Clinical application of Real-Time quantitative PCR in the differential diagnosis of sarcoidosis and atypical tuberculosis
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Objective To verify and evaluate the clinical application value of pre-established Real-Time quantitative PCR to detect Mycobacterium tuberculosis DNA in the differential diagnosis of sarcoidosis and atypical tuberculosis.Method Forty-nine patients with granulomatosis disciformis but no final diagnosis as sarcoidosis or tuberculosis were enrolled into this study from Jun 2008 to Jun 2009.Real-Time quantitative PCR pre-established were used to detect Mycobacterium tuberculosis DNA(TB-PCR)in 49 patients' paraffin blocks with the optimal cut-off value 1.14×10~3 copies/ml. Clinical,imaging,pathology and other data were combined to make differential diagnosis and give guidance of therapy.The clinical appearance,imaging changes,therapeutic effects were followed up till Aug 1st,2009 and the clinical application value of this method were evaluated.Result Ten samples of TB-PCR were positive(2.01×10~3 -7.98×10~4 copy/ml),the positive rate was 20.41%,39 samples were negative. Combined with clinical data and TB-PCR results,33 cases were diagnosed as sarcoidosis,2 were tuberculosis. The percentage of definite diagnosis achieved was 72%(35/49).Patients with definite diagnosis were followed up for 2～14 months in which 33 patients with sarcoidosis and 2 cases with tuberculosis got stable or improved after the therapy,none of them worsen.Conclusion The Real-Time quantitative PCR for detection of Mycobacterium tuberculosis DNA in paraffin-embedded tissues can sensitively and effectively detect Mycobacterium tuberculosis from tuberculosis proliferative granulomatous lesions.It is effective to help to differentiat sarcoidosis and atypical tuberculosis.Cite
To evaluate the clinical value of polymerase chain reaction (PCR) technique in diagnosis of bone tuberculosis.PCR, standard light-microscopy and standard culture technique were used to detect Mycobacterium tuberculosis in samples obtained from 60 patients with bone tuberculosis and 20 patients without bone tuberculosis. In the meantime, some factors affecting PCR result were analysed and methods to deal with them were discussed.In the group of 60 patients with bone tuberculosis, the positive rate was 83% in PCR technique, 3% in standard light-microscope technique and 7% in the standard culture technique. A statistically obvious difference was seen (P < 0.005). In the group of 20 patients without bone tuberculosis, 2 cases showed positive in PCR technique, none in the other methods. Specificity of PCR technique in a blind comparison study indicated 100%. The whole process of PCR amplification is fully automatic and can be finished within several hours, and the detection time is considerably reduced.PCR technique is a rapid, specific, sensitive and simple method for detection of mycobacterium tuberculosis in sample of bone tuberculosis, and it is of great value in the diagnosis of bone tuberculosis and differentiating bone tuberculosis from other bone diseases.
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Aims: Use of the polymerase chain reaction for the detection of Mycobacterium tuberculosis (TB PCR) as a basis for making clinical decisions on the initiation of antituberculosis treatment was studied. Methods: A retrospective study involving a cohort of 155 patients being investigated for tuberculosis in an infectious disease consultation service was undertaken. TB PCR was performed on pulmonary and extrapulmonary specimens from these patients. The sensitivity of TB PCR was analysed. Results: Of the 155 patients, 144 fitted the clinical diagnosis of tuberculosis, and 112 of them were culture positive for M tuberculosis . Sixty (58.3%) patients with clinical features suggestive of tuberculosis received antituberculosis treatment based on positive TB PCR alone. Of 224 clinical specimens (138 pulmonary and 86 extrapulmonary) sent for TB PCR, 148 (99 pulmonary and 49 extrapulmonary) were positive in 117 patients. Of the 690 clinical specimens sent for culture, 279 were positive for M tuberculosis in 112 patients. The diagnostic sensitivity of TB PCR was 75.9% (85 of 112) and 81.3% (117 of 144) in patients with culture confirmed and clinically diagnosed tuberculosis, respectively. Using culture as the gold standard, the overall sensitivity of TB PCR was 78.3%, and for pulmonary and extrapulmonary specimens it was 82.3% and 72.0%, respectively. Conclusions: TB PCR is a rapid and reliable test in the diagnosis and management of tuberculosis.
Extrapulmonary tuberculosis
Gold standard (test)
Tuberculosis diagnosis
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Objective To estimate the presence of Mycobacterium tuberculosis DNA in tissues from tuberculosis and sarcoidosis patients.and to evaluate the value of nested polymerase chain reaction(nested-PCR) in distinguishing tuberculosis and sarcoidosis.Methods 68 paraffin-embedded tissue biopsies corresponding to cases of tuberculosis(n=37) and asarcoidosis(n=31) were analyzed by means of nested-PCR using primers corresponding to the insertion element IS6110 of M.tuberculosis complex.Results M.tuberculosis DNA was present in 36 out of 37 tuberculosis biopsies(97.3%),in 8 out of 31 sarcoidosis biopsies(25.8%)(P 0.01),and none was found in negative controls from unborn mice,and all was found in positive controls from H37RV standard strain.The positivity of nestedPCR was obviously higher than general PCR.Conclusion Nested-PCR may be a reliable tool in distinguish tuberculosis and sarcoidosis.
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Polymerase chain reaction (PCR) using primers targeting the IS6110 repetitive sequence was employed to detect Mycobacterium tuberculosis in 228 samples from patients with tuberculosis or other pulmonary diseases and controls, and the results were compared with culture and clinical findings. None of culture negative samples from 17 healthy controls were PCR positive. Of 109 active tuberculosis patients under chemotherapy, 88 (80.7%) were PCR positive and were significantly higher than 63 (57.8%) positive by culture. Fifty-nine (93.7) of 63 culture positive and 29 (63.0%) of 46 culture negative specimens contained M. tuberculosis detectable by PCR. In 41 specimens from inactive tuberculosis patients who visited to the chest clinic because of chest problems, 16 (39.0%) also gave PCR positive results. In addition, 14 (46.7%) of 30 specimens submitted for M. tuberculosis culture from patients with pulmonary diseases were PCR positive. Presumptive diagnosis of these PCR positive patients was bronchitis, pneumonia, bronchial asthma, etc. Therefore, this study suggests that PCR is sensitive and specific in detecting M. tuberculosis in clinical specimens. However, the interpretation of the PCR results in specimens from patients with pulmonary diseases should be done cautiously in areas with a high prevalence of tuberculosis.
Chronic bronchitis
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Abstract Background Cutaneous tuberculosis is especially difficult to distinguish from other granulomatous dermatoses. We used polymerase chain reaction (PCR) to evaluate the incidence of cutaneous tuberculosis and atypical mycobacterial infection in formalin‐fixed, paraffin‐embedded tissues with unspecified granulomatous inflammation and negative results for acid‐fast bacilli (AFB), and analyzed the pattern of cutaneous tuberculosis in this group of patients. Methods A total of 38 specimens which had been collected from 36 patients and fulfillled the criteria for tissues described above were used in this study. Two different primer pairs targeting the gene encoding for 16S ribosomal RNA (common to all mycobacteria) and the insertion sequence IS6110 (specific for M. tuberculosis complex) were used in the PCR assays. The clinical characteristics, histopathologic findings, and culture results of the patients were also analyzed. Results Four specimens were excluded from the analysis due to the lack of internal control testing. Of the remaining 34 specimens, 22 were PCR positive for the 16S rRNA gene. Among them, 18 specimens were PCR positive for both the 16S rRNA gene and IS6110. Cutaneous tuberculosis could be diagnosed in these 18 cases (56.2%). Out of the 18 cases, there were 8 women and 10 men. The age range was 15–77 years (mean: 44.2 years). After reviewing their clinical presentation, 11 cases were considered as tuberculosis verrucosa cutis, 6 cases as lupus vulgaris, and 1 case as erythema induratum. The remaining 4 cases (12.5%) positive only for 16S rRNA gene were considered as possible atypical mycobacteria infection. Conclusions These results show that in paucibacillary form of cutaneous tuberculosis with unclassical clinical and histological presentation, this PCR system provides rapid and sensitive detection of M. tuberculosis DNA in formalin‐fixed, paraffin‐embedded specimens. Cutaneous tuberculosis represents a significant proportion in specimens showing granulomatous inflammation. In areas like Taiwan, where prevalence of pulmonary tuberculosis is still high, tuberculosis verrucosa cutis and lupus vulgaris are common forms of cutaneous tuberculosis and are seen more frequently than atypical mycobacterial infection.
Granulomatous Inflammation
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Objective To evaluate the prospect of real-time PCR in the clinical diagnosis and efficiency evaluation of the pulmonary tuberculosis treatment.Methods Real-time PCR was established to detect M.tuberculosis specific sequence IS6110 in the sputum samples taken from suspected and diagnosed tuberculosis patients.The result was compared with acid-fast staining and cultivation Result The sensitivity of real-time PCR was 4 copies/reaction,that was significantly higher than acid-fast staining and cultivation.The positive rate of real-time PCR,acid-fast staining and cultivation.was 64.29%,35.12% and 12.5%.The copy of TB diminished obviously in the treatment follow-up.Conclusion The real-time PCR has high sensitivity and specificity in the detection of M.tuberculosis.The method could feedback the efficiency rapidly and in time,and provide evidence for the further treatment from doctors.
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To evaluate the role of mycobacterial infection in the pathogenesis of sarcoidosis by examination of mycobacterial DNA in tissue samples of sarcoidosis and tuberculosis, and to examine the value of quantitative real-time polymerase chain reaction (PCR) in the differentiation of the two diseases.Mycobacterium tuberculosis DNA was measured by quantitative real-time PCR from formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes and lung tissues from 31 patients with sarcoidosis, 30 patients with tuberculosis and 15 patients with other diseases (as the control samples) in Shanghai Pulmonary Hospital from January 1998 to December 2003. Lung tissues from 15 normal embryonic mice served as the negative control.The positive rate of mycobacterial DNA in the tuberculosis samples (30/30) was higher than that of the sarcoidosis samples (6/31) and of the control samples (2/15). The difference between sarcoidosis and normal samples showed no statistical significance. The absolute and relative copies of mycobacterial DNA in the tuberculosis samples were significantly higher than those in the sarcoidosis and the control samples; while there was no statistical difference between the sarcoidosis and the control samples. There was no positive result in the lung tissues of the embryonic mice.The results do not show any relationship between mycobacterial infection and sarcoidosis. Quantitative PCR may be a reliable method for the differentiation of sarcoidosis from tuberculosis.
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The role of polymerase chain reaction (PCR) in the diagnosis of cutaneous tuberculosis in clinical practice has not been defined as no PCR assay has been tested in a large-scale clinical study. The objective of this study was to test the clinical utility of a PCR assay in the diagnosis of different types of cutaneous tuberculosis and tuberculids.Analysis of archival biopsy specimens by a nested PCR assay targeting IS6110 of Mycobacterium tuberculosis (M. tb) DNA was performed in a tertiary-care skin hospital in Singapore. PCR results were compared with cultures and concordance with final diagnosis. PATIENTS AND SPECIMENS: One hundred and nineteen skin biopsies from 105 patients comprising 58 cases of confirmed or highly probable cutaneous tuberculosis, ranging from multibacillary infections to paucibacillary forms and 47 cases of possible tuberculids were analysed. Twenty-four subjects with non-tuberculous granulomas and normal skin controls were included.In 14 immunocompromised patients with multibacillary mycobacterial infections (AFB+ on biopsy), PCR was positive in 9 patients. Correlating PCR results with the final diagnosis, the PCR technique was 100% sensitive and specific in this group. In paucibacillary tuberculosis, PCR positivity rates were 55% for tuberculosis verrucosa cutis (38 cases) and 60% for lupus vulgaris (5 cases). When confirmed cases of tuberculosis were considered, the overall sensitivity was 73%. In 26 cases of erythema induratum, PCR was positive in 54% and correlated with a documented response to anti-tuberculous treatment in 80%.The use of PCR in the routine diagnostic panel for cutaneous tuberculosis should take into consideration the differential sensitivities for different clinical types. In the setting of an immunocompromised patient with AFB+ lesions, PCR has a definite role in rapid diagnosis and in differentiating atypical mycobacterial infection from tuberculosis. Where paucibacillary tuberculosis is suspected, clinical decision should not be based on PCR results alone. In erythema induratum, we found some correlation between PCR results and response to anti-tuberculous therapy.
Concordance
Skin biopsy
Cutis
Lupus vulgaris
Cutaneous tuberculosis
Mycobacterium tuberculosis complex
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Increasing evidence indicates that mycobacteria may be involved in the aetiology and pathophysiology of sarcoidosis.To investigate the association between Mycobacterium tuberculosis complex infection and sarcoidosis.Mediastinal lymph node biopsy specimens (formalin-fixed, paraffin-embedded) from 52 Danish patients with sarcoidosis, 50 patients with mediastinal lymphadenopathy of other non-mycobacterial causes (negative controls) and 12 patients with histologically and/or culture-verified mycobacteriosis (positive controls) were included in the study. Biopsy samples were analysed for the presence of Mycobacterium tuberculosis complex by strand displacement assay and a subset of specimens were examined for bacterial rRNA by fluorescent in situ hybridisation using an eubacterial probe with general bacterial specificity (EUB338).One patient with sarcoidosis displayed a positive M. tuberculosis complex test. All negative controls were negative in the test and 5/12 patients with mycobacteriosis were positive in the test. We detected M. tuberculosis complex DNA in 10-year-old biopsy samples. Thirty-six samples were tested with the eubacterial probe; of these, 67% were positive with no difference between patients and controls.Our results do not support the hypothesis that M. tuberculosis complex infection is involved in the pathogenesis of sarcoidosis. However, we stress the importance of excluding mycobacteriosis in the diagnostic workup of sarcoidosis patients.
Mycobacterium tuberculosis complex
Etiology
Mediastinal lymphadenopathy
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Summary Background There is a need for rapid diagnosis of pulmonary tuberculosis. We have previously used a PCR to detect circulating Mycobacterium tuberculosis DNA in blood samples from patients (mostly HIV-infected) with pulmonary tuberculosis. We have now prospectively investigated the role of this blood-based PCR assay for diagnosis of this disease in a clinical setting. Methods Our PCR assay is specific for the IS6110 insertion element of the M tuberculosis complex of organisms. We used it to test peripheral blood from 88 consecutive patients admitted to a chest ward with suspected pulmonary tuberculosis. Personnel who carried out the assay did not know the results of any clinical investigations and ultimate diagnosis, and clinicians did not know the PCR results. Results of the PCR assay were compared with the final clinical diagnosis. A subgroup of 15 patients had blood samples assayed serially to track the PCR signal over time. Findings 41 patients had a final clinical diagnosis of tuberculosis, and the cases were typical of those seen at our hospital: HIV infection was common, and most cases were not sputum-smear positive for acid-fast bacilli. The PCR assay correctly identified 39 of 41 patients with proven pulmonary tuberculosis, 26 (63%) of whom were sputum-smear negative. There were five patients in whom a positive PCR result did not accord with the final clinical diagnosis, and two of the 44 negative PCR results were classified as false negatives. The overall sensitivity and specificity of the PCR assay for a diagnosis of tuberculosis was 95% and 89%, respectively. In 15 patients with pulmonary tuberculosis and a positive blood assay, the PCR result remained positive after 1 month of therapy, but had reverted to negative in 13 of the 15 by 4 months of therapy. Interpretation We conclude that peripheral-blood-based PCR detection for the diagnosis of tuberculosis is a technically feasible approach that has a potentially important role in the diagnosis of pulmonary tuberculosis.
Tuberculosis diagnosis
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