Comparison of Amplicor, In-House Polymerase Chain Reaction, and Conventional Culture for the Diagnosis of Tuberculosis in Children

Abstract

A total of 251 clinical specimens (235 gastric aspirates and 16 bronchoalveolar lavages) from 88 children were prospectively tested in a blinded manner for the presence of Mycobacterium tuberculosis complex, by use of the Amplicor M. tuberculosis test and by means of in-house polymerase chain reaction (PCR). The results were compared with those obtained by conventional culture and by direct microscopy. All of the children underwent extended follow-up to verify or exclude the clinical diagnosis of tuberculosis. The results of the different tests, when compared to the final clinical diagnosis, were a sensitivity of 60% and a specificity of 96.8% for in-house PCR, 44% and 93.7% respectively for the Amplicor test, 44% and 100% for mycobacterial culture and 12% and 100% for microscopy. Amplicor tests presented false-positive findings in children without tuberculous infection. We conclude that both in-house PCR and the Amplicor test are rapid methods that can be helpful for difficult or urgent diagnosis of tuberculosis in children. However, efforts should be aimed toward improvement of the sensitivity and specificity of an easy-to-use PCR kit.

Classic and New Diagnostic Approaches to Childhood Tuberculosis

“…The diagnosis of tuberculosis in children is complicated mainly because (i) TB can mimic many common childhood diseases, including pneumonia, generalized bacterial and viral infections, malnutrition, and HIV [18]; (ii) the absence of a practical reference test or gold standard [12, 24]; (iii) the inability of preadolescent patients to expectorate sputum [14, 22]; (iv) the nonspecific clinical presentation [9, 11, 19, 22]; (v) the lower bacillary load often smear negative [3, 14, 19, 22, 25–27]; (vi) confirmation by culture of Mycobacterium tuberculosis , the gold standard of diagnosis in adult TB, rarely exceeds 30–40% sensitivity [3] (although it may be considerably higher in children with advanced disease) [19] even when using gastric aspirates, induced sputum, liquid media, and polymerase chain reaction (PCR) [6, 14, 22] and, in addition, the distinction between recent primary infection and active disease is highly problematic [28] even the gastric aspirates continue to be the best specimens for testing for suspected pulmonary TB in children [29] with 30–40% sensitivity [19]. Symptoms of childhood TB are nonspecific, and up to 50% of children may be asymptomatic in early stages of the disease.…”

“…The isolation of Mycobacterium tuberculosis takes several weeks. Consequently, the diagnosis of TB in children is often supported only by epidemiological, clinical, and radiographic findings in the presence of a positive tuberculin skin test [29]. …”

Advances in the Diagnosis of Pulmonary Tuberculosis in HIV-Infected and HIV-Uninfected Children

“…In adults, commercial assays display high specificity (pooled specificity, 97% [24]) but poor sensitivity for smear-negative samples (pooled estimate, 66% [25]). Although there have been no large studies evaluating the performance of these commercial assays in children, in one study, the sensitivity of the Roche Amplicor MTB test was disappointing (44%) [26]. The performance of in-house assays has been highly variable [27], probably because of variability in patient populations, DNA extraction and amplification protocols, and operator experience.…”

Relevance of Commercial Amplification Methods for Direct Detection of Mycobacterium tuberculosis Complex in Clinical Samples

“…When extrapulmonary specimens or mixtures of respiratory and extrapulmonary specimens were evaluated, sensitivities ranged from 27.3 to 85%, from 87.5 to 100% and from 17.2 and 70.8% for overall, smear-positive and smear-negative specimens, respectively. Disappointing results were obtained (11,29,43,53) when testing pleural fluids, gastric aspirates, lymph nodes, and cerebrospinal fluids. On the other hand, Gamboa et al (27) did not find any significant difference in sensitivities and specificities between the COBAS Amplicor with the IAC and the manual version, as none of the 755 specimens showed inhibition.…”