Low cost in-house PCR for the routine diagnosis of extra-pulmonary tuberculosis.
METHODS
Conventional methods for the identification of mycobacteria are slow and labour intensive. DNA amplification methods offer rapid sensitive and specific diagnosis.


OBJECTIVE
To determine the feasibility of an in-house polymerase chain reaction (PCR) method to detect Mycobacterium tuberculosis in clinical samples.


METHODS
The present study focused mainly on diagnosing extra-pulmonary tuberculosis (EPTB) using an in-house PCR method in 465 clinical samples. This study also compared the efficacy of a standard phenol-chloroform (PC) extraction procedure and the guanidine thiocyanate with diatomaceous silica (GTCS) method of DNA extraction and purification. A subsample of patients was used for the validation of results based on the final diagnosis.


RESULTS
Among 373 patients with suspected EPTB, 75 specimens were positive by PCR, four by microscopy and six by culture. Of the 25 PCR-positive patients, 95% had a final diagnosis of TB. Globally, the GTCS method was found to be superior to the PC method for DNA extraction and removal of inhibitors from clinical specimens.


CONCLUSIONS
The DNA amplification method was found to be significantly more sensitive and rapid compared to culture and microscopy for a reliable final diagnosis of EPTB.
Magana-Arachchi
D
D
Department of Microbiology, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka. nayomam@yahoo.com
Perera
J
J
Gamage
S
S
Chandrasekharan
V
V
D004269:DNA, Bacterial / Q000302:isolation & purification; D005240:Feasibility Studies; D006146:Guanidines; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000191:economics* / Q000379:methods*; D013048:Specimen Handling; D013188:Sri Lanka; D013861:Thiocyanates; D014376:Tuberculosis / Q000175:diagnosis*
D004269:DNA, Bacterial; D006146:Guanidines; D013861:Thiocyanates; C054436:guanidine thiocyanate