Smita Sarma

A multicentre study of antifungal susceptibility patterns among 350 Candida auris isolates (2009–17) in India: role of the ERG11 and FKS1 genes in azole and echinocandin resistance

Background Candida auris has emerged globally as an MDR nosocomial pathogen in ICU patients. Objectives We studied the antifungal susceptibility of C. auris isolates (n = 350) from 10 hospitals in India collected over a period of 8 years. To investigate azole resistance, ERG11 gene sequencing and expression profiling was conducted. In addition, echinocandin resistance linked to mutations in the C. auris FKS1 gene was analysed. Methods CLSI antifungal susceptibility testing of six azoles, amphotericin B, three echinocandins, terbinafine, 5-flucytosine and nystatin was conducted. Screening for amino acid substitutions in ERG11 and FKS1 was performed. Results Overall, 90% of C. auris were fluconazole resistant (MICs 32 to ≥64 mg/L) and 2% and 8% were resistant to echinocandins (≥8 mg/L) and amphotericin B (≥2 mg/L), respectively. ERG11 sequences of C. auris exhibited amino acid substitutions Y132 and K143 in 77% (n = 34/44) of strains that were fluconazole resistant whereas WT genotypes, i.e. without substitutions at these positions, were observed in isolates with low fluconazole MICs (1-2 mg/L) suggesting that these substitutions confer a phenotype of resistance to fluconazole similar to that described for Candida albicans. No significant expression of ERG11 was observed, although expression was inducible in vitro with fluconazole exposure. Echinocandin resistance was linked to a novel mutation S639F in FKS1 hot spot region I. Conclusions Overall, 25% and 13% of isolates were MDR and multi-azole resistant, respectively. The most common resistance combination was azoles and 5-flucytosine in 14% followed by azoles and amphotericin B in 7% and azoles and echinocandins in 2% of isolates.

Comparison of DNA extraction protocols for Mycobacterium Tuberculosis in diagnosis of tuberculous meningitis by real-time polymerase chain reaction

Background: Several nucleic acid amplification techniques are available for detection of Mycobacterium tuberculosis (MTB) in pulmonary and extrapulmonary samples, but insufficient data are available on the diagnostic utility of these techniques in tubercular meningitis where bacilli load is less. The success of final amplification and detection of nucleic acid depends on successful extraction of DNA from the organism. Aims: We performed this study to compare four methods of extraction of MTB DNA from cerebrospinal fluid (CSF) samples so as to select one method of DNA extraction for amplification of nucleic acid from clinical samples. Materials and Methods: Four methods of extracting MTB DNA from CSF samples for testing by real-time polymerase chain reaction (PCR) were compared: QIAGENR protocol for DNA purification using QIAamp spin procedure (manual), AMPLICORR respiratory specimen preparation kit, MagNA PureR kit extraction, combined manual DNA extraction with automated extraction by MagNA PureR. Real-time PCR was performed on COBAS TaqMan 48 AnalyzerR with known positive and negative controls. Results: The detection limit for the combined manual and MagNA PureR extraction protocol was found to be 100 copies of MTB DNA per reaction as against 1,000 copies of MTB DNA per reaction by the QIAGENR, AMPLICORR, and the MagNA PureR extraction protocol. Conclusion: The real-time PCR assay employing the combination of manual extraction steps with MagNA PureR extraction protocol for extraction of MTB DNA proved to be better than other extraction methods in analytical sensitivity, but could not detect less than 102 bacilli /ml.

Laboratory diagnosis of tuberculous meningitis - Is there a scope for further improvement?

Aims: Tuberculous meningitis (TBM) still remains a diagnostic challenge because of inconsistent clinical presentation and lack of rapid, sensitive and specific tests. This study was carried out to diagnose TBM by a combination of direct microscopy on Ziehl-Neelsen (ZN) staining, culture by conventional Lowenstein Jensen (LJ) media and Bactec MGIT 960 system in clinically suspected cases, supported by laboratory parameters. Materials and Methods: A total of 164 cerebrospinal fluid (CSF) samples from suspected cases of TBM were processed for direct acid fast bacilli (AFB) smear examination, and culture on Bactec MGIT 960 and LJ media. Results: AFB were detected on direct smears in 13 of 164 (7.9%) specimens and Mycobacterium tuberculosis was isolated by at least one of the culture methods from 49 (29.8%) CSF samples of which 45 (27.4%) were detected positive for M. tuberculosis by MGIT 960 culture and 18 (10.9%) by culture on LJ medium. The mean time of detection in MGIT and LJ media for M. tuberculosis were 18 and 38 days, respectively. Conclusions: A combination of laboratory parameters like smear microscopy, conventional culture and automated method like Bactec MGIT increases the sensitivity of diagnosing TBM as compared to any single method.

Elizabethkingia meningosepticum : An emerging cause of septicemia in critically Ill patients

62 blood culture were sterile. Widal test was reactive up to a dilution of 1:320 for Salmonella typhi antigens (TO). A high vaginal swab collected under aseptic conditions grew Salmonella typhi, suggesting a probable transplacental leakage secondary to bacteremia. The patient gave a history of fever 1 month back for which she was treated with some antibiotics by the local physician, details of which were not known. In the absence of any other evidences of spontaneous miscarriage, Salmonellosis can be presumed to be the most likely cause. According to Brion et al., Salmonella typhi should be considered in a pregnant woman with fever.[4] Hicks and French reported a high rate (65–85%) of abortion or premature labor with Salmonella typhi.[5]

Mycobacterium abscessus bacteremia in an immunocompetent patient following a coronary artery bypass graft.

Sternal osteomyelitis with bacteremia due to Mycobacterium abscessus is rarely seen in immunocompetent hosts. Routine pyogenic cultures in these cases are often negative causing a delay in diagnosis and treatment. Clinicians and microbiologists should rule out the possibility of infection due to nontuberculous mycobacteria while managing cases of nonhealing culture-negative wounds with conventional antibiotic therapy. We report a case of bacteremia secondary to a nonhealing sternal wound due to M. abscessus. A combination of radical debridement and prolonged antimicrobial therapy helped in the complete eradication of the infection.

Renal Abscess Caused by Salmonella Typhi.

Salmonella typhi is a true pathogen, which is capable of causing both intestinal and extraintestinal infections. Unusual presentations of Salmonella should always be kept in mind as this organism can cause disease in almost any organ of the body. S. typhi has been reported to cause the life-threatening infections such as meningitis, endocarditis, myocarditis, empyema, and hepatic abscess. Renal involvement by S. typhi is a relatively rare presentation. We report a case of renal abscess caused by S. typhi in an afebrile, 10-year-old child who did not have any clinical history of enteric fever. To our knowledge, this is the first reported case of isolation of S. typhi from the renal abscess, and interestingly this isolate was found to be resistant to quinolones.

Evaluation of a new molecular method illumigene for detection of Clostridium difficile associated diarrhoea

1. Clinical Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty First Informational Supplement. CLSI Document M100-S21. Wayne, PA: CLSI; 2011. 2. Hornsey M, Phee L, Wareham DW. A novel variant, NDM-5, of the New Delhi metallo-β-lactamase in a multidrug-resistant Escherichia coli ST648 isolate recovered from a patient in the United Kingdom. Antimicrob Agents Chemother 2011;55:5952-4. 3. Nordmann P, Naas T, Poirel L. Global spread of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2011;17:1791-8. 4. Poirel L, Castanheira M, Carrër A, Rodriguez CP, Jones RN, Smayevsky J, et al. OXA-163, an OXA-48-related class D ß-lactamase with extended activity toward expanded-spectrum cephalosporins. Antimicrob Agents Chemother 2011;55:2546-51.

Epstein–Barr virus infection–Related hemophagocytic lymphohistiocytosis

We report a case of 27-year-old female diagnosed with hemophagocytic lymphohistiocytosis (HLH) following a recent Epstein–Barr virus (EBV) infection. A known case of relapsing remitting multiple sclerosis on corticosteroids for last 6 months presented to the critical care unit with fever, maculopapular rash and difficulty in breathing. A rapid and correct diagnosis with the precise treatment led to complete recovery of this patient. The HLH is a rare complication of primary EBV infection.

Unusual presentation of a culture-positive right atrial mass

We report a patient with a mass in the right atrium which led to pulmonary embolism. Postoperatively the mass was identified as a tuberculoma and it was culture-positive for Mycobacterium tuberculosis. Patient responded to modified antitubercular treatment and discharged from hospital in satisfactory condition.