Genetic diversity of Plasmodium falciparum AMA-1 antigen from the Northeast Indian state of Tripura and comparison with global sequences: implications for vaccine development.

BACKGROUND: Malaria continues to be a major public health problem in the Northeastern part of India despite the implementation of vector control measures and changes in drug policies. To develop successful vaccines against malaria, it is important to assess the diversity of vaccine candidate antigens in field isolates. This study was done to assess the diversity of Plasmodium falciparum AMA-1 vaccine candidate antigen in a malaria-endemic region of Tripura in Northeast India and compare it with previously reported global isolates with a view to assess the feasibility of developing a universal vaccine based on this antigen. METHODS: Patients with fever and malaria-like illness were screened for malaria and P. falciparum positive cases were recruited for the current study. The diversity of PfAMA-1 vaccine candidate antigen was evaluated by nested PCR and RFLP. A selected number of samples were sequenced using the Sanger technique. RESULTS: Among 56 P. falciparum positive isolates, Pfama-1 was successfully amplified in 75% (n = 42) isolates. Allele frequencies of PfAMA-1 antigen were 16.6% (n = 7) for 3D7 allele and 33.3% (n = 14) in both K1 and HB3 alleles. DNA sequencing revealed 13 haplotypes in the Pfama-1 gene including three unique haplotypes not reported earlier. No unique amino-acid substitutions were found. Global analysis with 2761 sequences revealed 435 haplotypes with a very complex network composition and few clusters. Nucleotide diversity for Tripura (0.02582 ± 0.00160) showed concordance with South-East Asian isolates while recombination parameter (Rm = 8) was lower than previous reports from India. Population genetic structure showed moderate differentiation. CONCLUSIONS: Besides documenting all previously reported allelic forms of the vaccine candidate PfAMA-1 antigen of P. falciparum, new haplotypes not reported earlier, were found in Tripura. Neutrality tests indicate that the Pfama-1 population in Tripura is under balancing selection. This is consistent with global patterns. However, the high haplotype diversity observed in the global Pfama-1 network analysis indicates that designing a universal vaccine based on this antigen may be difficult. This information adds to the existing database of genetic diversity of field isolates of P. falciparum and may be helpful in the development of more effective vaccines against the parasite.

Development & validation of scales to assess stigma related to COVID-19 in India.

Background & objectives: COVID-19 pandemic has triggered social stigma towards individuals affected and their families. This study describes the process undertaken for the development and validation of scales to assess stigmatizing attitudes and experiences among COVID-19 and non-COVID-19 participants from the community. Methods: COVID-19 Stigma Scale and Community COVID-19 Stigma Scale constituting 13 and six items, respectively, were developed based on review of literature and news reports, expert committee evaluation and participants' interviews through telephone for a multicentric study in India. For content validity, 61 (30 COVID-19-recovered and 31 non-COVID-19 participants from the community) were recruited. Test-retest reliability of the scales was assessed among 99 participants (41 COVID-19 recovered and 58 non-COVID-19). Participants were administered the scale at two-time points after a gap of 7-12 days. Cronbach's alpha, overall percentage agreement and kappa statistics were used to assess internal consistency and test-retest reliability. Results: Items in the scales were relevant and comprehensible. Both the scales had Cronbach's α above 0.6 indicating moderate-to-good internal consistency. Test-retest reliability assessed using kappa statistics indicated that for the COVID-19 Stigma Scale, seven items had a moderate agreement (0.4-0.6). For the Community COVID-19 Stigma Scale, four items had a moderate agreement. Interpretation & conclusions: Validity and reliability of the two stigma scales indicated that the scales were comprehensible and had moderate internal consistency. These scales could be used to assess COVID-19 stigma and help in the development of appropriate stigma reduction interventions for COVID-19 infected, and mitigation of stigmatizing attitudes in the community.

Long telomeres cooperate with p53, MDM2, and p21 polymorphisms to raise pediatric solid tumor risk.

BACKGROUND: While leukocyte telomere length has been linked with altered risk in adult cancer, limited information is available on its association with risk in pediatric solid tumors. We investigated the association of telomeric alterations with risk of pediatric solid tumors. We also investigated whether altered telomeres cooperated with the TP53 rs1042522, MDM2 rs2279744 and CDKN1A (p21cip1 ) rs1059234 single-nucleotide polymorphisms to modify cancer risk. METHODS: A total of 101 tumor patients and 202 controls were recruited for this age- and gender-matched case-control study. Relative telomere length (RTL) was determined in peripheral blood leukocytes using quantitative real-time polymerase chain reaction (PCR), and the polymorphisms were genotyped using PCR-restriction fragment length polymorphism. RESULTS: Using median RTL in the healthy controls as a cut-off, children with longer telomeres were at an increased risk of developing a solid tumor (OR, 2.70; P < 0.01). When participants were categorized according to control RTL quartiles, a significant dose-response relationship was observed (χ2  = 10.95; P < 0.001). The risk for tumors increased nearly threefold (P = 0.001) for the triple interaction RTL × TP53 rs1042522 × p21cip1 rs1059234 compared with the maximum effect of any single factor, although the interaction effect was less than additive. The MDM2 rs2279744 GG genotype reduced pediatric solid tumor risk significantly (OR, 0.51). CONCLUSION: Combined analysis of telomeres and genetic polymorphisms in the TP53 pathway can provide important clues to understanding pediatric solid tumor etiology.

Association of VDR gene polymorphisms and 22 bp deletions in the promoter region of TLR2Δ22 (-196-174) with increased risk of pulmonary tuberculosis: A case-control study in tea garden communities of Assam.

BACKGROUND: A high number of pulmonary tuberculosis (PTB) cases have been reported from tea garden communities of Assam. Till date, no molecular epidemiological study was performed to investigate the association of candidate gene(s) with the risk PTB in this region. The present case-control study was aimed to investigate the association of vitamin D receptor (VDR) gene polymorphisms and 22 bp deletion in the promoter region of toll-like receptor 2 (TLR2) gene with the risk of PTB in tea garden communities of Assam. METHODS: Genotyping of VDR polymorphisms and TLR2Δ22 (-196-174) gene was carried out for 169 PTB cases and 227 apparently healthy community controls using blood samples by PCR-RFLP followed by DNA sequencing. For association study, both univariate and multivariate logistic regression analyses were performed. RESULTS: This study has shown that BsmI and FokI polymorphisms of VDR gene significantly associated with an increased risk of PTB (AOR = 3.58, 95% CI = 1.64-7.80, P < .01 for B/b genotype of BsmI and AOR = 2.44, 95% CI = 1.40-4.24, P < .01 for F/f genotype of FokI polymorphism). No significant association of TaqI and ApaI polymorphism of VDR gene was found with the risk of PTB. Moreover, this study has revealed that person carrying deletion allele in their TLR2Δ22 (-196-174) gene is significantly associated with an increased risk of PTB having b/b or F/f genotypes in BsmI or FokI polymorphisms of VDR gene. CONCLUSION: This study has revealed that BsmI and FokI polymorphisms of VDR gene significantly associated with an increased risk of PTB.

Association of toll-like receptor 2 ∆22 and risk for gastric cancer considering main effects and interactions with smoking: a matched case-control study from Mizoram, India.

Toll-like receptors (TLRs) are evolutionary conserved cell surface receptors of the innate immune system. Smoking has significant immunological effects which are mediated via TLRs on various receptor-mediated innate response pathways. Polymorphisms of TLR genes are associated with susceptibility toward various malignancies. The present study was undertaken to examine the association between TLR2 ∆22 and gastric cancer. In this study, we also investigated the interaction between TLR2 ∆22 and smoking. A total of 133 histologically confirmed gastric cancer cases and 266 age-sex-matched controls were selected for this study. TLR2 ∆22 genotypes were determined by allele-specific polymerase chain reaction (PCR). Binary conditional logistic regression analysis was used to find the association of TLR2 ∆22 with risk of gastric cancer. Logistic regression using hierarchically well-formulated models was used for interaction analysis between smoking and TLR2 ∆22. Persons having TLR2 ∆22 heterozygous genotype had two times increased risk of gastric cancer in multivariate logistic regression model. The interaction analysis using hierarchical logistic regression models between smoking and TLR2 ∆22 by calculating separate X (2) for interaction model and only main effect model, the difference of X (2) 57.68-47.70 = 9.98 and degrees of freedom (df) 5-3 = 2, revealed significant (α = 0.05, df = 2) omnibus interaction. Our present study revealed TLR2 ∆22 to be significantly and independently associated with gastric cancer risk in Mizoram, and there is also evidence of significant interaction between smoking and TLR2 ∆22 with risk of gastric cancer.

TLR2∆22 (-196-174) significantly increases the risk of breast cancer in females carrying proline allele at codon 72 of TP53 gene: a case-control study from four ethnic groups of North Eastern region of India.

Breast cancer (BC) is the second most common cancer in women. In the North Eastern Region (NER) of India, BC is emerging as an important concern as evidenced by the data available from population and hospital-based cancer registries. Studies on genetic susceptibility to BC are important to understand the increase in the incidence of BC in NER. The present case control study was conducted to investigate the association between tumour suppressor gene TP53 codon 72 polymorphism and innate immune pathway gene TLR2∆22 (-196-174) polymorphism with BC in females of NER of India for the identification of novel biomarker of BC. Four hundred sixty-two histopathologically confirmed BC cases from four states of NER of India, and 770 healthy controls were included by organizing community surveys from the neighbourhood of cases. In our study, no significant association between TP53 codon 72 polymorphisms and the risk of BC was found. However, our study has shown that TP53 codon 72 polymorphism is an important effect modifier. In the present study it was found that females carrying 22 base-pair deletion in the promoter region of their TLR2 gene had two times (AOR= 2.18, 95 % CI 1.13-4.21, p=0.019 in dominant model; AOR= 2.17, 95 % CI 1.09-4.34, p=0.027 in co-dominant model) increased risk of BC whwn they also carry proline allele at codon 72 of their TP53 gene.

Declining prevalence of pulmonary paragonimiasis following treatment & community education in a remote tribal population of Arunachal Pradesh, India.

BACKGROUND & OBJECTIVES: In India, human pulmonary paragonimiasis is an important public health problem in the northeastern (NE) region. In 2005 we reported a hyperendemic focus of paragonimiasis in a remote tribal village in the hills of Changlang district in Arunachal Pradesh. The community was made aware of the disease and all active cases were treated. This study was aimed to assess the decline in the prevelance of paragonimiasis in the same area after a re-survey done in 2011 after a gap of six years. METHODS: Re-surveys were carried to determine the reduction in the prevalence of paragonimiasis. Community education was given to the villagers to raise their awareness about paragonimiasis. A total of 624 individuals including 301 children (age 15 yr) were included in the study. Sputum and stool samples were examined for eggs of lung flukes. Serum samples were screened for IgG antibodies against lung fluke antigen by ELISA. RESULTS: A significant (P<0.001) decline in the prevalence of paragonimiasis was found. There was decline in both ELISA positivity and egg positivity. Antibody positivity against excretory-secretary (ES) antigen in children (age 15 yr) fell down from earlier 51.7 to 15.9 per cent and in individuals 16 - 30 yr of age the serological prevalence fell down from 22.4 to 8.2 per cent and in individuals aged th > 31 yr, the decline in prevalence was from 15.3 to 3.7 per cent. Gender-wise analysis revealed that the decline in ELISA positivity was similar in both genders and fell down from 33.9 to 11.5 per cent in males and from 29.8 to 10.7 per cent in females. Similarly, there was a significant decline rate in egg positivity also. INTERPRETATION & CONCLUSIONS: The strategy of hotspot targeted active paragonimiasis case detection and treatment of infected cases together with community education appears to be feasible methods to achieve control of paragonimiasis in this region.

First Report of Anopheles annularis s.l., An. maculatus s.s., and An. culicifacies s.l. as Malaria Vectors and a New Occurrence Record for An. pseudowillmori and An. sawadwongporni in Alipurduar District Villages, West Bengal, India.

A comprehensive entomological survey was undertaken in Alipurduar District, West Bengal, from 2018 to 2020 and in 2022. This study was prompted by reported malaria cases and conducted across nine villages, seven Sub-Centres, and three Primary Health Centres (PHCs). Mosquitoes were hand-collected with aspirators and flashlights from human dwellings and cattle sheds during the daytime. Both morphological and molecular techniques were used for species identification. Additionally, mosquitoes were tested for Plasmodium parasites and human blood presence. Mosquito species such as An. barbirostris s.l., An. hyrcanus s.l., An. splendidus, and An. vagus were morphologically identified. For species like An. annularis s.l., An. minimus s.s., An. culicifacies s.l., and An. maculatus s.s., a combination of morphological and molecular techniques was essential. The mitochondrial cytochrome c oxidase gene subunit 1 (CO1) was sequenced for An. annularis s.l., An. maculatus s.s., An. culicifacies s.l., An. vagus, and some damaged samples, revealing the presence of An. pseudowillmori and An. fluviatilis. The major Anopheles species were An. annularis s.l., An. culicifacies s.l., and An. maculatus s.s., especially in Kumargram and Turturi PHCs. Plasmodium positivity was notably high in An. annularis s.l. and An. maculatus s.s. with significant human blood meal positivity across most species. Morphological, molecular, and phylogenetic analyses are crucial, especially for archived samples, to accurately identify the mosquito fauna of a region. Notably, this study confirms the first occurrence of An. pseudowillmori and An. sawadwongporni in West Bengal and implicates An. maculatus s.s., An. culicifacies s.l., and An. annularis s.l. as significant vectors in the Alipurduar region.

Presence of three distinct genotypes within the Paragonimus westermani complex in northeastern India.

The name Paragonimus westermani (Kerbert, 1878) is commonly applied to members of a species complex that includes the well-known Asian lung fluke of medical and veterinary importance. Unambiguous molecular and morphological evidence showing the presence of a member of the complex in India has recently been published. In the present study we report the occurrence of 2 more members of the P. westermani complex in northeastern (NE) India. Surveys of the freshwater crabs Maydelliatelphusa lugubris in NE India revealed 2 morphologically distinct types of lung fluke metacercariae. Phylogenetic analyses, using DNA sequences from ITS2, 28S and cox1 gene regions indicate that these lung metacercariae belong to P. westermani complex. Type 1 metacercariae have a more basal position within the complex whereas type 2 metacercariae are closely related to the relatively derived forms of P. westermani from NE Asia (Japan, Korea, China) and Vietnam. A third type of metacercaria (type 3), detected in another crab host, Sartoriana spinigera in Assam, was phylogenetically close to P. siamensis, also a member of the P. westermani group. Molecular evidence has demonstrated the existence of 3 genotypes of lung flukes within the Paragonimus westermani complex in NE India. Two of these were previously unknown.

Immunoblotting Identification of Diagnostic Antigens of Paragonimus westermani Type 1 for the Detection of Human Pulmonary Paragonimiasis in North East India.

Human pulmonary paragonimiasis, an emerging concern in North East India, frequently masquerades as pulmonary tuberculosis due to clinical and radiological similarities, leading to diagnostic challenges. This research aimed to harness the immunoblotting technique to discern immunodiagnostic protein antigens from both adult worm and excretory-secretory (ES) extracts of the prevalent Paragonimus westermani type 1 in Arunachal Pradesh, North East India. We studied the time kinetics of immunoreactive patterns in relation to the duration of infection in rodent models. Immunoblot analyses were also conducted using sera from ELISA-positive patients confirmed with paragonimiasis, facilitating the selection of antigenic extracts with diagnostic potential. Further, ES protein antigens were subjected to 2D immunoblot analysis and immunoreactive protein spots identified using MALDI-TOF MS. The immunoreactivity patterns of ES antigens with sera of paragonimiasis-positive patients were detailed, and specific immunoreactive protein antigens were pinpointed using peptide mass fingerprinting (MALDI-TOF). This work underscores the enhanced diagnostic accuracy when combining ELISA with immunoblotting for pulmonary paragonimiasis in regions like North East India, marked by co-existing helminth infections.

Molecular diversity of Mycobacterium tuberculosis isolates in Treatment-experienced Patients from Andhra Pradesh, India.

INTRODUCTION: To get a comprehensive idea about the transmission and epidemiology of TB globally and locally, the use of molecular typing methods has become imperative not only for understanding genetic diversity but also the population structure of Mycobacterium tuberculosis complex (MTBC). We aimed to investigate the drug resistance pattern and genetic diversity of MTBC among previously treated patients with sputum smear-positive pulmonary tuberculosis in a South Indian population. METHODOLOGY: 104 patients with sputum smear positivity and who had previously undergone treatment were selected. Drug susceptibility testing, Spoligotyping, MIRU-VNTR, and SNP typing were performed. RESULTS: Mono-resistance to isoniazid 16 (15.38%) was the highest among all drugs. Out of 104 isolates, 24 (23%) isolates were classified as MDR strains. The distributions of most common lineages were: EAI3-Ind-20 (19.23%), EAI5-13 (12.50%), Beijing-12 (11.54%), CAS1-Delhi- 9 (8.65%), and 7 (6.73%) each of T-H37rv, Unknown and Orphan types. MIRU-VNTR-based analysis revealed that there are two major groups: CAS1-Delhi and Beijing groups. Out of 104 isolates, 82 belonged to well-defined lineages and 6 clusters, and the remaining 22 were singletons. SNP analysis showed no mutations associated with five sets of genes in 33 strains. CONCLUSIONS: The occurrence of 11.54% Beijing strains in South India is an important finding. High frequency of Isoniazid mono resistance noticed. Spoligotyping along with MIRU-VNTR and SNP typing is the best approach to the identification of strain lineages. No mutation with Antigen85C gene represents, can be used for vaccine candidates.

First Report of Rubber Collection Bowls & Plastic and Bamboo Water Containers as the Major Breeding Source of Ae. albopictus with the Indigenous Transmission of Dengue and Chikungunya in Rural Forested Malaria-Endemic Villages of Dhalai District, Tripura, India: The Importance of Molecular Identification.

BACKGROUND: With the reports of indigenous cases of dengue and chikungunya in the forest-covered rural tribal malaria-endemic villages of Dhalai District, Tripura, India, an exploratory study was undertaken to identify the vector breeding sites. METHODS: From June 2021 to August 2022, mosquito larvae were collected from both natural and artificial sources in the villages, house premises, and their nearby forested areas outside of the houses. Other than morphological characterisation, Aedes species were confirmed by polymerase chain reaction targeting both nuclear (ITS2) and mitochondrial genes (COI) followed by bidirectional Sanger sequencing. RESULTS: Aedes albopictus was abundantly found in this area in both natural and artificial containers, whereas Ae. aegypti was absent. Among the breeding sources of molecularly confirmed Ae. albopictus species, rubber collection bowls were found to be a breeding source reported for the first time. Plastic and indigenously made bamboo-polythene containers for storing supply water and harvesting rainwater in the villages with a shortage of water were found to be other major breeding sources, which calls for specific vector control strategies. Natural sources like ponds and rainwater collected on Tectona grandis leaves and Colocasia axil were also found to harbour the breeding, along with other commonly found sources like bamboo stumps and tree holes. No artificial containers as a breeding source were found inside the houses. Mixed breeding was observed in many containers with other Aedes and other mosquito species, necessitating molecular identification. We report six haplotypes in this study, among which two are reported for the first time. However, Aedes aegypti was not found in the area. Additionally, rubber collection bowls, ponds, and water containers also showed the presence of Culex quinquefasciatus and Culex vishnui, known JE vectors from this area, and reported JE cases as well. Different Anopheles vector spp. from this known malaria-endemic area were also found, corroborating this area as a hotbed of several vectors and vector-borne diseases. CONCLUSIONS: This study, for the first time, reports the breeding sources of Aedes albopictus in the forested areas of Tripura, with rubber collection bowls and large water storage containers as major sources. Also, for the first time, this study reports the molecular characterisation of the Ae. albopictus species of Tripura, elucidating the limitations of morphological identification and highlighting the importance of molecular studies for designing appropriate vector control strategies. The study also reports the co-breeding of JE and malaria vectors for the first time in the area reporting these vector-borne diseases.

Vaccination coverage and breakthrough infections of COVID-19 during the second wave among staff of selected medical institutions in India.

India experienced the second wave of SARS-CoV-2 infection from April 3 to June 10, 2021. During the second wave, Delta variant B.1617.2 emerged as the predominant strain, spiking cases from 12.5 million to 29.3 million (cumulative) by the end of the surge in India. Vaccines against COVID-19 are a potent tool to control and end the pandemic in addition to other control measures. India rolled out its vaccination programme on January 16, 2021, initially with two vaccines that were given emergency authorization-Covaxin (BBV152) and Covishield (ChAdOx1 nCoV- 19). Vaccination was initially started for the elderly (60+) and front-line workers and then gradually opened to different age groups. The second wave hit when vaccination was picking up pace in India. There were instances of vaccinated people (fully and partially) getting infected, and reinfections were also reported. We undertook a survey of staff (front line health care workers and supporting) of 15 medical colleges and research institutes across India to assess the vaccination coverage, incidence of breakthrough infections, and reinfections among them from June 2 to July 10, 2021. A total of 1876 staff participated, and 1484 forms were selected for analysis after removing duplicates and erroneous entries (n = 392). We found that among the respondents at the time of response, 17.6% were unvaccinated, 19.8% were partially vaccinated (received the first dose), and 62.5% were fully vaccinated (received both doses). Incidence of breakthrough infections was 8.7% among the 801 individuals (70/801) tested at least 14 days after the 2nd dose of vaccine. Eight participants reported reinfection in the overall infected group and reinfection incidence rate was 5.1%. Out of (N = 349) infected individuals 243 (69.6%) were unvaccinated and 106 (30.3%) were vaccinated. Our findings reveal the protective effect of vaccination and its role as an essential tool in the struggle against this pandemic.

Gender differences in COVID-19 knowledge, risk perception, and public stigma among the general community: Findings from a nationwide cross-sectional study in India.

Introduction: Individual and community characteristics predictive of knowledge, perception, and attitude on COVID-19, specifically on gender, have not been adequately explored. Objective: To examine the gender differences in COVID-19 knowledge, self-risk perception and public stigma among the general community and to understand other socio-demographic factors which were predictive of them. Method: A nationally representative cross-sectional multi-centric survey was conducted among adult individuals(≥18 yrs) from the community member (N = 1978) from six states and one union territory of India between August 2020 to February 2021. The participants were selected using systematic random sampling. The data were collected telephonically using pilot-tested structured questionnaires and were analyzed using STATA. Gender-segregated multivariable analysis was conducted to identify statistically significant predictors (p < 0.05) of COVID-19-related knowledge, risk perception, and public stigma in the community. Results: Study identified significant differences between males and females in their self-risk perception (22.0% & 18.2% respectively) and stigmatizing attitude (55.3% & 47.1% respectively). Highly educated males and females had higher odds of having COVID-19 knowledge (aOR: 16.83: p < 0.05) than illiterates. Highly educated women had higher odds of having self-risk perception (aOR: 2.6; p < 0.05) but lower public stigma [aOR: 0.57; p < 0.05]. Male rural residents had lower odds of having self-risk perception and knowledge [aOR: 0.55; p < 0.05 & aOR: 0.72; p < 0.05] and female rural residents had higher odds of having public stigma [aOR: 1.36; p < 0.05]. Conclusion: Our study findings suggest the importance of considering thegender differentials and their background, education status and residential status in designing effective interventions to improve knowledge and reduce risk perception and stigma in the community about COVID-19.

Challenges in Accessing and Delivering Maternal and Child Health Services during the COVID-19 Pandemic: A Cross-Sectional Rapid Survey from Six States of India.

BACKGROUND/OBJECTIVES: Globally, the COVID-19 pandemic and its prevention and control policies have impacted maternal and child health (MCH) services. This study documents the challenges faced by patients in accessing MCH services, and the experiences of health care providers in delivering those services during the COVID-19 outbreak, explicitly focusing on the lockdown period in India. METHODS: A cross-sectional study (rapid survey) was conducted in 18 districts from 6 states of India during March to June, 2020. The sample size included 540 MCH patients, 18 gynaecologists, 18 paediatricians, 18 district immunisation officers and 108 frontline health workers. Bivariate analysis and multivariable analysis were used to assess the association between sociodemographic characteristics, and challenges faced by the patients. RESULTS: More than one-third of patients (n = 212; 39%) reported that accessing MCH services was a challenge during the lockdown period, with major challenges being transportation-related difficulties (n = 99; 46%) unavailability of hospital-based services (n = 54; 23%) and interrupted outreach health services (n = 39; 18.4%). The supply-side challenges mainly included lack of infrastructural preparedness for outbreak situations, and a shortage of human resources. CONCLUSIONS/RECOMMENDATIONS: A holistic approach is required that focuses on both preparedness and response to the outbreak, as well reassignment and reinforcement of health care professionals to continue catering to and maintaining essential MCH services during the pandemic.

High prevalence of neurocysticercosis among patients with epilepsy in a tertiary care hospital of Assam, India.

Background: Neurocysticercosis (NCC) is a parasitic disease of the central nervous system, which is caused by the metacestode of the pork tapeworm, Taenia solium. The present unicentric, hospital-based, cross-sectional study was undertaken to assess the contribution of NCC as a cause of active epilepsy among patients attending a tertiary health care center in Assam, India. Materials and Methods: Over a period of 2 years, 152 active epilepsy patients were investigated based on clinical, epidemiological, neuroimaging (contrast-enhanced computerized tomography), and immunological techniques to establish the diagnosis of NCC. A precoded questionnaire was administered to patients and/or guardians to collect detailed medical history. Results: Ninety-three cases (61.2%) fulfilled either definitive or probable diagnostic criteria for NCC. Anti-cysticercus immunoglobulin G antibodies were detected by ELISA and enzyme electro-immune transfer blot in 69 (45.4%) active epilepsy patients. Seroprevalence was higher in males, 46.6% (54/116); than in females, 41.7% (15/36), and increased significantly with age; peaking in the 20-39 years age group (36/76; χ2 = 5.64; P = 0.02). Among the seropositive cases, 54 (78.3%) were diagnosed with NCC. A significantly higher number of seropositive individuals were diagnosed with NCC in the 20-39 years age group as compared to the 40 years and above age group (χ2 = 6.28; P = 0.01). The association between seropositivity for NCC, and the number of lesions in the brain was statistically significant (χ2 = -8.33; P = 0.003). Conclusions: This study indicates that NCC is a major cause of active epilepsy in Assam. A high prevalence of pediatric NCC is also a major concern.

Reply to Deora et al. Multiplexing for Plasmodium spp.? Think Again! Comment on "Bhowmick et al. Dry Post Wintertime Mass Surveillance Unearths a Huge Burden of P. vivax, and Mixed Infection with P. vivaxP. falciparum, a Threat to Malaria Elimination, in Dhalai, Tripura, India. Pathogens 2021, 10, 1259".

We thank Deora et al. [...].