Bacteraemia associated with multiple septic localizations caused by Klebsiella pneumoniae sequence type ST660.

We report a case of Klebsiella pneumoniae bacteraemia in an 80-year-old man in France with no history of travel to Asia, complicated by endogenous endophthalmitis, multiple cerebral microbleeds and hepatic microabscesses, associated with a Bentall endocarditis. Hypervirulence pathotype was suggested based on clinical picture, bacterial isolate genomic sequence and hypermucoidy. Interestingly, the isolate had the non-K1/K2-capsular serotype locus KL113-like, carried a KpVP-1-like virulence plasmid, and belonged to the emerging sublineage SL660 (comprising the sequence type ST660).

Evaluation of commercial, customized microdilution plates for Ureaplasma parvum, Ureaplasma urealyticum, and Mycoplasma hominis antimicrobial susceptibility testing and determination of antimicrobial resistance prevalence in France.

Antimicrobial susceptibility testing (AST) of human mycoplasmas using microdilution is time-consuming. In this study, we compared the performance of MICRONAUT-S plates (Biocentric-Bruker) designed for AST of Ureaplasma parvum, Ureaplasma urealyticum, and Mycoplasma hominis with the results using the Clinical & Laboratory Standards Institute (CLSI) reference method. Then, we investigated the prevalence and mechanisms of resistance to tetracyclines, fluoroquinolones, and macrolides in France in 2020 and 2021. The two methods were compared using 60 strains. For the resistance prevalence study, U. parvum-, U. urealyticum-, and M. hominis-positive clinical specimens were collected for 1 month each year in 22 French diagnostic laboratories. MICs were determined using the MICRONAUT-S plates. The tet(M) gene was screened using PCR, and fluoroquinolone resistance-associated mutations were screened using PCR and Sanger sequencing. Comparing the methods, 99.5% (679/680) MICs obtained using the MICRONAUT-S plates concurred with those obtained using the CLSI reference method. For 90 M. hominis isolates, the tetracycline, levofloxacin, and moxifloxacin resistance rates were 11.1%, 2.2%, and 2.2%, respectively, with no clindamycin resistance. For 248 U. parvum isolates, the levofloxacin and moxifloxacin resistance rates were 5.2% and 0.8%, respectively; they were 2.9% and 1.5% in 68 U. urealyticum isolates. Tetracycline resistance in U. urealyticum (11.8%) was significantly (P < 0.001) higher than in U. parvum (1.2%). No macrolide resistance was observed. Overall, the customized MICRONAUT-S plates are a reliable, convenient tool for AST of human mycoplasmas. Tetracycline and fluoroquinolone resistance remain limited in France. However, the prevalence of levofloxacin and moxifloxacin resistance has increased significantly in Ureaplasma spp. from 2010 to 2015 and requires monitoring. IMPORTANCE: Antimicrobial susceptibility testing of human urogenital mycoplasmas using the CLSI reference broth microdilution method is time-consuming and requires the laborious preparation of antimicrobial stock solutions. Here, we validated the use of reliable, convenient plates designed for antimicrobial susceptibility testing that allows the simultaneous determination of the MICs of eight antibiotics of interest. We then investigated the prevalence and mechanisms of resistance of each of these bacteria to tetracyclines, fluoroquinolones, and macrolides in France in 2020 and 2021. We showed that the prevalence of levofloxacin and moxifloxacin resistance has increased significantly in Ureaplasma spp. from 2010 to 2015 and requires ongoing monitoring.

Improved transformation efficiency in Mycoplasma hominis enables disruption of the MIB-MIP system targeting human immunoglobulins.

The pathogenicity of Mycoplasma hominis is poorly understood, mainly due to the absence of efficient genetic tools. A polyethylene glycol-mediated transformation protocol was recently developed for the M. hominis reference strain M132 using the pMT85-Tet plasmid. The transformation efficiency remained low, hampering generation of a large mutant library. In this study, we improved transformation efficiency by designing M. hominis-specific pMT85 derivatives. Using the Gibson Assembly, the Enterococcus-derived tet(M) gene of the pMT85-Tet plasmid was replaced by that of a M. hominis clinical isolate. Next, the Spiroplasma-derived spiralin gene promoter driving tet(M) expression was substituted by one of three putative regulatory regions (RRs): the M. hominis arginine deiminase RR, the M. hominis elongation factor Tu RR, or the 68 bp SynMyco synthetic RR. SynMyco-based construction led to a 100-fold increase in transformation efficiency in M. hominis M132. This construct was also transformed into the M. hominis PG21 reference strain and three other clinical isolates. The transposon insertion locus was determined for 128 M132-transformants. The majority of the impacted coding sequences encoded lipoproteins and proteins involved in DNA repair or in gene transfer. One transposon integration site was in the mycoplasma immunoglobulin protease gene. Phenotypic characterization of the mutant showed complete disruption of the human antibody cleavage ability of the transformant. These results demonstrate that our M. hominis-optimized plasmid can be used to generate large random transposon insertion libraries, enabling future studies of the pathogenicity of M. hominis. IMPORTANCE Mycoplasma hominis is an opportunistic human pathogen, whose physiopathology is poorly understood and for which genetic tools for transposition mutagenesis have been unavailable for years. A PEG-mediated transformation protocol was developed using the pMT85-Tet plasmid, but the transformation efficiency remained low. We designed a modified pMT85-Tet plasmid suitable for M. hominis. The use of a synthetic regulatory region upstream of the antibiotic resistance marker led to a 100-fold increase in the transformation efficiency. The generation and characterization of large transposon mutagenesis mutant libraries will provide insight into M. hominis pathogenesis. We selected a transformant in which the transposon was integrated in the locus encoding the immunoglobulin cleavage system MIB-MIP. Phenotypic characterization showed that the wild-type strain has a functional MIB-MIP system, whereas the mutant strain had lost the ability to cleave human immunoglobulins.

Staphylococcus aureus Adhesion and Biofilm Formation on Vascular Polyester Grafts are Inhibited In Vitro by Triclosan.

OBJECTIVE: This study evaluated Staphylococcus aureus adhesion and biofilm formation on vascular grafts, which has seldom been investigated. METHODS: Adhesion and biofilm formation capabilities of three methicillin susceptible S. aureus strains (one biofilm forming reference strain and two clinical isolates) on five different vascular biomaterials were evaluated in vitro, including polyester (P), P + gelatin (PG), P + collagen (PC), PC + silver (PCS), and PCS + triclosan (PCST). Staphylococcus aureus adhesion on grafts was evaluated after one hour of culture and biofilm formation after 24 hours of culture by four different methods: spectrophotometry after crystal violet staining; sonicate fluid culture; metabolic assay; and scanning electron microscopy (SEM). Optical density was compared using Mann-Whitney pairwise test, and bacterial counts using Wilcoxon pairwise test. RESULTS: PCST grafts were most efficient in preventing S. aureus adhesion and biofilm formation, regardless of the method used. Bacterial counts and metabolic activity were significantly lower on PCST grafts after 24 hours (5.65 vs. 9.24 [PCS], 8.99 [PC], 8.82 [PG], and 10.44 log10 CFU/mL [P]; p < .015), and only PCST grafts were bactericidal. Biofilm formation was significantly diminished on PCST grafts compared with all other grafts (p < .001). Bacterial viability and metabolic activity after 24 hours were more impaired on PG compared with PC graft, and were surprisingly higher on PCS compared with PC grafts. Biofilm biomass formed after exposure to P, PG, PC, and PCS grafts was also reduced after 24 hours of incubation with PCST grafts (p < .001). After 24 hours, few bacteria were visible by SEM on PCST grafts, whereas bacterial biofilm colonies were clearly identified on other graft surfaces. CONCLUSION: Triclosan impregnated PCST grafts appeared to interfere with S. aureus adhesion from early stages of biofilm formation in vitro. Silver impregnation was not efficient in preventing biofilm formation, and collagen coating promoted S. aureus biofilm formation more than gelatin coating.

A Narrative Review of Experimental Assessment to Study Vascular Biomaterials Infections and Infectability.

Objective: Many experimental studies have been conducted to evaluate vascular and endovascular graft infections (VGEIs) and infectability in order to elaborate strategies to prevent or to treat their occurrence. A systematic literature search was conducted to collect and summarise key features of infection and infectability assessment techniques in VGEI experimental models. Methods: The literature search was conducted using the Medline and Cochrane databases, with no limit on the date of publication, until 10 August 2021. Ex vivo, in vitro, and in vivo animal studies on VGEIs, published in English or French, were selected. Cross references retrieved from selected articles on PubMed database were also included in the search. Data were collected on the techniques and the protocols performed for vascular graft infection and infectability assessment. Results: A total of 243 studies were included in the review: 55 in vitro studies, 169 animal studies, 17 combining the two models, and two ex vivo studies. Many experimental techniques were performed, with a lot of protocol discrepancies. The main experiments conducted were bacterial culture, with (n = 82 studies) or without sonication (n = 120), histopathology (n = 69), scanning electron microscopy (n = 36), and graft diffusion tests (n = 28). These techniques were used to answer different research questions corresponding to different graft infection steps, such as microbial adhesion and/or viability, biofilm biomass or organisation, human cell reaction, or antimicrobial activity. Conclusion: Many experimental tools are available to study VGEIs, but to improve their reproducibility and scientific reliability research protocols must be standardised and include sonication of grafts before microbiological culture. Moreover, the key role of the biofilm in VGEI physiopathology must be taken into account in future studies.

Prevalence of Mycoplasma penetrans in Urogenital Samples From Men Screened for Bacterial Sexually Transmitted Infections.

Mycoplasma penetrans prevalence was assessed in urogenital samples from men screened for Chlamydia trachomatis and Neisseria gonorrhoeae. Prevalence was 3.5% among men who have sex with men and 5.3% among human immunodeficiency virus (HIV)-positive patients, significantly higher than in HIV-negative individuals (0.4%, P = .0016). No association was found between M. penetrans and urogenital symptoms.

Severe Bacterial Non-AIDS Infections in Persons With Human Immunodeficiency Virus: The Epidemiology and Evolution of Antibiotic Resistance Over an 18-Year Period (2000-2017) in the ANRS CO3 AquiVih-Nouvelle-Aquitaine Cohort.

BACKGROUND: Severe non-AIDS bacterial infections (SBIs) are among the leading causes of hospital admissions among persons with human immunodeficiency virus (PWH) in regions with high antiretroviral therapy coverage. METHODS: This large prospective cohort study of PWH examined the types of infections, bacterial documentation, and evolution of antibiotic resistance among PWH hospitalized with SBIs over an 18-year period. RESULTS: Between 2000 and 2017, 459 PWH had at least 1 SBI with bacterial documentation. Among the 847 SBIs, there were 280 cases of bacteremia, 269 cases of pneumonia, and 240 urinary tract infections. The 1025 isolated bacteria included Enterobacteriaceae (n = 394; mainly Escherichia coli), Staphylococcus aureus (n = 153), and Streptococcus pneumoniae (n = 82). The proportion of S. pneumoniae as the causative agent in pneumonia and bacteremia decreased sharply over time, from 34% to 8% and from 21% to 3%, respectively. The overall antibiotic resistance of S. aureus and S. pneumoniae decreased progressively but it increased for Enterobacteriaceae (from 24% to 48% for amoxicillin-clavulanate, from 4% to 18% for cefotaxime, and from 5% to 27% for ciprofloxacin). Cotrimoxazole prophylaxis was associated with higher nonsusceptibility of S. pneumoniae to amoxicillin and erythromycin, higher nonsusceptibility of Enterobacteriaceae to ß-lactams and fluoroquinolones, and a higher risk of extended-spectrum ß-lactamase-producing Enterobacteriaceae. CONCLUSIONS: The bacterial resistance pattern among PWH between 2014 and 2017 was broadly similar to that in the general population, with the exception of a higher resistance profile of Enterobacteriaceae to fluoroquinolones. The use of cotrimoxazole as prophylaxis was associated with an increased risk of antibiotic resistance.

Prevalence of macrolide and fluoroquinolone resistance-associated mutations in Mycoplasma genitalium in metropolitan and overseas France.

OBJECTIVE: Limited macrolide and fluoroquinolone resistance data are available in France for Mycoplasma genitalium. We performed a multicentre cross-sectional study to investigate the prevalence of macrolide and fluoroquinolone resistance-associated mutations in M. genitalium-positive patients in metropolitan France between 2018 and 2020 and in overseas France in 2018 and 2019. METHODS: Each year, a 1-month prospective collection of M. genitalium-positive specimens was proposed to metropolitan French microbiology diagnostic laboratories, and a similar 3-month collection was proposed to overseas French laboratories. Resistance-associated mutations were detected using commercial kits and sequencing. RESULTS: A total of 1630 M. genitalium-positive specimens were analysed. In metropolitan France, the prevalence of macrolide resistance-associated mutations ranged between 34.7% (95% CI 29.4% to 40.4%) and 42.9% (95% CI 37.1% to 49.0%) between 2018 and 2020 and was significantly higher in men (95% CI 52.4% to 60.2%) than in women (95% CI 15.9% to 22.2%) (p<0.001). These prevalences were significantly higher than those of 6.1% (95% CI 3.7% to 10.3%) and 14.7% (95% CI 10.9% to 19.6%) observed in overseas France in 2018 and 2019 (p<0.001), where no difference between genders was noted. The prevalence of fluoroquinolone resistance-associated mutations was also significantly higher in metropolitan France (14.9% (95% CI 11.2% to 19.5%) to 16.1% (95% CI 12.1% to 21.2%)) than in overseas France (1.3% (95% CI 0.4% to 3.7%) and 2.6% (95% CI 1.3% to 5.3%) in 2018 and 2019, respectively) (p<0.001), with no difference between men and women regardless of the location. CONCLUSION: This study reports the high prevalence of macrolide and fluoroquinolone resistance-associated mutations in M. genitalium in metropolitan France and highlights the contrast with low prevalence in overseas France. In metropolitan France, macrolide resistance-associated mutation prevalence was three times higher in men than in women, which was likely to be driven by the proportion of men who have sex with men. This suggests that gender and sexual practice should also be taken into account for the management of M. genitalium infections.

Clinical Performance of Three Commercial Molecular Diagnostic Assays for the Detection of Fluoroquinolone Resistance-Associated Mutations in Mycoplasma genitalium.

The high prevalence of macrolide resistance in Mycoplasma genitalium results in an increased reliance on moxifloxacin, the second-line treatment; however, moxifloxacin resistance has also emerged. Because assays that can detect fluoroquinolone resistance-associated mutations will be useful for the management of macrolide-resistant M. genitalium infections, we evaluated the performance of three commercial assays (the Allplex MG & MoxiR Assay [Seegene], LightMix Modular parC kit [TIBMOLBIOL], and MGMO qPCR [NYtor) in comparison with parC gene Sanger sequencing used as the reference. Between January 2018 and December 2020, remnants of M. genitalium-positive clinical specimens received at the French National Reference Center for Bacterial Sexually Transmitted Infections were collected if a Sanger sequencing result was obtained for the parC gene. Overall, 368 M. genitalium-positive specimens were assessed. The clinical sensitivities for the detection of the ParC mutations that are likely of clinical significance were 91.8% (95% CI = 83.2 to 96.2), 98.6% (95% CI = 92.4 to 99.8), and 94.4% (95% CI = 86.6 to 97.8) for the Allplex MG & MoxiR, LightMix Modular parC, and MGMO qPCR kits, respectively, with no significant difference between the three kits. The clinical specificity of the Allplex MG & MoxiR and MGMO qPCR kits was 100% (95% CI = 97.7 to 100 and 98.7 to 100, respectively), which was significantly higher than the specificity of the LightMix Modular parC kit of 95.4% (95%CI = 92.3 to 97.3), for which the interpretation of melting curves may be misleading. These kits should be useful for the selection of antimicrobials in macrolide-resistant M. genitalium infections, although further developments may be necessary because parC mutations involved in fluoroquinolone resistance have not been precisely determined.

Molecular Typing Reveals Distinct Mycoplasma genitalium Transmission Networks among a Cohort of Men Who Have Sex with Men and a Cohort of Women in France.

Mycoplasma genitalium causes sexually transmitted infecti.ons in men and women. Treatment failures to macrolides and fluoroquinolones have been reported worldwide. Although the mgpB typing method has often been used in M. genitalium-infected men who have sex with men (MSM), limited typing data are available for M. genitalium-infected women. In this study, we aimed to investigate the genetic relationship between M. genitalium strains and their antibiotic resistance profile in a cohort of MSM (86.2% on HIV preexposure prophylaxis [PrEP], 13.8% HIV positive) and a large cohort of women using mgpB/MG309 typing. The mgpB types were determined in 374 samples from 305 women and 65 MSM. Three MSM and one woman had two concurrent or subsequent samples. Macrolide and fluoroquinolone resistance-associated mutations were searched in the 23S rRNA as well as parC and gyrA genes. The mgpB phylogenetic construction revealed three large clusters that differed according to sexual practices and geographical origin of patients. The prevalence of macrolide and fluoroquinolone resistance was significantly higher in MSM compared with women (95.4% vs. 14.1% and 30.6% vs. 7.2%, p < 0.001, respectively). The macrolide resistance spread was polyclonal in both populations, but clonal diffusion of two dual-resistant types was observed in PrEP users in association with high antibiotic pressure and dense connectivity in this population.

Metagenomic next-generation sequencing restores the diagnosis of a rare infectious complication of B cell depletion.

A 45-year-old female patient receiving rituximab for B cell non-Hodgkin follicular lymphoma presented unexplained recurrent fever, abdominal discomfort, and pollakiuria. We performed shotgun metagenomic sequencing from peri-kidney collection that identified a co-infection with Mycoplasma hominis and Ureaplasma urealyticum. The patient recovered with sequelae after appropriate antibiotic treatment was given.

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.

Prevalence of Mycoplasma genitalium and other sexually transmitted pathogens in male urethritis in a sexual health centre in New Caledonia.

BACKGROUND: The prevalence of sexually transmitted infections (STIs) is high in New Caledonia (NC), but there are no data on Mycoplasma genitalium (MG). However, the syndromic treatment of urethritis used in the territory includes a single dose of azithromycin, which could generate resistance in MG. METHODS: We recruited 217 men referred to the Noumea public medical centre (CMP) with signs of urethritis and meeting the inclusion criteria from May 2016 to March 2018. Each was tested for Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Trichomonas vaginalis (TV) and for the first time in NC for MG by polymerase chain reaction (PCR). RESULTS: The prevalence of MG was 10.1% (22/217). Azithromycin resistance of MG (mutation in the 23S rRNA gene) could only be assessed for 10 of the 22 strains. Only one (1/10; 10%) was resistant. The prevalence of other STIs tested was high, as CT, NG and/or TV were associated in 77.3% (17/22) of MG-positive cases. CONCLUSIONS: Although co-infections further justify syndromic management, the presence of MG in NC urethritis cases could call treatment guidelines into question.

Mycoplasma pneumoniae detections before and during the COVID-19 pandemic: results of a global survey, 2017 to 2021.

BackgroundMycoplasma pneumoniae respiratory infections are transmitted by aerosol and droplets in close contact.AimWe investigated global M. pneumoniae incidence after implementation of non-pharmaceutical interventions (NPIs) against COVID-19 in March 2020.MethodsWe surveyed M. pneumoniae detections from laboratories and surveillance systems (national or regional) across the world from 1 April 2020 to 31 March 2021 and compared them with cases from corresponding months between 2017 and 2020. Macrolide-resistant M. pneumoniae (MRMp) data were collected from 1 April 2017 to 31 March 2021.ResultsThirty-seven sites from 21 countries in Europe, Asia, America and Oceania submitted valid datasets (631,104 tests). Among the 30,617 M. pneumoniae detections, 62.39% were based on direct test methods (predominantly PCR), 34.24% on a combination of PCR and serology (no distinction between methods) and 3.37% on serology alone (only IgM considered). In all countries, M. pneumoniae incidence by direct test methods declined significantly after implementation of NPIs with a mean of 1.69% (SD ± 3.30) compared with 8.61% (SD ± 10.62) in previous years (p < 0.01). Detection rates decreased with direct but not with indirect test methods (serology) (-93.51% vs + 18.08%; p < 0.01). Direct detections remained low worldwide throughout April 2020 to March 2021 despite widely differing lockdown or school closure periods. Seven sites (Europe, Asia and America) reported MRMp detections in one of 22 investigated cases in April 2020 to March 2021 and 176 of 762 (23.10%) in previous years (p = 0.04).ConclusionsThis comprehensive collection of M. pneumoniae detections worldwide shows correlation between COVID-19 NPIs and significantly reduced detection numbers.

A Narrative Review of Experimental Models to Study Vascular Grafts Infections.

Background: Many experimental models have been developed to decipher the mechanisms of vascular graft and endograft infections (VGEIs), and to elaborate strategies to prevent or treat their occurrence. A systematic literature research was conducted to identify the most accurate models for studying VGEIs, depending on the research question. Methods: A narrative literature search was conducted using the MEDLINE and Cochrane databases, with no set limit on the date of publication, up to 10 August 2021. Ex vivo, in vitro, and in vivo animal studies on VGEIs, published in English or French, were selected. Cross references retrieved from selected articles on PubMed database were also included. Data on microorganisms and grafts studied, details of experimental models, and of graft implantation and removal in animal studies were collected. Results: A total of 243 studies were included in the review after reading the full length articles: 55 in vitro studies, 169 animal studies, 17 studies which used both in vitro and animal models, and two ex vivo studies. Many differences in model characteristics were seen. The main in vitro model was the incubation of a graft sample in a bacterial solution, used to study the first steps of infection. In animals, vascular large animal models (dogs and pigs) were the most commonly described but supplanted over time by extravascular and particularly subcutaneous mouse and rat models, which have been reported increasingly over the last few years. In animal models, antibiotic prophylaxis and therapy were rarely administered (27.4% and 19.9%, respectively), and vascular reconstruction after VGEIs even less frequently (9.8%). Conclusion: Despite protocol discrepancies, it was possible to dinstinguish three main experimental models (i.e., in vitro and in vivo vascular models, and extravascular models), which all remain of interest to study specific phases of VGEIs.

Detection and Prevalence of Macrolide and Fluoroquinolone Resistance in Mycoplasma genitalium in Badalona, Spain.

Macrolide and fluoroquinolone resistance (MLr/FQr) in Mycoplasma genitalium (MG) infections is concerning worldwide. Current guidelines recommend performing MLr detection in MG-positive cases to adjust antimicrobial therapy. We aimed to evaluate the usefulness of PCR followed by pyrosequencing for MLr detection in comparison with a one-step commercial assay and to assess the prevalence of MLr and FQr in Badalona, Spain. A total of 415 MG-positive samples by Allplex STI-7 (Seegene) were analyzed for MLr detection by pyrosequencing. From those, 179 samples were further analyzed for MG and MLr by ResistancePlus® MG kit (SpeeDx) and 100 of them also for fluoroquinolone resistance (FQr) by sequencing the parC gene. Regarding MG detection, Allplex and Resistance Plus® showed an overall agreement of 87%, but this value rose to 95.4% if we compare them for MLr detection. Prevalence of MLr was 23.1% in Badalona, but this rate increased to 73.7% in the HIV-positive patients cohort. FQr detection showed 3% of resistant strains. Pyrosequencing is a convenient and cheap technique for MLr detection, but one-step tools should be considered in high-throughput laboratories. Despite the fact that MLr remained moderate and FQr was low in our study, simultaneous MG and MLr detection would improve patient's management applying resistance-guided treatment strategies.

Clinical performance of four multiplex real-time PCR kits detecting urogenital and sexually transmitted pathogens.

OBJECTIVES: We evaluated the clinical performances of four multiplex real-time PCR commercial kits for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis: the STI PLUS ELITe MGB kit (ELITechGroup), N. gonorrhoeae/C. trachomatis/M. genitalium/T.vaginalis Real-TM kit (Sacace Biotechnologies), Allplex STI Essential kit (Seegene), and FTD Urethritis Plus kit (Fast-Track Diagnostics). METHODS: The kit performance for C. trachomatis, N. gonorrhoeae, M. genitalium and T. vaginalis detection was compared to that of the cobas CT/NG and TV/MG kits (Roche Diagnostics) using 425 samples, mainly urine and cervicovaginal, throat and rectal swabs. Detection of Ureaplasma parvum, U. urealyticum and Mycoplasma hominis were compared to that of in-house TaqMan PCRs. RESULTS: The four kits showed good performances for the detection of C. trachomatis. They all presented a low positive agreement for the detection of M. genitalium and T. vaginalis (ranges 63.3-74.1% and 51.2-68.4%, respectively) compared to the cobas MG/TV kit. The Seegene and Sacace kits showed additional low positive agreement for the detection of N. gonorrhoeae (71.2%, 95%CI 61.8-79.0 and 63.1%, 95%CI 53.5-71.8, respectively). We observed a slight but significant lower negative agreement for N. gonorrhoeae detection using the ELITechGroup kit (92.5%, 89.1-94.9) and for M. genitalium detection using the Fast-Track kit (93.2%, 89.6-95.7) compared to other kits. CONCLUSION: Multiplex real-time PCR kits are convenient methods for the detection of several pathogens associated with sexually transmitted infections (STIs) in a single step, but colonizing Ureaplasma spp. and M. hominis species should not be included in these kits. Users should be aware of the weak performance of some kits for the detection of M. genitalium and T. vaginalis.