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    A, Central India. B, Central India showing the Jabalpur and Panna districts.

  • 1

    Nair CP, 1973. Malaria in Kashmir province of Jammu and Kashmir State India. J Commun Dis 5 :22–46.

  • 2

    Bloland BP, Ruebush TK, McCormick J, Ayisi DA, Boriga AJ, Oloo R, Beach W, Hawley LA, Nahlen B, Udhayakumar V, Campbell CC, 1999. Longitudinal cohort study of the epidemiology of malaria infections in an area of intense malaria transmission. I description of study site, general methodology and study population. Am J Trop Med Hyg 60 :615–640.

    • Search Google Scholar
    • Export Citation
  • 3

    World Health Organization, 2000. Malaria Diagnosis: New Perspectives. Report of a Joint WHO/US AID, Informal Consultation, October 25–27, 1999. Geneva: World Health Organization.

  • 4

    Singh N, Valecha N, 2000. Evaluation of a rapid diagnostic test, “Determine™ malaria Pf”, in epidemic-prone, forest villages of Central India (Madhya Pradesh). Ann Trop Med Parasitol 94 :421–427.

    • Search Google Scholar
    • Export Citation
  • 5

    Singh N, Sharma VP, Mishra AK, Singh OP, 1989. Bioenvironmental control of malaria in a tribal area of Mandla district Madhya Pradesh. Indian J Malariol 26 :103–120.

    • Search Google Scholar
    • Export Citation
  • 6

    Singh N, Singh OP, Sharma VP, 1996. Dynamics of malaria transmission in forested and deforested region of Mandla district, central India, Madhya Pradesh. J Am Mosq Control Assoc 12 :225–234.

    • Search Google Scholar
    • Export Citation
  • 7

    Singh J, Bhattacharji LM, 1944. Rapid staining of malarial parasites by a water soluble stain. Indian Med Gaz 79 :102–104.

  • 8

    Singh N, Saxena A, Sharma VP, 2002. Usefulness of an inexpensive, Paracheck® test in detecting asymptomatic infectious reservoir of Plasmodium falciparum during dry season in an inaccessible terrain in Central India. J Infect 45 :165–168.

    • Search Google Scholar
    • Export Citation
  • 9

    Tjitra E, Suprianto S, Dyer M, Currie BJ, Anstey NM, 1999. Field evaluation of the ICT malaria Pf / Pv immunochromatographic test for detection of Plasmodium falciparum and Plasmodium vivax in patients with a presumptic clinical diagnosis of malaria in eastern Indonesia. J Clin Microbiol 37 :2412–2417.

    • Search Google Scholar
    • Export Citation
  • 10

    Makler MT, Palmer CJ, Ager AL, 1998. A review of practical techniques for the diagnosis of malaria. Ann Trop Med Parasitol 92 :419–433.

  • 11

    Bulter D, 1997. Time to put malaria control on the global agenda. Nature 386 :535–541.

  • 12

    Marsh K, English M, Peshu N, Crawley J, Snow R, 1996. Clinical algorithm for malaria in Africa (letter). Lancet 347 :1327.

  • 13

    Craig MH, Bredenkamp BL, Williams CHV, Rossouw EJ, Kelly VJ, Martineau KA, Henry GFJ, 2002. Field and laboratory comparative evaluation of ten rapid malaria diagnostic test. Trans R Soc Trop Med Hyg 96 :258–265.

    • Search Google Scholar
    • Export Citation
  • 14

    Guthmann JP, Ruiz A, Priotto G, Kiguli J, Bonte L, Legros D, 2002. Validity, reliability and ease of use in the field of five rapid tests for the diagnosis of Plasmodium falciparum malaria in Uganda. Trans R Soc Trop Med Hyg 96 :254–257.

    • Search Google Scholar
    • Export Citation
  • 15

    Huong NM, Davis TME, Hewitt S, Huong NV, Uyen TT, Nhan DH, Cong LD, 2002. Comparison of three antigen detection methods for diagnosis and therapeutic monitoring of malaria: a field study from Southern Vietman. Trop Med Int Health 7 :304–308.

    • Search Google Scholar
    • Export Citation
  • 16

    Shukla MM, Singh N, Singh MP, Tejwani BM, Srivastava DK, Sharma VP, 1995. Cerebral malaria in Jabalpur, India. Indian J Malariol 32 :70–75.

    • Search Google Scholar
    • Export Citation
  • 17

    Price R, Nosten F, Simprson JA, Luxemburger C, Phaipum L, Kuile F, van Vugt M, Chongsupuajaisiddhi T, White NJ, 1999. Risk factors for gametocyte carriage in uncomplicated falciparum malaria. Am J Trop Med Hyg 60 :1019–1023.

    • Search Google Scholar
    • Export Citation

 

 

 

 

USEFULNESS OF A RAPID ON-SITE PLASMODIUM FALCIPARUM DIAGNOSIS (PARACHECK® PF) IN FOREST MIGRANTS AND AMONG THE INDIGENOUS POPULATION AT THE SITE OF THEIR OCCUPATIONAL ACTIVITIES IN CENTRAL INDIA

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  • 1 Malaria Research Centre Field Station, Regional Medical Research Centre Complex, Jabalpur, Madhya Pradesh, India

Logistic, economic, and technical factors limit rapid access to microscopic confirmation of malaria in many tropical countries, including India. The occurrence of high-grade fever and three deaths during the hot summer months in some forest migrants created an emergency situation in Jabalpur in central India. A cheap and rapid malaria test, Paracheck® Pf, was tested in this group of migrants in parallel with microscopy. The indigenous population at the site of occupational activities of these migrants approximately 250 km from Jabalpur was also screened by both methods. The results of this field investigation are very encouraging. Among migrants, the test had a sensitivity of 100% and a specificity of 67%. The positive and negative predictive values were 94% and 100%, respectively. Among indigenous population, the corresponding values were 100%, 97.3%, 98.4%, and 100%, respectively, indicating the usefulness of test as a diagnostic tool for providing on-site confirmation of symptomatic diagnosis of Plasmodium falciparum malaria.

INTRODUCTION

Malaria is a focal disease with multitudinous variations in its epidemiologic pattern in relation to the local situation. The unevenness of receptivity is determined by the location of areas in proximity to breeding sites of vectors, the living habits of the inhabitants, and the absence, presence, and degree of infection in different areas.1 Consequently, infection with Plasmodium falciparum can present with a wide spectrum of signs and symptoms, ranging from a fatal disease to an apparently asymptomatic infection, and from a rapidly progressing fulminate illness to a chronic condition.2 In areas where resources for malaria diagnosis are unavailable, malaria diagnosis is often made only on the basis of clinical symptoms, although this is alarmingly inaccurate.3 The detection of P. falciparum histidine rich protein-2 (HRP-2) in a blood specimen is one method by which expeditious diagnosis of infection with P. falciparum can be made on the spot in emergency cases with fairly high sensitivity and specificity.4

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.

MATERIALS AND METHODS

The study was conducted from April 28 to May 31, 2003 in two districts (Jabalpur and Panna) in central India (Madhya Pradesh) (Figure 1). 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.

Thirty-nine persons traveled from Jabalpur to Pawai in the Panna District, which is located approximately 250 km from Jabalpur, in the last week of March to collect Mahua (Madhuca indica), a flower used for making liquor. They stayed in this forest area approximately three weeks and slept under Mahua trees. These people returned to their homes on April 10. Within two weeks, nearly all (35 of 37) of these individuals developed a high fever and two of them died.

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 (100× 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 × TN) − (FP × FN)]/[(TP + FN)(TN + FP)]. Mixed infections with P. vivax and P. falciparum was treated as P. falciparum infections for the purpose of analysis.

RESULTS

This investigation was carried out in two phases: first among the migrants in Panagar, Jabalpur, and then at the site of infection in Pawai, Panna.

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.

A comparison of parasitemia versus test sensitivity showed that the Paracheck® Pf assay had a sensitivity of 100% for P. falciparum asexual parasitemias ranging from 120 to 300,000/μL, with a geometric mean ± SD parasite density (GMPD) of 1,021.33 ± 10.5/μL. The patient with a parasitemia of 300,000/μL died the next day. Follow-up of 30 cases on day 14 post-treatment showed that seven (23%) were positive by blood smear (GMPD = 563.1 ± 1.67/μL) and by the rapid diagnostic test. They were treated with sulfadoxine-pyrimethamine (1,500 mg of sulfadoxine and 75 mg of pyrimethamine) as per the National Anti-Malaria Program. In 13% (4 of 30) of these subjects, the results of the Paracheck® Pf assay remained positive although the corresponding smears were negative. On day 28, none of 30 was positive for malaria by either method.

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.

DISCUSSION

Malaria is a global problem responsible for nearly three million deaths each year (an average of one person every 12 seconds) and is increasing worldwide.10 In addition to these high numbers of malaria-attributable fatalities, it is believed that an additional 300–500 million people contract the disease each year11 with unmeasured impact on local economics, human health in general, and longevity. Case management of malaria is further problematic because of non-specificity in the presentation of malaria disease. Clearly, rapid and accurate diagnosis of malaria is urgently required to control malaria.

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.

Table 1

Comparison of the results of the Paracheck® Pf for malaria with those of conventional microscopy among migrants at the primary health center in Panagar, Jabalpur, India

Results of Paracheck® Pf
Results of microscopyPlasmodium falciparumNegativeTotal
P. falciparum, asexual ± sexual31031
Negative246
Total33437
Table 2

Comparison of the results of the Paracheck® Pf for malaria with those of conventional microscopy among the indigenous population at the primary health center in Pawai, Panna, India

Results of Paracheck® Pf
Results of microscopyPlasmodium falciparumNegativeTotal
P. falciparum, asexual ± sexual58058
P. vivax, asexual ± sexual088
Mixed (P. vivax plus P. falciparum)505
Negative12829
Total6436100
Figure 1.
Figure 1.

A, Central India. B, Central India showing the Jabalpur and Panna districts.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.26

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@yahoo.co.in.

Acknowledgments: We thank Orchid Bio-Medical Systems (Goa, India) for providing the Paracheck® Pf test.

REFERENCES

  • 1

    Nair CP, 1973. Malaria in Kashmir province of Jammu and Kashmir State India. J Commun Dis 5 :22–46.

  • 2

    Bloland BP, Ruebush TK, McCormick J, Ayisi DA, Boriga AJ, Oloo R, Beach W, Hawley LA, Nahlen B, Udhayakumar V, Campbell CC, 1999. Longitudinal cohort study of the epidemiology of malaria infections in an area of intense malaria transmission. I description of study site, general methodology and study population. Am J Trop Med Hyg 60 :615–640.

    • Search Google Scholar
    • Export Citation
  • 3

    World Health Organization, 2000. Malaria Diagnosis: New Perspectives. Report of a Joint WHO/US AID, Informal Consultation, October 25–27, 1999. Geneva: World Health Organization.

  • 4

    Singh N, Valecha N, 2000. Evaluation of a rapid diagnostic test, “Determine™ malaria Pf”, in epidemic-prone, forest villages of Central India (Madhya Pradesh). Ann Trop Med Parasitol 94 :421–427.

    • Search Google Scholar
    • Export Citation
  • 5

    Singh N, Sharma VP, Mishra AK, Singh OP, 1989. Bioenvironmental control of malaria in a tribal area of Mandla district Madhya Pradesh. Indian J Malariol 26 :103–120.

    • Search Google Scholar
    • Export Citation
  • 6

    Singh N, Singh OP, Sharma VP, 1996. Dynamics of malaria transmission in forested and deforested region of Mandla district, central India, Madhya Pradesh. J Am Mosq Control Assoc 12 :225–234.

    • Search Google Scholar
    • Export Citation
  • 7

    Singh J, Bhattacharji LM, 1944. Rapid staining of malarial parasites by a water soluble stain. Indian Med Gaz 79 :102–104.

  • 8

    Singh N, Saxena A, Sharma VP, 2002. Usefulness of an inexpensive, Paracheck® test in detecting asymptomatic infectious reservoir of Plasmodium falciparum during dry season in an inaccessible terrain in Central India. J Infect 45 :165–168.

    • Search Google Scholar
    • Export Citation
  • 9

    Tjitra E, Suprianto S, Dyer M, Currie BJ, Anstey NM, 1999. Field evaluation of the ICT malaria Pf / Pv immunochromatographic test for detection of Plasmodium falciparum and Plasmodium vivax in patients with a presumptic clinical diagnosis of malaria in eastern Indonesia. J Clin Microbiol 37 :2412–2417.

    • Search Google Scholar
    • Export Citation
  • 10

    Makler MT, Palmer CJ, Ager AL, 1998. A review of practical techniques for the diagnosis of malaria. Ann Trop Med Parasitol 92 :419–433.

  • 11

    Bulter D, 1997. Time to put malaria control on the global agenda. Nature 386 :535–541.

  • 12

    Marsh K, English M, Peshu N, Crawley J, Snow R, 1996. Clinical algorithm for malaria in Africa (letter). Lancet 347 :1327.

  • 13

    Craig MH, Bredenkamp BL, Williams CHV, Rossouw EJ, Kelly VJ, Martineau KA, Henry GFJ, 2002. Field and laboratory comparative evaluation of ten rapid malaria diagnostic test. Trans R Soc Trop Med Hyg 96 :258–265.

    • Search Google Scholar
    • Export Citation
  • 14

    Guthmann JP, Ruiz A, Priotto G, Kiguli J, Bonte L, Legros D, 2002. Validity, reliability and ease of use in the field of five rapid tests for the diagnosis of Plasmodium falciparum malaria in Uganda. Trans R Soc Trop Med Hyg 96 :254–257.

    • Search Google Scholar
    • Export Citation
  • 15

    Huong NM, Davis TME, Hewitt S, Huong NV, Uyen TT, Nhan DH, Cong LD, 2002. Comparison of three antigen detection methods for diagnosis and therapeutic monitoring of malaria: a field study from Southern Vietman. Trop Med Int Health 7 :304–308.

    • Search Google Scholar
    • Export Citation
  • 16

    Shukla MM, Singh N, Singh MP, Tejwani BM, Srivastava DK, Sharma VP, 1995. Cerebral malaria in Jabalpur, India. Indian J Malariol 32 :70–75.

    • Search Google Scholar
    • Export Citation
  • 17

    Price R, Nosten F, Simprson JA, Luxemburger C, Phaipum L, Kuile F, van Vugt M, Chongsupuajaisiddhi T, White NJ, 1999. Risk factors for gametocyte carriage in uncomplicated falciparum malaria. Am J Trop Med Hyg 60 :1019–1023.

    • Search Google Scholar
    • Export Citation
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