Quality assurance of malaria rapid diagnostic tests: An aid in malaria elimination.

Background & objectives: India targets malaria elimination by 2030 in a phased manner, so malaria's assured diagnosis is crucial. Introduction of rapid diagnostic kits in India in 2010 has revolutionized malaria surveillance. The storage temperature of rapid diagnostic tests (RDTs), kit components and handling in transportations impact the results of RDTs. Therefore, quality assurance (QA) is required before it reaches end-users. The Indian Council of Medical Research-National Institute of Malaria Research (ICMR-NIMR) has a World Health Organization (WHO) recognized lot-testing laboratory facility to assure the quality of RDTs. Methods: The ICMR-NIMR receives RDTs from different manufacturing companies as well as various agencies such as National and State Programmes and Central Medical Services Society. The WHO standard protocol is followed to conduct all the tests, including long-term and post-dispatch testing. Results: A total of 323 lots tested during January 2014-March 2021 were received from different agencies. Amongst them, 299 lots passed the quality of test and 24 failed. In long-term testing, 179 lots were tested and only nine failed. A total of 7741 RDTs were received from end-users for post-dispatch testing of which 7540 qualified the QA test with a score of 97.4 per cent. Interpretation & conclusions: RDTs received for quality testing showed compliance with QA evaluation of malaria RDTs based on the protocol recommended by the WHO. However, continuous monitoring of the quality of RDTs is required under QA programme. Quality-assured RDTs have a major role, especially in areas where low parasitaemia of parasites persists.

Design and validation of multiplex polymerase chain reaction as a diagnostic tool for Plasmodium species.

BACKGROUND & OBJECTIVES: The highly sensitive method for a true understanding of malaria prevalence is of utmost importance for India's elimination strategy. The PCR reaction type with rapid detection, cost-effectiveness, and less workforce should be preferable. Multiplex PCR type accomplishes the present requirement by saving time and resources to find true surveillance data for malaria, especially in low-parasitemia/asymptomatic groups or populations. METHODS: The present study focuses on designing multiplex PCR (mPCR) to detect simultaneously Plasmodium genus (PAN) and two common Plasmodium species found in India. It is compared to standard nested PCR on 195 clinical samples to diagnose malaria. The mPCR was designed with a minimum number of primers, leading to less clogging and effective and enhanced detection. It contains one common reverse primer and three forward primers amplifying three targeted genes corresponding to P. falciparum, P. vivax, and Plasmodium genus. RESULTS: The sensitivity and specificity for mPCR were 94.06 and 95.74, respectively. The limit of detection for mPCR was 0.1 parasites/µl. The study has shown a ROC curve area for the mPCR of 0.949 for Plasmodium genus and P. falciparum and 0.897 for P. vivax with standard nPCR. INTERPRETATION & CONCLUSION: The mPCR is rapid in detecting species together, cost-effective, and requires fewer human resources than the standard nPCR. Therefore, the mPCR can be used as an alternative technique for the higher sensitive detection of the malaria parasite. It could also become a vital tool for determining malaria prevalence, facilitating the application of the most effective measures.

National Control Laboratories in quality healthcare-a roadblock for substandard biopharmaceuticals.

Substandard or spurious drugs are a global problem with respect to Health and Economic burden. The impact is higher when medicines are from the category of life-saving drugs, essential medicines or high cost targeted medical treatment. Biopharmaceuticals are one such class of drugs where Quality testing plays a pivotal role to stop substandard drugs from reaching the patient. This study of 17,451 samples has highlighted the trend of occurrence of substandard biologicals (2.34%) over a decade (2011-2021) and the importance of continuous and complete evaluation of such Biopharmaceuticals. More such National Control Laboratories (NCL) should be involved in cross-checking the quality of the increasing number of biopharmaceuticals present in the market which are released only on the basis of the onsite inspection and dossier reviews. This will help the Regulators to ensure the readiness for testing the newer biologicals, devise effective policies for better health care initiatives and keep the substandard biopharmaceuticals at bay.

Multiplex loop mediated isothermal amplification (m-LAMP) as a point of care technique for diagnosis of malaria.

Diagnosis of malaria is a prominent challenge due to the endemic nature of infection. Malaria poses a great threat to global public health. The disease can be diagnosed by several techniques out of which microscopy is a known gold standard. High sensitivity of molecular techniques is making them more reliable and popular as tools for diagnosis of malaria. However, new methods are required which can fulfill the criteria of being Point of Care Test (POCT) as defined by WHO. Loop-mediated isothermal amplification (LAMP) technique amplifies DNA in an isothermal condition, and surpasses the disadvantages of conventional molecular techniques such as polymerase chain reaction. Multiplex LAMP, a modification of LAMP may emerge as a new POC for malaria diagnosis. This review deals with the use of LAMP and multiplex LAMP in diagnosis of malaria and its prospective use as point of care techniques.

Comparative Assessment of Diagnostic Performance of Cytochrome Oxidase Multiplex PCR and 18S rRNA Nested PCR.

Malaria elimination and control require prompt and accurate diagnosis for treatment plan. Since microscopy and rapid diagnostic test (RDT) are not sensitive particularly for diagnosing low parasitemia, highly sensitive diagnostic tools are required for accurate treatment. Molecular diagnosis of malaria is commonly carried out by nested polymerase chain reaction (PCR) targeting 18S rRNA gene, while this technique involves long turnaround time and multiple steps leading to false positive results. To overcome these drawbacks, we compared highly sensitive cytochrome oxidase gene-based single-step multiplex reaction with 18S rRNA nested PCR. Cytochrome oxidase (cox) genes of P. falciparum (cox-III) and P. vivax (cox-I) were compared with 18S rRNA gene nested PCR and microscopy. Cox gene multiplex PCR was found to be highly specific and sensitive, enhancing the detection limit of mixed infections. Cox gene multiplex PCR showed a sensitivity of 100% and a specificity of 97%. This approach can be used as an alternative diagnostic method as it offers higher diagnostic performance and is amenable to high throughput scaling up for a larger sample size at low cost.

Triple artemisinin-based combination therapies versus artemisinin-based combination therapies for uncomplicated Plasmodium falciparum malaria: a multicentre, open-label, randomised clinical trial.

BACKGROUND: Artemisinin and partner-drug resistance in Plasmodium falciparum are major threats to malaria control and elimination. Triple artemisinin-based combination therapies (TACTs), which combine existing co-formulated ACTs with a second partner drug that is slowly eliminated, might provide effective treatment and delay emergence of antimalarial drug resistance. METHODS: In this multicentre, open-label, randomised trial, we recruited patients with uncomplicated P falciparum malaria at 18 hospitals and health clinics in eight countries. Eligible patients were aged 2-65 years, with acute, uncomplicated P falciparum malaria alone or mixed with non-falciparum species, and a temperature of 37·5°C or higher, or a history of fever in the past 24 h. Patients were randomly assigned (1:1) to one of two treatments using block randomisation, depending on their location: in Thailand, Cambodia, Vietnam, and Myanmar patients were assigned to either dihydroartemisinin-piperaquine or dihydroartemisinin-piperaquine plus mefloquine; at three sites in Cambodia they were assigned to either artesunate-mefloquine or dihydroartemisinin-piperaquine plus mefloquine; and in Laos, Myanmar, Bangladesh, India, and the Democratic Republic of the Congo they were assigned to either artemether-lumefantrine or artemether-lumefantrine plus amodiaquine. All drugs were administered orally and doses varied by drug combination and site. Patients were followed-up weekly for 42 days. The primary endpoint was efficacy, defined by 42-day PCR-corrected adequate clinical and parasitological response. Primary analysis was by intention to treat. A detailed assessment of safety and tolerability of the study drugs was done in all patients randomly assigned to treatment. This study is registered at ClinicalTrials.gov, NCT02453308, and is complete. FINDINGS: Between Aug 7, 2015, and Feb 8, 2018, 1100 patients were given either dihydroartemisinin-piperaquine (183 [17%]), dihydroartemisinin-piperaquine plus mefloquine (269 [24%]), artesunate-mefloquine (73 [7%]), artemether-lumefantrine (289 [26%]), or artemether-lumefantrine plus amodiaquine (286 [26%]). The median age was 23 years (IQR 13 to 34) and 854 (78%) of 1100 patients were male. In Cambodia, Thailand, and Vietnam the 42-day PCR-corrected efficacy after dihydroartemisinin-piperaquine plus mefloquine was 98% (149 of 152; 95% CI 94 to 100) and after dihydroartemisinin-piperaquine was 48% (67 of 141; 95% CI 39 to 56; risk difference 51%, 95% CI 42 to 59; p<0·0001). Efficacy of dihydroartemisinin-piperaquine plus mefloquine in the three sites in Myanmar was 91% (42 of 46; 95% CI 79 to 98) versus 100% (42 of 42; 95% CI 92 to 100) after dihydroartemisinin-piperaquine (risk difference 9%, 95% CI 1 to 17; p=0·12). The 42-day PCR corrected efficacy of dihydroartemisinin-piperaquine plus mefloquine (96% [68 of 71; 95% CI 88 to 99]) was non-inferior to that of artesunate-mefloquine (95% [69 of 73; 95% CI 87 to 99]) in three sites in Cambodia (risk difference 1%; 95% CI -6 to 8; p=1·00). The overall 42-day PCR-corrected efficacy of artemether-lumefantrine plus amodiaquine (98% [281 of 286; 95% CI 97 to 99]) was similar to that of artemether-lumefantrine (97% [279 of 289; 95% CI 94 to 98]; risk difference 2%, 95% CI -1 to 4; p=0·30). Both TACTs were well tolerated, although early vomiting (within 1 h) was more frequent after dihydroartemisinin-piperaquine plus mefloquine (30 [3·8%] of 794) than after dihydroartemisinin-piperaquine (eight [1·5%] of 543; p=0·012). Vomiting after artemether-lumefantrine plus amodiaquine (22 [1·3%] of 1703) and artemether-lumefantrine (11 [0·6%] of 1721) was infrequent. Adding amodiaquine to artemether-lumefantrine extended the electrocardiogram corrected QT interval (mean increase at 52 h compared with baseline of 8·8 ms [SD 18·6] vs 0·9 ms [16·1]; p<0·01) but adding mefloquine to dihydroartemisinin-piperaquine did not (mean increase of 22·1 ms [SD 19·2] for dihydroartemisinin-piperaquine vs 20·8 ms [SD 17·8] for dihydroartemisinin-piperaquine plus mefloquine; p=0·50). INTERPRETATION: Dihydroartemisinin-piperaquine plus mefloquine and artemether-lumefantrine plus amodiaquine TACTs are efficacious, well tolerated, and safe treatments of uncomplicated P falciparum malaria, including in areas with artemisinin and ACT partner-drug resistance. FUNDING: UK Department for International Development, Wellcome Trust, Bill & Melinda Gates Foundation, UK Medical Research Council, and US National Institutes of Health.

Optimization of Plasmodium vivax sporozoite production from Anopheles stephensi in South West India.

BACKGROUND: Efforts to study the biology of Plasmodium vivax liver stages, particularly the latent hypnozoites, have been hampered by the limited availability of P. vivax sporozoites. Anopheles stephensi is a major urban malaria vector in Goa and elsewhere in South Asia. Using P. vivax patient blood samples, a series of standard membrane-feeding experiments were performed with An. stephensi under the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA). The goal was to understand the dynamics of parasite development in mosquitoes as well as the production of P. vivax sporozoites. To obtain a robust supply of P. vivax sporozoites, mosquito-rearing and mosquito membrane-feeding techniques were optimized, which are described here. METHODS: Membrane-feeding experiments were conducted using both wild and laboratory-colonized An. stephensi mosquitoes and patient-derived P. vivax collected at the Goa Medical College and Hospital. Parasite development to midgut oocysts and salivary gland sporozoites was assessed on days 7 and 14 post-feeding, respectively. The optimal conditions for mosquito rearing and feeding were evaluated to produce high-quality mosquitoes and to yield a high sporozoite rate, respectively. RESULTS: Laboratory-colonized mosquitoes could be starved for a shorter time before successful blood feeding compared with wild-caught mosquitoes. Optimizing the mosquito-rearing methods significantly increased mosquito survival. For mosquito feeding, replacing patient plasma with naïve serum increased sporozoite production > two-fold. With these changes, the sporozoite infection rate was high (> 85%) and resulted in an average of ~ 22,000 sporozoites per mosquito. Some mosquitoes reached up to 73,000 sporozoites. Sporozoite production could not be predicted from gametocyte density but could be predicted by measuring oocyst infection and oocyst load. CONCLUSIONS: Optimized conditions for the production of high-quality P. vivax sporozoite-infected An. stephensi were established at a field site in South West India. This report describes techniques for producing a ready resource of P. vivax sporozoites. The improved protocols can help in future research on the biology of P. vivax liver stages, including hypnozoites, in India, as well as the development of anti-relapse interventions for vivax malaria.

A model for malaria elimination based on learnings from the Malaria Elimination Demonstration Project, Mandla district, Madhya Pradesh.

BACKGROUND: Malaria Elimination Demonstration Project (MEDP) was started as a Public-Private-Partnership between the Indian Council of Medical Research through National Institute of Research in Tribal Health, Govt. of Madhya Pradesh and Foundation of Disease Elimination and Control of India, which is a Corporate Social Responsibility (CSR) initiative of the Sun Pharmaceutical Industries Limited. The project's goal was to demonstrate that malaria can be eliminated from a high malaria endemic district along with prevention of re-establishment of malaria and to develop a model for malaria elimination using the lessons learned and knowledge acquired from the demonstration project. METHODS: The project employed tested protocols of robust surveillance, case management, vector control, and capacity building through continuous evaluation and training.  The model was developed using the learnings from the operational plan, surveillance and case management, monitoring and feedback, entomological investigations and vector control, IEC and capacity building, supply chain management, mobile application (SOCH), and independent reviews of MEDP. RESULTS: The MEDP has been operational since April 2017 with field operations from August 2017, and has observed: (1) reduction in indigenous cases of malaria by about 91 %; (2) need for training and capacity building of field staff for diagnosis and treatment of malaria; (3) need for improvement insecticide spraying and for distribution and usage of bed-nets; (4) need for robust surveillance system that captures and documents information on febrile cases, RDT positive individuals, and treatments provided; (5) need for effective supervision of field staff based on advance tour plan; (6) accountability and controls from the highest level to field workers; and (7) need for context-specific IEC. CONCLUSIONS: Malaria elimination is a high-priority public health goal of the Indian Government with a committed deadline of 2030. In order to achieve this goal, built-in systems of accountability, ownership, effective management, operational, technical, and financial controls will be crucial components for malaria elimination in India. This manuscript presents a model for malaria elimination with district as an operational unit, which may be considered for malaria elimination in India and other countries with similar geography, topography, climate, endemicity, health infrastructure, and socio-economic characteristics.

Prevalence of Pfhrp2/3 gene deletions among false negative rapid antigen test results in central India.

Background &objectives: The diagnosis of Plasmodium falciparum malaria is widely dependent on the P. falciparum histidine rich protein 2 (PfHRP2) antigens based rapid diagnostic tests. There are few possible factors like Pfhrp2 polymorphism, Pfhrp2 deletion and density of malaria parasite which can affect the sensitivity of the Pf-HRP2-based RDT. The primary objective of the investigation was to check whether the Pfhrp2 gene deletion is the primary cause of RDT false negative cases. METHODS: Febrile patients from three districts of Chhattisgarh, India were screened for malaria during 2016-2017 by microscopy and RDT. All microscopy P. falciparum positive samples were validated by PCR. Microscopy positive and RDT negative samples were analyzed for the presence of Exon 2, across Exon 1-2, upstream and downstream of both the Pfhrp2 and Pfhrp3 genes fragment by PCR. RESULTS: Out of 203 screened samples, 85 were detected positive for P. falciparum malaria based on microscopy and PCR. Among these 85 P. falciparum positive samples, 4 samples were observed Pf-HRP2 RDT negative. Although, it signified that the RDTs used were reliable with sensitivity of 95.3% (81/85). 3/4 PfHRP2-RDT negative samples of the P. falciparum isolates exhibited complete deletion of Pfhrp2 and Pfhrp3 genes and one sample was found RDT false negative due to high parasite density. INTERPRETATION & CONCLUSION: Pfhrp2 and Pfhrp3 deletions that result in false negative RDTs were uncommon in our setting. The continued monitoring of RDTS which results in false negative tests due to Pfhrp2/3 gene deletion is the need of the hour for an effective malaria elimination strategy.

The risk of Plasmodium vivax parasitaemia after P. falciparum malaria: An individual patient data meta-analysis from the WorldWide Antimalarial Resistance Network.

BACKGROUND: There is a high risk of Plasmodium vivax parasitaemia following treatment of falciparum malaria. Our study aimed to quantify this risk and the associated determinants using an individual patient data meta-analysis in order to identify populations in which a policy of universal radical cure, combining artemisinin-based combination therapy (ACT) with a hypnozoitocidal antimalarial drug, would be beneficial. METHODS AND FINDINGS: A systematic review of Medline, Embase, Web of Science, and the Cochrane Database of Systematic Reviews identified efficacy studies of uncomplicated falciparum malaria treated with ACT that were undertaken in regions coendemic for P. vivax between 1 January 1960 and 5 January 2018. Data from eligible studies were pooled using standardised methodology. The risk of P. vivax parasitaemia at days 42 and 63 and associated risk factors were investigated by multivariable Cox regression analyses. Study quality was assessed using a tool developed by the Joanna Briggs Institute. The study was registered in the International Prospective Register of Systematic Reviews (PROSPERO: CRD42018097400). In total, 42 studies enrolling 15,341 patients were included in the analysis, including 30 randomised controlled trials and 12 cohort studies. Overall, 14,146 (92.2%) patients had P. falciparum monoinfection and 1,195 (7.8%) mixed infection with P. falciparum and P. vivax. The median age was 17.0 years (interquartile range [IQR] = 9.0-29.0 years; range = 0-80 years), with 1,584 (10.3%) patients younger than 5 years. 2,711 (17.7%) patients were treated with artemether-lumefantrine (AL, 13 studies), 651 (4.2%) with artesunate-amodiaquine (AA, 6 studies), 7,340 (47.8%) with artesunate-mefloquine (AM, 25 studies), and 4,639 (30.2%) with dihydroartemisinin-piperaquine (DP, 16 studies). 14,537 patients (94.8%) were enrolled from the Asia-Pacific region, 684 (4.5%) from the Americas, and 120 (0.8%) from Africa. At day 42, the cumulative risk of vivax parasitaemia following treatment of P. falciparum was 31.1% (95% CI 28.9-33.4) after AL, 14.1% (95% CI 10.8-18.3) after AA, 7.4% (95% CI 6.7-8.1) after AM, and 4.5% (95% CI 3.9-5.3) after DP. By day 63, the risks had risen to 39.9% (95% CI 36.6-43.3), 42.4% (95% CI 34.7-51.2), 22.8% (95% CI 21.2-24.4), and 12.8% (95% CI 11.4-14.5), respectively. In multivariable analyses, the highest rate of P. vivax parasitaemia over 42 days of follow-up was in patients residing in areas of short relapse periodicity (adjusted hazard ratio [AHR] = 6.2, 95% CI 2.0-19.5; p = 0.002); patients treated with AL (AHR = 6.2, 95% CI 4.6-8.5; p < 0.001), AA (AHR = 2.3, 95% CI 1.4-3.7; p = 0.001), or AM (AHR = 1.4, 95% CI 1.0-1.9; p = 0.028) compared with DP; and patients who did not clear their initial parasitaemia within 2 days (AHR = 1.8, 95% CI 1.4-2.3; p < 0.001). The analysis was limited by heterogeneity between study populations and lack of data from very low transmission settings. Study quality was high. CONCLUSIONS: In this meta-analysis, we found a high risk of P. vivax parasitaemia after treatment of P. falciparum malaria that varied significantly between studies. These P. vivax infections are likely attributable to relapses that could be prevented with radical cure including a hypnozoitocidal agent; however, the benefits of such a novel strategy will vary considerably between geographical areas.

Pregnancy outcomes and risk of placental malaria after artemisinin-based and quinine-based treatment for uncomplicated falciparum malaria in pregnancy: a WorldWide Antimalarial Resistance Network systematic review and individual patient data meta-analysis.

BACKGROUND: Malaria in pregnancy, including asymptomatic infection, has a detrimental impact on foetal development. Individual patient data (IPD) meta-analysis was conducted to compare the association between antimalarial treatments and adverse pregnancy outcomes, including placental malaria, accompanied with the gestational age at diagnosis of uncomplicated falciparum malaria infection. METHODS: A systematic review and one-stage IPD meta-analysis of studies assessing the efficacy of artemisinin-based and quinine-based treatments for patent microscopic uncomplicated falciparum malaria infection (hereinafter uncomplicated falciparum malaria) in pregnancy was conducted. The risks of stillbirth (pregnancy loss at ≥ 28.0 weeks of gestation), moderate to late preterm birth (PTB, live birth between 32.0 and < 37.0 weeks), small for gestational age (SGA, birthweight of < 10th percentile), and placental malaria (defined as deposition of malaria pigment in the placenta with or without parasites) after different treatments of uncomplicated falciparum malaria were assessed by mixed-effects logistic regression, using artemether-lumefantrine, the most used antimalarial, as the reference standard. Registration PROSPERO: CRD42018104013. RESULTS: Of the 22 eligible studies (n = 5015), IPD from16 studies were shared, representing 95.0% (n = 4765) of the women enrolled in literature. Malaria treatment in this pooled analysis mostly occurred in the second (68.4%, 3064/4501) or third trimester (31.6%, 1421/4501), with gestational age confirmed by ultrasound in 91.5% (4120/4503). Quinine (n = 184) and five commonly used artemisinin-based combination therapies (ACTs) were included: artemether-lumefantrine (n = 1087), artesunate-amodiaquine (n = 775), artesunate-mefloquine (n = 965), and dihydroartemisinin-piperaquine (n = 837). The overall pooled proportion of stillbirth was 1.1% (84/4361), PTB 10.0% (619/4131), SGA 32.3% (1007/3707), and placental malaria 80.1% (2543/3035), and there were no significant differences of considered outcomes by ACT. Higher parasitaemia before treatment was associated with a higher risk of SGA (adjusted odds ratio [aOR] 1.14 per 10-fold increase, 95% confidence interval [CI] 1.03 to 1.26, p = 0.009) and deposition of malaria pigment in the placenta (aOR 1.67 per 10-fold increase, 95% CI 1.42 to 1.96, p < 0.001). CONCLUSIONS: The risks of stillbirth, PTB, SGA, and placental malaria were not different between the commonly used ACTs. The risk of SGA was high among pregnant women infected with falciparum malaria despite treatment with highly effective drugs. Reduction of malaria-associated adverse birth outcomes requires effective prevention in pregnant women.

Delayed haemolysis after treatment with intravenous artesunate in patients with severe malaria in India.

BACKGROUND: Parenteral artesunate is the treatment of choice for severe malaria. It is safe, efficacious and well tolerated anti-malarial. However, delayed haemolysis has been reported in travellers, non-immune individuals and in African children. METHODS: A prospective, observational study was carried out in admitted severe malaria patients receiving parenteral artesunate. The patients were followed up until day 28 for monitoring clinical as well as laboratory parameters for haemolytic anaemia. RESULTS: Twenty-four patients with severe malaria receiving injection artesunate were enrolled in the study. Post-artesunate delayed haemolysis following parenteral artesunate therapy was observed in three of 24 patients (12.5%, 95% confidence interval 4.5-31.2%). Haemolysis was observed in two more patients possibly due to other reasons. The haemoglobin fall ranged from 13.6 to 38.3% from day 7 to day 28 in these patients. CONCLUSION: The possibility of delayed haemolysis should be considered while treating the severe malaria patients with parenteral artesunate. The study highlights the need for further studies in different epidemiological settings.

Plasmodium vivax liver stage assay platforms using Indian clinical isolates.

BACKGROUND: Vivax malaria is associated with significant morbidity and economic loss, and constitutes the bulk of malaria cases in large parts of Asia and South America as well as recent case reports in Africa. The widespread prevalence of vivax is a challenge to global malaria elimination programmes. Vivax malaria control is particularly challenged by existence of dormant liver stage forms that are difficult to treat and are responsible for multiple relapses, growing drug resistance to the asexual blood stages and host-genetic factors that preclude use of specific drugs like primaquine capable of targeting Plasmodium vivax liver stages. Despite an obligatory liver-stage in the Plasmodium life cycle, both the difficulty in obtaining P. vivax sporozoites and the limited availability of robust host cell models permissive to P. vivax infection are responsible for the limited knowledge of hypnozoite formation biology and relapse mechanisms, as well as the limited capability to do drug screening. Although India accounts for about half of vivax malaria cases world-wide, very little is known about the vivax liver stage forms in the context of Indian clinical isolates. METHODS: To address this, methods were established to obtain infective P. vivax sporozoites from an endemic region in India and multiple assay platforms set up to detect and characterize vivax liver stage forms. Different hepatoma cell lines, including the widely used HCO4 cells, primary human hepatocytes as well as hepatocytes obtained from iPSC's generated from vivax patients and healthy donors were tested for infectivity with P. vivax sporozoites. RESULTS: Both large and small forms of vivax liver stage are detected in these assays, although the infectivity obtained in these platforms are low. CONCLUSIONS: This study provides a proof of concept for detecting liver stage P. vivax and provide the first characterization of P. vivax liver stage forms from an endemic region in India.

Haplotype of RNASE 3 polymorphisms is associated with severe malaria in an Indian population.

Severe malaria (SM) caused by Plasmodium falciparum (Pf) infection has been associated with life-threatening anemia, metabolic acidosis, cerebral malaria and multiorgan dysfunction. It may lead to death if not treated promptly. RNASE 3 has been linked to Pf growth inhibition and its polymorphisms found associated with SM and cerebral malaria in African populations. This study aimed to assess the association of RNASE 3 polymorphisms with SM in an Indian population. RNASE 3 gene and flanking regions were amplified followed by direct DNA sequencing in 151 Indian patients who visited Wenlock District Government Hospital, Mangalore, Karnataka, India. Allele, genotype and haplotype frequencies were compared between patients with SM (n = 47) and uncomplicated malaria (UM; n = 104). Homozygous mutant genotype was only found for rs2233860 (+ 499G > C) polymorphism (< 1% frequency). No significant genetic associations were found for RNASE 3 polymorphism genotypes and alleles in Indian SM patients using the Fisher's exact test. C-G-G haplotype of rs2233859 (- 38C > A), rs2073342 (+ 371C > G) and rs2233860 (+ 499G > C) polymorphisms was correlated significantly with SM patients (OR = 3.03; p = 0.008) after Bonferroni correction. A haplotype of RNASE 3 gene was found associated with an increased risk of SM and confirming that RNASE 3 gene plays a role in susceptibility to SM.

Efficacy and safety of fixed dose combination of arterolane maleate and piperaquine phosphate in comparison with chloroquine phosphate in children with acute uncomplicated Plasmodium vivax malaria: A phase III, randomised, multicentric study.

BACKGROUND & OBJECTIVES: In India, the burden of Plasmodium vivax malaria has been projected to be highest in some areas. This study investigated the efficacy and safety of fixed dose combination (FDC) of arterolane maleate (AM) 37.5 mg and piperaquine phosphate 187.5 mg (PQP) dispersible tablets and (not with) chloroquine in the treatment of uncomplicated vivax malaria in pediatric patients. METHODS: This multicentric, open-label trial was carried out at 12 sites in India. A total of 164 patients aged 6 months to 12 years with P. vivax malaria were randomized in a ratio of 2:1 to AM-PQP (111 patients) or chloroquine (53 patients) arms. The duration of follow up was 42 days. RESULTS: At 72 hours, the proportion of a parasitaemic and afebrile patients was 100% in both treatment arms in per protocol (PP) population, and 98.2% and 100% [95% CI: -1.8 (-6.33 to 5.08)] in AM-PQP and chloroquine arms, respectively, in intent to treat (ITT) population. The efficacy and safety of AM-PQP was found to be comparable to chloroquine in the treatment of uncomplicated P. vivax malaria in pediatric patients. Overall, the cure rate at Day 28 and 42 was >95% for both AM-PQP or CQ. The commonly reported clinical adverse event was vomiting. No patient was discontinued for any QTc abnormality. INTERPRETATION & CONCLUSION: The efficacy and safety of FDC of arterolane maleate and piperaquine phosphate was found to be comparable to chloroquine for treatment of uncomplicated P. vivax malaria in pediatric patients.

C-reactive protein as a prognostic marker of Plasmodiumfalciparum malaria severity.

BACKGROUND & OBJECTIVES: Plasmodiumfalciparum malaria causes wide variety of clinical symptoms ranging from a mild febrile illness to life-threatening complications. For prevention of the severity and early diagnosis, evaluation of potential biomarkers is much needed. C-reactive protein (CRP) is an acute phase protein and well-recognized marker of inflammation in the body. It is synthesized by liver in response to pro-inflammatory responses and has correlation with complications associated with malaria. The study was aimed to assess, if it could serve as a predictive marker for malaria disease severity. METHODS: In the present study, 74 P. falciparum patients and 22 healthy controls were enrolled. Turbidimetric immunoassay was used to measure the CRP in serum samples of all the study participants. Mann-Whitney U-test for continuous data and chi-square test for categorical data were used to compare all malaria cases vs. healthy control group and uncomplicated vs. severe malaria groups. Using receiver operating characteristic (ROC) analysis, best threshold value was determined for CRP in severe malaria patients. RESULTS: CRP level was significantly elevated in all malaria case groups (1.6 mg/dl IQ 1-2.6) as compared to healthy controls (0.10 mg/dl IQ 0.1-0.20), with p-value <0.0001. Further, CRP level was significantly higher in the severe malaria group (2 mg/dl IQ 1.8-3.9) as compared to the uncomplicated malaria group (1.4 mg/dl IQ 1-2.47) and healthy control group (0.10 mg/dl IQ 0.10-0.20), with p-value <0.05. INTERPRETATION & CONCLUSION: The present study findings suggest that CRP level can be used to differentiate severe malaria from uncomplicated malaria. Elevated CRP level could be helpful in early prediction of the disease severity in patients infected with P. falciparum and may play an important role in diagnosis of falciparum malaria where improper initial test and clinical manifestations like fever may be absent even with a high load of parasite.

Methylene blue induced morphological deformations in Plasmodium falciparum gametocytes: implications for transmission-blocking.

BACKGROUND: Malaria remains a global health problem despite availability of effective tools. For malaria elimination, drugs targeting sexual stages of Plasmodium falciparum need to be incorporated in treatment regimen along with schizonticidal drugs to interrupt transmission. Primaquine is recommended as a transmission blocking drug for its effect on mature gametocytes but is not extensively utilized because of associated safety concerns among glucose-6-phosphate dehydrogenase (G6PD) deficient patients. In present work, methylene blue, which is proposed as an alternative to primaquine is investigated for its gametocytocidal activity amongst Indian field isolates. An effort has been made to establish Indian field isolates of P. falciparum as in vitro model for gametocytocidal drugs screening. METHODS: Plasmodium falciparum isolates were adapted to in vitro culture and induced to gametocyte production by hypoxanthine and culture was enriched for gametocyte stages using N-acetyl-glucosamine. Gametocytes were incubated with methylene blue for 48 h and stage specific gametocytocidal activity was evaluated by microscopic examination. RESULTS: Plasmodium falciparum field isolates RKL-9 and JDP-8 were able to reproducibly produce gametocytes in high yield and were used to screen gametocytocidal drugs. Methylene blue was found to target gametocytes in a concentration dependent manner by either completely eliminating gametocytes or rendering them morphologically deformed with mean IC50 (early stages) as 424.1 nM and mean IC50 (late stages) as 106.4 nM. These morphologically altered gametocytes appeared highly degenerated having shrinkage, distortions and membrane deformations. CONCLUSIONS: Field isolates that produce gametocytes in high yield in vitro can be identified and used to screen gametocytocidal drugs. These isolates should be used for validation of gametocytocidal hits obtained previously by using lab adapted reference strains. Methylene blue was found to target gametocytes produced from Indian field isolates and is proposed to be used as a gametocytocidal adjunct with artemisinin-based combination therapy. Further exploration of methylene blue in clinical studies amongst Indian population, including G6PD deficient patients, is recommended.

Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections.

BACKGROUND: The native gut microbiota of Anopheles mosquitoes is known to play a key role in the physiological function of its host. Interestingly, this microbiota can also influence the development of Plasmodium in its host mosquitoes. In recent years, much interest has been shown in the employment of gut symbionts derived from vectors in the control of vector-borne disease transmission. In this study, the midgut microbial diversity has been characterized among laboratory-reared adult Anopheles stephensi mosquitoes, from the colony created by rearing progeny of wild-caught mosquitoes (obtained from three different locations in southern India) for multiple generations, using 16S ribosomal RNA (rRNA) gene sequencing approach. Further, the influence of native midgut microbiota of mosquitoes on the development of rodent malaria parasite Plasmodium berghei in its host has been studied. METHODS: The microbial diversity associated with the midgut of An. stephensi mosquitoes was studied by sequencing V3 region of 16S ribosomal RNA (rRNA) gene. The influence of native midgut microbiota of An. stephensi mosquitoes on the susceptibility of the mosquitoes to rodent malaria parasite P. berghei was studied by comparing the intensity and prevalence of P. berghei infection among the antibiotic treated and untreated cohorts of mosquitoes. RESULTS: The analysis of bacterial diversity from the midguts of An. stephensi showed Proteobacteria as the most dominant population among the three laboratory-reared strains of An. stephensi studied. Major genera identified among these mosquito strains were Acinetobacter, Pseudomonas, Prevotella, Corynebacterium, Veillonella, and Bacillus. The mosquito infectivity studies carried out to determine the implication of total midgut microbiota on P. berghei infection showed that mosquitoes whose native microbiota cleared with antibiotics had increased susceptibility to P. berghei infection compared to the antibiotic untreated mosquitoes with its natural native microbiota. CONCLUSIONS: The use of microbial symbiont to reduce the competence of vectors involved in disease transmission has gained much importance in recent years as an emerging alternative approach towards disease control. In this context, the present study was aimed to identify the midgut microbiota composition of An. stephensi, and its effect on the development of P. berghei. Interestingly, the analysis of midgut microbiota from An. stephensi revealed the presence of genus Veillonella in Anopheles species for the first time. Importantly, the study also revealed the negative influence of total midgut microbiota on the development of P. berghei in three laboratory strains of An. stephensi, emphasizing the importance of understanding the gut microbiota in malaria vectors, and its relationship with parasite development in designing strategies to control malaria transmission.

Efficacy of two artemisinin-based combinations for the treatment of malaria in pregnancy in India: a randomized controlled trial.

BACKGROUND: In India, the recommended first-line treatment for malaria in the second and third trimester of pregnancy is artesunate + sulfadoxine-pyrimethamine (AS+SP). However, data on safety and efficacy of artemisinin-based combination therapy (ACT) in pregnancy is limited. This study assessed the safety and efficacy of AS+SP and artesunate + mefloquine (AS+MQ) for treatment of Plasmodium falciparum in pregnancy in India. METHODS: This open-label, randomized clinical trial was conducted from October 2010 to December 2013 at three sites in India (Ranchi and Jamshedpur in Jharkhand state, and Rourkela in Odisha state). Pregnant women in the second or third trimester who had P. falciparum mono-infection of any parasite density with or without fever were randomized to receive AS+SP or AS+MQ. Blood slides and filter paper samples for Polymerase Chain Reaction (PCR) were collected on days 0, 1, 2, 3, 14, 21, 28, 42 and 63 post treatment. Women were followed up at delivery and at day 42 postpartum. FINDINGS: Two hundred and forty-eight women of 7064 pregnant women (3.5%) who were screened at monthly antenatal clinics had a P. falciparum mono-infection and were randomized to receive AS+SP (125) or AS+MQ (123) and all of these women were included in the intention to treat (ITT) analysis. The primary endpoint of an adequate clinical and parasite response (ACPR) on day 63 was not available for 9 women who were counted as treatment failure in the ITT analysis. In the ITT population, the ACPR was 121/125 (96.8%; 95% Confidence interval (CI) 92.0-99.1%) in the AS+SP group and 117/123 (95.1%; 95% CI 89.7-98.2) in the AS+MQ group. Among the 239 women (121 from the AS+SP arm and 118 from the AS+MQ arm) who completed the day 63 follow up (per protocol analysis) the ACPR was 100% in the AS+SP group and 99.2% (117/118) in the AS+MQ group. There were five serious adverse events (SAE) among pregnant women (4 in the AS+SP group and 1 in the AS+MQ group) and 13 fetal/neonatal SAEs (7 in the AS+SP group and 6 in the AS+MQ) but none of them were related to the study drugs. A higher proportion of women in the AS+MQ arm reported vomiting within 7 days post-treatment than did women in the AS+SP arm (6.9 vs. 1.6%; p = 0.001). CONCLUSION: Both AS+SP and AS+MQ are safe and effective for treatment of uncomplicated falciparum malaria in pregnancy in India. Trial registration CTRI This study is registered with Clinical Trial Registry India (CTRI), number CTRI/2009/091/001055. Date of Registration 11 January 2010, http://ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=1185&EncHid=&userName=anvikar.

Insights into the early liver stage biology of Plasmodium.

Even though malaria is preventable and curable, it has become a serious threat to mankind. In 2016, there were an estimated 216 million cases of malaria across the world. The biology of its causative agent, i.e. Plasmodium parasite is full of complex mechanisms. There are five Plasmodium species responsible for malaria in humans, viz. Plasmodium falciparum, P. vivax, P. malariae, P. ovale and recently identified P. knowlesi that normally infect apes. In humans, malaria is spread by the injection of Plasmodium sporozoites through the bite of infectious Anopheles' female mosquito during their blood meal. From the time of entry into human skin till the development into the asexual forms, the parasite undergoes several transformations. This review attempts to understand the science behind the pre-erythrocytic liver stage of Plasmodium. Research articles explaining parasite biology, cell-traversal, transformation stages, cell-egress process, etc. were retrieved from PubMed and google scholar database. Various known and unknown mechanisms and strategies used by the malaria parasite P. berghei in rodent models have been discussed in this review. Limited or no information was available for humans, due to technical feasibility and complexity of parasite's life cycle. Hence, it was concluded that there is an urgent need to investigate the hepatic invasion, traversal and egress mechanism of P. falciparum and P. vivax for developing novel therapeutics to fight against malaria.