A LAMP-based assay for the molecular detection of group B Streptococcus.

PURPOSE: Streptococcus agalactiae remains a major pathogen in human health, especially in neonatal infection. Detection in pregnant women is essential to initiate intrapartum antibiotic prophylaxis. This study compared the HiberGene loop-mediated isothermal amplification (LAMP) assay to culture, the reference method, for the detection of group B Streptococcus (GBS) in pregnant women. METHODS: This was a prospective multicenter study conducted in four French hospitals. Three hundred fifty-four non-redundant routine care vaginal swabs were analyzed by both methods, LAMP assay and culture. Clinicians and patients were blinded to the results of the LAMP assay. RESULTS: Three hundred thirty-seven samples presented concordant results, 15 presented discordant results, and 2 were invalid using the LAMP assay (excluded from the study). Compared to culture, the LAMP assay had a sensitivity of 87.7%, a specificity of 98%, a negative predictive value of 97.6%, and a positive predictive value of 89.3%. CONCLUSION: The HiberGene GBS LAMP assay is an easy test that possesses good performances compared with the reference method, culture. It could be used in case of emergency when a quick result is needed.

Long-term surveillance of group B Streptococcus strains isolated from infection and colonization in pregnant women and newborns.

Introduction. Group B Streptococcus (GBS) remains the leading cause of bacterial neonatal infections worldwide, despite the spread of recommendations on vaginal screening and antibiotic prophylaxis.Hypothesis/Gap Statement. There is a need to evaluate the potential changes in GBS epidemiology over time following the introduction of such guidelines.Aim. Our aim was to perform a descriptive analysis of the epidemiological characteristics of GBS by conducting a long-term surveillance of strains isolated between 2000 and 2018, using molecular typing methods.Methodology. A total of 121 invasive strains, responsible for maternal infections (20 strains), fetal infections (8 strains) and neonatal infections (93 strains), were included in the study, representing all the invasive isolates during the period; in addition, 384 colonization strains isolated from vaginal or newborn samples were randomly selected. The 505 strains were characterized by capsular polysaccharide (CPS) type multiplex PCR assay and the clonal complex (CC) was assigned using a single nucleotide polymorphism PCR assay. Antibiotic susceptibility was also determined.Results. CPS types III (32.1 % of the strains), Ia (24.6 %) and V (19 %) were the most prevalent. The five main CCs observed were CC1 (26.3 % of the strains), CC17 (22.2 %), CC19 (16.2 %), CC23 (15.8 %) and CC10 (13.9 %). Neonatal invasive GBS diseases were predominantly due to CC17 isolates (46.3 % of the strains), which mainly express CPS type III (87.5 %), with a very high prevalence in late-onset diseases (76.2 %).Conclusion. Between 2000 and 2018, we observed a decrease in the proportion of CC1 strains, which mainly express CPS type V, and an increase in the proportion of CC23 strains, mainly expressing CPS type Ia. Conversely, there was no significant change in the proportion of strains resistant to macrolides, lincosamides or tetracyclines. The two molecular techniques used in our study provide almost as much information as classical serotyping and multilocus sequence typing, but are quicker, easy to perform, and avoid long sequencing and analysis steps.

Group B Streptococcus CRISPR1 Typing of Maternal, Fetal, and Neonatal Infectious Disease Isolates Highlights the Importance of CC1 in In Utero Fetal Death.

We performed a descriptive analysis of group B Streptococcus (GBS) isolates responsible for maternal and fetal infectious diseases from 2004 to 2020 at the University Hospital of Tours, France. This represents 115 isolates, including 35 isolates responsible for early-onset disease (EOD), 48 isolates responsible for late-onset disease (LOD), and 32 isolates from maternal infections. Among the 32 isolates associated with maternal infection, 9 were isolated in the context of chorioamnionitis associated with in utero fetal death. Analysis of neonatal infection distribution over time highlighted the decrease in EOD since the early 2000s, while LOD incidence has remained relatively stable. All GBS isolates were analyzed by sequencing their CRISPR1 locus, which is an efficient way to determine the phylogenetic affiliation of strains, as it correlates with the lineages defined by multilocus sequence typing (MLST). Thus, the CRISPR1 typing method allowed us to assign a clonal complex (CC) to all isolates; among these isolates, CC17 was predominant (60/115, 52%), and the other main CCs, such as CC1 (19/115, 17%), CC10 (9/115, 8%), CC19 (8/115, 7%), and CC23 (15/115, 13%), were also identified. As expected, CC17 isolates (39/48, 81.3%) represented the majority of LOD isolates. Unexpectedly, we found mainly CC1 isolates (6/9) and no CC17 isolates that were responsible for in utero fetal death. Such a result highlights the possibility of a particular role of this CC in in utero infection, and further investigations should be conducted on a larger group of GBS isolated in a context of in utero fetal death. IMPORTANCE Group B Streptococcus is the leading bacterium responsible for maternal and neonatal infections worldwide, also involved in preterm birth, stillbirth, and fetal death. In this study, we determined the clonal complex of all GBS isolates responsible for neonatal diseases (early- and late-onset diseases) and maternal invasive infections, including chorioamnionitis associated with in utero fetal death. All GBS was isolated at the University Hospital of Tours from 2004 to 2020. We described the local group B Streptococcus epidemiology, which confirmed national and international data concerning neonatal disease incidence and clonal complex distribution. Indeed, neonatal diseases are mainly characterized by CC17 isolates, especially in late-onset disease. Interestingly, we identified mainly CC1 isolates responsible for in utero fetal death. CC1 could have a particular role in this context, and such a result should be confirmed on a larger group of GBS isolated from in utero fetal death.

Genetic Diversity and Population Structure of Mycobacterium bovis at the Human-Animal-Ecosystem Interface in France: "A One Health Approach".

Mycobacterium bovis infects cattle and wildlife, and also causes a small proportion of tuberculosis cases in humans. In most European countries, M. bovis infections in cattle have been drastically reduced, but not eradicated. Here, to determine the M. bovis circulation within and between the human, cattle, and wildlife compartments, we characterized by spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing the genetic diversity of M. bovis isolates collected from humans, cattle, and wildlife in France from 2000 to 2010. We also assessed their genetic structure within and among the different host groups, and across time and space. The M. bovis genetic structure and its spatiotemporal variations showed different dynamics in the human and animal compartments. Most genotypes detected in human isolates were absent in cattle and wildlife isolates, possibly because in patients, M. bovis infection was contracted abroad or was the reactivation of an old lesion. Therefore, they did not match the genetic pool present in France during the study period. However, some human-cattle exchanges occurred because some genotypes were common to both compartments. This study provides new elements for understanding M. bovis epidemiology in France, and calls for increased efforts to control this pathogen worldwide.

Outcome of febrile neutropenic patients treated for bacteriuria in hematology.

INTRODUCTION: Positive urine sample is a frequent finding in post-chemotherapy febrile neutropenia (FN) and can lead to prolonged antibiotic therapy. The aim of this study was to assess the outcome of bacteriuria episodes in FN patients receiving targeted antibiotic therapy. MATERIALS AND METHODS: A multi-centric retrospective study was conducted over a four-year period (2014-2019) on systematic urinalysis. All consecutive first bacteriuria episodes (≤ 2 bacteria with at least ≥ 103 CFU/mL) during FN in hospitalized adult patients for hematological malignancies were included. Relapse and recurrence were defined by fever or urinary tract symptoms (UTS) with the same bacterial subspecies in urine occurring ≤ 7 days and ≤ 30 days, respectively, after antibiotic discontinuation. Mortality rate was determined at 30 days. Targeted antibiotic therapy ≤ 10 days for women and ≤ 14 for men was considered as short course. RESULTS: Among 97 patients, 105 bacteriuria episodes on systematic urinalysis were analyzed; 67.6% occurred in women, 41.9% in AML patients, 17.1% were bacteremic, 14.2% presented with UTS, and 61.9% were treated with short-course antibiotic treatment. One death was reported. In men, no relapse/recurrence was noted, even in the short-course antibiotic group. In women, 2.8% of episodes treated with short-course antibiotic led to relapse or recurrence. CONCLUSIONS: Relapse, recurrence, and mortality were uncommon events in FN patients experiencing bacteriuria episode, whatever the antibiotic duration. To distinguish asymptomatic bacteriuria from infection remained challenging in women. In men, systematic urinalysis at onset of FN could be useful.

Pooling Rectal, Pharyngeal, and Urine Samples to Detect Neisseria gonorrhoeae, Chlamydia trachomatis, and Mycoplasma genitalium Using Multiplex Polymerase Chain Reaction Is as Effective as Single-Site Testing for Men Who Have Sex With Men.

Background: Screening for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) at pharyngeal, urogenital, and anorectal sites is recommended for men who have sex with men (MSM). Pooling samples is a promising technique, but no data are available when pooled screening also includes Mycoplasma genitalium (MG). The main objective of this study was to examine the sensitivity of pooled samples for detecting CT, NG, and MG in MSM using nucleic acid amplification versus single-site testing. Methods: In this multicenter study, MSM with a positive result for CT, NG, or MG were recalled to the clinic for treatment and were asked to participate in this study. Separate samples were sent to a central virological department that proceeded to form the pooled samples. Testing was performed using the multiplex real-time polymerase chain reaction Allplex STI Essential Assay (Seegene, Seoul, Korea), which can simultaneously detect 7 pathogens. Results: A total of 130 MSM with at least 1 positive test for CT, NG, or MG were included. A total of 25.4% had a coinfection. The sensitivities of pooled-sample testing were 94.8% for CT, 97.0% for NG, and 92.3% for MG. Pooling failed to detect 8 infections, but pooled-sample analysis missed detecting only samples with a low bacterial load (cycle threshold >35). Conclusions: Pooling samples from MSM to detect CT, NG, and MG is as sensitive as individual-site testing for these 3 pathogens using the Allplex assay. Missed infections with a very low bacterial load could have a low impact on further transmission. Clinical Trials Registration. NCT03568695.

Multiplex PCR assay targeting Trichomonas vaginalis: need for biological evaluation and interpretation.

In a retrospective study, we used sequencing to investigate Trichomonas vaginalis-positive specimens (genital, rectal and pharyngeal) with the Allplex™ STI Essential or the Anyplex™-II-STI-7 assays. Our results confirm that majority of T. vaginalis-positive genital and pharyngeal specimens contained T. vaginalis DNA and actually T. tenax DNA, respectively.

Clinical Features and Outcome of Multidrug-Resistant Osteoarticular Tuberculosis: A 12-Year Case Series from France.

The optimal treatment for osteoarticular infection due to multidrug-resistant tuberculosis strains (MDR-OATB) remains unclear. This study aims to evaluate the diagnosis, management and outcome of MDR-OATB in France. We present a case series of MDR-OATB patients reviewed at the French National Reference Center for Mycobacteria between 2007 and 2018. Medical history and clinical, microbiological, treatment and outcome data were collected. Twenty-three MDR-OATB cases were reported, representing 3% of all concurrent MDR-TB cases in France. Overall, 17 were male, and the median age was 32 years. Six patients were previously treated for TB, including four with first-line drugs. The most frequently affected site was the spine (n = 16). Bone and joint surgery were required in 12 patients. Twenty-one patients (91%) successfully completed the treatment with a regimen containing a mean of four drugs (range, 2-6) for a mean duration of 20 months (range, 13-27). Overall, high rates of treatment success were achieved following WHO MDR-TB treatment guidelines and individualized patient management recommendations by the French National TB Consilium. However, the optimal combination of drugs, duration of treatment and role of surgery in the management of MDR-OATB remains to be determined.

Doxycycline versus azithromycin for the treatment of anorectal Chlamydia trachomatis infection in women concurrent with vaginal infection (CHLAZIDOXY study): a multicentre, open-label, randomised, controlled, superiority trial.

BACKGROUND: Anorectal infections with Chlamydia trachomatis are commonly found in women. Although the efficacy of doxycycline and azithromycin is comparable in the treatment of urogenital infection, their efficacies toward anorectal infection remain unclear. We therefore aimed to compare a single dose of azithromycin with a 7-day course of doxycycline for the treatment of anorectal C trachomatis infection in women with concurrent vaginal infection. METHODS: We did a multicentre, open-label, randomised, controlled, superiority trial involving four sexually transmitted infection screening centres and three pregnancy termination centres in France. We included sexually active adult women (≥18 years) with a positive C trachomatis vaginal swab who agreed to provide self-collected anorectal swabs for C trachomatis detection. Participants were randomly assigned (1:1), using block sizes of six and eight and stratification by each investigating centre, to orally receive either azithromycin (a single 1-g dose, with or without food) or doxycycline (100 mg in the morning and evening at mealtimes for 7 days [ie, 100 mg of doxycycline twice per day for 7 days]). All laboratory staff who did the bacteriological analyses, but not the participants and the investigators, were masked to the treatment groups. The primary outcome was the microbiological anorectal cure rate defined as a C trachomatis-negative nucleic acid amplification test (NAAT) result in anorectal specimens 6 weeks after treatment initiation among women who had a baseline C trachomatis-positive anorectal NAAT result. The primary analysis was done in the modified intention-to-treat population, with multiple imputation, which included all women who underwent randomisation and had a C trachomatis-positive vaginal and anorectal NAAT result at baseline. Adverse events were reported in all women who underwent randomisation. This study is registered with ClinicalTrials.gov, number NCT03532464. FINDINGS: Between Oct 19, 2018, and April 17, 2020, we randomly assigned a total of 460 participants to either the doxycycline group (n=230) or the azithromycin group (n=230). Four (1%) of 460 participants were excluded because they refused to take doxycycline or were found to be ineligible after randomisation. Among the 456 participants, 357 (78%) had a concurrent C trachomatis-positive anorectal NAAT result at baseline; 184 (52%) of 357 were in the doxycycline group and 173 (48%) were in the azithromycin group (ie, the modified intention-to-treat population). Microbiological anorectal cure occurred in 147 (94%) of 156 participants in the doxycycline group (28 missing values) versus 120 (85%) of 142 in the azithromycin group (31 missing values; adjusted odds ratio with imputation of missing values 0·43 [95% CI 0·21-0·91]; p=0·0274). Reported adverse events possibly related to treatment were notified in 53 (12%) of 456 women: 24 (11%) of 228 in the doxycycline group and 29 (13%) of 228 in the azithromycin group. Gastrointestinal disorders were the most frequently occurring, in 43 (9%) of 456 women: 17 (8%) of 228 in the doxycycline group and 26 (11%) of 228 in the azithromycin group. INTERPRETATION: The microbiological anorectal cure rate was significantly lower among women who received a single dose of azithromycin than among those who received a 1-week course of doxycycline. This finding suggests that doxycycline should be the first-line therapy for C trachomatis infection in women. FUNDING: French Ministry of Health. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Distribution, Diversity and Roles of CRISPR-Cas Systems in Human and Animal Pathogenic Streptococci.

Streptococci form a wide group of bacteria and are involved in both human and animal pathologies. Among pathogenic isolates, differences have been highlighted especially concerning their adaptation and virulence profiles. CRISPR-Cas systems have been identified in bacteria and many streptococci harbor one or more systems, particularly subtypes I-C, II-A, and III-A. Since the demonstration that CRISPR-Cas act as an adaptive immune system in Streptococcus thermophilus, a lactic bacteria, the diversity and role of CRISPR-Cas were extended to many germs and functions were enlarged. Among those, the genome editing tool based on the properties of Cas endonucleases is used worldwide, and the recent attribution of the Nobel Prize illustrates the importance of this tool in the scientific world. Another application is CRISPR loci analysis, which allows to easily characterize isolates in order to understand the interactions of bacteria with their environment and visualize species evolution. In this review, we focused on the distribution, diversity and roles of CRISPR-Cas systems in the main pathogenic streptococci.

Management of patients with pulmonary mycobacteriosis in France: a multicenter retrospective cohort study.

BACKGROUND: Recent studies report very low adherence of practitioners to ATS/IDSA recommendations for the treatment of nontuberculous mycobacteria pulmonary disease (NTM-PD), as well as a great variability of practices. Type of management could impact prognosis. METHODS: To evaluate management and prognosis of patients with NTM-PD cases with respect to ATS recommendations, we conducted a multicenter retrospective cohort study (18 sentinel sites distributed throughout France), over a period of six years. We collected clinical, radiological, microbiological characteristics, management and outcome of the patients (especially death or not). RESULTS: 477 patients with NTM-PD were included. Respiratory comorbidities were found in 68% of cases, tuberculosis sequelae in 31.4% of patients, and immunosuppression in 16.8% of cases. The three most common NTM species were Mycobacterium avium complex (60%), M. xenopi (20%) and M. kansasii (5.7%). Smear-positive was found in one third of NTM-PD. Nodulobronchiectatic forms were observed in 54.3% of cases, and cavitary forms in 19.1% of patients. Sixty-three percent of patients were treated, 72.4% of patients with smear-positive samples, and 57.5% of patients with smear-negative samples. Treatment was in adequacy with ATS guidelines in 73.5%. The 2-year mortality was 14.4%. In the Cox regression, treatment (HR = 0.51), age (HR = 1.02), and M. abscessus (3.19) appeared as the 3 significant independent prognostic factors. CONCLUSION: These findings highlight the adequacy between French practices and the ATS/IDSA guidelines. Treatment was associated with a better survival.

Inhaled amikacin versus placebo to prevent ventilator-associated pneumonia: the AMIKINHAL double-blind multicentre randomised controlled trial protocol.

INTRODUCTION: Pre-emptive inhaled antibiotics may be effective to reduce the occurrence of ventilator-associated pneumonia among critically ill patients. Meta-analysis of small sample size trials showed a favourable signal. Inhaled antibiotics are associated with a reduced emergence of antibiotic resistant bacteria. The aim of this trial is to evaluate the benefit of a 3-day course of inhaled antibiotics among patients undergoing invasive mechanical ventilation for more than 3 days on the occurrence of ventilator-associated pneumonia. METHODS AND ANALYSIS: Academic, investigator-initiated, parallel two group arms, double-blind, multicentre superiority randomised controlled trial. Patients invasively ventilated more than 3 days will be randomised to receive 20 mg/kg inhaled amikacin daily for 3 days or inhaled placebo (0.9% Sodium Chloride). Occurrence of ventilator-associated pneumonia will be recorded based on a standardised diagnostic framework from randomisation to day 28 and adjudicated by a centralised blinded committee. ETHICS AND DISSEMINATION: The protocol and amendments have been approved by the regional ethics review board and French competent authorities (Comité de protection des personnes Ouest I, No.2016-R29). All patients will be included after informed consent according to French law. Results will be disseminated in international scientific journals. TRIAL REGISTRATION NUMBERS: EudraCT 2016-001054-17 and NCT03149640.

CRISPR Typing Increases the Discriminatory Power of Streptococcus agalactiae Typing Methods.

We explored the relevance of a Clustered regularly interspaced short palindromic repeats (CRISPR)-based genotyping tool for Streptococcus agalactiae typing and we compared this method to current molecular methods [multi locus sequence typing (MLST) and capsular typing]. To this effect, we developed two CRISPR marker schemes (using 94 or 25 markers, respectively). Among the 255 S. agalactiae isolates tested, 229 CRISPR profiles were obtained. The 94 and 25 markers made it possible to efficiently separate isolates with a high diversity index (0.9947 and 0.9267, respectively), highlighting a high discriminatory power, superior to that of both capsular typing and MLST (diversity index of 0.9017 for MLST). This method has the advantage of being correlated with MLST [through analysis of the terminal direct repeat (TDR) and ancestral spacers] and to possess a high discriminatory power (through analysis of the leader-end spacers recently acquired, which are the witnesses of genetic mobile elements encountered by the bacteria). Furthermore, this "one-shot" approach presents the benefit of much-reduced time and cost in comparison with MLST. On the basis of these data, we propose that this method could become a reference method for group B Streptococcus (GBS) typing.

[Mycoplasma genitalium: Prevalence of macrolide and fluoroquinolone resistance at the University Hospital of Tours, and check of the S-DiaMGRes® (Diagenode Diagnostics) assay]. / Mycoplasma genitalium : prévalence de la résistance aux macrolides et fluoroquinolones au CHRU de Tours, et vérification de la trousse commerciale S-DiaMGRes® (Diagenode Diagnostics).

Mycoplasma genitalium is an emerging agent of sexually transmitted infections, associated with urethritis, cervicitis, and pelvic inflammatory disease. Over the past decade, a remarkable increase in macrolide-resistant M. genitalium, the first-line treatment, is observed all over the world. In some regions, an increase in fluoroquinolone-resistance, the second-line treatment, is also noticed. It therefore seems important to have a knowledge of the local epidemiology as well as the means of detecting these resistances in order to best adapt the treatment. Our study aimed to determine the prevalence of macrolide and fluoroquinolone-resistant Mycoplasma genitalium at the University Hospital of Tours in 2018, using a real-time PCR technique followed by Sanger sequencing. The prevalence of macrolide resistance in our population was 15.4 %. No FQ resistance-conferring mutations were observed in our study. Furthermore, we evaluated the performance of the commercial kit S-DiaMGRes® (Diagenode Diagnostics, Belgium), allowing the detection of 23S rRNA mutations conferring resistance to macrolides.

Functional Study of the Type II-A CRISPR-Cas System of Streptococcus agalactiae Hypervirulent Strains.

Nearly all strains of Streptococcus agalactiae, the leading cause of invasive infections in neonates, encode a type II-A clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. Interestingly, S. agalactiae strains belonging to the hypervirulent Sequence Type 17 (ST17) contain significantly fewer spacers in their CRISPR locus than other lineages, which could be the result of a less functional CRISPR-Cas system. Here, we revealed one large deletion in the ST17 cas promoter region and we evaluated its impact on the transcription of cas genes as well as the functionalities of the CRISPR-Cas system. We demonstrated that Cas9 interference is functional and that the CRISPR-Cas system of ST17 strains can still acquire new spacers, despite the absence of a regular cas promoter. We demonstrated that a promoter sequence upstream of srn036, a small RNA partially overlapping the antisense tracrRNA, is responsible for the ST17 CRISPR-Cas adaptation and interference activities.

Controlled Heat and Humidity-Based Treatment for the Reuse of Personal Protective Equipment: A Pragmatic Proof-of-Concept to Address the Mass Shortage of Surgical Masks and N95/FFP2 Respirators and to Prevent the SARS-CoV2 Transmission.

Background: The coronavirus infectious disease-2019 (COVID-19) pandemic has led to an unprecedented shortage of healthcare resources, primarily personal protective equipment like surgical masks, and N95/filtering face piece type 2 (FFP2) respirators. Objective: Reuse of surgical masks and N95/FFP2 respirators may circumvent the supply chain constraints and thus overcome mass shortage. Methods, design, setting, and measurement: Herein, we tested the effects of dry- and moist-air controlled heating treatment on structure and chemical integrity, decontamination yield, and filtration performance of surgical masks and FFP2 respirators. Results: We found that treatment in a climate chamber at 70°C during 1 h with 75% humidity rate was adequate for enabling substantial decontamination of both respiratory viruses, oropharyngeal bacteria, and model animal coronaviuses, while maintaining a satisfying filtering capacity. Limitations: Further studies are now required to confirm the feasibility of the whole process during routine practice. Conclusion: Our findings provide compelling evidence for the recycling of pre-used surgical masks and N95/FFP2 respirators in case of imminent mass shortfall.

Contribution of a molecular test for the diagnosis of genital infection with Trichomonas vaginalis and Mycoplasma genitalium.

In the present study, we assessed a recently-marketed molecular test, the S-DiaMGTV™ kit (Diagenode), which provides simultaneous detection of Mycoplasma genitalium and Trichomonas vaginalis in urogenital samples. Performance characteristics of the S-DiaMGTV™ kit were compared to an in-house PCR for detection of M. genitalium and, for first time, with direct observation of genital secretions in wet mounting microscopy for T. vaginalis, a routine laboratory method. For M. genitalium, out of 66 samples, two negative with the in-house PCR were found positive with the S-DiaMGTV™ kit and two positive with the in-house PCR were found negative with the kit. For T. vaginalis, four samples were found positive by the molecular test. Among them, two were previously tested by the wet mounting observation and only one was positive. The kit allows an increase of T. vaginalis detection even in a low incidence country. Performances of the kit are in favor of its use in routine laboratory practice.

Seven Years Leptospirosis Follow-Up in a Critical Care Unit of a French Metropolitan Hospital; Role of Real Time PCR for a Quick and Acute Diagnosis.

(1) Background: Leptospirosis infection can lead to multiple organ failure, requiring hospitalization in an intensive care unit for supportive care, along with initiation of an adapted antibiotic therapy. Achieving a quick diagnosis is decisive in the management of these patients. (2) Methods: We present here a review of leptospirosis cases diagnosed in the intensive care unit of our hospital over seven years. Clinical and biological data were gathered, and we compared the differences in terms of diagnostic method. (3) Results: Molecular biology method by Polymerase Chain Reaction (PCR) allowed quick and reliable diagnosis when performed in the first days after the symptoms began. Moreover, we identified that sampling blood and urine for PCR was more efficient than performing PCR on only one type of biological sample. (4) Conclusions: Our results confirm the efficiency of PCR for the quick diagnosis of leptospirosis and suggest that testing both blood and urine early in the disease might improve diagnosis.

Feature of Adhesins Produced by Human Clinical Isolates of Mycobacterium intracellulare, Mycobacterium intracellulare subsp. chimaera and Closely Related Species.

The Mycobacterium avium complex includes two closely related species, Mycobacterium avium and Mycobacterium intracellulare. They are opportunistic pathogens in humans and responsible for severe disease in a wide variety of animals. Yet, little is known about factors involved in their pathogenicity. Here, we identified, purified and characterized adhesins belonging to the heparin-binding hemagglutinin (HBHA) and laminin-binding protein (LBP) family from M. intracellulare ATCC13950 and examined clinical isolates from patients with different pathologies associated with M. intracellulare infection for the presence and conservation of HBHA and LBP. Using a recombinant derivative strain of M. intracellulare ATCC13950 producing green fluorescent protein and luciferase, we found that the addition of heparin inhibited mycobacterial adherence to A549 cells, whereas the addition of laminin enhanced adherence. Both HBHA and LBP were purified by heparin-Sepharose chromatography and their methylation profiles were determined by mass spectrometry. Patients with M. intracellulare infection mounted strong antibody responses to both proteins. By using PCR and immunoblot analyses, we found that both proteins were highly conserved among all 17 examined clinical M. intracellulare isolates from patients with diverse disease manifestations, suggesting a conserved role of these adhesins in M. intracellulare virulence in humans and their potential use as a diagnostic tool.