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1.
J Med Microbiol ; 69(2): 244-248, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31958047

RESUMO

Introduction. Mycoplasma genitalium is a sexually transmitted organism with high levels of resistance to the recommended first-line therapy, azithromycin. The ResistancePlus MG test concurrently detects M. genitalium, and the presence of macrolide-resistance mutations (MRM). European, UK and Australian guidelines recommend a diagnostic test that reports MRM to optimize treatment through resistance-guided therapy. Hence, for samples collected for use on other platforms, reflex testing using the ResistancePlus MG test would be beneficial.Aim. To validate the ResistancePlus MG assay using samples collected in Aptima buffer for testing on the Hologic Panther.Methodology. Positive (n=99) and negative (n=229) clinical samples collected in Aptima buffer were extracted on the MagNA Pure 96 (Roche Diagnostics), and tested with the ResistancePlus MG test on the LightCycler 480 II (Roche Diagnostics). Results were compared to matched samples collected using standard sample collection (urine or swab resuspended in PBS), with positive percent agreement (PPA), negative percent agreement (NPA) and Cohen's Kappa statistic.Results. The ResistancePlus MG test had high performance with a 200 µl input volume (PPA/NPA for M. genitalium detection, 92.9 % [95 % confidence interval (CI): 85.5-96.9]/100 % [95 % CI: 97.9-100], MRM detection, 96.9 % [95 % CI: 88.2-99.5]/85.7 % [95 % CI: 66.4-95.3]) and for 1 ml input volume (PPA/NPA for M. genitalium detection, 95.9%/96.6%, MRM detection, 98.4%/90.3%). Samples remained positive after storage at room temperature beyond the manufacturer-recommended storage of <60 days (mean storage time for 1 ml extraction: 129 days).Conclusion. Samples collected using Aptima collection kits are suitable for reflex testing using the ResistancePlus MG test, allowing detection of macrolide resistance.


Assuntos
Antibacterianos/farmacologia , Testes Diagnósticos de Rotina/métodos , Farmacorresistência Bacteriana , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/efeitos dos fármacos , Mycoplasma genitalium/isolamento & purificação , Austrália , Testes Diagnósticos de Rotina/instrumentação , Humanos , Macrolídeos/farmacologia , Infecções por Mycoplasma/diagnóstico , Mycoplasma genitalium/genética , Kit de Reagentes para Diagnóstico , Manejo de Espécimes
2.
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
3.
J Antimicrob Chemother ; 74(10): 2913-2915, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31340021

RESUMO

BACKGROUND: The emergence of drug-resistant Neisseria gonorrhoeae has prompted the development of rapid molecular assays designed to determine antimicrobial susceptibility. One common assay uses high-resolution melt analysis to target codon 91 of the gyrase A gene (gyrA) to predict N. gonorrhoeae susceptibility to ciprofloxacin. METHODS: We extracted DNA from remnant clinical specimens that had previously tested positive for N. gonorrhoeae using the Aptima Combo 2 for CT/NG assay (Hologic, San Diego, CA, USA). We selected DNA extracts from specimens with indeterminate, WT and mutant gyrA genotype results from a previous study using high-resolution melt analysis to detect the gyrA codon 91 mutation. We re-tested those specimens using the recently CE-marked ResistancePlus GC (beta) assay (SpeeDx, Sydney, Australia). RESULTS: Of 86 specimens with indeterminate gyrA genotypes on high-resolution melt analysis, the ResistancePlus GC (beta) assay (SpeeDx) identified 30 (35%) WT, 22 (26%) mutant and 34 (40%) indeterminate gyrA genotypes. CONCLUSIONS: The ResistancePlus GC (beta) assay showed improved N. gonorrhoeae gyrA genotype determination compared with a prior gyrA genotypic high-resolution melt assay.

4.
Sex Transm Dis ; 45(9): 632-635, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29509566

RESUMO

BACKGROUND: Mycoplasma genitalium is an important cause of bacterial sexually transmitted diseases. Diagnosis and susceptibility testing of M. genitalium are limited by the fastidious nature of the organism. Therefore, the prevalence of infection and azithromycin resistance are poorly studied. METHODS: We conducted an exploratory study on remnant clinical specimens. We collected remnant DNA from consecutive urine samples and clinical swabs (cervical/vaginal, rectal, and pharyngeal) previously tested for Neisseria gonorrhoeae and Chlamydia trachomatis using the Cobas 4800 CT/NG assay (Roche Molecular Systems, Pleasanton, CA) between March-April 2017 from across the University of California, Los Angeles Health System. We then retrospectively tested all specimens with the ResistancePlus MG (550) kit, a molecular assay for the detection of M. genitalium and genetic mutations associated with azithromycin resistance. RESULTS: Among 500 specimens, the prevalence of M. genitalium was 1.1% (95% confidence interval [CI], 0.04%-3.0%) in urine samples (n = 362), 17.4% (95% CI, 5.7%-39.6%) in rectal swabs (n = 23), and 1.9% (95% CI, 0.3%-7.3%) in cervical/vaginal swabs (n = 106). The prevalence of N. gonorrhoeae was 0.6% in urine samples and 4.3% in rectal swabs, whereas the prevalence of C. trachomatis was 2.2% in urine samples, 4.3% in rectal swabs and 3.8% in cervical/vaginal swabs. Of the 10 M. genitalium positive specimens, 8 (80.0%) had a mutation associated with azithromycin resistance. CONCLUSIONS: The prevalence of M. genitalium infection in our population varied by anatomic site of infection. Most M. genitalium infections had at least 1 mutation associated with azithromycin resistance.


Assuntos
Antibacterianos/farmacologia , Azitromicina/farmacologia , Farmacorresistência Bacteriana/genética , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/genética , Doenças Bacterianas Sexualmente Transmissíveis/microbiologia , Colo do Útero/microbiologia , DNA Bacteriano/urina , Feminino , Humanos , Los Angeles/epidemiologia , Mutação , Infecções por Mycoplasma/epidemiologia , Mycoplasma genitalium/efeitos dos fármacos , Faringe/microbiologia , Prevalência , Reto/microbiologia , Estudos Retrospectivos , Doenças Bacterianas Sexualmente Transmissíveis/epidemiologia
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