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| ABSTRACT |
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| INTRODUCTION |
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In this area of low transmission, treatment of malaria cases should ideally be based on biologic diagnosis because of the nonspecific nature of malaria symptoms,1 and the fact that infections with P. falciparum and P. vivax cannot be distinguished clinically, although different treatment is required. Detection of parasites in the blood by microscopy remains the most common method for the diagnosis of malaria in Colombia, but materials, supply lines, and trained staff are not sufficient in the isolated rural areas where MSF works nor easily applied in mobile clinics. Accurate malaria rapid diagnostic tests (RDTs) would greatly improve the quality of diagnosis and treatment of malaria in these remote settings.
Several rapid diagnostic test kits for malaria exist, which are fast, easy to perform, and can be carried out by relatively unskilled staff. The most commonly used tests for P. falciparum are based on the immuno-chromatographic detection of the histidine-rich protein-2 (HRP-2), a protein produced by asexual stages and young gametocytes of P. falciparum2 or of Plasmodium lactate dehydrogenase, pLDH.3 pLDH can be either species-specific antigens detecting P. falciparum or P. vivax or pan-malarial pLDH, detecting all four species of Plasmodium.4 In addition, there is another antigen, aldolase, which can detect all species of Plasmodium.5,6 The rapid tests we were interested in were (1) the Paracheck-Pf, a P. falciparum specific test, based on detection of parasite HRP-2, which has proven its accuracy and usefulness in many MSF-projects worldwide, (2) the Optimal-IT, a test that can detect P. falciparum as well as other Plasmodium species by Pf-specific PLDH and pan-malarial PLDH, and (3) the NOW malaria ICT, a test that combines Pf-specific HRP-2 with pan-malarial aldolase.
Most rapid tests have shown high accuracy in laboratory and field-based studies, though their sensitivity declines at low parasitemias (< 300500/µL).7,8 Test performance may vary for different geographical populations, levels of disease prevalence, and presence of different parasite species.9 It has been suggested that natural immunity in endemic areas may reduce the sensitivity, but this has not been proven.10 To determine the usefulness of RDTs in the specific situation of low-endemic, mixed P. falciparum and P. vivax malaria in southern American Colombia, we compared the diagnostic capacity of Optimal-IT and NOW Malaria ICT with the capacity of the MSF-standard, Paracheck test and that of expert microscopy, the latter considered as our gold standard. Additionally, the ease of use of the various tests was evaluated.
| MATERIALS AND METHODS |
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Patients. Patients of all ages with suspected malaria were recruited according to routine criteria of the health workers in the Malaria Center (i.e., fever or a history of fever and/or other complaints indicating a possible malaria infection). Persons who came for follow-up visits of an earlier episode of malaria or within 4 weeks after a (confirmed and treated) malaria infection were excluded. Patients were asked for their informed consent and when accepted, they had their blood sampled for blood slides and 3 RDTs. Patients whose results were positive for malaria (for any test) were treated according to the National Protocol with Amodiaquine + Sulfadoxine-Pyrimethamine + single dose Primaquine for P. falciparum and Chloroquine + 14 days Primaquine for P. vivax.
Sample size. The sample size was calculated assuming RDT sensitivities in the range of 7090%. A number of 140 positive patients had to be tested to reach a precision of 5% for a sensitivity of 90%, or 7% for a sensitivity of 80%, with alpha error = 0.05. For proper assessment of sensitivity of the Pf/Pv tests, this number was required for both P. falciparum and P. vivax. Applying similar calculations to the specificity, also 140 negative patients had to be tested. Recruitment was continued until the required number of P. falciparum patients (more rare than P. vivax or negative) was reached.
Data and sample collection. A patient form was filled with basic clinical and demographic information. The rapid test kits were opened only after the patient had been selected and interviewed by the medical staff. Capillary blood was collected by finger-prick, sampling a standard volume of blood for each test according to the manufacturers instructions, with the sampling device provided. Finger-pricking was repeated when needed to collect enough blood. Each selected patient had his/her blood examined by four methods: Optimal-IT, Paracheck-Pf, NOW malaria, and microscopy. The RDTs were compared by the bacteriologists scoring a list of issues on ease-of-use and other characteristics.
| RAPID DIAGNOSTIC TESTS |
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Rapid diagnostic tests were read by the same bacteriologist and confirmed by a second independent reader when needed, all according to the manufacturers instructions. The first person performed, read and recorded the results of the three tests and after that a second opinion was obtained from a second person reading again the same tests and recording the results. Each person read the RDT without knowing the result of the other reader or of the blood film. Results were compared and discussed to come to a consensus in case of different readings. At the end of this procedure, results were recorded on the patients individual record form.
Microscopy diagnosis. Two thick smears were taken on one slide and one thin smear on a separate slide. Thick smears were submerged in methylene blue for 1 second, washed with buffer solution and left to dry, thereafter stained horizontally with Field solution (one drop of solution A and one drop of solution B per 10 mL) in phosphate buffer B for 10 minutes, in accordance with nationally standard methods. Thin smears were fixed with methanol but not stained until necessary for species determination or better examination of the infection. Thick smears were evaluated by a well-trained, experienced microscopist, unaware of RDT results. A thick smear was considered negative if no parasites were seen in at least 200 fields. For positive smears, the number of parasites was counted in the number of fields needed to reach 200 white blood cells (WBCs) or 500 WBCs for low densities. Parasite density per µl was calculated assuming a standard of 8000 WBCs per µl of blood as per WHO guidelines.14 Presence of gametocytes or schizonts was also recorded. Thin smears were used for species verification.
Quality control. For internal quality control, a second independent reading was done by a different microscopist on about one third of the slides, especially low-density parasitemias and mixed infections. Slides with discordances between the two microscopists or between rapid tests and slide-reading (in terms of positivity and species determination), and a random sample of 20% of other slides, were sent to the University of Antioquia for external cross-checking. Disagreement results between the two were sent on to a third laboratory, of the National Health Institute in Bogota. In cases in which both reference laboratories agreed on one diagnosis different from ours, results were corrected accordingly.
The RDTs had a guaranteed history of proper storage (temperature 430°C, low humidity) and transport conditions, and were used within shelf life. Only tests from one batch were used.
Analysis.
The performance of Paracheck-Pf, NOW ICT Malaria, and Optimal-IT tests was expressed by calculating the sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV), for P. vivax and P. falciparum separately, taking microscopy results as the "gold standard". To assess the performance of the Optimal-IT test and NOW Malaria for diagnosis of P. vivax, cases with mixed infections with P. falciparum were excluded, because the pan-malaria antigen turns up positive due to P. falciparum infection. For performance on P. falciparum detection, mixed infections with P. falciparum were included. Slides with game-tocytes only were regarded as negative for further analyses. Data were analyzed in SPSS 12.0 (Chicago, IL) and Epi-info 6.04 (CDC, Atlanta, GA). Proportions were compared using the
2 test. Agreement (kappa statistic,
) between RDT and microscopy provided an estimation of the reliability of the RDT (
> 80% was considered as a measure of very good reliability).
Ethical considerations. The protocol was reviewed and approved by the Ethical Board of MSF (a committee of external experts) and the Ethical Board of the University of Antioquia and received approval from the National Institutes of Health, Bogotá. The provincial and local health authorities in Tierralta were informed of the plans of study and supported the study by spreading information to their staff in rural health centers and the local population. Selected patients or the caregivers of children under 15 years were asked for their informed, written consent. The patients in study were taken care of in exactly the same way as non-study patients, except for the few for whom an extra finger-prick was needed to collect enough blood.
| RESULTS |
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Rapid diagnostic tests versus microscopy.
The results of microscopy were in agreement in 93% of cases for Optimal-IT, 87% with NOW malaria ICT, and 98% of cases for Paracheck (P. falciparum only). Diagnosis by Optimal-IT gave 10 false positives (3 P. falciparum, 6 Pan-Plasmodium, and 1 Pf + Pan-Pl) and 46 false negatives (19 P. falciparum, 25 P. vivax, and 2 mixed). The NOW resulted in 47 false positives (44 P. falciparum, 1 pan-Pl, and 2 Pf + Pan-Pl) and 54 false negatives (4 P. falciparum and 48 P. vivax and 2 mixed). Paracheck gave 1 false P. falciparum and 15 false negatives (9 P. falciparum and 6 mixed). NOW and Paracheck gave a positive result for pure P. falciparum when microscopy indicated a pure P. vivax infection in four and three cases, respectively (Table 2
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values showed that for P. falciparum Paracheck was most reliable, with a
value of 0.92; the NOW test scored lower (0.71) and Optimal-IT intermediate (0.84). For non-P. falciparum, the
values of Optimal-IT and NOW were 0.91 and 0.84, respectively.
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| DISCUSSION |
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For P. falciparum detection the HRP2 test, the NOW malaria ICT, as well as Paracheck-Pf, appeared to be more sensitive than the pLDH test, Optimal-IT. The NOW test had a better capacity to detect lower density P. falciparum infections, but it gave a relatively high number of false-positive results (11% of all positives), so that its specificity and PPV were lower than that of the other two tests. For non-P. falciparum infections, here P. vivax, Optimal-IT (pLDH) was more sensitive than NOW malaria ICT (aldolase). Both Optimal-IT and NOW ICT revealed a relatively large number of P. vivax false negatives, missing respectively 9% and 17% of the infections. At lower P. vivax densities the tests performed less accurately than for P. falciparum. Doubtful, weakly colored test lines, found in positive as well as negative cases, were a problem encountered with NOW and Optimal-IT. Some of these, but not all, had low parasitemias.
The limitation of the study in this setting is the potential overestimation of the accuracy of microscopy. Hypothetically, the RDTs might be more sensitive than microscopy. If so, at least part of the 41 cases in which the NOW test indicated a P. falciparum infection as opposed to microscopy and the results of the other two RDTs, and the six cases in which Optimal-IT diagnosed P. vivax but microscopy, Paracheck, and NOW were all negative, might have been false-negative microscopy results rather than false-positive RDT results. Vice versa, some false-negative RDT results may have been false-positive microscopy results [e.g., slides read as very low density P. falciparum (N = 4), P. vivax (N = 23), or mixed infections (N = 2)] for which all three RDTs gave a negative result. Also, the few cases where microscopy detected P. vivax only and the RDTs indicated P. falicparum also (N = 5) may have been microscopy errors. We have applied maximum efforts to achieve expert reading in field conditions, with rigorous quality control procedures in place, such as double reading of difficult slides in our laboratory and blinded rereading of a considerable number of slides in two reference laboratories. Expert microscopy is judged by Moody to detect parasite densities down to 50 par/µl6 and remains the current universal gold standard, which is widely available.7,8 However, taking a blood sample on filter paper to confirm parasitemia by means of polymerase chain reaction can be considered for future studies.
Our results are in line with findings from other studies in areas of low to medium endemicity for malaria. Optimal was evaluated positively by most researchers but not all: in Latin America, sensitivities for P. falciparum averaged 82% (range 42100%) and for P. vivax 88% (65100), including studies from Colombia,1517 Honduras,4,18 Mexico,19 Peru,20 and Brazil;21 in Asia it showed about 87% (7994) sensitivity for P. falciparum and 80% (6595) for P. vivax (Afghanistan,22 Thailand,23 Pakistan,24 Kuwait8). The NOW test and its predecessor ICT Pf/Pv were reported to be very sensitive for P. falciparum, about 96% (range 89100) and a bit lower but acceptably sensitive for non-P. falciparum infections, about 87% (range 75100, Colombia [Mendoza and others, unpublished data], Indonesia,5 Thailand25,26). Paracheck Pf generally showed good diagnostic capacity, 96% (range 92100) in Thailand,27 Vietnam,28 and India.29
High-endemic versus low-endemic areas. Reports on the same rapid tests from high-endemic malaria areas are scarce, but they generally show a higher sensitivity: the older version of the NOW-test, ICT Pf/Pv 100% and Optimal 94% (Tanzania30), and Paracheck 97% (Uganda31). The hypothesis of Fryauff and others8that natural immunity against malaria might reduce the sensitivity of RDTsis not confirmed by this rough comparison. The main factor explaining the difference in sensitivity between high-endemic and low-endemic areas seems to be the parasite density. In our study group of symptomatic malaria patients, geometric mean parasite density was about 2300/µL for both species, whereas worldwide it is said to be 20,000 for P. vivax and 20,000 to 500,000 for P. falciparum.32 A total of 40% of P. falciparum infections and 38% of P. vivax infections had a parasite density below 2000 par/µl, and nearly 20% and 25% were below 500 par/µl. This proportion is higher than in high-endemic areas such as in Africa: in studies in DRC and Sudan (data from 3335) we saw that only 811% of P. falciparum infections of clinically ill children under 5 years were below 2000 par/µl. Hence, in areas of low and moderate malaria transmission, such as South America and Asia, rapid tests require a high sensitivity at lower densities of infection, to serve the non-immune populations that can suffer from clinical disease at much lower infection grades, as opposed to people in high-endemic areas in sub-Saharan Africa.
The PPV and NPV depend on the proportion of positive patients seen. The PPV reduces with lower prevalence, whereas the NPV increases.36 In the group of patients selected for study, 17% had a P. falciparum infection; however, of all patients visiting the Malaria Center in the period of study, only 10% were P. falciparum positive. This is higher than the annual parasite rates reported for Colombia.10 Health posts and mobile clinics where the RDT will be applied will probably see a lower positivity rate than in this specific Malaria Center where patients come for malaria diagnosis and treatment specifically. Thus, the PPV for P. falciparum of the NOW malaria ICT can be even lower than the 66% we reported here, related to a proportional increase in false positives among the few testing positive.
Implementation of rapid diagnostic tests. In Colombia, the tests that detect all Plasmodium species have an obvious added value above those detecting P. falciparum only. A test with HRP-2 for P. falciparum and pLDH for P. vivax detection would have given the best combined results, with both sensitivities over 90%; however, the tests available now combine HRP2aldolase (NOW ICT) and Pf pLDH-pan pLDH (Optimal-IT). The NOW test appears to be more sensitive for P. falciparum. It will however lead to more false- positive results. But if we accept some degree of overtreatment and prioritize P. falciparum over P. vivax, then NOW is the test of choice. The NOW test was considered easier to perform than the Optimal-IT, and as a card test is also very easy to read. The scoring of weak positive lines should be addressed in training.
In areas in Colombia where microscopy is in use and quality requirements of trained staff and proper equipment can be met, this is still the more accurate way to diagnose malaria in this zone of mixed Pf/Pv prevalence. The RDTs are quicker, but still far from perfect in the diagnosis of different Plasmodium species or mixed infections.
The disadvantage of the Pf/Pv combination rapid tests is that their price (US $2.5) is about 5 times more than the price of the Pf-only test (US $0.5), whereas microscopy is estimated to cost 0.12 to 0.40 US$ in endemic countries. The Colombian health system is privatized and health centers and hospitals often operate on a cost-recovery scheme; therefore a large proportion of the costs must be paid for by the patients themselves. RDTs should not replace microscopy in Colombia in areas where there is a good network of skilled technicians and where microscopy remains the best option. Nevertheless, RDTs will be a useful tool in remote, deprived settings.
Received April 8, 2006. Accepted for publication August 14, 2006.
Acknowledgments: The authors thank the Médecins sans Frontières (MSF) team in Tierralta who did all the work, and for input and feedback from other MSF staff in Monteria and Bogota. Support from Dr. Unni Karunakara and Dr. Ilse Broek, Health Advisors, MSF-Holland, Amsterdam was highly appreciated. We also thank Dr. Sylvia Blair and Dr. Jaime Carmona Fonseca of the University of Antioquia and Dr. Rubén Santiago Nicholls of the National Institute of Health, Bogotá, Colombia, for their help.
The authors have no conflict of interest to declare.
Financial support: The study was sponsored by MSF-Holland and its donors. The rapid tests were studied independently and purchased commercially from their manufacturers. The American Society of Tropical Medicine and Hygiene (ASTMH) assisted with publication expenses.
* Address correspondence to Ingrid van den Broek, Manson Unit, MSF-UK, 6774 Saffron Hill, EC1N 8QX London, UK, E-mail: Ingrid_vandenbroek{at}yahoo.com ![]()
Authors addresses: Ingrid van den Broek, Prudence Hamade, and Helen Counihan, Manson Unit, MSF-UK, 6774 Saffron Hill, EC1N 8QX London, UK, E-mail: Ingrid_vandenbroek{at}yahoo.com. Olivia Hill, Fabiola Gordillo, and Bibiana Angarita, MSF-Holland, Calle 37, 1664 Teusaquillo, Bogotá, Colombia. Jean-Paul Guthmann, Epicentre, 62 bis Boulevard Richard Lenoir, 75011, Paris, France.
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