Identification of the causative organism of tuberculous lymphadenitis in ethiopia by PCR

Abstract

Tuberculous lymphadenitis (TBLN) is a common form of extrapulmonary tuberculosis with multiple differential diagnoses. Demonstration of the etiologic agent by smear microscopy or culture of fine needle aspirate (FNA) specimens is often unsuccessful. FNA specimens from 40 patients presenting at a rural health center in South Ethiopia and diagnosed as positive for TBLN on the basis of clinical and cytological criteria were analyzed for mycobacterial DNA by PCR. Thirty (75%) had cervical lymphadenitis and 11 (27.5%) were seropositive for human immunodeficiency virus (HIV). Three primer sets were initially used to identify the causative agent at the genus (antigen 85 complex), complex (IS6110 insertion sequence), and species (pncA gene and allelic variation) levels. Among the forty TBLN cases, 35 (87.5%) were positive by PCR at the genus and complex levels. Based on PCR for detection of allelic variation at position 169, 24 (68.6%) of the 35 were positive for Mycobacterium tuberculosis and 6 (17.1%) were positive for M. bovis. These six were positive in additional PCR assays using the JB21-JB22 primer set, which is highly specific for M. bovis. Five (14.1%) showed amplification for both M. tuberculosis and M. bovis with the allele-specific primer set. Cooccurrence of pyrazinamide (PZA)-sensitive and -resistant M. tuberculosis in those five cases was indicated, since all were negative in assays with the JB21-JB22 primer set. This feature was seen in 3 of 11 HIV-positive and 2 of 29 HIV-negative individuals (P < 0.001).

Conclusion: among 35 PCR-positive cases of TBLN from southern Ethiopia, 29 (82.9%) were caused by M. tuberculosis and six (17.1%) were caused by M. bovis.

FIG. 1.

Analytic sensitivity of the multiprimer PCR system for M. tuberculosis DNA. Lane 1, 123 DNA ladder molecular weight markers; lane 2, 100-ng amplification of the 85a, 85b (506 bp), and IS6110 (984 bp) insertion element genes; lane 3, 10 ng; lane 4, 1 ng; lane 5, 100 pg; lane 6, 10 pg; lane 7, 1 pg.

FIG. 2.

Electrophoretic pattern of the amplified 85a, 85b, and IS6110 insertion element genes from clinical samples. Lane 1, 123 DNA ladder molecular weight markers; lanes 2 to 10, clinical samples; lane 11, positive control; lane 12, negative control.

FIG. 3.

Electrophoretic pattern of allelic PCR system amplified product of pncA genes (185 bp) from clinical samples in 1.5% agarose gel. Lane 1, 123 DNA ladder molecular weight markers; lanes 2 to 7, clinical samples positive for M. tuberculosis and negative for M. bovis; lanes 8 and 9, clinical sample negative for M. tuberculosis and positive for M. bovis, respectively; lanes 10 and 11, coamplified pncA positive for M. tuberculosis PZA-sensitive and -resistant strains, respectively; lanes 12 and 13, M. tuberculosis reference strain DNA tested for the pncA genes of M. tuberculosis and M. bovis, respectively (positive control); lanes 14 and 15, M. bovis reference strain DNA tested for the pncA genes of M. bovis and M. tuberculosis, respectively (positive control); lane 16, negative control.