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The Xpert MTB/RIF test (Xpert) can help in the accurate screening of tuberculosis, however, its widespread use is limited by its high cost and lack of accessibility. Pooling of sputum samples for testing is a strategy to cut expenses and enhance population coverage but may result in a decrease in detection sensitivity due to the dilution of Mycobacterium tuberculosis (Mtb) by sample mixing. We investigated how the mixing ratio affected the detection performance of Xpert. We used frozen sputum samples that had been kept after individual Xpert assays of the sputa from Mtb-confirmed TB patients and non-TB patients. Our results showed that the overall sensitivity of the Xpert pooling assay remained higher than 80% when the mixing ratio was between 1/2 and 1/8. When the mixing ratio was raised to 1/16, the positive detection rate fell to 69.0%. For patients with either a high sputum Mtb smear score ≥ 2+, a time-to-positive culture ≤ 10 days, or an Xpert test indicating a high or medium abundance of bacteria, the pooling assay positivity rates were 93.3%, 96.8%, and 100% respectively, even at a 1/16 mixing ratio. For participants with cavities and cough, the pooling assay positivity rates were 86.2% and 90.0% at a 1/8 ratio, higher than for those without these signs. Our results show that the Xpert pooled assay has a high overall sensitivity, especially for highly infectious patients. This pooling strategy with lower reagent and labor costs could support TB screening in communities with limited resources, thereby facilitating reductions in the community transmission and incidence of TB worldwide.
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Mycobacterium tuberculosis , Tuberculosis , Humanos , Esputo , Tos , BioensayoRESUMEN
BACKGROUND: Thalassemia is an inherited hemolytic blood disease, whose pathogenesis is an imbalance in the expression of hemoglobin. We report a case of a rare ß-globin gene intron mutation for thalassemia patient. METHODS: The blood routine test was performed with an automatic blood cell analyzer. Hb analysis was conducted by hemoglobin (Hb) analyzer. The common ß-thalassemia and α-thalassemia gene mutations were detected by Gap-PCR and fluorescence PCR melting curve, and the rare ß-thalassemia gene mutations were detected by DNA sequencing. RESULTS: A rare heterozygous mutation of ß-globin gene IVS-II-786 (T>A) was found in this case. Blood routine analysis showed the following values: Hb 92 g/L, RBC 4.1 x 1012/L, MCV 74.10 fL, MCH 22.4 pg, MCHC 303 g/L, HCT 0.304 L/L, and RET-He 22.7 pg. Hemoglobin analysis showed values of HbA2 2.2% and HbF < 2% by automatic capillary electrophoresis. The results of gene analysis and DNA sequencing showed that the ß-globin gene IVS-II-786 (T>A) mutation was heterozygous. CONCLUSIONS: The heterozygote of ß-globin gene IVS-II-786 (T>A) mutation was detected for the first time, and the clinical manifestation was moderate anemia. Hemoglobin analysis indicated that the level of HbA2 was decreased. This mutation is relatively rare and easy to misdiagnose in clinical practice. It will provide a new type of evidence and guidance for genetic counseling and clinical treatment of beta thalassemia.
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Talasemia beta , Humanos , Heterocigoto , Talasemia beta/diagnóstico , Talasemia beta/genética , Mutación , Hemoglobinas/análisis , Globinas beta/genéticaRESUMEN
BACKGROUND: The morbidity of rifampicin/multidrug-resistant tuberculous meningitis (RR/MDR-TBM) has shown an increasing trend globally. Its mortality rate is significantly higher than that of non-rifampicin/multidrug-resistant tuberculous meningitis (NRR/MDR-TBM). This article aimed to explore risk factors related to RR/MDR-TBM, and compare therapeutic effects of linezolid (LZD)- and non-linezolid-containing regimen for RR/MDR-TB patients in Shenzhen city. Furthermore, we aimed to find a better therapy for pathogen-negative TBM with RR/MDR-TBM related risk factors. METHODS: We conducted a retrospective study enrolling 137 hospitalized cases with confirmed TBM from June 2014 to March 2020. All patients were divided into RR/MDR-TBM group (12 cases) and NRR/MDR-TBM group (125 cases) based on GeneXpert MTB/RIF and (or) phenotypic drug susceptibility test results using cerebral spinal fluid (CSF). The risk factors related to RR/MDR-TBM were investigated through comparing clinical and examination features between the two groups. The mortality rate of RR/MDR-TBM patients treated with different regimens was analyzed to compare their respective therapeutic effects. A difference of P < 0.05 was considered statistically significant. RESULTS: Most patients (111/137, 81%) were from southern or southwestern China, and a large proportion (72/137, 52.55%) belonged to migrant workers. 12 cases were RR/MDR-TBM (12/137, 8.8%) while 125 cases were NRR/MDR-TBM (125/137, 91.2%). The proportion of patients having prior TB treatment history in the RR/MDR-TBM group was significantly higher than that of the NRR/MDR-TBM group (6/12 vs. 12/125, 50% vs. 10.5%, P < 0.01). No significant difference was observed on other clinical and examination features between the two groups. Mortality was significantly lower in RR/MDR-TBM patients on linezolid-containing treatment regimen than those who were not (0/7 versus 3/5, 0% versus 60%, P = 0.045). CONCLUSIONS: The main related risk factor of RR/MDR-TBM is the history of anti-tuberculosis treatment. Linezolid-containing regimen appears to lower mortality rate of RR/MDR-TBM significantly in our study. We think Linezolid should be evaluated prospectively in the treatment of RR/MDR-TBM.
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Mycobacterium tuberculosis , Tuberculosis Meníngea , Tuberculosis Resistente a Múltiples Medicamentos , Antituberculosos/uso terapéutico , China/epidemiología , Humanos , Linezolid/uso terapéutico , Estudios Retrospectivos , Rifampin/uso terapéutico , Tuberculosis Meníngea/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
Mycobacterium tuberculosis infection and nontuberculous mycobacteria (NTM) infections exhibit similar clinical symptoms; however, the therapies for these two types of infections are different. Therefore, the rapid and accurate identification of M. tuberculosis and NTM species is very important for the control of tuberculosis and NTM infections. In the present study, a Cas12a/guide RNA (gRNA)-based platform was developed to identify M. tuberculosis and most NTM species. By designing species-specific gRNA probes targeting the rpoB sequence, a Cas12a/gRNA-based platform successfully identified M. tuberculosis and six major NTM species (Mycobacterium abscessus, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium gordonae, and Mycobacterium fortuitum) without cross-reactivity. In a blind assessment, a total of 72 out of 73 clinical Mycobacterium isolates were correctly identified, which is consistent with previous rpoB sequencing results. These results suggest that the Cas12a/gRNA-based platform is a promising tool for the rapid, accurate, and cost-effective identification of both M. tuberculosis and NTM species.
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Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/genética , Endodesoxirribonucleasas/genética , Mycobacterium/clasificación , Sondas ARN , ARN Guía de Kinetoplastida/genética , Tuberculosis/diagnóstico , Sistemas CRISPR-Cas , Humanos , Mycobacterium/aislamiento & purificación , Infecciones por Mycobacterium no Tuberculosas/microbiología , Complejo Mycobacterium avium/genética , Complejo Mycobacterium avium/aislamiento & purificación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Micobacterias no Tuberculosas/genética , Micobacterias no Tuberculosas/aislamiento & purificación , ARN Ribosómico 16S/genética , Sensibilidad y Especificidad , Tuberculosis/microbiologíaRESUMEN
To understand functional role of PD-1-expressing MAIT cells during tuberculosis infection in humans, sorted PD-1+ and PD-1- MAIT cells from pleural effusions of patients with pleural tuberculosis were subjected to transcriptome sequencing. PD-1-expressing MAIT cells were analysed by flow cytometry and their phenotypic and functional features were investigated. Transcriptome sequencing identified 144 genes that were differentially expressed between PD-1+ and PD-1- MAIT cells from tuberculous pleural effusions and CXCL13 was the gene with highest fold difference. The level of PD-1-expressing MAIT cells was associated with extent of TB infection in humans. PD-1-expressing MAIT cells had increased production of CXCL13 and IL-21 as determined by flow cytometry. PD-1high CXCR5- MAIT cells were significantly expanded in pleural effusions from patients with pleural tuberculosis as compared with those from peripheral blood of both patients with tuberculosis and healthy controls. Although PD-1high CXCR5- MAIT cells from tuberculous pleural effusions had reduced IFN-γ level and increased expression of Tim-3 and GITR, they showed activated phenotype and had higher glucose uptake and lipid content. It is concluded that PD-1-expressing MAIT cells had reduced IFN-γ level but increased production of both CXCL13 and IL-21.
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Quimiocina CXCL13/biosíntesis , Células T Invariantes Asociadas a Mucosa/inmunología , Tuberculosis Pleural/inmunología , Adulto , Quimiocina CXCL13/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células T Invariantes Asociadas a Mucosa/metabolismo , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Tuberculosis Pleural/metabolismoRESUMEN
The genotyping of SNPs (single nucleotide polymorphisms) is a prerequisite for the analysis of many genetic diseases, including hereditary hearing-loss. However, the existing methods for SNP detection suffer from a long detection period, tedious operation, and a high risk of carryover contamination. To address these challenges, a microfluidic chip is constructed for rapid and efficient SNP genotyping by dividing the sample into many independent chambers for Kompetitive Allele Specific PCR in this study. Using this strategy, multiple detection can be easily accomplished and the challenge for the establishment of multiplex PCR is fundamentally overcome. The entire detection can be finished within 2 h in a fully sealed manner with this method, which is quite simple compared to SNaPshot and MassArray. After assessment of the basic performance, this chip was applied to screen 15 mutations, including SNPs and InDels (insertion-deletion markers), that can cover more than 80% of cases of hereditary hearing-loss in China. Over 40 clinical samples were analyzed with this microfluidic chip for SNP genotyping, and the results are consistent with that obtained by Sanger sequencing, demonstrating its practicability and potential in the application of genetic disease detection.
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Técnicas de Genotipaje , Pérdida Auditiva Sensorineural/genética , Técnicas Analíticas Microfluídicas , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple/genética , Genotipo , HumanosRESUMEN
BACKGROUND: For definitive diagnosis of cryptococcal meningitis, Cryptococcus neoformans and/or C. gattii must be identified within cerebral spinal fluid from the patients. The traditional methods for detecting Cryptococcus spp. such as India ink staining and culture are not ideal. Although sensitive and specific enough, detection of cryptococcal antigen polysaccharide has a high dose hook effect. Therefore, the aim of this study was to introduce a new rapid and simple detection method of Cryptococcus neoformans and C. gattii in cerebral spinal fluid. METHODS: The lateral flow strips combined with recombinase polymerase amplification (LF-RPA) assay was constructed to detect the specific DNA sequences of C. neoformans and C. gattii. The detection limit was evaluated using serial dilutions of C. neoformans and C. gattii genomic DNA. The specificity was assessed by excessive amount of other pathogens genomic DNA. The optimal detection time and amplification temperature were also analyzed. The diagnostic parameters were first calculated using 114 clinical specimens and then compared with that of other diagnostic method. A brief analysis and comparison of different DNA extraction methods was discussed, too. RESULTS: The LF-RPA assay could detect 0.64 pg of genomic DNA of C. neoformans per reaction within 10 min and was highly specific for Cryptococcus spp.. The system could work well at a wide range of temperature from 25 to 45 °C. The overall sensitivity and specificity were 95.2 and 95.8% respectively. As amplification template for LF-RPA assay, both cell lysates and genomic DNA produce similar experimental results. CONCLUSIONS: The LF-RPA system described here is shown to be a sensitive and specific method for the visible, rapid, and accurate detection of Cryptococcus spp. in cerebral spinal fluid and might be useful for clinical preliminary screening of cryptococcal meningitis.
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Criptococosis/diagnóstico , Cryptococcus gattii/aislamiento & purificación , Cryptococcus neoformans/aislamiento & purificación , Meningitis Criptocócica/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , ARN de Hongos/líquido cefalorraquídeo , Antígenos Fúngicos/líquido cefalorraquídeo , Antígenos Fúngicos/genética , Criptococosis/líquido cefalorraquídeo , Cryptococcus gattii/genética , Cryptococcus neoformans/genética , Cartilla de ADN/genética , Diagnóstico Precoz , Humanos , Límite de Detección , Meningitis Criptocócica/líquido cefalorraquídeo , Técnicas Analíticas Microfluídicas/métodos , ARN de Hongos/análisis , Recombinasas/genética , Sensibilidad y Especificidad , TemperaturaRESUMEN
The role of MAIT cells in immunity against Mycobacterium tuberculosis infection in humans is still largely unexplored. In this study, we investigated the functional role of 4-1BB on MAIT cells. We found that 4-1BB was highly up-regulated on MAIT cells from tuberculous pleural effusions following Mtb antigen stimulation and its level of expression correlated with IFN-γ and IL-17 production. 4-1BB expression on MAIT cells in response to Mtb antigens was partially dependent on IL-2 and was associated with common γ chain receptor. By transcriptome sequencing, we identified numerous differentially expressed genes between 4-1BB- and 4-1BB+ MAIT cells. GO enrichment and KEGG pathway analysis of differentially expressed genes identified enriched pathways that included T-cell receptor and NF-κB signaling pathways. It is concluded that 4-1BB has the potential to be used as a biomarker to identify MAIT cells with enhanced IFN-γ and IL-17 responses that might be associated with tuberculosis infection control.
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Células T Invariantes Asociadas a Mucosa/fisiología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/fisiología , Adulto , Biomarcadores/sangre , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Interferón gamma/inmunología , Interleucina-17/inmunología , Interleucina-2/inmunología , Masculino , Persona de Mediana Edad , Células T Invariantes Asociadas a Mucosa/metabolismo , Mycobacterium tuberculosis/inmunología , Tuberculosis/fisiopatología , Tuberculosis Pleural/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
To definitively diagnose active pulmonary Tuberculosis (TB), Mycobacterium tuberculosis complex (MTBC) bacilli must be identified within clinical specimens from patients. In this study, we introduced a rapid and visual detection method of MTBC using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strips. The LF-RPA assay, read results with naked eyes, could detect as few as 5 genome copies of M. tuberculosis H37Rv (ATCC 27294) per reaction and had no cross-reactions with other control bacteria even using excessive amount of template DNA. The system could work well at a broad range of temperature 25-45 °C and reach detectable level even within 5 min. When testing a total of 137 clinical specimens, the sensitivity and specificity of the LF-RPA assay were 100% (95% CI: 95.94%-100%) and 97.92% (95% CI: 88.93%-99.95%), respectively, compared to culture identification method. Therefore, the LF-RPA system we have demonstrated is a rapid, simple, robust method for MTBC detection which, subject to the availability of a suitable sample extraction method, has the potentiality to diagnose TB at the point-of-care testing.
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Mycobacterium tuberculosis/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Juego de Reactivos para Diagnóstico , Recombinasas/metabolismo , Humanos , Límite de Detección , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Temperatura , Factores de TiempoRESUMEN
High-risk human papillomavirus (hrHPV) has been identified as a key factor in the development of cervical cancer. Integration of viral DNA into the host genome has been postulated as an important etiological event during cervical carcinogenesis. High-risk HPV DNA integration frequently results in either the deletion or interruption of the large fragment of E1 and E2 region and the overexpression of oncogenes E6 and E7 in the viral genome, and the activation of oncogenes and the inactivation of tumor suppressors in host genome. Recent studies have showed that hrHPV integration can be used as a predictive biomarker in high-quality cervical lesion screening. Most effective diagnostic approaches are based on fluorescence in situ hybridization, real-time quantitative PCR and Sanger sequencing of hybrid captured viral DNA. This review highlights the primary mechanisms of hrHPV DNA integration associated with cervical carcinogenesis, illustrates recent advances in predictive biomarkers in cervical lesion screening and the development and popularization of prophylactic HPV vaccines, and summarizes the various methods of detecting hrHPV DNA integration.
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Alphapapillomavirus/genética , ADN Viral/genética , Neoplasias del Cuello Uterino/virología , Biomarcadores/metabolismo , Carcinogénesis/genética , Femenino , Humanos , Oncogenes/genéticaRESUMEN
Background: The currently used conventional susceptibility testing for drug-resistant Mycobacterium tuberculosis (M.TB) is limited due to being time-consuming and having low efficiency. Herein, we propose the use of a microfluidic-based method to rapidly detect drug-resistant gene mutations using Kompetitive Allele-Specific PCR (KASP). Methods: A total of 300 clinical samples were collected, and DNA extraction was performed using the "isoChip®" Mycobacterium detection kit. Phenotypic susceptibility testing and Sanger sequencing were performed to sequence the PCR products. Allele-specific primers targeting 37 gene mutation sites were designed, and a microfluidic chip (KASP) was constructed using 112 reaction chambers to simultaneously detect multiple mutations. Chip validation was performed using clinical samples. Results: Phenotypic susceptibility of clinical isolates revealed 38 rifampicin (RIF)-resistant, 64 isoniazid (INH)-resistant, 48 streptomycin (SM)-resistant and 23 ethambutol (EMB)-resistant strains, as well as 33 multi-drug-resistant TB (MDR-TB) strains and 20 strains fully resistant to all four drugs. Optimization of the chip-based detection system for drug resistance detection showed satisfactory specificity and maximum fluorescence at a DNA concentration of 1×101 copies/µL. Further analysis revealed that 76.32% of the RIF-resistant strains harbored rpoB gene mutations (sensitivity, 76.32%; specificity 100%), 60.93% of the INH-resistant strains had katG gene mutations (sensitivity, 60.93%; specificity, 100%), 66.66% of the SM-resistant strains carried drug resistance gene mutations (sensitivity, 66.66%; specificity, 99.2%), and 69.56% of the EMB-resistant strains had embB gene mutations (sensitivity, 69.56%; specificity, 100%). Further, the overall agreement between the microfluidic chip and Sanger sequencing was satisfactory, with a turnaround time of the microfluidic chip was approximately 2 hours, much shorter than the conventional DST method. Conclusion: The proposed microfluidic-based KASP assay provides a cost-effective and convenient method for detecting mutations associated with drug resistance in M. tuberculosis. It represents a promising alternative to the traditional DST method, with satisfactory sensitivity and specificity and a much shorter turnaround time.
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This study is a first report on the identification of multidrug-resistant (MDR) Acinetobacter bereziniae among non-baumannii acinetobacters that had previously escaped automated laboratory detection, and characterize their clinical courses of infection at two tertiary-care hospitals in Shenzhen city, China (2015-2017). Herein, definitive identification by PCR was performed with universal and species-specific primers targeting 16S rDNA and rpoB genes, respectively, followed by Sanger sequencing and blast analysis. Antimicrobial susceptibility of A. bereziniae isolates was assessed accordingly. Three of the five identified A. bereziniae isolates exhibited carbapenem-resistance and were subjected to a multiplex PCR assay to detect drug-resistance genes. Sequences of the rpoB amplicon were aligned with curated sequences from global databases for phylogenetic analysis on evolutionary relations. Five clinical isolates of A. bereziniae were thereby re-identified, whose infections were primarily nosocomial. Automated identification and susceptibility testing systems (Phoenix-100 and VITEK 2) proved insufficient for discriminating A. bereziniae from other acinetobacters such as Acinetobacter baumannii and Acinetobacter guillouiae. Among these isolates, three exhibited carbapenem-resistant phenotypes indistinguishable from that of carbapenem-resistant A. baumannii. The carbapenem-resistant A. bereziniae isolates were subsequently confirmed to carry a bla NDM-1 (New Delhi metallo-ß-lactamase-1) gene downstream of ISAba125. Phylogenetic analysis revealed that A. bereziniae isolates evolved slowly but independently in local habitats. A. bereziniae isolates are difficult to distinguish by traditional automated detection systems. PCR-based identification via amplification and sequencing of selected house-keeping genes provides sufficient resolution for discriminating the isolates.
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A saline gargle (SG) has proven to be an efficient method of sampling to detect SARS-CoV-2. The aim of this pilot study was to verify the efficiency of SG sampling in detecting the Omicron variant of SARS-CoV-2. Subjects were a total of 68 patients with COVID-19 (Omicron variant), and 167 pairs of samples were collected. A conventional oropharyngeal swab (OPS) was obtained and SG sampling was performed immediately afterward; both were subjected to RT-qPCR. A subgroup analysis of symptomatic and asymptomatic patients was performed. Results revealed no significant differences in the distribution of patients and cycle threshold (CT) values between the SG and OPS in overall data and data on days 1-3, 4-7, and 8-14. The subgroup analysis revealed no significant differences between the SG and OPS results in symptomatic patients. In asymptomatic patients, the CT values for the SG were significantly lower than those for the OPS, implying that SG sampling had better sensitivity in the context of the Omicron variant. These data indicate that the SG had satisfactory efficiency (vs. the OPS). An SG is a simple and less invasive method of sampling that is suited to mass, frequent, and repeated sampling to detect SARS-CoV-2.
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COVID-19 , SARS-CoV-2 , Humanos , Proyectos Piloto , COVID-19/diagnósticoRESUMEN
Imported malaria has become a major risk factor for malaria prevention and control in China. How to screen malaria quickly for people entering China is an urgent problem to be solved. Protein microarrays are widely used in high-throughput screening and diagnosis. In this study, surface plasmon resonance (SPR) technique for malaria detection was established by using the specific adsorption surface treated by polyethylene glycol polymer, and the malaria specific antigen HRP2 was used as capture probe. The optimal concentration of antigen, sensitivity and specificity of detection, as well as anti-interference ability of the chip were analyzed. The SPR protein chip was applied to detect specific antibodies of malignant malaria in serum with the advantage of label-free, instant and fast. Compared with fluorescence quantitative PCR, there were no significant difference in sensitivity and specificity between the two methods. This study lays a foundation for further development of protein microarray for malaria typing identification, and it is conducive to the rapid screening of malaria for people entering.
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Malaria , Resonancia por Plasmón de Superficie , Anticuerpos , China , Humanos , Malaria/diagnóstico , Análisis por Matrices de ProteínasRESUMEN
BACKGROUND: Sputum is commonly used for the diagnostic testing of pulmonary tuberculosis (PTB), but people living with HIV/AIDS (PLWHA) usually have little sputum. Moreover, the automated molecular test, Xpert MTB/RIF assay (Xpert), has a low sensitivity in PLWHA. We aimed to estimate the performance of Xpert Ultra on the detection of Mycobacterium tuberculosis (MTB) using bronchoalveolar lavage (BAL). METHODS: From February 5, 2018 to March 30, 2019, a total of 99 PLWHA with suspected PTB at the Third People's Hospital of Shenzhen, China, were recruited. The information on demographics and medical history, blood MTB antigen-specific interferon gamma enzyme-linked immunospot assay (T-SPOT.TB), T lymphocyte subsets, and plasma HIV RNA load were collected. Computed tomography (CT) and flexible bronchoscopy were performed, and BAL and blood samples were collected. Testing of acid-fast bacilli (AFB), tuberculosis real-time fluorescence quantitative PCR (TBDNA), Ultra, Xpert, and MTB culture were conducted. RESULTS: Compared to BAL MTB culture for tuberculosis diagnosis, Ultra, Xpert, T-SPOT.TB, TBDNA and AFB smear had the sensitivity of 0.96 (24/25), 0.80 (20/25), 0.84 (21/25), 0.44 (11/25), and 0.12 (3/25), respectively; and the specificity of 0.92 (68/74), 0.96 (71/74), 0.93 (69/74), 0.96 (71/74), and 0.99 (73/74), respectively. Our study found that the sensitivity of Ultra was higher than that of culture and Xpert (AUC 0.92, 0.86 and 0.84, respectively). The results also indicated that PLWHA with CD4 <200 cells/mm3 had reduced both sensitivity (from 1.00 and 0.86 to 0.94 and 0.78, respectively) and specificity (from 0.96 and 1.00 to 0.90 and 0.41, respectively) of Ultra and Xpert for the diagnosis of PTB. DISCUSSION: Our data supported an increased sensitivity of Ultra compared to that of Xpert on BAL samples of PLWHA, regardless of the CD4 counts and reference diagnosis standards.
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Mycobacterium kansasii can cause serious pulmonary disease. It belongs to a group of closely-related species of non-tuberculous mycobacteria known as the M. kansasii complex (MKC). Here, we report a population genomics analysis of 358 MKC isolates from worldwide water and clinical sources. We find that recombination, likely mediated by distributive conjugative transfer, has contributed to speciation and on-going diversification of the MKC. Our analyses support municipal water as a main source of MKC infections. Furthermore, nearly 80% of the MKC infections are due to closely-related M. kansasii strains, forming a main cluster that apparently originated in the 1900s and subsequently expanded globally. Bioinformatic analyses indicate that several genes involved in metabolism (e.g., maintenance of the methylcitrate cycle), ESX-I secretion, metal ion homeostasis and cell surface remodelling may have contributed to M. kansasii's success and its ongoing adaptation to the human host.
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Agua Potable/microbiología , Genoma Bacteriano/genética , Enfermedades Pulmonares/epidemiología , Infecciones por Mycobacterium no Tuberculosas/epidemiología , Mycobacterium kansasii/genética , Metabolismo Energético/genética , Variación Genética/genética , Genética de Población/métodos , Genómica , Humanos , Enfermedades Pulmonares/microbiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium kansasii/aislamiento & purificación , Virulencia/genética , Microbiología del AguaRESUMEN
CRISPR/Cas9 is a robust tool to manipulate genes in a wide range of species. Although several methods are introduced to identify the CRISPR/Cas9-induced mutations, they are labor-intensive, costly, and not easy to use or were sequence-limited. Moreover, few of them could identify the biallelic mutants that are the desired outcomes of targeted mutagenesis. Recently, a CRISPR/Cas12a-mediated biosensing platform was developed to detect nucleic acids based on the collateral DNA cleavage activity of Cas12a; it was highly sensitive, specific, rapid, and cost-efficient for genotyping, mutation detection, and single nucleotide polymorphism (SNP) identification, thereby deeming it as an innovative method for screening the CRISPR/Cas9-induced biallelic mutants. Thus, the CRISPR/Cas12a-based biosensing platform has been successfully utilized for screening 23 CRISPR/Cas9-induced biallelic mutants in Thp-1 cells, which were also confirmed by direct sequencing and ELISA. The precision and efficiency of CRISPR/Cas12a-based biosensing platform make it a promising tool for screening of CRISPR/Cas9-induced biallelic mutants in the future.
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Técnicas Biosensibles , Sistemas CRISPR-Cas/genética , Humanos , Mutación , ARN/genética , Células THP-1RESUMEN
Macrophage dysfunction is associated with increased tuberculosis (TB) susceptibility in patients with human immunodeficiency virus (HIV) infection. However, the mechanisms underlying how HIV infection impairs macrophage function are unclear. Here, we found that levels of autoantibodies against red blood cells (RBCs) were significantly elevated in patients with HIV as determined by direct antiglobulin test (DAT). DAT positivity was significantly associated with TB incidence in both univariate and multivariate analyses (odds ratio [OR] = 11.96 [confidence interval {CI}, 4.68 to 30.93] and 12.65 [3.33 to 52.75], respectively). Ex vivo analysis showed that autoantibodies against RBCs enhanced erythrophagocytosis and thus significantly impaired macrophage bactericidal function against intracellular Mycobacterium tuberculosis Mechanistically, autoantibody-mediated erythrophagocytosis increased heme oxygenase-1 (HO-1) expression, which inhibited M. tuberculosis-induced autophagy in macrophages. Silencing ATG5, a key component for autophagy, completely abrogated the effect of erythrophagocytosis on macrophage bactericidal activity against M. tuberculosis In conclusion, we have demonstrated that HIV infection increases autoantibody-mediated erythrophagocytosis. This process impairs macrophage bactericidal activity against M. tuberculosis by inhibiting HO-1-associated autophagy. These findings reveal a novel mechanism as to how HIV infection increases TB susceptibility.IMPORTANCE HIV infection significantly increases TB susceptibility due to CD4 T-cell loss and macrophage dysfunction. Although it is relatively clear that CD4 T-cell loss represents a direct effect of HIV infection, the mechanism underlying how HIV infection dampens macrophage function is unknown. Here, we show that HIV infection enhances autoantibody-mediated erythrophagocytosis, which dampens macrophage bactericidal activity against TB by inhibiting HO-1-associated autophagy. Our findings reveal a novel mechanism explaining how HIV infection increases susceptibility to TB. We propose that DAT could be a potential measure to identify HIV patients who are at high TB risk and who would be suitable for anti-TB chemotherapy preventive treatment.
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Autoanticuerpos/inmunología , Eritrocitos/inmunología , Infecciones por VIH/inmunología , Fagocitosis/inmunología , Tuberculosis/inmunología , Adulto , Autofagia , Proteína 5 Relacionada con la Autofagia , Femenino , Técnicas de Silenciamiento del Gen , Infecciones por VIH/complicaciones , Hemo-Oxigenasa 1/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Humanos , Modelos Logísticos , Macrófagos/microbiología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Mycobacterium tuberculosis/inmunología , Linfocitos T , Células THP-1 , Tuberculosis/complicacionesRESUMEN
Tuberculosis (TB) remains a major global public health problem. New immunization methods against TB are urgently needed. Plasmid DNA with a microneedle patch is a potentially attractive strategy to improve the immune effect. A DNA vaccine encoding the secreted protein Ag85B of Mycobacterium tuberculosis was immunized in the skin using microneedles, which can improve protective immunity compared to conventional intramuscular (IM) injection. There is no significant difference between microneedle patch (MNP) and IM immunization when the immunizing dose is low (4.2⯵g). However, the results for detecting humoral immunity showed MNP immunization could better provoke an antibody response than IM when the dose is high (12.6⯵g). A similar result was observed in cellular immune responses by measuring the cytokines in splenocytes. The effective protection of MNP can also be demonstrated by counting bacteria and analyzing the survival rate. This study indicated that DNA vaccination in the skin using dissolving microneedles may provide a new strategy against TB.
Asunto(s)
Aciltransferasas/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Tuberculosis/prevención & control , Vacunas de ADN/inmunología , Animales , Femenino , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Inmunogenicidad Vacunal/inmunología , Inmunogenicidad Vacunal/fisiología , Inmunogenicidad Vacunal/efectos de la radiación , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/patogenicidad , Vacunas de ADN/uso terapéuticoRESUMEN
Polysaccharide nucleic acid fractions of bacillus Calmette-Guérin, termed BCG-PSN, have traditionally been used as immunomodulators in the treatment of dermatitis and allergic diseases. While the sales of injectable BCG-PSN have shown steady growth in recent years, no reports of using BCG-PSN powder or its immunotherapeutic effects exist. Here, BCG-PSN powder was applied directly to the skin to evaluate the immunotherapeutic effects on mice infected with Mycobacterium tuberculosis (MTB). In total, 34 µg of BCG-PSN powder could be loaded into a microneedle patch (MNP). Mice receiving BCG-PSN powder delivered via MNP exhibited significantly increased IFN-γ and TNF-α production in peripheral blood CD4 + T cells and improved pathological changes in their lungs and spleens compared to control group mice. The immunotherapeutic effect of BCG-PSN powder delivered via MNP was better than that delivered via intramuscular injection to some extent. Furthermore, MNPs eliminate the side effects of syringes, and this study demonstrated that BCG-PSN can be clinically administrated in powder form.