Comparison of Loop-Mediated Isothermal Amplification, Microscopy, Culture, and PCR for Diagnosis of Pulmonary Tuberculosis

Abstract

A c c e p t e d M a n u s c r i p t SUMMARY: Diagnosis tuberculosis (TB) in endemic countries is challenging due to high caseloads and limited resources. A simple, cost-effective diagnostic test for rapid detection of Mycobacterium tuberculosis in clinical specimens is in need. In Thailand, we evaluated the performance of in-house loop-mediated isothermal amplification (LAMP) targeting M. tuberculosis 16s rRNA gene for TB diagnosis. A total of 252 sputum samples from pulmonary TB suspects were analyzed. The sensitivity of LAMP was 99.04% (103/104; 95% confidential interval [CI]: 94.76-99.98%) and 72.73% (16/22; 95% CI: 49.78-89.27) for smear-positive and smear-negative samples with TB-culture positivity, respectively. LAMP detected 20.69% (24/116) of TB-culture negative samples but those all were positive by conventional PCR. The sensitivity of LAMP was higher than that of sputum microscopy while the performance of LAMP was similar to PCR. None of the samples positive for nontuberculous mycobacteria by culture and PCR was positive by LAMP. Compared with TB culture, the positive predictive value (PPV), negative predictive value (NPV) and kappa coefficient of LAMP were 83.22%, 88.33% and 0.75 respectively. Based on the diagnostic performance, we proposed that this LAMP would be suitable as a potential diagnostic test for rapid TB diagnosis in resource-limited laboratory settings.

Pragmatic accuracy of an in-house loop-mediated isothermal amplification (LAMP) for diagnosis of pulmonary tuberculosis in a Thai community hospital

“…the majority of the previous in-house LAMP studies [11,15,[20][21][22][23]. Nonetheless, the specificity was comparable to other figures reported in the literature [11,12,15,21,22].…”

“…The LAMP test consists of three steps as follows: DNA extraction, isothermal amplification, and visual interpretation with fluorescence. In this study, we followed the TB Fast AMP technique, which was developed by the National Institute of Health of Thailand and was described in our previous studies [13,15,19]. The procedures were described as follow.…”

“…Based on the result of our study, we suggest that the in-house LAMP should not be a substitute to conventional smear methods, but should be done in parallel, which would result in a higher sensitivity with fewer false-negative TB cases. In the past, several studies reported a higher sensitivity of in-house LAMP tests, ranging from 90.0 to 100.0% [11,15,[20][21][22][23][24][25]. Most of these studies were reported from either university hospitals, TB-specialized centers or hospitals, or national TB-specialized laboratories, which were generally equipped with highly trained personnel and adequate infrastructural supports [17].…”

“…As financial resources are usually limited in countries with high TB prevalence, a commercial TB-LAMP could still be unattainable. An in-house LAMP assay may be more affordable to the commercial one [11][12][13][14][15]. However, it did require extra-training and skill of technicians to process the clinical specimens.…”

“…From the latest meta-analysis, the overall sensitivity and specificity of in-house LAMP tests was 93.0% (95%CI 88.9-95.7) and 91.8% (95%CI 86.4-95.1), respectively [17]. One recent study in Thailand reported the sensitivity and the specificity of the in-house LAMP at 94.4% (95%CI 88.9-97.7) and 94.3% (95%CI 87.2-98.1), respectively [15]. However, the reported accuracy could be overestimated if it is assessed in qualified laboratories with highly skilled technicians and sufficient resources where molecular tests are usually available [17].…”