A potential role for chlamydial infection in rheumatoid arthritis development.

OBJECTIVES: To assess the relationship between self-reported and serologic evidence of prior chlamydial infection, rheumatoid arthritis (RA)-related autoantibodies and risk of RA-development. METHODS: This is a nested study within a prospective Swiss-based cohort including all first-degree relatives of RA patients (RA-FDR) who answered a question on past chlamydial infections. Primary outcome was systemic autoimmunity associated with RA (RA-autoimmunity) defined as positivity for anti-citrullinated peptide antibodies (ACPA) and/or rheumatoid factor (RF). Secondary outcomes were high levels of RA-autoimmunity, RA-associated symptoms and RA-autoimmunity, and subsequent seropositive RA diagnosis. We conducted a nested case-control analysis by measuring the serological status against Chlamydia trachomatis' major outer membrane protein. We replicated our analysis in an independent United States-based RA-FDR cohort. RESULTS: Among 1231 RA-FDRs, 168 (13.6%) developed RA-autoimmunity. Prevalence of self-reported chlamydial infection was significantly higher in individuals with RA-autoimmunity compared with controls (17.9% vs 9.8%, OR = 2.00, 95%CI: 1.27-3.09, p < 0.01). This association remained significant after adjustments (OR = 1.91, 95%CI: 1.20-2.95). Stronger effect sizes were observed in later stages of RA development. There was a similar trend between a positive C. trachomatis serology and high levels of RA-autoimmunity (OR = 3.05, 95% CI: 1.10-8.46, p= 0.032). In the replication cohort, there were significant associations between chlamydial infection and RF positivity and incident RA, but not anti-CCP positivity. CONCLUSIONS: Self-reported chlamydial infections are associated with elevated RA-autoimmunity in at risk individuals. The differing association of chlamydial infections and ACPA/RF between cohorts will need to be explored in future studies but is consistent with a role of mucosal origin of RA-related autoimmunity.

Waddlia chondrophila, a Chlamydia-related bacterium, has a negative impact on human spermatozoa.

STUDY QUESTION: What is the impact of Waddlia chondrophila, an emerging Chlamydia-related bacterium associated with miscarriage, on human spermatozoa? SUMMARY ANSWER: W. chondrophila had a negative impact on human spermatozoa (decrease in viability and mitochondrial membrane potential) and was not entirely removed from infected samples by density gradient centrifugation. WHAT IS KNOWN ALREADY: Bacterial infection or colonization might have a deleterious effect on male fertility. Waddlia chondrophila was previously associated with miscarriage, but its impact on male reproductive function has never been studied. STUDY DESIGN SIZE, DURATION: An in vitro model of human spermatozoa infection was used to assess the effects of W. chondrophila infection. Controls included Chlamydia trachomatis serovar D and latex beads with similar size to bacteria. PARTICIPANTS/MATERIALS, SETTING, METHODS: Purified motile spermatozoa were infected with W. chondrophila (multiplicity of infection of 1). Immunohistochemistry combined with confocal microscopy was used to evaluate how bacteria interact with spermatozoa. The impact on physiology was assessed by monitoring cell viability, mitochondrial membrane potential and DNA fragmentation. MAIN RESULTS AND THE ROLE OF CHANCE: Using super-resolution confocal microscopy, bacteria were localized on spermatozoa surface, as well as inside the cytoplasm. Compared to controls, W. chondrophila caused a 20% increase in mortality over 72 h of incubation (P < 0.05). Moreover, higher bacterial loads significantly reduced mitochondrial membrane potential. Bacteria present on spermatozoa surface were able to further infect a cell-monolayer, indicating that sperm might vector bacteria during sexual intercourse. LIMITATIONS REASONS FOR CAUTION: The main limitation of the study is the use of an in vitro model of infection, which might be too simplistic compared to an actual infection. An animal model of infection should be developed to better evaluate the in vivo impact of W. chondrophila. WIDER IMPLICATIONS OF THE FINDINGS: Intracellular bacteria, including C. trachomatis, Mycoplasma spp. and Ureaplasma spp., are associated with male infertility. Waddlia chondrophila might represent yet another member of this group, highlighting the need for more rigorous microbiological analysis during investigations for male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work has been funded by the Department of Obstetrics and Gynecology, Lausanne University Hospital, Switzerland, and by the Swiss National Science Foundation (Grant nos. 310030-156169/1, 320030-169853/1 and 320030-169853/2 attributed to D.B.). D.B. is also supported by the 'Fondation Leenaards' through the 'Bourse pour la relève académique', by the 'Fondation Divesa' and by the 'Loterie Romande'. No conflicts of interest to declare.

Lausannevirus Encodes a Functional Dihydrofolate Reductase Susceptible to Proguanil.

Lausannevirus belongs to the family Marseilleviridae within the group of nucleocytoplasmic large DNA viruses (NCLDVs). These giant viruses exhibit unique features, including a large genome, ranging from 100 kb to 2.5 Mb and including from 150 to more than 2,500 genes, as well as the presence of genes coding for proteins involved in transcription and translation. The large majority of Lausannevirus open reading frames have unknown functions. Interestingly, a bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is encoded in the Lausannevirus genome. The enzyme plays central roles in DNA precursor biosynthesis. DHFR is the pharmacological target of antifolates, such as trimethoprim, pyrimethamine, and proguanil. First, the functionality of Lausannevirus DHFR-TS was demonstrated by the successful complementation of a DHFR-deficient Saccharomyces cerevisiae strain with a plasmid expressing the heterologous gene. Additionally, using this heterologous expression system, we demonstrated the in vitro susceptibility of Lausannevirus DHFR-TS to proguanil and its resistance to pyrimethamine and trimethoprim. Proguanil may provide a unique and useful treatment if Lausannevirus proves to be a human pathogen. To our knowledge, this is the first time that a DHFR-TS has been described and characterized in an NCLDV.

Bacterial genome sequencing in clinical microbiology: a pathogen-oriented review.

In recent years, whole-genome sequencing (WGS) has been perceived as a technology with the potential to revolutionise clinical microbiology. Herein, we reviewed the literature on the use of WGS for the most commonly encountered pathogens in clinical microbiology laboratories: Escherichia coli and other Enterobacteriaceae, Staphylococcus aureus and coagulase-negative staphylococci, streptococci and enterococci, mycobacteria and Chlamydia trachomatis. For each pathogen group, we focused on five different aspects: the genome characteristics, the most common genomic approaches and the clinical uses of WGS for (i) typing and outbreak analysis, (ii) virulence investigation and (iii) in silico antimicrobial susceptibility testing. Of all the clinical usages, the most frequent and straightforward usage was to type bacteria and to trace outbreaks back. A next step toward standardisation was made thanks to the development of several new genome-wide multi-locus sequence typing systems based on WGS data. Although virulence characterisation could help in various particular clinical settings, it was done mainly to describe outbreak strains. An increasing number of studies compared genotypic to phenotypic antibiotic susceptibility testing, with mostly promising results. However, routine implementation will preferentially be done in the workflow of particular pathogens, such as mycobacteria, rather than as a broadly applicable generic tool. Overall, concrete uses of WGS in routine clinical microbiology or infection control laboratories were done, but the next big challenges will be the standardisation and validation of the procedures and bioinformatics pipelines in order to reach clinical standards.

Importance of whole genome sequencing for the assessment of outbreaks in diagnostic laboratories: analysis of a case series of invasive Streptococcus pyogenes infections.

Outbreaks of Streptococcus pyogenes hypervirulent clones are constant public health threats. In western Switzerland, an increase of severe cases of S. pyogenes invasive infections was observed between December 2015 and March 2016. Our aim was (i) to investigate these cases by the use of Whole Genome Sequencing (WGS) and (ii) to determine the specific virulome and resistome of each isolate in order to undertake adequate public health measures. Eleven Streptococcus pyogenes strains isolated from 11 patients with severe invasive infections between December 13, 2015 and March 12, 2016 were included in our study. Practically, emm-typing, MLST and WGS were used to investigate the relatedness between the isolates. The presence of virulence and antibiotic resistance genes as well as mutations in transcriptional regulators of virulence and in genes encoding for antibiotic targets were assessed. Three and two groups of isolates shared the same emm-type and ST type, respectively. Single Nucleotide Polymorphism (SNP) analysis revealed 14 to 32 SNPs between the strains of the same emm-type group, ruling out the possibility of a clonal outbreak. Mutations found in covS and rocA could partially explain an increased virulence. As these reassuring results were obtained in less than 10 days, no specific hospital hygiene and no dedicated public health measures had to be undertaken. WGS is a powerful technique to discriminate between closely related strains, excluding an outbreak in less than 10 days. Moreover, WGS provided extensive data on the virulome and resistome of all these strains.

Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.

Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.

Visceral leishmaniasis in a lung transplant recipient: usefulness of highly sensitive real-time polymerase chain reaction for preemptive diagnosis.

We report the case of a lung transplant recipient in whom the diagnosis of visceral leishmaniasis (VL) was made by detection of parasites in a peripheral blood smear when the parasite load already reached 8.9 × 103 parasites/mL. We demonstrated that the VL diagnosis could have been done months before the development of symptoms by the use of Leishmania-specific real-time polymerase chain reaction (PCR), suggesting the role of preemptive PCR-based diagnosis in transplant recipients at risk for VL.

Impact of round-the-clock CSF Gram stain on empirical therapy for suspected central nervous system infections.

The impact of round-the-clock cerebrospinal fluid (CSF) Gram stain on overnight empirical therapy for suspected central nervous system (CNS) infections was investigated. All consecutive overnight CSF Gram stains between 2006 and 2011 were included. The impact of a positive or a negative test on empirical therapy was evaluated and compared to other clinical and biological indications based on institutional guidelines. Bacterial CNS infection was documented in 51/241 suspected cases. Overnight CSF Gram stain was positive in 24/51. Upon validation, there were two false-positive and one false-negative results. The sensitivity and specificity were 41 and 99 %, respectively. All patients but one had other indications for empirical therapy than Gram stain alone. Upon obtaining the Gram result, empirical therapy was modified in 7/24, including the addition of an appropriate agent (1), addition of unnecessary agents (3) and simplification of unnecessary combination therapy (3/11). Among 74 cases with a negative CSF Gram stain and without formal indication for empirical therapy, antibiotics were withheld in only 29. Round-the-clock CSF Gram stain had a low impact on overnight empirical therapy for suspected CNS infections and was associated with several misinterpretation errors. Clinicians showed little confidence in CSF direct examination for simplifying or withholding therapy before definite microbiological results.

Role of Chlamydia trachomatis and emerging Chlamydia-related bacteria in ectopic pregnancy in Vietnam.

In this case-control study, we investigated the seroprevalence and molecular evidence of Chlamydia trachomatis and Waddlia chondrophila in ectopic pregnancies (EP) and uneventful control pregnancies in 343 women from Vietnam. Whereas presence of C. trachomatis IgG was strongly associated with EP [adjusted odds ratio (aOR) 5·41, 95% confidence interval (CI) 2·58-11·32], its DNA remained undetected in all tubal lesions. We confirmed an independent association between antibodies against Waddlia and previous miscarriage (aOR 1·87, 95% CI 1·02-3·42). Further investigations are needed to understand the clinical significance of Waddlia's high seroprevalence (25·9% in control pregnancies) in this urban population.

Opportunistic testing for urogenital infection with Chlamydia trachomatis in south-western Switzerland, 2012: a feasibility study.

The feasibility of opportunistic screening of urogenital infections with Chlamydia trachomatis was assessed in a cross-sectional study in 2012, in two cantons of south-western Switzerland: Vaud and Valais. Sexually active persons younger than 30 years, not tested for C. trachomatis in the last three months, were invited for free C. trachomatis testing by PCR in urine or self-applied vaginal swabs. Of 2,461 consenting participants, 1,899 (77%) were women and all but six (0.3%) submitted a sample. Forty-seven per cent of female and 25% of male participants were younger than 20 years. Overall, 134 (5.5%) of 2,455 tested participants had a positive result and were followed up. Seven per cent of all candidates for screening were not invited, 10% of invited candidates were not eligible, 15% of the eligible candidates declined participation, 5% of tested participants testing positive were not treated, 29% of those treated were not retested after six months and 9% of those retested were positive for C. trachomatis. Opportunistic C. trachomatis testing proved technically feasible and acceptable, at least if free of charge. Men and peripheral rural regions were more difficult to reach. Efforts to increase testing and decrease dropout at all stages of the screening procedure are necessary.

Isolated mediastinal necrotizing granulomatous lymphadenopathy due to cat-scratch disease.

We report a patient suffering from cat-scratch disease limited to mediastinal lymphadenitis. Although rare, cat-scratch disease should be considered in the differential diagnosis of mediastinal lymphadenitis, especially when patients were exposed to cats.

[Microbiological diagnosis of pneumonia]. / Diagnostic microbiologique de la pneumonie.

Pneumonia is an importance cause of mortality and morbidity in adults. Two types of pneumonia are defined: community-acquired and nosocomial pneumonia with their corresponding etiology such as pneumococci or Haemophilus influenzae and Pseudomonas or enterobacteriaceae, respectively. However, the reality is more complex with aspiration pneumonia, pneumonia in immunocompromised patient, and pneumonia in ventilated patients. Culture in the case of nosocomial pneumonia is especially important to obtain the antibiotic susceptibility of the infectious agent and to adjust therapy. Moreover for immunocompromised patients, the differential diagnosis is much wider looking for viruses, filamentous fungi and Pneumocystis can be very informative, using new molecular assays.

[Diagnostic approach of intracellular bacteria and fastidious microorganisms]. / Approches diagnostiques des bactéries intracellulaires et des germes fastidieux.

Obligate or facultative intracellular bacteria are fastidious organisms that do not or poorly grow on conventional culture media. Some of them may be the cause of frequent and potentially severe infections, such as tuberculosis (Myco- bacterium tuberculosis), community-acquired respiratory infections (Legionella spp., Mycoplasma pneumoniae, Chlamydia pneumoniae) or blood culture-negative endocarditis (Coxiella burnetii, Bartonella spp., Tropheryma whipplei). The objective of this paper is to provide a comprehensive summary of the available and recommended diagnostic tests for the detection of these fastidious organisms in clinical practice.

[Rapid tests during respiratory infection: an aid to non-prescription of antibiotics? ]. / Tests rapides lors d'infection respiratoire: une aide à la non-prescription d'antibiotiques?

Due to overuse, we are about to reach the end of the antibiotic era. Each of us is responsible to limit their usage to a minimum. Respiratory infections are the first cause of antibiotic prescriptions. The use of new simple tests available at the bedside can be very useful in this context. The development of sophisticated molecular diagnostic tools, such as "multiorganism" panels, may revolutionize our approach to respiratory infections. The key will be to interpret the results correctly, with due consideration of the statement, "Treat patients, not lab results".

[ESBL and carbapenemases in Enterobacteriaceae]. / Bêtalactamases à spectre étendu et carbapénémases chez les Enterobacteriaceae.

Carbapenemase-producing enterobacteria (CPE) spread all over the world during the last years, causing serious infections with increasing frequency. Very few new drugs active against CPE are expected to be clinically available. Studies summarized in this review show that there is yet room to improve our therapeutic approaches, in the treatment of infections due to CPE.

[Which benefits for the clinician to implement MALDI-TOF/MS in the bacteriology laboratory?]. / Quels bénéfices pour les cliniciens de la mise en place du MALDI-TOF/MS dans te taboratoire de bactériologie?

Mass spectrometry (MALDI-TOF/MS) is a recent technology especially adapted to identify microbial pathogens. It has rapidly established itself as a must for most medical bacteriology laboratories. Its ease of use and speed of execution typically permit providing pathogen identification one day earlier to the clinicians. MALDI-TOF/MS facilitates identification of filamentous fungi and mycobacteria that require particular lab expertise when using conventional methods. This paper highlights the multiple advantages that MALDI-TOF/MS can bring to the physicians in their practice.

[Bacterial genomics and metagenomics: clinical applications and medical relevance]. / Génomique et métagénomique bactériennes: applications cliniques et importance médicale.

New sequencing technologies provide in a short time and at low cost high amount of genomic sequences useful for applications such as: a) development of diagnostic PCRs and/or serological tests; b) detection of virulence factors (virulome) or genes/SNPs associated with resistance to antibiotics (resistome) and c) investigation of transmission and dissemination of bacterial pathogens. Thus, bacterial genomics of medical importance is useful to clinical microbiologists, to infectious diseases specialists as well as to epidemiologists. Determining the microbial composition of a sample by metagenomics is another application of new sequencing technologies, useful to understand the impact of bacteria on various non-infectious diseases such as obesity, asthma, or diabetes. Genomics and metagenomics will likely become a specialized diagnostic analysis.

MRSA screening by the Xpert MRSA PCR assay: pooling samples of the nose, throat, and groin increases the sensitivity of detection without increasing the laboratory costs.

The performance of the Xpert MRSA polymerase chain reaction (PCR) assay on pooled nose, groin, and throat swabs (three nylon flocked eSwabs into one tube) was compared to culture by analyzing 5,546 samples. The sensitivity [0.78, 95 % confidence interval (CI) 0.73-0.82] and specificity (0.99, 95 % CI 0.98-0.99) were similar to the results from published studies on separated nose or other specimens. Thus, the performance of the Xpert MRSA assay was not affected by pooling the three specimens into one assay, allowing a higher detection rate without increasing laboratory costs, as compared to nose samples alone.

[Q fever, a zoonosis often overlooked]. / Fievre Q: une zoonose souvent méconnue.

Q fever is a zoonosis caused by an intracellular Gram-negative bacteria, Coxiella burnetii. Animals are the main reservoir and transmission to men generally is occurring by inhalation of contaminated aerosols. Acute Q fever generally is benign and usually resolves spontaneously. When symptomatic, the clinical presentation typically includes one of the following three syndromes: a flu-like illness, a granulomatous hepatitis or an atypical pneumonia. Individuals presenting risk factors such as patients with valvular heart diseases and vascular prostheses, as well as pregnant women and immuno-suppressed patients represent a population at risk of chronic infection, with endocarditis as the most common clinical form.

[Parasitic infections: when to think about it?]. / Parasitoses digestives autochtones: quelques bonnes raisons d'y penser.

When patients return from the Tropics, parasitic infections are immediately being considered when they present with digestive symptoms. Using three examples, we describe parasitic infections acquired in Switzerland simulating other more common community pathologies, which in each case resulted in delayed diagnosis. Awareness among primary care physicians of these rare diseases, whose treatment may be unfamiliar, should improve the care of patients and avoid such late presentations.