PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

<p>Abstract</p> <p>Background</p> <p><it>Anopheles culicifacies s.l</it>., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (<it>kdr</it>) is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of <it>kdr </it>mutation (L1014F) in a field population of <it>An. culicifacies s.l</it>. and three new PCR-based methods for <it>kdr </it>genotyping.</p> <p>Methods</p> <p>The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant <it>An. culicifacies s.l</it>. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F) in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR), an Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis-PCR (PIRA-PCR) were developed and tested for <it>kdr </it>genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped.</p> <p>Results</p> <p>The genotyping of this <it>An. culicifacies s.l</it>. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the <it>kdr </it>allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium.</p> <p>Conclusion</p> <p>The Leu-Phe mutation, which generates the <it>kdr </it>phenotype in many insects, was detected in a pyrethroid and DDT resistant <it>An. culicifacies s.l</it>. population. Three PCR-based methods were developed for <it>kdr </it>genotyping. All the three assays were specific. The ARMS method was refractory to non-specific amplification in non-stringent amplification conditions. The PIRA-PCR assay is able to detect both the codons for the phenylalanine mutation at <it>kdr </it>locus, i.e., TTT and TTC, in a single assay, although the latter codon was not found in the population genotyped.</p

Epidemiology and genetic aspects of multiple sclerosis in India

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system with a complex pathophysiology. Considered a rare disease in India in the past, studies over time suggest an increase in subjects with MS in India, although the observations are limited by the lack of formally conducted epidemiological studies and the absence of a nationwide registry. The current World Health Organization (WHO) Multiple Sclerosis International Federation (MSIF) "Atlas of MS" 2013 estimates a prevalence rate of 5-20 per 100,000, which also seems an underestimate. Although there have been reports of phenotypic differences between MS in Indians and the Western counterparts, recent studies report a reasonable similarity in disease types and characteristics. A few studies on the genetics of MS have been reported, including human leukocyte antigen (HLA) associations and non-major histopathology complex (MHC) disease loci. The current review discusses the pivotal studies of the past, newer observations on MS from India, and the need for a national registry

Toll like receptor 2 and 4 polymorphisms in malaria endemic populations of India

Toll like receptors (TLRs) play a pivotal role in recognizing the invading malaria parasite Plasmodium, thus genetic makeup of the exposed population can be of utmost importance for its predisposition to malaria. In this study 264 malaria patients from seven different eco epidemiological regions of India were genotyped for TLR2 and TLR4 polymorphisms using DNA sequencing methods. No variation was observed at residue positions 677 and 753 in TLR2 whereas residue positions 299 and 399 in TLR4 were highly polymorphic. The GC haplotype (Asp299Gly/Thr399Thr) was observed at the highest frequency in populations of East Singhbhum, Vizianagaram and North Goa and absent in Kolkata, Dakshin Kannada and Nicobar district. All polymorphisms were in Hardy Weinberg equilibrium. Populations of Kolkata, Nicobar district, Sundergarh and Dakshin Kannada were observed to be closely related. TLR2 polymorphism was absent in the Indian population and an overall heterogeneous pattern of TLR4 polymorphism can be attributed to genetic drift. However it can be inferred that GC haplotype is under the process of natural selection in the Indian population and one of the factors contributing to its selection could be predominance of Plasmodium falciparum in these regions

Early detection of tumor cells in bone marrow and peripheral blood in a fast‑progressing gastric cancer model.

Helicobacter&nbsp;pylori (H.&nbsp;pylori) infection is a major risk factor for the development of gastric cancer. The authors previously demonstrated that in mice deficient in myeloid differentiation primary response 88 (Myd88‑/‑), infection with Helicobacter&nbsp;felis (H.&nbsp;felis) a close relative of H.&nbsp;pylori, subsequently rapidly progressed to neoplasia. The present study examined circulating tumor cells (CTCs) by measuring the expression of cytokeratins, epithelial‑to‑mesenchymal transition (EMT)‑related markers and cancer stem cell&nbsp;(CSC) markers in bone marrow and peripheral blood from Myd88‑/‑ and wild‑type (WT) mice. Cytokeratins CK8/18 were detected as early as 4&nbsp;months post‑infection in Myd88‑/‑ mice. By contrast, cytokeratins were not detected in WT mice even after 7&nbsp;months post‑infection. The expression of Mucin‑1 (MUC1) was observed in both bone marrow and peripheral blood at different time points, suggesting its role in gastric cancer metastasis. Snail, Twist and ZEB were expressed at different levels in bone marrow and peripheral blood. The expression of these EMT‑related markers suggests the manifestation of cancer metastasis in the early stages of disease development. LGR5, CD44 and CD133 were the most prominent CSC markers detected. The detection of CSC and EMT markers along with cytokeratins does reinforce their use as biomarkers for gastric cancer metastasis. This early detection of markers suggests that CTCs leave primary site even before cancer is well established. Thus, cytokeratins, EMT, and CSCs could be used as biomarkers to detect aggressive forms of gastric cancers. This information may prove to be of significance in stratifying patients for treatment prior to the onset of severe disease‑related characteristics

A Bioluminescence-Based Drug Screen Identifies Activities of Fexinidazole and Its Metabolites against Helicobacter pylori.

Helicobacter pylori is responsible for a wide range of gastric diseases, including gastric cancer and gastritis. With half of the worlds population infected by H. pylori and the current standard of care associated with suboptimal outcomes, a search for more effective drugs is critical. To facilitate drug screening for H. pylori, we developed a microtiter plate-based compound screening method that is faster and can screen multiple compounds. We identified activities of fexinidazole and its sulfoxide and sulfone metabolites against H. pylori. Both fexinidazole and its metabolites exhibited equipotency against SS1, 60190, and G27 strains, which were about 3-6-fold more potent than the currently used metronidazole. We also determined the minimal inhibitory concentration (MIC) of metronidazole, fexinidazole, and its metabolites against these strains by a traditional agar plate-based method. While MIC values of fexinidazole and metronidazole were similar against all the strains, both sulfoxide and sulfone showed lower MIC values than metronidazole against SS1 and 60190. Given the recent FDA approval of fexinidazole, our data on the in vitro antibacterial activities of fexinidazole and its metabolites support further evaluation of this drug with the goal of producing an alternative nitro-based antimicrobial with good safety profiles for the treatment of H. pylori infection

Virulence factors of Aggregatibacter actinomycetemcomitans - A status update

Periodontitis is a chronic infectious inflammatory disease characterized by the destruction of tooth. The contribution of bacteria to the disease progression is poorly understood probably due to the multifactorial background of this disease. Aggregatibacter actinomycetemcomitans is part of the normal flora in many healthy individuals, but is also a major etiologic agent in some aggressive forms of periodontitis. The genetic diversity among different isolates of Aggregatibacter actinomycetemcomitans is great and its ability to express and release virulence factors varies. Aggregatibacter actinomycetemcomitans, a pathogen not only in periodontal but also in some nonoral infections, possesses several virulence determinants which contribute to its ability to colonize the oral cavity, persist in the periodontal pocket, resist and evade host defenses, cause destruction of soft and hard tooth-supporting tissues, and interfere with host tissue repair after infection. Authors conducted a comprehensive search through PubMed/Medline databases to compile the available literature till June 2014, for the purpose of detailed insight into the bacteria. The search was designed to identify appropriate articles related to virulence factors of Aggregatibacter actinomycetemcomitans, and the articles were independently screened for eligibility

A Bioluminescence-Based Drug Screen Identifies Activities of Fexinidazole and Its Metabolites against <i>Helicobacter pylori</i>

Helicobacter pylori is responsible for a wide range of gastric diseases, including gastric cancer and gastritis. With half of the world’s population infected by H. pylori and the current standard of care associated with suboptimal outcomes, a search for more effective drugs is critical. To facilitate drug screening for H. pylori, we developed a microtiter plate-based compound screening method that is faster and can screen multiple compounds. We identified activities of fexinidazole and its sulfoxide and sulfone metabolites against H. pylori. Both fexinidazole and its metabolites exhibited equipotency against SS1, 60190, and G27 strains, which were about 3–6-fold more potent than the currently used metronidazole. We also determined the minimal inhibitory concentration (MIC) of metronidazole, fexinidazole, and its metabolites against these strains by a traditional agar plate-based method. While MIC values of fexinidazole and metronidazole were similar against all the strains, both sulfoxide and sulfone showed lower MIC values than metronidazole against SS1 and 60190. Given the recent FDA approval of fexinidazole, our data on the in vitro antibacterial activities of fexinidazole and its metabolites support further evaluation of this drug with the goal of producing an alternative nitro-based antimicrobial with good safety profiles for the treatment of H. pylori infection

Microbiome Signatures in a Fast- and Slow-Progressing Gastric Cancer Murine Model and Their Contribution to Gastric Carcinogenesis.

Gastric cancer is the third most common cause of death from cancer in the world and infection with Helicobacter pylori (H. pylori) is the main cause of gastric cancer. In addition to Helicobacter infection, the overall stomach microbiota has recently emerged as a potential factor in gastric cancer progression. Previously we had established that mice deficient in myeloid differentiation primary response gene 88 (MyD88, Myd88-/- ) rapidly progressed to neoplasia when infected with H. felis. Thus, in order to assess the role of the microbiota in this fast-progressing gastric cancer model we investigated changes of the gastric microbiome in mice with different genotypic backgrounds: wild type (WT), MyD88-deficient (Myd88-/- ), mice deficient in the Toll/interleukin-1 receptor (TIR) domain-containing adaptor-inducing interferon-β (TRIF, Trif Lps2), and MyD88- and TRIF-deficient (Myd88-/- /Trif Lps2, double knockout (DKO)) mice. We compared changes in alpha diversity, beta diversity, relative abundance, and log-fold differential of relative abundance ratios in uninfected and Helicobacter infected mice and studied their correlations with disease progression to gastric cancer in situ. We observed an overall reduction in microbial diversity post-infection with H. felis across all genotypes. Campylobacterales were observed in all infected mice, with marked reduction in abundance at 3 and 6 months in Myd88-/- mice. A sharp increase in Lactobacillales in infected Myd88-/- and DKO mice at 3 and 6 months was observed as compared to Trif Lps2 and WT mice, hinting at a possible role of these bacteria in gastric cancer progression. This was further reinforced upon comparison of Lactobacillales log-fold differentials with histological data, indicating that Lactobacillales are closely associated with Helicobacter infection and gastric cancer progression. Our study suggests that differences in genotypes could influence the stomach microbiome and make it more susceptible to the development of gastric cancer upon Helicobacter infection. Additionally, increase in Lactobacillales could contribute to faster development of gastric cancer and might serve as a potential biomarker for the fast progressing form of gastric cancer