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1.
BMC Infect Dis ; 20(1): 110, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32033533

RESUMO

BACKGROUND: Men who have sex with men (MSM) are disproportionally affected by sexually transmitted infections (STI). STI are often extragenital and asymptomatic. Both can delay diagnosis and treatment. Approval of HIV pre-exposure prophylaxis (PrEP) might have influenced sexual behaviour and STI-prevalence of HIV- MSM. We estimated STI-prevalence and risk factors amongst HIV- and HIV+ MSM in Germany to plan effective interventions. METHODS: We conducted a nationwide, cross-sectional study between February and July 2018. Thirteen MSM-friendly STI-practices screened MSM for Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), Neisseria gonorrhea (NG), and Trichomonas vaginalis (TV) using self-collected rectal and pharyngeal swabs, and urine samples. APTIMA™ STI-assays (Hologic™ Inc., San Diego, USA) were used for diagnostics, and samples were not pooled. We collected information on socio-demographics, HIV-status, clinical symptoms, sexual behaviour within the last 6 months, and PrEP use. We combined HIV status and PrEP use for defining risk groups, and used directed acyclic graphs and multivariable logistic regression to identify risk factors for STI. RESULTS: Two thousand three hundred three MSM were included: 50.5% HIV+, median age 39 [18-79] years. Median number of male sex partners within the last 6 months was five. Sex without condom was reported by 73.6%, use of party drugs by 44.6%. 80.3% had a STI history, 32.2% of STI+ MSM reported STI-related symptoms. 27.6% of HIV- MSM used PrEP. Overall STI-prevalence was 30.1, 25.0% in HIV-/PrEP- MSM (CT:7.2%; MG:14.2%; NG:7.4%; TV:0%), 40.3% in HIV-/PrEP+ MSM (CT:13.8%; MG:19.4%; NG:14.8%; TV:0.4%), and 30.8% in HIV+ MSM (CT:10.1%; MG:18.4%; NG:8.6%; TV:0.1%). Being HIV+ (OR 1.7, 95%-CI 1.3-2.2), using PrEP (OR 2.0, 95%-CI 1.5-2.7), having > 5 sex partners (OR:1.65; 95%-CI:1.32-2.01.9), having condomless sex (OR:2.11.9; 95%-CI:1.65-2.86), and using party drugs (OR:1.65; 95%-CI:1.32-2.0) were independent risk factors for being tested positive for at least one STI. CONCLUSIONS: We found a high STI-prevalence in MSM in Germany, especially in PrEP users, frequently being asymptomatic. As a relevant proportion of PrEP users will not use a condom, counselling and comprehensive STI screening is essential and should be low threshold and preferably free of cost. Counselling of PrEP users should also address use of party drugs.


Assuntos
Infecções por Chlamydia/epidemiologia , Chlamydia trachomatis/genética , Gonorreia/epidemiologia , Infecções por HIV/epidemiologia , Homossexualidade Masculina , Infecções por Mycoplasma/epidemiologia , Mycoplasma genitalium/genética , Neisseria gonorrhoeae/genética , Profilaxia Pré-Exposição , Minorias Sexuais e de Gênero , Adolescente , Adulto , Idoso , Chlamydia trachomatis/isolamento & purificação , Preservativos , Aconselhamento , Estudos Transversais , Alemanha/epidemiologia , Gonorreia/diagnóstico , Gonorreia/microbiologia , Infecções por HIV/prevenção & controle , Humanos , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Mycoplasma genitalium/isolamento & purificação , Neisseria gonorrhoeae/isolamento & purificação , Prevalência , Fatores de Risco , Comportamento Sexual , Parceiros Sexuais , Adulto Jovem
2.
Nat Commun ; 11(1): 836, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32047145

RESUMO

In the future, entire genomes tailored to specific functions and environments could be designed using computational tools. However, computational tools for genome design are currently scarce. Here we present algorithms that enable the use of design-simulate-test cycles for genome design, using genome minimisation as a proof-of-concept. Minimal genomes are ideal for this purpose as they have a simple functional assay whether the cell replicates or not. We used the first (and currently only published) whole-cell model for the bacterium Mycoplasma genitalium. Our computational design-simulate-test cycles discovered novel in silico minimal genomes which, if biologically correct, predict in vivo genomes smaller than JCVI-Syn3.0; a bacterium with, currently, the smallest genome that can be grown in pure culture. In the process, we identified 10 low essential genes and produced evidence for at least two Mycoplasma genitalium in silico minimal genomes. This work brings combined computational and laboratory genome engineering a step closer.


Assuntos
Algoritmos , Simulação por Computador , Genoma Bacteriano , Mycoplasma genitalium/genética , Ontologia Genética , Genes Bacterianos/genética , Genes Essenciais/genética , Engenharia Genética/métodos , Tamanho do Genoma , Biologia Sintética/métodos
3.
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
4.
Emerg Microbes Infect ; 9(1): 5-19, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31859607

RESUMO

Transition metals participate in numerous enzymatic reactions and they are essential for survival in all living organisms. For this reason, bacterial pathogens have evolved dedicated machineries to effectively compete with their hosts and scavenge metals at the site of infection. In this study, we investigated the mechanisms controlling metal acquisition in the emerging human pathogen Mycoplasma genitalium. We observed a robust transcriptional response to metal starvation, and many genes coding for predicted lipoproteins and ABC-transporters were significantly up-regulated. Transcriptional analysis of a mutant strain lacking a metalloregulator of the Fur family revealed the activation of a full operon encoding a putative metal transporter system and a gene coding for a Histidine-rich lipoprotein (Hrl). We recognized a conserved sequence with dyad symmetry within the promoter region of the Fur-regulated genes. Mutagenesis of the predicted Fur operator within the hrl promoter abrogated Fur- and metal-dependent expression of a reporter gene. Metal starvation still impelled a strong transcriptional response in the fur mutant, demonstrating the existence of Fur-independent regulatory pathways controlling metal homeostasis. Finally, analysis of metal accumulation in the wild-type strain and the fur mutant by ICP-MS revealed an important role of Fur in nickel acquisition.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Mycoplasma genitalium/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Genética , 2,2'-Dipiridil/farmacologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Homeostase , Modelos Moleculares , Mycoplasma genitalium/genética , Regiões Promotoras Genéticas , Proteômica , Proteínas Repressoras/química , Transcrição Genética/efeitos dos fármacos
6.
BMC Infect Dis ; 19(1): 827, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547805

RESUMO

BACKGROUND: The worldwide expansion of macrolide-resistant Mycoplasma genitalium (MG) in cases of genital infections has led to an increased recurrence rate of these infections after first-line azithromycin treatment. By detecting the presence of azithromycin-resistant MG, the patient's antibiotic treatment can be targeted and the spread of resistance prevented. With this aim in mind, macrolide-resistance detection kits are helpful tools for the physician. METHODS: Azithromycin resistance mutations in MG are targeted using a four-color multiplex real-time RT-PCR assay. Tested targets include plasmid DNA (as positive controls) as well as macrolide-sensitive and macrolide-resistant genomic DNA from characterized cell lines and clinical samples. RESULTS: The analytical data presented here were generated from plasmid DNA and genomic RNA/DNA and include adaptation to an internal control, specificity between targets, specificity vs non-MG species, limit of detection (LoD) and interference studies (co-infection and endogenous substances). The clinical data were based on the application of the assay to clinical samples characterized by sequencing. CONCLUSIONS: A new NAAT (nucleic acid amplification test) prototype has been developed in collaboration with the Diagenode s.a. company, this prototype targets MG and azithromycin-resistance mutations in that pathogen.


Assuntos
Azitromicina/farmacologia , Farmacorresistência Bacteriana/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Mutação , Mycoplasma genitalium/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Antibacterianos/farmacologia , Humanos , Infecções por Mycoplasma/tratamento farmacológico , Infecções por Mycoplasma/microbiologia , Especificidade por Substrato
7.
Infect Dis Obstet Gynecol ; 2019: 8639510, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31379424

RESUMO

Background: Sexually transmitted infections (STIs) are highly prevalent in sub-Saharan Africa. Genital self-sampling may facilitate the screening of STIs in hard-to-reach remote populations far from large health care centers and may increase screening rates. The cross-sectional GYNAUTO-STI study was carried out to assess the performance of a novel genital veil (V-Veil-Up Gyn Collection Device, V-Veil-Up Pharma, Ltd., Nicosia, Cyprus) as a genital self-sampling device to collect genital secretions to diagnose STIs by molecular biology as compared to reference clinician-collected genital specimens, in adult African women. Methods: Adult women living in N'Djamena, the capital city of Chad, were recruited from the community and referred to the clinic for women's sexual health "La Renaissance Plus". A clinician obtained an endocervical specimen using flocked swab. Genital secretions were also obtained by self-collection using veil. Both clinician- and self-collected specimens were tested for common curable STIs (including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis) and genital Mycoplasma spp. by multiplex real-time PCR (Allplex™ STI Essential Assay, Seegene, Seoul, South Korea). Test positivities for both collection methods were compared by assessing methods agreement, sensitivity, and specificity. Results: A total of 251 women (mean age, 35.1 years) were prospectively enrolled. Only seven (2.8%) women were found to be infected with at least one common STIs [C. trachomatis: 3 (1.2%), N. gonorrhoeae: 1 (0.4%), M. genitalium: 4 (1.6%) and T. vaginalis: 1 (0.4%)], while the prevalence of genital mycoplasmas was much higher (54.2%) with a predominance of Ureaplasma parvum (42.6%). Self-collection by veil was non-inferior to clinician-based collection for genital microorganisms DNA molecular testing, with "almost perfect" agreement between both methods, high sensitivity (97.0%; 95%CI: 92.5-99.2%), and specificity (88.0%; 95%CI: 80.7-93.3%). Remarkably, the mean total number of genital microorganisms detected per woman was 1.14-fold higher in self-collected specimens compared to that in clinician-collected specimens. Conclusions: Veil-based self-collection of female genital secretions constitutes a convenient tool to collect in gentle way cervicovaginal secretions for accurate molecular detection of genital bacteria. Such sampling procedure could be easily implemented in STIs clinics in sub-Saharan Africa.


Assuntos
Reação em Cadeia da Polimerase Multiplex , Infecções por Mycoplasma/diagnóstico , Mycoplasma genitalium/isolamento & purificação , Doenças Sexualmente Transmissíveis/diagnóstico , Doenças Sexualmente Transmissíveis/microbiologia , Manejo de Espécimes/instrumentação , Adolescente , Adulto , África ao Sul do Saara/epidemiologia , Idoso , Estudos Transversais , Feminino , Humanos , Programas de Rastreamento , Pessoa de Meia-Idade , Infecções por Mycoplasma/epidemiologia , Mycoplasma genitalium/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Doenças Sexualmente Transmissíveis/epidemiologia , Manejo de Espécimes/métodos , Adulto Jovem
8.
J Appl Microbiol ; 127(4): 1219-1223, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31220405

RESUMO

AIMS: Mycoplasma genitalium causes a common, sexually transmitted bacterial infection. This study assessed the detection of M. genitalium in stored urine samples to understand the impact of sample storage on M. genitalium detection. METHODS: Aliquots of M. genitalium-positive urine (n = 20 patients) were stored at either room temperature (22°C) or 4°C, without a preservative. At weekly intervals, samples were tested using the commercial test ResistancePlus MG® (SpeeDx® , Australia). We report the analysis at 1 week, an acceptable collection-to-test turnaround time, with further analysis over 5 weeks to illustrate degradation trends. RESULTS: After storing at 4°C, the proportion of specimens that remained positive for M. genitalium was 100% after 1 week and 95% after 4 weeks. Storage at 22°C led to more rapid decline in detection in the first 4 weeks, with 95% detected after 1 week and 85% at 2 weeks onwards. At 5 weeks, samples stored at both temperatures had an 85% M. genitalium detection rate, with increase in crossing points (Cq) of 0·72 (95% confidence interval (CI) 0·01-1·43; P-trend = 0·027) at 4°C, and 1·75 ((95% CI 0·79-2·71), P-trend <0·001) at 22°C. CONCLUSIONS: Urine samples stored without preservative, and unfrozen, retained high M. genitalium detection levels over the short term (up to 5 weeks). To minimize degradation, storing at 4°C is recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: There is little known about the stability of clinical samples for M. genitalium detection. This study found that a high proportion (85-100%) of samples are still suitable for M. genitalium detection after storage for up to 5 weeks.


Assuntos
Tipagem Molecular , Infecções por Mycoplasma , Mycoplasma genitalium , Manejo de Espécimes , Urinálise , Austrália , Humanos , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/genética , Mycoplasma genitalium/isolamento & purificação
9.
Emerg Infect Dis ; 25(7): 1297-1303, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31211669

RESUMO

Mycoplasma genitalium infections of the urogenital tract are usually treated with azithromycin; however, for the past several years, rates of azithromycin treatment failure have increased. To document the occurrence and frequency of macrolide resistance-mediating mutations (MRMMs) in M. genitalium infections, we collected 894 M. genitalium-positive samples during April 2014-December 2017 and retrospectively tested them for MRMMs. We designated 67 samples collected within 6 weeks after a positive result as test-of-cure samples; of these, 60 were MRMM positive. Among the remaining 827 samples, the rate of MRMM positivity rose from 22.7% in 2014 and 22.3% in 2015 to 44.4% in 2016 but decreased to 39.7% in 2017. Because of these high rates of MRMMs in M. genitalium infections, we recommend that clinicians perform tests of cure after treatment and that researchers further explore the clinical consequences of this infection.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Macrolídeos/farmacologia , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/efeitos dos fármacos , Adulto , Antibacterianos/uso terapêutico , Feminino , História do Século XXI , Humanos , Macrolídeos/uso terapêutico , Masculino , Pessoa de Meia-Idade , Infecções por Mycoplasma/tratamento farmacológico , Infecções por Mycoplasma/história , Mycoplasma genitalium/genética , Países Baixos/epidemiologia , Vigilância em Saúde Pública , Estações do Ano , Doenças Bacterianas Sexualmente Transmissíveis/epidemiologia , Doenças Bacterianas Sexualmente Transmissíveis/microbiologia , Adulto Jovem
10.
Sex Transm Infect ; 95(5): 328-335, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31055469

RESUMO

BACKGROUND: Mycoplasma genitalium is increasingly seen as an emerging sexually transmitted pathogen, and has been likened to Chlamydia trachomatis, but its natural history is poorly understood. The objectives of this systematic review were to determine M. genitalium incidence, persistence, concordance between sexual partners and the risk of pelvic inflammatory disease (PID). METHODS: We searched Medline, EMBASE, LILACS, IndMed and African Index Medicus from 1 January 1981 until 17 March 2018. Two independent researchers screened studies for inclusion and extracted data. We examined results in forest plots, assessed heterogeneity and conducted meta-analysis where appropriate. Risk of bias was assessed for all studies. RESULTS: We screened 4634 records and included 18 studies; six (4201 women) reported on incidence, five (636 women) on persistence, 10 (1346 women and men) on concordance and three (5139 women) on PID. Incidence in women in two very highly developed countries was 1.07 per 100 person-years (95% CI 0.61 to 1.53, I2 0%). Median persistence of M. genitalium was estimated from one to three months in four studies but 15 months in one study. In 10 studies measuring M. genitalium infection status in couples, 39%-50% of male or female sexual partners of infected participants also had M. genitalium detected. In prospective studies, PID incidence was higher in women with M. genitalium than those without (risk ratio 1.73, 95% CI 0.92 to 3.28, I2 0%, two studies). DISCUSSION: Incidence of M. genitalium in very highly developed countries is similar to that for C. trachomatis, but concordance might be lower. Taken together with other evidence about age distribution and antimicrobial resistance in the two infections, M. genitalium is not the new chlamydia. Synthesised data about prevalence, incidence and persistence of M. genitalium infection are inconsistent. These findings can be used for mathematical modelling to investigate the dynamics of M. genitalium. REGISTRATION NUMBERS: CRD42015020420, CRD42015020405.


Assuntos
Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/isolamento & purificação , Adolescente , Adulto , Antibacterianos/administração & dosagem , Feminino , Humanos , Incidência , Masculino , Infecções por Mycoplasma/tratamento farmacológico , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/psicologia , Mycoplasma genitalium/classificação , Mycoplasma genitalium/efeitos dos fármacos , Mycoplasma genitalium/genética , Comportamento Sexual , Parceiros Sexuais , Adulto Jovem
11.
Sex Transm Infect ; 95(7): 522-528, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30981999

RESUMO

BACKGROUND: Mass drug administration (MDA) of 20 mg/kg (maximum 1 g in adults) azithromycin for ocular Chlamydia trachomatis (CT) infection is a key component of the WHO trachoma elimination strategy. However, this dose may be suboptimal in Mycoplasma genitalium infection and may encourage emergence of antimicrobial resistance (AMR) to azithromycin. OBJECTIVES: To determine the effect of MDA for trachoma elimination on M. genitalium prevalence, strain type and azithromycin resistance. METHODS: A secondary analysis of CT-negative vulvovaginal swabs from three outpatient antenatal clinics (Honiara, Solomon Islands) from patients recruited either pre-MDA, or 10 months post-MDA in two cross-sectional surveys was carried out. Swabs were tested for M. genitalium infection using Fast Track Diagnostics Urethritis Plus nucleic acid amplification assay. M. genitalium-positive samples were subsequently tested for azithromycin resistance by sequencing domain V of the 23S rRNA DNA region of M. genitalium and underwent phylogenetic analysis by dual locus sequence typing. RESULTS: M. genitalium prevalence was 11.9% (28/236) in women pre-MDA and 10.9% (28/256) 10 months post-MDA (p=0.7467). Self-reported receipt of azithromycin as part of MDA was 49.2% in women recruited post-MDA and 17.9% (5/28) in those who tested M. genitalium positive. Of samples sequenced (21/28 pre-MDA, 22/28 post-MDA), all showed a macrolide susceptible genotype. Strain typing showed that sequence types diverged into two lineages, with a suggestion of strain replacement post-MDA. CONCLUSION: A single round of azithromycin MDA in an island population with high baseline M. genitalium prevalence did not appear to impact on either prevalence or azithromycin resistance, in contrast to reported decreased genital CT prevalence in the same population. This may be due to limitations such as sample size, including CT-negative samples only, and low MDA coverage. Further investigation of the impact of multiple rounds of MDA on M. genitalium azithromycin AMR in antibiotic experienced and naïve populations is warranted.


Assuntos
Antibacterianos/efeitos adversos , Azitromicina/efeitos adversos , Farmacorresistência Bacteriana , Administração Massiva de Medicamentos/efeitos adversos , Infecções por Mycoplasma/epidemiologia , Mycoplasma genitalium/efeitos dos fármacos , Tracoma/tratamento farmacológico , Adolescente , Adulto , Antibacterianos/administração & dosagem , Azitromicina/administração & dosagem , Análise por Conglomerados , Estudos Transversais , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Feminino , Genótipo , Humanos , Melanesia/epidemiologia , Pessoa de Meia-Idade , Tipagem Molecular , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/classificação , Mycoplasma genitalium/genética , Mycoplasma genitalium/isolamento & purificação , Filogenia , Prevalência , RNA Ribossômico 23S/genética , Análise de Sequência de DNA , Tracoma/prevenção & controle , Adulto Jovem
13.
BMC Infect Dis ; 19(1): 148, 2019 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760230

RESUMO

BACKGROUND: Antimicrobial resistance in Mycoplasma genitalium is rising globally with resultant clinical treatment failure. We investigated the prevalence of mutations in the macrolide and fluoroquinolone resistance-determining regions of M. genitalium in Johannesburg, South Africa, and ascertained their association with HIV serostatus. METHODS: Stored M. genitalium positive specimens, collected from STI and HIV patients enrolled in the Gauteng STI National Microbiological Surveillance programme (2007-2014) and a large HIV outpatient clinic-based study (2007) in Johannesburg, were tested for antimicrobial resistance. RESULTS: We determined the prevalence of 23S rRNA gene mutations conferring macrolide resistance and mutations in the quinolone resistance-determining regions (QRDR) of the gyrA and parC genes in 266 M. genitalium positive DNA extracts. No macrolide resistance-associated mutations were detected in any of the specimens analysed. QRDR mutations with known M. genitalium-associated fluoroquinolone resistance were not detected in gyrA, however, one specimen (0.4%) contained a D87Y amino acid alteration in parC, which has been linked to fluoroquinolone treatment failure. The most common parC amino acid change detected, of unknown clinical significance, was P62S (18.8%). We found no significant association between QRDR mutations in M. genitalium and HIV-infection. CONCLUSIONS: Ongoing antimicrobial resistance surveillance in M. genitalium is essential, as macrolide resistance may emerge given the recent incorporation of azithromycin into the 2015 South African national STI syndromic management guidelines.


Assuntos
Farmacorresistência Bacteriana/genética , Mutação , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/efeitos dos fármacos , Mycoplasma genitalium/genética , Antibacterianos/uso terapêutico , DNA Girase/genética , DNA Topoisomerase IV/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Feminino , Fluoroquinolonas/uso terapêutico , Infecções por HIV/microbiologia , Humanos , Macrolídeos/uso terapêutico , Masculino , Infecções por Mycoplasma/tratamento farmacológico , Prevalência , RNA Ribossômico 23S/genética , África do Sul , Falha de Tratamento
14.
Clin Microbiol Infect ; 25(1): 35-47, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29729331

RESUMO

BACKGROUND: The vaginal microbiota may modulate susceptibility to human papillomavirus (HPV), Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium infections. Persistent infection with a carcinogenic HPV is a prerequisite for cervical cancer, and C. trachomatis, N. gonorrheae and M. genitalium genital infections are all associated with pelvic inflammatory disease and subsequent infertility issues. OBJECTIVES: To evaluate the association between these infections and the vaginal microbiota. DATA SOURCES: The search was conducted on Medline and the Web of Science for articles published between 2000 and 2016. STUDY ELIGIBILITY CRITERIA: Inclusion criteria included a measure of association for vaginal microbiota and one of the considered STIs, female population, cohort, cross-sectional and interventional designs, and the use of PCR methods for pathogen detection. METHODS: The vaginal microbiota was dichotomized into high-Lactobacillus vaginal microbiota (HL-VMB) and low-Lactobacillus vaginal microbiota (LL-VMB), using either Nugent score, Amsel's criteria, presence of clue cells or gene sequencing. A random effects model assuming heterogeneity among the studies was used for each STI considered. RESULTS: The search yielded 1054 articles, of which 39 met the inclusion criteria. Measures of association with LL-VMB ranged from 0.6 (95% CI 0.3-1.2) to 2.8 (95% CI 0.3-28.0), 0.7 (95% CI 0.4-1.2) to 5.2 (95% CI 1.9-14.8), 0.8 (95% CI 0.5-1.4) to 3.8 (95% CI 0.4-36.2) and 0.4 (95% CI 0.1-1.5) to 6.1 (95% CI 2.0-18.5) for HPV, C. trachomatis, N. gonorrhoeae and M. genitalium infections, respectively. CONCLUSIONS: Although no clear trend for N. gonorrhoeae and M. genitalium infections could be detected, our results support a protective role of HL-VMB for HPV and C. trachomatis. Overall, these findings advocate for the use of high-resolution characterization methods for the vaginal microbiota and the need for longitudinal studies to lay the foundation for its integration in prevention and treatment strategies.


Assuntos
Interações Microbianas , Microbiota , Vagina/microbiologia , Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/genética , Feminino , Gonorreia/diagnóstico , Humanos , Infecções por Mycoplasma/diagnóstico , Mycoplasma genitalium/genética , Neisseria gonorrhoeae/genética , Papillomaviridae/genética , Doença Inflamatória Pélvica/microbiologia , Doenças Sexualmente Transmissíveis/microbiologia , Doenças Sexualmente Transmissíveis/virologia
15.
Sex Transm Dis ; 46(1): 18-24, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29979336

RESUMO

BACKGROUND: Mycoplasma genitalium (MG) is a sexually transmitted pathogen associated with inflammatory syndromes in men and women. Macrolides and fluoroquinolones are recommended MG treatments. The frequency of MG strains with macrolide resistance-associated mutations (MRMs) and quinolone resistance-associated mutations (qRMs) is increasing worldwide, however these data are sparse in populations in the United States. METHODS: We investigated the prevalence of MG infections with MRMs and qRMs and MG infection concordance within African American couples in Birmingham, AL. We used a real-time polymerase chain reaction to detect MG and identify MRMs. quinolone resistance-associated mutations were detected using traditional polymerase chain reactions amplifying regions in gyrA, gyrB, parC, and parE. The MG concordance in couples was evaluated by MG positivity and MG genotypes. RESULTS: Oral, anal, urine, and/or vaginal specimens were tested from 116 couples. Twenty-eight (12.1%) participants comprising 22 couples tested MG-positive (11.2% in men and 12.9% in women). Macrolide resistance-associated mutations were detected in 17 (60.7%) MG-positive participants, with gender-specific resistance rates of 69.2% for men and 53.3% for women. quinolone resistance-associated mutations were detected in 3 (11.1%) MG-positive participants, all of whom also had MRMs. By MG positivity status, 27.3% of couples were concordant. If MG strain genotypes are also considered, then concordance was 20.0%. CONCLUSIONS: Among heterosexual African Americans with MG infection, about 60% had strains with MRMs and 11% had strains with both MRMs and qRMs, highlighting the potential for MG treatment failure to not only macrolides, but also quinolones. These findings may help to guide clinicians in MG testing and treatment decisions in the United States.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Infecções por Mycoplasma/etnologia , Mycoplasma genitalium/efeitos dos fármacos , Adolescente , Adulto , Afro-Americanos , Alabama/epidemiologia , DNA Bacteriano/genética , Feminino , Heterossexualidade , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/genética , Prevalência , Adulto Jovem
16.
BMC Infect Dis ; 18(1): 601, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30486786

RESUMO

BACKGROUND: The increasing prevalence of macrolide resistant Mycoplasma genitalium is a major concern worldwide. In Cuba, several cases of clinical treatment failure with 1 g single dose and extended azithromycin regimen have been detected and the aim of the present investigation was to retrospectively determine the prevalence of macrolide-resistance mediating mutations (MRMM) in M. genitalium-positive samples conserved at the Cuban National Reference Laboratory of Mycoplasma Research between 2009 and 2016. METHODS: A total of 280 positive DNA extracts were analysed by a 5' nuclease assay for detection of M. genitalium MRMM. Ten urogenital specimens from patients with azithromycin treatment failure and MRMM were inoculated in Vero cell to obtain the isolates for subsequent determination of antimicrobial susceptibility. RESULTS: The overall prevalence of MRMM was 32%. No MRMM was detected in samples collected between 2009 and 2013 but since 2014 a dramatic increase to 90% (95% CI, 76-96%) in 2016 was seen. Three new M. genitalium isolates were isolated in Vero cell cultures and confirmed phenotypic resistance to macrolides in a cell-culture assisted susceptibility test. Preliminary observations suggest that combination therapy with levofloxacin and doxycycline may represent an affordable option for treatment of macrolide resistant M. genitalium infections. CONCLUSIONS: This investigation showed the rapid emergence and high prevalence of MRMM in M. genitalium-infected patients in Cuba and confirmed the phenotypic resistance in isolates carrying MRMM. We suggest that Cuban guidelines for sexually transmitted infections are modified to include testing for M. genitalium and detection of MRMM in patients with failure of syndromic treatment, to ensure that in these cases, the treatment will be guided by etiologic diagnosis.


Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Macrolídeos/uso terapêutico , Infecções por Mycoplasma/tratamento farmacológico , Infecções por Mycoplasma/epidemiologia , Mycoplasma genitalium , Adulto , Animais , Cuba/epidemiologia , Erros de Diagnóstico/estatística & dados numéricos , Farmacorresistência Bacteriana/efeitos dos fármacos , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/efeitos dos fármacos , Mycoplasma genitalium/genética , Mycoplasma genitalium/isolamento & purificação , Prevalência , Estudos Retrospectivos , Doenças Bacterianas Sexualmente Transmissíveis/tratamento farmacológico , Doenças Bacterianas Sexualmente Transmissíveis/epidemiologia , Células Vero
17.
Nat Commun ; 9(1): 4471, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30367053

RESUMO

Adhesion of pathogenic bacteria to target cells is a prerequisite for colonization and further infection. The main adhesins of the emerging sexually transmitted pathogen Mycoplasma genitalium, P140 and P110, interact to form a Nap complex anchored to the cell membrane. Herein, we present the crystal structures of the extracellular region of the virulence factor P110 (916 residues) unliganded and in complex with sialic acid oligosaccharides. P110 interacts only with the neuraminic acid moiety of the oligosaccharides and experiments with human cells demonstrate that these interactions are essential for mycoplasma cytadherence. Additionally, structural information provides a deep insight of the P110 antigenic regions undergoing programmed variation to evade the host immune response. These results enlighten the interplay of M. genitalium with human target cells, offering new strategies to control mycoplasma infections.


Assuntos
Adesinas Bacterianas/metabolismo , Infecções por Mycoplasma/fisiopatologia , Mycoplasma genitalium/fisiologia , Receptores de Superfície Celular/metabolismo , Fatores de Virulência/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Sítios de Ligação/genética , Membrana Celular/metabolismo , Eritrócitos/metabolismo , Eritrócitos/microbiologia , Hemadsorção/genética , Humanos , Modelos Moleculares , Mutação , Infecções por Mycoplasma/metabolismo , Mycoplasma genitalium/genética , Potássio/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Superfície Celular/química , Fatores de Virulência/química , Fatores de Virulência/genética
18.
Eur J Clin Microbiol Infect Dis ; 37(11): 2137-2144, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30276584

RESUMO

Patients infected by Mycoplasma genitalium are often treated empirically with the macrolide azithromycin. Macrolide resistance is becoming quite common; empirical treatment is compromised. Sequencing was initially used to detected azithromycin resistance-associated mutations. As this was laborious, qPCRs have been developed for their detection. In the present study, we describe a fast, sensitive, and specific qPCR assay that enables routine testing of M. genitalium and macrolide resistance-associated mutations in a single assay. M. genitalium positive clinical samples were used to compare (i) the commonly used MgPa assay for the detection of M. genitalium infections (MgPa qPCR), (ii) a combined 23S rRNA gene PCR/sequencing assay (Mg23S qPCR/Sequencing) to identify macrolide resistance-associated mutations, and (iii) our newly developed probe-based melt curve qPCR for simultaneous detection of M. genitalium and macrolide resistance-associated mutations (Macrolide-R/MG ELITe MGB Kit, Elitech Bothel USA in short Mg MacrolideR qPCR). Specificity of the qPCR was tested using urogenital samples that were tested positive for a range of other micro-organisms. M. genitalium was detected in 196/236 (83.1%) samples by the MgPa qPCR, versus 172/236 (72.9%) by the combined Mg23S qPCR/Sequencing, and 202/236 (85.6%) by the Mg MacrolideR qPCR. The Mg MacrolideR qPCR showed high concordance to the Mg23S qPCR/Sequencing assay (201 vs 202 could be genotyped, respectively) for the detection of the macrolide resistant mutations. None of the other urogenital pathogens were tested positive in the Mg MacrolideR qPCR, indicating specificity. The Mg MacrolideR qPCR is fast, sensitive, specific, and can easily be implemented in the routine diagnostics.


Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Macrolídeos/farmacologia , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/efeitos dos fármacos , Mycoplasma genitalium/genética , Adolescente , Adulto , Antituberculosos/uso terapêutico , Feminino , Humanos , Macrolídeos/uso terapêutico , Masculino , Pessoa de Meia-Idade , Mutação , Infecções por Mycoplasma/tratamento farmacológico , RNA Ribossômico 23S/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Adulto Jovem
19.
Clin Lab ; 64(7): 1217-1224, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30146824

RESUMO

BACKGROUND: Genitourinary Mycoplasma (Ureaplasma urealyticum, Mycoplasma hominis, and Mycoplasma genitalium) is a common pathogen among women, which can cause funisitis, spontaneous abortion, and low birth weight. However, current laboratory testing methods for genitourinary mycoplasma normally need complex processes, expensive devices, and qualified staffs, and there are many limits for application. Up to now, the LAMP method is a rapidly developing field because of the significance for clinical application and commercial value. Few studies have reported the use of mLAMP to detect UU, MH, and MG. In this study, a multiplex loop-mediated isothermal amplification system was developed for rapid detection of UU, MH, and MG, concurrently. METHODS: Three sets of multiplex LAMP primers were designed to specifically target urease of UU, 16S rRNA of MH, and mgpa of MG. The ratio of primer concentration was optimized. The specificity and sensitivity of multiplex LAMP were explored. Twenty-nine clinical samples were successfully used with mLAMP. RESULTS: In this study, the primer concentration in the mLAMP system was set to 1.3 µmol/L which could maintain reaction efficiency and avoid non-specific reaction. Multiplex LAMP can test UU, MG, and MH simultaneously with high specificity. Meanwhile, the sensitivity of multiplex LAMP was found to be 100 pg for UU, 100 pg for MH and 1 ng for MG, which was much higher than that of conventional PCR. Furthermore, among the 29 clinical samples, there were two positive samples determined by mLAMP, which was consistent with the PCR and sequencing results. CONCLUSIONS: The multiplex LAMP assay can potentially facilitate simultaneous detection of three kinds of mycoplasma in a large number of samples in clinic, which could be used as a primary screening method and as a supplementary method for classical methods.


Assuntos
Doenças Urogenitais Femininas/diagnóstico , Infecções por Mycoplasma/diagnóstico , Mycoplasma/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções por Ureaplasma/diagnóstico , Adulto , Primers do DNA/genética , Feminino , Doenças Urogenitais Femininas/microbiologia , Humanos , Mycoplasma/classificação , Mycoplasma/fisiologia , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/genética , Mycoplasma genitalium/fisiologia , Mycoplasma hominis/genética , Mycoplasma hominis/fisiologia , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Infecções por Ureaplasma/microbiologia , Ureaplasma urealyticum/genética , Ureaplasma urealyticum/fisiologia , Urease/genética
20.
Ann Clin Microbiol Antimicrob ; 17(1): 25, 2018 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-29866110

RESUMO

BACKGROUND: Miscarriage and preterm delivery are the most important challenges of pregnancy. Different bacterial and viral infection may cause miscarriage and preterm delivery. Among bacterial factors, Mycoplasma genitalium and Chlamydia trachomatis have the most important role and human papilloma virus (HPV) is the leading viral factor in this regard. METHODS: First void urine samples were collected from 119 pregnant women who visited health centers for routine first-trimester screening (12-14 weeks gestation). About 10 ml of the sample was centrifuged at 3000×g for 20 min and 1-2 ml of the sediment was transferred to sterile microfuges and stored at - 20 °C until analysis. DNA extraction was conducted using A101211 kits imported by Pars Tous Biotechnology Company. The following commercial kits, imported by Pars Tous Biotechnology, were used for PCR. RESULTS: There is no significant association between urinary isolation of C. trachomatis and miscarriage (P = 0.93) and there is no significant association between urinary isolation of M. genitalium and miscarriage (P = 0.80). Regarding HPV, since all urine samples were PCR-negative, comparison was not possible. C. trachomatis was isolated from the urine samples of 6.72% of the pregnant women who underwent first-trimester screening in health centers using PCR. Previous studies reported a mean chlamydia isolation rate of 3% from urine specimens collected from pregnant women in general. T test showed no significant difference between the two groups (P = 0.10). Based on present study the mycoplasma isolation rate was 17.65% using PCR. Previous studies reported a mean mycoplasma isolation rate of 10% from urine specimens collected from pregnant women in general. T-test showed a significant difference between the two groups (P = 0.03). DISCUSSION: First void urine samples in pregnant women may be an appropriate sample for detection of C. trachomatis and M. genitalium; however, it is not a good method for HPV isolation therefore vaginal or cervical discharge specimens should be used instead for detection of HPV.


Assuntos
Infecções por Chlamydia/diagnóstico , DNA Bacteriano/urina , DNA Viral/urina , Infecções por Mycoplasma/diagnóstico , Infecções por Papillomavirus/diagnóstico , Complicações Infecciosas na Gravidez/diagnóstico , Aborto Espontâneo/microbiologia , Adulto , Infecções por Chlamydia/urina , Chlamydia trachomatis/genética , Chlamydia trachomatis/isolamento & purificação , DNA Bacteriano/genética , DNA Viral/genética , Feminino , Humanos , Infecções por Mycoplasma/urina , Mycoplasma genitalium/genética , Mycoplasma genitalium/isolamento & purificação , Trabalho de Parto Prematuro/microbiologia , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/urina , Reação em Cadeia da Polimerase/métodos , Gravidez , Complicações Infecciosas na Gravidez/microbiologia , Primeiro Trimestre da Gravidez
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