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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has seriously threatened global public health. Severe COVID-19 has been reported to be associated with an impaired IFN response. However, the mechanisms of how SARS-CoV-2 antagonizes the host IFN response are poorly understood. In this study, we report that SARS-CoV-2 helicase NSP13 inhibits type I IFN production by directly targeting TANK-binding kinase 1 (TBK1) for degradation. Interestingly, inhibition of autophagy by genetic knockout of Beclin1 or pharmacological inhibition can rescue NSP13-mediated TBK1 degradation in HEK-293T cells. Subsequent studies revealed that NSP13 recruits TBK1 to p62, and the absence of p62 can also inhibit TBK1 degradation in HEK-293T and HeLa cells. Finally, TBK1 and p62 degradation and p62 aggregation were observed during SARS-CoV-2 infection in HeLa-ACE2 and Calu3 cells. Overall, our study shows that NSP13 inhibits type I IFN production by recruiting TBK1 to p62 for autophagic degradation, enabling it to evade the host innate immune response, which provides new insights into the transmission and pathogenesis of SARS-CoV-2 infection.
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Autofagia , COVID-19/inmunología , ARN Polimerasa Dependiente de ARN de Coronavirus/fisiología , Interferón Tipo I/biosíntesis , Metiltransferasas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Helicasas/fisiología , SARS-CoV-2/fisiología , Proteína Sequestosoma-1/metabolismo , Proteínas no Estructurales Virales/fisiología , Beclina-1/antagonistas & inhibidores , Línea Celular , Regulación hacia Abajo , Humanos , Evasión Inmune , Inmunidad Innata , Inmunoprecipitación , Interferón Tipo I/genética , Complejos Multiproteicos , Agregado de Proteínas , Mapeo de Interacción de ProteínasRESUMEN
BACKGROUND: In this study, we aimed to evaluate the clinical utility of Metagenomic Next-Generation sequencing (mNGS) on bronchoalveolar lavage fluid (BALF) in diagnosis of Lower Respiratory Tract Infections (LRTIs). METHODS: In this study, we retrospectively analyzed 186 hospitalized patients who were suspected with LRTIs and performed mNGS (DNA) test of BALF simultaneously at The Fifth Affiliated Hospital of Sun Yat-Sen University from March 2023 to August 2023. Suspected LRTI was based on LRTI related clinical manifestations or imaging examination. Among them, 155 patients had undergone conventional culture and mNGS (DNA) testing simultaneously. Finally, 138 cases (89.03%,138/155) were diagnosed as LRTI and 17 cases (10.97%,17/155) were diagnosed as non-LRTI. Both detecting rate and diagnostic efficacy of mNGS and conventional culture were compared. RESULTS: The positive detection rates of pathogens between mNGS and conventional culture were significant different (81.29% VS 39.35%, P < 0.05). Compared with paired conventional culture result, the sensitivity of mNGS in diagnosis of LRTIs was more superior (88.41% VS 43.48%; P < 0.05), the specificity was opposite (76.47% VS 94.12%; P > 0.05). Furthermore, 77.54% and 35.51% of LRTI cases were being etiologically diagnosed by mNGS and culture respectively. Importantly, mNGS directly led to a change of treatment regimen in 58 (37.42%) cases, including antibiotic adjustment (29.68%) and ruling out active infection (7.74%). Moreover, treatment regimen remained unchanged in 97 (62.58%) cases, considering the current antibiotic therapy already covered the detected pathogens (36.13%) or empirical treatment was effective (11.61%). CONCLUSIONS: mNGS can identify a wide range of pathogens in LRTIs, with improved sensitivity and being more superior at diagnosing LRTIs etiologically. mNGS has the potential to enhance clinical outcomes by optimizing the treatment regimens.
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Líquido del Lavado Bronquioalveolar , Secuenciación de Nucleótidos de Alto Rendimiento , Metagenómica , Infecciones del Sistema Respiratorio , Humanos , Líquido del Lavado Bronquioalveolar/microbiología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Masculino , Estudios Retrospectivos , Femenino , Persona de Mediana Edad , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/microbiología , Metagenómica/métodos , Adulto , Anciano , Sensibilidad y Especificidad , Adulto JovenRESUMEN
The EmbCAB proteins have been considered a target for ethambutol (EMB). Mutations in embCAB are known to confer most EMB resistance. However, the knowledge about the effects of embCAB mutations on the EMB resistance level and about the role of mutation-mutation interactions is limited in China. Here, we sequenced embCAB among 125 Mycobacterium tuberculosis isolates from China and quantified their EMB MICs by testing growth at 10 concentrations. Furthermore, a multivariate regression model was established to assess the effects of both individual mutations and multiple mutations. Our results revealed that in China, 82.6% of EMB-resistant isolates (71/86 isolates) harbored at least one mutation within embCAB Most of the mutations were located in the embB and embA upstream region. Several individual mutations and multiple mutations within this region contributed to the different levels of EMB resistance. Their effects were statistically significant. Additionally, there was an association between high-level EMB resistance and multiple mutations.
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Antituberculosos/farmacología , Proteínas Bacterianas/genética , Etambutol/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , China , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Humanos , Análisis Multivariante , Mutación , Operón , Tuberculosis Pulmonar/microbiologíaRESUMEN
Ethambutol (EMB) plays a pivotal role in the chemotherapy of drug-resistant tuberculosis (TB), including multidrug-resistant tuberculosis (MDR-TB). Resistance to EMB is considered to be caused by mutations in the embCAB operon (embC, embA, and embB). In this study, we analyzed the embCAB mutations among 139 MDR-TB isolates from China and found a possible association between embCAB operon mutation and EMB resistance. Our data indicate that 56.8% of MDR-TB isolates are resistant to EMB, and 82.2% of EMB-resistant isolates belong to the Beijing family. Overall, 110 (79.1%) MDR-TB isolates had at least one mutation in the embCAB operon. The majority of mutations were present in the embB gene and the embA upstream region, which also displayed significant correlations with EMB resistance. The most common mutations occurred at codon 306 in embB (embB306), followed by embB406, embA(-16), and embB497. Mutations at embB306 were associated with EMB resistance. DNA sequencing of embB306-497 was the best strategy for detecting EMB resistance, with 89.9% sensitivity, 58.3% specificity, and 76.3% accuracy. Additionally, embB306 had limited value as a candidate predictor for EMB resistance among MDR-TB infections in China.
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Antituberculosos/farmacología , Proteínas Bacterianas/genética , Etambutol/farmacología , Proteínas de la Membrana/genética , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Pentosiltransferasa/genética , Tuberculosis Resistente a Múltiples Medicamentos/genética , Tuberculosis/microbiología , China , ADN Bacteriano/genética , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Objective: To explore the association between the variant mutations within embB and ubiA, and the degree of ethambutol (EMB) resistance of Mycobacterium tuberculosis (M. tuberculosis) isolates. Methods: A total of 146 M. tuberculosis isolates were used to determine the minimum inhibitory concentrations (MICs) of EMB with a 96-well microplate-based assay. The mutations within embB and ubiA among these isolates were identified with DNA sequencing. Moreover, a multivariate regression model and a computer model were established to assess the effects of mutations on EMB resistance. Results: Our data showed that overall 100 isolates exhibited 28 mutated patterns within the sequenced embB and ubiA. Statistical analysis indicated that embB mutations Met306Val, Met306Ile, Gly406Ala, and Gln497Arg, were strongly associated with EMB resistance. Of these mutations, Met306Val and Gln497Arg were significantly associated with high-level EMB resistance. Almost all multiple mutations occurred in high-level EMB-resistant isolates. Although the mutation within ubiA accompanied with embB mutation presented exclusively in EMB-resistant isolates, four single ubiA mutations (Ala39Glu, Ser173Ala, Trp175Cys, and Val283Leu) leading to protein instability were observed in EMB-susceptible isolates. Conclusion: This study highlighted the complexity of EMB resistance. Some individual mutations and multiple mutations within embB and ubiA contributed to the different levels of EMB resistance.
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Objective: To evaluate the diagnostic value of metagenomic next-generation sequencing (mNGS) in Vibrio vulnificus (V. vulnificus) infection. Methods: A retrospective analysis of patients with V. vulnificus infection at the Fifth Affiliated Hospital of Sun Yat-Sen University from January 1, 2020 to April 23, 2023 was conducted. 14 enrolled patients were diagnosed by culture or mNGS. The corresponding medical records were reviewed, and the clinical data analyzed included demographics, epidemiology laboratory findings, physical examination, symptoms at presentation, antibiotic and surgical treatment, and outcome. Results: In this study, 78.6% (11/14) patients had a history of marine trauma (including fish stab, shrimp stab, crab splints and fish hook wounds), 7.1% (1/14) had eaten seafood, and the remaining 14.3% (2/14) had no definite cause. Isolation of V. vulnificus from clinical samples including blood, tissue, fester and secreta. 9 cases were positive for culture, 5 cases were detected synchronously by mNGS and got positive for V. vulnificus. 85.7% (12/14) cases accepted surgical treatment, with 1 patient suffering finger amputated. 14 enrolled patients received appropriate antibiotic therapy, and all of them had recovered and discharged. 9 strains V. vulnificus isolated in this study were sensitive to most beta-lactam antibiotics, aminoglycosides, quinolones, etc. Conclusion: Vibrio vulnificus infection is a common water-exposed disease in Zhuhai, which requires identification of a number of pathogens. Of severe infections with unknown pathogen, mNGS can be used simultaneously, and the potential to detect multiple pathogens is of great help in guiding treatment.
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Vibriosis , Vibrio vulnificus , Animales , Humanos , Estudios Retrospectivos , Vibriosis/diagnóstico , Vibriosis/epidemiología , Vibrio vulnificus/genética , Antibacterianos/uso terapéutico , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Global control of the tuberculosis epidemic is threatened by increasing prevalence of drug resistant M. tuberculosis isolates. Many genome-wide studies focus on SNP-associated drug resistance mechanisms, but drug resistance in 5-30% of M. tuberculosis isolates (varying with antibiotic) appears unrelated to reported SNPs, and alternative drug resistance mechanisms involving variation in gene/protein expression are not well-studied. Here, using an omics approach, we identify 388 genes with lineage-related differential expression and 68 candidate drug resistance-associated gene pairs/clusters in 11 M. tuberculosis isolates (variable lineage/drug resistance profiles). Structural, mutagenesis, biochemical and bioinformatic studies on Rv3094c from the Rv3093c-Rv3095 gene cluster, a gene cluster selected for further investigation as it contains a putative monooxygenase/repressor pair and is associated with ethionamide resistance, provide insights on its involvement in ethionamide sulfoxidation, the initial step in its activation. Analysis of the structure of Rv3094c and its complex with ethionamide and flavin mononucleotide, to the best of our knowledge the first structures of an enzyme involved in ethionamide activation, identify key residues in the flavin mononucleotide and ethionamide binding pockets of Rv3094c, and F221, a gate between flavin mononucleotide and ethionamide allowing their interaction to complete the sulfoxidation reaction. Our work broadens understanding of both lineage- and drug resistance-associated gene/protein expression perturbations and identifies another player in mycobacterial ethionamide metabolism.
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Antituberculosos , Farmacorresistencia Bacteriana Múltiple , Etionamida , Mycobacterium tuberculosis , Antituberculosos/farmacología , Etionamida/farmacología , Mononucleótido de Flavina , Mycobacterium tuberculosis/genética , Farmacorresistencia Bacteriana Múltiple/genéticaRESUMEN
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and has led to a global coronavirus disease 2019 (COVID-19) pandemic. Currently, incomplete understanding of how SARS-CoV-2 arrogates the host cell to establish its life cycle has led to slow progress in the development of effective drugs. Results: In this study, we found that SARS-CoV-2 hijacks the host protein EWSR1 (Ewing Sarcoma breakpoint region 1/EWS RNA binding protein 1) to promote the activity of its helicase NSP13 to facilitate viral propagation. NSP13 is highly conserved among coronaviruses and is crucial for virus replication, providing chemical energy to unwind viral RNA replication intermediates. Treatment with different SARS-CoV-2 NSP13 inhibitors in multiple cell lines infected with SARS-CoV-2 effectively suppressed SARS-CoV-2 infection. Using affinity-purification mass spectrometry, the RNA binding protein EWSR1 was then identified as a potent host factor that physically associated with NSP13. Furthermore, silencing EWSR1 dramatically reduced virus replication at both viral RNA and protein levels. Mechanistically, EWSR1 was found to bind to the NTPase domain of NSP13 and potentially enhance its dsRNA unwinding ability. Conclusions: Our results pinpoint EWSR1 as a novel host factor for NSP13 that could potentially be used for drug repurposing as a therapeutic target for COVID-19.
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Objective: To assess the relationship between the variant rpoB mutations and the degree of rifampin (RIF)/rifabutin (RFB) resistance in Mycobacterium tuberculosis (M. tuberculosis). Methods: We analyzed the whole rpoB gene in 177 M. tuberculosis clinical isolates and quantified their minimum inhibitory concentrations (MICs) using microplate-based assays. Results: The results revealed that of the 177 isolates, 116 were resistant to both RIF and RFB. There were 38 mutated patterns within the sequenced whole rpoB gene of the 120 isolates. Statistical analysis indicated that mutations, S450L, H445D, H445Y, and H445R, were associated with RIF and RFB resistance. Of these mutations, S450L, H445D, and H445Y were associated with high-level RIF and RFB MIC. H445R was associated with high-level RIF MIC, but not high-level RFB MIC. D435V and L452P were associated with only RIF, but not RFB resistance. Q432K and Q432L were associated with high-level RFB MIC. Several single mutations without statistical association with rifamycin resistance, such as V170F, occurred exclusively in low-level RIF but high-level RFB resistant isolates. Additionally, although cross-resistance to RIF and RFB is common, 21 RIF-resistant/RFB-susceptible isolates were identified. Conclusion: This study highlighted the complexity of rifamycin resistance. Identification of the rpoB polymorphism will be helpful to diagnose the RIF-resistant tuberculosis that has the potential to benefit from a treatment regimen including RFB.
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The Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a global pandemic that seriously threatens health and socioeconomic development, but the existed antiviral drugs and vaccines still cannot yet halt the spread of the epidemic. Therefore, a comprehensive and profound understanding of the pathogenesis of SARS-CoV-2 is urgently needed to explore effective therapeutic targets. Here, we conducted a multiomics study of SARS-CoV-2-infected lung epithelial cells, including transcriptomic, proteomic, and ubiquitinomic. Multiomics analysis showed that SARS-CoV-2-infected lung epithelial cells activated strong innate immune response, including interferon and inflammatory responses. Ubiquitinomic further reveals the underlying mechanism of SARS-CoV-2 disrupting the host innate immune response. In addition, SARS-CoV-2 proteins were found to be ubiquitinated during infection despite the fact that SARS-CoV-2 itself didn't code any E3 ligase, and that ubiquitination at three sites on the Spike protein could significantly enhance viral infection. Further screening of the E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) library revealed four E3 ligases influencing SARS-CoV-2 infection, thus providing several new antiviral targets. This multiomics combined with high-throughput screening study reveals that SARS-CoV-2 not only modulates innate immunity, but also promotes viral infection, by hijacking ubiquitination-specific processes, highlighting potential antiviral and anti-inflammation targets.
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COVID-19 , SARS-CoV-2 , Antivirales , Humanos , Proteómica , Ubiquitina-Proteína Ligasas , Ubiquitinación/genéticaRESUMEN
Asymptomatic carriers contribute to the spread of Coronavirus Disease 2019 (COVID-19), but their clinical characteristics, viral kinetics, and antibody responses remain unclear. A total of 56 COVID-19 patients without symptoms at admission and 19 age-matched symptomatic patients were enrolled. RNA of SARS-CoV-2 was tested using transcriptase quantitative PCR, and the total antibodies (Ab), IgG, IgA, and IgM against the SARS-CoV-2 were tested using Chemiluminescence Microparticle Immuno Assay. Among 56 patients without symptoms at admission, 33 cases displayed symptoms and 23 remained asymptomatic throughout the follow-up period. 43.8% of the asymptomatic carriers were children and none of the asymptomatic cases had recognizable changes in C-reactive protein or interleukin-6, except one 64-year-old patient. The initial threshold cycle value of nasopharyngeal SARS-CoV-2 in asymptomatic carriers was similar to that in pre-symptomatic and symptomatic patients, but the positive viral nucleic acid detection period of asymptomatic carriers (9.63 days) was shorter than pre-symptomatic patients (13.6 days). There were no obvious differences in the seropositive conversion rate of total Ab, IgG, and IgA among the three groups, though the rates of IgM varied largely. The average peak IgG and IgM COI of asymptomatic cases was 3.5 and 0.8, respectively, which is also lower than those in symptomatic patients with peaked IgG and IgM COI of 4.5 and 2.4 (p < 0.05). Young COVID-19 patients seem to be asymptomatic cases with early clearance of SARS-CoV-2 and low levels of IgM generation but high total Ab, IgG, and IgA. Our findings provide empirical information for viral clearance and antibody kinetics of asymptomatic COVID-19 patients.
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OBJECTIVE: To investigate the mutations within the whole rpoB gene of Mycobacterium tuberculosis and analyze their effects on rifampin (RIF) resistance based on crystal structure. METHODS: We sequenced the entire rpoB gene in 175 tuberculosis isolates and quantified their minimum inhibitory concentrations using microplate-based assays. Additionally, the structural interactions between wild-type/mutant RpoB and RIF were also analyzed. RESULTS: Results revealed that a total of 34 mutations distributed across 17 different sites within the whole rpoB gene were identified. Of the 34 mutations, 25 could alter the structural interaction between RpoB and RIF and contribute to RIF resistance. Statistical analysis showed that S450L, H445D, H445Y and H445R mutations were associated with high-level RIF resistance, while D435V was associated with moderate-level RIF resistance. CONCLUSION: Some mutations within the rpoB gene could affect the interaction between RpoB and RIF and thus are associated with RIF resistance. These findings could be helpful to design new antibiotics and develop novel diagnostic tools for drug resistance in TB.
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Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the ongoing global pandemic that poses substantial challenges to public health worldwide. A subset of COVID-19 patients experience systemic inflammatory response, known as cytokine storm, which may lead to death. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important mediator of inflammation and cell death. Here, we examined the interaction of RIPK1-mediated innate immunity with SARS-CoV-2 infection. We found evidence of RIPK1 activation in human COVID-19 lung pathological samples, and cultured human lung organoids and ACE2 transgenic mice infected by SARS-CoV-2. Inhibition of RIPK1 using multiple small-molecule inhibitors reduced the viral load of SARS-CoV-2 in human lung organoids. Furthermore, therapeutic dosing of the RIPK1 inhibitor Nec-1s reduced mortality and lung viral load, and blocked the CNS manifestation of SARS-CoV-2 in ACE2 transgenic mice. Mechanistically, we found that the RNA-dependent RNA polymerase of SARS-CoV-2, NSP12, a highly conserved central component of coronaviral replication and transcription machinery, promoted the activation of RIPK1. Furthermore, NSP12 323L variant, encoded by the SARS-CoV-2 C14408T variant first detected in Lombardy, Italy, that carries a Pro323Leu amino acid substitution in NSP12, showed increased ability to activate RIPK1. Inhibition of RIPK1 downregulated the transcriptional induction of proinflammatory cytokines and host factors including ACE2 and EGFR that promote viral entry into cells. Our results suggest that SARS-CoV-2 may have an unexpected and unusual ability to hijack the RIPK1-mediated host defense response to promote its own propagation and that inhibition of RIPK1 may provide a therapeutic option for the treatment of COVID-19.
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COVID-19/patología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , SARS-CoV-2/fisiología , Enzima Convertidora de Angiotensina 2/genética , Animales , COVID-19/mortalidad , COVID-19/virología , ARN Polimerasa Dependiente de ARN de Coronavirus/genética , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Receptores ErbB/metabolismo , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Indoles/farmacología , Indoles/uso terapéutico , Pulmón/patología , Pulmón/virología , Ratones , Ratones Transgénicos , Mutación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismo , Tasa de Supervivencia , Transcriptoma/efectos de los fármacos , Carga Viral/efectos de los fármacos , Internalización del Virus , Tratamiento Farmacológico de COVID-19RESUMEN
Objective: To investigate the clinical characteristics of children with coronavirus disease 2019 (COVID-19) and identify the occurrence of viral shedding of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) during follow-up. Methods: We retrospectively retrieved data from pediatric patients with COVID-19 from the Shenzhen Third People's Hospital in China. The dynamics of SARS-CoV-2 and antibodies against SARS-CoV-2 were analyzed during hospitalization and after discharge. Results: From January 23 to March 15, 2020, a total of 28 pediatric patients were diagnosed with COVID-19 and were followed for at least 1 month. The median age was 7 years (IQR 3.5-10) and none of the children progressed to severe COVID-19 during hospitalization. Ten patients tested positive for SARS-CoV-2 1 month after discharge while four patients tested positive during the 2nd month after discharge. Only three of 12 children showed detectable immunoglobulin-M (IgM) on day 5, 18, and 21 after illness onset, respectively. Conclusions: COVID-19 disease was relatively mild among children while a number did test positive after discharge from the hospital. Public health initiatives should thus adapt control measures targeted toward children.
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BACKGROUND: The characteristics, significance and potential cause of positive SARS-CoV-2 diagnoses in recovered coronavirus disease 2019 (COVID-19) patients post discharge (re-detectable positive, RP) remained elusive. METHODS: A total of 262 COVID-19 patients discharged from January 23 to February 25, 2020 were enrolled into this study. RP and non-RP (NRP) patients were grouped according to disease severity, and the characterization at re-admission was analyzed. SARS-CoV-2 RNA and plasma antibody levels were measured, and all patients were followed up for at least 14 days, with a cutoff date of March 10, 2020. RESULTS: A total of 14.5% of RP patients were detected. These patients were characterized as young and displayed mild and moderate conditions compared to NRP patients while no severe patients were RP. RP patients displayed fewer symptoms but similar plasma antibody levels during their hospitalization compared to NRP patients. Upon hospital readmission, these patients showed no obvious symptoms or disease progression. All 21 close contacts of RP patients were tested negative for viral RNA and showed no suspicious symptoms. Eighteen out of 24 of RNA-negative samples detected by the commercial kit were tested positive for viral RNA using a hyper-sensitive method, suggesting that these patients were potential carriers of the virus after recovery from COVID-19. CONCLUSIONS: Our results indicated that young patients, with a mild diagnosis of COVID-19 are more likely to display RP status after discharge. These patients show no obvious symptoms or disease progression upon re-admission. More sensitive RNA detection methods are required to monitor these patients. Our findings provide information and evidence for the management of convalescent COVID-19 patients.
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BACKGROUND: MPT64 and PstS1 are the earliest known immune-dominant antigens of Mycobacterium tuberculosis and have been commonly used as candidates in the diagnosis of tuberculosis. METHODS: We constructed recombinant plasmids pET-32a-Rv0934 and pET-32a-Rv1980c to express both wild and mutant forms of MPT64 and PstS1 and purified them. From November 9 to December 9, 2016, and November 9 to December 10, 2017, 96 patients with tuberculosis, 53 patients without tuberculosis, and 96 healthy volunteers were enrolled in this study. We used the purified proteins as antigens to perform T-spot and enzyme-linked immunosorbent assay (ELISA) for samples obtained from healthy volunteers and tuberculosis patients. RESULTS: Regarding T-spot, the area under the curve (AUC) values for MPT64-wild protein (MPT64-H37Rv) and MPT64-mutant protein (MPT64-FJ05395) were 0.723 and 0.750, respectively. The AUC values for PstS1-H37Rv, PstS1-FJ05132, and PstS1-JL06035 were 0.817, 0.796, and 0.745, respectively. With regard to ELISA, the AUC values for MPT64-H37Rv and MPT64-FJ05395 were 0.525 and 0.528, respectively, while those for PstS1-H37Rv, PstS1-FJ05132, PstS1-JL06035 were 0.588, 0.509, and 0.560, respectively. There was no difference between wild and mutant proteins when we used them as antigens to perform T-spot and ELISA assays. CONCLUSION: MPT64 and PstS1 are likely candidate diagnostic antigens for M tuberculosis T-spot test, at least in combination with other proteins. Polymorphisms of MPT64 and PstS1 had little effect on cell-mediated and humoral immunity in the host.
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Transportadoras de Casetes de Unión a ATP/genética , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Mycobacterium tuberculosis/genética , Tuberculosis/diagnóstico , Tuberculosis/inmunología , Femenino , Humanos , Inmunoensayo/métodos , Masculino , Plásmidos , Polimorfismo GenéticoRESUMEN
BACKGROUND: Tuberculosis (TB) and non-tuberculous mycobacteriosis are serious threats to health worldwide. A simple non-sequencing method is needed for rapid diagnosis, especially in less experienced hospitals, but there is no specific biomarker commonly used for all mycobacteria. The ku gene of the prokaryotic error-prone non-homologous end joining system (NHEJ) has the potential to be a highly specific detection biomarker for mycobacteria. METHODS: A total of 7294 mycobacterial genomes and 14 complete genomes of other families belonging to Corynebacteriales with Mycobacteriaceae were downloaded and analyzed for the existence and variation of the ku gene. Mycobacterium tuberculosis complex (MTBC) and non-tuberculosis mycobacteria (NTM)- specific primers were designed and the actual amplification and identification efficiencies were tested with 150 strains of 40 Mycobacterium species and 10 kinds of common respiratory pathogenic bacteria. RESULTS: The ku gene of the NHEJ system was ubiquitous in all genome sequenced Mycobacterium species and absent in other families of Corynebacteriales. On the one hand, as a single gene non-sequencing biomarker, its specific primers could effectively distinguish mycobacteria from other bacteria, MTBC from NTM, which would make the clinical detection of mycobacteria easy and have great clinical practical value. On the other hand, the sequence of ku gene can effectively distinguish NTM to species level with high resolution. CONCLUSION: The Ku protein existed before the differentiation of Mycobacterium species, which was an important protein involved in maintaining of the genome's integrity and related to the special growth stage of mycobacteria. It was rare in prokaryotes. These features made it a highly special differential biomarker for Mycobacterium.
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OBJECTIVES: To explore the prevalence, risk and genetic characteristics of drug-resistant tuberculosis (TB) from a tertiary care TB hospital in China. PATIENTS AND METHODS: We carried out a retrospective study including isolates from 189 patients with pulmonary TB at Fuzhou Pulmonary Hospital. All isolates from these patients were subjected to drug susceptibility testing and genotyping. For drug-resistant isolates, DNA sequencing was used to investigate mutations in 12 loci, including katG, inhA, oxyR-ahpC, rpoB, rpsL, rrs 1 (nucleotides 388-1084 of rrs), embB, tlyA, eis, rrs 2 (nucleotides 1158-1674 of rrs), gyrA and gyrB. RESULTS: Among 189 isolates, 28.6% were resistant to at least one of the seven anti-TB drugs, including isoniazid (INH), rifampin (RIF), streptomycin (STR), ethambutol (EMB), capreomycin (CAP), kanzmycin (KAN) and ofloxacin (OFX). The proportion of multidrug-resistant TB and extensively drug-resistant TB isolates was 9.5% and 1.1%, respectively. Patients in rural areas as well as previously treated patients showed a significantly increased risk of developing drug resistance. In addition, among these isolates, 111 (58.7%) were Beijing genotype strains, 84 (75.7%) of which belonged to modern Beijing sublineage. There was no association between genotype and drug resistance. The most common mutations were katG315, rpo B531 rpsL43, embB306, rrs1401 and gyrA94. CONCLUSION: These findings provided additional information of drug-resistant TB in China. Previously treated patients and patients in rural areas should receive greater attention owing to their higher risk of developing drug resistance.
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Mycobacterium tuberculosis Beijing genotype strains are widespread globally. However, there has been no systematic study on the association between Beijing genotype and the characteristics of drug resistance. In this study, 359 M. tuberculosis isolates from south China were collected and their background information, genotype diversity and drug resistance was investigated. The results revealed that 66.0% of strains (237/359) were categorised as Beijing genotype. There was no statistical difference between Beijing and non-Beijing genotype strains in terms of patient sex, age, place of residence and treatment history. Drug resistance testing showed that 34.8% (125/359) of isolates were resistant to at least one of the seven drugs tested. The proportions of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis were 17.0% and 1.4%, respectively. Previously treated patients presented a significantly higher risk of developing drug resistance than new cases. Although the prevalence of drug resistance was higher in Beijing genotype than in non-Beijing genotype strains, there was no significant difference between these two genotypes in the multivariate analysis. Even in re-treated patients, the association of Beijing genotype with drug resistance was not significant. This study provides an insight into genotype diversity and demonstrates the characteristics of drug resistance in Beijing genotype strains, which will be useful in generating efficient tuberculosis prevention and control strategies in China.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana/genética , Genotipo , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/epidemiología , Tuberculosis/microbiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , China/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/genética , Adulto JovenRESUMEN
OBJECTIVES: Evaluating the activity of nineteen ß-lactams in combination with different ß-lactamase inhibitors to determine the most potent combination against Mycobacterium tuberculosis (MTB) in vitro. METHODS: Drug activity was examined by drug susceptibility test with 122 clinical isolates from China. Mutations of blaC and drug targets ldtMt1 , ldtMt2 , dacB2, and crfA were analyzed by nucleotide sequencing. RESULTS: Tebipenem (TBM) in combination with clavulanate (CLA) exhibited the highest anti-TB activity. The MIC of ß-lactam antibiotics was reduced most evidently in the presence of CLA, compared to avibactam (AVI) and sulbactam (SUB). Eight polymorphism sites were identified in blaC, which were not associated with ß-lactams resistance. Interestingly, one strain carrying G514A mutation in blaC was highly susceptible to ß-lactams regardless of the presence of inhibitors. The transpeptidase encoding genes, ldtMt1 , ldtMt2 , and dacB2, harboured three mutations, two mutations, and one mutation, respectively, but no correlation was found between these mutations and drug resistance. CONCLUSION: The activity of ß-lactams against MTB and different synergetic effect of ß-lactamase inhibitors were indicated. TBM/CLA exhibited the most activity and has a great prospect in developing novel anti-TB regimen; however, further clinical research is warranted. Moreover, the resistance to the ß-lactam antibiotics might not be conferred by single target mutation in MTB and requires further studies.