[Show abstract][Hide abstract] ABSTRACT: Variable number of tandem repeat (VNTR) typing with a panel of 24 loci is the current gold standard in the molecular typing of Mycobacterium tuberculosis complex. However, due to technical problems a part of the loci can often not be amplified in the multiplex PCRs. Therefore, a considerable number of single-locus PCR reactions have to be repeated for the loci with missing results, which impairs the laboratory workflow. Therefore, the original in-house method described by Supply et al. in 2006 was re-evaluated. We modified 7 primers and the PCR mastermix, resulting in a strongly optimized in-house 24-locus VNTR typing method. The percentage of instantly complete 24-locus VNTR patterns, detected in the routine flow of typing activities increased to 84.7% compared to 72.3% for the typing conducted with the commercially available MIRU-VNTR typing kit by GenoScreen. The analytical sensitivity of the optimized in-house method was assessed by serial dilutions of M. tuberculosis in broncho-alveolar lavage fluid (BALF). The 1:10 dilution of the different strains tested was the lowest dilution for the detection of a complete 24-locus VNTR pattern.The optimized in-house 24-locus VNTR typing method will reduce the turn-around-time of typing significantly and also the financial burden of these activities.
Journal of clinical microbiology 02/2014; · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 43-year-old female developed an Epstein-Barr virus (EBV)-positive post-transplant lymphoproliferative disorder (PTLD) in the central nervous system (CNS), 14 years after renal transplantation. One year prior to presentation, the patients' treatment regimen was altered from cyclosporine, azathioprine, and prednisone to mycophenolate mofetil and prednisone. Magnetic resonance imaging of the brain revealed lesions suspect for malignant lymphoma. The EBV real-time polymerase chain reaction (PCR) on peripheral blood was negative, but highly positive on cerebrospinal fluid. EBV-positive PTLD was confirmed using histological analysis of cerebral biopsies. Despite tapering of immune suppressive medication and treatment with rituximab and chemotherapy, the patient deceased 50 days after presentation. This case illustrates that vigilance is required when presented with a negative EBV PCR result on peripheral blood when PTLD of the CNS is suspected. This late presentation suggests a relation to the switch in immunosuppressive regimen 1 year earlier.
Transplant International 08/2012; 25(11):e113-6. · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A young female health professional was diagnosed with pulmonary tuberculosis caused by Mycobacterium bovis. Source finding and contact tracing was initiated by the regional municipal health service using both tuberculin skin test and QuantiFERON(®)-TB Gold (QFT-GIT (IGRA). The strain appeared near-identical to that of an elderly Dutch patient.
Medical Microbiology and Immunology 03/2012; 201(3):397-400. · 3.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Indonesia has a concentrated but rapidly growing HIV epidemic. We examined the effect of HIV on causative organisms, clinical features and prognosis of adult meningitis.
A prospective cohort study.
All adult patients at a referral hospital who underwent cerebrospinal fluid examination for suspected meningitis were examined for HIV and included in a prospective cohort study. Microbiological testing was done for common bacterial pathogens, mycobacteria and fungi. Patients were followed for at least 6 months, and logistic regression models were used to identify risk factors for mortality.
Among 185 patients who mostly presented with subacute meningitis, 60% were male and the median age was 30 years. HIV infection was present in 25% of the patients; almost two-thirds were newly confirmed, and all presented with severe immunosuppression (median CD4 cell count 13/microl, range 2-98). One-third of HIV-infected patients had cryptococcal meningitis whereas two-thirds suffered from tuberculosis. After 1 month, 41% of patients had died. HIV infection was strongly associated with 1-month mortality (adjusted odds ratio 12.15; 95% confidence interval 3.04-15.72) and death during extended follow-up (hazard ratio 2.48; 95% confidence interval 1.97-5.74).
Although HIV is still uncommon in the general population in Indonesia, its prevalence among adult meningitis cases already seems high. Mycobacterium tuberculosis and Cryptococcus neoformans are the main causes of meningitis in this setting, and mortality is very high, especially in HIV-infected patients. Our data suggest that adult meningitis cases in Indonesia should be screened routinely for HIV infection. Further studies are needed to address the high mortality.
AIDS (London, England) 09/2009; 23(17):2309-16. · 6.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Comparison of Mycobacterium tuberculosis genotype distributions in different areas might help to find determinants of the emergence of certain genotypes, such as the Beijing family. In this study, M. tuberculosis isolates originating from patients from two Indonesian islands were genotyped, and possible associations with patients' characteristics and drug resistance were explored. A high degree of genetic diversity was observed among the M. tuberculosis strains, and a significant difference was found in the geographical distribution of genotype families. The predominant Beijing genotype family was isolated from 268 of 813 patients from West Java (33.0%) versus 12 of 84 patients from Timor (14.3%) (P = 0.002). Family F (East African-Indian) (33.3%) and family D (Latin American and Mediterranean) (20.0%) were more prevalent in Timor. No significant associations were found between genotype families and age, vaccination with Mycobacterium bovis BCG, previous treatment, disease localization, or drug resistance. Possible explanations for the differences in the geographical distribution of the M. tuberculosis genotypes are discussed.
Journal of clinical microbiology 11/2008; 46(11):3639-45. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tuberculosis (TB) in Africa is increasing because of the human immunodeficiency virus (HIV) epidemic, and in HIV/AIDS patients it presents atypically. Pulmonary tuberculosis (PTB) in Africa is mainly diagnosed clinically, by chest radiograph or by sputum smear for acid fast bacilli (AFB).
We evaluated in 120 HIV-infected patients with chest infection the diagnostic accuracy of AFB smear of sputum and bronchoalveolar lavage (BAL) fluid, sputum Mycobacterium tuberculosis (MTB) culture, real-time PCR and MycoDot serological test, using MTB culture of BAL fluid as gold standard. We correlated PCR cycle threshold values (C(T)) to the culture results. Retrospectively, we evaluated the development of active TB in patients with positive PCR but negative culture.
Culture of BAL fluid identified 28 patients with PTB. Fifty-six patients could not produce adequate sputum. Sputum AFB smear and the serological test had sensitivities of 66.7% and 0%, respectively. PCR with C(T) 40 was positive in 73 patients, 27 of whom were also TB culture positive (96.4% sensitivity and 52.3% specificity of PCR). PCR with C(T) 32 had sensitivity of 85.7% and specificity of 90.9% to diagnose PTB in BAL. No patients with positive PCR but negative culture developed active TB during 18 months follow-up.
In these HIV-infected patients, AFB smear and serology had very low sensitivities. PCR of BAL with C(T) value 32 had improved specificity to diagnose active PTB. A prospective follow-up study is warranted in TB/HIV endemic settings, applying real time PCR to both sputum and BAL.
Tropical Medicine & International Health 11/2007; 12(10):1210-7. · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The etiology of Crohn's disease in humans is largely unknown. Clinical signs of Crohn's disease partly resemble the clinical picture of Johne's disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis. Because of the high prevalence of these bacteria in (products of) ruminants and their remarkable thermostability, concern has been raised about the possible role of these bacteria in the pathogenesis of Crohn's disease. In an attempt to develop a molecular typing method to facilitate meaningful comparative DNA fingerprinting of M. avium subsp. paratuberculosis isolates from the human and animal reservoirs, multilocus variable-number tandem-repeat analysis (MLVA) was explored and compared to IS900 restriction fragment length polymorphism (RFLP) typing. MLVA typing subdivided the most predominant RFLP type, R01, into six subtypes and thus provides a promising molecular subtyping approach to study the diversity of M. avium subsp. paratuberculosis.
Journal of Clinical Microbiology 12/2004; 42(11):5022-8. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium heckeshornense is a rare isolate in clinical specimens. We performed simultaneous 16S rRNA sequence analysis of a mycobacterium culture and a histopathology specimen to determine the relevance of M. heckeshornense infection in an immunocompetent patient initially presenting with pneumothorax.
Journal of Clinical Microbiology 10/2004; 42(9):4386-9. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tuberculosis was diagnosed in a student nurse. The source of infection was unknown and no positive culture was available.
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.
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.
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.
The Netherlands Journal of Medicine 10/2001; 59(3):152-7. · 2.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Clinical Microbiology 03/1999; 37(2):471-4. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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".
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Clinical Microbiology 08/1998; 36(7):1840-5. · 4.23 Impact Factor