Serological tests for visceral leishmaniasis

Visceral leishmaniasis is a parasitic disease transmitted by sandflies, with 0.5 million new cases annually.¹ It is most commonly seen in India, Bangladesh, Brazil, Sudan, and around the Mediterranean. About two cases are seen each year in the United Kingdom, and these usually originate from around the Mediterranean.²

Patients with visceral leishmaniasis present with fever, splenomegaly, and weight loss. It can be difficult to diagnose this disease in endemic settings as several causes of febrile splenomegaly exist, notably malaria. In this week's BMJ, a meta-analysis by Chappuis and colleagues compares the diagnostic performance of two serological tests in endemic settings, the direct agglutination test (DAT) and rK39 dipstick test.³ Outside endemic areas visceral leishmaniasis is often only considered after haematological malignancies have been excluded.²

In immunocompetent people visceral leishmaniasis can be treated with a 28 day course of a pentavalent antimonial, and the cure rate is 90-95%.⁴ In resource rich settings patients are treated with six to 10 days of liposomal amphotericin (an antifungal agent), and the cure rate is higher at 95-98%.⁴

The gold standard for diagnosis of visceral leishmaniasis is to identify parasites in smears of tissue aspirates (spleen, bone marrow, or lymph node). Splenic smears have a sensitivity of > 90%, but the procedure carries a small risk of intra-abdominal haemorrhage.⁵ Bone marrow aspiration is often done but is painful and less sensitive.

In endemic settings the resources needed to support tissue diagnosis—skilled technicians, good smears, proper stains, appropriately maintained and working microscopes—are often unavailable.⁵ Serological diagnosis is safer and two field tests have been developed, DAT and the Ks30 dipstick test.

DAT measures anti-leishmania antibody titres using a freeze dried antigen.^6-8 However, it requires test readings to be standardised, prolonged incubation, and the handling of multiple samples. In Sudan, DAT testing is done in field laboratories on filter paper blood samples. Médecins sans Frontières has developed a management strategy based on DAT titres in which patients with a titre of 1:6400 are treated, and those with titres between 1:400 and 1:6400 have a splenic aspirate. About 10% of suspected cases will need splenic aspirates. The K39 dipstick test is highly specific for visceral leishmaniasis and detects antibodies to a specific 39 amino acid sequence (K39). It has been developed as an immunochromatographic strip test dipstick.⁹ The test is easy to perform—a village health worker can be trained in few hours—the kit can be stored at ambient temperature, no equipment is needed, and it can be carried to remote areas.

A meta-analysis published in this week's BMJ evaluated the performance of both serological tests.³ Only studies with the gold standard of parasites seen on splenic aspirate were included, and data from 30 studies on the DAT test and 13 on the K39 dipstick test were analysed. The combined data set relates to 2817 patients with visceral leishmaniasis and 6552 controls. Both tests have high sensitivities, 94.8% for DAT and 93.9% for the K39 test. The authors were rigorous and calculated separate specificities when controls were patients with clinically suspected disease or healthy people; even so the overall specificities were 85.9% and 90.6%, respectively. This confirms that both tests performed well and either could be incorporated into national guidelines for diagnosing visceral leishmaniasis. There are interesting regional differences—the tests are more sensitive in South Asia than in Sudan—perhaps because Sudanese patients produce lower antibody titres. This highlights the importance of validating new diagnostic tests in endemic areas even though this is costly. Visceral leishmaniasis is an important coinfection in HIV positive people, particularly in areas where highly active antiretroviral therapy is not available, and this meta-analysis only considered data from HIV negative patients. Serology may be negative in up to half of HIV positive patients coinfected with visceral leishmaniasis.¹,¹⁰ Such coinfections will become common in endemic regions, and studies on the performance of the tests in this group are needed.

Both serological tests can remain positive for several years after cure and so cannot be used to detect relapse or reinfection. Furthermore, in areas with high transmission of visceral leishmaniasis, many people will be infected but only a minority will develop clinical illness. People who carry the infection test positive serologically and sometimes form up to 32% of the healthy population.⁸,¹¹ Tests are therefore needed that identify only active disease. A limitation of the dipstick test is that it is only positive or negative and the titre based screening strategy used by Médecins sans Frontières cannot be applied.

Other tests are being developed. The latex agglutination test (KAtex) detects a leishmanial antigen in boiled urine that disappears after cure. It has been tested in Asia and Africa, and specificity has ranged from 47% to 95%¹²,¹³; it may be developed as an immunochromatographic strip test for use with unboiled urine. Another potential test detects erythrocyte sialic acid, which is positive in patients with active disease but becomes negative after cure.¹⁴ The test warrants further clinical evaluation.

The World Health Organization could help in ensuring that these tests remain available for the people who need them. Two manufacturers have already stopped producing dipsticks (Arista Biological and Amrad). Academic studies are needed for evaluation, and public health action is needed to incorporate good tests into national policies while recognising the commercial needs of manufacturers. Progress in neglected diseases like leishmaniasis is often hampered by lack of commitment from industry, and diagnostics and drugs are abandoned when profits are insufficient.