|Year : 2016 | Volume
| Issue : 2 | Page : 96-101
Diagnosis of malaria: A comparison between microscopy and rapid diagnostic test among under-five children at Gusau, Nigeria
Bilkisu Ilah Garba1, Aminu Sakajiki Muhammad2, Abdullahi Musa3, Bassey Edem1, Ibrahim Yusuf1, Nura Kaura Bello1, Akeem Oladiran Adeniji1, Taofik Kolawole1
1 Department of Paediatrics, Yariman Bakura Specialist Hospital, Gusau, Zamfara, Nigeria
2 Department of Medicine, Yariman Bakura Specialist Hospital, Gusau, Zamfara, Nigeria
3 Department of Paediatrics, Ahmadu Bello University, Zaria, Nigeria
|Date of Submission||24-Jan-2016|
|Date of Acceptance||30-May-2016|
|Date of Web Publication||21-Jun-2016|
Dr. Bilkisu Ilah Garba
Department of Paediatrics, Yariman Bakura Specialist Hospital, Gusau, Zamfara
Background: Malaria remains a disease of public health concern in tropical countries. Diagnosis in such countries largely depends on clinical assessment, microscopy (as the gold standard) and recently by rapid diagnostic tests (RDTs). This study was conducted to document common presenting symptoms of malaria, compare microscopy and RDT in the diagnosis of malaria in children in Gusau, Nigeria. Materials and Methods: A cross-sectional, study of children under-five years with presumptive diagnosis of malaria that was managed at the pediatric outpatient department and emergency pediatric unit of Yariman Bakura Specialist Hospital, Gusau. All consecutive children that met the inclusion criteria were recruited over a 4 weeks period. Malaria thick film microscopy and RDTs were carried out on all children. Results: Of the 118 children enrolled, 61 (51.69%) were males while 57 (48.31%) were females. Fever and vomiting were the most common presenting complaints. Microscopy was positive in 55 (46.61%) while RDT was positive in 10 (8.47%) of the children. Only 5 (4.24%) were positive for both microscopy and RDT. Fifty-eight (49.15%) children had negative microscopy and RDT results. There was no significance when positivity of both tests were compared (χ2 = 0.050, P = 0.822). RDT had a sensitivity of 9.09%, a specificity of 92.06%, positive predictive value of 50.00%, and a negative predictive value of 53.70%. Conclusion: Our study observed higher positivity rate for microscopy than RDT. We recommend microscopy to be carried out on all patients suspected of having malaria where possible, even in the presence of negative RDT.
Keywords: Malaria, microscopy, rapid diagnostic test, under five children
|How to cite this article:|
Garba BI, Muhammad AS, Musa A, Edem B, Yusuf I, Bello NK, Adeniji AO, Kolawole T. Diagnosis of malaria: A comparison between microscopy and rapid diagnostic test among under-five children at Gusau, Nigeria. Sub-Saharan Afr J Med 2016;3:96-101
|How to cite this URL:|
Garba BI, Muhammad AS, Musa A, Edem B, Yusuf I, Bello NK, Adeniji AO, Kolawole T. Diagnosis of malaria: A comparison between microscopy and rapid diagnostic test among under-five children at Gusau, Nigeria. Sub-Saharan Afr J Med [serial online] 2016 [cited 2019 Oct 19];3:96-101. Available from: http://www.ssajm.org/text.asp?2016/3/2/96/184371
| Introduction|| |
Malaria remains a disease of public health concern in tropical countries.  The diagnosis of malaria in such countries largely depends on clinical assessment, microscopy and recently by rapid diagnostic tests (RDTs).  Microscopy is considered the gold standard for diagnosis of malaria.  Affordability, low-threshold sensitivity, identification of species, and parasite density gives it an edge over RDT. , However, long turnaround time, erratic power supply, and need for the technical expertise are some of the major challenges limiting its use in resource-limited countries where the burden of malaria is greatest. 
RDT, on the other hand, was developed to improve the sensitivity and objectivity of malaria diagnosis through less reliance on microscopy.  It is an immune-chromatographic capture procedure which targets antigens abundant in all asexual and sexual stages of the parasite. Current RDTs detect Histidine-Rich Protein 2 (PfHRP2) from Plasmodium falciparum and Parasite-Specific Lactate Dehydrogenase (pLDH) or Plasmodium aldolase from the parasite glycolytic pathway found in all species.  The tests that utilize PfHRP2 have been found to be more sensitive than pLDH based ones, especially at low parasite densities, with certain exceptions.  Both PfHRP2 and pLDH RDTs have been found to be more sensitive than aldolase-based tests. The PfHRP2 based RDTs can detect antigen when P. falciparum parasites are sequestered either in placental tissues or elsewhere, which makes them not to be present in peripheral blood for detection by microscopy. 
However, RDT performance can be affected by factors which include short shelf life because of temperature instability, especially during transportation from place of manufacturing or supply to final place of use, suboptimal sensitivity at low parasite densities and inability to identify parasite's species or quantify parasite density. ,,, Manufacturers recommend an optimal temperature range of 4-30°C., The pLDH-detecting RDTs produced a consistent level of parasite lactate when maintained at 4°C, but activity is reduced after cumulative exposure to temperatures likely to be encountered over a few months in a malaria-endemic area. 
Presumptive diagnosis of malaria based on clinical ground is the most commonly used method in endemic countries, the signs, and symptoms of which are nonspecific; mostly based on fever or history of fever.  However, the accuracy of clinical diagnosis varies with the level of endemicity, malaria season and age group,  and overdiagnosis;  thus RDT is still relevant for objective diagnosis of malaria.
The World Health Organisation (WHO) recommends that parasite-based diagnosis should be used in all cases of suspected malaria with the possible exception of children in high-prevalence areas and certain situations. , However, where microscopy is not available, WHO recommends RDT. 
There are few published studies have assessed the performance of RDTs among under-five children in Nigeria despite the high burden of malaria. ,,
Thus, this study was conducted to compare microscopy and RDT in the diagnosis of malaria in children under-five years seen at a specialist Hospital in Gusau.
| Materials and Methods|| |
This was a cross-sectional study of children under the age of 5 years with a presumptive diagnosis of malaria. Enrolled children included those that met the selection criteria while attending the pediatric outpatient department or were admitted to the emergency pediatric unit of the Yariman Bakura Specialist Hospital Gusau, Zamfara State, Nigeria. The study was conducted over a 4-week period in May 2014. Inclusion criteria were symptoms of malaria, presence of axillary temperature of ≥37.5°C and/or history of fever in the 24 h before presentation. Those with an obvious focus of infection, use of antimalarial therapy within 2 weeks before presentation, use of malaria prophylaxis and refusal of consent by parents or guardians were excluded. Informed consent was obtained from parents/guardians. Ethical approval was obtained from the Hospital's Ethical Committee.
A questionnaire was designed to capture age, sex, use of pretreatment antimalarial and presenting complaints. Malaria tests (microscopy for thick film and RDT) were simultaneously carried out on all blood samples from the studied population. Thin film microscopy was done at random to identify the Plasmodium specie since it is known that the most common specie in our environment is falciparum.
Smears for thick film were made from the fresh blood samples collected from all the children as all parents consented. An experienced medical laboratory scientist stained the slides with Giemsa using standard methods;  and parasites were counted against 200 white blood cells. The smears were examined with × 100 magnification under oil immersion objectives. A total of 100 fields were examined before declaring the slides as positive or negative. Two laboratory scientists independently examined the slides; the third reading of discordant results by another scientist was taken as final.
Rapid Diagnostic Test
RDT was performed on 5 mcl of blood using CareStart™ Malaria HRP2 (Access Bio, Inc., Somerset, New Jersey, USA) according to manufacturer's instructions by trained laboratory scientists. The test was considered positive when the antigen line was visible in the test window and negative when only the control band was visible. It has a sensitivity of 98% and specificity of 97.5% as reported in the test kit pamphlet. The test is only limited to the detection of antigens of P. falciparum. The entire test kits were within the shelf life (Manufacturing date December 2012 and expiry date May 2015) and were stored in the laboratory store which is air-conditioned though not on a 24 h basis. Hence, the temperature was variable. Recommended storage temperature by the manufacturer is between 1°C and 40°C.
The microscopy and RDT were done by different laboratory personnel. Those with positive results were given antimalarial free of charge which was provided by a nongovernmental organisation, however, parents/guardians were not informed that antimalarial would be given free so as not to influence participation.
Data were entered into Statistical Package for Social Sciences (SPSS) version 16 (SPSS Inc Chicago, IL, USA) for cleaning and analysis using standard methods. Quantitative variables were summarized using mean and standard deviation. Categorical variables were summarized using frequency and percentages.
Sensitivity, specificity, negative and positive predictive values (NPV and PPV) were calculated using standard formulae. Chi-square test and Fisher's exact were used for association between categorical variables. P < 0.05 was considered statistically significant.
| Results|| |
A total of 118 children were studied, of whom 61 (51.69%) were males with a male to female ratio of 1.07:1. The mean age of the studied population was 28.56 ± 18.42 months, with a range of 4 to 59 months. Majority of the children (31.40%) were aged 12 months and below; males predominated in the 13-24 months age group as shown in [Table 1].
All the children presented with a history of fever while 52.54% had vomiting, 39.83% had cough, 18.64% had a headache, 30.00% had abdominal pain, whereas 5.93% had convulsion. None of the symptoms on presentation was associated with the positivity of either microscopy or RDT as shown in [Table 2]. The mean duration of symptoms was 5.23 ± 2.99 days, with a range of one to 15 days before presenting at the hospital.
|Table 2: Association between presenting symptoms with microscopy and rapid diagnostic tests |
Click here to view
Sixteen (13.56%) of the children had pretreatment with an antimalarial, which were all artemether-lumefantrine combination. The frequency of gender and pretreatment antimalarial with microscopy and RDT are shown in [Table 3]. Pretreatment with antimalarial affect neither microscopy (χ2 = 1.88, P = 0.17) nor RDT (Fishers exact = 1.00).
|Table 3: Frequency of some variables with microscopy and rapid diagnostic tests |
Click here to view
Microscopy was positive in 55 (46.61%), and RDT was positive in only 10 (8.47%) of the children. Only 5 (4.23%) were positive for both microscopy and RDT while 58 (49.15%) children had both negative microscopy and RDT as shown in [Table 4]. The prevalence of malaria in this group of febrile children with a clinical diagnosis of malaria, using the microscopy test (Gold standard), was 46.61% where 55 of the 118 subjects were positive.
|Table 4: Comparison between rapid diagnostic tests and microscopy results |
Click here to view
Comparison between positivity of microscopy and RDT was not significant (χ2 = 0.05, P = 0.82). There were five true positives, 50 false positives, 58 true negatives, and five false negatives. The RDT when compared with the gold standard, which is microscopy; had a sensitivity of 9.09% (5/55) and a specificity of 92.06% (58/63). The PPV was 50.00% (5/10) and NPV of 53.70% (58/108).
Thirty-one (56.36%) of the children with positive microscopy were males, while 24 (43.64%) were females. Gender was not significantly associated with positivity of microscopy (χ2 = 0.39, P = 0.34). Five (50%) of the children with positive RDT were males while the remaining 5 (50%) were females. This was also not associated with gender (χ2 = 0.01, P = 0.91).
The RDT positivity rate was low, and as such did not increase as the age advances, however, it was not significant as shown in [Table 5]. However, microscopy positivity progressively declined with advancing age even though it was also not significant as shown in [Table 5].
|Table 5: Association and outcome of malaria rapid diagnostic tests and microscopy according to age group |
Click here to view
Twenty-four slides were examined at random for species identification, only 1 (4.17%) had trophozoite of Plasmodium malariae, while the remaining 23 (95.83%) were ring forms of P. falciparum.
| Discussion|| |
The prevalence of malaria among febrile children presumptively diagnosed to have malaria in our study was 46.61%. We found a higher positivity rate for microscopy than RDT, with a low sensitivity but higher specificity rate. Malaria still remains a common cause of morbidity and mortality, especially in children under the age of 5 years  in whom immunity to malaria has not yet developed, hence the need for proper case definition and management. For diagnosis, positive microscopy or RDT should preferably be obtained before commencing treatment. 
We did not observe an increase in positivity rates with age of the children for both RDT and microscopy. Acheampong et al.  and Abeku et al.  similarly did not observe an increase in RDT positivity with age. This is not consistent with findings by Elechi et al.  where they observed an increase in sensitivity with advancing age. Reasons for the variations may be due to differences in sample size and age group studied. There was no significant association between gender and RDT similar to what was obtained by Elechi et al. 
Our microscopy positivity rate was similar to what was obtained by Uzochukwu et al.,  Ojurongbe et al.  and Sani et al.  but lower than the report by Azikiwe et al. and Harchut et al. However, our RDT positivity rate was quite lower than what was obtained in these studies but higher than 2.3% obtained by Elechi et al.  at Maiduguri. Our positivity rate for both microscopy and RDT was also lower than the 100% obtained by Elechi et al.,  35.1% by Uzochukwu et al.,  and 59% by Azikiwe et al.  Harchut et al.  in Uganda reported a 78% positivity for microscopy and only 14% for RDT. Reasons for these variations may be attributable to different sample size, methodology, and types of RDT kits used.
The sensitivity, specificity, PPV and NPV were 9.09%, 92.06%, 50.00%, and 53.70% respectively. Most studies that have been carried out to compare microscopy and RDT showed both to be sensitive in the diagnosis of malaria. ,,, Batwala et al.  in Uganda found RDT to be not only sensitive but also more feasible than microscopy. Our study got a low sensitivity and specificity as against the manufacturer's specifications. This is not in consonance with the findings in Angola  and Uganda  where RDT was found to have higher sensitivity and specificity. However, recently, Elechi et al.  found RDT not to be a reliable test in under five children with acute uncomplicated malaria as their sensitivity of 8.3% was also low which is comparable to our finding. The sensitivity to RDT was lower than the 69.6%, and 82.0% reported from Lagos and Enugu, respectively. ,
Variations in sensitivity between the different studies may be due to differences in the types of RDTs used or test methodology and skill of the microscopists. Other reasons for our low positivity rate using RDT could probably be due to low parasite density as RDT does not detect low parasite density of < 50 mcl. However, this was not examined as it is not the aim of our study. Another reason could be due to the presence of other species of Plasmodium as our specie identification was done at random, even though only one child had trophozoites of P. malariae.
We obtained quite a high number of both negative RDT and microscopy, which may suggest children were not infected with malaria parasite. This may mean that a high proportion of children are still over-diagnosed with malaria; which is in consonance with what was obtained by Uzochukwu et al.,  Ojurongbe et al.,  and Azikiwa et al.  and Batwala et al. 
The reasons for the low positivity rates may be due to external factors that may have affected the stability of the RDT such as exposure to extreme temperatures which has been found to be a major contributor to poor performance of RDTs, especially during transport as well as storage. ,, The pLDH-detecting RDTs produced a consistent level of parasite lactate when maintained at 4°C, but activity is reduced after cumulative exposure to temperatures likely to be encountered over a few months in a malaria-endemic area.  High humidity has also been found to rapidly degrade RDTs.  The environmental temperature in Gusau can reach up to 35°C or more,  and high temperature can degrade the RDT kits. Maintaining a cold chain from the point of arrival of the RDTs into the country, storage and during transportation to point of care cannot be ascertained to be optimal; however this was not looked at in our study.
RDT technical problems can be from quality performance or assurance, test quality and accuracy, and the packaging of the kits.  RDTs are more expensive relative to microscopy and also the intensity of test band varies with amount of antigen present at low parasite densities which may lead to reader variation in test results. ,
| Conclusion|| |
Our study showed that microscopy performed better than RTD in the diagnosis of malaria in children under the age of 5 years. We suggest microscopy, if available and possible to be done for all patients suspected of having malaria with negative RDT as it remains the gold standard for malaria diagnosis. We recommend more studies using a large sample size to be carried out comparing microscopy and RDT in the diagnosis of malaria in children.
We wish to acknowledge Murtala Rabiu, Husaini Umar Bako and Salisu Lawal, medical laboratory scientists at Yariman Bakura Specialist Hospital, Gusau for their contributions in carrying out the microscopy and rapid diagnostic testing.
Financial Support and Sponsorship
Conflicts of Interest
There are no conflicts of interest.
| References|| |
Wongsrichanalai C, Barcus MJ, Muth S, Sutamihardja A, Wernsdorfer WH. A review of malaria diagnostic tools: Microscopy and rapid diagnostic test (RDT). Am J Trop Med Hyg 2007;77 6 Suppl:119-27.
Amexo M, Tolhurst R, Barnish G, Bates I. Malaria misdiagnosis: Effects on the poor and vulnerable. Lancet 2004;364:1896-8.
Makler MT, Palmer CJ, Ager AL. A review of practical techniques for the diagnosis of malaria. Ann Trop Med Parasitol 1998;92:419-33.
Beadle C, Long GW, Weiss WR, McElroy PD, Maret SM, Oloo AJ, et al.
Diagnosis of malaria by detection of Plasmodium falciparum
HRP-2 antigen with a rapid dipstick antigen-capture assay. Lancet 1994;343:564-8.
Abanyie FA, Arguin PM, Gutman J. State of malaria diagnostic testing at clinical laboratories in the United States, 2010: A nationwide survey. Malar J 2011;10:340.
Mouatcho JC, Goldring JP. Malaria rapid diagnostic tests: Challenges and prospects. J Med Microbiol 2013;62(Pt 10):1491-505.
Moody A. Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev 2002;15:66-78.
Jorgensen P, Chanthap L, Rebueno A, Tsuyuoka R, Bell D. Malaria rapid diagnostic tests in tropical climates: The need for a cool chain. Am J Trop Med Hyg 2006;74:750-4.
Albertini A, Lee E, Coulibaly SO, Sleshi M, Faye B, Mationg ML, et al.
Malaria rapid diagnostic test transport and storage conditions in Burkina Faso, Senegal, Ethiopia and the Philippines. Malar J 2012;11:406.
Lon CT, Alcantara S, Luchavez J, Tsuyuoka R, Bell D. Positive control wells: A potential answer to remote-area quality assurance of malaria rapid diagnostic tests. Trans R Soc Trop Med Hyg 2005;99:493-8.
Stephens JK, Phanart K, Rooney W, Barnish G. A comparison of three malaria diagnostic tests, under field conditions in North-West Thailand. Southeast Asian J Trop Med Public Health 1999;30:625-30.
Shillcutt S, Morel C, Goodman C, Coleman P, Bell D, Whitty CJ, et al.
Cost-effectiveness of malaria diagnostic methods in sub-Saharan Africa in an era of combination therapy. Bull World Health Organ 2008;86:101-10.
Uzochukwu BS, Obikeze EN, Onwujekwe OE, Onoka CA, Griffiths UK. 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 2009;8:265.
Elechi HA, Rabasa AI, Bashir MF, Gofama MM, Ibrahim HA, Askira UM. Uncomplicated malaria in children: The place of rapid diagnostic test. Niger Med J 2015;56:85-90.
Ben-Edet AE, Lesi FE, Mafe AG, Grange AO. Diagnosis of falciparum malaria in children using the immunochromatographic technique. Niger J Paediatr 2004;31:71-8.
Adesanmi TA, Okafor HU, Okoro AB, Mafe AG. Diagnosis of malaria parasitemia in children using a rapid diagnostic test. Niger J Clin Pract 2011;14:195-200.
Marianne L. Giemsa staining of thick or thin blood film. In: Kristen M, Inger L, Heduig P, Artur S, Mats W, editors. Methods in Malaria Research. 5 th
ed. Paris: Bio-MalPar; 2008. p. 17.
Ojurongbe O, Adegbosin OO, Taiwo SS, Alli OA, Olowe OA, Ojurongbe TA, et al.
Assessment of clinical diagnosis, microscopy, rapid diagnostic tests, and polymerase chain reaction in the diagnosis of Plasmodium falciparum
in Nigeria. Malar Res Treat 2013;2013:308069.
Acheampong DO, Appiah MG, Boamponsem LK, ABoampong JN, Afoakwah R. The efficacy of rapid diagnostic test (rdt) in diagnosing Plasmodium falciparum
malaria in some selected health facilities in the capecoast metropolis of Ghana. Adv Appl Sci Res 2011;2:348-56.
Abeku TA, Kristan M, Jones C, Beard J, Mueller DH, Okia M, et al.
Determinants of the accuracy of rapid diagnostic tests in malaria case management: Evidence from low and moderate transmission settings in the East African highlands. Malar J 2008;7:202.
Sani UM, Jiya NM, Ahmed H. Evaluation of a malaria rapid diagnostic test among febrile children in Sokoto, Nigeria. Int J Med Med Sci 2013;3:434-40.
Azikiwe CC, Ifezulike CC, Siminialayi IM, Amazu LU, Enye JC, Nwakwunite OE. A comparative laboratory diagnosis of malaria: Microscopy versus rapid diagnostic test kits. Asian Pac J Trop Med 2012;2:307-10.
Harchut K, Standley C, Dobson A, Klaassen B, Rambaud-Althaus C, Althaus F, et al.
Over-diagnosis of malaria by microscopy in the Kilombero Valley, Southern Tanzania: An evaluation of the utility and cost-effectiveness of rapid diagnostic tests. Malar J 2013;12:159.
Nwachukwu C. Evaluation of BID ®
Plasmodium Lactate Dehydrogenase (pLDH) Rapid Test for Detections of Malaria Parasite in Calabar, Nigeria. Institute of Tropical Disease Research and Prevention, University of Calabar Teaching Hospital; Calabar: 2006. Available from: www2.sciencewithamission.org/pdf_instructions/malaria_clinical-evaluation-report.pdf. [Last accessed on 2014 Jun 04].
Reyburn H, Mbakilwa H, Mwangi R, Mwerinde O, Olomi R, Drakeley C, et al.
Rapid diagnostic tests compared with malaria microscopy for guiding outpatient treatment of febrile illness in Tanzania: Randomised trial. BMJ 2007;334:403.
Batwala V, Magnussen P, Nuwaha F. Comparative feasibility of implementing rapid diagnostic test and microscopy for parasitological diagnosis of malaria in Uganda. Malar J 2011;10:373.
Fançony C, Sebastião YV, Pires JE, Gamboa D, Nery SV. Performance of microscopy and RDTs in the context of a malaria prevalence survey in Angola: A comparison using PCR as the gold standard. Malar J 2013;12:284.
Hopkins H, Bebell L, Kambale W, Dokomajilar C, Rosenthal PJ, Dorsey G. Rapid diagnostic tests for malaria at sites of varying transmission intensity in Uganda. J Infect Dis 2008;197:510-8.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]