Evaluation Of The Efficacy Of The Carestart Malaria HRP2 And pLDH/HRP2 Combo Compared To Microscopy In The Diagnosis Of Malaria

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Evaluation Of The Efficacy Of The Carestart Malaria HRP2 And pLDH/HRP2 Combo Compared To Microscopy In The Diagnosis Of Malaria

Chapter One

1.0 Introduction

Malaria is a life-threatening illness, that has continued to pose public health challenges. It affects millions of people all around the globe especially, in Africa, Asia and South America. Malaria is currently endemic in over 100 countries with 3 billion people at risk of infection and around 225 million cases in 2009, leading to approximately 781,000 deaths (WHO, 2010). Malaria has remained a major public health problem in Nigeria, and is responsible for 30% childhood and 11% maternal mortality (FMoH, 2005). It accounts for 300,000 deaths each year and about 60% of outpatient visits (President’s Malaria Iniative, 2011). Together Nigeria, and the Democratic Republic of Congo account for over 40% the estimated total malaria burden and deaths globally (WHO, 2012). It is caused by the asexual form of the parasitic protozoan know as Plasmodium. The species incriminated arePlasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale which is found humans and Plasmodium knowlesi which found in non-humans. Among these parasites, Plasmodium falciparum and Plasmodium vivax are the most widespread and common causes of mixed-species malaria, which is defined as co-infection with more than one species or genotype of Plasmodium (Mayxay et al., 2004).

Most cases of malaria are uncomplicated, commonly presenting with fever and sometimes with other non-specific symptoms including headache, and aches and pains elsewhere in the body (Gilles, 1991; WHO, 2003). Mtoni and Senosi (2007) noted that early diagnosis and treatment are key to addressing morbidity and mortality due to malaria. Proper management of malaria cases within the first 24 hours of onset is considered to be the best way to reduce its morbidity and mortality (Singh et al., 2013). This would be adequately achieved if most of the patients have access to laboratory facilities (Kamugisha et al., 2008). Most victims of malaria still die, because the disease is not diagnosedin time by health workers (Uzochukwu et al., 2009). Microscopy is the gold standard for laboratory diagnosis of malaria in many developing countries, though expertise may be lacking in both endemic and non-endemic settings (Moody, 2002), especially in Nigeria. However, in situations lacking reliable microscopic diagnosis, rapid diagnostic tests (RDTs) may offer a useful alternative to microscopy (Nour et al., 2009).

In general, RDTs are fast, easy to perform and relatively cheap (Lubell et al., 2007). A lot of research and development has been going on to develop alternative methods for laboratory diagnosis of malaria. Rapid diagnostic tests have been developed, validated and field tested. It was introduced in the nineties, but has now undergone many improvements (Martha et al., 2010). Malaria rapid diagnostic test plays a key role in malaria control and elimination programmes in order to avoid unnecessary anti-malarial therapy, to prevent drug resistance and to enhance case finding (Eibach et al., 2013). The RDTs are based on the principle of immunochromatography, which require finger prick blood and detect malaria specific antigen. There are three different RDTs that are available commercially; one of them is specific for detecting P. falcipraum antigens, while the other two detects one or more of the three human malaria species. The RDTs provide quick results, are reliable, and require less skilled persons as compared to microscopic diagnosis. They do not require electricity or any equipment. It promotes patient’s confidence as well as health services.

More than 60 RDT brands and over 200 different products have been developed. Of these, the WHO and Foundation for Innovative New Diagnostics (FIND) evaluated 70 from 26 manufacturers (WHO, 2008; 2009). Of these products, 39 are three-band tests that detect and differentiate P. falciparum from non falciparum species (Martha et al., 2010). The CareStart™ Malaria HRP-2/ pLDH (Pf/pan) Combo Test and the SD Bioline Ag pf/pan, HRP-2 and pan-pLDH are both a three-band RDT detecting HRP-2 and pan-pLDH. This present study is focused on evaluating the efficacy of two of the many RDTs; SD Bioline and CareStart™ Malaria kits using it microscopy test as the gold standard for the diagnosis of malaria.

SD Bioline (Ag pf/pan, Cassette, RDT, kit) is a one step differential diagnosis by detecting HRP-II antigen from Plasmodium falciparum and pLDH antigen from other species (P. vivax, P. malariae, P. ovale) in human whole blood. The CareStart (Combo, dev., RDT) is a test designed for the differential diagnosis between Plasmodium falciparum and other Plasmodium species such as Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. Though, the gold standard for malaria testing remains microscopy, but the limitations associated with this technique could affect the speed of delivery of quality services to the patients (Ameh et al., 2012).

1.1 Statement of the Problem

Microscopy has been in use for over 100 years and is inexpensive, rapid and relatively sensitive when used appropriately (Laveran, 1891). Microscopy is regarded as the ‘gold standard’ for malaria diagnosis (WHO, 1999). However, the lack of skilled scientists in medical facilities in affected areas often leads to poor interpretation of data. In addition, microscopy is time consuming, labour intensive, and cannot detect sequestered P. falciparum parasites (Leke et al., 1999). It is less reliable at low-density parasitaemia that is, 50 parasites (ml blood) (Kilian et al., 2000; Bell et al., 2005). Even though microscopy is cheap, reliable and available on an instant base, it has limitations. For instance, in resource-limited centres, there are problems of equipment, training manpower, and workload, whereas in non-endemic countries, laboratory staff may lack sufficient exposure to malaria positive samples resulting in low expertise (Moody, 2002; Hanscheid, 2003).

In Nigeria, RDTs are still new to the people, and they are unsure of the efficacy, accuracy and authenticity. It has been 7 years since the launching of malaria RDTs in Nigeria but the populace know little or nothing about Malaria RDTs due to poor promoting from the part of manufacturers. In addition, the implementation of RDTs also faces many difficulties such as logistics; transport and continuous supply, limited shelf life and the need of proper storage rooms. RDTs are quickly affected by humidity and extreme temperatures (Wongsrichanalai et al., 2007). They are not able to quantify parasitaemia and may give false positive results owing to the persistence of antigens that can remain in the circulation of a patient after treatment (Wongsrichanalai et al., 2007).

1.2 Significance of the Study

The essence of continuous research and development is to find a way to improve the lives of people around the globe. Thus, finding an alternatively cheap, fast, convenient and effective way to diagnosis malaria is a key to control malaria. This study is therefore significant in many ways:

  1. The finding of this study will be useful and helpful to the Federal and State Government with regard to malaria eradication in making decisions on implementation of RDTs for routine diagnosis in the Nigeria, especially in rural areas.
  2. The findings of this study will provide an alternative, effective and reliable diagnosis of malaria patients in both those that are asymptomatic and symptomatic.
  3. RDTs are fast, easy to perform and relatively cheap and can easily be used by both the trained and untrained.

1.3 Research Questions

  1. What is the efficacy of SD Bioline and Carestart when compared to microscopy?
  2. Can RDTs such as SD Bioline and Carestart be alternative for the gold standard (microscopy) in the diagnosis of malaria.

1.4 Research Hypothesis

  • HA; RDTs are more efficient in the detecting of malaria cases than microscopy
  • HO; Microscopy is more efficient in defecting malaria than RDTs

1.5 Aims and Objectives of the Study

The aims and objectives of this study were to:

  1. Evaluate the efficacy of the Carestart Malaria HRP2 and pLDH/HRP2 Combo compared to microscopy in the diagnosis of malaria.
  2. Determine the sensitivity, specificity, positive and negative predictive values of the malaria RDTs to microscopy.
  3. Determine the relationship between malaria parasite density and results of malaria RDTs.
  4. Correlate results of negative malaria detection rate by microscopy to results of malaria RDTs.

Chapter Five

Discussion, Conclusion and Recommendations

5.1 Discussion

On the experimental study a total of 185 patients were recruited for the study within a period of 2 months (60 days); blood sample were collected as patients visits, results of test conducted were recorded immediate after each test. Microscopic analysis showed that 89.18% (165/185) of the blood slides were positive for malaria parasitaemia. Hence, microscopy was used as the control for the study. While the malaria rapid diagnostic tests (RDTs) that were used are CareStart AgPf/Pan and SD Bioline AgPf/Pan. It is expected that typical malaria RDTs should have high sensitivity 95% and specificity 97%, and ability to detect low parasite density infections (WHO, 2003). The finding of this study is slightly contrary as the results from CareStartTM and SD Bioline, indicate otherwise, compare to the World Health Organizations standardization for RDTs.

In Table 4.7 CareStartTM AgPf/Pan showed 157/165 positive, with 8/165 that is False Positive (2). CareStartTM AgPf/Pan has a sensitivity of 95.15% with Confidence Interval 91.87% to 98.43%, and specificity of 90.00% with Confidence Interval of 76.85% to 103.15%. The efficiency showed 94.59% with a confidence interval of 91.33% to 97.85%. Also the Positive Predictive Value (PPV) indicated 98.74%, while Negative Predictive Value (NPV) 69.23% was observed.

In the same vain, SD Bioline AgPf/Pan test kits used, showed 155/165, 10/165, that is the False Negative is 10, a false positive 3/20 was calculated. The study showed that SD Bioline AgPf/Pan has a sensitivity of 93.94% with Confidence Interval of 90.30% to 97.58%, and specificity was calculated to 85.00% with Confidence Interval of 69.35% to 100.65%. The efficiency showed 92.97% with a confidence interval of 89.29% to 96.65%. Also the Positive Predictive Value (PPV) showed 98.10%, while Negative Predictive Value (NPV) was 62.96%.

The findings in this study are in agreement with other studies which found sensitivity and specificity of CareStartTM falls within the ranges (82.9-100%) and 56.0-96.4%) respectively (Guthmann et al.,2002; Mboera et al., 2006; Swarthout et al., 2007; Kamugisha et al.,2008; Nigussie et al.,2008; Abeku et al.,2008; Baboo et al., 2008; Wilcox et al., 2009; Sharew et al.,2009; Gerstl et al., 2010;). The study results also agrees with recent findings of some researchers with a sensitivity for the SD Bioline tests of 83.3–100% and a specificity of 46.8–98.9% (Ogouyemi-Hounto et al., 2013; Kosack et al., 2013; Kim et al., 2013;).

The specificity of the test reported falls within the acceptable range when compared with previous studies as earlier mentioned and may allow us to diagnose patients as truly negative for falciparum malaria, but the low negative predictive value for SD Bioline of 62.96%, do not agree with previous studies in the range of 90 to 98% (Ashton et al., 2010; Singh et al., 2010), and this provided the risk of missing an infected individual. Reasons for this low specificity in SD Bioline RDT used could not be ascertained but the truth will not be far fetch from what Ameh and colleagues (2012) reported in their study, they stated that the loss in specificity as per WHO recommendation could be as a result of self-treatment. It is often observed in our setting where most patients treat themselves for malaria before presenting for themselves to the healthcare facility for diagnosis (Ameh et al., 2012). Hence, in their study they recorded false negative (FN) and false positive (FP). It is therefore important for the laboratorians to understand the likely causes of false negative results in malaria testing using malaria RDT, especially in an endemic setting like Nigeria. In malaria endemic areas like Nigeria the first line of diagnosis for most outpatients department is malaria is microscopic, but RDTs could be considered useful in most cases, however, the possibility of missing an individual infected with malaria may be risky and unacceptable.

However, the high positive predictive value 98.74% for CareStartTM AgPf/Pan and 98.10% for SD Bioline recorded in this study is higher than values obtained from previous studies which ranges from 64 to 90.2% (Ashton et al., 2010; Singh et al., 2010; Ameh et al., 2012) and this outcome provides an argument to the use of RDT as screening as the platform consider patients with a positive result as truly malaria infected patients and the clinicians can therefore proceed with the treatment (Ameh et al., 2012).

The evaluation described in this study has demonstrated that both CareStart and SD Bioline RDTs can be used as screening test for malaria diagnosis in a health based facility with laboratory unit, especially during the time of high transmission rate of malaria. The use of the RDT in a setting like ours (Nigeria) is useful to reduce the TAT of the malaria diagnosis. Indeed, laboratories in a resource constraint setting have to face the fact that reliable microscopy is not always available due to insufficient trained and experienced microscopists

In addition, the presence of only a single microscope that competes for several other microscopic examinations can be a limitation. Overall, while the gold standard for malaria testing remains microscopy, but the limitations associated with this technique could affect the speed of delivery of quality services to the patients. Hence, the RDT can become the first screening test for the diagnosis of malaria in the laboratory. However, the cost of RDT analysis can be a burden to patients in a setting like ours, but this can be averted by interventions from the government and non-profit organizations considering its contribution to patient’s care.

Recent studies have highlighted the variability in sensitivity and reliability of RDTs at both high and low levels of parasitaemia (Kim et al.,2013). Some pLDH tests showed sensitivities as low as 37.5% at parasitaemias <=1000 P/μL (Tagbo, 2007). The variability in sensitivity of these tests is of concern if early treatment intervention is to be based on diagnosis by RDT. Currently, the World Health Organization recommends a lower sensitivity limit of detection for a non-microscopic rapid diagnostic test for P. falciparum of 95% at a parasitaemia of 100P/μL (WHO, 2010). The development of such capability presents a challenge to scientists and product manufacturers. Some recent reviews have examined the technical limitations of malaria RDTs, and highlighted several shortcomings of current tests.

Finally, from the hypothesis tested, calculated value X2 of 44.7 was recorded at 95% level of confidence, with critical table value of 3.83, hence, the null hypothesis was rejected and the alternative accepted. The study proves that Microscopy is better than RDTs, in the diagnosis of Malaria.

5.2 Conclusion

This study showed that the efficacy and performance of CareStart Ag-Pf/pan and SD Bioline malaria Ag-Pf/pan. RDTs are rapid and simple to perform and the results can be interpreted by anyone or healthcare workers. CareStart Ag-Pf/pan and SD Bioline malaria Ag-Pf/pan is a useful test which has shown desirable results to alleviate the diagnostic challenges by many people who cannot access microscopic services for malaria diagnosis in Nigeria. The results of this study also indication that RDTs should be evaluated in each new setting before they are deployed, in view of possible variations in performance in different populations.

Overall, while the gold standard for malaria testing remains microscopy, but the limitations associated with this technique could affect the speed of delivery of quality services to the patients especially in endemic areas. RDTs will indisputably help to target malaria treatment in areas where microscopy is poor; on-going evaluations of field use accuracy and further studies on potential factors affecting the sensitivity and specificity of RDTs are proposed. Hence, the RDT can become a good screening test for the diagnosis of malaria in the laboratory. However, the cost of RDT analysis can be a burden to patients in a setting like ours, but this can be averted by interventions from the government and non-profit organizations considering its contribution to patient’s care.

5.3 Recommendations

Based on the findings, discussion, and conclusion of this study, it is imperative to highlight the relevance of malaria RDTs in diagnosing malaria, especially in endemic areas like Nigeria. The study therefore makes the following recommendations to improve on the use, understanding of RDTS in Nigeria, especially in tackling this dreadful disease called malaria.

  1. It is important to adopt a standard malaria testing algorithm for RDTs in the laboratory. Also that negatives malaria RDTs tests must be confirmed by microscopy before communication to the clinician, while positive results by RDTs can be reported without confirmation by microscopy.
  2. Malaria RDTs can be used for the diagnosis of malaria. As malaria is a multisystem disorder which pose major health threat and could mimic many diseases. Clinicians, especially those in endemic areas, should be aware of the varied manifestations and maintain a high index of suspicion for the disease so that the diagnosis and treatment are timely and morbidity and mortality minimized.
  3. Malaria RDTs could be used as alternative where microscopy services are not operational (for instance as a result of power failure, or in the evenings, weekends, and during public holidays) or as a primary diagnostic tool for rural and remote areas without microscopy services, but should not be regarded as a first-line diagnostic test.

5.4 Limitations of the Study

This study has several limitations. The data were collected as part of routine programmatic activities and were unlinked to patient records. Thus, it was impossible to collect clinical information, such as recent intake of antimalarials or the presence of other factors, which may have influenced the results. Another limitations is that the + system has been used to grade parasitaemia instead of a parasite count. This less accurate estimation may have influenced the results on sensitivity especially in the stratified analysis of parasitaemia levels. It is important to note that the described data were obtained in true field conditions, that is conditions that were suboptimal in terms of RDTs storage and handling and staff expertise, and non-standardized specimens may have been present. It is known that such factors influence RDTs performance, but it is difficult to optimize all these variables under difficult real-life conditions.

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