Analysis of the insecticide resistance mechanism in Anopheles culicifacies sensu lato from a malaria-endemic state in India.

BACKGROUND: Understanding the dynamics and mechanisms of insecticide resistance in malaria vectors is crucial for vector control activities. The present study investigates the level of insecticide resistance in Anopheles culicifacies and explores the role of two main mechanisms in conferring resistance target site insensitivity and metabolic resistance. METHODS: A. culicifacies mosquitoes were collected and the voltage-gated sodium channel (VGSC) gene was amplified and sequenced to analyse the knockdown resistance (kdr) mutations. Further, a non-experimental homology model was generated to investigate the effect of kdr mutations on the conformation of protein. Metabolic resistance was determined using bioassay-based resistant and susceptible mosquitoes and the expression levels of the genes CYP6Z1 and GSTe2 were compared between the two groups. RESULTS: Sequence analysis of the VGSC gene revealed the presence of L1014F (n=48 [17%]), L1014S and V1010L (n=5 [1.7%]) mutations in the study area. In gene expression studies, a significant upregulation of CYP6Z1 in deltamethrin-resistant (fold change 243.62; p=0.02) mosquitoes and that of GSTe2 in dichlorodiphenyltrichloroethane (fold change 403.45; p=0.01) and alpha-cypemethrin resistant (fold change 217.51; p=0.0005) mosquitoes was observed. CONCLUSIONS: The study revealed that expression of the genes (CYP6Z1 and GSTe2) conferring metabolic resistance play a key role in insecticide resistance in A. culicifacies populations in central India. However, mutations L101F, L10104S and V10101L also have a role to some extent in spreading resistance. GeneBank accession numbers: MW559058, MW559059 and MW559060 Cover Image: Workflow of Chimera-Modeller interface. In the top window of Chimera's multi-align viewer the sequence alignment of VGSC proteins of human (pdb id_6AGF), cockroach (pdb id 5XOM) and A. culicifacies (ACT176122.1) is shown. The dialog box in the middle is of the comparative modelling tool of Modeller. The A. culicifacies sequence is designated as the target while human and cockroach sequences are templates. Upon selection of the template sequences in the dialog box, the structures of the respective proteins are displayed in the Chimera window. As the run is completed, the results are displayed in the form of a list of models with their scores in a table.

Therapeutic efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in four malaria endemic states of India.

BACKGROUND: Malaria is a major public health problem in India and accounts for about 88% of malaria burden in South-East Asia. India alone accounted for 2% of total malaria cases globally. Anti-malarial drug resistance is one of the major problems for malaria control and elimination programme. Artemether-lumefantrine (AL) is the first-line treatment of uncomplicated Plasmodium falciparum in north eastern states of India since 2013 after confirming the resistance against sulfadoxine-pyrimethamine. In the present study, therapeutic efficacy of artemether-lumefantrine and k13 polymorphism was assessed in uncomplicated P. falciparum malaria. METHODS: This study was conducted at four community health centres located in Koraput district of Odisha, Bastar district of Chhattisgarh, Balaghat district of Madhya Pradesh and Gondia district of Maharashtra state. Patients with uncomplicated P. falciparum malaria were administered with fixed dose combination (6 doses) of artemether-lumefantrine for 3 days and clinical and parasitological response was recorded up to 28 days as per World Health Organization protocol. Nucleotide sequencing of msp1 and msp2 gene was performed to differentiate between recrudescence and reinfection. Amplification and sequencing of k13 propeller gene region covering codon 450-680 was also carried out to identify the polymorphism. RESULTS: A total 376 malaria patients who fulfilled the enrolment criteria as well as consented for the study were enrolled. Total 356 patients were followed up successfully up to 28 days. Overall, the adequate clinical and parasitological response was 98.9% and 99.4% with and without PCR correction respectively. No case of early treatment failure was observed. However, four cases (1.1%) of late parasitological failure were found from the Bastar district of Chhattisgarh. Genotyping of msp1 and msp2 confirmed 2 cases each of recrudescence and reinfection, respectively. Mutation analysis of k13 propeller gene showed one non-synonymous mutation Q613H in one isolate from Bastar. CONCLUSIONS: The study results showed that artemether-lumefantrine is highly effective in the treatment of uncomplicated P. falciparum malaria among all age groups. No functional mutation in k13 was found in the study area. The data from this study will be helpful in implementation of artemether-lumefantrine in case of treatment failure by artesunate plus sulfadoxine-pyrimethamine.

Population Dynamics and Insecticide Susceptibility of Anopheles culicifacies in Malaria Endemic Districts of Chhattisgarh, India.

A study was undertaken in the villages of Korea and Bastar district (Chhattisgarh) during the years 2012-2015 to investigate the bionomics of malaria vectors and the prevalence of their sibling species complexes. Entomological surveys carried out every month included indoor resting collections, pyrethrum spray catches, light trap catches, and insecticide susceptibility status of Anopheles culicifacies using World Health Organization (WHO) methods. Anopheles culicifacies and Anopheles fluviatilis species were assayed by polymerase chain reaction (PCR) for the detection of malaria parasite, and sibling species were identified using PCR and DNA sequencing. A total of 13,186 samples of Anopheles comprising 15 species from Bastar and 16 from Korea were collected. An. Culicifacies was recorded as the most dominant species and also the only active vector at both sites. This species was found to be resistant to dichlorodiphenyltrichloroethane (DDT) and Malathion, showing signs of emerging resistance against pyrethroids. Among the sibling species of An. culicifacies, the group BCE was found in maximum numbers, while sibling species T of the An. fluviatilis was recorded to be dominant among its complex. The study provides a comprehensive view of the vector bionomics in the highly malarious regions of India that may have importance in developing vector control strategies.

Prevalence of malaria in two highly endemic Community Health Centers in the Bastar district, Chhattisgarh showing mixed infections with Plasmodium species.

Malaria is a major public health problem in India and in the Chhattisgarh state. The diagnosis of malaria presents a major challenge in remote areas The prevalence of malaria in Darbha and Kilepal Community Health Centers (CHCs) of the Jagdalpur district, Chhattisgarh affected by conflict was determined using microscopy and polymerase chain reaction (PCR). In the year 2015, 29.4% and 21.5% cases were found to be positive for malaria at the Darbha and Kilepal CHCs, respectively, by microscopy, and 7.4% and 1.6% of cases had mixed infections, respectively. Among the suspected cases of mixed infections and doubtful diagnoses, 21% had mixed infections with two or more species at the Darbha CHC, and 17% from the Kilepal CHC, as determined by PCR. Both the P. vivax subspecies Pv210 (56%) and Pv247 (44%) and the P. ovale curtisi subspecies were found in this area. The high proportion of mixed malaria parasitic infections detected in this study indicate the need to adequately train health staff involved in diagnosing malaria. This study showed that there is a need for site-specific data to understand the epidemiological picture and to develop appropriate intervention strategies and management guidelines for controlling and eliminating malaria in India.