Disseminated mycobacterium genavense infection with central nervous system involvement in an HIV patient: a case report and literature review.

BACKGROUND: Immunodeficient patients, particularly HIV patients, are at risk of opportunistic infections. Nontuberculous mycobacteria can cause severe complications in immunodeficient patients. CASE PRESENTATION: We describe a 57-year-old HIV patient, primarily presented with coughs and constitutional symptoms, with a unique Mycobacterium genavense abdominal, pulmonary, and central nervous system infection, accompanied by intracranial masses. CONCLUSION: The diagnosis of NTM, including M. genavense, must always be considered by clinicians in immunodeficient patients, especially those with HIV, who have a compromised immune system.

Implementation fidelity of tuberculosis preventive therapy for under five children exposed to sputum smear positive pulmonary tuberculosis in Kaski district, Nepal: An implementation research.

INTRODUCTION: In line with the WHO recommendation, Nepal has started implementing Tuberculosis prevention therapy (TBPT) for under five children exposed to Sputum Smear Positive Pulmonary Tuberculosis, as one of the strategies for prevention, care and control of TB. Implementation fidelity study is important to assess on what extent preventive program is being implemented. The objective of the study measured the implementation fidelity of TBPT program Kaski district, Nepal. METHODS: We used a mixed-method explanatory sequential design study. Quantitative data were collected through retrospective review of records from April 2018 to May 2019 and level of adherence was established. Moderating factors influencing the implementation of TBPT were TBPT were assessed qualitatively. Sixteen in-depth interviews and a focus group discussion was conducted purposively with responsible stakeholders. The study was guided by the Conceptual Framework for Implementation Fidelity (CFIR) developed by Carroll. RESULTS: The majority of the components of the TBPT program were found to be implemented with a moderate level of fidelity. The proportion of under five years children initiate and complete the full course of TBPT was 72.5% and 75.86% respectively. The proportion of index cases traced for household contact, contact tracing within two months and timely initiation of therapy within two months were 54.19%, 82.73% and 86.20%. Moderating factors identified in the implementation of the program were contact tracing and enrollment, partnership and ownership, training resources, medication, awareness and information dissemination. CONCLUSION: The TBPT program is being moderately implemented in Kaski districts. Addressing the key challenges identified in contact tracing, partnership/ownership, incentives, training and knowledge of health workers results in more identification of children eligible for TBPT.

Reasons for loss to follow-up (LTFU) of pulmonary TB (PTB) patients: A qualitative study among Saharia, a particularly vulnerable tribal group of Madhya Pradesh, India.

BACKGROUND: Loss to follow-up (LTFU) among pulmonary tuberculosis (PTB) patients is a significant challenge for TB control. However, there is a dearth of information about the factors leading to LTFU among marginalized communities. This study highlights the factors associated with LTFU in Saharia, a tribe of Madhya Pradesh having high tuberculosis (TB) prevalence. METHODS: A qualitative study was carried out during January-April 2020 among twenty-two pulmonary TB patients, recorded as LTFU in NIKSHAY, with ten treatment supporters and ten patient's family members. Semi-structured personal interview tools were used to collect the information on the history of anti-tuberculosis treatment, adverse drug events (ADE), social cognitive, behaviors, myths, and misbeliefs. The interviews were transcribed and thematically analysed to examine underlying themes. RESULTS: The study explored various social, behavioral factors leading to loss to follow-up among PTB patients. Drug side effects, alcoholism, social stigma, lack of awareness of the seriousness of the diseases and poor counseling are the main barriers to treatment adherence in this community. CONCLUSIONS: The study highlights the need to address the issues related to LTFU during TB treatment. The enhanced efforts of treatment supporters, health staff, and family & community persons must motivate and support the patients.

A simple assay to quantify mycobacterial lipid antigen-specific T cell receptors in human tissues and blood.

T cell receptors (TCRs) encode the history of antigenic challenge within an individual and have the potential to serve as molecular markers of infection. In addition to peptide antigens bound to highly polymorphic MHC molecules, T cells have also evolved to recognize bacterial lipids when bound to non-polymorphic CD1 molecules. One such subset, germline-encoded, mycolyl lipid-reactive (GEM) T cells, recognizes mycobacterial cell wall lipids and expresses a conserved TCR-ɑ chain that is shared among genetically unrelated individuals. We developed a quantitative PCR assay to determine expression of the GEM TCR-ɑ nucleotide sequence in human tissues and blood. This assay was validated on plasmids and T cell lines. We tested blood samples from South African subjects with or without tuberculin reactivity or with active tuberculosis disease. We were able to detect GEM TCR-ɑ above the limit of detection in 92% of donors but found no difference in GEM TCR-ɑ expression among the three groups after normalizing for total TCR-ɑ expression. In a cohort of leprosy patients from Nepal, we successfully detected GEM TCR-ɑ in 100% of skin biopsies with histologically confirmed tuberculoid and lepromatous leprosy. Thus, GEM T cells constitute part of the T cell repertoire in the skin. However, GEM TCR-ɑ expression was not different between leprosy patients and control subjects after normalization. Further, these results reveal the feasibility of developing a simple, field deployable molecular diagnostic based on mycobacterial lipid antigen-specific TCR sequences that are readily detectable in human tissues and blood independent of genetic background.

Case report of the first cured patient with Mycobacterium Chimaera infection following cardiac valve replacement in the mainland of China.

BACKGROUND: Mycobacterium chimaera infections subsequent to cardiac surgery are related to contaminated heater-cooler devices, with high mortality. Nevertheless, few studies have been reported in Asia. CASE PRESENTATION: We described the case of a 55-year-old man with Mycobacterium chimaera infection following cardiac surgery in the mainland of China. He was diagnosed with endocarditis caused by Mycobacterium chimaera subsequent to open heart surgery. Metagenomic next-generation sequencing (mNGS) and 16S rRNA gene PCR analysis were used to identify potential pathogens. The patient underwent redo valve replacement surgery and received combination therapy with azithromycin, ethambutol, linezolid, and amikacin. No signs of relapse were observed during the 11-month follow-up visit. CONCLUSIONS: This is the first documented case of Mycobacterium chimaera infection following cardiac surgery in the mainland of China and the first documented transnational imported case worldwide. Moreover, mNGS is a novel diagnostic technology that can guide antimicrobial therapy prior to obtaining fluid/tissue culture results for Mycobacterium chimaera, providing a new approach for the detection of potential Mycobacterium chimaera infection.

Bacteriological and molecular characterization of Mycobacterium bovis isolates from tuberculous lesions collected among slaughtered cattle, Northwest Ethiopia.

BACKGROUND: In Ethiopia, the distribution of bovine tuberculosis (BTB) has long been known and documented as a major problem of animal health. However, the burden of circulating M. bovis strains is poorly understood in the country. Therefore; this study aimed to identify and characterize the mycobacterial isolates responsible for BTB in Northwest Ethiopia. METHODS: A cross-sectional study was conducted on tuberculous lesions that had been collected from slaughtered cattle between September 2018 to June 2019. Collected lesions were cultured and tested for tuberculous bacilli. The MPT64 assay and Genotype line probe assay (LPA) were used for identification of mycobacterial isolates, and region of deletion 4 (RD4) typing and spoligotyping were used to characterize the M. bovis strains. RESULTS: Of the total 1458 examined slaughtered cattle, only 62 (4.3, 95%CI; 0.0328-0.0542) had tuberculous lesions. The highest number of gross tuberculous lesions were observed from the lymph nodes of the thoracic cavity; at the mediastinal (40.3%, 25/62) and bronchial (22.6%, 14/62) lymph nodes. Of the 62 collected tuberculous lesions; 18 (29.0%) were culture positive for mycobacterium isolates, and only five isolates were confirmed for M. tuberculosis complex (MTBc) by the MPT64 assay and LPA. All the five MTBc isolates were positive for RD4 typing of M. bovis with a PCR product size of 446 bp, and no isolate was noticed to have M. tuberculosis. The detected M. bovis strains displayed five spoligotypes; with the common SB1176 and SB0133 M. bovis strains, although the two spoligotypes had not been previously reported. CONCLUSION: The present study shows that BTB in North Gondar, Ethiopia, is caused by M. bovis strains SB1176 and SB0033, with low frequency. Thus, the finding highlights the importance of continuous surveillance for mycobacterial strains in cattle populations.

Management of patients with pulmonary mycobacteriosis in France: a multicenter retrospective cohort study.

BACKGROUND: Recent studies report very low adherence of practitioners to ATS/IDSA recommendations for the treatment of nontuberculous mycobacteria pulmonary disease (NTM-PD), as well as a great variability of practices. Type of management could impact prognosis. METHODS: To evaluate management and prognosis of patients with NTM-PD cases with respect to ATS recommendations, we conducted a multicenter retrospective cohort study (18 sentinel sites distributed throughout France), over a period of six years. We collected clinical, radiological, microbiological characteristics, management and outcome of the patients (especially death or not). RESULTS: 477 patients with NTM-PD were included. Respiratory comorbidities were found in 68% of cases, tuberculosis sequelae in 31.4% of patients, and immunosuppression in 16.8% of cases. The three most common NTM species were Mycobacterium avium complex (60%), M. xenopi (20%) and M. kansasii (5.7%). Smear-positive was found in one third of NTM-PD. Nodulobronchiectatic forms were observed in 54.3% of cases, and cavitary forms in 19.1% of patients. Sixty-three percent of patients were treated, 72.4% of patients with smear-positive samples, and 57.5% of patients with smear-negative samples. Treatment was in adequacy with ATS guidelines in 73.5%. The 2-year mortality was 14.4%. In the Cox regression, treatment (HR = 0.51), age (HR = 1.02), and M. abscessus (3.19) appeared as the 3 significant independent prognostic factors. CONCLUSION: These findings highlight the adequacy between French practices and the ATS/IDSA guidelines. Treatment was associated with a better survival.

LAMP-CRISPR-Cas12-based diagnostic platform for detection of Mycobacterium tuberculosis complex using real-time fluorescence or lateral flow test.

A CRISPR-based nucleic acid detection platform, termed LACD (loop-mediated isothermal amplification coupled with CRISPR-Cas12a-mediated diagnostic) has been developed. In the LACD system, the core primer used in conventional LAMP (forward inner primer or backward inner primer) was engineered to contain a PAM (protospacer adjacent motif) site (TTTT) at the linker region. As a result, the LAMP amplicons contained a specific PAM site for CRISPR-Cas12a recognition. At the CRISPR-mediated detection stage, the resulting LAMP products can activate the corresponding CRISPR-Cas12a effector upon the formation of the CRISPR-Cas12a/gRNA/target DNA complex. The single-strand DNA (ssDNA) reporter molecules are then rapidly cleaved due to the CRISPR-Cas12a's trans-enzyme activity. The ssDNA degradation can then be visualized on a lateral flow biosensor or measured  by a real-time fluorescence instrument. Our LACD assay allows any target sequence to be detected (even targets which do not contain any PAM sites) as long as they met the design requirement for LAMP. The feasibility of the LACD methodology for nucleic acid detection was validated on the Mycobacterium tuberculosis complex (MTC). This proof-of-concept assay can be reconfigured to detect a variety of target sequences by redesigning the engineered LAMP primers.

Validation, Implementation, and Clinical Utility of Whole Genome Sequence-Based Bacterial Identification in the Clinical Microbiology Laboratory.

The application of next-generation sequencing extends from microbial identification to epidemiologic insight and antimicrobial resistance prediction. Despite this potential, the roadblock for clinical laboratories lies in implementation and validation of such complex technology and data analysis. Herein, a validation study used whole-genome sequencing (WGS) for pan-bacterial identification (ID) in a clinical laboratory, and assessed its clinical relevance. A diverse set of 125 bacterial isolates, including a subset without genus (25) and/or species (10) ID, were analyzed by de novo assembly and reference genome mapping. The 16S rRNA, rpoB, and groEL genes were used for ID. Using WGS, 100% (89 of 89) and 89% (79 of 89) of isolates were identified at the genus and species-levels, respectively. WGS also provided improved results for 71% (25/35) isolates originally reported with genus-only or descriptive IDs. Chart review identified cases in which improved genus and/or species-level ID by WGS may have had a positive impact on patient care. Reasons included the use of an ineffective antibiotic owing to unclear ID, use of antibiotics when not clinically indicated, and help with an outbreak investigation. The implementation of next-generation sequencing in a clinical microbiology setting is a challenging but necessary task. This study provides a model for the validation and implementation of bacterial ID by WGS in such a setting.

Emerging MDR-Mycobacterium avium subsp. avium in house-reared domestic birds as the first report in Egypt.

BACKGROUND: Avian tuberculosis is a chronic and zoonotic disease that affects a wide variety of birds, mammals, and humans. This study aimed to estimate the frequency of Mycobacterium avium subsp. avium in some domestic birds based on molecular diagnosis, antibiogram profile, and PCR-based detection of inhA, rpoB, rpsL, and otrB antibiotic resistance-related genes. METHODS: A total of 120 fecal samples were collected from small flocks of house-reared domestic birds at Ismailia Governorate, Egypt. The collected samples were processed and subjected to the bacteriological examination. The antimicrobial susceptibility testing of the recovered isolates was performed using the broth microdilution method for the detection of minimum inhibitory concentrations (MICs). The genetic detection of the IS901confirmatory gene, inhA, rpoB, rpsL, and otrB genes was carried out using PCR. RESULTS: The frequency of M. avium subsp. avium was 4.1% (5/120); 10% (4/40) in ducks, and 2.5% (1/10) in geese. The identification of the recovered isolates was confirmed using PCR, where all the tested isolates were positive for IS901confirmatory gene. The results of the broth microdilution method revealed that most of the recovered isolates exhibited multidrug resistance (MDR) to isoniazid, rifampicin, streptomycin, oxytetracycline, and doxycycline, and harbored the inhA, rpoB, rpsL, and otrB genes. CONCLUSION: In brief, to the best of our knowledge this is the first report that emphasized the emergence of avian tuberculosis in house-reared domestic birds in Egypt. The emergence of MDR- M. avium subsp. avium is considered a public health threat. Emerging MDR-M. avium subsp. avium in domestic birds are commonly harbored the IS901, inhA, rpoB, rpsL, and otrB genes. Azithromycin and clofazimine revealed a promising in-vitro antibacterial activity against M. avium subsp. avium.

Culturing Mycobacteria.

Building upon the foundational research of Robert Koch, who demonstrated the ability to grow Mycobacterium tuberculosis for the first time in 1882 using media made of coagulated bovine serum, microbiologists have continued to develop new and more efficient ways to grow mycobacteria. Presently, all known mycobacterial species can be grown in the laboratory using either axenic culture techniques or in vivo passage in laboratory animals. This chapter provides conventional protocols to grow mycobacteria for diagnostic purposes directly from clinical specimens, as well as in research laboratories for scientific purposes. Detailed protocols used for production of M. tuberculosis in large scale (under normoxic and hypoxic conditions) in bioreactors and for production of obligate intracellular pathogens such as Mycobacterium leprae and "Mycobacterium lepromatosis" using athymic nude mice and armadillos are provided.

A Mycobacterium species for Crohn's disease?

In ruminants Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of a chronic granulomatous inflammatory bowel disease called Johne's disease (JD). Some researchers have hypothesised that MAP is also associated with Crohn's disease (CD), an inflammatory bowel disease in humans that shares some histological features of JD. Despite numerous attempts to demonstrate causality by researchers, direct microbiological evidence of MAP involvement in CD remains elusive. Importantly, it has not been possible to reliably and reproducibly demonstrate mycobacteria in the tissue of CD patients. Past attempts to visualise mycobacteria in tissue may have been hampered by the use of stains optimised for Mycobacterium tuberculosis complex (MTB) and the lack of reliable bacteriological culture media for both non-tuberculous mycobacteria (NTM) and cell-wall-deficient mycobacteria (CWDM). Here we describe a Ziehl-Neelsen (ZN) staining method for the demonstration of CWDM in resected tissue from patients with Crohn's disease, revealing the association of CWDM in situ with host tissue reactions, and posit this as a cause of the tissue inflammation. Using the ZN stain described we demonstrated the presence of CWDM in 18 out of 18 excised tissue samples from patients diagnosed as having Crohn's disease, and in zero samples out of 15 non-inflammatory bowel disease controls.

Hematologic values in domestic pigeons naturally infected with Mycobacterium avium subsp. avium.

Avian mycobacteriosis is an important disease of birds and is most often caused by Mycobacterium avium or Mycobacterium genavense. However, little information on the hematologic changes associated with this infectious disease in pigeons has been published. The aim of this investigation was to compare the hematologic parameters of domestic pigeons (Columba livia var. domestica) naturally infected with M avium subsp. avium (MAA) with clinically healthy pigeons. Blood samples were collected from 12 pigeons with suspected mycobacteriosis and 12 clinically healthy pigeons. All the birds with suspect infections were necropsied, and affected organs were cultured and examined on histopathology for mycobacteriosis. Total leukocyte and erythrocyte counts were performed on each blood sample with the Natt and Herrick method using a Neubauer hemocytometer. White blood cell (WBC) differential counts were performed on Giemsa-stained blood smears. Packed cell volumes (PCVs) were measured using the microhematocrit technique. Hemoglobin concentrations were measured with a spectrophotometer using the cyanomethemoglubin method. Mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentrations (MCHCs), and mean cell volumes (MCVs) were calculated manually. All of the infected birds had typical histopathologic findings of avian mycobacteriosis, which were confirmed using microbiologic and molecular methods to detect MAA. The hematologic data from the two groups were compared. The total WBC, heterophil, lymphocyte, and monocyte counts were significantly higher, and the PCV, HGB, MCH, and MCHC values were significantly lower in the infected birds compared with the clinically healthy pigeons.

Ocular tuberculosis in a calf.

BACKGROUND: Bovine tuberculosis is a chronic inflammatory disease that causes granuloma formation mainly in retropharyngeal, tracheobronchial, mediastinal lymph nodes and lungs of bovines. The presence of these lesions in other tissues such as the eyeball is very rare and difficult to diagnose. This study describes macroscopic and microscopic pathological findings in a calf with ocular and meningeal tuberculosis. CASE PRESENTATION: March 2019, an eight-month-old Holstein Friesian calf was identified in a dairy farm located in central Mexico with a clinical cough, anorexia, incoordination, corneal opacity and vision loss. At necropsy, pneumonia, lymphadenitis, meningitis, and granulomatous iridocyclitis were observed. The histopathological examination revealed granulomatous lesions in lung tissue, lymph nodes, meninges and eyes with the presence of acid-fast bacilli associated with Mycobacterium spp. CONCLUSION: To the best of our knowledge, this is the first report that describes macroscopic and microscopic pathological findings of ocular tuberculosis in cattle. This report highlights the importance of considering bovine tuberculosis in the differential diagnosis of corneal opacity and loss of vision in cattle.

Diagnosis of cutaneous mycobacterial spindle cell pseudotumor in the absence of typical inflammatory cells: A case report.

Mycobacterial spindle cell pseudotumor (MSP) is a non-neoplastic condition that is characterized by spindle-shaped histiocytes colonized by mycobacteria. MSP is most commonly diagnosed in the immunocompromised and, while MSP can occur throughout the body, the most common sites of MSP involvement are the lymph nodes and the skin. To diagnose MSP, histopathological analysis typically demonstrates the presence of inflammatory cells, in addition to spindle cells and the unequivocal mycobacteria, which guides the diagnosis away from potential neoplasms. If properly diagnosed and treated with appropriate antibiotic therapy, patients tend to experience almost complete resolution of their symptoms. MSP is a rare condition; to our knowledge, there have only been 11 documented cases of cutaneous MSP, including the one introduced in this report. Here, we present a unique case of a 50-year-old female on chronic immunosuppressive therapy diagnosed with cutaneous MSP in the absence of inflammatory cells on pathology.

Mycobacterium vicinigordonae sp. nov., a slow-growing scotochromogenic species isolated from sputum.

A slow-growing, scotochromogenic mycobacterial strain (24T) was isolated from the sputum of a Chinese male human. Phylogenetic analysis using the 16S rRNA gene assigned strain 24T to the Mycobacterium gordonae complex, which includes Mycobacterium gordonae and Mycobacterium paragordonae. The phenotypic characteristics, unique mycolic acid profile and the results of phylogenetic analysis based on hsp65 and rpoB sequences strongly supported the taxonomic status of strain 24T as a representative of a species distinct from the other members of the M. gordonae complex. The genomic G+C content of strain 24T was 65.40mol%. Genomic comparisons showed that strain 24T and M. gordonae ATCC 14470T had an average nucleotide identity (ANI) value of 81.00 % and a DNA-DNA hybridization (DDH) value of 22.80 %, while the ANI and DDH values between strain 24Tand M. paragordonae 49 061T were 80.98 and 22.80 %, respectively. In terms of phylogenetic, phenotypic and chemotaxonomic features, strain 24T is distinguishable from its closest phylogenetic relatives and represents a novel species of the genus Mycobacterium, therefore the name Mycobacterium vicinigordonae sp. nov. is proposed. The type strain is 24T (=CMCC 93559T=DSM 105979T).

Spatial patterns of pulmonary tuberculosis (TB) cases in Zimbabwe from 2015 to 2018.

INTRODUCTION: Accurate mapping of spatial heterogeneity in tuberculosis (TB) cases is critical for achieving high impact control as well as guide resource allocation in most developing countries. The main aim of this study was to explore the spatial patterns of TB occurrence at district level in Zimbabwe from 2015 to 2018 using GIS and spatial statistics as a preamble to identifying areas with elevated risk for prioritisation of control and intervention measures. METHODS: In this study Getis-Ord Gi* statistics together with SaTscan were used to characterise TB hotspots and clusters in Zimbabwe at district level from 2015 to 2018. GIS software was used to map and visualise the results of cluster analysis. RESULTS: Results show that TB occurrence exhibits spatial heterogeneity across the country. The TB hotspots were detected in the central, western and southern part of the country. These areas are characterised by artisanal mining activities as well as high poverty levels. CONCLUSIONS AND RECOMMENDATIONS: Results of this study are useful to guide TB control programs and design effective strategies which are important in achieving the United Nations Sustainable Development goals (UNSDGs).

Isolation of Mycobacterium talmoniae from a patient with diffuse panbronchiolitis: a case report.

BACKGROUND: Mycobacterium (M) talmoniae isolated from a patient with cystic fibrosis was first described in 2017, and cases of M. talmoniae remain exceedingly rare. CASE PRESENTATION: A 51-year-old woman had respiratory symptoms for 10 years. Diffuse panbronchiolitis (DPB) was detected at the first visit at our hospital. A cavity lesion in the apex of the left lung was found, and sputum and bronchoalveolar lavage fluid were acid-fast bacillus (AFB) smear- and culture-positive besides Pseudomonas aeruginosa. M. talmoniae was finally identified, and the standard combination therapy for non-tuberculous mycobacteria (NTM) was administered for 2 y referring to the drug-susceptibility test. Thereafter, the AFB culture was negative, the wall thickness of the lung cavity was ameliorated, and oxygen saturation improved. CONCLUSIONS: We encountered a rare case of M. talmoniae with DPB, for which standard combination therapy was effective. M. talmoniae may be considered a potential pathogen of lung disease, especially in patients with bronchiectatic lesions.