PCR-based assays for the diagnosis of tuberculosis.
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DNA, Bacterial↗
Biomedical subjects
A H Kolk
Publications and source records attributed to A H Kolk.
Molecular diagnostic laboratory design--The Amsterdam experience.
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Facility Design and Construction↗
Interleukin-1 signaling is essential for host defense during murine pulmonary tuberculosis.
Interleukin (IL)-1 signaling is required for the containment of infections with intracellular microorganisms, such as Listeria monocytogenes and Leishmania major. To determine the role of IL-1 in the host response to tuberculosis, we infected IL-1 type I receptor-deficient (IL-1R(-/-)) mice, in which IL-1 does not exert effects, with Mycobacterium tuberculosis. IL-1R(-/-) mice were more susceptible to pulmonary tuberculosis, as reflected by an increased mortality and an enhanced mycobacterial outgrowth in lungs and distant organs, which was associated with defective granuloma formation, containing fewer macrophages and fewer lymphocytes, whereas granulocytes were abundant. Lymphocytes were predominantly confined to perivascular areas, suggesting a defective migration of cells into inflamed tissue in the absence of IL-1 signaling. Impaired host defense in IL-1R(-/-) mice was further characterized by a decrease in the ability of splenocytes to produce interferon-gamma. Analysis of these data suggests that IL-1 plays an important role in the immune response to M. tuberculosis.
Animals↗
Development of antigen detection assay for diagnosis of tuberculosis using sputum samples.
The rising incidence of tuberculosis worldwide means an increasing burden on diagnostic facilities, so tests simpler than Ziehl-Neelsen staining are needed. Such tests should be objective, reproducible, and have at least as good a detection limit as 10(4) bacteria/ml. A capture enzyme-linked immunosorbent assay (ELISA) was developed for detection of lipoarabinomannan (LAM) in human sputum samples. As a capture antibody, we used a murine monoclonal antibody against LAM, with rabbit antiserum against Mycobacterium tuberculosis as a source of detector antibodies. The sensitivity of the capture ELISA was evaluated by using purified LAM and M. tuberculosis whole cells. We were able to detect 1 ng of purified LAM/ml and 10(4) M. tuberculosis whole cells/ml. LAM could also be detected in culture filtrate of a 3-week-old culture of M. tuberculosis. The culture filtrate contained approximately 100 microgram of LAM/ml. The detection limit in sputum pretreated with N-acetyl-L-cysteine and proteinase K was 10(4) M. tuberculosis whole cells per ml. Thirty-one (91%) of 34 sputum samples from 18 Vietnamese patients with tuberculosis (32 smear positive and 2 smear negative) were positive in the LAM detection assay. In contrast, none of the 25 sputum samples from 21 nontuberculous patients was positive. This specific and sensitive assay for the detection of LAM in sputum is potentially useful for the diagnosis of tuberculosis.
Antibodies, Bacterial↗
Polymerase chain reaction based method for the detection of BCG retention after intravesical instillation in guinea pig bladders.
OBJECTIVE: In intravesical Bacille bilié de Calmette-Guérin (BCG) immunotherapy of superficial bladder cancer, a T cell mediated immunological reaction is associated with the antitumor activity. To gain insight into the approximate number of BCG bacteria retained in the normal, noninjured, urinary bladder after intravesical application responsible for induction of the immune reaction, the utility of a sensitive polymerase chain reaction (PCR) based assay was investigated in a guinea pig model. METHODS: After one single or six subsequent weekly instillations with 1x10(7) CFU of BCG, the bladders were resected and processed for BCG determination with PCR. The bladders were resected 24 h after instillation, aiming at (semi)quantifying the number of BCG organisms able to resist the natural voiding washout of the bladder. The PCR was based on amplification of a 249 base pair fragment of the insertion element IS6110 and is specific for bacteria belonging to the Mycobacterium tuberculosis complex which includes Mycobacterium bovis BCG. RESULTS: After one single instillation no detectable BCG retention was found. However, after six weekly instillations, BCG bacteria could be demonstrated in 2 out of 5 guinea pig bladders, indicating that the number of adhering BCG organisms was around the detection limit of the assay (600-1,000 BCG bacteria per bladder). CONCLUSIONS: The data suggest that after six instillations, the retention of BCG in the guinea pig bladder is enhanced as compared with one single instillation. This finding is suggestive of a role of the inflammatory process that is, besides immune system mediated reactions, associated with intravesical BCG instillations. The nature of the molecules involved in enhanced BCG retention after repeated instillations remains to be investigated.
Administration, Intravesical↗
Generalized mycobacterium genavense infection in HIV-infected patients: detection of the mycobacterium in hospital tap water.
We describe 3 HIV-infected patients with disseminated M. genavense infection. The use of corticosteroids possibly favoured colonization and dissemination of atypical mycobacteria in these patients with low CD4 cell counts and may have masked symptoms of infection. The fact that these patients were treated with highly active antiretroviral therapy (HAART) together with antimycobacterial therapy may explain that 1 patient was free from mycobacteria 16 months after the end of specific treatment. Hospital tap water contained M. genavense at a concentration of >10 bacteria/l as examined by PCR. This species caused 12% of cases of non-tuberculous disseminated mycobacteriosis in HIV-infected patients at our hospital.
AIDS-Related Opportunistic Infections↗
[Mycobacterium genavense infection in 2 HIV seropositive patients in Amsterdam].
Two patients with an HIV-I infection, a man aged 47 with confusion, aphasia and diarrhoea, and a man aged 32 with dysphagia, a non-productive cough and diarrhoea, were diagnosed as having a disseminated Mycobacterium genavense infection. Both had low counts of CD4+ T lymphocytes. They responded to antimycobacterial treatment. M. genavense was recognized in Geneva in the early nineties as a causative agent of disseminated mycobacterial infections in HIV-seropositive patients with poor cellular immunity. The clinical picture resembles that of a generalized infection with M. avium-intracellulare. M. genavense is a slowly growing mycobacterium which can be isolated and identified using enriched nutrient media and molecular-biological techniques. The infection probably begins in the gastrointestinal tract after oral contamination. DNA of M. genavense can be demonstrated in 25% of the intestinal biopsy samples of non-HIV-seropositive patients.
AIDS-Related Opportunistic Infections↗
Localization of cell division protein FtsQ by immunofluorescence microscopy in dividing and nondividing cells of Escherichia coli.
The localization of cell division protein FtsQ in Escherichia coli wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in ftsQ1(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the gamma form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane.
Antibodies, Monoclonal↗
Stabilized, freeze-dried PCR mix for detection of mycobacteria.
We report here the development of a freeze-drying procedure allowing stabilization at ambient temperature of preoptimized, premixed, and predispensed PCR mixes aimed at the detection of mycobacteria in clinical materials. The freeze-dried mixes retained activity at 4 degrees C and at 20 degrees C for 1 year and for 3 months at 37 degrees C, as judged by their performance with 50 and 500 fg of purified Mycobacterium bovis BCG target DNA.
DNA, Bacterial↗
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.
Adult↗
Clinical utility of the polymerase chain reaction in the diagnosis of extrapulmonary tuberculosis.
This study examines the diagnostic utility of the polymerase chain reaction (PCR) in 156 patients (five human immunodeficiency virus (HIV) seropositive) suspected of extrapulmonary tuberculosis. The results of PCR in 226 samples from 11 different sites were compared with the results of microscopy and culture. Positive culture results were predicted in 86% of samples by PCR but in only 31% by microscopy. Specificity of PCR was 92%. In cases with culture-proven tuberculosis, PCR identified all 11 microscopy positive cases and 19 of 24 (79%) of the microscopy-negative cases. In four patients, PCR excluded the diagnosis of tuberculosis in microscopy-positive samples, which were later shown to contain mycobacteria other than Mycobacterium tuberculosis or laboratory contaminants. In 20 patients (microscopy, PCR and culture negative) a trial of antituberculous drugs was given, but patients showed no improvement and treatment was stopped. In 17 patients, all culture negative (in nine PCR was positive, three of whom also had positive microscopy) the diagnosis was probable tuberculosis based on clinical findings and response to treatment. This polymerase chain reaction has a much higher sensitivity than microscopy and can facilitate therapeutic decisions for those with suspected extrapulmonary tuberculosis.
AIDS-Related Opportunistic Infections↗
Multiplex PCR assay for immediate identification of the infecting species in patients with mycobacterial disease.
Rapid identification of infecting mycobacterial species enables appropriate medical care decisions to be made. Our aim was to demonstrate the clinical usefulness of the multiplex PCR assay, a test based on PCR, which permits direct identification of 12 mycobacterial species in clinical specimens. A total of 259 specimens from 177 patients who had clinical symptoms of mycobacterial disease but for whom there were difficulties in diagnosis were tested. Specimens were analyzed within 48 h of receipt of the sample. Mycobacteria were identified in 102 specimens; 66 specimens contained nontuberculous mycobacteria, and 36 specimens contained Mycobacterium tuberculosis complex mycobacteria. The PCR assay identified the mycobacterial species in 43 (97.7%) of 44 microscopy- and culture-positive specimens and in 15 (93.8%) of 16 culture-positive, microscopy-negative specimens. It also permitted species identification in infections caused by more than one mycobacterial species. For 56 (96.5%) of the 58 specimens from patients with infections caused by opportunistic mycobacteria, the organisms were identified with the PCR assay. The test was useful also for the identification of fastidious mycobacteria, e.g., M. genavense, and those that cannot be cultured, e.g., M. leprae. After resolution of discrepant results, the sensitivity of the PCR assay was 97.9%, the specificity was 96.9%, the positive predictive value was 95.0%, and the negative predictive value was 98.7%. For culture these values were 60.8, 100, 100, and 81.0%, respectively. Thus, the multiplex PCR assay enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.
AIDS-Related Opportunistic Infections↗
The potential contribution of the polymerase chain reaction to the diagnosis of tuberculous meningitis.
OBJECTIVE: To examine diagnostic utility of polymerase chain reaction (PCR) on cerebrospinal fluid (CSF) in tuberculous meningitis (TBM). DESIGN: Comparison study. SETTING: Referral center for tuberculosis diagnosis and treatment in Ho Chi Minh City, Vietnam, and research laboratory in Amsterdam, the Netherlands. PATIENTS: One hundred thirty-six consecutive patients, aged 4 months to 85 years, with features compatible with TBM seen during a 12-month period. MEASUREMENTS: Clinical examination; cytology; Gram, india ink, and Ziehl-Neelsen staining; culture of CSF for bacteria, mycobacteria, fungi, and viruses; and CSF chloride, protein, and glucose. All these tests were performed in Vietnam. The PCR on CSF was performed in the Netherlands. RESULTS: Patients were managed in Vietnam without knowledge of PCR results. Based on clinical grounds and the results of initial CSF microscopy, antituberculous treatment was given to 104 patients, 66 of whom had evidence of extraneural tuberculosis. Among the 39 patients with confirmed TBM (ie, positive Ziehl-Neelsen staining or culture or PCR results for Mycobacterium tuberculosis), PCR detected 32 patients (82%), 1 case was proven positive through microscopy and 17 (44%) had positive culture results. There were no false-positive PCR results. In 99 patients with a final diagnosis of confirmed or probable TBM (ie, clinical features of TBM and response to antituberculous treatment), PCR had a sensitivity of 32%; culture, 17% and microscopy, 1%. CONCLUSIONS: Many patients who respond to treatment for TBM do not have M tuberculosis in the CSF identifiable by microscopy, PCR, or culture. Polymerase chain reaction on CSF is the best method for the laboratory diagnosis of TBM. Polymerase chain reaction is especially useful for the early diagnosis of TBM in those without active extraneural tuberculosis.
Adolescent↗
PCR detection of mycobacteraemia in tanzanian patients with extrapulmonary tuberculosis.
In 191 Tanzanian patients admitted to hospital with suspected extrapulmonary tuberculosis (TB), TB was diagnosed in 158 patients; the remaining 33 patients had neither microbiological nor clinical evidence of TB. Mycobacterium tuberculosis was detected in the blood of 25 patients, in 92% by a polymerase chain reaction (PCR) technique and in 52% by culture of buffy coat cells. The presence of mycobacterial DNA or Mycobacterium tuberculosis bacteria in peripheral blood (positive culture) was significantly associated with HIV infection; it was detected in 22 (21.4%) of 103 HIV-seropositive patients compared to only 3 (3.5%) of 55 HIV-seronegative patients (p < 0.009). In two-thirds of the patients with mycobacteraemia, TB can be detected by simple smears from other organ sites. In patients with suspected extrapulmonary tuberculosis in whom smears from the infected site are negative or not available, PCR on blood will confirm the diagnosis within 24 hours in one third of the cases.
Adult↗
Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: an international collaborative quality control study among 30 laboratories.
Nucleic acid amplification to detect Mycobacterium tuberculosis in clinical specimens is increasingly used as a laboratory tool for the diagnosis of tuberculosis. However, the specificity and sensitivity of these tests may be questioned, and no standardized reagents for quality control assessment are available. To estimate the performance of amplification tests for routine diagnosis, we initiated an interlaboratory study involving 30 laboratories in 18 countries. We prepared blinded panels of 20 sputum samples containing no, 100, or 1,000 mycobacterial cells. Each laboratory was asked to detect M. tuberculosis by their routine method of nucleic acid amplification. Only five laboratories correctly identified the presence or absence of mycobacterial DNA in all 20 samples. Seven laboratories detected mycobacterial DNA in all positive samples, and 13 laboratories correctly reported the absence of DNA in the negative samples. Lack of specificity was more of a problem than lack of sensitivity. Reliability was not found to be associated with the use of any particular method. Reliable detection of M. tuberculosis in clinical samples by nucleic acid amplification techniques is possible, but many laboratories do not use adequate quality controls. This study underlines the need for good laboratory practice and reference reagents to monitor the performance of the whole assay, including pretreatment of clinical samples.
Bacterial Typing Techniques↗
Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control.
A microwell hybridization assay was developed for the detection of the PCR products from both Mycobacterium tuberculosis complex bacteria and the recombinant Mycobacterium smegmatis strain 1008 that is used as an internal control to monitor inhibition in the PCR based on the M. tuberculosis complex-specific insertion sequence IS6110. The test is based on specific detection with digoxigenin-labeled oligonucleotide probes of biotinylated PCR products which are captured in a microtiter plate coated with streptavidin. The captured PCR products are hybridized separately with two probes, one specific for the PCR product from IS6110 from M. tuberculosis complex and the other specific for the PCR fragment from the modified IS6110 fragment from the recombinant M. smegmatis 1008. The microwell hybridization assay discriminates perfectly between the two types of amplicon. The amount of PCR product that can be detected by this assay is 10 times less than that which can be detected by agarose gel electrophoresis. The test can be performed in 2 h. It is much faster and less laborious than Southern blot hybridization. Furthermore, the interpretation of results is objective. The assay was used with 172 clinical samples in a routine microbiology laboratory, and the results were in complete agreement with those of agarose gel electrophoresis and Southern blot hybridization.
DNA, Bacterial↗
Aetiology of uveitis in Sierra Leone, west Africa.
BACKGROUND: In 1992, non-onchocercal uveitis caused 9% of blindness, 8% of visual impairment, and 11% of uniocular blindness among patients visiting an eye hospital in Sierra Leone, west Africa. The aim of this study was to determine the aetiology of uveitis in this population. METHODS: General and ophthalmic examination complemented by serum and aqueous humour analyses for various infectious agents was performed for 93 uveitis patients and compared with serum (n = 100) and aqueous humour (n = 9) analysis of endemic controls. RESULTS: At the initial examination, 45 patients (48%) proved to be severely visually handicapped. After clinical and laboratory analyses, an aetiological diagnosis was established for 49 patients (52%). Toxoplasma gondii was the most important cause of uveitis (40/93; 43%). Anti-toxoplasma IgM antibodies were detected in serum samples of seven of 93 patients (8%) compared with one of 100 controls (1%, p < 0.05). At least six patients (15%) with ocular toxoplasmosis had acquired the disease postnatally. Antibodies against Treponema pallidum were detected in 18 of 92 patients (20%) and in 21 controls (21%). Other causes of uveitis were varicella zoster virus (one patient), herpes simplex virus (two patients), and HLA-B27 positive acute anterior uveitis with ankylosing spondylitis (one patient), while one patient had presumed HTLV-I uveitis. CONCLUSIONS: In a hospital population in Sierra Leone, west Africa, uveitis was associated with severe visual handicap and infectious diseases. Toxoplasmosis proved to be the most important cause of the uveitis. Although the distribution of congenital versus acquired toxoplasmosis in this population could not be determined, the results indicate an important role of postnatally acquired disease. The results further suggested minor roles for HIV, tuberculosis, toxocariasis, and sarcoidosis as causes of uveitis in this population.
Adolescent↗
Granulomatous hepatitis caused by Bacillus Calmette-Guerin (BCG) infection after BCG bladder instillation.
BACKGROUND: Bladder instillations with Bacillus Calmette-Guerin (BCG) are commonly used as immunotherapy for bladder carcinoma. Sometimes patients experience serious systemic side effects, such as sepsis or pneumonitis. Granulomatous hepatitis is a rare serious side effect, which has been considered a hypersensitivity reaction to BCG. PATIENT: The first case of granulomatous hepatitis after BCG bladder instillation in which mycobacteria were identified by staining techniques and mycobacterial DNA was detected in liver tissue using the polymerase chain reaction is reported. CONCLUSION: The granulomatous hepatitis was caused by BCG infection of the liver after haematogenous dissemination of BCG, rather than hypersensitivity.
Administration, Intravesical↗