Laboratories performing Strongyloides IFA testing 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 Strongyloides Serology testing in Australia. Validation of method Uncertainty Difficulty of antigen production Standardisation/Proficiency testing
Parasite Isolations W.A Blast. hominis G. intestinalis Ent. coli Endolimax. nana Crypt. parvum H. nana S. stercoralis Iodam. buetschlii Hookworm Chilomastix mesnili Ent. hartmanni Ent. polecki Ent. spp S. mansoni T. hominis E. vermicularis T. trichiura A. lumbricoides Ent. histolytica Pathogenic organisms
Serosurveys W.A. Location Strongyloides Strongyloides Toxocara total Nos No/Pos% canis No/ Pos% Kalumburu /11.4* Billiluna /37.0 Kalumburu 1986** 44 2/5.0 One Arm Point / 4.5 7/10.4 Looma /58.0 6/10.5 Lombardina / /40 Beagle Bay /17.8 4/14.3 * 9% pos Melioidosis **1986 surveys age range LaTrobe 1989 n=372 NE Arnhem Pop.Strongy 62%+ Toxocara 41%+
S.stercoralis Isolations W.A Year 1st Qtr 2nd Qtr 3rd Qtr 4th Qtr Total * * * excluding repeats
S.stercoralis Isolations W.A & 2002 source Region Year Kimberley Pilbara 11 2 Goldfields 3 2
Perth Bililuna Kalumbaru Looma One Arm Point Beagle Bay Lombadina W.A. Community Locations
Toxocara Absorption. Lynch et al 1988 Parasite Immunology. (Example of EIA pre- absorption vs competitive inhibition)
Competitive inhibition of Toxocara (TcESA) ELISA. Lynch et al 1988 Parasite Immunology Extract % sera Inhibited* % Inhibition Ascaris ** / Necator/Ancylostoma /- 7.1 Trichuris/Enterobius /- 5.3 Strongyloides*** 0 0 Dirofilaria /- 1.1 Schistosoma/Taenia /- 5.8 Entamoeba/Giardia /- 9.4 Leishmania/Trypanosoma /- 1.0 *48 TcESA pos sera with wide range of values. ** suum. ***ratti.
Lynch’s investigation of helminth cross reactions in the Toxocara E/S EIA suggested results in the equivocal/low positive area were more likely to be caused by cross reaction.
Pre-absorption study with S.ratti EIA - PathCentre. (Aust Northern Indigenous Group)
Pre-absorption study with S.ratti EIA - PathCentre (Amazon Indian Group)
Pre-absorption study with S.ratti EIA - PathCentre (Aust WWII Group)
Cross reactivity Study on 5 Positive Strongyloides IgG EIA sera from Northern Australian Indigenous Population. (PathCentre EIA S.ratti 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 Strongyloides stercoralis Ab. Sato et al 1990 Trans of Royal Soc Trop Med and Hygiene.
Increasing the specificity of the Indirect EIA. Conway et al 1993 Trans of Royal Soc Trop Med and Hygiene.
Distribution and frequency of antigenic bands. Sato et al 1990 Trans of Royal Soc Trop Med and Hygiene.
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
Sensitivity & Specificity: The percentage of people with the disease that give a positive result. The percentage of people without the disease that give a negative result.
Pos/Neg S.ratti EIA study. Carroll et al 1981, vol 75 Trans RSTMH
Predictive value (positive): The predictive value of a positive result is defined as the percentage of positive results that are true positives when the test is applied to a population containing both healthy and diseased subjects.The predictive value of a positive result is defined as the percentage of positive results that are true positives when the test is applied to a population containing 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% Prevalence 10%
Prevalence effect on PV when Sens and Spec are 90%
Prevalence effect on PV when EIA Sens 100% and Spec 94% (Very good if real) Ref: Lindo et al 1994 prospective eval S.stercoralis EIA with 41kD Ag
Predictive Value with Strongyloides 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: Dreyer et al JCM Oct 1996, vol 34 no 10 Patterns of Detection of Strongyloides stercoralis in stool specimens: Implications for Diagnosis and clinical trials.
Prevalence effect on PV when Sens is 27.7% and Spec 100% (Faeces examination, 25gm - Baermann Method)
Advantages EIA with S.ratti Antigen 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: EIA’s will continue to be improved. Strongyloides antigen or PCR assays are required with high positive predictive values.