Assessing the colony morphotypes and antibiotic susceptibility profile of Malaysian clinical Burkholderia pseudomallei to support the use of EUCAST disk diffusion breakpoints to determine antibiotic resistance.

Burkholderia pseudomallei is intrinsically resistant to many antibiotics. This study aimed to assess bacterial colony morphotypes and the validity of using disk diffusion method (DD) to determine antibiotic resistance in Malaysian clinical B. pseudomallei isolates for ceftazidime (CAZ), meropenem (MEM), amoxicillin-clavulanate (AMC) and doxycycline (DOX). DD produced good categorical agreements exhibiting concordance of 100% with reference method, broth microdilution for CAZ and DOX, 98.6% for MEM and 97.2% for AMC. Smooth-centred colonies were most frequently observed. EUCAST DD interpretative criterion is suitable to interpret B. pseudomallei CAZ, MEM, AMC and DOX resistance. Increasing AMC MIC in B. pseudomallei is a concern.

Secondary and Co-Infections in Hospitalized COVID-19 Patients: A Multicenter Cross-Sectional Study in Malaysia.

Bacterial and fungal secondary and co-infections are commonly identified with viral respiratory infections. This study was undertaken to determine the incidence and factors associated with bacterial and fungal infections in patients with COVID-19 as well as antibiotics prescription patterns within the first and second waves of the outbreak in Malaysia. Clinical records of 3532 COVID-19 patients admitted to hospitals in Malaysia between 4 February and 4 August 2020 were analyzed. Co-morbidities, clinical features, investigations, treatment, and complications were captured using the REDCap database. Culture and sensitivity test results were retrieved from the WHONET database. Univariate and multivariate regression analyses were used to identify associated determinants. A total of 161 types of bacterial and fungal infections were found in 81 patients, i.e., 2.3%. The most common bacterial cultures were Gram-negative, i.e., Pseudomonas aeruginosa (15.3%) and Klebsiella pneumoniae (13.9%). The most common fungal isolate was Candida albicans (41.2%). Augmentin, ceftriaxone, tazocin, meropenem, and azithromycin were the five most frequently prescribed antibiotics. The latter four were classified under the "Watch" category in the WHO AwaRe list. Our data showed that bacterial and fungal secondary and co-infections were frequently found in severely ill COVID-19 patients and were associated with a higher mortality rate.

Vero CCL-81 and Calu-3 Cell Lines as Alternative Hosts for Isolation and Propagation of SARS-CoV-2 Isolated in Malaysia.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the etiologic agent for the pneumonia outbreak that started in early December 2019 in Wuhan City, Hubei Province, China. To date, coronavirus disease (COVID-19) has caused almost 6 million deaths worldwide. The ability to propagate the virus into a customizable volume will enable better research on COVID-19 therapy, vaccine development, and many others. In the search for the most efficient replication host, we inoculated three (3) local SARS-CoV-2 isolates of different lineages (Clade L/Lineage B Wuhan, Clade GR/Lineage B.1.1.354, and Clade O/Lineage B.6.2) into various clinically important mammalian cell lines. The replication profile of these isolates was evaluated based on the formation of cytopathic effects (CPE), viral load (Ct value and plaque-forming unit (pfu)), as well as observation by electron microscopy (EM). Next-generation sequencing (NGS) was performed to examine the genomic stability of the propagated SARS-CoV-2 in these cell lines. Our study found that Vero E6 and Vero CCL-81 cell lines posed similar capacities in propagating the local isolates, with Vero CCL-81 demonstrating exceptional potency in conserving the genomic stability of the Lineage B Wuhan isolate. In addition, our study demonstrated the utility of Calu-3 cells as a replication host for SARS-CoV-2 without causing substantial cellular senescence. In conclusion, this study provides crucial information on the growth profile of Malaysian SARS-CoV-2 in various mammalian cell lines and thus will be a great source of reference for better isolation and propagation of the SARS-CoV-2 virus isolated in Malaysia.

Prevalence of Antibiotic-Resistant Pathogenic Bacteria and Level of Antibiotic Residues in Hospital Effluents in Selangor, Malaysia: Protocol for a Cross-sectional Study.

BACKGROUND: Antimicrobial resistance (AMR) has emerged as a major global public health challenge due to the overuse and misuse of antibiotics for humans and animals. Hospitals are among the major users of antibiotics, thereby having a large contribution to AMR. OBJECTIVE: The aim of this study is to determine the prevalence of antibiotic-resistant pathogenic bacteria and the level of antibiotic residues in the hospital effluents in Selangor, Malaysia. METHODS: A cross-sectional study will be performed in the state of Selangor, Malaysia. Tertiary hospitals will be identified based on the inclusion and exclusion criteria. The methods are divided into three phases: sample collection, microbiological analysis, and chemical analysis. Microbiological analyses will include the isolation of bacteria from hospital effluents by culturing on selective media. Antibiotic sensitivity testing will be performed on the isolated bacteria against ceftriaxone, ciprofloxacin, meropenem, vancomycin, colistin, and piperacillin/tazobactam. The identification of bacteria will be confirmed using 16S RNA polymerase chain reaction (PCR) and multiplex PCR will be performed to detect resistance genes (ermB, mecA, blaNDM-L, blaCTX-M, blaOXA-48, blaSHV, VanA, VanB, VanC1, mcr-1, mcr-2, mcr-3, Intl1, Intl2, and qnrA). Finally, the level of antibiotic residues will be measured using ultrahigh-performance liquid chromatography. RESULTS: The expected outcomes will be the prevalence of antibiotic-resistant Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter (ESKAPE) bacterial species from the hospital effluents, the occurrence of antibiotic resistance genes (ARGs) from the isolated ESKAPE bacteria, and the level of antibiotic residues that may be detected from the effluent. Sampling has been conducted in three hospitals. Data analysis from one hospital showed that as of July 2022, 80% (8/10) of E. faecium isolates were resistant to vancomycin and 10% (1/10) were resistant to ciprofloxacin. Further analysis will be conducted to determine if the isolates harbor any ARGs and effluent samples are being analyzed to detect antibiotic residues. Sampling activities will be resumed after being suspended due to the COVID-19 pandemic and are scheduled to end by December 2022. CONCLUSIONS: This study will provide the first baseline information to elucidate the current status of AMR of highly pathogenic bacteria present in hospital effluents in Malaysia. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/39022.

Prevalence of Antibiotic-Resistant Bacteria and Antibiotic-Resistant Genes and the Quantification of Antibiotics in Drinking Water Treatment Plants of Malaysia: Protocol for a Cross-sectional Study.

BACKGROUND: Antimicrobial resistance is a known global public health threat. In addition, it brings serious economic consequences to agriculture. Antibiotic resistance in humans, animals, and environment is interconnected, as proposed in the tricycle surveillance by the World Health Organization. In Malaysia, research and surveillance of antimicrobial resistance are mainly performed in clinical samples, agricultural settings, and surface waters, but no surveillance of the drinking water systems has been performed yet. Hence, this policy-driven study is a combined effort of microbiologists and engineers to provide baseline data on the magnitude of antimicrobial resistance in the drinking water systems of Malaysia. OBJECTIVE: The aim of this study was to study the baseline level of antibiotic-resistant bacteria in the drinking water distribution systems of Malaysia by collecting samples from the pretreatment and posttreatment outlets of water treatment plants in a selected state of Malaysia. We aimed to determine the prevalence of antibiotic-resistant bacteria, the occurrence of antibiotic-resistant genes, and the level of antibiotics present in the drinking water systems. METHODS: This is a laboratory-based, cross-sectional study in a selected state of Malaysia. Water samples from 6 drinking water treatment plants were collected. Samples were collected at 3 sampling points, that is, the intake sampling station, service reservoir outlet station, and the distribution system sampling station. These were tested against 7 types of antibiotics in triplicates. Samples were screened for antibiotic-resistant bacteria and antibiotic-resistant genes and quantified for the level of antibiotics present in the drinking water treatment plants. RESULTS: We will show the descriptive statistics of the number of bacterial colonies harvested from water samples grown on Reasoner's 2A agar with or without antibiotics, the occurrence of antibiotic-resistant genes, and the level of antibiotics detected in the water samples. The sampling frame was scheduled to start from November 2021 and continue until December 2022. Data analysis is expected to be completed by early 2023, and the results are expected to be published in mid-2023. CONCLUSIONS: This study provides baseline information on the status of the antimicrobial-resistant bacteria, the presence of resistance genes as contaminants, and the level of antibiotics present in the drinking water systems of Malaysia, with the aim of demonstrating to policymakers the need to consider antimicrobial resistance as a parameter in drinking water surveillance. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/37663.

Predominance of the East-Asian Beijing genotype in a Mycobacterium tuberculosis drug-resistant population in Central Malaysia.

OBJECTIVES: Previous diversity studies on local Mycobacterium tuberculosis (MTB) isolates, with or without antibiotic resistance, showed predominance of Indo-Oceanic East African-Indian (EAI) strains. This study focused specifically on a drug-resistant MTB population from Central Malaysia and aimed to investigate the genotypes and resistance patterns involved. METHODS: Whole-genome sequencing was performed on 56 local MTB isolates with known rifampicin resistance or multidrug resistance towards 13 anti-TB agents. Analysis of each genome sequence was performed using the widely recognized online MTB genotyping platforms, TBProfiler and Mykrobe, to determine lineage and genotypic drug resistance profiles. RESULTS: Forty (71.4%) isolates were identified as East-Asian Beijing strains. Phenotypic to genotypic antibiotic resistance patterns differed in 33 isolates (58.9%), with one isolate showing extensive drug-resistance (XDR) previously not detected by conventional drug-susceptibility testing. CONCLUSION: This drug resistance population study demonstrated predominance of the East-Asian Beijing strains and a newly detected extensively drug-resistant MTB (XDR-TB) isolate in Malaysia. Information regarding the association between lineage and drug-resistant TB in Malaysia is scarce, and more studies are needed to determine the significance of such association, if any, in our local settings.

Multidrug-Resistant Gram-Negative Bacteria and Extended-Spectrum ß-Lactamase-Producing Klebsiella pneumoniae from the Poultry Farm Environment.

The indiscriminate use and overuse of various antibiotics have caused the rapid emergence of antibiotic-resistant bacteria (ARB) in poultry products and the surrounding environment, giving rise to global public health issues. This study aimed to determine the prevalence of multidrug-resistant (MDR) Gram-negative bacteria (GNB) found in the environment of poultry farms and to evaluate the risk of contamination in these farms based on multiple antibiotic resistance (MAR) index values. Soil and effluent samples were collected from 13 poultry farms. The VITEK 2 system was used for bacterial identification and susceptibility testing of the isolates. The identified Gram-negative isolates were Acinetobacter spp., Aeromonas spp., Enterobacter spp., Klebsiella pneumoniae, Proteus spp., Providencia spp., Pseudomonas spp., and Sphingomonas paucimobilis. The results showed that Enterobacter spp., Aeromonas spp., and Providencia spp. exhibited the highest MDR rates and MAR indices; 14% of K. pneumoniae isolates (3/21 isolates) were resistant to 13 antibiotics and found to be extended-spectrum ß-lactamase (ESBL)-producing bacteria. As for the tested antibiotics, 96.6% of the isolates (28/29 isolates) demonstrated resistance to ampicillin, followed by ampicillin-sulbactam (55.9% [33/59 isolates]) and cefazolin (54.8% [57/104 isolates]). The high percentage of MDR bacteria and the presence of ESBL-producing K. pneumoniae strains suggested the presence of MDR genes from the poultry farm environment, which poses an alarming threat to the effectiveness of the available antibiotic medicines to treat infectious diseases. Therefore, the use of antibiotics should be regulated and controlled, while studies addressing One Health issues are vital for combating and preventing the development and spread of ARB. IMPORTANCE The occurrence and spread of ARB due to high demand in poultry industries are of great public health concern. The widespread emergence of antibiotic resistance, particularly MDR among bacterial pathogens, poses challenges in clinical treatment. Some pathogens are now virtually untreatable with current antibiotics. However, those pathogens were rarely explored in the environment. In alignment with the concept of One Health, it is imperative to study the rate of resistance in the environment, because this domain plays an important role in the dissemination of bacteria to humans, animals, and other environmental areas. Reliable data on the prevalence of MDR bacteria are crucial to curb the spread of bacterial pathogens that can cause antimicrobial-resistant infections.

Virulence Gene Profile, Antimicrobial Resistance and Multilocus Sequence Typing of Salmonella enterica Subsp. enterica Serovar Enteritidis from Chickens and Chicken Products.

This study was undertaken to determine the virulence, antimicrobial resistance and molecular subtypes of Salmonella in the Central Region of Peninsular Malaysia. A total of 45 Salmonella Enteritidis were detected from live chicken (cloacal swab), and chicken products (fresh and ready-to-eat meat) samples upon cultural isolation and serotyping. Similarly, an antimicrobial susceptibility test based on the Kirby Bauer disk diffusion method as well as antimicrobial resistance AMR genes, virulence determinants and multilocus sequence typing (MLST) typing were conducted after the Whole Genome Sequencing and analysis of the isolates. The results indicate that sequence types ST1925 (63.7%), and ST11 (26.5%) were the predominant out of the seven sequence types identified (ST292, ST329, ST365, ST423 and ST2132). The phenotypic antimicrobial profile corresponds to the genotypic characterization in that the majority of the isolates that exhibited tetracycline, gentamycin and aminoglycoside resistance; they also possessed the tetC and blaTEM ß-Lactam resistance genes. However, isolates from cloacal swabs showed the highest number of resistance genes compared to the chicken products (fresh and ready-to-eat meat) samples. Furthermore, most of the virulence genes were found to cluster in the Salmonella pathogenicity island (SPI). In this study, all the isolates were found to possess SPI-1, which codes for the type III secretion system, which functions as actin-binding proteins (SptP and SopE). The virulence plasmid (VP) genes (spvB, spvC) were present in all genotypes except ST365. The findings of this study, particularly with regard to the molecular subtypes and AMR profiles of the Salmonella Enteritidis serotype shows multidrug-resistance features as well as genetic characteristics indicative of high pathogenicity.

Hirudotherapy in Wound Healing.

Leeches are hermaphrodite, bloodsucking parasitic worms usually found in places with fresh water. Leech therapy existed 3000 years, and it is being used at a different scope. Several species of leeches have been used in medicine, and the most common species used is Hirudo medicinalis. Leeches suck the excess blood, reduce the swelling in the tissues, and promote healing by allowing fresh oxygenated blood to reach the area until normal circulation can be restored. Pain relief from leech therapy is rapid, effective, and long-lasting in many conditions. The aim of this study was to evaluate the efficacy and duration of healing utilizing sterile medicinal leeches, Hirudinaria manillensis, in the management of pain and wound healing. Leech was taken out from its sterile tube by using a pair of non-tooth sterile plastic forceps and gloved hands. Each leech was left in place for as long as it was feeding. Leeches were removed only after they became detached from the patient. The specimen jars containing the used leeches were sealed in either a biohazard bag or in a small yellow clinical waste bin liner securely fastened with a cable tie. The leech was killed by using 70% alcohol prior to disposal into a yellow hazard bin, which undergoes incineration. All 3 patients had improvements in their condition, especially in terms of reduction in the pain and improvement in their sense of balance. All the wounds healed well. Therefore, leech therapy is effective in reducing pain and increasing perfusion to allow the wounds to heal quickly. However, a more robust trial is needed to show significance as the sample size is small.

Discerning the Antimicrobial Resistance, Virulence, and Phylogenetic Relatedness of Salmonella Isolates Across the Human, Poultry, and Food Materials Sources in Malaysia.

Salmonella enterica subspecies enterica serovar Enteritidis is one of the major foodborne zoonotic pathogens globally. It has significantly impacted human health and global trade. In this investigation, whole-genome sequencing was employed to determine the antimicrobial resistance (AMR) pattern of a collection of Salmonella Enteritidis isolated from humans, poultry, and food sources. The study also investigated the virulence genes profile of the isolates as well as the phylogenetic relationships among strains. Illumina NextSeq technology was used to sequence the genome of 82 Salmonella Enteritidis strains isolated over 3 years (2016-2018) in Peninsular Malaysia. The pattern of resistance showed that tetracycline had the highest frequency (37/82, 45.12%), and isolates from food samples showed the highest rate of 9/18 (50.00%), followed by human 17/35 (48.57%) and then poultry 11/29 (37.93%). The second drug with the highest resistance rate is ampicillin with 5/29 (17.24%) for poultry, 4/35 (11.43%) for human, and 0/18 (0.00%) for food isolates respectively. Similarly, a total of 19 antimicrobial resistance (AMR) genes corresponding to the nine drugs used in the disc diffusion assay were evaluated from the whole genome sequence data. The aminoglycoside resistance gene aac(6')-ly was detected in 79 of the 82 isolates (96.34%). While the phylogenetic analysis revealed distinct lineages isolated, the three sources indicating possible cross-contamination. In conclusion, the results showed that the genomic profile of Salmonella Enteritidis isolated from humans, poultry, and food samples share genetic traits, hence the need to institute measures at controlling the continuous spread of these resistant pathogens.

Seroprevalence of diphtheria toxoid IgG antibodies in the Malaysian population.

BACKGROUND: Despite high childhood immunization coverage, sporadic cases of diphtheria have been reported in Malaysia in recent years. This study aims to evaluate the seroprevalence of diphtheria among the Malaysian population. METHODS: A total of 3317 respondents age 2 years old to 60 years old were recruited in this study from August to November 2017. Enzyme-linked immunosorbent assay (ELISA) was used to measure the level of IgG antibody against the toxoid of C. diphtheriae in the blood samples of respondents. We classified respondent antibody levels based on WHO definition, as protective (≥0.1 IU/mL) and susceptible (< 0.1 IU/mL) to C. diphtheriae infection. RESULTS: Among the 3317 respondents, 57% were susceptible (38.1% of children and 65.4% of adults) and 43% (61.9% of children and 34.6% of adults) had protective antibody levels against diphtheria. The mean antibody level peaked among individuals aged 1-2 years old (0.59 IU/mL) and 6-7 years old (0.64 IU/mL) but generally decreased with age, falling below 0.1 IU/mL at around 4-6 years old and after age 20 years old. There was a significant association between age [Children: χ2 = 43.22(df = 2),p < 0.001)], gender [Adults: χ2 = 5.58(df = 1),p = 0.018] and ethnicity [Adults: χ2 = 21.49(df = 5),p = 0.001] with diphtheria toxoid IgG antibody level. CONCLUSIONS: About 57% of the Malaysian population have inadequate immunity against diphtheria infection. This is apparently due to waning immunity following childhood vaccination without repeated booster vaccination in adults. Children at age 5-6 years old are particularly vulnerable to diphtheria infection. The booster vaccination dose normally given at 7 years should be given earlier, and an additional booster dose is recommended for high-risk adults.

COVID-19 screening test by using random oropharyngeal saliva.

An optimal clinical specimen for accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by minimizing the usage of consumables and reduce hazard exposure to healthcare workers is an urgent priority. The diagnostic performance of SARS-CoV-2 detection between healthcare worker-collected nasopharyngeal and oropharyngeal (NP + OP) swabs and patient performed self-collected random saliva was assessed. Paired NP + OP swabs and random saliva were collected and processed within 48 h of specimen collection from two cohort studies which recruited 562 asymptomatic adult candidates. Real-time reverse-transcription polymerase chain reaction targeting Open reading frame 1a (ORF1a) and nucleocapsid (N) genes was performed and the results were compared. Overall, 65 of 562 (28.1%) candidates tested positive for COVID-19 based on random saliva, NP + OP swabs, or both testing techniques. The detection rate of SARS-CoV-2 was higher in random saliva compared to NP + OP testing (92.3%; 60/65 vs. 73.8%; 48/65; p < .05). The estimated sensitivity and specificity of random saliva were higher than NP + OP swabs (95.0; 99.9 vs. 72.2; 99.4). The Ct  values of ORF1a and N genes were significantly lower in random saliva compared to NP + OP swabs specimens. Our findings demonstrate that random saliva is an alternative diagnostic specimen for the detection of SARS-CoV-2. Self-collected random oropharyngeal saliva is a valuable specimen that provides accurate SARS-CoV-2 surveillance testing of a community.

Comparing Nasopharyngeal Swab and Early Morning Saliva for the Identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

BACKGROUND: The ideal severe acute respiratory syndrome coronavirus 2 (SARs-CoV-2) testing method would be accurate and also be patient-performed to reduce exposure to healthcare workers. The aim of this study was to compare patient-performed testing based on a morning saliva sample with the current standard testing method, healthcare worker-collected sampling via a nasopharyngeal swab (NPS). METHODS: This was a prospective single center study which recruited 217 asymptomatic adult male participants in a coronavirus disease 2019 (COVID-19) quarantine center who had tested positive for SARS-CoV-2 8-10 days prior to isolation. Paired NPS and saliva specimens were collected and processed within 5 hours of sample collection. Real time reverse transcription polymerase chain reaction (RT-PCR) targeting Envelope (E) and RNA-dependent RNA polymerase (RdRp) genes was performed and the results were compared. RESULTS: Overall, 160 of the 217 (74%) participants tested positive for COVID-19 based on saliva, NPS, or both testing methods. The detection rate for SARS-CoV-2 was higher in saliva compared to NPS testing (93.1%, 149/160 vs 52.5%, 84/160, P < .001). The concordance between the 2 tests was 45.6% (virus was detected in both saliva and NPS in 73/160), whereas 47.5% were discordant (87/160 tested positive for 1 whereas negative for the other). The cycle threshold (Ct) values for E and RdRp genes were significantly lower in saliva specimens compared to NP swab specimens. CONCLUSIONS: Our findings demonstrate that saliva is a better alternative specimen for detection of SARS-CoV-2. Taking into consideration, the simplicity of specimen collection, shortage of PPE and the transmissibility of the virus, saliva could enable self-collection for an accurate SARS-CoV-2 surveillance testing.

Multi locus sequence typing of clinical Burkholderia pseudomallei isolates from Malaysia.

BACKGROUND: Melioidosis is a neglected tropical disease with rising global public health and clinical importance. Melioidosis is endemic in Southeast Asia and Northern Australia and is of increasing concern in Malaysia. Despite a number of reported studies from Malaysia, these reports are limited to certain parts of the country and do not provide a cohesive link between epidemiology of melioidosis cases and the nation-wide distribution of the causative agent Burkholderia pseudomallei. METHODOLOGY/PRINCIPLE FINDINGS: Here we report on the distribution of B. pseudomallei sequence types (STs) in Malaysia and how the STs are related to STs globally. We obtained 84 culture-confirmed B. pseudomallei from confirmed septicaemic melioidosis patients from all over Malaysia. Prior to performing Multi Locus Sequence Typing, the isolates were subjected to antimicrobial susceptibility testing and detection of the YLF/BTFC genes and BimA allele. Up to 90.5% of the isolates were sensitive to all antimicrobials tested while resistance was observed for antimicrobials typically administered during the eradication stage of treatment. YLF gene cluster and bimABp allele variant were detected in all the isolates. The epidemiological distribution patterns of the Malaysian B. pseudomallei isolates were analysed in silico using phylogenetic tools and compared to Southeast Asian and world-wide isolates. Genotyping of the 84 Malaysian B. pseudomallei isolates revealed 29 different STs of which 6 (7.1%) were novel. ST50 was identified as the group founder followed by subgroup founders ST376, ST211 and ST84. A low-level diversity is noted for the B. pseudomallei isolates described in this study while phylogenetic analysis associated the Malaysian STs to Southeast Asian isolates especially isolates from Thailand. Further analysis also showed a strong association that implicates agriculture and domestication activities as high-risk routes of infection. CONCLUSIONS/SIGNIFICANCE: In conclusion, MLST analysis of B. pseudomallei clinical isolates from all states in Malaysia revealed low diversity and a close association to Southeast Asian isolates.

Carriage of Haemophilus influenzae among children attending childcare centres in Kuala Lumpur, Malaysia in the post vaccination era: A cross-sectional study.

BACKGROUND: Haemophilus influenzae (H. influenzae) is a human upper respiratory tract colonizer which causes wide range of disease especially in children<5 years old and in the elderly. Although worldwide incidence in industrialised countries where Hib vaccination is commonly used has dropped sharply since implementation of H. influenzae type b (Hib) vaccination, there is limited data on the disease burden caused by H. influenzae in Malaysia post vaccination era. A change in predominant serotype from type b to non-b serotypes of H. influenzae in invasive diseases was reported worldwide. We investigated the carriage of H. influenzae post vaccination era among 2-4 years old. METHODOLOGY: Randomly, we collected 436 oropharyngeal swabs from healthy children aged 2-4 years in 30 registered childcare centres in Kuala Lumpur (August 2018-May 2019). Informed consent and written questionnaires were obtained from parents. H. influenzae was identified by standard microbiological methods. Univariable analysis was carried out to describe variables associated with colonization. All variables with p < 0.25 were included in multivariable logistic regression model. A p value < 0.05 was considered significant. RESULTS: A higher carriage rate was noted among the unvaccinated children (4/28; 14.3%) compared to vaccinated children (16/326; 4.9%) but were not statistically significant. The serotypes were type a (9; 37.5%), type b (5; 20.8%), type c (3; 12.5%), type d (2; 8%), type e (1; 4.2%) and type f (4; 16.7%). Variables like age, basic sanitation, immunization status, body mass index were included in multivariable logistic regression test since p values in univariate analysis were<0.25. Planned sewage system was found to be significant (Adjusted OR, 0.06; 95% CI, 0.01-0.46; p = 0.006). CONCLUSION: Fewer carriage rates were observed among children post Hib vaccination era. Hib carriage is still possible after vaccination. The presence non-b serotypes may imply emerging replacement serotypes.

Analysis of Salmonella enterica serovar Enteritidis isolates from chickens and chicken meat products in Malaysia using PFGE, and MLST.

BACKGROUND: Salmonella is a very important foodborne pathogen causing illness in humans. The emergence of drug-resistant strains also constitutes a serious worry to global health and livestock productivity. This study investigated Salmonella isolates from chicken and chicken meat products using the phenotypic antimicrobial screening as well as the molecular characteristics of Salmonella isolates. Upon serotyping of the isolates, the antimicrobial susceptibility profiling using a panel of 9 commonly used antimicrobials was done. Subsequently, the molecular profiles of all the isolates were further determined using Pulsed Field Gel Electrophoresis (PFGE) and the Whole Genome Multi-Locus Sequence Type (wgMLST) analysis in order to obtain the sequence types. RESULTS: The PFGE data was input into FPQuest software, and the dendrogram generated was studied for possible genetic relatedness among the isolates. All the isolates were found to belong to the Salmonella Enteritidis serotype with notable resistance to tetracycline, gentamycin, streptomycin, and sulfadimidine. The S. Enteritidis isolates tested predominantly subtyped into the ST11 and ST1925, which was found to be a single cell variant of ST11. The STs were found to occur in chicken meats, foods, and live chicken cloacal swabs, which may indicate the persistence of the bacteria in multiple foci. CONCLUSION: The data demonstrate the presence of S. Enteritidis among chickens, indicating its preference and reservoir status for enteric Salmonella pathogens.

Detection of Antimicrobial Resistance Genes Associated with Carbapenem Resistance from the Whole-Genome Sequence of Acinetobacter baumannii Isolates from Malaysia.

BACKGROUND: The spread of carbapenem-resistant A. baumannii (CrAb) is gaining worldwide attention. The spread of this pathogen is largely due to its ability to acquire various resistance genes of intrinsic and extrinsic origins that confer unpredictable susceptibility to ß-lactams. The aim of this study was to analyze ß-lactamase genetic compositions of CrAb in Malaysia. METHODS: Whole-genome sequencing (WGS) was carried out on 13 CrAb isolates from clinical samples in Malaysia from 2011 to 2016. RESULTS: Endotracheal aspirate was the dominant clinical sample source (n = 6), and only one isolate was obtained from wound swab. A total of 6 sequence types (STs) of the Oxford scheme were identified, including 4 reported STs and 2 novel STs. Eleven isolates were classified into clonal complex 92 (CC92/ICII), among which ST195 and ST208 were the most prevalent STs. All 13 CrAb isolates harbored multiple ß-lactamase genes. bla OXA-23 (n = 13) and bla OXA-66 (n = 11) were the dominant carbapenemase gene families found in these isolates. All isolates harbor bla ADC, bla OXA-51-like, and bla OXA-23-like genes. bla TEM (n = 7), bla NDM-1 (n = 3), bla CARB-8 (n = 1), and bla PER-3 (n = 1) are amongst other ß-lactamase genes found in this study. ISAba1 was found upstream to bla OXA-23 (n = 13), bla OXA-66 (n = 1), and bla ADC (n = 11). All bla NDM-1 isolates had ISAba125 (mobile genetic element) upstream to the genes. All isolates were positive for Tn2006/2008 and Tn2009 but were negative for Tn2007. CONCLUSION: Most of the isolates were grouped under the CC92 clonal complex which belongs to international clonal lineage 2. These findings predict that carriage of carbapenem-resistant genes possibly constitutes the underlying basis of high level of international clone II prevalence. Therefore, molecular surveillance and antimicrobial stewardship are essential in implementing policies to prevent and control the spread of CrAb in hospital settings.

Estimating the population health and economic impacts of introducing a pneumococcal conjugate vaccine in Malaysia- an economic evaluation.

Pneumococcal disease is a potentially fatal bacterial infection that is vaccine-preventable. Malaysia has yet to adopt a pneumococcal conjugate vaccine (PCV) into its national immunization program (NIP). In 2016, pneumonia was the 3rd leading cause of death in children under five in Malaysia, accounting for 3.8% of under-five deaths. Introducing a pneumococcal conjugate vaccine (PCV) is an effective strategy to reduce the disease burden. This study used a decision-analytic model to assess the potential impacts of introducing the available PCVs (13-valent and 10-valent) in Malaysia. Epidemiological and costs inputs were sourced from published literature. For each vaccination program, health outcomes and associated healthcare costs were estimated. The scenarios of initiating PCV13 vs. PCV10 and the status quo (no pneumococcal vaccine) were compared. Serotype trends of Finland and the U.K. were used to model the clinical impacts of PCV10 and PCV13 respectively. The base-case analysis used a societal perspective over a 5-year time horizon. Compared with PCV10, PCV13 was projected to avert an additional 190,628 cases of pneumococcal disease and 1126 cases of death. The acquisition of PCV13 was estimated to cost an incremental US$89,904,777, offset by a cost reduction of -US$250,219,914 on pneumococcal disease-related medical care and lost productivity. PCV13 demonstrated a higher cost-saving potential over PCV10. Compared with no vaccination, PCV13 was estimated as cost-saving. Results were robust across a series of sensitivity analyses. The introduction of PCV13 in a NIP was estimated to reduce a significant burden of disease and to be a cost-saving for the Malaysian health system.

Changes in serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates from adult patients in Asia: Emergence of drug-resistant non-vaccine serotypes.

This study was performed to investigate the serotype distribution and antimicrobial susceptibility of Streptococcus pneumoniae in Asian countries. A prospective surveillance study on S. pneumoniae collected from adult patients (≥50 years old) with invasive pneumococcal disease or community-acquired pneumonia was performed at 66 hospitals in Asian countries (Korea, China, Malaysia, Singapore, the Philippines, and Thailand) in 2012-2017. Serotyping and antimicrobial susceptibility tests of 850 pneumococcal isolates were performed. The proportions of isolates with serotypes covered by 13-valent pneumococcal conjugate vaccine (PCV13) were 37.0% in Korea, 53.4% in China, 77.2% in Malaysia, 35.9% in the Philippines, 68.7% in Singapore, and 60.2% in Thailand. Major serotypes were 19F (10.4%), 19A (10.1%), and 3 (8.5%) in 2012-2017, with different serotype distributions in each country. Macrolide resistance in pneumococci was high (66.8%) and prevalence of multidrug resistance (MDR) also remained high (50.8%). MDR non-PCV13 serotypes such as 11A, 15A, 35B, and 23A have emerged in Asian countries. This study showed the persistent prevalence of 19F and 19A with a noteworthy increase of certain non-PCV13 serotypes in Asian countries. High prevalence of macrolide resistance and MDR was also found in pneumococcal isolates. These data emphasize the need for continued surveillance of pneumococcal epidemiology in Asia in the post-pneumococcal vaccine era.

Multidrug-resistant Streptococcus pneumoniae causing invasive pneumococcal disease isolated from a paediatric patient.

The emergence of non-vaccine multidrug-resistant Streptococcus pneumoniae serotypes is on rise. This study was performed to investigate a highly resistant serotype 15A S. pneumoniae isolated from the blood specimen of a 20-month-old patient who died of her infection. The SS40_16 isolate was resistant to erythromycin, co-trimoxazole, tetracycline, and chloramphenicol, as well as to penicillin, ceftriaxone, and cefotaxime (using meningitis cut-off points, Clinical and Laboratory Standards Institute). The isolate belonged to sequence type 1591 (ST1591) and was related to CC81 clonal complex, suggesting the possibility of horizontal gene transfer. Scanning electron microscopy comparison between resistant and sensitive pneumococcal isolates also indicated similar phenotypic characteristics that confer high resistance. The emergence of highly resistant non-vaccine pneumococci is of great concern to public health and in the clinical setting. Pneumococcal surveillance programs represent a crucial tool, not only for determining the impact of pneumococcal conjugate vaccines, but also for monitoring the selective pressure of serotype replacement with regard to the treatment of invasive pneumococcal disease.