Monitoring the efficacy of antimalarial medicines in India via sentinel sites: Outcomes and risk factors for treatment failure.

BACKGROUND & OBJECTIVES: To combat the problem of antimalarial drug resistance, monitoring the changes in drug efficacy over time through periodic surveillance is essential. Since 2009, systematic and continuous monitoring is being done through nationwide sentinel site system. Potential early warning signs like partner drug resistance markers were also monitored in the clinical samples from the study areas. METHODS: A total of 1864 patients with acute uncomplicated malaria were enrolled in therapeutic efficacy studies of artesunate plus sulphadoxine-pyrimethamine (AS+SP) for Plasmodium falciparum; those infected with P. vivax were given chloroquine (CQ). Polymerase chain reaction (PCR) was used to distinguish post-treatment reinfection from treatment failures. Isolates of P. falciparum were also analysed for dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) gene mutations. RESULTS: Overall, 1687 (91.7%) patients completed the follow-up. In most of the falciparum patients the parasitaemia was cleared within 24 h of treatment, except 12 patients who remained parasite positive after 72 h. Presence of dhfr and dhps quintuple mutation was observed predominantly in treatment failure samples. A daily dose of artesunate of < 3 mg/kg of body weight, age of <5 yr, and fever at enrolment were associated with an increased risk of treatment failure. The AS+SP in P. falciparum was effective in > 95% cases in all the sentinel sites except in Northeastern region (NE). Chloroquine remained 100% efficacious in case of P. vivax infections. INTERPRETATION & CONCLUSION: Till 2012, India's national antimalarial drug resistance monitoring system proved highly efficacious and safe towards first-line antimalarials used in the country, except in Northeastern region where a decline in efficacy of AS+SP has been observed. This led to change in first-line treatment for P. falciparum to artemether-lumefantrine in Northeastern region.

Impact of changing over of insecticide from synthetic pyrethroids to DDT for indoor residual spray in a malaria endemic area of Orissa, India.

BACKGROUND & OBJECTIVES: Development of insecticide resistance in malaria vectors has been a major problem for achieving effective vector control. Due to limited availability of insecticides, the only option is management of resistance by judiciously using the insecticides and rotating them to maintain their effectiveness. This study was carried out in a malaria endemic area of Sundergarh district in Orissa where synthetic pyrethroids (SP) were in use for the last couple of years. The change-over from SP to DDT was done in one arm of study, and the other two arms remained on SP and insecticide-treated nets (ITN). Entomological and parasitological monitoring was done to assess the impact. METHODS: The study design comprised of three arms (i) two rounds of indoor residual spraying (IRS) with DDT 1g/m(2) as a change-over insecticide in areas previously under synthetic pyrethroids; (ii) two rounds of IRS with synthetic pyrethroid (alphacypermethrin, ACM) @ 25 mg/m(2) ; and (iii) an unsprayed area under ITN/long lasting insecticide nets (LNs). Indoor residual spraying was undertaken under strict supervision to maintain quality and coverage. Contact bioassays were conducted to know the persistence of insecticide on sprayed surfaces and adult vector density was monitored in fixed and randomly selected houses. Malaria incidence was measured through fortnightly domiciliary surveillance under primary health care system in all the study villages. RESULTS: The insecticide susceptibility tests showed that An.culicifacies was resistant to DDT but susceptible to malathion and ACM. However, An. fluviatilis was susceptible to all the three insecticides. ACM was effective in killing An. culicifacies on mud and wooden sprayed surfaces and maintained effective bioefficacy ranging from 92 to 100 per cent up to five months, whereas DDT failed to achieve effective mortality in An.culicifacies. However, there was significant decline in the density of An.culicifacies in ACM and DDT areas in comparison to ITNs/LNs. There was 61 per cent reduction in the slide positivity rate in ACM area in comparison to 48 and 51 per cent in DDT and ITN/LNs areas, respectively. The adjusted incidence rate of malaria cases per 1000 population in three study areas also showed significant declines within each group. INTERPRETATION & CONCLUSIONS: The present findings show that the change-over of insecticide from synthetic pyrethroids to DDT brings about the same epidemiological impact as envisaged from continuing SP spray or distributing insecticide treated nets/long-lasting insecticidal nets provided there is a good quality spray and house coverage.

Artesunate-amodiaquine fixed dose combination for the treatment of Plasmodium falciparum malaria in India.

BACKGROUND: Artemisinin-based combination therapy (ACT) has been recommended for the treatment of falciparum malaria by the World Health Organization. Though India has already switched to ACT for treating falciparum malaria, there is need to have multiple options of alternative forms of ACT. A randomized trial was conducted to assess the safety and efficacy of the fixed dose combination of artesunate-amodiaquine (ASAQ) and amodiaquine (AQ) for the treatment of uncomplicated falciparum malaria for the first time in India. The study sites are located in malaria-endemic, chloroquine-resistant areas. METHODS: This was an open label, randomized trial conducted at two sites in India from January 2007 to January 2008. Patients between six months and 60 years of age having Plasmodium falciparum mono-infection were randomly allocated to ASAQ and AQ arms. The primary endpoint was 28-day PCR-corrected parasitological cure rate. RESULTS: Three hundred patients were enrolled at two participating centres, Ranchi, Jharkhand and Rourkela, Odisha. Two patients in AQ arm had early treatment failure while there was no early treatment failure in ASAQ arm. Late treatment failures were seen in 13 and 12 patients in ASAQ and AQ arms, respectively. The PCR-corrected cure rates in intent-to-treat population were 97.51% (94.6-99.1%) in ASAQ and 88.65% (81.3-93.9%) in AQ arms. In per-protocol population, they were 97.47% (94.2-99.2%) and 88.30% (80-94%) in ASAQ and AQ arms respectively. Seven serious adverse events (SAEs) were reported in five patients, of which two were reported as related to the treatment. All SAEs resolved without sequel. CONCLUSION: The fixed dose combination of ASAQ was found to be efficacious and safe treatment for P. falciparum malaria. Amodiaquine also showed acceptable efficacy, making it a suitable partner of artesunate. The combination could prove to be a viable option in case India opts for fixed dose combination ACT. CLINICAL TRIAL REGISTRY: ISRCTN84408319.

Distinct cytokine profiles define clinical immune response to falciparum malaria in regions of high or low disease transmission.

The immune effector response to Plasmodium falciparum infection involves a finely-tuned interplay between different cell types and cytokines. However, the processes by which they mediate the development of clinical immunity, in areas of different endemicity, are poorly understood. We analyzed circulating levels of pro-inflammatory (TNF, IFN-γ, IL-12, IL-16) and anti-inflammatory (IL-4, IL-10, IL-13) cytokines in control and patient groups drawn from a P. falciparum-endemic and a non-endemic region of India. The endemic region control population exhibited a lower pro- to anti-inflammatory cytokine ratio, indicating a shift towards a high basal Th2 response. Levels of IL-10 contributed most towards the region-specific difference in basal cytokine response. IL-10 was also the strongest predictor of disease in the endemic region, while IL-12, along with IL-10 and IL-6, contributed most to disease outcome in the non-endemic region. A low, mean IFN-γ/IL-10 ratio was associated with disease severity in the endemic region (p < 0.0001). In contrast, a low mean IL-12/IL-10 ratio correlated with disease outcome in the non-endemic region (p < 0.0001). In the endemic region, IL-13 correlated negatively with IFN-γ in severe patients (Spearman's ρ: -0.49; p : 0.013), while in the non-endemic region, IL-13 correlated negatively with IL-6 in severe malaria patients (Spearman's ρ: -0.485; p : 0.001). In conclusion, levels of pro- and anti-inflammatory cytokines and the relative balance between the Th1 and Th2 response, illustrates how populations residing in areas of varying disease endemicity may respond to P. falciparum-induced immune challenge.

Wash-resistance and field evaluation of alphacypermethrin treated long-lasting insecticidal net (Interceptor) against malaria vectors Anopheles culicifacies and Anopheles fluviatilis in a tribal area of Orissa, India.

A field trial was conducted on the efficacy of Interceptor nets-a long-lasting insecticidal net (LLN) factory treated with alphacypermethrin 0.667% (w/w) corresponding to 200mg/m(2), against malaria vectors Anopheles culicifacies and Anopheles fluviatilis in one of the highly endemic areas of Orissa. The study area comprised 19 villages which were randomized into three clusters and designated as Interceptor net cluster, untreated net cluster, and no net cluster. Baseline studies showed that both the vector species An. culicifacies and An. fluviatilis were 100% susceptible to alphacypermethrin. Results of wash-resistance and bio-efficacy of Interceptor nets showed 100% mortality in An. culicifacies and An. fluviatilis even after 20 washings. Bioassays on the Interceptor nets while in use in the field conditions showed a knockdown effect on 70-90% mosquitoes during different months of intervention after 3 min of exposure and 100% mortality was recorded after 24h of recovery period. The median knockdown time for these species ranged between 4.10-5.25 min and 4.00-5.00 min respectively during intervention period. In Interceptor net study area, there was a significant reduction of 88.9, 96.3 and 90.6% in the entry rate of An. culicifacies, An. fluviatilis and other anopheline species respectively with an over all reduction of 87.5% in total mosquitoes. The overall feeding success rate of mosquitoes in the trial villages was only 12.8% in comparison to 35.0 and 78.8% in villages with untreated nets and no nets respectively. A significant reduction was also recorded in parity rate and human blood index of vector species in Interceptor net area. The results of the study showed that Interceptor nets are effective against the malaria vectors and may be used as a suitable intervention strategy in high-risk areas.

Efficacy of permethrin treated long-lasting insecticidal nets on malaria transmission and observations on the perceived side effects, collateral benefits and human safety in a hyperendemic tribal area of Orissa, India.

Studies were conducted on the efficacy of Olyset nets-a long-lasting insecticidal net (LLIN) factory treated with 2% (w/w) permethrin on malaria transmission in an area under the influence of pyrethroid susceptible vector species Anopheles culicifacies and A. fluviatilis in Sundargarh District, Orissa, India. The study area comprised 22 villages that were randomized into three clusters and designated as Olyset net, untreated net, and no net area. Malaria incidence in the study population was measured through longitudinal active surveillance at fortnightly intervals. There was a reduction of 65-70% in malaria incidence in Olyset net area as compared to the control areas. The attack rate of Plasmodium falciparum or number of episodes per person per year in different age groups also showed significant reduction in Olyset net area as compared to untreated net and no net areas. Cross-sectional point prevalence surveys showed 45.7% reduction of malaria prevalence in Olyset net users, whereas there was an increase of 33.3% and 51% in untreated net and no net villages respectively. The compliance rate of Olyset net usage in the study population was 80-98% during different months, whereas it was between 70% and 90% for untreated nets. There were minimal complains of skin irritation (4%), itching (8%) and eye irritation (1.2%). However, these effects were only transitory in nature lasting for few hours of the first usage. Olyset nets also provided collateral benefits in terms of relief not only from mosquitoes and malaria but also from other household pests such as head lice, bed bugs, cockroaches, ants and houseflies. The Olyset nets were found to be safe to humans as no adverse event was recorded in the net users that can be attributed to the use of net. The study showed that Olyset nets are effective personal protection tool that can be used in a community based intervention programme.

CR1 levels and gene polymorphisms exhibit differential association with falciparum malaria in regions of varying disease endemicity.

Complement receptor 1 (CR1/CD35) levels on erythrocytes and related CR1 polymorphisms have been associated with response to falciparum malaria in populations inhabiting malaria-endemic regions. Differences in disease association profiles of its low expression alleles have been observed in populations from different regions of the world. We analyzed the influence of CR1 levels and associated SNPs on susceptibility/resistance to falciparum malaria in Indian populations. Two CR1 SNPs [exon 22 (A/G) and intron 27 (A/T)] define the low expression (L) CR1 allele in populations inhabiting a Plasmodium falciparum-endemic and a nonendemic region of India. Populations of the endemic region have very low red blood cell surface CR1 levels and higher frequencies of the exon 22 and intron 27 mutant L alleles. Whereas low CR1 levels correlated with susceptibility to severe malaria in the nonendemic region, high CR1 levels were associated with manifestation of disease in the endemic region. In addition, the exon 22 L allele was a risk factor for severe malaria in the nonendemic region. Absence of correlation between levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 with CR1 levels in patients with severe disease indicated that RBC CR1 levels in individuals are not the major determinants of pro-inflammatory cytokine release during infection. Our results are interpreted in the context of differences in the pathogenesis of severe malaria in the malaria-endemic and nonendemic region.

Therapeutic efficacy of artemether-lumefantrine in uncomplicated falciparum malaria in India.

BACKGROUND: Artemisinin-based combination therapy (ACT) is the treatment of choice for uncomplicated falciparum malaria. Artemether-lumefantrine (AL), a fixed dose co-formulation, has recently been approved for marketing in India, although it is not included in the National Drug Policy for treatment of malaria. Efficacy of short course regimen (4 x 4 tablets of 20 mg artemether plus 120 mg lumefantrine over 48 h) was demonstrated in India in the year 2000. However, low cure rates in Thailand and better plasma lumefantrine concentration profile with a six-dose regimen over three days, led to the recommendation of higher dose globally. This is the first report on the therapeutic efficacy of the six-dose regimen of AL in Indian uncomplicated falciparum malaria patients. The data generated will help in keeping the alternative ACT ready for use in the National Programme as and when required. METHODS: One hundred and twenty four subjects between two and fifty-five years of age living in two highly endemic areas of the country (Assam and Orissa) were enrolled for single arm, open label prospective study. The standard six-dose regimen of AL was administered over three days and was followed-up with clinical and parasitological evaluations over 28 days. Molecular markers msp-1 and msp-2 were used to differentiate the recrudescence and reinfection among the study subjects. In addition, polymorphism in pfmdr1 was also carried out in the samples obtained from patients before and after the treatment. RESULTS: The PCR corrected cure rates were high at both the sites viz. 100% (n = 53) in Assam and 98.6% (n = 71) in Orissa. The only treatment failure case on D7 was a malnourished child. The drug was well tolerated with no adverse events. Patients had pre-treatment carriage of wild type codons at positions 86 (41.7%, n = 91) and 184 (91.3%, n = 91) of pfmdr1 gene. CONCLUSION: AL is safe and effective drug for the treatment of acute uncomplicated falciparum malaria in India. The polymorphism in pfmdr1 gene is not co-related with clinical outcome. However, treatment failure can also occur due to incomplete absorption of the drug as is suspected in one case of failure at D7 in the study. AL can be a viable alternative of artesunate plus sulphadoxine/pyrimethamine (AS + SP), however, the drug should be used rationally and efficacy needs to be monitored periodically.

Variations in host genes encoding adhesion molecules and susceptibility to falciparum malaria in India.

BACKGROUND: Host adhesion molecules play a significant role in the pathogenesis of Plasmodium falciparum malaria and changes in their structure or levels in individuals can influence the outcome of infection. The aim of this study was to investigate the association of SNPs of three adhesion molecule genes, ICAM1, PECAM1 and CD36, with severity of falciparum malaria in a malaria-endemic and a non-endemic region of India. METHODS: The frequency distribution of seven selected SNPs of ICAM1, PECAM1 and CD36 was determined in 552 individuals drawn from 24 populations across India. SNP-disease association was analysed in a case-control study format. Genotyping of the population panel was performed by Sequenom mass spectroscopy and patient/control samples were genotyped by SNaPshot method. Haplotypes and linkage disequilibrium (LD) plots were generated using PHASE and Haploview, respectively. Odds-ratio (OR) for risk assessment was estimated using EpiInfotrade mark version 3.4. RESULTS: Association of the ICAM1 rs5498 (exon 6) G allele and the CD36 exon 1a A allele with increased risk of severe malaria was observed (severe versus control, OR = 1.91 and 2.66, P = 0.02 and 0.0012, respectively). The CD36 rs1334512 (-53) T allele as well as the TT genotype associated with protection from severe disease (severe versus control, TT versus GG, OR = 0.37, P = 0.004). Interestingly, a SNP of the PECAM1 gene (rs668, exon 3, C/G) with low minor allele frequency in populations of the endemic region compared to the non-endemic region exhibited differential association with disease in these regions; the G allele was a risk factor for malaria in the endemic region, but exhibited significant association with protection from disease in the non-endemic region. CONCLUSION: The data highlights the significance of variations in the ICAM1, PECAM1 and CD36 genes in the manifestation of falciparum malaria in India. The PECAM1 exon 3 SNP exhibits altered association with disease in the endemic and non-endemic region.

Building small dams can decrease malaria: a comparative study from Sundargarh District, Orissa, India.

The adverse health effect of environmental changes brought about with the construction of large and small dams has often been reported. Here, we present results of a 5-year (2001-2005) study documenting the positive effect of such developmental projects in reducing malaria in an area where malaria transmission is mainly due to the highly efficient anthropophagic vector Anopheles fluviatilis with some contribution from Anopheles culicifacies. The former breeds exclusively in the slow-flowing streams and the latter breeds in a variety of habitats. The study was conducted in San Dulakudar village and comparisons were made with two control villages situated near the stream with similar topography and malaria transmission pattern. Epidemiological data was collected through longitudinal weekly surveillance and cross-sectional surveys in all the study villages. The mean annual malaria incidence rates due to Plasmodium falciparum in children of 1-5 years age group during 2001 before construction of dam was 1304.3 and 785.7 cases/1000 population in dam site village and control villages, respectively. However, after construction of dam, there was gradual reduction in the malaria cases in dam site village and during 2005 the incidence was significantly reduced to 181.8 (P<0.01) whereas it was increased to 1000 in control villages without any significant change in comparison to baseline year (P>0.05). A significant reduction in malaria incidence and parasite rate was also recorded in all the age groups in dam site village without registering any significant change in control villages. The construction of a small dam in the study village altered the water flow above and below the dam thereby making it unfavourable for the breeding of A. fluviatilis which in turn brought about significant impact on malaria transmission.

Polymorphism and epitope sharing between the alleles of merozoite surface protein-1 of Plasmodium falciparum among Indian isolates.

BACKGROUND: The C-terminal region of merozoite surface protein-1 (MSP-1) is one of the leading candidates for vaccination against the erythrocytic stages of malaria. However, a major concern in the development of MSP-1 based malaria vaccine is the polymorphism observed in different geographical Plasmodium falciparum isolates. To explore whether the sequence heterogeneity of PfMSP-1 leads to variation in naturally acquired anti-MSP-119 antibodies, the present study was undertaken to study PfMSP-119 sequence polymorphism in malaria-endemic villages in eastern India and also carried out a competition enzyme-linked immunosorbent assay using three PfMSP-119 variant forms. METHODS: The sequence variations in the C-terminal region of PfMSP-119 were determined in a malaria endemic region. Three PfMSP-119 variants were produced in Escherichia coli (PfMSP119QKNG-L, PfMSP119EKNG-L and PfMSP119ETSR-F) and an immunodepletion assay was carried out using the corresponding patients' sera. RESULTS: Results revealed predominance of PfMAD20 allele among Indian field isolates. Seven PfMSP-119 variant forms were isolated in a singe geographical location. Three of PfMSP-119 variant forms when expressed in E. coli showed presence of cross-reaction as well as variant specific antibodies in malaria infected patient sera. CONCLUSION: The present study demonstrates the existence of allele specific antibodies in P. falciparum-infected patient sera, however their role in protection requires further investigation. These results thereby, suggest the importance of a multi-allelic PfMSP-119 based vaccine for an effective malaria control.

Pfcrt haplotypes and in-vivo chloroquine response in Sundergarh district, Orissa, India.

The Plasmodium falciparum chloroquine resistance transporter (Pfcrt) K76T mutation and haplotype (amino acids 72-76) were analyzed as markers of chloroquine (CQ) resistance in the blood samples of patients from two sites of different intensities of malaria transmission (high, n=70; low, n=68) in Sundergarh district of Orissa, India and correlated with the in-vivo response. Early treatment failure (ETF) was significantly more frequent in the high endemic area (32.9 vs. 7.4%, P<0.001), with children below 5 years suffering more. A high frequency of pfcrt K76T mutation was observed in both the areas (87.1 vs. 79.4%, P=0.22). Patients carrying pfcrt 76T were the most likely to develop ETF (odds ratio 36; 95% CI 3.35-1653.3; P<0.001). The ratio of 76T:K76 was 22:9 and 11:14, respectively, in high and low endemic areas (odds ratio 3.1; 95% CI 0.9-11.03; P=0.04), which may be used as a measure of drug pressure. Sequences of pfcrt codons 72-76 showed 16 of the CQ-resistant haplotypes to be SVMNT, 5 CVMNT and 12 CVIET. The CQ-sensitive haplotypes were mostly CVMNK in 10 samples; CVIEK in 2 samples. Both Southeast Asian and South American haplotypes were present, with the latter predominating.

Epidemiology of malaria transmission in forest and plain ecotype villages in Sundargarh District, Orissa, India.

A study of the epidemiology of malaria transmission was undertaken in 13 tribal villages located in forest and plain areas of Sundargarh District of Orissa state, India, from January 2001 to December 2003. In forest areas, intense transmission of malaria is attributed to the highly anthropophagic vector Anopheles fluviatilis sibling species S and is complemented by A. culicifacies sibling species C. In plain areas, A. culicifacies sibling species C is responsible for malaria transmission. The entomological inoculation rate in the forest and plain areas was 0.311 and 0.014 infective bites/person/night, respectively, during 2003. Malaria transmission is perennial both in forest and plain areas but is markedly low in the plain area compared with the forest area. Plasmodium falciparum accounted for 85.0% of the total malaria cases during the study period. In forest and plain areas, the number of P. falciparum cases per 1000 population per year was 284.1 and 31.2, respectively, whereas the parasite rate was 14.0% and 1.7%, respectively. In forest areas, clinical malaria occurs more frequently in children aged 0-5 years and declines gradually with increasing age. The study showed that villages in forest and plain areas separated by short geographical distances have distinct epidemiology of malaria transmission.

Epidemiology of malaria transmission and development of natural immunity in a malaria-endemic village, San Dulakudar, in Orissa state, India.

We describe the epidemiology of malaria in San Dulakudar, a village in Sundargarh District in the state of Orissa in eastern India. Malaria transmission is perennial with Plasmodium falciparum, accounting for greater than 80% of malaria cases. Transmission intensity varies with season with high transmission after the monsoon rains in autumn and winter, low transmission in summer, and intermediate transmission in spring. The anthropophagic mosquito Anopheles fluviatilis was identified as the main vector for malaria transmission. Based on observations of spleen rates and supported by data on malaria parasite prevalence and malaria incidence, San Dulakudar can be classified as a hyperendemic area for P. falciparum malaria. Parasite prevalence and malaria incidence rates decrease with age, suggesting that residents of San Dulakudar develop immunity to malaria. The study demonstrates the presence of regions in the Indian subcontinent such as Sundargarh District where P. falciparum is the primary cause of malaria and where malaria transmission rates are comparable to those found in many parts of Africa.

Assessment of therapeutic efficacy of chloroquine and sulphadoxine-pyrimethamine in uncomplicated falciparum malaria.

A standardised protocol has been developed by World Health Organization (CDS/RBM/2002) to assess the efficacy of common antimalarials in the treatment of clinically manifested infection with uncomplicated P. falciparum malaria for areas with low to moderate transmission. The therapeutic efficacy protocol is based on clinical and parasitological responses of the patients and it has the purpose of determining the practical efficacy of the drug regimen in study areas with the ultimate objective of ascertaining its continued usefulness or the necessity for replacing it in the routine treatment. Present study has been conducted at seven sites--Kathiatali and Simonabasti of District Nowgaon, Assam; Sonapur and Boko of District Kamrup, Assam; Keonjhar Town, Padampur and Basudebpur of District Keonjhar, Orissa. In order to reduce the patient recruitment time, health centre close to well-defined community was identified to conduct the activities at peak malaria season by selecting local pockets and organising mobile clinics. Microscopically confirmed cases of P. falciparum were enrolled according to the criteria for inclusion and exclusion. Treatment with recommended drug was given under supervision and a follow-up schedule at various intervals for 28 days was maintained. In chloroquine (CQ) study areas, wherever patients showed treatment failure, they were treated with second line drug--sulphadoxine-pyrimethamine (SP) combination and then followed-up as per study protocol. It was observed that 30% cases showed treatment failure to CQ in District Nowgaon, where revised drug policy has already been introduced. In Kamrup district, treatment failure with CQ was found to be less than 25%, which denotes the said regimen is still effective. Almost all the patients from Padampur and Basudebpur of District Keonjhar responded to CQ, treatment failure was noticed only in two patients (3%). The antifolate combination found to be fully effective as second line and also as first line wherever revised drug policy has been introduced.