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1.
Nat Commun ; 11(1): 6430, 2020 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-33353937

RESUMEN

The trp operon of Chlamydia trachomatis is organized differently from other model bacteria. It contains trpR, an intergenic region (IGR), and the biosynthetic trpB and trpA open-reading frames. TrpR is a tryptophan-dependent repressor that regulates the major promoter (PtrpR), while the IGR harbors an alternative promoter (PtrpBA) and an operator sequence for the iron-dependent repressor YtgR to regulate trpBA expression. Here, we report that YtgR repression at PtrpBA is also dependent on tryptophan by regulating YtgR levels through a rare triple-tryptophan motif (WWW) in the YtgCR precursor. Inhibiting translation during tryptophan limitation at the WWW motif subsequently promotes Rho-independent transcription termination of ytgR, thereby de-repressing PtrpBA. Thus, YtgR represents an alternative strategy to attenuate trpBA expression, expanding the repertoire for trp operon attenuation beyond TrpL- and TRAP-mediated mechanisms described in other bacteria. Furthermore, repurposing the iron-dependent repressor YtgR underscores the fundamental importance of maintaining tryptophan-dependent attenuation of the trpRBA operon.


Asunto(s)
Proteínas Bacterianas/metabolismo , Chlamydia trachomatis/genética , Hierro/metabolismo , Operón/genética , Triptófano/metabolismo , Secuencias de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Chlamydia trachomatis/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Indoles/farmacología , Modelos Biológicos , Regiones Promotoras Genéticas , Biosíntesis de Proteínas/efectos de los fármacos , Dominios Proteicos , ARN de Transferencia de Triptófano/metabolismo , Transcripción Genética/efectos de los fármacos , Proteínas de Unión al GTP rho/metabolismo
2.
Zhonghua Yu Fang Yi Xue Za Zhi ; 54(10): 1133-1140, 2020 Oct 06.
Artículo en Chino | MEDLINE | ID: mdl-33115201

RESUMEN

Objective: To investigate the performance of ZNA(ZIP Nucleic Acid) probes and its application in the quantitative detection of Chlamydia trachomatis (CT)nucleic acid. Methods: Use CT positive plasmids to compare the PCR amplification curves of ZNA probes coupled with different ZIP numbers. Compare ZNA probes with other three sets of probes [namely, 29mer ordinary Taqman probes (long-DNA probe), 20mer ordinary Taqman probes (short-DNA probe) and MGB probes] for stability in PCR amplification curves and repeated freezing and thawing, and the difference in the detection rate of low-concentration plasmids. Use CT positive clinical samples to compare the difference in amplification curves between ZNA probes, long-DNA probe, short-DNA probe and MGB probes, and the detection rate of low-concentration samples. Results: (1) The Ct value and fluorescence value of the probes coupled with 5ZIP units are both better than those coupled with a smaller number of ZIPs. And the difference is biggest when compared with only coupled with 1 ZIP unit: Ct value increased by 1.34 (sensitivity increased by 2.37 times), and fluorescence value increased by 30%. (2) The amplification efficiency of the ZNA probe coupled with 5 ZIPs is 2.14-2.64 times that of the preferred ordinary Taqman probe and MGB probe, and the fluorescence value is 17%-90% higher. (3) The probe freeze-thaw stability results show that the ZNA probe has the best stability, and the lowest concentration of Ct value has the smallest deviation (CV% = 1.4), which is better than the other three sets of probes (CV%=1.7-3.7). (4) Using 35 CT positive clinical samples to compare the PCR amplification performance, compared with other three sets of probes, the amplification sensitivity of ZNA probes was increased by 1.60, 0.99 and 1.06 times respectively. And the results of the consistency analysis of the Ct value show that compared with short-DNA probe and MGB probes, ZNA probes have better detection performance for clinical samples. (5) Use low concentration plasmid template (200, 100, 50 and 10 copies/mL respectively) to compare the amplification sensitivity of the four sets of probes, the detection rate of ZNA probe is the best. Especially, at the lowest concentration 10 copies/mL, the detection rate of the other sets of probes is only 15%-20%, but the ZNA probe is still 30%. (6) In 20 clinical samples with different low concentrations (200, 150, 100, and 50 copies/mL), the detection rate of ZNA probes was the highest, which were 100%, 95%, 90%, and 70%, respectively. Conclusions: Through testing of the amplification efficiency, fluorescence value, freeze-thaw stability, the amplification performance of clinical samples and the detection sensitivity of low-concentration samples, ZNA probes coupled with 5 ZIPs show better performance than ordinary Taqman probes and MGB probes. As a new probe technology with flexible design and easy synthesis, ZNA probe can further improve detection sensitivity of low concentration samples in the field of gene expression.


Asunto(s)
Chlamydia trachomatis , Técnicas de Amplificación de Ácido Nucleico , Chlamydia trachomatis/genética , Plásmidos , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad
3.
PLoS Pathog ; 16(9): e1008878, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32946535

RESUMEN

As an obligate intracellular pathogen, host cell invasion is paramount to Chlamydia trachomatis proliferation. While the mechanistic underpinnings of this essential process remain ill-defined, it is predicted to involve delivery of prepackaged effector proteins into the host cell that trigger plasma membrane remodeling and cytoskeletal reorganization. The secreted effector proteins TmeA and TarP, have risen to prominence as putative key regulators of cellular invasion and bacterial pathogenesis. Although several studies have begun to unravel molecular details underlying the putative function of TarP, the physiological function of TmeA during host cell invasion is unknown. Here, we show that TmeA employs molecular mimicry to bind to the GTPase binding domain of N-WASP, which results in recruitment of the actin branching ARP2/3 complex to the site of chlamydial entry. Electron microscopy revealed that TmeA mutants are deficient in filopodia capture, suggesting that TmeA/N-WASP interactions ultimately modulate host cell plasma membrane remodeling events necessary for chlamydial entry. Importantly, while both TmeA and TarP are necessary for effective host cell invasion, we show that these effectors target distinct pathways that ultimately converge on activation of the ARP2/3 complex. In line with this observation, we show that a double mutant suffers from a severe entry defect nearly identical to that observed when ARP3 is chemically inhibited or knocked down. Collectively, our study highlights both TmeA and TarP as essential regulators of chlamydial invasion that modulate the ARP2/3 complex through distinct signaling platforms, resulting in plasma membrane remodeling events that are essential for pathogen uptake.


Asunto(s)
Proteínas Bacterianas , Membrana Celular/metabolismo , Chlamydia trachomatis , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/genética , Membrana Celular/patología , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/patogenicidad , Células HeLa , Humanos , Mutación , Dominios Proteicos , Seudópodos/genética , Seudópodos/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genética
4.
PLoS Negl Trop Dis ; 14(9): e0008647, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32877398

RESUMEN

The transmission of trachoma, caused by repeat infections with Chlamydia trachomatis, and many enteropathogens are linked to water quantity. We hypothesized that children living further from a water source would have higher exposure to C. trachomatis and enteric pathogens as determined by antibody responses. We used a multiplex bead assay to measure IgG antibody responses to C. trachomatis, Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae in eluted dried blood spots collected from 2267 children ages 0-9 years in 40 communities in rural Ethiopia in 2016. Linear distance from the child's house to the nearest water source was calculated. We derived seroprevalence cutoffs using external negative control populations, if available, or by fitting finite mixture models. We used targeted maximum likelihood estimation to estimate differences in seroprevalence according to distance to the nearest water source. Seroprevalence among 1-9-year-olds was 43% for C. trachomatis, 28% for S. enterica, 70% for E. histolytica, 54% for G. intestinalis, 96% for C. jejuni, 76% for ETEC and 94% for C. parvum. Seroprevalence increased with age for all pathogens. Median distance to the nearest water source was 473 meters (IQR 268, 719). Children living furthest from a water source had a 12% (95% CI: 2.6, 21.6) higher seroprevalence of S. enterica and a 12.7% (95% CI: 2.9, 22.6) higher seroprevalence of G. intestinalis compared to children living nearest. Seroprevalence for C. trachomatis and enteropathogens was high, with marked increases for most enteropathogens in the first two years of life. Children living further from a water source had higher seroprevalence of S. enterica and G. intestinalis indicating that improving access to water in the Ethiopia's Amhara region may reduce exposure to these enteropathogens in young children.


Asunto(s)
Anticuerpos Antibacterianos/sangre , Anticuerpos Antiprotozoarios/sangre , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/inmunología , Criptosporidiosis/sangre , Cryptosporidium/inmunología , Entamebiasis/sangre , Giardiasis/sangre , Niño , Preescolar , Infecciones por Chlamydia/sangre , Infecciones por Chlamydia/epidemiología , Chlamydia trachomatis/genética , Estudios Transversales , Criptosporidiosis/epidemiología , Criptosporidiosis/parasitología , Cryptosporidium/genética , Entamoeba histolytica/genética , Entamoeba histolytica/inmunología , Entamebiasis/epidemiología , Entamebiasis/parasitología , Etiopía/epidemiología , Femenino , Agua Dulce/parasitología , Giardia lamblia/genética , Giardia lamblia/inmunología , Giardiasis/epidemiología , Giardiasis/parasitología , Humanos , Masculino , Estudios Seroepidemiológicos
5.
New Microbiol ; 43(3): 115-120, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32656570

RESUMEN

Chlamydia trachomatis and Neisseria gonorrhoeae are the most common agents of bacterial sexually transmitted infections (STIs) worldwide. Here, we evaluated genital and extra-genital C. trachomatis and N. gonorrhoeae infection prevalence in a cohort of young women attending an STI Outpatients Clinic in Italy. From May 2019 to December 2019, 134 women aged 18-26 years were enrolled. A vaginal, a pharyngeal and a rectal swab for the molecular detection of C. trachomatis and N. gonorrhoeae were collected from each patient. Chlamydia-positive samples underwent a molecular genotyping based on pmpH gene. Total prevalence of C. trachomatis and N. gonorrhoeae infections was 17.9% and 11.2%, respectively. Chlamydial infections were prevalent in the urogenital (16.4%) and rectal (13.4%) sites, whereas N. gonorrhoeae predominated in the genital (7.4%) and pharyngeal (6%) mucosa. Overall, 5.2% of cases would have been missed if extra-genital sites had not been tested. Notably, 60% of women with a rectal infection did not report anal sex. A history of sexual contacts with a positive partner (P=0.03) and a history of ≥3 partners in the last 6 months (P=0.0075) were highly predictive of a bacterial STI. No cases of lymphogranuloma venereum were found. These data could help set up effective strategies to prevent the spread of STIs.


Asunto(s)
Infecciones por Chlamydia , Gonorrea , Enfermedades de Transmisión Sexual , Adolescente , Adulto , Infecciones por Chlamydia/epidemiología , Chlamydia trachomatis/genética , Chlamydia trachomatis/aislamiento & purificación , Femenino , Genitales , Gonorrea/epidemiología , Humanos , Italia/epidemiología , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/aislamiento & purificación , Prevalencia , Adulto Joven
6.
PLoS Negl Trop Dis ; 14(7): e0008449, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32667914

RESUMEN

BACKGROUND: The presence of Chlamydia trachomatis (Ct) DNA at non-ocular sites suggests that these sites may represent plausible routes of Ct transmission in trachoma. However, qPCR cannot discriminate between DNA from viable and non-viable bacteria. Here we use a propodium monoazide based viability PCR to investigate how long Ct remains viable at non-ocular sites under laboratory-controlled conditions. METHODS: Cultured Ct stocks (strain A2497) were diluted to final concentrations of 1000, 100, 10 and 1 omcB copies/µL and applied to plastic, woven mat, cotton cloth and pig skin. Swabs were then systemically collected from each surface and tested for the presence Ct DNA using qPCR. If Ct DNA was recovered, Ct viability was assessed over time by spiking multiple areas of the same surface type with the same final concentrations. Swabs were collected from each surface at 0, 2, 4, 6, 8 and 24 hours after spiking. Viability PCR was used to determine Ct viability at each timepoint. RESULTS: We were able to detect Ct DNA on all surfaces except the woven mat. Total Ct DNA remained detectable and stable over 24 hours for all concentrations applied to plastic, pig skin and cotton cloth. The amount of viable Ct decreased over time. For plastic and skin surfaces, only those where concentrations of 100 or 1000 omcB copies/µL were applied still had viable loads detectable after 24 hours. Cotton cloth showed a more rapid decrease and only those where concentrations of 1000 omcB copies/µL were applied still had viable DNA detectable after 24 hours. CONCLUSION: Plastic, cotton cloth and skin may contribute to transmission of the Ct strains that cause trachoma, by acting as sites where reservoirs of bacteria are deposited and later collected and transferred mechanically into previously uninfected eyes.


Asunto(s)
Chlamydia trachomatis/genética , ADN Bacteriano/aislamiento & purificación , Fómites/microbiología , Reacción en Cadena de la Polimerasa/métodos , Tracoma/microbiología , Tracoma/transmisión , Humanos
7.
J Vis Exp ; (160)2020 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-32597859

RESUMEN

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. The non-replicative elementary body (EB) initiates infection of the host. Once inside, the EB differentiates into the reticulate body (RB). The RB then undergoes multiple rounds of replication, before differentiating back to the infectious EB form. This cycle is essential for chlamydial survival as failure to switch between cell types prevents either host invasion or replication. Limitations in genetic techniques due to the obligate intracellular nature of Chlamydia have hampered identification of the molecular mechanisms involved in the cell-type development. We designed a novel dual promoter-reporter plasmid system that, in conjunction with live-cell microscopy, allows for the visualization of cell type switching in real time. To identify genes involved in the regulation of cell-type development, the live-cell promoter-reporter system was leveraged for the development of a forward genetic approach by combining chemical mutagenesis of the dual reporter strain, imaging and tracking of Chlamydia with altered developmental kinetics, followed by clonal isolation of mutants. This forward genetic workflow is a flexible tool that can be modified for directed interrogation into a wide range of genetic pathways.


Asunto(s)
Chlamydia trachomatis/genética , Chlamydia trachomatis/aislamiento & purificación , Genómica/métodos , Mutación/genética , Chlamydia trachomatis/crecimiento & desarrollo , Análisis de Datos , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Humanos , Cinética , Mutagénesis/genética , Fenotipo , Regiones Promotoras Genéticas/genética , Reproducibilidad de los Resultados
8.
PLoS One ; 15(6): e0233990, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32497069

RESUMEN

OBJECTIVES: Typing of Chlamydia trachomatis (CT) is traditionally performed by characterising the ompA gene, resulting in more than a dozen different genovars, A to L. Type L is associated with Lymphogranuloma venereum (LGV) and commonly screened for using PCR, targeting the chromosomal pmpH gene. We aimed to develop and validate a new CT/LGV plasmid-based typing assay targeting the pgp3 gene, to increase sensitivity and thus reduce the number of non-typeable results. METHODS: The new pgp3 PCR assay using LNA probes to detect point mutations was analytically and prospectively validated in a routine diagnostic laboratory setting. For the analytical tests, quantified nucleotide constructs (gBlocks) were used to perform limit of detection analyses. Quality control panel samples from 2018 and 2019 for CT were also tested. For the clinical study patient samples which were collected in two months in 2018 were tested simultaneously using the pmpH PCR and the pgp3 PCR. RESULTS: Analytically, the assay proved to be 100% specific relative to the previously used LGV typing assay targeting the single copy pmpH gene but it was much more sensitive to detect non-LGV CT. In the quality control panel 2 nonLGV samples and 7 LGV samples were solely positive with the pgp3 PCR and not with the pmpH PCR. None of the samples from analytical specificity panels were positive, indicating 100% specificity. In a prospective panel of 152 clinical samples, 142 (93%) were successfully typed with the pgp3 PCR compared to 78% with the pmpH PCR. The pgp3 PCR was fully concordant with the pmpH PCR to identify all LGV subtypes and detected an increased number of clinical samples of non-LGV subtype. CONCLUSION: We developed and validated a sensitive and specific plasmid-based typing assay to discriminate LGV from non-LGV CT subtypes. This is useful in a clinical setting to quickly determine the optimal treatment for Chlamydia trachomatis infections.


Asunto(s)
Chlamydia trachomatis/genética , Linfogranuloma Venéreo/microbiología , Plásmidos/genética , Reacción en Cadena de la Polimerasa/métodos , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana/métodos , Genes Bacterianos , Humanos
9.
BMC Infect Dis ; 20(1): 419, 2020 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-32546213

RESUMEN

BACKGROUND: Four new variants of Chlamydia trachomatis (nvCTs), detected in several countries, cause false-negative or equivocal results using the Aptima Combo 2 assay (AC2; Hologic). We evaluated the clinical sensitivity and specificity, as well as the analytical inclusivity and exclusivity of the updated AC2 for the detection of CT and Neisseria gonorrhoeae (NG) on the automated Panther system (Hologic). METHODS: We examined 1004 clinical AC2 samples and 225 analytical samples spiked with phenotypically and/or genetically diverse NG and CT strains, and other potentially cross-reacting microbial species. The clinical AC2 samples included CT wild type (WT)-positive (n = 488), all four described AC2 diagnostic-escape nvCTs (n = 170), NG-positive (n = 214), and CT/NG-negative (n = 202) specimens. RESULTS: All nvCT-positive samples (100%) and 486 (99.6%) of the CT WT-positive samples were positive in the updated AC2. All NG-positive, CT/NG-negative, Trichomonas vaginalis (TV)-positive, bacterial vaginosis-positive, and Candida-positive AC2 specimens gave correct results. The clinical sensitivity and specificity of the updated AC2 for CT detection was 99.7 and 100%, respectively, and for NG detection was 100% for both. Examining spiked samples, the analytical inclusivity and exclusivity were 100%, i.e., in clinically relevant concentrations of spiked microbe. CONCLUSIONS: The updated AC2, including two CT targets and one NG target, showed a high sensitivity, specificity, inclusivity and exclusivity for the detection of CT WT, nvCTs, and NG. The updated AC2 on the fully automated Panther system offers a simple, rapid, high-throughput, sensitive, and specific diagnosis of CT and NG, which can easily be combined with detection of Mycoplasma genitalium and TV.


Asunto(s)
Infecciones por Chlamydia/diagnóstico , Chlamydia trachomatis/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Análisis de Secuencia de ARN/métodos , Candida/genética , Candidiasis/diagnóstico , Candidiasis/microbiología , Infecciones por Chlamydia/microbiología , Reacciones Cruzadas , Femenino , Gonorrea/diagnóstico , Gonorrea/microbiología , Humanos , Neisseria gonorrhoeae/genética , ARN Bacteriano/genética , ARN Ribosómico 23S/genética , Sensibilidad y Especificidad , Tricomoniasis/diagnóstico , Tricomoniasis/parasitología , Trichomonas vaginalis/genética
10.
BMC Infect Dis ; 20(1): 375, 2020 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-32460721

RESUMEN

BACKGROUND: Sexually transmitted infections (STIs) cause a major public health problem that affect both men and women in developing and developed countries. The aim of the study was to estimate the prevalence of 11 STIs among women who voluntarily participated in the study, while seeking gynecological checkup. The existence of an association between the presence of pathogens and symptoms and various sociodemographic risk factors was assessed. METHODS: A total of 505 vaginal and cervical specimens were collected from women above 18 years of age, with or without symptoms related to gynecological infections. Nucleic acid was extracted and samples were tested by real-time PCR for the following pathogens: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Urealplasma parvum, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma girerdii, Gardnerella vaginalis, Candida albicans and Human Papillomavirus (HPV). Positive HPV samples underwent genotyping using a microarray system. RESULTS: Of the 505 samples, 312 (62%) were screened positive for at least one pathogen. Of these, 36% were positive for Gardnerella vaginalis, 35% for Ureaplasma parvum, 8% for Candida albicans, 6.7% for HPV, 4.6% for Ureaplasma urealyticum, 3.6% for Mycoplasma hominis, 2% for Trichomonas vaginalis, 0.8% for Chlamydia trachomatis, 0.4% for Mycoplasma girerdii, 0.2% for Mycoplasma genitalium and 0.2% for Neisseria gonorrhoeae. Lack of symptoms was reported in 187 women (37%), among whom 61% were infected. Thirty-four samples were HPV positive, with 17 high risk HPV genotypes (HR-HPV); the highest rates being recorded for types 16 (38%), 18 (21%) and 51 (18%). Out of the 34 HPV positives, 29 participants had HR-HPV. Association with various risk factors were reported. CONCLUSIONS: This is the first study that presents data about the presence of STIs among women in Lebanon and the MENA region by simultaneous detection of 11 pathogens. In the absence of systematic STI surveillance in Lebanon, concurrent screening for HPV and PAP smear is warranted.


Asunto(s)
Enfermedades de Transmisión Sexual/epidemiología , Adulto , Cuello del Útero/microbiología , Cuello del Útero/parasitología , Cuello del Útero/virología , Chlamydia trachomatis/genética , Chlamydia trachomatis/aislamiento & purificación , Estudios Transversales , Femenino , Gardnerella vaginalis/genética , Gardnerella vaginalis/aislamiento & purificación , Humanos , Líbano/epidemiología , Masculino , Epidemiología Molecular , Infecciones por Mycoplasma/epidemiología , Mycoplasma genitalium/genética , Mycoplasma genitalium/aislamiento & purificación , Mycoplasma hominis/genética , Mycoplasma hominis/aislamiento & purificación , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/aislamiento & purificación , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Factores de Riesgo , Enfermedades de Transmisión Sexual/microbiología , Enfermedades de Transmisión Sexual/parasitología , Enfermedades de Transmisión Sexual/virología , Trichomonas vaginalis/genética , Trichomonas vaginalis/aislamiento & purificación , Ureaplasma/genética , Ureaplasma/aislamiento & purificación , Vagina/microbiología , Vagina/parasitología , Vagina/virología , Frotis Vaginal , Adulto Joven
11.
Sex Transm Infect ; 96(6): 402-407, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32447324

RESUMEN

OBJECTIVES: Test of cure (TOC) for Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) infection is an important tool in the public health management of STIs. However, there are limited data about the optimal time to perform TOC using nucleic acid amplification tests (NAATs) for NG and CT infections. A study was performed to assess the feasibility of a larger study to determine the optimal time to TOC using NAATS. METHODS: The Sexually Transmitted Bacteria Reference Unit at Public Health England undertook testing of gonococcal and chlamydial nucleic acids within neat urine stored in different conditions over 25 days to provide evidence of the stability of the nucleic acid prior to recruitment. Individuals diagnosed with uncomplicated NG or CT infection were recruited from three sexual health clinics. Individuals were asked to return nine self-taken samples from the site of infection over a course of 35 days. Survival analyses of time to first negative NAAT result for NG and CT infection and univariate regression analysis of factors that affect time to clearance were undertaken. RESULTS: At room temperature, chlamydial DNA in urine is stable for up to 3 weeks and gonococcal DNA for up to 11 days. We analysed data for 147 infections (81 NG and 66 CT). The median time to clearance of infection was 4 days (IQR 2-10 days) for NG infection and 10 days (IQR 7-14 days) for CT infection. Vaginal CT infections took longer to clear (p=0.031). NG infection in men who have sex with men took longer to clear (p=0.052). CONCLUSION: Chlamydial and gonococcal nucleic acids are stable in urine before addition of preservatives, longer than recommended by the manufacturer. The TOC results suggest that it may be possible to undertake TOC for NG and CT infections earlier than current guidelines suggest and that anatomical site of infection may affect time to clearance of infection.


Asunto(s)
Antibacterianos/uso terapéutico , Infecciones por Chlamydia/diagnóstico , Infecciones por Chlamydia/tratamiento farmacológico , Gonorrea/diagnóstico , Gonorrea/tratamiento farmacológico , Adulto , Anciano , Azitromicina/uso terapéutico , Ceftriaxona/uso terapéutico , Chlamydia trachomatis/genética , Doxiciclina/uso terapéutico , Estudios de Factibilidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neisseria gonorrhoeae/genética , Técnicas de Amplificación de Ácido Nucleico , Faringitis/diagnóstico , Faringitis/tratamiento farmacológico , Proctitis/diagnóstico , Proctitis/tratamiento farmacológico , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Tiempo , Resultado del Tratamiento , Uretritis/diagnóstico , Uretritis/tratamiento farmacológico , Vulvovaginitis/diagnóstico , Vulvovaginitis/tratamiento farmacológico , Adulto Joven
12.
BMC Infect Dis ; 20(1): 314, 2020 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-32345231

RESUMEN

BACKGROUND: Mycoplasma genitalium is an emerging sexually transmitted infection, with increasing rates of resistance to fluroquinolones and macrolides, the recommended treatments. Despite this, M. genitalium is not part of routine screening for Sexually Transmitted Infections (STIs) in many countries and the prevalence of infection and patterns of disease remain to be determined in many populations. Such data is of particular importance in light of the reported rise in antibiotic resistance in M. genitalium isolates. METHODS: Urine and urethral swab samples were collected from the primary public sexual health clinic in Singapore and tested for C. trachomatis (CT) or N. gonorrhoeae (NG) infection and for the presence of M. genitalium. Antibiotic resistance in M. genitalium strains detected was determined by screening for genomic mutations associated with macrolide and fluroquinolone resistance. RESULTS: We report the results of a study into M. genitalium prevalence at the national sexual health clinic in Singapore. M. genitalium was heavily associated with CT infection (8.1% of cases), but present in only of 2.4% in CT negative cases and not independently linked to NG infection. Furthermore, we found high rates of resistance mutations to both macrolides (25%) and fluoroquinolones (37.5%) with a majority of resistant strains being dual-resistant. Resistance mutations were only found in strains from patients with CT co-infection. CONCLUSIONS: Our results support targeted screening of CT positive patients for M. genitalium as a cost-effective strategy to reduce the incidence of M. genitalium in the absence of comprehensive routine screening. The high rate of dual resistance also highlights the need to ensure the availability of alternative antibiotics for the treatment of multi-drug resistant M. genitalium isolates.


Asunto(s)
Antibacterianos/farmacología , Infecciones por Chlamydia/diagnóstico , Chlamydia trachomatis/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple , Infecciones por Mycoplasma/diagnóstico , Mycoplasma genitalium/efectos de los fármacos , Instituciones de Atención Ambulatoria , Antibacterianos/uso terapéutico , Infecciones por Chlamydia/complicaciones , Infecciones por Chlamydia/tratamiento farmacológico , Chlamydia trachomatis/genética , Chlamydia trachomatis/aislamiento & purificación , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Fluoroquinolonas/farmacología , Fluoroquinolonas/uso terapéutico , Humanos , Macrólidos/farmacología , Macrólidos/uso terapéutico , Infecciones por Mycoplasma/complicaciones , Infecciones por Mycoplasma/tratamiento farmacológico , Infecciones por Mycoplasma/epidemiología , Mycoplasma genitalium/genética , Mycoplasma genitalium/aislamiento & purificación , Prevalencia , ARN Ribosómico 23S/química , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/metabolismo , Análisis de Secuencia de ADN , Singapur/epidemiología , Uretra/microbiología
13.
PLoS Negl Trop Dis ; 14(3): e0008120, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32130213

RESUMEN

BACKGROUND: Trachoma elimination efforts are hampered by limited understanding of Chlamydia trachomatis (Ct) transmission routes. Here we aimed to detect Ct DNA at non-ocular sites and on eye-seeking flies. METHODS: A population-based household survey was conducted in Oromia Region, Ethiopia. Ocular and non-ocular (faces, hands, clothing, water containers and sleeping surfaces) swabs were collected from all individuals. Flies were caught from faces of children. Flies, ocular swabs and non-ocular swabs were tested for Ct by quantitative PCR. RESULTS: In total, 1220 individuals in 247 households were assessed. Active trachoma (trachomatous inflammation-follicular) and ocular Ct were detected in 10% and 2% of all-ages, and 21% and 3% of 1-9-year-olds, respectively. Ct was detected in 12% (95% CI:8-15%) of tested non-ocular swabs from ocular-positive households, but in none of the non-ocular swabs from ocular-negative households. Ct was detected on 24% (95% CI:18-32%) of flies from ocular-positive households and 3% (95% CI:1-6%) of flies from ocular-negative households. CONCLUSION: Ct DNA was detected on hands, faces and clothing of individuals living in ocular-positive households suggesting that this might be a route of transmission within Ct infected households. In addition, we detected Ct on flies from ocular-positive households and occasionally in ocular-negative households suggesting that flies might be a vector for transmission within and between Ct infected and uninfected households. These potential transmission routes may need to be simultaneously addressed to suppress transmission.


Asunto(s)
Infecciones por Chlamydia/diagnóstico , Infecciones por Chlamydia/transmisión , Chlamydia trachomatis/aislamiento & purificación , Infecciones Comunitarias Adquiridas/diagnóstico , Infecciones Comunitarias Adquiridas/transmisión , Transmisión de Enfermedad Infecciosa , Adolescente , Adulto , Animales , Niño , Preescolar , Chlamydia trachomatis/genética , Vestuario , Estudios Transversales , Dípteros/microbiología , Etiopía , Heces/microbiología , Femenino , Fómites/microbiología , Mano/microbiología , Humanos , Lactante , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto Joven
14.
APMIS ; 128(6): 440-444, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32202687

RESUMEN

Chlamydia trachomatis infection is the most common bacterial sexually transmitted infection globally, and nucleic acid amplification tests (NAATs) are recommended for highly sensitive and specific diagnosis. In early 2019, the Finnish new variant of Chlamydia trachomatis (FI-nvCT) was identified. The FI-nvCT has a C1515T mutation in the 23S rRNA gene, making it escaping detection in the Aptima Combo 2 (AC2; Hologic) NAAT, and the FI-nvCT has been subsequently reported in Sweden and Norway. In the present study, we investigated the presence of the FI-nvCT and other AC2 diagnostic-escape CT mutants in July-September 2019 in Denmark. The FI-nvCT was present but rare in Denmark. However, another AC2 diagnostic-escape CT mutant (with a 23S rRNA G1523A mutation) was found to be widespread across Denmark, accounting for 95% (76/80) of AC2 diagnostic-escape nvCT samples from five Danish CT-diagnostic laboratories. This nvCT-G1523A has previously only been detected in one single sample in the United Kingdom and Norway, respectively. It is vital to monitor the continued stability of the NAAT targets in local, national and international settings and monitor as well as appropriately analyse incidence, unexplained shifts in diagnostics rates and/or annual collections of samples diagnosed as negative/equivocal using NAATs with different target(s). Furthermore, diagnostic CT NAATs with dual target sequences are crucial, and fortunately, an updated Hologic AC2 assay including one additional target sequence is in advanced development.


Asunto(s)
Infecciones por Chlamydia/diagnóstico , Chlamydia trachomatis/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Ribosómico 23S/genética , Análisis de Secuencia de ARN/métodos , Infecciones por Chlamydia/microbiología , Dinamarca/epidemiología , Femenino , Gonorrea/diagnóstico , Gonorrea/microbiología , Humanos , Masculino , Neisseria gonorrhoeae/genética , ARN Bacteriano/genética , Sensibilidad y Especificidad
15.
Infect Immun ; 88(5)2020 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-32152196

RESUMEN

The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia's ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.


Asunto(s)
Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/genética , Chlamydia trachomatis/patogenicidad , Mutagénesis/genética , Actinas/genética , Alelos , Animales , Proteínas Bacterianas/genética , Línea Celular Tumoral , Fluorescencia , Eliminación de Gen , Células HeLa , Humanos , Ratones , Ratones Endogámicos C3H , Fosfoproteínas/genética , Virulencia/genética
16.
Int J Infect Dis ; 96: 121-127, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32173573

RESUMEN

OBJECTIVE: The aim of this study was to investigate the relationships between treatment outcomes of patients with urogenital Chlamydia trachomatis infections and minimum inhibitory concentrations (MICs) and drug resistance genes. METHODS: The clinical data of 92 patients diagnosed with Chlamydia trachomatis (C. trachomatis) infections were collected. Of these patients, 28 received regular treatment with azithromycin and 64 received minocycline. All patients underwent three monthly follow-ups after the completion of treatment. The microdilution method was used for the in vitro susceptibility tests. The acquisition of 23S rRNA mutations and presence of the tet(M) gene were detected by gene amplification and sequencing. RESULTS: The MICs of azithromycin, clarithromycin, erythromycin, tetracycline, doxycycline, and minocycline were comparable for isolates from the treatment failure and treatment success groups. Higher detection rates of 23S rRNA gene mutations and tet(M) were found in the treatment failure group (57.14% and 71.43%, respectively) than in the treatment success group (14.29% and 30.23%, respectively) (p < 0.05). The A2057G, C2452A, and T2611C gene mutations of 23S rRNA were detected in eight clinical isolates from the azithromycin treatment failure group, while the T2611C gene mutation was detected in one clinical strain from the treatment success group. CONCLUSIONS: The detection of resistance genes could better explain the high treatment failure rate than the MIC results in patients with urogenital C. trachomatis infections, highlighting the need for genetic antimicrobial resistance testing in infected patients.


Asunto(s)
Infecciones por Chlamydia/tratamiento farmacológico , Chlamydia trachomatis/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Adulto , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Azitromicina/farmacología , Azitromicina/uso terapéutico , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/genética , Chlamydia trachomatis/aislamiento & purificación , Femenino , Enfermedades de los Genitales Femeninos/tratamiento farmacológico , Enfermedades de los Genitales Femeninos/microbiología , Enfermedades de los Genitales Masculinos/tratamiento farmacológico , Enfermedades de los Genitales Masculinos/microbiología , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Minociclina/farmacología , Minociclina/uso terapéutico , ARN Ribosómico 23S/genética , Insuficiencia del Tratamiento , Infecciones Urinarias/tratamiento farmacológico , Infecciones Urinarias/microbiología , Adulto Joven
17.
Infect Immun ; 88(5)2020 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-32094256

RESUMEN

Peptidoglycan, the sugar-amino acid polymer that composes the bacterial cell wall, requires a significant expenditure of energy to synthesize and is highly immunogenic. To minimize the loss of an energetically expensive metabolite and avoid host detection, bacteria often recycle their peptidoglycan, transporting its components back into the cytoplasm, where they can be used for subsequent rounds of new synthesis. The peptidoglycan-recycling substrate binding protein (SBP) MppA, which is responsible for recycling peptidoglycan fragments in Escherichia coli, has not been annotated for most intracellular pathogens. One such pathogen, Chlamydia trachomatis, has a limited capacity to synthesize amino acids de novo and therefore must obtain oligopeptides from its host cell for growth. Bioinformatics analysis suggests that the putative C. trachomatis oligopeptide transporter OppABCDF (OppABCDF Ct ) encodes multiple SBPs (OppA1 Ct , OppA2 Ct , and OppA3 Ct ). Intracellular pathogens often encode multiple SBPs, while only one, OppA, is encoded in the E. coli opp operon. We hypothesized that the putative OppABCDF transporter of C. trachomatis functions in both oligopeptide transport and peptidoglycan recycling. We coexpressed the putative SBP genes (oppA1Ct , oppA2Ct , oppA3Ct ) along with oppBCDFCt in an E. coli mutant lacking the Opp transporter and determined that all three chlamydial OppA subunits supported oligopeptide transport. We also demonstrated the in vivo functionality of the chlamydial Opp transporter in C. trachomatis Importantly, we found that one chlamydial SBP, OppA3 Ct , possessed dual substrate recognition properties and is capable of transporting peptidoglycan fragments (tri-diaminopimelic acid) in E. coli and in C. trachomatis These findings suggest that Chlamydia evolved an oligopeptide transporter to facilitate the acquisition of oligopeptides for growth while simultaneously reducing the accumulation of immunostimulatory peptidoglycan fragments in the host cell cytosol. The latter property reflects bacterial pathoadaptation that dampens the host innate immune response to Chlamydia infection.


Asunto(s)
Chlamydia trachomatis/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Oligopéptidos/metabolismo , Peptidoglicano/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico/genética , Transporte Biológico/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Pared Celular/genética , Pared Celular/metabolismo , Infecciones por Chlamydia/metabolismo , Chlamydia trachomatis/genética , Citosol/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos/genética , Células HeLa , Humanos , Inmunidad Innata/genética , Proteínas de Transporte de Membrana/genética , Oligopéptidos/genética , Operón/genética , Peptidoglicano/genética
18.
Mikrobiyol Bul ; 54(1): 135-143, 2020 Jan.
Artículo en Turco | MEDLINE | ID: mdl-32050884

RESUMEN

Sexually transmitted infections (STIs) are important as a public health problem all over the world. There are some difficulties in prevention and control programs of STIs due to clinical and laboratory diagnostic problems.The most common STIs are Chlamydia trachomatis infections, trichomoniasis and gonorrhea. The study aimed to investigate the direct microscopic examination, culture and polymerase chain reaction (PCR) tests in the diagnosis of Trichomonas vaginalis infection; to determine other microbiological agents that may cause vaginal discharge and to evaluate the various social variables in women with vaginal discharge admitted to the outpatient clinic of Obstetrics and Gynecology in Akdeniz University Hospital. Two hundred and fifteen patients were enrolled in the study. The socio-demographic features of the patients were recorded. Vaginal/endocervical swab specimens taken from patients were evaluated by microscopic examination. Swab specimens were inoculated into blood agar, MacConkey agar and chocolate agar for bacterial culture. Modified Trichosel broth with 5% horse blood (Becton Dickinson, USA) was used for Trichomonas spp. culture. The presence of C.trachomatis, Neisseria gonorrhoeae, and T.vaginalis in swab samples were investigated by multiplex PCR assay (BD Max CT/GC/TV, Becton Dickinson, USA). At least one pathogen was detected among 65 (30.3%) samples. T.vaginalis was detected by microscopic examination and PCR in four of 215 (1.9%) patients. Existence of yeast morphology was observed in 21 (9.8%) specimens by microscopic examination. Twenty four (11.2%) patients were diagnosed as bacterial vaginosis microscopically according to Nugent score system. Candida species grew in 32 (14.9%) and Streptococcus agalactiae grew in 2 (0.9%) of the specimens. C.trachomatis was detected in 2 (0.9%) samples and N.gonorrhoeae in 1 (0.5%) sample by PCR. In this study, 95.3% of the patients were married and 96.7% had only one sexual partner in the mean time. The rate of detection of pathogens were statistically higher in women who have had two or more pregnancies (p<0.05). In our study, T.vaginalis together with N.gonorrhoeae and C.trachomatis were investigated by PCR method in women with vaginal discharge. The use of multiplex PCR test allowed simultaneous investigation of multiple pathogens in the patient samples.


Asunto(s)
Infecciones por Chlamydia , Técnicas y Procedimientos Diagnósticos , Gonorrea , Tricomoniasis , Vaginitis por Trichomonas , Técnicas de Cultivo de Célula , Infecciones por Chlamydia/diagnóstico , Chlamydia trachomatis/genética , Técnicas y Procedimientos Diagnósticos/normas , Femenino , Gonorrea/diagnóstico , Humanos , Microscopía/normas , Reacción en Cadena de la Polimerasa Multiplex , Neisseria gonorrhoeae/genética , Embarazo , Tricomoniasis/diagnóstico , Tricomoniasis/parasitología , Vaginitis por Trichomonas/diagnóstico , Trichomonas vaginalis/genética
19.
Euro Surveill ; 25(5)2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32046818

RESUMEN

The Finnish new variant of Chlamydia trachomatis (FI-nvCT) is escaping diagnostics in Finland, Norway and Sweden. We have developed and validated an Aptima-format nucleic acid amplification test (NAAT) designed specifically to detect the FI-nvCT. This NAAT has high sensitivity (100%) and specificity (100%) for the FI-nvCT strain, enabling further investigation of the geographic distribution, prevalence and transmission of this diagnostic-escape mutant in screening populations in Europe.


Asunto(s)
Infecciones por Chlamydia/diagnóstico , Chlamydia trachomatis/genética , Variación Genética/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Análisis de Secuencia de ARN/métodos , Infecciones por Chlamydia/epidemiología , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/aislamiento & purificación , Finlandia/epidemiología , Humanos , Reacción en Cadena de la Polimerasa , ARN Bacteriano/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
20.
Gastroenterology ; 158(6): 1546-1547, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32017908

Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Coinfección/diagnóstico , Granuloma/diagnóstico , Proctitis/diagnóstico , Enfermedades de Transmisión Sexual/diagnóstico , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida/inmunología , Antibacterianos/administración & dosificación , Antivirales/administración & dosificación , Ceftriaxona/administración & dosificación , Chlamydia trachomatis/genética , Chlamydia trachomatis/aislamiento & purificación , Coinfección/tratamiento farmacológico , Coinfección/inmunología , Coinfección/microbiología , Colonoscopía , Citomegalovirus/aislamiento & purificación , ADN Bacteriano/aislamiento & purificación , Doxiciclina/administración & dosificación , Quimioterapia Combinada/métodos , Granuloma/tratamiento farmacológico , Granuloma/inmunología , Granuloma/microbiología , Humanos , Mucosa Intestinal/diagnóstico por imagen , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/aislamiento & purificación , Proctitis/tratamiento farmacológico , Proctitis/inmunología , Proctitis/microbiología , Recto/diagnóstico por imagen , Recto/microbiología , Recto/patología , Enfermedades de Transmisión Sexual/tratamiento farmacológico , Enfermedades de Transmisión Sexual/inmunología , Enfermedades de Transmisión Sexual/microbiología , Resultado del Tratamiento , Valganciclovir/administración & dosificación
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