Manas K. Sen

Visual Format for Detection of Mycobacterium tuberculosis and M. bovis in Clinical Samples Using Molecular Beacons

A real-time polymerase chain reaction (PCR) assay for the direct identification of Mycobacterium tuberculosis and M. bovis using molecular beacons was developed. The assay was modified for use in regular thermal cyclers. Molecular beacons that were specific for M. tuberculosis (Tb-B) and M. bovis (Bo-B) were designed. The fluorescence of the target PCR product-molecular beacon probe complex was detected visually using a transilluminator. The results were then compared with those of conventional multiplex PCR (CM-PCR) assays and biochemical identification. The detection limit of Tb-B and Bo-B beacons was 500 fg and 50 fg by the visual format and real-time PCR assay, respectively, compared with 5 pg by CM-PCR assay. Pulmonary and extrapulmonary samples were examined. The agreement between culture and the two assays was very good in sputum samples and fair in extrapulmonary samples. The agreement between clinical diagnoses with the two assays was moderate in extrapulmonary samples. There was very good agreement between CM-PCR and visual format assays for all samples used in the study. Concordance in the identification of isolates by the visual, CM-PCR assay, and biochemical identification was seen. Hence, the use of molecular beacon detection of M. tuberculosis and M. bovis in clinical samples is feasible by setting up two asymmetric PCRs concurrently. The assay is sensitive, specific, simple to interpret, and takes less than 3 hours to complete.

Assessment of the N-PCR Assay in Diagnosis of Pleural Tuberculosis: Detection of M.tuberculosis in Pleural Fluid and Sputum Collected in Tandem

Background The nonspecific clinical presentation and paucibacillary nature of tuberculous pleuritis remains a challenge for diagnosis. Diagnosis of tuberculous pleural effusion depends on the demonstration of the presence of tubercle bacilli in the sputum, pleural fluid, or pleural biopsy specimen, or demonstration of granuloma in pleura by histological examination. We examined the clinical utility of the diagnosis of pleural tuberculosis using the in house N-PCR assay, AFB smear microscopy and culture. Besides pleural fluid the inclusion of sputum in the efficacy of diagnosis of pleural tuberculosis was scrutinized. Methodology/Principal Findings Pleural fluid and sputum samples of 58 tuberculous and 42 non-tuberculous pleural effusion patients were processed for AFB smear microscopy, culture and the N-PCR assay. Mycobacteria were detected exclusively in tuberculous pleural effusion samples. None of the non-tuberculous pleural effusion samples were positive for mycobacteria. Comparative analysis showed that the N-PCR assay had the highest sensitivity. Inclusion of sputum along with pleural fluid increased N-PCR sensitivity from 51.7 to 70.6% (p<0.0001).This improved sensitivity was reflected in AFB smear microscopy and isolation by culture. The sensitivity enhanced on inclusion of sputum from 3.4 (p = 0.50) to 10.3% (p = 0.038) for AFB smear microscopy and for isolation of mycobacteria from 10.3(p = 0.03) to 22.4% (p = 0.0005). Thirteen isolates were obtained from 58 pleural tuberculosis patients. Eleven mycobacterial isolates were identified as M.tuberculosis and two as M.fortuitum and M.chelonae. Complete concordance was seen between the biochemical identification of isolates and the N-PCR identification of mycobacterial species prior to isolation. Conclusions/Significance To the best of our knowledge this is the first PCR based report on utility of sputum for diagnosis of pleural tuberculosis. The present study demonstrates that a combination of pleural fluid with sputum sample and N-PCR improved the diagnosis of pleural tuberculosis.

Outcome of adenotonsillectomy for children with sleep apnea

BACKGROUND AND OBJECTIVES The prevalence of obstructive sleep apnea syndrome is about 1%-4.9% in children aged 2-18 years. This is a prospective study carried out to evaluate the role of adenotonsillectomy (AT) in pediatric sleep apnea. METHODS Fifty children aged less than 15 years presenting with the chief complaints of snoring, mouth breathing, recurrent upper respiratory infections, and adenotonsillar hypertrophy were included in the study. Physical examination included body mass index (BMI) z-score, orodental and nasal examination, modified Mallampati scoring; whole-night level I polysomnography was conducted and repeated after three to six months of AT. RESULTS The mean preoperative BMI z-score was -0.76, which improved significantly to -0.15 (p < 0.001) after AT. A negative correlation was seen between respiratory distress index (RDI) and pre surgery BMI z-score. As per pre-operative RDI, OSA was classified mild in 6.7% children (31.1% as per apnea-hypopnea index [AHI]), moderate in 35.6% (31.1% as per AHI), and severe in 57.8% (37.8% as per AHI). The average RDI value reduced significantly from 16.2 ± 10.7 to 6.46 ± 4.8 (p < 0.001) and AHI from 8.5 (SD ± 5.7) to 1.3 (SD ± 1) post-operatively. Only 6.7% children could be cured with surgery, of whom none belonged to moderate or severe category. Multivariate analysis shows that initial severity of disease, modified Mallampati scores III and IV, high-arched palate, and age above eight years were associated with significant residual disease after AT. CONCLUSION AT was associated with a statistically significant change in RDI and AHI. However, complete resolution of OSA could be seen in a small percentage of patients with a mild degree of disease.

Metabolic changes in normal- and underweight children with obstructive sleep-disordered breathing

OBJECTIVE This study evaluates the metabolic profile of normal- and underweight children with sleep-disordered breathing (SDB) due to adenotonsillar hypertrophy. METHODS A total of 39 children aged 3-15 years with SDB and 28 age- and gender-matched controls were included in the study. Body mass index z score, blood pressure, and fasting serum levels of triglycerides (TGs), high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol, very-low-density lipoprotein (VLDL), blood glucose, plasma insulin, and homeostatic model assessment (HOMA) were determined in both case patients and controls. RESULTS We observed significantly lower levels of fasting blood glucose (p = 0.015) and higher levels of HDL (p = 0.002), LDL (p = 0.002), and cholesterol (p = 0.001) in case patients than in controls. The mean values of fasting insulin and HOMA were higher in case patients (6.42 ± 6.47 and 1.40 ± 1.48) than in controls (5.31 ± 3.40 and 1.20 ± 0.84) respectively. No direct correlation between indices of severity of SDB and various metabolic and blood pressure parameters was found. When the effect of body weight was studied by subgrouping case patients according to normal weight and underweight, significant increases in the levels of fasting insulin (p = 0.039), HOMA (p = 0.017), and fasting blood glucose (p = 0.021) were observed. Also, a significant correlation was observed between the duration of illness and fasting insulin (p = 0.023), HOMA (p = 0.020), fasting glucose (p = 0.004), and diastolic blood pressure (p = 0.030). CONCLUSION This study shows an independent effect of body weight and duration of illness on various metabolic and blood pressure parameters in normal- and underweight children with SDB.

Diagnostic utility of endobronchial ultrasound features in differentiating malignant and benign lymph nodes

Endobronchial ultrasound (EBUS) features have been shown to be useful in predicting etiology of enlarged malignant lymph nodes. However, there is dearth of evidence especially from developing countries. We assessed the EBUS characteristics across various mediastinal and hilar lymphadenopathies. In this prospective study, all patients with mediastinal and hilar lymphadenopathy on CT Chest and who were planned for EBUS-FNA (Fine Needle Aspiration) were included. EBUS features of lymph nodes studied were shape, size, margins, echogenicity, central hilar structure (CHS), coagulation necrosis sign and colour power doppler index (CPDI). These were scored and compared between benign and malignant lymphadenopathies. A total of 86 lymph nodes in 46 patients were prospectively studied of which 23 (26.7%) were malignant, 27 (31.3%) tuberculosis and 36 (41.8%) sarcoidosis. There was significant difference between malignant and benign lymph nodes in terms of CHS [central hilar structutre] (p=0.011), margins (p=0.036) and coagulation necrosis sign (p<0.001). On comparison of features of malignancy and tuberculosis, there were significant differences in margins (p=0.016) and coagulation necrosis sign (p 0.001). However, when malignancy and sarcoidosis was compared, there were differences in echogenicity (p=0.002), CHS (p=0.009) and coagulation necrosis sign (p<0.001). Only coagulation necrosis sign was found to be highly consistent with malignant lymph nodes. The other features cannot be used to distinguish malignant from benign lymph nodes, especially in a developing country like India where tuberculosis is a common cause of mediastinal lymphadenopathy.

Comparison of diagnostic yield and safety profile of radial endobronchial ultrasound-guided bronchoscopic lung biopsy with computed tomography-guided percutaneous needle biopsy in evaluation of peripheral pulmonary lesions: A randomized controlled trial

Background: Peripheral pulmonary lesions (PPLs) pose a diagnostic challenge, and the optimal investigation in many such cases remains unclear. Computed tomography (CT)-guided percutaneous needle biopsy (CT-PNB) has been the modality of choice for such lesions with a high diagnostic accuracy but with high rates of pneumothorax. Endobronchial ultrasound (EBUS) with a radial probe is an alternate diagnostic modality with increased diagnostic yield of bronchoscopy in the evaluation of PPL. We conducted a randomized controlled trial comparing the diagnostic accuracy and complication rates of radial EBUS with CT-guided lung biopsy for the evaluation of PPL. Methods: Fifty patients with PPL surrounded by lung parenchyma on all sides were randomly assigned to either radial EBUS or CT-PNB group (25 each). Results: Both groups had similar clinicoradiologic characteristics. The diagnostic accuracy of radial EBUS was comparable to CT-PNB with no statistically significant difference (72 vs. 84%; P = 0.306). However, the yield was significantly lower in right upper lobe lesions (20% vs. 83.3%; P = 0.03). CT-PNB group had significantly higher pneumothorax rates than radial EBUS (20% vs. 0%; P = 0.03). The lesions that were more than 2 cm, those with ultrasound feature of continuous hyperechoic margin around the lesion (P = 0.007), and the position of the ultrasound probe within the lesion (P < 0.001) were associated with a higher diagnostic yield with radial EBUS. Conclusion: Our findings suggest that radial EBUS is a safer investigation than CT-PNB with a comparable diagnostic accuracy for PPL not abutting the chest wall (CTRI/2017/02/007762).

Cavitating lung disease due to concomitant drug resistant tuberculosis and invasive pulmonary Aspergillosis in a post-partum patient: A case report.

Many disorders can present as cavitating lesions in the lung. In this case report, a case of mixed infection with drug resistant tuberculosis and invasive pulmonary aspergillosis in a post-partum patient has been presented.

Ptosis of the lung

83 A young male patient of 17 years presented to the Emergency after sustaining blunt trauma to the chest as result of a road traffic accident. At the Emergency he was found to have severe hypoxemia. The chest examination was suggestive of right‐sided tension pneumothorax, for which an intercostal drainage (ICD) catheter was inserted and the patient was intubated and ventilated. There were large air‐leaks noted in the ICD bag and the pneumothorax persisted even after ICD placement. A chest x‐ray (CXR) was done, which is shown in Figure 1.

Cutaneous tuberculosis due to multidrug-resistant tubercle bacilli and difficulties in clinical diagnosis

This report describes 6 HIV-negative patients including 5 children with scrofuloderma and an adult with lupus vulgaris, out of a total of 303 cases of cutaneous tuberculosis seen during a 4½-year period, who showed a positive tuberculin test and granulomatous histopathology, but failed to respond to first-line antitubercular therapy. They were suspected to have multidrug-resistant infection as no other cause could be ascertained. Tissue aspirate or biopsy was sent for histopathology and culture. Mycobacterium tuberculosis was isolated from the aspirate in three patients and sputum in one with associated pulmonary tuberculosis. Drug susceptibility tests showed that all isolates were resistant to rifampicin and isoniazid, and one each additionally to streptomycin and ethambutol, respectively. In two, culture was unsuccessful. All were administered second-line antitubercular drugs. Clinical improvement was appreciable within 2 months as weight gain, and regression of ulcers, swellings and plaques. Two completed the recommended 24 months of therapy. Multidrug-resistant cutaneous tuberculosis should be suspected in patients with no response to first-line drugs, with clinical deterioration, and where other causes of treatment failure are not forthcoming. Owing to poor isolation rates on culture and low sensitivity of molecular tests, in such cases, a trial of second-line anti-tubercular drugs may be justified for a reasonable period of 2 months. Where facilities permit, culture and drug sensitivity tests should be done before starting treatment. Culture positivity is better from aspirated material.

Cystic lung disease in tuberculosis: An unusual presentation.

Cysts in the lung can arise due to large number of causes out of which tuberculosis is very rare, We report a case of tuberculosis in a young female presenting as a febrile illness and respiratory failure with radiological features of cystic lung disease. With treatment,fever and respiratory distress subsided and cysts in the lungs showed partial regression. We highlight the need to consider tuberculosis in the differential diagnoses of cystic lung disease under appropriate circumstances.