Real-time loop-mediated isothermal amplification (real-time LAMP) assay for rapid diagnosis of amoebic liver abscess.

Among the parasitic diseases, amoebic liver abscess (ALA) ranks second to malaria in terms of mortality. Due to the poor sensitivity of conventional diagnostic methods, there is a need for the development of effective and rapid diagnostic methods for ALA. Thus, the purpose of this work was to develop a real-time loop-mediated isothermal amplification (RT-LAMP) assay specific to Entamoeba histolytica. Further, we compared the performance of real-time LAMP with conventional and real-time PCR (RT-PCR) targeting 18S small subunit ribosomal RNA (18S SSU rRNA) gene of E. histolytica in patients with ALA. A total of 126 liver samples were obtained for the study. Of these, 96 aspirated pus samples were obtained from patients suffering from an ALA (serology confirmed, anti-amoebic immunoglobulin IgG positive), 19 aspirated pus samples from patients with pyogenic liver abscess (PLA, 16S RNA gene positive) and 11 autopsy liver tissues. The results showed that the DNA of E. histolytica was detected in 81 samples by conventional PCR, 93 by RT-PCR and 95 by RT-LAMP. The analytical sensitivity of the RT-LAMP assay was much higher than the other two techniques. RT-LAMP assay was able to amplify up to one copy of the targeted gene of E. histolytica while conventional PCR and RT-PCR could amplify up to 103 and 102 copies of the targeted gene of E. histolytica, respectively. In conclusion, RT-LAMP proved to be a sensitive, specific and rapid test which can be utilised as an effective tool for the diagnosis of ALA.

A rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for detection of Entamoeba histolytica and Giardia duodenalis.

We developed a rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for two common intestinal parasites-Entamoeba histolytica and Giardia duodenalis, where early detection may be helpful. The mLAMP assay was optimized for the detection of DNA of E. histolytica (18S rRNA gene) and G. duodenalis (Elongation factor 1 alpha gene) from standard strains by using six specific primers FIP (forward inner primer), BIP (backward inner primer), F3 (forward outer primer), B3 (backward outer primer), loopF (forward loop primer), and loopB (backward loop primer) for each gene target. The amplification time was 16-26 min for E. histolytica and 10-15 min for G. duodenalis, and the parasites could be distinguished based on melting-curve analysis for specific annealing temperatures (Tm) of 84°C-86°C and 88°C-90°C for E. histolytica and G. duodenalis, respectively. The analytical sensitivity was one fg, and no cross-reactivity with other intestinal pathogens was observed. Thus, the mLAMP assay could detect and clearly distinguish E. histolytica and G. duodenalis with a rapid turnaround time and excellent analytical sensitivity and specificity.

Impact of Community Based Nutrition Intervention in the Prevention and Control of Anaemia Among Reproductive Aged Women of India.

INTRODUCTION: Maternal and child under-nutrition is particularly widespread in low and middle-income nations, increasing the overall disease burden due to poor nutritional status. The aim of this study was to develop nutrition intervention for the prevention and control of anaemia among women of reproductive age. METHODS: Community-based intervention study was conducted among 443 women of reproductive age group (15-49 years) to determine the effectiveness of a 6-month nutrition intervention package. The nutrition intervention was developed by using Precede-Proceed model and the trans-theoretical model of behavior change. Multi-channel communication approach was adopted and nutrition intervention package was provided. Assessment of haemoglobin, red blood cells, platelet, ferritin, folate, vitamin B12, haematocrit test, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red cell distribution width and total leucocyte count was compared at the baseline and endline after the intervention among the participants. The chi-square test of independence and t-test were performed. RESULTS: The only mean ferritin level shows significant improvement (p < 0.001). A significant decrease (~ 15%, p = 0.027) in anaemia was observed after the intervention. CONCLUSIONS: Improvement in anaemic status of women was observed. National schemes and programs require a more robust strategical implementation like food fortification/bio fortification and behaviour change communication at village level to enhance the availability and accessibility of fortified food.

Comparative efficacy of natural aquatic predators for biological control of mosquito larvae: A neglected tool for vector control.

Mosquitoes are a dominant fraction of dipteran fauna, occupying a variety of niches. The most common method deployed for their control is the use of insecticides. Throughout their life cycle they are exposed to a wide range of predators in different habitats, thus biological control of mosquitoes by using aquatic predators has been suggested. Therefore, the present study was carried out to explore the type of natural predators coexisting with the mosquito larvae in still water bodies and to determine their efficacy as predators for mosquito larvae. A coexistence of different predators with mosquito larvae was observed in 27 standing water bodies of Chandigarh, India. The predation efficiency of tadpoles of frog was comparable to Gambusia fish, as 97% of the mosquito larvae of all instars of the medically important mosquito genera Anopheles, Aedes, Culex and Armigeres were preyed. The toad tadpoles were found to be least effective and their predation rate was found to be negligible. Further studies on larval source management by frog tadpoles in combination with insecticides or stand-alone would be worthwhile.

Dynamics of Plasmodium berghei NK-65 parasitaemia and CD3+CD4+CD25+Fox-p3+ T-regulatory cells in experimentally induced malaria during early, mid, and late-pregnancy in BALB/c mice.

Introduction: Malaria in pregnancy causes a dual brunt on the mother as well as the foetus. Upregulation of T-regulatory cells (Tregs) during pregnancy allows tolerance towards the growing foetus, their suppression predisposes the mother to infections. This study analyzed the levels of CD3+CD4+CD25+Fox-p3+ Tregs, parasitaemia, maternal and foetal outcomes in BALB/c mice infected with P. berghei NK65 during early-, mid-, and late-pregnancy. Methodology: Total of 114 mice, non-pregnant non-infected (n = 6), non-pregnant infected (n = 12), pregnant non-infected (n = 48) and pregnant infected (n = 48) were included in the study. Infected groups were inoculated intra-peritoneally with 1 × 106 P. berghei infected RBCs during early-, mid-, and late- pregnancy (D6, D10, and D14 respectively). Six mice from each stage were sacrificed on the 5th and 7th day post-infection (DPI) to evaluate parasitaemia (staining) and Tregs from splenocytes (by flow cytometry). Results: The parasitaemia was significantly higher among early pregnancy infected mice (≥ 70%) than mid-pregnancy infected (40-70%), late pregnancy infected (50-65%), and non-pregnant infected mice (≤ 50%) (p < 0.05). The level of Tregs was significantly higher among non-pregnant infected mice as compared to non-pregnant non-infected mice (%Tregs 0.86 vs. 0.44). Among pregnant mice, the levels of Tregs in infected mice were lower than in non-infected mice during all stages of pregnancy. None of the mice infected during early- and mid-pregnancy survived at 6DPI and 7DPI, respectively, and those infected during late-pregnancy delivered premature pups. Conclusion: In contrast to non-pregnant mice, the levels of Tregs among pregnant mice decrease when malaria infection is acquired thereby leading to adverse pregnancy outcomes. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01089-2.

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.

Global scenario of Plasmodium vivax occurrence and resistance pattern.

Malaria caused by Plasmodium vivax is comparatively less virulent than Plasmodium falciparum, which can also lead to severe disease and death. It shows a wide geographical distribution. Chloroquine serves as a drug of choice, with primaquine as a radical cure. However, with the appearance of resistance to chloroquine and treatment has been shifted to artemisinin combination therapy followed by primaquine as a radical cure. Sulphadoxine-pyrimethamine, mefloquine, and atovaquone-proguanil are other drugs of choice in chloroquine-resistant areas, and later resistance was soon reported for these drugs also. The emergence of drug resistance serves as a major hurdle to controlling and eliminating malaria. The discovery of robust molecular markers and regular surveillance for the presence of mutations in malaria-endemic areas would serve as a helpful tool to combat drug resistance. Here, in this review, we will discuss the endemicity of P. vivax, a historical overview of antimalarial drugs, the appearance of drug resistance and molecular markers with their global distribution along with different measures taken to reduce malaria burden due to P. vivax infection and their resistance.

In silico study to predict promiscuous T cell and B cell-epitopes derived from the vaccine candidate antigens of Plasmodium vivax binding to MHC class-II alleles.

Malaria is one of the major causes of health and disability globally, even after tremendous efforts to eradicate it. Till date no highly effective vaccine is available for its control. The primary reason for the low efficacy of vaccines is extensive polymorphism in potential vaccine candidate antigen genes and HLA polymorphisms in the human population. This problem can be resolved by developing a vaccine using promiscuous peptides to combine the number of HLA alleles. This study predicted T and B cell epitopes (promiscuous peptides) by targeting PPPK-DHPS and DHFR-TS proteins of Plasmodium vivax, using different in silico tools. Selected peptides were characterized as promiscuous peptides on the basis of their immunogenicity, antigenicity and hydrophobicity. Furthermore, to confirm their immunogenicity, these peptides were utilized for molecular modelling and docking analysis. For determining the requisite affinity with distinct HLA Class-I, and HLA Class-II alleles, only five peptides for DHFR-TS and 3 peptides for PPPK-DHPS were chosen as promiscuous peptides. The D1 peptide has the maximum binding energy with HLA alleles, according to HLA-peptide complex modelling and binding interaction analyses. These findings could lead to the development of epitope-based vaccinations with improved safety and efficacy. These epitopes could be major vaccine targets in P. vivax as they possess a higher number of promiscuous peptides. Also, the B cell epitopes possess maximum affinity towards different alleles as analyzed by docking scores. However, further investigation is warranted in vitro and in vivo.

Distribution pattern of amino acid mutations in chloroquine and antifolate drug resistance associated genes in complicated and uncomplicated Plasmodium vivax isolates from Chandigarh, North India.

BACKGROUND: The increasing antimalarial drug resistance is a significant hindrance to malaria control and elimination programs. For the last six decades, chloroquine (CQ) plus pyrimethamine remains the first-line treatment for P. vivax malaria. Regions where both P. falciparum and P. vivax co-exist, P. vivax is exposed to antifolate drugs due to either misdiagnosis or improper treatment that causes selective drug pressure to evolve. Therefore, the present study aims to estimate antimalarial drug resistance among the complicated and uncomplicated P. vivax patients. METHODS: A total of 143 P. vivax malaria positive patients were enrolled in this study, and DNA was isolated from their blood samples. Pvcrt-o, Pvmdr-1, Pvdhps, and Pvdhfr genes were PCRs amplified, and drug resistance-associated gene mutations were analyzed. Statistical analysis of the drug resistance genes and population diversity was performed using MEGA vs. 7.0.21 and DnaSP v software. RESULTS: Among the CQ resistance marker gene Pvcrt-o, the prevalence of K10 insertion was 17.5% (7/40) and 9.5% (7/73) of complicated and uncomplicated P vivax group isolates respectively. In Pvmdr-1, double mutant haplotype (M958/L1076) was found in 99% of the clinical isolates. Among the pyrimethamine resistance-associated gene Pvdhfr, the double mutant haplotype I13P33F57R58T61N117I173 was detected in 23% (11/48) in complicated and 20% (17/85) in uncomplicated group isolates. In the sulphadoxine resistance-associated Pvdhps gene, limited polymorphism was observed with the presence of a single mutant (D459A) among 16 and 5% of the clinical isolates in the complicated and uncomplicated group respectively. CONCLUSION: The study presents the situations of polymorphism in the antimalarial drug resistance-associated genes and emphasizes the need for regular surveillance. It is imperative for the development of suitable antimalarial drug policy in India.

Role of Polymerase Chain Reaction in Stool and Duodenal Biopsy for Diagnosis of Giardiasis in Patients with Persistent/Chronic Diarrhea.

BACKGROUND: Giardia duodenalis is a common cause of chronic diarrhea especially in tropical countries. Diagnosis is based on microscopy (three stool samples) for trophozoites/cysts. Role of stool or duodenal biopsy PCR as a diagnostic method needs to be defined. We conducted a prospective study to determine the diagnostic characteristics of G. duodenalis stool and duodenal biopsy PCR in comparison to stool microscopy (reference standard). Later, we compared other techniques with stool PCR, considering it as new reference standard and characterized the type of Giardia assemblage. METHODS: G. duodenalis stool nested PCR was first evaluated using 40 positive controls and 50 negative controls considering stool microscopy as reference standard. Patients with chronic diarrhea (n = 100) were evaluated by stool microscopy and nested PCR. In 30 patients in whom upper gastrointestinal endoscopy was performed, duodenal biopsy samples were obtained and evaluated by histopathology, imprint cytology, and nested PCR. The type of Giardia assemblage was detected by assemblage-specific PCR. RESULTS: Stool nested PCR was found to have sensitivity and specificity of 100% and 94%, respectively, compared to stool microscopy. In patients with chronic diarrhea, 48% had evidence of Giardia infection. Stool microscopy detected 65%, stool PCR detected an additional 27%, and duodenal biopsy PCR detected an additional 8% of cases. The commonest assemblage found was assemblage B. Clinical and demographic characteristics were similar in patients harboring either assemblage A or B. CONCLUSION: Stool PCR is more sensitive than stool microscopy. By utilizing stool microscopy, stool nested PCR, and duodenal biopsy PCR in sequential manner, diagnostic yield can be increased.

Designing peptide-based vaccine candidates for Plasmodium falciparum erythrocyte binding antigen 175.

Plasmodium falciparum leads to a virulent form of malaria. Progress has been achieved in understanding the mechanisms involved in the malarial infection, still there is no effective vaccine to prevent severe infection. An effective vaccine against malaria should be one which can induce immune responses against multiple epitopes in the context of predominantly occurring HLA alleles. In this study, an integrated approach was employed to identify promiscuous peptides of a well-defined sequence of erythrocyte binding antigen-175 and promiscuous peptides for HLA alleles were designed using bioinformatics tools. A peptide with 15 amino acids (ILAIAIYESRILKRK) was selected based on its high binding affinity score and synthesized. This promiscuous peptide was used as stimulating antigen in lymphoproliferative responses to evaluate the cellular immune response. It was observed this peptide evokes lymphoproliferative and cytokine responses in individuals naturally exposed to the malaria parasite. The intensity of PBMCs proliferation was observed to be higher in sera obtained from P. falciparum exposed as compared to unexposed healthy individuals, suggesting earlier recognition of peptide of this region by T cells. Furthermore, the binding mode of HLA-peptide complex and their interaction may lead to a rational and selective peptide-based vaccine candidate design approach which can be used as a malaria prophylaxis.

Exploration of genetic diversity of Plasmodium vivax circumsporozoite protein (Pvcsp) and Plasmodium vivax sexual stage antigen (Pvs25) among North Indian isolates.

BACKGROUND: Malaria is one of the important vector-borne diseases with high fatality rates in tropical countries. The pattern of emergence and spread of novel antigenic variants, leading to escape of vaccine-induced immunity might be factors responsible for severe malaria. A high level of polymorphism has been reported among malarial antigens which are under selection pressure imposed by host immunity. There are limited reports available on comparative stage-specific genetic diversity among Plasmodium vivax candidate genes in complicated vivax malaria. The present study was planned to study genetic diversity (Pvcsp and Pvs25) among complicated and uncomplicated P. vivax isolates. METHODS: Pvcsp and Pvs2-specific PCRs and DNA sequencing were performed on P. vivax PCR positive samples. Genetic diversity was analysed using appropriate software. RESULTS: The present study was carried out on 143 P. vivax clinical isolates, collected from Postgraduate Institute of Medical Education and Research, Chandigarh. Among the classic and variant types of Pvcsp, the VK210 (99%; 115/116) was found to be predominant in both complicated and uncomplicated group isolates. Out of the various peptide repeat motifs (PRMs) observed, GDRADGQPA (PRM1) and GDRAAGQPA (PRM2) was the most widely distributed among the P. vivax isolates. Whereas among the Pvs25 isolates, 100% of double mutants (E97Q/I130T) in both the complicated (45/45) as well as in the uncomplicated (81/81) group was observed. CONCLUSION: An analysis of genetic variability enables an understanding of the role of genetic variants in severe vivax malaria.

In vitro anti-malarial efficacy of chalcones: cytotoxicity profile, mechanism of action and their effect on erythrocytes.

BACKGROUND: Malaria extensively leads to mortality and morbidity in endemic regions, and the emergence of drug resistant parasites is alarming. Plant derived synthetic pharmaceutical compounds are found to be a foremost research to obtain diverse range of potent leads. Amongst them, the chalcone scaffold is a functional template for drug discovery. The present study involves synthesis of ten chalcones with various substitution pattern in rings A and B and assessment of their anti-malarial efficacy against chloroquine sensitive and chloroquine resistant strains as well as of their cytotoxicity and effect on haemozoin production. METHODS: The chalcones were synthesized by Claisen-Schmidt condensation between equimolar quantities of substituted acetophenones and aryl benzaldehydes (or indole-3-carboxaldehyde) and were screened for anti-malarial activity by WHO Mark III schizont maturation inhibition assay. The cytotoxicity profile of a HeLa cell line was evaluated through MTT viability assay and the selectivity index (SI) was calculated. Haemozoin inhibition assay was performed to illustrate mode of action on a Plasmodium falciparum strain. RESULTS: The IC50 values of all compounds were in the range 0.10-0.40 µg/mL for MRC-2 (a chloroquine sensitive strain) and 0.14-0.55 µg/mL for RKL-9 (a chloroquine resistant strain) of P. falciparum. All the chalcones showed low cellular toxicity with minimal haemolysis. The statistically significant reduction (p < 0.05) in the haemozoin production suggests a similar mechanism than that of chloroquine. CONCLUSIONS: Out of ten chalcones, number 7 was found to be a lead compound with the highest potency (IC50 = 0.11 µg/mL), as compared to licochalcone (IC50 = 1.43 µg/mL) and with high selectivity index of 85.05.

A strategic approach to the synthesis of ferrocene appended chalcone linked triazole allied organosilatranes: Antibacterial, antifungal, antiparasitic and antioxidant studies.

A series of ferrocene appended chalcone allied triazole coupled organosilatranes (FCTSa 7-FCTSa 12) were synthesised with the aim of amalgamating the pharmacological action of the constituting moieties into a single molecular scaffold. All the synthesised silatranes were well characterized by various spectroscopic techniques like IR, 1H NMR, 13C NMR and elemental analysis. Organosilatranes were then evaluated for their biological alacrity against bacterial and fungal strains compared with the standard drugs Rifampicin and Amphotericin B respectively. The ferrocene conjugates were found to be only moderately effective against the tested microbes. However, the organosilatranes conceded excellent efficacy against parasite G. lamblia with FCTSa 11 arraying the leading results. On the other hand against another parasite T. vaginalis, FCTSa 8 has emerged as an outstanding composite. Further, Total Antioxidant Assay (TAA) with 2,2'-azino-bis-3-(ethylbenzothiazoline-6-sulphonic acid) revealed FCTSa 10 to be the best claimant for radical scavenging activity. Along these lines, introducing some different substituents in the synthesised hybrids may act as a useful strategy for increasing the biological profile of the drugs.

Screening and identification of potential novel biomarker for diagnosis of complicated Plasmodium vivax malaria.

BACKGROUND: In the recent years Plasmodium vivax has been reported to cause severe infections associated with mortality. Clinical evaluation has limited accuracy for the early identification of the patients progressing towards the fatal condition. Researchers have tried to identify the serum and the plasma-based indicators of the severe malaria. Discovery of MicroRNA (miRNA) has opened up an era of identification of early biomarkers for various infectious and non-infectious diseases. MicroRNAs (miRNA) are the small non-coding RNA molecules of length 19-24 nts and are responsible for the regulation of the majority of human gene expressions at post transcriptional level. METHODS: We identified the differentially expressed miRNAs by microarray and validated the selected miRNAs by qRT-PCR. We assessed the diagnostic potential of these up-regulated miRNAs for complicated P. vivax malaria. Futher, the bioinformtic analysis was performed to construct protein-protein and mRNA-miRNA networks to identify highly regulated miRNA. RESULTS: In the present study, utility of miRNA as potential biomarker of complicated P. vivax malaria was explored. A total of 276 miRNAs were found to be differentially expressed by miRNA microarray and out of which 5 miRNAs (hsa-miR-7977, hsa-miR-28-3p, hsa-miR-378-5p, hsa-miR-194-5p and hsa-miR-3667-5p) were found to be significantly up-regulated in complicated P. vivax malaria patients using qRT-PCR. The diagnostic potential of these 5 miRNAs were found to be significant with sensitivity and specificity of 60-71% and 69-81% respectively and area under curve (AUC) of 0.7 (p < 0.05). Moreover, in silico analysis of the common targets of up-regulated miRNAs revealed UBA52 and hsa-miR-7977 as majorly regulated hubs in the PPI and mRNA-miRNA networks, suggesting their putative role in complicated P. vivax malaria. CONCLUSION: miR-7977 might act as a potential biomarker for differentiating complicated P. vivax malaria from uncomplicated type. The elevated levels of miR-7977 may have a role to play in the disease pathology through UBA52 or TGF-beta signalling pathway.

Protein profiling of Acanthamoeba species using MALDI-TOF MS for specific identification of Acanthamoeba genotype.

Acanthamoeba spp. are ubiquitous in the environment and have the potential to cause severe infections. The different genotypes of Acanthamoeba have been shown to influence the severity of the disease and response to therapy. Characterizing Acanthamoeba spp. upto genotype can aid in infection control practices. Twenty-five Acanthamoeba isolates, characterized by 18S rDNA sequencing, were subjected to MALDI-TOF MS analysis by creating a database for the individual genotypes. The differentiating features of the various spectra were observed; the coded samples were then tested against the created database. The results of identification were compared with sequencing. Five different genotypes were obtained-T3, T4, T5, T10, and T11. Spectral analysis revealed genus-specific and genotype-specific peaks. The peak patterns for individual genotype were discrete and reproducible. Clinical isolates produced different peaks from the environmental isolate of the same genotype. A concordance of 92% was obtained with MALDI-TOF MS in comparison with 18sDNA sequencing. MALDI-TOF MS, once optimized, has the potential to reliably identify the genotype of Acanthamoeba spp. and to differentiate clinical isolate from mere contaminant.

Genetic diversity of Plasmodium falciparum merozoite surface protein-1 (block 2), glutamate-rich protein and sexual stage antigen Pfs25 from Chandigarh, North India.

OBJECTIVE: To elucidate the genetic diversity of Plasmodium falciparum in residual transmission foci of northern India. METHODS: Clinically suspected patients with malaria were screened for malaria infection by microscopy. 48 P. falciparum-infected patients were enrolled from tertiary care hospital in Chandigarh, India. Blood samples were collected from enrolled patients, genomic DNA extraction and nested PCR was performed for further species confirmation. Sanger sequencing was carried out using block 2 region of msp1, R2 region of glurp and pfs25-specific primers. RESULTS: Extensive diversity was found in msp1 alleles with predominantly RO33 alleles. Overall allelic prevalence was 55.8% for RO33, 39.5% for MAD20 and 4.7% for K1. Six variants were observed in MAD20, whereas no variant was found in RO33 and K1 alleles. A phylogenetic analysis of RO33 alleles indicated more similarity to South African isolates, whereas MAD20 alleles showed similarity with South-East Asian isolates. In glurp, extensive variation was observed with eleven different alleles based on the AAU repeats. However, pfs25 showed less diversity and was the most stable among the targeted genes. CONCLUSION: Our findings document the genetic diversity among circulating strains of P. falciparum in an area of India with low malaria transmission and could have implications for control strategies to reach the national goal of malaria elimination.

Molecular characterization of Plasmodium falciparum in Arunachal Pradesh from Northeast India based on merozoite surface protein 1 & glutamate-rich protein.

BACKGROUND & OBJECTIVES: Northeast (NE) India is one of the high endemic regions for malaria with a preponderance of Plasmodium falciparum, resulting in high morbidity and mortality. The P. falciparum parasite of this region showed high polymorphism in drug-resistant molecular biomarkers. However, there is a paucity of information related to merozoite surface protein 1 (msp-1) and glutamate-rich protein (glurp) which have been extensively studied in various parts of the world. The present study was, therefore, aimed at investigating the genetic diversity of P. falciparum based on msp-1 and glurp in Arunachal Pradesh, a State in NE India. METHODS: Two hundred and forty nine patients with fever were screened for malaria, of whom 75 were positive for P. falciparum. Blood samples were collected from each microscopically confirmed patient. The DNA was extracted; nested polymerase chain reaction and sequencing were performed to study the genetic diversity of msp-1 (block 2) and glurp. RESULTS: The block 2 of msp-1 gene was found to be highly polymorphic, and overall allelic distribution showed that RO33 was the dominant allele (63%), followed by MAD20 (29%) and K1 (8%) alleles. However, an extensive diversity (9 alleles and 4 genotypes) and 6-10 repeat regions exclusively of R2 type were observed in glurp. INTERPRETATION & CONCLUSIONS: The P. falciparum population of NE India was diverse which might be responsible for higher plasticity leading to the survival of the parasite and in turn to the higher endemicity of falciparum malaria of this region.

Circulating filarial antigen detection in brugian filariasis.

Human lymphatic filariasis (LF) is a major cause of disability globally. The success of global elimination programmes for LF depends upon effectiveness of tools for diagnosis and treatment. In this study on stage-specific antigen detection in brugian filariasis, L3, adult worm (AW) and microfilarial antigenaemia were detected in around 90-95% of microfilariae carriers (MF group), 50-70% of adenolymphangitis (ADL) patients, 10-25% of chronic pathology (CP) patients and 10-15% of endemic normal (EN) controls. The sensitivity of the circulating filarial antigen (CFA) detection in serum samples from MF group was up to 95%. In sera from ADL patients, unexpectedly, less antigen reactivity was observed. In CP group all the CFA positive individuals were from CP grade I and II only and none from grade III or IV, suggesting that with chronicity the AWs lose fecundity and start to disintegrate and die. Amongst EN subject, 10-15% had CFA indicating that few of them harbour filarial AWs, thus they might not be truly immune as has been conventionally believed. The specificity for antigen detection was 100% when tested with sera from various other protozoan and non-filarial helminthic infections.

Neurocysticercosis: An uncommon cause of drug-refractory epilepsy in North Indian population.

OBJECTIVE: Being a common cause of epilepsy in endemic areas, neurocysticercosis (NCC) is expected to account for a sizable proportion of patients with drug-refractory epilepsy (DRE) as well. However, data regarding prevalence of DRE in NCC are sparse. This study aimed to determine the prevalence of DRE as well as identification of clinical and radiologic factors that lead to DRE in patients with NCC. METHODS: This study was conducted in a tertiary-care postgraduate teaching institute in Northern India from July 2011 to July 2013. Two hundred patients with epilepsy due to NCC (definite [n = 59, 29.5%] or probable [n = 141, 70.5%]) based on diagnostic criteria by Del Brutto et al. were enrolled in the study in both a prospective (n = 51 [25.5%]) and a retrospective manner (n = 149 [74.5%]), and were followed for a minimum period of 1 year. RESULTS: Thirteen patients with NCC were found to be refractory to drug therapy. Prevalence of DRE was found to be 65 of 1,000 NCC patients with epilepsy in the present study. The risk factors associated with high risk of DRE were male sex (p = 0.035), older age (p = 0.016), pig-raising practices (p = 0.003), pork eating (p = 0.04), and presence of multiple (>2) (p = 0.0001) or mixed stage lesions (p = 0.007) on neuroimaging. On multivariate analysis, it was found that residing in an area where pig raising is prevalent (p = 0.01) and presence of multiple (>2) (p = 0.004) lesions on neuroimaging are associated with increased risk of DRE. SIGNIFICANCE: NCC is only rarely associated with the development of DRE. The common risk factors associated with increased chance of DRE include pig-rearing practices and presence of multiple (>2) lesions on neuroimaging.