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Abstract Chloroquine is still used as a first-line treatment for uncomplicated Plasmodium vivax malaria in India and resistance to this therapy can act as a major hurdle for malaria elimination. It is difficult to monitor drug-efficacy and drug resistance through in vivo and in vitro studies in case of Plasmodium vivax so analysis of molecular markers serves as an important tool to track resistance. Molecular methods that are currently in use for detecting single nucleotide polymorphisms in resistant genes including Polymerase chain reaction (PCR), Realtime-Polymerase chain reaction require highly sophisticated labs and are time consuming. So, with this background the study has been designed to optimize Loop Mediated Isothermal Amplification Assay to detect single nucleotide polymorphisms in chloroquine resistance gene of Plasmodium vivax in field settings. Eighty-eight Plasmodium vivax positive samples were collected. Pvmdr1 gene was amplified for all the samples and sequenced. Obtained sequences were analyzed for the presence of single nucleotide polymorphisms in the target gene. Further Loop Mediated Isothermal Amplification Assay primer sets were designed for the target mutants and the assay was optimized. Clinical as well as analytical sensitivity and specificity for the assay was calculated. Double mutants with variations at T958M and F1076L were detected in 100% of the Plasmodium vivax clinical isolates with haplotype M958 Y976 Y1028 L1076. Designed primers for Loop Mediated Isothermal Amplification Assay successfully detected both the mutants (T958M and F1076L) in 100% of the isolates and do not show cross-reactivity with other strains. So, the assay was 100% sensitive and specific for detecting single nucleotide polymorphisms in the target Pvmdr1 gene. Limit of detection was found to be 0.9 copies/µl and lowest DNA template concentration detected by designed assay was 1.5 ng/µL. Observed prevalence of single nucleotide polymorphisms in Pvmdr 1 gene is indicating a beginning of trend towards chloroquine resistance in Plasmodium vivax. The present study optimized LAMP for detecting single nucleotide polymorphisms in Plasmodium vivax cases in field settings, thus would help in finding significant hubs of emerging chloroquine drug resistance and ultimately helping in the management of suitable antimalarial drug policy.

Antibiotics like colistin can save patients infected with carbapenem-resistant Pseudomonas aeruginosa. However, patients can succumb to such infections even if they undergo colistin therapy. This prompted us to investigate the probable antimicrobial resistance mechanisms and virulence determinants involved in colistin- and carbapenem-resistant P. aeruginosa (CCRPA). Of the 448 P. aeruginosa clinical strains, 19 isolates were resistant to both colistin and carbapenem. Carbapenemases and efflux pump encoding genes were assessed by multiplex PCR and qPCR, respectively. blaVIM was detected among six CCRPA isolates and blaIMP in one strain. The expression levels of pmrA and phoP, as well as pmrB genes and their association with colistin resistance, were assessed by qPCR and semi-quantitate PCR, respectively. pmrA and phoP genes were significantly enhanced in three and nine CCRPA isolates, respectively. We also phenotypically evaluated biofilms, pyocyanin, and alginate production among CCRPA strains. Alginate production was observed in 15 isolates, followed by biofilm (n = 8) and pyocyanin (n = 5). Our results highlighted the coexistence of colistin and carbapenem resistance and biofilm formation among clinical isolates of CCRPA. Further studies are required to trace the source and the origin of colistin and carbapenem resistance in this specific environment.

Fretting wear is very common damage found in parts under small-amplitude motion or vibration. Nano-lubrication is a very efficient way to reduce the friction and wear of mating parts. Therefore, fretting study was conducted on magnesium alloy (AZ91) under lubricating conditions using poly alpha olefin grade 4 (PAO4) with Graphite nanoparticles (NPs) (GNP) and hexagonal boron nitride (h-BN) NPs as lubricant additives. The experimental results reveal that the coefficient of friction (COF) and wear volume were reduced by 76% and 28%, respectively, using GNP and by 28% and 20%, respectively, using h-BN NPs. At optimal concentration of NPs, the effect of load on COF and wear was also investigated in this study. Wear volume and COF increases with increasing load. The performance of GNP was observed to be better as compared to h-BN NPs, which suggests its use as a lubricant additive in PAO4 as lubricated fretting conditions for AZ91 alloy. However, h-BN NPs perform well under higher loads.

Abstract Objective: We aimed to assess risk of COVID-19 infection & seroprotection status in healthcare workers (HCWs) in both hospital and community settings following an intensive vaccination drive in India. Setting: Tertiary Care Hospital Methods: We surveyed COVID-19 exposure risk, personal protective equipment (PPE) compliance, vaccination status, mental health & COVID-19 infection rate across different HCW cadres. Elecsys® test for COVID-19 spike (Anti-SARS-CoV-2S; ACOVs) and nucleocapsid (Anti-SARS-CoV-2; ACOV) responses following vaccination and/or COVID-19 infection were measured in a stratified sample of 386 HCW. Results: We enrolled 945 HCWs (60.6% male, age 35.9 ± 9.8 years, 352 nurses, 211 doctors, 248 paramedics & 134 support staff). Hospital PPE compliance was 90.8%. Vaccination coverage was 891/945 (94.3%). ACOVs neutralizing antibody was reactive in 381/386 (98.7%). ACOVs titer (U/ml) was higher in the post-COVID-19 infection group (N =269; 242.1 ± 35.7 U/ml) than in the post-vaccine or never infected subgroup (N = 115, 204.1 ± 81.3 U/ml). RT PCR + COVID-19 infections were documented in 224/945 (23.7%) and 6 HCWs had disease of moderate severity, with no deaths. However, 232/386 (60.1%) of HCWs tested positive for nucleocapsid ACOV antibody, suggesting undocumented or subclinical COVID-19 infection. On multivariate logistic regression, only female gender [aOR 1.79, 95% CI 1.07–3.0, P = .025] and COVID-19 family contact [aOR 5.1, 95% CI 3.84–9.5, P < .001] were predictors of risk of developing COVID-19 infection, independent of association with patient-related exposure. Conclusion: Our HCWs were PPE compliant and vaccine motivated, with immunization coverage of 94.3% and seroprotection rate of 98.7%. There was no relationship between HCW COVID-19 infection to exposure characteristics in the hospital. Vaccination reduced disease severity and prevented death in HCW.

The Acanthamoeba genus comprises the free-living amoebae that are ubiquitously present as opportunistic pathogens. They cause serious human diseases—for instance, Acanthamoeba keratitis (AK), granulomatous amoebic encephalitis (GAE), cutaneous acanthamoebiasis and disseminated infections. The traditional method for classifying Acanthamoeba was based on the morphological examination of cysts. However, this method was less consistent as the morphology of cysts changes with the culture conditions. After the advent of molecular techniques, genotyping is considered an essential tool in accurately identifying Acanthamoeba at the species level and is further helpful in classification up to the sub-genotype level. The most recommended and currently used methods for Acanthamoeba genotyping are 18S and 16S rDNA gene sequencing. Based on these two genes, Acanthamoeba is classified into 23 genotypes. Out of these, it is the T4 genotype that is most commonly associated with clinical disease and isolation from environmental samples. The T4 genotype contains more than ten species within it. Differences in geographical distribution, virulence, pathogenesis and drug susceptibility profile have been observed among different genotypes. However, whether such differences exist within sub-genotypes/species under T4 is yet unknown. In the present study, 11 Acanthamoeba isolates, which were already characterized as the T4 genotype by the hypervariable region of diagnostic fragment 3 (DF3) of the 18S rDNA, were sub-genotyped using the 16S rDNA mitochondrial sequence. Nine of these were isolated from patients with AK and two from water samples. Phylogenetic analysis of these isolates attributed them to four sub-genotypes (T4a (n = 6), T4b (n = 1), T4Neff (n = 2) and T4d (n = 2)). The study highlights the potential use of 16S in the sub-genotyping of Acanthamoeba T4. The 16S rDNA sequences of two isolates, one from an Acanthamoebic keratitis (AK) patient and one environmental, were found to group with A. mauritaniensis (T4d). This group was believed to be a non-pathogenic environmental Acanthamoeba and the identification of the AK isolate may be confirmed by whole-genome sequencing.

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.

Abstract Background Malaria is a complex issue due to the availability of few therapies and chemical families against Plasmodium and mosquitoes. There is increasing resistance to various drugs and insecticides in Plasmodium and in the vector. Additionally, human behaviors are responsible for promoting resistance as well as increasing the risk of exposure to infections. Chalcones and their derivatives have been widely explored for their antimalarial effects. In this context, new derivatives of chalcones have been evaluated for their antimalarial efficacy. Methods BALB/c mice were infected with P. berghei NK-65. The efficacy of the three most potent chalcone derivations (1, 2, and 3) identified after an in vitro compound screening test was tested. The selected doses of 10 mg/kg, 20 mg/kg, and 10 mg/kg were studied by evaluating parasitemia, changes in temperature, body weights, organ weights, histopathological features, nitric oxide, cytokines, and ICAM-1 expression. Also, localization of parasites inside the two vital tissues involved during malaria infections was done through a transmission electron microscope. Results All three chalcone derivative treated groups showed significant (p

The aim of this study is to determine the ultimate bearing capacity of an unpaved road model under unsoaked and soaked conditions by using finite element method (FEM). The numerical methodology applied in this investigation to know the ultimate bearing capacity is subjected to vertical load, to apply the application of finite element method (FEM) using ABACUS software. The results reveal the variation in the magnitude of the stresses, displacement pattern and in bearing capacity by inclusion of untreated and treated coir geotextiles under unsoaked and soaked conditions. However, further validation of the results presented in this paper is recommended using an experimental study conducted on similar size unpaved road model. The proposed study can be useful for the Civil Engineering where a good quality rural road network is required.

Using the repeated or cyclic load test in a laboratory, the current study investigates the modulus of resilience of unpaved roads model reinforced with untreated/treated woven/non-woven coir geotextile. P-aminophenol, sodium periodate, and sodium hydroxide were the chemicals that used to treat the coir geotextiles. Untreated/treated coir geotextiles were placed at the interface under unsoaked/soaked conditions, and laboratory cyclic load testing for the sand layer overlaying clay was performed in a test tank. Models reinforced with untreated/treated woven coir geotextiles performed better than models reinforced with untreated/treated non-woven coir geotextiles, according to the study’s findings. Furthermore, in the unsoaked state, models reinforced with untreated/treated woven and non-woven coir geotextiles better than the models reinforced with soaked coir geotextiles in terms of modulus of resilience. Untreated/treated woven/non-woven coir geotextiles can increase the material’s maximum energy per volume that it can absorb in unpaved roads when placed at the interface of the sand layer overlying clay. This study is very beneficial, especially in situations where highway subgrade design necessitates a reasonably accurate estimation of strength and stiffness characteristics.

Friction and wear are two major elements that influence the life of a variety of equipment. According to estimates, as much as 30% of the energy used is dissipated as friction. However, this figure can be reduced by developing materials that enhance surfaces and apply lubricants appropriately. This study aims to analyze the impact of hexagonal boron nitride (h-BN) nanoparticles on the rheological and antiwear characteristics of four distinct oil varieties, namely Society of Automotive Engineers SAE-20W50, soybean, Polyalphaolefin PAO-4, and olive oils. The results of the tribological tests demonstrated a noteworthy enhancement in antiwear characteristics with the addition of 0.2 wt.% of h-BN nanolubricant. Among all nanolubricants, the maximum reduction in coefficient of friction (COF) and wear scar diameter was observed at 0.2 wt.% of h-BN in SAE-20W50 oil. Compared to the base oils, the wear scar diameter decreased by 17.93%, 8.80%, 22.65%, and 24.04% for (soybean oil and 0.2 wt.% of h-BN), (SAE20W50 and 0.2 wt.% of h-BN), (PAO4 and 0.2 wt.% of h-BN), and (olive oil and 0.2 wt.% of h-BN), respectively. Rheological test results indicated that with the addition of h-BN nanoparticles, the viscosity of the base oils significantly increased. The maximum viscosity was observed at 40 °C for SAE20W50 base nanolubricants, according to the rheological measurements.

Echinococcosis or hydatid disease is caused by the larval stage of the dog tapeworm, that is, Echinococcus granulosus, E. multilocularis, E. vogeli, or E. oligarthrus. Echinococcus granulosus causes cystic echinococcosis, which has a worldwide distribution. Liver is the most common site, affecting approximately two-third of the patients, whereas lung involvement is seen in approximately 25% of cases. This case is a very rare scenario of bilateral pulmonary hydatid cysts in a young child having exposure to a pet dog with negative hydatid serology and normal eosinophil count.

Abstract 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.

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.

Leishmaniasis is a vector-borne parasitic infection caused by the infective bite of female Phlebotomine sandflies. Treatment of leishmaniasis by conventional synthetic compounds is met by challenges pertaining to adverse effects which call for the discovery of newer anti-leishmanial molecules. This study was performed to evaluate the effect and modes of action of a sesquiterpene alcoholic molecule Farnesol on Leishmania major, the causative agent of Zoonotic CL. The cytotoxic effect of Farnesol against L.major promastigotes, amastigotes and macrophages was assessed by MTT test and counting. The IC50 on promastigotes by Farnesol on L.major was also evaluated by flow cytometry. In the findings, promastigotes were reduced at 167μM. The mean numbers of L.major amastigotes in macrophages were significantly decreased on exposure to Farnesol at 172μM. In addition, Farnesol induced significant apoptosis dose-dependent on L.major promastigotes. In silico protein-ligand_binding analyses indicated the effect of Farnesol in perturbation of the ergosterol synthesis pathway of Leishmania with attributes suggesting inhibition of Lanosterol-α-demethylase, the terminal enzyme of ergosterol synthesis machinery. Findings from flow cytometry reveal the role of Farnesol in apoptosis-induced killing in promastigotes. Farnesol was effective at very lower concentrations when compared to Paromomycin. Further studies are crucial to evaluate the therapeutic potential of Farnesol alone or in combination with other conventional drugs in animal models.

Leishmaniasis is a zoonotic disease transmitted by an obligate intra-macrophage protozoan of the genus Leishmania through the infective bite of a vector sandfly. This study investigated the therapeutic efficacy of farnesol, a sesquiterpene compound, for the treatment of cutaneous leishmaniasis (CL) using in vivo BALB/c mouse model. In this study, farnesol's efficacy was compared with the standard drug, paromomycin. It was observed that farnesol significantly reduced lesion sizes and footpad thickness compared to the control group (paromomycin). Lymph node size was also significantly reduced in farnesol-treated mice, indicating its ability to control infection spread. Combination therapy with farnesol and Paromomycin did not demonstrate synergistic effects. These results highlight the potential of farnesol as an alternative therapeutic agent for CL. Further investigations are required to elucidate its mechanism of action and assess potential off-target effects. Optimization of oral delivery methods should be explored to enhance bioavailability. Overall, our findings support farnesol's efficacy in CL treatment, offering promising prospects for improved disease management.

Background: Pathogenic free-living amoeba (FLA) such as Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris are causative agents of fatal amoebic encephalitis/meningoencephalitis. The diagnosis of such infections is challenging due to a lack of clinical suspicion and expertise in microscopic identification. We evaluated the performance of molecular assays for the timely and accurate detection of FLA-causing central nervous system (CNS) afflictions. Methods: This study included samples from 156 patients with suspected encephalitis/meningoencephalitis, including 149 cerebrospinal fluid (CSF) samples, 5 brain tissue biopsies, and 2 brain abscess samples. All the samples were subjected to PCR-based detection of Acanthamoeba spp., N. fowleri, and B. mandrillaris. The diagnostic characteristics and the inter-rater reliability scores were evaluated for parasite-specific polymerase chain reaction (PCR) using culture on non-nutrient agar (NNA)/microscopy or histopathological examination as a confirmatory test for Acanthamoeba spp. and N. fowleri and histopathology for B. mandrillaris. Results: We detected 11 samples positive for FLA, including 6 Acanthamoeba spp., 3 B. mandrillaris, and 2 N. fowleri. Furthermore, all 11 samples were positive according to the confirmatory tests, i.e., culture on NNA/microscopy/histopathology in the case of Acanthamoeba spp. and N. fowleri and histopathology of tissue biopsies for B. mandrillaris. The inter-rater reliability between the PCRs and the confirmatory tests for the detection of Acanthamoeba spp., N. fowleri, and B. mandrillaris was 100%. Conclusions: The PCR-based detection of FLA in patients suspected of encephalitis/meningoencephalitis was found to be fast, efficient, and reliable in our study. We suggest the use of these PCRs in laboratories to obtain additional data on their efficiency in diagnosing FLA infections of the CNS. The present study was conducted with a small sample size of 156 patient samples, and we found only six Acanthamoeba spp., three B. mandrillaris, and two N. fowleri. The present study should be conducted on a larger sample size for better evaluation of the primer pairs.