Imported diphyllobothriasis in Switzerland: molecular methods to define a clinical case of Diphyllobothrium infection as Diphyllobothrium dendriticum, August 2010

Following a first clinical case of infection by Diphyllobothrium dendriticum in Switzerland in 2006, we report a second case in the country. The species was identified by molecular methods. In the Swiss, French and Italian subalpine regions, human diphyllobothriasis has seen a comeback since the late 1980's, and Diphyllobothrium latum is usually considered the causative agent of the disease. In addition, several locally acquired and imported clinical infections due to allochthonous Diphyllobothrium species have been documented in the last years. Due to the colonisation potential of these parasites and their probably underestimated presence in the human population, there is a need for discriminating them at the medical laboratory level. Because the morphological characters are very similar among the different taxa, a correct identification requires the use of molecular methods. Molecular identification would improve diagnosis and help monitor the distribution of Diphyllobothrium species in Europe.

Imported diphyllobothriasis in Switzerland: molecular evidence of Diphyllobothrium dendriticum (Nitsch, 1824)

We report for the first time in Switzerland a clinical case because of Diphyllobothrium dendriticum, identified by molecular methods. We discuss the potential for this imported species to infect local intermediate hosts and thus to achieve autochthonous cyclic transmission.

Molecular identification and epidemiological tracing of Pasteurella multocida meningitis in a baby

We report a case of Pasteurella multocida meningitis in a 1-month-old baby exposed to close contact with two dogs and a cat but without any known history of injury by these animals. 16S rRNA gene sequencing of the isolate from the baby allowed identification at the subspecies level and pointed to the cat as a possible source of infection. Molecular typing of Pasteurella isolates from the animals, from the baby, and from unrelated animals clearly confirmed that the cat harbored the same P. multocida subsp. septica strain on its tonsils as the one isolated from the cerebrospinal fluid of the baby. This case stresses the necessity of informing susceptible hosts at risk of contracting zoonotic agents about some basic hygiene rules when keeping pets. In addition, this study illustrates the usefulness of molecular methods for identification and epidemiological tracing of Pasteurella isolates.

Standard methods for Nosema research

Methods are described for working with Nosema apis and Nosema ceranae in the field and in the laboratory. For fieldwork, different sampling methods are described to determine colony level infections at a given point in time, but also for following the temporal infection dynamics. Suggestions are made for how to standardise field trials for evaluating treatments and disease impact. The laboratory methods described include different means for determining colony level and individual bee infection levels and methods for species determination, including light microscopy, electron microscopy, and molecular methods (PCR). Suggestions are made for how to standardise cage trials, and different inoculation methods for infecting bees are described, including control methods for spore viability. A cell culture system for in vitro rearing of Nosema spp. is described. Finally, how to conduct different types of experiments are described, including infectious dose, dose effects, course of infection and longevity tests

Endocarditis due to Tropheryma whipplei: rapid detection, limited genetic diversity, and long-term clinical outcome in a local experience

The characteristic features of Whipple's disease include abdominal pain, diarrhoea, wasting, and arthralgias, with the causative agent, Tropheryma whipplei, being detected mainly in intestinal biopsies. PCR technology has led to the identification of T. whipplei in specimens from various other locations, including the central nervous system and the heart. T. whipplei is now recognized as one of the causes of culture-negative endocarditis, and endocarditis can be the only manifestation of the infection with T. whipplei. Although it is considered a rare disease, the true incidence of endocarditis due to T. whipplei is not clearly established. With the increasing use of molecular methods, it is likely that T. whipplei will be more frequently identified. Questions also remain about the genetic variability of T. whipplei strains, optimal diagnostic procedures and therapeutic options. In the present study, we provide clinical data on four new patients with documented endocarditis due to T. whipplei in the context of the available published literature. There was no clinical involvement of the gastrointestinal tract. Genetic analysis of the T. whipplei strains with DNA isolated from the excised heart valves revealed little to no genetic variability. In a selected case, we describe acridine orange staining for early detection of the disease, prompting early adaptation of the antibiotic therapy. We provide long-term follow-up data on the patients. In our hands, an initial 2-week course of intravenous antibiotics followed by cotrimoxazole for at least 1 year was a suitable treatment option for T. whipplei endocarditis.

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.

[First detection of "Candidatus Mycoplasma haemolamae" in South American Camelids of Switzerland and evaluation of prevalence]

Haemotrophic mycoplasmas (also known as haemoplasmas), small bacterias which parasite the surface of erythrocytes, have been described in several species. Recently, molecular methods were developed for the diagnosis of haemoplasma infection. The presented study describes the first detection and the investigation of prevalence of "Candidatus Mycoplasma haemolamae" in South American Camelids in Switzerland. A random sample of the latter population was tested for haemoplasma infections using real-time PCR. The infection was detected in 18.6% of the animals and was found both in indigenous and in imported camelids. Of the tested herds 39,1% harboured at least one animal positive for haemoplasmas in PCR. There was no difference in prevalence between male and female animals and llamas and alpacas, respectively. Furthermore, the prevalence of infection was not significantly different in diseased animals compared to healthy camelids. From the latter observation and the fact that the high prevalence was accompanied by an undetectable incidence, we concluded that the pathogenicity of "Candidatus Mycoplasma haemolamae" may be low.

Development and application of a real-time TaqMan((R)) qPCR assay for detection and quantification of 'Candidatus Mycoplasma haemolamae' in South American camelids

Two alpacas from a herd in southwest Switzerland died for unknown reasons. Necropsy revealed chronic weight loss and pale mucous membranes. Infection with hemotropic mycoplasmas was suspected and subsequently confirmed by molecular methods. In order to investigate the epidemiological situation in this herd, a real-time TaqMan((R)) qPCR assay for the specific detection and quantification of hemoplasma infection in South American camelids was developed. This assay was based on the 16S rRNA gene and amplified 'Candidatus Mycoplasma haemolamae' DNA, but not DNA from other hemoplasmas or non-hemotropic mycoplasma species. The lower detection limit was one copy/PCR, and the amplification efficiency was 97.4%. In 11 out of 24 clinically healthy herd mates of the two infected alpacas, 'Candidatus M. haemolamae' infection was confirmed. No correlation was found between bacterial load and clinical signs or anemia. The assay described herein enables to detect and quantify 'Candidatus M. haemolamae' and may be used in future studies to investigate the prevalence, pathogenesis and treatment follow-up of hemoplasma infections in South American camelids.

Characterisation and clinical features of Enterobacter cloacae bloodstream infections occurring at a tertiary care university hospital in Switzerland: is cefepime adequate therapy?

Despite many years of clinical experience with cefepime, data regarding the outcome of patients suffering from bloodstream infections (BSIs) due to Enterobacter cloacae (Ecl) are scarce. To address the gap in our knowledge, 57 Ecl responsible for 51 BSIs were analysed implementing phenotypic and molecular methods (microarrays, PCRs for bla and other genes, rep-PCR to analyse clonality). Only two E. cloacae (3.5%) were ESBL-producers, whereas 34 (59.6%) and 18 (31.6%) possessed inducible (Ind-Ecl) or derepressed (Der-Ecl) AmpC enzymes, respectively. All isolates were susceptible to imipenem, meropenem, gentamicin and ciprofloxacin. Der-Ecl were highly resistant to ceftazidime and piperacillin/tazobactam (both MIC₉₀≥256 μg/mL), whereas cefepime retained its activity (MIC₉₀ of 3 μg/mL). rep-PCR indicated that the isolates were sporadic, but Ecl collected from the same patients were indistinguishable. In particular, three BSIs initially due to Ind-Ecl evolved (under ceftriaxone or piperacillin/tazobactam treatment) into Der-Ecl because of mutations or a deletion in ampD or insertion of IS4321 in the promoter. These last two mechanisms have never been described in Ecl. Mortality was higher for BSIs due to Der-Ecl than Ind-Ecl (3.8% vs. 29.4%; P=0.028) and was associated with the Charlson co-morbidity index (P=0.046). Using the following directed treatments, patients with BSI showed a favourable treatment outcome: cefepime (16/18; 88.9%); carbapenems (12/13; 92.3%); ceftriaxone (4/7; 57.1%); piperacillin/tazobactam (5/7; 71.4%); and ciprofloxacin (6/6; 100%). Cefepime represents a safe therapeutic option and an alternative to carbapenems to treat BSIs due to Ecl when the prevalence of ESBL-producers is low.

Biological safety concepts of genetically modified live bacterial vaccines

Live vaccines possess the advantage of having access to induce cell-mediated and antibody-mediated immunity; thus in certain cases they are able to prevent infection, and not only disease. Furthermore, live vaccines, particularly bacterial live vaccines, are relatively cheap to produce and easy to apply. Hence they are suitable to immunize large communities or herds. The induction of both cell-mediated immunity as well as antibody-mediated immunity, which is particularly beneficial in inducing mucosal immune responses, is obtained by the vaccine-strain's ability to colonize and multiply in the host without causing disease. For this reason, live vaccines require attenuation of virulence of the bacterium to which immunity must be induced. Traditionally attenuation was achieved simply by multiple passages of the microorganism on growth medium, in animals, eggs or cell cultures or by chemical or physical mutagenesis, which resulted in random mutations that lead to attenuation. In contrast, novel molecular methods enable the development of genetically modified organisms (GMOs) targeted to specific genes that are particularly suited to induce attenuation or to reduce undesirable effects in the tissue in which the vaccine strains can multiply and survive. Since live vaccine strains (attenuated by natural selection or genetic engineering) are potentially released into the environment by the vaccinees, safety issues concerning the medical as well as environmental aspects must be considered. These involve (i) changes in cell, tissue and host tropism, (ii) virulence of the carrier through the incorporation of foreign genes, (iii) reversion to virulence by acquisition of complementation genes, (iv) exchange of genetic information with other vaccine or wild-type strains of the carrier organism and (v) spread of undesired genes such as antibiotic resistance genes. Before live vaccines are applied, the safety issues must be thoroughly evaluated case-by-case. Safety assessment includes knowledge of the precise function and genetic location of the genes to be mutated, their genetic stability, potential reversion mechanisms, possible recombination events with dormant genes, gene transfer to other organisms as well as gene acquisition from other organisms by phage transduction, transposition or plasmid transfer and cis- or trans-complementation. For this, GMOs that are constructed with modern techniques of genetic engineering display a significant advantage over random mutagenesis derived live organisms. The selection of suitable GMO candidate strains can be made under in vitro conditions using basic knowledge on molecular mechanisms of pathogenicity of the corresponding bacterial species rather than by in vivo testing of large numbers of random mutants. This leads to a more targeted safety testing on volunteers and to a reduction in the use of animal experimentation.

Intraocular microsporidiosis due to Encephalitozoon cuniculi in a patient with idiopathic CD4+ T-lymphocytopenia

Encephalitozoon cuniculi was documented to cause disseminated microsporidial infection including an iris tumor and endophthalmitis in an adolescent with idiopathic CD4+ T-lymphocytopenia. The diagnosis was established by microscopic, serologic and molecular methods. E. cuniculi (rabbit strain) was identified from the iris tumor, as well as from urine, conjunctival, corneal, and nasal swabs. Treatment with oral albendazole led to rapid improvement. This case raises the possibility of disseminated microsporidial infection in the context of idiopathic CD4+ T-lymphocytopenia and possibly advanced human immunodeficiency virus (HIV) infection, and above all the possibility of intraocular infection with E. cuniculi in humans.

Phenotypic and genetic characterization of Pasteurella multocida and related isolates from rabbits in Switzerland

Several bacteria belonging to the family Pasteurellaceae are potential pathogens in rabbits. In particular, Pasteurella multocida is considered to be important, and outbreaks caused by this species result in considerable economic losses in rabbitries. However, Pasteurellaceae spp. isolated from rabbits are poorly characterized, and thus, proper identification of P. multocida isolates from these animals is problematic and often unsatisfactory, thereby hampering epidemiological investigations. Therefore, 228 isolates from rabbit populations originating from a breeding and fattening organization with group management and postmortem cases with pasteurellosis from individual owners were phenotypically and genotypically analyzed using biochemical tests and repetitive extragenic palindromic polymerase chain reaction (REP-PCR). Furthermore, 41 samples representing observed phenotypes were selected for phylogenetic analysis using 16S ribosomal RNA and rpoB genes. The REP-PCR typing and phylogenetic analyses correlated well and appeared to be distinct molecular methods for characterization of rabbit isolates. Phenotyping, however, diverged from molecular recognition, reflecting the problematic conventional diagnosis of these strains. The fermentation of sorbitol appeared to be an imprecise indicator for P. multocida subspecies classification. According to REP-PCR and sequencing results, 82% of the isolates were characterized as P. multocida subsp. multocida, 3% as P. multocida subsp. septica, and 5% as P. multocida. Further, 5% were identified as Pasteurella canis. The other 5% represented a homogeneous group of unknown species belonging to the Pasteurellaceae. Samples obtained from individual postmortem cases demonstrated a higher phenotypic and genetic heterogeneity than samples from group management rabbits.

Tuberculosis caused by Mycobacterium microti in South American camelids

BACKGROUND: Infection with Mycobacterium microti can cause chronic disease in animals and threaten human health through its zoonotic potential. OBJECTIVE: To describe clinical findings, diagnostic investigations, necropsy, and epidemiology results in South American camelids (SAC) infected with M. microti, member of the Mycobacterium tuberculosis complex. ANIMALS: Eleven SAC with tuberculous lesions. METHODS: Description of 10 llamas and 1 alpaca, aged 4-18 years, from 6 herds with a history of wasting and weakness admitted to the Vetsuisse-Faculty of Berne over 8 years. RESULTS: Clinical signs included weight loss, recumbency, and anorexia in late stages of the disease. Respiratory problems were seen in 6 animals of 11. No consistent hematologic abnormalities were identified. Suspect animals were examined in detail by abdominal ultrasonography and thoracic radiology. Abnormal findings such as enlarged mediastinal, mesenteric, or hepatic lymph nodes were seen only in animals with advanced disease. Single comparative intradermal tuberculin test with bovine protein purified derivate (PPD) and avian PPD was negative in all animals. At necropsy, typical tuberculous lesions were found, and confirmed by bacteriological smear and culture, molecular methods, or both. CONCLUSIONS AND CLINICAL IMPORTANCE: Infection caused by M. microti should be considered a differential diagnosis in chronic debilitating disease with or without respiratory signs in SAC. Antemortem confirmation of the diagnosis remains challenging at any stage of infection. Because cases of M. microti infection have been reported in immunocompromized human patients, the zoonotic potential of the organism should be kept in mind when dealing with this disease in SAC.

Genotyping of Francisella tularensis strains by pulsed-field gel electrophoresis, amplified fragment length polymorphism fingerprinting, and 16S rRNA gene sequencing

We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

Microbial communities in the respiratory tract of patients with interstitial lung disease

Background Molecular methods based on phylogenetic differences in the 16S rRNA gene are able to characterise the microbiota of the respiratory tract in health and disease. Objectives Our goals were (1) to characterise bacterial communities in lower and upper airways of patients with interstitial lung disease (ILD) and (2) to compare the results with the microbiota of patients with Pneumocystis pneumonia (PCP) and normal controls. Methods We examined the upper and lower respiratory tract of 18 patients with ILD of whom 5, 6, and 7 had idiopathic interstitial pneumonia (IIP), non-IIP and sarcoidosis, respectively. In addition, six immune-compromised patients with PCP and nine healthy subjects were included as controls. Exclusion criteria were recent bacterial/viral respiratory tract infection, HIV-positivity and subjects receiving antibiotic therapy. Bronchoalveolar lavage fluid and oropharyngeal swabs were simultaneously collected, and microbiota was characterised by ultra-deep 16S rRNA gene sequencing. Results The microbiota in lower airways of the majority of patients (30; 90%) primarily consisted of Prevotellaceae, Streptococcaceae and Acidaminococcaceae. α and β diversity measurements revealed no significant differences in airway microbiota composition between the five different groups of patients. Comparison of bacterial populations in upper and lower respiratory tract showed significant topographical discontinuities for 7 (23%) individuals. Conclusions IIP, non-IIP and sarcoidosis are not associated with disordered airway microbiota and a pathogenic role of commensals in the disease process is therefore unlikely. Nevertheless, molecular analysis of the topographical microbiota continuity along the respiratory tract may provide additional information to assist management of individual patients.