Trypanosomiasis Diagnostics

Trypanosomiasis comprises human African trypanosomiasis (HAT, sleeping sickness, Trypanosoma brucei gambiense in West and Central Africa and T. b. rhodesiense in East and Southern Africa) and American trypanosomiasis (Chagas disease, T. cruzi).

Diagnosis of HAT rests on finding trypanosomes in blood, lymph node aspirate or CSF. Serological screening is used in the gambiense form (card agglutination test for trypanosomiasis, CATT, and rapid immunochromatographic tests). Staging by CSF examination decides treatment.

Chagas disease is diagnosed in the acute phase by direct parasitology (fresh blood film, thick and thin films, microhaematocrit concentration, Strout concentration) and PCR. In the chronic phase it is diagnosed by two different serological tests (enzyme immunoassay, indirect immunofluorescence, haemagglutination, or immunochromatography), with a third test when results disagree.

HAT has fallen to under 1000 reported cases a year, mostly gambiense in the Democratic Republic of Congo, with rhodesiense in Uganda, Malawi, Tanzania and Zimbabwe, but remains fatal if untreated. Chagas disease affects 6 to 7 million people, mostly in Latin America, with 30 000 new cases and 10 000 deaths each year, and cases now appear in non-endemic countries through migration. About 30% of chronically infected people develop cardiomyopathy and 10% megaoesophagus or megacolon.

Indications. HAT: fever, headache, lymphadenopathy (Winterbottom sign), pruritus, weight loss, sleep disturbance, behavioural change, or a chancre (rhodesiense) in a person from or returning from an endemic area.

Chagas disease:

• Fever with unilateral periorbital oedema (Romana sign) or an inoculation chagoma in an endemic area.
• Unexplained cardiomyopathy, arrhythmia or conduction block, dysphagia or megacolon in a person from Latin America.
• Screening of blood and organ donors, pregnant women from endemic areas, infants born to infected mothers, and immunosuppressed patients with reactivation.

Specimen. HAT:

• Capillary or venous blood for CATT (whole blood, plasma or serum), thick and thin films, microhaematocrit centrifugation technique, and mini anion exchange centrifugation technique (sensitivity 80 to 90%).
• Lymph node aspirate (gambiense); chancre aspirate (rhodesiense).
• CSF 1 to 2 mL examined within 15 minutes for white cells, trypanosomes (double centrifugation) and protein.

Chagas: EDTA blood for fresh film and PCR (acute), serum for serology (chronic), CSF or pericardial fluid and tissue biopsy in reactivation, and cord blood or infant blood at birth and at 8 to 10 months for congenital infection.

Reference values. No trypanosomes on any film, aspirate or CSF. CATT and serology negative. CSF white cells under 5 cells/microlitre and protein under 0.4 g/L.

Interpretation.

• HAT, trypanosomes found in blood, lymph or chancre: confirmed infection, with staging by CSF.
• Stage 1 (early haemolymphatic): CSF white cells 5 cells/microlitre or fewer and no trypanosomes. Stage 2 (late meningo encephalitic): CSF white cells over 5 cells/microlitre (some programmes use over 20) or trypanosomes in CSF, with raised IgM, Mott cells, and neuropsychiatric signs.
• Positive CATT in gambiense with no parasite found: serological suspect, confirmed by a second test (immune trypanolysis, trypanosomal PCR) and repeated parasitology; CATT sensitivity is 87 to 98% and specificity 95 to 99%, and cannot be used for rhodesiense.
• Chagas acute phase: trypanosomes (flagellated, C-shaped) in a fresh blood film or concentrated blood, or positive PCR, with fever, hepatosplenomegaly, myocarditis and Romana sign.
• Chagas chronic phase: positive results on two different serological tests (sensitivity and specificity over 98%) confirm infection; discordant tests need a third test or PCR.
• Chronic Chagas disease with negative parasitology is common, since parasitaemia is intermittent and PCR sensitivity is only 50 to 70%.
• Reactivation in HIV or transplant recipients: parasites in blood, CSF or tissue, and rising PCR load.
• Congenital Chagas: parasites in cord blood or infant blood in the first month, or positive serology after 8 months of age.
• Cross-reaction of Chagas serology with Leishmania, syphilis, malaria and other infections causes false positives.

Critical values and findings needing immediate action.

• Confirmed HAT with CSF involvement, confusion, seizures, coma or reversal of sleep rhythm: urgent treatment with specialist drugs (fexinidazole, eflornithine with nifurtimox, melarsoprol for rhodesiense), under supervision.
• Acute Chagas myocarditis, pericardial effusion, meningoencephalitis or heart failure: admission and benznidazole or nifurtimox, with cardiac monitoring.
• New complete heart block, ventricular tachycardia or syncope in chronic Chagas disease: pacing, implantable cardioverter defibrillator and anticoagulation as the picture requires.
• Chagas reactivation with immunosuppression: immediate antiparasitic therapy and review of immunosuppressive drugs.
• Pregnant woman with Chagas disease: screening of the newborn and treatment of the infant (treatment of the mother is delayed to after delivery).

Interferences and pitfalls.

• Parasitaemia in gambiense HAT is low and fluctuating, so repeated examinations and concentration methods are needed, and CSF is read promptly because white cells and trypanosomes degrade.
• CATT is falsely negative in strains lacking the variant antigen, and falsely positive from other infections and in blood with high haematocrit or poor reagent storage.
• Lumbar puncture in HAT after parasitological confirmation only, since contamination of CSF with blood trypanosomes affects staging.
• Rhodesiense is acute and rapidly fatal, and CATT is not used; a thin film and thick film are positive in most cases.
• Chagas disease serological tests have variable performance between regions, because of antigenic differences between parasite types, so two assays with different antigens are used.
• Blood donations in non-endemic countries are screened in at risk donors and organ donors.
• Benznidazole and nifurtimox cause dermatitis, neuropathy, gastrointestinal upset and bone marrow suppression, and need monitoring.
• Chronic cardiac disease needs ECG, echocardiography and Holter monitoring, regardless of treatment.
• Cure of Chagas disease after treatment is judged by seroreversion, which takes years to decades in adults.

Special groups.

• Children: congenital Chagas disease (transmission risk 1 to 5%) and HAT with behavioural change and school problems.
• Pregnancy: antiparasitic drugs for Chagas are deferred, and HAT treatment uses available drugs with specialist input.
• Immunosuppressed patients: reactivation of Chagas disease, with brain lesions (pseudotumour) and myocarditis.
• Travellers: HAT after game park visits in East Africa (rhodesiense), with chancre and fever.
• Migrants: Chagas disease in migrants from Bolivia, Argentina, Paraguay and Mexico, with screening programmes.
• Regions: Latin America for Chagas, with spread through triatomine bugs, oral outbreaks (contaminated juice), blood transfusion and mother to child; HAT in tsetse fly belts of sub-Saharan Africa.

Follow up tests.

• CSF cell count, protein, IgM and parasite examination (HAT staging), trypanolysis and PCR.
• Repeat CSF at 6, 12, 18 and 24 months after treatment for HAT.
• Full blood count, liver enzymes and creatinine.
• ECG, echocardiography, Holter monitoring and cardiac MRI for Chagas.
• Barium swallow and enema for megaviscera.
• HIV test and serology for other infections.
• PCR load during and after Chagas treatment, and family screening.