non-TB Patients Research Articles

Overview of TB Patient Compliance for Control during the Covid-19 Pandemic at the UGM RSA TB Clinic

Tuberculosis (TB) is a public health problem that is a global challenge and is the third leading cause of death in Indonesia and the first leading cause of death due to infectious diseases. Based on data from the Ministry of Health of the Republic of Indonesia, there has been a decrease in the number of TB cases found and treated. As one of the strategies to control risk factors for TB disease transmission, RSA UGM provides a special clinic for TB patients whose rooms meet national standards. However, several times researchers found that there were still TB patients who controlled outside the TB clinic schedule so that they mingled with non-TB patients, which resulted in increasing the risk of TB disease transmission to patients visiting the UGM Hospital. The World Health Organization (WHO) declared Covid-19 a pandemic on 9 March 2020. This has caused public fear to visit health facilities. This study aims to determine the level of compliance of TB patient visits during the Covid-19 pandemic at the UGM RSA TB Clinic. This descriptive observational study used secondary data, with a total sampling technique and analyses using univariate analysis. Of the 40 samples that met the inclusion and exclusion criteria, 92.5% of patients were in the productive age range (18-65 years), mostly male (70%). The proportion of patients with pulmonary TB was 62.5% and the remaining 37.5% were extra- pulmonary TB patients. There was an increase in adherence of TB patients to control at the TB clinic and on schedule from month 1 to the following months.

Factors associated with hospitalization and mortality in adult and pediatric extrapulmonary tuberculosis at a tertiary care hospital in Central India

Background Comorbidities complicate the management of tuberculosis (TB) and have become an essential part of the end TB strategy to eradicate TB. However, pulmonary TB has received the most attention, and little is known about the impact of comorbidities and other factors on outcomes in patients with extrapulmonary tuberculosis (EPTB). Objectives Our aim was to analyze the factors associated with hospitalization and mortality in EPTB at a hospital in Central India, using non-TB patients with similar clinical presentations as a comparison. Methods Patients with presumptive EPTB were prospectively enrolled and followed up until the end of treatment or for at least 6 months. Detailed demographic and clinical information was collected for all participants, and patients were categorized as TB or non-TB using a composite reference standard. Multivariate logistic regression was used to analyze the impact of various clinical findings and risk factors on hospitalization and mortality. Results A total of 276 patients were categorized as TB cases and 175 as non-TB cases. Factors associated with hospitalization in children were younger age and non-adenitis site of disease. In adults, factors associated with mortality were older age, non-adenitis site of disease and HIV infection regardless of TB diagnosis, while diabetes mellitus increased the odds of mortality in EPTB patients. Conclusion Our results show that comorbidities increase the odds of death in both TB and non-TB patients in low-resource settings. This argues for a shift away from the traditional vertical management of diseases in these areas and supports a continued focus on building robust healthcare systems.

Pulmonary dimorphic fungal infections among HIV/AIDS non-TB patients with chronic cough in Kampala, Uganda.

Dimorphic fungi cause infection following the inhalation of spores into the pulmonary system. In the lower respiratory tract, the conidia transform into yeasts, which are engulfed by alveolar macrophages and may be destroyed without disease manifestation. However, in some immunocompromised individuals, they may persist and cause active fungal disease characterized by formation of granulomas in the infected tissues, which may mimic Mycobacterium tuberculosis (MTB). To determine the prevalence of pulmonary dimorphic fungal infections among HIV/AIDS patients with non-TB chronic cough at Mulago National Referral and Teaching Hospital in Kampala, Uganda. Sputum samples were collected from 175 consented HIV/AIDS patients attending the immuno-suppression syndrome (ISS) clinic at the hospital. Upon Xpert MTB/RIF sputum testing, 21 patients tested positive for MTB, and these were excluded from further analysis. The other 154 sputum negative samples were then subjected to PCR for dimorphic fungi at MBN Clinical Laboratories. Singleplex PCR was used to detect the target sequences in selected respective genes of each dimorphic fungal species of interest. DNA amplicons were detected based on gel electrophoresis. Dimorphic fungi were detected in 16.2% (25/154) of the studied population. Of these 9.1% (14/154) had Blastomyces dermatitidis and 7.1% (11/154) had Talaromyces marneffei. The remaining 84% of the studied participants had no dimorphic fungi. Histoplasma capsulatum, Coccidioides immitis and Paracoccidioides brasiliensis were not detected in any of the participants. Dimorphic fungi (B. dermatitidis and T. marneffei) were found in 16.2% of the HIV/AIDS patients with non-TB chronic cough in Kampala, Uganda. We recommend routine testing for these pathogens among HIV/AIDS patients with chronic cough.

Tetrad metal ion mediated molecular switch aptamer sensor for fluorescence assay of plasma IP-10 in clinical tuberculosis

Interferon-γ-inducible protein 10 (IP-10) has demonstrated enormous potential as a novel biomarker for tuberculosis (TB), particularly in the monitoring of patient progression for active or latent tuberculosis infection (LTBI). Herein, we established a tetrad metal ion-mediated molecular switch aptamer sensor for the convenient detection of IP-10 in plasma and the diagnosis of TB. The simple sensor relied on the partially complementary IP-10 aptamer and four DNA hairpin strands rich in cytosine (C), which can form a relatively stable tetrad C-Ag+-C structure when combined with Ag+ without additional modification. Based on the specific recognition of IP-10 and aptamer, a large amount of Ag+ could be released in a short time. Combining with cadmium telluride quantum dots (CdTe QDs) as the signal reporter, we achieved superior analytical performance, with the limits of detection (LOD) reaching 3 fg/mL for IP-10 analysis in 1 h. Clinical practicality was confirmed by evaluating 38 clinical samples (19 TB-positive patients, 14 non-TB patients, and 5 latent TB infection patients). The results showed that the detected IP-10 levels were significantly different between the non-TB and TB/LTBI groups, and the results aligned with clinical computed tomography (CT) results, indicating that the method could represent a reliable diagnostic technique for TB.

Discovering novel biomarkers for diagnosis and treatment monitoring of active pulmonary tuberculosis by ion metabolism analysis

Tuberculosis (TB) is a highly lethal infectious disease that poses a global threat. Timely and accurate biomarker for TB diagnosis and treatment monitoring remains a pressing need. Ions, the crucial trace element for humans, may be potential targets for TB diagnosis and the forecasting of TB development. To explore the potential of ions as biomarkers, we measured and compared the levels of various ions in whole blood and plasma samples from healthy control (HC), pulmonary TB patients (TB), cured pulmonary TB patients (RxTB), and other non-TB pneumonia patients (PN) by using ultra-high performance liquid chromatography-tandem mass spectrometry. Our study demonstrated that Cu (AUC = 0.670), Pb (AUC = 0.660), and Zn (AUC = 0.701) in whole blood exhibited promising diagnostic performance for TB. Then we used a neural network (NNET) for TB prediction, the AUC values used to differentiate definite TB from HC or PN in plasma were 0.867 and 0.864, respectively. The AUC values used to differentiate definite TB from HC or PN in whole blood were 0.818 and 0.660, respectively. Our correlation analysis showed that Zn (r= 0.356, p=0.001) and Cu (r= 0.361, p=0.0004) in plasma are most closely related to disease severity. Additionally, six ions (Cu, Sb, V, Mn, Fe, Sr) in plasma and whole blood were altered following anti-TB therapy. These results showed that ions could be diagnostic biomarkers for TB. Furthermore, the level of particular ions can forecast the degree of lung damage and the success of the TB treatment. In conclusion, this study highlights the possibility of using ions from blood samples to enable rapid tuberculosis diagnosis.

Mycobacterium tuberculosis hijacks host macrophages-derived interleukin 16 to block phagolysosome maturation for enhancing intracellular growth

ABSTRACT The discovery of promising cytokines and clarification of their immunological mechanisms in controlling the intracellular fate of Mycobacterium tuberculosis (Mtb) are necessary to identify effective diagnostic biomarkers and therapeutic targets. To escape immune clearance, Mtb can manipulate and inhibit the normal host process of phagosome maturation. Phagosome maturation arrest by Mtb involves multiple effectors and much remains unknown about this important aspect of Mtb pathogenesis. In this study, we found that interleukin 16 (IL-16) is elevated in the serum samples of Tuberculosis (TB) patients and can serve as a specific target for treatment TB. There was a significant difference in IL-16 levels among active TB, latent TB infection (LTBI), and non-TB patients. This study first revealed that macrophages are the major source of IL-16 production in response to Mtb infection, and elucidated that IL-16 can promote Mtb intracellular survival by inhibiting phagosome maturation and suppressing the expression of Rev-erbα which can inhibit IL-10 secretion. The experiments using zebrafish larvae infected with M. marinum and mice challenged with H37Rv demonstrated that reducing IL-16 levels resulted in less severe pathology and improved survival, respectively. In conclusion, this study provided direct evidence that Mtb hijacks the host macrophages-derived interleukin 16 to enhance intracellular growth. It is suggesting the immunosuppressive role of IL-16 during Mtb infection, supporting IL-16 as a promising therapeutic target.

TB-CXRNet: Tuberculosis and Drug-Resistant Tuberculosis Detection Technique Using Chest X-ray Images

Tuberculosis (TB) is a chronic infectious lung disease, which caused the death of about 1.5 million people in 2020 alone. Therefore, it is important to detect TB accurately at an early stage to prevent the infection and associated deaths. Chest X-ray (CXR) is the most popularly used method for TB diagnosis. However, it is difficult to identify TB from CXR images in the early stage, which leads to time-consuming and expensive treatments. Moreover, due to the increase of drug-resistant tuberculosis, the disease becomes more challenging in recent years. In this work, a novel deep learning-based framework is proposed to reliably and automatically distinguish TB, non-TB (other lung infections), and healthy patients using a dataset of 40,000 CXR images. Moreover, a stacking machine learning-based diagnosis of drug-resistant TB using 3037 CXR images of TB patients is implemented. The largest drug-resistant TB dataset will be released to develop a machine learning model for drug-resistant TB detection and stratification. Besides, Score-CAM-based visualization technique was used to make the model interpretable to see where the best performing model learns from in classifying the image. The proposed approach shows an accuracy of 93.32% for the classification of TB, non-TB, and healthy patients on the largest dataset while around 87.48% and 79.59% accuracy for binary classification (drug-resistant vs drug-sensitive TB), and three-class classification (multi-drug resistant (MDR), extreme drug-resistant (XDR), and sensitive TB), respectively, which is the best reported result compared to the literature. The proposed solution can make fast and reliable detection of TB and drug-resistant TB from chest X-rays, which can help in reducing disease complications and spread.

CORRELATION BETWEEN MONOCYTE CHEMOATTRACTANT PROTEIN 2 (MCP-2) SERUM LEVELS WITH TUBERCULOSIS SEVERITY IN CHEST X-RAYS

Background and Objectives: Tuberculosis (TB) is a public health problem causing the death of children worldwide. Radiological features in children have distinctive characteristics that can be used as basis for diagnosis. The use of MCP-2 as a TB biomarker has been widely studied in adult patients but has not been used extensively in child tuberculosis. Objective: This study aims to determine the relationship between MCP-2 levels with severity of TB in children based on radiological features. Methods: Pediatric patients aged 18 years with suspected TB who had never undergone TB treatment in Saiful Anwar Hospital. The 60 participants who met the inclusion and exclusion criteria were analyzed for serum levels of MCP-2. The data is grouped into non-TB and TB variables and the latter were classified into mild, moderate and severe chest photos. Data analysis was done with normality and homogeneity test, then proceeded with correlation test (p 0,05). Result: The total participants are 60 subjects comprising of 30 TB patients; male 36 subjects; less body weight 17 subjects; TB scoring 6 27 subjects; TCM rif sensitive 16 subjects; Pulmonary TB 22 subjects; and moderate severity based on chest X-ray 18 subjects. The severity of the chest X-ray showed mild degree of MCP-2 level 167,97 pg/dl (102,3-568,62); moderate 284,49pg/dl (233,36-321,71); severe 594,12 pg/dl (439,07-692,03). Mann Whitney test results showed the MCP-2 level compared with severity of TB were significantly different between groups (P0,000) and there was a signficant correlation between MCP-2 levels and the severity of TB in children (P0,000) Conclusion: There are differences in MCP-2 levels in non-TB and TB patients, and there is a correlation between MCP-2 levels and the severity of TB in children.

Pooling sputum samples for the Xpert MTB/RIF assay: a practical screening strategy for highly infectious tuberculosis cases

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.

Reanalysis and validation of the transcriptional pleural fluid signature in pleural tuberculosis.

Pleural tuberculosis (PlTB), the most common site of extrapulmonary TB, is characterized by a paucibacillary nature and a compartmentalized inflammatory response in the pleural cavity, both of which make diagnosis and management extremely challenging. Although transcriptional signatures for pulmonary TB have already been described, data obtained by using this approach for extrapulmonary tuberculosis and, specifically, for pleural tuberculosis are scarce and heterogeneous. In the present study, a set of candidate genes previously described in pulmonary TB was evaluated to identify and validate a transcriptional signature in clinical samples from a Brazilian cohort of PlTB patients and those with other exudative causes of pleural effusion. As a first step, target genes were selected by a random forest algorithm with recursive feature elimination (RFE) from public microarray datasets. Then, peripheral blood (PB) and pleural fluid (PF) samples from recruited patients presenting exudative pleural effusion were collected during the thoracentesis procedure. Transcriptional analysis of the selected top 10 genes was performed by quantitative RT-PCR (RT-qPCR). Reanalysis of the public datasets identified a set of candidate genes (CARD17, BHLHE40, FCGR1A, BATF2, STAT1, BTN3A1, ANKRD22, C1QB, GBP2, and SEPTIN4) that demonstrated a global accuracy of 89.5% in discriminating pulmonary TB cases from other respiratory diseases. Our validation cohort consisted of PlTB (n = 35) patients and non-TB (n = 34) ones. The gene expressions of CARD17, GBP2, and C1QB in PF at diagnosis were significantly different between the two (PlTB and non-TB) groups (p < 0.0001). It was observed that the gene expressions of CARD17 and GBP2 were higher in PlTB PF than in non-TB patients. C1QB showed the opposite behavior, being higher in the non-TB PF. After anti-TB therapy, however, GBP2 gene expression was significantly reduced in PlTB patients (p < 0.001). Finally, the accuracy of the three above-cited highlighted genes in the PF was analyzed, showing AUCs of 91%, 90%, and 85%, respectively. GBP2 was above 80% (sensitivity = 0.89/specificity = 0.81), and CARD17 showed significant specificity (Se = 0.69/Sp = 0.95) in its capacity to discriminate the groups. CARD17, GBP2, and C1QB showed promise in discriminating PlTB from other causes of exudative pleural effusion by providing accurate diagnoses, thus accelerating the initiation of anti-TB therapy.

Spatial transcriptomic sequencing reveals immune microenvironment features of Mycobacterium tuberculosis granulomas in lung and omentum.

Granulomas are a key pathological feature of tuberculosis (TB), characterized by cell heterogeneity, spatial composition, and cellular interactions, which play crucial roles in granuloma progression and host prognosis. This study aims to analyze the transcriptome profiles of cell populations based on their spatial location and to understand the core transcriptome characteristics of granuloma formation and development. Methods In this study, we collected four clinical biopsy samples including Mycobacterium tuberculosis (Mtb) infected lung (MTB-L) and omentum tissues (MTB-O), as well as two lung and omentum biopsies from non-TB patients. The tissues were analyzed by spatial transcriptomics to create a spatial atlas. Utilizing cell enrichment scores and intercellular communication analysis, we investigated the transcriptome signatures of cell populations in various spatial regions and identified genes that may play a decisive role in the formation of pulmonary and omental tuberculosis granulomas. To validate our major findings, an in vitro TB model based on organoid-macrophage co-culture was established. Results Spatial transcriptomics mapped the cell composition and spatial distribution characteristics of tuberculosis granulomas in lung and omental tissues infected with Mtb. The characteristics and evolutionary relationships of major cell populations in granulomas reveal a shift in the immune microenvironment: from a predominance of B cells and fibroblasts in pulmonary granulomas to a predominance of myeloid cells and fibroblasts in omental granulomas. Furthermore, our data identified key differentially expressed genes across cell clusters and regions, showing that upregulation of collagen genes is a common feature of granulomas. Using an organoid-macrophage co-culture model, we demonstrated the notable efficacy of Thrombospondin-1 (THBS1) in reducing protein expression levels related to extracellular matrix remodeling. Conclusion These results provide insights into the pathogenesis and development of tuberculosis, enhancing our understanding of the composition and interactions of tuberculosis granuloma cells from a spatial perspective, and pave the way for novel adjuvant treatments for tuberculosis.

Outbreaks of COVID-19 in a tuberculosis treatment sanatorium on the Thailand-Myanmar border: a retrospective cohort analysis

Background Tuberculosis (TB) is a chronic condition, with overlapping symptoms to those of coronavirus disease 2019 (COVID-19). There has been inconsistent evidence on whether TB is a predisposing factor for developing severe COVID-19. The aim of this report is to explore whether TB influences the severity of COVID-19. Methods COVID-19 cases at two TB sanatoria on the Thailand-Myanmar border were reviewed. Demographic, clinical and laboratory data including TB treatment and co-morbidities, were analyzed. Characteristics and COVID-19 clinical outcomes were compared between two groups of patients: TB and those without TB (the caretakers and the medical personnel). Multivariable ordered logistic regression was conducted to compare the risk of severe COVID-19 between the two groups. Results Between September 2021 and March 2022, 161 COVID-19 cases were diagnosed. Over half of the COVID-19 patients were infected with TB (n= 104, 64.6%), and the rest were not (n=57, 35.4%). The median (interquartile range) age was 48 (33.5-57.0) and 27 (23-33) years in the TB and in the non-TB COVID-19 patients, respectively. Before COVID-19 infection, 67.1% (106/158) of patients had received at least one dose of COVID-19 vaccine. The median cycle threshold value at diagnosis was not different between TB (18.5, IQR 16.1-32.3) and non-TB patients (18.8, 15.1-30.0). Fever, gastrointestinal symptoms and ageusia were more common in non-TB patients. Six patients (3.8%, 6/156) all from the TB group became severe of which five (3.2%, 5/156) required oxygen therapy. One TB patient died (1/104, 0.96%) of lung cancer. After adjustment for potential confounders, the final clinical severity was not different between the two groups (adjusted odds ratio 1.40, 95% confidence interval 0.16–12.39). Conclusions TB was not associated with severe outcomes in the two TB sanatoria. The high uptake of COVID-19 vaccination and active screening could have impacted on disease progression and prevented unfavorable outcomes.

Prevalence of anaemia among symptomatic pulmonary tuberculosis patients at Weija Gbawe Municipal Hospital in Accra, Ghana

In Ghana, TB remains a substantial healthcare challenge, with an incidence rate of 136 cases per 100,000 people. Anaemia is prevalent among TB patients, impacting their health and recovery. Anaemia can lead to fatigue, weakness, and compromised immunity, all critical concerns for TB patients. This study aims to investigate the prevalence of anaemia among pulmonary TB patients at Weija Gbawe Municipal Hospital in Accra, Ghana. Among the participants, 22.6% were found to have pulmonary TB, with the majority not showing resistance to rifampicin (95.5%), a primary treatment drug. Females had a higher prevalence of TB (53.3%), possibly due to hormonal and social factors. Participants over 66 years had a high prevalence, likely due to weakened immune systems associated with aging. Anaemia was prevalent among participants, especially females, and it was more common among TB patients (68.8%) compared to non-TB patients. Normocytic anaemia was the most common type among TB patients, followed by microcytic anaemia, suggesting potential iron metabolism issues. This study provides valuable insights into the prevalence of anemia among TB patients and the distribution of different types of anaemia in this population. Understanding these factors can aid in better management and treatment of TB patients.

Three years’ experience of tuberculosis versus non-tuberculosis cases of bronchopleural fistula repair at Dr. Soetomo General Academic Hospital Surabaya

Link of Video Abstract: https://youtu.be/DVqGIPOyWR0 Background: Tuberculosis (TB) continues to be a significant health issue and causes various complications, especially in developing countries. Bronchopleural fistula (BPF) is a rare and potentially life-threatening complication of pulmonary tuberculosis that needs to be treated by surgical procedure. This research aimed to compare the outcome of post-operative BPF repair caused by Tuberculosis and non-Tuberculosis cases. Methods: In this retrospective review, we include patients from post-operative repair of BPF with Tuberculosis and non-Tuberculosis cases from January 2020 to December 2022 who were admitted to our primary center. Preoperative, operative and postoperative data were collected from medical records. We identified 36 patients and analyzed the differences between TB and non-TB groups. Results: There were 36 patients with BPF who underwent surgery. The mean age of the subjects was 47.940±15.288 years. Out of these, 25 subjects (69.4%) had TB, while 11 (30.6%) subjects did not. Among the subjects, 14 TB patients (38.9%) and 2 non-TB patients (56%) experienced recurrent pneumothorax before surgery. Statistical analysis showed a significant association between comorbid TB and the occurrence of secondary attack BPF before surgery (p = 0.035). Regarding the duration of post-operative drain placement, 22 TB patients (61.1%) had the drain placed for less than 7 days, while 3 subjects (8.3%) had it for more than 7 days. Among the non-TB patients, 10 subjects (27.8%) had the drain placed for less than 7 days, and only 1 subject (2.8%) had it for more than 7 days. The statistical analysis showed no association between the TB group and the duration of post-operative drain placement (p=0.798). Conclusion: There are numerous post-operative complications and morbidities following BPF repair surgery. Patients with TB who undergo BPF repair surgery are significantly associated with secondary attack BPF before surgery.

Accuracy of Nanopore Sequencing as a Diagnostic Assay for Pulmonary Tuberculosis versus Smear, Culture and Xpert MTB/RIF: A Head-to-Head Comparison.

Early diagnosis of pulmonary tuberculosis (PTB) is pivotal for achieving effective tuberculosis (TB) control. This study aimed to assess the effectiveness of nanopore sequencing of sputum, bronchoalveolar lavage fluid (BALF), and pleural fluid samples for achieving early PTB diagnosis and provided head-to-head comparisons of nanopore sequencing results versus results obtained using smear, culture, and Xpert MTB/RIF assays. Patients admitted from October 2021 to April 2023 were screened for PTB using diagnostic imaging and electronic medical records. A total of 172 patients (129 PTB, 43 non-TB patients) were included in the final analysis after the exclusion of patients who did not meet the study's inclusion criteria. PTB-positive rates were determined for each assay, and then, assay diagnostic efficacies were compared. The positive MTB-detection rates obtained using nanopore sequencing were 86.8% for all samples, 62.3% for BALF, and 84.6% for pleural fluid, all of which were significantly higher than the corresponding rates obtained using the other three assays. The overall sensitivity rates, specificity rates, and area under the curve (AUC) values obtained from smear testing were 5.4%, 95.3%, and 0.504, respectively, as compared to the respective results obtained via culture (18.6%, 100.0%, and 0.593), Xpert MTB/RIF (26.4%, 97.7%, and 0.620), and nanopore sequencing (85.3%, 95.4%, and 0.903). The diagnostic efficacy of nanopore sequencing surpassed the diagnostic efficacies of smear, culture, and Xpert MTB/RIF assays. Thus, nanopore sequencing holds promise as an alternative to Xpert MTB/RIF for early PTB detection, particularly for the testing of BALF and pleural fluid samples.

Development and preliminary assessment of a CRISPR-Cas12a-based multiplex detection of Mycobacterium tuberculosis complex.

Since the onset of the COVID-19 pandemic in 2020, global efforts towards tuberculosis (TB) control have encountered unprecedented challenges. There is an urgent demand for efficient and cost-effective diagnostic technologies for TB. Recent advancements in CRISPR-Cas technologies have improved our capacity to detect pathogens. The present study established a CRISPR-Cas12a-based multiplex detection (designated as MCMD) that simultaneously targets two conserved insertion sequences (IS6110 and IS1081) to detect Mycobacterium tuberculosis complex (MTBC). The MCMD integrated a graphene oxide-assisted multiplex recombinase polymerase amplification (RPA) assay with a Cas12a-based trans-cleavage assay identified with fluorescent or lateral flow biosensor (LFB). The process can be performed at a constant temperature of around 37°C and completed within 1h. The limit of detection (LoD) was 4 copies μL-1, and no cross-reaction was observed with non-MTBC bacteria strains. This MCMD showed 74.8% sensitivity and 100% specificity in clinical samples from 107 patients with pulmonary TB and 40 non-TB patients compared to Xpert MTB/RIF assay (63.6%, 100%). In this study, we have developed a straightforward, rapid, highly sensitive, specific, and cost-effective assay for the multiplex detection of MTBC. Our assay showed superior diagnostic performance when compared to the widely used Xpert assay. The novel approach employed in this study makes a substantial contribution to the detection of strains with low or no copies of IS6110 and facilitates point-of-care (POC) testing for MTBC in resource-limited countries.

Amino acid profiling as a screening and prognostic biomarker in active tuberculosis patients

BackgroundTuberculosis (TB) is one of the world's most deadly chronic infectious diseases; early diagnosis contributes to reducing disease transmission among populations. However, discovering novel diagnostic and prognostic biomarkers is still an important topic in the field of TB. Amino acid is the basic unit of protein composition, and its structure and physicochemical characteristics are more stable. Therefore, it is a potential target for TB diagnosis and the prediction of TB development. MethodsIn this study, the blood of healthy people (HC), TB patients (TB), cured TB (RxTB), and other non-TB pneumonia patients (PN) were collected to detect the levels of amino acids in whole blood and plasma using ultra-high performance liquid chromatography coupled with mass spectrometry. ResultsWe detected that the amino acid levels correlated with participants status (TB, HC, RxTB, or PN) and the degree of lung damage. The results showed that phenylalanine had a good effect on the screening of TB (AUC = 0.924). We then built a TB prediction model. The model, which was based on the ratio of plasma amino acid content to whole blood amino acid content, showed good performance for the screening of TB, with 84% (95% CI = 60–97) sensitivity and 97% (95% CI = 83–100) specificity. The result of correlation between the HRCT score and amino acid level indicated that the glutamine content of plasma was significantly inversely associated with disease severity. Additionally, ornithine levels in the plasma of RxTB group reduced and four amino acids of which the ratio in plasma to whole blood showed significantly changed. ConclusionsTaken together, amino acid profiling can be used for TB screening, and a multiparameter profiling model is better. The profiling can also reflect the severity of lung damage. Moreover, the amino acid profile is useful for reflecting the efficacy of TB treatment.

Xpert MTB/RIF Ultra Assay Using Stool: an Effective Solution for Bacilli Identification from Adult Pulmonary Tuberculosis Suspects without Expectorated Sputum.

This study evaluated the diagnostic performance of stool-based Xpert MTB/RIF Ultra assay (Xpert-Ultra, Cepheid, USA) against other tests using respiratory tract specimens (RTS) and stool for diagnosing adult pulmonary tuberculosis. A prospective study on patients with presumptive pulmonary tuberculosis was conducted in Beijing Chest Hospital from June to November 2021. The smear test, MGIT960 liquid culture, and Xpert MTB/RIF (Xpert, Cepheid, USA) were performed simultaneously on RTS, and smear, culture Xpert, and Xpert-Ultra were performed simultaneously using stool. Patients were grouped based on the outcomes of RTS examination and other tests. In total, 130 eligible patients were enrolled that included 96 pulmonary tuberculosis and 34 non-TB patients. The sensitivity of smear, culture, Xpert, and Xpert-Ultra using stool was 10.96%, 23.28%, 60.27%, and 79.45%, respectively. The specificities of Xpert and Xpert-Ultra using RTS and stool were all 100% (34/34). Notably, all five confirmed cases detected by bronchoalveolar lavage fluid (BALF) examination yielded Xpert-Ultra positive outcomes with the stool specimens. Xpert-Ultra assay on stool sample harbors comparable sensitivity with Xpert on RTS. Thus, the Xpert-Ultra testing on stool specimens could be a very promising and practical strategy to improve pulmonary tuberculosis (PTB) diagnosis, especially among patients who could not expectorate sputum. IMPORTANCE This study is aimed at assessing the value of Xpert MTB/RIF Ultra (Xpert-Ultra) in PTB on stool in adult in low HIV settings and Xpert-Ultra assay on stool sample harboring comparable sensitivity with Xpert MTB/RIF on respiratory tract specimens. Although the yield in stool samples by Xpert-Ultra is lower than RTS, it may be useful in detecting disease in presumptive TB patients who cannot expectorate sputum and are not open to BALF collection. In addition, Xpert-Ultra with a "trace call" on stool in adult was highly supportive of PTB.

Outbreaks of COVID-19 in a tuberculosis treatment sanatorium on the Thailand-Myanmar border: a retrospective cohort analysis

Background: Tuberculosis (TB) is a chronic condition, with overlapping symptoms to those of coronavirus disease 2019 (COVID-19). There has been inconsistent evidence on whether TB is a predisposing factor for developing severe COVID-19. The aim of this report is to explore whether TB influences the severity of COVID-19. Methods: COVID-19 cases at two TB sanatoria on the Thailand-Myanmar border were reviewed. Demographic, clinical and laboratory data including TB treatment and co-morbidities, were analyzed. Characteristics and COVID-19 clinical outcomes were compared between two groups of patients: TB and those without TB (the caretakers and the medical personnel). Multivariable ordered logistic regression was conducted to compare the risk of severe COVID-19 between the two groups. Results: Between September 2021 and March 2022, 161 COVID-19 cases were diagnosed. Over half of the COVID-19 patients were infected with TB (n= 104, 64.6%), and the rest were not (n=57, 35.4%). The median (interquartile range) age was 48 (33.5-57.0) and 27 (23-33) years in the TB and in the non-TB COVID-19 patients, respectively. Before COVID-19 infection, 78.7% (122/155) of patients had received at least one dose of COVID-19 vaccine. The median cycle threshold value at diagnosis was not different between TB (18.5, IQR 16.1-32.3) and non-TB patients (18.8, 15.1-30.0). Fever, gastrointestinal symptoms and ageusia were more common in non-TB patients. Six patients (3.8%, 6/156) all from the TB group became severe of which five (3.2%, 5/156) required oxygen therapy. One TB patient died (1/104, 0.96%) of lung cancer. After adjustment for potential confounders, the final clinical severity was not different between the two groups (adjusted odds ratio 1.21, 95% confidence interval 0.45–3.28). Conclusions: TB was not associated with severe outcomes in the two TB sanatoria. The high uptake of COVID-19 vaccination and active screening could have impacted on disease progression and prevented unfavorable outcomes.

Evaluation of the IP-10 mRNA release assay for diagnosis of TB in HIV-infected individuals.

HIV-infected individuals are susceptible to Mycobacterium tuberculosis (M.tb) infection and are at high risk of developing active tuberculosis (TB). Interferon-gamma release assays (IGRAs) are auxiliary tools in the diagnosis of TB. However, the performance of IGRAs in HIV-infected individuals is suboptimal, which limits clinical application. Interferon-inducible protein 10 (IP-10) is an alternative biomarker for identifying M.tb infection due to its high expression after stimulation with M.tb antigens. However, whether IP-10 mRNA constitutes a target for the diagnosis of TB in HIV-infected individuals is unknown. Thus, we prospectively enrolled HIV-infected patients with suspected active TB from five hospitals between May 2021 and May 2022, and performed the IGRA test (QFT-GIT) alongside the IP-10 mRNA release assay on peripheral blood. Of the 216 participants, 152 TB patients and 48 non-TB patients with a conclusive diagnosis were included in the final analysis. The number of indeterminate results of IP-10 mRNA release assay (13/200, 6.5%) was significantly lower than that of the QFT-GIT test (42/200, 21.0%) (P = 0.000026). IP-10 mRNA release assay had a sensitivity of 65.3% (95%CI 55.9% - 73.8%) and a specificity of 74.2% (95%CI 55.4% - 88.1%), respectively; while the QFT-GIT test had a sensitivity of 43.2% (95%CI 34.1% - 52.7%) and a specificity of 87.1% (95%CI 70.2% - 96.4%), respectively. The sensitivity of the IP-10 mRNA release assay was significantly higher than that of QFT-GIT test (P = 0.00062), while no significant difference was detected between the specificities of these two tests (P = 0.198). The IP-10 mRNA release assay showed a lower dependence on CD4+ T cells than that of QFT-GIT test. This was evidenced by the fact that the QFT-GIT test had a higher number of indeterminate results and a lower sensitivity when the CD4+ T cells counts were decreased (P < 0.05), while no significant difference in the number of indeterminate results and sensitivity were observed for the IP-10 mRNA release assay among HIV-infected individuals with varied CD4+T cells counts (P > 0.05). Therefore, our study suggested that M.tb specific IP-10 mRNA is a better biomarker for diagnosis of TB in HIV-infected individuals.