Molecular epidemiology of tuberculosis in Cambodian children.

SUMMARY We analysed Mycobacterium tuberculosis strains from children, hospitalized from January 2004 to July 2008 in the largest paediatric hospital complex in Cambodia. Specimens were tested for drug susceptibility and genotypes. From the 260 children, 161 strains were available. The East African-Indian genotype family was the most common (59.0%), increasing in frequency with distance from the Phnom Penh area, while the frequency of the Beijing genotype family strains decreased. The drug resistance pattern showed a similar geographical gradient: lowest in the northwest (4.6%), intermediate in the central (17.1%), and highest in the southeastern (30.8%) parts of the country. Three children (1.9%) had multidrug-resistant tuberculosis. The Beijing genotype and streptomycin resistance were significantly associated (P < 0.001). As tuberculosis in children reflects recent transmission patterns in the community, multidrug resistance levels inform about the current quality of the tuberculosis programme.

IGRA-positive patients and interferon-gamma/interleukin-2 signatures: can the Fluorospot assay provide further information?

A goal of testing for latent tuberculosis (TB) infection is to identify individuals who are at increased risk for the development of active TB. No laboratory tool is currently available to distinguish between individuals in the process of progressing from latent TB infection towards active disease and those who are not. Determination of the interferon-gamma and interleukin-2 T cell signature might provide an additional and rapid tool to evaluate treatment necessity and clinical management of a patient. Here, we present three cases of interferon-gamma release assay-positive patients with differing interferon-gamma and interleukin-2 signatures when analyzed by the Fluorospot assay.

Successful management of a Clostridioides difficile ribotype 027 outbreak with a lean intervention bundle.

BACKGROUND: In a 2015 point-prevalence study, Clostridioides difficile 027, a hypervirulent ribotype, was absent from healthcare institutions in Switzerland. In late 2016, we detected an outbreak of C. difficile infection (CDI) with ribotype 027 occurring across several hospitals in the same hospital network. METHODS: The first cases of CDI due to ribotype 027 triggered an outbreak investigation, including whole genome sequencing (WGS) to identify outbreak strains. FINDINGS: Twenty-eight patients with CDI caused by ribotype 027 between December 2016 and December 2017 were identified, out of which 20 were caused by a single clone. Commonalities among these patients were hospitalization in the same room or on the same ward, receiving care from the same healthcare workers, and shared toilet areas. In addition to the epidemiological links suggesting possible transmission pathways between cases, WGS confirmed the clonality of this C. difficile 027 outbreak. The outbreak was contained by isolation precautions, raising awareness among healthcare workers, harmonizing diagnostic algorithms, and switching to a sporicidal agent for environmental disinfection. Of note, neither default gowning and gloving nor hand washing with water and soap were implemented. CONCLUSION: This C. difficile 027 outbreak was recognized belatedly due to lack of screening for this ribotype in some hospitals, and was contained by a swift response with simple infection prevention measures and adapting the laboratory approach. In order to have a better understanding of C. difficile epidemiology, diagnostic approaches should be standardized, CDI declared notifiable, and longitudinal data on prevalent ribotypes collected in countries where this is not established.

Evaluating the potential of IP-10 and MCP-2 as biomarkers for the diagnosis of tuberculosis.

The aim of the present study was to evaluate the potential of diagnostic tests based on interferon-gamma inducible protein (IP)-10 and monocyte chemotactic protein (MCP)-2, and compare the performance with the QuantiFERON TB Gold In-Tube (QFT-IT; Cellestis, Carnagie, Australia) test. IP-10 and MCP-2 were determined in supernatants from whole blood stimulated with Mycobacterium tuberculosis-specific antigens. Samples were obtained from 80 patients with culture- and/or PCR-proven tuberculosis (TB), and 124 unexposed healthy controls: 86 high school students and 38 high school staff. IP-10 and MCP-2 test cut-offs were established based on receiver operating characteristic curve analysis. TB patients produced significantly higher levels (median) of IP-10 (2158 pg x mL(-1)) and MCP-2 (379 pg x mL(-1)) compared with interferon (IFN)-gamma (215 pg x mL(-1)). The QFT-IT, IP-10 and MCP-2 tests detected 81, 83 and 71% of the TB patients; 0, 3 and 0% of the high school students and 0, 16 and 3% of the staff, respectively. Agreement between tests was high (>89%). By combining IP-10 and IFN-gamma tests, the detection rate increased among TB patients to 90% without a significant increase in positive responders among the students. In conclusion, interferon-gamma inducible protein-10 and monocyte chemotactic protein-2 responses to Mycobacterium tuberculosis-specific antigens could be used to diagnose infection. Combining interferon-gamma inducible protein-10 and interferon-gamma may be a simple approach to increase the detection rate of the Mycobacterium tuberculosis-specific in vitro tests.

Development of real-time PCR for the differential detection and quantification of Ureaplasma urealyticum and Ureaplasma parvum.

Ureaplasma parvum and Ureaplasma urealyticum are recently recognized species of the genus Ureaplasma. In humans, Ureaplasma spp. can be found on mucosal surfaces, primarily in the respiratory and urogenital tracts. They have been implicated in various human diseases such as nongonococcal urethritis, intrauterine infections in association with adverse pregnancy outcome and fetal morbidity, and pneumonitis in immunocompromised hosts. We have developed two quantitative real-time PCR assays to differentially detect U. parvum and U. urealyticum. Based upon the sequence information of the urease gene (ureB), we designed two TaqMan primer and probe combinations specific for U. parvum and U. urealyticum, respectively. The assays did not react with nucleic acid preparations from 16 bacterial species commonly encountered in relevant clinical specimens, including seven urease-producing species. Each assay had a detection limit of approximately five copies per reaction of the respective gene target. The results suggest that these assays are both sensitive and specific for U. parvum and U. urealyticum. Further investigation of both assays using clinical specimens is appropriate.

Mycobacterium avium resists exposure to the acidic conditions of the stomach.

Organisms of the Mycobacterium avium complex are common pathogens in immunosuppressed patients such as individuals with AIDS. There is evidence that in AIDS patients, the main route for M. avium infection is the gastrointestinal tract. The stomach is a formidable barrier to pathogens and the ability to resist exposure to pH lower than 3 has been shown to be a virulence determinant of enteric pathogens. Incubation of three clinical isolates of M. avium under acidic pH revealed resistance of M. avium grown both to the exponential and stationary phase at pH 2.2 for 2 h. Inhibition of protein synthesis had no effect on the acid tolerance. When the duration of the incubation at pH 2.2 was extended to 24 h, bacteria grown to the stationary phase had a significantly greater tolerance to acid than exponential phase bacteria. M. avium incubated with acid in the presence of water was significantly more resistant to pH 2.2 than M. avium in the presence of buffer. Pre-adaptation in water prior to exposure to acidic conditions was also associated with increased resistance to pH 2.2. Isoosmolarity of Hank's balanced salt solution appears to be responsible for the impaired resistance to acid between 2 and 24 h of incubation. These findings indicate that M. avium is naturally tolerant to pH<3 and that pre-adaptation under conditions similar to the conditions where M. avium is found in the environment results in increased acid resistance.

[The prevalence of salmonella, yersinia and mycobacteria in slaughtered pigs in Switzerland]. / Verbreitung von Salmonellen, Yersinien und Mykobakterien bei Schlachtschweinen in der Schweiz.

Clinically healthy food animals can be reservoirs for various foodborne pathogens. In general, such animals do not have lesions that are visible during meat inspection. Pigs are considered to be carriers of salmonella, yersinia and mycobacteria, but the risk of transmission to humans is difficult to assess. The aim of this study was to estimate the actual prevalence of the three above mentioned pathogens in the Swiss pig population and to comment on their significance. A total of 570 samples each of tonsils and mesenteric lymphnodes, were collected at two slaughterhouses from carcasses of apparently healthy pigs and analyzed for the presence of salmonella, yersinia and mycobacteria. The prevalence of salmonella (0.9%) was found to be lower than--while that of yersinia (8.1%) and mycobacteria (12.8%) about equal to--results reported from other European countries. Yersinia typing showed that serotype O:9 of Yersinia enterocolitica (2.5%) was 6 to 7 times more frequent than serotype O:3 (0.4%)--formerly the most frequent serotype. Mycobacterium avium was the most frequent isolate (90.7%) among the mycobacteria isolated. Although all three pathogens are present in the Swiss pig population, we consider the risk of transmission to humans via consumption of pork as low. Appropriate preventive measures and quality management should contribute to keep the risk under control.

[Occurrence of Mycobacterium genavense in birds]. / Vorkommen von Mycobacterium genavense bei Vögeln.

A total of 253 birds were investigated to determine the presence of mycobacteria. Scrapings from various internal organs were stained according to Ziehl-Neelsen, and acid-fast bacilli were found in 26 birds (10.2%). Cultivation of mycobacteria was attempted from 22 livers, 12 spleens, 14 kidneys, 12 lungs, and 9 intestines from these 26 birds. Each sample was first decontaminated using a modified sodium dodecyl sulfate method, and three media were inoculated (Bactec 12 B; Löwenstein-Jensen, Herrold). Using a Polymerase-Chain-Reaction-Restriction-Enzyme-Analysis (PRA), M. genavense could be identified in 19 of 26 birds (73%). M. avium could be isolated from three birds and M. fortuitum from one bird. In total, mycobacteriosis was the primary diagnosis made in 24 of 26 birds (92%). A presumptive diagnosis of mycobacteriosis was already made macroscopically in 14 of these birds. In the remaining 10 cases, bacteriological and histological investigations with specific staining methods were necessary for diagnosis. Several different histological changes were found. We observed individual macrophages as well as epitheloid cell proliferation, with variable, relatively mild inflammatory and fibrous reactions. We could not find any correlation between infection with a mycobacterium species and a specific tissue reaction pattern. This report demonstrates that M. genavense is an important cause of avian mycobacteriosis especially in pet birds.

Laboratory diagnosis of tuberculosis in a large pediatric hospital in Cambodia.

SETTING: Tuberculosis laboratory in the Jayavarman VII Children's Hospital in Siem Reap, part of the Kantha Bopha Hospitals, the largest pediatric hospital complex in Cambodia. OBJECTIVE: To determine the efficiency of on-site microscopy and rRNA amplification in children with a clinical diagnosis of tuberculosis (TB) and specimen sampling for culture. RESULTS: From 1 July 2005 to 31 March 2006, 52,400 children were admitted to the hospital. Among these, 405 children had tuberculosis, including 91 (22.5%) laboratory-confirmed cases, or respectively 7.7 and 1.7 per 1000 admissions. Among cases confirmed by microscopy or rRNA assay, rRNA identified 91.2%. Among all culture-confirmed cases, rRNA identified 90.5%. Culture alone contributed 7.1% to all laboratory confirmed cases. The yield of culture from preserved specimens was not affected by shipment delay. For 97.4% of the children, the maximum turnaround time for the on-site laboratory result was 48 h. CONCLUSION: Implementation of a mycobacteriology service in a referral hospital is feasible, as the molecular technique is highly efficient. Storage of specimen aliquots allows subsequent culture without loss of viability due to shipment delay.

Accidental infection of veterinary personnel with Mycobacterium tuberculosis at necropsy: a case study.

Mycobacterium tuberculosis is the main cause of human tuberculosis. Infection in companion animals is mainly acquired from close contact to a diseased human patient and hence rarely diagnosed in countries with low tuberculosis incidence rates. Therefore the general awareness of the disease might be low. Here we report the potential risk of infection for veterinary personnel with M. tuberculosis during the clinical and pathological examination of a dog with unexpected disseminated tuberculosis. The dog had presented with symptoms of a central nervous system disease; rapid deterioration prevented a complete clinical workup, however. Post-mortem examination revealed systemic mycobacteriosis, and M. tuberculosis was identified by PCR amplification of DNA extracts from paraffin-embedded tissue sections and spoligotyping. Contact investigations among the owners and veterinary personnel using an IFN-γ release assay indicated that the index dog did not infect humans during its lifetime. Serological and IFN-γ release assay results of one of two cats in direct contact with the index dog, however, suggested that transmission of M. tuberculosis might have occurred. Importantly, all three pathologists performing the necropsy on the dog tested positive. Accidental infection was most likely due to inhalation of M. tuberculosis containing aerosols created by using an electric saw to open the brain cavity. As a consequence routine necropsy procedures have been adapted and a disease surveillance program, including tuberculosis, has been initiated. Our results highlight the importance of disease awareness and timely diagnosis of zoonotic infectious agents in optimizing work safety for veterinary personnel.

[Interferon-gamma release assays in tuberculosis diagnostics]. / Interferon-gamma-Release-Assays in der Tuberkulosediagnostik.

Two years ago, CE certified interferon-gamma release assays (IGRA) were launched on the German market (QuantiFERON-TB Gold In-Tube and T-SPOT-TB). Since this time, a multitude of studies have analysed these assays. Guidelines have been elaborated by national expert committees of England, the USA and Switzerland. However, standards of tuberculosis diagnostics and management may vary from country to country. This statement provides practice relevant recommendations for indications, pre-analytics and the interpretation of IGRA test results under different clinical conditions. The IGRA are integrated into existing guidelines for the management of tuberculosis.

[Clinical presentation and therapy of Mycobacterium marinum infection as seen in 12 cases]. / Klinische Präsentation und Therapie der Mycobacterium-marinum-Infektion anhand von 12 Fallbeispielen.

BACKGROUND AND OBJECTIVE: Mycobacterium marinum (M.m.) is the causative pathogen of skin infections that have been called "swimming pool granulomas". An increasing number of reports that deep structures are involved in these infections was the reason for studying the clinical presentation and response of the infection to different therapeutic regimens. PATIENTS AND METHODS: All patients (eight men, four women, age range 18-73 years) were included in whom, between january 1991 and February 1995, M.m. infection had been proven by culture. The clinical data of these patients were retrospectively obtained by standardized questionnaire. RESULTS: The infection was limited to the skin in four of the twelve patients, deep structures only were involved in three, and five had both. Infections limited to the skin were successfully treated with sulphamethoxazole and trimethoprim or with tetracyclines, while rifampicin, alone or in combination with ethambutol, was efficacious when deep structures were involved. No surgical intervention was--or should be--performed. CONCLUSIONS: Infections with M.m. often involve deep structures, even in the absence of the skin being involved. The term "swimming pool granuloma" is, therefore, misleading when the infection is limited to he skin. A history of a chronic and indolent course, frequent changes of doctor and striking polypharmacy in its treatment are pointers to this infection.

Screening of respiratory tract specimens for the presence of Mycobacterium tuberculosis by using the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test.

A prospective 2-month trial involving 617 respiratory tract specimens was conducted to compare sensitivity, specificity, and predictive values of the newly developed Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test kit (AMTDT; Gen-Probe, Inc., San Diego, Calif.) for the rapid detection of Mycobacterium tuberculosis and of fluorescent acid-fast staining versus combined BACTEC 12B and solid-medium cultures as the "gold standard." A total of 590 specimens were culture and AMTDT negative. Twenty-one (3.4%) cultures yielded M. tuberculosis. Of these, 15 (71.4%) were detected by AMTDT, whereas 6 (28.6%) were missed. M. tuberculosis did not grow in six (28.6%) of AMTDT-positive specimens derived from three patients under treatment for tuberculosis. AMTDT exhibited a sensitivity, a specificity, a negative predictive value, and a positive predictive value of 71.4, 99, 99, and 71.4%, respectively. In comparison, the same values for fluorescent microscopy were 66.7, 98.3, 98.8, and 58.3%, respectively. AMTDT was easy to perform and highly specific. However, a screening test would require an improved sensitivity and, when feasible, the implementation of an internal amplification control.

[Systemic Nocardia infection in an AIDS patient]. / Systemische Nokardiose bei einem Aids-Patienten.

We report the case of a patient with Aids, Kaposi's sarcoma and a systemic nocardial infection. After surgical drainage and adequate antibiotic treatment the infection was cured. Under secondary antimicrobial prophylaxis no relapse has occurred to date. Generalized N. asteroides infections have been rarely described in patients with Aids and the possible reasons for this are discussed.

[Rhodococcus erythropolis infection in HIV-associated immunodeficiency]. / Rhodococcus-erythropolis-Infektion bei HIV-assoziierter Immunschwäche.

We report the first case in the literature of human infection with Rhodococcus erythropolis in an HIV-positive patient. A 24-year-old bisexual flight attendant had severe HIV-associated immunodeficiency with a CD4 cell count of 0.02 G/l. He complained of multiple subcutaneous nodules at different sites on the extremities. Biopsy of one node at his left knee revealed granulomatous inflammation filled with acid-fast rods. These bacteria were identified as Rhodococcus erythropolis. The disseminated infection was restricted to the skin and showed a slow response to long-term therapy with amoxicillin/clavulanic acid. No relapse has been observed more than 6 months after discontinuation of antibiotic therapy. The etiology of a concomitant polyarthritis remains unknown; a relationship with the rhodococcus infection is possible as the arthritis responded well to the antibiotic therapy and did not reactivate after discontinuation of antibiotics. Due to the difficult isolation technic, this pathogen may be overlooked in routine diagnostic procedures. The implications in clinical practice are discussed.

Mycobacterium genavense infection in a patient with long-standing chronic lymphocytic leukaemia.

We describe the first case of disseminated infection with Mycobacterium genavense in an HIV-seronegative patient with a chronic haematological disorder. Our patient, an 80-year-old woman, had been under long-term treatment with chlorambucil (partially in combination with prednisone) for B-cell chronic lymphocytic leukaemia (B-CLL). When she developed general fatigue and progressive anaemia, as well as progressive lymphadenopathy and splenomegaly, bone marrow biopsy revealed granulomas with acid-fast bacilli, and cultures of both bone marrow and blood grew M. genavense. The patient's CD4+ cell count was approximately 100 microL(-1). Treatment with clarithromycin, ethambutol and rifabutin resulted in improvement of anaemia and general health as well as in regression of lymphadenopathy and splenomegaly.

Evaluation of the COBAS AMPLICOR MTB system.

A panel of 1,012 respiratory sediments was retrospectively tested by PCR amplification to detect Mycobacterium tuberculosis with the COBAS AMPLICOR MTB system. The sensitivities and specificities of COBAS and fluorescence microscopy compared to culture were 92.6 versus 95.6% and 99.6 versus 95.3%, respectively. Inhibition occurred in 48 (4.7%) specimens.

[Disseminated nontuberculous mycobacterial infections in AIDS patients]. / Disseminierte nicht-tuberkulöse Mykobakteriosen bei Aids-Patienten.

Disseminated nontuberculous mycobacteriosis is a frequent and late complication of HIV infection. All the 13 patients described here had CD4-lymphocyte counts < 20/mm3. The causative agent was mainly M. avium complex. But we also found, for the first time, a double infection with M. avium complex and M. "genavense" and one patient with growth of M. shimoidei in the blood culture. Clinical signs are nonspecific (fever, reduced performance, anemia). Positive cultures of blood or tissue biopsies are diagnostic. The therapeutic approach is the combination of new macrolides with other antimycobacterial agents. Prognosis is poor, mainly due to advanced immunodeficiency, but two of our patients survived more than one year after diagnosis. Prophylactic treatment should be considered in patients with CD4-counts less than 50/mm3.

[Current developments in Mycobacteria diagnosis]. / Neue Entwicklungen in der Mykobakteriendiagnostik.

The reemergence of tuberculosis in the industrialized countries has hastened the development of new laboratory techniques. Hence, well-known shortcomings of traditional techniques such as the lack of a rapid and specific detection system, the delayed availability of species identification and drug susceptibility results, and the lack of a reliable method for determining strain identity for epidemiological purposes, have become immediate targets for implementing molecular biology techniques. In particular, nucleic acid amplification techniques, restriction fragment-length polymorphism and single-strand conformation polymorphism analyses have dramatically improved diagnostic timeliness and accuracy of mycobacteriology laboratory results. Our paper reviews recent developments and comments on the diagnostic applications of the new tools as compared to traditional techniques.