Tushar Shaw

Antifungal Susceptibility Patterns, In Vitro Production of Virulence Factors, and Evaluation of Diagnostic Modalities for the Speciation of Pathogenic Candida from Blood Stream Infections and Vulvovaginal Candidiasis

Candida spp. have emerged as successful pathogens in both invasive and mucosal infections. Varied virulence factors and growing resistance to antifungal agents have contributed to their pathogenicity. We studied diagnostic accuracy of HiCrome Candida Differential Agar and Vitek 2 Compact system for identification of Candida spp. in comparison with species-specific PCR on 110 clinical isolates of Candida from blood stream infections (54, 49%) and vulvovaginal candidiasis (56, 51%). C. albicans (61%) was the leading pathogen in VVC, while C. tropicalis (46%) was prominent among BSIs. HiCrome Agar and Vitek 2 Compact had good measures of agreement (κ) 0.826 and 0.895, respectively, in comparison with PCR. We also tested these isolates for in vitro production of proteinase, esterase, phospholipases, and biofilms. Proteinase production was more among invasive isolates (P = 0.017), while phospholipase production was more among noninvasive isolates (P = 0.001). There was an overall increase in the production of virulence factors among non-albicans Candida. Identification of clinical isolates of Candida up to species level either by chromogenic agar or by Vitek 2 Compact system should be routinely done to choose appropriate therapy.

Unravelling the Molecular Epidemiology and Genetic Diversity among Burkholderia pseudomallei Isolates from South India Using Multi-Locus Sequence Typing.

There is a slow but steady rise in the case detection rates of melioidosis from various parts of the Indian sub-continent in the past two decades. However, the epidemiology of the disease in India and the surrounding South Asian countries remains far from well elucidated. Multi-locus sequence typing (MLST) is a useful epidemiological tool to study the genetic relatedness of bacterial isolates both with-in and across the countries. With this background, we studied the molecular epidemiology of 32 Burkholderia pseudomallei isolates (31 clinical and 1 soil isolate) obtained during 2006–2015 from various parts of south India using multi-locus sequencing typing and analysis. Of the 32 isolates included in the analysis, 30 (93.7%) had novel allelic profiles that were not reported previously. Sequence type (ST) 1368 (n = 15, 46.8%) with allelic profile (1, 4, 6, 4, 1, 1, 3) was the most common genotype observed. We did not observe a genotypic association of STs with geographical location, type of infection and year of isolation in the present study. Measure of genetic differentiation (FST) between Indian and the rest of world isolates was 0.14413. Occurrence of the same ST across three adjacent states of south India suggest the dispersion of B.pseudomallei across the south western coastal part of India with limited geographical clustering. However, majority of the STs reported from the present study remained as “outliers” on the eBURST “Population snapshot”, suggesting the genetic diversity of Indian isolates from the Australasian and Southeast Asian isolates.

The antibiotics of choice for the treatment of melioidosis in Indian set up

Therapeutic options for the treatment of melioidosis caused by Burkholderia pseudomallei are limited due to the inherent resistance conferred by this pathogen to various groups of antibiotics. Witnessing an increase in the number of microbiological culture-confirmed cases of melioidosis at our settings in the past few years, we undertook this study to estimate the minimum inhibitory concentrations of clinical isolates of B. pseudomallei against the four commonly employed antimicrobial agents in the patient management at our settings, namely, ceftazidime, meropenem, trimethoprim-sulfamethoxazole and doxycycline. All isolates were susceptible to the antibiotics tested, except for one isolate which showed resistance to doxycycline (minimum inhibitory concentration [MIC]: 32 μg/ml). MIC50 and 90 for all the four antibiotics were estimated. From this study, we conclude that the clinical isolates of B. pseudomallei from the southern part of India are well susceptible to the commonly employed antimicrobial agents for therapy.

Performance evaluation of Active Melioidosis Detect-Lateral Flow Assay (AMD-LFA) for diagnosis of melioidosis in endemic settings with limited resources

Melioidosis is a fatal infection caused by the soil saprophyte Burkholderia pseudomallei. Early diagnosis and befitting medical management can significantly influence the clinical outcomes among patients with melioidosis. Witnessing an annual increment in the number of melioidosis cases, over the past few years, mainly from the developing tropical nations, the present study was undertaken to evaluate the diagnostic utility of Active Melioidosis DetectTMLateralFlow Assay (AMD-LFA), in comparison with enrichment culture and PCR. A total of 206clinical specimens obtained from 175 patients with clinical suspicion of melioidosis were considered for the evaluation. Positivity for B.pseudomallei using enrichment culture, PCR and AMD-LFA were observed among 63 (30.5%), 55 (26.6%) and 63 (30.5%) specimens respectively. The AMD-LFA failed to detect melioidosis from 9 culture-confirmed cases (6 whole blood specimens, 2 pus samples, and one synovial fluid). Further the test gave faint bands from 9 urine samples which were negative by culture and PCR. AMD-LFA demonstrated a sensitivity, specificity, of 85.71%(CI:74.61% to 93.25%) and 93.62% (CI:88.23% to 97.04%), with positive predictive value of 85.71% (CI: 75.98% to 91.92%) and negative predictive value of 93.62% (CI:88.89% to 96.42%). The test needs further evaluation in view of the faint bands from negative urine samples, for incorporating the test as a point of care assay.In view of its rapidity and ease of testing AMD-LFA might be useful in early diagnosis of melioidosis at resource constraint settings.

Melioidosis in South Asia (India, Nepal, Pakistan, Bhutan and Afghanistan)

Despite the fact that South Asia is predicted to have the highest number of cases worldwide, melioidosis is a little-known entity in South Asian countries. It has never been heard of by the majority of doctors and has as yet failed to gain the attention of national Ministries of Health and country offices of the World Health Organization (WHO). Although a few centers are diagnosing increasing numbers of cases, and the mortality documented from these institutions is relatively high (nearly 20%), the true burden of the disease remains unknown. In India, most cases have been reported from southwestern coastal Karnataka and northeastern Tamil Nadu, although this probably simply reflects the presence of centers of excellence and researchers with an interest in the disease. As elsewhere, the majority of cases have type 2 diabetes mellitus and occupational exposure to the environment. Most present with community-acquired pneumonia and/or bacteremia, especially during heavy rainfall. The high seropositivity rate (29%) in Karnataka and isolation of B. pseudomallei from the environment in Tamil Nadu and Kerala confirm India as melioidosis-endemic, although the full extent of the distribution of the organism across the country is unknown. There are limited molecular epidemiological data, but, thus far, the majority of Indian isolates have appeared distinct from those from South East Asia and Australia. Among other South Asian countries, Sri Lanka and Bangladesh are known to be melioidosis-endemic, but there are no cases that have conclusively proved to have been acquired in Nepal, Bhutan, Afghanistan or Pakistan. There are no surveillance systems in place for melioidosis in South Asian countries. However, over the past two years, researchers at the Center for Emerging and Tropical Diseases of Kasturba Medical College, University of Manipal, have established the Indian Melioidosis Research Forum (IMRF), held the first South Asian Melioidosis Congress, and have been working to connect researchers, microbiologists and physicians in India and elsewhere in South Asia to raise awareness through training initiatives, the media, workshops, and conferences, with the hope that more patients with melioidosis will be diagnosed and treated appropriately. However, much more work needs to be done before we will know the true burden and distribution of melioidosis across South Asia.

Melioidosis: the great mimicker presenting as spondylodiscitis

Melioidosis, a syndrome with protean clinical manifestations, is caused by Gram-negative soil saprophyte Burkholderiapseudomallei. Among its diverse clinical presentations, the involvement of spine is a rare phenomenon and can mimic tuberculosis on presentation. A 65-year-old female with a known case of diabetes presented with fever with lower back pain. Blood culture grew Staphylococcus aureus, and as per sensitivity report, clindamycin and cefazolin were started. X-ray and MRI lumbosacral spine showed spondylodiscitis (likely Koch’s). Decompression and biopsy were done, and a sample was sent for microbiological investigations that showed no growth of any significant pathogen; furthermore, all tests for tuberculosis diagnosis also remained negative. Active Melioidosis Detect Lateral Flow Assay was used on the tissue sample, which was positive for B. pseudomallei Capsular Polysaccharide (CPS) antigen; the case was confirmed by typethree secretion system 1 PCR for melioidosis. Antibiotics were changed to parenteral ceftazidime for 2 weeks followed by oral cotrimoxazole. A dedicated team of microbiologists and physicians is required to identify and treat the disease.

Melioidosis: Reinfection Going Incognito as Relapse

Melioidosis has recently gained importance as an emerging disease in India. Recurrent melioidosis has been reported from different parts of the world and can be due to relapse or reinfection. Distinction between relapse and reinfection is important for epidemiology, investigation and management. Here, we present the data regarding rate of recurrence and utility of multilocus sequence typing (MLST) in differentiating relapse form reinfection amongst melioidosis patients from a tertiary care hospital in South India. Amongst the 31 patients who survived and underwent follow-up, 4 (13%) presented with recurrence. Three cases (75%) were identified as reinfection and one (25%) as relapse based on MLST. Re-exposure to environmental Burkholderia pseudomallei amongst patients with melioidosis in endemic areas is likely. In such a scenario, more often than not, recurrence of melioidosis can be attributed to reinfection.

Genome Sequence of a Burkholderia pseudomallei Clinical Isolate from a Patient with Community-Acquired Pneumonia and Septicemia

ABSTRACT Here, we report the draft genome sequence of Burkholderia pseudomallei CM_Manipal, the causative agent of melioidosis isolated from a diabetic patient in Manipal, southern India. The draft genome consists of 107 contigs and is 7,209,157 bp long. A total of 5,600 coding sequences (CDSs), 60 tRNAs, 12 rRNAs, and one noncoding RNA (ncRNA) were predicted from this assembly.