Immunodiagnostic Service

AMOEBIASIS   BABESIOSIS    CYSTICERCOSIS   FILARIASIS    ECHINOCOCCOSIS   LEISHMANIASIS   MALARIA  PARAGONIMIASIS   SCHISTOSOMIASIS    STRONGYLOIDIASIS   TRICHINELLOSIS   TRYPANOSOMIASIS   TOXOCARIASIS

¶Tests submitted to a cost recovery program
*Tests performed at the CDC, Atlanta
†Test performed at Mahidol University, Thailand
¥Test performed at the Quebec Provincial laboratory (LSPQ). It is a confirmatory assay requiring an IgM positivity. For serological confirmation a minimum of 1.3 ml is needed. The PCR must be accompanied with positive results of IgG and IgM.

AMOEBIASIS

Assay: Enzyme immunoassay (EIA)

Antigen: HK-9 soluble antigens of Entamoeba histolytica.

Indications: This assay facilitates the evaluation of abscesses, potentially caused by Entamoeba histolytica (liver, lung). It can be useful to evaluate colitis also. The gold standard for E. histolytica diagnosis remains the visualization of hematophagous trophozoites, in biopsy, matinal abscess aspirates, or stool specimens.

Interpretation: The sensitivity of this test varies with the disease process such that 90-100% of individuals with extraintestinal amoebiasis are positive while only 70-80% of those with invasive disease limited to the intestine are positive. It is unclear if asymptomatic cyst passage without local invasion can elicit a systemic IgG response. As a result, individuals infected with the non invasive, but morphologically identical Entamoeba dispar, would be expected to be negative. Although the reported specificity of this assay is high, only limited data are available using sera from individuals with diseases caused by other invasive Amoeba species (e.g. Acanthamoeba, Naegleria).

Significant antibody titers may take 10-14 days to develop even with documented invasive amoebiasis. Since positive titers may persist for many years after infection, a single test result must be interpreted with caution when prior infection is possible.

In some circumstances, it may be useful to determine the titers of anti-Entamoeba antibodies in paired specimens. Please contact the NCP(S) laboratory directly if this service is required.

BABESIOSIS

Assay: Indirect immunofluorescence assay (IFA)

Antigen: Fixed Babesia microti.

Indications: This test may be useful for diagnosis after parasitemia has cleared and for seroepidemiology. The identification of the parasite in blood smears is the gold standard.

Interpretation: Sensitivity is thought to be 100% for B. microti cases except AIDS patients. The specificity is estimated at 99%. There is little cross-reactivity with other Babesia sp., however, cross-reactivity with Plasmodium sp. has been observed. Titers of 1:64 or greater are indicative of infection with B. microti. Titers may persist for 6 to 12 months after acute illness. A titer range of 1:64-1:256 suggests an infection probably more than 6 months ago. However, a titer of 1024 or greater indicates a relatively recent infection. 

CYSTICERCOSIS

Assay: Immunoblot assay (IB)

Antigen: Chromatography affinity-purified Taenia solium cysticerci glycoprotein antigens derived from pigs.

Indications: This test facilitates the diagnosis of lesions (usually intracranial), that are compatible with Taenia solium cysts by CT or MRI scans. Although the gold standard test is a tissue biopsy, this procedure is seldom used in the diagnosis of cysticercosis.

Interpretation: The sensitivity of the IB depends upon the number and vitality of the cysts. Sensitivity is estimated to be 98% in biopsy proven cases of neurocysticercosis with 2 or more cysts. Sensitivity decreases in cases with single enhancing cyst or calcified cysts (range 28% to 72%). In patients with only subcutaneous cysts, the sensitivity is < 50%. The specificity of the test is thought to be 100% and no cross-reactivity is known.

Both serum and cephalospinal fluid (CSF) may be tested, but the highest sensitivity can be achieved in serum.

FILARIASIS

Assay: Enzyme immunoassay (EIA)

Antigen: Antigens extracted from adult Brugia malayi worms.

Indications: Investigation of several clinical syndromes suggesting a filarial etiology, and screening asymptomatic individuals whose sera and geographic origins or travel make a filarial infection possible. Settings in which testing could be appropriate include: generalized itchiness (onchocerciasis), lymphangitis (Wuchereria bancrofti, Brugia malayi), eosinophilic lung disease (W. bancrofti), history of intermittent swellings (Loa loa). This test is of little use in the diagnosis of chronic lymphadema (elephantiasis), hydrocoele and chylurea, all of which typically complicate "burned out" filarial disease. The gold standard for diagnosis of filarial disease remains visualization of adult worms (e.g. Loa loa eye worm, mobile worms by ultra son), or microfilaria (e.g. skin snip).

Interpretation: A positive titer (_1:128) indicates a filaria infection but gives no info with regard to disease state (e.g. acute, chronic, cured). Virtually all residents of filariasis-endemic areas are antibody positive. Anti-filaria antibodies may persist for years after infection. Therefore a single test result must be interpreted with caution when prior infection is possible. 

ECHINOCOCCOSIS (HYDATID DISEASE)

Assay: Enzyme immunoassay (EIA) followed by confirmatory Immunoblot assay (IB).

Antigen: EIA Echinococcus cyst soluble antigens and Immunoblot E. granulosis hydatid cyst fluid from camel.

Indications: This test plays an important role in the evaluation of suspected hydatid cysts (e.g. liver, lung). Given that aspiration of such cysts is contraindicated. The combination of positive serology and typical CT or MRI appearance of the cyst (e.g. internal septations, daughter cysts, lily pad) is often sufficient to make a secure diagnosis.

Interpretation: The sensitivity of the EIA test is high but its specificity is relatively low with 10-15% false-positive results due to cross-reactions in a wide variety of helminth infections (e.g. cysticercosis) and non-infectious conditions (e.g. cancer, pregnancy, autoimmune diseases). Furthermore, the high sensitivity refers to long-standing hepatic disease only. E. granulosus titers may be false negative in several situations; small cysts (e.g. early after infection), intact cysts (i.e.: no leakage of the immunogenic cyst fluid), cysts in extrahepatic locations and heavily calcified cysts (e.g. non viable). The results of this test should be interpreted with additional caution when an organism other than "classic" E. granulosus is suspected on the basis of clinical or epidemiological information (e.g. Arctic E. granulosus). 

LEISHMANIASIS

Assay: Indirect immunofluorescence assay (IFA)

Antigen: Fixed promastigotes of Leishamania donovani, L. tropica, or L. brasiliensis.

Indications: Evaluation of visceral or disseminated leishmaniasis. The gold standard remains the identification of Leishmania amastigotes in stained smears of material aspirated from cutaneous lesions, spleen, bone marrow or other sites. This involves biopsy histology, PCR and culture. (all done at this centre, the NCP)

Interpretation: IgG titers of 1:16 or greater are considered positive, but provide only suggestive support for the diagnosis of Leishmania. The sensitivity of the test is reasonable for visceral leishmaniasis. The specificity of this test is not ideal, with many potential cross-reactions (e.g. Chagas disease, leprosy, malaria, and schistosomiasis). 

MALARIA

Assay: Indirect immunofluorescence assay (IFA)

Antigen: Fixed Plasmodium falciparum .

Indications: Used only for the diagnosis of Ideopathic Tropical Splenomegaly Syndrome (Hypertrophic Malarial Splenomegaly). This test should not be used to diagnose malaria attacks. The gold standard for acute malaria remains the malaria smear (e.g. thin, thick, buffy coat) or antigen capture techniques or PCR.

Interpretation: Antibody titers may persist for years after therapy. Titers for tropical splenomegaly syndrome can be extraordinarily high (e.g. 1:125,000) and decline slowly after therapy.

PARAGONIMIASIS:

Assay: Immunoblot assay (IB)

Antigen: Antigens extracted from Paragonimus westermani adult worms.

Indications: Identification of eggs in sputum remains the diagnostic gold standard. Eggs in stool specimens are less conclusive because of their morphologic similarity to other fluke eggs. Serology may be useful for the diagnosis of stool and sputum negative paragonimiasis, Paragonimus cutaneous larval migrans and cerebral paragonimiasis although the sensitivity in these conditions is unknown. Also several species of Paragonimus infect humans. The sensitivity of the IB assay for infections caused by these other species is unknown.

Interpretation: The sensitivity of this assay is ~ 96% in proven cases of P. westermani in Southeast Asians. The specificity is estimated at 99% with low cross-reaction for Schistosoma haematobium. Antibodies are often undetectable until 3 weeks post-infection. Although Complement Fixation titers decline rapidly after treatment, no data for antibody persistence in the immunoblot assay are available. 

SCHISTOSOMIASIS

Assay: Enzyme immunoassay (EIA)

Antigen: A pooled extract of adult Schistosoma mansoni and Schistosoma hematobium.

Indications: This assay can be used to screen individuals who have travelled to or resided in endemic areas and have had freshwater contact and/or have unexplained eosinophilia.

Interpretation
1) This test uses antigens extracted from adult worms of S. mansoni and S. haematobium.
2) Sensitivity and specificity of the screening test (IgG-ELISA) is unknownas there have been insufficient studies within appropriatre populations. Claims of 90% should be suspected without solid evidence. This assay could be used to investigate eosinophilia, abnormal liver function tests, dysentery, hematuria or fever and hepatosplenomegaly. Positive reactions detected in a patient with definitive filaria, Taenia solium, Strongyloides or Echinoccocus infections may be cross-reactions.
3) An OD = 0.5 indicates schistosomea infection at some unknown time and the OD level can not be related to worm burden, egg production, clinical status, or prognosis. Anti-Schistosoma antibodies may persist for many months after infection; a single test result must be interpreted with caution when prior infection is possible. Serolopositivity may be negative early in a Katayama syndrome.
4) The performance of this test in individuals infected with related S. japonicum and S. megongi is not currently known.

STRONGYLOIDIASIS

Assay: Enzyme immunoassay (EIA)

Antigen: Recombinant antigen, NIE, developed by NIH.

Indications: Standard stool examinations have low sensitivity for who have lived or travelled in tropical areas. These tests can be used to screen persons with unexplained eosinophilia, epigastralgia or trunkal larva migrans. Screening is also appropriate for individuals who have lived or travelled in the tropics, who will undergo immuno-ablative therapy (transplants, chemotherapy, high dose corticosteroids).

Interpretation:
1) This test uses recombinant protein antigen (NIE)
2) Sensitivity and specificity of the screening test (IgG-EIA) is estimated at ~95%. However, only a small number of ‘gold-standard (stool positive)’ sera have been tested to date. Sensitivity with the recombinant antigen might be lower in subjects with lower parasite burdens (ie: true positive but stool negative). A positive reaction detected in a patient with definitive filaria, Schistosoma, or Echinoccocus infections may be cross-reactions, but tests of such cases suggests that that false positivity is less than 5 %.
3) In certain circumstance, positive samples are tested by immunoblot. This confirmatory assay shows a good specificity (98%) with a sensitivity equavalet to the EIA (~95%).
4) Antibody ODs are reported to decline by 50% in successfully treated cases when the crude antigen EIA is used. This decline has not yet been confirmed for the NIE antigen.

TRICHINELLOSIS:

Assay: Enzyme immunoassay (EIA)

Antigen: This test uses excretory-secretory antigen derived from Trichinella larvae of pigs.

Indications: The diagnosis of trichinellosis is based on a combination of epidemiology and clinical findings (muscle symptoms, diarrhea, eosinophilia, raised creatine kinase and a high positive or rising Trichinella antibody titre). A positive muscle biopsy for Trichinella larvae is the gold standard test but is rarely required because the sensitivity of the clinical and laboratory findings listed above is excellent.

Interpretation: The sensitivity and specificity estimates (93% and 85%, respectively) apply to infections with Trichinella spiralis. The performance of this test with other species (e.g. polar Trichinella nativa) appears to be similar. Cross-reactivity can occur with other helminth infections (e.g. filaria, Strongyloides).

The IgG response to T. spiralis and T. nativa infection tends to be slow and is proportional to the number of larvae ingested. Anti-Trichinella antibodies can be detected at 2 weeks in most individuals who have consumed a large number of cysts. Less heavily infected individuals may take up to 3-4 weeks to mount a detectable IgG response. Antibody titers peak 3-6 months after infection and then fall progressively even without treatment. Since low titers may persist for many years after infection, a single test result must be interpreted with caution when prior infection is possible.

TRYPANOSOMIASIS (CHAGA’S DISEASE)

Assay: Indirect immunofluorescence assay (IFA)

Antigen: Fixed Trypanosoma cruzi promastigotes.

Indications: The test is used to diagnosis both occult (e.g. screening of blood for transfusions), and overt (e.g. Chagas’Disease, cardiac and intestinal sequelae of chronic infections).

Interpretations: The sensitivity of the test varies with the stage of infection. For chronic trypanosomiasis, sensitivity is good and negative serology is helpful to exclude the diagnosis. In acute cases of T. cruzi, the sensitivity is lower because it takes from 2 to 8 weeks to develop a detectable antibody titer. The specificity of the test is reasonable but cross-reactions with leishmaniasis and syphilis were observed.

TOXOCARIASIS:

Assay: Enzyme immunoassay (EIA)

Antigen: Excretory-secretory antigen derived from Toxocara larvae.

Indications: This test remains the definitive test for the diagnosis of toxocaral visceral larva migrans, despite the lack of good studies of its sensitivity and specificity. The test can be used in the investigation of asymptomatic eosinophilia. The role of this assay in defining the etiology of suspected retinal toxocariasis is not clear.

Interpretation: There is no "gold standard" test with which the performance of serologic assays for Toxocara can be compared; larvae are very rarely identified in the human host. Furthermore, the titers obtained in individuals with clinical visceral larva migrans vary widely. The sensitivity and specificity estimates (86% and 98%, respectively) apply to infections with T. canis. The performance of this test in individuals infected with related species (e.g. T. leonis) is not currently know. Cross reactions can occur with other nematode infections (trichinellosis, filariasis, strongyloidiasis etc.). The IgG response Toxocara is likely to be proportional to the number of infecting larvae.