Nanopore sequencing for smear-negative pulmonary tuberculosis-a multicentre prospective study in China.

PURPOSE: In this prospective study, the diagnosis accuracy of nanopore sequencing-based Mycobacterium tuberculosis (MTB) detection was determined through examining bronchoalveolar lavage fluid (BALF) samples from pulmonary tuberculosis (PTB) -suspected patients. Compared the diagnostic performance of nanopore sequencing, mycobacterial growth indicator tube (MGIT) culture and Xpert MTB/rifampin resistance (MTB/RIF) assays. METHODS: Specimens collected from suspected PTB cases across China from September 2021 to April 2022 were tested then assay diagnostic accuracy rates were compared. RESULTS: Among the 111 suspected PTB cases that were ultimately diagnosed as PTB, the diagnostic rate of nanopore sequencing was statistically significant different from other assays (P < 0.05). Fleiss' kappa values of 0.219 and 0.303 indicated fair consistency levels between MTB detection results obtained using nanopore sequencing versus other assays, respectively. Respective PTB diagnostic sensitivity rates of MGIT culture, Xpert MTB/RIF and nanopore sequencing of 36.11%, 40.28% and 83.33% indicated superior sensitivity of nanopore sequencing. Analysis of area under the curve (AUC), Youden's index and accuracy values and the negative predictive value (NPV) indicated superior MTB detection performance for nanopore sequencing (with Xpert MTB/RIF ranking second), while the PTB diagnostic accuracy rate of nanopore sequencing exceeded corresponding rates of the other methods. CONCLUSIONS: In comparison with MGIT culture and Xpert MTB/RIF assays, BALF's nanopore sequencing provided superior MTB detection sensitivity and thus is suitable for testing of sputum-scarce suspected PTB cases. However, negative results obtained using these assays should be confirmed based on additional evidence before ruling out a PTB diagnosis.

Antibacterial activity of the novel compound Sudapyridine (WX-081) against Mycobacterium abscessus.

Objective: This study aimed to investigate sudapyridine (WX-081) antibacterial activity against Mycobacterium abscessus in vitro and its effect on in vivo bacterial growth and host survival using a zebrafish model of M. abscessus infection. Methods: WX-081 in vitro antibacterial activity was assessed based on growth inhibition of M. abscessus standard strain ATCC19977 and 36 clinical isolates. Maximum tolerated concentrations (MTCs) of WX-081, bedaquiline, and azithromycin and inhibition of M. abscessus growth were assessed in vivo after fluorescently labelled bacilli and drugs were injected into zebrafish. Bacterial counts were analysed using one-way ANOVA and fluorescence intensities of zebrafish tissues were analysed and expressed as the mean ± SE. Moreover, Kaplan-Meier survival analysis was conducted to assess intergroup differences in survival of M. abscessus-infected zebrafish treated with different drug concentrations using a log-rank test, with a p value <0.05 indicating a difference was statistically significant. Results: Drug sensitivity testing of M. abscessus standard strain ATCC19977 and 36 clinical isolates revealed MICs ranging from 0.12-0.96 µg/mL and MIC50 and MIC90 values of 0.48 µg/mL and 0.96 µg/mL, respectively. Fluorescence intensities of M. abscessus-infected zebrafish tissues was lower after treatment with the WX-081 MTC (62.5 µg/mL) than after treatment with the azithromycin MTC (62.5 µg/mL) and the bedaquiline MTC (15.6 µg/mL). When the concentration of WX-081 increased from 1.95µg/mL to 1/8 MTC(7.81µg/mL), the survival rate of zebrafish at 4-9 dpf decreased from 90.00% to 81.67%. Conclusion: WX-081 effectively inhibited M. abscessus growth in vitro and in vivo and prolonged survival of M. abscessus-infected zebrafish, thus indicating that WX-081 holds promise as a clinical treatment for M. abscessus infection.

Antibacterial activity of the novel oxazolidinone contezolid (MRX-I) against Mycobacterium abscessus.

Objective: To evaluate contezolid (MRX-I) antibacterial activity against Mycobacterium abscessus in vitro and in vivo and to assess whether MRX-I treatment can prolong survival of infected zebrafish. Methods: MRX-I inhibitory activity against M. abscessus in vitro was assessed by injecting MRX-I into zebrafish infected with green fluorescent protein-labelled M. abscessus. Thereafter, infected zebrafish were treated with azithromycin (AZM), linezolid (LZD) or MRX-I then maximum tolerated concentrations (MTCs) of drugs were determined based on M. abscessus growth inhibition using one-way ANOVA. Linear trend analysis of CFU counts and fluorescence intensities (mean ± SE values) was performed to detect linear relationships between MRX-I, AZM and LZD concentrations and these parameters. Results: MRX-I anti-M. abscessus minimum inhibitory concentration (MIC) and MTC were 16 µg/mL and 15.6 µg/mL, respectively. MRX-I MTC-treated zebrafish fluorescence intensities were significantly lower than respective LZD group intensities (whole-body: 439040 ± 3647 vs. 509184 ± 23064, p < 0.01); head: 74147 ± 2175 vs. 95996 ± 8054, p < 0.05). As MRX-I concentration was increased from 0.488 µg/mL to 15.6 µg/mL, zebrafish whole-body, head and heart fluorescence intensities decreased. Statistically insignificant differences between the MRX-I MTC group survival rate (78.33%) vs. corresponding rates of the 62.5 µg/mL-treated AZM MTC group (88.33%, p > 0.05) and the 15.6 µg/mL-treated LZD MTC group (76.67%, p > 0.05) were observed. Conclusion: MRX-I effectively inhibited M. abscessus growth and prolonged zebrafish survival when administered to M. abscessus-infected zebrafish, thus demonstrating that MRX-I holds promise as a clinical treatment for human M. abscessus infections.

[Genotypes of Toxoplasma gondii isolates from HIV positive patients in Yunnan Province].

One hundred and fifty serum samples of HIV positive patients were collected in western Yunnan Province from September 2011 to December 2012. Toxoplasma gondii B1 gene was amplified by nested PCR. Genotyping of T. gondii isolates were performed by restriction fragment length polymorphism (RFLP) with Pm1 I and Xho I. 13 samples were found positive with the B1 gene (530 bp) amplification and belonged to type I. The sequencing results showed that 4 T. gondii B1 gene positive samples were identical, with 3 nucleotide variation compared with T. gondii strain RH (type I) B1 gene (GenBank No. AF179871), and in the other sample a "G --> A" mutation at 230bp was detected. The results indicated that the genotype of Toxoplasma gondii in HIV positive patients in Yunnan Province is type I.

[PCR-derived technology in gene identification and typing of Toxoplasma gondii].

Different genotypes of Toxoplasma gondii show a great diversity in pathogenicity and drug sensitivity. Application of the PCR-derived technologies in gene identification and typing of T. gondii provides an important basis to clinical diagnosis and treatment. This article reviews the relevant technologies in gene identification and typing of T. gondii.

[Serological investigation of Toxoplasma gondii infection in HIV positive cases in Dali and Dehong of Yunnan].

Serum samples were collected from HIV positive cases (927) and HIV, negative ones (80) from June 2010 to August 2011 in Dali and Dehong Prefectures of Yunnan. Serum anti-Toxoplasma gondii IgG was detected by ELISA. The overall anti-Toxoplasma gondii IgG positive rate among HIV positive cases and HIV negative ones was 35.1% (325/927) and 23.8% (19/80), respectively. In HIV positive cases, the seropositive rate was 30.3% (178/588) in Dali and 43.4% (147/339) in Dehong. The seropositive rate was significantly different among ethnic groups (chi2 = 28.433, P < 0.05). No significant difference was found among age groups (chi2 = 4.248, P > 0.05), and the age group of 41-60 showed the highest positive rate (36.1%, 103/285). The seropositive rate was 35.6% (203/571) in males and 34.3% (122/356) in females (chi2 = 0.158, P > 0.05).