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author:("eaton, kaia")
1.  Microbiota Present in Cystic Fibrosis Lungs as Revealed by Whole Genome Sequencing 
PLoS ONE  2014;9(3):e90934.
Determination of the precise composition and variation of microbiota in cystic fibrosis lungs is crucial since chronic inflammation due to microorganisms leads to lung damage and ultimately, death. However, this constitutes a major technical challenge. Culturing of microorganisms does not provide a complete representation of a microbiota, even when using culturomics (high-throughput culture). So far, only PCR-based metagenomics have been investigated. However, these methods are biased towards certain microbial groups, and suffer from uncertain quantification of the different microbial domains. We have explored whole genome sequencing (WGS) using the Illumina high-throughput technology applied directly to DNA extracted from sputa obtained from two cystic fibrosis patients. To detect all microorganism groups, we used four procedures for DNA extraction, each with a different lysis protocol. We avoided biases due to whole DNA amplification thanks to the high efficiency of current Illumina technology. Phylogenomic classification of the reads by three different methods produced similar results. Our results suggest that WGS provides, in a single analysis, a better qualitative and quantitative assessment of microbiota compositions than cultures and PCRs. WGS identified a high quantity of Haemophilus spp. (patient 1) or Staphylococcus spp. plus Streptococcus spp. (patient 2) together with low amounts of anaerobic (Veillonella, Prevotella, Fusobacterium) and aerobic bacteria (Gemella, Moraxella, Granulicatella). WGS suggested that fungal members represented very low proportions of the microbiota, which were detected by cultures and PCRs because of their selectivity. The future increase of reads’ sizes and decrease in cost should ensure the usefulness of WGS for the characterisation of microbiota.
doi:10.1371/journal.pone.0090934
PMCID: PMC3944733  PMID: 24599149
2.  Effect of Mutation and Genetic Background on Drug Resistance in Mycobacterium tuberculosis 
Bacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistant Mycobacterium tuberculosis isolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains, katG mutations were associated with high-level and inhA promoter mutations with low-level drug resistance. Only katG(S315T) (65.6% of all isoniazid-resistant strains) and inhA promoter −15C/T (22.7%) were found in molecular clusters. M. tuberculosis lineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6; P < 0.0001). Lineage 1 was associated with inhA promoter −15C/T mutations (OR, 6.4; 95% CI, 2.0 to 20.7; P = 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages; P < 0.0001). In conclusion, M. tuberculosis drug resistance mutations were associated with various levels of drug resistance and transmission, and M. tuberculosis lineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds in M. tuberculosis drug resistance.
doi:10.1128/AAC.06460-11
PMCID: PMC3370767  PMID: 22470121
3.  Intestinal Spirochetosis mimicking inflammatory bowel disease in children 
BMC Pediatrics  2012;12:163.
Background
Intestinal spirochetosis is an unusual infection in children and its clinical significance in humans is uncertain. The presence of these microorganisms in humans is well-known since the late 1800’s and was first described in 1967 by Harland and Lee by electron microscopy.
Case presentation
This article reports the findings of one pediatric case, review of the current literature, and an overview of therapeutic options.
Conclusion
A high degree of suspicion is required in cases presenting with abdominal pain, chronic diarrhoea and/or hematochezia associated with a normal endoscopic examination, thus emphasizing the importance of multiple biopsies throughout the colon.
doi:10.1186/1471-2431-12-163
PMCID: PMC3480841  PMID: 23066991
Intestinal spirochetosis; Brachyspira aalborgi; Brachyspira pilosicoli; Inflammatory bowel disease
4.  Mycobacterium tuberculosis Transmission in a Country with Low Tuberculosis Incidence: Role of Immigration and HIV Infection 
Journal of Clinical Microbiology  2012;50(2):388-395.
Immigrants from high-burden countries and HIV-coinfected individuals are risk groups for tuberculosis (TB) in countries with low TB incidence. Therefore, we studied their role in transmission of Mycobacterium tuberculosis in Switzerland. We included all TB patients from the Swiss HIV Cohort and a sample of patients from the national TB registry. We identified molecular clusters by spoligotyping and mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) analysis and used weighted logistic regression adjusted for age and sex to identify risk factors for clustering, taking sampling proportions into account. In total, we analyzed 520 TB cases diagnosed between 2000 and 2008; 401 were foreign born, and 113 were HIV coinfected. The Euro-American M. tuberculosis lineage dominated throughout the study period (378 strains; 72.7%), with no evidence for another lineage, such as the Beijing genotype, emerging. We identified 35 molecular clusters with 90 patients, indicating recent transmission; 31 clusters involved foreign-born patients, and 15 involved HIV-infected patients. Birth origin was not associated with clustering (adjusted odds ratio [aOR], 1.58; 95% confidence interval [CI], 0.73 to 3.43; P = 0.25, comparing Swiss-born with foreign-born patients), but clustering was reduced in HIV-infected patients (aOR, 0.49; 95% CI, 0.26 to 0.93; P = 0.030). Cavitary disease, male sex, and younger age were all associated with molecular clustering. In conclusion, most TB patients in Switzerland were foreign born, but transmission of M. tuberculosis was not more common among immigrants and was reduced in HIV-infected patients followed up in the national HIV cohort study. Continued access to health services and clinical follow-up will be essential to control TB in this population.
doi:10.1128/JCM.05392-11
PMCID: PMC3264153  PMID: 22116153
5.  Fever of unknown origin in a Swiss-born child: don't miss tuberculosis! 
Clinics and Practice  2012;2(2):e36.
Tuberculosis incidence is low in Switzer land. We report here on a Swiss-born toddler. Tuberculosis manifested with a fever of unknown origin, mimicking an inflammatory or autoimmune disorder triggering a high dose of corticosteroid treatment. The disease went unrecognized for several weeks until development of a miliary tuberculosis with advanced central nervous system involvement. This case highlights the difficulties encountered in diagnosing tuberculosis and in identifying the origin of this case. It reminds us that this disease must never be forgotten when facing a child with persistent fever who must be screened for, before starting immunosuppressive therapy.
doi:10.4081/cp.2012.e36
PMCID: PMC3981286  PMID: 24765435
fever; Swiss; tuberculosis.
6.  Development of a New Chlamydiales-Specific Real-Time PCR and Its Application to Respiratory Clinical Samples ▿† 
Journal of Clinical Microbiology  2011;49(7):2637-2642.
Originally composed of the single family Chlamydiaceae, the Chlamydiales order has extended considerably over the last several decades. Chlamydia-related bacteria were added and classified into six different families and family-level lineages: the Criblamydiaceae, Parachlamydiaceae, Piscichlamydiaceae, Rhabdochlamydiaceae, Simkaniaceae, and Waddliaceae. While several members of the Chlamydiaceae family are known pathogens, recent studies showed diverse associations of Chlamydia-related bacteria with human and animal infections. Some of these latter bacteria might be of medical importance since, given their ability to replicate in free-living amoebae, they may also replicate efficiently in other phagocytic cells, including cells of the innate immune system. Thus, a new Chlamydiales-specific real-time PCR targeting the conserved 16S rRNA gene was developed. This new molecular tool can detect at least five DNA copies and show very high specificity without cross-amplification from other bacterial clade DNA. The new PCR was validated with 128 clinical samples positive or negative for Chlamydia trachomatis or C. pneumoniae. Of 65 positive samples, 61 (93.8%) were found to be positive with the new PCR. The four discordant samples, retested with the original test, were determined to be negative or below detection limits. Then, the new PCR was applied to 422 nasopharyngeal swabs taken from children with or without pneumonia; a total of 48 (11.4%) samples were determined to be positive, and 45 of these were successfully sequenced. The majority of the sequences corresponded to Chlamydia-related bacteria and especially to members of the Parachlamydiaceae family.
doi:10.1128/JCM.00114-11
PMCID: PMC3147821  PMID: 21562107
7.  Role of Chlamydia trachomatis in Miscarriage 
Emerging Infectious Diseases  2011;17(9):1630-1635.
TOC Summary: Women experiencing miscarriage should be screened for C. trachomatis.
To determine the role of Chlamydia trachomatis in miscarriage, we prospectively collected serum, cervicovaginal swab specimens, and placental samples from 386 women with and without miscarriage. Prevalence of immunoglobulin G against C. trachomatis was higher in the miscarriage group than in the control group (15.2% vs. 7.3%; p = 0.018). Association between C. trachomatis–positive serologic results and miscarriage remained significant after adjustment for age, origin, education, and number of sex partners (odds ratio 2.3, 95% confidence interval 1.1–4.9). C. trachomatis DNA was more frequently amplified from products of conception or placenta from women who had a miscarriage (4%) than from controls (0.7%; p = 0.026). Immunohistochemical analysis confirmed C. trachomatis in placenta from 5 of 7 patients with positive PCR results, whereas results of immunohistochemical analysis were negative in placenta samples from all 8 negative controls tested. Associations between miscarriage and serologic/molecular evidence of C. trachomatis infection support its role in miscarriage.
doi:10.3201/eid1709.100865
PMCID: PMC3322049  PMID: 21888787
Chlamydia trachomatis; abortion; adverse pregnancy outcome; placental infection; sexually transmitted disease; miscarriage; bacteria; research
8.  High Proportion of Wrongly Identified Methicillin-Resistant Staphylococcus aureus Carriers by Use of a Rapid Commercial PCR Assay Due to Presence of Staphylococcal Cassette Chromosome Element Lacking the mecA Gene▿ 
Journal of Clinical Microbiology  2011;49(2):722-724.
During a 9-month period, 217 patients were newly diagnosed as methicillin-resistant Staphylococcus aureus (MRSA) carriers by using a commercial rapid PCR-based test (GeneXpert). However, no MRSA was recovered by culturing the second swab in 61 of these patients. Further analyses showed that 28 (12.9%) of the patients harbored S. aureus isolates with a staphylococcal cassette chromosome element lacking the mecA gene and were thus incorrectly determined to be MRSA carriers.
doi:10.1128/JCM.01988-10
PMCID: PMC3043509  PMID: 21159945
9.  False-Negative PCR Result Due to Gene Polymorphism: the Example of Neisseria meningitidis ▿  
Journal of Clinical Microbiology  2010;48(12):4590-4591.
Early treatment of meningococcal meningitis is mandatory but may negate the cerebrospinal fluid culture. Etiological diagnosis then mainly relies on PCR. Here, we report a case of false-negative results for real-time PCR for a Neisseria meningitidis serogroup B isolate with a polymorphism in the ctrA gene.
doi:10.1128/JCM.01766-10
PMCID: PMC3008480  PMID: 20962143
10.  Mycoplasma hominis necrotizing pleuropneumonia in a previously healthy adolescent 
BMC Infectious Diseases  2010;10:335.
Background
Mycoplasma hominis is a fastidious micro-organism causing systemic infections in the neonate and genital infections in the adult. It can also be the cause of serious extra-genital infections, mainly in immunosuppressed or predisposed subjects.
Case Presentation
We describe a case of severe pneumonia and pericarditis due to Mycoplasma hominis in a previously healthy adolescent who did not respond to initial therapy.
Conclusions
Mycoplasma hominis could be an underestimated cause of severe pneumonia in immunocompetent patients and should be particularly suspected in those not responding to standard therapy.
doi:10.1186/1471-2334-10-335
PMCID: PMC3006422  PMID: 21106079
11.  New Diagnostic Real-Time PCR for Specific Detection of Mycoplasma hominis DNA 
Mycoplasma hominis is a fastidious micro-organism causing genital and extragenital infections. We developed a specific real-time PCR that exhibits high sensitivity and low intrarun and interrun variabilities. When applied to clinical samples, this quantitative PCR allowed to confirm the role of M. hominis in three patients with severe extragenital infections.
doi:10.1155/2010/317512
PMCID: PMC2913506  PMID: 20706532
12.  Mycobacterium tuberculosis aortic graft infection with recurrent hemoptysis: a case report 
Introduction
Mycobacterium tuberculosis may cause a large variety of clinical presentations due to its ability to disseminate by contiguity or hematogenously. Tuberculosis may remain undiagnosed for years due to the chronic course of the disease, with potentially life-threatening long-term complications.
Case presentation
In this case report, we describe a tuberculous aortic graft infection in a 72-year-old man documented by polymerase chain reaction and cultures. The patient presented with three episodes of hemoptysis following a remote history of miliary tuberculosis. The infection was treated by graft replacement and prolonged antimycobacterial therapy.
Conclusion
Tuberculous infection of a vascular graft is an uncommon complication, but should be considered in patients with an intravascular device and a history of previous tuberculosis, especially when hematogenous spread may have occurred a few months after surgery, or when an active mycobacterial infection is present in close proximity to the graft.
doi:10.1186/1752-1947-2-233
PMCID: PMC2491651  PMID: 18634553
13.  Low prevalence of Chlamydia trachomatis infection in asymptomatic young Swiss men 
Background
Prevalence and risk factors for Chlamydia trachomatis infection among young men in Switzerland is still unknown. The objective of the present study was to assess prevalence and risk factors for C. trachomatis infection in young Swiss men.
Methods
517 young Swiss men were enrolled in this cross-sectional study during their compulsory military recruitment. Participants completed a questionnaire and gave urine samples which were screened for C. trachomatis DNA by PCR. Genotyping of positive samples was done by amplification and sequencing the ompA gene.
Results
The prevalence of chlamydial infection among young Swiss male was 1.2% (95% confidence interval [95%CI], 0.4–2.5%). C. trachomatis infection was only identified among the 306 men having multiple sexual partner. Although frequent, neither unprotected sex (absence of condom use), nor alcohol and drug abuse were associated with chlamydial infection. Men living in cities were more frequently infected (2.9%, 95%CI 0.8–7.4%) than men living in rural areas (0.5%, 95%CI 0.1–1.9%, p = 0.046). Moreover, naturalised Swiss citizens were more often positive (4.9%, 95%CI 1.3–12.5%) than native-born Swiss men (0.5%, 95%CI 0.1–1.7%, p = 0.003).
Conclusion
In comparison with other countries, the prevalence of chlamydial infection in men is extremely low in Switzerland, despite a significant prevalence of risky sexual behaviour. C. trachomatis infection was especially prevalent in men with multiple sexual partners. Further research is required (i) to define which subgroup of the general population should be routinely screened, and (ii) to test whether such a targeted screening strategy will be effective to reduce the prevalence of chlamydial infection among this population.
doi:10.1186/1471-2334-8-45
PMCID: PMC2359751  PMID: 18405389
14.  First Case of Bacteremia and Multifocal Cellulitis Due to Helicobacter canis in an Immunocompetent Patient▿  
Journal of Clinical Microbiology  2006;44(12):4598-4600.
Bacteremia due to Helicobacter canis has been reported in a patient with X-linked hypogammaglobulinemia. Here we report on the first human case of H. canis bacteremia in an immunocompetent host. Identification of the organism was made by genetic and phylogenetic analyses of the complete 16S rRNA sequence.
doi:10.1128/JCM.01453-06
PMCID: PMC1698384  PMID: 17005753
15.  Bloodstream and endovascular infections due to Abiotrophia defectiva and Granulicatella species 
Background
Abiotrophia and Granulicatella species, previously referred to as nutritionally variant streptococci (NVS), are significant causative agents of endocarditis and bacteraemia. In this study, we reviewed the clinical manifestations of infections due to A. defectiva and Granulicatella species that occurred at our institution between 1998 and 2004.
Methods
The analysis included all strains of NVS that were isolated from blood cultures or vascular graft specimens. All strains were identified by 16S rRNA sequence analysis. Patients' medical charts were reviewed for each case of infection.
Results
Eleven strains of NVS were isolated during the 6-year period. Identification of the strains by 16S rRNA showed 2 genogroups: Abiotrophia defectiva (3) and Granulicatella adiacens (6) or "para-adiacens" (2). The three A. defectiva strains were isolated from immunocompetent patients with endovascular infections, whereas 7 of 8 Granulicatella spp. strains were isolated from immunosuppressed patients, mainly febrile neutropenic patients. We report the first case of "G. para-adiacens" bacteraemia in the setting of febrile neutropenia.
Conclusion
We propose that Granulicatella spp. be considered as a possible agent of bacteraemia in neutropenic patients.
doi:10.1186/1471-2334-6-9
PMCID: PMC1360077  PMID: 16426445
16.  Detection of Four Plasmodium Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays 
Journal of Clinical Microbiology  2004;42(12):5636-5643.
There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the “gold standard” in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.
doi:10.1128/JCM.42.12.5636-5643.2004
PMCID: PMC535226  PMID: 15583293
17.  Evaluation of MRSA-Screen, a Simple Anti-PBP 2a Slide Latex Agglutination Kit, for Rapid Detection of Methicillin Resistance in Staphylococcus aureus 
Journal of Clinical Microbiology  1999;37(5):1591-1594.
The MRSA-Screen test (Denka Seiken Co., Ltd., Tokyo, Japan), consisting of a slide latex agglutination kit that detects PBP 2a with a monoclonal antibody, was blindly compared to the oxacillin disk diffusion test, the oxacillin-salt agar screen, and PCR of the mecA gene for the detection of methicillin resistance in Staphylococcus aureus. A total of 120 methicillin-susceptible S. aureus (MSSA) and 80 methicillin-resistant S. aureus (MRSA) isolates, defined by the absence or presence of the mecA gene, respectively, were tested. The MRSA-Screen test, the oxacillin disk diffusion test, and the oxacillin-salt agar screening test showed sensitivities of 100, 61.3, and 82.5% and specificities of 99.2, 96.7, and 98.3%, respectively. We conclude that the MRSA-Screen is a very accurate, reliable, and fast test (15 min) for differentiation of MRSA from MSSA colonies on agar plates.
PMCID: PMC84841  PMID: 10203531

Results 1-17 (17)