Assignment of Streptococcus agalactiae isolates to clonal complexes using a small set of single nucleotide polymorphisms

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus agalactiae </it>(Group B Streptococcus (GBS)) is an important human pathogen, particularly of newborns. Emerging evidence for a relationship between genotype and virulence has accentuated the need for efficient and well-defined typing methods. The objective of this study was to develop a single nucleotide polymorphism (SNP) based method for assigning GBS isolates to multilocus sequence typing (MLST)-defined clonal complexes.</p> <p>Results</p> <p>It was found that a SNP set derived from the MLST database on the basis of maximisation of Simpsons Index of Diversity provided poor resolution and did not define groups concordant with the population structure as defined by eBURST analysis of the MLST database. This was interpreted as being a consequence of low diversity and high frequency horizontal gene transfer. Accordingly, a different approach to SNP identification was developed. This entailed use of the "Not-N" bioinformatic algorithm that identifies SNPs diagnostic for groups of known sequence variants, together with an empirical process of SNP testing. This yielded a four member SNP set that divides GBS into 10 groups that are concordant with the population structure. A fifth SNP was identified that increased the sensitivity for the clinically significant clonal complex 17 to 100%. Kinetic PCR methods for the interrogation of these SNPs were developed, and used to genotype 116 well characterized isolates.</p> <p>Conclusion</p> <p>A five SNP method for dividing GBS into biologically valid groups has been developed. These SNPs are ideal for high throughput surveillance activities, and combining with more rapidly evolving loci when additional resolution is required.</p

Rapid Detection of the H275Y Oseltamivir Resistance Mutation in Influenza A/H1N1 2009 by Single Base Pair RT-PCR and High-Resolution Melting

Introduction: We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR), high-resolution melting (HRM) assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses.Findings: A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of nonspecific primer-dimer was evident and affected the overall melting temperature (Tm) of the amplified products. Due to primer-dimer appearance at .30 cycles we found that if the cycle threshold (CT) for a dilution was .30, the HRM assay did not consistently discriminate mutant from wildtype. Where the CT was ,30 we noted an inverse relationship between CT and Tm and fitted quadratic curves allowed the discrimination of wildtype, mutant and 30:70 mutant:wildtype virus mixtures. We compared the CT values for a TaqMan H1N1 09 detection assay with those for the HRM assay using 59 clinical samples and demonstrated that samples with a TaqMan detection assay CT.32.98 would have an H275Y assay CT.30. Analysis of the TaqMan CT values for 609 consecutive clinical samples predicted that 207 (34%) of the samples would result in an HRM assay CT.30 and therefore not be amenable to the HRM assay.Conclusions: The use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting Tm against CT. Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations

Chlamydia trachomatis genotypes in a cross-sectional study of urogenital samples from remote Northern and Central Australia

his is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Abstract OBJECTIVES: The objective was to determine the frequency of trachoma genotypes of Chlamydia trachomatis-positive urogenital tract (UGT) specimens from remote areas of the Australian Northern Territory (NT). SETTING: The setting was analysis of remnants of C. trachomatis positive primarily UGT specimens obtained in the course of clinical practice. The specimens were obtained from two pathology service providers. PARTICIPANTS: From 3356 C. trachomatis specimens collected during May 2012-April 2013, 439 were selected for genotyping, with a focus on specimens from postpubescent patients, in remote Aboriginal communities where ocular trachoma is potentially present. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome measure was the proportion of successfully genotyped UGT specimens that were trachoma genotypes. The secondary outcome measures were the distribution of genotypes, and the frequencies of different classes of specimens able to be genotyped. RESULTS: Zero of 217 successfully genotyped UGT specimens yielded trachoma genotypes (95% CI for frequency=0-0.017). For UGT specimens, the genotypes were E (41%), F (22%), D (21%) and K (7%), with J, H and G and mixed genotypes each at 1-4%. Four of the five genotyped eye swabs yielded trachoma genotype Ba, and the other genotype J. Two hundred twenty-two specimens (50.6%) were successfully genotyped. Urine specimens were less likely to be typable than vaginal swabs (p<0.0001). CONCLUSIONS: Unlike in some other studies, in the remote NT, trachoma genotypes of C. trachomatis were not found circulating in UGT specimens from 2012 to 2013. Therefore, C. trachomatis genotypes in UGT specimens from young children can be informative as to whether the organism has been acquired through sexual contact. We suggest inclusion of C. trachomatis genotyping in guidelines examining the source of sexually transmitted infections in young children in areas where trachoma genotypes may continue to circulate, and continued surveillance of UGT C. trachomatis genotypes

Burkholderia pseudomallei Isolates from Sarawak, Malaysian Borneo, Are Predominantly Susceptible to Aminoglycosides and Macrolides

Melioidosis is a potentially fatal disease caused by the saprophytic bacterium Burkholderia pseudomallei. Resistance to gentamicin is generally a hallmark of B. pseudomallei, and gentamicin is a selective agent in media used for diagnosis of melioidosis. In this study, we determined the prevalence and mechanism of gentamicin susceptibility found in B. pseudomallei isolates from Sarawak, Malaysian Borneo. We performed multilocus sequence typing and antibiotic susceptibility testing on 44 B. pseudomallei clinical isolates from melioidosis patients in Sarawak district hospitals. Whole-genome sequencing was used to identify the mechanism of gentamicin susceptibility. A novel allelic-specific PCR was designed to differentiate gentamicin-sensitive isolates from wild-type B. pseudomallei. A reversion assay was performed to confirm the involvement of this mechanism in gentamicin susceptibility. A substantial proportion (86%) of B. pseudomallei clinical isolates in Sarawak, Malaysian Borneo, were found to be susceptible to the aminoglycoside gentamicin, a rare occurrence in other regions where B. pseudomallei is endemic. Gentamicin sensitivity was restricted to genetically related strains belonging to sequence type 881 or its single-locus variant, sequence type 997. Whole-genome sequencing identified a novel nonsynonymous mutation within amrB, encoding an essential component of the AmrAB-OprA multidrug efflux pump. We confirmed the role of this mutation in conferring aminoglycoside and macrolide sensitivity by reversion of this mutation to the wild-type sequence. Our study demonstrates that alternative B. pseudomallei selective media without gentamicin are needed for accurate melioidosis laboratory diagnosis in Sarawak. This finding may also have implications for environmental sampling of other locations to test for B. pseudomallei endemicity

Chlamydia trachomatis from Australian Aboriginal people with trachoma are polyphyletic composed of multiple distinctive lineages.

Chlamydia trachomatis causes sexually transmitted infections and the blinding disease trachoma. Current data on C. trachomatis phylogeny show that there is only a single trachoma-causing clade, which is distinct from the lineages causing urogenital tract (UGT) and lymphogranuloma venerum diseases. Here we report the whole-genome sequences of ocular C. trachomatis isolates obtained from young children with clinical signs of trachoma in a trachoma endemic region of northern Australia. The isolates form two lineages that fall outside the classical trachoma lineage, instead being placed within UGT clades of the C. trachomatis phylogenetic tree. The Australian trachoma isolates appear to be recombinants with UGT C. trachomatis genome backbones, in which loci that encode immunodominant surface proteins (ompA and pmpEFGH) have been replaced by those characteristic of classical ocular isolates. This suggests that ocular tropism and association with trachoma are functionally associated with some sequence variants of ompA and pmpEFGH

Rapid Detection of the H275Y Oseltamivir Resistance Mutation in Influenza A/H1N1 2009 by Single Base Pair RT-PCR and High-Resolution Melting

Introduction: We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR), high-resolution melting (HRM) assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses.Findings: A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of nonspecific primer-dimer was evident and affected the overall melting temperature (Tm) of the amplified products. Due to primer-dimer appearance at .30 cycles we found that if the cycle threshold (CT) for a dilution was .30, the HRM assay did not consistently discriminate mutant from wildtype. Where the CT was ,30 we noted an inverse relationship between CT and Tm and fitted quadratic curves allowed the discrimination of wildtype, mutant and 30:70 mutant:wildtype virus mixtures. We compared the CT values for a TaqMan H1N1 09 detection assay with those for the HRM assay using 59 clinical samples and demonstrated that samples with a TaqMan detection assay CT.32.98 would have an H275Y assay CT.30. Analysis of the TaqMan CT values for 609 consecutive clinical samples predicted that 207 (34%) of the samples would result in an HRM assay CT.30 and therefore not be amenable to the HRM assay.Conclusions: The use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting Tm against CT. Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations

Contaminated fingers: a potential cause of Chlamydia trachomatis-positive urine specimens

This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Objectives The detection of an STI agent in a urogenital tract (UGT) specimen from a young child is regarded as being indicative of sexual abuse. However, the probabilities of contamination events that could conceivably lead to STI positive specimens in the absence of sexual contact are unclear. The objective was to estimate the potential for fingers that have come in contact with Chlamydia trachomatis-positive urine to detectably contaminate C. trachomatis-negative urine. Methods The study design was based on self-experimentation. Dilutions of C. trachomatis elementary bodies (EBs) were prepared. A participant contacted an EB dilution then a urine surrogate specimen. The experiment was performed by three participants using three C. trachomatis isolates, of genotype E, F and B. Two surrogate urine contact methods were used to mimic contamination of a carer assisting with a child’s urine collection. All EB dilutions and urine surrogate specimens were subjected to C. trachomatis assay and quantification in a real-time PCR-based diagnostic system. Results The amplimer crossing point (Cq) for EB dilutions was 10.0±1.6 less than for corresponding finger contacted urine specimens, which corresponds to ~10 µL of EB suspension transferred. This was largely independent of participant identity, C. trachomatis strain or EB dilution. Hand decontamination led to large reductions in EBs transferred, but transfer remained consistently detectable. Recent Cq data from C. trachomatis-positive clinical urine specimens were collated, and 20% clearly contained sufficient C. trachomatis to detectably contaminate another specimen by finger-mediated transfer, as in this experiment. Conclusions This study directly demonstrated the potential for urine contaminated fingers to convert a C. trachomatis-negative urine specimen to C. trachomatis positive as a result of contact. Accordingly, procedures for urine specimen collection, particularly from children, need to be designed to prevent contamination