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VolumeVolume 3737 NumberNumber 11 MarchMarch 20162016

Parasitic infections Bringing more

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Culture mycobacteria specimens with a full range of mycobacteria media, including Thermo Lowenstein-Jensen, MacConkey Agar w/o Crystal Violet and © 2014 Thermo Fisher Scientifi c Inc. All rights reserved. © 2014 Thermo Fisher Scientifi Middlebrook Agar Media. The Australian Society for Microbiology Inc. OFFICIAL JOURNAL OF THE AUSTRALIAN SOCIETY FOR MICROBIOLOGY INC. 9/397 Smith Street Fitzroy, Vic. 3065 Tel: 1300 656 423 Volume 37 Number 1 March 2016 Fax: 03 9329 1777 Email: [email protected] www.theasm.org.au Contents ABN 24 065 463 274 From the Editorial Team 2 For Microbiology Australia correspondence, see address below. Jo, Ian, and Hayley Macreadie Editorial team Guest Editorial 3 Prof. Ian Macreadie, Mrs Jo Macreadie and Mrs Hayley Macreadie Parasitic infections: overlooked, under-diagnosed and under-researched 3 Harsha Sheorey and Richard S Bradbury Editorial Board Dr Chris Burke (Chair) Dr Gary Lum In Focus 4 Prof. Mary Barton Dr John Merlino Prof. Linda Blackall Prof. Wieland Meyer The laboratory diagnosis of Strongyloides stercoralis 4 Prof. Sharon Chen Prof. William Rawlinson Matthew R Watts, Gemma Robertson and Richard S Bradbury Prof. Peter Coloe Dr Paul Selleck Dr Narelle Fegan Dr David Smith Under the Microscope 10 Dr Geoff Hogg Ms Helen Smith Current WHO protocols for mass drug administration in helminth control 10 Prof.. Jonathan Iredell Dr Jack Wang Dr Ipek Kurtböke Richard S Bradbury and Patricia M Graves Subscription rates Zoonotic tissue parasites of Australian wildlife 12 Current subscription rates are available David M Spratt from the ASM Melbourne offi ce. Editorial correspondence Assessing enteric helminths in refugees, asylum seekers and new migrants 15 Prof. Ian Macreadie/Mrs Jo Macreadie Sarah Hanieh, Norbert Ryan and Beverley-Ann Biggs Tel: 0402 564 308 (Ian) Email: [email protected] Free living amoebae and human disease 20 Evan Bursle and Jennifer Robson Published four times a year in print and open access online by Tapeworm cysts in the brain: can we prevent it happening? 25 Marshall Lightowlers Seafood-borne parasitic diseases in Australia: how much do we know about them? 27 Shokoofeh Shamsi Unipark, Building 1, Level 1 195 Wellington Road, Clayton, Vic. 3168 Children, snails and worms: the Brachylaima cribbi story 30 http://microbiology.publish.csiro.au Andrew R Butcher Publishing enquiries Malaria: global challenges for malaria eradication 34 Jenny Foster Email: [email protected] Graham Brown and Stephen Rogerson Production enquiries Plasmodium knowlesi: an update 39 Helen Pavlatos Balbir Singh Email: [email protected] Advertising enquiries Diagnosis of human 43 Doug Walters Abdul Jabbar, Charles Gauci and Marshall W Lightowlers Tel: 03 9545 8505 Mobile: 0419 357 779 Lab Report 46 Email: [email protected] PCR: boon or bane 46 © 2016 The Australian Society for Microbiology Inc. Colin Pham and Harsha Sheorey The ASM, through CSIRO Publishing, reserve all rights to the content, artwork and photographs in Microbiology Australia. Permission to reproduce text, photos and ASM Affairs 50 artwork must be sought from CSIRO Publishing. The Australian Copyright Act 1968 and subsequent FT020 Water Microbiology Australian Standards Committee 50 amendments permit downloading and use of an article by an individual or educational institution for non- 2016 ASM Communication Ambassador Program 50 commercial personal use or study. Multiple reproduction of any Microbiology Australia article in a study block is Stopping dengue: recent advances and new challenges 51 governed by rights agreement managed by Copyright Agency Limited and fees may apply. Authors published in Microbiology Australia have the moral right under Australian law to be acknowledged as the creator. ISSN 1324-4272 eISSN 2201-9189 While reasonable effort has been made to ensure the accuracy of the content, the Australian Society for Microbiology, CSIRO, and CSIRO Publishing accept no responsibility for any loss or damage from the direct or indirect use of or reliance on the content. The opinions expressed in articles, letters, and advertisements in Microbiology Australia are not necessarily those of the Australian Society for Microbiology, the Editorial Board, CSIRO, and CSIRO Publishing. Cover image: Protoscolex of , showing row of hooklets; obtained from liver aspirate of a case of hydatid cyst. Courtesy of Associate Professor Rob Baird, Royal Darwin Hospital.

MICROBIOLOGY AUSTRALIA • MARCH 2016 1 From the Editorial Team

Welcome to the first issue of Microbiology Australia for 2016. comments to contributors to ensure the articles are appropriately presented. We also thank the authors for their contributions. They As members of The Australian Society for Microbiology know, our provide a great amount of current information about their field and electronic issues are freely available. To be notified of a new issue go seek to educate us all in the exciting developments of microbiology. to our publisher’s website http://microbiology.publish.csiro.au/ and register to get alerts when each issue becomes available. We thank The Australian Society for Microbiology and those who lead it. Inthe challenging world ofpublishing ithas taken boldness to ASM members receive print copies by mail if they have opted to enable Microbiology Australia to be produced as a publication that receive them that way. Contact the ASM Office if you wish to receive is freely available to all. We know from the download data provided print copies. Additional copies are often made for special issues. by CSIRO Publishing, that Microbiology Australia is highly accessed For example, ~1000 copies were made for delegates of the ISHAM around the world, and that the articles continue as an ongoing conference in Melbourne last year. The print copies are highly resource many years after their first publication. Articles published valued by many members and are great to share with others. more than a decade ago still enjoy thousands of downloads every The online issues are highly accessed as a reference and learning year. There is no doubt that they serve as resource material for resource. Individual articles as well as entire issues can be readily teachers, students and those seeking reliable scientific information downloaded from the above website. from experts. ’ Themes of issues and Guest Editors are chosen by the Editorial We trust that you enjoy your reading of this year s issues, which will Board who meet by teleconference five times each year. The Board cover: always values input from ASM members for future issues. All articles * Parasitic Infections * Education to enhance microbiology graduate employability are peer-reviewed and invited by the Guest Editors who have a deep * Diseases of Aquaculture understanding of the themes they present. Themes are chosen to * Microbiology of Travel [a special joint issue with The Microbiology Society in the UK]. be topical and of interest to ASM members: authors write to reach a broad audience. Jo, Ian, and Hayley Macreadie

On occasions urgent updates are needed and such updates are presented as Hot Topics. However, we are aware that there is a need to have an occasional non-themed issue. Please contact us if you have suggestions for articles that will have broad interest to ASM members.

We are grateful for the support the Editorial Board and for Guest Editors who are experts in their fields. Guest Editors present the latest knowledge in their field through the selection of contributors who write compact articles that are readable and informative to the ASM’s diverse membership. We also thank the reviewers, who provide valuable comments on each contribution. They also have expertise in the articles they review, and they provide valuable

Access to Microbiology Australia Online early articles: Articles appear on the Microbiology Australia website (http:// microbiology.publish.csiro.au/) when authors have approved the pdf of their article. Completed issues: Register at http://microbiology.publish.csiro.au/ to receive notification that an issue is complete. You will get an email showing the titles and abstracts of the completed issue. You will have one click access to any article or the whole issue. Print issue: ASM members are welcome to receive the print version of Microbiology Australia without charge. To receive the print version you need to notify the ASM National Office (http:// www.theasm.org.au/).

2 10.1071/MA16001 MICROBIOLOGY AUSTRALIA * MARCH 2016 Guest Editorial

Parasitic infections: overlooked, under-diagnosed and under-researched

Richard S Bradbury Harsha Sheorey School of Medical and Applied Microbiology Department Sciences St Vincent’s Hospital Melbourne Central Queensland University Fitzroy, Vic., Australia Rockhampton, Qld, Australia Email: [email protected] Email: [email protected]

Professor George Nelson (1924–2009) once stated that, ‘Parasitology situations, morphological and serological techniques still remain is the preserve of the diagnostically destitute’. Little has changed to relevant. thisday,withpotentiallyrelevantparasiticcausesof illnessesoftennot This edition also considers zoonotic parasitic infections, the treat- being considered early in the differential diagnoses of clinical pre- ment of parasitic infections, both from the current WHO recom- sentations. Parasitic infections are sometimes overlooked as causes mendations for mass drug administration in highly endemic settings of morbidity and (in some cases) mortality in both the medical and to the rise of resistance to anti-parasitic agents in protozoa such as veterinary fields. In Australia there remain significant problems malarial parasites and Giardia intestinalis. Whilst antimicrobial associated with , cryptosporidiosis, and resistance is greatly investigated in bacterial infections, its emer- other parasitic diseases, particularly in remote, underserved and gence and prevalence in parasitic infections ofhuman and veterinary tropical regions of the country and also in the immuno-compromised importance will require further investigation and attention in the individuals (HIV, immunosuppressive drugs etc.). The burden of future. many parasitic diseases is greater in tropical and sub-tropical areas of non-industrialised countries. With increasingly adventurous travel We hope that this edition of Microbiology Australia will update and dining, increasing numbers of Australians returning from travel knowledge and serve to inform all our readers of the importance and overseas with added souvenirs of common or exotic parasitoses relevance of . Whether one is involved in medical, every year and refugees and migrants arriving in Australia, these veterinary, food, environmental or other microbiological work, it is infections are becoming increasingly important. likely that aspects of your work will at some stage involve this important and sometimes neglected field of our scientific discipline. Recent advances in diagnostic techniques for the detection of As guest editors, we are grateful and excited to be involved in the parasitic infections have revolutionised how we undertake such planning and execution of this edition. We would like to thank the diagnostic investigations. These methodologies often provide editorial staff and all of the authors and reviewers who have kindly greater sensitivity as well as in some cases providing additional contributed their time and expertise into the preparation of this epidemiological data. As we increasingly move towards the use of edition. We hope that all members of the society will find it helpful, molecular methodologies and away from traditional morphological interesting and that it may spark the interest of many into this most diagnosis, new challenges have emerged for clinicians, veterinarians fascinating and under-researched area. and laboratory staff. A focus of several articles in this edition is consideration of the advantages and disadvantages of these new Biographies methods, in what circumstances they are best applied and how the The biography for Dr Harsha Sheorey is on page 49. results of such investigations should be best interpreted. Molecular methods are not without potential sources of error, hence in some The biography for Dr Richard Bradbury is on page 9.

MICROBIOLOGY AUSTRALIA * MARCH 2016 10.1071/MA16002 3 In Focus

The laboratory diagnosis of Strongyloides stercoralis

Matthew R Watts A,*, Gemma Robertson B,* and Richard S Bradbury C,D ACentre for Infectious Diseases and Microbiology, Pathology West – ICMPR, and Marie Bashir Institute, University of Sydney, Westmead Hospital, Westmead, Sydney, NSW, Australia, Tel: +61 2 9845 6255, Email: [email protected] BMelbourne Pathology, Collingwood and James Cook University, Tel: +61 3 9287 7700, Email: [email protected] CSchool of Medical and Applied Sciences, Central Queensland University, Rockhampton, Qld, Australia DCorresponding author. Email: [email protected]

It is estimated that over 30 million people worldwide are The gold standard for the diagnosis of strongyloidiasis is the infected by the , Strongyloides stercoralis1.Itis morphological identification of larvae in stool, tissue biopsies, and endemic in sub-tropical and tropical parts of Australia, with other clinical specimens such as bronchoalveolar lavage. However, high rates of infection documented in some indigenous in chronic infections, detection can be limited by low larval output in communities2. Due to the potential for chronic autoinfec- stool, leading to false negative results6. Consequently, in validation tion, that may persist for decades, migration leads to the studies for serological and nucleic acid tests there is a tendency presence of the infection in non-endemic areas1. Transmis- to define heavier infections as ‘true positives’. This affects serolog- sion to humans is generally through the penetration of larvae ical cut-offs, measurements of sensitivity and specificity, and through the skin, following contact with faecally contami- positive and negative predictive values7. Recognition of these nated soil1. Disease severity ranges from asymptomatic limitations is important for the interpretation of negative diagnostic chronic carriage to an overwhelming illness, where large test results, where clinical suspicion remains. Here, we will give an numbers spread throughout the body, usually triggered by overview of currently available conventional and molecular tests immunosuppression1. for the diagnosis of strongyloidiasis.

Clinicians are advised to consider strongyloidiasis in patients prior to immunosuppression, or with indicative symptoms, if there is a Stool microscopy and culture methods history of probable exposure in an endemic area, regardless of the Specimen transport and storage have a major impact on the efficacy elapsed time since exposure3,4. is not an accurate of culture techniques in the laboratory diagnosis of S. stercoralis marker of strongyloidiasis, with a retrospective study finding that from faecal samples. Fresh, unrefrigerated samples should be only a quarter of patients with Strongyloides infection had a raised delivered to the laboratory for culture as soon after collection as eosinophil count5. The detection of strongyloidiasis is optimised by possible, as the viability of larvae decreases incrementally with appropriate test ordering, clinical notes, specimen transportation, storage at 48C over a 72-h period8. Rhabditiform and filariform and processing by the receiving laboratory. larvae will be found along with free-living adults of S. stercoralis in

*These two authors contributed equally.

4 10.1071/MA16003 MICROBIOLOGY AUSTRALIA * MARCH 2016 In Focus older cultures (Figure 1). Larval stages must be differentiated from Serological diagnosis those of hookworms, which may also be recovered. Several tests for the serological diagnosis of strongyloidiasis have been described, using both crude and recombinant antigens. Two Microscopic methodologies such as examination of Kato-Katz pre- commercial ELISA kits employing somatic antigens are available parations, FLOTAC, and formalin/ethyl acetate concentrates have a from BORDIER (Strongyloides ratti antigen) and IVD Research low yield compared to culture9,10. A modified formalin/ethyl acetate (S. stercoralis antigen), respectively14. Recently, two recombinant method proposed by Anamnart et al. improved rates of detection9. antigens (32 kD recombinant antigen, called NIE and S. stercoralis Overall, however, microscopic techniques alone are insensitive and immmunoreactive antigen, SsIR) have been employed for serolog- not sufficient for the exclusion of strongyloidiasis. In one of these ical testing in both ELISA and luciferase immunoprecipitation sys- studies, though 30 of 254 participants were diagnosed with stron- tem assay (LIPS) platforms15. The reported sensitivity and specificity gyloidiasis by either agar plate culture (APC; Figure 2) or Baermann of various serological platforms ranges from 56-100% and 29-100%, culture techniques, no infections were identified by microscopy dependent upon the method, antigens, cut-offs, study populations, using the Kato-Katz technique11. and reference methods employed16. Strongyloides serology using a APC is possibly the easiest culture to perform in the context of high crude larval extract antigen was shown in one study to be less volume diagnostic testing. Results are available within two days, sensitive for the diagnosis of returned travellers (73%) compared although extended incubation up to four days increases yield8,11,12. to patients who have lived for an extended period in an endemic area Two studies comparing 48 h APC with Baermann culture found an (98%)17.Nodefinitive study of serological methods has been con- improved recovery of S. stercoralis larvae in APC11,12. Recovery rates ducted to date, and much of the available data is subject to flaws in improve markedly with multiple stool cultures6,11,13 methodology, particularly the use of microscopy only as a reference

(a) (b)

(c)

Figure 1. Life stages of Strongyloides stercoralis in agar plate culture: (a) rhabditiform ; (b) a free-living adult male with filariform larva adjacent; and (c) a gravid, free-living adult female with filariform larva adjacent.

MICROBIOLOGY AUSTRALIA * MARCH 2016 5 In Focus

Figure 2. Larval tracks of Strongyloides stercoralis on Koga agar plate culture.

standard for positive specimens and varying S. stercoralis exposure However, this effect is not universal and varies between studies3,17. rates amongst tested serum groups16. A 2010 study with a reference Immunosuppression was demonstrated to cause a reduction in standard of a combination of three culture methods and sedimen- serological sensitivity (62% vs previously determined 92%), when tation concentration found NIE LIPS had a sensitivity of 97.8% (cut- testing haematological patients on antineoplastic therapies18. NIE off 37.89 LU) in a study population with high endemicity but from LIPS did not cross-react with antigens from other parasites in the regions without filarial infection15. Lower sensitivity resulted when study by Bisoffi et al., whereas IFAT and the two commercial ELISAs testing the same samples by NIE ELISA(84%), NIE-SsIR LIPS (91.2) did yield false positives, particularly from and a S. stercoralis crude antigen extract ELISA (97%)18. All assays infection14.Such cross-reaction may bedecreased by pre-incubation tested showed 100% specificity in this study15. A more recent study of serum in an extract of Onchocerca gutterosa16. compared an in-house crude S. stercoralis filariform larvae immuno- fluorescent antibody test (IFAT) with the ELISAs from IVD Research, Nucleic acid tests Bordier, and a recombinant antigen NIE ELISA and LIPS14. This study Nucleic acid tests complement non-molecular methodologies for used reference samples identified as positive by culture as well as the diagnosis of S. stercoralis, and allow the use of refrigerated, microscopy, and also a composite reference standard of concordant frozen, or preserved specimens11,19,20. This simplifies specimen results in at least three of five serological tests14. The in-house IFAT transportation, particularly where collection occurs some distance was found to be the most sensitive (93.9%) when used in a test from the testing laboratory, and there is no risk of laboratory- subject group with no known previous exposure to S. stercoralis and acquired strongyloidiasis21. DNA extraction and amplification can using the composite reference standard, whilst NIE LIPS was found be performed within 1 day, however, laboratories may batch speci- to be the most specific test (100%)14. Furthermore, when tested mens according to demand. against subjects with potential previous exposure and using the composite reference standard, NIE LIPS was almost 100% specific DNA extraction and 84.6% sensitive (cut off value 1388 LU)14. In testing against the It is important that DNA extraction methods for stool specimens are same sample group, the Bordier and IVD ELISAs maintained a high effective at removing the numerous nucleic acid test inhibitors in specificity (almost 100%), but a lower sensitivity (70% and 79%, stool22. A comparison of 5 methods of DNA extraction demonstrated respectively) and the NIE ELISA showed the highest specificity that two column-based methods were the most effective for the PCR (99%), but a low sensitivity (45%) (cut-off 76.5 U/mL)14. detection of DNA from Strongyloides ratti that had been spiked into Seroreversion following treatment of many, but not all, patients human stool. These were the MoBio PowerSoil kit (MoBio Labora- was noted in a study using a Strongyloides ratti antigen ELISA3. tories, Carlsbad, CA, USA) and a method based on modifications of

6 MICROBIOLOGY AUSTRALIA * MARCH 2016 In Focus

the QiaAmp Tissue kit (Qiagen, Hilden, Germany) by Verweij et al., by Verweij et al.19. This has also allowed for the development of which has been successfully automated23. The comparison multiplexed PCR10,30,34,38. Some studies evaluating the diagnostic found that bead beating prior to the use of the NucliSens EasyMag accuracy of these PCR methods have used both morphological (BioMerieux, Marcy l’Etoile, France) was less effective, which indi- diagnosis and detection of PCR products as their reference stan- cates the method of sample pretreatment prior to automated dards, and are not reviewed here. Their methodology precludes the extraction will impact upon test sensitivity. Other investigators have calculation of sensitivity and specificity based on gold-standard, used a variety of different extraction methods for Strongyloides PCR, according to an FDA Guidance39. including in-house methods, the Qiagen stool kit (unmodified and modified), and the Nucleospin Soil kit (Macherey-Nagel, Duren, In the absence of a consistent gold standard in chronic infection, – Germany)10,24 30. positive nucleic acid test results, where conventional tests are negative, may be due to greater sensitivity or false positive results6. One of the inherent limitations of the molecular diagnosis of No PCR studies have reported false positive results when analytical S. stercoralis is the sampling error that can occur when relatively specificity has been tested using DNA extracted from bacteria, small amounts are extracted in the context of low larval output6. viruses, fungi, protozoa, and other helminths19,23,24,27,29,30. Studies For example, 2g of stool can be used for agar plate culture, whereas have also assessed the specificity of the PCR products by sequence 250 mg of specimen is recommended for the MoBio PowerSoil analysis, with all finding 100% sequence homology with the target kit31. Methods that concentrate larger amounts of stool prior to sequence of S. stercoralis.24,25,27,29. Sitta et al. found a number of DNA extraction have the potential to increase test sensitivity, if false positives, using published genus and species-specific primers, they remove inhibitors and retain larvae29. based on non-target sized bands on gel electrophoresis19,25. The PCR genus-specific primers amplified sequences that generated non- Current PCR methods most commonly target one of four regions: target bands on electrophoresis in specimens that contained the 18S rRNA small subunit (SSU); the internal transcribed spacer Blastocytis and other helminths on microscopy, and the species- region 1 (ITS-1); the 28S rRNA gene; or the cyclooxygenase gene specific primers amplified sequences that generated non-target – – (cox1)19,23 30,32 37. Published sensitivities and specificities for bands on electrophoresis in specimens positive for hookworm on Strongyloides PCR vary according to the reference methods and microscopy25. Similar accounts of cross-reactivity have not yet been are listed in Table 1. The majority of Strongyloides PCR publications reported, so further data will be useful to monitor the specificity of have used a real-time method with primers and probe published PCR in different populations.

Table 1. Sensitivity and specificity of stool PCR for human strongyloidiasis. Reference Target Sensitivity Specificity Reference method 19 18S 61.0% 92.4% Coproculture; Baermann

32 18S 58.6%/96.6%A ND McMaster

26 18S 61.0% 92.7% APC; Baermann

34 18S 100% 100% Direct microscopy

23 18S 33.0% 99.0% Harada-Mori

25 18S 84.8%/78.8%A ND APC

10 18S 11.6% 90.6% Baermann 83.3% 96.2% FLOTAC

29 18S 93.8% 86.5% FEAC; APC; Harada-Mori

36 18S 90.0% 85.7% APC

27 18S/cox1B 100% 91.6% FEC; APC 18S 84.7% 95.8%

AThe first value relates to a species-specific primer, the second to a genus-specific primer. BNested PCR. APC, agar plate culture; FEAC, formalin-ethyl acetate concentration; FEC, formalin-ether concentration.

MICROBIOLOGY AUSTRALIA * MARCH 2016 7 In Focus

LAMP 8. Inês, EdeJ. et al. (2011) Efficacy of parasitological methods for the diagnosis of Strongyloides stercoralis and hookworm in faecal specimens. Acta Trop. 120, Loop-mediated isothermal amplification (LAMP) is an additional 206–210. doi:10.1016/j.actatropica.2011.08.010 nucleic acid detection method. LAMP uses a DNA polymerase with 9. Anamnart, W. et al. (2010) Factors affecting the recovery of Strongyloides strand-displacement activity, so it doesn’t require the temperature stercoralis larvae: an approach to a newly modified formalin-ether concentra- tiontechnique for diagnosis of strongyloidiasis. J. Clin. Microbiol. 48,97–100. cycling of PCR, and can be performed with a simple source of doi:10.1128/JCM.01613-09 40 constant temperature such as a heating block . LAMP has been 10. Knopp, S. et al. (2014) Diagnostic accuracy of Kato-Katz, FLOTAC, Baermann, and successfully applied in resource limited-settings for the detection PCR methods for the detection of light-intensity hookworm and Strongyloides stercoralis infections in Tanzania. Am. J. Trop. Med. Hyg. 90,535–545. 40 of pathogens . doi:10.4269/ajtmh.13-0268 11. Steinmann, P. et al. (2007) Occurrence of Strongyloides stercoralis in Yunnan The Strongyloides LAMP assay uses primers that are genus specific Province, China, and comparison of diagnostic methods. PLoS Negl. Trop. Dis. 1, and bind to the 28S rRNA gene20. The reaction runs at 608C for e75. doi:10.1371/journal.pntd.0000075 8 12. Khieu, V. et al. (2013) Diagnosis, treatment and risk factors of Strongyloides 1 hour. Pre-heating of the reagents and DNA template to 95 C, prior stercoralis in schoolchildren in Cambodia. PLoS Negl. Trop. Dis. 7, e2035. to the addition of enzyme, increases the limit of detection and doi:10.1371/journal.pntd.0002035 eliminates the need to pre-heat the template and keep it at 48C20. 13. Hirata, T. et al. (2007) Short report: increased detection rate of Strongyloides stercoralis by repeated stool examinations using the agar plate culture method. A novel use of Syto-82 dye (Life Technologies, Carlsbad, CA, USA) Am. J. Trop. Med. Hyg. 77, 683–684. enables the detection of positive results in real-time or visually on 14. Bisoffi,Z.et al. (2014) Diagnostic accuracy of five serologic tests for Strongyloides stercoralis infection. PLoS Negl. Trop. Dis. 8, e2640. doi:10.1371/journal.pntd. completion of the reaction20. Analytical sensitivity and specificity are 0002640 19,20,23 comparable to PCR, according to the method of Verweij et al. . 15. Krolewiecki, A.J. et al. (2010) Improved diagnosis of Strongyloides stercoralis When 28 human stool specimens that were microscopy and PCR using recombinant antigen-based serologies in a community-wide study in northern Argentina. Clin. Vaccine Immunol. 17, 1624–1630. doi:10.1128/ positive for S. stercoralis were tested with the LAMP method, 27 CVI.00259-10 20 were positive . The negative specimen had a high cycle threshold 16. Requena-Méndez, A, Chiodini, P, Bisoffi, Z, Buonfrate, D, Gotuzzo, E and Munoz, J (38.44) on PCR20. Further validation of the LAMP assay with clinical (2013) The laboratory diagnosis and follow up of strongyloidiasis: a systematic review. PLoS Negl Trop Dis. 7, e2002. specimens is currently in progress. 17. Sudarshi, S. et al. (2003) Clinical presentation and diagnostic sensitivity of laboratory tests for Strongyloides stercoralis in travellers compared with immi- grants in a non-endemic country. Trop. Med. Int. 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Strongyloides stercoralis in stool. Am. J. Trop. Med. Hyg. 88, 1048–1051. 3. Page, W.A. et al. (2006) Utility of serological follow-up of chronic strongyloidiasis doi:10.4269/ajtmh.12-0437 afterantihelminthic chemotherapy. 100,1056–1062. Trans. R. Soc. Trop. Med. Hyg. 24. Repetto, S.A. et al. (2013) An improved DNA isolation technique for PCR doi:10.1016/j.trstmh.2005.12.006 detection of Strongyloides stercoralis in stool samples. Acta Trop. 126, 4. Bailey, M.S. et al. (2006) Helminth infections in British troops following an 110–114. doi:10.1016/j.actatropica.2013.02.003 – operation in Sierra Leone. Trans. R. Soc. Trop. Med. Hyg. 100, 842 846. 25. Sitta, R.B. et al. (2014) Conventional PCR for molecular diagnosis of human doi:10.1016/j.trstmh.2005.10.001 strongyloidiasis. Parasitology 141, 716–721. doi:10.1017/S0031182013002035 5. Naidu, P . (2013) Eosinophilia: a poor predictor of infection in et al Strongyloides 26. Schär, F. et al. (2013) Evaluation of real-time PCR for Strongyloides stercoralis – refugees. Can. J. Infect. Dis Med. Microbiol. 24,93 96. and hookworm as diagnostic tool in asymptomatic schoolchildren in Cambodia. 6. Dreyer, G. et al. (1996) Patterns of detection of Strongyloides stercoralis in stool Acta Trop. 126,89–92. doi:10.1016/j.actatropica.2012.12.012 specimens: implications for diagnosis and clinical trials. 34, J. Clin. Microbiol. 27. Sharifdini, M. et al. (2015) Comparison of nested polymerase chain reaction and – 2569 2571. real-time polymerase chain reaction with parasitological methods for detection of 7. Siddiqui, A.A. and Berk, S.L. (2001) Diagnosis of Strongyloidies stercoralis Strongyloides stercoralis in human fecal samples. Am. J. Trop. Med. Hyg. 93, infection. Clin. Infect. Dis. 33,1040–1047. doi:10.1086/322707 1285–1291.

8 MICROBIOLOGY AUSTRALIA * MARCH 2016 In Focus

28. Angal, L. et al. (2015) Determining intestinal parasitic infections (IPIs) in inmates 38. Basuni, M. et al. (2012) Detection of selected intestinal helminths and protozoa at from Kajang Prison, Selangor, Malaysia for improved prison management. BMC Hospital Universiti Sains Malaysia using multiplex real-time PCR. Trop. Biomed. Infect. Dis. 15, 467. doi:10.1186/s12879-015-1178-3 29, 434–442. 29. Saugar, J.M. et al. (2015) Application of real-time PCR for the detection of 39. U.S. Food and Drug Administration (2007) Statistical guidance on reporting results Strongyloides spp. in clinical samples in a reference center in Spain. Acta Trop. from studies evaluating diagnostic tests. http://www.fda.gov/RegulatoryInforma- 142,20–25. doi:10.1016/j.actatropica.2014.10.020 tion/Guidances/ucm071148.htm. 30. Janwan, P. et al. (2011) Rapid detection of and Strongy- 40. Mori, Y. et al. (2013) Loop-mediated isothermal amplification (LAMP): recent loides stercoralis in human fecal samples using a duplex real-time PCR and progress in research and development. J. Infect. Chemother. 19,404–411. melting curve analysis. Parasitol. Res. 109, 1593–1601. doi:10.1007/ doi:10.1007/s10156-013-0590-0 s00436-011-2419-z 31. Koga, K. et al. (1991) A modified agar plate method for detection of Strongyloides Biographies stercoralis. Am. J. Trop. Med. Hyg. 45,518–521. Matthew Watts is an Infectious Diseases Physician and Clinical 32. Marra, N.M. et al. (2010) Faecal examination and PCR to detect Strongyloides venezuelensis in experimentally infected Lewis rats. Mem. Inst. Oswaldo Cruz Microbiologist based at the Centre for Infectious Diseases and 105,57–61. doi:10.1590/S0074-02762010000100008 Microbiology, Pathology West-ICPMR and the Marie Bashir Institute, 33. Basuni, M. et al. (2011) A pentaplex real-time polymerase chain reaction assay for detection of four species of soil-transmitted helminths. Am. J. Trop. Med. Hyg. 84, University of Sydney, Westmead Hospital. His interests include 338–343. doi:10.4269/ajtmh.2011.10-0499 parasitic and zoonotic infections. 34. Mejia, R. et al. (2013) A novel, multi-parallel, real-time polymerase chain reaction Gemma Robertson is a final year microbiology trainee at Mel- approach for eight gastrointestinal parasites provides improved diagnostic capabilities to resource-limited at-risk populations. Am. J. Trop. Med. Hyg. 88, bourne Pathology. She has an interest in tropical medicine and – 1041 1047. doi:10.4269/ajtmh.12-0726 parasitology, and will be undertaking a PhD to continue her research 35. Zueter, A.M. et al. (2014) Detection of Strongyloides stercoralis infection among into soil-transmitted helminthiases in Aboriginal communities. cancer patients in a major hospital in Kelantan, Malaysia. Singapore Med. J. 55, 367–371. doi:10.11622/smedj.2014088 Dr Richard Bradbury is an Australian Parasitologist with an 36. de Paula, F.M. et al. (2015) Molecular diagnosis of strongyloidiasis in tropical areas: interest in all fields of parasitology. He was recently appointed as a comparison of conventional and real-time polymerase chain reaction with parasitological methods. Mem. Inst. Oswaldo Cruz 110, 272–274. doi:10.1590/ the Team Lead in the Parasite Diagnostics and Biology Laboratory 0074-02760140371 of the Centers for Disease Control and Prevention in Atlanta, USA. 37. Moghaddassani, H. et al. (2011) Molecular diagnosis of Strongyloides stercoralis He is writing this work in both his personal capacity and in his infection by PCR detection of specific DNA in human stool samples. Iran. J. Parasitol. 6,23–30. capacity as an adjunct academic at Central Queensland University.

MICROBIOLOGY AUSTRALIA * MARCH 2016 9 Under the Microscope

Current WHO protocols for mass drug administration in helminth control

Patricia M Graves Richard S Bradbury College of Public Health Medical and Veterinary Sciences School of Medical and Applied Division of Tropical Health and Sciences Medicine Central Queensland University James Cook University Rockhampton, Qld, Australia Cairns Qld, Australia Email: [email protected] Email: [email protected]

Soil transmitted helminths (STH), comprising Ascaris, For the insect borne helminths, and LF, the goal is Trichuris, Strongyloides and the hookworms remain a sig- transmission interruption and the entire community is eligible for nificant cause of morbidity amongst people in many parts of MDA. the world, including Australia. Other important helminth The frequency of MDA for STH in school-age children is dependent infections include lymphatic filariasis (LF), on the prevalence of infections in a given population (Table 1). and onchocerciasis. Preventive chemotherapy (mass drug Current WHO protocols for STH control recommend MDA with a administration [MDA]) campaigns are frequently conducted single oral dose of (400 mg), mebendazole (500 mg) or for these helminth infections in endemic areas, but the target levamisole (80 mg)1. Mebendazole is more effective than albenda- population groups, duration of campaigns, cointerventions zole for T. trichiura, whilst albendazole is slightly more effective (e.g. vector control) criteria for inclusion, drugs used and against hookworm than mebendazole. The two drugs have equally doses of drugs differ.

fi The bene ts of deworming individuals, especially children, who are Table 1. Current recommended regularity of mass drug administration A infected with soil-transmitted helminths and schistosomiasis, (MDA) for helminth (STH) infections in school-age children (adapted from WHO 2011)1. include reduction in anaemia and improved growth. Rarer, but PrevalenceB Regularity of MDA more severe presentations, such as intestinal obstruction with For control of soil-transmitted helminth (STH) infections A. lumbricoides and due to T. trichiura infection, will also be reduced1. Treatment for filariasis and onchcocerciasis !50% Twice per year, or every 4 months if at the in childhood will prevent the development of later severe conse- high end of prevalence quences of these diseases including lymphoedema, hydrocoele, !20 and <50% Once yearly elephantiasis and blindness. <20% Treat on case-by-case basis In situations of moderate to high endemicity, it has been considered For control of schistosomiasis more efficient and cost-effective to treat the entire eligible popu- !50% Once per year, or every 4 months if at the lation in particular age groups or communities for these diseases, high end of prevalence rather than first testing individuals to determine who is infected. The ! < B goals of such MDA are morbidity control in some cases and inter- 10 and 50% Once every 2 years ruption of transmission through vectors in others. For these rea- <10% Treat on case-by-case basis sons, MDA for STH is usually undertaken for school age children, AUsually defined as children between 5 and 14 years of age. while for schistosomiasis, MDA is performed either in children or BAs determined by parasitological methods; cut-off is !30% if based only on in eligible people of all ages, depending on the endemicity level. questionnaires for visible haematuria.

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greater benefit than a single dose4. Both the authors of this paper and others2 recommend further studies into the optimal dose schedules for ivermectin in the control of Strongyloidiasis within a larger cohort of participants.

Recently, there has been discussion about the outcomes and optimal age range for MDA programs to control STH and more – evidence on the impacts is required5 7; this controversy is outside the scope of this review. A novel concept of elimination of STH by MDA ‘one village at a time’ rather than by wide-scale MDA has been proposed for remote areas with low populations, such as many remote Australian Aboriginal communities. This approach is cur- rently being trialled in a remote area of the Solomon Islands8.It Figure 1. A Solomon Islander researcher undertaking a community wide advocates allowing individual communities to act as autonomous STH prevalence survey as part of an integrated STH control program (photograph by Richard Bradbury). units and to employ control options specifically tailored to the geographic, cultural, economic, aetiological and environmental high efficacy when used against Ascaris lumbricoides1. Despite factors influencing STH transmission in their own community8 these differences, in practice when administered biennially over a (Figure 1). number of years, either drug used on its own is effective for overall Deworming of school-age children has been a mainstay of helminth STH control1. control for many years. Discussion continues on the optimal meth- Onchocerciasis control and/or elimination requires annual or bian- od of MDA for this purpose. In some cases, such as where high rates nual treatment with ivermectin for many years (up to 20 in some of strongyloidiasis or onchocerciasis are present, the addition of cases), while lymphatic filariasis uses annual administration with other drugs may be warranted. Further consideration of new ther- albendazole and either ivermectin or diethylcarbamazine (DEC) apies, combination therapies, the reconsideration of use of the with at least 65% population coverage for at least five years. Thus use of ‘old’ anti-helminthic therapies have all been postulated as ivermectin and/or albendazole administration may occur in some mechanisms by which to improve absolute cure rates in MDA communities annually or biannually as part of onchocerciasis or LF programs and to reduce the possible development of antihelminthic elimination programs. Where this occurs, STH control programs resistance9. Ways to improve MDA participation, as well as paediatric should be harmonised to ensure that there is a 6 month delay formulations of for schistosomiasis prevention in pre- between albendazole administrations1. In communities with en- school children may also be added to this list. The current WHO demic schistosomiasis, the addition of praziquantel (40 mg/kg) is protocols for MDA provide an important baseline guide to those recommended (Table 1). Reductions in the frequency of MDA may undertaking MDA for helminth control in endemic areas. be considered after 5–6 years of consistent >75% population cov- erage, after testing of the residual prevalence of helminths in that population. Such a decision is based on several factors, specific References details of which may be found in the World Health Organization 1. World Health Organization (2011) Helminth control in school-age children: 1 a guide for managers of control programmes. 2nd edn. Geneva: World Health guide for managers of control programmes . Organization. 2. Bisoffi,Z.et al. (2011) Randomized clinical trial on ivermectin versus thiabendazole Reliance on albendazole and mebendazole in WHO recommenda- for the treatment of strongyloidiasis. PLoS Negl. Trop. Dis. 5, e1254. doi:10.1371/ tions for MDA will result in a lower impact on the clearance of journal.pntd.0001254 Strongyloides stercoralis, for which ivermectin and thiabendazole 3. Adams, M. et al. (2003) Strongyloidiasis: an issue in Aboriginal communities. Rural Remote Health 3, 152. are more effective drugs2. Thiabendazole was discontinued in 4. Suputtamongkol, Y. et al. (2011) Efficacy and safety of single and double doses of Australia in 2003 and due to the lower rate of side effects, ivermectin ivermectin versus 7-day high dose albendazole for chronic strongyloidiasis. PLoS Negl. Trop. Dis. 5, e1044. doi:10.1371/journal.pntd.0001044 has been recommended by some as the treatment of choice2. Due 5. Anderson, R.M. et al. (2015) Should the goal for the treatment of soil transmitted to the auto-infective cycle of this helminth, some authors have Helminth (STH) infections be changed from morbidity control in children to recommended re-treatment at one and two months to ensure community-wide transmission elimination? PLoS Negl. Trop. Dis. 9, e0003897. doi:10.1371/journal.pntd.0003897 elimination3. Only one randomised trial has been performed thus 6. Taylor-Robinson, D.C. et al. (2015) Deworming drugs for soil-transmitted intestinal far, in which treatment twice at 2 weeks apart was found to have no worms in children: effects on nutritional indicators, haemoglobin and school

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performance. Cochrane Database Syst. Rev. 7, CD000371. doi:10.1002/14651858. the Team Lead in the Parasite Diagnostics and Biology Laboratory CD000371.pub6 of the Centers for Disease Control and Prevention in Atlanta, USA. 7. Hicks, J.H. et al. (2015) The case for mass treatment of intestinal helminths in endemic areas PLoS Negl. Trop. Dis. 9, e0004214. doi:10.1371/journal.pntd.000 He is writing this work in both his personal capacity and in his 4214 capacity as an adjunct academic at Central Queensland University. 8. Harrington, H. et al. (2015) Prevalence of soil transmitted helminths in remote villages in East Kwaio, Solomon Islands. Western Pac. Surveill. Response J. 6,1–8. doi:10.5365/wpsar.2015.6.1.016 Dr Patricia Graves is a vector-borne disease epidemiologist with 9. Savioli, L. (2014) Preventive anthelmintic chemotherapy – expanding the arma- mentarium. N. Engl. J. Med. 370,665–666. doi:10.1056/NEJMe1312403 research, field, laboratory and consulting experience in the control and elimination of malaria and filariasis. She is Director of the Biographies JCU/WHO Collaborating Centre for Control of LF, STH and other Dr Richard Bradbury is an Australian Parasitologist with an NTDs in the Division of Tropical Health and Medicine, James interest in all fields of parasitology. He was recently appointed as Cook University.

Zoonotic tissue parasites of Australian wildlife

lizards), which feed on gastropods. Rats become infected by ingest- ing intermediate or paratenic hosts. In the rat, the nematode undergoes an obligatory migration through the spinal column and David M Spratt brain en route to the final site in the pulmonary arteries of the lungs. Australian National Wildlife Collection Humans become infected by accidentally or deliberately eating National Research Collections infected gastropods or paratenic hosts, or unwashed salad greens Australia, CSIRO GPO Box 1700 containing these. The parasite has been reported from domestic and Canberra, ACT 2601, Australia zoo animals, mammalian and avian wildlife and humans in Brisbane Tel: +61 2 6242 1648 – Email: [email protected] and Sydney2 4. The clinical signs of headache, vomiting, paralysis and sometimes death are induced as a consequence of the oblig- atory period of development of the parasite in the central nervous Increasing use of bushlands for recreational, commercial system. This occurs in young children who deliberately or acciden- and scientific activities fosters movement across the urban- – tally ingest snails or slugs containing infective larvae5 7, or foolish bushland interface. This may facilitate the transmission of young adults who do so for a bet8,9. parasitic diseases from wildlife to humans (zoonoses). The fashionable trend to consumption of game meats such as Muspiceoidosis feral pig and crocodile, and raw fish such as sushi, sashimi and pickled herring has exacerbated the zoonotic potential Haycocknema perplexum is a minute muspiceoid nematode living of parasites of wildlife. as adults inside individual skeletal muscle cells of humans in Australia10. Eight cases have been documented, 4 in Tasmania and – Transmission from wildlife to humans 4 in north Queensland11 13 Gasser (personal communication). Eight to twelve eggs hatch inside the uterus of the female, develop to third-stage infective larvae and burst from the head region killing the Angiostrongylus cantonensis is a nematode parasite of the pulmo- adult, an efficient mechanism for auto-re-infection. Escaped larvae nary arteries and right ventricle of Rattus rattus and R. norvegicus in invade uninfected muscle cells. The occurrence of H. perplexum in Australia1. It is the causative agent of eosinophilic meningoenceph- intramyofibres results in eosinophilic polymyositis but no reaction alitis, a zoonotic infection of humans. The life cycle includes an within the invaded cell itself11. Progressive myopathy occurs and obligatory period of larval development in terrestrial or aquatic infection becomes life threatening. Early human diagnosis by mus- snails and slugs, and also may involve a range of paratenic or cle biopsy is imperative in cases of progressive myopathy associated transport hosts (freshwater prawns, land crabs, planarians, frogs, with blood eosinophilia and elevated creatine kinase levels. Steroid

12 10.1071/MA16005 MICROBIOLOGY AUSTRALIA * MARCH 2016 Under the Microscope

Table 1. Potential zoonotic tissue parasite infections in Australia. Disease name Organism Type Source Reference Leishmania sp. Trypanosomatid Forcipomyia spp. 26,27

Protozoan

Sparganosis erinaceieuropaei Cestode (larval, plerocercoid) Eating raw/undercooked meat 28

Pentastomiasis Armillifer spp. Modified brachiuran Eating undercooked snake/ 29,30,31 mammal flesh, drinking Linguatula spp. Crustacean contaminated water

treatment of patients exacerbates their infections to a life-threat- behaviour, in particular the attenuation of predator odour aversion ening illness and may delay diagnosis by masking key diagnostic and anxiety. This facilitates ingestion of the intermediate host by the features. Treatment with albendazole, 400 mg twice daily for 8– cat definitive host and perpetuation of the life cycle17. 9 weeks, is recommended12. Haycocknema perplexum is consid- Humans become infected with T. gondii mainly by ingesting un- ered a zoonosis although the source of infection of humans – water, cooked meat containing viable tissue cysts or by ingesting food or soil, plants or animals – remains unknown. water contaminated with oocysts from the faeces of infected cats. fi Halicephalobus gingivalis Transplacental transmission may occur in women during their rst trimester of pregnancy and this infection is then passed to the Halicephalobus gingivalis formerly known as Micronema deletrix, developing embryo via the placenta. This frequently results insevere is a free-living nematode of soil, manure and decaying humus known brain and eye lesions in the newborn or death of the developing to cause opportunistic infections, primarily in horses but also in embryo. Research results from the past 15 years have shown that humans14.The majority of cases in horses have been fatal and usually T. gondii infection is associated with several neuropsychiatric not diagnosed before necropsy. All human cases have involved fatal diseases and behavioural changes in humans as well animals18. meningoencephalitis including the first human case in Australia, a Although the mechanisms are unknown a growing body of data 74-year-old woman from Eyre Penninsula, South Australia15.In indicates that they are complex, comprising humoral, immune, tissues, only ova, larvae and adult females are seen, reproduction neurotransmitter, epigenetic, genetic, and structural effects19. in the parasitic phase presumed to be by parthenogenesis16. It is not known how H. gingivalis infects humans or horses although expo- (hydatid disease) sure through an oromaxillary route may explain common neuro- Australia has, on average, >80 new cases of human hydatidosis per logical involvement15. annum caused by the larval stage of the cestode, Echinococcus granulosus20. The parasite was introduced into Australia with Transmission from domestic animals to humans domestic livestock and dogs. However, a cycle in wildlife is main- and wildlife tained through a predator/prey interaction between dingoes, wild Toxoplasmosis dogs and less importantly foxes and kangaroos and wallabies, less importantly feral pigs (Sus scrofa)21,22. The establishment of a Felids, domestic cats in particular, are the only definitive host of the dingo/wild dog-macropod cycle, which effectively maintains para- obligate intracellular protozoan parasite, Toxoplasma gondii17. site transmission, acts as a spill-back reservoir of infection for sheep Most species of mammals and birds are susceptible to infection and cattle23. This is a major problem for control strategies focussed and may act as intermediate hosts. Infection is usually systemic on human education and husbandry practices to break the domestic resulting in a short period of rapid multiplication in various tissues ‘dog-sheep’ cycle. In contrast to the situation in livestock, hydatid followed by the establishment of tissue cysts in the muscles and cysts occur primarily in the lungs rather than the liver of marsupials. brain. These are transmitted only if ingested by predation or Infection occurs predominantly in the eastern States but the parasite scavenging or if passed vertically across the placenta from mother has established recently in wildlife in water catchment and forestry to foetus. areas outside Perth21. Western grey kangaroos and feral pigs act as The localisation of T. gondii cysts in the forebrain of rats and mice intermediate hosts. This focus of transmission may have been together with the immune reaction to the cysts is related to altered initiated through E. granulosus-infected domestic pig hunting dogs

MICROBIOLOGY AUSTRALIA * MARCH 2016 13 Under the Microscope

from the eastern states. Transmission appears to be perpetuated by 17. Dubey, J.P. et al. (1998) Structures of Toxoplasma gondii tachyzoites, brady- zoites, and sporozoites and biology and development of tissue cysts. Clin. dogs of local pig hunters infected through being fed the offal of Microbiol. Rev. 11, 267–299. locally shot kangaroos21. Hydatid disease is also an important 18. Evans, A.K. (2014) Patterns of Toxoplasma gondii cyst distribution in the forebrain associated with individual variation in predator odor avoidance and conservation issue, especially for small endangered species and anxiety-related behavior in male Long-Evans rats. Brain Behav. Immun. 37, populations of Macropodidae by severely reducing effective lung 122–133. doi:10.1016/j.bbi.2013.11.012 volume24,25. Such reductions impact the fitness of the animals 19. Hinze-Selch, D. (2015) Toxoplasma gondii infection and neuropsychiatric dis- ease: current insight. Rep. Parasitol. 4,43–51. doi:10.2147/RIP.S52980 enhancing susceptibility to predation, thus ensuring perpetuation 20. Thompson, R.C.A. and McManus, D.P. (2002) Towards a taxonomic revision of of the cycle. the genus Echinococcus. Trends Parasitol. 18,452–457. doi:10.1016/S1471-4922 (02)02358-9 Several potential zoonotic tissue parasite infections in Australia are 21. Thompson, R.C.A. et al. (1988) Hydatid disease in urban areas of Western listed in Table 1. Australia: an unusual cycle involving western grey kangaroos (Macropus fuliginosus), feral pigs and domestic dogs. Aust. Vet. J. 65,188–190. doi:10.1111/j.1751-0813.1988.tb14298.x References 22. Jenkins, D.J. and Morris, B. (2003) Echinococcus granulosus in and around – 1. Mackerras, M.J. and Sandars, D.F. (1955) The life history of the rat lung-worm, the Kosciuszko National Park, south-eastern Australia. Aust. Vet. J. 81,8185. Angiostrongylus cantonensis (Chen) (Nematoda: Metastrongylidae). Aust. doi:10.1111/j.1751-0813.2003.tb11440.x J. Zool. 3,1–21. doi:10.1071/ZO9550001 23. Durie, P.H. and Riek, R.F. (1952) The role of the dingo and wallaby in the 2. Spratt, D.M. (2005a) Australian ecosystems, capricious food chains and parasitic of cattle with hydatids (Echinococcus granulosus (Batsch, 1786) – consequences for people. Int. J. Parasitol. 35, 717–724. doi:10.1016/j.ijpara.2005. Rudolphi, 1805) in Queensland. Aust. Vet. J. 28,249254. doi:10.1111/ 01.014 j.1751-0813.1952.tb13436.x 3. Spratt, D.M. (2005b) Neuroangiostrongyliasis: disease in wildlife and humans. 24. Johnson, P.M. et al. (1998) Mortality in wild and captive rock wallabies and nailtail Microbiol. Aust. 26,63–64. wallabies due to hydatid disease caused by Echinococcus granulosus. Aust. Mammal. 20, 419–423. 4. Ma, G. (2013) Tawny frogmouths and brushtail possums as sentinels for Angios- trongylus cantonensis, the rat lungworm. Vet. Parasitol. 192, 158–165. 25. Barnes, T.S. et al. (2008) Cystic echinococcosis in a wild population of the brush- doi:10.1016/j.vetpar.2012.11.009 tailed rock-wallaby (Petrogale penicillata), a threatened macropodid. Parasitol- ogy 135, 715–723. doi:10.1017/S0031182008004423 5. Prociv, P. and Tiernan, J.R. (1987) Eosinophilic meningoencephalitis with per- manent sequelae. Med. J. Aust. 147, 294–295. 26. Rose, K. (2004) in red kangaroos: isolation and charac- terisation of the causative organisms. Int. J. Parasitol. 34,655–664. doi:10.1016/ 6. Prociv, P. et al. (2000) Neuro-angiostrongyliasis: unresolved issues. Int. j.ijpara.2004.03.001 J. Parasitol. 30, 1295–1303. doi:10.1016/S0020-7519(00)00133-8 27. Dougall, A.M. et al. (2011) Evidence incriminating midges (Diptera: Ceratopogo- 7. Morton, N.J. et al. (2013) Severe haemorrhagic meningoencephalitis due to nidae) as potential vectors of Leishmania in Australia. Int. J. Parasitol. 41, Angiostrongylus cantonensis among young children in Sydney, Australia. Clin. 571–579. doi:10.1016/j.ijpara.2010.12.008 Infect. Dis. 57, 1158–1161. doi:10.1093/cid/cit444 28. Liu, Q. (2015) Human sparganosis, a neglected food borne zoonosis. Lancet Infect. 8. Senanayake, S.N. (2003) First case of human angiostrongyliasis acquired in Dis. 15, 1226–1235. doi:10.1016/S1473-3099(15)00133-4 Sydney. Med. J. Aust. 179, 430–431. 29. Paré, J.A. (2008) An overview of Pentastomiasis in reptiles and other vertebrates. 9. Blair, N.F. et al. (2013) Angiostrongylus meningoencephalitis: survival from J. Exot. Pet Med. 17, 285–294. doi:10.1053/j.jepm.2008.07.005 minimally conscious state to rehabilitation. Med. J. Aust. 198, 440–442. doi:10.5694/mja12.11085 30. Kelehear, C. et al. (2014) Pentastomids of wild snakes in the Australian tropics. Intl. J. Parasitol.: Para. Wldlfe. 3,20–31. [with online supplementary table] 10. Spratt, D.M. et al. (1999) Haycocknema perplexum n.g., n. sp. (Nematoda: – Robertdollfusidae): an intramyofibre parasite in man. Syst. Parasitol. 43, 31. Drabick, J.J. (1987) Pentastomiasis. Rev. Infect. Dis. 9, 1087 1094. doi:10.1093/ 123–131. doi:10.1023/A:1006158218854 clinids/9.6.1087 11. Dennett, X. et al. (1998) Polymyositis caused by a new nematode. Med. J. Aust. 168,226–227. 12. Basuroy, R. et al. (2008) Parasitic myositis in tropical Australia. Med. J. Aust. 188, Biography 254–256. 13. McKelvie, P. et al. (2013) A further patient with parasitic myositis due to Hay- Dave Spratt is an Honorary Fellow at the Australian National – cocknemaperplexum, a rare entity. J. Clin. Neurosci. 20, 1019 1022. doi:10.1016/ Wildlife Collection, National Research Collections Australia, CSIRO, j.jocn.2012.08.009 in Canberra. The major themes of Dr Spratt’s research are the study 14. Anderson, R.C. et al. (1998) Halicephalobus gingivalis (Stefanski, 1954) from a fatal infection of a horse in Ontario, Canada with comments on the validity of and understanding of the diseases of wildlife including disease H. deletrix and a review of the genus. Parasite 5, 255–261. doi:10.1051/parasite/ ecology, parasite taxonomy, helminth biodiversity and zoonoses. 1998053255 fi 15. Lim, C.K. et al. (2015) First human case of fatal Halicephalobus gingivalis Research topics of interest include metastrongyloid, larioid, trichi- meningoencephalitis in Australia. J. Clin. Microbiol. 53, 1768–1774. doi:10.1128/ nelloid and muspiceoid , pentastomes, and small mam- JCM.00032-15 mal succession and recolonisation of their helminth communities 16. Blunden, A.S. et al. (1987) Halicephalobus deletrix infection in a horse. Equine Vet. J. 19,255–260. doi:10.1111/j.2042-3306.1987.tb01399.x following wildfire.

14 MICROBIOLOGY AUSTRALIA * MARCH 2016 Under the Microscope

Assessing enteric helminths in refugees, asylum seekers and new migrants

Sarah Hanieh A,D,*, Norbert Ryan B,* and Beverley-Ann Biggs B,C AVictorian Infectious Diseases Reference Laboratory, Peter Doherty Institute for Immunity and Infection, Parkville, Vic. 3050, Australia BDepartment of Medicine, The University of Melbourne, Peter Doherty Institute for Immunity and Infection, Parkville, Vic. 3050, Australia CThe Victorian Infectious Diseases Service, Royal Melbourne Hospital, Parkville, Vic. 3050, Australia DCorresponding author. Department of Medicine, The University of Melbourne, Doherty Institute, Parkville, Vic. 3050, Australia, Tel: +61 3 8344 3257, Fax: +61 3 9347 1863, Email: [email protected]

Currently there are 59.5 million people forcibly displaced water, access to running water, access to footwear) and insufficient worldwide as a result of conflict, human rights violations, access to adequate health care and treatment may significantly generalised violence or persecution. Of these, 19.5 million increase the risk of exposure to intestinal parasitic disease in the are refugees and 1.8 million are asylum seekers. Each year refugee population1. Other practices such as the use of night-soil as Australia accepts 13 750 refugees through the offshore fertiliser, dietary habits, and past occupational exposures are likely Humanitarian program, and in 2016 that number will to play an important role in increasing the burden of disease. Soil almost double with the addition of 12 000 refugees from transmitted helminth (STH) infections are very common in those Syria and Iraq. Many refugees have complex medical needs living in resource constrained settings1. Treatment of refugees and and have reached Australia after a difficult journey, often asylum seekers is often empirical, in refugee camps, before depar- involving time in refugee camps and exposure to traumatic ture as part of the pre-departure health check, or after arrival in events including physical hardship and illness. Refugees Australia. More serious infections, such as strongyloidiasis, schisto- often come from parts of the world where parasitic and somiasis, opisthorchus and , require diagnosis tropical infectious diseases are prevalent and untreated. and specific treatment. Table 1 summarises the findings of recent This article provides a review of enteric helminth infections prevalence studies of strongyloidisis and schistosomiasis in refugee in refugees, including asylum seekers and those from a groups from Australia and overseas. refugee-like background.

Parasitic infections in refugees and new migrants reflect the under- Strongyloidiasis lying epidemiology of parasites in areas where refugees may have The highest prevalence of Strongyloides stercoralis infection been exposed, including the country of origin, migration journey to occurs in refugees from Africa and South-East Asia2. Of those Australia, and place of detention. Factors such as poverty, disruption arriving in the last decade, the Burmese groups (e.g. Karen, Chin) of basic services, poor sanitation/hygiene (e.g. quality of drinking have the highest prevalence (26.0%). Earlier data show an even

*Joint first authors.

MICROBIOLOGY AUSTRALIA * MARCH 2016 10.1071/MA16006 15 Under the Microscope

Table 1. Prevalence of strongyloidiasis and schistosomiasis in refugee and asylum seeker groups. Reference Sample size Country/region Country of Prevalence of Prevalence of of origin settlement Schistosomiasis Strongyloides International studies

21 700 Latin America, Sub- Spain 5.9% 56.1% saharan Africa

22 1063 Europe, Eastern Canada 15% 3% Mediterranean, Africa

23 208 Brazil USA 27.7% 5.8%

24 350 Africa (46%), Asia Canada N/A 4.6% (28.6%)

16 1376 Middle East, Africa, USA 0.8% (Africa) 2.0% (SE Asia) Asia 2.5% (Africa)

20 176 Southeast Asia (59%), USA 8% 24% Africa (27%), Middle East (14%)

Australian studies

21 1136 Burma (Karen Australia 7% 20.8% refugees)

21 187 Asia Australia 17% 5.7%

27 182 Africa Australia N/A N/A

7 234 Cambodia Australia N/A 35%

9 156 Burma Australia 5.4% 26%

10 239 Africa Australia 37% –

28 258 Sudan, Liberia Australia 12% 9%

3 361 Cambodia, East Africa Australia 11% East African 2% East African 42% Cambodian

29 135 East Africa Australia 2% 11%

5 95 Laos Australia N/A 23%

N/A, not available.

– higher prevalence in Lao and Cambodian refugees3 5. Infections present along the Yangtze River in China and the Philippines. may persist for decades after leaving an endemic area due to a S. mekongi is found in the Mekong river valley. continual cycle of auto-infection. Although many patients are infection may also be encountered in other areas such as parts of asymptomatic or have minimal clinical symptoms, they remain at Indonesia, the Caribbean, the Arabian Peninsula, Madagascar, the risk for subsequent hyperinfection if immunosuppressed6. Stron- Middle east and Turkey8. Prevalence of schistosomiasis infection in gyloides antibodies decline after effective treatment7. refugees in Australia, as determined by serology, has been shown to range from 5.4% in Burmese9 to 37% in Africans10.

Schistosomiasis Schistosoma antibodies are thought to persist in those from en- The highest prevalence of schistosomiasis is found in Africa, ac- demic areas despite prior treatment. A long-term study of schisto- counting for an estimated 95% of global cases. Species involved are somiasis serology post-treatment showed an immediate increase in S. mansoni, S. haematobium and S. intercalatum. S. japonicum is titre and then a fourfold decline in most travellers after 6–12 months.

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However, for immigrants, serology remained elevated even three species of tapeworm T. saginata, T. solium and T. asiatica three years after effective treatment in a proportion of patients11. co-exist in the human population13.

Considerations in the Syrian refugee population Soil-transmitted helminths (STH) In September 2015, it was announced that 12 000 Syrian and Iraqi Many refugees will have received empirical albendazole as part of refugees would be accepted to Australia as part of the Humanitarian the predeparture medical assessment conducted by the Interna- Program during 2016–17. The recent prevalence of enteric parasite tional Organisation of Migration on behalf of the Australian infections is not well documented in Syria. However, schistosomi- Government. This has significantly altered the prevalence and asis is considered to have low endemnicity in Iraq (0.1%) and patterns of intestinal helminths in refugees12. However, albendazole Syria (<10% prevalence in 2010)14. There is no information available has limited effectiveness against , and is not an on the prevalence of S. stercoralis in Syria; however, a hospital- effective treatment for some less common helminths found in based survey in Iraq reported a prevalence of 24.2%15. Chang et al. refugees and asylum seekers (see below). reported a low prevalence of other parasitic infections in refugees from Iran and Iraq16. Less common helminth infections The majority of Asian refugees currently entering Australia are from Diagnosis Myanmar, from camps on the Thai border. Within Thailand, espe- In Australia, the number of faecal microscopy tests performed has cially the north east, Opisthorchis viverrini is highly prevalent. fallen in some States (e.g. NSW), with more emphasis being placed Infection results from consumption of raw, uncooked or fermented on empirical treatment of STH and the use of serology for the fish containing metacerciae. Long-term infection may cause cho- diagnosis of S. stercoralis or Schistosoma spp. Current recom- langitis, obstructive jaundice, , periductal fibrosis and mended diagnostic tests for enteric parasites are shown in Table 2. cancer, contributing to a liver cancer rate in excess of 70 per 100 000 in NE Thailand. There is a paucity of data on faecal Challenges and limitations of testing for microscopy findings for refugees from Myanmar. However, the schistosoma and strongyloides infections presence of vector cyprinoid fish and substantial wetlands suggests in a refugee population that infection with Opisthorchis viverrini is also likely. Serology may overestimate the prevalence of disease due to cross- Other serious infections in refugees from Asia include Taenia reactivity with other nematode infections and there is difficulty solium (pork tapeworm) with the potential risk of for distinguishing recent from past (and cured) infections. Serological both patient and household members. In Thailand faecal tests titres (OD values) in the equivocal and low positive ranges are for helminth eggs have revealed a prevalence of Taenia spp of difficult to interpret. Follow-up serology should preferably be done 2.3–3.7% in communities with high migrant populations along the in the same laboratory and in parallel with previous specimens Thai border. In the remote western border area of Kanchanaburi where available. The interpretation of Schistosoma serology in

Table 2. Recommended diagnostic tests for enteric parasites. Enteric parasite Diagnosis Strongyloides stercoralis Positive serology Stool microscopy should be performed to rule out other enteric infections

Schistosoma spp. Positive serology Stool/urine microscopy for ova should be performed in those with positive serology

Hookworm ( and ) Microscopic finding of ova in faecal specimens Concentration methods are necessary to detect light infections

Ascaris lumbricoides Microscopic identification of ova in faecal specimens

Trichuris trichiura Microscopic identification of ova in faecal specimens

Taenia spp. Microscopic identification of ova and proglottids in faecal specimens

MICROBIOLOGY AUSTRALIA * MARCH 2016 17 Under the Microscope

Australia presents several challenges as test methodology varies from specialist Refugee Health Services. Guidelines for post-arrival between laboratories. VIDRL in Melbourne, use the Fumouze assessment for people of refugee-like background were published indirect haemagglutination test (IHA). This is based on an antigen by the Australian Society for Infectious Diseases in 2009 and are derived from adult worms of S. mansoni. Estimates in one com- currently being revised19. If possible, a full health assessment of new parison study show that the sensitivity of this test is 76.2% for arrivals is ideally conducted within one month of arrival in Australia, , and slightly lower for S. haematobium, with including serology for Strongyloides for all, and Schistosoma in a specificity of 99%17. However, in NSW at ICPMR, an ‘in house’ those who have lived or travelled through an endemic area. For ELISA assay is the preferred assay used, based on S. mansoni egg many of the clinics in suburbs with high migrant populations, antigen. Two commercial assays based on the use of a similar the special pathology requirements are met by private pathology antigen showed 71.4–85.7% sensitivity but reduced specificity of providers. 76.9–88.4%. Both ELISA assays showed cross-reactivity with ces- tode, nematode and trematode infections17. The sensitivity of these Summary assays for other species of Schistosoma, such as S. haematobium, Intestinal helminth infections in refugees are common and should S. intercalatum, S. mekongi and S. japonicum, is not specified; remain a high priority for health workers. These populations often however, it is likely to be reduced. have specific needs that should be considered in diagnosis and management of these infections. Burden of disease is likely to reflect The sensitivity and specificity of Strongyloides stercoralis serology the country of origin, journey of migration to Australia, pre-depar- is reported to be up to 94.6% and 99.6% respectively, depending ture treatment and place of detention. Other socio-economic and on the assay used18. However, as there is no gold standard test for cultural factors are also likely to play a significant role in exposure comparison, these are estimations only. Serological titres decline risk. Helminth infections may be chronic and persist in humans for with effective treatment over a 12 month period7,18. more than four decades resulting in serious morbidity and mortality Persistence of parasitic infections in refugee and highlighting the need for early diagnosis. populations Acknowledgements Several studies have demonstrated that serious intestinal parasitic We thank Ms Christalla Hajisava for technical assistance. infections may persist for many years after arrival in Australia. A 2002 study of Laotian refugees who had arrived in Australia during References 1974–91 showed that strongyloides serology was positive in 24% 1. Freeman, M.C. et al. (2015) Associations between school- and household-level water, sanitation and hygiene conditions and soil-transmitted helminth infection and 3 carried Opisthorchis, compared to respective prevalences among Kenyan school children. Parasit. Vectors 8, 412. doi:10.1186/ of 19.2% and 41%, on initial screening by faecal microscopy4.As s13071-015-1024-x liver flukes survive for approximately 7–10 years, the three cases of 2. Puthiyakunnon, S. et al. (2014) Strongyloidiasis–an insight into its global prev- alence and management. PLoS Negl. Trop. Dis. 8, e3018. doi:10.1371/journal. fi Opisthorchis identi ed may well represent reinfection on subse- pntd.0003018 quent visits to Laos. 3. Caruana, S.R. et al. (2006) Undiagnosed and potentially lethal parasite infections among immigrants and refugees in Australia. J. Travel Med. 13,233–239. In a second study of East African refugees, who arrived in Australia doi:10.1111/j.1708-8305.2006.00045.x – in the late 90s, screening for strongyloides and schistosoma 4. Ryan, N. et al. (1988) Parasitic infections of refugees. Med. J. Aust. 148,491 494. 5. de Silva, S. et al. (2002) Chronic Strongyloides stercoralis infection in Laotian serology was positive in 11% and 15%, respectively, of patients immigrants and refugees 7-20 years after resettlement in Australia. Epidemiol. some 16 years later in 20063. In a further aspect of the same study, Infect. 128, 439–444. doi:10.1017/S0950268801006677 42% of 234 Cambodians who had arrived in the late 80s still tested 6. Olsen, A. et al. (2009) Strongyloidiasis–the most neglected of the neglected tropical diseases? Trans. R. Soc. Trop. Med. Hyg. 103,967–972. doi:10.1016/ 3 positive for strongyloides serology . This compared with 7.8% of j.trstmh.2009.02.013 Cambodians who were found to be positive for strongyloides by 7. Biggs, B.A. et al. (2009) Management of chronic strongyloidiasis in immigrants and refugees: is serologic testing useful? Am. J. Trop. Med. Hyg. 80,788–791. microscopy at initial health screening4. 8. Steinmann, P. et al. (2006) Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infect. Post arrival health assessment for refugees Dis. 6, 411–425. doi:10.1016/S1473-3099(06)70521-7 and asylum seekers 9. Chaves, N.J. et al. (2009) Screening practices for infectious diseases among Burmese refugees in Australia. Emerg. Infect. Dis. 15, 1769–1772. doi:10.3201/ Community Health Centres and GP services in suburbs with high eid1511.090777 rates of migrant and refugee settlement are now responsible for 10. Sheikh, M. et al. (2009) The epidemiology of health conditions of newly arrived refugee children: a review of patients attending a specialist health clinic in Sydney. much of the post-arrival refugee health screening, with support J. Paediatr. Child Health 45, 509–513. doi:10.1111/j.1440-1754.2009.01550.x

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11. Yong, M.K. et al. (2010) Long-term follow-up of schistosomiasis serology post- and Immunity, The University of Melbourne. She has interests in treatment in Australian travelers and immigrants. J. Travel Med. 17,89–93. doi:10.1111/j.1708-8305.2009.00379.x child nutrition, infectious diseases and refugee health. Her current 12. Swanson, S.J. et al. (2012) Albendazole therapy and enteric parasites in United research aims to understand the nutritional and infectious causes of States-bound refugees. N. Engl. J. Med. 366,1498–1507. doi:10.1056/ impaired child growth and development in resource poor settings. NEJMoa1103360 13. Anantaphruti, M.T. et al. (2010) Molecular and serological survey on taeniasis and cysticercosis in Kanchanaburi Province, Thailand. Parasitol. Int. 59, 326–330. Norbert Ryan is a Senior Scientist in the Bacteriology Laboratory at doi:10.1016/j.parint.2010.03.007 Victorian Infectious Diseases Reference Laboratory, Doherty Insti- 14. Hotez, P.J. et al. (2012) Neglected tropical diseases of the Middle East and North tute, Melbourne. Areas of interest include parasitology, diagnosis of Africa: review of their prevalence, distribution, and opportunities for control. PLoS Negl. Trop. Dis. 6, e1475. doi:10.1371/journal.pntd.0001475 Legionnaires’ disease and sexually transmitted infections. The use of 15. Schär, F. et al. (2013) Strongyloides stercoralis: global distribution and risk factors. staining methods for diagnosis of protozoal infections including PLoS Negl. Trop. Dis. 7, e2288. doi:10.1371/journal.pntd.0002288 microsporidia has been a specialty field. He was involved in proces- 16. Chang, A.H. et al. (2013) Decreasing intestinal parasites in recent Northern California refugees. Am. J. Trop. Med. Hyg. 88,191–197. doi:10.4269/ajtmh.2012. sing of specimens during the systematic health screening of refugee 12-0349 and family reunion migrant groups conducted by the Victorian 17. Kinkel, H.F. et al. (2012) Evaluation of eight serological tests for diagnosis of Department of health during the late 80s-early 90s. imported schistosomiasis. Clin. Vaccine Immunol. 19, 948–953. doi:10.1128/ CVI.05680-11 18. Bisoffi,Z.et al. (2014) Diagnostic accuracy of five serologic tests for Strongyloides Professor Beverley-Ann Biggs heads the International and Im- stercoralis infection. PLoS Negl. Trop. Dis. 8, e2640. doi:10.1371/journal.pntd. migrant Health Group in the Department of Medicine, The Univer- 0002640 sity of Melbourne and is an Infectious Diseases Physician in the 19. Australian Society of Infectious Diseases (2009) Diagnosis, management and prevention of infections in recently arrived refugees Sydney, NSW: ASID. Victorian Infectious Diseases Service at the Royal Melbourne Hospital. She has a special interest in parasitic and other infectious Biographies diseases in refugees and immigrants living in Australia, and has Dr Sarah Hanieh is a paediatric infectious diseases physician and published extensively in this area. NHMRC Early Career Research Fellow in the Immigrant and Inter- national Health Group, at the Peter Doherty Institute for Infection

MICROBIOLOGY AUSTRALIA * MARCH 2016 19 Under the Microscope

Free living amoebae and human disease

Evan Bursle Jennifer Robson Sullivan Nicolaides Pathology Sullivan Nicolaides Pathology Whitmore Street Whitmore Street Taringa, Qld 4068, Australia Taringa, Qld 4068, Australia Email: [email protected] Email: [email protected]

Pathogenic FLA are ubiquitous protozoans and despite fre- wide range of temperature, pH and osmolarity9 and may be found in quent human contact remain a rare cause of often devastat- air, soil and water samples. They are one of the most commonly ing infection with poor prognosis. Given changes in climate, isolated FLA in the environment10, and the most common in human human encroachment into the environment, increasing im- infection2. The strongest risk factor for GAE or disseminated infec- munosuppression, and improving diagnostic capacity, it is tion is immunodeficiency, while keratitis most commonly affects likely we will see increased cases in the future. Early diag- immunocompetent contact lens wearers who often have a history of nosis is challenging but crucial to achieving a favourable poor lens hygiene. Exposure is thought to occur by inhalation, outcome. It is best facilitated by improved awareness of mucosal contact or direct inoculation10. Acanthamoeba species FLA disease, appropriate clinical suspicion and early diag- were previously classified based on morphology, but are now nostic testing. grouped into 17 genotypes based on 18S rRNA sequencing, with the majority of pathogenic species belonging to the T4 genotype9,11. Free living amoebae (FLA) are a cosmopolitan group of protozoan organisms that do not require a host to survive. Despite their may be found in rivers, lakes and soil but does ubiquitous nature, these organisms are uncommon human patho- not survive in sea water. As thermophiles, their presence in fresh gens. However, four genera contain species known to cause invasive water is related to temperature and they may even be recovered disease in humans: Acanthamoeba, Naegleria, Balamuthia and from thermally polluted waters at high latitudes12. Human exposure Sappinia. Acanthamoeba infections may present as granulomatous occurs through contact with intact or disrupted nasal mucosa, amoebic encephalitis (GAE), disseminated disease (e.g. cutaneous, commonly through recreational or nasal ablution practices13 and sinus or pulmonary infection) or keratitis, with Balamuthia man- contaminated drinking water has been implicated as a source of drillaris causing similar cutaneous infections and GAE. Naegleria infection in some cases14. Australia has featured prominently in fowleri is responsible for the rapidly progressive primary amoebic the history of Naegleria fowleri infection, with cases described in meningoencephalitis (PAM). In addition, a single case of human Queensland, New South Wales and Western Australia following the central nervous system (CNS) infection with Sappinia pedata has first description of the disease in 1965 by two South Australian been reported1, along with isolated cases of corneal infection with pathologists, Fowler and Carter15,16. This discovery was related to Vahlkampfia spp., Hartmannella spp. and Paravahlkamfia spp.2. an outbreak of 20 cases, attributed to a contaminated overland This review aims to provide an overview of human disease caused water pipeline which reached optimal temperatures for Naegleria by the three most common genera involved, Acanthamoeba, proliferation during the summer months. Cases continued from Naegleria and Balamuthia, including their laboratory diagnosis. 1947 to 1972 when public health measures including adequate chlorination were applied17. Several cases in Western Australia have Epidemiology also been associated with overland water pipelines, and Australian Pathogenic FLA are found worldwide and serological studies suggest drinking water guidelines suggest a Naegleria monitoring and – human exposure is common3 5. Three cases of Acanthamoeba GAE response protocol for water supplies that seasonally exceed 308C, – have been described in Australia6 8. Acanthamoeba sp tolerate a or 258C continually18. Today, despite significant advancement in

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knowledge regarding risk reduction measures, the incidence of with changes in taste or smell, followed by fever, nausea, vomiting, infection with Naegleria fowleri appears to be increasing world- photophobia and headache. The disease is fulminant, with rapid wide, with factors such as global warming, substandard water progression to coma and death. management and sanitation, and changing recreational practices Balamuthia mandrillaris likely involved13. Balamuthia mandrillaris causes GAE in immunocompromised Balmuthia mandrillaris is most commonly found in soil, though and immunocompetent individuals. The onset of meningoenceph- 4 it may also be recovered from water samples . Infection tends to alitis is often subacute or chronic, with symptoms developing over a occur in immunocompetent individuals, most commonly children, period of 2 weeks to 2 years27. It also has the propensity to cause probably through inhalation or nasal or cutaneous inoculation. cutaneous lesions that may precede CNS involvement and are 19 Transmission via organ transplantation has also been described . similar in appearance to those of Acanthamoeba sp. These lesions 20–24 Six human cases have been described in Australia (Western appear as poorly defined plaques and may be single or with Australia, Tasmania, Victoria and Queensland), with another being bordering satellite lesions4. They often involve the central face and 25 described in a Victorian dog . appear to be more common in South America4. Cutaneous disease generally progresses to CNS involvement; however, it may resolve Clinical manifestations with therapy4. Acanthamoeba sp. Laboratory diagnosis The predominant clinical manifestations of Acanthamoeba infection Diagnosis of FLA infection, particularly systemic disease, is chal- are disseminated disease (e.g. cutaneous, nasopharyngeal or pulmo- lenging: it may masquerade as bacterial or viral infection, exposure nary infection), GAE and keratitis. With the exception of rare case events may not be apparent and specialised diagnostic testing reports, GAE and disseminated disease occur in the immunocom- availability is limited. Unfortunately as a result, many CNS infections promised or debilitated. The incubation period is unknown, but are diagnosed post-mortem. Successful early diagnosis depends on thought to be weeks to months in duration. Cutaneous infection appropriate clinical suspicion and collection of suitable diagnostic usually begins as fibronodular lesions, which progress to non-healing material, usually tissue or CSF. ulcerated lesions over time10. While GAE may be fatal within days of symptom onset, it generally assumes a more chronic course, with Microscopy slow progression over weeks to months. Clinical features of GAE are myriad and include fever, symptoms of meningism, personality CSF samples in cases of PAM appear purulent, with no bacteria and mental status change and, later, focal neurological deficits, evident on gram stain, a polymorphonuclear pleocytosis, elevated coma and death. Imaging of the brain may show single or multiple protein and decreased glucose. Wet preparations may show motile space occupying lesions which can be ring enhancing. Naegleria fowleri trophozoites (Figure 1a), as there are usually large numbers of organisms in the CSF. These findings contrast with Acanthamoeba keratitis is usually a disease of immunocompetent those of GAE caused by Acanthamoeba or Balamuthia. While CSF patients, with the strongest risk factor being contact lens use and from cases of GAE also demonstrates elevated protein and lowered poor lens hygiene. The disease is usually unilateral, with symptoms glucose, these changes are more modest, and a mononuclear, rather including lacrimation, pain, photophobia and foreign body sensa- than polymorphonuclear inflammatory response is seen. Further- tion. Signs include a typical corneal ring infiltrate, stromal infiltrates, more, Acanthamoeba or Balamuthia trophozoites are not typically epitheliopathy and hypopyon26. In the absence of effective therapy, seen in CSF preparations. it may progress to corneal perforation and loss of vision. The clinical diagnosis of AK can be difficult, as lesions may resemble bacterial Trophozoites from all pathogenic FLA species can be difficult to or fungal disease, or the dendritic ulcer of HSV infection26. Further- differentiate from host inflammatory cells, especially in stained more the clinical course may be characterised by periods of tem- tissue sections. The nuclear characteristics of amoeba can be helpful porary remission, leading to false impressions of response to in differentiating these parasites from host cells, with Naegleria antibacterial or viral agents2. fowleri possessing a nucleus with a large, round, central nucleolus and Acanthamoeba and Balamuthia (Figure 1b) a rounded Naegleria fowleri nucleus with large, central nucleolus forming a halo. Polyclonal Naegleria fowleri causes primary amoebic meningoencephalitis. and monoclonal antibodies, with a secondary detecting fluorescent Symptoms generally occur 2–5 days after exposure and may begin anti-IgG antibody (such as FITC), may be used to identify and

MICROBIOLOGY AUSTRALIA * MARCH 2016 21 Under the Microscope

differentiate each of these amoebae in tissue specimens28 (as can surface in 1–2 days when incubated at 378C and their presence can molecular methods), though availability is limited to the CDC, be confirmed by examination of the plate with a plate microscope, Atlanta, USA. or by performing microscopy of a wet mount from the agar plate. Balamuthia do not appear to use bacteria as a food source and In Acanthamoeba keratitis, a diagnosis may be made by demon- therefore cannot be cultivated in the same fashion32. They may be strating trophozoites and/or cysts in corneal samples. It is possible successfully cultured using axenic and tissue culture methods32, to directly identify Acanthamoeba trophozoites within the cornea however with generation times of around 25 h, culture is a lengthy using confocal microscopy and in experienced hands this technique process and not part of routine diagnostic testing. is sensitive and specific29. Trophozoites and cysts may be revealed by staining the smear with H&E or Giemsa, while cysts are also readily identified using PAS and fluorescent stains, such as calcofluor Nucleic acid testing white and acridine orange30. On occasion, non-specific fluores- The use of molecular testing to diagnose and confirm infections cence or binding to fungi in mixed infections can lead to diagnostic with FLA has transformed diagnostics in this area. It allows more errors31, especially when used by inexperienced microscopists. rapid diagnosis, with greater sensitivity than other methods and reduces the requirement for specialist, experienced staff to discern Culture subtle microscopic features. Most molecular assays use ribosomal Samples intended for amoebic culture should be kept at room genes, such as the 18S rRNA or ITS repeat regions, as targets for temperature and processed as quickly as possible. Freezing should PCR. However, as requests are infrequent, these tests are generally be avoided, particularly for samples where Naegleria is suspected only offered by reference or research laboratories. Of particular (the cyst stage is more fragile), as this compromises organism note is a multiplex PCR, described by Qvarnstrom et al.33. This viability. Naegleria fowleri and Acanthamoeba sp. (Figure 1c) can assay uses 18S rRNA primer/probe sets to accurately identify be readily cultured using non-nutrient media containing live or Acanthamoeba to the genus level and Naegleria fowleri and killed non-mucoid bacteria (usually E. coli or Enterobacter sp)asa Balamuthia mandrillaris species. The sensitivity is reported at food source32. Acanthamoeba and Naegleria will cover the agar one amoeba per sample. Sullivan Nicolaides Pathology instituted

(a)(b)(c)

Figure 1. (a) Naegleria trophozoite in CSF wet prep (x400). (b) Balamuthia trophozoite in brain tissue (haematoxylin and eosin). (c) Acanthamoeba cysts in culture, wet prep (x400).

Table 1. Multiplex Free Living Amoebae PCR33 at Sullivan Nicolaides Pathology, November 2011 to July 2015A. Organism Eye CNS Skin Sinus Total Acanthamoeba spp 5 1 81

Balamuthia 163 mandrillaris

Naegleria fowleri 263

Total tests 52 24 4 1 81

ADuplicates excluded (same patient and site, within 2 months). CNS, central nervous system.

22 MICROBIOLOGY AUSTRALIA * MARCH 2016 Under the Microscope

this test in 2010 and our experience is summarised in Acanthamoeba keratitis Table 1. As a significant number of ocular specimens have been Like disseminated disease, success in treatment is dependent on tested against all three targets, a large number of negatives for early diagnosis and institution of therapy. Fortunately, success Naegleria fowleri and Balamuthia mandrillaris are expected. It is rates in treating Acanthamoebic keratitis are more promising, with also notable that we have recently reported a probable false negative cure rates in the literature generally greater than 75–85%38,39. Acanthamoeba result. The referred CSF specimen tested negative Topical chlorhexidine and polyhexamethylenebiguanide (PHMB) at our laboratory, but positive at the CDC (using the same are effective against trophozoites and cysts and form the mainstay multiplex PCR). It is possible the small volume of CSF received at of therapy. These are usually used in combination with diamidine, our laboratory contributed to this result (only 100 mL was received, propamidine or hexamidine, though other agents including keto- where 200 mL is the standard volume for extraction). conazole, itraconazole, voriconazole and topical imidazoles have been used. Surgical intervention, including enucleation, is some- Treatment and prognosis times required in severe cases40. Primary amoebic meningitis

There have been few survivors of primary amoebic meningitis References and the factors that have resulted in successful therapy are poorly 1. Qvarnstrom, Y. et al. (2009) Molecular confirmation of Sappinia pedata as a defined, though early diagnosis and institution of therapy appears causative agent of amoebic encephalitis. J. Infect. Dis. 199, 1139–1142. critical. The treatment of choice is the antifungal , doi:10.1086/597473 2. Mandell, G.L. et al. (2015) Mandell, Douglas, and Bennett’s principles and which is often used both intravenously and intrathecally. Other practice of infectious diseases. Philadelphia, PA: Churchill Livingstone/Elsevier. agents used in survivors include , sulfisoxazole, flucon- 3. Chappell, C.L. et al. (2001) Standardized method of measuring Acanthamoeba azole, miconazole and rifampicin. antibodies in sera from healthy human subjects. Clin. Diagn. Lab. Immunol. 8, 724–730. Granulomatous amoebic encephalitis 4. Bravo, F.G. and Seas, C. (2012) Balamuthia mandrillaris amoebic encephalitis: an emerging parasitic infection. Curr. Infect. Dis. Rep. 14,391–396. doi:10.1007/ Acanthamoeba s11908-012-0266-4 fi fi 5. Marciano-Cabral, F. et al. (1987) Speci city of antibodies from human sera for Suboptimal ef cacy of antimicrobial agents, the high morbidity of Naegleria species. J. Clin. Microbiol. 25, 692–697. patients affected and tendency to late diagnosis contribute to a 6. Carter, R.F. et al. (1981) A fatal case of meningoencephalitis due to a free-living poor prognosis in Acanthamoeba GAE, with a mortality rate of amoeba of uncertain identity–probably Acanthamoeba Sp. Pathology 13,51–68. doi:10.3109/00313028109086829 >90%34. In the few reported survivors of GAE or cutaneous 7. Harwood, C.R. et al. (1988) Isolation of Acanthamoeba from a cerebral abscess. infection, most have received combination therapy. The agents Med. J. Aust. 148, 47. used have included trimethoprim-sulfamethoxazole, flucytosine, 8. Azzam, R. et al. (2015) Acanthamoeba encephalitis: isolation of genotype T1 in mycobacterial liquid culture medium. J. Clin. Microbiol. 53,735–739. fl sulfadiazine, penicillin G, chloramphenicol, , ucona- doi:10.1128/JCM.02887-14 zole and itraconazole. More recently, it appears that the inclusion 9. Trabelsi, H. et al. (2012) Pathogenic free-living amoebae: epidemiology – of miltefosine in combination regimens may result in improved and clinical review. Pathol. Biol. (Paris) 60,399405. doi:10.1016/j.patbio. 2012.03.002 35 survival . 10. Marciano-Cabral, F. and Cabral, G. (2003) Acanthamoeba spp. as agents of disease in humans. Clin. Microbiol. Rev. 16,273–307. doi:10.1128/CMR.16. 2.273-307.2003 Balamuthia 11. Siddiqui, R. and Khan, N.A. (2012) Biology and pathogenesis of Acanthamoeba. Early recognition of cutaneous disease is critical to allow early Parasit. Vectors 5, 6. doi:10.1186/1756-3305-5-6 12. Kemble, S.K. et al. (2012) Fatal Naegleria fowleri infection acquired in therapy and prevent progression to CNS infection. Unfortunately, Minnesota: possible expanded range of a deadly thermophilic organism. Clin. the overall prognosis in Balamuthia CNS infection is extremely Infect. Dis. 54, 805–809. doi:10.1093/cid/cir961 poor. However, there are several case reports of survival in the 13. Siddiqui, R. and Khan, N.A. (2014) Primary amoebic meningoencephalitis caused by Naegleria fowleri: an old enemy presenting new challenges. PLoS 36 24 literature , including an Australian case . These cases have re- Negl. Trop. Dis. 8, e3017. doi:10.1371/journal.pntd.0003017 ceived varied combination therapy regimens, with agents including 14. Cooter, R. (2002) The history of the discovery of primary amoebic meningoen- cephalitis. Aust. Fam. Physician 31, 399–400. pentamidine, flucytosine, fluconazole, macrolides, sulfadiazine, 15. Fowler, M. and Carter, R.F. (1965) Acute pyogenic meningitis probably due to miltefosine, thioridazine, amphotericin B, albendazole and trimeth- Acanthamoeba sp.: a preliminary report. BMJ 2,734–742. doi:10.1136/bmj.2. oprim-sulfamethoxazole. Miltefosine, in particular, has demonstrat- 5464.734-a 16. Carter, R.F. (1968) Primary amoebic meningo-encephalitis: clinical, pathological ed amoebicidal activity in vitro37 and its inclusion in combination and epidemiological features of six fatal cases. J. Pathol. Bacteriol. 96,1–25. 35 regimens may offer a survival advantage . doi:10.1002/path.1700960102

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17. Dorsch, M.M. et al. (1983) The epidemiology and control of primary amoebic 32. Schuster, F.L. (2002) Cultivation of pathogenic and opportunistic free-living meningoencephalitis with particular reference to South Australia. Trans. R. Soc. amebas. Clin. Microbiol. Rev. 15, 342–354. doi:10.1128/CMR.15.3.342-354.2002 – Trop. Med. Hyg. 77, 372 377. doi:10.1016/0035-9203(83)90167-0 33. Qvarnstrom, Y. et al. (2006) Multiplex real-time PCR assay for simultaneous 18. National Health and Medical Research Council Australia (2011) Natural detection of Acanthamoeba spp., Balamuthia mandrillaris,andNaegleria Resource Management Ministerial C. Australian drinking water guidelines 2011: fowleri. J. Clin. Microbiol. 44, 3589–3595. doi:10.1128/JCM.00875-06 national water quality management strategy. Canberra: National Health and 34. Barratt, J.L.N. et al. (2010) Importance of nonenteric protozoan infections in Medical Research Council. immunocompromised people. Clin. Microbiol. Rev. 23,795–836. doi:10.1128/ 19. Schlessinger, S. et al. (2010) Balamuthia mandrillaris transmitted through CMR.00001-10 – organ transplantation Mississippi, 2009. Morbidity and Mortality Weekly 35. Cope, J. et al. (2013) Increased patient survival: miltefosine for treatment of – Report 59, 1165 1170. free-living ameba infections caused by Acanthamoeba and Balamuthia 20. Carter, R.F. et al. (1981) A fatal case of meningoencephalitis due to a free-living [Poster]. Council of State and Territorial Epidemiologists’ Annual Conference. amoeba of uncertain identity–probably Acanthamoeba sp. Pathology 13,51–68. Pasadena, California. doi:10.3109/00313028109086829 36. Deetz, T.R. et al. (2003) Successful treatment of Balamuthia amoebic 21. Reed, R.P. et al. (1997) Fatal granulomatous amoebic encephalitis caused by encephalitis: presentation of 2 cases. Clin. Infect. Dis. 37, 1304–1312. Balamuthia mandrillaris. Med. J. Aust. 167,82–84. doi:10.1086/379020 22. Hill, C.P. et al. (2011) Balamuthia amebic meningoencephalitis and mycotic 37. Schuster, F.L. et al. (2006) In-vitro activity of miltefosine and voriconazole on aneurysms in an infant. Pediatr. Neurol. 45,45–48. doi:10.1016/j.pediatrneurol. clinical isolates of free-living amebas: Balamuthia mandrillaris, Acanthamoeba 2011.05.003 spp., and Naegleria fowleri. J. Eukaryot. Microbiol. 53,121–126. doi:10.1111/ 23. Doyle, J.S. et al. (2011) Balamuthia mandrillaris successfully j.1550-7408.2005.00082.x treated with complete surgical excision and prolonged combination antimicrobial 38. Dart, J. K. et al. (2009) Acanthamoeba keratitis: diagnosis and treatment update therapy. J. Neurosurg. 114,458–462. doi:10.3171/2010.10.JNS10677 2009. Am. J. Ophthalmol. 148, 487–99e2. doi:10.1016/j.ajo.2009.06.009 24. Moriarty, P. et al. (2014) Balamuthia mandrillaris encephalitis: survival of a 39. Butler, T.K.H. et al. (2005) Six-year review of Acanthamoeba keratitis in New child with severe meningoencephalitis and review of the literature. J. Pediatric South Wales, Australia: 1997–2002. Clin. Experiment. Ophthalmol. 33,41–46. Infect. Dis. Soc. 3,e4–e9. doi:10.1093/jpids/pit033 doi:10.1111/j.1442-9071.2004.00911.x 25. Finnin, P.J. et al. (2007) Multifocal Balamuthia mandrillaris infection in a dog 40. Vemuganti, G.K. et al. (2005) Granulomatous inflammation in Acanthamoeba in Australia. Parasitol. Res. 100,423–426. doi:10.1007/s00436-006-0302-0 keratitis: an immunohistochemical study of five cases and review of literature. – 26. Patel, D.V. and McGhee, C.N. (2009) Acanthamoeba keratitis: a comprehensive Indian J. Med. Microbiol. 23, 231 238. photographic reference of common and uncommon signs. Clin. Experiment. Ophthalmol. 37, 232–238. doi:10.1111/j.1442-9071.2008.01913.x 27. Visvesvara, G.S. et al. (2007) Pathogenic and opportunistic free-living Biographies amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol. Med. Microbiol. 50,1–26. doi:10.1111/ Dr Evan Bursle, BSc, MBBS, is a microbiology and infectious j.1574-695X.2007.00232.x diseases registrar based at Sullivan Nicolaides Pathology. Although 28. da Rocha-Azevedo, B, Tanowitz, HB and Marciano-Cabral, F (2009) Diagnosis his microbiological tastes are broad and still being refined, he has a of infections caused by pathogenic free-living amoebae. Interdisciplinary perspectives on infectious diseases 2009, 251406. doi:10.1155/2009/251406 keen interest in parasitology. 29. Tu, E.Y. et al. (2008) The relative value of confocal microscopy and superficial corneal scrapings in the diagnosis of Acanthamoeba keratitis. Cornea 27, Dr Jenny Robson, MBBS (Hons I); FRACP, FRCPA, FACTM, is an 764–772. doi:10.1097/ICO.0b013e31816f27bf infectious disease physician and microbiologist who has worked for 30. Hahn, T.-W. et al. (1998) Acridine orange staining for rapid diagnosis of Acantha- moeba keratitis. Jpn. J. Ophthalmol. 42, 108–114. doi:10.1016/S0021-5155(97) the past 26 years at Sullivan Nicolaides Pathology. She has a broad 00127-5 range of interests, which includes travel and tropical medicine. 31. Grossniklaus, H.E. et al. (2003) Evaluation of hematoxylin and eosin and special stains for the detection of Acanthamoeba keratitis in penetrating keratoplasties. Am. J. Ophthalmol. 136, 520–526. doi:10.1016/S0002-9394(03)00322-2

Future issues of Microbiology Australia May 2016: Education to enhance microbiology graduate employability Guest Editor: Danilla Grando September 2016: Diseases of Aquaculture Guest Editor: Nicky Buller November 2016: Microbiology of Travel Guest Editor: Ipek Kurtbo¨ke; joint issue with the Society for General Microbiology March 2017: Bat-associated diseases Guest Editor: Glenn Marsh

24 MICROBIOLOGY AUSTRALIA * MARCH 2016 Under the Microscope

Tapeworm cysts in the brain: can we prevent it happening?

that people from poor countries can travel anywhere with their intestinal residents and deliver their tapeworm eggs to you or me, just as happened in the New York community referred to above. No point turning vego. Consider this: I have a worm and I am Marshall Lightowlers not particularly hygienic when I go to the toilet. I make your salad; – Facultyof Veterinaryand Agricultural bingo see you in the neurology clinic. Sciences The University of Melbourne T. solium is not an obscure parasite. The Food and Agriculture 250 Princes Highway Werribee, Vic. 3030, Australia Organization of the United Nations considers it to be the most Email: [email protected] important foodborne parasitic infection from a global perspective2. T. solium is the most frequent preventable cause of seizure dis- orders, being associated with 29% of people with epilepsy3. The Imagine the consternation; you are a member of an orthodox World Health Organization list T. solium as one of 16 Neglected Jewish family and you and another family member are Tropical Diseases, and is actively promoting efforts to reduce the ’ 4 diagnosed with larvae of a pork tapeworm in your brain. parasite s transmission . You have recurrent seizures as a result. Ridiculous? Not For those living in poor countries in which T. solium is endemic, for members of a Jewish community in New York where a help is at hand from, of all places, Australia. I say ‘of all places’ Mexican domestic worker harbouring a Taenia solium tape- because the parasite is not, and has never been known to be, worm had apparently contaminated the family’s food with endemic here. Nevertheless, a research program at the University eggs from her tapeworm1. of Melbourne with an original genesis way back in the 1970s led T. solium is a cestode parasite that is transmitted between pigs and eventually to the development of both the first effective non-living people. Pigs may harbour the parasite’s larval stage in their muscles vaccine against a eukaryotic parasite and eventually also to a vaccine (Figure 1). If we accidentally eat one of these larvae in poorly cooked that can stop pigs being infected with T. solium. The vaccine uses a pig meat, the adult tapeworm will develop in our . recombinant antigen known as TSOL18. Several independent ex- Humans are the only definitive host (the host in which the parasite perimental trials of TSOL18 have confirmed that it is extraordinarily 5 undergoes sexual reproduction) for this species of tapeworm. A effective .Afield trial of the vaccine was undertaken in which pairs person with the tapeworm releases the parasite’s eggs in their of young piglets were distributed to families living in a T. solium 6 faeces, and the lifecycle is completed if foods contaminated with endemic region of north-east Cameroon . One animal from each these eggs, or indeed the faeces themselves, are consumed by a pig. pair was vaccinated and one acted as a control. When the animals The disease is fully transmitted only where pigs roam freely and were of normal eating-age (~12 months), they were recovered humans defecate in areas where the pigs are free roaming. This does from the farmers and assessed for T. solium infection. About 20% not happen in New York. However, it does happen across large areas of the controls were infected but not a single parasite was found in of central and South America, Africa and east and south-east Asia. any of the 110 vaccinated animals.

In former times T. solium and the human brain disease that the So far, so good. However the field trial involved a single cohort of parasite causes, neurocysticercosis, were endemic throughout animals that was vaccinated. In a real life situation, new disease Europe and other parts of the current-day First World; however, susceptible piglets are born into the community more-or-less improved public sanitation and hygienic standards for raising pigs every day. Difficulties arise when we try to work out a feasible have seen the disease in pigs eliminated without any efforts directed and sustainable program to deliver vaccination regularly enough specifically to the disease. Likely the same will happen, in time, to prevent pigs becoming infected on an on-going basis. The through economic development in those areas of the world where ‘standard’ vaccination protocol calls for two immunizations the disease remains endemic today. Unfortunately, this is unlikely to about a month apart. It is difficult to give any veterinary care to occur in our lifetimes. Meanwhile, those living in the T. solium pigs in the communities where T. solium is transmitted; however endemic areas of the world continue to suffer epilepsy and death having to deliver two vaccines a month apart would be close to due to the presence of T. solium cysts in their brain. Keep in mind impossible.

MICROBIOLOGY AUSTRALIA * MARCH 2016 10.1071/MA16010 25 Under the Microscope

(a) contribute to reducing the incidence of neurocysticercosis in peo- ple. One major difficulty with implementation of pig vaccination to prevent transmission of T. solium is that the owners of the animals often have little incentive to undertake control measures because the infection does not often directly cause illness or death in pigs. In the future, combination vaccines that include TSOL18 and also antigens that can provide protection against pig deaths caused by Classical Swine Fever (CSF) in the Americas, or African Swine Fever (ASF) in African countries, would improve the acceptability of pig vaccination by providing an economic incentive for the animal owners to vaccinate. While the potential for a combination with CSF is something that can be explored immediately because com- mercial vaccines already exist, there is yet to be a commercial vaccine (b) for ASF.

As for the Jewish community in New York who use domestic staff sourced from T. solium endemic countries, and of course all the people who live permanently in those endemic countries, the risk of exposure to T. solium remains. Hopefully we will be able to overcome the practical difficulties around working with pigs in T. solium endemic areas so that implementation of pig vaccination can reduce T. solium transmission and decrease the incidence of human neurocysticercosis as a result.

References 1. Schantz, P.M. et al. (1992) Neurocysticercosis in an Orthodox Jewish community in Figure 1. (a) Taenia solium cysts (cysticerci) in the tongue of a naturally New York City. N. Engl. J. Med. 327, 692–695. doi:10.1056/NEJM199209033271004 infected pig (Photo: M. Donadeu). (b) Cysticerci in the muscles of a fi heavily infected pig. 2. Robertson, L.J. et al. (2013) Have foodborne parasites nally become a global concern? Trends Parasitol. 29, 101–103. doi:10.1016/j.pt.2012.12.004

’ 3. Ndimubanzi, P.C. et al. (2010) A systematic review of the frequency of neurocy- Using knowledge about the parasite s development in pigs and ticercosis with a focus on people with epilepsy. PLoS Negl. Trop. Dis. 4, e870. information from the successful Cameroonian field trial, various doi:10.1371/journal.pntd.0000870 scenarios involving vaccination in pigs were compared for their 4. Organization, W.H. (2015) Investing to overcome the global impact of neglected tropical diseases. Third WHO report on neglected tropical diseases. WHO/HTM/ predicted effectiveness to control transmission7. One T. solium NTD/2015.1. scenario that appeared relatively practical was to vaccinate at 5. Lightowlers, M.W. (2010) Eradication of Taenia solium cysticercosis: a role for 4-monthly intervals. While the scenario looked good theoretically, vaccination of pigs. Int. J. Parasitol. 40, 1183–1192. doi:10.1016/j.ijpara.2010.05. it could not be recommended because we had no information 001 6. Assana, E. et al. (2010) Elimination of Taenia solium transmission to pigs in a field about whether the vaccine would raise a protective response if trial of the TSOL18 vaccine in Cameroon. Int. J. Parasitol. 40,515–519. doi:10.1016/ the interval between primary and secondary injections was longer j.ijpara.2010.01.006 than 4 weeks. 7. Lightowlers, M.W. (2013) Control of Taenia solium taeniasis/cysticercosis: past practices and new possibilities. Parasitology 140, 1566–1577. doi:10.1017/S003 Recently we have completed an experiment in which pigs received 1182013001005 theirsecondaryimmunizationwithTSOL18at4,8,12,16or20weeks 8. Lightowlers, M.W. et al. (2016) Anamnestic responses in pigs to the Taenia solium TSOL18 vaccine and implications for control strategies. Parasitology, in press. after the first injection8. The results were very promising. Antibody responses to the vaccine generally increased beyond the ‘standard’ 4-week interval. Responses seen in the animals vaccinated at a Biography 12 week interval were the best, and field evaluation of T. solium Marshall Lightowlers has been a full-time research scientist interventions are about to begin in several endemic regions of Africa supported by medical research funding for more-or-less all of his that will involve vaccinations at 3 or 4 monthly intervals. working life. He currently holds appointments as Laureate Professor Despite the solid progress that has been made so far, much remains at The University of Melbourne’s Faculty of Veterinary and Agricul- to be achieved before we would be likely to see pig vaccination tural Sciences, and Principal Research Fellow with the NHMRC.

26 MICROBIOLOGY AUSTRALIA * MARCH 2016 Under the Microscope

Seafood-borne parasitic diseases in Australia: how much do we know about them?

Infection in humans (zoonosis) Anisakidosis results from accidental infection with one or more larvae of species of certain genera of anisakids, including spp, Contracaecum spp and Pseudoterranova spp. Infection with Shokoofeh Shamsi larvae of Anisakis and Pseudoterranova is of common occurrence, School of Animal and Veterinary whereas infection with other genera, such as Contracaecum has Sciences Charles Sturt University been reported less frequently. Humans usually become infected Wagga Wagga, NSW 2650, Australia with these parasites after eating raw, undercooked or improperly Tel: +61 2 6933 4887 fi Email: [email protected] processed sh or seafood (Figure 1). Clinically, several different types of human anisakidosis have been described based on the location of the parasite. This includes gastro-intestinal, visceral, 4 Fish are host to many parasites, some of which can cause oropharyngeal and transient luminal anisakidosis . The first two disease in humans. With the increase in cultural and culinary types are associated with severe symptoms such as vomiting, severe diversity and the increased popularity of eating raw or pain in lower abdomen and fever. slightly cooked seafood dishes in Australia it is speculated In recent years, it has been recognised that an allergic response can that seafood-borne parasitic infections in Australian consu- occur in humans due to live anisakids or food in which worms have mers may rise. Seafood-borne zoonotic parasites are recog- been killed by cooking or pasteurisation5. Other allergic disorders, nised as a significant public health concern worldwide. such as chronic urticaria may also result from parasitism6. There is In Australia there are few reports of infection in humans in anecdotal evidence in other countries (e.g. Spain) that some allergic the medical literature. Australian Government enforcement reactions to seafood are indeed allergic reaction to anisakids in agencies rate the risk of seafood-borne zoonosis as low; seafood. however, the prevalence of seafood-borne zoonoses may be under-reported in Australia due to misdiagnosis. Although food safety regulations and import controls for seafood The Australian scenario in Australia are strict, the focus is more on the control of In Australia our knowledge about these important parasites is poor. food-borne bacterial, viral and chemical contaminant rela- Although anisakid nematodes are commonly found in Australian ted illnesses rather than parasitic diseases. marine fish (Figure 2), such as mackerel, flathead, snapper and whiting2,7,8, all forms of seafood are available and popular in Aus- Increasing demand for raw and exotic seafood has significantly tralia and 40% of fish consumed raw in Australia have been consid- expanded the geographical and demographic limits of fish-borne ered as infected with Anisakis at the time of processing for trade1, parasitic infections. Medical literature on the regular consumption there are only a handful of documented confirmed cases acquired in of seafood is plentiful. It is heavily promoted for prolonging life, Australia. aiding in childhood development, increasing brain stimulation and even to help our pets. However, there is an increasing risk of Case 1: There has been only one documented confirmed case of contracting parasitic zoonoses from consuming raw and under- infection with anisakid nematodes acquired in Australia9. This case cooked seafood, which should be of concern in Australia. Zoonotic involved a 41-year-old South Australian woman of Tongan descent parasites are common in Australian seafood, and can pose a major who became ill after eating raw mackerel that had been caught health risk to people who consume seafood or work in a fishing locally. She was hospitalised with severe gastrointestinal pain, industry1. Among zoonotic parasites in seafood, Anisakid nema- diarrhoea and vomiting that progressively worsened for 3 weeks. todes are of the greatest significance due to their high prevalence in The diagnosis only occurred after a worm was passed in her faeces. wild caught fish2 and also due to the severity of the disease they The initial presumptive identification of the larva was an intestinal cause, which is known as anisakidosis. Other nematodes such as nematode, possibly a species of the or Ascaris and Angiostrongylus as well as platyhelminths such as genera. However, on further detailed microscopic examination, Diphylobothrium, Clonorchis and Paragonimus, can occasionally the larva was identified as a species of Contracaecum. Had the cause seafood-borne zoonotic infections3. worm not been found, or properly examined by a taxonomist,

MICROBIOLOGY AUSTRALIA * MARCH 2016 10.1071/MA16015 27 Under the Microscope

Definitive hosts

1st intermediate hosts

2nd intermediate/ paratenic hosts

Figure 1. General life cycle of anisakid nematodes. Adult nematodes inhabit stomach of definitive hosts, including marine mammals, piscivorous birds and large predatory fish in which they reproduce and lay eggs. Eggs pass through faeces to the water. Embryonated eggs or hatched larvae are ingested by first intermediate hosts, including a wide range of aquatic invertebrates. Larvae develop further when infected first intermediate hosts are predated upon by second intermediate or paratenic hosts, including a broad range of fish species. The larval stages of Anisakids are not host specific and a wide range of fish species can become infected as intermediate or paratenic hosts. This allows the parasite to be widely distributed by passing through several fish species and infect a wide range of marine mammals and fish-eating birds where they complete their life cycle and become adults. Thus infection with anisakids is not limited to one simple food chain but rather a wide network of species, increasing the impact and importance of these parasites. As shown in the picture humans become infected by consuming infected seafood (fish and invertebrates such as crustaceans).

misdiagnosis would have occurred. It is very rare for anisakid larvae to be found after passing through the gastro-intestinal tract as was the case in this Australian patient.

Since then the author has come across several suspected cases of anisakidosis, mostly among Australian travellers after returning home (unpublished cases). The cases could not be confirmed due to the lack of knowledge, clinical suspicion and a reliable diagnostic technique in Australia. In many countries, the disease is usually diagnosed by endoscopy, radiography, or surgery if the worm has embedded within the gastro-intestinal tract in patients with symp- toms and a history of consuming raw seafood. Serological tests to detect parasite allergens have also been recently developed10. However, in Australia there is no standard test available for diagnosis Figure 2. Anisakid larvae (circled) on the surface of the internal organs of these important parasites. General health practitioners are not of a fish caught in South Australia. If fish is not gutted immediately aware of the presence and high abundance of these parasites in after being caught these parasites migrate toward flesh of the fish (photograph by Shokoofeh Shamsi). Australian fish and diagnostic laboratory staff are not trained to

28 MICROBIOLOGY AUSTRALIA * MARCH 2016 Under the Microscope

identify these worms. As a result, the full extent of hidden cases of of consumers must be protected. Therefore further research is the disease in Australia remains unknown. required to fill in the current knowledge gaps of the biology and ecology of these parasites and the risk they pose to consumers and Case 2: Another confirmed case report of fish-borne parasitic workers. disease in humans in Australia occurred in 201111. In this case, a couple became infected with Gnathostoma species after consump- tion of ‘Black Bream’ (possibly Acanthopagrus berda or Hephaes- References tus jenkinsi), which they caught in the Calder River in Western 1. Anantanawat, S. et al. (2012) A semi-quantitative risk assessment of harmful parasites in Australian finfish. South Australian Research & Development Institute, Australia. They presented with recurring skin swellings. The fish 2012. fi had been cooked on a camp re, but the cooking time was unclear. 2. Shamsi, S. et al. (2011) Occurrence and abundance of anisakid nematode larvae in The diagnosis was based on eosinophilia and a positive serology. five species of fish from southern Australian waters. Parasitol. Res. 108,927–934. Humans become infected accidentally by consuming third-stage doi:10.1007/s00436-010-2134-1 3. Butt, A.A. et al. (2004) Infections related to the ingestion of seafood. Part II: larvae. Gnathostoma larvae are unable to mature inside the human parasitic infections and food safety. Lancet Infect. Dis. 4,294–300. doi:10.1016/ body, and instead migrate through visceral and cutaneous tissues, S1473-3099(04)01005-9 causing a variety of generalised, non-specific symptoms. Gnathos- 4. Smith, J.W. (1999) Ascaridoid nematodes and pathology of the alimentary tract tomiasis is generally endemic in parts of the world where seafood is and its associated organs in vertebrates, including man: a literature review. Helminthological Abstracts 68,49–96. consumed raw, such as in South-East Asia and Japan, but also more 5. Audicana, M.T. and Kennedy, M.W. (2008) Anisakis simplex: from obscure recently in Latin America, India and Africa, and in travellers returning infectious worm to inducer of immune hypersensitivity. Clin. Microbiol. Rev. from these areas12. 21, 360–379. doi:10.1128/CMR.00012-07 6. Daschner, A. and Pascual, C.Y. (2005) Anisakis simplex: sensitization and clinical Another aspect of Australia’s seafood consumption that must be allergy. Curr. Opin. Allergy Clin. Immunol. 5,281–285. doi:10.1097/01.all.0000 considered is the large volume of imported seafood that Australians 168795.12701.fd fi consume. The most important sources of seafood products are 7. Doupe, R.G. et al. (2003) Larval anisakid infections of some tropical sh species from north-west Australia. J. Helminthol. 77,363–365. Thailand, New Zealand, Vietnam and China (frdc.com.au/knowl- 8. Shamsi, S. et al. (2011) Mutation scanning-coupled sequencing of nuclear ribo- edge/Factsheets/Factsheet_Imported_Seafood_in_Australia.pdf). somal DNA spacers (as a taxonomic tool) for the specific identification of different The literature describes several zoonotic parasites affecting fish Contracaecum (Nematoda: Anisakidae) larval types. Mol. Cell. Probes 25,13–18. endemic to these countries13. CSIRO conducted a comprehensive 9. Shamsi, S. and Butcher, A.R. (2011) First report of human anisakidosis in Australia. Med. J. Aust. 194, 199–200. review of AQIS’s imported seafood testing protocols14 and con- 10. García, M. et al. (1997) The use of IgE immunoblotting as a diagnostic tool in cluded that imported seafood does not pose any greater health risk Anisakis simplex allergy. J. Allergy Clin. Immunol. 99,497–501. doi:10.1016/ to the consumer than locally produced seafood. Nevertheless S0091-6749(97)70076-9 proper precautions when preparing seafood in order to reduce 11. Jeremiah, C.J. et al. (2011) in remote northern Western Australia: the first confirmed cases acquired in Australia. Med. J. Aust. 195,42–44. the risk of illness or disease has been recommended. However, 12. Herman, J.S. and Chiodini, P.L. (2009) Gnathostomiasis, another emerging recommendations to eliminate or reduce parasites have not been imported disease. Clin. Microbiol. Rev. 22,484–492. doi:10.1128/CMR.00003-09 dealt with in details. 13. Chai, J.-Y. et al. (2005) Fish-borne parasitic zoonoses: status and issues. Int. J. Parasitol. 35, 1233–1254. doi:10.1016/j.ijpara.2005.07.013 Prevention 14. Moir, C. (2009) Review of the current testing protocols for imported seafood products. CSIRO Report reference number R-661-03-11. 90 pp. Gastro-intestinal anisakidosis can be prevented by properly cooking 15. Hochberg, N.S. and Hamer, D.H. (2010) Anisakidosis: perils of the deep. Clin. fish to an internal temperature of approximately 638C or freezing at Infect. Dis. 51, 806–812. or below À208C for 7 days15. This will most likely prevent most other parasites as well. Biography Consuming raw seafood in reputable restaurants only, where chefs Dr Shokoofeh Shamsi is a senior lecturer in veterinary Parasito- are trained to recognise infected seafood. logy. After completing a Masters Degree in Medical Parasitology (Tehran University of Medical Sciences) she gained her PhD in Conclusion Veterinary Parasitology from The University of Melbourne. She is a In the absence of standard diagnostic techniques it is difficult to taxonomist who couples conventional morphological techniques have a realistic estimation of occurrence of seafood borne parasitic with modern molecular technologies to diagnose infections and diseases in the country. As Chai et al.12 stated it appears that fish identify parasite species, especially aquatic parasites. This has borne parasitic disease in Australia remains a ‘public health orphan’ resulted in the discovery of several new pathogenic species and with very little research having been carried out on what the real strains, and their recognition as disease causing agents in humans risks are. The economic value of the fishing industry and health and animals in Australia and worldwide.

MICROBIOLOGY AUSTRALIA * MARCH 2016 29 Under the Microscope

Children, snails and worms: the Brachylaima cribbi story

which enabled the establishment of a laboratory life cycle using parasite eggs recovered from the stool of an infected human1. My long-term association, as an active member of the Australian Society for Microbiology and the special interest groups played a significant role in having a network of colleagues to help find Andrew R Butcher techniques and data to assist in the study of this parasite. SA Pathology Microbiology and Infectious Diseases fi SA, Australia The publication of the rst human Brachylaima sp. infections Email: followed the detection of a fluke worm egg, in the stool of two [email protected] South Australian children, who had never travelled overseas. The eggs were identified as belonging to a genus of trematode worm endemic in the local area2. After conversations with many local and Brachylaimids are parasitic trematode fluke worms that interstate colleagues, a veterinary parasitology colleague Michael have a terrestrial life cycle involving land snails and slugs O’Callaghan remembered the post-doctoral work of Thomas Cribb as the first and/or second intermediate hosts for the cercarial in South Australia. He had studied and published work on a and metacercarial larval stages. A wide range of mammals, brachylaimid in South Australia that infected mice3,4. This provided birds, reptiles and amphibians are the definitive hosts for the the data on a local trematode that had an egg morphology matching adult worm. Brachylaima spp. have been reported from the eggs detected in the children’s stools. most continents including Europe, Africa, Asia, North and However, was this a true human infection that resulted in the South America and Australia. There are over 70 described establishment of mature gravid worms producing the eggs detected species in the genus with seven species indigenous to Aus- in their stool or was it a spurious infection? The answer to this tralia. Although Brachylaima spp. are a cosmopolitan ter- question was resolved 18 months later when an elderly lady from the restrial trematode they have not been recorded to infect mid-north of South Australia presented with chronic diarrhoea. humans other than the three Brachylaima cribbi infections Microscopic examination of her stool detected Brachylaima eggs, reported in two children and an adult from South Australia. which matched the morphology of the eggs detected in the two The publication of a new species of brachylaimid, Brachylaima children. In addition, on the first day following treatment with cribbi by Butcher and Grove in 2001 was the culmination of a 12-year praziquantel a gravid degenerate adult Brachylaima worm was scientific journey that would not have been possible without a recovered from her stool5. This provided the evidence that a broad scientific network of colleagues and some ‘scientific luck’, Brachylaima sp. was infecting humans and completing its life cycle.

(a) (b)

Figure 1. (a) European helicid and hygromiid land snails aestivate over summer to escape the ground heat by attaching to fence posts, which presents an easy meal for natural definitive hosts like birds. (b) Fertile Brachylaima cribbi egg being smooth shelled with an inconspicuous operculum, an abopercular knob or thickening and measuring 26–32 mm (29.1 mm) long and 16–17.5 mm (16.6 mm) wide.

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However, due to the advanced state of decomposition of the worm techniques published for snail farming6.Myoffice in the diagnostic there was insufficient morphological detail to establish the species Microbiology laboratory had now become part snail culture labo- identification. ratory. These snails were used to study the larval stages in an attempt to understand how humans could have become infected. At the Previous to the finding of the third case described above I had same time I had obtained a copy of an article by Mas-Coma and commenced the cultivation of introduced European helicid and Montoliu detailing the life cycle of Brachylaima ruminae from the hygromiid land snails (Figure 1a) in homemade terrariums using of a rodent fromthe Mediterranean island of Formentera (Spain)7. This article provided methods for the establishment of a laboratory life cycle. With these methods in hand and my snail cultures thriving I set myself the task to attempt to establish a laboratory life cycle using eggs recovered from the stool of the third patient. Faecal sediment of the patient’s stool containing washed concentrated eggs was smeared on wet filter paper and was fed to laboratory snails in Petri dishes overnight. Eight weeks later, when the snails were exposed to moisture in a Petri dish, cercariae by the thousands were observed emerging from the snails (Figure 2). Cercariae were collected in water and used to infect the common brown garden snails Cornu aspersum (Helix aspersa) via the respiratory orifice. After 8–10 weeks mature metacercariae were Figure 2. Scanning electron microscopy ventral view of a Brachylaima harvested from kidneys of the snails. Mice were inoculated orally cribbi cercaria showing oral (os), ventral suckers (vs) and elongated sensory papilla (sp). Bar = 50 mm. with metacercariae. Seven weeks later eggs were detected in mouse

(a)

(b)(c)

Figure 3. Brachylaima cribbi adult worm. (a) Oral (os) and ventral suckers (vs) and genital pore (gp). Bar = 500 mm. (b) Ventral view of the oral and ventral suckers showing rows of tegumental spines. Bar = 100 mm. (c) Genital pore showing extended cirrus (c) releasing sperm (s). Bar = 100 mm.

MICROBIOLOGY AUSTRALIA * MARCH 2016 31 Under the Microscope

(e)

(a)

(d)

(b)

(c)

Figure 4. Life cycle of Brachylaima cribbi. The definitive host sheds eggs (a) in their faeces. The egg is eaten by introduced European land snails (b), which hatch in the snail’s gut to release the miracidium. The miracidium develops into a sporocyst in the snail’s digestive gland. Mature cercariae (c) released from the sporocyst emerge from snail passing into the environment. Cercariae infect other land snails and develop into metacercariae (d) in the snail’s kidney. The definitive host (mammals, birds and reptiles) (e) ingest infected snails, releasing metacercariae, which migrate to the small intestine, attach and develop into mature adult worms. Humans are incidental definitive hosts being infected after eating raw snails containing metacercariae.

faeces. Gravid adult Brachylaima worms were recovered from the Elizabeth Hospital (TQEH) Laboratories. Professor David Grove small intestine for characterisation and morphological identification was the Clinical Microbiologist Head of TQEH Microbiology (Figure 3). Eggs from mouse faeces and dissected worms were used Department and a world recognised expert clinical parasitologist. to continue the life cycle (Figure 4) in the laboratory to study the life At that time Professor Grove offered me the opportunity to join the cycle kinetics. TQEH Microbiology team under the amalgamation program to continue the Brachylaima study and commence a PhD under his At the same time as I was using my spare time to tinker with this life supervision. That is scientific luck, right place at the right time and cycle experiment the former Institute of Medical and Veterinary the rest is now history, with the following years being the most Science (IMVS) was undergoing amalgamation with the Queen simulating and productive science of my career.

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Human cases and western Victoria8. Therefore, the potential exists for further human infections, especially in children living in these regional Since the publication of the three human cases to my knowledge districts. there have been a further 12 laboratory confirmed human infections (A.R. Butcher and D.I. Grove, unpublished observations). Part of my PhD studies was the follow-up of all human infections with a References standard questionnaire to obtain clinical history. The main present- 1. Butcher, A.R. and Grove, D.I. (2001) Description of the life-cycle stages of Brachy- ing symptoms were mucoid, watery diarrhoea, abdominal pain, laima cribbi n. sp. (Digenea: Brachylaimidae) derived from eggs recovered from human faeces in Australia. Syst. Parasitol. 49,211–221. doi:10.1023/A:1010 anorexia and weight loss or poor weight gain in children. All 616920412 identified cases were in patients who lived in rural or semi-rural 2. Butcher, A.R. et al. (1996) Locally acquired Brachylaima sp. (Digenea: Brachylai- midae) intestinal fluke infection in two South Australian infants. 164, districts where helicid and hygromiid land snails infected with Med. J. Aust. 475–478. B. cribbi metacercariae were abundant. The likely infection source 3. Cribb, T.H. (1990) Introduction of a Brachylaima species (Digenea: Brachylaimi- was the ingestion of raw snails. The majority of infections (80%) were dae) to Australia. Int. J. Parasitol. 20, 789–796. doi:10.1016/0020-7519(90)90013-D ’ in children under the age of two years. At this age the mouthing of 4. Cribb, T.H. and O Callaghan, M. (1992) An unusual trematode infecting domestic chickens. Aust. Vet. J. 69,69–70. doi:10.1111/j.1751-0813.1992.tb07456.x objects is a common behavioural characteristic with parents of the 5. Butcher, A.R. et al. (1998) First report of the isolation of an adult worm of the genus infected children detailing their child’s consumption of snails. For Brachylaima (Digenea: Brachylaimidae), from the of a human. Int. J. Parasitol. 28, 607–610. doi:10.1016/S0020-7519(97)84372-X the three adults infected they had all eaten home-grown garden 6. Baker, G.H. (1991) Production of eggs and young snails by adult Theba pisana vegetables contaminated with snails. The duration of symptoms (Müller) and Cernuella virgata (da Costa) (Mollusca: Helicidae) in laboratory ranged from 1 month to 2 years with the majority of infections culture and field populations. Aust. J. Zool. 39,673–679. doi:10.1071/ZO9910673 7. Mas-Coma, S. and Montoliu, I. (1986) The life cycle of n. sp. diagnosed within 1–2 months of the first signs of symptoms. All Brachylaima ruminae (: Brachylaimidae), a parasite of rodents. Z. Parasitenkd. 72,739–753. patients were treated successfully with praziquantel with no adverse doi:10.1007/BF00925095 side-effects. The most commonly used dose in both adults and 8. Butcher, A.R. and Grove, D.I. (2003) Field prevalence and laboratory susceptibility of southern Australian land snails to Brachylaima cribbi sporocyst infection. children was 20 mg/kg once per day for 3 days. Parasite 10, 119–125. doi:10.1051/parasite/2003102119 Laboratory diagnosis Diagnosis of the infection relies on the detection of typical eggs in Biography the faeces which are asymmetrical, being ovoid, with one side Dr Andrew Butcher is a retired medical scientist with 38 years slightly flattened. They have a smooth shell, inconspicuous oper- experience as a diagnostic medical microbiologist with a special culum, an abopercular knob or thickening and measure 26–32 mm interest in parasitology. He has been an active member of the ASM by 16–17.5 mm. The eggs generally contain a well-developed mira- Parasitology and Tropical Medicine Special Interest Group for cidium (Fig. 1b). However, in chronic infections many of the eggs many years being national convenor for 7 years. He has been may be infertile, being of similar morphology but smaller size and involved in teaching diagnostic medical parasitology and one lacking a developed miracidium. Infected land snails have been of the founding committee members of the ASM Parasitology reported from coastal and inland Western Australia, South Australia Master Class.

MICROBIOLOGY AUSTRALIA * MARCH 2016 33 Under the Microscope

Malaria: global challenges for malaria eradication

Graham Brown Stephen Rogerson The University of Melbourne The University of Melbourne Vic. 3010, Australia Vic. 3010, Australia Email: [email protected] Email: [email protected]

The enormous decline in the annual morbidity and mortality predominantly on malaria control. Many factors contributed to this from malaria is the spectacular global public health success success from the many constituencies that make up the Roll Back of the past decade. This achievement results largely from Malaria Partnership2, but most of the progress in Africa is usually increased finance for investment in measures known to attributed to widespread distribution of bednets treated with long- prevent malaria: bednets treated with long-lasting insecti- lasting insecticides, combined with increased access to highly- cides, chemoprophylaxis, and rapid access to effective treat- effective drugs3. ment. Such has been the success of these measures that plans are being put in place to achieve the vision of a malaria-free The current situation world within the next three decades. Large financial and According to the latest information provided by the WHO, ~1.2 political commitments and ongoing research will be re- billion people are at risk of malaria with the major burden of disease quired to maintain the gains, overcome known and un- carried by young children and pregnant women living in endemic known challenges such as drug and insecticide resistance, areas.1 and to achieve those goals. Effective vaccines or methods for The burden of malaria is not evenly distributed across the world. reducing mosquito vectorial capacity would add enormous- About 90% of malaria deaths occur in sub-Saharan Africa and recent ly to the chance of achieving this goal. The aim of this article estimates suggest that 80% of malaria deaths occur in only 15 is to summarise the current status of malaria control, the countries. The large morbidity resulting from P. vivax is seen mainly recent research successes, the challenges being addressed, in the Asia Pacific region. Further general information on malaria is and the plan for progress to elimination of malaria in the available in the most recent World Malaria Report from WHO1.There longer term. has been great recent interest in the finding that infections with the simian malaria P. knowlesi have become increasingly important in Spectacular progress in reducing the burden Malaysia and surrounding countries. of malaria The strategy for malaria control leading Funding for major efforts in implementation of known effective preventive measures in the past 15 years has caused an almost 40% to elimination reduction in incidence of malaria disease episodes. These measures, The overall guiding strategy for addressing malaria is summarised in together with increased access to effective treatment, have led to the Global Technical Strategy for Malaria Elimination 2016–2030, a reduction in malaria death rates of 60% globally, and 71% in which was approved and adopted by the World Health Assembly in children under five1. It is estimated that between 2001 and 2015, May 20154. The strategy calls for accelerated action towards malaria ~6.2 million lives were saved by increasing the provision of these elimination in countries and regions, but does not set a time frame services. Approximately 110 countries are free from malaria, ~40 for global eradication. Notably this document recognises the con- have committed to an elimination timetable, while 70 focus tinuum of activities from malaria control to elimination, the

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importance of a multi-disciplinary approach, and the need for tailor- apply control measures locally, and search for the source of intro- made strategies for different situations, even within one country, in duced cases. other words, ‘One size does not fit all’. One area, for example a mountainous region, may have only the problem of introduced malaria occurring in people of all ages, whereas another highly MalERA (Malaria Eradication Research Agenda) endemic area may need to focus on children and pregnant women Following the call for malaria eradication, the Bill & Melinda Gates who remain at risk and suffer the consequences all year round. Foundation recognised the need for new and better tools for elimination, and new and better systems to guide delivery of services The Global Malaria Programme of WHO provides support to coun- to achieve those goals. Following extensive discussions by working tries, compiles the Annual Global Malaria Report, and through the groups, a research agenda was developed (MalERA)6 that included: Malaria Policy Advisory Committee provides a normative role in * Better and safer drugs for ease of administration to populations. reviewing new data or new problems to provide advice for policy The ‘dream’ product profile is ‘SERCAP’, (Single Encounter implementation at country level. Radical Cure And Prophylaxis), i.e. a single dose treatment to kill not only asexual blood-stage parasites but also liver and sexual This WHO strategy is complemented by the Roll Back Malaria stages. An intermediate goal would be an alternative and more effective drug to kill hypnozoites, the dormant liver stage respon- advocacy plan, Action and Investment to Defeat Malaria sible for relapse in P. vivax, but without the troublesome side- 2016–2030, that was approved in 20155. The publication outlines effects of primaquine, the only drug currently available for that purpose. both the health and economic benefits that would flow from malaria * Basic research enabling culture of all life cycle stages of all parasites elimination, and like the Global Technical Strategy was the result of infecting humans would be required for development of vaccines extensive consultative processes involving the participation of more and for production of better drugs for eradication of hypnozoites. This would entail investment in culture of hepatic stages of than 400 malaria experts from 70 countries. Ambitious but achiev- P. vivax, and achieving the dream of laboratory culture of spor- able global targets were established, including: ozoites from gametocytes. * The emphasis on elimination draws attention to P. vivax, com- * reducing malaria case incidence by at least 90% by 2030 mon outside Africa, and with separate challenges such as long- * reducing malaria mortality rates by at least 90% by 2030 term relapses from hypnozoites, and transmission by vectors * eliminating malaria in at least 35 countries by 2030 biting outdoors and during the daytime, which are therefore not * preventing a resurgence of malaria in all countries that are malaria- amenable to the standard interventions for vector control. free. Many considered that elimination would not be achieved without a The timeline of 2016–2030 is aligned with the 2030 Agenda for major breakthrough such as effective vaccines to reduce morbidity Sustainable Development, the new global development framework and transmission, or a technology for vast reduction in vectorial adopted by all UN Member States in September. capacity of Anopheles gambiae, the most efficient and important vector of P. falciparum in Africa.

Malaria elimination It is a source of optimism that since the MalERA agenda was The major progress already referred to is the result of implemen- proposed, funding was directed to address important gaps, a Malaria tation of known effective preventive measures, including vector Eradication Scientific Alliance (MESA) was established, and now control with long-lasting insecticide-treated nets and indoor resid- through its MESA Track database tool can capture relevant research ual spraying; chemoprophylaxis including intermittent preventive and development projects. By the end of October 2015, 685 projects treatment of malaria in pregnancy (IPTp); intermittent preventive from 647 institutions and 84 countries had been documented7. treatment of malaria in infants (IPTi) or malaria chemoprevention MESA is currently coordinating several expert groups to perform a during the season of malaria transmission (SMC); and provision of 5-year ‘refresh’ of the MalERA agenda. ready access to case management with rapidly-effective drugs, Progress is being made on many other fronts, including conference primarily artemisinin combination treatments (ACTs). reports of the in vitro culture of sporozoites from gametocytes; As countries, or areas within countries, move towards elimination, successful culture of hepatic stages of plasmodia; and development the emphasis changes from population level data and disease of new antimalarial drugs, through ‘Medicines for Malaria Venture’ control to intense surveillance for identification of individual infec- (MMV), a highly successful not-for-profit public-private partnership. tions, followed by planning and, most importantly, implementation The timeframe for initial testing of new drugs (or vaccines) has been of a response. For example, teams might test everyone in a com- dramatically shortened by the development of a blood stage chal- pound or a village surrounding a person presenting with illness, lenge model by Queensland-based researchers.

MICROBIOLOGY AUSTRALIA * MARCH 2016 35 Under the Microscope

Table 1. Some of the challenges for malaria control and elimination. replace the requirement for in vivo testing for the presence of Parasite and vector factors resistance. A warning sign, before emergence of clinical resistance, is Artemisinin resistance (and resistance to partner drugs) the detection of delayed parasite clearance from the blood over the first few days of treatment. Because of the fear of emergence P. vivax hypnozoites of resistance, combination therapy is essential, for example with Detecting infections at low parasitaemia piperaquine or lumefantrine, so the efficacy of partner drugs is also Vector resistance (chemical and behavioural) of great importance to ensure complete parasite clearance. Recent- ly, drug resistance has been associated with some but not all Substandard and counterfeit drugs mutations of the kelch gene in P. falciparum that will probably Societal and economic assist in mapping resistance8,9. Melbourne-based researchers have Inadequate health services to deliver interventions developed synthetic artemisinin-like compounds, which are prob- ably not susceptible to kelch-based resistance. Lack of financial commitment

Lack of political will

Cross border coordination Insecticide resistance

Underserved minorities at high risk Chemical resistance to insecticides is a well known threat. Strong movements against DDT that is cheap and effective for indoor Civil unrest residual spraying (because of past evidence of harmful environmen- tal effects when very widely distributed in agriculture) mean that there are few suppliers globally, and alternatives are very expensive. Table 2. Preparing for elimination. Biological approaches Behavioural resistance is another well known complication of the introduction of effective vector control directed at mosquitoes that SERCAP a drug for ‘Single Encounter Radical Cure And Prophylaxis’ bite indoors in the evening. Repeated studies have documented the Highly sensitive non-invasive point-of-care tests gradual increase in proportion of mosquitoes biting earlier in the

Highly effective malaria vaccines evening or later in the morning and resting outdoors where they are not susceptible. Novel vector control strategies Another huge challenge, particularly for control of P. vivax is that Societal and economic the majority of vectors for this parasite bite outdoors and during Political will for a long-term program at high cost per case the daytime. No satisfactory method for long-term control of these Financial commitment vectors is available.

Experienced work force

Technical and operational capacity Elimination as an approach to drug resistance Ongoing research in the Mekong region Where artemisinin has been widely used in the Mekong region, delayed parasite clearance has been noted in many countries, and it The challenges for malaria control is felt that in the absence of alternative drugs, the only appropriate and elimination approach is to eliminate malaria in the area where resistance has already been detected. Pilot studies have been initiated to test the See Table 1 for a more comprehensive list. Some of the biological, feasibility of this approach10. societal and economic requirements for elimination are listed in Table 2. Fortunately, delayed parasite clearance due to artemisinin resis- tance has not yet been seen in parasites from India and Africa, but Artemisinin resistance in the recent past, development of resistance to chloroquine The time and mode of action of artemisinin are not well understood, or sulphadoxine/pyrimethamine accompanied by ongoing use of and in vitro tests are not sufficiently developed and standardised to those drugs led to many unnecessary deaths.

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Funding shortfall achievement of elimination such as financing, or the safety and quality of medicines (to tackle the problem of counterfeit drugs The major advances of the past two decades are at great risk from and ensure that only certified effective products are available in the lack of funding. Not only could progress stall, but major epidemics public and private sectors). Recently, the Alliance endorsed a Malaria of malaria could return to cleared areas, as has been seen so often Elimination Roadmap towards a malaria-free Asia Pacific by 2030. in the past11, with serious effects on people of all ages whose immunity has declined through lack of boosting by recurrent Elimination to eradication sub-clinical infections. The malaria eradication campaign of 50 years ago succeeded in fi The case is now strong for the economic bene ts that will follow eliminating malaria from manycountries but did not achieve its goals 5 from the elimination of malaria . This message needs to reach the in areas of most intense transmission such as heartland Africa. fi highest levels of government to ensure nancial and political However, many important lessons were learned about the need commitment and the engagement not just of the health system, for community engagement, political will, intensive surveillance as a but all sectors of society. response, and the need for continued vigilance to prevent return of malaria to a now susceptible entire population that has benefited Strengthened Health Services from a few years of interrupted transmission. As transmission As the malaria map shrinks, the last pockets of transmission are often declines and malaria-specific services are phased out, it is critical found in marginalised poor or rural populations with least access to for awareness of the disease in all its manifestations to be retained at services. As episodes become infrequent, success will depend on a a high level in general services for detection, management, surveil- general health service with the capacity to recognise and diagnose lance and response to any new episodes. infections, then respond with appropriate increased surveillance Lessons of the eradication campaign are being refreshed, for ex- to detect the origin of the infection and interventions to prevent ample on mass drug administration to entire populations (MDA), further spread. and comparing this strategy with treatment only of those found with highly sensitive tests to be infected, thus avoiding the unnec- Regional initiatives essary use of drugs that could have side-effects, so called Mass APMEN Recognising the need for a partnership across regions and Screening and Treatment, or MSAT. Research on application of across borders, the Asia Pacific Malaria Elimination Network was more modern tools such as rapid diagnostic tests, including nucleic established with the support of the Australian government in 2008 to acid amplification or data transmission by mobile phones are facilitate collaboration amongst nations of the region that had made providing further data to guide strategy. For example, it is important commitments to achieve elimination within a defined time period. to decide whether the cost of a more sensitive but more expensive This network enables regional collaboration and facilitates capacity and technically difficult nucleic acid detection test adds significant building and knowledge transfer among nations of the region that benefit in elimination campaigns, or how PCR-based parasite share similar challenges such as artemisinin resistance, inadequate ‘barcoding’ (genetic epidemiology) can be used to provide rapid finance for malaria, or a vast market penetration of counterfeit analysis of the source of emergent infections, the spread of drug drugs. resistance, or the risk of the parasite’s appearance in susceptible areas. AICEM, the Australian Initiative for Control and Elimination of Malaria, is funded by the Australian government to strengthen Other major lessons of the eradication campaign were that research malaria control in the Solomon Islands and Vanuatu, and at the must continue throughout the programs to deal with new chal- same time establish pilot projects for elimination of malaria in lenges, and that every country needs to develop the human resource certain island populations. capacity to deal with the multi-disciplinary approach to this grand challenge. APLMA (www.aplma.org), the Asia Pacific Leaders Malaria Alli- ance, co-chaired by the Heads of Government of Australia and Recent vaccine breakthroughs; progress Vietnam, modelled loosely on the African Leaders Malaria Alliance, with P. falciparum but little progress with aims to bring together Heads of State, politicians, and opinion leaders of civil society to advocate for political and financial com- other species mitment to the goals of elimination of malaria. Special task forces Successful Phase 3 field trials of RTS,S vaccine in African are designed to address critical issues that could be barriers to children. Development of the RTS,S vaccine, which provides

MICROBIOLOGY AUSTRALIA * MARCH 2016 37 Under the Microscope

35% efficacy against clinical episodes of P. falciparum malaria, was References an important milestone for the malaria vaccine field. The vaccine 1. World Health Organization (2015) Malaria: fact sheet no. 94. http://www.who.int/ mediacentre/factsheets/fs094/en/ includes a fusion protein containing the immunogenic repeat 2. World Health Organization (2016) Roll back malaria partnership. http://www. regions of the circumsporozoite protein, T-cell epitopes of rollbackmalaria.org the same molecule and the B surface antigen that is 3. Bhatt, S. et al. (2015) The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature 526,207–211. doi:10.1038/nature15535 co-expressed with free hepatitis B virus surface antigen leading to 4. World Health Organization (2015) Global technical strategy for malaria 2016– formation of a viral-like particle known as RTS,S. In recent very large 2030. http://www.who.int/malaria/publications/atoz/9789241564991/en/. trials, participants had good access to malaria prevention and rapid 5. World Health Organization Secretariat, on behalf of the Roll Back Malaria Pro- gramme. (2015) Action and investment to defeat malaria 2016–2030. For a malaria- access to treatment, so mortality was low and no effect of vaccination free world. http://www.rollbackmalaria.org/about/about-rbm/aim-2016-2030 on mortality could be detected. There was a concerning, but 6. (2011) malERA – a research agenda for malaria eradication (PLoS medicine unexplained, slight increase in episodes of meningitis in the RTS,S monographic volume). http://collections.plos.org/malera?issue=info:doi/10.1371/ issue.pcol.v07.i13 arm, requiring further evaluation. A recent review of RTS,S by WHO 7. Malaria Eradication Scientific Alliance (2015) MESA track. http://www.malariaer- recommended that the next step should be to initiate 3–5 pilot adication.org/mesa-track implementation studies12. Further analysis of trial data showed that 8. Ariey, F. et al. (2014) A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505,50–55. doi:10.1038/nature12876 the vaccine had higher efficacy against strains homologous to the 9. Ashley, E.A. et al. (2014) Spread of artemisinin resistance in Plasmodium vaccine, suggesting both a strain-specific as well as a strain-inde- falciparum malaria. N. Engl. J. Med. 371, 411–423. doi:10.1056/NEJMoa1314981 pendent mechanism13. This vaccine is a very important first step but 10. World Health Organization (2015) Draft strategy for malaria elimination in the Greater Mekong subregion (2015–2030). http://www.who.int/malaria/areas/great- on its own, is unlikely to be the solution that is required. er_mekong/consultation-elimination-strategy/en/ 11. Cohen, J.M. et al. (2012) Malaria resurgence: a systematic review and assessment Attenuated sporozoite vaccines of its causes. Malar. J. 11, 122. doi:10.1186/1475-2875-11-122 12. World Health Organization (2015) Pilot implementation of first malaria vaccine The most impressive of all malaria vaccine trial results are those recommended by WHO advisory groups. http://www.who.int/mediacentre/news/ achieved with intravenous inoculation of radiation-attenuated releases/2015/sage/en/ whole sporozoites in which up to 100% protection has been 13. Neafsey, D.E. et al. (2015) Genetic diversity and protective efficacy of the RTS,S/ AS01 malaria vaccine. N. Engl. J. Med. 373, 2025–2037. doi:10.1056/NEJMoa 14 achieved in Phase 1 studies using multiple doses of vaccine . Field 1505819 studies in Mali and Tanzania have also demonstrated efficacy and 14. Richie, T.L. et al. (2015) Progress with Plasmodium falciparum sporozoite – further trials are planned to optimise dose and delivery schedule14. (PfSPZ)-based malaria vaccines. Vaccine 33, 7452 7461. doi:10.1016/j.vaccine. 2015.09.096 Conclusion With the spectacular reduction of the burden of malaria in the past Biographies 15 years, the time is ripe to re-double efforts both to prevent Graham Brown, AM Professor Emeritus at The University of resurgence and to increase resources with the goal of achieving Melbourne, currently serves as the Deputy Chair of the Board, the health and economic benefits that would result from disease and Chair of the Executive Committee of Roll Back Malaria. As a elimination. Major progress is being reported in developing the clinician-researcher, his interests include malaria vaccine develop- tools to add to current successful interventions from basic research ment, antigenic variation and clinical infectious diseases. into biology of hypnozoites and transition of gametocytes to spor- Stephen Rogerson is a Professor at The University of Melbourne. ozoites, to field trials of vaccines and innovations in surveillance and His research interests include the pathogenesis of malaria in preg- epidemiology. A multi-disciplinary response coupled with strong nant women and young children, and tools for prevention of community engagement, and ongoing political and financial sup- malaria in these high-risk groups. port will be required to maintain the current rate of decline in malaria, and hopefully achieve the ambitious goal of elimination.

Member of Australian Society for Microbiology (MASM) Want to upgrade from an associate to a full member? No points or cash required and only one supporting referee needed. Go to the ASM website, download and complete the form, and forward it with a CV to the National Office. We will do the rest.

38 MICROBIOLOGY AUSTRALIA * MARCH 2016 Under the Microscope

Plasmodium knowlesi: an update

samples from 208 malaria patients at Kapit Hospital were analysed by PCR assays, none were identified as P. malariae, although 141 had been diagnosed as P. malariae by microscopy. Fifty-eight Balbir Singh percent (120) were either single P. knowlesi infections or mixed Malaria Research Centre infections of P. knowlesi with P. falciparum and P. vivax. Misdi- University Malaysia Sarawak agnosis had occurred because the blood stages of P. knowlesi and 94300 Kota Samarahan 3 Sarawak, Malaysia P. malariae are morphologically indistinguishable . Tel: +6 082 581000 Fax: +6 082 665152 Email: [email protected] Epidemiology and risk factors of acquiring knowlesi malaria Human infections with P. knowlesi have been reported throughout Malaysia and in Thailand, Singapore, the Philippines, Vietnam, There were only four species of Plasmodium that were Cambodia, Indonesia, Brunei, Myanmar and in the Nicobar and thought to cause malaria in humans until a large number Andaman Islands of India4,5. In Malaysia, P. falciparum and P. vivax of human infections by Plasmodium knowlesi, a malaria cases have declined over the past five years and P. knowlesi has now parasite typically found in long-tailed and pig-tailed maca- become the most common cause of human malaria6,7. The true ques, were reported in 2004 in Malaysian Borneo. Since then, incidence of knowlesi malaria is not known in other parts of South- cases of knowlesi malaria have been reported throughout east Asia since not many large-scale studies have been undertaken South-east Asia and also in travellers returning from the with molecular detection assays. region. This article describes the molecular, entomological and epidemiological data which indicate that P. knowlesi is The geographical distribution of human P. knowlesi infections is an ancient parasite that is primarily zoonotic, and there are similar to that of the natural hosts of P. knowlesi, the long-tailed and three highly divergent sub-populations. It also describes the pig-tailed macaques8. Reports from 1931 to 1970 identified maca- detection methods for P. knowlesi, which is morphologicaly ques as hosts of P. knowlesi in Peninsular Malaysia, Singapore and similar to P. malariae, and the clinical features and treat- the Philippines9, and a banded leaf monkey (Presbytis melalophos) ment of this malaria parasite that is potentially fatal. in Peninsular Malaysia9. Since 2007, P. knowlesi infections detected by molecular methods have been described in macaques in Penin- Malaria parasites and discovery of large focus sular Malaysia, Malaysian Borneo, Singapore and Thailand4. of human knowlesi malaria cases The transmission of P. knowlesi in nature has been shown to be Malaria is caused by parasites that belong to the genus Plasmodium restricted to mosquitoes belonging to the Anopheles leucosphyrus and there are more than 150 species of Plasmodium that infect group10. The members of this forest-dwelling group of mosquitoes reptiles, birds and mammals1. These parasites, in general, tend to be that have been identified as vectors include An. latens (in Sarawak, host-specific. Long-tailed and pig-tailed macaques (Macaca fasci- Malaysian Borneo)11, An. balabacensis balabacensis (in Sabah, cularis and M. nemestrina respectively) are hosts to five species Malaysian Borneo), An. dirus (in Vietnam)12 and An. hackeri and (P. knowlesi, P. inui, P. cynomolgi, P. fieldi and P. coatneyi). Only An. cracens (in Peninsular Malaysia)1,13. four species of Plasmodium, namely P. falciparum, P. vivax, P. malariae and P. ovale, were thought to cause malaria in humans People that are at risk of acquiring knowlesi malaria are those that until a large number of human cases due to P. knowlesi were enter the habitat of the macaque reservoir hosts and the Anopheline reported in Sarawak, Malaysian Borneo over 11 years ago2. The vectors at dusk or later as this coincides with the peak biting time of study in Kapit was prompted by observations that cases diagnosed the vectors14,15. These include subsistence farmers, timber camp by microscopy as P. malariae had high parasitaemias, required workers, hunters, army personnel and also travelers to forests or hospitalization and that 95% of patients were adults. This was in forest-fringe areas. Visitors to South-east Asia from Australia, USA, contrast to P. malariae infections which typically are asymptomatic Finland, Sweden, Germany, France, New Zealand, Taiwan and Japan with low parasitaemia and occur in all age groups. When blood have acquired knowlesi malaria following holidays or working visits

MICROBIOLOGY AUSTRALIA * MARCH 2016 10.1071/MA16014 39 Under the Microscope

to Malaysian Borneo, Peninsular Malaysia, Brunei, Thailand, parasites multiply every 24 h in the blood while this erythrocytic Indonesia and the Philippines16. cycle is 72 h for P. malariae9.

Molecular detection methods are the most sensitive and accurate Molecular and whole genome studies techniques for identification of P. knowlesi. These include single In order to understand the molecular epidemiology and demo- and nested PCR assays, real-time PCR assays and loop-mediated graphic history of knowlesi malaria, the mitochondrial (mt) genome isothermal assays4. However, these assays are relatively expensive, 17 sequences of P. knowlesi were initially studied . Certain mt hap- not rapid and are not readily available in resource-poor laboratories lotypes were shared between humans and macaques and there where the majority of P. knowlesi infections are detected. Rapid were no haplotypes that were associated exclusively with either diagnostic tests (RDTs) for malaria are available, but the overall host; further evidence supporting P. knowlesi as a zoonotic parasite. sensitivity of detection of a small number of RDTs that have been Additional analyses indicated that P. knowlesi was as old as, if not evaluated against knowlesi malaria cases varied between 26–74% older than, P. falciparum and P. vivax, and that it underwent a and was even lower (0–45%) for parasitaemias below 1000 parasites/ – population expansion between 30,000 to 40,000 years ago. Maca- mL22 24. Due to the rapid multiplication rate of P. knowlesi in 18 ques colonized Asia over 5 million years ago and are probably the the blood of 24 h, sensitive RDTs capable of detecting knowlesi original hosts for P. knowlesi. A recent study, where 599 P. knowlesi malaria at the early phase of infection are urgently required for rural samples from Peninsular Malaysia and Malaysian Borneo were laboratories. analysed by a panel of ten microsatellite markers, showed there are two highly divergent sub-populations of P. knowlesi, and each of these subpopulations correspond with parasites from either long- Clinical and laboratory features tailed or pig-tailed macaques19. More recently, genome-wide se- of knowlesi malaria quence analysis of clinical P. knowlesi isolates from Malaysian P. knowlesi causes a wide spectrum of disease, from asymptomatic Borneo shows sub-population structure that matches the analysis – infections9,25 to fatal ones26 29. The most common presenting signs using microsatellite markers and also demonstrate there is a third and symptoms reported are fever with chills, followed by headache, sub-population of parasites, corresponding to laboratory strains myalgia, poor appetite, arthralgia, cough, abdominal pain and isolated over 50 years ago from Peninsular Malaysia and the diarrhoea27. These are not significantly different to those observed Philippines20. No signals of positive selection were observed in in patients with vivax and falciparum malaria. The majority of cases P. knowlesi around five orthologues of known P. falciparum (93.5%27 and 84.5%30) at district hospitals in Sarawak had uncom- drug resistance genes, indicating that the parasites in the reservoir plicated malaria with a fatality rate of 2%, whereas in a retrospective macaque hosts have not been under antimalarial drug selection, study in a referral hospital in Sabah, 61% of 56 cases were uncom- thereby providing further evidence that knowlesi malaria is a plicated and the fatality rate was 27%31. However, subsequently at zoonosis. the same referral hospital, the use of intravenous artesunate for severe malaria cases and artemisinin combination therapy for non- Diagnosis severe cases, resulted in no deaths among 130 knowlesi malaria In laboratories in malaria-endemic countries, malaria is diagnosed patients29. Typical complications of severe knowlesi malaria in by examination of blood films by microscopy. Under the micro- adults include jaundice, acute kidney injury, hypotension, acute scope, the early blood forms of P. knowlesi are identical to those respiratory distress syndrome and metabolic acidosis26,27,29,30,32.In of P. falciparum, while the other developmental stages, including adults, severe anaemia has not been observed and neither has the ‘band forms’, are similar to those of P. malariae3. There are cerebral malaria, while severe disease has not been noted in the minor morphological differences between these two species. The relatively small number of children with knowlesi malaria4,33. mature schizonts of P. knowlesi can contain up to 16 merozoites, Thrombocytopaenia is very common, occurring in 97.3 to 100% of whereas those of P. malariae have between 6–123. However, knowlesi malaria patients, and together with parasitaemia, corre- mature schizonts are not found in all blood films examined and in lates with severity of disease27,30,31. Following a case control study, diagnostic laboratories, where technologists are only trained to it was recommended that any patient with a platelet count of recognise P. falciparum, P. vivax, P. ovale and P. malariae, most <45 000/mL or parasitaemia of >35 000 parasites/mL should be P. knowlesi infections have been identified by microscopy as regarded at risk of developing complications and should be treated P. malariae2,4,21. Although morphologically similar, P. knowlesi for severe malaria30.

40 MICROBIOLOGY AUSTRALIA * MARCH 2016 Under the Microscope

5. Tyagi, R.K. et al. (2013) Discordance in drug resistance-associated mutation Treatment of knowlesi malaria patterns in marker genes of Plasmodium falciparum and Plasmodium knowlesi – Since knowlesi malaria is primarily a zoonosis, the parasites have during coinfections. J. Antimicrob. Chemother. 68,1081 1088. doi:10.1093/jac/ dks508 been under no antimalarial drug pressure and should be susceptible 6. William, T. et al. (2013) Increasing incidence of Plasmodium knowlesi malaria to all antimalarials. This has been observed in hospital-based studies following control of P. falciparum and P. vivax Malaria in Sabah, Malaysia. PLoS Negl. Trop. Dis. 7, e2026. doi:10.1371/journal.pntd.0002026 as well as case reports where several antimalarials have been used 7. Yusof, R. et al. (2014) High proportion of knowlesi malaria in recent malaria cases 4 successfully to treat knowlesi malaria patients . P. knowlesi para- in Malaysia. Malar. J. 13, 168. doi:10.1186/1475-2875-13-168 34 sites are highly sensitive to chloroquine but following an informal 8. Cox-Singh, J. and Singh, B. (2008) Knowlesi malaria: newly emergent and of public – consultation on the public health importance of knowlesi malaria health importance? Trends Parasitol. 24, 406 410. doi:10.1016/j.pt.2008.06.001 9. Coatney, G.R. et al. (1971) The primate malarias. US Department of Health, organised by the WHO in 2011, it was recommended that in areas Education and Welfare. where knowlesi malaria has been detected, all infections diagnosed 10. Collins, W.E. (2012) Plasmodium knowlesi: a malaria parasite of monkeys and – as P. malariae by microscopy should be treated and managed as humans. Annu. Rev. Entomol. 57, 107 121. doi:10.1146/annurev-ento-121510- 133540 for falciparum malaria35. Therefore, for uncomplicated knowlesi 11. Vythilingam, I. et al. (2006) Natural transmission of Plasmodium knowlesi to malaria cases in South-east Asia, artemisinin combination therapy is humans by Anopheles latens in Sarawak, Malaysia. Trans. R. Soc. Trop. Med. Hyg. 100, 1087–1088. doi:10.1016/j.trstmh.2006.02.006 recommended. For severe knowlesi malaria, intravenous antimalar- 12. Marchand, R.P. et al. (2011) Co-infections of Plasmodium knowlesi, ials should be administered and the use of artesunate in a tertiary P. falciparum,andP. vivax among humans and Anopheles dirus mosquitoes, referral hospital in Sabah was associated with zero mortality29. Southern Vietnam. Emerg. Infect. Dis. 17,1232–1239. doi:10.3201/eid1707. 101551 13. Vythilingam, I. et al. (2008) Plasmodium knowlesi in humans, macaques and Future directions mosquitoes in peninsular Malaysia. Parasit. Vectors 1,26.doi:10.1186/1756-3305- 1-26 The available molecular, entomological and epidemiological data 14. Tan, C.H. et al. (2008) Bionomics of Anopheles latens in Kapit, Sarawak, Malaysian strongly indicate that knowlesi malaria is primarily a zoonosis. Borneo in relation to the transmission of zoonotic simian malaria parasite Plasmodium knowlesi. Malar. J. 7, 52. doi:10.1186/1475-2875-7-52 However, human-to-human transmission has been demonstrated 15. Vythilingam, I. (2012) Plasmodium knowlesi and : their under experimental conditions9 and it is not known whether it is vectors and challenges for the future. Front. Physiol. 3, 115. doi:10.3389/ fphys.2012.00115 currently occurring. The reasons for the increase in the number of 16. Cramer, J.P. (2015) Plasmodium knowlesi malaria: overview focussing on travel- knowlesi malaria cases, particularly in Malaysian Borneo, are also associated infections. Curr. Infect. Dis. Rep. 17, 8. doi:10.1007/s11908-015-0469-6 unknown. Whether the increase is due to increased awareness, 17. Lee, K.S. et al. (2011) Plasmodium knowlesi: reservoir hosts and tracking the emergence in humans and macaques. PLoS Pathog. 7, e1002015. doi:10.1371/ changes in the feeding habits of the vectors, the destruction of the journal.ppat.1002015 natural habitats of the macaque reservoir, human migration to areas 18. Ziegler, T. et al. (2007) Molecular phylogeny and evolutionary history of Southeast close to macaque habitats, a recent adaptation of knowlesi malaria Asian macaques forming the M. silenus group. Mol. Phylogenet. Evol. 42,807–816. doi:10.1016/j.ympev.2006.11.015 parasites to humans, or to some other factors needs to be investi- 19. Divis, P.C. et al. (2015) Admixture in humans of two divergent Plasmodium gated. In addition, currently available methods of control of human knowlesi populations associated with different macaque host species. PLoS malaria involving the use of insecticide treated bednets and residual Pathog. 11, e1004888. doi:10.1371/journal.ppat.1004888 20. Assefa, S. et al. (2015) Population genomic structure and adaptation in the spraying of houses are ineffective against knowlesi malaria, where zoonotic malaria parasite Plasmodium knowlesi. Proc. Natl. Acad. Sci. USA transmission primarily occurs outdoors. Therefore, effective meth- 112, 13027–13032. doi:10.1073/pnas.1509534112 ods of preventionand controlneed tobe foundand implemented,in 21. William, T. et al. (2014) Changing epidemiology of malaria in Sabah, Malaysia: increasing incidence of Plasmodium knowlesi. Malar. J. 13, 390. doi:10.1186/ order to prevent P. knowlesi from establishing itself in the human 1475-2875-13-390 population. 22. Foster, D. et al. (2014) Evaluation of three rapid diagnostic tests for the detection of human infections with Plasmodium knowlesi. Malar. J. 13, 60. doi:10.1186/ 1475-2875-13-60 References 23. Barber, B.E. et al. (2013) Evaluation of the sensitivity of a pLDH-based and an aldolase-based rapid diagnostic test for diagnosis of uncomplicated and severe 1. Garnham, P.C.C. (1966) Malaria parasites and other haemosporidia. Blackwell malaria caused by PCR-confirmed Plasmodium knowlesi, Plasmodium falci- Scientific Publications. parum, and Plasmodium vivax. J. Clin. Microbiol. 51,1118–1123. doi:10.1128/ 2. Singh, B. et al. (2004) A large focus of naturally acquired Plasmodium knowlesi JCM.03285-12 infections in human beings. Lancet 363, 1017–1024. doi:10.1016/S0140-6736(04) 24. Grigg, M.J. et al. (2014) Combining parasite lactate dehydrogenase-based and 15836-4 histidine-rich protein 2-based rapid tests to improve specificity for diagnosis of 3. Lee, K.S. et al. (2009) Morphological features and differential counts of Plasmo- malaria due to Plasmodium knowlesi and other Plasmodium species in Sabah, dium knowlesi parasites in naturally acquired human infections. Malar. J. 8, 73. Malaysia. J. Clin. Microbiol. 52, 2053–2060. doi:10.1128/JCM.00181-14 doi:10.1186/1475-2875-8-73 25. Fornace, K.M. et al. (2015) Asymptomatic and submicroscopic carriage of 4. Singh, B. and Daneshvar, C. (2013) Human infections and detection of Plasmo- Plasmodium knowlesi malaria in household and community members of dium knowlesi. Clin. Microbiol. Rev. 26, 165–184. doi:10.1128/CMR.00079-12 clinical cases in Sabah, Malaysia. J. Infect. Dis. doi:10.1093/infdis/jiv475

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26. Cox-Singh, J. et al. (2008) Plasmodium knowlesi malaria in humans is widely 32. Cox-Singh, J. et al. (2010) Severe malaria – a case of fatal Plasmodium knowlesi distributed and potentially life threatening. Clin. Infect. Dis. 46, 165–171. infection with post-mortem findings: a case report. Malar. J. 9, 10. doi:10.1186/ doi:10.1086/524888 1475-2875-9-10 27. Daneshvar, C. et al. (2009) Clinical and laboratory features of human 33. Barber, B.E. et al. (2011) Plasmodium knowlesi malaria in children. Emerg. Infect. Plasmodium knowlesi infection. Clin. Infect. Dis. 49, 852–860. doi:10.1086/ Dis. 17, 814–820. doi:10.3201/eid1705.101489 605439 34. Daneshvar, C. et al. (2010) Clinical and parasitological response to oral chloro- 28. Rajahram, G.S. et al. (2012) Deaths due to Plasmodium knowlesi malaria in Sabah, quine and primaquine in uncomplicated human Plasmodium knowlesi infec- Malaysia: association with reporting as Plasmodium malariae and delayed tions. Malar. J. 9, 238. doi:10.1186/1475-2875-9-238 parenteral artesunate. Malar. J. 11, 284. doi:10.1186/1475-2875-11-284 35. WHO (2011) Informal consultation on the public health importance of Plasmo- 29. Barber, B.E. et al. (2013) A prospective comparative study of knowlesi, dium knowlesi. World Health Organization Regional Office for the Western Pacific falciparum, and vivax malaria in Sabah, Malaysia: high proportion with severe Press. disease from Plasmodium knowlesi and Plasmodium vivax but no mortality with early referral and artesunate therapy. Clin. Infect. Dis. 56, 383–397. doi:10.1093/cid/cis902 Biography 30. Willmann, M. et al. (2012) Laboratory markers of disease severity in Plasmodium Professor Balbir Singh is the Director of the Malaria Research knowlesi infection: a case control study. Malar. J. 11, 363. doi:10.1186/1475- 2875-11-363 Centre at University Malaysia Sarawak. His research interests 31. William, T. et al. (2011) Severe Plasmodium knowlesi malaria in a tertiary care include the epidemiology, pathogenesis and evolution of malaria – hospital, Sabah, Malaysia. Emerg. Infect. Dis. 17, 1248 1255. doi:10.3201/eid parasites. 1707.101017

42 MICROBIOLOGY AUSTRALIA * MARCH 2016 Under the Microscope

Diagnosis of human taeniasis

Abdul Jabbar, Charles Gauci and Marshall W Lightowlers Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Werribee, Vic. 3030, Australia, Email: [email protected]

Taenia solium, T. saginata and T. asiatica are taeniid tape- Therefore, differentiation of Taenia species becomes significant for worms that cause taeniasis in humans and cysticercosis in surveillance and control of human taeniasis. intermediate host animals. T. solium can also cause cysti- A number of diagnostic methods have been used to differentiate the cercosis in humans. A number of diagnostic methods have common human cestodes, T. saginata and T. solium; however, been developed to diagnose Taenia species that infect each method has its advantages and disadvantages, and careful humans. This article is aimed at providing an overview of attention should be paid to determining which particular test is currently available diagnostic methods for human taeniasis. best to use for differentiation of the two species4. The following Human taeniasis is an important zoonotic health problem and is sections provide a quick rundown on various diagnostic methods caused by adult stages of taeniid cestodes, including Taenia solium, available to differentiate Taenia spp. that infect humans. T. saginata, and T. asiatica. These parasites have indirect life cycles where humans act as definitive hosts whereas pigs (for T. solium and Microscopic diagnosis T. asiatica) and cattle (T. saginata) serve as intermediate hosts. Traditionally, diagnosis of taeniasis has been based on the detection Humans become infected by ingesting parasite cysts present in raw of eggs by microscopic examination but this method lacks or undercooked infected meat/liver; when the cyst reaches the sensitivity and specificity as T. solium and T. saginata eggs are human intestine, it develops into an adult tapeworm, releasing morphologically identical, making species identification impossi- segments and/or eggs in the stools or motile segments (e.g. ble5. However, morphological examination of gravid proglottids can T. saginata) are expelled actively. Intermediate hosts such as cattle allow the differentiation of T. solium and T. saginata provided the and pigs become infected when they ingest taeniid eggs via con- internal structures (i.e. uterine branches) are intact5. Sometimes, taminated feed or water and the larval stage (cysticercus) forms in even the morphological examination of proglottids does not allow muscular and sometimes other tissues1. The adult tapeworm stage the differentiation of T. solium and T. saginata, thus requiring of Taenia spp. is relatively innocuous and does not cause patho- alternate methods for the differentiation of human Taenia spp. genic effects in humans; however, the intermediate stage of T. solium can also develop in human brains causing neurocysticer- Immunodiagnosis cosis, a major cause of neurological disease in many developing The first method of coproantigen (parasite antigens in human stool) countries as well as other organs causing intramuscular, ocular, detection using enzyme-linked immunosorbent assay (ELISA) was subcutaneous and spinal cysticercoses2,3. T. saginata is endemic in developed by Allan et al.6. Although the test displayed a higher Australia, whereas T. solium and T. asiatica are exotic. However, sensitivity and specificity than microscopic diagnosis for the detec- people coming and/or returning to Australia from endemic coun- tion of Taenia spp.7, it did not allow differentiation of T. saginata tries can be infected with T. solium, leading to the possibility that and T. solium. Recently, Guezala et al.8 developed another coproan- infected individualsmay pass segmentsof the parasitein theirstools, tigen ELISA and successfully differentiated T. solium from which can serve as a source of infection for human cysticercosis. T. saginata. These tests were developed in individual labs using

MICROBIOLOGY AUSTRALIA * MARCH 2016 10.1071/MA16011 43 Under the Microscope

in-house reagents and have not been independently validated in been found to improve diagnostic sensitivity where Yamasaki different laboratories nor have they been widely used in diagnostic et al.13 showed that some proven egg-positive cases were negative laboratories. by PCR. Specificity of PCR is high with control faecal samples, including samples from patients with other parasitic infections, The first serological assay to detect specific antibodies against being almost always negative in PCR. T. solium infection in humans was developed by Wilkins et al.9 Subsequently, a number of studies reported various immunoassays To date, predominantly conventional and multiplex PCR, and PCR- for the diagnosis of human taeniasis, primarily caused by restriction fragment length polymorphism (PCR-RFLP) have utilized T. solium10,11 and these studies used either native excretory-secre- various markers, including internal transcribed spacer, mitochon- tory products collected from adult tapeworms9 or cloned and drial cytochrome c oxidase subunit I gene as well as 12S rDNA, expressed excretory-secretory products of adult T. solium10,11. Like cathepsin L-like cysteine peptidase, T. saginata-specific repetitive coproantigen ELISAs, the detection of Taenia species-specific sequence (HDP1) and cestode-specific sequence (HDP2) to dis- – antibodies are also more specific and sensitive than microscopic criminate between human Taenia spp13 21 (Table 1). Mayta et al.19 techniques. However, these tests have been found to have some developed a nested PCR utilizing two rounds of PCR amplification of degree of cross-reactivity in sera from patients with cystic echino- the Tso31 gene, which is more sensitive than conventional PCR but coccosis, , and schistosomiasis11. Furthermore, currently pose technical difficulties. A field DNA-based test known as loop available immunodiagnostic tests may give false positive results mediated isothermal amplification (LAMP) was developed by as specific circulating antibodies in taeniasis patients could possibly Nkouawa et al.20, which amplifies the cox1 gene. To date, only one remain detectable for some time either after treatment and recent real-time PCR to discriminate T. solium and T. saginata has been past infections4. developed which targets the internal transcribed spacer 1 of the nuclear ribosomal RNA21. The majority of DNA-based methods Molecular diagnosis have utilised DNA isolated from proglottids for Taenia species identification while relatively fewer studies isolated DNA from A number of molecular methods using PCR-based technologies stool samples (Table 1). In addition, almost all studies have been have been developed to either determine the presence of Taenia tested only on small numbers of usually known positive and negative species-specific DNA in human stools or differentiate Taenia samples. spp. (T. solium, T. saginata or T. asiatica) based on the analysis of DNA extracted directly from tapeworm12. PCR-based methods have higher sensitivity in the detection of taeniasis cases (i.e. the Conclusions and future perspectives detection of parasite DNA in human stools) than microscopy Human taeniasis is a worldwide parasitic disease and detection and alone. In addition, a combined use of PCR and microscopy have discrimination of T. solium, T. saginata and T. asiatica remains a

Table 1. Selected PCR-based assays for detection and characterization of human Taenia spp. Taenia species Molecular method Target(s) Sample type(s) Reference T. saginata PCR Gel HDP1 Proglottids 14

T. solium, T. saginata Multiplex PCR HDP2 Proglottids 15

T. solium, T. saginata Multiplex PCR-RFLP HDP2 Proglottids 16

T. solium, T. saginata, PCR-RFLP Mitochondrial 12S rDNA Proglottids 17 T. asiatica

T. solium, T. saginata, Multiplex PCR cox1 Proglottids, stool 13 T. asiatica

T. solium, T. saginata PCR-RFLP cox1 Stool 18

T. solium Nested PCR Tsol31 Stool 19

T. solium, T. saginata, LAMP Clp, cox1 Proglottids, cysticerci, stool 20 T. asiatica

T. solium, T. saginata Real-time multiplex PCR ITS1 Stool 21

RFLP, restriction fragment length polymorphism; ITS, internal transcribed spacer; cox1, mitochondrial cytochrome coxidase subunit I gene; Clp, cathepsin L-like cysteine peptidase; HDP1, T. saginata-specific repetitive sequence; HDP2, cestode-specific sequence. Adapted from Verweij and Stensvold12.

44 MICROBIOLOGY AUSTRALIA * MARCH 2016 Under the Microscope

public health concern. Microscopic, immunological and molecular 11. Levine, M.Z. et al. (2007) Development of an enzyme-linked immunoelectro- transfer blot (EITB) assay using two baculovirus expressed recombinant antigens methods have been used to detect and differentiate Taenia spp., for diagnosis of Taenia solium taeniasis. J. Parasitol. 93,409–417. doi:10.1645/ and a combination of two or more methods appears to provide GE-938R.1 higher sensitivity13,21. Almost all of the immunological and molec- 12. Verweij, J.J. and Stensvold, C.R. (2014) Molecular testing for clinical diagnosis and epidemiological investigations of intestinal parasitic infections. Clin. Microbiol. ular methods developed so far have not been independently tested Rev. 27, 371–418. doi:10.1128/CMR.00122-13 and validated on controlled negative and positive samples as well as 13. Yamasaki, H. et al. (2003) Cysticercosis/taeniasis: recent advances in serological field samples. Among existing molecular methods, the nested PCR19 and molecular diagnoses. Southeast Asian J. Trop. Med. Public Health 34,98–102. provides the highest sensitivity and specificity of those methods that 14. Gonzalez, L.M. (2000) Differential diagnosis of and Taenia solium infection by PCR. J. Clin. Microbiol. 38,737–744. have been developed and validated using unselected faecal samples 15. González, L.M. et al. (2002) Differential diagnosis of Taenia saginata and Taenia from parasitological proven taeniasis carriers. Although the method solium infections: from DNA probes to polymerase chain reaction. Trans. R. Soc. is technically challenging and could be expected to be relatively Trop. Med. Hyg. 96, S243–S250. doi:10.1016/S0035-9203(02)90083-0 expensive to use, the reagents required are available worldwide 16. González, L.M. et al. (2002) PCR tools for the differential diagnosis of Taenia saginata and Taenia solium taeniasis/cysticercosis from different geographical and laboratories capable of undertaking PCR competently can run locations. Diagn. Microbiol. Infect. Dis. 42,243–249. doi:10.1016/S0732-8893(01) 4 this PCR . 00356-X 17. Rodriguez-Hidalgo, R. et al. (2002) Comparison of conventional techniques to To the best of our knowledge, no standardised PCRs are available in differentiate between Taenia solium and Taenia saginata and an improved Australia to discriminate human Taenia spp. Future studies should polymerase chain reaction-restriction fragment length polymorphism assay using – 19 a mitochondrial 12S rDNA fragment. J. Parasitol. 88, 1007 1011. doi:10.1645/ focus on independent validation of the nested PCR and the LAMP 0022-3395(2002)088[1007:COCTTD]2.0.CO;2 20 technique as these two DNA-based tests offer higher sensitivity 18. Nunes, C.M. et al. (2005) Taenia saginata: differential diagnosis of human and a user-friendly option without sophisticated equipment, re- taeniasis by polymerase chain reaction-restriction fragment length polymorphism assay. Exp. Parasitol. 110, 412–415. doi:10.1016/j.exppara.2005.03.008 spectively. In addition, future studies should be aimed at extracting fi fi 19. Mayta, H. et al. (2008) Nested PCR for speci c diagnosis of Taenia solium DNA from sodium acetate-acetic acid-formalin (SAF) xed proglot- taeniasis. J. Clin. Microbiol. 46, 286–289. doi:10.1128/JCM.01172-07 tids using differentmethods as the current DNA extraction protocols 20. Nkouawa, A. et al. (2009) Loop-mediated isothermal amplification method for do not provide reliable and consistent DNA yield for PCRs (Jabbar, differentiation and rapid detection of Taenia species. J. Clin. Microbiol. 47, 168–174. doi:10.1128/JCM.01573-08 unpublished data). To date only one study has reported the extrac- 22 21. Praet, N. et al. (2013) Bayesian modelling to estimate the test characteristics of tion of DNA from long-term stored Taenia specimens in formalin coprology, coproantigen ELISA and a novel real-time PCR for the diagnosis of and this has not been independently verified. taeniasis. Trop. Med. Int. Health 18, 608–614. doi:10.1111/tmi.12089 22. Jeon, H.-K. et al. (2011) Molecular identification of Taenia specimens after long- term preservation in formalin. Parasitol. Int. 60,203–205. doi:10.1016/j.parint. References 2010.12.001 1. Roberts, L.S. et al. (2013) Foundations of Parasitology, 9th edn. McGraw-Hill Education, USA. 2. Mahanty, S. et al. (2010) Cysticercosis and neurocysticercosis as pathogens Biographies affecting the nervous system. Prog. Neurobiol. 91, 172–184. doi:10.1016/ j.pneurobio.2009.12.008 Dr Abdul Jabbar is a Senior Lecturer in Veterinary Parasitology at 3. García, H.H. et al. (2003) Taenia solium cysticercosis. Lancet 362, 547–556. The University of Melbourne. His main research interests cover doi:10.1016/S0140-6736(03)14117-7 epidemiology and diagnosis of parasites of socioeconomic impor- 4. Lightowlers, M.W. et al. (2015) Monitoring the outcomes of interventions against tance using next-generation molecular tools. Taenia solium: option and suggestion. Parasite Immunol. In press. doi:10.1111/ pim.12291 Dr Charles Gauci is a Senior Research Fellow at The University of 5. Chapman, A. et al. (1995) Isolation and characterization of species-specific Melbourne. He has worked with Prof Marshall Lightowlers through- DNA probes from Taenia solium and Taenia saginata and their use in an egg detection assay. J. Clin. Microbiol. 99, 1283–1288. out his career and his research interests focus on recombinant 6. Allan, J.C. et al. (1990) Immunodiagnosis of taeniasis by coproantigen detection. vaccines for prevention of transmission of the parasite causing – Parasitology 101,473 477. doi:10.1017/S0031182000060686 neurocysticercosis and the related parasite that causes hydatid 7. Allan, J.C. et al. (1996) Field trial of the coproantigen-based diagnosis of Taenia disease. solium taeniasis by enzymelinked immunosorbent assay. Am. J. Trop. Med. Hyg. 54,352–356. Professor Marshall W Lightowlers has been a full-time research 8. Guezala, M.C. et al. (2009) Development of a species-specific coproantigen ELISA for human Taenia solium taeniasis. Am. J. Trop. Med. Hyg. 81, 433–437. scientist supported by medical research funding for more-or-less all 9. Wilkins, P.P. et al. (1999) Development of a serologic assay to detect Taenia of his working life. He currently holds appointments as Laureate – solium taeniasis. Am. J. Trop. Med. Hyg. 60, 199 204. Professor at the University of Melbourne’s Faculty of Veterinary 10. Levine, M.Z. et al. (2004) Characterization, cloning, and expression of two and Agricultural Sciences, and Principal Research Fellow with the diagnostic antigens for Taenia solium tapeworm infection. J. Parasitol. 90, 631–638. doi:10.1645/GE-189R NHMRC.

MICROBIOLOGY AUSTRALIA * MARCH 2016 45 Lab Report

Protozoa PCR: boon or bane

Colin Pham Harsha Sheorey St Vincent’s Hospital St Vincent’s Hospital Melbourne, Vic., Australia Melbourne, Vic., Australia Email: [email protected] Email: [email protected]

Parasite detection in faeces has traditionally been performed designed to detect Giardia intestinalis, Cryptosporidium species, by microscopy, a procedure that is labour-intensive and and , four human gas- highly specialised. In addition, identification by microscopy trointestinal protozoan pathogens3. Blastocystis was not included as a based on morphological features alone is subjective and target inthismultiplex assay,as theprimers donotdifferentiate the 17 prone to wide variability. Although enzyme immunoassays differentsubtypesnowknown4,manybeingofanimaloriginandmost (EIA) of high sensitivity have been developed1 they can of doubtful clinical significance. detect only a limited range of pathogens. Given these factors the introduction of Polymerase Chain Reaction Since the commencement of testing by PCR, the rates of detection (PCR) into the routine diagnostic laboratory has improved have improved when compared with results obtained based on parasite detection rates2. The ability to multiplex has en- the previous protocol that included microscopy and Giardia/ abled the detection of multiple targets from a single sample Cryptosporidium antigen testing (Table 1). Comparison over a and provides an objective alternative to identification by three month period showed that the range of common parasites morphology. detected in the population remains similar with D. fragilis accounting for the majority of the additional parasites detected Results (Figure 1). The reduction of Cryptosporidium cases from 18 (0.8%) In February 2014, St Vincent’s Pathology, Melbourne, introduced a to 4 (0.4%) was not statistically significant and is thought to relate new testing algorithm making use of a commercial multiplex PCR to seasonal variation in prevalence. The additional E. histolytica (LightMix Parasite Kit, Roche Molecular Systems, cases were detected from microscopy-positive specimens referred Pleasanton, California, USA) for parasite detection. This assay is from other laboratories. In addition, the assay was positive for

Table 1. Three months comparison between pre and post implementation of protozoa PCR testing. Quarter 2: 2013 Quarter 2: 2014 Statistical significance Pre PCR Post PCR Samples 2198 1096 P < 0.001

G. intestinalis 24 (1.1%) 33 (3.0%) P < 0.001

C. parvum 18 (0.8%) 4 (0.4%) P = 0.132

E. histolytica – 4 (0.4%) P = 0.005

D. fragilis 47 (2.1%) 163 (14.9%) P < 0.001

Total positives 89 (4%) 204 (19%) P < 0.001

46 10.1071/MA16007 MICROBIOLOGY AUSTRALIA * MARCH 2016 Lab Report

5 180 moshkovskii . During the initial validation period, 10 Entamoeba fi 160 species identi ed by microscopy were tested by PCR and only two of these samples were positive for E. histolytica. The ability to 140 G. intestinalis Cryptosporidium spp. differentiate between pathogenic E. histolytica and non-pathogenic 120 fi E. histolytica Entamoeba species is clinically signi cant. Results concur with a

100 D. fragilis prevalence study of Entamoeba in immigrants in Spain that showed

80 that the majority of cyst-positive samples detected by microscopy 5

Total positives were E. dispar and do not require treatment . 60 ‘ ’ 40 On the other hand, PCR seemed to become a bane by detecting a large number of D. fragilis in asymptomatic people, especially 20 children6,7. Numerous parents of ‘well’ children get anxious about 0 ‘ ’ Quarter 2 – 2013: Pre PCR Quarter 2 – 2014: Post PCR this so called bad bug that does not go away . Many of these ‘ ’ Figure 1. Three months comparison of positive parasite cases pre and worried well get treated with various drugs and post implementation of protozoa PCR. combinations (some of which are known to imbalance the normal gut flora). They ‘doctor shop’ and are being referred to specialists Table 2. Protozoal infections detected by multiplex PCR February 2014 to May 2015. such as paediatricians, gastroenterologists and infectious diseases Total positive % of all physicians (communications with General Practitioners and (percentage) positives Australia and New Zealand Paediatric Infectious Diseases Group). D. fragilis 828 (15.3%) 78.0 The reliance on morphological diagnosis by microscopy of fixed G. intestinalis 183 (3.4%) 17.2 stained-smears and the recognition that parasite shedding may be intermittent, suggests that prior to PCR diagnosis, the true preva- Cryptosporidium 34 (0.6%) 3.2 spp. lence of this protozoon has been underestimated. A review of D. fragilis carriage has shown that prevalence ranges from 0.2% to E. histolyticaA 13 (0.24%) 1.22 82% in numerous geographical settings, utilising a variety of detec- Total positives 1062 (19.7%) 100 tion methods8. In this review, the authors conclude that in Total tested 5384 – ‘symptomatic patients who harbor Dientamoeba and no other pathogen, it should be considered as the etiological agent and AAdditional four liver aspirates were positive by PCR. treated’. However, they also mention that evidence is only circum- stantial. There has been debate about the clinical significance of D. fragilis given such wide variation in prevalence estimates coupled E. histolytica in liver aspirates from four cases of suspected amoebic with the observation that the protozoa may be found in symptomatic hepatitis. and asymptomatic individuals and variable responses to antipara- The detection of protozoa by molecular technology at St Vincent’s sitic drugs. A recent case-controlled study by Krogsgaard et al.6 Pathology, Melbourne, over the 15 month period since implemen- demonstrated that D. fragilis and Blastocystis were detected in a tation of the multiplex PCR assay is shown in Table 2. The rate of greater proportion of faecal samples from the asymptomatic back- D. fragilis detection increased from 2.1% by microscopy to 15.3% ground population in Denmark than from subjects with irritable with PCR; this represents the most significant finding, accounting bowel syndrome. Röser et al.7 presented data from four years of for 78% of all parasites detected by this test. routine PCR testing and found that from a total of 22,484 samples, 43% were positive for D. fragilis. With the introduction of PCR Discussion into routine testing within our laboratory, our data suggest that the prevalence of asymptomatic D. fragilis carriage is higher than The advent of PCR has definitely become a ‘boon’ for our laboratory previously reported. in providing a rapid, less labour-intensive method for detecting the three pathogenic protozoa G.intestinalis,Cryptosporidium species Of all D. fragilis detected in our study period, 40% of faeces were and E. histolytica. PCR can detect low numbers easily missed by formed, 58% were semi-formed or unformed and only 2% were microscopy and also can differentiate pathogenic E. histolytica from loose/liquid as observed in the laboratory. The clinical notes pro- morphologically similar, non-pathogenic Enta-moeba dispar/ vided on pathology request forms ranged from diarrhoea to vague

MICROBIOLOGY AUSTRALIA * MARCH 2016 47 Lab Report

50

45

40

35

30 positives

25

20 D. fragilis

15 Total

10

5

0 1 1121314151617181 Age Figure 2. Age distribution of patients positive for D. fragilis by PCR in our study.

IBS like symptoms such as abdominal pain or discomfort, altered G. intestinalis and Cryptosporidium diagnosis and superior to bowel habit and others unrelated to gastrointestinal tract. Samples microscopy for D. fragilis and E. histolytica detection. Our new that were positive for D. fragilis by PCR were predominantly in testing algorithm for parasite detection utilising PCR has shown to children below 10 years with a second peak in young adults in age improve sensitivity and specificity. It has significantly reduced the group 30–40 years (Figure 2), a similar distribution to that described time taken to stain fixed smears and microscopy, allowing for a more by Röser et al.7. efficient use of labour, whilst improving parasite detection rates. However, laboratories should consider the limitations of reporting With the more widespread adoption of faecal parasite multiplex parasites of doubtful clinical significance such as D. fragilis and PCR assays, the higher positive rate of D. fragilis by PCR reported Blastocystis species, and should alert physicians of the possibility of by laboratories (especially when diagnosed in those with non- this being non-pathogenic in immunocompetent people. specific symptoms), is confusing and of concern to clinicians. Many patients get unnecessary courses of antiparasitic drugs with more Summary of current faecal protozoa molecular testing: than half not clearing the parasite. A double-blinded, placebo- * 9 Very good at detecting low numbers of known protozoan parasites such as controlled study by Röser et al. suggests that E. histolytica, Cryptosporidium species and Giardia intestinalis treatment is not associated with better clinical outcomes. Apart * Useful for discriminating between invasive E. histolytica and from optimal therapy not being known and a highly variable re- non-pathogenic Entamoeba species sponse to treatment, the pathogenicity of D. fragilis has not been * Detects protozoa with doubtful pathogenicity such as D. fragilis at a higher reliably demonstrated. rate when compared to microscopy, leading to confusion, anxiety and unnecessary treatment

It must be noted that the use of molecular technology is limited to * Cannot differentiate animal subtypes of Blastocystis (non-pathogenic) from human subtypes (of debatable pathogenicity) the targeted screening of only a relatively small number of parasites in a low prevalence population. Microscopy still plays a key role in less common parasite identification, especially in specific patient groups where parasites other than those targeted by this multiplex are suspected. For parasite examination in populations from regions References where there is high parasite endemicity, microscopy remains the 1. Vidal, A.M. and Catapani, W.R. (2005) Enzyme-linked immunosorbent assay (ELISA) immunoassaying versus microscopy: advantages and drawbacks for diagnosing ‘ ’ gold standard with the capacity to screen for the majority of giardiasis. Sao Paulo Med. J.123, 282–285. doi:10.1590/S1516-31802005000600006 pathogens. 2. Bruijnesteijn van Coppenraet, L.E. et al. (2009) Parasitological diagnosis combining an internally controlled real-time PCR assay for the detection of four protozoa in In summary, molecular testing can offer accurate results for the stool samples with a testing algorithm for microscopy. Clin. Microbiol. Infect. 15, 869–874. doi:10.1111/j.1469-0691.2009.02894.x detection of the four commonly seen parasites in low prevalence 3. Slack, A. (2012) Parasitic causes of prolonged diarrhoea in travellers – diagnosis and populations. It has been shown to be comparable to EIA for management. Aust. Fam. Physician 41,782–786.

48 MICROBIOLOGY AUSTRALIA * MARCH 2016 Lab Report

4. Stensvold, C.R. (2013) Blastocystis: genetic diversity and molecular methods for training at St Vincent’s Melbourne in 2003, and started his career – diagnosis and epidemiology. Trop. Parasitol. 3,2634. doi:10.4103/2229-5070. in Microbiology at the Monash Medical Centre. Since re-joining 113896 ’ 5. Gutiérrez-Cisneros, M.J. et al. (2010) Application of real-time PCR for the differ- St Vincent s, he has specialised in molecular testing and emerging entiation of Entamoeba histolytica and E. dispar in cyst-positive faecal samples molecular technology. He completed a Masters of Applied Science – from 130 immigrants living in Spain. Ann. Trop. Med. Parasitol. 104, 145 149. ‘ doi:10.1179/136485910X12607012373759 (Medical Science) at RMIT in 2014, with his thesis titled Impact 6. Krogsgaard, L.R. et al. (2015) The prevalence of intestinal parasites is not greater of molecular testing on the laboratory diagnosis of Giardia among individuals with : a population-based case-control intestinalis, Cryptosporidium parvum, Dientamoeba fragilis and study. Clin. Gastroenterol. Hepatol. 13, 507–513. Entamoeba histolytica from clinical stool samples’. 7. Röser, D. et al. (2013) Dientamoeba fragilis in Denmark: epidemiological expe- rience derived from four years of routine real-time PCR. Eur. J. Clin. Microbiol. Infect. Dis. 32,1303–1310. doi:10.1007/s10096-013-1880-2 Dr Harsha Sheorey, is a Clinical Microbiologist at St Vincent’s 8. Barratt, J.L. et al. (2011) A review of Dientamoeba fragilis carriage in humans: Hospital in Melbourne. His special interest is in clinical parasitology several reasons why this organism should be considered in the diagnosis of – gastrointestinal illness. Gut Microbes 2,3–12. doi:10.4161/gmic.2.1.14755 and tropical medicine and is the author of Clinical Parasitology 9. Röser, D. et al. (2014) Metronidazole therapy for treating dientamoebiasis in a handbook for medical practitioners and microbiologists:a children is not associated with better clinical outcomes: a randomized, double- second edition has recently been published. He is an invited writer blinded and placebo-controlled clinical trial. Clin. Infect. Dis. 58,1692–1699. doi:10.1093/cid/ciu188 for the Nematodes chapter in ASM Manual of Clinical Microbiology. He coordinates the Victorian branch of Parasitology & Tropical Biographies Medicine SIG and is actively involved in organisation of the Austra- Colin Pham, is a Medical Scientist with over 10 years’ experience lian Parasitology and Tropical Medicine Master Class. in bacteriology, serology and molecular testing. He underwent his

MICROBIOLOGY AUSTRALIA * MARCH 2016 49 ASM Affairs

FT020 Water Microbiology Australian Standards Committee

Robin Woodward Email: [email protected]

I am the Australian Society of Microbiology representative on the the committee at a meeting. The discussion outcome is also docu- FT020 Water Microbiology Australian Standards Committee. This mented and any changes required are made and eventually the committee meets when there are Australian Standards (AS) that are Standard is printed. It is not an easy process. Writing an unambig- up for review (approximately every 5 years) or when a new Standard uous Standard takes time and a lot of discussion. Think back to those has been proposed and requires work. Before I became a member tasks you had at TAFE or university when you had to write instruc- on this committee I thought AS were put together by an elite group tions on how to cook a piece of toast. It is often the little things that of people and that was all they did. The Committee is made up of can be overlooked, the things you may take for granted. There are elite members, (of course that goes without saying ) but we are certain guidelines that have to be followed and are set out by people who work in the industry and most still work in the laboratory Standards Australia. The method steps are there to follow so that or have at some time in our career. There is the Standards Australia everyone using the Standard is doing the same thing therefore secretary who ensures the meeting and Standards follow the correct creating a uniform procedure that should give the same results no protocols, the Chair who runs the meeting, a NATA representative matter where you are. Standard procedures are important and it is and members comprising people like myself who are in a supervi- the basis of the quality systems in laboratories. sory role but still perform bench work in the real world. I still do bench work and that is why I enjoy the challenge of being on So please comment if you feel there is something you need to bring the Committee. It entails reading all the Standards, assessing their up when a Standard goes out for Public Comment. Even if it just a relevance and at meetings discussing any issues that may arise from typographical error or something you think is wrong-COMMENT. the testing side. These issues can be industry driven changes, Your company can subscribe to StandardsWatch so you are alerted changes to the type of testing required, the introduction and when a Standard relevant to your area is released or open for public availability of new methods or consumables, feedback from users comment. of the Standard or personal experience when using the method in the laboratory. The Standards are reviewed and it is then the Thank you to the ASM and my employer (ALS) for supporting me decision must be made on what to do with the Standard. They can be to be a committee member on FT020 Water Microbiology. It is reconfirmed (no changes), withdrawn or revised (the Committee appreciated and it is only through organisations like the ASM evaluates and revises the Standard). and generous employers that Standards Australia committees can operate. All committee members are volunteers and must have When a Standard is released for public comment it is crucial that the relevant qualifications and knowledge to ensure that these the users of the Standard read it and make any relevant comments. Standards can continue to be developed and maintained. All of these comments are documented and must be discussed by

2016 ASM Communication Ambassador Program

The ASM is looking for teams of student and ECR members to Interest (maximum 200 words) to [email protected] by 31 volunteer as part of a roster of Communication Ambassadors within March 2016 and highlight the following: the society, who will: * Your name, institution, qualifications, area of interest within microbiology, and ASM membership status; (1) Be in charge of ASM’s online communication channels together * Your strategy for broadening ASM’s online presence and collab- with a team of ASM members, bolstering the society’s networks orating with other communication ambassadors; by sharing your story with scientists, industry, media members * What conferences, scientific meetings, or events you can provide and policy makers; social media coverage for in 2016. (2) Effectively represent the society by using these channels to showcase your work, reveal personal insights into your scientific Successful applicants for the 2016 Communication Ambassador career, and serve as a public advocate for microbiology. program will receive a $100 discount for registration to the 2016 We are looking for enthusiastic members who are keen to contrib- ASM Annual Scientific Meeting (3–6 July 2016, Perth Convention & ute. To apply for the 2016 program, please submit an Expression of Exhibition Centre).

50 10.1071/MA16016 MICROBIOLOGY AUSTRALIA * MARCH 2016 ASM Affairs

Stopping dengue: recent advances and new challenges

Gayathri Manokaran, Kirsty McPherson and Cameron P Simmons

Symposium 16 November 2015 described the novel finding of a viral RNA-host protein interaction that allows dengue virus to evade the host immune response- this Peter Doherty Institute for Infection and Immunity, work was recently published in the prestigious journal, Science. Melbourne A highlight of this symposium was the ‘Public Health’ session, which Dengue remains a major problem throughout the world with an was the first of its kind in Australia, with speakers from various estimated 30% of the world’s population at risk of infection. In the companies including Sanofi Pasteur and Takeda, coming together to past few years, major advances have been made across virology, discuss their respective dengue vaccines, thereby providing atten- clinical insights, vaccines and mosquito control strategies. The first dees with a useful summary of all the dengue vaccines currently in of its kind to be ever held in Australia, the International Dengue clinical development. Symposium 2015 showcased some of the best science and clinical/ public health research being undertaken in the field. Novel and highly innovative medical technologies were next dis- ‘ ’ Proudly organised by The Peter Doherty Institute for Infection and cussed in the Clinical Research session, with Dr Jenny Low Immunity, under the leadership of Professor Cameron Simmons, explaining how in vivo imaging technologies such as positron this symposium brought together 130 national and international emission tomography are being optimised presently to track den- dengue researchers. The organising committee acknowledges the gue virus progression in a mouse model. Dr Sophie Yacoub gave a support of ASM as an important partner to this meeting, together summary on changes in microvasculature of dengue patients upon with generous support from The University of Melbourne, The infection and how these disturbances might precede the develop- Royal Melbourne Hospital, the Oxford University Clinical Research ment of severe disease.

Unit, Vietnam and the DUKE-NUS Graduate Medical School, In the next session, Dr Laura Rivino discussed their work on fi Singapore. Industry sponsors included Sano Pasteur, BioTools, elucidating the human cellular responses during dengue infection fi VWR International, In Vitro Technologies, Paci c Lab Products, and how both CD4+ and CD8+ T-cell subsets remain enriched in Interpath Services and Alere Global. the skin of dengue patients up to 5 months post infection. Professor The formal symposium was opened by Professor Cameron Cameron Simmons then described an interesting dengue neutra- Simmons. This was followed by a series of 21 brief talks spanning lisation assay developed in Vietnam using patients’ viraemic blood across four main themes of dengue research: Virology, Public and infected mosquitoes as a readout of the efficacy of monoclonal Health, Clinical Research and Immunology/Pathogenesis. Short antibodies to efficiently neutralise dengue virus. Next, Dr David breaks for networking were scheduled in between each of these Muller introduced the concept of nanopatches that allow vaccine four sessions. delivery in a needle-free manner.

All of the invited speakers had considerable international profiles in In closing, the sponsors were wholeheartedly acknowledged for dengue and hence the quality of the presentations was very high. their contributions by Professor Cameron Simmons and their Associate Professor Sheemei Lok discussed the structural basis support was instrumental in allowing this symposium to be a behind how a dengue-2-specific human monoclonal antibody success. Not only was the stellar line-up of talks highly educational effectively protects mice from dengue infection due to its ability but importantly, this symposium provided a networking opportu- to ‘lock’ the dengue envelope proteins while blocking the binding nity for everyone interested in meeting the challenge of reducing of enhancing antibodies. Following that, Professor Ooi Eng Eong dengue burden throughout the world.

MICROBIOLOGY AUSTRALIA * MARCH 2016 10.1071/MA16017 51

Confirmed Plenary speakers

Professor Peter Professor Dan Assoc Prof Susan Lynch Dr Brian Conlon Professor Anna Hawkey Andersson University of California Northeastern Durbin University of Upsalla University San Francisco University, Boston Johns Hopkins Birmingham Environmental pollution , Crohn's Disease Drug discovery in Dengue and vaccines Nosocomial by antibiotics and its and Microbiome soil bacteria infection control and role in the evolution of Research antibiotic resistance resistance

As with previous years, ASM 2016 will be co-run with NOW CONFIRMED! EduCon 2016: Microbiology Educators’ Conference 2016 Rubbo Oration Watch this space for more details on the scientific and Professor Anne Kelso social program, speakers, ASM Public Lecture, workshops, CEO NHMRC ASM awards, student events, travel awards, abstract deadlines and much more..

Perth, WA A vibrant and beautiful city located on the banks of the majestic Swan river. Come stay with us in WA and experience our world class wineries and restaurants, stunning national parks, beaches and much more..

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