Tuberculosis Caused by Mycobacterium orygis in Dairy Cattle and Captured Monkeys in Bangladesh: a New Scenario of Tuberculosis in South Asia.

Mycobacterium orygis, commonly known as the oryx bacillus and a newly proposed Mycobacterium tuberculosis complex subspecies, was isolated from 18 cattle in a dairy farm and two captured rhesus monkeys in a zoo in Bangladesh. All the infected animals had tuberculosis lesions in their lungs, suggesting transmission and infection with M. orygis by an airborne route. The 20 isolates were analysed using a range of conventional and molecular typing methods, and RD-deletion typing and sequencing of selected genes confirmed the isolates as M. orygis. Multiple-locus variable-number tandem repeat analysis (MLVA) allowed the isolates to be divided into three clusters based on the relatedness of their MLVA profiles. The two monkey isolates shared the same MLVA pattern with 15 of the cattle isolates, whereas the remaining three cattle isolates had different patterns, even though the latter animals had been kept in the same dairy farm. The diversity observed among isolates may suggest the bacteria have been established in this area for a long period. This study along with other recent findings that report the detection of M. orygis from animals as well as humans originating from South Asia potentially indicate endemic distribution of M. orygis in South Asia.

Case-control comparison of bacterial and protozoan microorganisms associated with gastroenteritis: application of molecular detection.

The introduction of molecular detection of infectious organisms has led to increased numbers of positive findings, as observed for pathogens causing gastroenteritis (GE). However, because little is known about the prevalence of these pathogens in the healthy asymptomatic population, the clinical value of these additional findings is unclear. A case-control study was carried out in a population of patients served by general practitioners in the Netherlands. A total of 2710 fecal samples from case and matched control subjects were subjected to multiplex real-time PCR for the 11 most common bacterial and four protozoal causes of GE. Of 1515 case samples, 818 (54%) were positive for one or more target organisms. A total of 49% of the controls were positive. Higher positivity rates in cases compared to controls were observed for Campylobacter spp., Salmonella spp., Clostridium difficile, enteroinvasive Escherichia coli/Shigella spp., enterotoxigenic E. coli, enteroaggregative E. coli, atypical enteropathogenic E. coli (EPEC), Cryptosporidium parvum/hominis, and Giardia lamblia. However, Dientamoeba fragilis and Shiga-like toxigenic E. coli were detected significantly less frequent in cases than in controls, while no difference in prevalence was found for typical EPEC and enterohemorrhagic E. coli. The association between the presence of microorganisms and GE was the weakest in children aged 0 to 5 years. Higher relative loads in cases further support causality. This was seen for Campylobacter spp., Salmonella spp., enterotoxigenic E. coli, and C. parvum/hominis, and for certain age categories of those infected with C. difficile, enteroaggregative E. coli, and atypical EPEC. For D. fragilis and Shiga-like toxigenic E. coli/enterohemorrhagic E. coli, pathogen loads were lower in cases. Application of molecular diagnostics in GE is rapid, sensitive and specific, but results should be interpreted with care, using clinical and additional background information.

Localised pyogranulomatous dermatitis due to Mycobacterium abscessus in a cat: a case report.

A case of pyogranulomatous dermatitis, caused by Mycobacterium abscessus, an unusual opportunistic Mycobacterium spp., is described in a cat. Histopathological examination of the affected skin confirmed the diagnosis and Ziehl-Neelsen staining revealed acid-fast rods. A rapidly growing mycobacterium was found after culture on a Löwenstein-Jensen medium. Real-time polymerase chain reaction for the 16S rDNA (434bp) sequence and the sequence of the rpoB gene (359bp) revealed 99% and 100% matches, respectively, with M. abscessus. This is the first report of a feline infection caused by this organism in Europe.

Isolation of Mycobacterium mageritense from the lung of a harbor porpoise (Phocoena phocoena) with severe granulomatous lesions.

Post-mortem investigation of a harbor porpoise (Phocoena phocoena) found dead on the beach of the island of Vlieland, The Netherlands, revealed severe granulomatous changes in the right lung lobe. Ziehl Neelsen staining demonstrated relatively large acid-fast rods. Mycobacterial culture yielded a fast-growing mycobacterium, which was identified by molecular biological methods as Mycobacterium mageritense. Autolysis prevented histopathology. It was tentatively concluded that the granulomatous changes were the cause of porpoise's death and that M. mageritense was the causative agent. This is the first report of the isolation and molecular identification of this mycobacterium in a nonhuman animal species and the first association with the marine environment.

Single nucleotide polymorphism profiling assay to exclude serum sample mix-up.

BACKGROUND AND OBJECTIVES: Sample mix-ups are a threat to the validity of clinical laboratory test results. To detect serum sample mix-ups we developed a single nucleotide polymorphism (SNP) profiling test. SNPs are frequent sequence variations in the human genome. Each individual has a unique combination of these nucleotide variations. MATERIALS AND METHODS: Predeveloped SNP amplification assays are commercially available. We recently discovered that these SNP assays could be applied to serological samples, which is not self-evident because a key step in serum preparation is removal of white blood cells, the major source of DNA, from blood. DNA was extracted from serum samples. Real-time polymerase chain reaction (PCR) analysis of the purified DNA using a selection of 10 SNP assays provided SNP profiles. RESULTS: The applicability of the SNP profiling test was demonstrated by means of a case where hepatitis E virus serological determinations of four serum samples of one patient seemed inconsistent. SNP profiling of the samples demonstrated that this was due to the enzyme-linked immunosorbent assay test instead of sample mix-up. CONCLUSION: We have developed an SNP profiling assay that provides a way to link human serum samples to a source, without post-PCR processing. The chance for two randomly chosen individuals to have an identical profile is 1 in 18 000. Solving potential serum sample mix-ups will secure downstream evaluations and critical decisions concerning the patients involved.

Specific and sensitive detection of Neisseria gonorrhoeae in clinical specimens by real-time PCR.

Early diagnosis of Neisseria gonorrhoeae infections is important with regard to patients' health and infectivity. We report the development of a specific and sensitive TaqMan assay for the detection of N. gonorrhoeae in clinical samples. The target sequence is a 76-bp fragment of the 5' untranslated region of the opa genes that encode opacity proteins. A panel of 448 well-defined N. gonorrhoeae isolates was used to evaluate and optimize the assay. The method employs two minor-groove binding probes, one of them recognizing a newly identified sequence in the opa genes. Testing a large panel of related and unrelated microorganisms revealed that other Neisseria strains and other microorganisms tested negative in the opa test. With a lower detection limit of one genome per reaction, the opa test appeared more sensitive than both the COBAS AMPLICOR (Roche Diagnostics Nederland BV, Almere, The Netherlands) and a LightCycler 16S rRNA test. Analysis of a panel of 122 COBAS AMPLICOR-positive samples revealed that 68% were negative in both the 16S rRNA test and the opa assay (confirming that the COBAS AMPLICOR test produces false positives), while 30% were positive in both assays. Three samples were opa positive and 16S rRNA negative, which may be due to the higher sensitivity of the opa assay. We conclude that the opa gene-based real-time amplification assay offers a sensitive, specific, semiquantitative, and reliable assay suitable for the detection of N. gonorrhoeae in clinical specimens and/or for confirmation of less specific tests.

Use of DNA extracts from Ziehl-Neelsen-stained slides for molecular detection of rifampin resistance and spoligotyping of Mycobacterium tuberculosis.

Multidrug resistance among new cases of tuberculosis (TB) is increasingly becoming a significant problem in countries with a high prevalence of TB and with inadequate therapies for TB. Rifampin resistance is widely used as a marker for multidrug-resistant (MDR) TB; therefore, a new approach to the retrospective measurement of rifampin resistance without the need of a viable culture has been introduced. In many developing countries culture is unavailable and diagnosis relies on clinical manifestations and the results of Ziehl-Neelsen staining of sputum smears. We determined rifampin resistance directly with DNA extracts from Ziehl-Neelsen-stained slides by identification of mutations in the rpoB gene using reverse line blot hybridization and DNA sequencing. Analysis of the rpoB gene revealed that samples containing rifampin-resistant Mycobacterium tuberculosis carried altered codons representing amino acid positions 516, 526, and 531 of the RNA polymerase. Although the sensitivities of both methods were equal (84%), sequencing of the rpoB gene was more accurate in identifying mutations in the core region of the rpoB gene. Sequence analysis of the rpoB gene in extracts from Ziehl-Neelsen-stained slides may be used to quantify more precisely the magnitude of MDR TB and, more importantly, provide information on trends in the development of resistance on a global scale. The nature of rifampin resistance and the genotype can be determined by analysis of Ziehl-Neelsen-stained slides in a laboratory equipped for sequencing and spoligotyping without the need to ship biohazardous materials.

Improvement of differentiation and interpretability of spoligotyping for Mycobacterium tuberculosis complex isolates by introduction of new spacer oligonucleotides.

The direct repeat (DR) region in Mycobacterium tuberculosis complex strains is composed of multiple well-conserved 36-bp DRs interspersed with nonrepetitive DNA spacer sequences of similar size. Clinical isolates show extensive polymorphism in this DR region, and this has led to the development of a 43-spacer reversed line blot methodology: spoligotyping. Although this method has contributed significantly to the molecular epidemiology of tuberculosis in the last decade, the discriminatory power and the readability of this method were not found to be optimal. In order to improve the discriminatory power, the usefulness of 43 redesigned oligonucleotides and the usefulness of 51 new spacer oligonucleotides were evaluated. For 314 M. tuberculosis complex strains isolated in the central part of The Netherlands over a 5-year period, 264 different IS6110 RFLP types could be distinguished, and 160 different spoligotype patterns were identified by traditional spoligotyping. After the introduction of 51 new spacer oligonucleotides, 14 additional spoligotypes were recognized. This enabled us to split 11 clusters of isolates identified by the traditional spoligotyping. Furthermore, on the basis of the new spacer oligonucleotides a dichotomy was found among the Beijing genotype isolates. Among 76 Mycobacterium bovis strains, 20 patterns were found by traditional spoligotyping and 30 patterns were found by novel probe spoligotyping, respectively. Nine M. bovis subsp. caprae isolates yielded six patterns by traditional spoligotyping and eight patterns by novel probe spoligotyping. A part of the redesigned oligonucleotides slightly improved the reading of spoligotype patterns. The reproducibility of spoligotyping, based on internal control probes, invariably yielded a high score; only 4 (1%) of the 314 patient isolates gave discrepant results. Analysis of a set of 31 duplicate M. tuberculosis complex strains demonstrated a 10% error rate for the identification of blinded duplicate samples. In a redundancy analysis, 40 essential spacer oligonucleotides of the 94-spacer sequences were selected, yielding the same number of spoligotype patterns. We propose to leave the traditional commercialized first-generation membrane for spoligotyping unchanged for current applications and to introduce a second-generation spoligotyping membrane whenever extended discrimination is required, e.g., for low-copy-number IS6110 strains or for phylogenetic studies of Beijing genotype strains.

Mycobacterium tuberculosis Beijing genotype strains associated with febrile response to treatment.

DNA fingerprinting has demonstrated predominance of the Beijing genotype among Mycobacterium tuberculosis strains isolated in Southeast Asia. We prospectively examined the occurrence of Beijing genotype strains in tuberculosis patients in Indonesia. Early in treatment, patients infected with Beijing genotype strains more often had fever unrelated to disease severity, toxicity, or drug resistance, indicating that Beijing genotype strains may have specific pathogenic properties.

Nosocomial transmission of tuberculosis to a nurse demonstrated by means of spoligotyping of a formalin-fixed bronchial biopsy.

BACKGROUND: Tuberculosis was diagnosed in a student nurse. The source of infection was unknown and no positive culture was available. METHODS: The diagnosis of tuberculous bronchitis was established on the grounds of a positive Mantoux test, the pathology of a bronchial biopsy and the results of a CT scan of the thorax. Spoligotyping of, for example, formalin-fixed tissue makes it possible to establish the diagnosis in a later phase after all. RESULTS: Cultures for Mycobacterium tuberculosis were not performed for the student nurse and Ziehl-Neelsen staining of the formalin-fixed bronchial biopsy was negative. The final tuberculosis diagnosis was confirmed by a PCR fingerprint technique, i.e., spoligotyping of a formalin-fixed biopsy specimen. By means of contact investigation and identification of the strain via spoligotyping, comparison of the spoligo patterns made it possible to treat both the patient and those infected by this person correctly. CONCLUSIONS: When there is a pronounced suspicion of tuberculosis and a microbiological culture is not available, it is recommended that supplementary spoligotyping of clinical specimens be carried out. The purpose is to confirm the diagnosis, trace the presumed source case and indirectly to provide information on the drug susceptibility of the relevant M. tuberculosis strain.

Retrospective analysis of the Beijing family of Mycobacterium tuberculosis in preserved lung tissues.

Direct repeat spoligotyping of 85 paraffin-embedded lung biopsies was used to investigated the occurrence around Beijing of the Beijing family of Mycobacterium tuberculosis. Samples ranged in time from 1956 to 1990. Hybridization patterns were found with 49 (58%) samples, and 45 (92%) produced typical Beijing family patterns extending over the 34-year period.

Diagnosis of Mycobacterium microti infections among humans by using novel genetic markers.

As a result of DNA typing of Mycobacterium microti isolates from animals in the United Kingdom and The Netherlands, we diagnosed four human M. microti infections. These are the first M. microti infections among humans to be reported. Three of the patients were immunocompromised and suffered from generalized forms of tuberculosis. The fourth patient was a 34-year-old immunocompetent male with a persistent cough and undefined X-ray abnormalities. Two of the M. microti infections were recognized by their IS6110 restriction fragment length polymorphism (RFLP) patterns, which showed a high degree of similarity with those of M. microti strains isolated from a pig and a ferret in The Netherlands. The two other human M. microti infections were recognized by using the recently developed DNA fingerprinting method, "spoligotyping," directly on clinical material. All M. microti isolates from the United Kingdom and The Netherlands were found to contain an exceptionally short genomic direct repeat region, resulting in identical two-spacer sequence reactions in spoligotyping. In contrast, the highly similar IS6110 RFLP patterns of the vole strains from the United Kingdom differed considerably from the RFLPs of all M. microti strains isolated in The Netherlands, suggesting that geographic isolation led to divergent strains in the United Kingdom and on the continent.

Simultaneous detection and strain differentiation of Mycobacterium tuberculosis complex in paraffin wax embedded tissues and in stained microscopic preparations.

AIMS: To detect and differentiate Mycobacterium tuberculosis simultaneously by polymerase chain reaction (PCR) in clinical samples prepared for histopathological analysis and for microscopic detection of acid fast bacteria. METHODS: Paraffin wax embedded tissue samples and Ziehl-Neelsen (ZN) and auramine stained microscopic preparations from culture positive tuberculosis patients were subjected to DNA extraction and amplification by PCR. PCR was performed with primers specific for direct repeats and the product was detected by hybridisation to a set of 43 different oligonucleotides, a procedure designated as "spoligotyping". RESULTS: Mycobacterium tuberculosis complex DNA was detected in all of the 23 paraffin wax embedded tissues analysed. Strain differentiation was possible in 20 of the 23 paraffin wax embedded tissues. Mycobacterium tuberculosis complex DNA was also detected and typed in eight of 10 ZN stained microscopic preparations. The hybridisation patterns obtained from virtually all of these samples were identical to those obtained from DNA extracted from cultures. CONCLUSION: Simultaneous detection and strain differentiation of M. tuberculosis complex bacteria is possible in clinical samples prepared by current methods for microscopic and histopathological analysis, without the need to culture. The methodology described opens the way to rapid disclosure of outbreaks in high risk settings, such as hospitals and prisons, where dissemination of tuberculosis might be very fast as a result of a high prevalence of human immunodeficiency virus infected patients.

A rapid method for identification of Mycobacterium species by polyacrylamide gel electrophoresis of soluble cell proteins.

The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of an easily and rapidly prepared soluble protein fraction were used in conjunction with conventional techniques to identify different strains of Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. avium, M. kansasii, M. marinum, M. gastri, M. simiae and M. malmoense. Complete concordance of results from both methods was obtained with all species except those of the M. tuberculosis complex. With the SDS-PAGE technique, all strains of the M. tuberculosis complex were recognised as belonging to one species. By visual analysis of the SDS-PAGE polypeptide profiles, only minor differences between strains of the same species were seen and each species showed a characteristic polypeptide profile. Quantitation of the data by calculation of the Dice coefficient of similarity of the band positions obtained by densitometry indicated that the similarity between different strains of one species was 90-100% and the similarity between the species was in the range 30-45%. The results indicate that SDS-PAGE is a simple and rapid method for identifying mycobacterial strains.