Informed decision-making before changing to RDT: a comparison of microscopy, rapid diagnostic test and molecular techniques for the diagnosis and identification of malaria parasites in Kassala, eastern Sudan
Corresponding Author Petra Mens, Royal Tropical Institute, Meibergdreef 39, 1105AZ Amsterdam, The Netherlands.
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Objectives Rapid diagnostic tests (RDTs) are promoted for the diagnosis of malaria in many countries. The question arises whether laboratories where the current method of diagnosis is microscopy should also switch to RDT. This problem was studied in Kassala, Sudan where the issue of switching to RDT is under discussion.
Methods Two hundred and three blood samples were collected from febrile patients suspected of having malaria. These were subsequently analysed with microscopy, RDT (SD Bioline P.f/P.v) and PCR for the detection and identification of Plasmodium parasites.
Results Malaria parasites were detected in 36 blood samples when examined microscopically, 54 (26.6%) samples were found positive for malaria parasites by RDT, and 44 samples were positive by PCR. Further analysis showed that the RDT used in our study resulted in a relatively high number of false positive samples. When microscopy was compared with PCR, an agreement of 96.1% and k = 0.88 (sensitivity 85.7% and specificity 100%) was found. However, when RDT was compared with PCR, an agreement of only 81.2 and k = 0.48 (sensitivity 69% and specificity 84%) was found.
Conclusion PCR has proven to be one of the most specific and sensitive diagnostic methods, particularly for malaria cases with low parasitaemia. However, this technique has limitations in its routine use under resource-limited conditions, such as our study location. At present, based on these results, microscopy remains the best option for routine diagnosis of malaria in Kassala, eastern Sudan.
Prendre des décisions éclairées avant de passer au TDR: Une comparaison de la microscopie, du test de diagnostic rapide et des techniques moléculaires pour le diagnostic et l’identification des parasites de la malaria à Kassala, dans l’est du Soudan
Objectifs: Les tests de diagnostic rapide (TDR) sont promus pour le diagnostic de la malaria dans de nombreux pays. La question se pose de savoir si les laboratoires où la méthode habituelle de diagnostic est la microscopie devraient également passer au TDR. Ce problème a étéétudiéà Kassala, au Soudan, où la question du passage au TDR est en cours de discussion.
Méthodes: 203 échantillons de sang ont été prélevés chez des patients fébriles suspectés de malaria. Ils ont été ensuite analysés par microscopie, TDR (SD Bioline Pf/Pv) et PCR pour la détection et l’identification des parasites du genre Plasmodium.
Résultats: Les parasites de la malaria ont été détectés dans 36 échantillons de sang lorsque examinés au microscope, 54 (26,6%) échantillons ont été trouvés positifs pour les parasites de la malaria par TDR et 44 échantillons étaient positifs par PCR. Une analyse supplémentaire a révélé que le TDR utilisé dans notre étude a menéà un nombre relativement élevé d’échantillons faux positifs. Lorsque la microscopie a été comparée à la PCR, une concordance de 96,1% avec un k = 0,88 (sensibilité: 85,7%, spécificité: 100%) a été trouvée. Toutefois, lorsque le TDR a été comparéà la PCR, une concordance de seulement 81,2% avec un k = 0,48 (sensibilité: 69%, spécificité: 84%) a été trouvée.
Conclusion: La PCR s’est avérée être l’une des méthodes les plus spécifiques et sensibles pour le diagnostic, en particulier pour les cas de malaria avec une parasitémie faible. Cependant, cette technique a des limites dans son utilisation de routine dans des conditions de ressources limitées, telles que dans notre lieu d’étude. À l’heure actuelle, sur base de ces résultats, la microscopie reste la meilleure option pour le diagnostic de routine de la malaria à Kassala, dans l’est du Soudan.
Mots-clés: malaria, diagnostic, Soudan, microscopie, TDR
Toma de decisión informada antes de cambiar a una prueba de diagnóstico rápido: Una comparación de microscopía, prueba de diagnóstico rápido y técnicas moleculares para el diagnóstico y la identificación de parásitos de malaria en Kassala, nororiente de Sudán
Objetivos: Las pruebas de diagnóstico rápidas (PDR) son promovidas como medio para el diagnóstico de la malaria en muchos países. La pregunta es si aquellos laboratorios en los que existe actualmente microscopía deberían también cambiarse a las PDR. Este problema ha sido estudiado en Kassala, Sudán, en donde se está discutiendo actualmente la cuestión sobre si cambiar o no a las PDR.
Métodos: Se recogieron 203 muestras de sangre de pacientes con fiebre y sospecha de malaria. Estos fueron analizados mediante microscopía, PDR (SD Bioline P.f/P.v) y PCR para la detección e identificación de parásitos de Plasmodium.
Resultados: Se detectaron parásitos de malaria en 36 muestras de sangre examinadas mediante microscopía, 54 (26.6%) muestras dieron positivas para parásitos de malaria mediante PDR, y 44 muestras dieron positivas por PCR. Análisis subsecuentes mostraron que las PDR utilizadas en nuestro estudio daban un número relativamente alto de falsos positivos. Cuando se comparó la microscopía con las PCR, se observó una concordancia del 96.1%, k=0.88 (sensibilidad del 85.7%, especificidad del 100%). Sin embargo, cuando se compararon las PDR con las PCR, se observó una concordancia de tan solo 81.2%, k=0.48 (sensibilidad 69%, especificidad 84%).
Conclusión: La técnica de la PCR ha demostrado ser uno de los métodos diagnósticos más específicos y sensibles, particularmente para los casos de malaria con baja parasitemia. Sin embargo, esta técnica tiene limitaciones en su uso rutinario bajo condiciones con recursos limitados, tales como el emplazamiento de nuestro estudio. Por el momento, y basándose en estos resultados, la microscopía continúa siendo la mejor opción para el diagnóstico rutinario de malaria en Kassala, Sudán.
Palabras clave: Malaria, Diagnóstico, Sudán, Microscopía, prueba de diagnóstico rápido.
Malaria causes between 7.5 and 10 million clinical cases and 35 000 deaths every year in Sudan (Elamin et al. 2005; Salah et al. 2006). Therefore, in this country just as any other malaria-endemic country, early diagnosis and effective treatment with an appropriate drug are essential and the main components of the World Health Organization’s strategy to reduce malaria-related mortality (WHO 2010). The gold standard for diagnosing malaria is demonstration of Plasmodium parasites in Giemsa-stained smears of peripheral blood. Expert microscopy is highly sensitive and specific and allows differentiation of the four malaria parasite species capable of infecting humans: P. falciparum, P. vivax, P. ovale and P. malariae (Rafael et al. 2006). However, microscopy may also have its limitations. Microscopic diagnosis can lead to a delay in making proper decisions on antimalaria drug treatment, in particular if many slides have to be examined during epidemics or at low parasitaemia. Furthermore, this method can sometimes be misleading in identifying Plasmodium species, especially in cases with low-level parasitaemia and mixed parasite infection.
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). Moreover, RDTs do not rely on specialized equipment or electricity and are therefore suitable for use in resource poor settings. However, the implementation of RDTs also faces many difficulties such as logistics of 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). In addition, RDTs 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). Molecular diagnosis, PCR in particular, has proven to be one of the most specific and sensitive diagnostic methods, particularly for malaria cases with low parasitemia or mixed infection (Barker et al. 1992; Brown et al. 1992; Tangpukdee et al. 2009). PCR was also more sensitive than various dipstick assays (Tham et al. 1999; Mens et al. 2007; Satoguina et al. 2009) but is, at the moment, not useful for routine diagnosis in many clinical settings because of technical requirements, expensive equipment and dependency on electricity.
Currently, many National Malaria Control Programmes are considering switching from microscopy to the use of RDTs for the routine diagnosis of malaria. This discussion is in part driven by the recommendation of the WHO to use RDTs for the diagnosis of malaria in areas where there is no or very poor microscopy available (WHO 2006, 2010). This study was undertaken to assess the quality of routine microscopy in Kassala (East Sudan) and to see whether RDT might be a suitable alternative for microscopy.
This study is therefore designed to aid local health authorities to make decisions on implementation of RDTs for routine diagnosis in the region. Its main objective was to compare the quality of routine expert microscopy to RDT performance and PCR for the detection and identification of Plasmodium parasites in patients from Kassala, eastern Sudan.
Study site, patients and ethical review
The study was carried out at EL Kuwaiti Hospital in Kassala, eastern Sudan. The Kassala area is mesoendemic for malaria and characterized by high seasonal malaria incidence during the rainy season from August to November. The area has a predominance of P. falciparum and P. vivax infection with an occasional P. ovale infection (Red Cressent 2003). Patients recruited into the study were selected based on the following criteria: suspected of uncomplicated malaria, having fever at the time of examination (temperature ≥ 37.5 °C) and willing to participate in the study. Pregnant women were excluded from participation. All participants were evaluated by physical examination and laboratory investigations.
The purpose of this work was explained to the health authorities at the federal Ministry of Health in Kassala, and the study was also discussed with the directors of the participating EL Kuwaiti Hospital. The study received ethical clearance from the Research Board at the Faculty of Medical Laboratory Sciences, University of Gezira, Sudan. Verbal consent for participation in the study was obtained from all participants and in the case of children from their guardian or parents. Verbal consent was taken because of the high illiteracy rate in the area.
Thick and thin blood films were prepared from venous blood collected in tubes containing EDTA. The slides were stained with Giemsa and screened for the presence of parasites and parasite species. Stained blood films were examined with (×100) oil immersion lens. The parasite density was determined by counting the parasites and leucocytes, assuming 6000 leucocytes per microlitre. Blood films were examined by an expert microscopist who was ‘blinded’ to the results of additional diagnostic testing. Smears were considered negative if no parasite was seen in 100 oil immersion fields, on a thick blood film. Parasite density was calculated by determining the number of parasites per 200 white blood cells (WBC) in a thick blood film. All the slides were double checked blindly by a second independent expert microscopist. There was no additional reading for the discrepant results between RDT, microscopy and PCR.
The RDT used in this study, purchased from Standard Diagnostics (SD Bioline P.f/P.v Biostandard Diagnostics, Gurgaon, Korea), is able to specifically detect P. falciparum and P. vivax. Plasmodium falciparum is detected by Histadine Rich Proteine-2 and P. vivax by the detection of vivax-specific lactate dehydrogenase. Five microlitres of whole blood was added to the card pad, and three drops of specific lyzing agent were added. The RDT result was read after 5 min according to the manufacturer’s instructions and immediately recorded.
The test was considered valid when there was a red control line on the immuno chromatographic test strip at the control line position. The test was considered positive if next to the control line, an additional line (red line number1: for P. falciparum, number 2 for P. vivax) was visible. If no line appeared at all, then the test was considered a test failure. The reader of RDT tests was blinded to the results of microscopy.
Blood samples were collected on Whatman Grade No. 3 paper and air-dried at room temperature until further use. Samples were shipped to the Netherlands where further PCR analysis was performed. DNA was isolated following the procedure described by Boom et al. (1990), which was followed by a generic duplex PCR for the detection of Plasmodium species on the 18s rRNA gene of the Plasmodium genus and human GAPDH gene as internal control for amplification.
The PCR was performed in a total volume of 25 μl comprising a mixture of 2 μl of extracted DNA solution, 4 mm MgCl2, 100 μm dNTPs, 200 μm dUTP, forward primer 5′-TCAGATACCGTCGTAATCTTA-3′, reverse primer 5′-AACTTTCTCGCTTGCGCGAA-3′, GAPDH forward primer 5′-GAA-GGT-GAA-GGT-CGG-AGT-C-3′, reverse primer 5′-GTT-CAC-ACC-CAT-GAC-GAA-CA-3′, 2% DMSO, 1 U/reaction UNG, 0.5 U/reaction Taq-polymerase and MQ water up to volume 25 μl per reaction. The following PCR protocol was employed: 10 min at 50 °C, 10 min at 95 °C followed by 40 cycles of 45 s at 94 °C, 45 s at 58 °C, 90 s at 72 °C and completed by 10 min at 72 °C. If there was no GAPDH amplification product visible on an ethidium bromide–stained gel, then DNA from that particular sample was isolated and processed again.
To identify the Plasmodium species present in the positive reactions as identified by the generic PCR, a species-specific PCR was performed as described by Snounou et al. (1993).
Data were recorded on individual case record forms and thereafter entered into Excel (Microsoft office) for analysis. To calculate sensitivity and specificity, 2 × 2 tables were made, and percentages were calculated as follows: specificity = TN/(TN + FP) and sensitivity = TP/(TP + FN). TN is true negatives (both reference and index test negative), TP is true positives (both reference test and index test positive), FP is false positive and FN is false negatives. The agreement between microscopy, RDT and conventional PCR was assessed using Epi Info version 6.04 (Centers for Disease Control and Prevention, Atlanta, GA). κ Values expressed the agreement beyond chance (Altman 1991) and were calculated with a 95% confidence interval (CI). A κ value of 0.21–0.60 is moderate, a κ value of 0.61–0.80 is good, and a κ value over 0.80 is an almost perfect agreement beyond chance.
This is a prospective, cross-sectional, hospital-based study. Six hundred patients were screened for eligibility for inclusion into the study. The population included in the study comprised 203 febrile patients, 100 (49.3%) men and 103 (50.7%) women, 144 (70.9%) adults (14 years old) and 59 (29.1%) children up to 14 years of age. The median age was 25 (SD: 14.5); the youngest child was 1 year and the oldest participant was 58 year old. Other symptoms and diseases that were frequently seen were malaise, headache, vomiting, restlessness and cough. Also, 107 cases of the study population (52.7%) were anaemic (Hb < 12.5 g/dl).
The majority (n = 167; 82.3%) of the patients included in the study were microscopically negative for malaria. Malaria parasites were detected in 36 blood samples (17.7%) when examined microscopically, 26 slides were identified as P. falciparum (12.8%), three were P. vivax (1.5%), six were P. ovale (3.0%), and one sample was mixed infection (P. falciparum and P. ovale) (0.5%). The geometric mean parasite count in the P. falciparum samples was 15 630 parasites/μl. (range: 1100 parasites/μl to 60 000 parasites/μl). The geometric mean parasitaemia in the P. ovale samples was 11 750 parasites/μl and of the P. vivax samples 6349 parasites/μl.
The result of RDT testing revealed the following: 54 samples were found positive, of which 45 (22.2%) were P. falciparum positive, 1 (0.5%) case was P. vivax, and 8 (3.9%) cases were mixed infections of P. falciparum and P. vivax. In total, two test failures were found, which were not repeated. Twenty-two RDT-positive samples gave the same results as the microscopy data. However, four P. falciparum–positive microscopy samples were found negative with RDT. The parasitaemia of all these four samples was above 6000 parasites/μl. Moreover, 20 microscopy-negative samples were found positive for P. falciparum infection with RDT employed. In addition, seven microscopy-negative samples were found to be RDT positive for P. vivax and P. falicparum. Of the six microscopically positive P. ovale samples, three were negative with RDT, but the other three were found to be RDT positive for P. falciparum. It is of interest to note that of the three microscopically P. vivax positive samples, two were negative by RDT, and one was identified by RDT as a P. falciparum infection.
All samples were successfully amplified by PCR as demonstrated by the presence of a GAPDH amplification control band in all samples. Forty-four samples (21.7%) were positive for Plasmodium when the generic pan-Plasmodium PCR was carried out.
When PCR was performed for Plasmodium identification, 28 samples (13.8%) were identified as P. falciparum, eight samples (3.9%) were P. vivax, one sample was P. ovale (0.5%), and none of the samples was positive for P. malariae. Nine samples were not successfully amplified in the species differentiation assay because of the small amount of DNA present.
Comparison of the tests
One hundred and thirty-six samples were found negative with all three tests employed. All 36 microscopy-positive samples were positive with PCR, and an additional eight samples were positive by PCR whilst being negative with microscopy (Table 1). Compared to RDT, 13 samples were PCR positive whilst RDT negative, but 25 RDT-positive samples were found negative with PCR (Table 2). With respect to sensitivity and specificity based on a positive or negative result regardless of the species, microscopy has a 100% specificity and 85.7% sensitivity (Table 1). Compared to PCR, the RDT is not performing well with an agreement of only 81.2%; κ = 0.48.
Table 1. Comparison between microscopy and PCR
Table 2. Comparison between RDTs and PCR
When species identification is taken into consideration, 22 microscopically identified P. falciparum samples were in concordance with PCR, and four were negative with microscopy for P. falciparum whilst being positive for this species with PCR. The three P. vivax samples were all positive with PCR, but two were identified indeed as P. vivax and one as P. falciparum. This last sample was also scored as P. falciparum positive with RDT. However, one sample that was considered positive for P. falciparum by both RDT and microscopy was identified as P. vivax by species-specific PCR (Table 3).
Table 3. Results of microscopy and RDT on samples identified as Plasmodium vivax positive by species-specific PCR
|Plasmodium falciparum||P. falciparum|
|Plasmodium ovale||P. falciparum|
|P. ovale||P. falciparum|
Three microscopically identified P. ovale samples that were negative with RDT were identified as P. falciparum with PCR. Two of these had a mixed infection with P. vivax, and two microscopic P. ovale samples contained P. vivax mono infection. When these six samples are used in a comparison between RDT and PCR, no concordance at all could be found (Table 4). Although microscopical suspicion for P. ovale was found in 3.0% of all the included cases, only one of the samples showed this in PCR.
Table 4. Results of PCR and RDT resting on the microscopically identified Plasmodium ovale samples
|P. falciparum & P. vivax||Failed||Failed|
Although microscopy is still considered the gold standard for diagnosis of malaria, many countries consider shifting their diagnostic policy for malaria to implementing RDT as standard diagnosis. Sudan has chosen to recommend through its National Malaria Control Programme implementation of RDTs in those settings where no expert microscopy is available, and maintain microscopical examination in those places where microscopy is of adequate level. This strategy, which is supported by WHO (WHO 2010), originates from the fact that in many areas, proper microscopy is not possible because of lack of electricity or equipment, although electricity issues can now be solved by the use of solar rechargeable LED microscopes (Kuhn et al. 2010), or is facing quality issues (improper staining or limited expertise of the microscopist). In other areas, another reason is that the incidence of malaria is at such a low level that keeping expertise at a sustainable level is getting difficult.
Several studies have shown that in some hospitals in Sudan, malaria diagnosis is of poor quality. For example, a study performed among pregnant women in Medani Maternity Hospital showed that only 21 (8.6%) of 243 pregnant women admitted as malaria cases after an initial microscopic test by general microscopists were actually blood film positive for malaria when retrospective examination of the slides was performed by expert microscopists (Elhassan et al. 2010).
Malaria RDTs of good quality can, when appropriately used, provide a rapid and reliable way to demonstrate the presence or absence of malaria parasites at all levels of the health service. RDTs are thus considered a good alternative for microscopy but, owing to its own limitations, need to be carefully considered when implemented. In this study, we examined the quality of microscopy of a specific hospital in Kassala to establish whether this laboratory should also switch to RDTs.
The results of the RDT in this study were disappointing. The sensitivity and specificity of the RDT were 77.8% and 84.9%, respectively, when compared with microscopy, and when the RDT results were compared with PCR, the sensitivity and specificity were 69% and 84.5%, respectively. Thirteen negative samples were considered positive with RDT and would thus lead to unnecessary treatment, whereas 25 were missed that could potentially lead to under or no treatment. Our findings differ from those of a study carried out in Wad Medani, Sudan, which showed that RDT was sensitive for the diagnosis of acute P. falciparum malaria infection with a parasitaemia more than 100 parasites/μl blood (Nour et al. 2009).
The importance of implementing and testing a good-quality RDT may also play a role. The RDT employed in this study was P.f/P.v from Biostandard Diagnostics, Gurgaon, Korea. This RDT together with four other RDTs for malaria diagnosis from this company have recently been tested in the WHO/FIND evaluation with differing results: three were highly sensitive for P. falciparum and one had poor sensitivity. The fifth RDT was not able to detect P. falciparum and was not tested for this species. This test was performed together with 3 others including the test used in this study for the detection of P. vivax, and they showed differing results from very good to very poor. However, the P.f/P.v test used in the current study was very sensitive and specific in the WHO/FIND evaluation (WHO 2010). Interestingly, the tests performed in Wad Medani that used the Paracheck test to detect HRPII (Lot No. CB041A; Orchid Biomedical System, Verna/Goa, India) and the optiMAL test to detect PLDH (Batch No. 41670.11.1; DiaMed AG, Cressier, Switzerland) do not score so well in the WHO/FIND evaluation (WHO 2010). Another interesting observation is that the SD bioline P.f/p.v also gave varying results in other studies. In a study carried out in Nigeria, the test also gave a low (47%) sensitivity (Jeremiah et al. 2007), whereas in a non-endemic setting it performed very well (Gillet et al. 2009). Transmission intensity is known to influence the sensitivity and specificity, so this could be an explanation for the observed differences.
Another issue explaining the conflicting results could be the storage and handling of the test, although all care was taken to ensure that the RDTs used in the current study were stored well. These findings highlight that it is essential to carefully evaluate the RDT that is planned to be implemented in the setting that you want to implement it in. Measures to ensure adequate and proper quality control must be undertaken. Otherwise, the implementation of RDTs may not be beneficial.
The National Malaria Control Programme of Sudan does not have a shortlist of adequately tested and good-quality RDTs for recommendation to its health facilities at the moment. Most health services do their own procurement and decide on the RDT they want to use. Centralizing these decisions by providing a recom-mendation and providing quality control services via NMCP for good-quality RDTs may avoid an abundance of potentially poor-quality RDTs in health centres, which would not improve diagnostics but may make quality of services worse.
Molecular biological techniques are appropriate for research laboratories and are useful for species identification as well as for quantifying parasite density in clinical samples with low parasitemia. Implementing molecular diagnostics is at present no option for the routine diagnosis of malaria in a district hospital in a place like Kassala. However, analysis with PCR has shown that (i) microscopy detection was of good quality in this hospital but (ii) species identification was difficult. P. ovale was diagnosed by the microscopist in a few cases, but molecular analysis showed that this was actually P. vivax infection. In conclusion, these observations stress the need for continuous monitoring of the quality of the microscopy and training of the operators (in particular in species identification) to assure and maintain the current quality. A switch to RDT should be considered only when microscopy is absent or very poor.
This work was supported by The Ministry of Higher Education and Scientific Research, in Sudan. The authors are very grateful to the patients who participated in the study; also they thank the local heath authority in Kassala State, Sudan. In addition, we are very grateful to Dr Henk Schallig for his hospitality at the Royal Tropical Institute, Amsterdam, the Netherlands, to facilitate the molecular work and his support and advice on the manuscript.