Characteristics of TB cases without documented sputum culture in the United States, 2011-2021.

Culture-based diagnostics are the gold standard for diagnosing pulmonary TB (PTB). We characterized culture practices by comparing cases with documented sputum culture to those without.Using multivariable logistic regression, we examined associations between PTB case characteristics and no documented sputum culture reported to the U.S. National TB Surveillance System during 2011-2021.Among 69,538 PTB cases analyzed, no sputum culture attempt was documented for 5,869 (8%). Non-sputum culture specimens were documented for 54%, 80%, and 89% of cases without documented sputum culture attempts among persons aged <15 years, 15-64, and 65+ years, respectively; bronchial fluid and lung tissue were common non-sputum specimens among cases in persons >15 years old. Having no documented sputum culture was associated with age <15 years (aOR 23.84, 99% CI 20.09-28.27) or ≥65 years (aOR 1.22, 99% CI 1.07-1.39), culture of a non-sputum specimen (aOR 6.57, 99% CI 5.93-7.28), residence in a long-term care facility (aOR 1.58, 99% CI 1.23-2.01), and receiving TB care outside of a health department (aOR 1.79, 99% CI 1.61-1.98).Inability to obtain sputum from children and higher diagnostic suspicion for disease processes that require tissue-based diagnostics could explain these findings..

Community Voices and Whole-Genome Sequencing for Tuberculosis: Storytelling and the Importance of Listening.

One of the primary public health functions of a tuberculosis (TB) program is to arrest the spread of infection. Traditionally, TB programs have relied on epidemiological information, gathered through contact tracing, to infer that transmission has occurred between people. The ability of drawing such inferences is extensively context dependent. Where epidemiological information has been strong, such as 2 cases of TB occurring sequentially within a single household, confidence in such inferences is high; conversely, public health authorities have been less certain about the significance of TB cases merely occurring in the same wider social group or geographic area. Many current laboratory tests for TB used globally may be sufficient to confirm a diagnosis and guide appropriate therapy but still be insufficiently precise for distinguishing two strains reliably. In short, drawing inferences regarding a chain of transmissions has always been as much art as science.

Miniaturized Rapid Electrochemical Immunosensor Based on Screen Printed Carbon Electrodes for Mycobacterium tuberculosis Detection.

In 2019, over 21% of an estimated 10 million new tuberculosis (TB) patients were either not diagnosed at all or diagnosed without being reported to public health authorities. It is therefore critical to develop newer and more rapid and effective point-of-care diagnostic tools to combat the global TB epidemic. PCR-based diagnostic methods such as Xpert MTB/RIF are quicker than conventional techniques, but their applicability is restricted by the need for specialized laboratory equipment and the substantial cost of scaling-up in low- and middle-income countries where the burden of TB is high. Meanwhile, loop-mediated isothermal amplification (LAMP) amplifies nucleic acids under isothermal conditions with a high efficiency, helps in the early detection and identification of infectious diseases, and can be performed without the need for sophisticated thermocycling equipment. In the present study, the LAMP assay was integrated with screen-printed carbon electrodes and a commercial potentiostat for real time cyclic voltammetry analysis (named as the LAMP-Electrochemical (EC) assay). The LAMP-EC assay was found to be highly specific to TB-causing bacteria and capable of detecting even a single copy of the Mycobacterium tuberculosis (Mtb) IS6110 DNA sequence. Overall, the LAMP-EC test developed and evaluated in the present study shows promise to become a cost-effective tool for rapid and effective diagnosis of TB.

Aplicación de PCR a partir de cultivo en la identificación de subespecies del complejo Mycobacterium Tuberculosis / The use of culture-based PCR for the identification of subspecies of the mycobacterium tuberculosis complex

Los métodos diagnósticos clásicos de tuberculosis (TB) se basan en la utilización de baciloscopía y cultivo. La identificación del agente etiológico desde la positivización del cultivo requiere entre 10 y 15 días, mientras que el empleo de la reacción en cadena de la polimerasa (PCR) disminuye el tiempo a 24 h, lo que permite no solo identificar las subespecies del complejo Mycobacterium tuberculosis (CMTB) sino también diferenciarlas de otras especies ambientales clínicamente importantes (MOTT) facilitando el diagnóstico y tratamiento. El objetivo del presente trabajo fue determinar la utilidad de la PCR en la identificación temprana de las micobacterias pertenecientes al CMTB, a partir de cultivos positivos, de pacientes con sospecha de TB, atendidos en un hospital pediátrico de alta complejidad, durante un período de cuatro años. A cada muestra, se le realizó baciloscopía y cultivo en medio líquido. A los cultivos positivos, una inmunocromatografía lateral (TBIDR) y luego PCR. El 4,6% del total de muestras (510/11.162) pertenecientes a 198 pacientes presentó cultivos positivos. Cuatrocientos veintiseis (84%) correspondieron a muestras respiratorias. El rendimiento de la baciloscopía directa fue del 41% (194/470). Cuatrocientos treinta y ocho (86%) resultaron M. tuberculosis, 21 (4%) Mycobacterium bovis, 7 (1%), M. bovis-BCG y 44 (9%) MOTT. La utilización de medios de cultivos líquidos junto con el empleo de PCR favorecen una rápida orientación microbiológica y constituye una estrategia útil para optimizar el manejo clínico de estas infecciones, desde el punto de vista terapéutico y epidemiológico, especialmente en pediatría (AU)

Classical diagnostic methods for tuberculosis (TB) are based on the use of smear microscopy and culture. The identification of the etiological agent from positive culture requires 10 to 15 days, while the use of the polymerase chain reaction (PCR) reduces the time to 24 h, which allows not only to identify the subspecies of the Mycobacterium tuberculosis complex (MTC) but also to differentiate them from clinically important environmental mycobacteria other than tuberculosis (MOTT), facilitating diagnosis and treatment. The aim of this study was to determine the usefulness of PCR in the early identification of mycobacteria belonging to the MTC, from positive cultures of patients with suspected TB seen in a pediatric tertiary hospital over a 4-year period. For each sample, smear microscopy and culture in liquid medium was performed. Positive cultures were subjected to lateral immunochromatography (TBIDR) and then PCR. Of the total number of samples (510/11,162) belonging to 198 patients, 4.6% showed positive cultures; 426 (84%) were respiratory samples. The direct smear microscopy yield was 41% (194/470). Overall, 438 (86%) were found to be M. tuberculosis, 21 (4%) Mycobacterium bovis, 7 (1%), M. bovis-BCG, and 44 (9%) MOTT. The use of liquid culture media together with the use of PCR favors a rapid microbiological orientation and is a useful strategy to optimize the clinical management of these infections, from a therapeutic and epidemiological point of view, especially in children (AU)

Novel Mutations in MPT64 Secretory Protein of Mycobacterium tuberculosis Complex.

Tuberculosis (TB) is a global health problem caused by the Mycobacterium tuberculosis complex (MTBC). These bacteria secrete various proteins involved in the pathogenesis and persistence of MTBC. Among the secretory proteins, MPT64 (Rv1980C) is highly conserved and is also known as a major culture filtrate that is used in rapid diagnosis of MTBC. In the current study, we aimed to find the mutation in this highly conserved protein in isolates from the Pashtun-dominant province of Pakistan. We analyzed 470 M. tuberculosis whole-genome sequences of Khyber Pakhtunkhwa Province. Mutations in the MPT64 gene were screened through TB-Profiler and BioEdit software tools. The DynaMut web server was used to analyze the impact of the mutation on protein dynamics and stability. Among 470 MTB genomes, three non-synonymous mutations were detected in nine isolates, and one synonymous mutation (G208A) was found in four isolates. Mutation G211T (F159L), which was detected at the C-terminal domain of the protein in six isolates, was the most prominent. The second novel mutation, T480C (I70V), was detected in two isolates at the C-terminal side of the protein structure. The third novel mutation, A491C (L66R), was detected in a single isolate at the N-terminal side of the MPT64 protein. The effect of these three mutations was destabilizing on the protein structure. The molecular flexibility of the first two mutations increased, and the last one decreased. MPT64 is a highly conserved secretory protein, harboring only a few mutations. This study provides useful information for better managing the diagnosis of MTB isolates in high TB-burden countries.

GenoType MTBDRsl for detection of second-line drugs and ethambutol resistance in multidrug-resistant Mycobacterium tuberculosis isolates at a high-throughput laboratory.

We assessed the performance of MTBDRsl for detection of resistance to fluoroquinolones, aminoglycosides/cyclic peptides, and ethambutol compared to BACTEC MGIT 960 by subjecting simultaneously to both tests 385 phenotypically multidrug-resistant-Mycobacterium tuberculosis isolates from Sao Paulo, Brazil. Discordances were resolved by Sanger sequencing. MTBDRsl correctly detected 99.7% of the multidrug-resistant isolates, 87.8% of the pre-XDR, and 73.9% of the XDR. The assay showed sensitivity of 86.4%, 100%, 85.2% and 76.4% for fluoroquinolones, amikacin/kanamycin, capreomycin and ethambutol, respectively. Specificity was 100% for fluoroquinolones and aminoglycosides/cyclic peptides, and 93.6% for ethambutol. Most fluoroquinolone-discordances were due to mutations in genome regions not targeted by the MTBDRsl v. 1.0: gyrA_H70R and gyrB_R446C, D461N, D449V, and N488D. Capreomycin-resistant isolates with wild-type rrs results on MTBDRsl presented tlyA mutations. MTBDRsl presented good performance for detecting resistance to second-line drugs and ethambutol in clinical isolates. In our setting, multidrug-resistant. isolates presented mutations not targeted by the molecular assay.

Tuberculosis treatment failure associated with evolution of antibiotic resilience.

The widespread use of antibiotics has placed bacterial pathogens under intense pressure to evolve new survival mechanisms. Genomic analysis of 51,229 Mycobacterium tuberculosis (Mtb)clinical isolates has identified an essential transcriptional regulator, Rv1830, herein called resR for resilience regulator, as a frequent target of positive (adaptive) selection. resR mutants do not show canonical drug resistance or drug tolerance but instead shorten the post-antibiotic effect, meaning that they enable Mtb to resume growth after drug exposure substantially faster than wild-type strains. We refer to this phenotype as antibiotic resilience. ResR acts in a regulatory cascade with other transcription factors controlling cell growth and division, which are also under positive selection in clinical isolates of Mtb. Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.

Pericarditis purulenta por mycobacterium tuberculosis y streptococcus pneumoniae: revisión del tema, a propósito de un caso / Purulent pericarditis due to mycobacterium tuberculosis and streptococcus pneumoniae: case report and review

La pericarditis purulenta es una patología poco frecuente pero que conlleva alta mortalidad. En la era pre antibióticos, se observaba en pacientes con neumonía complicada y las cocáceas gram positivas eran los gérmenes frecuentemente involucrados. Por otro lado, la pericarditis tuberculosa representa el 1% del total de casos de tuberculosis, aunque es frecuente zonas endémicas, principalmente asociada a la infección por el virus de la inmunodeficiencia humana (VIH). Presentamos el caso de un paciente de 19 años, en situación calle, infectado con VIH, con diagnóstico de pericarditis purulenta, donde se demostró la co-infección de Mycobacterium tuberculosis y Streptecoccus pneumoniae en el pericardio. La pericarditis purulenta polimicrobiana es poco frecuente y la co-infección por los gérmenes mencionados es anecdótica. A pesar del tratamiento antimicrobiano, el aseo quirúrgico, los esteroides y la fibrinolisis intrapericárdica, esta patología tiene un pronóstico ominoso, en parte, debido a la condición basal de los enfermos que la padecen.

Purulent pericarditis is a rare disease with a high mortality rate. In the pre-antibiotic era it was observed as a complication in patients with pneumonia. Gram-positive coccaceae were the most commonly implicated bacteria. Tuberculous pericarditis represents 1% of all tuberculosis (TBC) cases, although it is common in endemic areas, associated with human immunodeficiency virus (HIV) infection. We present the case of a 19-year-old homeless, admitted with HIV and malnutrition, diagnosed with purulent pericarditis. Mycobacterium tuberculosis and Streptococcus pneumoniae were found as a cause of purulent pericarditis. Polymicrobial purulent pericarditis is a rare condition and co-infection with the bacteria previously mentioned is merely anecdotal. Despite antimicrobial treatment, surgical management, steroids, and intrapericardial fibrinolysis, this pathology has an ominous prognosis, due in part to the pre-existing condition of these patients.

Optimization of nitrofuranyl calanolides for the fluorescent detection of Mycobacterium tuberculosis.

Tuberculosis is a chronic lethal infectious disease caused by a single pathogen, Mycobacterium tuberculosis (Mtb). Previous studies developed nitrofuranyl calanolide (NFC) compounds as new class of Mtb diagnostic reagent, such as NFC-Tre-5. Through structure-fluorescence relationships (SFR) investigation, in this article, a new fluorescent probe, 3-vinylcoumarin (17a) with a 20-fold increase in the fluorescent fold change (FFC) value, was gained. Taking the advantage of specific trehalose metabolic mechanism of Mtb, 17a was further cooperated with a trehalose motif through modification of 4-propyl group to obtain 17a-Tre. The 17a-Tre could label the live infected mycobacteria in signal murine macrophage such as M. smeg, specifically mark the living Mtb in single cell from other typical species of bacteria, and detect the Mtb cells under microscope from the sputum of patients.

El dilema en los laboratorios de Koch, ¿Cultivar o no Cultivar? / The Dilemma in Koch's Laboratories, To Cultivate or Not to Cultivate?

La nueva norma técnica para el control y la eliminación de la tuberculosis es un gran avance para el diagnóstico de este microorganismo en Chile. Actualmente la principal técnica microbiológica para el diagnóstico de laboratorio es la biología molecular, que reduce el tiempo del resultado a tan solo un par de horas. La normativa actual indica que en el paciente caso presuntivo de tuberculosis (CPT) la técnica exclusiva a realizar es Biología molecular. La literatura indica que la detección a través de amplificación de material genético de la micobacteria tiene un límite de detección de 15,6 UFC/ ml, por tanto, todas las muestras bajo ese límite umbral potencialmente podrían no ser diagnosticadas bajo esta estructura emanada por el ministerio de Salud en Chile. Nuestra recomendación es continuar con el estudio de cultivo en medios líquidos o sólidos para todas las muestras hasta obtener literatura que avale lo contrario

The new technical standard for the control and elimination of tuberculosis in Chile is a great advance for the diagnosis of this microorganism. Currently the main microbiological technique for laboratory diagnosis is PCR, which reduces the time to result to just a couple of hours. The current regulations indicate that in the patient with a presumptive case of tuberculosis (CPT) t he exclusive technique to be performed is PCR. The literature indicates that the detection through amplification of genetic material of the mycobacterium has a detection limit of 15.6 CFU/ml, therefore, all samples under this threshold limit could potentially not be diagnosed under this structure emanated by the Ministry of Health in Chile. Our recommendation is to continue with the study of culture in liquid or solid media for all samples until literature confirms otherwise

Estimation of tuberculosis incidence at subnational level using three methods to monitor progress towards ending TB in India, 2015-2020.

OBJECTIVES: We verified subnational (state/union territory (UT)/district) claims of achievements in reducing tuberculosis (TB) incidence in 2020 compared with 2015, in India. DESIGN: A community-based survey, analysis of programme data and anti-TB drug sales and utilisation data. SETTING: National TB Elimination Program and private TB treatment settings in 73 districts that had filed a claim to the Central TB Division of India for progress towards TB-free status. PARTICIPANTS: Each district was divided into survey units (SU) and one village/ward was randomly selected from each SU. All household members in the selected village were interviewed. Sputum from participants with a history of anti-TB therapy (ATT), those currently experiencing chest symptoms or on ATT were tested using Xpert/Rif/TrueNat. The survey continued until 30 Mycobacterium tuberculosis cases were identified in a district. OUTCOME MEASURES: We calculated a direct estimate of TB incidence based on incident cases identified in the survey. We calculated an under-reporting factor by matching these cases within the TB notification system. The TB notification adjusted for this factor was the estimate by the indirect method. We also calculated TB incidence from drug sale data in the private sector and drug utilisation data in the public sector. We compared the three estimates of TB incidence in 2020 with TB incidence in 2015. RESULTS: The estimated direct incidence ranged from 19 (Purba Medinipur, West Bengal) to 1457 (Jaintia Hills, Meghalaya) per 100 000 population. Indirect estimates of incidence ranged between 19 (Diu, Dadra and Nagar Haveli) and 788 (Dumka, Jharkhand) per 100 000 population. The incidence using drug sale data ranged from 19 per 100 000 population in Diu, Dadra and Nagar Haveli to 651 per 100 000 population in Centenary, Maharashtra. CONCLUSION: TB incidence in 1 state, 2 UTs and 35 districts had declined by at least 20% since 2015. Two districts in India were declared TB free in 2020.

Novel rrs mutations in second-line injectable drug-resistant clinical isolates of Mycobacterium tuberculosis from the Punjab province of Pakistan.

INTRODUCTION: Phenotypic drug susceptibility testing is the most common approach to assess drug-resistant isolates; however, molecular methods of drug susceptibility testing are fast, accurate hence, offer less time for transmission during the diagnosis period. As data on the molecular methods regarding injectable drug resistance in the Punjab province of Pakistan is limited, therefore in this study, we aimed to analyze the mutations in the rrs gene behind second-line injectable drug resistance. MATERIAL AND METHODS: Mycobacterium tuberculosis isolates were collected from the sputum of 5362 TB suspects. The strains confirmed for resistant to injectable drugs through drug susceptibility testing were further proceeded. The 1537bp rrs gene was amplified with the help of three sets of primers with overlapping regions and DNA sequencing was performed. Obtained sequences were aligned with reference sequence to find mutations. RFLP-PCR method was also optimized for rapid detection of a common (143bp and 205bp) rrs gene mutation. RESULTS: Among 172 rifampicin resistance isolates, 163(95%) were resistant to both rifampicin and isoniazid, and 9 (5%) were resistant to only rifampicin. Among the resistant samples, 12 (6.9%) samples were resistant to all three injectable drugs. Sixty out of 172 (34.9%) samples showed resistance to at least one drug and 10 (5.8%) samples were resistant to two drugs among the 3 s-line drugs. Sequencing analysis showed novel mutations in different samples at positions 443InsC, 19DelT, 29G>A, 48C>T, 50G>C, 265InsT, 423T>G, 476InsA, 446A>G, 563DelA, 695G>A, 805DelA, 900G>A, and 1510A>G, while some already reported mutations at position 1401A>G, 1402A>G, and 1484G>T were also observed. MIC of novel rrs gene mutations in KAN, CAP, and AMK resistant isolates were found between 2.5 mg/L-3.05 mg/L, 2.08 mg/L-3.0 mg/L, and 2.1 mg/L-2.7 mg/L respectively. CONCLUSION: Novel mutations in the rrs gene reported in this study may confer second-line injectable drugs resistance in Mtb. This molecular insight into second-line injectable drug resistance is useful for better management of resistance Mtb in high burden countries.

Preparedness of tertiary care hospitals to implement the national TB infection prevention and control guidelines in Bangladesh: A qualitative exploration.

In high tuberculosis (TB) burden countries, health settings, including non-designated TB hospitals, host many patients with pulmonary TB. Bangladesh's National TB Control Program aims to strengthen TB infection prevention and control (IPC) in health settings. However, there has been no published literature to date that assessed the preparedness of hospitals to comply with the recommendations. To address this gap, our study examined healthcare workers knowledge and attitudes towards TB IPC guidelines and their perceptions regarding the hospitals' preparedness in Bangladesh. Between January to December 2019, we conducted 16 key-informant interviews and four focus group discussions with healthcare workers from two public tertiary care hospitals. In addition, we undertook a review of 13 documents [i.e., hospital policy, annual report, staff list, published manuscript]. Our findings showed that healthcare workers acknowledged the TB risk and were willing to implement the TB IPC measures but identified key barriers impacting implementation. Gaps were identified in: policy (no TB policy or guidelines in the hospital), health systems (healthcare workers were unaware of the guidelines, lack of TB IPC program, training and education, absence of healthcare-associated TB infection surveillance, low priority of TB IPC, no TB IPC monitoring and feedback, high patient load and bed occupancy, and limited supply of IPC resources) and behavioural factors (risk perception, compliance, and self and social stigma). The additional service-level gap was the lack of electronic medical record systems. These findings highlighted that while there is a demand amongst healthcare workers to implement TB IPC measures, the public tertiary care hospitals have got key issues to address. Therefore, the National TB Control Program may consider these gaps, provide TB IPC guidelines to these hospitals, assist them in developing hospital-level IPC manual, provide training, and coordinate with the ministry of health to allocate separate budget, staffing, and IPC resources to implement the control measures successfully.

Combination of Xpert® MTB/RIF and DetermineTM TB-LAM Ag improves the diagnosis of extrapulmonary tuberculosis at Jimma University Medical Center, Oromia, Ethiopia.

BACKGROUND: Ethiopia is one of the high burden countries for extrapulmonary tuberculosis (EPTB); however, the prompt diagnosis of EPTB remains challenging. This study is aimed to evaluate the diagnostic performance of Xpert MTB/RIF and DetermineTM TB-LAM Ag (TB-LAM) for the prompt diagnosis of EPTB in Ethiopia. METHODS: A total of 147 presumptive EPTB patients, including 23 HIV- positive participants were enrolled. Extra-pulmonary samples were collected from all presumptive EPTB cases and tested for Mycobacterium tuberculosis complex (MTBC) using fluorescent microscopy, Xpert MTB/RIF, and culture. Additionally, urine samples were also collected from 126 participants and were tested by DetermineTM TB-LAM Ag (Alere Inc, Waltham, USA). The Sensitivity and specificity of Xpert and TB- LAM tests were calculated by comparing with a composite reference standard (CRS), which comprises smear microscopy, culture and response to empirical anti-TB treatment. RESULTS: Of 147 patients, 23 (15.6%) were confirmed EPTB cases (culture-positive), 14 (9.5%) were probable EPTB (clinically, radiologically or cytologically positive and received anti-TB treatment with good response), and 110 (74.8%) were classified as "non- TB" cases. Compared to the composite reference standard (CRS), the overall sensitivity and specificity of Xpert MTB/RIF were 43.2% and 100%, respectively with the highest sensitivity for Lymph node aspirate (85.7%) and lower sensitivity for pleural fluid (14.3%) and 100% specificity for all specimen types. The sensitivity and specificity of TB-LAM were 33.3% and 94.4% respectively with the highest sensitivity for HIV co-infected participants (83.3%). The sensitivity of the combination of Xpert MTB/RIF and TB-LAM tests regardless of HIV status was 61.1% whereas the sensitivity was improved to 83.3% for HIV-positive cases. CONCLUSION: TB-LAM alone has low sensitivity for EPTB diagnosis; however, the combination of TB-LAM and Xpert MTB/RIF improves the diagnosis of EPTB particularly for countries with high EPTB and HIV cases.

Drug-Resistant Characteristics, Genetic Diversity, and Transmission Dynamics of Rifampicin-Resistant Mycobacterium tuberculosis in Hunan, China, Revealed by Whole-Genome Sequencing.

To gain a deep insight into the additional drug-resistant profiles, genetic diversity, and transmission dynamics of rifampicin-resistant tuberculosis (RR-TB) circulating in Hunan province, drug susceptibility testing and whole-genome-sequencing were performed among RR-TB strains collected from Jan. 2013 to Jun. 2018 in Hunan province. A total of 124 RR-TB strains were recovered successfully and included into the final analysis. Lineage 2.2.1 was the dominant sublineage, accounting for 72.6% (90/124), followed by lineage 4.5 (11.3%, 14/124), lineage 4.4 (8.1%, 10/124), lineage 4.2 (6.5%, 8/124) and lineage 2.2.2 (1.6%, 2/124). Overall, 83.1% (103/124) and 3.2% (4/124) of RR-TB were MDR-TB and XDR-TB, respectively. Nearly 30% of RR-TB isolates were resistant to fluoroquinolones, and 26.6% (33/124) were pre-XDR-TB. Moreover, 30.6% (38/124) of RR-TB strains were identified as phenotypically resistance to pyrazinamide. Totally, 17 clusters containing 48 (38.7%, 48/124) RR-TB strains were identified, ranging in size from 2 to 10 isolates. No significant difference was detected in clustering rate between lineage 2 and lineage 4 (χ2 = 0.027, P = 0.870). Our study revealed the complexity of RR-TB strains circulating in Hunan province with complex additional drug-resistant profile and relatively higher clustering rates. Comprehensive information based on WGS should be used to guide the design of treatment regimens and tailor public interventions. IMPORTANCE Comprehensive information such as genetic background and drug-resistant profile of MTB strains could help to tailor public interventions. However, these data are limited in Hunan province, one of the provinces with high-TB burden in China. So, this study aimed to provide us with deep insight into the molecular epidemiology of RR-TB isolates circulating in Hunan province by combining phenotypic drug susceptibility testing and whole-genome sequencing. To our knowledge, this is the first study to use whole-genome sequencing data of RR-TB strains spanning more than 5 years for molecular epidemiology analysis in Hunan province, which allows us to identify genetic background information and clustered strains more accurately. Our study revealed the complexity of RR-TB strains circulating in Hunan province with complex additional drug-resistant profile and relatively higher clustering rates. Comprehensive information based on WGS should be used to guide the design of treatment regimens and tailor public interventions.

Accurate and rapid prediction of tuberculosis drug resistance from genome sequence data using traditional machine learning algorithms and CNN.

Effective and timely antibiotic treatment depends on accurate and rapid in silico antimicrobial-resistant (AMR) predictions. Existing statistical rule-based Mycobacterium tuberculosis (MTB) drug resistance prediction methods using bacterial genomic sequencing data often achieve varying results: high accuracy on some antibiotics but relatively low accuracy on others. Traditional machine learning (ML) approaches have been applied to classify drug resistance for MTB and have shown more stable performance. However, there is no study that uses deep learning architecture like Convolutional Neural Network (CNN) on a large and diverse cohort of MTB samples for AMR prediction. We developed 24 binary classifiers of MTB drug resistance status across eight anti-MTB drugs and three different ML algorithms: logistic regression, random forest and 1D CNN using a training dataset of 10,575 MTB isolates collected from 16 countries across six continents, where an extended pan-genome reference was used for detecting genetic features. Our 1D CNN architecture was designed to integrate both sequential and non-sequential features. In terms of F1-scores, 1D CNN models are our best classifiers that are also more accurate and stable than the state-of-the-art rule-based tool Mykrobe predictor (81.1 to 93.8%, 93.7 to 96.2%, 93.1 to 94.8%, 95.9 to 97.2% and 97.1 to 98.2% for ethambutol, rifampicin, pyrazinamide, isoniazid and ofloxacin respectively). We applied filter-based feature selection to find AMR relevant features. All selected variant features are AMR-related ones in CARD database. 78.8% of them are also in the catalogue of MTB mutations that were recently identified as drug resistance-associated ones by WHO. To facilitate ML model development for AMR prediction, we packaged every step into an automated pipeline and shared the source code at https://github.com/KuangXY3/MTB-AMR-classification-CNN .

Treatment outcomes of patients with MDR-TB and its determinants at referral hospitals in Ethiopia.

BACKGROUND: There is limited empirical evidence in Ethiopia on the determinants of treatment outcomes of patients with multidrug-resistant tuberculosis (MDR-TB) who were enrolled to second-line anti-tuberculosis drugs. Thus, this study investigated the determinants of treatment outcomes in patients with MDR-TB at referral hospitals in Ethiopia. DESIGN AND METHODS: This study was underpinned by a cross-sectional quantitative research design that guided both data collection and analysis. Data is collected using structured questionnaire and data analyses was performed using the Statistical Package for Social Sciences. Multi-variable logistic regression was used to control for confounders in determining the association between treatment outcomes of patients with MDR-TB and selected predictor variables, such as co-morbidity with MDR-TB and body mass index. RESULTS: From the total of 136 patients with MDR-TB included in this study, 31% had some co-morbidity with MDR-TB at baseline, and 64% of the patients had a body mass index of less than 18.5 kg/m2. At 24 months after commencing treatment, 76 (69%), n = 110), of the patients had successfully completed treatment, while 30 (27%) died of the disease. The odds of death was significantly higher among patients with low body mass index (AOR = 2.734, 95% CI: 1.01-7.395; P<0.048) and those with some co-morbidity at baseline (AOR = 4.260, 95%CI: 1.607-11.29; p<0.004). CONCLUSION: The higher proportion of mortality among patients treated for MDR-TB at Adama and Nekemte Hospitals, central Ethiopia, is attributable to co-morbidities with MDR-TB, including HIV/AIDS and malnutrition. Improving socio-economic and nutritional support and provision of integrated care for MDR-TB and HIV/AIDS is recommended to mitigate the higher level of death among patients treated for MDR-TB.

Detection of Mycobacterium tuberculosis multiple strains in sputum samples from patients with pulmonary tuberculosis in south western Uganda using MIRU-VNTR.

Infections with multiple strains of Mycobacterium tuberculosis are now widely recognized as a common occurrence. Identification of patients infected with multiple strains provides both insight into the disease dynamics and the epidemiology of tuberculosis. Analysis of Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeats (MIRU-VNTR) has been shown to be highly sensitive in detecting multiple M. tuberculosis strains even in sputum. The goal of this study was to identify cases of multiple M. tuberculosis strain infections among patients diagnosed with pulmonary tuberculosis in Southwestern Uganda and assessment of factors associated with multiple strain infections. DNA extracted directly from 78 sputum samples, each from an individual patient, was analyzed using the standard 24 loci MIRU-VNTR typing. Five (6.4%) of the 78 patients were infected with multiple strains of M. tuberculosis with all of them being the newly diagnosed cases while two-thirds of them were co-infected with HIV. Exact regression analysis projected that the natives were more likely to harbor multiple strains (OR; 0.981, 95% CI 0-7.926) as well as those with a high microbial load (OR; 0.390, 95% CI 0-3.8167). Despite these findings being not statistically significant due to the small sample size, this points to a critical component of disease dynamics that has clinical implications and emphasizes a need for a study using a larger cohort. It is also essential to study the potential factors associated with higher risk of exposure to newly diagnosed and HIV positive patients at the community level. In addition, our ability to detect multiple M. tuberculosis strains using the standard 24 loci MIRU-VNTR typing especially with allelic diversity in loci 2059 and 3171, which are excluded from the 15-locus MIRU-VNTR, lead us to recommend the use of this genotyping technique, especially in areas with tuberculosis endemicity similar to this study.