Specific PCRs Research Articles

Using a practical molecular capsular serotype prediction strategy to investigate Streptococcus pneumoniae serotype distribution and antimicrobial resistance in Chinese local hospitalized children.

BackgroundChina is one of ten countries with the highest prevalence rate of pneumococcal infections. However, there is limited serotype surveillance data for Streptococcus pneumoniae, especially from the community or rural regions, partly due to limited serotyping capacity because Quellung serotyping is only available in few centers in China. The aim of this study was to develop a simple, practical and economic pneumococcal serotype prediction strategy suitable for future serotype surveillance in China.MethodsIn this study, 193 S. pneumoniae isolates were collected from hospitalized children, 96.9 % of whom were < 5 years old. The cpsB sequetyping, complemented by selective and modified USA CDC sequential multiplex-PCR, was performed on all the isolates, and serotypes 6A-6D specific PCRs were done on all serogroup 6 isolates. Based on systematic analysis of available GenBank cpsB sequences, we established a more comprehensive cpsB sequence database than originally published for cpsB sequetyping. Antibiotic susceptibility of all isolates was determined using the disk diffusion or E-test assays.ResultsWe built up a comprehensive S. pneumoniae serotype cpsB sequetyping database for all the 95 described serotypes first, and then developed a simple strategy for serotype prediction based on the improved cpsB sequetyping and selective multiplex-PCR. Using the developed serotype prediction strategy, 191 of 193 isolates were successfully “serotyped”, and only two isolates were “non-serotypeable”. Sixteen serotypes were identified among the 191 “serotypeable” isolates. The serotype distribution of the isolates from high to low was: 19 F (34.7 %), 23 F (17.1 %), 19A (11.9 %), 14 (7.3 %), 15B/15C (6.7 %), 6B (6.7 %), 6A (6.2 %), 9 V/9A (1.6 %); serotypes 6C, 3, 15 F/15A, 23A and 20 (each 1.1 %); serotypes 10B, 28 F/28A and 34 (each 0.5 %). The prevalence of parenteral penicillin resistance was 1.0 % in the non-meningitis isolates and 88.6 % in meningitis isolates. The total rate of multidrug resistance was 86.8 %.ConclusionsThe integrated cpsB sequetyping supplemented with selective mPCR and serotypes 6A-6D specific PCRs “cocktail” strategy is practical, simple and cost-effective for use in pneumococcal infection serotype surveillance in China. For hospitalized children with non-meningitis penicillin-susceptible pneumococcal infections, clinicians still can use narrow-spectrum and cheaper penicillin, using the parenteral route, rather than using broader-spectrum and more expensive antimicrobials.Electronic supplementary materialThe online version of this article (doi:10.1186/s12887-016-0589-7) contains supplementary material, which is available to authorized users.

Application of a multiplex PCR assay for the detection of gastrointestinal pathogens in a rural African setting.

BackgroundDespite high morbidity and mortality, the laboratory diagnosis of gastrointestinal infections is largely neglected in tropical African settings. This study aims to apply the Luminex multiplex PCR assay for the diagnosis of gastrointestinal pathogens in rural Ghana to evaluate its usefulness as a routine method.MethodsA case–control study was conducted at the Agogo Presbyterian Hospital in Ghana. Stool samples were collected from children below 6 years of age with (cases) and without (controls) diarrhoea. Samples were screened for 15 different diarrhoeal pathogens by the Luminex xTAG GPP assay and associations between diarrhoea and gastrointestinal infections and fractions attributable to diarrhea (AF) were determined.ResultsThe Luminex PCR assay identified organisms in 96.6 % (n = 428) of 443 cases and in 92.5 % (n = 221) of 239 selected controls. A mean of 2.5 (standard deviation [SD]: ±1.3) and 2.3 (SD: ±1.3) organisms per sample were detected in cases and controls respectively. An association with diarrhoea was found for rotavirus (adjusted odds ratio [aOR] = 7.2; 95 % confidence interval [CI]: 2.9–18.1), norovirus (aOR = 2.7; 95 % CI: 1.4–5.3) and Shigella spp. (aOR = 1.7; 95 % CI: 1.2–2.4) with respective AFs of 12.5 % (95 % CI: 9.6–15.3), 7.9 % (95 % CI: 3.8–11.7) and 16.9 % (95 % CI: 6.9–25.9).ConclusionThe high proportion of pathogen-positive stool samples with a high number of co-infections in cases and controls suggests a substantial amount of transient or colonizing microorganisms for which treatment is not necessarily implicated. The use of sequential diagnostic algorithms with pathogen specific or quantitative PCRs might be most appropriate for diagnosing gastrointestinal infections.

Non-Functional ("haploinsufficient"), but Not Dominant Negative Clonal IKZF1 Deletions Confer an Adverse Prognosis in Adult BCR-ABL-Negative Acute Lymphoblastic Leukemia

Background: IKAROS (IKZF1) is a key transcription factor for B-cell development. The IKZF1 protein exerts its functions as a dimer (homodimer or heterodimer with other IKAROS family members). Various IKZF1 intragenic deletions have been identified as recurrent aberrations in acute lymphoblastic leukemia: those resulting in the loss of transcription of one allele or at least loss of the dimerization domain ("haploinsufficient", "non-functional") and those involving only the DNA-binding domain but retaining the dimerization domain of the transcription factor, leading to so-called "dominant negative" isoforms.Several studies in pediatric ALL have confirmed the negative prognostic impact of IKZF1 alterations although there is still no consensus if they are independent risk factors in a multivariate analysis. The situation in adult ALL is less clear and few larger studies have addressed this issue, in particular the prognostic impact of different types of IKZF1 alterations.Objectives: We investigated archived patient samples obtained in the context of the German Multicenter ALL Study Group (GMALL) therapy studies for different IKZF1 alterations to assess their prognostic implications and their molecular pattern.Methods: We used RT-PCR to determine the presence of aberrant IKZF1 transcript variants and additionally investigated all patients by DNA-based PCRs for the IKZF1 aberrations D4-7, D2-7, D4-8, D2-8. Rare transcript variants, such as D2-3, D2, D5-7 were molecularly characterized on the DNA level by specific PCRs. IKZF1 aberrations were quantified using gel densitometry and/or quantitative real time PCR such that clonal and subclonal aberrations could be distinguished. All chromosomal breakpoints were molecularly characterized.Results: In total, 482 B cell precursor ALL patients aged between 15 and 65 years were included in the analysis. Samples were obtained at primary diagnosis and tested BCR-ABL -negative. Other molecular aberrations were tested according to the GMALL standards: 39 MLL-AF4-positive, 4 MLL-ENL, 1 MLL-AF9, 30 TCF3-PBX1, 3 ETV6-RUNX1. The immunophenotypes were the following: 424 pre B/common, 58 pro B.One hundred and twenty-eight out of 482 (27%) patients carried at least one IKZF1 deletion, with 37 patients carrying more than one deletion. Altogether there were 175 IKZF1 deletions, 71 cases of D4-7, 47 with D2-7, 26 with D4-8, 19 with D2-3, 10 with D2-8, and 1 with D5-7 and D2 each. Taken together 56 patients (12%) carried only non-functional mutations while 50 (10%) had dominant-negative mutations only. There was a group of 22 patients with both types of mutations (5%).Evaluating the prognostic impact, we considered the effect of non-functional and dominant-negative mutations separately. Patients with a non-functional IKZF1 mutation had a reduced overall survival (OS at 5 ys 37% vs 59% in IKZF1-unmutated patients, N=78/404, p=0.001) while dominant-negative mutations had no effect on OS (OS at 5 ys 54% compared to 56%, N=72/410, p=0.95). However, solely present subclonal non-functional mutations did not affect survival (OS at 5 ys 59% compared to 59%, N=23/404). In the dominant-negative group, clonality had no influence on OS (p=0.56, N=45/73/351). Taken together, patients with clonal non-functional IKZF1 had a significantly worse OS compared to those without (0.28 vs 0.59, N=53/427, p<.0001). This was true for patients with standard risk as defined by GMALL risk stratification (0.37 vs 0.68, N=24/243, p.0002). High risk patients also tended to do worse, which however did not reach significance (0.26 vs 0.46, N=30/184, p=.06).With regard to molecular analysis 193 chromosomal fusion sites were characterized. In the great majority (>95%) putative cryptic recombination sites were identified in the near vicinity thus underlining the causal role of the VDJ recombination machinery in the generation of these aberrations.Conclusions: We conducted a detailed and large-scale molecular analysis of intragenic IKZF1 deletions and identified clonal non-functional IKZF1 deletions as adverse prognostic factor in adult and adolescent B cell precursor ALL patients treated according to the GMALL protocols. Subclonal, or dominant negative IKZF1 mutations had no prognostic effect. It remains open for further analysis whether clonal non-functional IKZF1 deletions represent an independent prognostic factor in MRD-based risk stratification. DisclosuresWäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Widespread dispersion of the resistance element tet(B)::ISCR2 in XDR Acinetobacter baumannii isolates.

Acinetobacter baumannii is a significant nosocomial pathogen often associated with extreme drug resistance (XDR). In Argentina, isolates of A. baumannii resistant to tetracyclines have accounted for more than 40% of drug-resistant isolates in some hospitals. We have previously reported the dispersion of the tet(B) resistance element associated with the ISCR2 transposase in epidemiologically unrelated A. baumannii isolates recovered from 1983 to 2011. This study extends this surveillance to 77 recent (2009-2013) XDR A. baumannii isolates with different levels of minocycline susceptibility. Isolates were examined by a pan-PCR assay, which showed six different amplification patterns, and specific PCRs were used for the confirmation of the the ΔISCR2-tet(B)-tet(R)-ISCR2 element. The tet(B) gene was present in 66 isolates and the ISCR2 element in 68 isolates; the tet(B) gene was associated with ISCR2 in all tet(B)-positive isolates. We conclude that this element is widespread in XDR A. baumannii isolates from Argentina and could be responsible for the emergence of tetracycline resistance in recent years.

How common are chlamydia-related bacteria in early pregnancy and are they associated with miscarriage or preterm birth? Community-based prospective cohort study

BackgroundSerological case-control studies suggest that certain chlamydia-related bacteria (Chlamydiales) that cause cows to miscarry might do the same in human beings. Included in the order Chlamydiales are Waddlia chondrophila, Chlamydophila abortus, and Chlamydia trachomatis. We aimed to investigate the prevalence of Chlamydiales in pregnant women, and possible associations with miscarriage or preterm birth. MethodsWe tested stored urine samples from a carefully characterised cohort of 847 pregnant women recruited at mean 49·3 (SD 10·1) days’ gestation from 37 general practices in London, UK. Previous repeat testing of samples positive for Mycoplasma genitalium confirmed bacterial DNA integrity after storage. W chondrophila and pan-Chlamydiales specific real-time PCRs targeting the 16s rRNA gene were used to test samples. Samples positive on either of the two PCRs were subjected to DNA sequencing and Chlamydia trachomatis PCR. Outcomes were compared between infected and uninfected women with Fisher's exact test. Ethics review was conducted by Wandsworth, Croydon, and Riverside Research Ethics Committees. FindingsThe overall prevalence of Chlamydiales was 4·3% (36/847, 95% CI 3·0–5·8). Prevalence of W chondrophila was 0·6% (5/847, 0·2–1·4), Chlamydia trachomatis 1·7% (14/847, 0·9–2·8), and other Chlamydiales species 2·0% (17/847, 1·2–3·2). Infection with Chlamydia trachomatis (but not with other Chlamydiales or W chondrophila) was more common in women younger than 25 years, of black ethnicity, or with bacterial vaginosis. Follow-up was 99·9% (846/847) at 16 weeks’ gestation and 89·6% (759/847) at term. No infection was significantly associated with miscarriage (prevalence 10%, 84/827) or spontaneous preterm birth before 37 weeks’ (4%, 23/628) but numbers were small. One of three (33%) followed-up women infected with W chondrophila had a preterm birth compared with 22 (4%) of 625 uninfected women (p=0·11). Sequencing of 25 samples that were positive on W chondrophila or pan-Chlamydiales PCR revealed seven samples (28%) positive for Chlamydiales bacterium sequences that have been associated with respiratory tract infections in children. Interpretation4% of newly pregnant women tested positive for Chlamydiales, including species known to be pathogenic in mothers and neonates. Higher rates might have been found in vaginal samples and the study lacked power to test associations. Although W chondrophila might be associated with preterm birth, the prevalence was very low in this urban, community-based sample, suggesting that screening is unlikely to be cost-effective in such a population. FundingMedical Research Council.

Novel MicroRNA signatures in HPV-mediated cervical carcinogenesis in Indian women.

This study aimed to investigate the role of miRNAs in HPV-mediated cervical pre-cancer and cancer cases in Indian population. We analysed the HPV infection and its genotypes in uterine cervical pre-cancer (n = 80), cancer (n = 200) and normal cervical samples (n = 150) by consensus sequence PCR followed by type specific PCRs. Also, microRNA profiling was done in a subset of cervical pre-cancer (n = 20), cancer cases (n = 50) and normal samples (n = 30) by real-time quantitative PCR (qRT-PCR). The prevalence of HPV infection in pre-cancer was found to be 81% (65/80) and 94% (188/200) in cancer cases, with most predominant high-risk HPV type-16 (HR-HPV-16) in 83% of cancer and 91% of pre- cancer cases, respectively. Whereas in controls, the HPV infection was found to be very low (5%). The miRNA profiling revealed that in cervical pre-cancer, 100 miRNAs were significantly (p < 0.001) differentially expressed with 70 miRNAs upregulated and 30 miRNAs downregulated. In cervical cancer cases, 383 miRNA were found to be differentially expressed (p < 0.001), of which 350 miRNAs were upregulated and 33 miRNAs were downregulated. We also observed that 182 miRNAs were differentially expressed (p < 0.001) in HPV-16/18-positive (SiHa/HeLa) cell lines compared with HPV-negative (C33A) cell line. In addition, we identified the novel microRNAs such as miR-892b, miR-500, miR-888, miR-505 and miR-711 in cervical precancerous lesions and cervical cancer cases in Indian population. Taken together, the study demonstrates a crucial role of microRNAs in cervical cancer, which may serve as potential early diagnostic markers for cervical carcinogenesis.

Practical Prediction of Ten Common Streptococcus pneumoniae Serotypes/Serogroups in One PCR Reaction by Multiplex Ligation-Dependent Probe Amplification and Melting Curve (MLPA-MC) Assay in Shenzhen, China.

Background Streptococcus pneumoniae has more than 95 distinct serotypes described to date. However, only certain serotypes are more likely to cause pneumococcal diseases. Thus serotype surveillance is important for vaccine formula design as well as in post-vaccine serotype shift monitor. The goal of this study was to develop a practical screening assay for ten Shenzhen China common pneumococcal serotypes/serogroups in one molecular reaction.MethodsA molecular assay, based on multiplex ligation-dependent probe amplification (MLPA) and melting curve (MC) analysis, was developed in an integrated approach (MLPA-MC) for the detection of ten capsular serotypes/serogroups 4, 6 (6A/6B/6C/6D), 9V/9A, 14, 15F/15A, 15B/15C, 18 (18F/18A/18B/18C), 19F, 19A and 23F. We designed serotype/serogroup-specific MLPA probes and fluorescent detection probes to discriminate the different serotypes/serogroups in one molecular reaction. The three steps of MLPA-MC assay are continuous reactions in one well detected by LightCycler 480. A total of 210 S. pneumoniae isolates from our local Maternity and Child Health Hospital were randomly chosen to evaluate the assay against published multiplex PCR assays.ResultsOur results showed that 198 (94.3%) of S. pneumoniae isolates were type-able by our assays and the results were in complete concordance with the published multiplex PCRs. Using the MLPA-MC assay, 96 S. pneumoniae isolates could be typed within 3 hours with limited hands-on time. This serotype/serogroup-screening assay can be easily modified or extended by modification of the serotype/serogroup-specific MLPA probes combinations according to the needs of different laboratories.ConclusionsWe recommend use of this assay as a starting point for screening serotype/serogroup frequencies. There is a need for this assay to be combined with other molecular typing assays, like published serotype specific PCRs, or even the Quellung reaction for serotype confirmation.

PTH-228 Genotyping of rotavirus isolates prior to the introduction of the rotavirus vaccine in scotland and early indications of the impact of the vaccine

<h3>Introduction</h3> Rotaviruses (RV) are the leading cause of severe gastroenteritis in children less than five years of age worldwide. Rotarix®, a live attenuated monovalent vaccine containing a RV strain of G1P[8] specificity has been included in the routine childhood immunisation schedule from June 2013 in Scotland and is offered to infants under the age of four months in two doses. This study aimed to characterise the prevalent rotavirus strains in Scotland prior to the introduction of the vaccine. <h3>Method</h3> Rotavirus positive faecal samples from various Scottish regional virology laboratories covering the years 2012–2013, before the introduction of the vaccine, were genotyped. Viral RNA was extracted from faecal suspensions using guanidine isothiocyanate – silica gel extraction method. VP7 and VP4 gene specific multiplex hemi-nested PCRs were used for genotyping. Surveillance systems were established by Health Protection Scotland to monitor the impact of the vaccine. <h3>Results</h3> A total of 387 samples were genotyped from the regional virology laboratories in Aberdeen, Dundee, Inverness, Glasgow and Edinburgh. The commonest strain in Scotland was G1P[8] accounting for 72.1% of cases. The other strains that were identified were G2P[4] in 7.2%, G4P[8] in 6.9%, G9P[8] in 3.4% and G3P[8] in a further 2.3% of cases. Single cases of G12P[8] and G9P[4] were also found (0.26% respectively). Mixed infections were seen in 5.4% of cases. The provisional uptake for the first dose of Rotarix® vaccine in Scotland for children born between 1^stof July to 30^thSept 2013 was 92.7%% for the completed course by 12 months. The laboratory confirmed cases of RV reported to HPS via ECOSS (The Electronic Communication of Surveillance in Scotland) showed a marked reduction in confirmed reports of RV compared to a three year average of the years 2011–13. Surrogate markers, e.g. calls to NHS24 and GP consultations with childhood diarrhoea and vomiting, have also shown a downward trend. <h3>Conclusion</h3> The pre-vaccine surveillance of RV strains confirms that G1P[8] is the predominant strain in Scotland. There has been a promising reduction in laboratory confirmed cases of RV in Scotland following the introduction of the vaccine. The baseline genotyping data will be used to ascertain cross protection against strains and also identify vaccine induced RV strain shifts and an overall evaluation of the programme. <h3>Disclosure of interest</h3> None Declared.

A real time genotyping PCR assay for polyomavirus BK

BackgroundPolyomavirus BK (BKV) may cause nephropathy in renal transplant recipients and hemorrhagic cystitis in bone marrow recipients. We developed real-time PCRs (RT-PCR) to determine easily and rapidly the different BKV genotypes (BKGT) (I–IV). MethodsOn the VP1 gene a duplex of RT-PCRs was developed and validated to differentiate the four main BKGT. 212 BKV positive samples (21 plasma, 191 urine) were tested with these specific PCRs. Of these 212 samples, 55 PCR results were additionally confirmed by sequencing a VP1 gene fragment (nucleotide 1630–1956). ResultsFor every genotype, a highly specific, precise and internally controlled assay was developed with a limit of detection of log 3 copies per ml. In 18 (8.5%) of these samples genotyping was not successful due to a low viral load. By sequence analysis, the genotype of 46 out of 55 and 2 out of 4 samples with double infection could be confirmed. ConclusionsThis study describes RT-PCRs for detection of the main BKGT. It proved to be rapid, cheap and sensitive compared to sequencing. Double infections can also be detected. This method will be of value to investigate the role of BKV infection in relation to the genotype.

Investigation of the molecular epidemiology of Acinetobacter baumannii isolated from patients and environmental contamination.

The objective of this work was to investigate correlations between Acinetobacter baumannii isolates from neurosurgical intensive care unit patients and its environment. This is a prospective, observational study. The minimal inhibitory concentrations of antimicrobial agents against 27 clinical and 28 environmental isolates were determined by the agar dilution method. Molecular genotyping was performed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The presence of carbapenemase and metallo-β-lactamase genes were analyzed by specific PCRs and DNA sequencing. From the clinical A. baumannii isolates, 25.9% were found resistant to minocycline, 51.9% to cefoperazone-sulbactam, 59.3% to imipenem and 70% resistant to other antimicrobial agents. Environmental isolates were more sensitive compared with clinical isolates (P<0.05). Twenty-seven clinical isolates comprised three ERIC-PCR genotypes, four major PFGE pulsotypes and five distinct MLST sequence types (STs) (ST208, ST368, ST191, ST195, ST540), all belonging to CC92 with only one locus (gpi) difference among them. Twenty-eight environmental isolates showed more diverse genetic types than clinical isolates and comprised six ERIC-PCR groups, nine PFGE groups and two main STs (ST208, ST229). Four clinical and 15 environmental isolates could not be identified by MLST and were assigned to non-clonal STs. We identified the presence of the blaOXA-23 carbapenemase encoding gene in most of the clinical (21/27) but fewer in the environmental isolates (3/28). The A. baumannii strains isolated from patients were genetically similar to the environmental strains, with CC92 members as the major fraction but with different antibiotic susceptibilities.

Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs.

To verify whether porcine deltacoronavirus infection induces disease, we inoculated gnotobiotic pigs with 2 virus strains (OH-FD22 and OH-FD100) identified by 2 specific reverse transcription PCRs. At 21–120 h postinoculation, pigs exhibited severe diarrhea, vomiting, fecal shedding of virus, and severe atrophic enteritis. These findings confirm that these 2 strains are enteropathogenic in pigs.

Implication of high risk human papillomavirus HR-HPV infection in prostate cancer in Indian population--a pioneering case-control analysis.

Prostate cancer is the second most common cancer with sexual history as a consistent risk factor. This is the pioneering study that evaluates the frequency of HPV infection in prostate cancer in India. Ninety five (95) histopathologically confirmed cancer and fifty five (55) BPH from Indian population were analyzed for HPV infection using a pair of consensus sequence primer followed by type specific PCRs for both high-risk and low-risk HPV types. The data demonstrate HPV infection in 41% of prostate tumor biopsies and 20% in BPH. Subsequent PCR- based HPV typing using type - specific primers revealed 32% were infected with HPV type 16 whereas 6% were found to be positive for HPV type 18, while in BPH controls only 5% of the BPH controls were infected with HPV 16 and this difference was highly significant (p = 0.0004). Significant proportion of HPV infected (74%) cases belonged to stage III and IV (p < 0.001) with a high Gleason score ≥8 (p = 0.003). The study represents for the first time the incidence of HPV infection in prostate cancer in Indian population and strengthens the hypothesis that HPV infection could be one of the co factor associated with progression of prostate cancer.

Helicobacteraceae in Bulk Tank Milk of Dairy Herds from Northern Italy.

Helicobacter pylori is responsible for gastritis and gastric adenocarcinoma in humans, but the routes of transmission of this bacterium have not been clearly defined. Few studies led to supposing that H. pylori could be transmitted through raw milk, and no one investigated the presence of other Helicobacteraceae in milk. In the current work, the presence of Helicobacteraceae was investigated in the bulk tank milk of dairy cattle herds located in northern Italy both by direct plating onto H. pylori selective medium and by screening PCR for Helicobacteraceae, followed by specific PCRs for H. pylori, Wolinella spp., and “Candidatus Helicobacter bovis.” Three out of 163 bulk milk samples tested positive for Helicobacteraceae, but not for the subsequent PCRs. H. pylori was not isolated in any case. However, given similar growth conditions, Arcobacter butzleri, A. cryaerophilus, and A. skirrowii were recovered. In conclusion, the prevalence of Helicobacteraceae in raw milk was negligible (1.8%), and H. pylori was not identified in any of the positive samples, suggesting that, at least in the farming conditions of the investigated area, bovine milk does not represent a potential source of infection.

Molecular profiling of single circulating tumor cells with diagnostic intention.

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance.

Whole-Genome Sequencing Identifies Patient-Specific DNA Minimal Residual Disease Markers in Neuroblastoma

PCR-based detection of minimal residual disease (MRD) in neuroblastoma is currently based on RNA markers; however, expression of these targets can vary, and only paired-like homeobox 2b has no background expression. We investigated whether chromosomal breakpoints, identified by whole-genome sequencing (WGS), can be used as patient-specific DNA MRD markers. WGS data were used to develop large numbers of real-time PCRs specific for tumors of eight patients. These PCRs were used to quantify chromosomal breakpoints in primary tumor and bone marrow samples. Finally, the DNA breakpoints with the highest abundance were compared with a panel of RNA markers. By WGS we identified 42 chromosomal breakpoints in tumor samples from eight patients and developed specific quantitative real-time PCRs for each breakpoint. The tumor-specific breakpoints were all present in bone marrow at diagnosis. For one patient slight clonal selection was observed in response to treatment. Positivity of DNA MRD markers preceded disease progression in four of five patients; in one patient the RNA markers remained negative. For 16 of 22 samples MRD levels determined by RNA and DNA were comparable and in 6 of 22 samples higher MRD levels were detected by DNA markers. DNA breakpoints used as MRD targets in neuroblastoma are reliable and stable markers. In addition, this technique might be applicable for detecting tumor cells in other types of cancer.

Pathogenic eukaryotes in gut microbiota of western lowland gorillas as revealed by molecular survey.

Although gorillas regarded as the largest extant species of primates and have a close phylogenetic relationship with humans, eukaryotic communities have not been previously studied in these populations. Herein, 35 eukaryotic primer sets targeting the 18S rRNA gene, internal transcribed spacer gene and other specific genes were used firstly to explore the eukaryotes in a fecal sample from a wild western lowland gorilla (Gorilla gorilla gorilla). Then specific real-time PCRs were achieved in additional 48 fecal samples from 21 individual gorillas to investigate the presence of human eukaryotic pathogens. In total, 1,572 clones were obtained and sequenced from the 15 cloning libraries, resulting in the retrieval of 87 eukaryotic species, including 52 fungi, 10 protozoa, 4 nematodes and 21 plant species, of which 52, 5, 2 and 21 species, respectively, have never before been described in gorillas. We also reported the occurrence of pathogenic fungi and parasites (i.e. Oesophagostomum bifurcum (86%), Necator americanus (43%), Candida tropicalis (81%) and other pathogenic fungi were identified). In conclusion, molecular techniques using multiple primer sets may offer an effective tool to study complex eukaryotic communities and to identify potential pathogens in the gastrointestinal tracts of primates.

Occurrence, diversity, and host association of intestinal Campylobacter, Arcobacter, and Helicobacter in reptiles.

Campylobacter, Arcobacter, and Helicobacter species have been isolated from many vertebrate hosts, including birds, mammals, and reptiles. Multiple studies have focused on the prevalence of these Epsilonproteobacteria genera in avian and mammalian species. However, little focus has been given to the presence within reptiles, and their potential zoonotic and pathogenic roles. In this study, occurrence, diversity, and host association of intestinal Epsilonproteobacteria were determined for a large variety of reptiles. From 2011 to 2013, 444 cloacal swabs and fecal samples originating from 417 predominantly captive-held reptiles were screened for Epsilonproteobacteria. Campylobacter, Arcobacter, and Helicobacter genus specific PCRs were performed directly on all samples. All samples were also cultured on selective media and screened for the presence of Epsilonproteobacteria. Using a tiered approach of AFLP, atpA, and 16S rRNA sequencing, 432 Epsilonproteobacteria isolates were characterized at the species level. Based on PCR, Campylobacter, Arcobacter, and Helicobacter were detected in 69.3% of the reptiles; 82.5% of the chelonians, 63.8% of the lizards, and 58.0% of the snakes were positive for one or more of these genera. Epsilonproteobacteria were isolated from 22.1% of the reptiles and were isolated most frequently from chelonians (37.0%), followed by lizards (19.6%) and snakes (3.0%). The most commonly isolated taxa were Arcobacter butzleri, Arcobacter skirrowii, reptile-associated Campylobacter fetus subsp. testudinum, and a putative novel Campylobacter taxon. Furthermore, a clade of seven related putative novel Helicobacter taxa was isolated from lizards and chelonians. This study shows that reptiles carry various intestinal Epsilonproteobacteria taxa, including several putative novel taxa.

Plasmid-mediated qnrA1 in Klebsiella pneumoniae ST147 in Recife, Brazil

Qnr-mediated quinolone resistance is increasingly detected worldwide, but few studies have been carried out so far in Brazil. The aim of this study was to test for qnr determinants in isolates of ciprofloxacin-resistant Klebsiella pneumoniae. Fifteen ciprofloxacin-resistant isolates from urine cultures of hospitalized patients at a university hospital in North-East Brazil were investigated. Specific PCRs were performed for blaCTX-M and blaTEM, qnr, and class 1 integrons. Plasmid analyses and sequence type (ST) determination were performed, as described previously. The KP930 isolate showed qnrA1 and blaTEM-1, together with dfrA12 and aadA2 in a class 1 integron. The qnr gene was located in a 133-kb plasmid. Multilocus sequence typing classified the isolate as ST147. We identified the combination of qnr with ST147 in Brazil; this is a clone that has disseminated widely and successfully in Latin America. The purpose of describing Qnr-mediated quinolone resistance in North-East Brazil is to draw attention to the spread of this mechanism in the country.

Characterization of methicillin-resistant coagulase-negative staphylococci in milk from cows with mastitis in Brazil.

Staphylococci are one of the most prevalent microorganisms in bovine mastitis. Staphylococcus spp. are widespread in the environment, and can infect animals and humans as opportunistic pathogens. The objective of this study was to determine the frequency of methicillin-resistance (MR) among coagulase-negative staphylococci (CoNS) previously obtained from milk of mastitic cows in Brazil and to characterize the antimicrobial resistance phenotype/genotype and the SCCmec type of MRCoNS isolates. Identification of MRCoNS was based on both biochemical and molecular methods. Susceptibility testing for eleven antimicrobials was performed by disk-diffusion agar. Antimicrobial resistance genes and SCCmec were investigated by specific PCRs. Twenty-six MRCoNS were detected (20% of total CoNS), obtained from 24 animals, and were identified as follows: S. epidermidis (7 isolates), S. chromogenes (7), S. warneri (6), S. hyicus (5) and S. simulans (1). All MRCoNS isolates carried mecA while the mecC gene was not detected in any CoNS. The SCCmec IVa was demonstrated in nine MRCoNS, while the remaining 17 isolates harbored non-typeable SCCmec cassettes. In addition to oxacillin and cefoxitin resistance, MRCoNS showed resistance to tetracycline (n=7), streptomycin (n=6), tobramycin (n=6), and gentamicin (n=4), and harbored the genes tet(K) (n=7), str (n=3), ant(4') (n=6) and aac(6')-aph(2″) (n=4), respectively. In addition, seven strains showed intermediate resistance to clindamycin and two to streptomycin, of which two harboured the lnu(B) and lsa(E) genes and two the aad(E) gene, respectively. One isolate presented intermediate erythromycin and clindamycin resistance and harbored an erm(C) gene with an uncommon 89-bp deletion rendering a premature stop codon. MRCoNS can be implicated in mastitis of cows and they constitute a reservoir of resistance genes that can be transferred to other pathogenic bacteria.

Genetic elements associated with antimicrobial resistance among intestinal bacteria.

Background:Integrons are the major reasons of multidrug resistance (MDR) among enteropathogenic bacteria. Occurrence of horizontal gene transfer between integron-carrying microorganisms and other enteric bacteria may increase the rate of emergence of integron-associated antibiotic resistance.Objectives:The objective of this study was to investigate class 1 integrons among members of enteropathogenic bacteria isolated from patients in Iran.Materials and Methods:A total of 120 enteropathogenic bacterial isolates from diarrhoeal patients were included in this study. Identities of the isolates were investigated by biochemical tests and confirmed by genus or species specific PCRs. Antimicrobial susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Presence of class 1 integron among the isolates was investigated using primers specific for the integrase gene conserved region.Results:The result of this study showed the highest resistance to trimethoprim and cotrimoxazole, especially in enteropathogenic Escherichia coli (EPEC) (100%), Shigella sonnei (93.7%) and Vibrio cholerae (95%). The results showed that 16 (57.1%) of the 28 EPEC, 9 (25%) of the 36 Salmonella enterica, 32 of the 13 (40.6%) Sh. sonnei, and only 1 (4.2%) of the 24 V. cholerae isolate harbored class 1 integron.Conclusions:The data obtained in the present study suggested that class 1 integrons are widely distributed among members of Enterobacteriaceae. The resistance patterns of our E. coli, S. sonnei, and S. enterica isolates were nearly identical, suggesting the same genetic elements involved in attainment of multi-drug resistance.