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Indication - Neglected tropical diseases
Visceral leishmaniasis direct agglutination test
Facility level:
2. Laboratory
Assay formats
Agglutination assay
Status history
First added in 2020
Purpose type
Aid to diagnosis
Purpose
To aid in the diagnosis of clinically suspected visceral leishmaniasis (kala-azar)
Specimen types
Serum, Dried blood spots (check validation)
WHO prequalified or recommended products
N/A
WHO supporting documents
Control of the leishmaniases: report of a meeting of the WHO Expert Committee on the Control of Leishmaniases. Geneva: World Health Organization; 2010 (WHO Technical Report Series, No 949) https://apps.who.int/iris/handle/10665/44412
Codes
ICD11 code: 1F9Z

Summary of evidence evaluation

There is evidence that the accuracy of the DAT is comparable to the rK39 and suitable for use as a diagnostic test in endemic regions, as is recommended in WHO and many national guidelines. Estimates of accuracy are only available from reviews more than 10 years old that suggest sensitivity of 95% (CI: 93–96%) and specificity of 86% (CI: 72–93%). The full evidence review for this test category is available online at: https://www.who.int/medical_devices/diagnostics/selection_in-vitro/selection_in-vitro-meetings/new-prod-categories_3

Summary of SAGE IVD deliberations

As well as being supported by WHO guidelines for the diagnosis of VL, the DAT is a non-invasive, cost-effective test compared with alternative tests based on microscopy of bone marrow or lymph node aspirates or spleen puncture. It has a higher diagnostic accuracy compared with rK39 RDT in eastern Africa; and a higher sensitivity, negative predictive value (NPV) and PPV compared with rK39 for screening and diagnosis among HIV-infected patients. Its higher NPV among HIV-infected patients could help avoid unnecessary treatment with toxic medications in non-infected people. Importantly, the DAT can be used in test-and-treat strategies targeting VL in LMICs, where the disease is often endemic, because it is easy to perform and requires minimum laboratory skills; it can be done on DBSs that are collected in primary care settings and sent to higher care levels.

SAGE IVD recommendation

SAGE IVD recommended including the visceral leishmaniasis direct agglutination test in the third EDL: • as a disease-specific IVD for use in clinical laboratories (EDL 3, Section II.b, Neglected tropical diseases); • in an agglutination assay format; • to aid in the diagnosis of clinically suspected visceral leishmaniasis. The group noted the need to ensure that the entry for this test category in EDL 3 is clearly linked to WHO guidelines stating that the VL DAT must be used in combination with a clinical definition.

Details of submission from 2020

Background

Note: This submission was made in response to a recommendation by SAGE IVD during its second annual meeting in March 2019. Disease condition and impact on patients The leishmaniases are a group of diseases caused by protozoan parasites from more than 20 Leishmania species that are transmitted by the bite of infected female phlebotomine sandflies (1). The disease affects some of the poorest people in the world, and is associated with malnutrition, population displacement, poor housing, a weak immune system and lack of financial resources. There are three main forms of leishmaniases: visceral, also known as kala-azar and the most serious form of the disease; cutaneous (CL), the most common; and mucocutaneous, the most destructive. VL is a life-threatening systemic disease and is considered the second-largest parasitic killer disease after malaria. It is almost always fatal in more than 95% of cases without adequate and timely treatment. The disease occurs in poverty-stricken remote areas and affects the poorest people, and is associated with malnutrition, population displacement, poor housing conditions and lack of financial resources. The typical L. donovani complex is the causative agent of VL. The disease is characterized by prolonged fever, weight loss, enlargement of the spleen, liver and lymph nodes, anaemia and low blood cell count. In children, other symptoms include diarrhoea, cough, abdominal distension and growth retardation. If the disease remains untreated, it progresses to debilitation, bleeding and secondary infections, resulting in death. The mode of transmission is anthroponotic and zoonotic. Human-to-human transmission is predominant in astern Africa and the Indian subcontinent; zoonotic transmission (through dogs) is found in the Mediterranean region and the Americas (2). In endemic areas, a significant proportion of healthy people are exposed to infection but remain asymptomatic. However, the precise ratio between clinical cases and asymptomatic individuals varies from country to country: 1 : 5 in Kenya (3), 1 : 8 in Brazil (4), 1 : 5.6 in Ethiopia (5), 1 : 8.9 in India and Nepal (6), and 1 : 11 in Sudan (7). Does the test meet a medical need? The signs and symptoms of VL are non-specific, sharing clinical presentation with various other tropical diseases such as malaria, TB and enteric fever. This makes it mandatory to follow a diagnostic algorithm in a clinically suspected case of VL. The gold standard for diagnosis is microscopic detection of the parasite in specimens. The most sensitive of these techniques is the splenic puncture followed by bone marrow and lymph node aspirates with decreasing order of sensitivity, respectively. These are invasive methods that require expertise and equipment and are only available at the referral level. Splenic aspirate carries the risk of fatal bleeding as a complication of the technique. Kosack et al. 2017 (8) suggest that the ideal diagnostic test for VL-endemic settings should be available at the level where they are needed most and be accurate, sensitive, specific, user-friendly, robust and equipment-free (i.e. it should meet the ASSURE criteria). The development of diagnostic tests to improve VL case management has been rated as one of the most pressing needs to tackle infectious diseases in LMICs. It is important to diagnose VL correctly not only because untreated disease can be fatal but also because treatments are limited, expensive and have toxic side effects. Diagnostic tests should not give false-negative results that might result in death; nor should they give false-positive results that may cause people without VL to receive toxic treatments (9). The invasive methods of diagnosis and associated complications have driven development of non-invasive serological tests like the DAT (10–12). How the test is used The DAT is a semi-quantitative test that uses microplates in which increasing dilutions of a patient’s serum or blood are mixed with stained killed promastigotes of L. donovani. The DAT is a freeze-dried suspension of trypsin-treated fixed and stained culture of L. donovani promastigotes. If antibodies against the parasite are present in the sample, agglutination is visible with the naked eye. Although the test requires minimal laboratory set-up, skilled laboratory personnel and a moderate level of training are needed to perform the test and interpret its results (13). During infection with VL, circulating antibodies are produced against the surface antigens of the invading parasites. The DAT detects antibodies to L. donovani in the blood or serum of those infected by means of direct agglutination. In the absence of antibodies to Leishmania, the DAT antigen accumulates at the bottom of the plate to form a dark blue spot. If antibodies to Leishmania are present, the antigen forms a pale blue film over the well, which constitutes a positive result (13). The DAT test procedure requires an overnight incubation period, so results are not immediately available. That is why DATs are recommended at the district hospital level. Despite these limitations, DAT has excellent clinical accuracy and high precision in diagnosing VL cases. By enabling early and correct diagnosis and timely start of treatment, it can significantly impact patient outcomes, including curing the disease and thus helping to control VL in the endemic community.

Public health relevance

Prevalence and socioeconomic impact Out of the 194 countries and six territories reporting to WHO, 75 (38%) are currently considered endemic for VL: most of these are in South-East Asia, eastern Africa and Brazil. In Asia and eastern Africa, the disease is caused by L. donovani; in Brazil (and elsewhere in Latin America, Europe and North Africa), it is caused by L. infantum (14). There are an estimated 300 000 new cases of VL and 20 000–30 000 associated deaths each year, of which only 25–45% are reported to WHO. In 2017, more than 95% of cases were reported from just 10 countries: Bangladesh, Brazil, China, Ethiopia, India, Kenya, Nepal, Somalia, South Sudan and Sudan (15). The disease has a huge economic impact on affected populations and communities. For example, in Bihar, India, 83% of households in communities with high attack rates of VL were also among the poorest 40%. Evidence is arguably most complete for VL, with studies from multiple countries showing that even when diagnosis and medicines are provided free of charge, between 25% and 75% of households of sufferers experience some type of financial catastrophe.

WHO or other clinical guidelines relevant to the test

The WHO Expert Committee on the Control of the Leishmaniases (16) issued international guidelines on controlling VL in 2010. Several countries where VL is endemic also have national guidelines for diagnosing, treating and preventing the disease, including Ethiopia (17), Kenya (18), South Sudan (20), Sudan (19) and Uganda (21).

Evidence for diagnostic accuracy

DAT is an easy-to-perform test that is widely applicable with high sensitivity (90–100%) and specificity (95–100%) and that has routinely been used in some regions for the past 2 decades (22–25). The test can be carried out using plasma, serum or even urine samples, making it suitable for both field and laboratory application (26–32). There have been several validation studies on the clinical accuracy of DAT. A meta-analysis by Chappuis et al. (32) found DAT to be almost 1% more sensitive and 2% more specific than the rK39 strip test. A systematic review with meta-analysis of the rK39 strip test compared with DAT and IIF test and ELISA showed that sensitivity was 94.23% and specificity 89.97% and that the likelihood ratio of a positive test was 9.39 (33). Another study in Sudan by Abdullah et al. (30) evaluated the DAT for serodiagnosis of VL based on freeze-dried L. donovani antigen and found it comparable with standard liquid antigen by testing serum and blood samples. The freeze-dried DAT was found to have a sensitivity of 96.8% and a specificity of 96.2%.

Evidence for clinical usefulness and impact

There are no systematic reviews of the test’s clinical utility or impact on patient management and care. There are, however, several studies available to show that early diagnosis and complete treatment positively impact the control of VL in very high endemic settings. In one study, about 6.2% of normal persons in VL endemic areas were found to be reactive to DAT, with 3.6% becoming seropositive in a year’s time (34). In another study, the same author showed a strong association between serological status and probability of progression to clinical VL in prospective cohort studies in India and Nepal by using DAT titres, thereby demonstrating the utility of DAT not only as a diagnostic test but also for field surveillance (35).

Evidence for economic impact and/or cost–effectiveness

Boelaert et al. (31) show that the availability of DAT has proved convenient for use in field conditions. Since the available options are invasive techniques (splenic, bone marrow or lymph node aspirations) or serologic tests (DAT or rK39), the latter are clearly more cost-effective.

Ethical issues, equity and human rights issues

Availability of serological tests for VL have greatly improved access to diagnosis and treatment.
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