Comparative evaluation of nested PCR and conventional smear methods for the detection of Mycobacterium tuberculosis in sputum samples.
BACKGROUND
With 1.8 million new cases each year, India carries 20% of the global burden of tuberculosis, a situation that is now further exacerbated with the emergence of drug resistance. The current diagnostic technique suggested by the Government of India's Revised National Tuberculosis Control Programme is Ziehl-Neelsen staining of a sputum smear. This technique is known to be inadequate.


OBJECTIVE
The aim of this study was to evaluate nested PCR (nPCR) in the detection of pulmonary tuberculosis in sputum samples in comparison with conventional smear findings, in an effort to improve detection rates from those obtained by the smear-alone approach.


METHODS
Patients attending a tertiary-care hospital (situated in a rural area of Vellore district) with clinical suspicion of pulmonary tuberculosis were prospectively recruited from mid-April 2009 to mid-December 2009 and investigated. The sputum samples were stained by Ziehl-Neelsen staining for smear examination. DNA extracted from concentrated sputum was tested by nPCR, targeting the IS6110 sequence in the Mycobacterium tuberculosis genome.


RESULTS
Among 84 patients tested (median age 45.5 years), 80.95% were from the rural community and 19.05% were from the peri-urban community. Seventeen patients (20.24%; mid-p 95% CI 31.5, 52.4) tested positive by the smear examination and 35 (41.67%; mid-p 95% CI 12.7, 29.8) tested positive by nPCR. The difference in detection rates was statistically significant (chi(2) = 9.02; p = 0.002). The kappa coefficient between smear findings and nPCR findings was 0.47, which was a statistically significant agreement (Z = 4.91; p < 0.0001).


CONCLUSIONS
This report describes the molecular detection of M. tuberculosis in patients' sputum samples tested by the nPCR format, using IS6110 as a target sequence. A high prevalence of pulmonary tuberculosis was identified by the nPCR assay, which was shown to have a significantly higher detection rate than conventional smear staining.
Sankar
Sathish
S
Division of Biomedical Research, Sri Narayani Hospital and Research Centre, Thirumalaikodi, Sripuram, Vellore, Tamil Nadu, India. sathish3107@gmail.com
Balakrishnan
Babu
B
Nandagopal
Balaji
B
Thangaraju
Kannaki
K
Natarajan
Subapriya
S
D000328:Adult; D004251:DNA Transposable Elements; D005260:Female; D006801:Humans; D007194:India; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015995:Prevalence; D011446:Prospective Studies; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000453:epidemiology
D004251:DNA Transposable Elements
Vet Microbiol. 2004 Nov 15;103(3-4):209-17
15504592
J Exp Med. 1915 Jan 1;21(1):38-42
19867850
Pak J Biol Sci. 2009 Feb 15;12(4):373-7
19579972
J Bras Pneumol. 2009 Jul;35(7):690-7
19669008
J Clin Microbiol. 2001 Sep;39(9):3222-7
11526154
J Clin Microbiol. 2000 Mar;38(3):1166-9
10699014
J Clin Microbiol. 1993 Jul;31(7):1777-82
8349753
Clin Infect Dis. 2009 Mar 15;48(6):725-32
19207077
J Clin Microbiol. 2004 Sep;42(9):4307-9
15365029
Mol Cell Probes. 1998 Aug;12(4):207-11
9727196
J Clin Microbiol. 2001 Oct;39(10):3799-800
11574623
Tuber Lung Dis. 1995 Oct;76(5):425-30
7496004
J Clin Microbiol. 2006 Apr;44(4):1594-5
16597905
J Clin Microbiol. 2002 Dec;40(12):4785-8
12454197
Indian J Med Microbiol. 2010 Jul-Sep;28(3):227-32
20644311
Southeast Asian J Trop Med Public Health. 2001 Mar;32(1):114-25
11485071
Indian J Med Microbiol. 2006 Apr;24(2):124-6
16687864
J Clin Microbiol. 2002 Dec;40(12):4646-51
12454166
Indian J Tuberc. 2007 Jul;54(3):130-5
17886701
J Clin Microbiol. 1993 Jun;31(6):1435-8
8314982
J Clin Microbiol. 2006 Mar;44(3):1029-39
16517891