Rapid Diagnosis of Extrapulmonary Tuberculosis by PCR: Impact of Sample Preparation and DNA Extraction

Abstract

In cases of suspected extrapulmonary tuberculosis, rapid and accurate laboratory diagnosis is of prime importance, since traditional techniques of detecting acid-fast bacilli have limitations. The major difficulty with mycobacteria is achieving optimal cell lysis. Buffers used in commercial kits do not allow this complete lysis in a number of clinical specimens. A comparison of two sample preparation methods, pretreatment with proteinase K (PK-Roche) and complete DNA purification (cetyltrimethylammonium bromide [CTAB]-Roche), was conducted on 144 extrapulmonary specimens collected from 120 patients to evaluate the impact on the CobasAmplicor method. Thirty patients were diagnosed with tuberculosis, with 15 patients culture positive for Mycobacterium tuberculosis. Amplification and detection of the amplicons were impaired by a high number of inhibitory specimens (39 to 52%). CTAB-Roche allowed the detection of more culture-positive specimens by PCR than PK-Roche. Comparison with the final diagnoses of tuberculosis confirmed that CTAB-Roche produced the best sensitivity (53.8%) compared to culture (43.3%), PK-Roche (16%), and smear (13%). However, the specificity of the PCR assay with CTAB-Roche-extracted material was always lower (78.8%) than those with culture (100%) and PK-Roche (96.5%). False-positive specimens were lung biopsy material, lymph node biopsy material and aspirate, or bone marrow aspirate, mainly from immunocompromised patients. Despite the efficiency of complete DNA extraction for the rapid diagnosis by PCR of extrapulmonary tuberculosis, the false-positive results challenge our understanding of PCR results.

Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

“…In the diagnostic testing of highly prevalent infectious diseases, inhibitors responsible for false-negative results represent a serious problem. The specimens that contained PCR inhibitors, characterized by the absence of the targeted diagnostic fragments in positive samples, reinforced the need to overcome this problem by using an IC to effectively monitor the reaction (Almeda et al 2000, Honoré-Bouakline et al 2003. The use of the same primer sequences to amplify the M. tuberculosis diagnostic fragment and the IC fragment allowed the IC to be used without increasing the laboratory workload (Soini et al 1992, Almeda et al 2000.…”

Diagnosis of Osteoarticular - Solution is in Peripheral Blood!!

“…(2) A PCR result is of great value in early diagnosis, particularly in infections of certain body systems where disease progression is rapid and detection by culture method is time-consuming. [14][15][16][17] PCR can also detect very low levels AFB in clinical specimens and results are available within two days. However, misleading results can occur, such as the smallest amount of contaminating DNA can be amplified.…”

Desempenho da técnica nested PCR na detecção específica do complexo Mycobacterium tuberculosis em amostras sanguíneas de pacientes pediátricos

“…(5) The sensitivity of the detection was more efficient than that observed in other studies using the same system. (19,20) Most patients with initial suspicion of TB were diagnosed as an active or latent case, probably due to the fact that the diagnoses were made at hospitals or outpatient clinics that were referral centers for TB.…”