Visceral leishmaniasis(VL) or Kala-Azar is a potentially fatal disease caused by infection with various species of the intercellular protozoan parasite. It is spread by the bite of the female sandfly and is endemic to many regions in the world including north eastern India, northern Brazil and sub-Saharan Africa. The infection is also known to be endemic around the Mediterranean basin where the reservoir of the infection is dogs, as it is in many other parts of the world. Aside from the great degree of morbidity caused by this disease the WHO has concern that in the regions where VL is endemic HIV is beginning to spread and in Spain it has been found that approximately 50% of patients with HIV were also co-infected with VL. Most recently this problem has also emerged in parts of Ethiopia.
Typical clinical signs include fever, anaemia, muscle wastage, and swelling of the liver and spleen. Though few of those infected develop the full range of symptoms, the swollen spleen is virtually universal. Correct diagnosis is important as the treatment is of long duration and can be dangerous. The availability of accurate laboratory tests is therefore, essential.
Problems of current diagnostic tests
The current, definitive method involves microscopic detection of parasites in stained smears obtained from biopsies of spleen, liver or lymph node. This can be hazardous for the patient, requires good laboratory facilities and the sensitivity depends on the competence of the microscopist. Serology tests detect antibodies to leishmania and, though not standardized, are sensitive and widely used. However they remain falsely positive long after successful treatment and also give false negatives in patients with VL/HIV co-infections.
This varies between regions but consists of multiple injections with either pentavalent antimony compounds such as sodium stibogluconate or more recently with Amphoteracin B. Neither of these drugs is without potentially harmful side effects. More recently, liposomal preparations of Amphoteracin B are proving to be most efficacious. However, the cost of these drugs is a major consideration and may preclude their use in some regions where the disease is endemic.
Leishmaniasis patients are highly susceptible to contracting HIV and in HIV patients the presence of leishmania accelerates the onset of AIDS by cumulative immunosupression and stimulation of virus replication. Leishmaniasis is spreading in several areas of the world as a result of epidemiological changes which sharply increase the overlapping of AIDS and VL. So far, 33 countries worldwide have reported co-infections.
Intravenous drug users have been identified as the main population at risk in southern European countries such as France, Italy, Portugal and, in particular, Spain. In these countries 20-70% of adult cases of VL are associated with HIV while up to 9% of those with AIDS also have VL. Diagnosis of VL in HIV co-infections is particularly difficult as the clinical signs are frequently absent and over 40% are VL antibody negative. In reality VL/HIV patients have parasitemia and are highly infectious both to sandflies and, via needles and syringes, to other humans.
For more details about the co-infections, which are considered to be a real emerging disease in south-western Europe, see the Leishmania HIV co-infections: south-western Europe 1990-1998
In response to this situation, WHO and UNAIDS have set up a surveillance system through a network of 28 institutions worldwide. All members of the network use the same guidelines for diagnosis and a computerized case report form, both endorsed by WHO.
The KAtex kit
Kalon Biological's KAtex kit is a unique latex agglutination test that detects a stable, non protein, disease specific antigen in the urine of patients with an active infection. It gives a result in two minutes and is both simple to use and is diagnostically accurate. Samples of urine to be tested are immersed briefly in boiling and cooled to ambient before testing - this step eliminates cross reacting antigens. In the accepted cotton rat animal model, the concentration of this urinary antigen was found to increase progressively following experimental infection and to decrease rapidly following treatment. Recent indications from field trials are that in humans also the antigen rapidly disappears from the urine following successful treatment.
KAtex was developed in collaboration with the Liverpool School of Tropical Medicine and was trialed at the University of Khartoum in 1999 on urines from the Gedarif state of Sudan. A panel of 73 patients was tested by KAtex - for antigen - and at least four other serological tests - i.e. detecting serum antibody - which were: -
- Direct agglutination test (DAT)
- Commercial ELISA
- Immunofluorescent antibody test (IFA)/LI>
- rK39 dipstick test (Corixa Corpn, Seattle, Washinton, USA)
A combined serological score was used as there was not complete agreement between these tests. This combined score was compared with the KAtex result. By correlation of the KAtex results with the combined serological score plus microscopy it was shown that this latex agglutination test performed better than any other serological test in predicting positivity. Combining KAtex and DAT results gave a particularly good predictive value.
It was noted that 41/47 microscopy negative patients were also KAtex negative while 15/15 microscopy positives were also positive by the latex test. In a separate study on 52 samples from the Yemen a sensitivity of 86% and specificity of 100% was obtained. Testing a small panel from VL/HIV co-infections it was found that these urines have very high concentrations of the antigen detected by KAtex. The agglutination produced by these patients was faster and more pronounced than seen to date in any patients with the single infection.
Studies of kala-azar infection in eastern Nepal - using 155 parasitologically confirmed positive and 77 confirmed negative samples - showed that KAtex had a sensitivity of 47.7% with a specificity of 98.7%.
Comparison of the KAtex kit against the "gold standard" bone marrow aspirant method for leishmaniasis diagnosis concluded that the Kalon test is a sensitive and specific, non-invasive diagnosis tool. Sensitivity of 100% and specificity of 96% - in a study group of 168 patients, with 83 showing some symptoms of VL - was reported. A similar study reported electivity of 87% and specificity of 99% in a sample of VL patients through treament.
As noted above, HIV/VL co-infection is increasing globally, and accurate VL diagnosis in HIV positive patients is often difficult due to a lack of clinical symptoms. The KAtex kit was evaluated for its suitability for VL diagnosis in the treatment of 49 co-infected patients in Barcelona. A sensitivity of 85.7% and selectivity of 100% was reported. The study concluded that KAtex is appropriate for diagnosis of primary VL, for monitoring treatment efficacy and subclinical infection detection. Studies in Nepal show that KAtex sensitivity is, in fact, increased by the presence of co-infection, due to the increased parasite load in these patients. The Nepal study reports a sensitivity of 57% and a specificity of 98% in a study group of 124 patients.