Clinical Laboratory Standards Institute Guidelines Research Articles

Bullet-related bacterial wound infections among injured personnel at emergency site hospitals in Bahir Dar: prevalence, antimicrobial susceptibility and associated factors.

Bullet-related bacterial wound infection can be caused by high-velocity bullets and shrapnel injuries. In Ethiopia, significant injuries were reported that may cause severe wound infections, persistent systemic infections and may lead to amputation and mortality. The magnitude, antimicrobial susceptibility profiles, and factors associated with bacterial wound infections among patients with bullet-related injuries are not yet studied particularly at health facilities in Bahir Dar, Northwest Ethiopia. Therefore, this study was aimed to determine the prevalence, bacterial profiles, antimicrobial susceptibility profiles, and factors associated with bacterial infections among patients with bullet-related injuries at referral health facilities in Bahir Dar, Northwest Ethiopia. A Hospital-based cross-sectional study was conducted among patients with bullet-related injuries at three referral health facilities in Bahir Dar from May 25 to July 27, 2022. A total of 384 patients with bullet-related injuries were included in the study. Sociodemographic and clinical data were collected using a structured questionnaire. Wound swabs were collected aseptically and cultured on Blood and MacConkey agar following bacteriological standards. Biochemical tests were performed to differentiate bacteria for positive cultivation and antimicrobial susceptibility profiles of the isolates were done on Muller Hinton agar using the Kirby-Bauer disk diffusion technique according to the 2021 Clinical Laboratory Standard Institute (CLSI) guideline. The data were entered using Epi-Info version 7.3 and analyzed using SPSS version 25. Descriptive data were presented using frequency, percentages, figures, and charts. Logistic regression was carried out to identify factors associated with bacterial wound infections. P-value < 0.05 was considered statistically significant. The prevalence of bullet-related bacterial wound infection among three referral hospitals in Bahir Dar city was 54.7%. The most commonly isolated Gram-negative organism was Klebsiella spps 49 (23.3%) while among Gram-positive organism, Staphylococcus aureus 58 (27.6%) and coagulase-negative staphylococci (CONS) 18 (8.6%). Contamination, hospitalization and smoking habit were significantly associated with the presence of bullet-related bacterial wound infections. Over 97% multidrug resistant (MDR) bacterial isolates were identified and of theses, E. coli, Proteus species, Citrobactor, and Staphylococcus aureus were highly drug resistant. Increased prevalence of bullet-related bacterial wound infection was noticed in this study. S. aureus followed by Klebsiella species were most commonly isolated bacteria. High frequency of resistance to Ampicillin, Oxacillin, Cefepime, Ceftriaxone, Ceftazidime, Vancomycin, and Norfloxacin was observed. Therefore, proper handling of bullet injuries, prompt investigation of bacterial infections, monitoring of drug sensitivity patterns and antibiotic usage are critical.

Antibiotic susceptibility profile of Pseudomonas species isolated from clinical specimens to access, watch and reserve drugs across various hospital settings at a tertiary care hospital of central India

Background and Objectives: Over the last decade, hospital-acquired infections, particularly in the critical care setting, have become more common, with Gram-negative bacterial infections having the highest prevalence. This study aims to determine the prevalence and antibiotic susceptibility pattern of Pseudomonas species to WHO’s, aware class of antibiotics, which are commonly prescribed across various ICU’s, medical and surgical wards of our tertiary care teaching hospital.&#x0D; Materials and Methods: This prospective study conducted from January 2021 to June 2022 at a tertiary care centre of cen- tral India identified Pseudomonas species from clinical samples using standard procedures and antimicrobial susceptibility testing performed as per Clinical Laboratory Standards Institute (CLSI) guidelines (M100; 32th Edition).&#x0D; Results: A total of 1490 non duplicate Pseudomonas species isolates were grown from 21,019 culture positive clinical sam- ples, of which 1247 were Pseudomonas aeruginosa. Out of these 1247 Pseudomonas aeruginosa 384 were MDR (30.7%). Pseudomonas aeruginosa were most commonly isolated from the pus samples (85%). ICU isolates were significantly more resistant to antibiotics than those from other units. P. aeruginosa strains from ICUs showed the highest rates of resistance to ceftazidime (93.9%). Reserve drug colistin showed good susceptibility (98.2%). All the 18 colistin resistant strains were found to be negative for plasmid mediated mcr-1,2,3 genes.&#x0D; Conclusion: The study shall help to generate and disseminate the data so that proper antibiotic policy can be made for judi- cious use of Access, Watch and Reserve antibiotics and antibiotic de-escalation plan can be put forth.

Molecular epidemiology of carbapenem-resistant gram-negative bacilli in Ecuador

IntroductionCarbapenem-resistant gram-negative bacilli are a worldwide concern because of high morbidity and mortality rates. Additionally, the increasing prevalence of these bacteria is dangerous. To investigate the extent of antimicrobial resistance and prioritize the utility of novel drugs, we evaluated the molecular characteristics and antimicrobial susceptibility profiles of carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii in Ecuador in 2022.MethodsNinety-five clinical isolates of carbapenem non-susceptible gram-negative bacilli were collected from six hospitals in Ecuador. Carbapenem resistance was confirmed with meropenem disk diffusion assays following Clinical Laboratory Standard Institute guidelines. Carbapenemase production was tested using a modified carbapenemase inactivation method. Antimicrobial susceptibility was tested with a disk diffusion assay, the Vitek 2 System, and gradient diffusion strips. Broth microdilution assays were used to assess colistin susceptibility. All the isolates were screened for the blaKPC,blaNDM,blaOXA-48,blaVIM and blaIMP genes. In addition, A. baumannii isolates were screened for the blaOXA-23, blaOXA-58 and blaOXA-24/40 genes.ResultsCarbapenemase production was observed in 96.84% of the isolates. The blaKPC, blaNDM and blaOXA-48 genes were detected in Enterobacterales, with blaKPC being predominant. The blaVIM gene was detected in P. aeruginosa, and blaOXA-24/40 predominated in A. baumannii. Most of the isolates showed co-resistance to aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole. Both ceftazidime/avibactam and meropenem/vaborbactam were active against carbapenem-resistant gram-negative bacilli that produce serin-carbapenemases.ConclusionThe epidemiology of carbapenem resistance in Ecuador is dominated by carbapenemase-producing K. pneumoniae harbouring blaKPC. Extensively drug resistant (XDR) P. aeruginosa and A. baumannii were identified, and their identification revealed the urgent need to implement strategies to reduce the dissemination of these strains.

Frequency of drug-resistant bacterial isolates among pregnant women with UTI in maternity and children’s hospital, Bisha, Saudi Arabia

Urinary tract infections (UTIs) are one of the most prevalent bacterial infections affecting humans, with a higher incidence among women. Pregnant women are at an increased risk of developing UTIs, which can have detrimental consequences for both the mother and fetus. UTIs can be caused by various bacteria, and the prevalence of drug-resistant UTIs in maternity and children’s hospitals is a cause for concern due to the potential for severe complications if left untreated. The primary objective of the current study was to determine the distribution of UTI-causing bacteria and investigate the antibiotic sensitivity patterns of isolated cultures obtained from pregnant women with UTIs at the Maternity and Children’s Hospital, Bisha, Saudi Arabia. This cross-sectional study was conducted from October 2021 to October 2023, involving the analysis of urine samples collected from 321 participants who acquired UTIs during pregnancy. Using biochemical tests and standard cultures, the urine samples were examined for pathogenic bacteria and their anti-microbial sensitivity patterns. The study analyzed susceptibility results according to the Clinical Laboratory Standards Institute guidelines (M100, 28th Edition, 2018). Bacterial strains demonstrating resistance to three or more antibiotics were classified as multidrug-resistant (MDR). This study revealed the distribution of UTI-causing bacteria to be as follows: Escherichia coli, 57.01%; Klebsiella pneumoniae, 24.61%; Pseudomonas aeruginosa, 4.36%; Proteus mirabilis and Enterobacter cloacae, 3.74%; Streptococcus agalactiae, 3.11%; Enterococcus faecalis, 2.18%; and Staphylococcus aureus, 1.24%. Antimicrobial susceptibility testing varied among gram-positive and gram-negative bacteria. Gentamicin demonstrated the highest sensitivity among both gram-positive and gram-negative bacteria; piperacillin-tazobactam was the second most effective drug against gram-negative bacteria. The bacterial isolates showed varying susceptibility to different antibiotics, with Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa being mainly sensitive to gentamicin, piperacillin-tazobactam, and ciprofloxacin, respectively. The strategies for reducing the risk of UTIs need to be improved to limit the spread of MDR bacteria. These strategies may include promoting hygienic practices and administering appropriate antibiotics to prevent the emergence and spread of drug-resistant bacteria. Further research is required to monitor the trends in antibiotic resistance among UTI-causing bacteria and develop effective strategies for managing this public health menace.

The antimicrobial effect of different vitamin D compounds on Streptococcus mutans and their impact on glycosyltransferase expression

ABSTRACT Background Streptococcus mutans is a virulent microorganism associated with dental caries. This in vitro study aimed to investigate the antimicrobial effects of Cholecalciferol (D3) and Doxercalciferol (D2), against S. mutans and on glycosyltransferase gene expression. Methods Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of D3 and D2 for S. mutans were determined according to the Clinical Laboratory Standards Institute guidelines. The effect of the compounds on environmental pH in 1% w/v and 5% w/v sucrose broth cultures after 24 hours were assessed colorimetrically. Additionally, their impact on glycosyltransferases gene expression (GtfB, GtfC, GtfD) in 5% w/v sucrose culture was evaluated using quantitative real-time PCR. Results The MBCs of D3 and D2 were 83 µg/ml and 166 µg/ml respectively. Both compounds were effective in preventing the local pH drop <5.5 at ≥166 µg/ml in sucrose supplemented cultures. However, the compounds did not inhibit pH drop at MIC values. Notably, D2 upregulated GtfD expression significantly (p < 0.05) and downregulated GtfB and GtfC. Conclusion Vitamin D2 and D3 inhibited S. mutans mediated pH drop in sucrose supplemented cultures and altered glycosyltransferase expression, suggesting potential therapeutic roles in dental caries prevention. Further research is needed to assess their full impact on S. mutans survival under environmental stresses.

Determination of Inducible Clindamycin Resistance and Correlation with Vitek2 Inducible Clindamycin Resistance Test in Staphylococcus aureus Isolated from Clinical Samples.

To compare the performance of Vitek2 with the gold standard D test in terms of inducible clindamycin resistance (ICR) detection. Descriptive study. Place and Duration of the Study: Indus Hospital and Health Network Karachi, Pakistan, from November 2021 to April 2022. Standard operating procedures of the laboratory were followed for processing of clinical samples. Methicillin-resistant Staphylococcus aureus (MRSA) isolates were included. The isolates from the same patient within two-week time period were excluded. Clinical laboratory standards institute guidelines were followed for performing and interpreting D test. The results of the D test were compared with Vitek2 results for ICR. A total of 313 isolates were MRSA, of which 93 isolates tested positive for ICR on both the D test and Vitek2. Nine isolates were positive for ICR on Vitek2 and negative on the Kirby-Bauer disk diffusion method. One isolate tested positive on the disk method and negative on Vitek2. Vitek 2 appeared to give false positive results. Reporting false susceptibility of clindamycin can cause therapeutic failure which can markedly affect the patient's outcome. This discordance needs to be investigated further with a large sample size and stringent observation of D-test results to pick laboratory error. Methicillin-resistant Staphylococcus aureus, Inducible clindamycin resistance, D zone, Vitek2.

Bacterial Isolation from Wound Swab and Pus with their Antibiotic Susceptibility Pattern in a Tertiary Care Hospital of Bangladesh

Background: Wound infection is a global health problem, plays an important role in development of chronicity, delaying wound healing associated with long hospital stay. Objective: This study was aimed to identify the bacterial pathogens present in infected wounds and characterize their resistance profile to the most common antibiotics used in the therapy. Methods: This observational study was conducted from January, 2023 to June, 2023 in a tertiary care hospital in Dhaka, Bangladesh. A total of 220 wound swabs and pus samples were collected from the outpatient and inpatient department of this hospital with skin and soft tissue infection. Samples were inoculated on appropriate media and cultured and the isolates were identified by standard procedure as needed. Antimicrobial susceptibility testing was done by disc diffusion method according to ‘The Clinical Laboratory Standard Institute Guidelines’. Results: Out of 220 cases 165(75%) were Male and 55(25%) were female. Majority of the patients 77(35%) were in the age group of 21-31 years. Of the total 220 isolates, 156(70.91%) were culture positive cases. Among the isolated organisms, predominant bacteria was Pseudomonas spp 76(48.22%) followed by Klebsiella 27(17.31%), Escherichia coli 19(12.18%), Proteus 13(8.33%), Staphylococcus aureus 12(7.69%) and Acinetobacter 9(5.77%). Among the gram negative isolates, Pseudomonas was highly sensitive to colistin(88.15%), followed by piperacillin-Tazobactam(77.63%)and Imipenem(50%) and low sensitivity found in ceftriaxone(14.47%), Amoxiclav(13.16) and Clotrimoxazole (13.16%). Klebsiella found sensitive to colistin (90.47%), Piperacillin-Tazobactam (85.71%), Imipenem (76.19%), Gentamycin (71.43%). Escherichia coli shows low sensitivity to almost all the drug except Imipenem (94.74%), Piperacillin-Tazobactam (84.21%) and colistin(84.21%). Stapylococcus aureus show sensitivity to linezolid (100%), vancomycin (91.67%) and Ciprofloxacin (61.67%). Conclusion: Antibiotic sensitivity pattern of various isolates will guide for appropriate selection of the antibiotic against wound infection and reduce the spread of resistance bacteria. Bangladesh Heart Journal 2024; 39(1): 38-43

Coagulation Testing in Real-World Setting: Insights From a Comprehensive Survey.

The objective of this survey was to gain a real-world perspective on coagulation testing by evaluating the availability of various coagulation laboratory tests, assessing specific analytic and postanalytic steps in clinical laboratories in Korea.Participants were surveyed using a 65-question questionnaire specifically focused on their coagulation testing practices related to prothrombin time (PT), activated partial thromboplastin time (aPTT), plasma-mixing studies, lupus anticoagulant (LA) tests, platelet function tests, coagulation factor assays, and the composition of hemostasis and thrombosis test panels. The survey was performed between July and September 2022.The survey achieved a 77.9% (81 of 104) response rate. PT or aPTT tests were performed directly at all participating institutions, followed by D-dimer and fibrinogen tests, platelet function test, and plasma-mixing studies in order of frequency. Variations existed in the performance of mixing test and LA assessment. Patterns of coagulating testing differed depending on the size of the hospital. The survey revealed that most laboratories conducted coagulation tests following the international guidelines such as Clinical Laboratory Standards Institute guidelines and the Korean Laboratory Certification system. However, some coagulation tests, including mixing test and LA tests, are yet to be standardized in Korea.Continuous education on coagulation test methods and internal and external quality control are required to encourage laboratories to enhance the performance of coagulation testing.

Performance verification and clinical application evaluation of D-dimer assay

Objective: To evaluate the clinical application performance of a domestic D-dimer assay reagent (ADX D-dimer). Methods: A total of 546 residual sodium citrate anticoagulated plasma samples (530 of which were used for comparability validation and 16 for sample preparation of other validation components) were selected after the completion of clinical testing at Peking Union Medical College Hospital from Jun 2022 to May 2023. According to the American Clinical Laboratory Standards Institute (CLSI) guidelines, national health industry standards and relevant references, the performance of ADX D-dimer used in Sysmex CS 5100 fully automated coagulation analyzer which included accuracy, precision, linear range, carryover rate, interference resistance capability and reference interval were validated and the agreement compared with two mainstream imported detection reagents (reagent A: Vidas D-dimer reagent; reagent B: Innovance D-dimer detection reagent) was evaluated. The clinical diagnostic efficacy of the ADX D-dimer was evaluated using the ELISA D-dimer (reagent A) test results as criteria. Results: The linear correlation coefficient of the 6-point calibrated absorbance and target value was 0.998, the bias of accuracy met the requirements (-2.8%-8.4%), and the coefficient of variation (CV) of within-run and between-day precision of the two levels were 1.0%-2.7% and 2.7%-4.1%, respectively, which were less than the requirements of the manufacturer's statement and the national health industry standard. The linear range within 0.33-9.69 mg/L FEU was verified and the carryover rate was 0. There was no significant interference with the assay results at bilirubin F≤0.22 g/L, bilirubin C≤0.22 g/L, hemoglobin≤5.5 g/L and celiac≤2 800 FTU. The manufacturer's reference interval≤0.5 mg/L FEU was verified suitable for this laboratory. For 358 samples without suspicious heterophilic antibody whose D-dimer levels range from 0.06 to143.63 mg/L FEU, the correlation between ADX D-dimer and another two assay was good, with r values being 0.968 and 0.975, respectively, the percentage of deviation and relative deviation beyond the 95% confidence interval was 3.4%-4.5% and 5.3%-7.0%. The correlation between ADX D-dimer and ELISA D-dimer was better than that of reagent B in the concentration range of 0.06-1.00 mg/L FEU (r=0.858, 0.134). For 172 samples with heterophilic antibody, the correlation between ADX D-dimer and ELISA D-dimer was still good(r=0.827), with the percentage of deviation and relative deviation being 6.4% (11/172). The diagnostic efficacy was evaluated using 530 samples, and the sensitivity, specificity, positive predictive value, negative predictive value of ADX D-dimer was 97.4%, 77.6%, 91.9%, 91.9%. The area under the curve was 0.976 (95%CI: 0.964-0.987, P<0.001). Conclusion: The ADX D-dimer reagent has superior assay and diagnostic performance, and can meet the needs of clinical laboratories.

Escherichia coli dominance and antimicrobial resistance in urinary tract infections among diabetic patients: Insights from Birnin Kebbi Metropolis, Nigeria

Background: The global rise in antibiotic-resistant urinary tract infections (UTIs) is a growing concern, particularly among diabetic patients. This study examines the antibiotic resistance patterns of bacterial uropathogens in diabetic patients at Sir Yahaya Memorial Hospital in Birnin Kebbi.&#x0D; Methods: A purposive sampling approach was used to collect 51 mid-stream urine samples in sterile containers. Cultural and biochemical methods were employed for the isolation and identification of uropathogenic bacteria. Antibiotic sensitivity testing was performed using the disc diffusion method, with results interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines.&#x0D; Results: UTIs were prevalent in 23.5% (12/51) of the samples. Escherichia coli was the most prevalent uropathogen, accounting for 41.3% (7/17) of cases, followed by Klebsiella pneumoniae at 23.5% (4/17). Staphylococcus aureus and Proteus mirabilis each contributed to 17.6% (3/17) of cases. Notably, E. coli and K. pneumoniae exhibited 100% resistance to chloramphenicol and sparfloxacin, respectively.&#x0D; Conclusion: These findings underscore the need for further molecular research to characterize these uropathogens and identify the genes contributing to antibiotic resistance.

Patterns of antibiotics sensitivity in culture positive cases of urinary tract infections in children.

Objective: To determine the effective narrow spectrum antibiotics for treatment of UTI in children in order to control the rapidly increasing antibiotic resistance to broad spectrum antibiotics. Study Design: Prospective Clinical study. Setting: Department of Pediatric, Akbar Niazi Teaching Hospital, Bhara Kahu-Pakistan. Period: January 2021 to December 2021. Material &amp; Methods: 361 children with clinical suspicion of UTI Urine samples were collected. Antimicrobial sensitivity test was applied on Samples on which bacterial growth was identified using Clinical Laboratory Standards Institute (CLSI) guidelines. The data was analyzed using SPSS version 25. Results: Total 361 patients were enrolled in the study according to inclusion criteria, out of which, 30 (8.3%) were infants (&lt;1 years old), 178 (49.3%) were aged 1 to 5 years and 153 (42.4%) were aged from 5 to 12 years of age. Out of 361 patients, 125 (34.6%) patients were males and 236 (65.4%) were females. Out of 124 patients, whose urine culture was positive, most common pathogen was E. coli that was observed in 92 (25.5%) cases, followed by Klebsialla in 9 (2.5%) and enterococcus in 9 (2.5%) patients, Pseudomonas in 5 (1.4%), Proteus in 4 (1.1%), Mirabilis in 4 (1.1%), Acinobacter in 2 (0.6%), and enterobacter in 2 (0.6%) cases. Spectrum of sensitivity of 17 drugs was studied. The most sensitive drug was Imipenem, (29.9%), followed by Nitrofurantoin, Fosfomycin (25.8%), Amikacin (24.1%), Piperacilin / Tazobactam (22.7%). Conclusion: Thus E. coli was the most common pathogen and the most sensitive drug was Imipenem, followed by Nitrofurantoin, Fosfomycin, Amikacin, and Piperacilin / Tazobactam.

Age specific reference intervals for plasma biomarkers of neurodegeneration and neurotrauma in a Canadian population

IntroductionIn this study, we aimed to create reference intervals (RI) using a large Canadian population-based cohort, for plasma protein biomarkers with potential utility to screen, diagnosis, prognosticate and manage a variety of neurological diseases and disorders. RIs were generated for: the ratio of amyloid beta 42 over 40 (Aβ42/40), phosphorylated tau-181 (p-tau-181), neurofilament light (NfL), and glial fibrillary acidic protein (GFAP). Methods900 plasma specimens from male and female participants aged 3–79 years old were obtained from the Statistics Canada Biobank, which holds specimens from the Canadian Health Measures Survey. Analysis of Aβ42/40, p-tau-181, NfL and GFAP was performed on the Quanterix Simoa HD-X analyzer using the Neurology 4-plex E and p-tau-181 assays. Discrete RIs were produced according to Clinical Laboratory Standards Institute guidelines (EP28-A3c). Continuous RIs were created using quantile regression. ResultsFor discrete RIs, significant age partitions were determined for each biomarker. No significant sex partitions were found. The following ranges and age partitions were determined: Aβ42/40: 3–<55y = 0.053–0.098, 55–<80y = 0.040–0.090; p-tau-181: 3–<12y = 1.4–5.6 pg/ml, 12–<60y = 0.8–3.1 pg/ml, 60–<80y = 0.9–4.0 pg/ml; NfL: 3–<40y = 2.6–11.3 pg/ml, 40–<60y = 4.6–17.7 pg/ml, 60–<80y = 8.1–47.1 pg/ml; GFAP; 3–<10y = 47.0–226 pg/ml, 10–<60y = 21.2–91.9 pg/ml, 60–<80y = 40.7–228 pg/ml. Continuous RIs produced smooth centile curves across the age range, from which point estimates for each year of age were calculated. ConclusionsDiscrete and continuous RIs for neurological plasma biomarkers will help refine normative cut-offs across the lifespan and improve the precision of interpretating biomarker levels. Continuous RIs are recommended for use in age groups, such as pediatrics and older adults, that experience rapid concentration changes by age.

Using Protein Fingerprinting for Identifying and Discriminating Methicillin Resistant Staphylococcus aureus Isolates from Inpatient and Outpatient Clinics.

In hospitals and other clinical settings, Methicillin-resistant Staphylococcus aureus (MRSA) is a particularly dangerous pathogen that can cause serious or even fatal infections. Thus, the detection and differentiation of MRSA has become an urgent matter in order to provide appropriate treatment and timely intervention in infection control. To ensure this, laboratories must have access to the most up-to-date testing methods and technology available. This study was conducted to determine whether protein fingerprinting technology could be used to identify and distinguish MRSA recovered from both inpatients and outpatients. A total of 326 S. aureus isolates were obtained from 2800 in- and outpatient samples collected from King Faisal Specialist Hospital and Research Centre in Riyadh, Saudi Arabia, from October 2018 to March 2021. For the phenotypic identification of 326 probable S. aureus cultures, microscopic analysis, Gram staining, a tube coagulase test, a Staph ID 32 API system, and a Vitek 2 Compact system were used. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), referred to as protein fingerprinting, was performed on each bacterial isolate to determine its proteomic composition. As part of the analysis, Principal Component Analysis (PCA) and a single-peak analysis of MALDI-TOF MS software were also used to distinguish between Methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA. According to the results, S. aureus isolates constituted 326 out of 2800 (11.64%) based on the culture technique. The Staph ID 32 API system and Vitek 2 Compact System were able to correctly identify 262 (80.7%) and 281 (86.2%) S. aureus strains, respectively. Based on the Oxacillin Disc Diffusion Method, 197 (62.23%) of 326 isolates of S. aureus exhibited a cefoxitin inhibition zone of less than 21 mm and an oxacillin inhibition zone of less than 10 mm, and were classified as MRSA under Clinical Laboratory Standards Institute guidelines. MALDI-TOF MS was able to correctly identify 100% of all S. aureus isolates with a score value equal to or greater than 2.00. In addition, a close relationship was found between S. aureus isolates and higher peak intensities in the mass ranges of 3990 Da, 4120 Da, and 5850 Da, which were found in MRSA isolates but absent in MSSA isolates. Therefore, protein fingerprinting has the potential to be used in clinical settings to rapidly detect and differentiate MRSA isolates, allowing for more targeted treatments and improved patient outcomes.

Flow Cytometry Method Validation Protocols.

Analytical method validation provides a means to ensure that data are credible and reproducible. This article will provide a brief introduction to analytical method validation as applied to cellular analysis by flow cytometry, along with practical procedures for four different types of validation. The first, Basic Protocol 1 (the limited validation protocol), is recommended for research and non-regulated laboratories. Next, Basic Protocol 2) presents a reasonable, fit-for-purpose validation approach appropriate for biopharma and research settings. Basic Protocol 3 addresses the type of validation performed in clinical laboratories for moderate-risk tests developed in house. Finally, Basic Protocol 4 describes the process that should be applied whenever a method is being transferred from one facility to another. All four validation plans follow the fit-for-purpose validation approach, in which the validation parameters are selected based on the intended use of the assay. These validation protocols represent the minimal requirement and may not be applicable for every intended use such as high-risk clinical assays or data to be used as a primary endpoint in a clinical trial. The recommendations presented here are consistent with the white papers published by the American Association of Pharmaceutical Scientists and the International Clinical Cytometry Society, as well as with Clinical Laboratory Standards Institute Guideline H62: Validation of Assays Performed by Flow Cytometry (CLSI, 2021). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Limited validation Basic Protocol 2: Fit-for-purpose validation for biopharma and research settings Basic Protocol 3: Validation for moderate clinical risk laboratory developed tests Basic Protocol 4: Transfer validation.

Are we missing the silver lining of COVID-19 pandemic: An analytical study to determine effects of three COVID-19 peaks on antimicrobial susceptibility of Staphylococcus aureus isolates

ABSTRACT Background: Given the evolving nature of COVID-19, for better understanding of its effect on antimicrobial resistance of Staphylococcus aureus (S. aureus), it becomes crucial that we follow the resistance patterns across different surges of COVID-19 cases. Methods: This prospective surveillance study extended over two years from January 2020-March 2022 and was conducted in a healthcare center of North India. Susceptibility patterns of Staphylococcus aureus during January-March 2020 were considered as prepandemic patterns. Processing of clinical specimens, identification of S. aureus, and in-vitro antibiotic susceptibility testing were performed in accordance with standard microbiological testing procedures and Clinical Laboratory Standard Institute guidelines. Results: Lowest prevalence (38.9%) of Methicillinresistant S. aureus was reported during January-March 2021 and July-September 2021. More than 50% S. aureus isolates were susceptible to linezolid, cotrimoxazole, tetracycline, and gentamicin in January-March 2020. In January-March 2021, ≥50% of S. aureus isolates from clinical specimens were additionally susceptible to clindamycin and erythromycin. Antibiotic agents of linezolid, tetracycline, clindamycin, and cotrimoxazole were susceptible in ≥50% of S. aureus isolates in January-March 2022. Conclusions: This study reveals a sharp decline in overall resistance to commonly prescribed antibiotic agents for S. aureus isolates after first peak of COVID-19 cases. However, same trend was not observed in subsequent peaks and probably we are approaching the same resistance levels that were seen prior to COVID-19 pandemic.

Prevalence of asymptomatic bacteriuria and its antibacterial susceptibility pattern among pregnant women

Background and Aim: The condition of asymptomatic bacteriuria in pregnancy is common, and up to 40% of women developing pyelonephritis in pregnancy may suffer low birth weight and membrane’s premature rupture. The present study intended to investigate the incidence of asymptomatic bacteriuria and its antibacterial susceptibility pattern among pregnant women. Patients and Methods: This cross-sectional study was carried out on 183 pregnant women attending Obstetrics and Gynecology Department of Ayub Teaching Hospital, Abbottabad from December 2021 to May 2022. Pregnant women with asymptomatic bacteriuria attended the ANC services and willing to provide written consent were enrolled. Standard bacteriological procedures were followed for bacterial isolates identification using biochemical tests, colony characteristics, and Gram-staining. Kirby- Bauer disk diffusion technique was used for Antimicrobial susceptibility test on Muller-Hinton agar medium and Clinical Laboratory Standard Institute guidelines was used for interpretation of zone of inhibition diameter. Midstream urine samples were collected from each study participant and standard microbiological methods to culture urine, identify the pathogen, and conduct sensitivity tests. Data analysis was done in SPSS version 27. Results: The prevalence of asymptomatic bacteriuria was 14.2% (n=26) among pregnant women.

Emergence of Multidrug-Resistant Non-Fermenting Gram-Negative Bacilli in a Tertiary Care Teaching Hospital of Central India: Is Colistin Resistance Still a Distant Threat?

Purpose Multidrug-resistant (MDR) organisms are being increasingly reported from India. This study aimed to determine the antibiotic susceptibility pattern of non-fermenting Gram-negative bacilli (NF-GNB) isolated from all the clinical samples to estimate the prevalence of MDR MDR NF-GNB and to screen for colistin-resistance genes among all colistin-resistant strains. Materials and methods This prospective study conducted from January 2021 to July 2022 at a tertiary care teaching hospital in central India identified MDR NF-GNB from clinical samples using standard procedures and antimicrobial susceptibility testing conducted as per Clinical Laboratory Standards Institute (CLSI) guidelines. Colistin-resistant strains identified by broth microdilution were further subjected to detection of plasmid-mediated colistin-resistant genes (mcr-1, mcr-2, mcr-3) by polymerase chain reaction (PCR). Results A total 2,106 NF-GNB were isolated from 21,019 culture positive clinical samples, of which 743 (35%) were MDR. Majority of MDR NF-GNB isolated were from pus (45.50%) followed by blood (20.50%). Out of 743 non-duplicate MDR non-fermenters,the most common were Pseudomonas aeruginosa (51.7%), Acinetobacter baumannii (23.4%),and others (24.9%).Around5.2% Pseudomonas aeruginosa and 2.3% Acinetobacter baumannii were resistant to colistin, and 88.2% were resistant to ceftazidime. Burkholderia cepacia complexwas 100% susceptible to minocycline and least susceptible to ceftazidime (28.6%). Out of 11, 10 (90.9%) Stenotrophomonas maltophilia were susceptible to colistin and least susceptible to ceftazidime and minocycline (27.3%). All 33 colistin-resistant strains (minimal inhibitory concentration ≥ 4 µg/mL) were found to be negative for mcr-1, mcr-2, and mcr-3 genes. Conclusion Our study showed a significantly wide variety of NF-GNB, ranging from Pseudomonas aeruginosa (51.7%), Acinetobacter baumannii (23.4%),to Acinetobacter haemolyticus (4.6%), Pseudomonas putida (0.9%), Elizabethkingia meningoseptica (0.7%), Pseudomonas luteola (0.5%), and Ralstonia pickettii (0.4%), which have not been commonly reported in literature. Of all the non-fermenters isolated in the present study, 35.28% were MDR, raising the concern for rationalizing antibiotic use and improving infection control measures to avert or slow the emergence of antibiotic resistance.

Antibiotic Susceptibility, Biofilm-Forming Ability, and Prevalence of Extended-Spectrum Beta-Lactamase (ESBL)- and Biofilm-Associated Genes Among Klebsiella pneumoniae Isolates from Hospitalized Patients in Northwest of Iran

Klebsiella pneumoniae is an opportunistic bacterium, which is globally recognized for its high prevalence and antimicrobial resistance (AMR). Biofilm-forming capability, susceptibility testing, and phenotypic confirmatory test for extended-spectrum beta-lactamase (ESBL)-producing isolate recognition of 104K. pneumoniae isolates were performed according to the Clinical Laboratory Standard Institute (CLSI) guidelines. The prevalence of ESBL-associated genes bla-VIM, bla-NDM, and bla-OXA-48, as well as biofilm-associated genes luxS, fimH1, wza, and mrkD, was determined by multiplex PCR. The highest resistance rate was against ampicillin (100.0%). Among the 104K. pneumoniae isolates, 52 (50.0%) and 31 (29.8%) isolates were determined as multi- and extensively drug resistant (MDR, XDR), respectively. Moreover, 21 (40.4%) isolates were determined as ESBL producing. Among 50 biofilm-producing K. pneumoniae isolates, 7 (14.0%), 15 (30.0%), and 28 (56.0%) isolates exhibited high, moderate, and weak levels of biofilm formation, respectively. A number of 41 (78.8%) isolates were susceptible to colistin, and 10 (19.2%) were resistant. AMR was significantly higher (P < 0.05) in the biofilm-forming isolates compared with non-biofilm formers.

Antimicrobial resistance pattern of avian pathogenic Escherichia coli with detection of extended-spectrum β-lactamase-producing isolates in broilers in east Algeria.

Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis, one of the most prevalent bacterial diseases responsible for significant economic losses in the poultry industry worldwide. This study aimed to assess the antimicrobial resistance (AMR) patterns of APEC isolates recovered from poultry in east Algeria and estimate the prevalence of extended-spectrum β-lactamase (ESBL)-producing isolates. In the slaughterhouse of Batna City (Algeria), livers indicating colibacillosis were sampled from 204 suspected carcasses with growth retardation and generalized congestion. Escherichia coli isolation and identification were performed on MacConkey agar using conventional methods and the API 20E system. Antimicrobial resistance susceptibility was tested by the disk diffusion method according to the Clinical Laboratory Standards Institute Guidelines. Extended-spectrum β-lactamase detection was carried out using the double-disk confirmation test. One hundred sixty E. coli isolates were recovered (one isolate per sample). Avian pathogenic Escherichia coli isolates showed high levels of resistance to ampicillin and tetracycline (100%), nalidixic acid (95%), ofloxacin (93.75%), doxycycline (91.87), ciprofloxacin (87.50%), trimethoprim/sulfamethoxazole (62.50%), gentamycin (32.50%), chloramphenicol (27.50%), amoxicillin/clavulanic acid (16.25%), colistin (14.37%), and nitrofurantoin (10.62%). All strains were multidrug-resistant to at least three antibiotics, and more than half (52.52%) of the isolates were resistant to at least seven antibiotics. All isolates were susceptible to ceftriaxone, ceftazidime, and aztreonam. Two E. coli isolates were ESBL producers (1.25%). Avian pathogenic Escherichia coli resistance to most antimicrobial agents used in poultry may lead to antimicrobial therapy failure.

FOSFOMYCIN SUSCEPTIBILITY IN BACTERIAL ISOLATES FROM UROPATHOGENS WITH SPECIAL REFERENCE TO EXTENDED SPECTRUM B-LACTAMASE(ESBL) PRODUCERS AND MULTIDRUG RESISTANT(MDR) ORGANISMS

Introduction: Urinary tract infection (UTI) is one of the most common clinical entities encountered by physicians globally. Most of the uropathogens exhibit Multiple Drug Resistance(MDR) mechanisms especially in the members of Enterobacteriaceae. As there is increase in resistance rates, there is necessity in opting alternative treatment Strategies in cases of UTI like Fosfomycin which seems to be a good option attracting the clinicians interest globally. Objective: To evaluate the Fosfomycin susceptibility in bacterial isolates from uropathogens. Methodology: A total of 624 urine samples were included in the study, out of which 84 isolates satisfying Kass concept were processed and identified by standard protocol and all the isolates were tested for Fosfomycin susceptibility by using Kirby Bauer disc diffusion and E-test, interpreted as per Clinical Laboratory Standards Institute(CLSI) guidelines. Results:Among the isolates majority were Escherichia coli(60.71%) followed by Klebsiella species(26.19%), Enterococcus species(8.33%), Staphylococcus saprophyticus(2.38%) and Proteus species(2.38%) out of which 54.76% were Extended-spectrum-beta-lactamase-producing(ESBL) organisms, 40.47% were Multi Drug Resistant Enterobacteriaceae(MDRE) and 15.47% were Carbapenem Resistant Organisms(CRO). Fosfomycin susceptibility was observed highest in Staphylococcus saprophyticus(100%), Proteus species(100%) followed by E.coli(96.7%) Klebsiella species(95.45%) and Enterococcus species(85.71%). Conclusion: Our study mainly focussed on Fosfomycin susceptibility in various uropathogens where Fosfomycin was found to have a promising role in treating UTI with both Gram positive and Gram negative organisms and can be considered as a drug of choice for empirical oral treatment in cases of UTI.