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Detection of microsporidia in clinical specimens has relied on electron microscopy, histology, or staining. This article describes further alterations to the modified trichrome staining method which make it easier to identify microsporidial spores. The changes are a decrease in the phosphotungstic acid level and the substitution of a colorfast counterstain, aniline blue, for the fast green of the original stain. The modified stain provides good contrast between microsporidial spores and background material including human and fungal cells. Stool specimens from 139 human immunodeficiency virus-seropositive patients revealed that 5 patients were infected with Enterocytozoon bieneusi and 6 patients had larger spores. Thin-section electron microscopy of the larger spores showed a structure consistent with that of either Encephalitozoon or Septata species. Three of the patients with Encephalitozoonor Septata-like species had disseminated infection, with spores detected in nasopharyngeal aspirates and urine samples.

Background A fatal case of Japanese encephalitis (JE) occurred in a resident of the Tiwi Islands, in the Northern Territory of Australia in February 2021, preceding the large JE outbreak in south-eastern Australia in 2022. This study reports the detection, whole genome sequencing and analysis of the virus responsible (designated JEV/Australia/NT_Tiwi Islands/2021). Methods Reverse transcription quantitative PCR (RT-qPCR) testing was performed on post-mortem brain specimens using a range of JE virus (JEV)-specific assays. Virus isolation from brain specimens was attempted by inoculation of mosquito and mammalian cells or embryonated chicken eggs. Whole genome sequencing was undertaken using a combination of Illumina next generation sequencing methodologies, including a tiling amplicon approach. Phylogenetic and selection analyses were performed using alignments of the Tiwi Islands JEV genome and envelope (E) protein gene sequences and publicly available JEV sequences. Results Virus isolation was unsuccessful and JEV RNA was detected only by RT-qPCR assays capable of detecting all JEV genotypes. Phylogenetic analysis revealed that the Tiwi Islands strain is a divergent member of genotype IV (GIV) and is closely related to the 2022 Australian outbreak virus (99.8% nucleotide identity). The Australian strains share highest levels of nucleotide identity with Indonesian viruses from 2017 and 2019 (96.7–96.8%). The most recent common ancestor of this Australian-Indonesian clade was estimated to have emerged in 2007 (95% HPD range: 1998–2014). Positive selection was detected using two methods (MEME and FEL) at several sites in the E and non-structural protein genes, including a single site in the E protein (S194N) unique to the Australian GIV strains. Conclusion This case represents the first detection of GIV JEV acquired in Australia, and only the second confirmed fatal human infection with a GIV JEV strain. The close phylogenetic relationship between the Tiwi Islands strain and recent Indonesian viruses is indicative of the origin of this novel GIV lineage, which we estimate has circulated in the region for several years prior to the Tiwi Islands case.