Objective. The study aims to speciate clinical Candida isolates and detect their biofilm-forming ability and antifungal resistance. Methods. All the Candida spp. isolated from different clinical samples like pus, urine, blood, and body fluid were included in the study. Biofilm production was tested by the microtiter plate method. Antifungal susceptibility was studied by the disk diffusion method. Patient’s demographic details such as age, sex, and clinical information were collected. Presence of other risk factors such as diabetes mellitus, history of antibiotic use, and any urinary tract instrumentations was also recorded. Results. Among 90 Candida species isolated, most predominant species was found to be C. albicans (45.5%) followed by C. tropicalis (28.88%), C. krusei (20%), C. glabrata (3.33%), and C. parapsilosis (2.22%). Candida spp. were isolated from urine (43%), BAL/sputum (18.88%), high vaginal swab (8.88%), suction tips (7.77%), blood and wound swabs (6.66%), pus (3.33%), bile aspirate (2.22%), and deep tissue (1.11%). A larger number of females were affected than males, and the age group of 51 to 60 years was more susceptible to candidiasis. A higher number of C. albicans isolates produced biofilm followed by C. parapsilosis, C. tropicalis, and C. krusei. However, C. glabrata showed no biofilm production in our study. All Candida isolates were 100% sensitive to amphotericin B. Voriconazole was the next effective drug with 81.11% susceptibility. 24.44% of strains were resistant to fluconazole. Conclusion. Speciation of Candida isolates, detection of ability to form the biofilm, and monitoring of antifungal susceptibility testing are necessary for appropriate treatment.

Rapid identification of Mycobacterium tuberculosis (MTB), its resistance to rifampicin and differentiation of MTB from nontuberculous mycobacteria (NTM) is necessary in the management of mycobacterial diseases. Culture, the "gold standard" for the detection of MTB, is time consuming. In spite of its rapidity and low cost, smear microscopy has poor sensitivity for the detection of acid-fast bacilli (AFB). A real-time PCR based rapid diagnostic method like GeneXpert MTB/RIF assay can simultaneously detect M. tuberculosis and rifampicin (RIF) resistance. Hence, we aim to compare the performance of GeneXpert MTB/RIF assay with smear microscopy and culture. In this descriptive cross-sectional study, we compared the performance of GeneXpert in pulmonary (N=127) and extrapulmonary (N=48) clinical specimens with other diagnostic methods like culture, Auramine O (AO), and Ziehl Neelsen (ZN) staining. Rifampicin resistance was detected only by GeneXpert. Demographic data and clinical history of the subjects were collected from the patient’s hospital records. AO and ZN staining when compared with mycobacterial growth indicator (MGIT) culture showed the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of 68.6, 95.7, 80, 92.4, 90.3% and 65.7, 95.7, 79.3, 91.8, 89.7%, respectively. The sensitivity, specificity, PPV, NPV and accuracy of GeneXpert was 88.6, 93.6, 77.5, 97.0 and 92.6%, respectively. GeneXpert is the best available rapid diagnostic method as it can detect MTB and rifampicin resistance gene simultaneously. Accuracy and negative predictive value of GeneXpert was found to be better than AFB staining. Thus, a negative GeneXpert test can rule out TB. Further, a negative GeneXpert and a positive smear microscopy results indicate the presence of NTM. However, GeneXpert is expensive and needs sophisticated instrument when compared to smear microscopy.

Enterococcus is a commensal in the intestine and is now emerging as a drug-resistant pathogen. It produces different virulence factors. Enterococcus surface protein (esp) is a virulence factor that helps in the adhesion, but its role in biofilm formation is still contradictory. Moreover, in many bacterial species, strong biofilm producer exhibits multidrug resistance. Hence, this study is done to know the antimicrobial susceptibility pattern of Enterococcus spp. and to correlate the drug resistance with biofilm production and esp gene. Enterococcal isolates were collected from various clinical specimens. Antibiotic susceptibility testing was done by disc diffusion, and biofilm production was performed by microtiter plate method. PCR was performed for detection of esp gene. Two E. faecium strains resistant to vancomycin and high-level aminoglycoside (HLAR) were non-biofilm-producers and did not harbor esp gene. However, other biofilm-producing E. faecium harbored esp gene, and this association was found to be statistically significant (p=0.024). It was observed that there was no significant association between biofilm formation and presence of esp gene in E. faecalis. Moreover, a significant correlation was not found between drug resistance and biofilm production in both Enterococcus species. Thus, biofilm formation is not always associated with the presence or absence of esp gene and or drug resistance in Enterococcus spp.

A fatal case of primary amebic meningoencephalitis (PAM) in a 5-month-old infant is described. The disease may have been contracted during bathing. The source of water was from an artificial well. The clinical presentation, the isolation of the ameba from the cerebrospinal fluid, the poor response to amphotericin B, and the ultimate fatal outcome are all consistent with the diagnosis of PAM. On the basis of its ability to grow at temperatures above 30°C, the morphology of the trophozoite, and the presence of flagellate forms, the ameba was identified as Naegleria fowleri . Pathogenic N. fowleri amebae were recovered from samples of water from the well. To our knowledge this case represents the second case of PAM in an infant in the absence of the history of swimming.

In resource-limited tuberculosis-endemic countries, Mycobacterium tuberculosis in sputum is mainly detected by acid-fast bacillus (AFB) staining and the identification of sputum-derived cultures. PCR techniques are practical only in well-resourced laboratories. This study investigated the application of a rapid, simple, and inexpensive fluorescence in situ hybridization (FISH) assay to identify and differentiate M. tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) in sputum. The Mycobacterium/Nocardia Genus (MN Genus)-MTBC FISH assay performed in this study utilizes two different DNA probes labeled with different fluorescent molecules that hybridize respectively with 16S rRNA of the genus Mycobacterium and 23S rRNA of MTBC. The assay was tested on 202 patient sputum samples in Mangaluru, Karnataka State, India. Sputa were first liquefied and bacteria concentrated before performing the FISH assay and parallel culturing and AFB staining. The identities of cultured bacteria from DNA sequencing were compared with FISH assay findings from corresponding sputa. Of the 202 sputum samples tested, 67 reacted with both MN Genus-specific and MTBC-specific probes, none reacted only with the MTBC-specific probe, and 22 reacted only with the MN Genus-specific probe. The FISH assay yielded results in 2h and had a limit of detection of 2.2×104CFU/ml in sputum spiked with cultured M. tuberculosis. The diagnostic sensitivity, specificity, and positive and negative predictive values of the FISH assay for MTBC in patient sputa were 89.7%, 95.5%, 88.0%, and 92.6%, respectively. NTM were a significant cause of tuberculosis-like infections in Mangaluru. The MN Genus-MTBC dual probe fluorescence FISH assay previously applied to cultures can also be utilized in resource-limited tuberculosis-endemic countries for rapidly identifying and differentiating MTBC and NTM in sputum samples.