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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Data of National Anti-Malaria Program (NAMP) indicated that in 1990 only three parasitologically confirmed cases of malaria were reported in this region. By 1995, the number of malaria cases had reached 26, and by 1998, the number of cases exceeded 1,400 (a 489-fold increase). Since there have been no systematic studies to determine the reason(s) for this steady increase in malaria infection, this fever outbreak from 1998 to 2000 was investigated to better understand malaria epidemiology and transmission dynamics in forest villages of Mohkhed Primary Health Center in Chhindwara.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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All study villages have a perennial stream running through them. The stream, with several seasonal tributaries, is characterized by the presence of rocky pools, pits, and seepages that criss-cross the villages and provides numerous breeding zones for Anopheles mosquitoes. The villages are located off the main road and are inaccessible during the rainy season. Primitive agriculture is the main activity on which the tribes of these villages subsist. A typical tribal house consists of a combined living room and kitchen. The doors are low and small, and windows are seldom present. Domestic animals (cattle, goats, and chickens) are sheltered in the houses. The villagers are scantly clothed and work primarily in fields, forest nurseries, road construction, and road maintenance.
Malaria surveillance. Active case detection was done by conducting door-to-door searches during each survey. Blood smears were prepared from all current fever cases and people who reported fever during the preceding 14 days. Blood smears were stained with Jaswant Singh and Bhattacharji (JSB)3 and examined in the field so that malaria treatment could be provided within two hours, according to the recommendations of the NAMP.4 Adults with P. vivax malaria were given 600 mg of chloroquine (CQ) as a single dose, followed by 15 mg of primaquine (PQ) a day for five days. Adults with P. falciparum malaria were given 1,500 mg of CQ over a three-day period (600 mg on day 1, 600 mg on day 2, and 300 mg on day 3) and 45 mg of PQ as a single dose. Children were given proportionately lower doses according to age. For children with P. falciparum malaria, the doses were 187.5 mg of CQ for those less than one year of age, 375 mg of CQ and 7.5 mg of PQ for those 1 to < 4 years of age, 750 mg of CQ and 15 mg of PQ for those 4 to < 8 years of age, and 1,125 mg of CQ and 30 mg of PQ for those 8 to < 14 years of age (CQ was administered in three divided doses). For children with P. vivax malaria, the doses were 75 mg of CQ for those less than one year of age, 150 mg of CQ and 2.5 mg of PQ for those 1 to < 4 years of age, 300 mg of CQ and 5 mg of PQ for those 4 to < 8 years of age, and 450 mg of CQ and 10 mg of PQ for those 8 to < 14 years of age. The same doses of PQ were administered over the next four days in each group. Infants less than one year of age and pregnant women were not given PQ. Pregnant women in this region generally do not allow blood to be obtained for preparation and examination of slides because they avoid medications. Similarly, most (>50%) of the women did not allow blood samples to be obtained from their infants. Spleen enlargement was determined by Hackett’s method in children between two and nine years of age.5
In vivo test.
Only patients with P. falciparum malaria (> 1 year of age with a parasite density 
1,000 asexual parasites per microliter of blood on a thick blood smear) with no history of antimalarial drug intake during the previous seven days were eligible for this study. Verbal informed consent was obtained from all adults or parents or guardians of children who participated in the study. Patients with severe signs or symptoms of malaria were excluded.6 Patients were initially given CQ (25 mg/kg of body weight) under supervision in three divided doses. The in vivo test was carried out according to the method of Rieckmann.7 Urine tests were conducted on day 2 to confirm the absorption of CQ. Thick blood films were stained with Giemsa. Asexual parasites were counted against 200 white blood cells assuming a leukocyte count of 8,000/µL of blood. Response to treatment was evaluated by the measurement of asexual parasitemias during the first 48 hours and then on day 7. Temperature and clinical symptoms were recorded at each follow-up visit. No follow-up was done after day 7. Blood samples could not be collected for measurement of CQ levels. Patients who failed treatment with CQ were treated with Fansidar® (F. Hoffmann La Roche, Basel, Switzerland) (three tablets containing 1,500 mg of sulfadoxine plus 75 mg of pyrimethamine) and 45 mg of PQ in a single dose. Children were given proportionately lower doses. The study protocol was reviewed and approved by the Institutional Review Committee of Malaria Research Centre, New Delhi and conducted in accordance with the Ministry of Health and National Anti-Malarial Treatment Policy Recommendations for in vivo drug efficacy testing.8 The study was also reviewed and approved by Institutional Ethics Committee of the Malaria Research Center in New Delhi.
Mosquito sampling. Indoor resting Anopheles mosquitoes inside four designated houses located in different parts of the village (two human dwellings and two cattle sheds) were sampled in the early morning (6:00 AM) for 15 minutes each by a team of two insect collectors with flashlights and mouth aspirators. All houses were treated with DDT in June and August each year. Anopheline mosquitoes were identified using taxonomic keys.9
Light trap catches (indoor-outdoor) were made in February, April, and July in four villages other than those selected for indoor resting collections. Traps were always placed at a constant height of 5.5 feet at fixed locations inside human dwellings and outside near occupied human dwellings (18 locations outside and 18 inside). Each trap was emptied manually at hourly intervals until morning. The human dwellings had no windows but did have many eaves, holes, and large cracks in the walls and roofs. All the houses in these experiments were occupied during the nights of observations. Mosquitoes collected were identified in the laboratory.
Anophelines attracted to animal baits were sampled from dusk to dawn at fixed stations (one inside the house and one outside the house) for 15 minutes per hour. Human bait catches were not done because of ethical reasons.
Vector and malaria parasite identification. An enzyme-linked immunosorbent assay (ELISA) for circumsporozoite protein was performed according to the method of Wirtz and others.10 Head and thoraces of An. culicifacies and An. fluviatilis were isolated and dried. These specimens were stored at -80°C until the ELISA could be done. The absorbances were read visually and at 414 nm 60 minutes after adding substrate. Anopheles culicifacies and An. fluviatilis were also dissected microscopically.
Retrospective data analysis.
Before beginning the investigation, malaria data for the last 10 years were obtained from the district malaria officer/Primary Health Centre Medical Officer (Table 1
). It was observed that spraying with DDT had been conducted in Chhindwara. However, from 1988 to 1997, the Mohkhed Primary Health Center had not been sprayed with DDT. From 1993 to 1997, entire region was not sprayed with any insecticide because the reported annual parasite incidence was less than 2. Since 1998, this district is part of the World Bank–Assisted Enhanced Malaria Control Program of the NAMP, which recommended the ending of regular spraying in most villages, and replacing it with one round of focal spraying whenever this was believed to be appropriate and early detection and prompt treatment of malaria. However, intensive intervention measures were undertaken after the outbreak of fever was reported, i.e., spraying of houses with DDT and prompt treatment to combat malaria infection.
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RESULTS |
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. Further analysis showed that both sexes had equal parasite rates in all age groups; thus, the data were pooled. Children between 1 and 4 years of age showed the highest rate of parasite positivity. A decrease in malaria prevalence occurred in those between the ages of 4 and 8 and 8 and 14. An additional decrease in malaria prevalence was seen in adults (> 14 years old). This decrease in adults was statistically significant when compared with that in children (
2 = 28.3, degrees of freedom [df] = 3, P < 0.0001). Although more P. vivax malaria was observed in children 1–< 4 years and more P. falciparum malaria was observed in children 8–< 14 years old, these findings were not statistically significant. Mixed infections with P. falciparum and P. vivax were seen in all age groups except those 1–< 4 years of age. Gametocytes of P. falciparum were observed in blood smears of 15%, 9%, 13%, and 15% of subjects 1–4, > 4–8, > 8–14, and > 14 years old, respectively. The differences in the prevalence of gametocyte carriers between age groups were not statistically significant (2 = 5.0, df = 3, P > 0.05).
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). The gametocyte rate was highest in November 1999 (16%) and lowest in August 2000 (5%). This difference was statistically significant (P < 0.05). Gametocytes of only P. falciparum were seen in 4.0% of the cases, with a minimum of 2.5% in February 1999 and a maximum of 7% in November 1999.
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). These results were not statistically significant. The geometric mean parasite density (GMPD) in infants was 1,956.7/µL (95% confidence interval [CI] = 1,222.06–2,691.36) for P. falciparum and 2,682.7/µL (95% CI = 1,542.8–3,822.57) for P. vivax. This difference was statistically significant (P < 0.0001). Among pregnant women, the prevalence of malaria in December 1998 (50%) was significantly higher (P < 0.05) than that in August 2000 (18%). Parity stratification showed that rate of malaria infection was identical in all three parity groups. Only two postnatal cases were found. Of which one was infected with P. falciparum, her two-day-old neonate, was negative for malaria parasites. None of the pregnant women harbored gametocytes of only P. falciparum. The GMPD in pregnant women was 2,411/µL (95% CI = 1,647.16–3,175.2) for P. falciparum and 4,733/µL, (95% CI = 901.35–8,565.3) for P. vivax. This difference was also statistically significant (P< 0.0001).
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Monitoring of CQ resistance by the in vivo test showed that four of 73 patients (5.4%) infected with P. falciparum showed a reduction of 25% or less in their asexual parasitemias during the first 48 hours of treatment, but they did not clear their parasitemias by day 7. This suggested infection with P. falciparum strains resistant to CQ at the RIII level. Two other patients (2.7%) showed a reduction of less than 75% in their parasitemias after 48 hours of treatment, indicating resistance to CQ at the RII level. Another 11% patients (who were aparasitemic after 48 hours) became parasitemic by day 7, indicating RI resistance. All of these patients were asymptomatic. The remaining patients who cleared asexual parasites by day 7 were not examined further and could either have harbored sensitive parasites or have had a delayed recrudescence (RI). Furthermore, age-specific analysis showed that only one patient with RI resistance and another with RII resistance were less than four years of age. Maximum resistance to CQ was seen in those 4–< 14 years of age (five with RI and four with RIII). Only six resistant cases were seen in those > 14 years of age (five with RI and one with RII).
Entomologic results.
The mean number of hand catches of indoor resting individuals (2,655) is shown in Table 5. These findings indicated the presence of four species of anophelines, of which two were known vectors (An. culicifacies and An. fluviatilis). The former species was the predominant species, accounting for more than 85% of all anophelines collected by hand catch, and the latter species accounted for more than 5% of the specimen collected. The other species found were An. subpictus and An. annularis. Most of these species were found breeding in streams, stream bed pools, rocky pits, and ditches.
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). Other species found in small numbers were An. fluviatilis, An. theobaldi, An. varuna, An. vagus, and An. splendidus. Analysis by trap location showed that although the numbers of An. culicifacies, An. fluviatilis, and other species obtained outdoors were higher than the number collected in all indoor collections, this difference was not statistically significant (by the chi-square test).
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Vector incrimination studies (indoor collections) showed that of 286 and 65 An. culicifacies and 56 and 53 An. fluviatilis collected in August 1999 and February 1999, respectively, only one An. culicifacies had salivary glands positive for sporozoites. This mosquito was captured in August 1999. Of 58 and 456 An. culicifacies and 10 and 4 An. fluviatilis captured in April 1999 and July 1999, respectively, none showed the presence of sporozoites of P. vivax or P. falciparum.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Age-specific analysis of the data indicated that all age groups showed a high positivity for malaria, particularly for P. falciparum malaria. A slightly decreasing prevalence with age was also seen, as was observed in Orissa12 and Assam.13 However, the gametocyte rate did not decrease with age. Infant positivity was detected in all seasons and in all surveys, indicating active transmission. However, a decrease was observed in August 2000 in all age groups when compared with December 1998. This decrease was most likely the result of intensive intervention measures. Interestingly, although the burden of malaria morbidity is high in this region, the sporozoite rates are rather low. The problem is compounded by the presence of a moderate level of resistance to CQ. Furthermore, it is possible that some recrudescent infections did not become apparent until after seven days, the cutoff value we used to estimate the RI level. The prevalence of P. falciparum malaria was so high during the study period that it is difficult to distinguish reinfection from recrudescence or relapse. By excluding these resistant cases, we may have underestimated the true incidence of CQ-resistant malaria in the population. The epidemiologic consequence of resistance can be assessed from the fact that in April 1999, during hot dry season (before the beginning of the monsoon season) when the prevalence of P. falciparum malaria would be expected to be virtually zero, 12% harbored gametocytes and 23% harbored trophozoites of P. falciparum. This is an impressive burden despite comprehensive programs of vector control, surveillance, and treatment. This finding is further supported by the fact that active transmission was not corroborated by the entomologic results (e.g., during April 1999, no infective mosquitoes were found). Therefore, the use of PQ to eliminate gametocytes should aid in reducing transmission. However, malaria has continued to flourish in this region.
Interestingly, of the two known vectors in this region (An. culicifacies2,14 and An. fluviatilis15), only An. culicifacies has been incriminated as a vector of malaria. Although only a small number of An. fluviatilis were identified, the results indicate that An. fluviatilis is unlikely to have played an important role in malaria transmission. Therefore, An. culicifacies, which was found in the highest proportions in all surveys, appears to be the primary vector, although it was also collected in large numbers from animal baits, indicating some degree of zoophilic behavior. Furthermore, the vectorial capacity of An. culicifacies is known to be very low.16 Therefore, the inhabitants of these communities would have been exposed to very few infectious bites during each malaria season. This finding is an epidemiologically important one for policy makers and program implementators.
The pattern of malaria in infants and pregnant women, the most vulnerable groups, suggests that individuals acquire some sort of protection against malaria. This is supported by the finding that many malaria infections in infants and pregnant women show a mild or nearly asymptomatic course, and that parasite densities were low. Fifty percent of all P. falciparum infections in infants and pregnant women had parasitemias less than 2,000 parasites/µL. The occurrence of asymptomatic infections with low asexual parasitemias has also been observed in infants and pregnant women in other parts of Madhya Prasesh.17 Since the natural history of malaria is highly influenced by the level of transmission, malaria-related morbidity based upon the parasite density must be specific for each geographic area. For example in Pakistan, where both P. vivax and P. falciparum are prevalent and An. culicifacies is the main vector, patients frequently have malaria morbidity associated with parasitemias less than 1,000 parasites/µL.18 This has also been observed in Vanuatu.19 Although there are limitations in the interpretation of our results, it is possible that these results may not represent all pregnancy outcomes in Chhindwara because of the small sample size. Thus, it was not possible to reach statistically valid conclusions. However, these results provide information that may be relevant to future studies.
Data collected by the Ministry of Health in Madhya Pradesh have shown that the number of parasitologically confirmed cases of P. falciparum malaria increased from 151 in 1990 to more than 9,000 in 2000 (a 60-fold increase). Furthermore, 22-fold increase in malaria incidence from 0.31 per 1,000 in 1990 to 6.75 per 1,000 in 2000 was also observed. However, no deaths were reported until 1996. Eight deaths due to malaria were reported in 2000.20 Malaria positivity consistently increased in a nearly linearly fashion during the study period in Chhindwara, as well as in the Mohkhed Primary Health Center. Underestimation of the malaria prevalence in this region resulted in inadequate vector control measures and an increased parasite reservoir. The situation was aggravated further between 1988 and 1997 because the Mohkhed Primary Health Center was not sprayed with insecticides. Between 1993 and 1997, Chhindwara was not sprayed with insecticides. In addition, insecticide susceptibility tests showed that An. culicifacies was resistant to DDT. The corrected percent mortality was only 25% on paper impregnated with 4% DDT after an exposure of one hour and a 24-hour post-recovery period.21 The use of mosquito repellents, coils, and electronic insecticide vaporizers were beyond the means of most residents of the forest villages, thus rendering these measures impractical.22 Insecticide-treated bed nets were not very successful among these individuals because of cultural reasons, i.e., outdoor sleeping, collection of forest produce, fishing, and hunting during hours when mosquitoes are active.23
Control of malaria in this region was based on two powerful tools: use of DDT and treatment with CQ. Their use is now very problematic. It is difficult to quantify the effect of a single factor or its importance in control of malaria. Indoor spraying requires large amounts of DDT for adequate coverage. However, resistance to insecticides is still increasing. The risks of DDT on human health and the environment have resulted in a reduction in spraying with DDT and an increase in the use of chemotherapy, thus changing the strategy from the vector control to malaria control.24,25 Nevertheless, resistance to CQ is also increasing.
The primary purpose of a malaria drug policy is to ensure prompt, effective, and safe treatment. Although many factors need to be considered in the development of a national anti-malarial drug policy, effective alternative drugs exist and can be used in a way that minimizes the selection pressure for drug resistance,26 thus delaying the emergence of resistance. Chloroquine and sulfadoxine-pyrimethamine are still very effective in most regions of India, and their usefulness can be extended by combination with artemisinin derivatives or other antimalarials. Therefore, we conclude that improved access to treatment facilities, combination therapy, and vector control using an effective insecticide appear to be the most promising methods for controlling malaria.
Received April 20, 2002. Accepted for publication November 14, 2002.
Acknowledgment: We thank Dr. S. K. Subbarao (Director of the Malaria Research Centre in New Delhi) for her help with many aspects of this study.
Authors’ address: Neeru Singh, A. K. Mishra, M. M. Shukla, and S. K. Chand, Malaria Research Centre, Field Station, Medical College Building, Jabalpur 482003, Madhya Pradesh, India, Telephone: 91-761-2370900, Fax: 91-761-2370935, E-mails: oicmrc{at}yahoo.com or nsmcr{at}hotmail.com
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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