Linezolid-resistant Enterococcus faecium clinical isolates from Pakistan: a genomic analysis.

BACKGROUND: Linezolid-resistant Enterococcus faecium (LRE) is a global priority pathogen. Thirteen LRE were reported from clinical specimens between November 2021 and April 2023 at two laboratories in Karachi, Pakistan. We aimed to investigate the strain types and genes associated with linezolid resistance among these isolates. Whole genome sequencing (WGS) was performed and analyzed by multilocus sequence typing (MLST). The presence of linezolid resistance genes was identified using ResFinder v4.1.11 and the LRE-finder tool. RESULTS: Twelve isolates belonged to clonal complex 17 (CC17); ST80 (n = 10), ST612 (n = 1) and ST1380 (n = 1). Six isolates showed the presence of optrA gene and G2576T mutations in the 23S rRNA gene, while six showed poxtA and cfr(D) genes. One isolate showed the combination of optrA, cfr(D) and poxtA genes. CONCLUSION: Our findings show the circulation of CC17 sequence types with a known outbreak potential and we identified molecular mechanisms of resistance that were not previously reported from Pakistan.

Open Online Courses for Strengthening Laboratory-Based Detection of Antimicrobial Resistance in Pakistan.

Introduction: Quality-assured antimicrobial susceptibility testing (AST) depends upon the knowledge and skills of laboratory staff. In many low- and middle-income countries (LMICs), including Pakistan, such types of knowledge and skills are limited. Therefore, the objective of this study was to use openaccess online courses to improve the knowledge of laboratory staff involved in the detection and reporting of antimicrobial resistance (AMR). Methodology: Seven online modules comprising 22 courses aimed at strengthening the laboratory detection of Antimicrobial resistance (AMR) were developed. The courses were uploaded onto the website www.parn.org.pk. Participants had an option of selecting courses of their interest. Online registration and completion of a pre-course assessment (pre-test) were essential for enrolment. However, participation in post-course assessment (post-test) was optional. The number of registered participants and the proportion of participants who completed each course were computed. A paired t-test was used to assess the increase between mean pre- and post-test scores. The association between the participants working in public vs. private laboratories and course completion rates were determined using the chi-square test. Results: A total of 227 participants from Pakistan (March 2018 to June 2020) were registered. The largest number of registered participants and the highest completion rate were noted for AST and biosafety courses, while quality-related courses attracted a lower interest. A comparison of pre- and post-test performance using the paired mean score for the individual courses showed a statistically significant (the value of p < 0.05) improvement in 13/20 assessed courses. A higher course completion rate was observed in participants from public vs. private sector laboratories (56.8 vs. 30.8%, the value of p = 0.005). Conclusions: Our study suggests a promising potential for open online courses (OOCs) toward addressing knowledge gaps in laboratory practice in resource limited settings.

Micromechanical Intervention in Sandwich Restoration.

OBJECTIVE: To evaluate the sealing ability of two different types of Glass Ionomer Cements (GICs) used for sandwich restorations and assess the effect of acid etching of GIC on microleakage at GIC-resin composite interface. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: Department of Operative, DIEKIOHS (DUHS) and NED University, Karachi, from February to June 2011. METHODOLOGY: Eighty cavities were prepared on the proximal surfaces of 40 permanent human premolars (2 cavities per tooth), assigned to 4 groups (n=20) and restored as follows: Group CIE - Conventional GIC (CI) was applied onto the axial and cervical cavity walls, allowed setting for 5 minutes and acid etched (E) along the cavity margins with 37% phosphoric acid for 15 seconds, washed for 30 seconds and dried; the adhesive system was applied and light cured for 10 seconds, completing the restoration with composite resin light cured for 40 seconds; Group CIN - same as Group CIE, except for acid etching of the CI surface; Group RME - same as CIE, but using a resin modified GIC (RMGIC); Group RMN - same as Group RME, except for acid etching of the RMGIC surface. Specimens were soaked in 1% methylene blue dye solution at 37ºC for 24 hours, rinsed under running water for 15 minutes, bisected mesiodistally and dye penetration was measured following the ISO/TS 11405-2003 standard. Kruskal Wallis and post Hoc tests significant differences in the microleakage among all the four groups. RESULTS: There was a significant difference between the two groups of GICs (RMGIC and CI, p=0.001). There was no significant difference in between the two sub-groups that is between CIN and CIE (p=0.656), and between Groups RME and RMN (p=0.995). CONCLUSION: Phosphoric acid etching of GIC, prior to the placement of composite resin, does not improve the sealing ability of sandwich restorations. RMGIC was more effective in preventing dye penetration at the GIC-resin composite dentine interfaces than CI.

Species identification of invasive yeasts including Candida in Pakistan: limitations of phenotypic methods.

OBJECTIVE: To compare phenotypic and genotypic methods of yeast identification. METHODS: The in-vitro cross-sectional study was conducted from January 2006 to May 2009. Invasive yeasts isolated at the clinical microbiology laboratory at the Aga Khan University (AKU), Karachi, Pakistan, were identified. Speciation by phenotypic and molecular methods was compared. All yeasts isolated during the study period from blood and other invasive sites were identified using standard methods. Isolates were shipped to Mycotic Diseases Branch, Centres for Disease Control and Prevention, Atlanta, Georgia, USA, for identification by Luminex flow cytometric multianalyte profiling (xMAP) system. Ribosomal ITS2 DNA sequencing was performed on isolates not identified by Luminex. RESULT: Of the 214 invasive yeasts evaluated, Candida species were 209 (97.7%) while the frequency of non-Candida species was 5 (2.3%). Overall agreement between phenotypic and molecular identification was 81.3%, 90.3% amongst the more common Candida species, and only 38.8% amongst the uncommon yeasts. CONCLUSION: Phenotypic methods of identification proved adequate for common Candida species, but were deficient in recognising rare Candida and non-Candida yeasts, highlighting the importance of molecular methods for identification.

Rhinocladiella mackenziei as an emerging cause of cerebral phaeohyphomycosis in Pakistan: a case series.

Six cases of Rhinocladiella mackenziei cerebral phaeohyphomycosis are being reported for the first time in Pakistan. Identification was confirmed by DNA sequencing (isolates and fixed tissue). Diabetes, head trauma, immunosuppressive treatment, and postpartum state were present in 4 cases. Two survivals and 3 fatalities occurred, with 1 patient lost to follow-up.