Combined IFN-γ and IL-2 release assay for detect active pulmonary tuberculosis: a prospective multicentre diagnostic study in China.

BACKGROUND: We performed a prospective multicentre diagnostic study to evaluate the combined interferon-γ (IFN-γ) and interleukin-2 (IL-2) release assay for detect active pulmonary tuberculosis (TB) in China. METHODS: Adult patients presenting symptoms suggestive of pulmonary TB were consecutively enrolled in three TB-specialized hospitals. Sputum specimens and blood sample and were collected from each participant at enrolment. The levels of Mycobacterium tuberculosis (MTB)-specific antigen-stimulated IFN-γ and IL-2 were determined using enzyme-linked immunosorbent assay (ELISA). RESULTS: Between July 2017 and December 2018, a total of 3245 patients with symptoms suggestive of pulmonary TB were included in final analysis. Of 3245 patients, 2536 were diagnosed as active TB, consisting of 1092 definite TB and 1444 clinically diagnosed TB. The overall sensitivity and specificity of IFN-γ were 83.8% and 81.5%, respectively. In addition, compared with IFN-γ, the specificity of IL-2 increased to 94.3%, while the sensitivity decreased to 72.6%. In addition, the highest sensitivity was achieved with parallel combination of IFN-γ/IL-2, with a sensitivity of 87.9%, and its overall specificity was 79.8%. The sensitivity of series combination test was 68.5%. Notably, the sensitivity of series combination test in definite TB (72.1%) was significantly higher than that in clinically diagnosed TB (65.8%). CONCLUSION: In conclusion, we develop a new immunological method that can differentiate between active TB and other pulmonary diseases. Our data demonstrates that the various IFN-γ/IL-2 combinations provides promising alternatives for diagnosing active TB cases in different settings. Additionally, the diagnostic accuracy of series combination correlates with severity of disease in our cohort.

Retrospective Analysis of False-Positive and Disputed Rifampin Resistance Xpert MTB/RIF Assay Results in Clinical Samples from a Referral Hospital in Hunan, China.

Concerns about the specificity of the Xpert MTB/RIF (Xpert) assay have arisen, as false-positive errors in the determination of Mycobacterium tuberculosis complex (MTBC) infection and rifampin (RIF) resistance in clinical practice have been reported. Here, we investigated 33 cases where patients were determined to be RIF susceptible using the Bactec MGIT 960 (MGIT) culture system but RIF resistant using the Xpert assay. Isolates from two of these patients were found not to have any mutations in the rifampin resistance determining region (RRDR) region of rpoB and had good treatment outcomes with first-line antituberculosis (anti-TB) drugs. The remaining 31 patients included 5 new cases and 26 previously treated patients. A large number of well-documented disputed mutations, including Leu511Pro, Asp516Tyr, His526Asn, His526Leu, His526Cys, and Leu533Pro, were detected, and mutations, including a 508 to 509 deletion and His526Gly, were described here as disputed mutations for the first time. Twenty-one (81%) of the 26 previously treated patients had poor treatment outcomes, and isolates from 19 (90%) of these 21 patients were resistant to isoniazid (INH) as determined using the MGIT culture system. Twenty-seven of the 31 isolates with disputed rpoB mutations were phenotypically resistant to INH, 21 (78%) being predicted by GenoType MTBDRplus to have a high level of INH resistance. Most (77.4%) of the isolates with disputed mutations were of the Beijing lineage. These findings have implications for the interpretation of false-positive and disputed rifampin resistance Xpert MTB/RIF results in clinical samples and provide guidance on how clinicians should manage patients carrying isolates with disputed rpoB mutations.

Prevalence and molecular characteristics of drug-resistant Mycobacterium tuberculosis in Hunan, China.

To determine the prevalence and molecular characteristics of drug-resistant tuberculosis in Hunan province, drug susceptibility testing and spoligotyping methods were performed among 171 M. tuberculosis isolates. In addition, the mutated characteristics of 12 loci, including katG, inhA, rpoB, rpsL, nucleotides 388 to 1084 of the rrs gene [rrs(388-1084)], embB, pncA, tlyA, eis, nucleotides 1158 to 1674 of the rrs gene [rrs(1158-1674)], gyrA, and gyrB, among drug-resistant isolates were also analyzed by DNA sequencing. Our results indicated that the prevalences of isoniazid (INH), rifampin (RIF), streptomycin (SM), ethambutol (EMB), pyrazinamide (PZA), capreomycin (CAP), kanamycin (KAN), amikacin (AKM), and ofloxacin (OFX) resistance in Hunan province were 35.7%, 26.9%, 20.5%, 9.9% 15.2%, 2.3%, 1.8%, 1.2%, and 10.5%, respectively. The previously treated patients presented significantly increased risks for developing drug resistance. The majority of M. tuberculosis isolates belonged to the Beijing family. Almost all the drug resistance results demonstrated no association with genotype. The most frequent mutations of drug-resistant isolates were katG codon 315 (katG315), inhA15, rpoB531, rpoB526, rpoB516, rpsL43, rrs514, embB306, pncA96, rrs1401, gyrA94, and gyrA90. These results contribute to the knowledge of the prevalence of drug resistance in Hunan province and also expand the molecular characteristics of drug resistance in China.

Bacillus velezensis WB induces systemic resistance in watermelon against Fusarium wilt.

BACKGROUND: Our previous findings indicated that Bacillus velezensis WB could control Fusarium wilt by changing the structure of the microbial community in the watermelon rhizosphere. However, there are few studies on its mechanism in the pathogen resistance of watermelon. Therefore, in this study, we determined the mechanism of B. velezensis WB-induced systemic resistance in watermelon against Fusarium wilt through glasshouse pot experiments. RESULTS: The results showed that B. velezensis WB significantly reduced the incidence and disease index of Fusarium wilt in watermelon. B. velezensis WB can enhance the basal immunity of watermelon plants by: increasing the activity of phenylalanine ammonia-lyase (PAL), peroxidase (POD), superoxide dismutase (SOD) and ß-1,3-glucanase; accumulating lignin, salicylic acid (SA) and jasmonic acid (JA); reducing malondialdehyde (MDA) concentrations; and inducing callus deposition in watermelon plant cells. RNA-seq analysis showed that 846 watermelon genes were upregulated and 612 watermelon genes were downregulated in the WF treatment. This process led to the activation of watermelon genes associated with auxin, gibberellin, SA, ethylene and JA, and the expression of genes in the phenylalanine biosynthetic pathway was upregulated. In addition, transcription factors involved in plant resistance to pathogens, such as MYB, NAC and WRKY, were induced. Gene correlation analysis showed that Cla97C10G195840 and Cla97C02G049930 in the phenylalanine biosynthetic pathway, and Cla97C02G041360 and Cla97C10G197290 in the plant hormone signal transduction pathway showed strong correlations with other genes. CONCLUSION: Our results indicated that B. velezensis WB is capable of inducing systemic resistance in watermelon against Fusarium wilt. © 2023 Society of Chemical Industry.

Complete genome sequence of Bacillus velezensis WB, an isolate from the watermelon rhizosphere: genomic insights into its antifungal effects.

OBJECTIVES: Here, we report the complete genome of strain WB, isolated from the rhizosphere of a healthy watermelon from a Fusarium wilt diseased field, which possesses antifungal activity against Fusarium oxysporum f. sp. niveum (Fon) and reduces the incidence of Fusarium wilt in watermelon. METHODS: Genome sequences were determined using the Illumina HiSeq and PacBio platforms. Genome assembly was performed by Unicycler software. Gene clusters responsible for secondary metabolite biosynthesis were predicted using antiSMASH. RESULTS: The size of the genome was 3 896 799 base pairs, and there were 3977 coding DNA sequences (CDSs). The G+C content of the circular genome was 46.65%, and there were 27 rRNAs and 86 tRNAs. Strain WB was finally designated Bacillus velezensis based on phylogenomic analyses. In addition, 13 gene clusters were related to the synthesis of antimicrobial secondary metabolites, including surfactin, fengycin, iturin, bacillibactin, bacilysin, bacillaene, and butirosin. CONCLUSION: The complete genome sequence of strain WB reported here will be a valuable reference for studying the biocontrol mechanisms of Fusarium wilt in watermelon.

[Effect of fluorouracil combined with FK228 on the proliferation, apoptosis and Fas mRNA level in HepG2 hepatoma cell lines].

OBJECTIVE: To explore the effect of fluorouracil (FU) combined with epigenetic drug FK228 (depsipeptide FR901228) on the proliferation, apoptosis and Fas mRNA level in HepG2 hepatoma cell lines. METHODS: There were 4 groups in this experiment: an untreated group, an FK228 treated group, a FU treated group,and a FU combined with FK228 group.Tetrazolium salt colorimetry (MTT) assay was used to assess the cell inhibition rate. Q value of Kingos formula and multi-factor analysis of variance (ANOVA ) were used to judge the combination treatment effect,and flow cytometry was used to detect the apoptosis rate. Fas mRNA level was analyzed by RT-PCR. RESULTS: FK228 or FU would inhibit the growth of HepG2 cells in a concentration-dependent and time-dependent manner. Cell inhibition rates of HepG2 were significantly enhanced in the FU combined with FK228 group, compared with that in the FU treated group alone (P<0.05). Both Q values were more than 1, the 2 drug combinations showed interaction,and FU combined with FK228 had synergistic effect.Compared with the FK228 treated group and the FU treated group, apoptosis rate of HepG2 cells was significantly increased (P<0.05), and the Fas mRNA level was up-regulated in HepG2 cells in FU combined FK228 group (P<0.05). CONCLUSION: Combination of FK228 and FU can enhance the proliferation inhibition and apoptosis induction of FU in hepatoma cell lines, up-regulate the Fas mRNA level, and increase the sensitivity of hepatoma cell lines to FU.

Multicenter clinical evaluation of three commercial reagent kits based on the interferon-gamma release assay for the rapid diagnosis of tuberculosis in China.

OBJECTIVE: To evaluate the performance of three interferon-gamma release assay (IGRA) kits in detecting Mycobacterium tuberculosis infection in China. METHODS: A multicenter clinical trial was used to compare the effectiveness and application of the three kits. A total of 1026 participants were enrolled at three hospitals, including 597 tuberculosis (TB) patients diagnosed clinically (517 patients with pulmonary TB and 80 patients with extrapulmonary TB) and 429 negative controls (244 patients with pulmonary disease but not TB, or with non-tuberculosis mycobacterial lung diseases, and 185 healthy people). Detection performance indicators including sensitivity, specificity, and the Youden index (YI) were used to evaluate performance. RESULTS: Through bacterial culture evaluation, 224 of the 517 pulmonary TB patients were positive and all 429 negative controls were negative. When the gold standard bacterial methods were used, the sensitivity, specificity, and YI were 89.7% (201/224), 91.1% (391/429), and 0.81 for T-SPOT.TB, 86.2% (193/224), 87.2% (374/429), and 0.73 for QB-SPOT, and 83.9% (188/224), 88.6% (380/429), and 0.73 for TB-IGRA, respectively. There were no significant differences in the sensitivity and specificity of the three kits. CONCLUSIONS: The results showed that the three kits had very high sensitivity and specificity and exhibited a good performance for the detection of M. tuberculosis infection.

Evaluation of the Xpert MTB/RIF assay for diagnosis of tuberculosis and rifampin resistance in county-level laboratories in Hunan province, China.

BACKGROUND: The Xpert MTB/RIF showed high sensitivity and specificity in previous studies carried out in different epidemiological and geographical settings and patient populations in high-burden tuberculosis (TB) countries. However, there were little data obtained by validation or demonstration study of the assay in China. In this study, the performance of Xpert MTB/RIF was investigated in two county-level laboratories in Hunan Province, China. METHODS: Consecutive patients with suspected pulmonary tuberculosis (PTB) and suspicion for multidrug-resistant tuberculosis (MDR-TB) were enrolled. For each patient suspected to have PTB, three sputum specimens (one spot sputum, one night sputum, and one morning sputum) were collected and each sputum was tested with smear microscopy, Löwenstein-Jensen (LJ) culture, and Xpert MTB/RIF test. For comparison across subgroups and testing methods, 95% confidence intervals were calculated. All analyses were done with SPSS 16.0, and P < 0.05 was regarded as significant. RESULTS: For case detection, the sensitivity of Xpert MTB/RIF was 100% for smear- and culture-positive TB and 88.6% for smear-negative and culture-positive TB; the overall sensitivity was 94.5% for all culture-positive patients. The specificity was 99.8%. The sensitivity of Xpert MTB/RIF assay was 22.0% in clinical TB patients and the specificity reached 100.0% in the group of patients who are infected with nontuberculous mycobacteria. For the detection of rifampin resistance, the sensitivity of MTB/RIF RIF-resistance detection was 92.9%, and the specificity was 98.7%. Of the 26 Xpert MTB/RIF-positive and RIF-resistant patients confirmed by LJ proportion tests, 20 (76.9%) patients were infected by MDR-TB. CONCLUSIONS: The Xpert MTB/RIF assay is a highly sensitive and specific method for diagnosis of TB and RIF resistance, which will enable it to have the potential to be used in county-level laboratories and lead to the reduction of the infectious pool and improvements in TB control in China. Further evaluations in county-level laboratories for implementing the assay are still required.

Multicenter research on the BACTEC MGIT 960 system for the second-line drugs susceptibility testing of Mycobacterium tuberculosis in China.

The reliability of the BACTEC MGIT 960 system for the second-line drugs (capreomycin [CPM], kanamycin [KAN], ofloxacin [OFX] and ethionamide [ETH]) susceptibility testing (DST) of Mycobacterium tuberculosis (M. tuberculosis) was compared to that of traditional Lowenstein-Jensen (L-J) proportion method (PM) among four different sites in China. After resolution of discrepant results by retesting the strains using both methods in the National Reference Laboratory of tuberculosis, the overall concordance values between the 2 systems were 99.7% (kappa value: 0.97) for CPM, 99.7% (kappa value: 0.97) for KAN, 100.0% (kappa value: 1.00) for OFX, and 98.6% (kappa value: 0.95) for ETH. The average turnaround time with BACTEC MGIT 960 system among four sites was 8.9 ± 1.7 days, significantly shorter than 28 days with the traditional L-J PM. Therefore, the BACTEC MGIT 960 system is a reliable and rapid method for the second-line drug susceptibility testing of tuberculosis in China. Notably, a stricter quality control program should be routinely carried out when clinical laboratories perform the second-line DST with BACTEC MGIT 960 system.

Evaluation of a novel biphasic culture medium for recovery of mycobacteria: a multi-center study.

BACKGROUND: Mycobacterial culture and identification provide a definitive diagnosis of TB. Culture on Löwenstein-Jensen (L-J) medium is invariably delayed because of the slow growth of M. tuberculosis on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients. METHODS AND FINDINGS: The biphasic medium consisted of 7 ml units of L-J slant medium, 3 ml units of liquid culture medium, growth indicator and a mixture of antimicrobial agents. The decontamination sediments of sputum specimens were incubated in the biphasic culture medium at 37°C. Mycobacterial growth was determined based on the appearance of red granule sediments and the examination using acid-fast bacilli (AFB). The clinical sputum specimens were cultured in the biphasic medium, on L-J slants and in the Bactec MGIT 960 culture system. Among smear-positive specimens, the mycobacteria recovery rate of the biphasic medium was higher than that of the L-J slants (P<0.001) and similar to that of MGIT 960 (P>0.05). Among smear-negative specimens, the mycobacterial recovery rate of the biphasic medium was higher than that of L-J slants (P<0.001) and lower than that of MGIT 960 (P<0.05). The median times to detection of mycobacteria were 14 days, 20 days and 30 days for cultures grown in MGIT, in biphasic medium, on L-J slants for smear negative specimens, respectively (P<0.001). CONCLUSIONS: The biphasic culture medium developed in this study is low-cost and suitable for mycobacterial recovery. It does not require any expensive detection instrumentation, decreases the time required for detection of M. tuberculosis complex, and increases the detection rate of M. tuberculosis complex.

[Population dynamics of pests and their enemies in different cultivated rice fields].

Through a whole year field investigation, this paper analyzed the population dynamics of pests and their enemies in organic and normally cultivated rice fields. The results indicated that the population of main pests varied more lightly in organic than in normally cultivated rice field, while subordinated pest species had little difference between these two fields. The individuals of enemies were more in organic than in normally cultivated rice field. It is suggested that the pest-control effect of enemies could be restored and became stronger when no chemical pesticides were applied.