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.

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.

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.

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.

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.

A Fatal Case of Candida auris and Candida tropicalis Candidemia in Neutropenic Patient.

We report a fatal case of Candida auris that was involved in mixed candidemia with Candida tropicalis, isolated from the blood of a neutropenic patient. Identification of both isolates was confirmed by amplification and sequencing of internal transcribed spacer and D1/D2 domain of large subunit in rRNA gene. Antifungal susceptibility test by E-test method revealed that C. auris was resistant to amphotericin B, anidulafungin, caspofungin, fluconazole, itraconazole and voriconazole. On the other hand, C. tropicalis was sensitive to all antifungal tested. The use of chromogenic agar as isolation media is vital in detecting mixed candidemia.

Potential serum biomarkers associated with mild and severe leptospirosis infection: A cohort study in the Malaysian population.

Leptospirosis is an emerging zoonotic infectious disease in Malaysia. The symptoms of leptospirosis vary from mild nonspecific flu-like illness to a severe condition which is usually associated with serious complication and fatality. To study the protein expression profile of mild and severe leptospirosis, 15 paired sera were collected from the patients who were mildly infected and following that progressed to severe stage. The proteome profiles of mild and severe cases were studied using 2DE analysis in combination with LC-MS/MS. The expression of proteins that were significantly different and had a fold difference of at least 2 had been identified and then validated using Western blot. Our study demonstrated apolipoprotein A-I (APOA-I), serum amyloid A (SAA), transferrin (TF), haptoglobin (HP) and transthyretin (TTR) have significantly different expression between mild and severe leptospirosis. The Ingenuity Pathway Analysis software suggested the expression of these five proteins were modulated by acute phase response signaling pathway. Besides that, a functional network of lipid metabolism, molecular transport and small molecule biochemistry that interconnects these five proteins with interactomes also had been predicted by this software. In conclusion, this finding supports the potential of these five proteins to be the biomarkers for mild and severe human leptospirosis.

First isolation of Candida wangnamkhiaoensis from the blood of immunocompromised paediatric patient.

Candida wangnamkhiaoensis is a species clustered under the Hyphopichia clade has not ever been isolated from any clinical specimens. To the best of our knowledge, this is the first report of C. wangnamkhiaoensis associated with fungaemia in immunocompromised paediatric patient. The isolate was assigned a strain name as UZ1679/14, in which the identification was confirmed by a polymerase chain reaction-sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) regions of the rRNA gene. Antifungal susceptibility pattern showed that the isolate was sensitive to anidulafungin, caspofungin, fluconazole and voriconazole. The patient clinically improved after the antifungal treatment with caspofungin.

Subcutaneous Infection Associated with Trichosporon ovoides: A Case Report and Review of Literature.

Trichosporon species are opportunistic yeasts which can cause infections, especially in immunocompromised patients. This is a report of Trichosporon ovoides that caused subcutaneous infection in a patient with underlying ischemic heart disease. The identification of fungal isolate was confirmed by PCR sequencing of ITS and large subunit regions in rRNA gene. In vitro susceptibility study showed that the isolate was susceptible to amphotericin B, fluconazole and voriconazole, and resistant to caspofungin, anidulafungin and itraconazole. The lesion improved after treatment with oral fluconazole and topical miconazole.

First Two Cases of Fungal Infections Associated with Multi-drug Resistant Yeast, Fereydounia khargensis.

The number of new fungal pathogens is increasing due to growing population of immunocompromised patients and advanced identification techniques. Fereydounia khargensis is a yeast and was first described in 2014 from environmental samples. As far as we know, this is the first report of human infections associated with F. khargensis. The yeasts were isolated from blood of a HIV-positive patient and pleural fluid of chronic renal failure patient. Amplification and sequencing of the internal transcribed spacer and the large subunit regions confirmed the identity of the isolates. Both isolates showed multi-drug resistance to antifungal agents tested.

DETECTION OF BURKHOLDERIA PSEUDOMALLEI FROM POST-FLOOD SOIL SAMPLES IN KELANTAN, MALAYSIA.

Burkholderia pseudomallei is an important causative organism of fatal community bacteremia especially in Southeast Asia and northern Australia. Outbreaks of melioidosis have been reported post-floods and -typhoons. A cross sectional study was conducted in January 2015, following a major flood in Kelantan, Malaysia to detect presence of B. pseudomallei from soil. A total of 89 soil samples were cultured for B. pseudomallei on Ashdown agar. Putative colonies underwent further staining and biochemical testing prior to confirmation by PCR. Rate of detection was 1%, although low, it nevertheless indicated a risk for melioidosis among flood victims in Kelantan. Flood affected individuals should be made aware of symptoms of melioidosis and healthcare providers must have a high index of suspicion of patients presenting with fever. Such subjects should be screened for the possibility of melioidosis and given prompt treatment to avoid preventable death.

Multiple-locus variable-number tandem-repeat analysis (MLVA) genotyping of human Brucella isolates in Malaysia.

BACKGROUND: Brucellosis is one of the most common zoonotic diseases worldwide. It can cause acute febrile illness in human and is a major health problem. Studies in human brucellosis in Malaysia is limited and so far no genotyping studies has been done on Brucella isolates. The aim of the study was to determine the genetic diversity among Brucella species isolated from human brucellosis, obtained over a 6-year period (2009-2014). METHODS: In this study, the genotypic characteristics of 43 human Brucella melitensis isolates were analysed using multiple-locus variable-number tandem-repeat analysis (MLVA) which consisted of eight minisatellite loci (panel 1) and eight microsatellite loci; panels 2A (3 microsatellite loci) and panel 2B (5 microsatellite loci). Two human Brucella suis isolates were also investigated using the MLVA assay. RESULTS: Using panel 1 (MLVA8), two genotypes namely genotype 43 and 44 were obtained from the 43 B. melitensis isolates. Using the combination of panels 1 and 2A loci (MLVA11), two genotypes were obtained while using the complete panels 1, 2A and 2B, nine genotypes were obtained. The polymorphisms in using the complete panels (MLVA16) were observed in three loci from panel 2B, which showed a diversity index higher than 0.17. All B. melitensis isolates were closely related to the East Mediterranean group. For B. suis isolates, only genotype 6 and genotype 33 were obtained using panel 1 and MLVA11 respectively. CONCLUSION: In conclusion, the results of the present study showed a low genetic diversity among B. melitensis and B. suis isolates from human patients. Based on the MLVA16 assay, B. melitensis belonging to the East Mediterranean group is responsible for the vast majority of Brucella infections in our Malaysian patients. To our knowledge, this is the first genotyping study of human Brucella isolates in Malaysia.

Chaetomium globosum Cutaneous Fungal Infection Confirmed by Molecular Identification: A Case Report from Malaysia.

An 11-year-old girl presented with multiple blisters on her the right foot complicated with cellulitis. The conventional and molecular identification were performed on the culture. The internal transcribed spacer (ITS) region in rRNA gene of the isolate was amplified by PCR. The sequence of the amplified ITS region matched 99 % with that of Chaetomium globosum in the GenBank. This is the first report describing C. globosum causing cutaneous infection in Malaysia.

In-house ELISA screening using a locally-isolated Leptospira in Malaysia: determination of its cut-off points.

BACKGROUND: Leptospirosis is a zoonotic disease caused by Leptospira species and is distributed globally. Microscopic agglutination test (MAT) is the serological 'gold standard' for diagnosis of leptospirosis but it is time-consuming and labour-intensive. An alternative serological method that is rapid, sensitive and specific is important for early treatment to reduce morbidity and mortality. The use of local Leptospira isolation may improve the sensitivity and specificity of the test because it may varies from one geographical region to another region. The objective of this study was to determine the sensitivity, specificity and cut-off points for an in-house Immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) using a locally isolated Leptospiral strain IMR/175 as the antigen for the detection of anti-Leptospiral IgM. METHODS: Serum samples from 270 patients with clinical symptoms of leptospirosis were subjected to the in-house IgM ELISA, MAT and Leptospirosis rapid test. The optimal cut-off values for positivity and negativity of the IgM ELISA were determined by Receiver Operating Characteristic curves and mean ± 2 standard deviation (SD) analyses of the ELISA values. RESULTS: The area under the curve (AUC) which indicates the diagnostic performance of the in-house IgM ELISA was 0.953 (95% Confidence Interval, CI: 0.928, 0.978). The sensitivity and specificity of 90.38% and 87.72% respectively were obtained with the cut-off point of 0.55. A higher sensitivity (96.15%) was obtained when the cut-off point was set at 0.45. CONCLUSIONS: The in-house IgM ELISA assay using local Leptospira isolation was shown to be sensitive and may be suitable to use for the serological diagnosis of leptospirosis for our local hospital setting.

Community-acquired methicillin-resistant Staphylococcus aureus in a Malaysian tertiary centre.

Abstract. Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is a pathogen recognized to be distinct in both phenotype and genotype from hospital-acquired MRSA. We have identified CA-MRSA cases in Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia, including their antibiotic susceptibility patterns and genotypic characteristics. Cases were identified during January to December 2009 from routine clinical specimens, where culture and antibiotic susceptibility results yielded pauci-resistant MRSA isolates suspected as being CA-MRSA. The patients' clinical data were collected and their specimens were sent for molecular confirmation and analysis. Five cases of CA-MRSA were identified, which had a multi-sensitive pattern on antibiotic susceptibility tests and were resistant to only penicillin and oxacillin. All cases were skin and soft-tissue infections, including diabetic foot with gangrene, infected scalp hematoma, philtrum abscess in a healthcare worker, thrombophlebitis complicated with abscess and infected bedsore. All five cases were confirmed MRSA by detection of mecA. SCCmec typing (ccr and mec complex) revealed SCCmec type IV for all cases except the infected bedsore case. Panton-Valentine leukocidin gene was positive in all isolates. As clinical features among methicillin-sensitive Staphylococcus aureus, CA-MRSA and "nosocomial CA-MRSA" are indistinct, early recognition is necessary in order to initiate appropriate antibiotics and infection control measures. Continual surveillance of pauci-resistant MRSA and molecular analysis are necessary in order to identify emerging strains as well as their epidemiology and transmission, both in the community and in healthcare setting.

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.

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.

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.

Characterization of Shigella sonnei in Malaysia, an increasingly prevalent etiologic agent of local shigellosis cases.

BACKGROUND: Shigellosis is a major public health concern worldwide, especially in developing countries. It is an acute intestinal infection caused by bacteria of the genus Shigella, with a minimum infective dose as low as 10-100 bacterial cells. Increasing prevalence of Shigella sonnei as the etiologic agent of shigellosis in Malaysia has been reported. As there is limited information on the genetic background of S. sonnei in Malaysia, this study aimed to characterize Malaysian S. sonnei and to evaluate the prospect of using multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) for subtyping of local S. sonnei. METHODS: Forty non-repeat clinical strains of S. sonnei isolated during the years 1997-2000, and 2007-2009 were studied. The strains were isolated from stools of patients in different hospitals from different regions in Malaysia. These epidemiologically unrelated strains were characterized using biotyping, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) and MLVA. RESULTS: The two biotypes identified in this study were biotype a (n = 29, 73%) and biotype g (n = 11, 27%). All the 40 strains were sensitive to kanamycin, ceftriaxone and ciprofloxacin. Highest resistance rate was observed for streptomycin (67.5%), followed by tetracycline (40%) and trimethoprim-sulfamethoxazole (37.5%). All the S. sonnei biotype g strains had a core resistance type of streptomycin - trimethoprim-sulfamethoxazole - tetracycline whereas the 29 biotype a strains were subtyped into eight resistotypes. All the strains were equally distinguishable by PFGE and MLVA. Overall, PFGE analysis indicated that S. sonnei biotype a strains were genetically more diverse than biotype g strains. Cluster analysis by MLVA was better in grouping the strains according to biotypes, was reflective of the epidemiological information and was equally discriminative as PFGE. CONCLUSIONS: The S. sonnei strains circulating in Malaysia throughout the period studied were derived from different clones given their heterogeneous nature. MLVA based on seven selected VNTR loci was rapid, reproducible and highly discriminative and therefore may complement PFGE for routine subtyping of S. sonnei.