Pulmonary nocardiosis: Risk factors and species distribution from a high burden centre.

Nocardiosis is a clinical and diagnostic challenge. This was a retrospective study carried out on cases of pulmonary nocardiosis presenting over 15 years. Clinical data was retrieved using the electronic patient records. Vitek MS 3.2 (MALDI TOF MS) was carried out on 22 isolates and sequencing on another 9 isolates. Of 71 patients presenting with pulmonary nocardiosis, 58 (81.6%) were on immunosuppressant therapy, 26 (46%) had a previous lung pathology, 11 (8%) were HIV associated. Disseminated disease was seen in 6 (8.4%). There were 8 (11.26%) deaths in this cohort of patients. Of 31/71 identified to species, the most common were Nocardia cyriacigeorgica (n â€‹= â€‹11) followed by Nocardia farcinica (n â€‹= â€‹9).

Cryptic aspergillosis: a rare entity and a diagnostic challenge.

Introduction: Cryptic aspergillosis, caused by cryptic species of Aspergillus, is increasingly reported in humans and causes significant morbidity and mortality in immunocompromised individuals. The main aim of this study was to describe the occurrence of this entity at a large tertiary care centre and analyse the challenges in identifying them in a routine diagnostic laboratory. Methods: This was a retrospective case review of all patients diagnosed with cryptic Aspergillus species from April 2019 to February 2020. The isolates were identified using conventional microbiological techniques, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF MS), 28S rRNA and internal transcribed spacer (ITS) sequencing. Results: The species identified were Aspergillus tamarii, Aspergillus lentulus and Aspergillus sydowii. Identification by MALDI- TOF MS and sequencing was concordant for all except A. sydowii, with MALDI- TOF MS misidentifying it as Aspergillus thermomutans. All isolates showed low minimum inhibitory concentrations (MICs) for the panel of antifungal drugs. Conclusion: Aspergillosis caused by cryptic Aspergillus species presents a diagnostic challenge. This study confirms the importance of molecular methods for accurate identification.

Accuracy of Xpert MTB/RIF Ultra for the diagnosis of tuberculosis in adult patients: a retrospective cohort study.

OBJECTIVES: The value of the "trace" result in Xpert Ultra for diagnosing active tuberculosis (TB) remains unclear. Our study evaluated the diagnostic accuracy of Xpert MTB/RIF Ultra (Xpert Ultra) (Cepheid, Sunnyvale, USA) over Xpert MTB/RIF (Xpert) (Cepheid, Sunnyvale, USA) and mycobacterial culture when compared with a composite reference standard (CRS). METHODS: A retrospective single-center observational study was conducted in a tertiary care hospital in South India. Over three months, patients (aged ≥15 years) data on Xpert Ultra tests and mycobacterial culture of pulmonary and extrapulmonary samples were extracted from their electronic medical records. Patients were defined as TB cases based on the CRS criteria. Sensitivity, specificity, positive and negative predictive values of diagnostic tests were calculated by comparing them to the CRS. RESULTS: Xpert Ultra was more sensitive (87.8%) than Xpert (72.1%) and culture (44.1%). The specificity of Xpert Ultra was lower (98.1%) than those of Xpert (100%) and culture (100%). The sensitivity (92%) and specificity (100%) of Xpert Ultra were highest when performed on pus samples. CONCLUSIONS: Xpert Ultra with the trace category is superior to the conventional Xpert, and mycobacterial culture in identifying TB.

Candida utilis: a rare cause of septicemia in children.

Candida utilis is an emerging fungal pathogen in blood. The main aim of this study was to describe the prevalence, methods of speciation and antifungal susceptibility of Candida utilis at a tertiary care centre. METHODS: This was a retrospective study carried out at a tertiary care centre in South India. Over a period of 1 year, three Candida utilis were isolated from blood culture identified by MALDI-TOF MS Version 3.2 and were confirmed by ITS sequencing. Susceptibility testing was carried out by micro broth dilution. RESULTS: All three patients had a common risk factor of prolonged ICU stay but the source of infection could not be identified. Candida utilis isolates were identified by MALDI-TOF and confirmed by ITS sequencing. They were pansusceptible to all tested antifungal drugs. Among these, two patients who were treated in hospital had good clinical outcome and response to antifungal drugs. A third patient was lost to follow up. CONCLUSION: Candida utilis was predominantly seen between 0-3 month olds. Conventional methods of speciation were unable to identify C. utilis to species level. Rapid identification was done by MALDI-TOF MS and confirmed by sequencing. Rapid identification leads to prompt treatment and favours a good clinical outcome.

Cryptococcosis with Tuberculosis: Overlooked Coinfections.

Tuberculosis and cryptococcosis are important opportunistic pathogens causing significant morbidity and mortality in immunocompromised individuals. Concurrent infections of these two agents are rarely reported. We report five cases of culture-proven coinfection of Mycobacterium tuberculosis and Cryptococcus neoformans during inpatient admission at a tertiary care hospital in southern India between 2007 and 2019. Four patients were persons living with HIV infection and one was on immune suppression for chronic renal disease. Maintaining a high degree of clinical suspicion will ensure early diagnosis and appropriate management of coinfections in the immunocompromised individual.

Challenges in the management of slowly growing non-tuberculous mycobacteria causing pulmonary disease: Perspectives from a high burden country.

PURPOSE: There is a dearth of data on epidemiology, diagnosis and management of slow growing non tuberculous mycobacteria(NTM) in India, despite being a TB endemic country. This study aims to describe the geographic distribution, risk factors, and the challenges in management of slow growing NTM causing pulmonary infections. METHODS: Over a period of 3 years, all slow growing NTM received from pulmonary specimens at a tertiary care centre were further studied from electronic hospital records to correlate non tuberculous mycobacteria species with demographics, geographic location, describe comorbidities including immunosuppression, radiologic findings and treatment regimes. RESULTS: M.intracellullare was found in the majority of isolates with significant geographic variation and M.simiae the second commonest found exclusively in southern states. Common comorbidities included a previous history of treatment for tuberculosis, structural lung disease as well as systemic risk factors. Disseminated disease only occurred in immunocompromised hosts as was expected, but at a high rate of 44%. Treatment completion and outcomes were difficult to attain in our population. CONCLUSION: The burden of NTM infection and its management in India remain a challenge. Ensuring it is made a notifiable disease may improve the current situation.

Clinical implications of high-risk mutations in drug resistant tuberculosis (DR-TB): An observational cohort study.

Genotype MTBDRsl [SL-LPA] was endorsed as a tool for early diagnosis of fluoroquinolones (FQ) and injectable second-line TB drugs (SLID) resistance in DR-TB. Correlation between specific genetic mutations using this tool and clinical outcome has not hitherto been studied in India. We conducted a observational cohort study to evaluate the predictive value of specific mutations for bad outcome. Our study identified 15 different types of gyrA mutations, commonest being A90V and D94G. Poor outcome was associated with mutations D94G and D94N/D94Y.Most XDR-TB patients harbored the high risk mutation of A1401G. Hence information of specific mutations using SL-LPA can help prognosticate and design appropriate treatment regimens.

Differential gene expression profile of Shigella dysenteriae causing bacteremia in an immunocompromised individual.

AIM: Shigella species has varying levels of virulence gene expression with respect to different sites of infection. In this study, the differential gene expression of S. dysenteriae in response to its site of infection was analyzed by transcriptomics. METHODS: This study includes four clinical Shigella isolates. Transcriptomics was done for the stool and blood samples of a single patient. Isolates were screened for the presence of antimicrobial resistance genes. RESULTS: The majority of genes involved in invasion were highly expressed in the strain isolated from the primary site of infection. Additionally, antimicrobial resistance (dhfr1A, sulII, bla OXA. bla CTX-M-1 and qnrS) genes were identified. CONCLUSION: This study provides a concise view of the transcriptional expression of clinical strains and provides a basis for future functional studies on Shigella spp.

Candida auris: Clinical profile, diagnostic challenge and susceptibility pattern: Experience from a tertiary-care centre in South India.

OBJECTIVES: Candida auris is an emerging, multidrug-resistant yeast transmitted in healthcare settings. Conventional methods of speciation are unable to identify C. auris to species level. Three methods, namely VITEK® MS v.3.0, VITEK®2 and target sequencing of the internal transcribed spacer (ITS) region and 28S rRNA gene, were evaluated. Antifungal susceptibility testing (AFST) was also performed, and risk factors for acquisition of C. auris candidaemia were studied. METHODS: Between November 2016 and November 2017, 203 Candida spp. were isolated from blood cultures, of which 11 isolates that were unidentifiable by conventional methods were further tested by VITEK® MS v.3.0 and VITEK®2 and were confirmed by sequencing. AFST was carried out on all 11 isolates by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Clinical and epidemiological data of all patients retrieved from electronic patient records were reviewed. RESULTS: Of the 11 isolates identified as C. auris both by ITS and 28S rRNA sequencing, VITEK®2 identified only 5 as C. auris and VITEK® MS v.3.0 was not able to identify any of them as C. auris. Ten isolates (91%) were resistant to fluconazole, whereas all isolates were susceptible to amphotericin B and caspofungin. CONCLUSION: Candida auris can be misidentified in routine microbiology laboratories. Sequencing remains the gold standard if commercial identification systems are not updated.

Robustness of the Line Probe Assay for the Rapid Diagnosis and Characterization of Mutations in Extensively Drug-Resistant Tuberculosis.

Introduction: Extensively drug-resistant tuberculosis (XDRTB) is a public health concern. We evaluated the diagnostic accuracy of Genotype® MTBDRsl for detection of resistance to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs) and characterized mutations seen. Materials and Methods: MTBDRsl was carried out either directly on sputum samples or indirectly on culture isolates (n = 100) from known multidrug-resistant tuberculosis (MDRTB) patients from July 2015 to September 2017. Diagnostic accuracy for the detection of resistance to FQs and SLIDs was calculated in comparison with conventional culture-based drug susceptibility testing. Mutations at the gyrA and rrs loci, as well as discrepant phenotypic and genotypic results, were studied. A subset of isolates underwent pyrosequencing. Results: Out of 100 MDRTB samples/isolates tested, 59% were pre-XDRTB and 7% were XDRTB. The sensitivity and specificity for the detection of resistance to FQs were 96.6% [95% confidence interval (CI): 88.3-99.6] and 80% [95% CI: 64.4-90.9] and those for SLIDs were 70% [95% CI: 34.8-93.3] and 100% [95% CI: 95.9-100]. The most frequent mutations were the absence of wild type 3 with corresponding mutation 3c (20/66) at the gyrA locus, and absence of wild type 1 and corresponding mutation 1 (6/7) at the rrs locus. The absence of a wt2 band with a corresponding mutation at the gyrA locus was seen in four of eight patients with discrepant genotypic and phenotypic results for FQ resistance. All isolates tested by pyrosequencing (n = 5) were concordant with the line probe assay for FQ resistance with identical mutations (D94G) and four of five isolates were concordant with SLIDs with identical mutations (A1401G). Conclusion: The MTBDRsl is a useful test for accurate diagnosis of XDRTB and may help to tailor therapy.

The diagnostic utility of line probe assays for multidrug-resistant tuberculosis.

Owing to the burden of multidrug-resistant tuberculosis, molecular techniques have been approved by the WHO for the rapid diagnosis of the same. The objectives of this prospective, diagnostic study, conducted at Christian Medical College, a tertiary care center in South India, were to compare the performance of line probe assay (GenoTypeMTBDRplus) with culture, as well as the Xpert MTB/Rif assay on sputum samples. Ninety-one consecutive suspects of multidrug-resistant pulmonary tuberculosis patients from January 2013 to June 2013 were enrolled in this study and the results of line probe assay compared to culture and Xpert MTB/Rif. Compared to culture, the assay demonstrated a sensitivity and specificity of 81.5% (95%CI 67.4-91.1%) and 87.5% (95%CI 71-96.5%) for the detection of tuberculosis, with sensitivity and specificity of 100% (95%CI 85.2-100%) and 93.8% (95%CI 69.8-99.8%), respectively, for rifampicin resistance. For isoniazid resistance, sensitivity and specificity were 89.3% (95%CI 71.8-97.7%) and 100% (95%CI 71.5-100%), respectively. Compared to Xpert MTB/Rif assay, the assay showed a sensitivity of 80% (95%CI 68.2-88.9%) and specificity of 100% (95%CI 85.8-100%) for the detection of tuberculosis a sensitivity of 94.3% (95%CI 80.8-99.3%) and specificity of 94.1% (95%CI 71.3-99.9%) for rifampicin resistance was attained. This assay performed well on smear positive samples, but poorly on smear negative and scanty samples, and can serve as a rapid diagnostic tool, particularly in isoniazid monoresistant cases of tuberculosis, which are not diagnosed by Xpert MTB/Rif.

Extended spectrum ß-lactamase producing Shigella flexneri serotype-2 causing bacteremia in a patient with uncontrolled diabetes mellitus.

We report a case of Shigella flexneri serotype-2 causing bacteremia in an elderly gentleman with uncontrolled diabetes mellitus, who had no other apparent risk factors. Antibiotic susceptibility testing revealed that the organism was a multidrug resistant extended spectrum beta-lactamase producing straian, which was confirmed by molecular characterization. This rare case alerts both the clinician and microbiologist to a previously unaddressed risk factor of Shigella spp. causing bacteremia, as well as emerging resistant strains that are on the rise in immunocompromised patients.

Xpert MTB/Rif for the diagnosis of extrapulmonary tuberculosis--an experience from a tertiary care centre in South India.

OBJECTIVE: The Xpert MTB/Rif, with a detection limit of 131 CFU/ml, plays a valuable role in the diagnosis of extrapulmonary tuberculosis, both susceptible and resistant. This study aims at evaluating the Xpert MTB/Rif for the same, at a tertiary care centre in south India, assessing it against both culture and a composite gold standard (CGS). METHODS: We tested consecutive samples from patients suspected of extrapulmonary tuberculosis with Xpert MTB/Rif, evaluated its sensitivity and specificity against solid and/or liquid culture and CGS. An individual analysis of different sample types (tissue biopsies, fluids, pus, lymph node biopsies and CSF) given an adequate sample size, against both culture and CGS, was also performed. RESULTS: In total, 494 samples were analysed against culture. Compared to culture, the sensitivity of Xpert MTB/Rif was 89% (95% CI 0.81-0.94) and its specificity was 74% (95% CI 0.70-0.78). When Xpert MTB/Rif was compared to the CGS, pooled sensitivity was 62% (95% CI 0.56-0.67) and specificity was 100% (95% CI 0.91-1.00). CONCLUSION: This assay performs better than the currently available conventional laboratory methods. The rapidity with which results are obtained is an added advantage, and its integration into a routine diagnostic protocol must be considered.