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1.
BMC Infect Dis ; 19(1): 571, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266450

RESUMO

BACKGROUND: Carbapenemase-producing organisms (CPOs) have emerged as antibiotic-resistant bacteria of global concern. Here we assessed the performance of the Carba (beta) assay, a multiplex real-time PCR assay developed by SpeeDx for the detection of key carbapenemase-encoding genes: KPC, NDM, OXA-48-like, IMP-4-like, and VIM. METHODS: DNA extracts of 180 isolates were tested with the Carba (beta) assay, using previously validated in-house TaqMan probe assays for the relevant carbapenemase genes as the reference standard. The Carba (beta) assay was then directly used to screen 460 DNA extracts of faecal specimens, with positive results subjected to the aforementioned in-house assays plus Sanger sequencing. RESULTS: The Carba (beta) assay correctly identified the presence of the respective carbapenemase genes in 154 of 156 isolates and provided negative results for all 24 non-CPO isolates. Two isolates provided positive results for OXA-48-like carbapenemase by the Carba (beta) assay only. The Carba (beta) assay had sensitivities of 100% for all targets, and specificities of 100% for KPC, NDM, IMP-4-like, and VIM targets, and 98.5% for OXA-48-like targets. When applied directly to faecal specimens, eight samples were positive by the Carba (beta) assay, two of which were confirmed by in-house TaqMan probe PCR or DNA sequencing. CONCLUSIONS: The Carba (beta) assay is highly sensitive and specific for detecting key carbapenemase genes in isolates. Further testing is required to assess this assay's suitability for direct screening of clinical specimens.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , beta-Lactamases/genética , Antibacterianos , Bactérias/efeitos dos fármacos , Técnicas Bacteriológicas/métodos , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Humanos , Sensibilidade e Especificidade
2.
J Antimicrob Chemother ; 74(8): 2214-2219, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31170281

RESUMO

OBJECTIVES: Our aim was to investigate the emergence and spread of ciprofloxacin resistance in clinical Neisseria gonorrhoeae isolates in New South Wales, Australia, from the first reported case in 1991 until ciprofloxacin resistance was sustained at or above the WHO threshold for treatment change of 5% (1999), to inform future strategies for controlling gonococcal antimicrobial resistance. METHODS: The index isolate and all subsequent clinical isolates of ciprofloxacin-resistant N. gonorrhoeae in New South Wales from 1991 to 1999 were genotyped using a previously described method on the Agena MassARRAY iPLEX platform. Region of acquisition data, where available, were used to determine whether cases were travel associated. RESULTS: In New South Wales, of the 325 ciprofloxacin-resistant N. gonorrhoeae isolates reported from 1991 to 1999, 98% (320/325) were able to be recovered and 100% (320/320) were genotyped. There were 66 different genotypes, comprising 1-99 isolates each. Notably no single clone was found to account for ciprofloxacin resistance being sustained in the population, with considerable variability in genotype prevalence observed throughout the study period. A total of 65% (209/320) of genotyped isolates had information regarding the likely place of acquisition; of these, 44% (93/209) were associated with overseas travel or sexual contact with an overseas visitor. The first ciprofloxacin-resistant N. gonorrhoeae in New South Wales was associated with travel to Thailand. Index cases of each resistant genotype were significantly more likely to have been acquired overseas (51.5%), predominantly in Asia (45%, 30/66). CONCLUSIONS: The continued importation of multiple genotypes, rather than the expansion of a single genotype, led to ciprofloxacin-resistant N. gonorrhoeae being established in New South Wales.

3.
J Antimicrob Chemother ; 74(7): 1820-1824, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30897201

RESUMO

OBJECTIVES: To evaluate the performance of the ResistancePlus GC (beta) assay for the simultaneous detection of Neisseria gonorrhoeae and gyrA S91 markers of resistance (S91F) and susceptibility (WT) to ciprofloxacin, from both clinical specimens and isolates. METHODS: Performance was assessed on several sample banks, including N. gonorrhoeae isolates (n = 822), non-gonococcal isolates (n = 110), N. gonorrhoeae-positive clinical specimens (n = 402) and N. gonorrhoeae-negative specimens (n = 290). Results were compared with previous testing data, including S91 genotyping and phenotypic resistance profiles. RESULTS: Overall, the assay demonstrated 100% sensitivity for N. gonorrhoeae detection in clinical isolates. For gyrA S91 mutation detection in clinical isolates, the assay showed 100% sensitivity/specificity compared with the genotype, and >99%/>97% sensitivity/specificity when compared with phenotype. For positive clinical specimens, the assay demonstrated >96% sensitivity for N. gonorrhoeae detection and 100% sensitivity/specificity for gyrA S91 mutation detection. The assay demonstrated >99% specificity for N. gonorrhoeae detection against non-gonococcal isolates and 100% specificity for negative clinical specimens. CONCLUSIONS: The ResistancePlus GC (beta) assay is suitable for the detection of N. gonorrhoeae and gyrA markers associated with resistance/susceptibility to ciprofloxacin directly in clinical samples. This assay could be implemented for the individualized treatment of gonorrhoea infections as well as to enhance current antimicrobial resistance surveillance methods.

4.
mSphere ; 3(5)2018 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-30305321

RESUMO

Resistance to last-line polymyxins mediated by the plasmid-borne mobile colistin resistance gene (mcr-1) represents a new threat to global human health. Here we present the complete genome sequence of an mcr-1-positive multidrug-resistant Escherichia coli strain (MS8345). We show that MS8345 belongs to serotype O2:K1:H4, has a large 241,164-bp IncHI2 plasmid that carries 15 other antibiotic resistance genes (including the extended-spectrum ß-lactamase bla CTX-M-1) and 3 putative multidrug efflux systems, and contains 14 chromosomally encoded antibiotic resistance genes. MS8345 also carries a large ColV-like virulence plasmid that has been associated with E. coli bacteremia. Whole-genome phylogeny revealed that MS8345 clusters within a discrete clade in the sequence type 95 (ST95) lineage, and MS8345 is very closely related to the highly virulent O45:K1:H4 clone associated with neonatal meningitis. Overall, the acquisition of a plasmid carrying resistance to colistin and multiple other antibiotics in this virulent E. coli lineage is concerning and might herald an era where the empirical treatment of ST95 infections becomes increasingly more difficult.IMPORTANCE Escherichia coli ST95 is a globally disseminated clone frequently associated with bloodstream infections and neonatal meningitis. However, the ST95 lineage is defined by low levels of drug resistance amongst clinical isolates, which normally provides for uncomplicated treatment options. Here, we provide the first detailed genomic analysis of an E. coli ST95 isolate that has both high virulence potential and resistance to multiple antibiotics. Using the genome, we predicted its virulence and antibiotic resistance mechanisms, which include resistance to last-line antibiotics mediated by the plasmid-borne mcr-1 gene. Finding an ST95 isolate resistant to nearly all antibiotics that also has a high virulence potential is of major clinical importance and underscores the need to monitor new and emerging trends in antibiotic resistance development in this important global lineage.


Assuntos
Antibacterianos/farmacologia , Colistina/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/classificação , Infecções por Escherichia coli/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Plasmídeos/genética , beta-Lactamases/genética
5.
J Med Microbiol ; 67(6): 846-853, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29664716

RESUMO

PURPOSE: The molecular epidemiology and resistance mechanisms of carbapenem-resistant Pseudomonas aeruginosa (CRPA) were determined in hospitals in the countries of the Gulf Cooperation Council (GCC), namely, Saudi Arabia, the United Arab Emirates, Oman, Qatar, Bahrain and Kuwait. METHODOLOGY: Isolates were screened for common carbapenem-resistance genes by PCR. Relatedness between isolates was assessed using previously described genotyping methods: an informative-single nucleotide polymorphism MassARRAY iPLEX assay (iPLEX20SNP) and the enterobacterial repetitive intergenic consensus (ERIC)-PCR assay, with selected isolates being subjected to multilocus sequence typing (MLST). Ninety-five non-repetitive isolates that were found to be resistant to carbapenems were subjected to further investigation.Results/Key findings. The most prevalent carbapenemase-encoding gene, blaVIM-type, was found in 37/95 (39 %) isolates, while only 1 isolate (from UAE) was found to have blaIMP-type. None of the CRPA were found to have blaNDM-type or blaKPC-type. We found a total of 14 sequence type (ST) clusters, with 4 of these clusters being observed in more than 1 country. Several clusters belonged to the previously recognized internationally disseminated high-risk clones ST357, ST235, ST111, ST233 and ST654. We also found the less predominant ST316, ST308 and ST823 clones, and novel MLST types (ST2010, ST2011, ST2012 and ST2013), in our collection. CONCLUSION: Overall our data show that 'high-risk' CRPA clones are now detected in the region and highlight the need for strategies to limit further spread of such organisms, including enhanced surveillance, infection control precautions and further promotion of antibiotic stewardship programmes.


Assuntos
Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Carbapenêmicos/uso terapêutico , Infecções por Pseudomonas/epidemiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , beta-Lactamases/genética , Barein/epidemiologia , DNA Bacteriano/genética , Hospitais , Kuweit/epidemiologia , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Omã/epidemiologia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Catar/epidemiologia , Risco , Arábia Saudita/epidemiologia , Emirados Árabes Unidos/epidemiologia
6.
Sci Rep ; 8(1): 1503, 2018 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-29367612

RESUMO

Increasing rates of gonorrhoea have been observed among women within the Australian state of New South Wales. Here, we applied whole genome sequencing (WGS) to better understand the associated networks and transmission dynamics. Ninety-four isolates of a particular N. gonorrhoeae genotype (G122) associated with women (years 2012 to 2014) underwent phylogenetic analysis using core single nucleotide polymorphisms. WGS data revealed five main clusters, all of which were heterogeneous in terms of patient age and site of infection. The relatively high cervical/vaginal infections in each cluster was indicative of transmission in the general heterosexual population, noting that there is typically high rates of condom use for vaginal sex among local commercial sex workers. WGS also enabled the identification of groups of individuals belonging to tighter transmission chains within clusters, and hence may present a new tool for targeting public health interventions. The enhanced resolution of WGS provides a ready means of confirming suspected changes in N. gonorrhoeae epidemiology, but also enables key features to be identified or new questions to be raised regarding the composition of the associated sexual networks.


Assuntos
Transmissão de Doença Infecciosa , Variação Genética , Técnicas de Genotipagem , Gonorreia/epidemiologia , Neisseria gonorrhoeae/classificação , Neisseria gonorrhoeae/genética , Sequenciamento Completo do Genoma , Cidades/epidemiologia , Análise por Conglomerados , Feminino , Genótipo , Gonorreia/microbiologia , Gonorreia/transmissão , Humanos , Epidemiologia Molecular , Neisseria gonorrhoeae/isolamento & purificação , New South Wales/epidemiologia , Filogenia
7.
J Med Microbiol ; 66(10): 1451-1453, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28893363

RESUMO

The macrolide azithromycin is recommended for treatment of Mycoplasma genitalium infection; however, M. genitalium strains possessing macrolide resistance-mediating mutations (MRMMs) are increasingly being reported. Here, we used the SpeeDx ResistancePlus MG kit, which provides simultaneous detection of M. genitalium and MRMMs, to assess MRMM carriage among M. genitalium infections in Queensland, Australia. Performance characteristics of the ResistancePlus MG kit for M. genitalium detection were compared to in-house PCR. Available M. genitalium PCR-positive (n=67) and negative (n=281) samples from the years 2011 to 2017 were tested using the SpeeDx ResistancePlus MG kit. In total, 63.6 % M. genitalium-positive samples were indicated to harbour MRMMs. The ResistancePlus MG method provided sensitivity and specificity of 97 and 99.6 % respectively compared to in-house PCR for M. genitalium detection. Such high levels of macrolide-resistant M. genitalium raise further concerns over future use of azithromycin for treatment of M. genitalium infection.


Assuntos
Antibacterianos/farmacologia , Azitromicina/farmacologia , Macrolídeos/farmacologia , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/efeitos dos fármacos , Feminino , Humanos , Masculino , Queensland/epidemiologia
8.
Emerg Infect Dis ; 23(9): 1478-1485, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28820128

RESUMO

Neisseria gonorrhoeae antimicrobial resistance (AMR) is a globally recognized health threat; new strategies are needed to enhance AMR surveillance. The Northern Territory of Australia is unique in that 2 different first-line therapies, based primarily on geographic location, are used for gonorrhea treatment. We tested 1,629 N. gonorrhoeae nucleic acid amplification test-positive clinical samples, collected from regions where ceftriaxone plus azithromycin or amoxicillin plus azithromycin are recommended first-line treatments, by using 8 N. gonorrhoeae AMR PCR assays. We compared results with those from routine culture-based surveillance data. PCR data confirmed an absence of ceftriaxone resistance and a low level of azithromycin resistance (0.2%), and that penicillin resistance was <5% in amoxicillin plus azithromycin regions. Rates of ciprofloxacin resistance and penicillinase-producing N. gonorrhoeae were lower when molecular methods were used. Molecular methods to detect N. gonorrhoeae AMR can increase the evidence base for treatment guidelines, particularly in settings where culture-based surveillance is limited.


Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Gonorreia/epidemiologia , Neisseria gonorrhoeae/genética , Vigilância em Saúde Pública , Adulto , Amoxicilina/uso terapêutico , Azitromicina/uso terapêutico , Ceftriaxona/uso terapêutico , Ciprofloxacino/uso terapêutico , Feminino , Gonorreia/tratamento farmacológico , Gonorreia/microbiologia , Gonorreia/transmissão , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/isolamento & purificação , Northern Territory/epidemiologia , Penicilinas/uso terapêutico
10.
J Antimicrob Chemother ; 72(3): 705-711, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27999043

RESUMO

Objectives: To examine how gonococcal genotypes and associated changes over time influence rates of Neisseria gonorrhoeae antimicrobial resistance. Methods: All available N. gonorrhoeae isolates collected in New South Wales, Australia in the first half of both 2012 and 2014 were genotyped using the Agena MassARRAY iPLEX platform. Genotypic data were compared with phenotypic antimicrobial resistance profiles over time. We focused on penicillin and ciprofloxacin as significant increases in resistance to both antibiotics were observed over this time period. Results: Genotyping data were obtained for 760 and 782 isolates in 2012 and 2014, respectively. A total of 162 distinct genotypes were identified in the study, including 36 (22.2%) genotypes present in both years ( persisting genotypes), 54 (33.3%) observed in 2012 only and 72 (44.4%) observed in 2014 only (s ingle-year genotypes). Overall, persisting genotypes comprised 15 of the 20 most common genotypes, 8 of which showed a significant change in proportion from 2012 to 2014. Persisting genotypes also comprised the majority (>70%) of ciprofloxacin- and penicillin-resistant isolates in both years. Significant fluctuations in the most common persisting genotypes accounted for the majority of observed increases in both ciprofloxacin and penicillin resistance. Single-year genotypes contributed to ∼20% of ciprofloxacin and penicillin resistance in each year. Conclusions: The results show that the gonococcal genotypes persisting in the study population fluctuated significantly within a 3 year period, with numerous other genotypes appearing or disappearing. It is the net effect of these changes that determines N. gonorrhoeae antimicrobial resistance levels within the population.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Gonorreia/epidemiologia , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Austrália/epidemiologia , Ceftriaxona/farmacologia , Ciprofloxacino/farmacologia , DNA Bacteriano/genética , Genótipo , Gonorreia/microbiologia , Testes de Sensibilidade Microbiana , Resistência às Penicilinas/genética , Penicilinas/farmacologia , Espectinomicina/farmacologia , Resistência a Tetraciclina/genética
11.
J Antimicrob Chemother ; 72(2): 407-409, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27707989

RESUMO

OBJECTIVES: Previous studies have shown that mixed-strain gonococcal infections can occur. However, it remains unclear whether such infections impact upon the reliability of Neisseria gonorrhoeae antimicrobial resistance (AMR) surveillance. In this study, we aimed to resolve this question by intensively sampling isolates from gonorrhoea-positive specimens in a high-risk population in Sydney, Australia. METHODS: A total of 615 N. gonorrhoeae isolates, originating from 63 clinical samples (31 rectal swabs and 32 throat swabs), were characterized. All isolates were subject to N. gonorrhoeae identification, antimicrobial susceptibility testing and genotyping by SNP-based MLST. RESULTS: Only 2 of the 63 (3.2%) samples provided evidence of mixed-strain infections. These comprised two rectal swabs that harboured isolates of different SNP-based MLST genotypes; however, the AMR susceptibility profiles of the different genotypes from these samples were indistinguishable. Within-sample differences in the AMR susceptibility profiles were observed for a further seven samples; however, the differences were not considered significant; MIC values were typically within a 2-fold difference or were close to test breakpoints. CONCLUSIONS: Results of this study provide further evidence that mixed-strain gonococcal infections do occur, although at low prevalence. Our data indicate that at a population level such infections are unlikely to impact significantly upon N. gonorrhoeae AMR surveillance.


Assuntos
Coinfecção/microbiologia , Farmacorresistência Bacteriana , Gonorreia/microbiologia , Neisseria gonorrhoeae/classificação , Neisseria gonorrhoeae/isolamento & purificação , Austrália/epidemiologia , Coinfecção/epidemiologia , Monitoramento Epidemiológico , Feminino , Gonorreia/epidemiologia , Humanos , Masculino , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética
13.
Clin Infect Dis ; 63(12): 1591-1598, 2016 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-27682063

RESUMO

BACKGROUND: Antimicrobial resistance (AMR) by Neisseria gonorrhoeae is considered a serious global threat. METHODS: In this nationwide study, we used MassARRAY iPLEX genotyping technology to examine the epidemiology of N. gonorrhoeae and associated AMR in the Australian population. All available N. gonorrhoeae isolates (n = 2452) received from Australian reference laboratories from January to June 2012 were included in the study. Genotypic data were combined with phenotypic AMR information to define strain types. RESULTS: A total of 270 distinct strain types were observed. The 40 most common strain types accounted for over 80% of isolates, and the 10 most common strain types accounted for almost half of all isolates. The high male to female ratios (>94% male) suggested that at least 22 of the top 40 strain types were primarily circulating within networks of men who have sex with men (MSM). Particular strain types were also concentrated among females: two strain types accounted for 37.5% of all isolates from females. Isolates harbouring the mosaic penicillin binding protein 2 (PBP2)-considered a key mechanism for cephalosporin resistance-comprised 8.9% of all N. gonorrhoeae isolates and were primarily observed in males (95%). CONCLUSIONS: This large scale epidemiological investigation demonstrated that N. gonorrhoeae infections are dominated by relatively few strain types. The commonest strain types were concentrated in MSM in urban areas and Indigenous heterosexuals in remote areas, and we were able to confirm a resurgent epidemic in heterosexual networks in urban areas. The prevalence of mosaic PBP2 harboring N. gonorrhoeae strains highlight the ability for new N. gonorrhoeae strains to spread and become established across populations.


Assuntos
Antibacterianos/uso terapêutico , Gonorreia/epidemiologia , Neisseria gonorrhoeae/efeitos dos fármacos , Estudos Transversais , Feminino , Técnicas de Genotipagem , Gonorreia/tratamento farmacológico , Gonorreia/microbiologia , Homossexualidade Masculina , Humanos , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Neisseria gonorrhoeae/genética , Polimorfismo de Nucleotídeo Único , Especificidade da Espécie
14.
J Antimicrob Chemother ; 71(11): 3090-3095, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27494921

RESUMO

OBJECTIVES: The objective of this study was to develop a real-time PCR assay targeting the gonococcal porB gene (PorB-PCR) for predicting susceptibility of Neisseria gonorrhoeae to penicillin. This complements a previously described PCR assay for detecting penicillinase-producing N. gonorrhoeae (PPNG) developed by our laboratory (PPNG-PCR). METHODS: The PorB-PCR assay was designed using six probes to characterize various combinations of amino acids at positions 101 and 102 of the PorB1b class protein, including the WT G101/A102 and mutant G101K/A102D, G101K/A102N and G101K/A102G sequences, as well as the PorB1a sequence. The ability of these sequences to predict penicillin susceptibility was initially assessed using 2307 N. gonorrhoeae isolates from throughout Australia for which phenotypic susceptibility data were available. The assay was then applied to N. gonorrhoeae-positive clinical specimens (n = 70). Specificity was assessed by testing commensal Neisseria strains (n = 75) and N. gonorrhoeae-negative clinical specimens (n = 171). RESULTS: Testing of the 2307 N. gonorrhoeae isolates using PorB-PCR to detect G101/A102 and PorB1a sequences identified a total of 78.4% (61.2% and 17.2%, respectively) of penicillin-susceptible isolates with specificities of 97.4% and 99.3% and positive predictive values of 98.8% and 98.9%, where PPNG strains were simultaneously identified and excluded. Similar performance data were obtained when the PorB-PCR assay was applied to the N. gonorrhoeae-positive clinical specimens. No false-positive results were observed for the N. gonorrhoeae-negative samples and no cross-reactions were observed with the non-gonococcal species. CONCLUSIONS: When used in parallel with the previously described PPNG-PCR, the PorB-PCR approach has the potential to facilitate individualized treatment of gonorrhoea using penicillin.


Assuntos
Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Penicilinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Austrália , Técnicas de Genotipagem/métodos , Humanos , Sondas de Oligonucleotídeos/genética , Porinas/genética , Valor Preditivo dos Testes , Sensibilidade e Especificidade
15.
J Antimicrob Chemother ; 71(2): 353-6, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26538505

RESUMO

OBJECTIVES: The objective of this study was to develop a real-time PCR method for specific detection of the gonococcal GyrA amino acid 91 locus directly in clinical samples so as to predict Neisseria gonorrhoeae ciprofloxacin susceptibility. METHODS: The real-time PCR assay, GyrA91-PCR, was designed using two probes, one for detection of the WT S91 sequence and the other for detection of the S91F alteration. The performance of the assay was initially assessed using characterized N. gonorrhoeae isolates (n = 70), a panel of commensal Neisseria and Moraxella species (n = 55 isolates) and clinical samples providing negative results by a commercial N. gonorrhoeae nucleic acid amplification test (NAAT) method (n = 171). The GyrA91-PCR was then applied directly to N. gonorrhoeae NAAT-positive clinical samples (n = 210) from the year 2014 for which corresponding N. gonorrhoeae isolates with susceptibility results were also available. RESULTS: The GyrA91-PCR accurately characterized the GyrA 91 locus of all 70 N. gonorrhoeae isolates (sensitivity = 100%, 95% CI = 94.9%-100%), whereas all non-gonococcal isolates and N. gonorrhoeae NAAT-negative clinical samples gave negative results by the GyrA91-PCR (specificity = 100%, 95% CI = 98.4%-100%). When applied to the 210 N. gonorrhoeae NAAT-positive clinical samples, the GyrA91-PCR successfully characterized 195 samples (92.9%, 95% CI = 88.5%-95.9%). When compared with the corresponding bacterial culture results, positivity by the GyrA91-PCR WT probe correctly predicted N. gonorrhoeae susceptibility to ciprofloxacin in 161 of 162 (99.4%, 95% CI = 96.6%-99.9%) samples. CONCLUSIONS: The use of a PCR assay for detection of mutation in gyrA applied directly to clinical samples can predict ciprofloxacin susceptibility in N. gonorrhoeae.


Assuntos
Antibacterianos/farmacologia , Ciprofloxacino/farmacologia , DNA Girase/genética , Farmacorresistência Bacteriana , Genótipo , Neisseria gonorrhoeae/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Humanos , Moraxella/efeitos dos fármacos , Moraxella/genética , Neisseria gonorrhoeae/genética
16.
PLoS One ; 10(12): e0144022, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26633539

RESUMO

In cystic fibrosis (CF), Pseudomonas aeruginosa undergoes intra-strain genotypic and phenotypic diversification while establishing and maintaining chronic lung infections. As the clinical significance of these changes is uncertain, we investigated intra-strain diversity in commonly shared strains from CF patients to determine if specific gene mutations were associated with increased antibiotic resistance and worse clinical outcomes. Two-hundred-and-one P. aeruginosa isolates (163 represented a dominant Australian shared strain, AUST-02) from two Queensland CF centres over two distinct time-periods (2001-2002 and 2007-2009) underwent mexZ and lasR sequencing. Broth microdilution antibiotic susceptibility testing in a subset of isolates was also performed. We identified a novel AUST-02 subtype (M3L7) in adults attending a single Queensland CF centre. This M3L7 subtype was multi-drug resistant and had significantly higher antibiotic minimum inhibitory concentrations than other AUST-02 subtypes. Prospective molecular surveillance using polymerase chain reaction assays determined the prevalence of the 'M3L7' subtype at this centre during 2007-2009 (170 patients) and 2011 (173 patients). Three-year clinical outcomes of patients harbouring different strains and subtypes were compared. MexZ and LasR sequences from AUST-02 isolates were more likely in 2007-2009 than 2001-2002 to exhibit mutations (mexZ: odds ratio (OR) = 3.8; 95% confidence interval (CI): 1.1-13.5 and LasR: OR = 2.5; 95%CI: 1.3-5.0). Surveillance at the adult centre in 2007-2009 identified M3L7 in 28/509 (5.5%) P. aeruginosa isolates from 13/170 (7.6%) patients. A repeat survey in 2011 identified M3L7 in 21/519 (4.0%) P. aeruginosa isolates from 11/173 (6.4%) patients. The M3L7 subtype was associated with greater intravenous antibiotic and hospitalisation requirements, and a higher 3-year risk of death/lung transplantation, than other AUST-02 subtypes (adjusted hazard ratio [HR] = 9.4; 95%CI: 2.2-39.2) and non-AUST-02 strains (adjusted HR = 4.8; 95%CI: 1.4-16.2). This suggests ongoing microevolution of the shared CF strain, AUST-02, was associated with an emerging multi-drug resistant subtype and possibly poorer clinical outcomes.


Assuntos
Fibrose Cística/microbiologia , Genes Bacterianos , Variação Genética , Pseudomonas aeruginosa/genética , Adulto , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Austrália , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Resultado do Tratamento , Adulto Jovem
17.
J Antimicrob Chemother ; 70(12): 3244-9, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26338048

RESUMO

OBJECTIVES: Surveillance for Neisseria gonorrhoeae azithromycin resistance is of growing importance given increasing use of ceftriaxone and azithromycin dual therapy for gonorrhoea treatment. In this study, we developed two real-time PCR methods for direct detection of two key N. gonorrhoeae 23S rRNA mutations associated with azithromycin resistance. METHODS: The real-time PCR assays, 2611-PCR and 2059-PCR, targeted the gonococcal 23S rRNA C2611T and A2059G mutations, respectively. A major design challenge was that gonococcal 23S rRNA sequences have high sequence homology with those of commensal Neisseria species. To limit the potential for cross-reaction, 'non-template' bases were utilized in primer sequences. The performance of the methods was initially assessed using a panel of gonococcal (n = 70) and non-gonococcal (n = 28) Neisseria species. Analytical specificity was further assessed by testing N. gonorrhoeae nucleic acid amplification test (NAAT)-negative clinical samples (n = 90), before being applied to N. gonorrhoeae NAAT-positive clinical samples (n = 306). RESULTS: Cross-reactions with commensal Neisseria strains remained evident for both assays; however, cycle threshold (Ct) values were significantly delayed, indicating reduced sensitivity for non-gonococcal species. For the N. gonorrhoeae NAAT-negative clinical samples, 7/21 pharyngeal samples provided evidence of cross-reaction (Ct values >40 cycles); however, the remaining urogenital and rectal swab samples were negative. In total, the gonococcal 2611 and 2059 23S rRNA nucleotides were both successfully characterized in 266/306 (87%) of the N. gonorrhoeae NAAT-positive clinical specimens. CONCLUSIONS: Real-time PCR detection of gonococcal 23S rRNA mutations directly from clinical samples is feasible and may enhance culture- and non-culture-based N. gonorrhoeae resistance surveillance.


Assuntos
Antibacterianos/farmacologia , Azitromicina/farmacologia , Farmacorresistência Bacteriana , Técnicas de Genotipagem/métodos , Gonorreia/microbiologia , Neisseria gonorrhoeae/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Gonorreia/diagnóstico , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação , Neisseria gonorrhoeae/genética , RNA Ribossômico 23S/genética
18.
Sex Health ; 12(5): 439-44, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26145099

RESUMO

UNLABELLED: Background Mixed gonococcal infections within the one anatomical site have been recognised but questions remain over how often they occur. In this study, the aim was to estimate the prevalence of mixed gonococcal infections using novel real-time polymerase chain reaction (PCR) methods that were developed and validated, targeting the gonococcal porB gene. METHODS: Neisseria gonorrhoeae strains were categorised into three different porB groups, based on sequence data derived from N. gonorrhoeae multi-antigen sequence typing (NG-MAST) analyses of local isolates. Specific PCR methods for each group were then developed and these PCR methods were used to test clinical samples (n=350) that were positive for gonorrhoea as determined by nucleic acid amplification test (NAAT) diagnostic screening. RESULTS: Initial validation using isolates showed the group PCR methods proved 100% sensitive and 100% specific for their respective porB groups. When applied to the clinical specimens, 298/350 (85%) provided positive results by the group PCR methods. Of these, four specimens showed evidence of mixed infections, supported by subsequent DNA sequencing of the PCR products. CONCLUSIONS: The data provide further evidence of mixed gonococcal infections at the same anatomical site, but show that such infections may be relatively infrequent (1.3%; 95% confidence interval 0.01-2.6%) in a general screening population.

19.
J Clin Microbiol ; 53(8): 2706-8, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25994166

RESUMO

A multitarget PCR was developed for the direct detection of penicillinase-producing Neisseria gonorrhoeae (PPNG). The assay was validated by testing 342 PPNG isolates and 415 clinical samples. The method is suitable for routine detection of PPNG strains. Its multitarget approach reduces the potential for false-negative results caused by sequence variations.


Assuntos
Farmacorresistência Bacteriana , Gonorreia/microbiologia , Neisseria gonorrhoeae/enzimologia , Penicilinase/genética , Reação em Cadeia da Polimerase/métodos , Monitoramento Epidemiológico , Gonorreia/epidemiologia , Humanos , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética
20.
Pathology ; 47(3): 219-26, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25714587

RESUMO

In the last 20 years, nucleic acid amplification tests (NAATs) have gradually replaced traditional methods for the detection of sexually transmitted infections. NAAT technology comes with some considerable benefits for diagnosis, including increased sensitivity, rapid result turnaround and suitability for high throughput screening of asymptomatic individuals using more-readily available specimens. However, the transition to NAAT has not come without its problems. False-negative and false-positive results have been reported owing to various technical issues. Furthermore, increased reliance on NAATs for diagnosis have created the need to develop NAAT-based methods to inform treatment, being an area that presents its own set of challenges. In this review article, we explore NAAT-based detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis. In doing so, we consider the benefits and limitations of NAAT-based technology and highlight areas where further research and development is in need.


Assuntos
Técnicas Microbiológicas , Técnicas de Diagnóstico Molecular , Doenças Sexualmente Transmissíveis/diagnóstico , Doenças Sexualmente Transmissíveis/microbiologia , Humanos
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