International Journal of Recent Advances in Multidisciplinary Topics 85

Volume 2, Issue 10, October 2021

https://www.ijramt.com | ISSN (Online): 2582-7839

Sensitivity of Rapid Diagnostic Test and

Microscopy in Malaria Diagnosis in Iva-Valley
Suburb, Enugu
Nwosu Chinemelum Joyce1*, Ekwunife Chinyelu2, Nkwocha Elaine Adaku3
1,2
Department of Parasitology and Entomology, Nnamdi Azikiwe University, Awka, Nigeria
3
Department of Microbiology, Alex Ekwueme Federal University, Ndufu-Alike Ikwo, Nigeria

Abstract: Malaria disease is still of public health importance. For effective management of malaria infection, prompt and
For effective management and control, an alternative parasite- accurate diagnosis is very essential.7 The use of antigen-
based diagnosis is paramount. This study focused on determining detecting rapid diagnostic test kits (RDTs) forms a vital part of
the sensitivity of rapid diagnostic tests and microscopy in malaria
diagnosis in Iva-valley suburb, Enugu State, Nigeria. A total of 379 providing parasite-based diagnosis in areas where good quality
blood samples were collected from five communities and examined microscopy cannot be maintained.3 Also Rapid diagnostic test
for malaria parasites using microscopy and rapid diagnostic test. do not require laboratory support, are easily read and can reach
Out of the 379 blood samples, 166 (43.80%) were positive for sensitivity similar to that commonly achieved by well-
malaria parasites using the CareStart test kit, while 169 (44.6%) performed microscop.8 Other diagnostic methodologies have
were positive using microscopy. Using microscopy as the gold also risen to overcome the inefficient malaria diagnosis such as
standard, the sensitivity and specificity of the CareStart RDT were
89% and 92.86% respectively, while the positive and negative Polymerase Chain Reaction (PCR) based genetic tests.
predictive values were 90.96% and 91.55% respectively. Females
had the highest prevalence (69.2 %,) while males had the least 2. Materials and Methods
(30.8%). However, the difference in gender prevalence was not 1) Study Area
significant (X2 calc = 1.939, X2 tab=3.841, P>0.05). The age group
11-15 years had the highest prevalence (27.8%), while the 21-25
Iva valley is a sub-urban settlement in Enugu Metropolis in
years age group had the least prevalence (1.18%). This study the capital city of Enugu State of Nigeria. It has geographical
showed that the accuracy of CareStart RDT is comparable to the co-ordinates of approximately 60.14” and 60.18” North latitude
gold standard microscopy, making it a suitable diagnostic tool for and 70.5oC and 70.09” East longitude. It is located in the
any local health staff in remote endemic areas. tropical rainforest zone, although it has derived savanna
vegetation. It has two marked seasons, the dry and wet seasons.
Keywords: Malaria, CareStart RDT and Microscopy
There are about 8 months (April – November) of wet season
and four months (November – March) of dry season. It has a
1. Introduction
relative humidity of 70% reaching 80% during rainy season and
Malaria a seasonal disease remains a major public health an annual rainfall of about 2000mm. The daily temperature
problem in both tropical and subtropical countries in Africa, ranges from 26oC – 35oC during the dry season stretching from
major incidence occurs during the rainy season. 1-2 Malaria November to March, and from 220C –30oC during the wet
remains the leading cause of mortality and morbidity in sub- season stretching from April to November. The inhabitants are
Saharan Africa, 3 an estimated cases of 229 million and 409 mainly Igbos’. The settlers are retired coal miners, civil
000 estimated death in 2019.4 Nigeria is known for high servants, traders, craftsmen, educated people and farmers. With
prevalence of malaria and the disease remains one of the leading the creation of Anambra State in 1991, and the establishment of
causes of childhood and maternal morbidity and mortality, low Government machineries’ and institutions of higher learning,
productivity and reduced school attendance in Nigeria [5]. over 60% of the populations are civil servants and students
Malaria diagnosis involves identifying malaria parasites or while the remaining 40% are farmers, traders and other
antigens/products in patient blood. Diagnosis of malaria occupations. This structure is still the same when Enugu State
infection based on clinical symptoms alone is unreliable was carved out of Anambra State.
because the symptoms of malaria are non-specific and often 2) Community mobilization
overlaps with other febrile diseases.6 Lack of precise diagnosis A letter of intent to carry out the study at Iva valley was
remains an important obstacle to the treatment adherence, collected from the Head of Department of Parasitology and
effectiveness and clinical managements of severe cases. Entomology addressed to the community leader, for permission

*Corresponding author: [email protected]

N. C. Joyce et al. International Journal of Recent Advances in Multidisciplinary Topics, VOL. 2, NO. 10, OCTOBER 2021 86

and mobilization of the people of Iva valley. The study in field’s stain A (eosin) for 5 seconds. It was washed off gently
population were properly sensitized on the mode and intent of in clean water and then dipped in field’s stain B (methyl azure)
the study. for 5 seconds and washed again in clean water. The stained
3) Study design and sampling films were examined under a microscope using X100 objective
The work was a cross-sectional study of the population for the presence of malaria parasites.
(carried out during the month of November) to ensure that every 2) Care-Start rapid diagnostic testing
group was represented, in order to determine malaria infection Malaria Rapid Diagnostic Test kit (Care Start Tm Malaria
across all age groups. HRP2 One Step Rapid Test) was used in the field to detect
4) Malaria Prevalence Study malaria infections. Whole blood (5μl) was added into sample
The participants in the study were apparently healthy wells and 60μl (2 drops) of assay buffer were added into assay
individuals who did not show any of the common signs of buffer wells. The blood-buffer mixture was allowed to run
malaria. Their biodata such as names, ages, sex and occupations toward the test and control window. Result was read within 20
were collected through oral interview and recorded in a field minutes. The presence of two colour bands (One on the control
note book. and one on the test) indicated positive result. The presence of
5) Blood sample collection only one band (the control line) within the result window
Blood sample was collected using venipuncture technique. 9, indicated negative result while the presence of only one band
10 Soft tubing tourniquet was fastened to the upper-arm of the on the test line indicate an invalid result.
patient to enable the index finger feel a suitable vein. The 3) Statistical analysis of data
puncture site was then cleansed with methylated spirit Data on blood samples and mosquitoes collected were
(methanol) and venipuncture made with the aid of a 21g needle analyzed using Social Sciences Statistical Package (SPSS)
attached to a 5 ml syringe. When 2ml blood had been collected, version 17.0 and chi square. The sampling error was taken to be
the tourniquet was released and the needle removed 95% Confidence interval.
immediately while the blood was transferred into an EDTA
bottle [10]. 4. Results
Collection of blood sample starts with the cleaning of a A total of 379 persons examined in Iva valley sub-urban area,
patient’s finger is cleaned with 70% ethyl alcohol, allowed to 169 (44.6%) were positive for malaria parasite using
dry and then the fingertip is pricked with a sharp sterile lancet microscopy while CareStart RDT recorded 43.80% malaria
and two drops of blood are placed on a glass slide. The thick prevalence in the area (Table 1). In the different study location
blood film is prepared by placing a blood spot which is stirred in the in Iva valley, Camp one recorded the highest prevalence
in a circular motion with the corner of the slide, taking care not of 46 (27.2%) while Forest hill recorded the lowest prevalence
make the preparation too thick, and allowed to dry without rate of 22 (13.0%). The difference between the various
fixative. After drying, the spot was stained with diluted Giemsa residential location was however not significant (X2 calc =
(1: 20, vol/vol) for 20 min, and washed by placing the film in 2.8447, X2 tab = 9.837, P>0.05).
buffered water for 3 min. The slide is allowed to air-dry in a table
vertical position and examination using a light micro- scope. As
they are unfixed, the red cells lyse when a water-based stain is Table 1
Prevalence of malaria in the study area according to communities
applied. A thin blood film is prepared by immediately placing Location No Positive (Microscopy) Positive (RDT)
the smooth edge of a spreader slide in a drop of blood, adjusting examined (%) (%)
the angle between slide and spreader to 45 and then smearing Camp One 100 46 (27.2) 42 (25.3)
the blood with a swift and steady sweep along the surface. The Camp two 51 25 (14.8) 22(13.2)
Valley 100 38 (22.5) 42(25.30)
film is then allowed to air-dry and is fixed with absolute road
methanol. After drying, the sample is stained with diluted Forest Hill 44 22 (13) 20(12.04)
Giemsa (1: 20, vol/vol) for 20 min and washed by briefly Pottery 84 38 (22.5) 40(24.09)
Total 379 169 (44.6) 166 (43.8)
dipping the slide in and out of a jar of buffered water (excessive
washing will decolorize the film). The slide is then allowed to Table 2
air-dry in a vertical position and examined under a light Prevalence of Malaria in relation to Gender in the Study areas
microscope [11]. Gender Number Number Positive by Positive by
Examined Microscopy (%) RDT (%)
Female 248 117(69.2) 108(65.1)
3. Identification of Malaria Parasites
Male 131 52(30.8) 58(51.2)
1) Microscopy Total 379 169 (44.6) 166 (43.8)
Thick-blood films were prepared according to the technique
outlined by Baker12, 13, 10. A drop of each blood sample was
placed in the centre of a grease-free clean glass slide. The blood Malaria prevalence in gender and age groups in the study
was homogenously spread out in a circular motion using an area using microscopy. Females have higher prevalence of
edge of a spreader slide to make an even smear. The slide was 69.2% than males with prevalence of 30.8% (Table 2).
kept for air-drying and staining with field’s stain. The slide was However the difference in gender prevalence was not
held with the dried thick film side facing downward and dipped significant (X2 calc = 1.939, X2 tab=3.841, P>0.05). The study
N. C. Joyce et al. International Journal of Recent Advances in Multidisciplinary Topics, VOL. 2, NO. 10, OCTOBER 2021 87

showed that the age group 11 -15 years had the highest attributed to health seeking and treatment behavior often
prevalence rate of 27.8%, followed by 16- 20 with 15.98%. The displayed by the female group. The higher prevalence recorded
age group 21 - 25 years had the least prevalence rate of 1.18%. among the female can be found in other studies reported in
The mean age of the sampled population was 16.7+ 21.47 years Awka and Abeokuta. 14 However, at 5% level of significance,
(Table 3). the difference was not statistically significant (P>0.05). This
agrees with other findings who reported that sex did not affect
Table 3
Prevalence in relation to Age in the Study Areas using microscopy
malaria prevalence among individuals [15]. There was also
Age Group Number Examined Number Positive (%) unequal representation in the age group, the highest number of
1-5 26 14 (8.3) participants were of the age group 11 – 15 years, 103 (27.2%).
6-10 75 26 (15.4) This may be attributed to the fact that most of the women who
11-15 103 47 (27.8) participated in the study came with their children.
16-20 52 27 (16.0)
21-25 14 2 (1.2) A high prevalence of malaria 169 (44.6%) for microscopy
26-30 12 4 (2.4) and 166 (43.8%) for RDT was observed among the study
31-35 13 9 (5.3) participants. The total prevalence of 44.6% reported in this
36-40 25 8 (4.7)
41-45 10 5 (3.0) study is higher than other studies. For instance, prevalence of
46-50 14 10 (5.9) 17% was reported in Eastern Nigeria 16, 27.3% prevalence in
51-55 18 5 (3.0) Sokoto.17 Although the prevalent is lower when compared to
56-60 9 7 (4.1)
>60 8 5 (3.0) other studies: 46% prevalence in Nnewi, 18 76% in Azia,
Total 379 169 Anambra State, 19 72% prevalence in Osogbo, 20and 80%
prevalence in Awka.14 From the findings, it shows that malaria
Table 4
Evaluation of CareStart RDT using microscopy as gold standard
is prevalent in Iva valley sub-urban although the population
Test Malaria +ve Malaria –ve Total involved in the study were obviously healthy individuals at the
RDT +ve 151 15 166 (43.80%) time of the study. Malaria infection was recorded from all the
RDT –ve 18 195 213 (56.20%) five communities with Camp one recording the highest
Total 169(44.6%) 210 (55.4%) 379
prevalence of 46 (27.2%) while Forest hill recorded the lowest
Table 5
prevalence rate of 22 (13.0%). The difference between the
Sensitivity, Specificity and the Predictive Value of Care-start RDT various residential location was however not significant (X2
Test Malaria +ve Malaria –ve calc = 2.8447, X2 tab = 9.837, P>0.05). This is an indication of
RDT +ve 151 15 widespread end emicity of malaria in the community.
RDT –ve 18 195 Furthermore, a higher percentage prevalence of malaria
Sensitivity 89.35% using microscopy was recorded (44.6%) than that of RDT
Specificity 92.86%
Positive Predictive Value 90.96%
(43.8%). The sensitivity of 89.35% of CareStart RDT testkit
Negative Predictive Value 91.55% was fairly reasonable while specificity of 92.86% was
significantly high. The finding in the current study was
Malaria Prevalence using Care-start RDT and microscopy as consistent with the findings in China with a sensitivity of
gold standard. Care Start RDT kit indicated that 43.80% 89.68% 21though slightly higher than the findings from other
(166/379) of the sampled population were infected with malaria studies conducted in Northern Nigeria with a sensitivity of
parasite and 44.6% (169) were positive by microscopy (Table 78.4%, 22 82% sensitivity and 91% specificity. 23 The false
4). Of those positive by microscopy (n=18) were negative by positives recorded can be attributed to the fact that both children
RDT (false negatives), while 9.0% of those positive by RDT and adult involved in the study may have received some anti-
(n=15) were negative by microscopy (false positives). malaria before presentation were not excluded in this study, and
Determination of the Sensitivity, Specificity and the HRP-2 is known to persist in the blood for a few weeks after
Predictive Value of Care-start RDT. treatment. Thus the test may still remain positive even when the
The sensitivity of the CareStart RDT test kit was 89.35%, the parasites have been cleared. The positive predictive value of
specificity was 92.86%, while the positive and negative 90.96% recorded in this study meant that the kit has the
predictive values were 90.96% and 91.55%, respectively when capability of confirming malaria with a precision of 91%, while
compared to microscopic examination of blood films as gold the negative predictive value of 91.55% means that the RDT is
standard for detection of malaria (Table 5). NB: Sensitivity = good in ruling out malaria, thus giving the clinician the
True positive/ (True positive + False negative) x (100); confidence that a negative test excluded malaria in about 92%
Specificity = True negative / (True negative + False positive) x of cases. The data collected in this study was not linked to
(100); Positive predictive Value = True positive/ (True positive patients’ medical records. Therefore, it was impossible to
+ False positive) x (100); Negative predictive value= True collect clinical information, such as recent intake of
negative/ true negative +false negative x 100. antimalarials or presence of other factors, which may have
influence on the results. However it is important to note that the
5. Discussion data described were obtained in true field conditions, i.e.,
There was unequal representation of the participants from the conditions that were suboptimal in terms of RDT storage and
communities (131 Males, 248 Females). This could be handling and staff expertise. It is known that such factors
N. C. Joyce et al. International Journal of Recent Advances in Multidisciplinary Topics, VOL. 2, NO. 10, OCTOBER 2021 88

influence RDT performance [24, 25]. Several factors such as [10] Epidi, T. T., Nwani, C. D., and Ugorji, N. P. Prevalence of malaria in
blood donors in Abakaliki Metropolis, Nigeria. Scientific Research and
the manufacturing process, environmental conditions may Essay 3, pp. 162-164, 2008.
affect RDT performance.26, 27 Manufacturers usually [11] Chotivanich, K., Silamut, K., and Day, N.P.J., (2006). Laboratory
recommend 4°–30°C as the optimal temperature range. In diagnosis of malaria infection-a short review of methods. Aust J Med Sci;
vol. 27, pp. 11-15.
practice, exposure of RDTs to > 70% humidity and/or > 30°C [12] Baker, F. J., Silverton, R. E. and Pallister, C. J. (2001). Cellular pathology
frequently occurs in the tropics. Although the figures reported and Introduction to Histology. In: Baker’s & Silverton’s Introduction to
in this study was less than the 95% recommended by the World Medical Laboratory Technology. 7th ed., Martins of Berwick, Britain.
Health Organization (WHO), and though microscopy is the pp.. 173-243.
[13] Cheesebrough, M. (2004). District laboratory practice in tropical
gold standard there is still possibility of human error and countries. Part 2. Cambridge University Press. pp. 357.
technical problem of microscopic identification of parasites [14] Ibekwe, A. C, Okonko, I, O., Onunkwo, A. I., Ogun, A. A., Udeze, A.O.
which could explain the disparity between the two diagnostic (2009). Comparative Prevalence Level of Plasmodium in Freshmen (First
Year Students) of Nnamdi Azikwe University in Awka, South-Eastern,
methods. Nigeria. Malaysian J. Microbiol. vol. 5, no. 1, pp. 51-54.
[15] Mbanugo, J.I. and Ejim, D.O. Plasmodium infections in children aged 0–
6. Conclusion 5years in Awka metropolis, Anambra State Nigeria. Nigerian Journal of
Parasitology; vol. 21, pp. 55– 59, 2000.
CareStart TM HRP2 Rapid Diagnostic Test (RDT) kit has [16] Anumudu, C. I., Adepoju, A., Adeniran, M., Adeoye, O., Kassim, A.,
good sensitivity and specificity when compared with the gold Oyewole, I., Nwuba, R. I. (2006). Malaria prevalence and treatment
seeking behaviour of young Nigerian adults. Ann. Afr. Med. vol. 15, pp.
standard Light Microscopy. The use of CareStart HRP-2 RDT 82-88.
in health facilities especially in areas without access to [17] Abdullahi, K., Abubakar, U., Daneji, A.I., Adamu, T., Abdullahi, Y.M.,
microscopy or where standard laboratories and trained Bandiya, H.M. and Hassan, S.W. Anopheles gambiae in Sokoto north
western Nigeria. Nigerian Journal of Parasitology, vol. 31, no. 2, pp. 118-
microscopist are not available should be encouraged rather than 122, 2010.
depending on presumptive treatment. The government likewise [18] Umeanaeto, P. U. and Ekejindu, I. M. Prevalence and intensity of malaria
can encourage diagnosis by subsidizing the various RDTs that in blood donors at Nnamdi Azikwe University Teaching Hospital
(NAUTH) Nwewi, Anambra State, Nigeria. Nig. J. Parasitol. vol. 27, pp.
have good sensitivity and specificity. Its deployment for use at
11- 15, 2006.
the community level and patent medicine stores should be [19] Aribodor, D.N., Njoku, O.O., Eneanya, C.I. and Onyali, I.O. Studies on
encouraged as people patronize them more than the the prevalence of malaria and management practices in Azia community
comprehensive health facilities. in Ihiala L.G.A. Anambra state Southeast Nigeria. Nigeria Journal of
Parasitology, vol. 24, pp. 33-38, 2003.
[20] Adefioye OA, Adeyeba OA, Hassan WO, Oyeniran OA (2007).
References Prevalence of malaria parasite infection among pregnant women in
Osogbo, Southwest Nigeria.Am. Eurasian J. Sci. Res. vol. 2, no. 1, pp. 43-
[1] Eneanya C.I. Seasonal variation in malaria episodes among residents in a
45.
semi-urban Community in South-East Nigeria. The Nigerian Journal of
[21] Xiaodong, S., Tambo, E., Chun, W., Zhibin, C., Yan, D., Jian, W., Jiazhi,
Parasitology, vol. 19, pp. 39-43, 1998.
W., Xiaonong, Z. (2013). Diagnostic performance of CareStartTM
[2] Oesterholt, M.J., Bousema, J.T., Mwerinde, O.K., Harris, C., Lushino, P.,
malaria HRP2/pLDH (Pf/pan) combo test versus standard microscopy on
Masokoto, A., Mwerinde, H., Mosha, F.W. and Drakeley, C.J. Spatial and
falciparum and vivax malaria between China-Myanmar endemic borders.
temporal variation in malaria transmission in a low endemicity area in
Malar J.
northern Tanzania. Malaria Journal, vol. 5, pp. 1475- 2875, 2006.
[22] Sheyin, Z., and Bigwan, I.E. (2013). Comparison of CARE START
[3] World Health Organisation (WHO) (2009). World malaria Report.
HRP¬2 rapid malaria test with light microscopy for guiding patient’s
Geneva, WHO/HTM/GMP
treatment of fever in Nigerian endemic areas. J. Med. Med. Sci, vol. 4, no.
[4] World Health Organization (WHO, 2020). World Malaria Report-
9, pp. 353.
World/Relief Web.
[23] Adesanmi, T., Okafor, H., Okoro, A., Mafe A.G. (2011) Diagnosis of
[5] Aribodor, D. N., Nwaorgu, O. C., Eneanya, C. I., Aribodor O. B. Malaria
malaria parasitemia in children using a rapid diagnostic test. Niger. J.
among primigravid women attending antenatal clinic in Awka, Anambra
Clinical Practice, vol. 14, pp. 195-20.
State, South-east Nigeria. Niger. J. Parasitol, vol. 28, no. 1, pp. 25-27,
[24] Jorgensen, P., Chanthap, L., Rebueno, A., Tsuyuoka, R., Bell D: Malaria
2007.
rapid diagnostic test in tropical climates: the need for a cool chain. Am J
[6] D'Acremont, V., Lengeler, C., Mshinda, H., Mtasiwa, D., Tanner, M.,
Trop Med Hyg, vol. 74, pp. 750-754, 2006.
Genton, B. (2009). Time to move from presumptive malaria treatment to
[25] Chiodini, P. L., Bowers, K., Jorgenssen, P., Barnwell, J. W., Grady, K.
laboratory-confirmed diagnosis and treatment in African children with
K., Luchavez ,J. (2007). The Heat Stability of Plasmodium Lactate
fever. PLoS Med.; vol. 6, no. 1, e252.
Dehydrogenase-based and Histidine–rich Protein 2-based Malaria Rapid
[7] Tangpukdee, N., Duangdee, C., Wilairatana, P., and Krudsood, S. Malaria
Diagnostic Tests. Transac. Royal Soc. of Trop. Med. and Hyg, vol. 101,
Diagnosis: A Brief Review. Korean J. Parasitol. vol. 47, no. 2, pp. 93–
pp. 331-337.
102, 2009.
[26] Bell D., Wongsrichanalai, C., and Barnwell, J.W. (2006). Ensuring
[8] Murray, C.K., Gasser, R.A. Jr. Magill, A.J., and Miller, R.S. (2008).
quality and access for malaria diagnosis: how can it be achieved? Nat Rev
Update on rapid diagnostic testing for malaria. Clin Microbiol Rev, vol.
Microbiol, vol. 4, pp. 682-695.
21, pp. 97–110.
[27] Mboera, E.M., Schander, G., Sambou, I., and Greenwood, B.M. (2006).
[9] Okocha, E.C., Ibeh, C.C., Ele P.U. and Ibeh, N.C. The prevalence of
Suppression of cell- mediated immune response to malaria antigen in
malaria parasitaemia in blood donors in an Nigerian teaching hospital.
pregnant Tanzanian women. American Journal of Tropical Medicine and
Journal of Vector Borne Diseases, vol. 42, pp. 21-24, 2005.
Hygiene, vol. 40, pp. 141 – 144.