Evaluation of an in-house loop-mediated isothermal amplification for Mycobacterium tuberculosis detection in a remote reference laboratory, Thailand

ABSTRACT Loop-mediated isothermal amplification (LAMP) is a simple and efficient nucleic acid amplification method for the rapid diagnosis of infectious diseases. This study assessed the performance of an in-house LAMP for tuberculosis (TB) diagnosis at a remote reference laboratory in the endemic setting of Thailand. As part of the routine service, 1,882 sputum samples were processed for mycobacterial culture in Lowenstein-Jensen and MGIT media. The DNA was extracted from the remaining decontaminated samples after the culture procedure for real-time polymerase chain reaction (PCR) analysis using Anyplex plus MTB/NTM detection kit. 785 (40.28%) were positive by mycobacterial culture. Of these, 222 DNA remnants were available and subjected to LAMP analysis. Based on culture as reference (Mycobacterium tuberculosis; MTB= 209/ non-tuberculous mycobacteria; NTM= 13), the overall sensitivity of LAMP and Anyplex plus assays for MTB detection were 89.95% (188/209; 95% confidential interval [CI]: 85.05-93.67%) and 96.65% (202/209; 95% CI: 93.22-98.64%), and the accuracy values were 88.74% (197/222; 95% CI: 83.83-92.58) and 96.40% (214/222; 93.02-98.43%), respectively. The sensitivity and accuracy of the in-house LAMP and the Anyplex plus real-time PCR assay were high in comparison to culture results. The high sensitivity and accuracy suggested that this in-house LAMP was promising and might be useful for early TB diagnosis.

Evaluation of the Anyplex BRAF V600E Real-Time Detection Assay Using Dual-Priming Oligonucleotide Technology in Fine-Needle Aspirates of Thyroid Nodules

BACKGROUND: Several molecular assays have been developed to detect the BRAF V600E mutation in fine needle aspirates (FNAs) for the diagnosis of papillary thyroid cancer. Using a multiplex PCR technique, we evaluated the Anyplex BRAF V600E Real-time Detection (Anyplex) assay and compared its efficacy with that of the Seeplex BRAF V600E ACE Detection (Seeplex) method. METHODS: We tested 258 consecutive FNA specimens using the Seeplex and Anyplex assays. Any conflicting results between the two assays were confirmed by using mutant enrichment with 3'-modified oligonucleotide (MEMO) sequencing. The limits of detection (LODs) and reproducibility for each assay were evaluated with serially diluted DNA from a BRAF V600E-positive cell line. RESULTS: The BRAF V600E mutation was detected in 36.4% (94/258) FNA specimens by either the Seeplex or Anyplex assay. Results for the two assays showed 93.4% (241/258) agreement, with a kappa value of 0.861 (95% confidence interval, 0.798-0.923). Of the eight specimens that were BRAF V600E-positive by the Anyplex assay but not by the Seeplex assay, five were found to be BRAF V600E-positive by MEMO sequencing. The mutation detection rate of the Seeplex and Anyplex assays was 79.0% and 84.0%, respectively, in the FNA specimens diagnosed as malignant (n=81). The LOD as determined by probit analysis was 0.046% (95% confidence interval, 0.019-0.532%). CONCLUSIONS: The Anyplex assay performed better than the Seeplex assay with respect to the detection of the BRAF V600E mutation.

Performance Evaluation of Anyplex Plus MTB/NTM and MDR-TB Detection Kit for Detection of Mycobacteria and for Anti-Tuberculosis Drug Susceptibility Test / 대한임상미생물학회지

BACKGROUND: The Anyplex plus MTB/NTM and MDR-TB Detection kits (Seegene, Korea) a real-time PCR assays for direct detection of Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) and for identification of rifampin (RIF) and isoniazid (INH) resistance of MTB in various specimens. We evaluated the diagnostic performance of the Anyplex plus MTB/NTM and MDR-TB Detection kit. METHODS: To determine the ability of the kit to detect MTB and NTM, 557 samples were tested. The diagnostic performance of the Anyplex plus MTB/NTM Detection kit was determined based on the results of culture, nucleic acid amplification tests (NAAT), radiologic analysis, and clinical features suggestive of mycobacterial infection. The performance of the kit was compared with those of other real-time PCR kits. For the drug susceptibility test (DST), 51 MTB isolates were tested. The diagnostic performance of the Anyplex plus MDR-TB Detection kit was determined based on the conventional DST and compared with other molecular DST kits. RESULTS: Sensitivity and specificity for MTB detection of the Anyplex plus MTB/NTM Detection kit were 82.9% (63/76) and 99.4% (478/481), respectively, while those for NTM detection were 76.5% (13/17) and 89.6% (484/540). Sensitivity and specificity for RIF resistance detection of the Anyplex plus MDR-TB Detection kit were 100% (3/3) and 97.9% (47/48), respectively, while those for INH resistance detection were 83.3% (5/6) and 100% (45/45). CONCLUSION: The Anyplex plus MTB/NTM Detection kit showed good diagnostic performance for detection of MTB and NTM. Especially in MTB-positive cases, the Anyplex plus MDR-TB Detection kit provided rapid and reliable results of drug resistance to RIF and INH.