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The rapid identification of bacterial pathogens in clinical samples like blood, urine, pus, and sputum is the need of the hour. Conventional bacterial identification methods like culturing and nucleic acid-based amplification have limitations like poor sensitivity, high cost, slow turnaround time, etc. Raman spectroscopy, a label-free and noninvasive technique, has overcome these drawbacks by providing rapid biochemical signatures from a single bacterium. Raman spectroscopy combined with chemometric methods has been used effectively to identify pathogens. However, a robust approach is needed to utilize Raman features for accurate classification while dealing with complex data sets such as spectra obtained from clinical isolates, showing high sample-to-sample heterogeneity. In this study, we have used Raman spectroscopy-based identification of pathogens from clinical isolates using a deep transfer learning approach at the single-cell level resolution. We have used the data-augmentation method to increase the volume of spectra needed for deep-learning analysis. Our ResNet model could specifically extract the spectral features of eight different pathogenic bacterial species with a 99.99% classification accuracy. The robustness of our model was validated on a set of blinded data sets, a mix of cultured and noncultured bacterial isolates of various origins and types. Our proposed ResNet model efficiently identified the pathogens from the blinded data set with high accuracy, providing a robust and rapid bacterial identification platform for clinical microbiology.

Ongoing need of alternative strategies for SARS-CoV-2 detection is undeniable. Self-collected samples without viral transport media (VTM), coupled with simple nucleic acid extraction methods for SARS-CoV-2 PCR are beneficial.To evaluate results of SARS-CoV-2 PCR using simple nucleic acid extraction methods from self -collected saliva and oral swabs without VTM.A cross-sectional single-centre study was conducted on 125 participants (101 SARS-CoV-2 positive cases and 24 controls). PCR was performed following five simple nucleic acid extraction methods on self -collect saliva and oral swabs without VTM and results were compared with gold standard PCR. For saliva, kit-based extraction (SKE), Proteinase K and Heat extraction (SPHE), only Heat extraction (SHE) methods and for dry oral swabs, Proteinase K and Heat extraction (DPHE) and only Heat extraction (DHE) was performed.SARS-CoV-2 was detected in self-collected saliva and oral swabs. 93.07% were correctly classified as positive by SKE, 69.31% by SHE, 67.33% by SPHE, 67.33% by DPHE and 55.45% by DHE. Discriminant power of SKE was significantly higher than other methods (p-value0.001) with good- fair agreement of alternate extraction methods against gold standard.Combination of self-collected saliva/ oral-swab without VTM and alternative RNA extraction methods offer a simplified, economical substitute strategy for SARS-CoV-2 detection.

Background: Pseudomonas aeruginosa is an important cause of multidrug-resistant nosocomial infections. The knowledge of resistance mechanisms in Pseudomonas is an important issue for antimicrobial treatment. Therefore, the objective was to detect carbapenems resistance in P. aeruginosa by phenotypic methods and genes (blaIMP and blaVIM) coding for carbapenemase resistance. Materials and Methods: The study conducted in the Department of Microbiology, BMCRI, Bengaluru. 91 samples from the patients hospitalised for 48 hours and more were processed. Carbapenem resistant P. aeruginosa (CRPA) were identified by biochemical tests and by Kirby Bauer Disk diffusion method as per CLSI guidelines. Those carbapenem resistant isolates were further subjected to two MBL detecting phenotypic tests- Modified Hodge Test (MHT) and Combined disk Test (CDT) by using imipenem and imipenem/ EDTA disk and MBL genes (blaIMP and blaVIM) were identified by PCR. Results: 91 clinical isolates were identified as CRPA, 92.3% isolates were positive by CDT whereas only 13.2% of isolates showed positive by MHT method indicating MBL production. Among 91 strains, 19.04% strains were harbouring blaVIM and 1.2% strain harbouring blaIMP gene. Conclusion: The detection of MBL-producing P. aeruginosa help in appropriate antimicrobial therapy and avoid development and dissemination of these strains. Hence routine detection of MBL production in P. aeruginosa should be undertaken. All CRPA isolates should be routinely screened for MBL production using CDT and positive isolates should be confirmed by PCR. MHT test had low sensitivity. To understand the epidemiology, there is a need of genetic analysis and typing of MBL enzymes. Keywords: Metallobetalactamases, Carbapenemase, Pseudomonas aeruginosa.

Uropathogenic

The neonatal skin microbiome consists of all the genomes and genetic products of microorganisms harboring on an infant’s skin. Host and the microbiota develop a harmonious environment resulting in symbiosis. Any disruption of this environment could lead to pathological disease. This study was conducted to understand the neonatal skin microbiome of very preterm neonates (under 32 weeks) admitted to the Neonatal Intensive Care Unit(NICU) at a tertiary healthcare setting before and after kangaroo mother care (KMC), using next-generation sequencing (NGS). Skin swabs were collected on two different occasions and analyzed using the NGS technique after amplification via polymerase chain reaction. The results showed relative abundance for Mycobacterium tuberculosis in 83.33% and 66.67% (p = 0.29) and Mycobacteroides abscessus in 100% and 93.33% (p = 0.30) of the very preterm neonates on the skin microbiome before and after KMC, respectively as an incidental finding. The mere presence of these bacilli as commensals or as potential pathogens is alarming due to the risk of early exposure and incidence of tuberculosis from birth. These findings, in our view, are the first findings to be established in such a setting.

Scrub typhus has re-emerged as an important cause of acute febrile illness in India. There is a dearth of information on strain diversity of Orientia tsutsugamushi from Karnataka, India, hence the present study sought to address this issue. One hundred clinically suspected cases of scrub typhus/rickettsiosis (as per the DHR-ICMR guidelines) were included. Nested-polymerase chain reaction (PCR) for 56-kDa gene and phylogenetic analysis was performed. PCR was positive in 22 cases and phylogenetic analysis showed the presence of different strains, with predominance of clustering (57%) with Gilliam-type for the first time in Karnataka. Knowledge of genetic diversity has implications in development of diagnostics and vaccine.

The neonatal skin microbiome consists of all the genomes and genetic products of microorganisms harbouring on the skin of babies. Host and the microbiota develop a harmonious environment resulting in symbiosis. Any disruption of this environment could lead to pathological disease. We conducted this study to understand the neonatal skin microbiome of very preterm neonates admitted to NICU at a tertiary health care setting before and after Kangaroo Mother Care. Next Generation sequencing showed relative abundance for Mycobacterium tuberculosis in 83.33% & 66.67% (p0.285) and Mycobacteroides abscessus in 100% & 93.33%( p0.303) of the very preterm neonates on the skin microbiome before and after KMC respectively. The mere presence of Mycobcaterium species as commensals or as potential pathogens, is alarming due to the risk of early exposure and incidence of tuberculosis right from birth. These findings, in our view, are the first findings to be established in such a setting.

Scrub typhus has re-emerged as an important cause of acute febrile illness in India. There is a dearth of information on strain diversity of Orientia tsutsugamushi from Karnataka, India, hence the present study sought to address this issue. One hundred clinically suspected cases of scrub typhus/rickettsiosis (as per the DHR-ICMR guidelines) were included. Nested-polymerase chain reaction (PCR) for 56-kDa gene and phylogenetic analysis was performed. PCR was positive in 22 cases and phylogenetic analysis showed the presence of different strains, with predominance of clustering (57%) with Gilliam-type for the first time in Karnataka. Knowledge of genetic diversity has implications in development of diagnostics and vaccine.

Purpose. Fungaemia is associated with substantial morbidity and mortality in neonates admitted to neonatal intensive care units (NICUs). We report an outbreak of fungaemia in a NICU due to rare yeast, Pichia kudriavzevii (a teleomorph of Candida krusei). To the best of our knowledge, this is the first report of neonatal sepsis due to P. kudriavzevii. Methodology. Between August and September 2014, blood cultures from nine neonates diagnosed with late-onset sepsis in the NICU yielded yeast-like organisms. The molecular identification and typing of these isolates was performed by sequencing the D1/D2 region of 26S rDNA and fluorescent amplified fragment length polymorphism (FAFLP) respectively. Antifungal susceptibility was tested by broth microdilution as per the Clinical Laboratory Standards Institute (CLSI) guidelines. Sampling from environmental sources and the hands of healthcare workers (HCWs) in the NICU was performed. Results. Of the nine neonates, eight were preterm and six had very low birth weight (VLBW). Thrombocytopenia was present in two neonates. Sequencing identified all the isolates as P. kudriavzevii and FAFLP showed their clonal origin. Antifungal susceptibility testing revealed the susceptibility of all isolates to the antifungals tested. Treatment with voriconazole was advised. However, only seven neonates were treated successfully and discharged after improvement, whereas two were lost for follow-up. Cultures from the environment and the hands of HCWs were negative. The outbreak was controlled by the strict implementation of infection control practices. Conclusion. This study emphasizes the importance of accurate identification of the aetiological agent of sepsis and vigilant monitoring for the possibility of an outbreak in NICUs.

Purpose: To study the virulence factors, serotypes of uropathogenic Escherichia coli (UPEC) and antimicrobial suspectibility pattern. Methods: A total of 200 Escherichia coli from symptomatic cases of urinary tract infections and 50 stool samples from apparently healthy individuals were included. UPEC were screened for virulence factors namely haemolysin, mannose resistant, mannose sensitive haemagglutination (MRHA, MSHA) and serum resistance by recommended methods. Antimicrobial susceptibility testing done by Kirby Bauer disc diffusion method as per NCCLS guidelines. Results: Among 200 Escherichia coli 42(21%) were haemolytic, 60(30%) showed MRHA and 72(36%) MSHA, 99(49.5%) were serum resistant. In 50 controls 8(16%) were haemolytic, 2(4%) showed MRHA and 4(8%) MSHA, 16(32%) were serum resistant. The difference between cases and controls for MRHA and serum resistance were significant (p

Oropharyngeal candidiasis (OPC) continues to be a common opportunistic infection in patients infected with Human Immunodeficiency Virus (HIV) and is predictive of increasing immunosuppression. Though Candida albicans remains the predominant isolate, a rise in the frequency of isolation of non-albicans Candida (NAC) species is being observed. The levels of virulence and the sensitivities to available antifungal drugs vary among these species. Of 340 HIV seropositive patients in this study, 132 (38.8%) had oral lesions suggestive of candidiasis. Samples were collected from the lesion using sterile cotton swabs. Isolation and speciation were done by standard techniques. Antifungal drug susceptibility testing was done by macro broth dilution. The total number of Candida isolates was 135, of which, 45 (33.3%) were NAC species and 90 were C.albicans (66.6%). Of the NAC species, C. dubliniensis was the predominant pathogen (22,48.9%). Antifungal susceptibility testing showed that 14 (31.1%) of the NAC species and 11 (12.2%) of C. albicans were resistant to fluconazole (MIC8 microg/ml). A very high MIC of32 microg/ml was noted among the NAC species resistant to fluconazole.

Context: Candida dubliniensis, an opportunistic yeast that has been implicated in oropharyngeal candidiasis (OPC) in patients infected with Human Immunodeficiency Virus (HIV) may be under-reported due to its similarity with Candida albicans. Resistance to Fluconazole is often seen in C. dubliniensis isolates from clinical specimens. Aims: To know the prevalence of C. dubliniensis in OPC in patients infected with HIV and their antifungal susceptibility pattern. Settings and Design: One hundred and thirty-two HIV seropositive individuals and 50 healthy controls were included in the study. Materials and Methods: Two oral swabs were collected from the site of the lesion from 132 HIV-infected patients. Oral rinse was obtained from 50 healthy controls. Samples were inoculated on Sabouraud's dextrose agar (SDA) medium and on HiCrome Candida Differential Agar (CHROM agar) medium. Isolates were speciated by standard tests. Dark green-colored, germ tube positive isolates, which failed to grow at 420C and negative for xylose assimilation were identified as C. dubliniensis. Antifungal susceptibility test was performed by Macro broth dilution technique (National Committee for Clinical Laboratory Standards guidelines). Results and Conclusions: From 132 patients, 22 (16.3%) C. dubliniensis were isolated; samples from healthy controls did not reveal their presence. Antifungal susceptibility test showed higher resistance among C. dubliniensis isolates to azoles compared to C. albicans. Five (22.7%) isolates of C. dubliniensis were resistant to Fluconazole followed by four (18.2%) to Ketoconazole. This study emphasizes the importance of identification and antifungal susceptibility testing of C. dubliniensis in HIV-infected patients.