Pulmonary tuberculosis in the West Bank, Palestinian Authority: molecular diagnostic approach

Abstract

Summaryobjective To compare the effectiveness and feasibility of an insertion sequence (IS6110)-based polymerase chain reaction (PCR) assay with conventional methods of detecting Mycobacterium tuberculosis and to analyse mutations present in the hot spot region of the RNA polymerase B subunit (rpoB) gene associated with rifampin resistance by DNA sequencing.methods Ninety-five sputum samples from 84 clinically suspected cases of tuberculosis were tested for mycobacterial infections by Ziehl Neelsen smear examination, Lowenstein-Jensen culture and IS6110-based PCR assay.results Sensitivity and specificity of the PCR were 94%; the sensitivity of culture was 65%, and of smear tests, 59%. Both smear microscopy and culture had 100% specificity. DNA sequencing data of the 305-bp fragment of the rpoB gene for nine clinical isolates revealed one point mutation at position I572F and double mutations at position S531F in two isolates obtained from two patients who did not respond to the anti-tuberculosis therapy.conclusion IS6110-based PCR can be used routinely in clinical laboratories for rapid detection of Mycobacterium tuberculosis and thus allow early diagnosis and treatment of any contacts by the cheapest method currently available in the Palestinian Authority region. Rapid detection of rifampin resistance isolates will enable efficient treatment of patients and assist in eradication of the disease in the Palestinian territories.

Detection of Mycobacterium tuberculosis complex by nested polymerase chain reaction in pulmonary and extrapulmonary specimens

“…In those blood samples, smear microscopy and culture were positive in only 1 and 2 samples, respectively; this might be due to the small number of bacilli in the circulation. ( 6 , 7 ) In fact, the sensitivity of PCR in peripheral blood mononuclear cells, as used here, has been reported to be better than is that of culture, both having similar specificity. ( 11 , 16 , 29 ) In addition, clinical specimens, such as cerebrospinal fluid, blood, and sputum, have been described as good substrates for PCR( 26 ) with good concordance (kappa = 0.6867; p = 0.625; McNemar's test), as in our results, in which the level of positivity in extrapulmonary samples was higher when nested PCR was used (p = 0.0042), thus corroborating the findings of Noussair et al( 21 ) Different rates of sensitivity were described using the molecular methodology for this type of sample in studies conducted in France( 21 ) and India( 12 ) (86.6% and 90%, respectively).…”

“…This false-negative result in a cerebrospinal fluid sample (Table 1) might be related to the absence of copies of the IS 6110 gene, as previously described in studies conducted in southeast Asia, Denmark, Tunisia, India, and Vietnam, indicating the need to incorporate additional targets, such as TRC4, in order to improve molecular detection. ( 7 , 10 ) However, to our knowledge, no study to date has reported the absence of this element in M. tuberculosis strains in Brazil, and various studies have reported that this sequence is the most sensitive in order to detect the M. tuberculosis complex. ( 9 , 17 ) Another factor could be the presence of molecular reaction inhibitors in up to 18.6% of extrapulmonary specimens.…”

“…(5,8) The molecular diagnosis of tuberculosis by polymerase chain reaction (PCR) and primers with high specificity (98%), with high variations in sensitivity (20-100%), has been used in order to identify genetic targets in the bacillus. ( 7 , 11 , 12 )…”

“…( 5 , 8 ) O diagnóstico molecular da tuberculose por polymerase chain reaction (PCR, reação em cadeia da polimerase) com primers de alta especificidade (98%), com amplas variações de sensibilidade (20-100%), tem sido utilizado para a identificação de alvos genéticos no bacilo. ( 7 , 11 , 12 )…”

IS6110 the Double-Edged Passenger

“…Some authors concluded that IS6110-based PCR could be used routinely in clinical laboratories for rapid detection of M. tuberculosis, in sputum samples allowing early diagnosis and treatment (Ereqat et al, 2011). Evaluation of in-house PCR showed that variability in sensitivity and specificity is high (Cho et al, 2007).…”