Utility of multiplex real-time PCR in the diagnosis of extrapulmonary tuberculosis

Abstract Objective The diagnosis of extrapulmonary tuberculosis is still a challenge because of its pauci-bacillary nature. The aim of the study was to evaluate the role of a multiplex PCR assay in the diagnosis of extrapulmonary tuberculosis and to compare the efficiency of two targets, IS6110 and MPB64 to detect Mycobacterium tuberculosis. Methods 150 extrapulmonary samples (61 pus/aspirate, 46 tissue, 32 body fluids, and 11 urine) from clinically suspected cases of tuberculosis were included in the study. All the samples were subjected to direct fluorescent microscopy, TB culture (BacT/ALERT 3D, biomerieux, Durham, North Carolina, USA) and a Multiplexed Tandem PCR targeting two mycobacterial DNA sequences, IS6110 and MPB64. Master-Mix reagents and primers were prepared by AusDiagnostics Pvt. Ltd (Alexandria, New South Wales, Australia). The performance of the assay was assessed using a composite gold standard, which included clinical characteristics, microbiology smear as well as culture, histopathology, cytology, radiology, and response to antitubercular therapy. Results 20.3%, 23.6%, and 45.3% of specimens were positive by smear, culture, and PCR, respectively. The sensitivity and specificity of the multiplex PCR was 91.9% and 88.4%, respectively, using the composite gold standard. Positive and negative predictive values of the PCR were estimated as 85.1% and 93.8%, respectively. Higher positivity was observed with target IS6110 (44.6%) as compared to target MPB64 (18.9%). The sensitivities of IS6110 and MPB64 individual targets were 90.3% and 64.5%, respectively, and specificities were 88.4% and 97.7%, respectively. Conclusion PCR can play an important role in rapid and accurate diagnosis of extrapulmonary tuberculosis. IS6110 alone is an effective target in our part of the country.

Multidrug-resistant pulmonary & extrapulmonary tuberculosis: A 13 years retrospective hospital-based analysis.

Background & objectives: Multidrug-resistant tuberculosis (MDR-TB) is a public health problem of great significance in India. In the present study an attempt was made to analyse the progression of MDR-TB pattern during a course of 13 years (2000-2012) among the patient population at a tertiary care centre in New Delhi, India. Methods: Mycobacterial isolates obtained on Lowenstein-Jensen (L-J) medium/BacT/ALERT 3D were identified using AccuProbe molecular identification system, routine biochemical tests or GenoType Mycobacteria CM. Antimycobacterial susceptibility testing was performed using resistance ratio method on L-J medium (2000-2004) and one per cent proportion method on BacT/ALERT 3D system (2005-2012). Results: Of the total 14,849 samples subjected to mycobacterial culture, 6569 pulmonary and 8280 extrapulmonary, 2364 were detected positive for mycobacteria. The average percentage positivity rate was 15.9 per cent (18.9 and 13.6% in case of pulmonary and extrapulmonary samples, respectively). Our study revealed a significant increase (P<0.001) in multidrug resistance by 12 per cent (4.7% in 2000 to 19.8% in 2012). MDR-TB was more in case of pulmonary (28.2%) than extrapulmonary (11.6%) TB (P<0.001). Only 6.5 per cent (154) of mycobacterial isolates were non-tuberculous mycobacteria and rapid growers represented by Mycobacterium fortuitum and M. abscessus were the most commonly isolated species. Interpretation & conclusions: Increase in prevalence of MDR-TB by 12 per cent in the past 13 years is alarming. Policy modifications may have to be done to strengthen the existing TB control programmes to encourage contact tracing and culture and drug susceptibility testing for all smear positive pulmonary cases to ensure early and appropriate therapy.

Plasma Epstein-Barr Virus (EBV) DNA as a Biomarker for EBV-associated Hodgkin lymphoma.

Objective: To assess plasma Epstein-Barr virus (EBV) DNA as a biomarker of tumour burden at diagnosis and during therapy in children with Hodgkin lymphoma. Design: Case-control study, with prospective follow-up of the Hodgkin lymphoma cohort (2007-2012). Setting: Pediatric Hematology Oncology unit of a tertiary care hospital in Delhi. Patients: Thirty children with Hodgkin lymphoma and 70 sex and age-matched controls (benign lymphadenopathy 19, non-lymphoid malignancy 29, Burkitt lymphoma 5, healthy children 17). Intervention: Positive EBV-staining on immunohistochemistry was defined as EBV-associated Hodgkin lymphoma. Plasma EBV real-time quantitative polymerase chain reaction (PCR) was tested at presentation, after first and last chemotherapy cycles, and on follow-up. Main outcome measures: Plasma EBV quantitative PCR was compared between cases and controls. Its kinetics was assessed during and after chemotherapy. Results: EBV quantitative PCR was positive in 19 (63%) Hodgkin lymphoma cases (range 500–430,000 copies/mL), with 87.5% accuracy (kappa=0.69) as compared with EBVimmunohistochemistry. Sensitivity and specificity of the quantitative PCR were 87.5% and 81.8%, respectively. Only boys showed positive EBV immunohistochemistry and/or quantitative- PCR positivity. All controls were quantitative-PCR negative. All quantitative-PCR positive cases with follow-up blood sample showed EBV clearance after the first cycle. A quantitative-PCR negative case in long-term remission became positive at relapse. EBV status did not influence survival. Conclusion: Plasma EBV-DNA, detectable in EBV-associated Hodgkin lymphoma, becomes undetectable early after initiating therapy. It can be used as a biomarker of treatment response in EBV-associated Hodgkin lymphoma.

Ecology of blood stream infection and antibiotic resistance in intensive care unit at a tertiary care hospital in North India

OBJECTIVE: To analyse the prevalent microorganisms and their antimicrobial resistance among intensive care unit patients in a tertiary care centre in New Delhi. METHODS: A retrospective study of all consecutive blood cultures from various intensive care unit patients in the hospital during four years (January 2008 to December 2011). Antibiotic consumption data in the intensive care units were also analysed during the same period. RESULTS: Out of the total 22,491 blood cultures processed, 2846 samples were positive and 3771 microorganisms were isolated. The blood culture positivity was estimated as 12.7% of which 67.5% were monomicrobial and 32.5% polymicrobial infections. Gram negative bacilli, Gram positive cocci, and fungi were isolated in 49%, 33%, and 18% cases, respectively. Coagulase negative staphylococcus was the commonest single isolate followed by Candida spp. A drastic shift in the distribution of Candida spp. towards nonalbicans along with high resistance to azole group of antifungals suggest echinocandins for the empiric therapy of candidemia. High penicillin resistance in Gram positive isolates suggest vancomycin, linezolid and tigecycline as the options for empiric therapy, whereas tigecycline and colistin are the only options remaining for highly resistant Gram negative isolates. Aminoglycosides were observed to have better sensitivity and reduced usage when compared with cephalosporins and ß-lactam + ß-lactam inhibitor combinations. CONCLUSIONS: High frequencies of multidrug resistant organisms were observed in intensive care units which is a warning as to use the only few effective antimicrobials wisely to reduce selective pressure on sensitive strains. .

Non-albicans Candida species in blood stream infections in a tertiary care hospital at New Delhi, India.

Background & objectives: During recent decades, there has been a change in the epidemiology of Candida infections, characterized by a progressive shift from a predominance of Candida albicans to non-albicans Candida species. This study was undertaken to analyze the change in the epidemiology of candidaemia and antifungal use at tertiary care hospital in New Delhi, India, over a period of 10 years. Methods: A retrospective review of candidaemia between 1999 and 2008 and antifungal use from 2000 to 2008 was performed at Sir Ganga Ram Hosptial, New Delhi. Initially (1999-2005), isolates were differentiated as C. albicans and non- albicans Candida species. Between 2006-2008, these were identified to the species level and antifungal susceptibility was performed. Results: The occurrence of candidaemia and total antifungal use increased significantly. Candidaemia due to non-albicans species increased and this was correlated with an increasing use of fluconazole. There was emergence and increased isolation of a novel species C. haemulonii with decreased susceptibility to both amphotericin B and azoles. Overall, sensitivities of 89.6, 90.9, 88.6, 68.8 and 54.3 per cent to amphotericin B, 5 flucytosine, voriconazole, fluconazole and itraconazole, respectively were observed. Cross-resistance or reduced susceptibility to both fluconazole (MIC >16 μg/ml) and voriconazole was observed in 11.3 per cent isolates. Interpretation & conclusions: The study demonstrates a shift to non-albicans Candida species causing fungaemia and the emergence of amphotericin B and azole resistant novel species, C. haemulonii. Decreased susceptibility to fluconazole, as well as the threat of emergence of cross-resistance to voriconazole in the background of high azole consumption may limit the use of these agents as a presumptive therapy for Candida blood stream infections (BSI).

A ten year analysis of multi-drug resistant blood stream infections caused by Escherichia coli & Klebsiella pneumoniae in a tertiary care hospital.

Background & objectives: Extensive use of antibiotics has added to the escalation of antibiotic resistance. This study was undertaken to evaluate the association, if any between antibiotic use and resistance in a hospital setting, and also detect the predominant mechanism of antibiotic resistance in Escherichia coli and Klebsiella pneumoniae over a period of 10 years. Methods: In a retrospective study of 10 years, a total of 77,618 blood culture samples from 2000 to 2009 from indoor patients were screened and those yielding E. coli and K. pneumoniae were included in the study. Antibiotic susceptibility records as well as the percentage of ESBL producers were noted. A total of 423 isolates of 2009 were also screened for AmpC and carbapenemase production. Antibiotic consumption data of 10 years were analysed. Results: ESBL producing E. coli increased from 40 per cent in 2002 to 61 per cent in 2009, similarly there was a significant (P<0.05) rise in resistance to cefotaxime (75 to 97%), piperacillin-tazobactum (55- 84%) and carbapenem (2.4-52%) in K. pneumoniae. A significant (P<0.05) association was observed between resistance and consumption of carbapenem and piperacillin and tazobactum consumption in K. pneumonia. Interpretation & conclusions: Our study demonstrated a rise in consumption and resistance to broad spectrum antimicrobial agents and also established an association between consumption and resistance to these antibiotics. Over a period of 10 years, the emergence of pan-resistance in K. pneumoniae could be due to the production of carbapenemases whereas ESBL production was the common mechanism of resistance in E. coli. This study warrants a directed effort towards continued surveillance and antibiotic stewardship to minimize selection pressure and spread.

Improving bacteriological diagnosis of tuberculosis.

This review is to summarize recent developments in the field of mycobacteriology since the diagnosis of tuberculosis remains elusive in spite of our best efforts and great scientific advances. Progress has been made in further improving upon the age old, time tested traditional techniques like microscopy (Auramine-Rhodamine Fluorescent staining and peptide nucleic acids), culture and sensitivity techniques (solid, liquid, radiometric, and non-radiometric systems) that still remain as the gold standard for its diagnosis. Development of rapid methods [(high performance liquid chromatography, thin layer chromatography, RNA sequencing and polymerase chain reaction (PCR), nucleic acid sequence based amplification assay (NASBA), Transcription mediated assay (TMA) and Ligase chain reaction (LCR)] have paved the way for its rapid detection and treatment. It is interesting to see the role of molecular assays appearing more often in literature now. The molecular amplification systems (PCR, NASBA, TMA, LCR) besides identifying Mycobacterium tuberculosis (MTB) as well as non-tuberculous mycobacteria (NTM), directly from the sample can also identify Rifampin (rpoB gene)/Isoniazide (katG gene) resistant strain. Molecular assays have been found useful particularly in smear positive sputum with high sensitivity and specificity whereas variable sensitivity for sputum negative and extra pulmonary specimens has been observed. Representative specimen and its quality affect the performance of these assays. Emphasis should given to proper collection and transportation of the representative specimen for appropriate evaluation.