Detection of mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains inhibiting wild type probe hybridization in the MTBDR plus assay by DNA sequencing directly from clinical specimens.

BACKGROUND: The potential of genetic testing for rapid and accurate diagnosis of drug-resistant Mycobacterium tuberculosis strains is vital for efficient treatment and reduction in dissemination. MTBDR plus assays rapidly detect mutations related to drug resistance and wild type sequences allied with susceptibility. Although these methods are promising, the examination of molecular level performance is essential for improved assay result interpretation and continued diagnostic development. Therefore this study aimed to determine novel mutations that were inhibiting wild type probe hybridization in the Line probe assay by DNA sequencing. Using data collected from Line Probe assay (GenoType MTBDRplus assay) the contribution of absent wild type probe hybridization to the detection of rifampicin resistance was assessed via comparison to a reference standard method i.e. DNA sequencing. RESULTS: Sequence analysis of the rpoB gene of 47 MTB resistant strains from clinical specimens showed that 37 had a single mutation, 9 had double mutations and one had triple mutations in the ropB gene. CONCLUSIONS: The absence of wild type probe hybridization without mutation probe hybridization was mainly the result of the failure of mutation probe hybridization and the result of the novel or rare mutations. Additional probes are necessary to be included in the Line probe assay to improve the detection of rifampicin-resistant Mycobacterium tuberculosis strains.

Should vitamin D be routinely checked for all chronic obstructive pulmonary disease patients?

AIMS AND OBJECTIVES: This study aimed to compare the vitamin D levels between chronic obstructive pulmonary disease (COPD) patients and healthy controls and to describe the correlation between vitamin D levels and lung functions. METHODS: Fifty COPD patients (cases) and 30 healthy volunteers (controls) were recruited and their serum vitamin D level was measured together with lung function (forced vital capacity and forced expiratory volume in 1 s [FEV1]) by spirometry. vitamin D was categorized as ≤20 nmol/l: deficient, 21-50 nmol/l: inadequate, and ≥51 nmol/l as sufficient. RESULTS: In this case-control cross-sectional study, lower vitamin D levels were associated with lower lung function in both cases as well as controls, the effect being more pronounced in cases. Mean FEV1 at vitamin D ≤20 nmol/l (0.98 ± 0.40 vs. controls 1.93 ± 0.24 with P = 0.006), mean FEV1 at vitamin D 21-50 nmol/l (1.55 ± 0.54 vs. 2.20 ± 0.31 with P = 0.000), and mean FEV1 at vitamin D ≥51 nmol/l (2.06 ± 0.54 vs. 2.20 ± 0.31 with P = 0.002). Moreover, the severity of predicted postbronchodilator FEV1% was also much lower among COPD cohort versus healthy volunteers (mean FEV1%: cases 47.88 ± 14.22 vs. controls 58.76 ± 15.05 with P = 0.002). CONCLUSIONS: Importantly, lung function in both the groups was affected by decreased vitamin D level; decrease in FEV1 was more pronounced among COPD patients compared to controls showing more expiratory airflow limitation. Vitamin D levels are associated with changes in lung function in cases of COPD as well as healthy controls. Larger studies to confirm the association in Indian context are required and routine assessment of vitamin D may be undertaken to obviate the effects of low vitmain D level on lung function.

Rapid detection of drug-resistant Mycobacterium tuberculosis directly from clinical specimens using allele-specific polymerase chain reaction assay.

Background & objectives: Rapid detection of drug resistance in Mycobacterium tuberculosis (MTB) is essential for the efficient control of tuberculosis. Hence, in this study a nested-allele-specific (NAS) PCR, nested multiple allele-specific PCR (NMAS-PCR) and multiple allele-specific (MAS) PCR assays were evaluated that enabled detection of the most common mutations responsible for isoniazid (INH) and rifampicin (RIF) resistance in MTB isolates directly from clinical specimens. Methods: Six pairs of primers, mutated and wild type, were used for the six targets such as codon 516, 526 and 531 of rpoB, codon 315 of katG and C15-T substitution in the promoter region of mabA-inhA using allele-specific (AS) PCR assays (NAS-PCR, NMAS-PCR and MAS-PCR). The performance of AS PCR method was compared with phenotypic drug susceptibility testing (DST). Results: The usefulness of AS PCR assays was evaluated with 391 clinical specimens (251 Acid fast bacilli smear positive and MTB culture positive; 93 smear negative and MTB culture positive; 47 smear positive and MTB culture negative) and 344 MTB culture positive isolates. With culture-based phenotypic DST as a reference standard, the sensitivity and specificity of the NAS-PCR, NMAS-PCR and MAS-PCR assay for drug resistance-related genetic mutation detection were 98.6 and 97.8 per cent for INH, 97.5 and 97.9 per cent for RIF and 98.9 and 100 per cent for multidrug resistance (MDR). Interpretation & conclusions: The performance of AS PCR assays showed that those could be less expensive and technically executable methods for rapid detection of MDR-TB directly from clinical specimens.

Association of Risk Factors and Drug Resistance Pattern in Tuberculosis Patients in North India.

CONTEXT: India is one of the high tuberculosis (TB) burden countries in the world. Improper implementation in the guidelines for the management of TB and high rate of defaults on the part of the patients are most important risk factors for the development of multi-drug resistant TB. AIMS: This study examines the drug resistance profile and the effect of demographic, clinical and behavioral risk factors on the prevalence of TB and multidrug resistance (MDR) in north India. SETTINGS AND DESIGN: This was a prospective, observational study carried out from May 2012 to February 2014 in tertiary care hospital of Varanasi. SUBJECTS AND METHODS: The study was performed on 721 pulmonary and extrapulmonary specimens of suspected TB patients based on history, was subjected for the Ziehl-Neelsen staining and culture on Lowenstein-Jensen (LJ) media. STATISTICAL ANALYSIS: The features of groups were compared by Chi-square (χ2) and odds ratio. RESULTS: Out of 721 clinically suspected pulmonary and extrapulmonary TB patients, 222 (30.8%) patients were smear positive for acid-fast bacilli and 244 (38.3%) were positive for Mycobacterium species cultured on LJ medium. The prevalence of resistance to at least one anti-TB drug was 71.1% and MDR was 53.5%. Age, gender, HIV status, nature of TB, smoking, and alcohol consumption risk factors were significantly associated with TB prevalence; while prior history of TB infection, pervious household exposure, smoking, and alcohol consumption were significantly associated with MDR. CONCLUSION: This study showed a high prevalence of drug resistance TB in this region. It also provides evidence in our circumstance, of the role of prior history of TB infection, alcohol and smoking in increasing the risk of developing TB and MDR-TB. Therefore, it is necessary for the public health community to incorporate and strengthen alcohol and smoking nonparticipation interference in TB control program.

Performance of nested multiplex PCR assay targeting MTP40 and IS6110 gene sequences for the diagnosis of tubercular lymphadenitis.

The conventional methods for diagnosis of tubercular lymphadenitis (TBLN) such as - fine needle aspiration cytology, Ziehl-Neelsen staining and culture have limitations of low sensitivity and/or specificity. So, it becomes essential to develop a rapid, sensitive, and specific method for an early diagnosis of TBLN. Therefore, the present study was conducted to evaluate nested multiplex polymerase chain reaction (nMPCR) targeting MTP40 and IS6110 gene sequences of Mycobacterium tuberculosis and Mycobacterium tuberculosis complex, respectively in 48 successive patients of TBLN and 20 random patients with non-tubercular lymph node lesions. Out of the 48 cases of TBLN, 14 (29.2%) were found to be positive by Ziehl-Neelsen staining, 15 (31.2%) were positive by culture and 43 (89.6%) cases were positive after first round of PCR while 48 (100%) cases were positive by nMPCR assay. The sensitivity and specificity of nMPCR was found to be 100% for the diagnosis of TBLN. The results thus obtained indicate that nMPCR assay is a highly sensitive and specific tool for the diagnosis of TBLN.

Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments.

BACKGROUND: Control of the global burden of tuberculosis is obstructed due to lack of simple, rapid and cost effective diagnostic techniques that can be used in resource poor-settings. To facilitate the early diagnosis of TB directly from clinical specimens, we have standardized and validated the use of nested multiplex PCR, targeting gene fragments IS6110, MTP40 and 32kD α-antigen encoding genes specific for Mycobacterium tuberculosis complex and non-tubercular mycobacteria (NTM), in comparison to smear microscopy, solid culture and single step multiplex PCR. The results were evaluated in comparison to a composite reference standard (CRS) comprising of microbiological results (smear and culture), clinical, radiological and cytopathological findings, clinical treatment and response to anti-tubercular therapy. METHODS: The nested multiplex PCR (nMPCR) assay was evaluated to test its utility in 600 (535 pulmonary and 65 extra-pulmonary specimens) clinically suspected TB cases. All specimens were processed for smear, culture, single step multiplex PCR and nested multiplex PCR testing. RESULTS: Out of 535 screened pulmonary and 65 extra-pulmonary specimens, 329 (61.5%) and 19 (29.2%) cases were culture positive for M. tuberculosis. Based on CRS, 450 patients had "clinical TB" (definitive-TB, probable-TB and possible-TB). Remaining 150 were confirmed "non-TB" cases. For culture, the sensitivity was low, 79.3% for pulmonary and 54.3% for extra-pulmonary cases. The sensitivity and specificity results for nMPCR test were evaluated taken composite reference standard as a gold standard. The sensitivity of the nMPCR assay was 97.1% for pulmonary and 91.4% for extra-pulmonary TB cases with specificity of 100% and 93.3% respectively. CONCLUSION: Nested multiplex PCR using three gene primers is a rapid, reliable and highly sensitive and specific diagnostic technique for the detection and differentiation of M. tuberculosis complex from NTM genome and will be useful in diagnosing paucibacillary samples. Nested multiplex PCR assay was found to be better than single step multiplex PCR for assessing the diagnosis of TB.

Cutaneous Cryptococcus: marker for disseminated infection.

Cryptococcosis is an infection caused by the encapsulated yeast, Cryptococcus neoformans, a dimorphic fungus recovered from pigeon excreta, soil, dust and human skin. After a primary infection in the lungs, the disease can disseminate via a haematogenous route to various organs, including the central nervous system and skin, in susceptible individuals. Cryptococcosis can present with a variety of skin and soft tissue manifestations including acneiform lesions, purpura, vesicles, nodules, abscesses, ulcers, granulomas, pustules, draining sinuses and cellulitis. We present a case of a young man with HIV infection who developed molluscum-like cutaneous lesions secondary to pulmonary cryptococcosis. The diagnosis was confirmed by Indian ink preparation of the cutaneous lesions. Primary cutaneous infection occurs rarely due to direct inoculation. Cutaneous lesions are usually secondary and act as a key marker of disseminated infection, especially in patients with impaired cell-mediated immunity, such as those infected with HIV, solid-organ transplant recipients, and those on chronic corticosteroid therapy.

Acute silicosis with bilateral pneumothorax.

We present a case of acute silicosis with bilateral pneumothorax of a 28-year-old man working at a stone crusher factory for 1 year. He presented to the emergency department with cough, respiratory distress and diffuse chest pain. The patient was managed with bilateral intercostal tube drainage under water seal, oxygen inhalation and conservative therapy. On follow-up he showed improvement of resting dyspnoea and was doing well. This case is being reported because of the rare complications of acute silicosis as bilateral pneumothorax.

Unilateral pulmonary artery agenesis with vertebral anomaly.

We report a two-and-half-year-old boy who presented with recurrent respiratory tract infections. He had cortriatum of right atrium, spina bifida occulta, hemivertebra and dysplastic right thumb. On CT of chest, he had also unilateral pulmonary artery agenesis. The case is being reported because of common manifestations of rare disease and its associated cardiac and skeletal abnormalities.

Adrenocortical insufficiency in smear positive pulmonary tuberculosis.

We studied 40 patients with pulmonary tuberculosis. All were positive for acid-fast bacilli (AFB) in the sputum. Their mean age was 30 yrs (range 10-50 yrs) and the duration of illness was 26.3 +/- 2.3 months. Radiologically minimal, moderately advanced and far advanced lesions were present in 7 (17.5%), 9 (22.5%) and 23 (57.5%) patients respectively. One patient with endobronchial lesion had no radiological evidence of pulmonary tuberculosis. Clinically, 14 patients (35%) had one or the other features of adrenocortical insufficiency. Postural hypotension was the commonest feature and was present in 11 patients (27.5%), followed by nausea and vomiting (20%), loss of axillary hair and libido (10%), skin and mucosal pigmentation in 7.5% of the cases. ACTH stimulation revealed incomplete adrenocortical insufficiency (partially responsive adrenal) in 5 patients (12.5%) and complete adrenocortical insufficiency (non-responsive adrenal) in 2 patients (5%). Patients with features of adrenal insufficiency had significantly longer duration of illness (p < 0.001) but there was no correlation with extent or type of lesion.

Oesophago-pleuro-cutaneous fistula.

We describe a case of oesophago-pleuro-cutaneous fistula occurring as a complication of tuberculous pyopneumothorax in a young adult.

Study of blood coagulation in experimental diabetes.

In experimental alloxan diabetes of rats a twofold increase in fibrinogen level was observated at 48 and 144 hrs following alloxanization. Other coagulation parameters did not show any alteration.