Rapid detection of carbapenem resistance among gram-negative organisms directly from positive blood culture bottles.

Background: Carbapenemase producing gram-negative bacteria (GNB) has become a huge problem in majority of tertiary care centers worldwide. They are associated with very high morbidity and mortality rates, especially when they cause invasive infections. Therefore, rapid detection of these organisms is very important for prompt and adequate antibiotic therapy as well as infection control. The aim of this study was rapid detection of carbapenemase genes and thereby likely carbapenem resistance, 24-48 hours in advance, directly from the positive-flagged blood culture bottles using CHROMagar and Xpert® Carba-R. Methods: Aspirate from positively flagged blood culture bottles was subjected to differential centrifuge. All gram-negative bacilli on gram stain from the deposit were processed in Xpert® Carba-R and inoculated on CHROMagar. The presence of genes and growth on CHROMagar was compared with carbapenem resistance on VITEK-2 Compact. Results: A total of 119 GNB isolates were processed. One or more of the carbapenemase genes were detected in 80 isolates. On comparison with VITEK-2 result, 92 samples showed concordance for carbapenem resistance 48 hours in advance. There was discordance in 21 isolates with 12 major errors and 09 minor errors. The sensitivity of direct Xpert® Carba-R test for rapid detection of carbapenem resistance, 48 hours in advance, was 81.42%. The sensitivity of direct CHROMagar test for accurate detection of carbapenem resistance, 24 hours in advance, was 92.06%. Conclusion: The ability to detect carbapenem resistance with very high accuracy, 48 hours in advance, helps in appropriate antibiotic therapy and implementation of effective infection control practices.

Anti-mycobacterial activity of heat and pH stable high molecular weight protein(s) secreted by a bacterial laboratory contaminant.

BACKGROUND: Tuberculosis currently stands as the second leading cause of deaths worldwide due to single  infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis. RESULTS: We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at - 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins. CONCLUSIONS: The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis.

Evaluation of the effects of 0.05% sodium hypochlorite and 0.12% chlorhexidine gluconate twice daily rinse on periodontal parameters and gingival crevicular fluid HSV1 and CMV levels in patients with chronic periodontitis: a multicentric study.

Background: Mechanical debridement of periodontal pockets remains the mainstay of therapy in all forms of periodontitis. There is 47% greater reduction in plaque amount when sodium hypochlorite (NaOCl) is used as an adjunct when compared with water rinsing. The aim of this study was to evaluate the effects of 0.05% NaOCl and 0.12% chlorhexidine gluconate twice daily rinse on periodontal parameters and gingival crevicular fluid (GCF) HSV1 and CMV levels in chronic periodontitis. Methods: Patients assigned to group A were prescribed 0.05% NaOCl mouthwash for twice daily rinse. Patients in group B were prescribed 0.12% chlorhexidine gluconate mouthwash to be used twice daily. Evaluation of periodontal parameters was done at baseline and after six months following therapy. GCF HSV1 and CMV levels were evaluated using a polymerase chain reaction. Results: A statistically significant difference was noted in the improvement in periodontal parameters between both groups, when evaluated six months following therapy with greater reduction in group A vis-a-vis group B. Conclusion: NaOCl when prescribed as a twice daily mouthwash can be recommended as a part of the home care regime in patients with chronic periodontitis. It is more cost-effective, easily available and can be beneficial to the troops in difficult terrains and extremes of climates, where oral healthcare facilities are not easily accessible.

Detection of dengue virus serotypes by single-tube multiplex RT-PCR and multiplex real-time PCR assay.

Background: All four dengue serotypes cause infection, with one of them predominantly reported from a particular geographical region. Coinfection by more than one serotype is reported from hyperendemic regions. These coinfections are clinically more severe than infection with a single serotype. This study was carried out to detect the predominant dengue serotype and presence of coinfections. Methods: Acute-phase serum samples of patients suffering from dengue infection were collected. They were screened for the presence of IgM, IgG and NS1Ag by a rapid test. Conventional multiplex reverse transcriptase polymerase chain reaction (RT-PCR) and multiplex real-time RT-PCR assays were carried out for detection and serotyping of the dengue virus. Results: A total of 196 samples were positive by the rapid card test. Of these, 139 were NS1Ag positive, 40 were positive only for IgM, 5 were positive only for IgG and 12 samples were positive for different combinations of antigen and antibodies. All four serotypes were detected in these samples by PCR. DENV-3 was found to be most common circulating serotype. A total of 22 cases were found to have coinfection with more than one dengue serotypes. Samples having only antibodies and no antigen on rapid card test were also positive for virus by PCR. Conclusion: Prevalence of dengue co-infections is increasing. Moreover, it is important to screen for dengue virus in those samples also which do not show NS1Ag on rapid tests and have either one or both the antibodies. Real-time multiplex RT-PCR is found to be more sensitive in detecting coinfection than conventional multiplex RT-PCR.

Prevalence of non tuberculous mycobacterial infection in surgical site infections and their antibiotic susceptibility profile.

BACKGROUND: Surgical site infections (SSIs) are one of the leading causes of hospital-acquired infections contributing to about 20% of all cases, thereby causing an increase in morbidity and financial burden. Causative organisms associated with SSIs have not changed greatly over the last 10-15 years; however, the proportions of different types of causative organisms have changed with an increase in case reports of rare organisms such as non-tuberculous mycobacteria (NTM). METHODS: Samples received from patients with SSI were simultaneously cultured for the isolation of NTM along with routine bacteriological examination. On isolation of NTM, identification was carried out by biochemical tests, and further antibiotic susceptibility profile was determined by using RAPMYCO kit. RESULTS: SSI occurred in 3.95% of the 7675 surgeries performed during the study period of which 10.9% were caused owing to NTM. Only rapidly growing NTM were isolated of which, Mycobacterium fortuitum was the most common (51.51%) and had least resistance to drugs. Other isolates were Mycobacterium abscessus and Mycobacterium chelonae having high degree of antimicrobial resistance. CONCLUSION: NTM are an important cause of SSI having delayed presentation, are difficult to diagnose and often not treated correctly. Identification and susceptibility testing is important as different species respond differently to antimicrobial agents.

A Novel Lineage of Ceftriaxone-resistant Salmonella Typhi From India That Is Closely Related to XDR S. Typhi Found in Pakistan.

Two MDR Salmonella Typhi isolates from India were found by whole genome sequencing to be closely related to the 2016 XDR S. Typhi outbreak strain from Pakistan. The Indian isolates have no chromosomal antimicrobial resistance cassette but carry the IncY plasmid p60006. Both isolates are susceptible to chloramphenicol, azithromycin, and carbapenems.

ß-d-Glucan and Aspergillus Galactomannan assays in the diagnosis of invasive fungal infections.

BACKGROUND: With an increase in the number of patients who are immunosuppressed or immunocompromised there has been an increase in invasive fungal infection (IFI) in the past few decades with its associated high morbidity and mortality, ranging from 60% to 90%. The critical problem is the identification of the causative fungus and initiation of appropriate therapy. Hence, there is a requirement for better diagnostic methods for IFI. Detection of markers for the presence of fungi during early stage of the infection, such as constituents of the cell wall or fungal DNA, is essential for timely diagnosis of IFI. Galactomannan (GM) which is a cell wall surface antigen is the most studied diagnostic marker, followed by 1,3 ß-d-Glucan (BG) which is seen in deep layers of cell wall. METHODS: We have assessed the effectiveness of Galactomannan/ß-d-Glucan for the early diagnosis of IFI in immunosuppressed patients in our tertiary care setting. RESULTS: The sensitivity, specificity, positive predictive value and negative predictive value of GM assay were 45%, 93%, 86% and 63% respectively, while the BG assay showed a sensitivity of 78%, specificity of 85%, Positive predictive value (PPV) 84% and Negative predictive value (NPV) 79%. CONCLUSION: BG assay is better for detection of IFI in patients with immunosuppression. However, a combination of both BG and GM assays would be the best approach as BG assay is highly sensitive, while the GM assay is highly specific for diagnosing IFI.

Speciation of fungi using real time PCR with molecular beacons: Can we solve the enigma of diagnosis of invasive fungal disease?

BACKGROUND: Invasive fungal diseases (IFDs) are difficult to diagnose and associated with high mortality rates, especially in the immunosuppressed. Species of Aspergillus and Candida are the cause of majority of invasive fungal disease however IFDs are also caused by Fusarium, Zygomycetes, Trichosporon, etc. Early detection is crucial for appropriate antifungal therapy. Blood cultures usually fail to isolate filamentous fungi, while detection of circulating beta-d-glucan or galactomannan antigens show variable sensitivity and specificity. There is a need of reliable, sensitive and specific diagnostic tests for IFDs. METHODS: A real-time Polymerase Chain Reaction (PCR) assay with a universal primer/molecular beacon system was developed for detecting and speciating most of the pathogenic fungi implicated in IFD. A single-reaction assay was designed targeting a carefully selected region of the ITS2 and ITS5 subunits of the fungal rDNA gene along with four molecular beacons capable of differential hybridization to the amplicons of different species. This generated a signature set of melting temperatures using the standard strains. The assay was tested on clinical specimens from patients with suspected invasive fungal disease. RESULTS: The assay was tested on 72 clinical samples and 72 healthy controls. Of these, 22 clinical samples (6/8 proven; 13/29 probable; 3/35 possible IFD, classified by the EORTC/MSG criteria) were positive by PCR and generated a set of melting temperatures enabling identification of the causative fungus. The assay was negative in all healthy controls. CONCLUSION: The molecular beacon assay is a promising tool providing a rapid method for detection and monitoring of invasive fungal disease in immunosuppressed patients.

Direct antimicrobial susceptibility testing from positive blood culture bottles in laboratories lacking automated antimicrobial susceptibility testing systems.

BACKGROUND: Timely initiation of appropriate antimicrobial can improve the outcome in terms of reduced morbidity and mortality in addition to reduced health-care costs. Availability of early preliminary Antimicrobial Susceptibility Test (AST) report will be useful in directing antimicrobial therapy. The aim of the study was to correlate AST by disc diffusion method, directly from positively flagged blood culture bottles, with the AST by automated method. METHODS: A total of 144 aerobic blood culture bottles flagged positive by the automated blood culture system were processed. The bacteria were pelleted by two-step centrifugation of the broth from the bottle and used to make a smear for Gram stain as well as an inoculum for antimicrobial sensitivity testing by Kirby Bauer disc diffusion method. Automated identification and AST were also carried out. RESULTS: On direct staining, 94 samples showed gram-negative bacilli, 39 showed gram-positive cocci, and 11 showed yeasts or polymicrobial growth. In the case of gram-negative bacteria, there was 99% categorical agreement between direct sensitivity testing and automated sensitivity testing with 1% disagreement. Among the gram-positive cocci, there was 96% categorical agreement with 4% disagreement between the two methods. CONCLUSION: High degree of agreement between the two methods is promising and applicable to situations where automated sensitivity testing is not available. Even if the systems are available, this method would prove useful as an adjunct to standard AST reporting. This sensitivity report can be generated earlier than the conventional AST, enabling choice of appropriate antimicrobial.

Molecular characterization and phylogenetic analysis of Chikungunya virus from Delhi, India.

BACKGROUND: Chikungunya virus is an alpha virus with high similarity to Dengue and Zika viruses, both in transmission cycle and in clinical presentation. Chikungunya is a re-emerging mosquito-borne infection known to cause small to very large outbreaks/epidemics at frequent intervals. In 2016, India witnessed a large outbreak of Chikungunya infection affecting more than 58,000 people. This study was undertaken to look at the genotypic phylogeny to know the relatedness with previously reported strains. METHODS: During the 2016 outbreak, samples from all patients clinically suspected to have Chikungunya were collected and subjected to testing for IgM antibody by ELISA and viral RNA detection by RT-PCR. Sequencing of the E1 gene segment was done to create a phylogenetic tree comparison with other strains. RESULTS: Serum samples were collected from 142 patients of clinically suspected Chikungunya infection. Majority of the patients were in the age group of 31-50 years accounting for more than 35% of the total cases. Twenty eight samples were positive for IgM antibody. Thirty seven samples were positive for viral RNA by RT-PCR. Only 06 cases were positive by both tests. Mutations in the amino acids K211E, M269V and D284E in the E1 gene segment of the Chikungunya virus were observed in the seven strains that were sequenced. On phylogeny tree, all the strains were found to belong to the ECSA genotype. CONCLUSION: Actively searching for the potential epidemic causing mutations and reporting of novel mutations may help in better understanding and probably forecasting of future CHIKV outbreaks and its nature.

Seroprevalence of influenza A H1N1 (swine) infection in the human population in a cantonment.

BACKGROUND: Various serosurveys and studies were conducted globally on pandemic influenza. H1N1 virus reported so far provides ample evidence of differing perspectives, regarding its epidemiology especially with regard to prevalence, populations groups, and behaviour related to vaccine acceptance. A multigroup, cross-sectional survey among 658 healthy subjects was carried out, in Pune among students, health-care workers (HCWs), and soldiers to assess the seroprevalence of pandemic influenza H1N1 virus and its associated factors. METHODS: The total sample size, based on forecasted prevalence of 33%, worked out to be 640. We studied 658 subjects including 103 students, 201 HCWs, and 354 serving soldiers. The sample for each group was selected from the respective study population by simple random sampling using a random number table. Haemagglutination inhibition test was carried out at the National Institute of Virology. RESULTS: The overall seroprevalence of pandemic influenza H1N1 (2009) virus was found to be 46.5% (95% confidence interval 42.6-50.4) which was adjusted to 39.4% after excluding those vaccinated. The availability of vaccine for high-risk group such as HCWs did not find much favour with the HCWs who did not accept vaccine for various reasons. Whereas only one student was vaccinated, 21.4% of HCWs and 32.5% of soldiers were vaccinated. CONCLUSION: Based on high seroprevalence of antibodies against H1N1 virus during pandemic, vaccination of general population is not recommended. However, high-risk groups and HCWs need to be protected with flu vaccine. There is a need to encourage HCWs for accepting vaccination.

Evaluation of three DNA extraction methods from fungal cultures.

Polymerase chain reaction (PCR) based assays have been developed to amplify DNA of fungal pathogens as culture-based detection methods show low sensitivity. In order to perform a sensitive, specific, and reliable PCR based assay, the availability of pure DNA as well as an easy-to-perform DNA extraction protocol is essential. The existing protocols for DNA extraction used for bacteria or viruses show poor release of fungal DNA. In this study, we evaluated three different methods of DNA extraction and compared their efficacy in the extraction of DNA from filamentous fungi, yeasts, and dermatophytes commonly isolated in our laboratory. It was found that the Fungi/Yeast Genomic DNA Isolation Kit (Norgen Biotek Corp, Ontario, Canada) demonstrated satisfactory extraction of DNA from all the fungi analyzed as compared to that of the Qiamp DNA extraction kit (Qiagen GmbH, Dusseldorf, Germany) or the Phenol Chloroform Isoamyl alcohol extraction method which failed to extract amplifiable DNA from many of the fungal species. Thus, we recommend the use of Fungi/Yeast Genomic DNA Isolation Kit (Norgen) with modifications for the extraction of DNA from fungal cultures.

Utility of a lateral flow assay for culture confirmation of Mycobacterium tuberculosis complex.

BACKGROUND: Therapy for the clinical management of patients with Mycobacterium tuberculosis complex (MTBC) and non tuberculous mycobacteria (NTM) is different. Prompt detection and discrimination is necessary for administration of suitable therapy. METHODS: The aim of the study was to evaluate the performance of a Immunochromatographic Test (ICT) in rapid differentiation of MTBC from NTM grown in Lowenstein-Jensen medium and MGIT broth in comparison to molecular methods. RESULTS: Of the 106 isolates in this study, 96 and 95 were identified as MTBC by 16S rRNA PCR and MPT64 respectively. The sensitivity and specificity of MPT64 was found to be 99% and 100% respectively. CONCLUSION: The Lateral Flow Assay Test is a useful and specific tool in rapid differentiation of M. tuberculosis complex from culture. Therefore proper identification avoids unnecessary ATT to patients infected with NTM.

Detection of glycopeptide resistance genes in enterococci by multiplex PCR.

BACKGROUND: Vancomycin Resistant Enterococci (VRE) are a major cause of nosocomial infections. There are various phenotypic and genotypic methods of detection of glycopeptide resistance in enterococci. This study utilizes multiplex PCR for reliable detection of various glycopeptides resistance genes in VRE. METHOD: This study was conducted to detect and to assess the prevalence of vancomycin resistance among enterococci isolates. From October 2011 to June 2013, a total of 96 non-repetitive isolates of enterococci from various clinical samples were analyzed. VRE were identified by Kirby Bauer disc diffusion method with Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) of all isolates for vancomycin and teicoplanin was determined by E-test. Multiplex PCR was carried out for all enterococci isolates using six sets of primers. RESULTS: Out of 96 isolates, 14 (14.6%) were found to be resistant to vancomycin by vancomycin E-test method (MIC ≥32 µg/ml). Out of these 14 isolates, 13 were also resistant to teicoplanin (MIC ≥16 µg/ml). VanA gene was detected in all the 14 isolates by Multiplex PCR. One of the PCR amplicons was sent for sequencing and the sequence received was submitted in the GenBank (GenBank accession no. KF181100). CONCLUSION: Prevalence of VRE in this study was 14.6%. Multiplex PCR is a robust, sensitive and specific technique, which can be used for rapid detection of various glycopeptide resistance genes. Rapid identification of patients infected or colonized with VRE is essential for implementation of appropriate control measures to prevent their spread.

Use of immunization as strategy for outbreak control of varicella zoster in an institutional setting.

BACKGROUND: Outbreaks of varicella gets reported often in India. However, outbreak in health care providers living in closed institutional setting and role of vaccination as post exposure prophylaxis for control of outbreak has not been studied extensively. This paper presents epidemiological investigation and control strategy undertaken in such scenario. METHODS: This is an epidemiological investigation of chickenpox in nursing students which highlights role of early identification and appropriate control strategy to prevent explosive outbreak in high risk vulnerable population. Vaccination of all susceptible in addition to isolation of cases, quarantine of suspects and proper screening for new cases was the major control strategy adopted. RESULTS: The index case was imported and all eight cases occurred within the incubation period of the case. Two cases occurred in students previously vaccinated for chickenpox. No second or third wave of infection occurred showing vaccination as effective tool in outbreak control strategy. CONCLUSION: Early identification of cases and vaccination of all susceptible contributed to effective control of the outbreak.

Evaluation of ceftriaxone-sulbactam-disodium edetate adjuvant combination against multi-drug resistant Gram-negative organisms.

BACKGROUND: Multi-drug resistant (MDR) Gram-negative bacteria are an emerging threat, both in hospital and community settings. As very few antibiotics are effective against such infections, the need of the hour is a new antibiotic or drug combination which can overcome the effect of extended-spectrum ß-lactamases (ESBL) and metallo ß-lactamases (MBL). A new antibiotic combination of ceftriaxone, sulbactam and disodium edetate (CSE) has recently been proposed to tackle the MDR organisms. OBJECTIVE: Our study was carried out to assess the susceptibility of ESBL- and MBL-producing Gram-negative organisms to CSE. METHODS: The study was conducted in a tertiary-care hospital in Delhi, India, from February 2017 to June 2017. A total of 179 MDR (85 ESBL + 94 MBL) Gram-negative isolates from various clinical samples, identified by an automated system (Vitek 2) were tested against CSE using the Kirby-Bauer disc diffusion method. Susceptibility to CSE was recorded based on interpretative zone sizes of ceftriaxone as per 2017 Clinical and Laboratory Standards Institute guidelines. RESULTS: The most common isolate was Escherichia coli (76/179; 42.4%) followed by Klebsiella pneumoniae (53/179; 29.6%) and Acinetobacter baumanii (27/179; 15.1%). The in vitro susceptibility of ESBL- and MBL-producing Gram-negative isolates to CSE was found to be 58/85 (68.2%) for ESBL and 37/94 (39.4%) for MBL. CONCLUSION: The in vitro susceptibility results obtained for CSE against ESBL-producing organisms is promising. It has the potential to emerge as a carbapenem-sparing antibiotic, active against ESBL-producing strains. Further clinical studies are required to establish the clinical efficacy of CSE against MDR pathogens.

Analysis of Aminoglycoside Modifying Enzyme Genes Responsible for High-Level Aminoglycoside Resistance among Enterococcal Isolates.

Enzymatic modification results in high-level resistance to aminoglycoside (HLAR), which eliminates the synergistic bactericidal effect of combined exposure to a cell wall-active agent and an aminoglycoside. So aim of the study was to determine prevalence of HLAR enterococcal isolate and to study distribution of aminoglycoside modifying enzyme genes in them. A total of 100 nonrepeat isolates of enterococci from various clinical samples were analyzed. As per Clinical and Laboratory Standards Institute guidelines enterococci were screened for HLAR by Kirby-Bauer disc diffusion method. Minimum inhibitory concentration of all isolates for gentamicin and streptomycin was determined by E-test. Multiplex polymerase chain reaction (PCR) was carried out for HLAR enterococcal isolates to identify aminoglycoside modifying enzymes genes responsible for resistance. 60% isolates were found to be high-level gentamicin resistant (HLGR) whereas 45% isolates were found to be high-level streptomycin resistant (HLSR). By multiplex PCR 80% HLGR isolates carried bifunctional aminoglycoside modifying enzyme gene aac(6')-Ie-aph(2'')-Ia whereas 18 out of 45 high-level streptomycin resistant, that is, 40%, isolates carried aph(3')-IIIa. However, aph(2'')-Ib, aph(2'')-Ic, aph(2'')-Id, and ant(4')-Ia genes which encode other aminoglycosides modifying enzymes were not detected. Bifunctional aminoglycoside modifying enzyme gene aac(6')-Ie-aph(2'')-Ia is the predominant gene responsible for HLAR.