Tick-tock, beat the clock: comparative analysis of disc diffusion testing with 6-, 10-, and 24-h growth for accelerated antimicrobial susceptibility testing and antimicrobial stewardship.

Disc diffusion testing by Kirby-Bauer technique is the most used method for determining antimicrobial susceptibility in microbiological laboratories. The current guidelines by The Clinical and Laboratory Standards Institute (CLSI) 2022 specify using an 18- to 24-h growth for testing by disc diffusion. We aim to determine if using an early growth (6 h and 10 h) would produce comparable results, thus ultimately leading to reduced turnaround time. Six-hour, 10-h, and 24-h growths of 20 quality control strains and 6-h and 24-h growths of 48 clinical samples were used to perform disc diffusion testing using a panel of appropriate antimicrobial agents. Disc diffusion zone sizes were interpreted for all and comparative analyses were performed to determine categorical agreement, minor errors (mE), major errors (ME), and very major errors (VME) according to CLSI guidelines. On comparing with the standard 24 h of incubation, disc diffusion from 6-h and 10-h growths of quality control strains showed 94.38% categorical agreement, 5.10% mE, 0.69% MEs, and no VMEs. Disc diffusion testing for the additional 40 clinical samples yielded a similarly high level of categorical agreement (98.15%) and mE, ME, and VME of 1.29%, 1.22%, and 0% respectively. Disc diffusion testing using early growth is a simple and accurate method for susceptibility testing that can reduce turnaround time and may prove to be critical for timely patient management.

Clinical and molecular epidemiology of vancomycin-resistant Enterococcus faecium bacteremia from an Indian tertiary hospital.

We determined the clinical and molecular epidemiology of emerging nosocomial vancomycin-resistant Enterococcus faecium (VREfm)-causing serious bloodstream infections (BSIs) and the correlations between antibiotic resistance and virulence determinants among isolates. All isolates were confirmed by molecular methods (16SrRNA and E. faecium ddl genes) and tested for disk diffusion. PCR was used to detect aac(6')-aph(2″), vanA and vanB resistance genes, and asa1, cylA, ace, esp, gelE and hyl virulence genes. VREfm and high-level gentamicin-resistant (HLGR) representative isolates were selected to characterize by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Of 173 isolates, 73 (42.2%), 146 (84.4%), and 0 (0.0%) were vanA-containing VREfm, aac(6')-aph(2″)-positive HLGR, and vanB-positive. Independent predictors of VREfm infection were hematological malignancies (P = 0.001) and previous hospitalizations (P = 0.007). Observed mortality rate was 34.7%. Independent predictors of BSI-related mortality were endotracheal intubations (P < 0.001), gastrointestinal diseases (P = 0.002), and pulmonary disease (P < 0.001). All VREfm were resistant to vancomycin, teicoplanin, ciprofloxacin, and erythromycin. The esp, hyl, ace, asa1, cylA, and gelE genes were detected at 55.9, 22.5, 2.9, 2.3, 1.7, and 1.2%, respectively. The esp gene was significantly associated with VREfm compared to VSEfm (P = 0.001). PFGE analysis revealed 23 clones, with 7 major clones. The MLST analysis revealed the following five sequence types: ST80, ST17, ST117, ST132, and ST280, all belonging to CC17. The emergence and expansion of VREfm CC17 with limited antibiotic options in our hospital present a serious public health menace and represent challenges to infection control.

Corynebacterium striatum: An emerging nosocomial skin and soft-tissue pathogen.

The genus Corynebacterium is composed of Gram-positive, aerobic, non-motile, non-spore-forming bacilli that are widely distributed throughout the environment. They are usually found as commensals on the skin and are often considered as mere contaminants when isolated from clinical samples. We describe a patient with skin and soft-tissue infections due to Corynebacterium striatum following exploratory laparotomy identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The clinical importance and pathogenic potential of Corynebacterium species, especially C. striatum, cannot be underestimated. This report is a reminder to physicians of the possible pathogenicity of non-diphtherial Corynebacteria.

Distribution of Chlamydia trachomatis omp A genotypes in patients attending a sexually transmitted disease outpatient clinic in New Delhi, India.

Background & objectives: Limited data are available on the typing of Chlamydia trachomatis in India. Serovars D to K of C. trachomatis are chiefly responsible for urogenital infections. Thus, this study was conducted to determine the distribution of C. trachomatis serovars in patients with urogenital infections and to characterize omp A gene of the detected C. trachomatis isolates by sequence analysis. Presence of other co-infections was also evaluated. Methods: Endocervical swabs were collected from 324 women and urethral swabs/urine were collected from 193 men attending the sexually transmitted diseases outpatient clinic. The samples were screened for C. trachomatis by cryptic plasmid PCR and omp A gene PCR. Genotyping was performed by PCR-restriction fragment length polymorphism (RFLP) and sequencing of the omp A gene. Samples were screened for genital mycoplasmas, Neisseria gonorrhoeae, Treponema pallidum and human immunodeficiency virus (HIV). Results: C. trachomatis was found in 15.0 per cent men and 10.8 per cent women. Serovar D was the most prevalent followed by serovars E, F, I and G. Twenty two C. trachomatis isolates were selected for omp A gene sequencing. No mixed infection was found. Variability in omp A sequences was seen in 31.8 per cent cases. Both PCR-RFLP and omp A gene sequencing showed concordant results. The presence of Ureaplasma spp. and Mycoplasma hominis was observed in 18.7 and 9.5 per cent patients, respectively. Co-infection of C. trachomatis was significantly associated with Ureaplasma urealyticum and HIV. Interpretation & conclusions: The high occurence of C. trachomatis infections warrants its screening in addition to other sexually transmitted infections namely U. urealyticum and HIV. Genotyping of the omp A gene may provide additional information for vaccine development.

Staged Reimplantation of a Total Hip Prosthesis After Co-infection with Candida tropicalis and Staphylococcus haemolyticus: A Case Report.

Fungal prosthetic joint infection is a rare complication in total joint arthroplasty. There are no established guidelines for management of these infections. We present a case of a 53-year-old male with a hip joint prosthesis co-infected with Candida tropicalis and Staphylococcus haemolyticus. A two-stage exchange arthroplasty was performed. The patient underwent implant removal, debridement, irrigation with saline solution and application of cement spacer impregnated with vancomycin followed by aggressive antimicrobial treatment in first stage. Complete eradication of infection was demonstrated by negative culture of sonicated cement spacer fluid and negative 16S rRNA and 18S rRNA gene PCR of sonicate fluid, synovial fluid and periprosthetic tissue samples. He underwent second-stage revision hip arthroplasty after 9 months of the first stage. At the latest follow-up, there was no evidence of recurrence of infection. This case illustrates the utility of sonication of biomaterials and molecular techniques for microbiological confirmation of absence of infection in staged surgeries which is required for a successful outcome.

Diagnostic efficacy of a real time-PCR assay for Chlamydia trachomatis infection in infertile women in north India.

BACKGROUND & OBJECTIVES: Little is known about the prevalence of Chlamydia trachomatis infection in Indian women with infertility. To improve the diagnosis of C. trachomatis infection in developing countries, there is an urgent need to establish cost-effective molecular test with high sensitivity and specificity. This study was conducted to determine the diagnostic utility of a real time-PCR assay for detention of C. trachomatis infection in infertile women attending an infertility clinic in north India. The in house real time-PCR assay was also compared with a commercial real-time PCR based detection system. METHOD: Endocervical swabs, collected from 200 infertile women were tested for C. trachomatis by three different PCR assays viz. in-house real time-PCR targeting the cryptic plasmid using published primers, along with omp1 gene and cryptic plasmid based conventional PCR assays. Specimens were also subjected to direct fluorescence assay (DFA) and enzyme immunoassay (EIA) Performance of in-house real time-PCR was compared with that of COBAS Taqman C. trachomatis Test, version 2.0 on all in-house real time-PCR positive sample and 30 consecutive negative samples. RESULTS: C. trachomatis infection was found in 13.5 per cent (27/200) infertile women by in-house real time-PCR, 11.5 per cent (23/200) by cryptic plasmid and/or omp1 gene based conventional PCR, 9 per cent (18/200) by DFA and 6.5 per cent (7/200) by EIA. The in-house real time-PCR exhibited a sensitivity and specificity of 100 per cent, considering COBAS Taqman CT Test as the gold standard. The negative and positive predictive values of the in-house real time-PCR were 100 per cent. The in-house real time-PCR could detect as low as 10 copies of C. trachomatis DNA per reaction. INTERPRETATION & CONCLUSIONS: In-house real time-PCR targeting the cryptic plasmid of C. trachomatis exhibited an excellent sensitivity and specificity similar to that of COBAS Taqman CT Test, v2.0 for detection of C. trachomatis infection in women attending an infertility clinic. In an effort to prevent Chlamydia infection associated infertility, we recommend screening of women with infertility due to C. trachomatis infection by in-house molecular method as a cost-effective solution in resource limited settings.

Macrolide and fluoroquinolone resistance associated mutations in Mycoplasma genitalium in men who have sex with men attending STI clinic: A pilot study from India.

Background Increasing rates of macrolide and fluroquinolone resistance in Mycoplasma genitalium (MG) are being reported worldwide with resultant treatment failure. Aim We aimed to determine the level of antibiotic resistance of MG in men who have sex with men (MSM) attending a sexually transmitted infections (STIs) clinic in New Delhi, India. Methods Real-time polymerase chain reaction (PCR) assays targeting MgPa and pdhD genes were performed to detect MG rectal, urogenital or oropharyngeal infections in 180 MSM between January 2022 and June 2023. Macrolide resistance-associated mutations (MRM) and quinolone resistance-associated mutations (QRM) were detected by specific amplification of domain V of 23SrRNA gene and appropriate regions of parC and gyrA genes respectively followed by sequencing. PCR-based screening for Chlamydia trachomatis (CT) infection was also performed. Results A total of 13 (7.2%) MSM were positive for MG infection. The most common site of infection was anorectum (8/13; 61.5%) followed by the urethra (5/13; 38.5%). None of the patients had infection at both the sites, and no oropharyngeal MG infection was detected. CT infection was detected in 37 (20.6%) MSM. Of the 13 MG-infected MSM, 6 (46.2%) were co-infected with CT. MRM and QRM were found in five (46.2%) and two (15.4%) strains, respectively. Both Quinolone resistance mutation (QRM)-harbouring strains also harboured MRM. All the five MG isolates carried the MRM A2071G. Both the QRM isolates co-harboured the parC and gyrA single-nucleotide polymorphisms. There was no correlation between the presence of antibiotic resistance and co-infection with CT (P = 0.52). Limitation Because all patients in the study were MSM, the high rate of resistance to macrolides and fluoroquinolones could not be extrapolated for non-MSM patients. Conclusion This is a report of an initial survey of antibiotic resistance to MG in a country where its diagnosis and treatment are not routinely available. We found a high prevalence of MG-carrying MRM, QRM and dual-class resistance in MSM in the absence of antibiotic exposure. This study mandates the need for both screening and detection of antimicrobial resistance against MG.

Chronic sexually acquired reactive arthritis secondary to Chlamydiatrachomatis.

Reactive arthritis is included in the spectrum of seronegative spondyloarthritides, occurring secondary to triggers of genitourinary and gastrointestinal tract infections. We describe two cases of sexually acquired reactive arthritis secondary to genital infection by Chlamydia trachomatis, diagnosed by in-house polymerase chain reaction performed on the first void urine. Both patients were managed with a combined approach of short course antibiotics, immunosuppressive agents, biologicals and surgical intervention.

Detection of Aminoglycoside Modifying Enzyme (AME) genes in Acinetobacter baumannii isolates and the inhibitory effect of efflux pump activity on drug susceptibility pattern.

INTRODUCTION: The development of aminoglycoside modifying enzymes (AMEs) and increased efflux activity are considered important aminoglycosides resistance mechanisms. AIM: This study is focused on the detection of the AMEs gene and assessing the effect of efflux pump inhibitor on the reversal of A. baumannii drug susceptibility. METHODOLOGY: Bacterial DNA was amplified using AMEs gene-specific primers. Isolates were also investigated for efflux pump activity using efflux pump inhibitor (EPI) i.e. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and the impact of both mechanisms was analyzed. RESULTS: Among A. baumannii isolates, 55% isolates (n â€‹= â€‹22/40) were identified to have aminoglycoside modifying enzymes genes; ant(3')-I gene (50%, 11/22), aac(6')-Ib gene (45.4%, 10/22), aph(3')-I gene (18.1%, 4/22) and aac(3)-I (9.1%, 2/22). Total 70% isolates have shown MIC alteration in different classes of drugs in response to EPI-CCCP. Such alteration was found in 100% amikacin sensitive and 58.6% amikacin resistant, 93.7% and 57.1% gentamicin sensitive and resistant isolates respectively. CONCLUSION: The presence of aminoglycosides modifying enzymes was frequent among aminoglycosides resistant A. baumannii isolates and the coexistence of efflux pumps activity also plays an important role to increase drug resistance. REPOSITORIES: Genbank and their accession numbers are MT903331[aac(3)-I], MT903332 MT903333 [ant(3')-I], MT903334, MT903335 [aph(3')-I)] and MT903336, MT940242 [ aac(6')-Ib].

A Quality Improvement (QI) initiative in diagnostic stewardship of urine culture in an era of Laboratory Information Systems (LIS).

Standard urine culture is the gold standard for diagnosing urinary tract infections (UTIs) but fails to differentiate true UTI from asymptomatic bacteriuria, which is important to prevent the overuse of antibiotics. Correlation with the presence or absence of pyuria can be helpful in giving a hint of the true situation. With the help of Laboratory Information System (LIS), patients' urinalysis reports can be conveniently accessed and compared simultaneously with appropriate reports. In our study, a quality improvement initiative was planned for appropriate reporting of urine culture and antimicrobial susceptibility testing using information obtained through LIS.

Antimicrobial efficacy of eravacycline against emerging extensively drug-resistant (XDR) Acinetobacter baumannii isolates.

PURPOSE: Drug-resistant Acinetobacter baumannii is an emerging threat. This study has been conducted to observe the efficacy of eravacycline along with the RND-efflux pump system. METHODS: A cross-sectional study was done collecting 48 clinical isolates of Acinetobacter baumannii. MICs of 15 antibiotics were detected along with BMD of tigecycline and eravacycline. PCR products of drug-resistant regulatory genes were sequenced and analyzed. RESULTS: Of the total 48 Isolates, 35 (72.91%) were XDR and 13 (27.08%) were MDR. Out of all, 60.41% of isolates were found to be susceptible to eravacycline by BMD according to both FDA and EUCAST guidelines. A 2-fold decline of MIC50/90 was observed with the use of eravacycline compared to tigecycline. RND-efflux genes like AdeC in 30 (62.5%) isolates and Regulatory gene AdeS in 29 (60.41%) isolates were detected, explaining the existing resistance mechanism. CONCLUSIONS: XDR Acinetobacter poses an escalating threat due to its resistance to multiple antibiotics, raising serious concerns in healthcare settings. Eravacycline is an encouraging new drug for empirical use in severe infection caused due to the same. Molecular investigation and strict antimicrobial stewardship should be followed to control the emergence, and a better understanding of mechanisms of resistance to prevent the spread of drug-resistant isolates.

Strategy for identification & characterization of Bartonella henselae with conventional & molecular methods.

BACKGROUND & OBJECTIVES: Bartonella henselae is a fastidious gram-negative bacterium usually causing self limiting infections in immunocompetent individuals but often causes potentially life threatening infection, such as bacillary angiomatosis in immunocompromised patients. Both diagnosis of infections and research into molecular mechanisms of pathogenesis have been hindered by lack of appropriate and reliable diagnostic techniques. We undertook this study to standardize methods to characterize B. henselae in clinical samples to diagnose Bartonella infection correctly. METHODS: B. henselae ATCC 49882 strain was procured from American type culture collection, USA. This strain was revived and maintained in the laboratory, and identification and characterization of this strain was done by conventional and molecular techniques, which included culture on various media, staining by different methods including electron microscopy, biochemical analysis by conventional methods and API, polymerase chain reaction (PCR) for amplification of citrate synthase gene followed by restriction fragment length polymorphism (RFLP). RESULTS: This organism was biochemically inert due to slow growth and generated unique identification code with API. The amplification of the citrate-synthase gene with primers yielded a 381 bp product followed by specific RFLP profile for B. henselae. INTERPRETATION & CONCLUSIONS: Bartonella is fastidious and fragile organism and should be handled carefully. Extra effort and careful observation are required to isolate and characterize this organism.

Prospective study estimating healthcare associated infections in a paediatric hemato-oncology unit of a tertiary care hospital in North India.

BACKGROUND & OBJECTIVES: Healthcare associated infections (HAIs) are responsible for morbidity and mortality among immunocompromised and critically ill patients. We undertook this study to estimate the burden of HAIs in the paediatric cancer patients in a tertiary care hospital in north India. METHODS: This prospective, observational study, based on active surveillance for a period of 11 months was undertaken in a 4-bedded isolated, cubicle for paediatric cancer patients. Patients who stayed in the cubicle for ≥48 h, were followed prospectively for the development of HAIs. RESULTS: Of the 138 patients, 13 developed 14 episodes of HAIs during the study period. Patient-days calculated were 1273 days. Crude infection rate (CIR) and incidence density (ID) of all HAIs were 9.4/100 patients and 11/1000 patient-days, respectively. Of the 14 episodes of HAIs, seven (50%) were of blood stream infections (HA-BSI), five (36%) of pneumonia (HAP) and two (14%) urinary tract infections (HA-UTI). The CIRs of HA-BSI, HAP and HA-UTI were 5.1, 3.6 and 1.4/100 patients, respectively. The corresponding IDs were 5.5, 3.9 and 1.6/1000 patient-days, respectively. Mean length of stay was significantly higher in patients who developed an HAI [13.8 (range 7-30), median (Interquartile range) 12 (11-14)] vs 7.5 days [range 2-28, median (interquartile range) 7 (5-9); P<0.0001]. Also mortality was significantly higher in patients who developed an HAI [23% (3/13) vs 3% (4/125), P<0.05]. INTERPRETATION & CONCLUSIONS: The incidence of HAIs in the paediatric cancer patients in the study was 11/1000 patient days, of which HA-BSIs were the commonest. HAIs were associated with an increase in morbidity and mortality amongst this high risk patient population.

Laboratory detection of bacterial pathogens and clinical and laboratory response of syndromic management in patients with cervical discharge: A retrospective study.

Background Cervical discharge as part of cervicitis and pelvic inflammatory disease is a cause of significant morbidity in sexually active women worldwide. Non-gonococcal and non- chlamydial bacterial pathogens are becoming more prevalent. Aims This study aims to determine bacterial pathogens causing cervical discharge using culture and/or polymerase chain reaction and assess the clinical and laboratory response to the conventional syndromic kit regimen established by the World Health Organisation. Methods A retrospective review of records of women with cervical discharge over one year period. Culture and/or polymerase chain reaction results of endocervical swabs of various bacterial pathogens at baseline and after four weeks of treatment with syndromic kit regimen were recorded. Results A total of 70 case records were reviewed for clinical details, out of which results of bacterial culture and polymerase chain reaction were available for 67 cases. Infectious aetiology was found in 30 (44.7%) patients with Ureaplasma species being the most common organism isolated on culture (18, 26.8%) and polymerase chain reaction (25, 37.3%), respectively. Polymerase chain reaction for Chlamydia trachomatis and Mycoplasma hominis was positive in ten (14.9%) and four (6%) cases, respectively. None of the patients showed positive culture for Neisseria gonorrhoeae. Coinfection was seen in eight (11.9%) patients with the majority showing Chlamydia trachomatis and Ureaplasma spp. coinfection (five patients). Forty one cases (58.5%) received tab. cefixime 400 mg and tab. azithromycin one gram stat (kit 1), while 29 cases (43.3%) received tab. cefixime 400 mg stat, tab. metronidazole 400 mg and cap. doxycycline 100 mg, both twice daily for 14 days (kit 6). Minimal to no clinical improvement with treatment was seen in 14 out of 32 cases (44%) at the end of four weeks with the conventional kit regimen. Post-treatment culture and/or polymerase chain reaction were positive in nine out of 28 cases (32.1%) with Ureaplasma spp. being the most common. Limitations Retrospective study design, small sample size and fewer cases with follow-up data were the main limitations. Conclusion Ureaplasma spp. was the most common infectious cause of cervical discharge in our patients. Treatment given as part of syndromic management led to a clinical and microbiological response in around half and two-third cases, respectively.

Impact of Bundled Intervention on Outcomes of Patients Undergoing Clean Orthopedic Surgeries With Hardware Implants: Small Prospective Randomized Controlled Trial.

Background: Prosthesis-related infections (PRIs) and surgical site infections (SSIs) remain one of the most devastating complications among patients undergoing clean orthopedic surgeries. Prevention strategies are critical to reduce infection rates in orthopedic surgeries. The current study aimed to determine the effectiveness of a set of evidence-based practices (bundled intervention) in reducing the incidence of PRIs and SSIs among patients undergoing clean orthopedic surgeries with hardware implants. Patients and Methods: A prospective, interventional randomized controlled trial was conducted for a period of three years. A total of 597 patients were enrolled, and depending on their Staphylococcus aureus carrier status were categorized into carrier group (n = 98) and non-carrier group (n = 499). Only carrier group patients were analyzed for effectiveness of bundled interventions, after being randomly assigned to two subgroups: interventional carrier group (ICG; n = 50) and non-interventional carrier group (NICG; n = 48). Results: Of the 597 patients, 98 (16.4%) were colonized with Staphylococcus aureus, among whom 9 (19.4%) had methicillin resistance. During follow-up, overall infection rate of 1.1% was observed (PRI, 0.3%; SSI, 0.8%). There was no case of PRI/SSI in the ICG. However, in the NICG, one patient developed SSI because of methicillin-resistant Staphylococcus aureus. An endogenous source of infection was demonstrated by pulsed field gel electrophoresis (PFGE). The SSI rate was higher in the NICG (p = 0.002). In the non-carrier group (n = 499), SSIs/PRIs occurred among 1.2% of the patients, because of organisms other than Staphylococcus aureus. Conclusions: Benefit of bundle intervention approach could be demonstrated. Further studies assessing the effectiveness of the individual components of the bundle can inform clinical practice greatly.