Novel Multipurpose Methodology for Detection of Mycobacteria in Pulmonary and Extrapulmonary Specimens by Smear Microscopy, Culture, and PCR

Abstract

A novel, robust, reproducible, and multipurpose universal sample processing (USP) methodology for highly sensitive smear microscopy, culturing on solid and liquid media, and inhibition-free PCR which is suitable for the laboratory diagnosis of both pulmonary and extrapulmonary tuberculosis (TB) has been developed. This method exploits the chaotropic properties of guanidinium hydrochloride for sample processing and involves incubating the specimen with USP solution, concentrating bacilli by centrifugation, and using the processed specimen for smear microscopy, culture, and PCR. The detection limit for acid-fast bacilli in spiked sputum by smear microscopy is approximately 300 bacilli per ml of specimen. USP solution-treated specimens are fully compatible with culturing on solid and liquid media. High-quality, PCR-amplifiable mycobacterial DNA can be isolated from all types of clinical specimens processed with USP solution. The method has been extensively validated with both pulmonary and extrapulmonary specimens. Furthermore, the USP method is also compatible with smear microscopy, culture, and PCR of mycobacteria other than tubercle bacilli. In summary, the USP method provides smear microscopy, culture, and nucleic acid amplification technologies with a single sample-processing platform and, to the best of our knowledge, is the only method of its kind described to date. It is expected to be useful for the laboratory diagnosis of TB and other mycobacterial diseases by conventional and modern methods.Tuberculosis (TB) kills more people in India and Southeast Asia than any other infectious disease-more than human immunodeficiency virus, sexually transmitted diseases, malaria, and tropical diseases combined. In India, 1 person dies every minute and 500,000 people die per year from TB (37). The rapid diagnosis of TB is central to minimizing the risk of disease transmission, especially in the wake of the emergence of drug-resistant TB and its severe implications for human immunodeficiency virus-infected patients (37). Although culturing of the etiologic agent remains the accepted "gold standard" for diagnosing TB, direct smear microscopy is by far the most popular among all the methods currently employed worldwide for TB diagnosis. In view of the enormous utility of smear microscopy, numerous efforts have been made to improve its sensitivity (11,13,18,27,31,36), with varying success rates. In recent times, however, maximum attention has been devoted to developing nucleic acid amplification (NAA) diagnostic technologies owing to their rapidity and sensitivity. Numerous gene targets (10, 29, 35) and several methods for isolating mycobacterial DNA from clinical specimens have been reported (2,9,19,23,26,32). Several automated and semiautomated kits for mycobacterial culture and NAA are available and are being extensively evaluated (3,5,14,15,20,30).Despite the availability of a plethora of tests, there are certain drawbacks associated with the existing diagnostic methodologies. Direct smear microscopy lacks sensitivity a...

Rapid Detection of Mutation in RRDR of rpo B Gene for Rifampicin Resistance in MDR-Pulmonary Tuberculosis by DNA Sequencing

“…Mycobacterial DNA were extracted from sputum sample by using Universal Sample Processing (USP) method [10,11].…”

TRC4 Gene Based PCR Assay in Diagnosis of Tuberculous Meningitis

“…This low yield of AFB smear and culture may be due to the paucibacillary nature of CSF samples. Furthermore, inadequate volume of samples for a number of tests, like cytology, biochemistry, microbiology and PCR, gives low positive result 9 . AFB smear negativity might be due to the low concentration of mycobacteria in those samples i.e.…”

Comparison of IS6110 and ‘short fragment’ devR (Rv3133c) gene targets with phenotypic methods for diagnosis of Mycobacterium tuberculosis

“…11 Short fragment devR (Rv3133c) is more sensitive and detects 250-500 genome equivalents while 'long fragment' devR (Rv3133c) is less sensitive and detects 500-1000 genome equivalents. 10 Assays targeting single copy genes or amplifying longer fragments are less sensitive than those targeting repetitive sequences or shorter fragments particularly in paucibacillary specimens and samples containing degraded DNA. 16 IS6110 is at least 50 fold more sensitive than 'short fragment' devR (Rv3133c) assay using purified DNA.…”

“…Short fragment devR (Rv3133c) specific PCR was performed in 40 ml reaction mixture containing 0.5 mM concentration (each) of forward primer devR (Rv3133c) f2 (5 0 -TGGCAACGGCA-TTGAACTGT-3 0 ) and reverse primer devR (Rv3133c) r2 (5 0 -TAAGCAGGCCCAGTAGCGT-3 0 ), 1x Taq Polymerase Buffer, 1.5 mM MgCl 2 , 0.2 mM each dNTP, 1 ml/40 ml Taq DNA Polymerase, water and 10 ml specimen DNA. 10 Denaturation was carried at 94 C for 10 min, annealing at 52 C for 1 min (45 cycles), extension at 72 C for 30 s and final extension at 72 C for 7 min. 10 ml water as negative control and 5 ml MTB DNA (2e5 ng) as positive control were put for both PCRs.…”