In situ hybridization to detect and identify Burkholderia pseudomallei in human melioidosis.

Burkholderia pseudomallei causes a potentially fatal infection called melioidosis. We have developed a nonfluorescent, colorimetric in situ hybridization assay using a specific probe to target 16s rRNA of B. pseudomallei in formalin-fixed, paraffin-embedded infected tissues for diagnostic purposes and to study infectious disease pathology. A 63-base pair DNA probe was synthesized and labeled with digoxigenin by PCR. Probe specificity was confirmed by BLAST analysis and by testing on appropriate microbial controls. The in situ hybridization assay was specifically and consistently positive for B. pseudomallei, showing strongly and crisply stained, single bacillus and bacilli clusters in mainly inflamed tissues in seven human acute melioidosis cases and experimentally infected mouse tissues. Intravascular and extravascular bacilli were detected in both intracellular and extracellular locations in various human organs, including lung, spleen, kidney, liver, bone marrow, and aortic mycotic aneurysm, particularly in the inflamed areas. Intravascular, intracellular bacteria in melioidosis have not been previously reported. Although the identity of infected intravascular leukocytes has to be confirmed, extravascular, intracellular bacilli appear to be found mainly within macrophages and neutrophils. Rarely, large intravascular, extracellular bacillary clusters/emboli could be detected in both human and mouse tissues. B. cepacia and non-Burkholderia pathogens (16 microbial species) all tested negative. Nonpathogenic B. thailandensis showed some cross-hybridization but signals were less intense. This in situ hybridization assay could be usefully adapted for B. pseudomallei identification in other clinical specimens such as pus and sputum.

Perinatal postmortem: factors influencing uptake and subsequent outcomes in an Asian population.

AIM: To assess uptake of perinatal postmortems (PM) among mothers experiencing perinatal deaths. Subjective assessment of factors influencing uptake was studied. Analysis of perinatal PM outcomes and its impact on cause analyses of intrauterine fetal demise was made. METHOD: 2-year prospective audit on all mothers who had experienced stillbirths at a tertiary centre. Couples returning for their postnatal consultation following their stillbirths were offered a subjective questionnaire on issues pertaining to the request of PM and their decisions on the requests. Outcome of their PMs was collated. RESULT: 71 of 87 (81.6%) women with stillbirths in the 2008-2009 (24 months) period were offered PM examination of their babies. The preliminary uptake of perinatal PM was 24 (33.8%) at counseling and but only 12 (16.9%) finally had PMs performed. Perinatal PMs clinched a diagnosis in 5 of 12 cases (42%). Discrepancies existed between external examination by on-site obstetric doctors and subsequent examination by a perinatal pathologist. CONCLUSION: Perinatal PM is a valuable tool in the assessment of intrauterine fetal demise. Increasing the perinatal PM uptake may improve care of women with stillbirths. Factors affecting parental decisions are discussed.

Severe malaria - a case of fatal Plasmodium knowlesi infection with post-mortem findings: a case report.

BACKGROUND: Zoonotic malaria caused by Plasmodium knowlesi is an important, but newly recognized, human pathogen. For the first time, post-mortem findings from a fatal case of knowlesi malaria are reported here. CASE PRESENTATION: A formerly healthy 40 year-old male became symptomatic 10 days after spending time in the jungle of North Borneo. Four days later, he presented to hospital in a state of collapse and died within two hours. He was hyponatraemic and had elevated blood urea, potassium, lactate dehydrogenase and amino transferase values; he was also thrombocytopenic and eosinophilic. Dengue haemorrhagic shock was suspected and a post-mortem examination performed. Investigations for dengue virus were negative. Blood for malaria parasites indicated hyperparasitaemia and single species P. knowlesi infection was confirmed by nested-PCR. Macroscopic pathology of the brain and endocardium showed multiple petechial haemorrhages, the liver and spleen were enlarged and lungs had features consistent with ARDS. Microscopic pathology showed sequestration of pigmented parasitized red blood cells in the vessels of the cerebrum, cerebellum, heart and kidney without evidence of chronic inflammatory reaction in the brain or any other organ examined. Brain sections were negative for intracellular adhesion molecule-1. The spleen and liver had abundant pigment containing macrophages and parasitized red blood cells. The kidney had evidence of acute tubular necrosis and endothelial cells in heart sections were prominent. CONCLUSIONS: The overall picture in this case was one of systemic malaria infection that fit the WHO classification for severe malaria. Post-mortem findings in this case were unexpectedly similar to those that define fatal falciparum malaria, including cerebral pathology. There were important differences including the absence of coma despite petechial haemorrhages and parasite sequestration in the brain. These results suggest that further study of knowlesi malaria will aid the interpretation of, often conflicting, information on malaria pathophysiology in humans.

53-year-old man with rapid cognitive decline.

Kufs' disease (adult neuronal ceroid lipofuscinosis) is a rare form of neurodegenerative lysosomal storage disease, the genetic basis of which remains obscure. We present a case of a 53-year-old man with a long history of adult onset epilepsy who presented with confusion and amnesia, and subsequently underwent rapidly progressive cognitive decline associated with myoclonic jerks. The clinical diagnosis was Creutzfeldt Jakob disease. However, autopsy brain examination revealed changes of Kufs' disease (Adult onset neuronal ceroid lipofuscinosis, or ANCL, also known as CLN4). No specific gross abnormalities were found but light microscopic examination revealed widespread neuronal ballooning and histochemical staining showed neuronal accumulation of PAS-positive material. Ultrastructural examination demonstrated excessive and abnormal lipopigment lysosomes typical of neuronal ceroid lipofuscinosis. The typical clinical and pathologic features of the Kufs' disease are discussed and the classification of neuronal ceroid lipofuscinosis is reviewed.