A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India.

A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy

Bovine origin Staphylococcus aureus: A new zoonotic agent?

Aim: The study aimed to assess the nature of animal origin Staphylococcus aureus strains. The study has zoonotic importance and aimed to compare virulence between two different hosts, i.e., bovine and ovine origin. Materials and Methods: Conventional polymerase chain reaction-based methods used for the characterization of S. aureus strains and chick embryo model employed for the assessment of virulence capacity of strains. All statistical tests carried on R program, version 3.0.4. Results: After initial screening and molecular characterization of the prevalence of S. aureus found to be 42.62% in bovine origin samples and 28.35% among ovine origin samples. Meanwhile, the methicillin-resistant S. aureus prevalence is found to be meager in both the hosts. Among the samples, only 6.8% isolates tested positive for methicillin resistance. The biofilm formation quantified and the variation compared among the host. A Welch two-sample t-test found to be statistically significant, t=2.3179, df=28.103, and p=0.02795. Chicken embryo model found effective to test the pathogenicity of the strains. Conclusion: The study helped to conclude healthy bovines can act as S. aureus reservoirs. Bovine origin S. aureus strains are more virulent than ovine origin strains. Bovine origin strains have high probability to become zoonotic pathogen. Further, gene knock out studies may be conducted to conclude zoonocity of the bovine origin strains

Improving prosthetic prognosis by connective tissue ridge augmentation of alveolar ridge

The contour of edentulous ridge should be carefully evaluated before a fixed partial denture is undertaken. The ideal ridge height and width allows placement of a natural looking pontic which facilitates maintenance of plaque-free environment. The localized alveolar ridge defect refers to the volumetric deficit of the limited extent of bone and soft tissue within the alveolar process. Such type of ridge defects can be corrected by surgical ridge augmentation that can be accomplished by the addition of either soft or hard tissues. This article describes a procedure of surgical connective tissue augmentation of a localized deficient alveolar ridge in the maxilla, followed by fixed partial denture, enhancing the esthetics, function and health

Epidemiology and zoonotic potential of Livestock-associated Staphylococcus aureus isolated at Tamil Nadu, India

Abstract Background Staphylococcus aureus is part of normal flora and also an opportunistic pathogen responsible for a wide range of infections in both humans and animals. Livestock-associated S. aureus (LA-SA) has gained importance in recent years due to its increased prevalence in recent years, becoming a worry in public health view. This study aimed to study the epidemiology of LA-SA strains in Madurai district, Tamil Nadu, India. Methods A total of 255 samples were collected from bovine and other small ruminants like goats and sheep nares (n = 129 and n = 126 respectively). Nasal swab samples were collected from study animals with sterile sample collecting cotton swabs (Hi-Media, Mumbai). Samples were transported to the lab in Cary-Blair Transport media for further analysis. The samples were tested for S. aureus using antibiotic selection and PCR-based assays. The pathogenicity of the bacteria was assessed using chicken embryo models and liver cross-sections were used for histopathology studies. Results The prevalence rate in bovine-associated samples was 42.63% but relatively low in the case of small ruminants associated samples with 28.57% only. The overall prevalence of S. aureus is found to 35.6% and MRSA 10.98% among the study samples. The antibiogram results that LA-SA isolates were susceptible to aminoglycosides and tetracyclines but resistant to β-lactam drugs. The biofilm formation results showed that the LA-SA isolates are weak to high-capacity biofilm formers. The enterotoxigenic patterns revealed that most of the isolated strains are enterotoxigenic and possess classical enterotoxins. The survival analysis of chicken embryos suggested that the Bovine-associated strains were moderately pathogenic. Conclusion The study concluded that economically important livestock animals can act as reservoirs for multi-drug resistant and pathogenic which in-turn is a concern for public health as well as livestock health

Translational repression of the cpw-wpc gene family in the malaria parasite Plasmodium

© 2016 Elsevier Ireland Ltd. All rights reserved.The technical challenges of working with the sexual stages of the malaria parasite Plasmodium have hindered the characterization of sexual stage antigens in the quest for a successful malaria transmission-blocking vaccine. One such predicted and largely uncharacterized group of sexual stage candidate antigens is the CPW-WPC family of proteins. CPW-WPC proteins are named for a characteristic domain that contains two conserved motifs, CPxxW and WPC. Conserved across Apicomplexa, this family is also present earlier in the Alveolata in the free-living, non-parasitophorous, photosynthetic chromerids, Chromera and Vitrella. In Plasmodium falciparum and Plasmodium berghei blood stage parasites, the transcripts of all nine cpw-wpc genes have been detected in gametocytes. RNA immunoprecipitation followed by reverse transcriptase-PCR reveals all P. berghei cpw-wpc transcripts to be bound by the translational repressors DOZI and CITH, and thus are likely under translational control prior to transmission from the rodent host to the mosquito vector in P. berghei. The GFP tagging of two endogenous P. berghei genes confirmed translational silencing in the gametocyte and translation in ookinetes. By establishing a luciferase transgene assay, we show that the 3' untranslated region of PF3D7_1331400 controls protein expression of this reporter in P. falciparum gametocytes. Our analyses suggest that cpw-wpc genes are translationally silenced in gametocytes across Plasmodium spp. and activated during ookinete formation and thus may have a role in transmission to the mosquito.Jorge M. Santos was supported by Fundação para a Ciência e a Tecnologia (FCT) of the Portuguese Ministry of Science, Technology Ph.D. Fellowship (SFRH/BD/63849/2009); Dr. Gunnar R. Mair was supported by FCT projects PTDC/BIA-BCM/105610/2008 and PTDC/SAU-MIC/122082/2010. Dr. Thomas Templeton was supported by the NIH/NIAID grant 1R01AI080754-01A1, the William Randolph Hearst Foundation, and a visiting professorship to the Institute of Tropical Medicine, Nagasaki University, Japan.info:eu-repo/semantics/publishedVersio

Functional complementation of yeast ribosomal P0 protein with Plasmodium falciparum P0

A complex of three phosphoproteins (P0, P1 and P2) constitutes the stalk region at the GTPase center of the eukaryotic large ribosomal subunit, amongst which the protein P0 plays the most crucial role. Earlier studies have shown the functional complementation of the conditional P0-null mutant of Saccharomyces cerevisiae (W303dGP0) with orthologous P0 genes from fungal and mammalian organisms, but not the protozoan parasite Leishmania infantum. In this paper we show that the PfP0 gene from the protozoan malaria parasite Plasmodium falciparum can functionally complement the conditional P0-null W303dGP0 mutant of S. cerevisiae. Unlike the above orthologous genes, PfP0 gene could also rescue the D67dGP0 strain, which in addition to being a conditional null for ScP0 gene, is a null-mutant for both ScP1α and β genes. However, under stress conditions such as high temperature, salt and osmolarity, PfP0 gene could not rescue D67dGP0 strain. Ribosomes purified from W303dGP0 carrying PfP0 gene did not contain ScP1 protein, indicating a lack of binding of ScP1 to PfP0 protein. Yeast 2-hybrid analysis further confirmed the lack of binding of ScP1 to PfP0 protein. The polymerizing activities of ribosomes with ScP0 or PfP0 protein, in the absence of ScP1 protein, were found to be about 40-45% that of ribosomes with all the yeast P-proteins. In its sensitivity to the inhibitor sordarin, PfP0 was similar to the P0 protein from the fungus Aspergillus fumigatus. These results indicate a closer functional relationship of P. falciparum P0 gene to fungal P0 genes