A host blood transcriptional signature differentiates multi-drug/rifampin-resistant tuberculosis (MDR/RR-TB) from drug susceptible tuberculosis: a pilot study.

BACKGROUND: Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis is one of the top thirteen causes of death worldwide. The major challenge to control TB is the emergence of drug-resistant tuberculosis (DR-TB); specifically, multi-drug resistant TB which are resistant to the most potent drugs; rifampin and isoniazid. Owing to the inconsistencies of the current diagnostic methods, a single test cannot identify the whole spectrum of DR-TB associated mutations. Recently, host blood transcriptomics has gained attention as a promising technique that develops disease-specific RNA signatures/biomarkers. However, studies on host transcriptomics infected with DR-TB is limited. Herein, we intended to identify genes/pathways that are differentially expressed in multi-drug/rifampin resistant TB (MDR/RR-TB) in contrast to drug susceptible TB. METHOD AND RESULTS: We conducted blood RNA sequencing of 10 pulmonary TB patients (4; drug susceptible and 6; DR-TB) and 55 genes that were differentially expressed in MDR/RR-TB from drug-susceptible/mono-resistant TB were identified. CD300LD, MYL9, VAMP5, CARD17, CLEC2B, GBP6, BATF2, ETV7, IFI27 and FCGR1CP were found to be upregulated in MDR/RR-TB in all comparisons, among which CLEC2B and CD300LD were not previously linked to TB. In comparison pathway analysis, interferon alpha/gamma response was upregulated while Wnt/beta catenin signaling, lysosome, microtubule nucleation and notch signaling were downregulated. CONCLUSION: Up/down-regulation of immunity related genes/pathways speculate the collective effect of hosts' attempt to fight against continuously multiplying DR-TB bacteria and the bacterial factors to fight against the host defense. The identified genes/pathways could act as MDR/RR-TB biomarkers, hence, further research on their clinical use should be encouraged.

Respiratory Bacterial Microbiota and Individual Bacterial Variability in Lung Cancer and Bronchiectasis Patients.

Respiratory bacterial microbiota plays a key role in human health. Lung cancer microbiome is a significant yet an understudied area while bronchiectasis microbiome is often studied. We assessed the bacterial microbiota in the upper and lower respiratory tract of the patients with lung cancer and bronchiectasis against a healthy group and their variations in individuality. 16S rRNA gene based metagenomic sequencing was used to detect entire bacterial community along with conventional aerobic bacterial culturing. In comparison to healthy, increased bacterial diversity was observed in diseased population. Abundance of more than 1% was considered and bacteria were identified in 97% similarity. Only lung cancer patients exhibited bacteria specific to the disease: Corynebacterium tuberculostearicum and Keratinibaculum paraultunense. However, Enterococcus faecalis and Delftia tsuruhatensis were also observed limited to lung cancer and bronchiectasis respectively, in less than 1% but supported with bacterial culturing. In conclusion the disease condition and intra-group variability should be considered in future with larger cohorts to understand individual patient variability highlighting the social habits and gender of the individual.

Real time PCR for the rapid identification and drug susceptibility of Mycobacteria present in Bronchial washings.

BACKGROUND: Mycobacteria have a spectrum of virulence and different susceptibilities to antibiotics. Distinguishing mycobacterial species is vital as patients with non-tuberculous mycobacterial (NTM) infections present clinical features that are similar to those of patients with tuberculosis. Thus, rapid differentiation of Mycobacterium tuberculosis complex from NTM is critical to administer appropriate treatment. Hence the aim of the study was to rapid identification of mycobacterial species present in bronchial washings using multiplex real time Polymerase Chain Reaction (PCR) and to determine the drug susceptibility in identified mycobacterial species. METHODS: Sputum smear negative bronchoscopy specimens (n = 150) were collected for a period of one year, from patients attending the General Hospital Kandy, Sri Lanka. The specimens were processed with modified Petroff's method and were cultured on Löwenstein- Jensen medium. DNA, extracted from the mycobacterial isolates were subjected to a SYBR green mediated real time multiplex, PCR assay with primers specific for the M. tuberculosis complex, M. avium complex, M. chelonae-M.abscessus group and M. fortuitum group. DNA sequencing was performed for the species confirmation, by targeting the 16S rRNA gene and the drug susceptibility testing was performed for the molecularly identified isolates of M. tuberculosis and NTM. RESULTS: The optimized SYBR Green mediated multiplex real-time PCR assay was able to identify the presence of genus Mycobacterium in 25 out of 26 AFB positive isolates, two M. tuberculosis complex, three M. avium complex and two isolates belonging to M. chelonae-M. abscessus group. DNA sequencing confirmed the presence of M. tuberculosis, M. chelonae-M. abscessus, M. intracellulare, M. avium, Rhodococcus sp. and M. celatum. Remaining isolates were identified as Mycobacterium sp. All the NTM isolates were sensitive to amikacin and seven were resistant to ciproflaxacin. Twenty two of the NTM isolates and the isolate Rhodococcus was resistant to clarithromycin. The two isolates of M. tuberculosis were sensitive to all first line anti tuberculosis drugs. CONCLUSION: The optimized SYBR Green mediated multiplex real time PCR assay could be an effective tool for the rapid differentiation of pathogenic M. tuberculosis complex from the opportunistic nontuberculous mycobacteria and also it confirmed the presence of NTM in 15.3 % of the study population.

Exploring bilateral pneumothorax leading to a rare diagnosis in a young lady.

Bilateral pneumothorax is a rare occurrence and vigilant clinical examination is mandatory to suspect that during the presentation. This case illustrates a young lady presented with bilateral pneumothorax, new identification of lung cysts and chylous pleural effusion leading to diagnosis of lymphangioleiomyomatosis.

Is International Travel an Emerging Issue on Transmission of Beijing Lineage Mycobacterium tuberculosis?

Mycobacterium tuberculosis belonging to Beijing sublineage (BL) is associated with high tuberculosis (TB) transmission, multidrug resistance, and adverse treatment outcomes. Sri Lanka experiences an increase in the number of travellers/workers to and from high TB-burden countries, and there is risk of getting BL strains imported into the country. In this context, a cohort study was conducted to assess the prevalence of BL strains among pulmonary tuberculosis (PTB) patients in the Kandy district of Sri Lanka (a popular tourist destination) and its association with patients' sociodemographic and clinical characteristics. The study population included sputum smear-positive PTB patients diagnosed from February 2018-July 2019. Fresh sputum samples were collected for culturing and conducted polymerase chain reaction using BL-specific primers. Among the 101 patients recruited, presence of BL strains could be ascertained in 94 patients of which 24 (26%; 95% CI: 18%-35%) had BL strains. Prevalence of BL strains was higher among those with high sputum smear grades (2+ and 3+) (P < 0.05) and those who had travelled abroad (P < 0.05). The prevalence was also higher among young people (aged <35 years). Treatment success rates were similar in patients with (83%) and without BL strains (83% vs. 81%; P value = 0.8375). The prevalence of BL strains in Kandy, Sri Lanka, was high compared to previously reported figures in Sri Lanka, and the percentage drives closer to the countries in South East Asia. International travel raises itself as an emerging issue in BL transmission urging the need of policies and practices in immigration/emigration strategies. The study findings have the potential to alter the TB epidemiology in the country and might represent the situation in other underexplored countries as well. Therefore, it is important to monitor the trends and factors related to the prevalence of Beijing strains globally and make decisions as a whole.

Evaluation of the 15 and 24-loci MIRU-VNTR genotyping tools with spoligotyping in the identification of Mycobacterium tuberculosis strains and their genetic diversity in molecular epidemiology studies.

BACKGROUND: The transmission dynamics of Mycobacterium tuberculosis (Mtb) using various genotyping tools has been studied globally and a particular tool for genotyping Mtb is the mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR). Tuberculosis (TB) remains an important public health problem worldwide and Sri Lanka being a country of tourist destination; because of major development projects undergoing, it has a high proportion of tourists and immigrants from Asia and Europe that are characterized with highest TB incidences and drug-resistant clinical isolates. Hence, in order to address the question of Mtb genetic diversity, we investigated the discriminatory power of both MIRU-VNTR typing of 15 and 24 loci with spoligotyping to differentiate Mtb isolates. METHOD: Acid-fast bacilli positive sputum samples (n = 150) from first visit patients were collected. Decontamination of sputum and extraction of genomic DNA were carried out using standard techniques. The isolates were characterized by MIRU-VNTR for both the 15 and 24 loci and spoligotyping. RESULTS: In our study population, MIRU-VNTR 15 and 24 loci did not show a significant difference among the identified M. tuberculosis strains. However, MIRU 24 loci yielded an additional strain LAM, which is of T1 origin. 15 loci strain grouping had more clusters of strains grouped together while 24 loci differentiated the same cluster of strains into distinct strain types. CONCLUSION: We conclude that the use of 15-locus MIRU-VNTR typing is sufficient for a first-line epidemiological study to genotype M. tuberculosis, but the additional discriminatory power of 24 loci MIRU-VNTR has been able to differentiate samples within highly homologous groups.

Insight into genetic diversity of Mycobacterium tuberculosis in Kandy, Sri Lanka reveals predominance of the Euro-American lineage.

OBJECTIVE: Sri Lanka is a country where the molecular epidemiology of Mycobacterium tuberculosis (MTB) is poorly explored. Therefore, this study was performed to identify circulating lineages/sub-lineages of MTB and their transmission patterns. METHODS: DNA was extracted from 89 isolates of MTB collected during 2012 and 2013 from new pulmonary tuberculosis patients in Kandy, Sri Lanka and analyzed by spoligotyping, large sequence polymorphism (LSP), mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing, and drug resistance-associated gene sequencing. RESULTS: The predominant lineage was lineage 4 (Euro-American, 45.9%), followed by lineage 1 (Indo-Oceanic, 29.4%), lineage 2 (East-Asian, 23.5%), and lineage 3 (Central-Asian, 1.2%). Among 26 spoligotype patterns, eight were undesignated or new types and seven of these belonged to lineage 4. Undesignated lineage 4/SIT124 (n=2/8) and SIT3234 (n=8/8) clustered together based on 24-locus MIRU-VNTR typing. The dominant sub-lineage was Beijing/SIT1 (n=19), with the isoniazid resistance katG G944C mutation (Ser315Thr) detected in two of them. CONCLUSIONS: The population structure of MTB in Kandy, Sri Lanka was different from that in the South Asian region. The clonal expansion of locally evolved lineage 4/SIT3234 and detection of the pre-multidrug resistant Beijing isolates from new tuberculosis patients is alarming and will require continuous monitoring.

Characteristics of stable chronic obstructive pulmonary disease patients in the pulmonology clinics of seven Asian cities.

BACKGROUND AND OBJECTIVES: Chronic obstructive pulmonary disease (COPD) is responsible for significant morbidity and mortality worldwide. We evaluated the characteristics of stable COPD patients in the pulmonology clinics of seven Asian cities and also evaluated whether the exposure to biomass fuels and dusty jobs were related to respiratory symptoms, airflow limitation, and quality of life in the COPD patients. METHODS: This cross-sectional observational study recruited 922 COPD patients from seven cities of Asia. The patients underwent spirometry and were administered questionnaires about their exposure to cigarette smoking, biomass fuels, and dusty jobs in addition to respiratory symptoms and health related quality of life. RESULTS: Of the patients, there appeared to be variations from city to city in the history of exposure to biomass fuels and dusty jobs and also in respiratory symptoms of cough, phlegm, wheeze, and dyspnea. These symptoms were more frequent in those COPD patients with a history of exposure to biomass fuels than without and those with a history of exposure to dusty jobs than without (P < 0.01 for all comparisons). Airflow limitation was more severe in those COPD patients with a history of exposure to biomass fuels than without (52.2% predicted versus 55.9% of post-bronchodilator forced expiratory volume in 1 second [FEV(1)], P = 0.009); quality of life was poorer in those with exposure to biomass fuels than without (40.4 versus 36.2 of the St George's Respiratory Questionnaire [SGRQ] total score, P = 0.001). Airflow limitation was more severe in those COPD patients with a history of exposure to dusty jobs than without (51.2% predicted versus 57.3% of post-bronchodilator FEV(1), P < 0.001); quality of life was poorer in those with dusty jobs than without (41.0 versus 34.6 of SGRQ score, P = 0.006). CONCLUSION: In Asian cities, the characteristics of COPD patients vary and the history of exposure to biomass fuels or dusty jobs was related to frequency of symptoms, severe airflow limitation, and poor quality of life.

An unusual case of fatal pulmonary hemorrhage in pregnancy.

Rickettsial diseases are common in Srilanka. The spotted fever group of rickettsiae presents in many ways, including very severe disease causing significant morbidity and mortality. A regional variation of the Rickettsia conorii subspecies and differences in clinical presentations are reported. This case describes disseminated Rickettsia conorii infection in a pregnant woman presenting with endocarditis.