A comparative assessment of Rapid Diagnosis Test (RDT) versus microscopy for malaria diagnosis in health care facilities

Background: Management of malaria requires prompt diagnosis of malaria by microscopy, Rapid Diagnostic Tests (RDTs), or other available tools. The objective of this study was to determine the best approach on malaria diagnosis in detection of malaria parasite in the Northwest region, of Cameroon among different population groups. Materials and method: The cross sectional study was conducted on 60 febrile patients who were directed to the laboratory department for blood screening at the Bamenda Regional Hospital after showing signs and symptoms of malaria infection. Blood sample were collected aseptically and dispensed into an EDTA container where RDTs and microscopic examination were performed to assess the presence of malaria parasites. Results from the rapid diagnostic kits were analyzed and compared to those obtained by general microscopy. Results : Of the 60 samples enrolled 37 (61.7%) were found to be positive with blood lms examination while in rapid diagnostic test 27 (45%) were positive. Based on frequency of infection by age 20-35 years had 13 (35.1%) as the most vulnerable group, followed by 0 -5 year with 11 (29.7%), 6-19 had 6 (16.2%) while age group of 36-59 had 5 (16.0%) and lastly the age group of 60 and above had least value of 2 (5.4%). The prevalence of malaria obtained through microscopy (62%) was signicantly higher than in RDT (45%). Considering microscopy as the gold standard, RDT exhibited high specicity (100%) and sensitivity (73%) with positive predictive and negative predictive values of 100% and 70%, respectively. The sensitivity of RDT increased signicantly with increase in P. falciparum parasitaemia which was the plasmodium specie detected in all positive cases. Conclusion: The study, therefore, highlights that the routine microscopy test demonstrated a superior sensitivity compared to RDT method of malaria diagnosis, however, RDT could be a useful tool in individuals suspected to show high degree of disease spectrum for quick intervention in order to avert danger associated with delayed diagnosis.


Introduction
Malaria has been known to mankind for thousands of years. Around 7,000-12,000 years ago with increase in temperatures in Africa, rise in humidity creating new water sources and the start of agriculture in the Middle East and North East Africa which brought about several water bodies and pools of water.
This led to a favorable climate and an area for breeding and transmission of malaria parasites and its carrier, the mosquito [1] The World Health Organization (WHO) estimated that, 3.2 billion people are at risk of being infected with malaria and according to the latest estimates, 219 million cases of malaria occurred globally in 2017 and the disease led to 407,000 deaths [2]. Sub-Saharan Africa bears the highest burden of malaria, accounting for an estimated 90% of all malaria deaths worldwide of which more than 78% of malaria deaths occur in children aged <5 years. Over 90% Cameroonians are at risk of malaria infection, and about 41% have at least one episode of malaria each year [3]. The main strategy for malaria control as recommended by the WHO is a prompt and accurate diagnosis followed by effective treatment [4,5]. The con rmation of suspected malaria cases by microscopy or a rapid diagnostic test (RDT) before treatment [5] will result in a signi cant reduction of morbidity and mortality related to misdiagnosis. Although PCR may represent the best diagnostic method with high sensitivity and speci city [6], it is still costly and not very useful for routine diagnosis. Hence, microscopy and RDTs continue to be the primary choices for diagnosing malaria in most endemic countries. Microscopy is still considered the "gold standard" for malaria diagnosis in endemic countries because has a sensitivity of 50-500 parasites/μl [7], is inexpensive, and allows the identi cation of species and parasite density [8,9], however the implementation of microscopy as diagnosis tool, especially in remote areas has been reported to be cumbersome and to requires trained personnel, microscopes and a source of electricity [10]. In remote areas of sub-Saharan Africa, RDTs have become the primary tool for the parasitological diagnosis or con rmation of malaria [11].The most widely used RDTs for malaria are based on the detection of parasite histidine-rich protein II (HRP2) and Plasmodium lactate dehydrogenase (pLDH) [12]. The major constraint of RDTs are false positives, because HRP2 persists in the blood for several days after infection clearance [13], and false negatives due to gene deletions [11]. Moreover, RDTs are thought to be more speci c to P. falciparum infections [14]. Another drawback of RDTs is the occurrence of, falsenegative results. This situation may delay treatment process and increase the number of persons capable of infecting mosquitoes in the community. Variation of performance in the diagnosis method has been shown to have a great impact on individual treatment and epidemiological surveillance [15]., and some health professionals in endemic countries have lose con dence in test results and resolved to treat systematically all fever cases as malaria [10,16]., This may result the over-or under-diagnosis of malaria, with excessive use of anti-malarial drugs or negligent treatment, which invariably contributes to malaria morbidity and the development of resistance [12,17]. Clinical diagnosis is imprecise but remains the basis of therapeutic care for the majority of febrile patients in malaria endemic areas, where laboratory support is often out of reach. Scienti c quanti cation or interpretation of the effects of malaria misdiagnosis on the treatment decision, epidemiologic records, or clinical studies has not been adequately investigated [18]. Despite an obvious need for improvement, malaria diagnosis is the most neglected area of malaria research, accounting for less than 0.25% ($700,000) of the U.S.$323 million investment in research and development in 2004 [18]. Rational therapy of malaria is essential to avoid non-target effects, to delay the advent of resistance, and to save cost on alternative drugs. Accurate diagnosis is the only way of effecting rational therapy. Con rmatory diagnosis before treatment initiation recently regained attention, partly in uenced by the spread of drug resistance and thus the requirement of more expensive drugs unaffordable to resource-poor countries [19].. The objective of the present study was to assess the performance of RDT compare to microscopy, the two main techniques used in Cameroon for the diagnosis of malaria.
2 Research Methodology

Study area
This study was conducted at the Regional hospital Bamenda located in the North West region found in Mezam division and in Mankon subdivision. It's a three-star high centered hospital created by the government for the entire population of the people of Bamenda. It incorporates a great number of patients a day coming from different areas of the region. Also having a quali ed staff determined for the well-being of its patients

Sample size and selection
The cross-sectional study was conducted for 60 patients within Mezam division. A total of 60 patients consisting of 30 males and 30 females, varying of ages were enrolled from the 30th of Novembre to the 15th December 2016 at the regional hospital in Bamenda. These patients were admitted for malaria RDT and microscopy diagnosis after showing signs and symptoms of malaria infection. Patients were of a variety of age groups presented at medical consultation with signs and symptoms of malaria infection and con rmed by parasitological test.

Sample collection
Blood samples were collected by venipuncture into containers containing EDTA. After mixing, the blood samples were used to determine the hematocrit, for the preparation of thick and thin slides for microscopy, and for the RDTs assay.

Microscopy analysis
When making thick lm, a drop of blood was placed at the center of the cream grease free slide and spread with the edge of another slide in a repeated coil shape to a diameter approximately 2 cm.The slide was labelled and left horizontally for drying and are kept well to prevent them from dust and damage. It was then stained with 2 ml of a 10% Giemsa solution and then washed after 10 min using clean water. As for the thin lm, a drop of blood was placed at the center of the cream grease free slide the lm is then spread using a smooth edged slide spreader. 2 ml of methanol is applied on to it and allowed for xation for a period of 2minutes. After xation 2 ml of a 10% giemsa solution is then applied on the xed smear and then washed after 10 min using clean water. A drop of immersion oil was applied on dried stained slides (thin and thick lms) and examined microscopically for malaria parasites using 100X objective lens and result was recorded [20] .

RDT Analysis
Test device, buffer and specimen were allowed to equilibrate at room temperature (10ºC -30ºC) prior to testing. The test cassette was removed from the foil pouch by tearing at the notch and then placed on level surface. 5 µl of whole blood were slowly added into the sample well. Then 3 drops of clearing buffer was added to the buffer well. As the test begins to work, a purple colour moving across the result window in the center of the test device. A result was read after 25 minutes.

Data Analysis
Data was carefully entered in Excel spread sheet (version 2013) and analyzed using the statistical package for social sciences (SPSS) version 20 software. Proportions were calculated and the diagnostic performance was determined by calculating the test sensitivity, speci city, and predictive values.

Results
The prevalence of positive test in both microscopy and RDT diagnosis was highest (33.4% and 35.1% respectively) among the 20 to 35 years age group (Fig. 1) and the least prevalence was seen in both diagnosis was seen in 60 and above age group (5.4% in microscopy and 3.7% in RDT). Figure 1: Prevalence of diagnosis with respect to age group As regards to age, it was noticed that a greater proportion of getting positive patients with RDT diagnosis within the age ranges of 20 to 35 hence having patients of this age group most sensitive to RDT, by which may be due to the large number of patients diagnosed within this age group followed 0 to 5 age group 6 to 19, 20 to 35 and 60 and above age groups respectively.
The overall prevalence of malaria parasites obtained through microscopy (62%) was signi cantly higher than in RDT (45%). Species of malaria parasite identi ed in all study participants was Plasmodium falciparum. The parasite density ranged from 40-400 per µL of blood. The number of subjects with true/false negative and true/false positive RDT's results was presented in Table 1.  In this study routine microscopic examination of stained blood lms which is considered as the gold standard for malaria diagnosis had a sensitivity of 100% and was able to detect more parasites than the RDT (sensitivity 73%). Though the speci city of microscopy and that of RDT was 100%, microscopy has a high sensitivity and has the ability to quantify parasitemia which is a good advantage.
The sensitivity of RDT is seen to be highest in age group of 20 to 35 (53%) having number of parasites ranging from 60 to 2720 (Table 3), followed by 6 to 19 (27%) with parasites ranging from 65 to 1200, 36 to 59 (10.5%) with parasites ranging from 68 to 720, 0 to 5 (6.6%) with parasites ranging from 128 to 2600 and least in age group of 60 and above (2.9%) with parasites number ranging from 100 to 650. The optimal function of the immune system is mostly between the age ranges of 20 to 35 years, hence been able to detect the least amount of antigen in the system hence a great sensitivity in RDT. Whereas infants (ranging from 0 to 5 years) which have a developing immune system is not highly sensitive to antigens in the system hence a low sensitivity in RDT. The elderly (60 and above) have a suppressed immune system due to aging leads to less sensitivity of the test.

Discussion
Microscopy and RDT, recommended by WHO, revealed to be the most reliable tools for prompt and accurate diagnosis prior to an effective treatment of malaria suspected cases [5]. The sensitivity reported in this study does not attain the 95% recommended World Health Organization value [21]. This low sensitivity is disadvantageous as it will impair control intervention since a fraction of the infected population will be left untreated especially if RDT is the only available diagnostic tool. This could have important implications in health, transmission, and possibly mortality. The sensitivity of RDT reported in this study is lower than previous reported in Nigeria [22], Zambia, Zanzibar and Thailand [20,23,24]. Importantly, this study recorded the speci city of 100% for RDT which is far higher than the one reported in Burkina Faso [25], but similar to the observation made among the Nigerian isolates [26]. The di culties and challenges faced by current RDTs have been reported showing a considerably varying speci cities, sensitivities, numbers of false positives, numbers of false negatives and temperature tolerances of these tests [27]. False negative cases in this study were recorded for RDT using microscopy as diagnosis tool of reference.. The high proportion of false negative test resulted in the low sensitivity recorded in this study. This false negative could be explained by the effect of PfHRP2 sequence variation on the binding of speci c mAbs [11,28,29,30,31]. In contrast, this sequence variation of PfHRP2 DNA obtained from isolates from African and South American was not found to be linked to varying sensitivities of RDTs [32]. Moreover, a prozone effect revealed to occur in PfHRP2 RDT and not with pLDH or aldolase based RDTs [33,34]. The storage temperature the RDT product by the marketers could also explain the observed low sensitivity. Several factors, including exposure to high temperature, storage and transportation conditions and training of health workers may likely in uence test performance [25,27,35]. Interestingly, our study showed no false positive case and the absence of false positive test in this study is a characteristic of a good test. This is interesting because accurate diagnosis is the basis for an adequate disease control and for delaying the emergence and spread of antimalarial drugs resistance [15]. However, the rheumatoid factor cross-reacting in the blood could generate a false positive test line but this issue is now resolved by developing new products in which IgG is being replaced by IgM [36]. Also, cross-reactivity with heterophile antibodies may be another cause of false positive test [37]. False positives have been recorded due to infection with Schistosoma mekongi [38]. False positives are also caused by the persistence of PfHRP2 in the blood for long periods after parasite clearance [7,39,40].
Overall, the use of microscopy in diagnosis of malarial infection in adult population that comprises pregnant women can pose some challenges due to placental sequestration of parasites thus reducing the sensitivity of microscopy. However, the detection of peripheral blood HRP-2 genes is possible with malaria parasites, making RDT very useful in such condition [41]. The ability to detect placental infection by antigen detection when microscopy does not identify parasitaemia could have a signi cant impact on material and foetal health care. Besides, the di culties associated with RDTs, such as genetic variability including polymorphisms in the PfHRP2 gene, and the persistence of antigens in the bloodstream following the elimination of parasites, highlights the importance for the development of alternative RDTs to the PfHRP2-based RDT. Several alternative malarial diagnostic targets have been identi ed and explored including heat-shock protein 70, dihydrofolate reductase-thymidylate synthase, haemdetoxi cation protein, glutamate-rich protein, hypoxanthine phosphoribosyltransferase and phosphoglycerate mutase [42,43]. Again, a promising alternative to RDTs is the PCR [6, 15], a molecular technique that has proven to be the most sensitive and speci c method available as it allows the detection of parasitemia as low as 2-5 parasites/µl [3] However, it is expensive, complex, and not appropriate for eld use; Again, it has to be validated by the WHO before its implementation in health facilities.

Conclusions
Stained blood lm microscopy and rapid diagnostic test each with its characteristics, strength and the limitation together present the best hope for diagnosis as a key component of successful malaria control hence rapid diagnostic test does not eliminate the need for stained blood lm malaria microscopy therefore all negative rapid diagnostic test must be followed with stained blood lm microscopy to con rm the result. Microscopy is the more reliable method in areas where malaria is most prevalent. RDT offers a good alternative, being an easy and rapid method that does not require an experienced laboratory technician. However, there is a need for the development of alternative RDTs to the PfHRP2-based RDT. Therefore, while waiting for the implementation of these new techniques, when PfHRP2-based RDT is used alone, it should be followed with stained blood lm microscopy to ascertain in the degree of infection and to know the malaria specie involved for proper treatment. The present study demonstrates that RDTs can act as a diagnostic tool to manage malaria in resource poor settings with limited access to expert microscopy as they are easy to use and perform.

Declarations
Ethical Approval and consent to participate This study was approved by the Ethics Committees of the Ministry of Public Health Cameroon and was registered with controlled-trials.com in the website: https://clinicaltrials.gov/ct2/show/NCT02974348 under the registration number NCT02974348.

Funding
This study was sponsored in part sponsored by the Chinese National Science and Technology Project grant No. 2012ZX10004-220 and by the Ministry of Higher Education through annual small research grant offered by the minister to support research activities in government owned Universities

Study Limitations
The number of participants enrolled was determined by the short period of recruitment which is two weeks from the 15 th of April to the 30th of April 2016. The trend of the outcome might have a slight change with an increased number of participants.
Authors' contributions MTN and ZXN designed and organized the study. CTF and PFG carried out the eld and laboratory work. CTF and MTN wrote the manuscript. CTF and PFG analysed data and wrote the manuscript. All authors read and approved the nal manuscript.

Figure 1
Frequency of diagnosis approach with respect to age group

Supplementary Files
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