Low prevalence of antimalarial-resistance mutations in India during 2014-15: Impact of combining first-line therapy with primaquine.

BACKGROUND: Antimalarial drug resistance surveillance and containment are crucial for countries aiming to eliminate malaria. Monitoring resistance evolution through studies before and after treatment policy changes is crucial. METHOD: A total of 939 P. falciparum-positive blood samples were collected between 2014 and 2015 across ten sites in India, categorized into four geographic clusters. PCR-amplified products were sequenced to identify point mutations at drug-resistance-conferring genes (Pfdhfr, Pfdhps, Pfmdr1, Pfk13). RESULT: Triple Pfdhfr mutants were found only in northeast India bordering Myanmar, while the wildtype was dominant in central India. Pfdhps wildtypes were prevalent in all areas, and no double mutants were found. Except in Northwest India, Pfmdr1 wildtype was dominant in all clusters. Nonsynonymous double mutations were only found in northwest India. Only synonymous mutations occurred in Pfk13. These were found in Central India at low frequency. The pattern of linkage disequilibrium and principal component analysis reflects low pressure for drug resistance and heterogeneity between the geographic clusters. CONCLUSION: Resistance levels were highest in Northeast India, close to the Myanmar border, where resistance is common. Primaquine has been widely used as a gametocidal and schizonticidal drug, has likely contributed to maintaining low drug resistance levels and preventing strong selection for resistance.

Malaria hemozoin: A target for point-of-care diagnosis.

The challenges in malaria diagnosis continue to threaten the malaria elimination goal in India and other malaria-endemic countries. A rapid diagnostic test (RDT) kit is widely used in resource-constrained areas where microscopy and molecular methods are not easily deployable. Considering the problems associated with the currently available RDT kit, such as histidine-rich protein 2 gene deletion and prolonged stability of the protein in the blood, it suggests that new potential biomarkers are urgently needed. Hemozoin (Hz) is an important biomarker for malaria diagnosis, which is the by-product of a detoxification mechanism in the malaria parasite. This article highlights the importance of "Hz" for point-of-care malaria diagnosis when India and other countries are moving toward the goal of malaria elimination.

A computational strategy for systematic virtual screening of plasmodium falciparum heme detoxification protein inhibitors from the Drugbank database.

Antimalarial drug resistance poses one of the greatest threats to malaria treatment, resulting in increased morbidity and mortality. Heme Detoxification Protein (HDP) is among the essential hemoglobinases of P. falciparum (Pf), a vital molecular target for the treatment of malaria. In this study, we utilized the virtual screening workflow tool of the Schrodinger suite to find the best hits for the PfHDP from the DrugBank library. A total of 14,942 compounds were identified against the PfHDP. The top compounds with the highest docking scores and least energy scores were subjected to molecular simulations for 500 nanosecond to check the stability of the protein-drug complexes. The top three DrugBank compounds were found to be stable over 500 ns, namely DB09298 (silibinin), DB07426 (1-Hydroxy-2-(1,1':3',1''-Terphenyl-3-Yloxy) Ethane-1,1-Diyl] Bis (Phosphonic Acid), and DB07410 [(2-(3-Dibenzofuran-4-yl-Phenyl)-1-Hydroxy-1-Phosphono-Ethyl]-Phosphonic Acid). Overall analysis suggests that the top three compounds, DB09298, DB07426, and DB07410, have good stability for 500 ns. Their scaffolds can be used to design and develop new analogs of the target HDP protein. Silibinin, the anti-cancer drug, was found to be highly stable for the entire simulation period as compared to the other compounds. DB07426 shows its therapeutic effect on bones, especially in the treatment of osteoporosis, and DB07410 has anti-tumor, antibacterial, anti-oxidative, and anti-viral activities. All three compounds can be considered for repurposing as antimalarial drugs to evaluate the binding capacity or inhibition potential of these compounds. Further in-vivo and in-vitro analysis against the PfHDP protein should be conducted.Communicated by Ramaswamy H. Sarma.

Malaria Slide Bank to Strengthen and Improve the Quality of Malaria Diagnosis: A National Slide Repository in India.

Malaria elimination is one of the top health care priorities in India, necessitating accessible and accurate diagnosis for effective treatment. A malaria slide bank in India is a collection of quality-controlled malaria-positive and -negative slides and is considered a vital asset for quality diagnosis. The collection of blood samples, preparation of blood smears, staining, quality control, molecular characterizations, and slide validation were carried out according to standard operating procedures in accordance with the WHO reference laboratory. The true count and parasite density per microliter were computed in accordance with WHO guidelines. Over 27 months, 48 batches (8,196 slides) were prepared. Overall, the majority of slide batches were Plasmodium vivax (45.9%; 22/48), followed by Plasmodium falciparum (25%; 12/48), malaria-negative infections (25%; 12/48), and mixed infections (4.1%; 2/48). All 48 batches passed internal validation by WHO-certified level-1 microscopists. For a batch, the true count was the median of the validators' counts (range, 111-280,795 parasites/µL). Except for mixed infections, the PCR results agreed with the verified microscopy results. Malaria slide bank slides would be a valuable tool for quality control, assurance, and microscopist training.

Monoclonal antibody: future of malaria control and prevention.

Monoclonal antibodies (mAbs) are extremely specialized proteins that are cloned from B cells and bind to pathogen epitopes. There are currently no known prophylactic immune-based strategies or efficient, widespread treatments to stop the spread of malaria. In order to lower the prevalence of malaria and its associated mortality, we need mAbs that are capable of offering immediate passive protection against the disease. mAbs have become more crucial in the treatment or prevention of several other infectious diseases. Recently, mAb development for malaria prevention and control has greatly evolved and widespread use in public health settings is now a possibility.

Malaria and malnutrition together jeopardizes India's tribal community and disease elimination.

Malaria and malnutrition are major public health problems in India, especially in the rural and tribal communities, and also remain primary causes of morbidity and mortality among children younger than five years. Both diseases are synergistic with each other. It is essential to have a better understanding of the intricate relationships between malnutrition and malaria to target interventions in areas where both diseases coexist. This article highlights the synergistic relationship between malnutrition and malaria, and how malnutrition and malaria play a significant role in disease severity and eventually hinder the elimination of these diseases by 2030. The government and several private sectors have made a substantial dent through various programmes and schemes. However, supplementing nutrition-sensitive measures, including easy accessibility to a healthy balanced diet, safe drinking water and improved sanitation, is necessary. Therefore, if India really aims to achieve its dream of disease elimination (malaria and all forms of malnutrition) by 2030, it is imperative that tribal regions are given more attention and all possible strategies are applied in the country's remotest corners.

Limited genetic diversity and expression profile of Plasmodium falciparum haem detoxification protein: a possible diagnostic target.

BACKGROUND: Haem detoxification protein (HDP) is a significant protein in the erythrocytic stage of the Plasmodium lifecycle. HDP could be of paramount interest as a diagnostic biomarker for accurate diagnosis of malaria. We thus explored HDP genetic variation, expression levels of HDP and immune response. METHODS: Phylogenetic analysis was carried out using Pfhdp orthologues sequences of various Plasmodium species. Blood samples were collected from patients in central India. Pfhdp gene was amplified, and sequenced by sanger DNA sequencing. B-cell epitopes were identified in PfHDP using Bepipred Linear Epitope Prediction 2.0, and median-joining network was constructed using global PfHDP sequences. Pfhdp expression levels during erythrocytic stage were assessed using real-time qPCR at 4-h intervals. An IgG immune response against synthetic PfHDP peptides was analysed using ELISA. RESULTS: Phylogenetic analysis revealed the conserved nature of Pfhdp gene. Diversity analysis revealed one non-synonymous mutation (F91L) among all isolates. Neutrality tests indicated negative selection for Pfhdp gene. HDP was expressed throughout the erythrocytic cycle, and comparatively, high expression was observed in the late trophozoite and schizont stages. High IgG response against both peptides was observed, and no polymorphism was seen in any of the seven predicted B-cell epitopes. CONCLUSIONS: Findings of the present study indicate the possibility of HDP being exploited as a diagnostic biomarker for Plasmodium falciparum malaria after proteomic validation studies.

Identification of Potential Antimalarial Drug Candidates Targeting Falcipain-2 Protein of Malaria Parasite-A Computational Strategy.

Falcipain-2 (FP-2) is one of the main haemoglobinase of P. falciparum which is an important molecular target for the treatment of malaria. In this study, we have screened alkaloids to identify potential inhibitors against FP-2 since alkaloids possess great potential as anti-malarial agents. A total of 340 alkaloids were considered for the study using a series of computational pipelines. Initially, pharmacokinetics and toxicity risk assessment parameters were applied to screen compounds. Subsequently, molecular docking algorithms were utilised to understand the binding efficiency of alkaloids against FP-2. Further, oral toxicity prediction was done using the pkCSM tool, and 3D pharmacophore features were analysed using the PharmaGist server. Finally, MD simulation was performed for Artemisinin and the top 3 drug candidates (Noscapine, Reticuline, Aclidinium) based on docking scores to understand the functional impact of the complexes, followed by a binding site interaction residues study. Overall analysis suggests that Noscapine conceded good pharmacokinetics and oral bioavailability properties. Also, it showed better binding efficiency with FP-2 when compared to Artemisinin. Interestingly, structure alignment analysis with artemisinin revealed that Noscapine, Reticuline, and Aclidinium might possess similar biological action. Molecular dynamics and free energy calculations revealed that Noscapine could be a potent antimalarial agent targeting FP-2 that can be used for the treatment of malaria and need to be studied experimentally in the future.

Unreported mixed Plasmodium species infection may increase vivax malaria in India: a challenge for malaria elimination.

BACKGROUND: In India, there are several malaria-endemic regions where non-falciparum species coexist with Plasmodium falciparum. Traditionally, microscopy and rapid diagnostic tests are used for the diagnosis of malaria. Nevertheless, microscopy often misses the secondary malaria parasite in mixed-infection cases due to various constraints. Misdiagnosis/misinterpretation of Plasmodium species leads to improper treatment, as the treatment for P. falciparum and Plasmodium vivax species is different, as per the national vector-borne disease control program in India. METHODS: Blood samples were collected from malaria-endemic regions (Jharkhand, Madhya Pradesh, Chhattisgarh, Maharashtra, Odisha, Assam, Meghalaya, Mizoram and Telangana) of India covering almost the entire country. Molecular diagnosis of Plasmodium species was carried out among microscopically confirmed P. falciparum samples collected during a therapeutic efficacy study in different years. RESULTS: The polymerase chain reaction analysis revealed a high prevalence (18%) of mixed malaria parasite infections among microscopically confirmed P. falciparum samples from malaria patients that are either missed or left out by microscopy. CONCLUSIONS: Deployment of molecular tools in areas of mixed species infection may prove vital for accurate diagnosis and treatment of malaria. Further, it will help in achieving the goal of malaria elimination in India.

Digital Health Care Services to Control and Eliminate Malaria in India.

In the rural and tribal areas of India, poor healthcare services for malaria are posing a great challenge to malaria control and elimination. Digitisation in malaria healthcare services, including surveillance, diagnosis, and treatment, may be helpful in malaria control and, subsequently, may move towards the elimination goal of India by 2030.

Malaria Elimination in India: Bridging the Gap Between Control and Elimination.

India observed a significant reduction in malaria cases in the previous year, reaffirming our trust and efficiency of the existing tools to achieve malaria elimination. On 25 April, 2019, countries around the world marked World Malaria Day under the theme "Zero malaria starts with me". This provides an opportunity to rejoice the success and re-evaluate ongoing challenges in the fight against this preventable and treatable parasitic disease. We highlight the potential gaps in the malaria elimination program, and underscore potential solutions and strategies to implement, improve and intensify the success of the national goal of malaria elimination by 2030.

Sequence variation in Plasmodium falciparum Histidine Rich Proteins 2 and 3 in Indian isolates: Implications for Malaria Rapid Diagnostic Test Performance.

Commercial malaria rapid diagnostic tests (RDTs) detect P. falciparum histidine rich protein 2 (PfHRP2) and cross react with PfHRP3, a structural homologue. Here, we analysed natural variations in PfHRP2 and PfHRP3 sequences from Indian isolates and correlated these variations with RDT reactivity. A total 1392 P. falciparum positive samples collected from eight endemic states were PCR amplified for Pfhrp2 and Pfhrp3 genes and were sequenced. The deduced protein sequences were analysed for repeat variations and correlated with RDT reactivity. Out of 1392 PCR amplified samples, a single sample was Pfhrp2 negative and two samples were Pfhrp3 negative. Complete Pfhrp2 and Pfhrp3 sequences were obtained for 769 samples and 750 samples, respectively. A total of 16 distinct repeat motifs were observed for Pfhrp2 and 11 for Pfhrp3, including some new repeat types. No correlation was found between variations in the size of Pfhrp2 repeat types 2 and 7, nor between any combinations of repeat motifs, and performance of a commercial RDT at low parasite densities. The findings suggest that sequence diversity in Pfhrp2 and Pfhrp3 genes in Indian isolates is not likely to negatively influence performance of currently used PfHRP2 RDTs.