A visualized hybrid PCR-LAMP assay for the detection of human Plasmodium species.

Combining the advantages of PCR and LAMP, we described a new technique, namely PCR-LAMP, for malaria diagnosis. The whole process of DNA amplification can be completed in 35 min. This hybrid amplification technique markedly improved the sensitivity of detection compared to the classic single PCR or LAMP assay alone. PCR-LAMP assay had a detection limit of 1 copy/µL for P. knowlesi and P. ovale, 0.1 copy/µL for P. vivax, P. falciparum and P. malariae, respectively. To facilitate the endpoint detection, xylenol orange was added. Positive samples were indicated in orange while negative reactions were violet. The inclusion of xylenol orange into the LAMP reaction mix significantly reduces the post-amplification workload. Without relying on the use of specific instruments, the color changes of the amplicons could be visualized directly through the naked eye. In conclusion, PCR-LAMP poses the potential to be developed as a new malaria molecular diagnosis tool.

ACORN (A Clinically-Oriented Antimicrobial Resistance Surveillance Network) II: protocol for case based antimicrobial resistance surveillance.

Background: Antimicrobial resistance surveillance is essential for empiric antibiotic prescribing, infection prevention and control policies and to drive novel antibiotic discovery. However, most existing surveillance systems are isolate-based without supporting patient-based clinical data, and not widely implemented especially in low- and middle-income countries (LMICs). Methods: A Clinically-Oriented Antimicrobial Resistance Surveillance Network (ACORN) II is a large-scale multicentre protocol which builds on the WHO Global Antimicrobial Resistance and Use Surveillance System to estimate syndromic and pathogen outcomes along with associated health economic costs. ACORN-healthcare associated infection (ACORN-HAI) is an extension study which focuses on healthcare-associated bloodstream infections and ventilator-associated pneumonia. Our main aim is to implement an efficient clinically-oriented antimicrobial resistance surveillance system, which can be incorporated as part of routine workflow in hospitals in LMICs. These surveillance systems include hospitalised patients of any age with clinically compatible acute community-acquired or healthcare-associated bacterial infection syndromes, and who were prescribed parenteral antibiotics. Diagnostic stewardship activities will be implemented to optimise microbiology culture specimen collection practices. Basic patient characteristics, clinician diagnosis, empiric treatment, infection severity and risk factors for HAI are recorded on enrolment and during 28-day follow-up. An R Shiny application can be used offline and online for merging clinical and microbiology data, and generating collated reports to inform local antibiotic stewardship and infection control policies. Discussion: ACORN II is a comprehensive antimicrobial resistance surveillance activity which advocates pragmatic implementation and prioritises improving local diagnostic and antibiotic prescribing practices through patient-centred data collection. These data can be rapidly communicated to local physicians and infection prevention and control teams. Relative ease of data collection promotes sustainability and maximises participation and scalability. With ACORN-HAI as an example, ACORN II has the capacity to accommodate extensions to investigate further specific questions of interest.

Socio-Demographic Factors and Public Knowledge of Antibiotic Resistance.

(1) Background: Antibiotic resistance is a serious health issue, and raising public awareness of it is crucial to combating it. This study aimed to assess the socio-demographic factors associated with knowledge of antibiotics and antibiotic resistance in Malaysia. (2) Methods: A cross-sectional study was carried out between April 2022 and March 2023. Malaysian adults aged ≥18 years old and able to understand English or Malay were recruited. During data collection, the WHO questionnaire "Antibiotic Resistance, Multi-Country Public Awareness Survey" was used. Data were collected across 14 states in Malaysia. (3) Results: A total of 517 participants completed the questionnaire. Most participants were females (67.9%), aged 30-49 (46%), and from central Malaysia (69.8%). Most participants (98.5%) reported taking antibiotics. A misconception presented was that sore throats, fevers, colds, and flu can be treated with antibiotics. A total of 58.8% of participants had high knowledge of antibiotic usage (scores 12-15), while 64% had high knowledge of antibiotic resistance (scores 9-14). Findings indicate that increasing age, income, and education were associated with higher knowledge. (4) Conclusions: This study highlights the knowledge deficiency of antibiotic resistance among Malaysians. Educational programs should engage a younger and lower socio-economic population to increase awareness.

Evaluation of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia: Epidemiology, clinical characteristics, and outcomes in the older patients in a tertiary teaching hospital in Malaysia.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is a major concern in the global healthcare system. However, data from Asian regions dealing with the singularity of this infection in older persons is lacking. We aimed to identify the differences in the clinical characteristics and outcomes of MRSA bacteremia between adults aged 18-64 and ≥ 65 years. METHODS: A retrospective study cohort was conducted at the University Malaya Medical Centre (UMMC) on cases of MRSA bacteremia from 2012 to 2016. Patient demographic and clinical data were collected for risk factors analyses. RESULTS: New cases of MRSA bacteremia showed a trend of increase from 0.12 to 100 admissions in 2012 to 0.17 per 100 admissions in 2016 but a drop was observed in 2014 (0.07 per 100 admissions). Out of the 275 patients with MRSA bacteremia, 139 (50.5%) patients were aged ≥ 65 years old. Co-morbidities and severity at presentation were significantly higher among older adults, including diabetes mellitus (p = 0.035), hypertension (p = 0.001), and ischemic heart disease (p < 0.001), as well as higher Charlson Comorbidity Index (p < 0.001) and Pitt bacteremia scores (p = 0.016). Central line-associated bloodstream infections were more common among younger patients (37.5% vs. 17.3% in older patients, p < 0.001), while skin and soft tissue infections are more frequent among older adults (20.9% vs. 10.3% in younger patients, p = 0.016). All-cause mortality and in-hospital mortality were significantly higher in older patients (82.7% and 56.1% vs. 63.2% and 28.7% in younger patients, p < 0.001). Multivariate analysis revealed age ≥ 65 years (adjusted odds ratio: 3.36; 95% confidence interval: 1.24-9.13), Pitt score ≥ 3 (2.15; 1.54-3.01), hospital (6.12; 1.81-20.72) and healthcare (3.19; 1.30-7.81) acquisition of MRSA, indwelling urinary catheters (5.43; 1.39-21.23), inappropriate targeted treatment (8.08; 1.15-56.86), lack of infectious disease team consultation (2.90; 1.04-8.11) and hypoalbuminemia (3.31; 1.25-8.79), were significant risk factors for 30-day mortality. CONCLUSION: Older patients' risk of mortality from MRSA bacteremia was three times higher than younger patients. Our data will contribute to developing and validating a robust scoring system for risk-stratifying patients to achieve better management and improved clinical outcomes.

Factors Associated With Nosocomial COVID-19 Infection Among Health Care Workers in an Urban-Setting Malaysia Hospital.

This single-center study aimed to explore the factors associated with coronavirus disease (COVID-19) transmission in a hospital. All laboratory-confirmed COVID-19 cases among health care workers (HCWs) in a tertiary hospital in Malaysia were analyzed cross-sectionally from January 25, 2020, to September 10, 2021. A total of 897 HCWs in the hospital had laboratory-confirmed COVID-19 infection during the study period. Around 37.4% of HCWs were suspected to acquire COVID-19 infection from the hospital workplace. Factors associated with lower odds of workplace COVID-19 transmission were being females, ≥30 years old, fully vaccinated, and working as clinical support staff. Involvement in COVID-19 patient care was significantly associated with higher odds (adjusted odds ratio = 3.53) of workplace COVID-19 transmission as compared with non-workplace transmission. Most HCWs in the tertiary hospital acquired COVID-19 infection from non-workplace settings. During a pandemic, it is important to communicate with HCWs about the risk of both workplace and non-workplace COVID-19 transmission and to implement measures to reduce both workplace and non-workplace COVID-19 transmission.

Socio-Demographic Factors Associated with Antibiotics and Antibiotic Resistance Knowledge and Practices in Vietnam: A Cross-Sectional Survey.

(1) Background: Antibiotic resistance (ABR) has been escalating to seriously high levels worldwide, accelerated by the misuse and overuse of antibiotics, especially in Vietnam. In this work, we investigated the Vietnamese public socio-demographic and knowledge factors associated with inappropriate practices of antibiotics to better understand the country's antibiotic use and ABR. (2) Methods: To this end, a cross-sectional survey was conducted among Vietnamese people aged 18−60 years via Computer Assisted Telephone Interviews (CATIs) from May 2019 to November 2019. (3) Results: Among 3069 responses distributed equally in all 63 provinces in Vietnam, 1306 respondents completed the survey (response rate of 42.5%). Socio-demographically, most participants were male (56.4%), 18−25 years old (40.4%), located in Southern Vietnam (67.1%), highly educated (93.7%), and possessed medical insurance (95.3%). Respondents with higher education levels (college and above) had 2.663 times higher knowledge scores than those with lower education levels (p < 0.001). High-income respondents possessed more knowledge than low-income respondents (OR = 1.555, CI 95% 0.835−2.910, p = 0.024). Students, non-skilled workers, skilled workers, and professionals and managers had 0.052, 0.150, 0.732, and 0.393 times lower practice scores than the unemployed group, respectively (p < 0.001). Furthermore, respondents with higher/adequate knowledge scores had higher practice scores than those with inadequate knowledge scores (p < 0.05). (4) Conclusions: The findings indicate that socio-demographic differences in knowledge and practices exist, and focusing on these issues should be the priority in forthcoming interventions. The research data also provide information for policy makers to raise the community's awareness of ABR.

The feasibility and generalizability of assessing the appropriateness of antimicrobial prescribing in hospitals: a review of the Australian National Antimicrobial Prescribing Survey.

The National Antimicrobial Prescribing Survey (NAPS) is a web-based qualitative auditing platform that provides a standardized and validated tool to assist hospitals in assessing the appropriateness of antimicrobial prescribing practices. Since its release in 2013, the NAPS has been adopted by all hospital types within Australia, including public and private facilities, and supports them in meeting the national standards for accreditation. Hospitals can generate real-time reports to assist with local antimicrobial stewardship (AMS) activities and interventions. De-identified aggregate data from the NAPS are also submitted to the Antimicrobial Use and Resistance in Australia surveillance system, for national reporting purposes, and to strengthen national AMS strategies. With the successful implementation of the programme within Australia, the NAPS has now been adopted by countries with both well-resourced and resource-limited healthcare systems. We provide here a narrative review describing the experience of users utilizing the NAPS programme in Canada, Malaysia and Bhutan. We highlight the key barriers and facilitators to implementation and demonstrate that the NAPS methodology is feasible, generalizable and translatable to various settings and able to assist in initiatives to optimize the use of antimicrobials.

Utility and Applicability of Rapid Diagnostic Testing in Antimicrobial Stewardship in the Asia-Pacific Region: A Delphi Consensus.

Rapid diagnostic tests (RDTs) facilitate fast and accurate identification of infectious disease microorganisms and are a valuable component of multimodal antimicrobial stewardship (AMS) programs but are currently underutilized in the Asia-Pacific region. An experienced group of infectious diseases clinicians, clinical microbiologists, and a clinical pharmacist used a modified Delphi consensus approach to construct 10 statements, aiming to optimize the utility and applicability of infection-related RDTs for AMS in the Asia-Pacific region. They provide guidance on definition, types, optimal deployment, measuring effectiveness, and overcoming key challenges. The Grading of Recommendations Assessment, Development, and Evaluation system was applied to indicate the strength of the recommendation and the quality of the underlying evidence. Given the diversity of the Asia-Pacific region, the trajectory of RDT development will vary widely; the collection of local data should be prioritized to allow realization and optimization of the full benefits of RDTs in AMS.

Factors influencing inappropriate use of antibiotics: Findings from a nationwide survey of the general public in Malaysia.

Antibiotic resistance is one of the biggest threats to global public health. Misuse of antibiotics has never been investigated on a nationwide scale among the general public in Malaysia. This study aimed to identify sociodemographic and knowledge factors associated with inappropriate use of antibiotics in the Malaysian context to inform the development of interventions to mitigate inappropriate antibiotic use. We conducted computer-assisted telephone interviews (CATI) between June 2019 and December 2019. The telephone numbers were randomly generated from the electronic residential telephone directory of all 13 states and 3 Federal Territories in Malaysia. The survey consisted of questions on demographics, knowledge about antibiotics and antibiotic resistance (53 items), and practices of antibiotic use (16 questions). A total of 864 complete responses were received. Pronounced erroneous beliefs that antibiotics are effective against infections caused by viruses and that antibiotics can speed up recovery from coughs and colds were evident. The proportions that were aware of the terms 'drug resistance', 'antimicrobial resistance', and 'superbugs' were low. The mean and standard deviation (SD) for the antibiotic knowledge score was 23.7 (SD ±9.25; range 0 to 50) out of a possible score of 52. Regarding antibiotic practices, a considerable proportion reported non-adherence to recommended doses. The mean and SD for the antibiotic practices score was 37.9 (SD ± 6.5; range 17 to 47) out of a possible score of 48. Participants who earned an average monthly household income of MYR1001-3000 (OR 1.61, 95% CI 1.04-2.50) were more likely to report higher antibiotic practice scores than those with

Investigation of biofilm formation in methicillin-resistant Staphylococcus aureus associated with bacteraemia in a tertiary hospital.

Biofilm formation is an important physiological process in Staphylococcus aureus (S. aureus) that can cause infections in humans. In this study, the ability of 36 methicillin-resistant S. aureus (MRSA) clinical isolates to form biofilm was studied based on genotypic and phenotypic approaches. These isolates were genotyped based on the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and biofilm-associated genes (icaAD) via polymerase chain reactions. Phenotyping was performed based on the determination of the strength of biofilm formation of MRSA isolates in vitro. The most prevalent MSCRAMMs and biofilm-associated genes were clfA, eno, and icaD, followed by clfB. The fnbB (38.9%) and ebpS (11.1%) occurred less frequently among the MRSA isolates, while bbp and fnbA genes were absent from all isolates. The MRSA isolates were mostly moderate to strong biofilm formers, despite the heterogeneity of the MSCRAMM profiles. MRSA isolates from different infection sources (primary, catheter-related bloodstream, or secondary infections) were capable of forming strong biofilms. However, persistent bacteraemia was observed only in 19.4% of the MRSA-infected individuals. This study suggested that persistent MRSA bacteraemia in patients might not be associated with the biofilm-forming ability of the isolates.

Genetic mechanisms and correlated risk factors of antimicrobial-resistant ESKAPEE pathogens isolated in a tertiary hospital in Malaysia.

BACKGROUND: Knowledge on the epidemiology, genotypic and phenotypic features of antimicrobial-resistant (AMR) ESKAPEE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli) and their association with hospital-acquired infections (HAIs) are limited in Malaysia. Therefore, we evaluated the AMR features and resistance mechanisms of the ESKAPEE pathogens collected in a tertiary hospital located in the capital of Malaysia. METHODS: A total of 378 AMR-ESKAPEE strains were obtained based on convenience sampling over a nine-month study period (2019-2020). All strains were subjected to disk diffusion and broth microdilution assays to determine the antimicrobial susceptibility profiles. Polymerase chain reaction (PCR) and DNA sequence analyses were performed to determine the AMR genes profiles of the non-susceptible strains. Chi-square test and logistic regression analyses were used to correlate the AMR profiles and clinical data to determine the risk factors associated with HAIs. RESULTS: High rates of multidrug resistance (MDR) were observed in A. baumannii, K. pneumoniae, E. coli, and S. aureus (69-89%). All organisms except E. coli were frequently associated with HAIs (61-94%). Non-susceptibility to the last-resort drugs vancomycin (in Enterococcus spp. and S. aureus), carbapenems (in A. baumannii, P. aeruginosa, and Enterobacteriaceae), and colistin (in Enterobacteriaceae) were observed. Both A. baumannii and K. pneumoniae harbored a wide array of extended-spectrum ß-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaOXA). Metallo-ß-lactamase genes (blaVEB, blaVIM, blaNDM) were detected in carbapenem-resistant strains, at a higher frequency compared to other local reports. We detected two novel mutations in the quinolone-resistant determining region of the gyrA in fluoroquinolone-resistant E. coli (Leu-102-Ala; Gly-105-Val). Microbial resistance to ampicillin, methicillin, and cephalosporins was identified as important risk factors associated with HAIs in the hospital. CONCLUSION: Overall, our findings may provide valuable insight into the microbial resistance pattern and the risk factors of ESKAPEE-associated HAIs in a tertiary hospital located in central Peninsular Malaysia. The data obtained in this study may contribute to informing better hospital infection control in this region.

One-Step Differential Detection of OXA-48-Like Variants Using High-Resolution Melting (HRM) Analysis.

OXA-48-like carbapenemase gene remains a hidden threat, as different OXA-48 variants have varying presentations of susceptibility to antibiotics that might affect the treatment decisions. Rapid detection and differentiation of OXA-48-like carbapenemase genes are critical for targeted treatment and infection control. In this study, we aimed to develop high-resolution melting (HRM) analysis for the differentiation of OXA-48 variants. HRM analysis is a post-polymerase chain reaction (post-PCR) method for identification of small variations in nucleic acid sequences based on the PCR dissociation curve. A total of 82 bacterial strains, which consisted of Enterobacteriaceae and non-Enterobacteriaceae, were collected from a tertiary teaching hospital. The sensitivity and specificity of the assay were determined, and the developed assay was evaluated using the collected isolates against conventional-sequencing method. Overall, the developed assay was able to detect isolates that harboured OXA-48 and OXA232/OXA-181 by showing two distinct peaks at 81.1 ± 0.2 °C and 82.1 ± 0.2 °C, respectively. The detection limit of the assay was 1.6 x 10-6 ng/µl for OXA-48 and 1.8 x 10-7 ng/µl for OXA-232/OXA-181. This assay showed 100% specificity when evaluated on a panel of 37 isolates comprised of different species of bacteria and yeasts. When the assay with isolates collected in the year 2016 was first evaluated, the assay showed comparable results with conventional PCR-sequencing method where 34 OXA-48 and OXA-232/OXA-181 were detected. By using HRM analysis, the presence of OXA-48-like variants could be easily identified within 3 hours from the pure culture.

Atypical Presentation of Methicillin-Susceptible Staphylococcus aureus Infection in a Dengue-Positive Patient: A Case Report with Virulence Genes Analysis.

Concurrent bacteraemia in patients with dengue fever is rarely reported. We report a case of a patient who initially presented with symptoms typical of dengue fever but later succumbed to septic shock caused by hypervirulent methicillin-susceptible Staphylococcus aureus (MSSA). A 50-year-old female patient with hypertension and diabetes mellitus presented with typical symptoms of dengue fever. Upon investigation, the patient reported having prolonged fever for four days prior to hospitalization. Within 24 hours post-admission, the patient developed pneumonia and refractory shock, and ultimately succumbed to multiple-organs failure. Microbiological examination of the blood culture retrieved a pan susceptible MSSA strain. Genomic sequence analyses of the MSSA strain identified genes encoding staphylococcal superantigens (enterotoxin staphylococcal enterotoxin C 3 (SEC3) and enterotoxin-like staphylococcal enterotoxins-like toxin L (SElL)) that have been associated with toxic shock syndrome in human hosts. Genes encoding important toxins (Panton-Valentine leukocidins, alpha-haemolysin, protein A) involved in the development of staphylococcal pneumonia were also present in the MSSA genome. Staphylococcus aureus co-infections in dengue are uncommon but could be exceptionally fatal if caused by a toxin-producing strain. Clinicians should be aware of the risks and signs of sepsis in dengue fever, thus allowing early diagnosis and starting of antibiotic treatment in time to lower the mortality and morbidity rates.

Antibiotics in surgical wards: use or misuse? A newly industrialized country's perspective.

INTRODUCTION: Studies exploring the appropriateness of therapeutic antibiotic use among surgical patients are limited, particularly in developing countries. Therefore, the aim of our study was to determine the appropriateness of antibiotics prescribed in a surgical setting in Malaysia. METHODOLOGY: A prospective observational study was conducted in two surgical wards at a tertiary hospital in Malaysia from November 2012-July 2013. Data was collected using a case report form. The appropriateness of antibiotic therapy was based on compliance with either the Malaysian National Antibiotic Guidelines 2008 or International Clinical Practice Guidelines and determined by an expert panel (consisting of two infectious disease consultants and a pharmacist). RESULTS: Over the study period, a total of 593 antibiotic courses were prescribed for 129 patients (4.6±3.4 antibiotics/patient). Only 34 (26.4%) patients received appropriate antibiotic therapy, whilst 95 (73.6%) patients received at least one course of inappropriate antibiotic therapy. The prevalence of inappropriate antibiotic use was 214 (66.3%) and 55 (42.0%) for prophylactic and therapeutic purposes, respectively. The most common causes of inappropriate prophylactic antibiotics were inappropriate timing 20 (36.4%) and inappropriate duration of prophylaxis 19 (34.5%). In cases of inappropriate timing, 9 (45%) were administered too late while 6 (30%) were too early. In contrast, inappropriate choice of antibiotics (42.1%) and inappropriate indication (40.7%) were the most common reasons encountered for inappropriate therapeutic antibiotics. CONCLUSION: Our study suggests considerable inappropriate use of both prophylactic and therapeutic antibiotics in the surgical wards; highlighting an urgent need for antibiotic stewardship initiatives in this setting.