QuantiFERON-TB Gold Plus and QuantiFERON-TB Gold In-tube assays for detecting latent tuberculosis infection in Thai healthcare workers

Detecting latent tuberculosis infection (LTBI) is important, especially in high-risk populations including healthcare workers (HCWs). QuantiFERON-TB Gold Plus (QFT-Plus) is a new version of the interferon-gamma release assays (IGRAs) to replace the QuantiFERON-TB Gold In-tube (QFT-GIT). However, data on the use of QFT-Plus for LTBI detection in high TB-burden countries are limited. This study was conducted in a TB-endemic setting in Thailand. HCWs were enrolled in the study and underwent both tests during the annual health screening. The testing results were compared and the concordance was determined. Of 102 HCWs, 11 (10.78%) were positive according to both tests, and 15 (14.71%) were positive according to QFT-Plus. The overall agreement between assays was 96.08%, with Cohen’s kappa coefficient (k) at 0.82. All four discordant results occurred with QFT-GIT negative and QFT-Plus positive. The comparison between QFT-GIT and QFT-Plus based on each antigen tube (TB1 or TB2) exhibited similar concordance with 99.02% and 95.10% agreement, respectively. The intra-comparison between TB1 and TB2 of QFT-Plus also showed good concordance at 96.08%. Among this group of HCWs, the LTBI prevalence of any positive results in both tests was low. Overall, the study showed good agreement between QFT-Plus and QFT-GIT (k = 0.82) with a minimal difference, suggesting similar assay performance to that mainly carried out in TB-low incidence countries. The results support the use of QFT-Plus for detecting LTBI in a format similar to QFT-GIT

The performance of an in-house loop-mediated isothermal amplification for the rapid detection of Mycobacterium tuberculosis in sputum samples in comparison with Xpert MTB/RIF, microscopy and culture

Simple, low-cost and effective diagnostic tests for tuberculosis (TB) are needed especially in TB-high burden settings. The present study evaluated the performance of an in-house loop-mediated isothermal amplification (LAMP) for diagnosing TB by comparing it to Xpert MTB/RIF, microscopy and culture. In Thailand, a total of 204 excess sputum samples volume after the processing of cultures were used for Mycobacterium tuberculosis (MTB) detection by Xpert MTB/RIF and LAMP. Based on culture results as the gold standard, the overall sensitivity of LAMP and Xpert MTB/RIF were 82.1% (126/153; 95% confidential interval [CI]: 75.4-88.98%) and 86.9 % (133/153; 95% CI: 80.5-90.8%) respectively, and the specificity of both tests was 100% (51/51; 95% CI: 93.0-100.0%). In comparison with Xpert MTB/RIF, the sensitivity and specificity of LAMP were 94.7% (126/133; 95% CI: 89.5-97.9%), and 100.0% (73/73; 95% CI: 94.9-100.0%), respectively. The average threshold cycle (Ct) of Xpert MTB/RIF detection for positive and negative LAMP results was statistically different, of 18.4 and 27.0, respectively (p < 0.05). In comparison with the acid-fast staining technique, and analyzing LAMP and Xpert MTB/RIF in smear-negative/ culture-positive specimens, there was an increase of the detection rate by 47.7% (21/44) and 54.6% (24/44). The diagnostic sensitivity and specificity of LAMP appeared to be comparable to those of Xpert MTB/RIF. We claim that this LAMP has potential to provide a sensitive diagnostic test for the rapid TB diagnosis. It allowed a fast detection of MTB before the cultures and it could be used in resource-limited laboratory settings

QuantiFERON-TB Gold In-Tube test in active tuberculosis patients and healthy adults

Interferon-gamma (IFN-γ) release assays have improved latent tuberculosis (TB) detection and have been considered promising for the diagnosis of TB disease. However, diagnosis efficacy data is limited in high burden countries. The aim of this study was to determine the diagnostic potential of the QuantiFERON-TB Gold In-Tube (QFT-GIT) test for the diagnosis of active TB in an endemic setting for TB. A cross-sectional study was conducted in a group of 102 Thai patients with clinical symptoms and chest x-ray findings suggesting of active pulmonary TB and a group of 112 healthy adults. Testing was carried out using sputum microscopy, mycobacterial culture and QFT-GIT test. Of these patients, QFT-GIT was positive in 73 (71.57%), negative in 27 (26.47%), and undetermined in 2 (1.96%) cases. Among healthy controls, QFT-GIT was positive in 18 (16.07%), negative in 93 (83.04%), and undetermined in 1 (0.89%) person. Based on TB culture results, the sensitivity of QFTGIT for diagnosing active TB was 84.21% (95% confidence interval (CI); 72.13-92.52). The positive and negative predictive values were 65.75% (95% CI; 59.26-71.70) and 66.67% (95% CI; 49.94-80.04), respectively. The median IFN-γ level in culture-confirmed TB patients was 3.91 compared to 0.03 IU/mL of the healthy group. QFT-GIT appears to be a useful indirect test for TB diagnosis in Thailand and its use is recommended in association with clinical and radiological assessments for identifying active or latent TB

3,3′-Dibutanoyl-1,1′-(o-phenyl­ene)dithio­urea

The mol­ecular conformation of the title compound, C16H22N4O2S2, is stabilized by two intramolecular N—H⋯O hydrogen bonds. The crystal packing shows N—H⋯O and N—H⋯S hydrogen bonds

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

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

The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae

Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02 Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae

Evaluation of qPCR-Based Assays for Leprosy Diagnosis Directly in Clinical Specimens

The increased reliability and efficiency of the quantitative polymerase chain reaction (qPCR) makes it a promising tool for performing large-scale screening for infectious disease among high-risk individuals. To date, no study has evaluated the specificity and sensitivity of different qPCR assays for leprosy diagnosis using a range of clinical samples that could bias molecular results such as difficult-to-diagnose cases. In this study, qPCR assays amplifying different M. leprae gene targets, sodA, 16S rRNA, RLEP and Ag 85B were compared for leprosy differential diagnosis. qPCR assays were performed on frozen skin biopsy samples from a total of 62 patients: 21 untreated multibacillary (MB), 26 untreated paucibacillary (PB) leprosy patients, as well as 10 patients suffering from other dermatological diseases and 5 healthy donors. To develop standardized protocols and to overcome the bias resulted from using chromosome count cutoffs arbitrarily defined for different assays, decision tree classifiers were used to estimate optimum cutoffs and to evaluate the assays. As a result, we found a decreasing sensitivity for Ag 85B (66.1%), 16S rRNA (62.9%), and sodA (59.7%) optimized assay classifiers, but with similar maximum specificity for leprosy diagnosis. Conversely, the RLEP assay showed to be the most sensitive (87.1%). Moreover, RLEP assay was positive for 3 samples of patients originally not diagnosed as having leprosy, but these patients developed leprosy 5–10 years after the collection of the biopsy. In addition, 4 other samples of patients clinically classified as non-leprosy presented detectable chromosome counts in their samples by the RLEP assay suggesting that those patients either had leprosy that was misdiagnosed or a subclinical state of leprosy. Overall, these results are encouraging and suggest that RLEP assay could be useful as a sensitive diagnostic test to detect M. leprae infection before major clinical manifestations

Oleic Acid Biosynthesis in Plasmodium falciparum: Characterization of the Stearoyl-CoA Desaturase and Investigation as a Potential Therapeutic Target

BACKGROUND:Plasmodium falciparum parasitization of erythrocytes causes a substantial increase in the levels of intracellular fatty acids, notably oleic acid. How parasites acquire this monounsaturated fatty acid has remained enigmatic. Here, we report on the biochemical and enzymatic characterization of stearoyl-CoA desaturase (SCD) in P. falciparum. METHODOLOGY/PRINCIPAL FINDINGS:Metabolic labeling experiments allowed us to demonstrate the production of oleic acid from stearic acid both in lysates of parasites incubated with [(14)C]-stearoyl-CoA and in parasite-infected erythrocytes labeled with [(14)C]-stearic acid. Optimal SCD activity was detected in schizonts, the stage of maximal membrane synthesis. This activity correlated with a late trophozoite stage-specific induction of PFE0555w transcripts. PFE0555w harbors a typical SCD signature. Similar to mammalian SCDs, this protein was found to be associated with the endoplasmic reticulum, as determined with PFE0555w-GFP tagged transgenic P. falciparum. Importantly, these parasites exhibited increased rates of stearic to oleic acid conversion, providing additional evidence that PFE0555w encodes the plasmodial SCD (PfSCD). These findings prompted us to assess the activity of sterculic acid analogues, known to be specific Delta9-desaturase inhibitors. Methyl sterculate inhibited the synthesis of oleic acid both with parasite lysates and infected erythrocytes, most likely by targeting PfSCD. This compound exhibited significant, rapid and irreversible antimalarial activity against asexual blood stages. This parasiticidal effect was antagonized by oleic acid. CONCLUSION/SIGNIFICANCE:Our study provides evidence that parasite-mediated fatty acid modification is important for blood-stage survival and provides a new strategy to develop a novel antimalarial therapeutic based on the inhibition of PfSCD