Phylogeographic characterization of Burkholderia pseudomallei isolated from Bangladesh.

BACKGROUND: Burkholderia pseudomallei possesses a diverse set of genes which encode a vast array of biological functions reflecting its clinical, ecological and phenotypic diversity. Strain variation is linked to geographic location as well as pattern of land uses. This soil-dwelling Gram-negative pathogen causes melioidosis, a tropical disease endemic in northern Australia and Southeast Asian regions including Bangladesh. Phylogeographic analyses of B. pseudomallei isolates by molecular typing techniques could be used to examine the diversity of this organism as well as to track melioidosis epidemics. METHODS: In this study, 22 B. pseudomallei isolates, of which 20 clinical and two soil isolates were analyzed, utilizing Real-time PCR assay and multilocus sequence typing (MLST). The sequences were then submitted to PubMLST database for analysis and construction of phylogenetic tree. FINDINGS: A total of 12 different sequence types (STs) that includes four novel STs were identified for the first time. Strains having STs 1005, 1007 and 56 were the most widespread STs frequently isolated in Bangladesh. ST 1005, ST 56, ST 1007 and ST 211 have been detected not only in Bangladesh but are also present in many Southeast Asian countries. SIGNIFICANCE: ST 1005 was detected in both soil and clinical samples of Gazipur. Most prevalent, ST 56 has been previously reported from Myanmar, Thailand, Cambodia and Vietnam, confirming the persistence of the genotype over the entire continent. Further large-scale study is necessary to find out the magnitude of the infection and its different reservoirs in the environment along with phylogeographic association.

Salmonellacidal antibody response to Salmonella enterica serovar Typhi in enteric fever and after vaccination with Vi capsular polysaccharide.

OBJECTIVES: Serum salmonellacidal (bactericidal) antibody could be used to detect functional capacity of antibody in patients with enteric fever and after typhoid vaccination. METHODS: Salmonellacidal antibody response was measured by colorimetric serum salmonellacidal assay from 70 acute and 11 convalescence sera of patients infected with Salmonella Typhi and Paratyphi A and also from 15 control and 6 Vi capsular polysaccharide vaccinated volunteer's sera. RESULTS: Sera from patients with typhoid and paratyphoid A showed significant (p < 0.05) levels of salmonellacidal antibody titer (549.9 ± 108.5 and 528.7 ± 187.3) compared with control (0.133 ± 0.1). Moreover, this titer increased significantly (p <0.05) in sera collected between 7 and 10 days and between 11 and 25 days of fever (titer 535.7 ± 119.2 and 794.6 ± 235.6) compared with sera collected from patients with fever for less than 7 days (136.4 ± 52.7). The mean titer significantly (p < 0.05) decreased to 5.5 ± 2.1 after 6-8 weeks onset of illness. Although, very low salmonellacidal titers (2.5 ± 1.5 and 2.3 ± 1.5) were detected after Vi CPS vaccine among the human volunteers, but mean titer was raised 15-fold from pre- to postvaccinated sera (0.166-2.5). CONCLUSION: The serum salmonellacidal antibody by colorimetric salmonellacidal assay could be used to detect acute typhoidal cases and also to monitor immune response of typhoid vaccine.

Parasitic infections in Malaysian aborigines with pulmonary tuberculosis: a comparative cross-sectional study.

The geographical distribution of tuberculosis (TB) overlaps with various parasitic infections. Uncovering the characteristics of coinfecting parasites that potentially affect the host susceptibility to TB is pertinent as it may provide input to current TB therapeutic and prophylactic measures. The present study was aimed at examining the types of parasitic infections in TB patients and healthy TB contacts (HC) in Orang Asli, Malaysian aborigines, who dwelled in the co-endemic areas. Stool and serum samples were collected from Orang Asli who fulfilled the selection criteria and provided written informed consents. Selected parasitic infections in the two study groups were determined by stool examination and commercial serum antibody immunoassays. The prevalence of parasitic infections in TB and HC participants were 100% (n = 82) and 94.6% (n = 55) respectively. The parasitic infections comprised toxocariasis, trichuriasis, amoebiasis, toxoplasmosis, hookworm infection, ascariasis, strongyloidiasis, and brugian filariasis, in decreasing order of prevalence. Overall, helminth or protozoa infection did not show any significant association with the study groups. However, when the species of the parasite was considered, individuals exposed to trichuriasis and toxoplasmosis showed significant odds reduction (odds ratio (OR) 0.338; 95% confidence interval (CI) 0.166, 0.688) and odds increment (OR 2.193; 95% CI 1.051, 4.576) to have active pulmonary TB, respectively. In conclusion, trichuriasis and toxoplasmosis may have distinct negative and positive associations respectively with the increase of host susceptibility to TB.

Novel PCR primers to diagnose visceral leishmaniasis using peripheral blood, spleen or bone marrow aspirates.

OBJECTIVE: To establish a suitable method of diagnosis of visceral leishmaniasis (VL) using peripheral blood, spleen or bone marrow aspirates. METHODS: Peripheral blood, bone marrow and spleen aspirate samples were collected from clinically suspected VL patients (n = 26). A new PCR primer pair (MK1F/R) was designed targeting kinetoplast mini circle DNA sequences of Leishmania donovani, and Leishmania infantum, and was used to diagnose VL along with some other established primers for VL in polymerase chain reactions. Test was validated by comparing with several other diagnostic methods. RESULTS: The designed primer set showed 100% specificity and 98% sensitivity in detecting VL using blood samples, when compared with more invasive samples: bone marrow or spleen aspirates. CONCLUSIONS: The newly designed primer MK1F/R could be a better alternative for PCR based diagnosis of VL using less invasive sample, peripheral blood instead of bone marrow or spleen aspirates.

Antimicrobial susceptibility pattern of clinical isolates of Burkholderia pseudomallei in Bangladesh.

BACKGROUND: Melioidosis an infectious disease, caused by a Gram negative bacterium called Burkholderia pseudomallei, is endemic in Bangladesh. This organism is sensitive to limited number of antimicrobial agents and need prolonged treatment. There is no comprehensive data on the antimicrobial susceptibility profile of B. pseudomallei isolated in Bangladesh over last several years. The present study aimed to determine the antimicrobial susceptibility pattern of B. pseudomallei isolated in a tertiary care hospital of Dhaka city from 2009 to 2015. METHODS: All B. pseudomallei isolated from melioidosis patients over a period of 7 years (2009-2015) in the Department of Microbiology of a 725-bed tertiary care referral hospital in Dhaka city, Bangladesh were included in the study. B. pseudomallei was identified by Gram stain, culture, specific biochemical tests, serology and PCR using specific primers constructed from 16s rRNA region of B. pseudomallei. Antimicrobial susceptibility to specific agents was determined by disk diffusion and minimum inhibitory concentration methods. RESULTS: A total of 20 isolates of B. pseudomallei which were isolated from patients coming from different geographic locations of Bangladesh were included in the study. All the isolates were uniformly sensitive (100%) to ceftazidime, imipenem, piperacillin-tazobactam, amoxicillin-clavulanic acid and tetracycline by both disk diffusion and MIC methods. Two strains were resistant to trimethoprim-sulfamethoxazole by disk diffusion method but were sensitive by MIC method. The MIC50 and MIC90 values of the above antimicrobial agents were almost similar. All the isolates were resistant to amikacin by both MIC and disk diffusion methods. CONCLUSION: The results of the study suggest that B. pseudomallei prevalent in Bangladesh were still susceptible to all recommended antimicrobial agents used for the treatment of melioidosis. However, regular monitoring is needed to detect any emergence of resistance and shifting of MIC50 and MIC90 values.

Burkholderia pseudomallei: Its Detection in Soil and Seroprevalence in Bangladesh.

BACKGROUND: Melioidosis, caused by Burkholderia pseudomallei, is an endemic disease in Bangladesh. No systematic study has yet been done to detect the environmental source of the organism and its true extent in Bangladesh. The present study attempted to isolate B. pseudomallei in soil samples and to determine its seroprevalence in several districts in Bangladesh. METHODOLOGY AND RESULTS: Soil samples were collected from rural areas of four districts of Bangladesh from where culture confirmed melioidosis cases were detected earlier. Multiple soil samples, collected from 5-7 sampling points of 3-5 sites of each district, were cultured in Ashdown selective media. Suspected colonies of B. pseudomallei were identified by biochemical and serological test, and by polymerase chain reaction (PCR) using 16s rRNA specific primers. Blood samples were collected from 940 healthy individuals of four districts to determine anti- B. pseudomallei IgG antibody levels by indirect enzyme linked immunosorbent assay (ELISA) using sonicated crude antigen. Out of 179 soil samples, B. pseudomallei was isolated from two samples of Gazipur district which is located 58 km north of capital Dhaka city. Both the isolates were phenotypically identical, arabinose negative and showed specific 550bp band in PCR. Out of 940 blood samples, anti- B. pseudomallei IgG antibody, higher than the cut-off value (>0.8), was detected in 21.5% individuals. Seropositivity rate was 22.6%-30.8% in three districts from where melioidosis cases were detected earlier, compared to 9.8% in a district where no melioidosis case was either detected or reported (p<0.01). Seropositivity increased with the advancement of age from 5.3% to 30.4% among individuals aged 1-10 years and > 50 years respectively. The seropositivity rates were 26.0% and 20.6% in male and female respectively, while it was 20-27% among different occupational groups. No significant association was observed with gender (χ2 = 3.441, p = 0.064) or any occupational group (χ2 = 3.835, p = 0.280). CONCLUSION: This is the first study demonstrating the presence of B. pseudomallei in the environmental (soil) samples of Bangladesh. It also suggested that a large proportion of people, residing in these districts, were exposed to the organism.

Detection and genetic characterization of PVL-positive ST8-MRSA-IVa and exfoliative toxin D-positive European CA-MRSA-Like ST1931 (CC80) MRSA-IVa strains in Bangladesh.

Severe skin lesions caused by Staphylococcus aureus infection are associated with production from bacterial cells of Panton-Valentine leukocidin (PVL), a typical virulence factor of community-acquired methicillin-resistant S. aureus (CA-MRSA), as well as other toxins represented by exfoliative toxins. Through a retrospective study of 26 S. aureus strains isolated from skin lesions of diabetic patients admitted to a hospital in Bangladesh, 2 PVL-gene-positive MRSA-IVa strains and 8 PVL-negative, exfoliative toxin D (ETD) gene (etd)-positive MRSA-IVa strains were isolated. A PVL-positive MRSA-IVa strain had a type I arginine catabolic mobile element (ACME), belonged to ST8/agr-type I/spa-type t121 (a variant of t008), and harbored blaZ, tet(K), msrA, and aph(3')-IIIa, which are mostly typical characteristics found in USA300, a predominant CA-MRSA clone in the United States. Another PVL-positive MRSA strain, belonging to ST1929 (CC88)/agr-type III/spa-type t3341, was negative for ACME, but possessed blaZ and tet(K). The etd-positive MRSA-IVa strains possessed the epidermal cell differentiation inhibitor B (EDIN-B)-encoding gene (edinB) and belonged to ST1931 (CC80)/agr-type III/spa-type t11023 (a variant of t044), which was genetic trait similar to that of the European CA-MRSA ST80 clone. However, unlike the European ST80 strains, the etd-positive MRSA strains detected in the present study harbored seb, sek, and seq, while they were negative for tet(K), aph(3')-IIIa, and fusB, showing susceptibility to fusidic acid. These findings suggested that etd-positive ST1931 MRSA strains belong to the same lineage as the European ST80 MRSA clone, evolving from a common ancestral clone via acquisition of a different pathogenicity island. This is the first report of a USA300-like MRSA-IV strain, PVL-positive ST1929 (CC88) MRSA-IV, and European ST80 CA-MRSA-like etd-positive ST1931 (CC80) MRSA-IV strains isolated in Bangladesh.

Detection of extended spectrum ß-lactamase in Pseudomonas spp. isolated from two tertiary care hospitals in Bangladesh.

BACKGROUND: Extended spectrum ß-lactamases (ESBLs) represent a major group of lactamases responsible for resistance, mostly produced by gram-negative bacteria, to newer generations of ß-lactam drugs currently being identified in large numbers worldwide. The present study was undertaken to see the frequency of ESBL producing Pseudomonas spp. isolated from six hundred clinical specimens (wound, pus, aural, urine, sputum, throat and other swabs) collected over a period of three years from two tertiary care hospitals in Bangladesh. FINDINGS: Aerobic bacterial culture was performed on aseptically collected swabs and only growth of Pseudomonas was considered for further species identification and ESBL production along with serotyping of Pseudomonas aeruginosa. Antimicrobial susceptibility testing was carried out using the Kirby-Bauer agar diffusion method and ESBL production was detected on Mueller Hinton agar by double-disk synergy technique using Amoxicillin-Clavulanic acid with Ceftazidime, Cefotaxime, Ceftriaxone and Aztreonam. Culture yielded 120 Pseudomonas spp. and 82 of them were biochemically characterized for species. Pseudomonas aeruginosa was found to be the predominant (90.2%) species. Of 82 isolates tested for ESBL, 31 (37.8%) were ESBL positive with 29 (93.5%) as Pseudomonas aeruginosa, the remaining 2 (6.5%) were Stenotrophomonas maltophilia and Ralstonia pickettii. Antibiogram revealed Imipenem as the most effective drug (93.3%) among all antimicrobials used against Pseudomonas spp. followed by Aminoglycosides (63.7%). CONCLUSION: ESBL producing Pseudomonas spp. was found to be a frequent isolate from two tertiary care hospitals in Bangladesh, showing limited susceptibility to antimicrobials and decreased susceptibility to Imipenem in particular, which is a matter of great concern.