PCR múltiplex para el diagnóstico de microorganismos atípicos transmitidos sexualmente / Multiplex PCR testing for the atypical microorganisms diagnosis sexually transmitted

Resumen Antecedentes: Las infecciones de transmisión sexual son un problema de salud pública mundial. El análisis rutinario incluye solo pruebas microbiológicas y serológicas para el diagnóstico de patógenos. Los microorganismos atípicos como Chlamydia trachomatis y micoplasmas no son identificados debido a los requerimientos. Además, no es incluida Gardnerella vaginalis, aunque se asocia a la vaginosis bacteriana. Objetivo: Desarrollar una PCR múltiplex para el diagnóstico de C. trachomatis, micoplasmas y G. vaginalis. Método: Se estandarizó la PCR múltiplex utilizando oligonucleótidos para C. trachomatis (gen ompA, orf6 plasmídico), Mycoplasma/Ureaplasma y G. vaginalis (genes rRNA16s). Resultados: Se estandarizaron pruebas de PCR múltiplex para los microorganismos estudiados, optimizándose las concentraciones y condiciones de las reacciones múltiplex. Se obtuvieron PCR dúplex para C. trachomatis (ompA, orf6), Chlamydia/Gardnerella y Chlamydia/micoplasmas y tríplex para Chlamydia/Mycoplasma/Ureaplasma. También un cuádruplex para Chlamydia/Mycoplasma/Ureaplasma/Gardnerella. Los resultados fueron verificados por PCR e hibridación automática (HybriSpot 12) y análisis in silico. Conclusión: Se desarrollaron pruebas de PCR múltiplex con una alta sensibilidad y especificidad para la identificación de C. trachomatis, micoplasmas y G. vaginalis.

Abstract Background: Sexually transmitted infections are a global public health problem. Routine analysis includes microbiological and serological tests for the diagnosis of pathogens. Atypical microorganisms such as Chlamydia trachomatis and mycoplasmas are not determined due to the requirements for their identification. Furthermore, Gardnerella vaginalis is not included despite being associated with bacterial vaginosis. Objective: To develop a multiplex PCR to diagnose Chlamydia, mycoplasmas, and Gardnerella. Method: Standardization of multiplex PCR tests was carried out using oligonucleotides for the identification of Chlamydia (ompA gene, plasmid orf6), Mycoplasma/Ureaplasma and Gardnerella (rRNA16s genes). Results: Multiplex PCR tests were standardized for the microorganisms studied, optimizing the concentrations and conditions of the multiplex reactions. Duplex PCR was obtained for Chlamydia (ompA, orf6), Chlamydia/Gardnerella, and Chlamydia/mycoplasmas, and triplex PCR for Chlamydia/mycoplasmas. Also, a quadruplex for Chlamydia, Mycoplasma/Ureaplasma and Gardnerella. PCR and automatic hybridization verified the results obtained (HybriSpot 12) and in silico analysis. Conclusion: Multiplex PCR tests with high sensitivity and specificity were developed to identify C. trachomatis, mycoplasmas, and G. vaginalis.

Incidence and Prevalence of Vaginal Infections in Women of Reproductive Age in North Macedonia.

In the available literature on this subject there are many studies which describe the effects of sexually transmitted infections on pregnancy and fertility of women. Because of the frequency of the infections with the atypical bacteria of the Ureaplasma Spp., Mycoplasma Spp., Chlamydia Trachomatis, as well as HPV infections in women of reproductive age, it is easy to underestimate their importance when establishing the basis of the genital health of women of reproductive age. In this prospective analysis, conducted from 2014 to 2018 in the laboratory for HPV and Molecular diagnostics at the University Clinic of Gynaecology and Obstetrics in Skopje, North Macedonia, we analysed the results of 10,387 patients of all ages, of which 973 patients were of reproductive age. A Panel analysis was also conducted (including the above-mentioned pathogens). An HPV analysis was also conducted on 643 patients in this group. Within the group of 643 patients, there was a positive result for HPV in 26.7% of them, while in 40.9% there was a positive result for one or more pathogens on the Panel analysis of bacterial pathogens. The statistical analysis of the results showed that the most frequent of all bacterial pathogens within the Macedonian population of women of reproductive age is Ureaplasma Spp, with an incidence of 33%, followed by Mycoplasma Spp., with 7.8%, while Chlamydia Trachomatis was present in 6.4% of the cases. We should highlight that a co-infection with HPV was present in 18.5% of all the patients where there was analysis of both diagnostic procedures. The analysis of the results in the patients co-infected with HPV and at least one bacterial pathogen on the Panel Analysis, showed a very high statistical correlation (p<001).

Detection of Putative Virulence Genes alr, goiB, and goiC in Mycoplasma hominis Isolates from Austrian Patients.

In Mycoplasma hominis, two genes (alr and goiB) have been found to be associated with the invasion of the amniotic cavity, and a single gene (goiC) to be associated with intra-amniotic infections and a high risk of preterm birth. The syntopic presence of Ureaplasma spp. in the same patient has been shown to correlate with the absence of goiC in M. hominis. The aim of our study was to investigate the presence of alr, goiB, and goiC genes in two groups of M. hominis isolates collected from symptomatic and asymptomatic male and non-pregnant female patients attending an Outpatients Centre. Group A consisted of 26 isolates from patients with only M. hominis confirmed; group B consisted of 24 isolates from patients with Ureaplasma spp. as the only co-infection. We extracted DNA from all M. hominis isolates and analysed the samples for the presence of alr, goiB, and goiC in a qPCR assay. Additionally, we determined their cytotoxicity against HeLa cells. We confirmed the presence of the alr gene in 85% of group A isolates and in 100% of group B isolates; goiB was detected in 46% of the samples in both groups, whereas goiC was found in 73% of group A and 79% of group B isolates, respectively. It was shown that co-colonisation with Ureaplasma spp. in the same patient had no effect on the presence of goiC in the respective M. hominis isolate. We did not observe any cytotoxic effect of the investigated isolates on human cells, regardless of the presence or absence of the investigated genes.

Retrospective Analysis of the Ureaplasma spp. Prevalence with Reference to Other Genital Tract Infections in Women of Reproductive Age.

Ureaplasma spp. are frequently isolated from the genital tract of women of reproductive age. To date, it remains unclear whether they are commensal or pathogenic. In our study, we assessed the prevalence of Ureaplasma spp. in a group of 1,155 women of childbearing age. In addition, we assessed how often women with positive Ureaplasma spp. develop genital tract co-infections and how the vaginal pH changes. This study showed a relationship between colonization by Ureaplasma spp. and presenting symptoms. In fact, we showed that colonization of the genital tract by Ureaplasma spp. can affect the occurrence of co-infections such as Gardnerella vaginalis. We also observed a relationship between increased pH values and the presence of Ureaplasma spp.

Prevalence and antibiotics resistance of Ureaplasma species and Mycoplasma hominis in Hangzhou, China, from 2013 to 2019.

Ureaplasma spp. and Mycoplasma hominis, frequent colonizers in the lower urogenital tract, have been implicated in various infections, with antibiotic resistance growing and varying regionally. This study aims to investigate the prevalence and antibiotic resistance profiles of Ureaplasma spp. and M. hominis in outpatients in Hangzhou, China, from 2013 to 2019. A total of 135,263 outpatients were examined to determine the prevalence of Ureaplasma spp. and M. hominis, including 48,638 males and 86,625 females. Furthermore, trends in antibiotic susceptibility of Ureaplasma spp. and M. hominis during 1999-2019 were analyzed. The cultivation, identification, and antibiotic susceptibility of the bacteria (ofloxacin, ciprofloxacin, erythromycin, clarithromycin, azithromycin, josamycin, tetracycline, doxycycline, and pristinamycin) were determined using the Mycoplasma IST2 kit. Our study indicated that the overall prevalence of total Ureaplasma spp./M. hominis was 38.1% from 2013 to 2019. Ureaplasma spp. were the most frequently isolated species (overall prevalence, 31.3%), followed by Ureaplasma spp./M. hominis coinfection (6.0%) and single M. hominis infection (0.8%). The prevalence of Ureaplasma spp. and M. hominis was significantly higher in females than in males, and the highest positive rates of total Ureaplasma spp./M. hominis were observed in both female and male outpatients aged 14-20 years. During 2013-2019, josamycin, tetracycline, doxycycline, and pristinamycin maintained exceptionally high activity (overall resistance rates, <5%) against both Ureaplasma spp. and M. hominis, but ofloxacin and ciprofloxacin showed limited activity (overall resistance rates, >70%). During 1999-2019, the rates of resistance to ofloxacin and ciprofloxacin increased against both Ureaplasma spp. and M. hominis but decreased to erythromycin, clarithromycin, azithromycin, tetracycline, and doxycycline against Ureaplasma spp. In conclusion, our study demonstrates a high prevalence of Ureaplasma spp. compared to M. hominis and Ureaplasma spp./M. hominis, and their distribution was associated with sex and age. Josamycin, doxycycline, and tetracycline are promising antibiotics that have remarkable activity against Ureaplasma species and M. hominis.

Case Report: Hyperammonemic Encephalopathy Linked to Ureaplasma spp. and/or Mycoplasma hominis Systemic Infection in Patients Treated for Leukemia, an Emergency Not to Be Missed.

Background: Hyperammonemic encephalopathy caused by Ureaplasma spp. and Mycoplasma hominis infection has been reported in immunocompromised patients undergoing lung transplant, but data are scarce in patients with hematological malignancies. Case Presentation: We describe the cases of 3 female patients aged 11-16 years old, developing initially mild neurologic symptoms, rapidly evolving to coma and associated with very high ammonia levels, while undergoing intensive treatment for acute leukemia (chemotherapy: 2 and hematopoietic stem cell transplant: 1). Brain imaging displayed cerebral edema and/or microbleeding. Electroencephalograms showed diffuse slowing patterns. One patient had moderate renal failure. Extensive liver and metabolic functions were all normal. Ureaplasma spp. and M. hominis were detected by PCR and specific culture in two patients, resulting in prompt initiation of combined antibiotics therapy by fluoroquinolones and macrolides. For these 2 patients, the improvement of the neurological status and ammonia levels were observed within 96 h, without any long-term sequelae. M. hominis was detected post-mortem in vagina, using 16S rRNA PCR for the third patient who died of cerebral edema. Conclusion: Hyperammonemic encephalopathy linked to Ureaplasma spp. and M. hominis is a rare complication encountered in immunocompromised patients treated for acute leukemia, which can lead to death if unrecognized. Combining our experience with the few published cases (n=4), we observed a strong trend among female patients and very high levels of ammonia, consistently uncontrolled by classical measures (ammonia-scavenging agents and/or continuous kidney replacement therapy). The reversibility of the encephalopathy without sequelae is possible with prompt diagnosis and adequate combined specific antibiotherapy. Any neurological symptoms in an immunocompromised host should lead to the measurement of ammonia levels. If increased, and in the absence of an obvious cause, it should prompt to perform a search for Ureaplasma spp. and M. hominis by PCR as well as an immediate empirical initiation of combined specific antibiotherapy.

In Vitro Activity of Delafloxacin and Finafloxacin against Mycoplasma hominis and Ureaplasma Species.

The in vitro activity of two new fluoroquinolones, delafloxacin and finafloxacin, were evaluated against M. hominis and Ureaplasma spp. The MICs of delafloxacin, finafloxacin, and two classical fluoroquinolones (moxifloxacin and levofloxacin) were tested against 29 M. hominis and 67 Ureaplasma spp. isolates using the broth microdilution method. The molecular mechanisms underlying fluoroquinolone resistance were also investigated. Delafloxacin exhibited low MICs against M. hominis and Ureaplasma spp., including the levofloxacin-resistant isolates. For M. hominis, delafloxacin showed low MIC90 value of 1 µg/mL (MIC range, <0.031 -1 µg/mL) compared to 8 µg/mL for finafloxacin, 16 µg/mL for moxifloxacin, and 32 µg/mL for levofloxacin. For U. parvum and U. urealyticum, delafloxacin had low MIC90 values (U. parvum, 2 µg/mL; U. urealyticum, 4 µg/mL) compared to 16 -32 µg/mL for finafloxacin, 16 µg/mL for moxifloxacin, and 32 - >32 µg/mL for levofloxacin. The two mutations GyrA S153L and ParC S91I were commonly identified in fluoroquinolone-resistant M. hominis, and ParC S83L was the most frequent mutation identified in fluoroquinolone-resistant Ureaplasma spp. Delafloxacin displayed lower MICs against fluoroquinolone-resistant isolates of both M. hominis and Ureaplasma spp. that have mutations in the quinolone resistance determining regions (QRDRs) than the two classical fluoroquinolones. Delafloxacin is a promising fluoroquinolone with low MICs against fluoroquinolone-resistant M. hominis and Ureaplasma spp. Our study confirms the potential clinical use of delafloxacin in treating antimicrobial-resistant M. hominis and Ureaplasma spp. infections. IMPORTANCE Fluoroquinolone resistance in Mycoplasma hominis and Ureaplasma spp. is on the rise globally, which has compromised the efficacy of the currently available antimicrobial agents. This study evaluated the antimicrobial activity of two new fluoroquinolones, delafloxacin and finafloxacin, for the first time, against M. hominis and Ureaplasma spp. clinical isolates. Delafloxacin and finafloxacin displayed different antimicrobial susceptibility profiles against M. hominis and Ureaplasma spp. in vitro. Delafloxacin was found to be more effective against M. hominis and Ureaplasma spp. than three classical fluoroquinolones (finafloxacin, moxifloxacin, and levofloxacin). Finafloxacin displayed activity similar to moxifloxacin but superior to levofloxacin against M. hominis and Ureaplasma spp. Our findings demonstrate that delafloxacin is a promising fluoroquinolone with outstanding activity against fluoroquinolone-resistant M. hominis and Ureaplasma spp.

Determination of the prevalence of Mycoplasma hominis and Ureaplasma species in Bacterial vaginosis patients in association with antibiotic resistance profile in Franceville, Gabon.

BACKGROUND: Genital mycoplasma are only considered pathogenic at a certain level and are often associated with other pathological situations such as bacterial vaginosis (BV). They may lead to infertility as well as other gynaeco-obstetrical and neonatal problems. Despite numerous reported resistances, macrolides are required to treat pregnant women while non-pregnant women are managed with tetracyclines and fluoroquinolones. This study aimed to establish the prevalence and resistance rates of Mycoplasma hominis (Mh) and Ureaplasma spp. (Uu) in BV positive (BV+) women. MATERIAL AND METHODS: Vaginal secretions were collected from women aged 14-56 years consulting for a cytobacteriological examination of the vaginal swab associated with a simultaneous search for genital mycoplasma in the medical analysis laboratory of the Research and Medical Analysis Unit (URAM) of CIRMF in Franceville, Gabon. BV was diagnosed using the Nugent score while genital mycoplasma identification and antibiotic susceptibility testing were performed using the Mycoplasma IST 2 kit. RESULTS: Of the 462 women included in this study, 60.18% (278/462, p = 0.00002) were both BV+ and genital mycoplasma carriers, including 5.19% (24/462) pregnant women. Overall mycoplasma carriage was 33.12% (153/462) for Uu, 1.95% for Mh and 25.11% (116/462) for mixed infections (Uu + Mh). The BV + patients most affected by mycoplasma were those whose age varied from 25 to 35 years with 27.49% (127/462, p = 0.980), those not using condoms with 39.40% (182/462, p = 0.014, OR = 2.35), those non-pregnant but capable of bearing children with 53.90% (249/462, p = 0.967, OR = 1.02). In the overall population, 83.66% and 51.63% of Uu strains were highly resistant to Ciprofloxacin and Azithromycin respectively; 100% and 55.56% of Mh strains were resistant to Azithromycin and Tetracycline respectively; while strong resistance has been observed in mixed infections to Ciprofloxacin (97.41%), Azithromycin (81.90%), Ofloxacin (69.83%) and Tetracycline (68.97%). CONCLUSION: The prevalence of genital mycoplasma infections is very high in women with bacterial vaginosis. Given the numerous emerging resistance rates to most classes of antibiotics available for the treatment of genital mycoplasma infections in our study, it would be advisable for therapeutic prescriptions to be made based on laboratory results.

Determination of In Vitro Antimicrobial Susceptibility for Lefamulin (Pleuromutilin) for Ureaplasma Spp. and Mycoplasma hominis.

Lefamulin is the first of the pleuromutilin class of antimicrobials to be available for therapeutic use in humans. Minimum inhibitory concentrations of lefamulin were determined by microbroth dilution for 90 characterised clinical isolates (25 Ureaplasma parvum, 25 Ureaplasma urealyticum, and 40 Mycoplasma hominis). All Mycoplasma hominis isolates possessed lefamulin MICs of ≤0.25 mg/L after 48 h (MIC50/90 of 0.06/0.12 mg/L), despite an inherent resistance to macrolides; while Ureaplasma isolates had MICs of ≤2 mg/L after 24 h (MIC50/90 of 0.25/1 mg/L), despite inherent resistance to clindamycin. Two U. urealyticum isolates with additional A2058G mutations of 23S rRNA, and one U. parvum isolate with a R66Q67 deletion (all of which had a combined resistance to macrolides and clindamycin) only showed a 2-fold increase in lefamulin MIC (1-2 mg/L) relative to macrolide-susceptible strains. Lefamulin could be an effective alternative antimicrobial for treating Ureaplasma spp. and Mycoplasma hominis infections irrespective of intrinsic or acquired resistance to macrolides, lincosamides, and ketolides. Based on this potent in vitro activity and the known good, rapid, and homogenous tissue penetration of female and male urogenital tissues and glands, further exploration of clinical efficacy of lefamulin for the treatment of Mycoplasma and Ureaplasma urogenital infections is warranted.

Phenotypic Antimicrobial Susceptibility and Genotypic Characterization of Clinical Ureaplasma Isolates Circulating in Shanghai, China.

There is a growing global concern regarding the rise of antimicrobial resistance among Ureaplasma spp. isolates. However, studies on the antimicrobial susceptibility profiles, resistance mechanisms, and clonality of Ureaplasma spp. clinical isolates are still limited and cover only some geographic regions. Firstly, Ureaplasma species from the urogenital tracts of patients in Shanghai, China, were isolated by using the culture medium (A8 and 10B broth), and identified the genotype by polymerase chain reaction (PCR). Secondly, the antimicrobial susceptibility tests were determined by using broth microdilution assay. Then, the resistance genetic determinants to fluoroquinolones (FQs), macrolides, and tetracyclines were investigated through PCR/DNA sequencing. Finally, the molecular epidemiology of Ureaplasma species was studied by multilocus sequence typing (MLST). Among 258 isolates, Ureaplasma parvum (UPA) and Ureaplasma urealyticum (UUR) were found in 226 (87.60%) and 32 (12.40%) isolates, respectively. The minimum inhibitory concentrations (MICs) of 258 Ureaplasma spp. strains ranged from 0.015 to 64µg/ml for all 11 kinds of antimicrobials. Regardless of species, the isolates were most sensitive to AZI (1.94%), JOS (3.49%), and CLA (4.23%). Among them, there were 39 (15.12%) multidrug-resistant (MDR) strains, including 32 UPA isolates. The resistance rates of UPA to CIP (91.59%), and ROX (36.28%) were significantly higher than those of UUR. Twenty six FQ-resistant isolates had amino acid substitutions in gyrA and in parC (Ser83Leu). Mutations were detected in genes encoding ribosomal proteins L4 (Thr84Ile) and L22 (Ser81Pro) in macrolide-resistant isolates. Tet(M) was found in four UPA isolates. These mutations were mainly found in UPA isolates. Sequence type 1 (ST1) was the predominant ST, which contained 18 isolates. In conclusion, this study showed a higher resistance rate (especially to ROX and CIP), higher substitution rate, and higher MDR rate among UPA strains. The most active antimicrobial agents were AZI, JOS, and CLA. Identifying UPA or UUR in clinical isolates could help clinicians to choose appropriate drugs for treatment. The main resistance mechanisms may involve gene substitution of Ser83Leu in parC and Ser81Pro in L22. ST1 was the predominant ST of Ureaplasma isolates with MDR to FQs and macrolides in Shanghai, China.

Maternal Vaginal Ureaplasma spp. Colonization in Early Pregnancy Is Associated with Adverse Short- and Long-Term Outcome of Very Preterm Infants.

Vaginal colonization with Ureaplasma (U.) spp. has been shown to be associated with adverse pregnancy outcome; however, data on neonatal outcome are scarce. The aim of the study was to investigate whether maternal vaginal colonization with U. spp. in early pregnancy represents a risk factor for adverse short- or long-term outcome of preterm infants. Previously, 4330 pregnant women were enrolled in an observational multicenter study, analyzing the association between vaginal U. spp. colonization and spontaneous preterm birth. U. spp. colonization was diagnosed via PCR analysis from vaginal swabs. For this study, data on short-term outcome were collected from medical records and long-term outcome was examined via Bayley Scales of Infant Development at 24 months adjusted age. Two-hundred-and-thirty-eight children were born <33 weeks gestational age. After exclusion due to asphyxia, malformations, and lost-to-follow-up, data on short-term and long-term outcome were available from 222 and 92 infants, respectively. Results show a significant association between vaginal U. spp. colonization and severe intraventricular hemorrhage (10.4% vs. 2.6%, p = 0.03), retinopathy of prematurity (21.7% vs. 10.3%, p = 0.03), and adverse psychomotor outcome (24.3% vs. 1.8%, OR 13.154, 95%CI 1.6,110.2, p = 0.005). The data suggest an association between vaginal U. spp. colonization in early pregnancy and adverse short- and long-term outcome of very preterm infants.

[Clinical implications of the genus Mycoplasma]. / Implicaciones clínicas de las especies del género Mycoplasma.

Within Mycoplasma genus, M. pneumoniae, M. genitalium, M. hominis or U. urealyticum are the main species that have been traditionally linked to infectious processes. However, there are many other species involved in these conditions and that are, frequently, unfamiliar to healthcare professionals. The aim of this review is to identify all Mycoplasma genus species that have been isolated in human beings and to determine their involvement in infectious pathology.

Impact of Screening and Treatment of Ureaplasma species on Hyperammonemia Syndrome in Lung Transplant Recipients: A Single Center Experience.

BACKGROUND: Infection with Ureaplasma species (spp) has been linked to fatal hyperammonemia syndrome (HS) in lung transplant recipients. We sought to characterize the epidemiology of Ureaplasma spp in candidates and donors and describe outcomes of antimicrobial therapy in preventing and treating HS. METHODS: Candidate testing for Ureaplasma spp was performed with urine culture and polymerase chain reaction (PCR) pretransplant. Positive candidates were treated with levofloxacin. Donor testing was performed with bronchoalveolar lavage (BAL) culture and PCR intraoperatively. From 7/2014 to 2/2017 patients were treated according to results; from 2/2017 to 10/2018 recipients received empiric levofloxacin and azithromycin at transplant until testing returned negative. HS was defined as new onset altered mental status after transplant with ammonia > 200 µmol/L. RESULTS: In total, 60 patients who underwent lung transplant were included. And 80% (n = 48) of patients had negative screening tests in donor and candidate pre-lung transplant, 8.3% (n = 5) of recipients had positive Ureaplasma spp testing in urine pre-transplant, and 13.3% (n = 8) had positive donor BAL testing at the time of lung transplant. Three patients developed HS a median of 7 days posttransplant; 2 died of HS. Recipients of organs with Ureaplasma spp who received empiric therapy did not develop HS. Donors with Ureaplasma spp were younger and more sexually active. CONCLUSIONS: Donor-derived Ureaplasma spp in lung transplant was associated with HS. Screening lung donors for Ureaplasma spp might allow for targeted therapy to reduce risk for development of HS, but future confirmatory studies are needed.

A specific bacterial DNA signature in the vagina of Australian women in midpregnancy predicts high risk of spontaneous preterm birth (the Predict1000 study).

BACKGROUND: Intrauterine infection accounts for a quarter of the cases of spontaneous preterm birth; however, at present, it is not possible to efficiently identify pregnant women at risk to deliver preventative treatments. OBJECTIVE: This study aimed to establish a vaginal microbial DNA test for Australian women in midpregnancy that will identify those at increased risk of spontaneous preterm birth. STUDY DESIGN: A total of 1000 women with singleton pregnancies were recruited in Perth, Australia. Midvaginal swabs were collected between 12 and 23 weeks' gestation. DNA was extracted for the detection of 23 risk-related microbial DNA targets by quantitative polymerase chain reaction. Obstetrical history, pregnancy outcome, and demographics were recorded. RESULTS: After excluding 64 women owing to losses to follow-up and insufficient sample for microbial analyses, the final cohort consisted of 936 women of predominantly white race (74.3%). The overall preterm birth rate was 12.6% (118 births); the spontaneous preterm birth rate at <37 weeks' gestation was 6.2% (2.9% at ≤34 weeks' gestation), whereas the preterm premature rupture of the membranes rate was 4.2%. No single individual microbial target predicted increased spontaneous preterm birth risk. Conversely, women who subsequently delivered at term had higher amounts of Lactobacillus crispatus, Lactobacillus gasseri, or Lactobacillus jensenii DNA in their vaginal swabs (13.8% spontaneous preterm birth vs 31.2% term; P=.005). In the remaining women, a specific microbial DNA signature was identified that was strongly predictive of spontaneous preterm birth risk, consisting of DNA from Gardnerella vaginalis (clade 4), Lactobacillus iners, and Ureaplasma parvum (serovars 3 and 6). Risk prediction was improved if Fusobacterium nucleatum detection was included in the test algorithm. The final algorithm, which we called the Gardnerella Lactobacillus Ureaplasma (GLU) test, was able to detect women at risk of spontaneous preterm birth at <37 and ≤34 weeks' gestation, with sensitivities of 37.9% and 44.4%, respectively, and likelihood ratios (plus or minus) of 2.22 per 0.75 and 2.52 per 0.67, respectively. Preterm premature rupture of the membranes was more than twice as common in GLU-positive women. Adjusting for maternal demographics, ethnicity, and clinical history did not improve prediction. Only a history of spontaneous preterm birth was more effective at predicting spontaneous preterm birth than a GLU-positive result (odds ratio, 3.6). CONCLUSION: We have identified a vaginal bacterial DNA signature that identifies women with a singleton pregnancy who are at increased risk of spontaneous preterm birth and may benefit from targeted antimicrobial therapy.

The Role of HPV and Non-HPV Sexually Transmitted Infections in Patients with Oropharyngeal Carcinoma: A Case Control Study.

Background: Certain high-risk (hr) types of human papillomavirus (HPV) can cause cervical cancer in women and penile cancer in men. Hr-HPV can also cause cancers of the oropharynx and anus in both sexes. In the anal and cervical region, a contribution of co-infections with Ureaplasma spp. on the persistence of the hr-HPV infection by a profound inflammatory state is suggested. Here, we investigated if non-HPV sexually transmitted infections are associated with oropharyngeal carcinoma (OPC). Materials and Methods: In this case-control study, a brush test directly from the tumor surface of OPC patients (study group) and from the oropharynx of healthy volunteers (control group), both groups matching in age and sex, was performed. HPV subtypes were detected using a commercially available test kit. For non-HPV sexually transmitted infections (Ureaplasma spp., Chlamydia trachomatis, Mycoplasma hominis, and Mycoplasma genitalium), a multiplex nucleic acid amplification approach was performed. Results: In the study group, 96 patients (23 female/73 male), with histologically confirmed OPC and in the control group 112 patients (19 female/93 male), were included. Oropharyngeal hr-HPV-positivity was detected in 68% (65/96 patients) of the study group and 1.8% (2/112 patients) of the control group (p < 0.001). In three patients in the study group, Ureaplasma spp. was detected, whereas no patient was Ureaplasma spp. positive in the control group (p = 0.097). Chlamydia trachomatis, Mycoplasma hominis, and Mycoplasma genitalium were negative in both groups. Conclusion: Based on the current study, the prevalence of oropharyngeal Ureaplasma spp. among patients with OPC is low and does not support a role in oropharyngeal cancer. However, the detection of the pathogen only among OPC patients but not in the healthy individuals might indicate a potential role and needs further elucidation.

Antimicrobial Resistance in Clinical Ureaplasma spp. and Mycoplasma hominis and Structural Mechanisms Underlying Quinolone Resistance.

Antibiotic resistance is a global concern; however, data on antibiotic-resistant Ureaplasma spp. and Mycoplasma hominis are limited in comparison to similar data on other microbes. A total of 492 Ureaplasma spp. and 13 M. hominis strains obtained in Hangzhou, China, in 2018 were subjected to antimicrobial susceptibility testing for levofloxacin, moxifloxacin, erythromycin, clindamycin, and doxycycline using the broth microdilution method. The mechanisms underlying quinolone and macrolide resistance were determined. Meanwhile, a model of the topoisomerase IV complex bound to levofloxacin in wild-type Ureaplasma spp. was built to study the quinolone resistance mutations. For Ureaplasma spp., the levofloxacin, moxifloxacin, and erythromycin resistance rates were 84.69%, 51.44%, and 3.59% in U. parvum and 82.43%, 62.16%, and 5.40% in U. urealyticum, respectively. Of the 13 M. hominis strains, 11 were resistant to both levofloxacin and moxifloxacin, and five strains showed clindamycin resistance. ParC S83L was the most prevalent mutation in levofloxacin-resistant Ureaplasma strains, followed by ParE R448K. The two mutations GyrA S153L and ParC S91I were commonly identified in quinolone-resistant M. hominis A molecular dynamics-refined structure revealed that quinolone resistance-associated mutations inhibited the interaction and reduced affinity with gyrase or topoisomerase IV and quinolones. The novel mutations S21A in the L4 protein and G2654T and T2245C in 23S rRNA and the ermB gene were identified in erythromycin-resistant Ureaplasma spp. As fluoroquinolone resistance in Ureaplasma spp. and Mycoplasma hominis remains high in China, the rational use of antibiotics needs to be further enhanced.

Prevalence of Cervico-vaginal High-risk HPV Types and Other Sexually Transmitted Pathogens in Anogenital Warts Patients.

AIM: To investigate the prevalence of cervico-vaginal co-infection with high-risk (HR) HPV types and other sexually transmitted pathogens (STPs) in women with anogenital warts (AGWs). PATIENTS AND METHODS: In this cross-sectional study, cervico-vaginal smears of women with AGWs were examined with real-time polymerase chain reaction for the presence of HR-HPV types and common STPs. Women with recent cervical HPV infection and general population were used for comparisons. RESULTS: A total of 689 women participated in the study. Among the examined groups, higher rates of cervico-vaginal co-infection with HR-HPV types and other STPs collectively were recorded in women with AGWs (p=0.0049 and p<0.004, respectively). Within the AGWs group, cervical co-infection with HR-HPV types was detected more often in women with recurrent disease (p<0.001). CONCLUSION: The higher rates of cervico-vaginal co-infection with HR-HPV types and common STPs in women with AGWs may affect their risk for cervical carcinogenesis and the natural course of their disease.

Ureaplasma species and preterm birth: current perspectives.

Preterm birth is the leading cause of neonatal morbidity and mortality worldwide, and the human Ureaplasma species are most frequently isolated from the amniotic fluid and placenta in these cases. Ureaplasma colonisation is associated with infertility, stillbirth, histologic chorioamnionitis, and neonatal morbidities, including congenital pneumonia, bronchopulmonary dysplasia, meningitis and perinatal death. The human Ureaplasma spp. are separated into Ureaplasma urealyticum and Ureaplasma parvum with 14 known serotypes. The small genome has several genes, which code for surface proteins; most significantly the Multiple Banded Antigen (MBA) where an antigenic C-terminal domain elicits a host antibody response. Other genes code for various virulence factors such as IgA protease and urease. Ureaplasma spp. infection is diagnosed by culture and polymerase chain reaction (PCR) and commercial assays are available to improve turnaround time. Microbroth dilution assays are routinely used to test antimicrobial susceptibility of clinical Ureaplasma spp. especially against doxycycline, azithromycin, ofloxacin and josamycin. Resistance to macrolides, fluoroquinolones and tetracyclines has been reported. A concise review of Ureaplasma spp. and their role in pregnancy outcomes, especially preterm birth, offers insight into the early diagnosis and appropriate antibiotic therapy to prevent long-term complications of Ureaplasma spp. infections.

Ureaplasma parvum causes hyperammonemia presenting as refractory status epilepticus after kidney transplant.

PURPOSE: Alert intensivists about the diagnostic pitfalls arising from hyperammonemia due to Ureaplasma infections in post-transplant patients. MATERIALS AND METHODS: Clinical observation of one patient. CASE REPORT: A 65-year-old female with a medical history of semi-recent kidney transplant was admitted to the Intensive Care Unit for refractory status epilepticus. There were no lesions on brain imaging. Bacterial cultures and viral PCR of cerebrospinal fluid were negative. The first blood ammonia level measured on day 2 was 13 times the normal level, but biological liver tests were normal. The persistence of elevated ammonia levels led to the initiation of symptomatic ammonia lowering-treatments and continuous renal replacement therapy, which led to its decrease without normalization. An Ureaplasma spp infection was then diagnosed. Levofloxacin and doxycyline were administered resulting in normalization of ammonia levels within 48 h. However repeat MRI showed diffuse cortical cytotoxic edema and the patient remained in a minimally conscious state. She eventually died 4 months later from a recurrent infection. CONCLUSION: Ureaplasma infection must be suspected in cases of neurological symptoms associated with hyperammonemia without liver failure, following an organ transplant. Only urgent treatment could improve the prognosis and prevent severe neurological damage or death.

Spread of multidrug resistance among Ureaplasma serovars, Tunisia.

Background: Ureaplasma spp. have been implicated in a variety of clinical conditions and certain serovars are likely to be disease-associated. Hence, the ascending trend of Ureaplasma spp. resistance to antimicrobials should deserve more attention. Here we assessed the extent of antimicrobial resistance of Ureaplasma serovars in Tunisia, and investigated the underlying molecular basis. Methods: This study included 101 molecularly typed Ureaplasma spp. clinical strains isolated over a 12-year time period (2005-2017). The antimicrobial susceptibility was tested against nine antibacterial agents using the broth microdilution method. Neighbor-joining tree was constructed to establish the phylogenetic relationships among isolates. Results: We found that all ureaplasma isolates were resistant to ciprofloxacin and erythromycin, intermediately resistant to azithromycin, and susceptible to doxycycline, moxifloxacin and josamycin. Ofloxacin and levofloxacin resistance was found in 73.27 and 17.82%, respectively, while 37.62% of isolates proved resistant to tetracycline. Consequently, we detected an elevated multidrug resistance rate among ureaplasma isolates (37.62%), particularly among serovars 2, 5, 8, and 9 (77.77% overall), as well as serovars 4, 10, 12, and 13 (52.63% overall). In most cases, drug resistance was found to be associated with known molecular mechanisms, yet we have identified two novel mutations in the L22 protein, which might be associated with macrolide-resistance. Conclusion: To our knowledge, this is the first study that reports the widespread expansion of multidrug resistance among Ureaplasma serovars, a finding of importance in terms of both surveillance and antimicrobial usage.