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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In response to a report in the last week of April 2003 (extremely dry hot weather) that fever cases were occurring in some families in the village of Raipura (Panagar Primary Health Center) in the Jabalpur District of central India (Madhya Pradesh) resulting in two deaths, an emergency medical assessment team was sent to investigate the cause of the high fever rate and deaths. The Paracheck® Pf (Orchid Bio-Medical Systems, Goa, India) rapid diagnostic test was used in the field in a group of highly febrile patients clinically suspected of having malaria for which there was no epidemiologic evidence.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Jabalpur, with an area of 5,600 km2, is the third largest town in Madhya Pradesh (14% of the area contains forests). The population of this district is 2.1 million, 14.9% of which are various ethnic tribal groups. Malaria is low to mesoendemic in this region. The peak transmission seasons are July–January for P. falciparum and February–June for P. vivax.5,6 The village of Raipura (Panagar Primary Health Center) has a population of 2,000. The houses of 14 affected families were located on the sides of the roads and were accessible throughout the year.
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An emergency medical team arrived in Jabalpur on April 28. The medical team suspected malaria, although this is not the season for this disease in this part of central India because of the extremely dry weather. A finger prick blood sample was collected by a team of two field workers from all migrants after obtaining informed consent. This sample was used to prepare thick and thin blood smears and for testing with the Paracheck® Pf assay. Before staining, hemoglobin was removed by immersing the smears in water. All blood smears were stained with Jaswant Singh Battacharya (JSB) stain.7 Thin smears were fixed in methanol, immersed in JSB solution II (1 gram of eosin yellow in 500 mL of distilled water) for 4–5 seconds, rinsed briefly in phosphate buffer (0.79 grams of KH2PO4 and 1.0 grams of Na2HPO4 in one liter of distilled water), immersed in JSB solution I (0.5 grams of methylene blue, 3 mL of sulfuric acid, 0.5 grams of potassium dichromate, and 3.5 grams of disodium hydrophosphate in 500 mL of distilled water) for 30–40 seconds, and rinsed thoroughly in water to remove excess stain. After drying, the slides were read by an experienced technician in the field laboratory using an oil-immersion lens (100x magnification). This technician was unaware of the Paracheck® Pf assay results. To ensure that low-grade infections were not missed, each smear was examined for 15 minutes before a negative result was made. Simultaneously, the Paracheck® Pf assay was performed by field workers according to the manufacturer’s instructions, taking approximately 15 minutes to provide a result without reference to the results of the blood smear. Details of the Paracheck® Pf assay have been previously described.8 The blood smears were re-examined by a different technician at a reference laboratory. This technician was blinded to the results of the first reading to eliminate false-negative results or mixed infections with P. vivax and P. falciparum. The results of the second examination were considered final. Parasites were counted against 200 white blood cells and converted into parasites/microliter assuming an average of 8,000 leukocytes/µL. Simultaneously, blood samples from individuals with and without fever in neighboring houses were also collected for analysis by the rapid diagnostic method and microscopy to assess the prevalence of P. falciparum infection among the indigenous population.
Another medical team was sent to Pawai in the Panna District where they accessed occupational activities to obtain epidemiologic information on malaria in this region. The Panna District has an area of 7,100 km2 (57% of the area contains forests) and a population of 0.9 million, 15% of which are various ethnic tribal groups. The terrain in this district is highly undulating and inaccessible for three months during the rainy season. After the rainy reason, perennial streams and their tributaries form small water pools that serve as potential breeding sites for two potential mosquito vectors (Anopheles culicifacies and An. fluviatilis) during the rest of the dry season. The team surveyed three villages and blood samples were collected from all individuals with a fever for testing by the Paracheck® Pf assay and microscopy.
All persons who tested positive were treated with 1,500 mg of chloroquine, followed by 45 mg of primaquine, as per the guidelines of the National Anti-Malaria Program. Children were given proportionately lower doses. Pregnant women were not given primaquine.
Ethical clearance. The study was reviewed and approved by the ethics committee of the Malaria Research Center (Delhi, India).
Data analysis. The data were recorded and analyzed using SPSS version 10.0 statistical software (SPSS Inc., Chicago, IL). Once all samples had been tested, specificity, sensitivity, predictive values, and accuracy were estimated for the Paracheck® Pf assay using microscopy as the gold standard.9 Briefly, sensitivity was calculated as TP/(TP + FN), specificity as TN/(TN + FP), positive predictive value (PPV) as TP/(TP + FP), negative predictive value (NPV) as TN/(TN + FN), and accuracy as (TP + TN)/number of all tests, where TP = true positive, FN = false negative, TN = true negative, and FP = false positive. The J index, the overall measure of reliability of a diagnostic test was calculated as [(TP x TN) – (FP x FN)]/[(TP + FN)(TN + FP)]. Mixed infections with P. vivax and P. falciparum was treated as P. falciparum infections for the purpose of analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Panagar Primary Health Center (Jabalpur).
Blood samples were obtained from 37 of 39 migrants (two died). They ranged in age from 4 to 46 years old. Microscopic examination of thick films detected 31 P. falciparum-positive slides. The Paracheck® Pf assay detected all 31 patients that were positive for P. falciparum infection by microscopy, plus two additional cases. The results of parasite detection by microscopy and the Paracheck® Pf assay are compared in Table 1. Using microscopy as the gold standard, the Paracheck® Pf assay was found to be highly sensitive (100%) and specific (67%) for the diagnosis of P. falciparum infection, with a PPV of 94% and an NPV of 100%. The accuracy of the test was 95% and the J index was 0.7%. A survey of neighboring houses showed that none of 150 blood smears was positive for malaria by the rapid diagnostic test or by microscopy.
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Pawai Primary Health Center (Panna).
Of the 100 patients screened in three villages, 71 were smear positive, 58 for P. falciparum, 8 for P. vivax, and 5 for mixed infections with P. vivax and P. falciparum. The Paracheck® Pf assay detected 64 patients and included all P. falciparum infections or mixed infections with P. vivax and P. falciparum by microscopy (Table 2). Thus, the Paracheck® Pf assay detected one false-positive subject who was negative by microscopy. There was excellent concordance between microscopy and the Paracheck® Pf assay results. The sensitivity, specificity, PPV, NPV, and accuracy were 100%, 97.3%, 98.4%, 100%, and 99%, respectively. The J index was 0.97. The asexual parasite density for P. falciparum ranged from 120 to 890,000/µL with a GMPD of 1,676.72 ± 9.61/µL. The patient with a parasitemia of 890,000/µL died four hours later while going to the hospital. Mass surveys in these three villages by traditional microscopy showed that 30% of 450 blood smears showed infections with P. falciparum. Four of the individuals with these infections died.
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DISCUSSION |
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The results show that the Paracheck® Pf assay was highly sensitive, but not as specific, among migrants from Jabalpur. In this situation, sensitivity is more important then specificity because although treatable, malaria is potentially fatal.12,13 Furthermore, the test was used when some patients had already taken drugs, and it is therefore likely that the specificity of test would be increased if the evaluation had been conducted with subjects presenting symptoms without any history of drug intake, as in the Panna District. It should also be noted that because of the presence of fever in almost all patients, there were no cases in which the parasite density was < 120/µL, although the highest parasitemia was 890,000/µL. Since a considerable body of literature already exists covering these areas,14,15 they shall not be discussed further here.
Almost all of the migrants became ill on their arrival in Pawai in the Panna District, and their severe and lethal malaria was characterized by symptoms that could be fatal if not properly treated immediately. Therefore, prompt and accurate diagnosis of infection and initiation of appropriate treatment are essential to improve the outcome.16 Microscopic detection of parasites is a time-, labor-, and equipment-intensive procedure. The main benefit of rapid tests is that they are quick to perform and easy to interpret, and that they require no laboratory facilities.3 Not only will early and appropriate treatment of malaria reduce morbidity and the likelihood of developing life-threatening complications, but such treatment may also decrease the proportion of the population with gametocytes, thereby decreasing transmission rates.17
This malaria outbreak in Panna was one of the most severe ever recorded during the summer in central India. This demonstrates the local/focal nature of malaria, which further justifies the use of a rapid test because it requires only short training, even for unskilled individuals. In this situation, the rapid test clearly has an advantage over microscopy, since it does not depend on one person, but can be performed by all staff members of the health facility. A patient who must go to a hospital or private clinic because the microscopist is not on duty waste times and is at increased risk of a fatal outcome. In these circumstances, rapid tests are more useful and cost-effective than microscopy because the time factor is favored by a rapid test. However, we do not suggest that this HRP-2-based test should replace microscopy as a diagnostic tool. Microscopy, when operationally feasible, is the best method of malaria diagnosis.
In conclusion, in developing countries there are many individual and structural barriers to effective and timely treatment of malaria. Migrant laborers in malaria-endemic areas are at a particularly high occupational risk of acquiring malaria. Thus, it is very important to ensure that they should be screened for malaria on a priority basis by a rapid test because this test can play a key role in diagnosis of severe cases of P. falciparum malaria.
Received August 19, 2003. Accepted for publication July 7, 2004.
Acknowledgments: We thank Orchid Bio-Medical Systems (Goa, India) for providing the Paracheck® Pf test.
Authors’ address: Neeru Singh and Ajay Saxena, Malaria Research Centre Field Station, Regional Medical Research Centre Complex, Nagpur Road, Jabalpur 482003, Madhya Pradesh, India, Telephone: 91-761-237-2597, Fax: 91-761-237-0900, E-mail: oicmrc{at}yahoo.co.in.
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REFERENCES |
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