Inhibition of the polymerase chain reaction by sputum samples from tuberculosis patients after processing using a silica-guanidiniumthiocyanate DNA isolation procedure

Abstract

With the objective to evaluate PCR-mediated detection of Mycobacterium tuberculosis DNA as a diagnostic procedure for diagnosis of tuberculosis in individuals attending ambulatory services in Primary Health Units of the City Tuberculosis Program in Rio de Janeiro, Brazil, their In 1995, 94,870 new cases of tuberculosis (TB) were reported in Brazil, being 76,840 pulmonary TB; the incidence of the disease was 58.2/100.000 (Ruffino Netto 1999). The situation is more severe in big cities like São Paulo and Rio de Janeiro, where co-infection with HIV and multi-drug resistant strains of Mycobacterium tuberculosis complicates efficient use of general control programs. Rapid diagnosis and adequate treatment of TB is the most efficient way to avoid spread of TB. During the last couple of years, several studies were performed to compare recently developed commercial and "in house" PCR systems with conventional diagnostic procedures such as detection of acid-fast bacilli by microscopy, culture and X-ray (Eing et al. 1998, Tortoli et al. 1999 speed, sensitivity and specificity, nucleic acid amplification systems are promising diagnostic tools; however, double blind studies have shown that the use of "in house" PCR as a routine procedure for diagnosis of TB is still controversial (Noordhoek et al. 1994, Suffys et al. 2000. More studies are needed to evaluate the sensitivity of the methods on paucibacillary material and for evaluation of feasibility and cost-effectiveness when used as a routine procedure in TB control programs, especially in developing countries (Roos et al. 1998).One of the bottle necks of PCR for diagnostic purpose is the extraction of parasite DNA from clinical samples (Noordhoek et al. 1994). During the last decade, several methods have been published, including a very promising procedure using guanidiniumthiocyanate (GuSCN) for isolation of different types of nucleic acids from cell-rich sources and pathogenic bacteria (Boom et al. 1990). For evaluation of PCR as a diagnostic procedure of pulmonary TB in individuals attended at 14 ambulatory services in Rio de Janeiro, sputum samples were collected locally, stored at 4°C for a maximum of 4 days and further processed at the State Reference Laboratoroy (Central Laboratory Noel Nutels- Lacen, RJ). The laboratory methods for processing cultures and smears as well as for the identification of mycobacteria were standard procedures (Kent & Kubica 1985). Briefly, an aliquot of 8-10 ml of sputum was processed using using NaOH-Nacetyl-L-cysteine, resuspended in 2.5 ml phosphatebuffered saline and a fraction was processed according to Boom et al. (1990), slightly modified. Briefly, 0.2 ml of the sample was added to 0.2 ml of 0.1 mm glass beads (Biospec products, Bartlesville, OK, USA) and 0.9 ml of L6 lysis buffer (Boom et al. 1990), vortexed during 10 min, submitted to two cycles of heat shock (5 min 65°C and 5 min -170°C) after which 40 µl of celite were added and the solution mixed gently at room temperature during 30 min. Supernatant was discarded and the pell...

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

“…Despite the use of various approaches (1,4,12,39), most methods do not consistently remove PCR inhibitors from clinical material (12,17,33,34,43). Even commercial PCR kits like the Amplicor M. tuberculosis kit are vulnerable to PCR inhibition (4,30).…”

Development and Validation of a Multiplex PCR for Detection of Scedosporium spp. in Respiratory Tract Specimens from Patients with Cystic Fibrosis

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Nested-PCR do gene que codifica o antígeno b aplicada ao diagnóstico da tuberculose pulmonar

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