Background: Nontuberculous mycobacteria (NTM) are easily misdiagnosed as multidrug-resistant tuberculosis (MDR-TB), and treatment drugs are very limited. The main objective of our study was to evaluate the minimal inhibitory concentration (MIC) in vitro of bedaquiline (BDQ), clofazimine (CFZ), linezolid (LZD), delamanid (DLM), and pretomanid (PA-824) for treatment of M. abscessus and M. intracellulare. Furthermore, we determined whether MAB_1448, MAB_4384, MAB_2299c, MAB_1483, MAB_0540, rplD, rplC, and rrl were related to drug resistance to provide an experimental basis for the use of these five drugs in the treatment of NTM. Methods: We identified sample characteristics of epidemics in 550 patients with suspected NTM infection in Nanjing from 2019 to 2021 using the PCR-reverse spot hybrid method. Furthermore, we evaluated the MIC of BDQ, CFZ, DLM, LZD, and PA-824 against 155 clinical isolates of NTM using the microbroth dilution method. The resistant isolates were sequenced using Sanger sequencing. Results: The top three dominant species of NTM distributed in Nanjing were M. intracellulare, M. avium, and M. abscessus. Notably, the proportion of M. abscessus infections increased. The proportion of M. abscessus increased from 12% in 2019 to 18% in 2021. Demographic analysis showed that female infection rates were substantialy greater than male for M. abscessus (P=0.017, < 0.05). Our results demonstrate that NTM are highly sensitive to bedaquiline and clofazimine in vitro. However, delamanid and pretomanid had little effect on M. abscessus and M. intracellulare. In addition, we found 30– 41 nucleotide deletion mutations and some novel point mutations in the MAB_0540 gene of M. abscessus that are resistant to clofazimine. Conclusion: Bedaquiline, clofazimine, and linezolid were more successful in vitro treatments against M. abscessus and M. intracellulare. The MAB_0540 mutation may be associated with resistance of M. abscessus to clofazimine.

Background: Transmitted drug resistance (TDR) is a major challenge in the clinical management of acquired immunodeficiency syndrome (AIDS). Therefore, this study aimed to investigate the epidemic characteristics of and risk factors for human immunodeficiency virus (HIV)-1 TDR in Nanjing from 2018 to 2021 to provide support for clinical management. Methods: The HIV-1 Pol gene was amplified by nested reverse transcription polymerase chain reaction from venous blood of 1190 HIV-infected patients who did not receive antiviral therapy, and the amplified product was sequenced using an in-house sequencing method. The sequencing result was compared with the HIV drug resistance database from Stanford University to elucidate the rates of antiviral drug resistance and distribution of drug-resistant mutation sites. Factors associated with TDR were evaluated using a logistic regression model. Results: Detection of drug resistance at the gene level was successful in 1138 of 1190 HIV-1-infected patients (95.6%), and the overall 4-year drug resistance rate was 8.2% (93/1138). The drug resistance rate was higher for non-nucleoside reverse transcriptase inhibitors (NNRTIs; 6.7%) than for nucleoside reverse transcriptase inhibitors (NRTIs; 2.5%) or protease inhibitors (PIs; 0.1%) (χ² = 83.907, P< 0.0001). The most common NNRTI-related mutation was V179D/E followed by K103N. M184V was the dominant NRTI-associated mutation, and M46L/I was the most prevalent PI-associated mutation. A CD4⁺ T cell count of < 50 cells/μL was significantly associated with an increased risk of TDR (OR=3.62, 95% CI: 1.38– 9.51, P=0.009). Conclusion: The prevalence of TDR in the city of Nanjing from 2018 to 2021 was at a moderate epidemic risk according to World Health Organization standards. Continuous monitoring of TDR can inform clinical diagnosis and treatment. Patients with advanced disease and a low CD4⁺ T lymphocyte count are more likely to have TDR in Nanjing.

Early and accurate etiological diagnosis is very important for improving the prognosis of central nervous system (CNS) infections in human immunodeficiency virus (HIV)-infected patients. The goal is not easily achieved by conventional microbiological tests. We developed a nanopore targeted sequencing (NTS) platform and evaluated the diagnostic performance for CNS infections in HIV-infected patients, with special focus on cryptococcal meningitis (CM). We compared the CM diagnostic performance of NTS with conventional methods and cryptococcal polymerase chain reaction (PCR). This study included 57 hospitalized HIV-infected patients with suspected CNS infections from September 2018 to March 2022. The diagnosis established during hospitalization includes 27 cases of CM, 13 CNS tuberculosis, 5 toxoplasma encephalitis, 2 cytomegalovirus (CMV) encephalitis and 1 Varicella-zoster virus (VZV) encephalitis. The 2 cases of CMV encephalitis also have co-existing CM. Target-specific PCR amplification was used to enrich pathogen sequences before nanopore sequencing. NTS was performed on stored cerebrospinal fluid (CSF) samples and the results were compared with the diagnosis during hospitalization. 53 (93.0%) of the patients were male. The median CD4 cell count was 25.0 (IQR: 14.0–63.0) cells/uL. The sensitivities of CSF culture, India ink staining, cryptococcal PCR and NTS for CM were 70.4% (95%CI: 51.5 − 84.1%), 76.0% (95%CI: 56.6 − 88.5%), 77.8% (59.2 − 89.4%) and 85.2% (95%CI: 67.5 − 94.1%), respectively. All those methods had 100% specificity for CM. Our NTS platform could identify Cryptococcus at species level. Moreover, NTS was also able to identify all the 5 cases of toxoplasma encephalitis, 2 cases of CMV encephalitis and 1 VZV encephalitis. However, only 1 of 13 CNS tuberculosis cases was diagnosed by NTS, and so did Xpert MTB/RIF assay. NTS has a good diagnostic performance for CM in HIV-infected patients and may have the ability of simultaneously detecting other pathogens, including mixed infections. With continuing improving of the NTS platform, it may be a promising alterative microbiological test for assisting with the diagnosis of CNS infections.

Medroxyprogesterone acetate (MPA) is one of the most widely used contraceptives in the world. Epidemiologic studies have uncovered a possible link between the use of MPA and an increased risk of HIV-1 transmission. However, the understanding of the mechanism is still limited. Our previous publication demonstrated that the lysosomal activity in human vaginal epithelial cells attenuated the trafficking of viral particles during HIV-1 transcytosis. In this study, we show that treating human primary cervical epithelial cells with MPA led to a reduction in lysosomal activity. This reduction caused an increase in the intracellular HIV-1 accumulation and, consequently, an increase in viral release. Our study uncovers a novel mechanism by which MPA enhances HIV-1 release in primary cervical epithelial cells, thus providing vital information for HIV intervention and prevention.