Genetic characterization of methicillin-resistant / susceptible Staphylococcus aureus (MRSA/MSSA) and Staphylococcus argenteus clinical isolates in Bangladesh: Dominance of ST6-MRSA-IV/t304 and detection of cfr/fexA in ST8-MSSA/t008.

Objectives: Coagulase-positive staphylococcus (CoPS), represented by Staphylococcus aureus, is a major cause of infections in humans. This study aimed to investigate molecular epidemiological characteristics, antimicrobial resistance, and their trends of CoPS in Bangladesh. Methods: Clinical isolates of CoPS were collected from two medical institutions in Bangladesh for a 2-year period and analyzed for their species, genotypes, virulence factors, antimicrobial susceptibility, and resistance determinants. Results: 172 CoPS isolates collected were identified as S. aureus or S. argenteus (170 and two, respectively). Methicillin-resistant S. aureus (MRSA) accounted for 36% (n = 61), having Staphylococcal cassette chromosome mec (SCCmec)-IV (82%) or V (18%). Panton-Valentine leukocidin (PVL) genes were detected at higher rate in methicillin-susceptible S. aureus (MSSA) (62%) than MRSA (26%). MRSA comprised 11 STs, including a dominant type ST6 (46%) associated with mostly SCCmec-IVa/spa-t304, and one isolate had genetic features of the USA300 clone (ST8/SCCmec-IVa/coa-IIIa/spa-t008/ACME-I/ΦSa2USA). STs of CC1, CC88, and CC398 were common in MSSA, with CC88 showing the highest PVL-positive rate. One MSSA isolate (ST8/spa-t008) harbored fexA and cfr showing susceptibility to linezolid. S. argenteus was methicillin-susceptible and belonged to ST2250/coa-XId. Conclusions: Genetic characteristics of current MRSA/MSSA in Bangladesh were revealed, with first identification of S. argenteus at low prevalence.

Epidemiological Features of Leptospirosis and Identification of Leptospira wolffii as a Persistently Prevailing Species in North-Central Bangladesh.

Leptospirosis is considered to be the most widespread, yet neglected, re-emerging zoonotic disease caused by infection with a pathogenic species of the genus Leptospira. Although this disease is prevalent in Bangladesh, the recent epidemiological status has not yet been well documented. In this study, we aimed to determine the prevalence of leptospirosis among febrile patients using different diagnostic methods and to characterize the epidemiological features and species of Leptospira in Mymensingh, north-central Bangladesh. Among the blood samples of 186 patients with suspected leptospirosis who met the inclusion criteria, including having a fever for more than 5 days (November 2021-June 2022), 88 samples (47%) were Leptospira-positive according to IgM LAT, IgM ELISA, or nested PCR (positivity rates: 38%, 37%, and 42%, respectively). Nested PCR showed a significantly higher positivity rate (54%) in patients with a short fever (5-10 day) than the other methods did, with lower rates among those with a longer fever. Leptospirosis cases were more common in males (68%), those 16-45 years of age (70%), residents of rural areas (81%), and farmers (41%). In addition to a fever, myalgia and jaundice were found in more than 70% of the patients, while variable symptoms were observed. The 16S rRNA sequencing analysis revealed that the Leptospira species in all the 22 samples tested were L. wolffii, belonging to the pathogenic subclade P2. This study showed the recent epidemiological features of leptospirosis in Bangladesh, indicating the presumptive predominance of L. wolffii since 2019.

The Prevalence, Risk Factors, and Antimicrobial Resistance Determinants of Helicobacter pylori Detected in Dyspeptic Patients in North-Central Bangladesh.

Chronic infection of Helicobacter pylori represents a key factor in the etiology of gastrointestinal diseases, with high endemicity in South Asia. The present study aimed to determine the prevalence of H. pylori among dyspeptic patients in north-central Bangladesh (Mymensingh) and analyze risk factors of infection and antimicrobial resistance (AMR) determinants in the pathogen. Endoscopic gastrointestinal biopsy samples were collected from dyspeptic patients for a one-year period from March 2022 and were checked for the presence of H. pylori via the rapid urease test and PCR and further analyzed for the status of virulence factors vacA/cagA and genetic determinants related to AMR via PCR with direct sequencing or RFLP. Among a total of 221 samples collected, 80 (36%) were positive for H. pylori, with the vacA+/cagA+ genotype being detected in almost half of them. H. pylori was most prevalent in the age group of 41-50-year-olds, with it being more common in males and rural residents with a lower economic status and using nonfiltered water, though the rates of these factors were not significantly different from those of the H. pylori-negative group. Relatively higher frequency was noted for the A2147G mutation in 23S rRNA, related to clarithromycin resistance (18%, 7/39). Amino acid substitutions in PBP-1A (T556S) and GyrA (N87K and D91N) and a 200 bp deletion in rdxA were detected in samples from some patients with recurrence after treatment with amoxicillin, levofloxacin, and metronidazole, respectively. The present study describes the epidemiological features of H. pylori infection in the area outside the capital in Bangladesh, revealing the spread of AMR-associated mutations.

Genetic Characterization of the Dengue Virus Type 3 Genotype I Prevailing in Dhaka, Bangladesh, 2021.

Background: In Bangladesh, dengue has been prevalent since its resurgence in 2018, and the dominant causative virus in 2019 was considered dengue virus serotype 3 (DENV-3). However, limited information is available for DENV serotype/genotype circulating after 2020. Materials and Methods: Viral RNA was extracted from NS1 antigen-positive blood samples of febrile patients in Dhaka, in 2021. DENV gene was detected by semi-nested RT-PCR, and sequences of envelope (E) gene and C-prM gene were determined by direct sequencing of RT-PCR products for genetic analysis. Results: Among 172 NS1-positive samples collected, 91 samples were assigned to DENV-3 and DENV-2 (88 and 3 samples, respectively) by RT-PCR targeting the C-prM gene. Phylogenetic analysis of the E gene for the 17 representative DENV-3 samples showed that all the viruses belonged to genotype I, forming a cluster (B-cluster) with those of DENV-3 reported in Bangladesh in 2017. Analysis of the deduced amino acid sequences of E protein revealed 16 amino acid substitutions, including two novel ones (G221W, L285P), and a substitution T223I that was specifically found in DENV-3 B-cluster. Conclusion: This study showed the persistent predominance of DENV-3 genotype I in Bangladesh having unique genetic traits in the E gene. (Approval number: MMC/IRB/2022/468).

Clonal Diversity of Candida auris, Candida blankii, and Kodamaea ohmeri Isolated from Septicemia and Otomycosis in Bangladesh as Determined by Multilocus Sequence Typing.

Candida auris, Candida blankii, and Kodamaea ohmeri have been regarded as emerging fungal pathogens that can cause infections with high mortality. For genotyping of C. auris, a multilocus sequence typing (MLST) scheme based on four locus sequences has been reported, while there is no typing scheme for C. blankii and K. ohmeri. In the present study, the existing MLST scheme of C. auris was modified by adding more locus types deduced from sequence data available in the GenBank database. Furthermore, MLST schemes of C. blankii and K. ohmeri were developed using the four cognate loci (ITS, RPB1, RPB2, D1/D2) and similar sequence regions to those of C. auris. These MLST schemes were applied to identify the ST (sequence type) of clinical isolates of C. auris (n = 7), C. blankii (n = 9), and K. ohmeri (n = 6), derived from septicemia or otomycosis in Bangladesh in 2021. All the C. auris isolates were classified into a single ST (ST5) and clade I, having a Y132F substitution in ERG11p, which is associated with azole resistance. Similarly, all the C. blankii isolates belonged to a single type (ST1). In contrast, six K. ohmeri isolates were assigned to five types (ST1-ST5), suggesting its higher genetic diversity. These findings revealed the availability of MLST schemes for these three fungal species for understanding their clonal diversity among clinical isolates.

Prevalence and Antifungal Susceptibility of Clinically Relevant Candida Species, Identification of Candida auris and Kodamaea ohmeri in Bangladesh.

Candida species are major fungal pathogens in humans. The aim of this study was to determine the prevalence of individual Candida species and their susceptibility to antifungal drugs among clinical isolates in a tertiary care hospital in Bangladesh. During a 10-month period in 2021, high vaginal swabs (HVSs), blood, and aural swabs were collected from 360 patients. From these specimens, Candida spp. was isolated from cultures on Sabouraud dextrose agar media, and phenotypic and genetic analyses were performed. A total of 109 isolates were recovered, and C. albicans accounted for 37%, being derived mostly from HVSs. Among non-albicans Candida (NAC), C. parapsilosis was the most frequent, followed by C. ciferrii, C. tropicalis, and C. glabrata. Three isolates from blood and two isolates from aural discharge were genetically identified as C. auris and Kodamaea ohmeri, respectively. NAC isolates were more resistant to fluconazole (overall rate, 29%) than C. albicans (10%). Candida isolates from blood showed 95% susceptibility to voriconazole and less susceptibility to fluconazole (67%). Two or three amino acid substitutions were detected in the ERG11 of two fluconazole-resistant C. albicans isolates. The present study is the first to reveal the prevalence of Candida species and their antifungal susceptibility in Bangladesh.

Nationwide Distribution of Dengue Virus Type 3 (DENV-3) Genotype I and Emergence of DENV-3 Genotype III during the 2019 Outbreak in Bangladesh.

Bangladesh is an endemic region of dengue fever and experienced an unprecedented large outbreak with more than 100,000 confirmed cases in 2019. To understand the prevalence of dengue antibody in patients and molecular epidemiological characteristics of dengue virus (DENV) in this outbreak, a total of 179 blood samples were collected from patients in 10 districts (seven divisions) covering nearly the whole country from August to December 2019. DENV NS-1 was detected in 162 samples, among which DENV-specific IgM was positive in 119 samples (73.5%), including 60.5% samples also positive for DENV-specific IgG. Sequencing of the partial C-prM gene and its phylogenetic analysis revealed predominance of DENV type 3 genotype I, accounting for 93% of samples examined. DENV-3 genotype III was identified in two samples from separate districts, and only one DENV-2 cosmopolitan genotype was found in the capital city, Dhaka. These findings suggest the predominance of DENV-3 genotype I and occurrence of DENV-3 genotype III, associated with increased incidence of recent secondary infection in Bangladesh in 2019.

Drug Resistance Determinants in Clinical Isolates of Enterococcus faecalis in Bangladesh: Identification of Oxazolidinone Resistance Gene optrA in ST59 and ST902 Lineages.

Enterococcus faecalis is one of the major causes of urinary tract infection, showing acquired resistance to various classes of antimicrobials. The objective of this study was to determine the prevalence of drug resistance and its genetic determinants for E. faecalis clinical isolates in north-central Bangladesh. Among a total of 210 E. faecalis isolates, isolated from urine, the resistance rates to erythromycin, levofloxacin, and gentamicin (high level) were 85.2, 45.7, and 11.4%, respectively, while no isolates were resistant to ampicillin, vancomycin and teicoplanin. The most prevalent resistance gene was erm(B) (97%), and any of the four genes encoding aminoglycoside modifying enzyme (AME) were detected in 99 isolates (47%). The AME gene aac(6')-Ie-aph(2")-Ia was detected in 46 isolates (21.9%) and was diverse in terms of IS256-flanking patterns, which were associated with resistance level to gentamicin. Tetracycline resistance was ascribable to tet(M) (61%) and tet(L) (38%), and mutations in the quinolone resistance-determining region of both GyrA and ParC were identified in 44% of isolates. Five isolates (2.4%) exhibited non-susceptibility to linezolide (MIC, 4 µg/mL), and harbored the oxazolidinone resistance gene optrA, which was located in a novel genetic cluster containing the phenicol exporter gene fexA. The optrA-positive isolates belonged to ST59, ST902, and ST917 (CC59), while common lineages of other multiple drug-resistant isolates were ST6, ST28, CC16, and CC116. The present study first revealed the prevalence of drug resistance determinants of E. faecalis and their genetic profiles in Bangladesh.

Comparative study between rectally administered misoprostol as a prophylaxis versus conventional intramuscular oxytocin in post partum hemorrhage.

Post partum hemorrhage is a major cause of maternal death in developing country. A prospective, Observational comparative study was carried out in the department of Obstetrics and Gynaecology of Dhaka Medical College Hospital during the period from July 2003 to December 2003 to compare the effectiveness of rectally administered misoprostol with intramuscular oxytocin in prevention of post partum hemorrhage. A total number of 200 women were studied among whom 100 women were received 10 units of oxytocin intramuscularly & 100 women were given 600 microgram misoprostol per rectally. The incidence of PPH and side effects were examined. In 94% cases (using misoprostol) additional oxytocin was not required, only in 6% cases bleeding was more than average and additional oxytocin was given intramuscularly, in 98% cases (using oxytocin) additional oxytocin was not required. No patient had blood loss more than 1000ml and none required blood transfusion in either group. The main side effects of misoprostol were shivering and rise of temperature. So it can be suggested that per rectally administered misoprostol may be effective in the prevention of PPH as an alternative to conventional intramuscular oxytocin.

Molecular Epidemiological Characterization of Methicillin-Susceptible and -Resistant Staphylococcus aureus Isolated from Skin and Soft Tissue Infections in Bangladesh.

Genetic background and molecular characteristics of Staphylococcus aureus collected from patients with skin and soft tissue infections were studied in the North-Central region of Bangladesh from 2015 to 2016. Among 430 clinical isolates, methicillin-resistant S. aureus (MRSA) accounted for 31% having SCCmec type IV (73%) and V (14%), and belonged mostly to coagulase (coa) genotypes IIa, IIIa, IVb, and XIa, while dominant coa type in methicillin-susceptible S. aureus (MSSA) was IIIa, followed by Va, IIa, and VIa. Panton-Valentine Leukocidin genes (pvl) were detected at higher rate in MSSA (54%) than in MRSA (24%). Based on multilocus sequence typing, pvl-positive MRSA isolates were classified into clonal complex 88 (CC88) (ST88, ST2884, ST4345), CC6 (ST6, ST4350), and CC1 (ST1, ST772), while pvl-negative MRSA into CC5, CC22, CC80, CC121, and CC672. The pvl-negative ST80 MRSA isolates had SCCmec-IVa (agr-III/coa-XIc, etd/edinB-positive, fusB-negative), indicating that they belong to the novel CC80 clade related to the European community-acquired MRSA clone. Among MSSA, genotypes ST121/spa-t645/coa-Va and ST2884 (CC88)/spa-t2393/coa-IIIa were identified in both pvl-positive and negative isolates, and all the ST772 isolates harbored pvl. All the ST121 isolates had a variant of elastin-binding protein gene (ebpS-v) with internal 180-nucleotide deletion. The present study suggested that CC88 (ST88, ST2884) and ST772 are the putative dominant lineages of pvl-positive MRSA/MSSA, while novel CC80 clade is one of the main pvl-negative MRSA lineages distributed endemically in Bangladesh.

Prevalence and Molecular Epidemiology of Clinical Isolates of Escherichia coli and Klebsiella pneumoniae Harboring Extended-Spectrum Beta-Lactamase and Carbapenemase Genes in Bangladesh.

Spread of Gram-negative bacteria producing extended-spectrum beta-lactamases (ESBLs) and carbapenemases constitutes a growing challenge in control of bacterial infections. In this study, prevalence and genetic characteristics of Escherichia coli and Klebsiella pneumoniae harboring ESBL and/or carbapenemase genes, with other beta-lactamase/resistance genes, were investigated for a total of 375 clinical isolates in Mymensingh located in north-central Bangladesh. The major ESBL gene was blaCTX-M-1 group, which was detected in 33.9% and 51.4% of E. coli and K. pneumoniae, respectively, with CTX-M-15 gene being dominant. SHV-type beta-lactamase genes, including newly identified alleles (SHV-201 and SHV-202) were detected at higher rate in K. pneumoniae (27%). Nine isolates of E. coli (3.9%) harbored carbapenemase genes; blaNDM-1 (phylogenetic group A-sequence type 2104 (A-ST2104), B2-ST73), blaNDM-5 (A-ST167, B2-ST38/ST2659-related STs), and blaNDM-7 (B1-ST101/ST224, D-ST6682). AmpC beta-lactamase genes (blaCMY-2 and blaCMY-42) were detected in E. coli, which mostly harbored blaCTX-M-15 and plasmid-mediated quinolone resistance (PMQR) determinants (aac6'-Ib-cr, qnrB, qnrS, qepA, and oqxAB). A new CMY allele (CMY-160) belonging to CMY-2 group was identified in phylogenetic group D E. coli. Among K. pneumoniae, carbapenemase gene was detected in three isolates (2%); blaNDM-1 in ST11 and ST1322, and blaOXA-181 in ST43 isolate. As well as higher rate of aac6'-Ib-cr in K. pneumoniae (39%), PMQR gene oqxAB was also commonly found among isolates analyzed. These findings indicated spread of blaNDM genes to diverse E. coli clones and emergence of blaOXA-181 in K. pneumoniae, with increased prevalence of ESBLs represented by CTX-M-15 in Bangladesh.

Distribution of two distinct rotavirus B (RVB) strains in the north-central Bangladesh and evidence for reassortment event among human RVB revealed by whole genomic analysis.

Human rotavirus B (RVB), a rare cause of diarrhea in several Asian countries, has been reported to be genetically highly conserved. However, 14 RVB strains with two distinct RNA electropherotypes E1 and E2 (11 and 3 strains, respectively) were detected in adult patients with diarrhea, in Mymensingh in the north-central Bangladesh in 2014. In this study, VP7 gene sequences of all the 14 strains and nearly full-length sequences of all the 11 RNA segments of four RVB (two strains each representing E1 and E2 types) were determined and analyzed phylogenetically. For all the gene segments, sequence identities among strains with the same RNA pattern were higher (99%-100%) than those between strains with different RNA patterns (94-98%). Although all the gene segments of RVB strains were grouped into Indian-Bangladeshi lineage, VP1-3, VP6, VP7, NSP1, NSP2 and NSP5 genes of strains with E1 and E2 types were assigned to distinct sublineages S1 and S2, respectively. E1-strains clustered with Bangladeshi RVB strains reported previously (e.g., Bang117), while E2-strains with those from India (e.g., NIV-1048101), Myanmar, and Nepal. In contrast, VP4, NSP3 and NSP4 genes of both E1 and E2 RVB strains were classified into sublineage S2. These findings indicated that two genetically distinct RVB strains were simultaneously circulating in Mymensingh, Bangladesh. RVB strains with E1 electropherotype were suggested to be reassortants acquiring three gene segments (VP4, NSP3 and NSP4 genes) from the foreign RVB in the genetic background of indigenous Bangladeshi RVB represented by the strain Bang117.