Molecular phylogeny of Australian isolates of Sporothrix schenckii sensu lato. David New Microbiology Registrar, PathWest

Transcription

1 Molecular phylogeny of Australian isolates of Sporothrix schenckii sensu lato David New Microbiology Registrar, PathWest

2 Background Sporothrix schenckii causes a cutaneous mycosis Sporotrichosis (aka rose gardener s disease) The fungus is found world-wide and lives in the soil. Infection is via inoculation of soil, plant matter or other organic material, most commonly through a cut or scrape In Brazil, infection is transmitted by the bite or scratch of cats (ie zoonosis) Subacute-chronic infection Cutaneous and subcutaneous tissues Can infect other anatomical sites (joints, lungs, meninges) diabetes, HIV, alcoholism and COPD Painless Systemic symptoms absent s/skin-disorders/sporotrichosisrose-gardeners-disease/

3 Diagnosis and Treatment Clinical Confirmation through culture Grows readily on Sabouraud s dextrose agar Incubate at room temperature Growth within 5 days Dimorphic fungus yeast like at 37 C Species confirmation through ITS sequencing Histopathology granulomatous. Often very few organisms Serology not used Itraconazole is the drug of choice for skin disease Continued for 2-4 weeks after lesions resolve (normally a 3-6 month course) mg/day Alternatives: Potassium iodide (5 drops in juice tds) SE + (N+V, metallic taste, fever) but cheap Fluconazole mg/day Local hyperthermia Amphotericin if disseminated

4 Sporothrix in Australia Sporothrix is endemic is some parts of Australia South West WA Hunter regions of NSW Tropical QLD Northern Territory Outbreaks in QLD, Northern Territory and Western Australia Conias S, Wilson P. Epidemic cutaneous sporotrichosis: Report of 16 cases in Queensland due to mouldy hay. Australas J Dermatol Feb 1;39(1):34 7. McGuinness SL, Boyd R, Kidd S, McLeod C, Krause VL, Ralph AP. Epidemiological investigation of an outbreak of cutaneous sporotrichosis, Northern Territory, Australia. BMC Infect Dis [Internet] Jan 13 [cited 2016 Mar 7];16. Available from: Feeney KT, Arthur IH, Whittle AJ, Altman SA, Speers DJ. Outbreak of Sporotrichosis, Western Australia. Emerg Infect Dis Aug;13(8):

5 Sporotrichosis in Australia Northern Territory Outbreak McGuinness et al, BMC Infect Dis, cases, all within a 60km radius of Darwin in 2014 All patients had exposure to mouldy hay which originated from a single supplier All were gardeners All responded well to itraconazole therapy (one relapse, responded well to repeat treatment) All Sporothrix schenckii sensu stricto Western Australian outbreak Feeney et al, Emerg, Infect Dis, patients between in SW of WA 9 patients had hay exposure, one after a camping trip, one after gardening NB: Both outbreaks led to a public health response with interventions including destruction of hay and improved storage practices

6 Marimon et al 2007 Work by Marimon et al on the calmodulin gene sequence and carbohydrate assimilation, established that S. schenckii existed as a group of six species, four of which were thought to be clinically significant 1)S. schenckii sensu stricto, 2) S. braziliensis, 3) S. globosa and 4) S. luriei Others S. albicans and S. mexicana (case report of infection)

7 Pictures S. schenckii sensu stricto S. globosa

8 Pictures S. schenkii sensu stricto S. globosa

9 Aim and Methods Aim: To establish the species of Sporothrix that exist in Australia in clinical specimens Methods: 53 clinical isolates were sourced from laboratories around Australia 24 were chosen to give the widest geographical spread Fungal DNA was extracted using OmniPrep for Fungi TM DNA quantification using both spectrophotometry (NanoDrop) and Spectrofluorimetry (Qubit 2.0 hsdna) Only 22 samples had sufficient DNA after extraction to proceed Whole genome sequencing using both the MiSeq and NextSeq platforms occurred after library preparation (Nextera XT)

10 Methods continued Six genes chosen: chitin synthase, beta-tubulin, calmodulin, internal transcribed spacer (ITS) and terminal elongation factors 1a and 3 (TEF1a, TEF3) Species identification was confirmed by BLAST alignment searches Four Sporothrix whole genome sequences were found on GenBank and were used as reference strains S. schenckii strain , S. pallida SPA8, S. brasiliensis 5110 and S. globosa CBS A set of four reference pseudocontigs was then created using each target gene listed above, separated by 500 Ns Phylogenetic trees Model selection by JModel test HKY+G model Trees built using MEGA7 programme Three trees in total 6 gene tree, ITS only, calmodulin only with international gene sequences

11 Tree 1- Australian isolates 6 genes

12 Tree 2 Australian isolates, ITS only

13 Tree 3 Calmodulin gene only Australian and International isolates The two Northern Territory and one Queensland isolate (PWQ3035) align most closely with Clade IIb. All other S. schenckii align in Clade IIa. The NSW isolates align most closely to isolates from Venezuela and Argentina; however, they exist on a separate branch from those South American isolates. The two QLD isolates, PWQ3034, PWQ3030, align with different Venezuelan isolates AM and AM All Western Australian isolates align most closely to isolates from the United States. Our S. globosa isolates from NSW align most closely to isolates from India, China, and Spain and all appear to be genetically indistinguishable at the level investigated.

14

15

16 Summary of findings Two species in Australia S. schenckii sensu stricto and S. globosa S. globosa is found only in NSW ITS sequencing is sufficient in distinguishing between the subspecies Each state appears to have its own genetic group: Clonal outbreaks? Sample selection bias? The difference in the three QLD isolates unexplained No accurate geographic data Perhaps isolate 3035 originated from near the NT border? Areas of further research Whole genome analysis could be more sensitive and specific about inter-state relationships of isolates Virulence differences between species

17 Thank you