Serological Diagnosis Serological Diagnosis of of Strongyloides Strongyloides

stercoralisstercoralis infection. infection.

Ian SampsonIan SampsonDr David. W. SmithDr David. W. SmithBrian MacKenzieBrian MacKenzie

July 2003July 2003

Copyright, 1996 © Dale Carnegie & Associates, Inc.

Laboratories performing Laboratories performing Strongyloides IFA testing Strongyloides IFA testing in Australia. in Australia.

•ICPMR - Rogan Lee - also maintains life cycle of S.ratti•PathCentre Ian Sampson•QML - Robyn Wood (ICPMR Ag)•VIDRL - Jennie Leydon (PathCentre Ag)

Problems associated with Problems associated with Strongyloides Serology Strongyloides Serology testing in Australia. testing in Australia.

•Validation of method•Uncertainty•Difficulty of antigen production•Standardisation/Proficiency testing

ParasiteParasite Isolations W.A. Isolations W.A. 20022002

Blast. hominis 258 228 248 230 964G. intestinalis 201 170 182 154 707Ent. coli 75 56 63 46 240Endolimax. nana 83 41 57 30 211Crypt. parvum 60 28 33 46 167H. nana 47 42 31 20 140S. stercoralis 25 31 18 14 88Iodam. buetschlii 29 18 24 14 85Hookworm 26 7 13 11 57Chilomastix mesnili 23 13 11 8 55Ent. hartmanni 8 7 6 9 30Ent. polecki 12 6 5 4 27Ent. spp 3 4 7 9 23S. mansoni 6 0 8 4 18T. hominis 3 1 4 10 18E. vermicularis 3 1 4 5 13T. trichiura 6 1 3 3 13A. lumbricoides 10 1 0 0 11Ent. histolytica 4 1 3 2 10

Pathogenic organisms

Serosurveys W.A.Serosurveys W.A.Location Strongyloides Strongyloides Toxocara total Nos No/Pos% canis No/ Pos%

Kalumburu 1999 229 26/11.4*Billiluna 1990 98 36/37.0Kalumburu 1986** 44 2/5.0One Arm Point 1986 67 3/ 4.5 7/10.4Looma 1986 57 33/58.0 6/10.5Lombardina 1986 30 3/10.0 12/40Beagle Bay 1986 28 5/17.8 4/14.3

* 9% pos Melioidosis **1986 surveys age range 5-18. LaTrobe 1989 n=372 NE Arnhem Pop.Strongy 62%+ Toxocara 41%+

S.stercoralisS.stercoralis Isolations W.A. Isolations W.A. 1998-021998-02

Year 1st Qtr 2nd Qtr 3rd Qtr 4th Qtr Total1998 31 32 23 15 1011999 25 29 26 23 1032000 29 75 39 27 170 139*

2001 34 29 26 32 1212002 25 31 18 14 88 55*

* excluding repeats

S.stercoralisS.stercoralis Isolations W.A. Isolations W.A. 2000 & 2002 source2000 & 2002 source

Region Year

2000 2002

Kimberley 125 51

Pilbara 11 2

Goldfields 3 2

Perth

Bililuna

Kalumbaru

Looma

One Arm Point

Beagle Bay

Lombadina

W.A. Community LocationsW.A. Community Locations

Toxocara Absorption. Lynch et al 1988 Toxocara Absorption. Lynch et al 1988 Parasite Immunology. (Example of EIA Parasite Immunology. (Example of EIA pre-absorption vs competitive pre-absorption vs competitive inhibition)inhibition)

Competitive inhibition of Competitive inhibition of ToxocaraToxocara (TcESA) ELISA . Lynch et al 1988 (TcESA) ELISA . Lynch et al 1988 Parasite ImmunologyParasite Immunology

Extract % sera Inhibited* % InhibitionAscaris ** 75 24.5 +/- 13.6Necator/Ancylostoma 38 16.0 +/- 7.1Trichuris/Enterobius 20 14.0 +/- 5.3Strongyloides*** 0 0Dirofilaria 11 10.0 +/- 1.1Schistosoma/Taenia 19 16.7 +/- 5.8Entamoeba/Giardia 31 17.8 +/- 9.4Leishmania/Trypanosoma 25 11.7 +/- 1.0

*48 TcESA pos sera with wide range of values.** suum. ***ratti.

Lynch’s investigation of helminth Lynch’s investigation of helminth cross reactions in the Toxocara cross reactions in the Toxocara E/S EIA suggested results in the E/S EIA suggested results in the equivocal/low positive area were equivocal/low positive area were more likely to be caused by cross more likely to be caused by cross reaction.reaction.

Pre-absorption study with Pre-absorption study with S.rattiS.ratti EIA - PathCentre. (Aust Northern EIA - PathCentre. (Aust Northern Indigenous Group)Indigenous Group)

Abs or % inhib change <10% not considered significant.Absorbed with OD

1.18 1.00 1.76 0.59 0.85S.ratti 0.03 0.03 0.07 0.04 0.03Dirofilaria 1.10 1.00 1.73 0.55 0.85Ancylostoma 0.88 0.96 1.50 0.49 0.82Toxocara 0.92 0.97 1.67 0.54 0.82Schistosoma 1.10 0.95 1.64 0.57 0.79Taenia 1.02 0.97 1.70 0.54 0.83Ascaris 1.05 0.96 1.52 0.47 0.85

% Inhib Av % InhibS.ratti 97.5 97.0 96.0 93.2 96.5 96.0Dirofilaria 6.8 0.0 1.7 6.8 0.0 3.1Ancylostoma 25.4 4.0 14.8 16.9 3.5 12.9Toxocara 22.0 3.0 5.1 8.5 3.5 8.4Schistosoma 6.8 5.0 6.8 3.4 7.1 5.8Taenia 13.6 3.0 3.4 8.5 2.4 6.2Ascaris 11.0 4.0 13.6 20.3 0.0 9.8

Pre-absorption study with Pre-absorption study with S.rattiS.ratti EIA - PathCentre (Amazon Indian EIA - PathCentre (Amazon Indian Group)Group)

Absorb Ag OD

Unabsorbed 1.78 1.60 1.63 1.40 1.10S.ratti 0.10 0.10 0.08 0.17 0.06Dirofilaria 1.57 1.60 1.47 1.20 1.10Ancylostoma 1.71 1.56 1.47 1.10 1.00Toxocara 1.54 1.56 1.53 1.30 1.04Schistosoma 1.66 1.43 1.42 1.23 0.92Taenia 1.56 1.40 1.44 1.32 0.97Ascaris 1.50 1.37 1.32 1.30 0.93

% Inhib Av % InhibS.ratti 94.7 93.8 95.1 87.9 94.5 93.2Dirofilaria 11.8 0.0 9.8 14.3 0.0 7.2Ancylostoma 3.9 2.5 9.8 21.4 9.1 9.4Toxocara 13.5 2.5 6.1 7.1 5.5 6.9Schistosoma 6.7 10.6 12.9 12.1 16.4 11.8Taenia 12.4 12.5 11.7 5.7 11.8 10.8Ascaris 15.7 14.4 19.0 7.1 15.5 14.3

Pre-absorption study with Pre-absorption study with S.rattiS.ratti EIA - PathCentre (Aust WWII EIA - PathCentre (Aust WWII Group)Group)

Absorb Ag OD

Unabsorbed 1.30 1.37 0.96 1.22 1.49S.ratti 0.04 0.03 0.01 0.03 0.08Dirofilaria 1.30 1.37 0.96 1.10 1.49Ancylostoma 1.30 1.37 0.87 1.16 1.33Toxocara 1.28 1.37 0.91 1.13 1.40Schistosoma 1.10 1.29 0.79 1.13 1.34Taenia 1.14 1.18 0.88 1.09 1.31Ascaris 1.24 1.29 0.92 1.07 1.32

% Inhib Av % InhibS.ratti 96.9 97.8 99.0 97.5 94.6 97.2Dirofilaria 0.0 0.0 0.0 9.8 0.0 2.0Ancylostoma 0.0 0.0 9.4 4.9 10.7 5.0Toxocara 1.5 0.0 5.2 7.4 6.0 4.0Schistosoma 15.4 5.8 17.7 7.4 10.1 11.3Taenia 12.3 13.9 8.3 10.7 12.1 11.4Ascaris 4.6 5.8 4.2 12.3 11.4 7.7

Cross reactivity Study on 5 Positive Cross reactivity Study on 5 Positive Strongyloides IgG EIA sera from Strongyloides IgG EIA sera from Northern Australian Indigenous Northern Australian Indigenous Population. (PathCentre EIA Population. (PathCentre EIA S.rattiS.ratti test) test)

•Cross reactions due to other helminths that may cause an OD increase of >26% were not found in this study. •Cross reactive antibody due to Hookworm, Toxocara or Taenia was found in some patients (May cause an OD increase up to 25% in the routine test - more commonly 10-17%) •No significant cross reactive antibody due to Filaria or Schistosoma detected. •Though 3/5 patients had some inhibition with Ascaris Ag this infection is not endemic in Australia so is not due to Ascaris. •Where OD increases of 10-25% may occur this should be considered for Strongyloides EIA results in the equivocal range and low positive patients.

Immunoblot analysis Immunoblot analysis Strongyloides Strongyloides stercoralisstercoralis Ab . Sato et al 1990 Trans Ab . Sato et al 1990 Trans of Royal Soc Trop Med and Hygiene.of Royal Soc Trop Med and Hygiene.

Increasing the specificity of the Increasing the specificity of the Indirect EIA. Conway et al 1993 Indirect EIA. Conway et al 1993 Trans of Royal Soc Trop Med and Trans of Royal Soc Trop Med and Hygiene.Hygiene.

Increasing the specificity of the Increasing the specificity of the Indirect EIA. Conway et al 1993 Indirect EIA. Conway et al 1993 Trans of Royal Soc Trop Med and Trans of Royal Soc Trop Med and Hygiene.Hygiene.

Distribution and frequency of Distribution and frequency of antigenic bands. Sato et al 1990 antigenic bands. Sato et al 1990 Trans of Royal Soc Trop Med and Trans of Royal Soc Trop Med and Hygiene.Hygiene.

Strongyloides Serology:Strongyloides Serology:•Western blots show there is considerable variation in the specificity of Ab between different patients with Strongyloides infection.•Cross-reactivity hard to assess in published articles due to variation in methods/samples. Co-infection with Strongyloides often not excluded.•Cross-reactivity caused by Ascaris*, Schistosoma or Filaria is unlikely to be a problem in Australia as these diseases are not endemic.•Immunosuppressed patients may have false negative EIA results.•Preabsorption of sera with dirofilaria Ag decreases cross reactivity by several types of helminths. *Toxocara infection possible

Strongyloides diagnostic Strongyloides diagnostic algorithm: Garcia Diag Med algorithm: Garcia Diag Med Parasitology 4th edition 2001Parasitology 4th edition 2001

Sensitivity & Specificity:Sensitivity & Specificity:

The percentage of people with The percentage of people with the disease that give a positive the disease that give a positive result.result.

The percentage of people The percentage of people without the disease that give a without the disease that give a negative result.negative result.

Pos/Neg Pos/Neg S.rattiS.ratti EIA study. EIA study. Carroll et al 1981, vol 75 Trans Carroll et al 1981, vol 75 Trans RSTMHRSTMH

Predictive value (positive):Predictive value (positive):

• The predictive value of a positive result is The predictive value of a positive result is defined as the percentage of positive defined as the percentage of positive results that are true positives when the results that are true positives when the test is applied to a population containing test is applied to a population containing both healthy and diseased subjects. both healthy and diseased subjects.

• = TP/TP+FP x 100= TP/TP+FP x 100

• ref Galen and Gambinoref Galen and Gambino

PV for Sens/Spec 90% PV for Sens/Spec 90% Prevalence 10%Prevalence 10%

Pos test Neg test TotalsSick 9000 1000 10000

Not sick 9000 81000 90000100000

POSITIVE PREDICTIVE VALUE = 50.0

NEGATIVE PREDICTIVE VALUE = 98.8

Prevalence effect on PV Prevalence effect on PV when Sens and Spec are when Sens and Spec are 90%90%

Disease Prev % Pos PV Neg PV %0.1 0.9 1001 8.3 99.95 32.1 99.420 69.2 97.350 90 90

Prevalence effect on PV Prevalence effect on PV when EIA Sens 100% and when EIA Sens 100% and Spec 94% (Very good if Spec 94% (Very good if real)real)Ref: Lindo et al 1994 prospective evalRef: Lindo et al 1994 prospective evalS.stercoralisS.stercoralis EIA with 41kD Ag EIA with 41kD Ag

Disease Prev % Pos PV Neg PV %

1 14.4 10020 80.6 100

Predictive Value with Predictive Value with Strongyloides EIA test in AustraliaStrongyloides EIA test in Australia

•This data suggests (With EIA sens/spec 90%) that in areas where the disease prevalence is <10% the test is best used to exclude disease with a negative result. The diagnosis should be considered in patients with a positive result. •Positive results of >0.8 (PathCentre test) would have a much higher positive predictive value as our preliminary data indicates false positives due to cross reaction rarely occur at this level if at all.•Though “better” antigens published in the literature promise tests of higher sensitivity and specificity the improvement in terms of predictive value still requires consideration.

Sensitivity of Baermann technique:Sensitivity of Baermann technique:Dreyer et al JCM Oct 1996, vol 34 no 10Dreyer et al JCM Oct 1996, vol 34 no 10 Patterns Patterns of Detection of of Detection of Strongyloides stercoralisStrongyloides stercoralis in stool in stool specimens: Implications for Diagnosis and specimens: Implications for Diagnosis and clinical trials.clinical trials.

Prevalence effect on PV Prevalence effect on PV when Sens is 27.7% and when Sens is 27.7% and Spec 100% Spec 100% (Faeces (Faeces examination, 25gm - Baermann examination, 25gm - Baermann Method)Method)

Disease Prevalence Pos Neg PVs1 100 99.35 100 96.320 100 84.7

False negatives become a concern withmicroscopy in areas of high diseaseprevalence. Incorrect transport of faeces orsimpler forms of microscopy may decreasethe sensitivity further.Dreyer et al JCM Oct 1996

Advantages EIA with Advantages EIA with S.rattiS.ratti AntigenAntigen

•Cheap to perform•Large numbers can be tested quickly• Give quantitative results. Post Tx changes in Ab levels can be monitored.•Not as technically demanding as IFA on live larvae•Does not require full time maintenance of life cycle

The future:The future:EIA’s will continue to be EIA’s will continue to be improved.improved.Strongyloides antigen or Strongyloides antigen or PCR assays are required PCR assays are required with high positive with high positive predictive values.predictive values.