Durability, household usage and washing pattern of DuraNet© and Interceptor® long-lasting insecticidal nets in long-term field trials in India.

BACKGROUND & OBJECTIVES: Universal coverage of population with long-lasting insecticidal nets (LLINs) living in endemic areas inhabited by ethnic tribal communities or in difficult to reach areas was found effective for disease control where other interventions such as indoor residual spraying have limited success because of operational issues. Evaluation of different LLINs with varied insecticides and fabrics are being evaluated to meet the demand of new products. This study was undertaken on two brands of LLINs, DuraNet© and Interceptor® that varied in fabric and manufacturing technologies to assess the usability in field conditions for atleast three years. METHODS: In large-scale field trials DuraNet©, alpha cypermethrin incorporated polyethylene net, was evaluated in Odisha state while, Interceptor® net, alpha cypermethrin coated polyester net was evaluated in Chhattisgarh and Gujarat states for a period of three years following WHO guidelines. Durability, usage pattern and washing behavior were monitored through periodic surveys and physical examination of nets. RESULTS: Survivorship of both the nets was 84-100% and every night usage rate was >62% in all seasons. Washing frequency was largely within the prescribed limits. The proportion of DuraNet© with holes was 26.7% in year one 74% in year three. In Gujarat, proportion of Interceptor® nets with holes at six months was 33.3% increased to 87% in year three and in Chhattisgarh, 6.7% after six months to 93.3% after three years of use respectively. INTERPRETATION & CONCLUSION: Both the nets revealed a useful life of three years under the field condition.

Burden of asymptomatic malaria among a tribal population in a forested village of central India: a hidden challenge for malaria control in India.

OBJECTIVE: Chhattisgarh in India is a malaria-endemic state with seven southern districts that contributes approximately 50-60% of the reported malaria cases in the state every year. The problem is further complicated due to asymptomatic malaria cases which are largely responsible for persistent transmission. This study was undertaken in one of the forested villages of the Keshkal subdistrict in Kondagaon district to ascertain the proportion of the population harbouring subclinical malarial infections. STUDY DESIGN: Community-based cross-sectional study. METHODS: Mass blood surveys were undertaken of the entire population of the village in the post-monsoon seasons of 2013 and 2014. Fingerprick blood smears were prepared from individuals of all ages to detect malaria infections in their blood. Individuals with fever at the time of the survey were tested with rapid diagnostic tests, and parasitaemia in thick blood smears was confirmed by microscopy. Malaria-positive cases were treated with anti-malarials in accordance with the national drug policy. RESULTS: Peripheral blood smears of 134 and 159 individuals, including children, were screened for malaria infection in 2013 and 2014, respectively. Overall, the malaria slide positivity rates were 27.6% and 27.7% in 2013 and 2014, respectively, and the prevalence rates of asymptomatic malaria were 20% and 22.8%. This study showed that, for two consecutive years, the prevalence of asymptomatic malaria infection was significantly higher among children aged ≤14 years (34.4% and 34.1% for 2013 and 2014, respectively) compared with adults (15.2% and 18.2% for 2013 and 2014, respectively; P = 0.023 and 0.04, respectively). CONCLUSION: The number of asymptomatic malaria cases, especially Plasmodium falciparum, is significant, reinforcing the underlying challenge facing the malaria elimination programme in India.

Comparison of three PCR-based assays for the non-invasive diagnosis of malaria: detection of Plasmodium parasites in blood and saliva.

The conventional molecular diagnosis of malaria uses 18S rRNA-based PCR assay employing blood samples. This assay presents limitation in terms of long turnaround time and increased chances of false-positive results. Here, we evaluated one-step singleplex or multiplex PCR assay based on high copy species-specific consensus repeat sequences (CRS) along with standard 18S rRNA nested PCR (18S n-PCR) assay to detect P. falciparum and P. vivax infection using blood and saliva samples from Indian febrile patients. Out of 327 patients, 187 were found to be positive for malaria parasites by microscopic examination of peripheral blood smears. Among these 130 were P. vivax and 57 were P. falciparum cases. The 18S n-PCR assay and CRS PCR assay identified 186 out of 187 cases (99.4 %). Multiplex CRS PCR assay detected Plasmodium in 176 out of 187 cases (94.1 %). Both singleplex and multiplex CRS PCR assay identified 6 mixed infection cases, while 18S n-PCR assay detected 10 mixed infection cases of P. vivax and P. falciparum, which were not recognized by microscopy. Non-invasive Plasmodium detection rate with DNA derived from saliva samples was highest for 18S n-PCR (87.36 %), followed by singleplex CRS (81 %) and multiplex CRS PCR assay (70.5 %). Specificity for P. vivax and P. falciparum detection for all assays was 98.48 % and 100 % respectively. Detection rate for P. vivax in saliva correlated with parasite density for CRS target-based assays. The species-specific CRS PCR, either as a singleplex or multiplex assay, can have an impact on diagnosis and epidemiological studies in malaria.

Standardization of a bottle assay--an indigenous method for laboratory and field monitoring of insecticide resistance and comparison with WHO adult susceptibility test.

The WHO adult susceptibility test is in use for insecticide resistance monitoring. Presently, materials are being imported from the Universiti Sains Malaysia, Malaysia and sometimes it is cost prohibitive. As an alternative, we present here a method of bottle bioassay using indigenous material. Different aspects related to the assay were studied and validated in the field. Bottle assay was standardized in the laboratory by using locally sourced material and laboratory-maintained insecticide-susceptible Anopheles stephensi and Aedes aegypti strains against technical grade deltamethrin and cyfluthrin insecticides dissolved in ethanol in a range of different concentrations. The frequency of use of the deltamethrin-coated bottles and shelf-life were determined. Discriminating dose for deltamethrin and cyfluthrin was 10 µg against An. stephensi and 2 µg against Ae. aegypti females. Insecticide-coated bottles stored at 25 to 35 °C can be used for three exposures within 7 days of coating. The study carried out in the laboratory was validated on wild caught An. culicifacies in the states of Odisha and Chhattisgarh against deltamethrin-coated bottles in comparison to WHO adult susceptibility test. Results of the study indicated that deltamethrin-coated bottles were effective up to three exposures within 7 days of coating for field population and 100% mortality was recorded within 35 min as observed in laboratory studies for field collected susceptible population. Also in the WHO adult susceptibility test, 100% knock-down within 35 min and 100% mortality after 24 h holding period were observed in susceptible population, while in it was 50% knock-down in 1 h and 64% mortality after 24 h holding period for resistant population (50% mortality in bottle assay in 60 min). The bottle assay can be used as an alternative to the WHO adult susceptibility test both in the laboratory and field for monitoring insecticide resistance in mosquito vectors using locally sourced material.

Genetic profiling of the Plasmodium falciparum population using antigenic molecular markers.

About 50% of malaria infections in India are attributed to Plasmodium falciparum but relatively little is known about the genetic structure of the parasite populations. The molecular genotyping of the parasite populations by merozoite surface protein (msp1 and msp2) and glutamate-rich protein (glurp) genes identifies the existing parasite population in the regions which help in understanding the molecular mechanisms involved in the parasite's drive for survival. This study reveals the genetic profile of the parasite population in selected regions across the country with varying degree of endemicity among them. We also report the prevalence of Pfcrt mutations in this parasite population to evaluate the pattern of drug resistance development in them.

Monitoring antimalarial drug resistance in India via sentinel sites: outcomes and risk factors for treatment failure, 2009-2010.

OBJECTIVE: To describe India's National Antimalarial Drug Resistance Monitoring System, measure the efficacy of first-line malaria treatments, and determine risk factors for treatment failure. METHODS: In 2009-2010, prospective studies with 28 days of follow-up were conducted at 25 sentinel sites. Patients infected with Plasmodium falciparum were given artesunate plus sulfadoxine-pyrimethamine (AS+SP); those infected with P. vivax were given chloroquine. Polymerase chain reaction was used to distinguish post-treatment reinfection from treatment failure. Isolates of P. falciparum were checked for dhfr and dhps mutations. FINDINGS: Overall, 1664 patients were enrolled. Kaplan-Meier survival analysis showed an efficacy of 98.8% for AS+SP. Most patients with P. falciparum parasitaemia cleared their parasitaemias within 24 hours of treatment initiation, but six, including four with treatment failure, remained parasitaemic after 72 hours. Double mutants in dhfr were found in 68.4% of the genotyped isolates. Triple or quadruple mutants in dhfr and mutations in dhps were rare. A daily dose of artesunate of < 3 mg per kg of body weight, age of less than 5 years, and fever at enrolment were associated with an increased risk of treatment failure. Chloroquine remained 100% efficacious and generally cleared P. vivax parasitaemias within 48 hours. Vomiting (seen in 47 patients) was the most common adverse event. CONCLUSION: India's National Antimalarial Drug Resistance Monitoring System provides wide coverage. The first-line antimalarials used in the country remain safe and efficacious. The treatment of malaria in young children and the relative benefits of age- and weight-based dosing need further exploration.

In vitro assessment of drug resistance in Plasmodium falciparum in five States of India.

BACKGROUND & OBJECTIVES: In vitro assays are an important tool to assess baseline sensitivity and monitor the drug response of Plasmodium falciparum over time and place and, therefore, can provide background information for the development and evaluation of drug policies. This study was aimed at determining the in vitro sensitivity of P. falciparum isolates to antimalarials. METHODS: The in vitro activity of 108 P. falciparum isolates obtained from five States of India was evaluated using WHO microtest (Mark III) to chloroquine, monodesethylamodiaquine, dihydroartesunate and mefloquine. Samples were collected from the States of Orissa, Jharkhand, Karnataka, Goa and Chhattisgarh from September 2007 to August 2009. In addition, representative samples from different States of India cryopreserved and culture adapted in the Malaria Parasite Bank of National Institute of Malaria Research, New Delhi, were also evaluated. RESULTS: The proportion of isolates resistant to chloroquine and monodesethylamodiaquine was 44.4 and 25 per cent, respectively. Of the 27 isolates resistant to monodesethylamodiaquine, 16 (59.3%) were cross-resistant to chloroquine. No isolate showed resistance to dihydroartesunate and mefloquine. Isolates from Orissa showed the highest degree of resistance to chloroquine and amodiaquine followed by Jharkhand. Forty two isolates were genotyped for pfcrt T76K chloroquine resistant mutation; mutations were seen in 38 (90.47%) isolates. INTERPRETATION & CONCLUSIONS: The Indian P. falciparum isolates showed a high degree of resistance to chloroquine followed by monodesethylamodiaquine. No resistance was recorded to mefloquine and dihydroartesunate.

Transcriptional profiling and genes involved in acquired thermotolerance in Banana: a non-model crop.

Banana is a non- model crop plant, and one of the most important crops in the tropics and sub tropics. Heat stress is the major abiotic stress affecting banana crop production because of its long growth period and is likely to become a threat due to global warming. To understand an acquired thermotolerance phenomenon at the molecular level, the RNA-seq approach was employed by adapting TIR method. A total of 136.38 million high quality reads were assembled. Differentially expressed genes under induction (I) was 3936, I + L was 2268 and lethal stress was 907 compared to control. Gene ontology and DGE analysis showed that genes related to heat shock factors, heat shock proteins, stress associated proteins, ROS scavenging, fatty acid metabolism, protein modification were significantly up regulated during induction, thus preparing the organism or tissue at molecular and cellular level for acquired thermotolerance. KEGG pathway analysis revealed the significant enrichment of pathways involved in protein processing, MAPK signaling and HSPs which indicates that these processes are conserved and involved in thermo tolerance. Thus, this study provides insights into the acquired thermotolerance phenomena in plants especially banana.

Activity of a crude extract formulation in experimental hepatic amoebiasis and in immunomodulation studies.

The activity of a crude extract formulation was evaluated in experimental amoebic liver abscess in golden hamsters and in immunomodulation studies. The formulation comprises the following five plants-Boerhavia diffusa, Tinospora cordifolia, Berberis aristata, Terminalia chebula and Zingiber officinale. The formulation had a maximum cure rate of 73% at a dose of 800 mg/kg/day in hepatic amoebiasis reducing the average degree of infection (ADI) to 1.3 as compared to 4.2 for sham-treated controls. In immunomodulation studies humoral immunity was enhanced as evidenced by the haemagglutination titre. The T-cell counts remained unaffected in the animals treated with the formulation but cell-mediated immune response was stimulated as observed in the leukocyte migration inhibition (LMI) tests.

The antiamoebic effect of a crude drug formulation of herbal extracts against Entamoeba histolytica in vitro and in vivo.

The antiamoebic effect of a crude drug formulation against Entamoeba histolytica was studied. In the traditional system of medicine in India, the formulation has been prescribed for intestinal disorders. It comprises of five medicinal herbs, namely, Boerhavia diffusa, Berberis aristata, Tinospora cordifolia, Terminalia chebula and Zingiber officinale. The dried and pulverized plants were extracted in ethanol together and individually. In vitro amoebicidal activity was studied to determine the minimal inhibitory concentration (MIC) values of all the constituent extracts as well as the whole formulation. The formulation had a MIC of 1000 micrograms/ml as compared with 10 micrograms/ml for metronidazole. In experimental caecal amoebiasis in rats the formulation had a curative rate of 89% with the average degree of infection (ADI) reduced to 0.4 in a group dosed with 500 mg/kg per day as compared with ADI of 3.8 for the sham-treated control group of rats. Metronidazole had a cure rate of 89% (ADI = 0.4) at a dose of 100 mg/kg per day and cured the infection completely (ADI = 0) when the dosage was doubled to 200 mg/kg per day. There were varying degrees of inhibition of the following enzyme activities of crude extracts of axenically cultured amoebae: DNase, RNase, aldolase, alkaline phosphatase, acid phosphatase, alpha-amylase and protease.

Prophylactic therapy of Salmonella typhi septicemia in mice with a traditionally prescribed crude drug formulation.

A crude drug formulation comprised of eight medicinal herb extracts was studied for in vitro and in vivo effect against Salmonella typhi. The formulation displayed inhibitory action against the test organism in vitro. Subsequently mice were challenged with a virulent strain of Salmonella typhi (Ty2) and the protective effect of the formulation was evaluated with post-infective, pre-infective, single and multiple dose schedules, administered either orally or subcutaneously. A schedule that included multiple divided doses prophylactically administered had a significant therapeutic effect.

Field evaluation of biolarvicides in Surat city, India.

BACKGROUND & OBJECTIVES: Two bacterial larvicide (bio-larvicide) formulations--Bacticide and VectoBac containing viable endospores and delta endotoxin of Bacillus thuringiensis var israelensis H-14 were evaluated in 2001 for their mosquito larvicidal efficacy under the operational conditions of urban malaria control programme in Surat city, India. METHODS: Larvicides were applied at the recommended dose in selected breeding habitats of Anopheles (An. stephensi), Aedes (Ae. aegypti) and Culex (Cx. quinquefasciatus) and reductions in the densities of III and IV instars were compared with that of untreated matched controls. RESULTS: At the construction sites in cemented tanks/chambers VectoBac produced reduction in the density of III and IV instar larvae of An. stephensi (98-100%) and Ae. aegypti (100%) in the first week of application whereas Bacticide produced 71-100% reduction in An. stephensi and 100% in Ae. aegypti. Re-application of VectoBac on Day 10 caused better control up to Day 20 when compared with Bacticide. In stagnant water pools, VectoBac produced 27.6-85.3% reduction in the larvae of An. subpictus and 18.5-83.8% in those of Cx. quinquefasciatus whereas Bacticide produced 23.3-30.3% and 39-97.2% reduction in An. subpictus and Cx. quinquefasciatus larval densities in the first week post application, respectively. Bacticide application gave better impact on Cx. quinquefasciatus larvae in the second week after re-application as compared to VectoBac. In storm water drains, VectoBac caused respectively 6.2-100% and 6.4-97.6% reduction in An. subpictus and Cx. quinquefasciatus larvae in the first week of application whereas Bacticide produced 100% and 13.3-98.8% reduction in An. subpictus and Cx. quinquefasciatus larval densities, respectively. INTERPRETATION & CONCLUSION: Both the formulations were equally effective on An. subpictus and Cx. quinquefasciatus larvae after a second application. The results showed that application of these biolarvicides would be required at 7-10 day intervals. The health workers engaged in the application of biolarvicides reported a better ease of handling and application of the liquid formulation (VectoBac) than the wettable powder formulation (Bacticide).

Sensitivity of mosquito-pathogenic bacterial strains to various antibodies.

Four strains of Bacillus sphaericus, 1593, 2362, 9001 and 9002, B. thuringiensis H-14 and B. thuringiensis neoleonensis were tested for sensitivity against 18 antibiotics. The results revealed that all the four strains of B. sphaericus are resistant to colistin, nalidixic acid, polymyxin B and streptomycin. However, B. thuringiensis H-14 was resistant to 9 antibiotics, viz. ampicillin, cephalexin, carbenicillin, co-trimoxazole, colistin, cloxacillin, penicillin, nitrofurantoin and polymyxin B whereas B. thuringiensis neoleonensis was found to be resistant to 8 antibiotics. These results may help in isolation of potential and resistant mosquito pathogenic bacteria.

A quantitative survey of anophelines in six villages of Kheda District, Gujarat.

Sampling of anophelines from six villages of Kheda district, Gujarat was done using four different methods viz. indoor resting, outdoor resting, bovine bait trap and immature collections. A total of 113,487 specimens representing 17 species were collected. An. subpictus (66.51 per cent) was most abundant species followed by An. culicifacies (14.12 per cent) and An. tessellatus (5.24 per cent). Bovine bait collections were found most productive yielding maximum species and highest number of anophelines per unit of collection efforts. Indoor resting collections yielded poorest diversity and greater number of specimens per unit of collection effort than outdoor resting collections. Maximum diversity was observed in outdoor collections. Most of the species exhibited unimodal prevalence.

An epidemiological study of G-6-PD deficiency, sickle cell haemoglobin, and ABO blood groups in relation to malaria incidence in Muslim and Christian communities of Kheda, Gujarat, (India).

783 blood samples for the study of distribution of ABO blood groups and sickle cell haemoglobin in relation to malaria, from both the sexes of Muslim and Christian populations of Kheda district were screened. 414 blood samples from male individuals were screened for G-6-PD deficiency. High frequency of G-6-PD deficiency was observed in Christians (5.9%) and low in Muslim (1.8%) population, whereas sickle cell haemoglobin in Muslim population was 1.5% and absent in Christians. Blood group B was dominant in both the communities. Significant association of ABO polymorphs with P. falciparum and total malaria cases was observed.

Genetic deletion of HRP2 and HRP3 in Indian Plasmodium falciparum population and false negative malaria rapid diagnostic test.

Genetic polymorphisms in diagnostic antigens are important factors responsible for variable performance of rapid diagnostic tests. Additionally, the failure of antigen expression due to gene deletion may also contribute to variable performance. We report Indian Plasmodium falciparum field isolates lacking both Pfhrp2 and Pfhrp3 genes leading to false negative results of rapid diagnostic tests. The study highlights need to determine the prevalence of P. falciparum isolates lacking these genes in larger field populations in India.