Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for rapid identification of micro-organisms in the routine clinical microbiology laboratory.

The study evaluates the utility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) Vitek MS for identification of microorganisms in the routine clinical microbiology laboratory. From May 2013 to April 2014, microbial isolates recovered from various clinical samples were identified by Vitek MS. In case of failure to identify by Vitek MS, the isolate was identified using the Vitek 2 system (bioMerieux, France) and serotyping wherever applicable or otherwise by nucleic acid-mediated methods. All the moulds were identified by Lactophenol blue mounts, and mycobacterial isolates were identified by molecular identification systems including AccuProbe (bioMerieux, France) or GenoType Mycobacterium CM (Hain Lifescience, Germany). Out of the 12,003 isolates, the Vitek MS gave a good overall ID at the genus and or species level up to 97.7% for bacterial isolates, 92.8% for yeasts and 80% for filamentous fungi. Of the 26 mycobacteria tested, only 42.3% could be identified using the Saramis RUO (Research Use Only) database. VITEK MS could not identify 34 of the 35 yeast isolates identified as C. haemulonii by Vitek 2. Subsequently, 17 of these isolates were identified as Candida auris (not present in the Vitek MS database) by 18S rRNA sequencing. Using these strains, an in-house superspectrum of C. auris was created in the VITEK MS database. Use of MALDI-TOF MS allows a rapid identification of aerobic bacteria and yeasts in clinical practice. However, improved sample extraction protocols and database upgrades with inclusion of locally representative strains is required, especially for moulds.

Microbial identification and automated antibiotic susceptibility testing directly from positive blood cultures using MALDI-TOF MS and VITEK 2.

The study addresses the utility of Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight mass spectrometry (MALDI-TOF MS) using VITEK MS and the VITEK 2 antimicrobial susceptibility testing (AST) system for direct identification (ID) and timely AST from positive blood culture bottles using a lysis-filtration method (LFM). Between July and December 2014, a total of 140 non-duplicate mono-microbial blood cultures were processed. An aliquot of positive blood culture broth was incubated with lysis buffer before the bacteria were filtered and washed. Micro-organisms recovered from the filter were first identified using VITEK MS and its suspension was used for direct AST by VITEK 2 once the ID was known. Direct ID and AST results were compared with classical methods using solid growth. Out of the 140 bottles tested, VITEK MS resulted in 70.7 % correct identification to the genus and/ or species level. For the 103 bottles where identification was possible, there was agreement in 97 samples (94.17 %) with classical culture. Compared to the routine method, the direct AST resulted in category agreement in 860 (96.5 %) of 891 bacteria-antimicrobial agent combinations tested. The results of direct ID and AST were available 16.1 hours before those of the standard approach on average. The combined use of VITEK MS and VITEK 2 directly on samples from positive blood culture bottles using a LFM technique can result in rapid and reliable ID and AST results in blood stream infections to result in early institution of targeted treatment. The combination of LFM and AST using VITEK 2 was found to expedite AST more reliably.

Pulmonary coccidiomycosis in New Delhi, India.

Coccidioidomycosis is an endemic disease of the western hemisphere. In cases occurring in non-endemic areas, eliciting a history of exposure as well as a high index of suspicion is imperative for timely and accurate diagnosis. In this case, a 65-year-old male presented to our hospital with fever, dry cough and malaise, and on X-ray chest, was found to have a lower lobe consolidation left lung with nodular lesions in both lungs and necrotic mediastinal lymphadenopathy. He lived in Arizona, USA, for 6 months before admission. Pulmonary coccidioidomycosis was confirmed by the isolation of Coccidioides spp. in pure culture from both broncho-alveolar lavage and lung biopsy specimens. The identity of the isolate was confirmed as C. posadasii by gene sequencing. The patient improved after being treated with fluconazole.

Irrational use of antibiotics and role of the pharmacist: an insight from a qualitative study in New Delhi, India.

WHAT IS KNOWN AND OBJECTIVE: The overall volume of antibiotic consumption in the community is one of the foremost causes of antimicrobial resistance. In developing countries like India, pharmacists often dispense 'prescription-only' drugs, like antibiotics, to patients who do not have a prescription. Not much data is available regarding detailed information on behaviour of antibiotic use by community pharmacists which is of particular significance to develop a suitable and sustainable intervention programme to promote rational use of antibiotics. A qualitative study was conducted to understand the dispensing practices and behaviour of community pharmacists to develop policy interventions that would improve the use of antibiotics at the community level. METHODS: Focus group discussions (FGDs) were held for five municipal wards of Delhi with retail pharmacists, public sector pharmacists and the office bearers of pharmacists' associations. Data on antibiotic use and resistance were collected earlier from these five wards. FGDs (n = 3 with 40 pharmacists) were analysed through grounded theory. RESULTS AND DISCUSSION: Four broad themes identified were as follows: prescribing and dispensing behaviour; commercial interests; advisory role; and intervention strategies for rational use of antibiotics. FGDs with pharmacists working in the public sector revealed that, besides the factors listed above, overstock and near-expiry, and under-supply of antibiotics promoted antibiotic misuse. Suggestions for interventions from pharmacists were the following: (i) education to increase awareness of rational use and resistance to antibiotics; (ii) involving pharmacists as partners for creating awareness among communities for rational use and resistance to antibiotics; (iii) developing an easy return policy for near-expiry antibiotics in public sector facilities; and (iv) motivating and showing appreciation for community pharmacists who participate in intervention programmes. WHAT IS NEW AND CONCLUSIONS: Inappropriate antibiotic dispensing and use owing to commercial interests and lack of knowledge about the rational use of antibiotics and antibiotic resistance were the main findings of this in-depth qualitative study. Community pharmacists were willing to participate in educational programme aimed at improving use of antibiotics. Such programmes should be initiated within a multidisciplinary framework including doctors, pharmacists, social scientists, government agencies and non-profit organizations.

Prescription auditing and antimicrobial resistance at a tertiary care hospital in New Delhi, India.

This paper reports the antibiotic consumption data of Sir Ganga Ram Hospital, New Delhi and bacterial resistance over a seven-year period. Cephalosporins, penicillins and fluoroquinolones were the most widely prescribed antibiotics. A correlation was seen between Escherichia coli resistance to third-generation cephalosporins and increased cephalosporin use, as well as resistance to co-amoxyclav and its use.

Utility of routine real time quantitative PCR monitoring of HCV infection in haemodialysis patients.

BACKGROUND: Hepatitis C virus (HCV) infection causes significant morbidity and mortality in patients of end stage renal disease (ESRD) on haemodialysis (HD). Stringent screening methods can help in its early diagnosis. OBJECTIVE: The study addresses the utility of real-time quantitative polymerase chain reaction (RQ-PCR) in the diagnosis and monitoring of HCV infection especially on seronegative and normal serum alanine aminotransferase (ALT) HD patients. MATERIAL AND METHODS: This retrospective study was carried out from January 2010 to December 2012. Patients of ESRD on maintenance HD and on whom all the three assays HCV antibody serology, PCR and ALT were done were included in the study (n=123). Group 1 (n=57), comprised of patients with negative serology and normal ALT, and Group 2 (n=66), had either raised ALT and or a positive or equivocal serology. RESULTS: Out of the 123 cases studied, HCV serology was positive in 36.5% (45), ALT raised in 18.6% (23) and PCR positive in 67.4% (83) cases. PCR positivity was significantly higher than serology and raised ALT. Group 2 had a significantly higher PCR positivity than Group 1 (P=0.0004), but 50.9% patients of Group 1, were also PCR positive and 69% of them had a high viral count of >8×10(5) IU/ml at the time of detection. CONCLUSION: Regular routine screening of HCV by RQ-PCR in ESRD patients can help in early diagnosis of HCV infection in patients with low index of suspicion.

Comparative evaluation of two rapid Salmonella-IgM tests and blood culture in the diagnosis of enteric fever.

PURPOSE: Enteric fever is a major public health problem in developing countries like India. An early and accurate diagnosis is necessary for a prompt and effective treatment. We have evaluated the diagnostic accuracy of two Rapid Salmonella-IgM tests (Typhidot-IgM and Enteroscreen-IgM) as compared to blood culture in rapid and early diagnosis of enteric fever. MATERIALS AND METHODS: A total of 2,699 patients' serum samples were tested by Rapid Salmonella-IgM tests and blood culture. Patients were divided into two groups. Test group - patients with enteric fever and blood culture positives for Salmonella Typhi; and three types of Controls, i.e. patients with non-enteric fever illnesses, normal healthy controls and patients positive for S. Paratyphi- A. In addition to this we have also evaluated the significance of positive Salmonella-IgM tests among blood culture-negative cases. RESULTS: The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Typhidot-IgM test and Enteroscreen-IgM test considering blood culture as gold standard were 97.29% and 88.13%, 97.40% and 87.83%, 98.18% and 92.03%, 96.15% and 82.27%, respectively. Typhidot-IgM test was found to be significantly more sensitive and specific as compared to Enteroscreen-IgM. Among blood culture-negative patients, Rapid Salmonella-IgM tests detected 72.25% additional cases of enteric fever. Although the Rapid Salmonella-IgM tests are meant to diagnose S. Typhi only, but these tests detect S. Paratyphi- A also. Thirty-eight patients who were blood culture-positive for S. Paratyphi- A were also positive by Rapid Salmonella-IgM tests. CONCLUSION: Rapid Salmonella-IgM tests offer an advantage of increased sensitivity, rapidity, early diagnosis and simplicity over blood culture.

Impact of informational feedback to clinicians on antibiotic-prescribing rates in a tertiary care hospital in Delhi.

CONTEXT: Antimicrobial use has been associated with increasing antimicrobial resistance. There is an urgent need for judicious use of antimicrobials. Informational feedback has been shown to result in changes in behavioural practices of physicians in certain healthcare settings. We conducted this study to see if the passive informational feedback can reduce in antimicrobial usage in a tertiary care centre. AIMS: The study was undertaken to evaluate if the feedback to clinicians on their own antibiotic prescription results in any change in their antibiotic prescription habits. SETTINGS AND DESIGN: The study was conducted at a tertiary care setting involving 33 units of different specialties. These units were split into 10 groups based on specialty and were allocated randomly to the control (16 units) and intervention (17 units) arms of the study. This study was a prospective intervention to assess the effect of prescribing feedback on clinical prescribing practices. MATERIALS AND METHODS: In the intervention arm, information on resistance rates and antibiotic-prescribing patterns was provided to all doctors. Behavioural change was assessed by comparing baseline prescribing rates of each unit with prescribing rates after the intervention. In the control arm, only information on monthly resistance rates was provided. STATISTICAL ANALYSIS: Change in the antimicrobial prescribing rates in the treatment group was assessed by using a Student's t-test. RESULTS: The mean antibiotic use for all the specialties was 189 DDDs/100BDs. The prospective intervention did not elicit any effect on the antibiotic prescribing practices of the physicians. Low prescribers continued to prescribe antibiotics at a low rate, and high prescribers continued to prescribe at a high rate. CONCLUSIONS: In view of unfavourable results of passive intervention in the above study, active intervention may be more effective.

Evaluation of human cellular immune function in echinococcosis.

Forty nine individuals (11 patients with surgically proved hydatid disease, 23 individuals with non hydatid disease and 15 normal healthy adults) were investigated for specific and nonspecific cellular immune status using in vitro blast transformation assay. Patients with hydatid disease had suppressed cellular immune function to a nonspecific T-cell mitogen (PHA) when compared to the relevant controls. Hydatid antigen directly inhibited in vitro blast transformation from a concentration of 0.1 microgram protein/ml onwards in all the three groups of subjects, irrespective of whether they had hydatidosis or not. However, hydatid antigen did not act as a T-cell mitogen in our study.

An outbreak of multidrug resistant Salmonella typhimurium in Delhi (India).

A total of 85 patients with multidrug resistant S. typhimurium were isolated between May and September 1991 at the Sir Ganga Ram Hospital, New Delhi, India. Fifty eight (72.5%) patients out of 80 stool culture positives suffered from enteritis and 23 (39.6%) of them settled with oral rehydration therapy alone. All strains were sensitive to 4 aminoquinolones (oflaxcin) but five were resistant to third generation cephalosporin (Cefotaxime; MIC between 50-75 micrograms/ml) whereas 88-96 per cent isolated were resistant to most of the other antibiotics. The convalescent carrier rate was prolonged with the use of antibiotics. The phage type of S. typhimurium isolated from the index and other cases was 178 and multidrug resistance strains had seven plasmids (1.2 to 16 kb). Barrier nursing and sodium hypochlorite disinfection helped in limiting the outbreak.

Cutaneous cryptococcosis.

We report a case of an apparently immunocompetent male, who presented with a painless nodule over the upper abdominal wall. He gave a history of exposure to pigeon droppings. Cryptococcus neoformans was isolated from the lesion and no underlying disorder could be detected. He improved on treatment with oral Fluoconazole.

LH750 hematology analyzers to identify malaria and dengue and distinguish them from other febrile illnesses.

INTRODUCTION: Tropical febrile illnesses such as malaria and dengue are challenging to differentiate clinically. Automated cellular indices from hematology analyzers may afford a preliminary rapid distinction. METHODS: Blood count and VCS parameters from 114 malaria patients, 105 dengue patients, and 105 febrile controls without dengue or malaria were analyzed. Statistical discriminant functions were generated, and their diagnostic performances were assessed by ROC curve analysis. RESULTS: Three statistical functions were generated: (i) malaria-vs.-controls factor incorporating platelet count and standard deviations of lymphocyte volume and conductivity that identified malaria with 90.4% sensitivity, 88.6% specificity; (ii) dengue-vs.-controls factor incorporating platelet count, lymphocyte percentage and standard deviation of lymphocyte conductivity that identified dengue with 81.0% sensitivity and 77.1% specificity; and (iii) febrile-controls-vs.-malaria/dengue factor incorporating mean corpuscular hemoglobin concentration, neutrophil percentage, mean lymphocyte and monocyte volumes, and standard deviation of monocyte volume that distinguished malaria and dengue from other febrile illnesses with 85.1% sensitivity and 91.4% specificity. CONCLUSIONS: Leukocyte abnormalities quantitated by automated analyzers successfully identified malaria and dengue and distinguished them from other fevers. These economic discriminant functions can be rapidly calculated by analyzer software programs to generate electronic flags to trigger-specific testing. They could potentially transform diagnostic approaches to tropical febrile illnesses in cost-constrained settings.

Variables affecting the performance of galactomannan assay in high-risk patients at a tertiary care centre in India.

BACKGROUND: Diagnosis of invasive aspergillosis (IA) in immunocompromised patients using galactomannan ELISA (GM-ELISA) has shown variable sensitivity and specificity. OBJECTIVES: To assess the diagnostic performance of GM-ELISA and analyze the effect of decreasing the cut off value, neutropenia, antifungals and piperacillin-tazobactam (PTZ). Prognostic value using 30 day all-cause mortality was also determined. MATERIALS AND METHODS: Serum samples from 81 patients categorized into "proven," "probable," and "possible," categories based on revised EORTC/MSG definitions were tested by GM-ELISA. RESULTS: Sensitivity of GM-ELISA in proven, probable and possible cases was 91.7%, 84.6% and 83.3% respectively. At an index cut-off value of 0.5 an increased sensitivity with minimal loss of specificity was observed. Use of antifungals demonstrated a decrease in sensitivity in proven and possible cases whereas it remained unaffected in probable category. Specificity increased from 75% to 100% with a positivity criterion of >2 consecutive samples. Although an increase in specificity was observed in patients not receiving PTZ, it was not statistically significant. Serial GM index values increased significantly in neutropenic patients and were associated with a poor prognosis. CONCLUSIONS: GM-ELISA may be a useful diagnostic and prognostic modality for the detection of IA in high risk patients.

Utility of GenoType MTBDRplus assay in rapid diagnosis of multidrug resistant tuberculosis at a tertiary care centre in India.

PURPOSE: Molecular methods which allow rapid detection of tuberculosis as well as drug resistance directly from clinical samples have become the most popular diagnostic methodology with the emergence of multidrug resistant tuberculosis. The aim of the present study was to evaluate the performance of a line probe assay, GenoType MTBDRplus for the rapid detection of Mycobacterium tuberculosis and mutations causing rifampicin and INH resistance directly in smear positive pulmonary specimens and also in M. tuberculosis isolates grown from various clinical specimens. MATERIALS AND METHODS: The MTBDRplus assay was done directly on 37 smear positive pulmonary specimens and also on 69 M. tuberculosis isolates obtained by rapid automated culture using Bact/Alert 3D. The results were compared with phenotypic drug susceptibility testing (1% proportion method) using Bact/Alert 3D. RESULTS: The sensitivity and specificity for detection of resistance to rifampicin was 100% and 97.3%, and to INH was 91.9% and 98.4%, respectively, in comparison with the phenotypic drug susceptibility testing. CONCLUSION: MTBDRplus assay had good sensitivity and specificity with turn around time of less than 48 hours. It may be a useful tool for rapid detection of multidrug resistant tuberculosis at a tertiary care centre.

Use of John Cunningham virus polymerase chain reaction in the diagnosis of progressive multifocal leucoencephalopathy - a rare presenting manifestation in an HIV-positive patient.

John Cunningham virus infection is an important cause of progressive multifocal leucoencephalopathy (PML) in the context of advanced human immunodeficiency virus infection. Limited data are available regarding the true incidence of PML as a presenting manifestation of HIV. We report one such case and also highlight the effective use of polymerase chain reaction in confirming its diagnosis.

Clinical Pulmonary Infection Score to diagnose ventilator-associated pneumonia in children.

BACKGROUND: There is a need to validate and suggest easy clinical method for diagnosis of ventilator-associated pneumonia (VAP) in developing countries. OBJECTIVES: To validate the use of simplified Clinical Pulmonary Infection Score (CPIS) for the diagnosis of VAP. DESIGN: Prospective study. SETTING: Pediatric intensive care unit of a tertiary care teaching hospital. SUBJECTS: 30 children receiving mechanical ventilation for more than 48 hours and with simplified CPIS=6. METHODS: All patients underwent flexible bronchoscopy to obtain bronchoalveolar lavage which was analyzed quantitatively. Colony count = 10(4) cfu/mL was considered reference standard for definite VAP. RESULTS: Of the five variables used for simplified CPIS, only patients temperature (P=0.013) and PaO2/FiO2 ratio were significant (P<0.001) to differentiate the presence of definite VAP. Patients with definite VAP (BAL colony count = 10(4) cfu/mL) had CPIS of 8.4 while in no definite VAP group it was 6.4 (P=0.007). CPIS of 8 was found to have sensitivity of 80%, specificity 80%, PPV 86.9%, NPV 70.5% and accuracy 80%. The area under Receiver operating characteristic curve of CPIS against reference standard was 0.81± 0.069 (P=0.001). CONCLUSION: Simplified CPIS is useful in patients on mechanical ventilation to diagnose ventilator-associated pneumonia.

Dengue NS1 antigen detection: a useful tool in early diagnosis of dengue virus infection.

PURPOSE: This study was undertaken to evaluate the efficacy of NS1 antigen (Ag) assay as an early marker for dengue virus (DV) infection. MATERIALS AND METHODS: Group I evaluated the performance of NS1 antigen (Ag) assay in comparison to MAC-ELISA and their detection rate when performed together in a single sample. Six hundred acute/early convalescent sera were screened by both the assays. Group II evaluated the efficacy of a single assay in 30 acute phase sera of paediatric OPD patients screened only by NS1 Ag assay. Group III evaluated the specificity of NS1 assay in comparison to MAC-ELISA on 40 samples included as controls. RESULTS: In Group I, 140 (23.3%) and 235 (39.1%) samples were positive by NS1 assay and MAC-ELISA respectively. The detection rate increased to 320 (53.3%) when both the assays were used together on a single sample. NS1 Ag positivity varied from 71.42% to 28.4% in acute and early convalescent sera, conversely IgM detection rate was 93.61% and 6.38% in early convalescent and acute phase sera respectively (P < 0.0001). In Group II, 66.66% (20) samples were positive by NS1 assay. All the samples in Group III were negative showing 100% specificity of both the assays. CONCLUSION: NS1 Ag assay holds promise in early diagnosis of dengue infection. When used in combination with MAC-ELISA on a single sample it significantly improves the diagnostic algorithm without the requirement of paired sera.