No Evidence for Widespread Babesia Microti Transmission in Australia

Faddy Helen (Orcid ID: 0000-0002-3446-8248)

Viennet Elvina (Orcid ID: 0000-0002-1418-1426)

No evidence for widespread Babesia microti transmission in Australia

Helen M Faddy1,2, Kelly M Rooks1, Peter J Irwin3, Elvina Viennet1, Andrea Paparini3, Clive R Seed4,

Susan L Stramer5, Robert J Harley6, Hiu-Tat Chan7, Peta M Dennington8, Robert LP Flower1

1Research and Development, Australian Red Cross Blood Service, Brisbane, Queensland, Australia

2School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia

3Murdoch University, Perth, Western Australia, Australia

4Clinical Services and Research, Australian Red Cross Blood Service, Perth, Western Australia,

Australia

5American Red Cross Scientific Affairs, Gaithersburg, Maryland, USA

6Clinical Services and Research, Australian Red Cross Blood Service, Brisbane, Queensland, Australia

7Clinical Services and Research, Australian Red Cross Blood Service, Melbourne, Victoria, Australia

8Clinical Services and Research, Australian Red Cross Blood Service, Sydney, New South Wales,

Australia

Corresponding author: Helen Faddy; 44 Musk Avenue, Kelvin Grove, Queensland, Australia, 4059;

TEL: +617 3838 9262 FAX: +617 3838 9428; [email protected]

Source of support: Australian governments fund the Australian Red Cross Blood Service to provide

blood, blood products and services to the Australian community.

Conflict of interest: All authors report no conflicts of interest.

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/trf.15336

Word count (excluding abstract, references and illustrations): 3668

BACKGROUND: A fatal case of autochthonous Babesia microti infection was reported in Australia in

2012. This has implications for Australian public health, and, given babesiosis is transfusion- transmissible, has possible implications for Australian blood transfusion recipients. We investigated the seroprevalence of antibodies to B. microti in Australian blood donors and in patients with clinically- suspected babesiosis.

STUDY DESIGN AND METHODS: Plasma samples (n = 7,000) from donors donating in at risk areas and clinical specimens from patients with clinically-suspected babesiosis (n = 29) were tested for B. microti IgG by immunofluorescence assay (IFA). IFA initially reactive samples were tested for B. microti IgG and IgM by immunoblot and B. microti DNA by PCR.

RESULTS: Although five donors were initially reactive for B. microti IgG by IFA, none was confirmed for B. microti IgG (zero estimate; 95% CI: 0–0.05%) and all were negative for B. microti

DNA. None of the patient samples had B. microti IgG, IgM or DNA.

CONCLUSIONS: This study does not provide evidence for widespread exposure to B. microti in

Australian blood donors at local theoretical risk, nor does it provide evidence of B. microti infection in

Australian patients with clinically-suspected babesiosis. Given that confirmed evidence of previous exposure to B. microti was not seen, these data suggest transmission of this pathogen is currently uncommon in Australia, and unlikely to pose a risk to transfusion safety at present.

Key words: babesiosis, Babesia microti, transmission, Australia, transfusion, safety

INTRODUCTION

Babesiosis is an emerging tick-borne zoonotic infectious disease.1-4 The first human case of autochthonous babesiosis in Australia, a fatality associated with Babesia microti infection, was reported in 2012.5 This case was a 56 year old male with no history of transfusion, intravenous drug use or travel to countries endemic for babesiosis. 5 The infection was therefore believed to have been from a tick bite.5 This has implications for Australian public health and blood transfusion safety, as transfusion-transmitted (TT) babesiosis has been documented.6 Furthermore, this finding prompted questions in relation to the scale of autochthonous babesiosis in Australia.

Babesiosis presents as a blood-borne haemolytic disease caused by intracellular tick-borne protozoan parasites of the genus Babesia. Over 100 Babesia spp. have been identified worldwide, with a subset being pathogenic in humans. Infection in humans can be low-grade and persistent; however, can be more severe in the immunosuppressed, asplenic, and elderly. Human babesiosis occurs sporadically in

Europe, Asia, Africa and the Americas, with endemic transmission in the northeastern and upper midwestern regions of the USA. In the USA, the rodent species B. microti is the most common species associated with human disease,6 as well as being the species implicated in the only autochthonous

Australian case to date.5 In Europe, the majority of cases in humans are due to the cattle species B. divergens.4 Other species of Babesia can infect animals; indeed babesiosis is well known as an introduced disease in Australian cattle and dogs, and as an endemic infection in native mammals.7-10

The life cycle of B. microti involves a reservoir vertebrate host and a vector tick. Transmission to humans and other mammalian hosts is usually through the bite of an infected ixodid tick.6 A myriad of

Ixodes ticks reside in Australia, the most common and perhaps medically relevant being Ixodes holocyclus, which can be found along the east coast.11,12 Ixodes holocyclus has been implicated in tick paralysis and transmission of rickettsial infections.13-15 To date, no Australian ticks have been shown to transmit B.microti.

The report of locally acquired babesiosis in Australia5 heightened public and governmental concern in relation to this disease. Thus, the risks posed by this parasite to Australian blood transfusion safety and public health needed to be assessed. This study investigated the prevalence of B. microti in blood donors residing in areas with theoretical risk, based on proximity to where the Australian case resided and also within the distribution range of I. holocyclus, the most common and perhaps medically relevant tick within Australia and in a cohort of Australian patients with clinically-suspected babesiosis. Although other species of Babesia are capable of causing disease in humans, we focused on

B. microti as this organism was responsible for the majority of historical TT cases (159/162 cases between 1979-2009 in the USA),16 and was the cause of the only known case of autochthonous babesiosis in Australia,5 the identification of which was the impetus for this study.

MATERIALS AND METHODS

Blood donor samples

This was a de-linked cross-sectional study of Australian blood donors aged 16 to 80 years inclusive, donating in areas considered ‘at-theoretical risk’ for potential exposure to B. microti. To the best of our knowledge there has been no previous investigation of seroprevalence of antibodies to Babesia spp. in

Australia, therefore sample size calculations were based on overseas estimates available when the study was designed. Although not ideal, as such estimates are from areas endemic for babesiosis and were based on unconfirmed results, they were used to provide some insight into an approximate required sample size. Assuming a similar seroprevalence to that recorded for B. microti in endemic regions of the USA (1.1%),17 testing ~7,000 samples would give a 95% confidence interval (CI) that was considered acceptable and fundable. ‘At-theoretical risk’ regions were defined as those in the

North/South Coast of New South Wales (NSW; region where the Australian case resided and also within the distribution range of I. holocyclus) or in coastal Queensland (QLD; also areas enzootic for I. holocyclus).5,18 Samples were collected between 29 December 2012 and 26 September 2013 from donors residing in the following areas and their surrounds: Cairns (n = 162), Townsville (n = 498),

Mackay (n = 261), Rockhampton (n = 586), Gladstone (n = 244), Fraser Coast (n = 503), Sunshine

Coast (n = 522), Toowoomba (n = 257), and Ipswich (n = 458) in QLD (n = 3,491 total); as well as

Lismore (n = 323), Coffs Harbour (n = 118), Port Macquarie (n = 232), Newcastle (n = 577), Gosford

(n = 310), Wollongong (n = 827), Camden (n = 383), Bateman’s Bay (n = 480) and Bega (n = 259) in

NSW (n = 3,509 total). Where possible, samples were collected from mobile collection facilities in the above areas; for many centres/mobile sites, particularly those with small collection numbers, these samples represented all available donors/samples during the study period.

Samples were collected into 6 mL dipotassium ethylenediaminetetraacetic acid (K2EDTA) sample preparation tubes (Becton Dickinson) and centrifuged (1,000 g, 10 min) within 72 h of collection.

Samples were sourced from those remaining after routine infectious disease testing was complete and where adequate volume remained. Plasma was aliquoted and stored at -30 ºC. Demographic information (age, sex, postcode, residence state and a basic travel history) was obtained for each sample prior to de-identification. Ethical approval was obtained from the Australian Red Cross Blood

Service (Blood Service) Human Research Ethics Committee.

Patient samples

Samples from patients with clinically-suspected babesiosis were sourced via their medical practitioners, along with a basic clinical history. Some patients had been tested for other tick borne illnesses (TBIs), however, any samples positive for antibodies to Babesia duncani or Borrelia spp. were excluded given the focus on B. microti and given no reported autochthonous cases of B. duncani or Borrelia spp. in Australia to date. Ethical approval was obtained from Murdoch University.

B. microti serological testing

All samples (n = 7,000 donor; n = 29 patient; n = 1 from the Australian case) were tested for B. microti

IgG with an indirect immunofluorescence assay (IFA; Imugen, Inc.). This assay had a reported 99.5% sensitivity and > 99.98% specificity in North American populations.16,19,20 Samples with a titre <64 were considered negative, while those ≥64 were considered initially reactive and tested by immunoblot

(IgM and IgG separately). Samples that tested IgG positive by immunoblot were considered confirmed positive. Serological testing was performed by Imugen, Inc. in the USA, as per standard screening protocols and as published elsewhere.19,20

B. microti molecular testing

Genomic DNA was purified from B. microti IFA initially reactive samples, whole blood/EDTA (200

µL) from patient samples (n = 29) and molecular grade water (extraction blanks), using the MasterPure

Purification Kit V.2 (Epicentre Biotechnologies). Purified DNA was resuspended in TE buffer (50 µL) and checked by spectrophotometry by Nanodrop (ThermoFisher Scientific). Replicate nested amplifications of piroplasmid-specific DNA targeting the small subunit ribosomal RNA gene (18S rDNA) used the primer pairs BT F1/R1 and BT F2/R2.21 PCR positive controls included Babesia gibsoni-positive canine genomic DNA. To rule out false negative results due to PCR amplification inhibition, piroplasm-positive canine DNA (0.5 µL) was added to random human test samples, prior to

PCR (spike analyses). Human DNA was also amplified at multiple dilutions. No template controls consisted of molecular-grade water. PCR products (15 µL/lane) were run on a 1% agarose gel containing SYBR Safe Gel Stain (Invitrogen), and visualized with a dark reader trans-illuminator

(Clare Chemical Research).

Anti-Plasmodium testing

To explore potential cross-reactivity, samples testing IFA initially reactive were tested with the Trinity

Biotech Captia Malaria Total Antibody EIA (Trinity Biotech)), as described previously.22 Briefly, this sandwich EIA incorporates four recombinant blood-stage antigens (three for P. falciparum and one for

P. vivax) and can detect malaria-specific IgG, IgM and IgA.

Mapping

To visualize the spread of donor samples, the number of samples collected from each postcode was mapped and overlayed with the location of I. holocyclus occurrences,12 and the location of the single

Australian B. microti case.5 All geographic layers were processed using ArcGIS 10.4 (ESRI).

Statistical analysis

Exact CIs were calculated for individual proportions (B. microti IgG initial reactivity).

RESULTS

To ensure the suitability of the IFA, we first confirmed that this assay was able to detect antibody in a sample from the only autochthonous B. microti case in Australia.5 A small aliquot was made available for this study and had a titre of 64 on the IFA. Unfortunately, there was insufficient volume to allow for immunoblot, however, given the observed titre on the B. microti IFA, and extensive prior testing,5,23 this sample was interpreted to have detectable B. microti IgG on this assay.

Samples were collected from donors donating along the east coast of Australia in theoretical risk areas

(Figure 1; Table 1). Five donors were initially reactive for B. microti IgG by IFA; however, all were negative for B. microti IgG by immunoblot, with one positive for B. microti IgM (Table 2). Given none of the initially reactive samples were confirmed for B. microti IgG, a zero estimate was assumed, with a one-sided 95% CI (95% CI: 0–0.05%). B. microti DNA was not detected in any initially reactive sample, all of which had no detectable anti-Plasmodium antibodies and all were from donors who had reported prior overseas travel.

Samples from Australian patients with clinically-suspected babesiosis were tested with the same assays

(Table 3). These patients were classified as suspected babesiosis by their medical practitioners. All were negative for B. microti IgG (IFA and immunoblot) and IgM (immunoblot). This included one patient with B. microti IgM on previous testing. B. microti DNA was not detected in any patient samples.

DISCUSSION

In response to the reported case of B. microti infection in Australia,5 we undertook this study to assess the scale of endemic babesiosis in Australia and investigate risk for public health and blood transfusion safety. We observed no convincing evidence of confirmed B. microti infection in donors or patients with clinically-suspected babesiosis. Our study provides additional supporting information that babesiosis due to B. microti infection is currently a rare event in Australia. The rarity is additionally supported by the lack of further cases of locally acquired babesiosis in the 6 years since the only autochthonous case was reported. This finding correlates with the absence of the known B. microti vector, Ixodes scapularis, in Australia. Collectively our study supports the hypothesis made by

Senanayake et al 5 that the single reported case of locally-acquired B. microti infection in Australia was an isolated event.23

To facilitate testing of donors with a greater potential risk of exposure to B. microti, we targeted collections from donors donating in theoretical risk areas, defined by proximity to the autochthonous case and the presence of a tick, I. holocyclus, well known to bite people.5,18 Five donors were initially reactive for B. microti IgG, however, four of the five had low/extremely low titres (64-128; the USA centres for disease control and prevention state laboratory supportive evidence for B. microti includes an IFA IgG antibody titre ≥1:256)24. Moreover, we were unable to confirm the presence of this antibody in any of the samples by IgG immunoblot, including in one high-titre (≥1,024) sample, highlighting the importance of confirmatory testing in such studies. As with any test used for screening

(rather than diagnosing) infections with a low incidence or in healthy individuals, false positivity is a potential concern, and the positive predictive value of a test can be low even when the specificity and sensitivity of a test are high. In addition, all initially reactive samples had undetectable B. microti DNA indicating none were from individuals actively infected at a level that could be detected by this assay.

One low-titre IgG initially reactive unconfirmed sample had detectable IgM on immunoblot; while true

B. microti IgM positivity is a possibility, IgM tests are well known to lack specificity and in the

absence of B. microti DNA this result was considered unconfirmed. Regardless of the unconfirmed status of these initially reactive samples, it is reassuring that all were from donors who had previously travelled overseas, however, the exact travel destination was unknown. Moreover, as we targeted collections from donors donating in theoretical risk areas, the prevalence among the total donor population would no doubt be lower. While we examined for potential malaria antibody cross- reactivity, the potential for cross-reactivity with other pathogens, including TBIs, cannot be excluded.

Australia has a large number of endemic ticks associated with native wildlife, and three introduced tick species of cattle and dogs.26 Given current interest among the Australian general public and also the medical community into TBIs, evidenced by parliamentary hearings and patient advocacy groups, large-scale studies are needed to understand what pathogens Australian ticks carry and whether they are capable of causing human disease.

We did not detect B. microti antibodies or DNA in samples from patients with clinically-suspected babesiosis, which included one patient with detectable B. microti IgM from previous testing. It should be highlighted that although babesiosis was suspected by their referring medical practitioner, none had laboratory-confirmed babesiosis infection. This highlights the need for appropriate confirmatory testing, and also the need to use only accredited laboratories for testing suspected TBI patients, as has been discussed extensively.15 Currently, with the exception of rickettsiosis and Coxiellosis, specific

TBIs have not been consistently identified in Australia. Until such organisms are identified, serological tests using antigens from overseas TBIs only contributes to uncertainty about autochthonous TBIs in

Australia. B. microti infection should therefore continue only to be investigated in patients with a travel history to areas endemic for this parasite, and diagnoses made only after repeat and comprehensive testing at accredited facilities.

Given the lack of evidence for exposure to B. microti in this cohort of donors and patients, autochthonous transmission of this parasite is of limited risk to the safety of Australia’s blood supply.

However, there remains a possible risk of TT babesiosis from overseas acquired-infections. The Blood

Service has taken a precautionary approach using risk mitigation strategies, including a permanent donation restriction for individuals with a history of the disease and medical review of any donor with systemic illness after a tick bite. In addition, fresh blood components are recalled if a donor becomes unwell after a tick bite or is diagnosed with babesiosis, donates within four weeks prior to their illness and notifies the Blood Service. However, given the high rate of asymptomatic infection in donors and the duration of parasitaemia,16 these measures would have minimal effectiveness in preventing potentially infectious donors donating as observed in the USA where the risk of reported TT-babesiosis outside ‘at-risk’ states from travel is less than 1 in 10 million.16 Since the risk of TT from overseas- acquired infection in Australia is expected to be lower than the non-endemic risk in the USA, such risk is considered negligible. Given the results from this study, we feel this current approach for the management of TT B. microti is appropriate, however, the Blood Service will continue to monitor this area and, should local epidemiology change, modify this management strategy accordingly. A similar approach may be suitable for other countries with an analogous situation.

Our study is not without limitations. Due to the de-linked nature of this study we were unable to trace study results back to individual donors and thus not able to retrospectively question donors regarding a history of tick-bite or travel to countries/regions endemic for babesiosis nor able to take follow-up samples. We attempted to target collections from donors residing in rural/peri-urban areas of Australia including the region where the autochthonous Australian case resided, based on the location of the donor centre they donated at, in an attempt to identify donors with an increased risk of exposure to ticks. It is possible our sampling approach may not have achieved our goal. It is also possible that our sample size was too small to accurately estimate low prevalence considering that sample size estimates were determined from the highest prevalence areas in the USA, using screening results in the absence of confirmation. Subsequent studies, using the same test used in this study, in these high prevalent areas in the USA, gave a confirmed-positive rate of 0.38%.16 For logistical purposes, testing was performed on frozen samples; it is possible that antibody or DNA stability may have been affected, however, this is unlikely as previously documented.20.

This was the first B. microti prevalence study in Australia. Together with the lack of further autochthonous cases, our study provides no evidence for widespread B. microti transmission in

Australia and supports the assumption that autochthonous babesiosis due to infection with this parasite is infrequent in Australia at this time and unlikely to currently pose a significant risk to the safety of the

Australian blood supply.

Acknowledgments

We thank Blood Service staff who assisted with this study, especially K.Winter and S.Tan for selecting

NSW samples; J.Fryk for assisting with sample management; the NSW testing team for performing malaria EIA testing; A.Keller, P. Kiely and H.Yang for assisting with study design; and V.Hoad for reviewing the manuscript. We also thank M.Busch and E.Bloch for providing advice on study design,

S.Senanayake and staff from the USA CDC for facilitating access to the sample from the 2012 reported case, and K.Weeks, V.Berardi, and D.Krysztof for facilitating transport and testing.

REFERENCES

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19. O'Brien SF, Delage G, Scalia V, Lindsay R, Bernier F, Dubuc S, Germain M, Pilot G, Yi QL, Fearon MA. Seroprevalence of Babesia microti infection in Canadian blood donors. Transfusion 2016;56: 237-43. 20. Moritz ED, Winton CS, Johnson ST, Krysztof DE, Townsend RL, Foster GA, Devine P, Molloy P, Brissette E, Berardi VP, Stramer SL. Investigational screening for Babesia microti in a large repository of blood donor samples from nonendemic and endemic areas of the United States. Transfusion 2014;54: 2226-36. 21. Jefferies R, Ryan UM, Irwin PJ. PCR-RFLP for the detection and differentiation of the canine piroplasm species and its use with filter paper-based technologies. Vet Parasitol 2007;144: 20-7. 22. Faddy HM, Seed CR, Faddy MJ, Flower RL, Harley RJ. Malaria antibody persistence correlates with duration of exposure. Vox Sang 2013;104: 292-8. 23. Paparini A, Senanayake SN, Ryan UM, Irwin PJ. Molecular confirmation of the first autochthonous case of human babesiosis in Australia using a novel primer set for the beta-tubulin gene. Exp Parasitol 2014;141: 93-7. 24. system CfDCaPnnds. Available from: https://wwwn.cdc.gov/nndss/conditions/babesiosis/case-definition/2011/ 25. Shrestha AC, Flower RL, Seed CR, Stramer SL, Faddy HM. A Comparative Study of Assay Performance of Commercial Hepatitis E Virus Enzyme-Linked Immunosorbent Assay Kits in Australian Blood Donor Samples. J Blood Transfus 2016;2016: 9647675. 26. Greay TL, Oskam CL, Gofton AW, Rees RL, Ryan UM, Irwin PJ. A survey of ticks (Acari: Ixodidae) of companion animals in Australia. Parasit Vectors 2016;9: 207.

FIGURE LEGENDS

Figure 1. Geographic spread of donors included in study and recorded I. holocyclus occurrences.

Donor numbers represented by shading; I. holocyclus recorded occurrence represented by green/blue dots (data obtained from 12), location of single locally acquired Australia case of B. microti infection delimitated by dotted circle.5

TABLES

Table 1: Demographic characteristics of donors included in study.

Age group (years) Total Proportion with <25 25-34 35-44 45-54 55-64 >65 n (%) overseas travel

80% Female 457 350 496 818 916 327 3,364 (48%)

78% Male 381 377 492 868 964 554 3,636 (52%)

79% Total 838 727 988 1,686 1,880 881 7,000

Table 2: Testing results and demographics of the five B. microti IgG initially reactive, but unconfirmed, donor samples.

Study Age Sex State Region Overseas B. microti testing Malaria # travel EIA IFA - IgG Immunoblot PCR Interpretation Titer Result IgM IgG 102 55 F NSW Bega Yes 64 IR N N N Unconfirmed N 446 44 F NSW Batemans Bay Yes 64 IR N N N Unconfirmed N 3074 66 M NSW Newcastle Yes 128 IR N N N Unconfirmed N 3527 54 F QLD Rockhampton Yes ≥1024 IR N N N Unconfirmed N 6971 53 F QLD Bundaberg Yes 64 IR P N N Unconfirmed N IR: initial reactive; N: negative; P: positive NSW: New South Wales; QLD: Queensland

Table 3: Characteristics of patients with clinically-suspected babesiosis included in study.

Patient State Age Sex Symptoms Babesiosis Overseas Blood IV # diagnosis travel transfusion Drugs method Tick bite

1 WA 51 F Chest tightness, Clinical only No Australia No No shortness of breath. dark urine, night sweats, vivid dreams, truncal haemangiomata, ecchymoses. 4 NSW 52 F Air hunger, fatigue Clinical only Yes Australia No No 5 NSW 22 F Skin rash, air hunger, Clinical only Yes Unknown No No fatigue 6 NSW 55 F Breathlessness, chest Clinical only No Australia No No pains, sternum pain, visual dreams, night sweats, haemangiomas 7 NSW 38 F Nodes under skin, Clinical only Yes Unknown No No vision problems 8 NSW 31 F Fatigue, myalgia, brain Clinical only No Australia No No fog, GI pain, poor concentration, memory loss, irritability 9 NSW 41 F Neurological signs, Clinical only Yes Unknown No No chronic pain, fatigue, night sweats, patches on skin, vertigo, right- sided paralysis 10 NSW 45 F Weakness, rash, Clinical only Yes Unknown No No fatigue, swollen glands, bowel signs 11 NSW 22 M Chronic fatigue Clinical only Yes Australia No No syndrome 12 VIC 39 M Cranial symptoms, Clinical only Yes Australia Yes No burning feet, brain fog, migratory pains 13 WA 52 F Headache, shortness of Clinical only Yes Unknown No No breath, palpitations, breast bone pain 14 WA 48 F Intermittent cough, B. microti IgM No Unknown Yes No back pain, heart (titre 40)* palpitations, skin B. microti DNA rashes, GI issues, brain negative* fog, swollen glands, headaches, visual issues, chronic fatigue 15 WA 40 F None provided Not sighted NA Unknown No No

16 NSW 41 M Crawling on skin Not sighted Yes Unknown No No 17 NSW 23 M Headaches, joint pain, Clinical only No No No No lack of coordination, confusion 18 NSW 23 F Multiple brain lesions, Clinical only No Australia No Yes fatigue, drop foot, numbness, migraines, lack of coordination, loss of sight (and more) 19 NSW 34 F Extreme chronic Clinical only No Unknown No No fatigue, extreme pain (and more)

20 VIC 48 F Many indicative signs Clinical only Yes Unknown No No

22 WA 15 M Tourette’s syndrome Clinical only Yes Australia No No since 2010 24 NSW 32 F Chest pain, chronic Clinical only No Australia No No fatigue, creaky neck, night sweats, malaise 25 NSW 43 M Respiratory symptoms Clinical only Yes Unknown No NA (shortness of breath, heavy breathing), sore throat, malaise, mild sore joints, chills, sweats, chronic fatigue 26 NSW 28 F Fevers, nausea, Clinical only Yes Australia No No vomiting, diarrhoea, night/day sweats, haemolytic anaemia, weight loss, muscle pain, weakness, petechiae, loss of appetite, joint pain, air hunger, respiratory failure, light sensitivity 27 WA 53 F Headaches, joint Clinical only Yes Australia No No aches, chest pain, vomiting, diarrhoea 30 NA F Vivid dreams, dark Clinical only No Australia No No urine, excessive bruising 31 NSW 39 M Light-headedness, Clinical only Yes Australia Yes No chest and jaw pain, (specialist in thirst, night sweats, USA) shaky vision, muscle weakness and fatigue, insomnia 32 WA 47 M Shortness of breath, Clinical only Yes Australia Yes No palpitations, vivid dreams, haematuria, night sweats 34 NA M Vivid dreams, night Clinical only Yes Australia NA NA sweats, haemangiomata, dark urine 35 WA 32 F Short of breath on Clinical only Yes Unknown No No minimal exertion, high sternal chest weight, vivid dreams, dark urine, ecchymoses 36 WA 38 F Memory loss, joint Clinical only Yes Australia No Yes aches, hair loss, and palsy?, nausea, fatigue, Overseas skin reactions, sun light sensitivity, noise sensitivity, food intolerances. NA not available (unknown); GI: gastrointestinal

*Testing performed at IGeneX, Inc., Palo Alto, California