Visceral Leishmaniasis (VL) or Kala-azar is the second most lethal, neglected tropical disease (NTD) endemic in India, Bangladesh, East-Africa, in Mediterranean countries and Brazil [1,2]. In Sudan VL is transmitted to humans and animals through the bite of sandfly Phlebotomus orientalis [3,4].

Sudan is one of the VL endemic countries in East Africa with the highest VL burden in the world, with 2000-7000 new infections per year, contributing 57% of the global annual cases (50,000-90,000) [5]. Most of these cases were reported in rural areas in Gedaref and White-Nile State. Even though, VL is treatable and curable, 52 and 46 people died from the disease in Sudan in 2019 and 2020, respectively [6].

Poverty, malnutrition and immunodeficiency are known risks for developing VL, and migration due to food shortages, civil unrest and war has led to a high incidence and prevalence of VL in this region [7,8].

Therefore, early and accurate diagnosis and treatment are critically important to control and manage VL in East-Africa. At rural VL-endemic areas in Sudan, diagnosis of this disease is primarily based on microscopic detection of amastigote Leishmania or rK39 based RDTs. Both methods are applicable at primary health care centres and rural settings. Microscopic detection of parasites remains the reference standard for VL diagnosis because of its high specificity. However, the sensitivity is generally low in East Africa and depends on the parasitemia and skills of the person that carries out the work. Aspiration of infected tissues such as lymph nodes, spleen or bone marrow is invasive and bears the risk of infection and other complications. It is increasingly recognized that in East Africa, both microscopy and rK39-based RDTs are not appropriate due to low sensitivity and variable specificity [9]. Therefore, new reliable and non-invasive diagnostic methods are urgently needed in resources-poor countries [10]. Accordingly, the WHO has prioritized the development of better RDTs for VL in East Africa in the 2021-2030 roadmap for NTDs [11].

Recently, a new recombinant kinesin antigen (rKLi8.3) from a Sudanese L. infantum strain (MHOM/SD/82/GILANI) was developed to improve VL serodiagnosis in East Africa and evaluated in a retrospective study with sera from Sudan and other endemic countries [12]. Here, a prospective diagnostic accuracy study was performed to diagnose VL suspects at a health centre in the VL endemic area of Gedaref, Eastern Sudan, by comparing INgezim Leishma-CROM with IT-LEISH.

107 VL suspects were included in a diagnostic accuracy study at a health care centre in Gedaref. The mean age of VL suspects was 22 ± 17.4 years (median, 15); 66 persons were male (61.7%) and 41 females (38.3%). Major clinical signs and symptoms were fever (100%), joint pain (99.1%), loss of appetite (95.3%), weight loss (88.8%), lymphadenopathy (72.9%), splenomegaly (67.3%), and hepatomegaly (62.6%) (Table 1). Following parasitological examination, VL suspects were divided into 77 smear positive and 30 smear negative cases, (Fig 1). The latter group included infections other than VL (malaria, typhoid fever, brucellosis) and 2 individuals without typical clinical signs of VL (cured VL) who had been treated with AmBisome and 3 symptomatic VL (VL relaps) who despite treatment with AmBisome showed typical clinical signs at the time of the study. 8 VL suspects were co-infected with malaria, none of the patients had a history of cutaneous Leishmaniasis (CL) or HIV infection. Independent of the parasitological outcome, INgezim Leishma-CROM was compared head-to-head with IT-LEISH for serodiagnosis of all VL suspects.

Sensitivity was determined using 69 VL, 8 VL-Malaria co-infected and 3 VL relaps cases and specificity was calculated on 25 non-VL cases (13 malaria, 9 typhoid, 3 brucellosis) and 2 cured VL cases. Accordingly, INgezim Leishma-CROM showed a sensitivity of 98.75% (95% CI 93.23-99.97%) and specificity of 92.59% (95% CI 75.71-99.09%) and IT-LEISH achieved a sensitivity of 92.5% (95% CI 84.39-97.2%) and specificity of 92.59% (95% CI 75.71-99.09%) (Table 2). Within the 77 smear positive group which included 8 VL-Malaria co-infected patients, INgezim Leishma-CROM and IT-LEISH detected 76 and 72 VL patients, respectively. While all VL-Malaria co-infections were detected by both RDTs, no cross-reactivity was observed within the 25 patients suffering from Malaria, Typhoid and Brucellosis. Within the smear negative group, 5 well documented patients previously received VL treatment. Two patients showed clinical improvement after treatment and were considered cured but showed positive results in both RDTs. Three patients showed typical VL clinical signs but were smear negative, all of them were positive with INgezim Leishma-CROM and 2 of them with IT-LEISH.

The PPV, NPV and AC of INgezim Leishma-CROM were 97.53% (91.23% to 99.34%), 96.15% (78.04% to 99.43%) and 97.20% (92.02% to 99.42%). For IT-LEISH the PPV value was 97.37% (90.69% to 99.29%), NPV 80.65% (65.71% to 90.06%) and AC 92.52% (85.80% to 96.72%).

The Kappa index (κ) values (95% CI) for IT-LEISH was 0.801 and for INgezim Leishma-CROM 0.925.

Although several RDTs for VL exist, IT LEISH and Kalazar Detect are most commonly used in Sudan. Both rely on the rK39 kinesin antigen, showing a varying sensitivity by region. Despite the high sensitivity in India (98.8%) and Brazil (92%), IT LEISH has lower sensitivity in East Africa (87.2%) due to regional polymorphisms of the rK39 antigen [17–19]. To address this, the rKLi8.3 antigen, based on a conserved kinesin sequence from a Sudanese L. infantum strain, was developed and showed superior diagnostic performance compared to IT LEISH in a retrospective study in Sudan [12]. Here, INgezim Leishma-CROM and IT-LEISH were compared in prospective accuracy study in Gedaref, Sudan.

Among smear-positive patients, INgezim Leishma-CROM had a sensitivity of 98.7%, while IT-LEISH had 92.5%. Both tests also correctly identified 8 VL patients co-infected with malaria. In smear-negative patients, none of the RDTs gave false positives for malaria, typhoid, or brucellosis, though two VL patients with past AmBisome treatment were false positive, likely due to persistent antibodies [20–22].

Three symptomatic but smear-negative patients that have been treated with AmBisome were tested positive with INgezim Leishma-CROM and two with IT-LEISH. They were classified as VL true positives. Presence of VL-specific antibodies together with typical clinical signs after treatment indicates ongoing infection, due to incomplete elimination of the parasite or relapse of VL, particularly in immunocompromised individuals [23,24].

Test accuracy depends on the composition of the population and the reliability of the reference test, which may vary in sensitivity (52-65%) for LNA in Sudan [25–28]. Molecular diagnosis, while ideal, is not feasible in East Africa [29].

In Gedaref, INgezim Leishma-CROM demonstrated a sensitivity of 98.75% and a specificity of 92.59%, meeting WHO guidelines for the diagnosis of VL with a sensitivity of greater than 95% [9].

Although VL/HIV co-infection complicates diagnosis, war conditions and its low prevalence in eastern Sudan, did not allow testing for HIV co-infection [30,31].

A common issue for all serological tests is the persistence of specific antibodies up to several years. Therefore antibody-based tests for VL diagnosis must always be used in combination with a standardized clinical definition or other diagnostic methods [22,29].

The limitations of our study was the restricted sample size, which was constrained by the onset of war in Sudan. With only 107 patients enrolled, the power of our study to detect smaller differences in diagnostic performance may be limited. Despite this, the study provides valuable insights into the performance of the novel POC test for VL in Sudan. Further studies with larger sample size are recommended to validate our findings.

In conclusion, a previous retrospective - and the current prospective study demonstrate that INgezim Leishma-CROM is suitable for field diagnosis and show enhanced diagnostic performance in East Africa, providing an improved alternative to the currently used RDTs in this endemic area.