RESUMEN
Compared to cervical cancer, little is known about human papillomavirus (HPV)-driven oropharyngeal cancer and their cofactors. Here, we investigated potential associations between Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) with oral HPV and HPV persistence, which are known cofactors in cervical carcinogenesis, and also play a role in HPV-driven oropharyngeal cancer. Saliva samples (n = 547) from 312 people were tested for CT and NG and whom had previously been tested for oral HPV infection in a longitudinal study. Eight participants were positive for CT (2.6%) and one for NG (0.3%). Six of these nine participants were also positive for oral HPV in at least one of their samples. We found no significant associations between HPV, CT, or NG infection in the saliva samples analyzed. These preliminary data suggest CT and NG have little influence on oral HPV-positivity and persistence in a general population. However, larger studies focusing on 'at risk' population cohorts are necessary to assess potential associations between oral sexually transmissible infections and oral HPV infections, and their outcomes.
Asunto(s)
Coinfección , Neoplasias Orofaríngeas , Infecciones por Papillomavirus , Humanos , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/epidemiología , Neisseria gonorrhoeae , Chlamydia trachomatis , Coinfección/epidemiología , Estudios Longitudinales , Virus del Papiloma HumanoRESUMEN
We investigated the potential effects of COVID-19 public health restrictions on the prevalence and distribution of Neisseria gonorrhoeae (NG) genotypes in our Queensland isolate population in the first half of the year 2020. A total of 763 NG isolates were genotyped to examine gonococcal strain distribution and prevalence for the first 6 months of 2020, with 1 January 2020 to 31 March 2020 classified as 'pre' COVID-19 restrictions (n = 463) and 1 April 2020 to 30 June 2020 classified as 'post' COVID-19 restrictions (n = 300). Genotypes most prevalent 'pre' restrictions remained proportionally high 'post' restrictions, with some significantly increasing 'post' restrictions. However, genotype diversity was significantly reduced 'post' restrictions. Overall, it seems public health restrictions (9-10 weeks) were not sufficient to affect rates of infection or reduce the prevalence of well-established genotypes in our population, potentially due to reduced access to services or health-seeking behaviours.
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COVID-19 , Gonorrea , Neisseria gonorrhoeae , Genotipo , Gonorrea/epidemiología , Queensland/epidemiología , PrevalenciaRESUMEN
BACKGROUND: Neisseria gonorrhoeae (NG) can lead to serious reproductive and sexual health outcomes, and the annual number of NG notifications in Australia increased steadily from 10329 in 2010 to 29549 by 2020. Australian populations most affected are urban men who have sex with men and First Nations peoples living in remote areas, and a resurgence in urban heterosexuals has been observed since 2012. METHODS: A case series analysis of Queensland NG isolates (2010-15) exploring temporal trends and antimicrobial resistance by demographic and geographic distribution and genotype was performed. Proportions describe age, sex, strain, genogroup (NG multi-antigen sequence typing), region, swab site, antimicrobial sensitivity and isolate rates per 100000 population. Dominant genogroups were identified. RESULTS: Among 3953 isolates, the median age was 25years (IQR 20-34years) and most (n =2871/3915, 73%) were men. Brisbane city (68.8) and Far North Queensland (54.1) excluding Cairns showed the highest rates. Forty-six genogroups were documented, seven (G2992, G6876, G1415, G4186, G5, G1407 and G6937) comprised half of all isolates. The predominant male genogroup was G2992 (16%), and G6876 (20%) for females; G5 was predominantly male from 2010 to 2011, but equal in both sexes from 2012 to 2015. CONCLUSION: Considerable temporal, geographical and demographical diversity was observed in Queensland NG isolates, which has public health implications. Certain genogroups are more transient than others, and evidence suggests bridging from male-dominant networks to heterosexual networks. Molecular surveillance can enhance tracking the epidemiology and movement of NG in Australia, highlighting the necessity of genotyping to expose potentially prevalent strains circulating in undetected or underrepresented networks by current screening methods.
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Gonorrea , Minorías Sexuales y de Género , Femenino , Humanos , Masculino , Adulto Joven , Adulto , Neisseria gonorrhoeae , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Gonorrea/tratamiento farmacológico , Gonorrea/epidemiología , Homosexualidad Masculina , Queensland/epidemiología , Epidemiología Molecular/métodos , Farmacorresistencia Bacteriana/genética , Australia , GenotipoRESUMEN
Carbapenemase-producing organisms (CPOs) pose a serious clinical threat and rapid detection tools are essential to aid in patient management. We developed rapid and simple molecular tests to detect blaNDM-type and blaVIM-type carbapenemase genes using recombinase polymerase amplification (RPA) combined with a lateral flow detection. The tests could provide results in approximately 15 min when using DNA extracts, with limits of detection of 9.2 copies/µl for the blaNDM-type assay and 7.5 copies/µl for blaVIM-type assay, and successfully detected all isolates harbouring the carbapenemase encoding genes in a panel of 57 isolates. These RPA tests may be suitable for use in low-resource settings to tailor rapid implementation of infection control precautions and antibiotic stewardship.
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Bacterias/enzimología , Proteínas Bacterianas/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , beta-Lactamasas/genética , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/genética , Proteínas Bacterianas/metabolismo , Cartilla de ADN/genética , Farmacorresistencia Bacteriana , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Recombinasas/metabolismo , beta-Lactamasas/metabolismoRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Ciprofloxacina/farmacología , Girasa de ADN/genética , Farmacorresistencia Bacteriana , Técnicas de Genotipaje , Pruebas de Sensibilidad Microbiana/métodos , Neisseria gonorrhoeae/efectos de los fármacos , Femenino , Genotipo , Gonorrea/diagnóstico , Gonorrea/microbiología , Humanos , Masculino , Neisseria gonorrhoeae/genética , Sensibilidad y EspecificidadRESUMEN
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.
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Antibacterianos/farmacología , Ciprofloxacina/farmacología , Farmacorresistencia Bacteriana , Gonorrea/epidemiología , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/genética , Estudios de Cohortes , Femenino , Genotipo , Gonorrea/microbiología , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Neisseria gonorrhoeae/clasificación , Nueva Gales del Sur/epidemiología , ViajeRESUMEN
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.
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Bacterias/genética , Proteínas Bacterianas/genética , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , beta-Lactamasas/genética , Antibacterianos , Bacterias/efectos de los fármacos , Técnicas Bacteriológicas/métodos , Proteínas de Escherichia coli/genética , Heces/microbiología , Humanos , Sensibilidad y EspecificidadRESUMEN
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.
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Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana/genética , Gonorrea/epidemiología , Neisseria gonorrhoeae/genética , Vigilancia en Salud Pública , Adulto , Amoxicilina/uso terapéutico , Azitromicina/uso terapéutico , Ceftriaxona/uso terapéutico , Ciprofloxacina/uso terapéutico , Femenino , Gonorrea/tratamiento farmacológico , Gonorrea/microbiología , Gonorrea/transmisión , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Epidemiología Molecular , Tipificación de Secuencias Multilocus , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/aislamiento & purificación , Northern Territory/epidemiología , Penicilinas/uso terapéuticoRESUMEN
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.
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Coinfección/microbiología , Farmacorresistencia Bacteriana , Gonorrea/microbiología , Neisseria gonorrhoeae/clasificación , Neisseria gonorrhoeae/aislamiento & purificación , Australia/epidemiología , Coinfección/epidemiología , Monitoreo Epidemiológico , Femenino , Gonorrea/epidemiología , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/genéticaRESUMEN
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 â¼20% 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.
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Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Gonorrea/epidemiología , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/genética , Australia/epidemiología , Ceftriaxona/farmacología , Ciprofloxacina/farmacología , ADN Bacteriano/genética , Genotipo , Gonorrea/microbiología , Pruebas de Sensibilidad Microbiana , Resistencia a las Penicilinas/genética , Penicilinas/farmacología , Espectinomicina/farmacología , Resistencia a la Tetraciclina/genéticaRESUMEN
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.
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Antibacterianos/uso terapéutico , Gonorrea/epidemiología , Neisseria gonorrhoeae/efectos de los fármacos , Estudios Transversales , Femenino , Técnicas de Genotipaje , Gonorrea/tratamiento farmacológico , Gonorrea/microbiología , Homosexualidad Masculina , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Epidemiología Molecular , Neisseria gonorrhoeae/genética , Polimorfismo de Nucleótido Simple , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Ciprofloxacina/farmacología , Girasa de ADN/genética , Farmacorresistencia Bacteriana , Genotipo , Neisseria gonorrhoeae/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Humanos , Moraxella/efectos de los fármacos , Moraxella/genética , Neisseria gonorrhoeae/genéticaRESUMEN
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 (nâ=â70). Specificity was assessed by testing commensal Neisseria strains (nâ=â75) and N. gonorrhoeae-negative clinical specimens (nâ=â171). 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.
Asunto(s)
Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/genética , Penicilinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Australia , Técnicas de Genotipaje/métodos , Humanos , Sondas de Oligonucleótidos/genética , Porinas/genética , Valor Predictivo de las Pruebas , Sensibilidad y EspecificidadRESUMEN
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.
Asunto(s)
Farmacorresistencia Bacteriana , Gonorrea/microbiología , Neisseria gonorrhoeae/enzimología , Penicilinasa/genética , Reacción en Cadena de la Polimerasa/métodos , Monitoreo Epidemiológico , Gonorrea/epidemiología , Humanos , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/genéticaRESUMEN
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 (nâ=â70) and non-gonococcal (nâ=â28) Neisseria species. Analytical specificity was further assessed by testing N. gonorrhoeae nucleic acid amplification test (NAAT)-negative clinical samples (nâ=â90), before being applied to N. gonorrhoeae NAAT-positive clinical samples (nâ=â306). 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.
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Antibacterianos/farmacología , Azitromicina/farmacología , Farmacorresistencia Bacteriana , Técnicas de Genotipaje/métodos , Gonorrea/microbiología , Neisseria gonorrhoeae/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Gonorrea/diagnóstico , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mutación , Neisseria gonorrhoeae/genética , ARN Ribosómico 23S/genéticaRESUMEN
Penicillinase-producing Neisseria gonorrhoeae (PPNG) carrying the blaTEM-135 gene is of particular concern, as it is considered a stepping stone toward resistance to extended-spectrum cephalosporins. Here, we sought to characterize plasmid types and the occurrence of the blaTEM-135 gene for N. gonorrhoeae clinical isolates from Australia. We found that blaTEM-135 was prevalent in Australian PPNG and was detected on all three major plasmid types.
Asunto(s)
Resistencia a las Cefalosporinas/genética , Neisseria gonorrhoeae/genética , Resistencia a las Penicilinas/genética , Penicilinasa/genética , Plásmidos/química , Antibacterianos/farmacología , Australia , Técnicas de Tipificación Bacteriana , Cefalosporinas/farmacología , Gonorrea/microbiología , Gonorrea/transmisión , Humanos , Pruebas de Sensibilidad Microbiana , Neisseria gonorrhoeae/clasificación , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/aislamiento & purificación , Penicilinas/farmacología , Plásmidos/clasificaciónRESUMEN
OBJECTIVES: Neisseria gonorrhoeae antimicrobial resistance (AMR) is a global problem heightened by emerging resistance to ceftriaxone. Appropriate molecular typing methods are important for understanding the emergence and spread of N. gonorrhoeae AMR. We report on the development, validation and testing of a Sequenom MassARRAY iPLEX method for multilocus sequence typing (MLST)-style genotyping of N. gonorrhoeae isolates. METHODS: An iPLEX MassARRAY method (iPLEX14SNP) was developed targeting 14 informative gonococcal single nucleotide polymorphisms (SNPs) previously shown to predict MLST types. The method was initially validated using 24 N. gonorrhoeae control isolates and was then applied to 397 test isolates collected throughout Queensland, Australia in the first half of 2012. RESULTS: The iPLEX14SNP method provided 100% accuracy for the control isolates, correctly identifying all 14 SNPs for all 24 isolates (336/336). For the 397 test isolates, the iPLEX14SNP assigned results for 5461 of the possible 5558 SNPs (SNP call rate 98.25%), with complete 14 SNP profiles obtained for 364 isolates. Based on the complete SNP profile data, there were 49 different sequence types identified in Queensland, with 11 of the 49 SNP profiles accounting for the majority (nâ=â280; 77%) of isolates. AMR was dominated by several geographically clustered sequence types. Using the iPLEX14SNP method, up to 384 isolates could be tested within 1 working day for less than Aus$10 per isolate. CONCLUSIONS: The iPLEX14SNP offers an accurate and high-throughput method for the MLST-style genotyping of N. gonorrhoeae and may prove particularly useful for large-scale studies investigating the emergence and spread of gonococcal AMR.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Neisseria gonorrhoeae/clasificación , Neisseria gonorrhoeae/genética , Polimorfismo de Nucleótido Simple , Antibacterianos/farmacología , Biología Computacional , Farmacorresistencia Bacteriana/genética , Genotipo , Gonorrea/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Neisseria gonorrhoeae/efectos de los fármacos , QueenslandRESUMEN
Rapid and cost-effective diagnosis of Neisseria gonorrhoeae (NG) are important measures for the control and management of gonococcal infection. Current diagnostic tools such as nucleic acid amplification tests and bacterial culture are not feasible in many resource-poor settings, and so syndromic patient management is commonplace. Alternative cost-effective diagnostic tools are therefore needed. Here, we sought to explore the utility and feasibility of Near Infrared Spectroscopy (NIRS) to (1) identify and differentiate NG from Neisseria commensals and (2) to differentiate fully susceptible NG from resistant NG. NIRS correctly classified NG from Neisseria commensals (R2= 0.89; SECV 0.164) and to a lesser capacity, susceptible NG from resistant (R2 = 0.60; SECV 0.32). To the best our knowledge, this is the first proof of concept study in the field. Further evaluations are now warranted to enhance capacity and accuracy of this diagnostic approach.
Asunto(s)
Antiinfecciosos , Gonorrea , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Gonorrea/diagnóstico , Gonorrea/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Neisseria , Neisseria gonorrhoeae , Espectroscopía Infrarroja CortaRESUMEN
OBJECTIVES: Gonorrhoea caused by Neisseria gonorrhoeae is the second most notified sexually transmitted infection (STI) in Australia and the case numbers for this STI have been increasing globally. Progressive gonococcal infection may lead to disseminated gonococcal infection (DGI), which causes significant morbidity among patients. This study aims to examine the genetic diversity of N. gonorrhoeae isolates collected in Queensland from January 2010 to August 2015 and to determine factors associated with DGI in Queensland. DESIGN: Retrospective surveillance study for epidemiological purposes. SETTING: All gonorrhoeae isolates referred by private and public pathology laboratories to the state of Queensland, Australia Neisseria reference laboratory. METHODS: Between January 2010 and August 2015, 3953 N. gonorrhoeae isolates from both metropolitan and regional Queensland infections were typed with NG-MAST (N. gonorrhoeae multiantigen sequence typing) to assess the genetic diversity between strains. Whole-genome sequencing (WGS) was used to investigate strain-related factors associated with DGI. RESULTS: ST6876 was the most common NG-MAST type, detected in 7.6% of the isolates. DGI was significantly more likely in females <30 years (OR 13.02, p<0.0001) and in older males >30 years (OR 6.04, p<0.0001), with most cases originating from North Queensland (OR 8.5, p<0.0001). Strains harbouring PIA class of porB type were associated with DGI (OR 33.23, p<0.0001). CONCLUSION: Genotyping techniques, such as NG-MAST and WGS, are proving instrumental in providing an insight into the population structure of N. gonorrhoeae, and genetic mechanisms of pathogenesis, such as for DGI.