndiop, Senegal. Malar J. 2010, : Robert V, Macintyre K, Keating J, et al. Malaria transmission in urban sub-saharan Africa. Am J Trop Med Hyg. 2003, 68: Zeno B, Sodiomon B.S, Joris M, et al. Accuracy of a rapid diagnostic test on the diagnosis of malaria infection and of malaria - attributable fever during low and high transmission season in Burkina Faso. Malar J. 2010, : Hopkins H, Kambale W, Kamya MR, et al. Comparison of HRP2- and pldh-based rapid diagnostic tests for malaria with longitudinal follow-up in Kampala, Uganda. Am J Trop Med Hyg. 2007, 76: Ochola LB, Vounatsou P, Smith T, et al. The reliability of diagnostic techniques in the diagnosis and management of malaria in the absence of a gold standard. Lancet Infect Dis. 2006, 6: Wongsrichanalai C, Barcus MJ, Muth S, et al. A review of malaria diagnostic tools: microscopy and rapid diagnostic test (RDT). Am J Trop Med Hyg. 2007, 77: Harani MS, Beg MA, Khaleeq L, et al. Role of ICT malaria immunochromatographic test for rapid diagnosis of malaria. J Pak Med Assoc. 2006, 56: Hopkins H, Bebell L, Kambale W, et al. Rapid diagnostic tests for malaria at sites of varying transmission intensity in Uganda. J Infect Dis. 2008, 17:

Outcome of Presumptive Ukwaja K. et al 179

Outcome of Presumptive Ukwaja K. et al 179 ORIGINAL ARTICLE OUTCOME OF PRESUMPTIVE VERSUS RAPID DIAGNOSTIC TESTS-BASED MANAGEMENT OF CHILDHOOD MALARIA PNEUMONIA OVERLAP IN URBAN NIGERIA: A PILOT QUASI-