METHODS: We used transmission electron microscopy (TEM) to investigate post-mortem tissue sections of patients with clinical melioidosis to identify the localisation of a recently identified gut microbiome, B. pseudomallei within host cells. The intranuclear presence of B. pseudomallei was confirmed using transmission electron microscopy (TEM) of experimentally infected guinea pig spleen tissues and Live Z-stack, and ImageJ analysis of fluorescence microscopy analysis of in vitro infection of A549 human lung epithelial cells.
RESULTS: TEM investigations revealed intranuclear localization of B. pseudomallei in cells of infected human lung and guinea pig spleen tissues. We also found that B. pseudomallei induced actin polymerization following infection of A549 human lung epithelial cells. Infected A549 lung epithelial cells using 3D-Laser scanning confocal microscopy (LSCM) and immunofluorescence microscopy confirmed the intranuclear localization of B. pseudomallei.
CONCLUSION: B. pseudomallei was found within the nuclear compartment of host cells. The nucleus may play a role as an occult or transient niche for persistence of intracellular pathogens, potentially leading to recurrrent episodes or recrudescence of infection.
METHODS: A part prospective, part retrospective study of children aged <15 years with culture-confirmed melioidosis was conducted in the 3 major public hospitals in Central Sarawak between 2009 and 2014. We examined epidemiological, clinical and microbiological characteristics.
FINDINGS: Forty-two patients were recruited during the 6-year study period. The overall annual incidence was estimated to be 4.1 per 100,000 children <15 years, with marked variation between districts. No children had pre-existing medical conditions. Twenty-three (55%) had disseminated disease, 10 (43%) of whom died. The commonest site of infection was the lungs, which occurred in 21 (50%) children. Other important sites of infection included lymph nodes, spleen, joints and lacrimal glands. Seven (17%) children had bacteremia with no overt focus of infection. Delays in diagnosis and in melioidosis-appropriate antibiotic treatment were observed in nearly 90% of children. Of the clinical isolates tested, 35/36 (97%) were susceptible to gentamicin. Of these, all 11 isolates that were genotyped were of a single multi-locus sequence type, ST881, and possessed the putative B. pseudomallei virulence determinants bimABp, fhaB3, and the YLF gene cluster.
CONCLUSIONS: Central Sarawak has a very high incidence of pediatric melioidosis, caused predominantly by gentamicin-susceptible B. pseudomallei strains. Children frequently presented with disseminated disease and had an alarmingly high death rate, despite the absence of any apparent predisposing risk factor.
OBJECTIVE: We sought to review case reports of melioidosis from Malaysia.
METHODS: We conducted a computerized search of literature resources including PubMed, OVID, Scopus, MEDLINE and the COCHRANE database to identify published case reports from 1975 to 2015. We abstracted information on clinical characteristics, exposure history, comorbid conditions, management and outcome.
RESULTS: Overall, 67 cases were reported with 29 (43%) deaths; the median age was 44 years, and a male preponderance (84%) was noted. Forty-one cases (61%) were bacteremic, and fatal septic shock occurred in 13 (19%) within 24-48 hours of admission; nine of the 13 cases were not specifically treated for melioidosis as confirmatory evidence was available only after death. Diabetes mellitus (n = 36, 54%) was the most common risk factor. Twenty-six cases (39%) had a history of exposure to contaminated soil/water or employment in high-risk occupations. Pneumonia (n = 24, 36%) was the most common primary clinical presentation followed by soft tissue abscess (n = 22, 33%). Other types of clinical presentations were less common-genitourinary (n = 5), neurological (n = 5), osteomyelitis/septic arthritis (n = 4) and skin (n = 2); five cases had no evidence of a focus of infection. With regard to internal foci of infection, abscesses of the subcutaneous tissue (n = 14, 21%) was the most common followed by liver (18%); abscesses of the spleen and lung were the third most common (12% each). Seven of 56 males were reported to have prostatic abscesses. Mycotic pseudoaneurysm occurred in five cases. Only one case of parotid abscess was reported in an adult. Of the 67 cases, 13 were children (≤ 18 years of age) with seven deaths; five of the 13 were neonates presenting primarily with bronchopneumonia, four of whom died. Older children had a similar presentation as adults; no case of parotid abscess was reported among children.
CONCLUSIONS: The clinical patterns of cases reported from Malaysia are consistent for the most part from previous case reports from South and Southeast Asia with regard to common primary presentations of pneumonia and soft tissue abscesses, and diabetes as a major risk factor. Bacteremic melioidosis carried a poor prognosis and septic shock was strong predictor of mortality. Differences included the occurrence of: primary neurological infection was higher in Malaysia compared to reports outside Malaysia; internal foci of infection such as abscesses of the liver, spleen, prostate, and mycotic pseudoaneurysms were higher than previously reported in the region. No parotid abscess was reported among children. Early recognition of the disease is the cornerstone of management. In clinical situations of community-acquired sepsis and/or pneumonia, where laboratory bacteriological confirmation is not possible, empirical treatment with antimicrobials for B. pseudomallei is recommended.
METHODOLOGY/PRINCIPAL FINDINGS: We obtained patient demographic and residential information and clinical presentation and medical history data from 254 confirmed melioidosis cases and 384 matched controls attending Hospital Sultanah Bahiyah (HSB), the main tertiary hospital of Alor Setar, the capital city of Kedah, during the period between 2005 and 2011. Crude and adjusted odds ratios employing conditional logistic regression analysis were used to assess if melioidosis in this region is related to risk factors connected with socio-demographics, various behavioural characteristics, and co-occurring diseases. Spatial clusters of cases were determined using a continuous Poisson model as deployed in SaTScan. A land cover map in conjunction with mapped case data was used to determine disease-land type associations using the Fisher's exact test deploying simulated p-values. Crude and adjusted odds ratios indicate that melioidosis in this region is related to gender (males), race, occupation (farming) and co-occurring chronic diseases, particularly diabetes. Spatial analyses of disease incidence, however, showed that disease risk and geographic clustering of cases are related strongly to land cover types, with risk of disease increasing non-linearly with the degree of human modification of the natural ecosystem.
CONCLUSIONS/SIGNIFICANCE: These findings indicate that melioidosis represents a complex socio-ecological public health problem in Kedah, and that its control requires an understanding and modification of the coupled human and natural variables that govern disease transmission in endemic communities.
METHODOLOGY/PRINCIPAL FINDINGS: A persistent infection was generated using a small-colony variant (SCV) and a wild-type (WT) B. pseudomallei in BALB/c mice via intranasal administration. Infected mice that survived for >60 days were sacrificed. Lungs, livers, spleens, and peripheral blood mononuclear cells were harvested for experimental investigations. Histopathological changes of organs were observed in the infected mice, suggestive of successful establishment of persistent infections. Moreover, natural killer (NK) cell frequency was increased in SCV- and WT-infected mice. We observed programmed death-1 (PD-1) upregulation on B cells of SCV- and WT-infected mice. Interestingly, PD-1 upregulation was only observed on NK cells and monocytes of SCV-infected mice. In contrast, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) downregulation was seen on NK cells of WT-infected mice, and on monocytes of SCV- and WT-infected mice.
CONCLUSIONS/SIGNIFICANCE: The SCV and the WT of B. pseudomallei distinctly upregulated PD-1 expression on B cells, NK cells, and monocytes to dampen host immunity, which likely facilitates bacterial persistence. PD-1/PD-L1 pathway appears to play an important role in the persistence of B. pseudomallei in the host.
Method: In this study, we developed a rapid, sensitive and specific insulated isothermal Polymerase Chain Reaction (iiPCR) targeting bimA gene (Burkholderia Intracellular Motility A; BPSS1492) for the identification of B. pseudomallei. A pair of novel primers: BimA(F) and BimA(R) together with a probe were designed and 121 clinical B. pseudomallei strains obtained from numerous clinical sources and 10 ATCC non-targeted strains were tested with iiPCR and qPCR in parallel.
Results: All 121 B. pseudomallei isolates were positive for qPCR while 118 isolates were positive for iiPCR, demonstrating satisfactory agreement (97.71%; 95% CI [93.45-99.53%]; k = 0.87). Sensitivity of the bimA iiPCR/POCKIT assay was 97.52% with the lower detection limit of 14 ng/µL of B. pseudomallei DNA. The developed iiPCR assay did not cross-react with 10 types of non-targeted strains, indicating good specificity.
Conclusion: This bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen.
METHODOLOGY/PRINCIPAL FINDINGS: With the availability of the B. pseudomallei whole genome sequence, we undertook to identify genes encoding the known immunogenic outer membrane protein A (OmpA). Twelve OmpA domains were identified and ORFs containing these domains were fully annotated. Of the 12 ORFs, two of these OmpAs, Omp3 and Omp7, were successfully cloned, expressed as soluble protein and purified. Both proteins were recognised by antibodies in melioidosis patients' sera by Western blot analysis. Purified soluble fractions of Omp3 and Omp7 were assessed for their ability to protect BALB/c mice against B. pseudomallei infection. Mice were immunised with either Omp3 or Omp7, subsequently challenged with 1x10(6) colony forming units (cfu) of B. pseudomallei via the intraperitoneal route, and examined daily for 21 days post-challenge. This pilot study has demonstrated that whilst all control unimmunised mice died by day 9 post-challenge, two mice (out of 4) from both immunised groups survived beyond 21 days post-infection.
CONCLUSIONS/SIGNIFICANCE: We have demonstrated that B. pseudomallei OmpA proteins are immunogenic in mice as well as melioidosis patients and should be further assessed as potential vaccine candidates against B. pseudomallei infection.