https://orcid.org/0000-0002-0845-4539
This is an uncorrected proof.
Figures
Abstract
Background
Visceral leishmaniasis (VL) remains a major public health challenge in East Africa, particularly in Ethiopia. Clinical trials on VL often exclude patients with severe comorbidities or laboratory abnormalities, limiting the generalizability of evidence used to guide real-world management. We comprehensively characterised the clinical profile, treatment outcomes, and mortality of VL patients treated in a referral centre.
Methodology and principal findings
This prospective cohort study enrolled patients at the Leishmaniasis Research and Treatment Center (LRTC), University of Gondar (02/2023-06/2024). All VL cases fulfilling eligibility criteria underwent detailed clinical, laboratory, and radiological assessments. VL treatment followed WHO guidelines. Outcomes, adverse events, and supportive care measures were recorded during treatment and over a 12-month follow-up period. Patients meeting at least one exclusion criterion (e.g., comorbidities, clinical signs of severe VL) of standard phase III VL treatment trials were defined as trial-ineligible patients and compared with trial-eligible patients. A total of 314 VL patients, mostly young adult males (median age 22 years), were enrolled. Of these, 21 (6.7%) had HIV co-infection; 204 (65%) met ≥ 1 key exclusion criterion typically used in VL clinical trials. Trial-ineligible patients had high parasitemia, more concurrent infections, more pronounced clinical and laboratory abnormalities and a higher need of supportive care measures including systemic antibiotics and blood transfusion. Overall cure rate was 90.1%. Mortality after the first VL treatment course was 6.4%, ranging from 1.8% in trial-eligible patients to 8.8% in trial-ineligible patients. Leading causes of death included severe bacterial infections, acute liver failure and haemorrhagic complications.
Conclusions
These findings underscore the severity and complexity of VL in routine care in our study population, and highlight the limitations of current trial populations to inform broader clinical practice. Strengthening supportive care and expanding research inclusivity designed to respond to the needs of this patient population are critical to achieve VL elimination targets.
Author summary
Visceral leishmaniasis is a life-threatening disease that continues to affect many people in East Africa, especially in Ethiopia. However, many of the patients we see in everyday clinical practice are very different from the carefully selected patients who take part in clinical trials. Trials often exclude people who are severely ill or who have other medical problems, which means that the results may not fully represent the patients who most need treatment. In this study, we followed people treated for visceral leishmaniasis at a referral centre to better understand how they present, how they respond to treatment, and what challenges they face. Most of the patients we treated would not have been eligible for typical clinical trials. These patients were more severely ill, had more infections, and required more intensive supportive care, such as antibiotics or blood transfusions. While most people were cured, those with more severe illness had a higher risk of death, often due to serious infections or bleeding. By describing the real-world situation of patients with visceral leishmaniasis, we show that current research does not fully capture the needs of the most vulnerable groups. We highlight the importance of improving supportive care and making future studies more inclusive.
Citation: Ayele E, Henten Sv, Ayalew DG, Atnafu S, Mereed AE, Yohannes H, et al. (2026) Severe visceral leishmaniasis in Ethiopia: Outcomes, co-infections and mortality in a prospective real-world cohort. PLoS Negl Trop Dis 20(6): e0013878. https://doi.org/10.1371/journal.pntd.0013878
Editor: Elise M. O'Connell, National Institute of Allergy and Infectious Diseases Division of Intramural Research, UNITED STATES OF AMERICA
Received: December 18, 2025; Accepted: May 26, 2026; Published: June 5, 2026
Copyright: © 2026 Ayele et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are in the manuscript and its supporting information files.
Funding: This study was funded by the Directorate-General for Development Cooperation and Humanitarian Aid (DGD), Belgium (Project Reference: BE-BCE/KBO-0410057701-PRG2022-5-ET) (EA, MSA, WA, MP, JvG). The funders had no role in the study design; collection, analysis, or interpretation of data; writing of the report; or the decision to submit the paper for publication.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Visceral leishmaniasis (VL) is a disseminated protozoan infection transmitted by sandflies. It is caused by intracellular parasites belonging to the Leishmania (L.) donovani complex. Globally, around 30,000–90,000 cases are estimated to occur annually [1,2]. VL is caused by zoonotic L. infantum in Latin America and the Mediterranean region, and by anthroponotic L. donovani in the Indian subcontinent and East-Africa. Currently, East-Africa carries 73% of the global VL burden, with Sudan, South Sudan and Ethiopia most affected [2].
The World Health Organization (WHO) recently launched a framework for the elimination of VL as a public health problem, with reducing case fatality in primary VL to below 1% as a main indicator in the Neglected Tropical Diseases (NTD) road map [3]. Ethiopia, one of the highest burden countries for VL in East Africa, plays a critical role in these elimination efforts [4,5]. The elimination strategy emphasizes early diagnosis, effective treatment, and comprehensive patient management to reduce transmission and mortality as one of the main pillars to achieve VL elimination.
However, reducing case fatality requires prompt access to diagnosis and treatment, but also a clear understanding of the clinical and laboratory features of VL patients in routine care. This knowledge is crucial for improving patient care and outcomes, thereby contributing to reduced mortality—an essential component of the elimination strategy. In East-Africa, studies describing clinical and laboratory features in detail are mostly clinical trials, which usually involve highly selected patient populations and controlled environments, while data on more general patient populations are often limited to basic socio-demographic variables. As such, available information may not fully reflect the realities of standard clinical care. Hence, data from routine hospital care, where patients present with a wide range of comorbidities and varying degrees of disease severity, are essential for identifying key shortcomings in care provision and guide the design of interventions adapted for the different patient populations [5,6].
This prospective cohort study aims to fill this critical gap by comprehensively describing clinical and laboratory features of VL in a tertiary hospital in North-West Ethiopia, and characterizing the prevalence of common co-infections and co-morbidities in VL-endemic regions. Additionally, we provide an overview of the supportive care provided and describe the main causes of death. We also describe differences in the clinical presentation, management and outcomes of typical clinical trial patients versus those generally excluded from clinical trials.
Results
Baseline charateristics, medical history and diagnostic work-up
During the recruitment period, a total of 916 VL suspects were seen, of which 558 were diagnosed with VL (Fig 1). Of these, 454 VL cases were treated at LRTC, of which 314 (69.2%) were included in this study. A total of 140 VL cases were not included, due to inclusion in another study, unable to adhere to the follow-up visits, unable or refusing to provide informed consent and having started Leishmania treatment for more than 3 days.
LRTC: Leishmaniasis Research and Treatment Center. FU: follow-up.
All but one of the 314 study participants included were male; the median age was 22 years (IQR 20–28) (Table 1). The median duration of symptom onset was 41 days (IQR 30–70); only 71 (22.7%) participants presented within 30 days of symptom onset. Twenty (6.4%) participants were previously diagnosed with HIV, of which all were on antiretroviral treatment (ART) and four were on TB treatment. Fourteen (4.5%) participants had previously been treated for VL. Almost half (47%) of the participants reported to have taken traditional medicine for the current disease episode prior to attending LRTC.
With the revised MF/PM trial exclusion criteria, 204 (65.0%) of the 314 cases met at least one exclusion criterion and were considered “trial-ineligible patients” while 110 (35%) were “trial- eligible patients”. Trial-ineligible patients were more likely to be on TB treatment at enrolment and have a history of VL.
Symptoms and clinical signs
Besides fever and weight loss, vomiting, diarrhea, dyspnea and cough were commonly reported by the patients (Table 2). On clinical examination at baseline, oedema, ascites, jaundice and spontaneous bleeding were commonly observed. On clinical examination, abnormal respiratory findings (mainly crepitation and absent air entry) were found in 14%. The median body mass index was 16.9 kg/m2.
Several findings were more common in ‘trial-ineligible patients’, such as the presence of cough, diarrhea, ascites, jaundice, oedema, bleeding tendencies, respiratory or cardiac abnormalities on examination, and severe weakness at enrolment (Table 2).
Laboratory and radiological evaluations
Laboratory results are shown in Table 3. While hematological abnormalities were common, severe abnormalities were also seen in around a third of patients such as hemoglobin < 7 g/dL in 25.2%, platelets < 20.000/µL in 9.3% and severe neutropenia in 37.6%. Severe abnormalities in liver enzyme tests such as alanine transaminase five times above the limit of normal occurred in 11.8% of the cases, while creatinine levels were generally normal. Hypokalemia was seen in 48% cases. Albumin levels were decreased in all but one patient. INR was increased in all but 11 patients; eight individuals had INR values > 8. CRP-levels were increased (≥ 3 mg/L) in all but 4, with a median of 42 mg/L (IQR 19–79), ranging from 1.9 up to 302 mg/L. Levels > 100 mg/L were seen in 56 (18.1%) and < 10 mg/L in 40 (12.9%). Leishmania blood PCR was positive in 299 (95.2%), with a median cycle threshold (Ct) value of 23 cycles (IQR 21–27).
Co-infections
Amongst the 294 individuals tested for HIV infection at the time of VL diagnosis, one was newly diagnosed with HIV during the study period (Table 3). Combined with the 20 previously diagnosed HIV patients (all on ART), the prevalence of VL-HIV coinfection was 6.7% (21/314). Two patients had Plasmodium falciparum parasites identified microscopically in their blood. Using urine microscopy, hematuria was found in 13%, proteinuria in 53.5% and pyuria in 4.4% (Table 4).
Chest X-ray analyses showed that pneumonia was diagnosed in 49 (17.8%) participants, and TB was reported to be likely in one. Other abnormalities included pleural effusions and cardiomegaly. Abdominal ultrasound was done based on clinical suspicion of TB in 130 patients. Based on the ultrasound findings, disseminated TB was considered likely in 23 (17.7%) participants.
Most laboratory and radiological abnormalities were more frequently observed in trial-ineligible patients, including severe thrombocytopenia, high INR values (including all eight with values > 8), severe liver test abnormalities, severe hypoalbuminemia and chest X-ray abnormalities.
VL treatment and treatment outcomes
Overall, 217 (69.3%) patients with VL were started on SSG&PM and 93 (29.7%) on L-AmB-based treatment, the latter exclusively in trial-ineligible patients (Table 1). Three were started on MF&PM, as L-AmB was not available. One patient with severe VL died before VL treatment could be initiated. After the first VL treatment course, 271 (86.3%) were cured and 14 (4.5%) individuals had treatment failure, of which 12 were cured after additional treatment, one was referred to another center and one defaulted. At discharge (outcomes of the VL episode, including additional therapy for initial failures), 283 (90.1) were cured, 24 (7.6%) had died, six (1.9%) had defaulted and one (0.3%) was referred (Table 5).
Mortality (10.8%) and failure rates (6.4%) were higher in the trial-ineligible patients. While mortality was highest (11.5%; 21/183) in HIV-negative trial-ineligible patients, failure rates were relatively high in HIV patients (14.3%; 3/21). In primary VL cases, mortality was 7.7% (23/300).
Treatment was changed due to treatment-related adverse events in 17 (5.4%) participants, of which 16 individuals had received SSG&PM. Amongst these, the main adverse events leading to treatment change were due to renal (n = 8), liver (n = 6) and cardiac toxicity (n = 2). One individual started on MF&PM was changed to L-AmB due to worsening of liver enzymes. None of the 93 patients started on L-AmB-based therapy had to change treatment due to drug-related adverse events.
Outcome information was available by six months for 191 (60.8%) and by 12 months for 133 (43.4%) participants. Loss to follow-up was substantial and primarily related to ongoing conflict and instability in the region during the study period, including road closures and communication disruptions, which made patient tracing and long-term follow-up challenging. After the VL episode, 14 relapses were documented during follow-up, of which two died during treatment. Six were seen in non-HIV-coinfected individuals and ten relapses occurred within six months after treatment. Three patients died during follow-up due to events apparently not related to VL (one due to acute watery diarrhea, one due to meningitis, one due to a gunshot). Overall, 29 (9.2%) of the 314 study participants were reported to have died.
Use of antibiotics and other supportive care measures
Out of the 314 study participants, systemic therapeutic antibiotics were given to 156 (49.7%) during the treatment period, with pneumonia as the most common indication (Table 6). While ceftriaxone (with or without azithromycin) was most commonly used (36.3%), ceftazidime & vancomycin combination therapy was not uncommon (10.5%).
Antibiotic prescription was frequent across all baseline CRP categories, with little apparent discrimination by CRP level. Among participants with CRP < 10 mg/L at baseline, 32.5% received antibiotics within 48 hours of VL diagnosis, while 37.5% of those with CRP > 100 mg/L received antibiotics in the same time window (S3 Table). As CRP testing was done solely for study purposes and was not available to treating clinicians, these data suggest that early antibiotic initiation was largely independent of the degree of systemic inflammation as reflected by CRP.
TB was diagnosed during the VL episode in 15 (4.8%) patients, which was defined as disseminated TB in 11, pulmonary TB in two, central nervous system TB in one, and pericardial TB in one. Only in two cases, the diagnosis was bacteriologically confirmed by sputum analysis.
Twenty (6.4%) study participants were transfused with whole blood transfusion, ten (3.2%) received platelets and 13 (4.1%) fresh frozen plasma (Table 7). All but one blood products were administered to trial-ineligible patients. Potassium supplementation was given to 125 (39.8%) predominantly to trial-ineligible patients and vitamin K to 20 (6.4%), exclusively to trial-ineligible patients. Inotropes were prescribed to 9 (2.9%); corticosteroids were given to 19 (6.1%). Ten (3.2%) were referred to the intensive care unit but for five transfer was not possible (due to financial or logistical reasons).
Trial-ineligible patients more frequently received systemic antibiotics, TB treatment and other supportive measures including blood transfusion, corticosteroids and inotropics.
Causes of death
Table 8 summarizes the information on the main cause of death for 24 patients that died while residing in hospital for VL treatment, as provided on the death report by the treating physician (see S2 Table for the information listed for each individual). Severe bacterial infections - with or without other conditions - were mentioned in 13 patients, predominantly pneumonia. Acute liver failure was defined as cause of death in eight individuals. Acute renal failure was mentioned for four cases, and severe bleeding disturbances for three. Two patients died presumably due to fatal pulmonary embolism.
Discussion
In this VL cohort, including VL cases across the full spectrum of severity, a wide range of organs and systems were affected, some leading to pronounced pulmonary, hepatic, renal and coagulation dysfunction and abnormalities. The need for supportive care was high, including high therapeutic use of antibiotics, cardiovascular support and transfusion of blood products. Important reported causes of death included severe bacterial infection (particularly pulmonary), liver and renal failure, fatal hemorrhages and cardiac arrest. As the most comprehensive characterization of VL in East-Africa, this study can provide avenues for improving supportive care in VL and define areas for future research.
Patients with varying degrees of VL severity were included in this study. Importantly, the majority (65%) of VL cases would typically not be included in phase III clinical trials, especially those with signs of severe disease - requiring AmBisome therapy - and HIV co-infected patients. Mortality was highest in HIV-negative trial-ineligible patients, at 11.5%. Phase III clinical trials are designed to identify safe and effective antileishmanial treatments, but not to inform the evidence-base for improved management of complicated VL cases. With the ambition to reduce case fatality to below 1% and to decentralize VL care, this gap will have to be filled. Additional research is needed to optimize VL care for severe VL cases, to define which patients can be safely managed at the lower health care level, which patients would require referral to secondary and tertiary care levels, and what package of care needs to be available at the different levels of health care facilities.
Despite antileishmanial treatment, VL can potentially progress to multi-organ failure resembling sepsis. Some authors have suggested that VL could drive a protracted sepsis-like syndrome - referred to as leishmanial sepsis - mediated by dysregulated immune response which further increases the susceptibility to invasive bacterial infections and bacterial sepsis further aggravating the inflammatory process [11]. In the same study site, we previously found culture-proven bloodstream infections in 19% of VL cases [11,12].
In our cohort, severe bacterial infections were the leading cause of death, being reported in 54% (13/24) of deceased participants. This is consistent with data from Brazil, where bacterial infection was also the main cause of death in VL, followed by bleeding, liver failure, and renal failure [13]. Together, these observations highlight that timely recognition and treatment of bacterial co-infection is critical to prevent clinical deterioration and death in VL.
Appropriate antibiotic use in VL is, however, challenging because clinical manifestations of VL and bacterial sepsis overlap. In most patients in our study, therapeutic antibiotics were initially withheld, as the presenting fever was often judged to be VL-related. In this context, host inflammatory markers such as CRP could be valuable adjuncts to guide decisions on when to start antibiotics, particularly when the clinical picture is equivocal.
CRP and other acute phase proteins are likely upregulated in VL itself [13,15–17], and the optimal thresholds to indicate concomitant bacterial infection remain uncertain [14]. Nevertheless, baseline CRP values in our cohort showed considerable variability, and antibiotic prescribing did not meaningfully track this variation. With clinicians unaware of the CRP results, 32.5% of patients with CRP < 10 mg/L received early antibiotics, whereas 62.5% of those with CRP > 100 mg/L did not. These patterns suggest that, if prospectively validated thresholds were available, CRP (alone or combined with other host biomarkers) might help reduce unnecessary antibiotic use in patients with very low CRP values and, conversely, prompt earlier targeted treatment among patients with markedly elevated CRP.
Further studies are therefore needed to define CRP- and biomarker-based algorithms for antibiotic initiation in VL and to evaluate their impact on patient outcomes and antimicrobial stewardship. Such tools may be particularly useful in lower-level health facilities, where VL expertise and diagnostic capacity are limited. In better resourced centers, systematic use of blood cultures could complement biomarker-based approaches by identifying the causative pathogen and its antibiotic susceptibility.
Spontaneous bleeding was commonly observed on admission, and some patients died due to fatal bleedings. To what extent the increased bleeding tendency in VL cases is related to low or dysfunctional platelets, coagulation disturbances or both remains poorly understood [13]. In our study, INR values were increased in 96.5% of patients, but rarely markedly. Baseline thrombocytopenia was common, but < 20.000/µL occurred in less than 10%. Outside of Africa, data suggest that disseminated intravascular coagulation occurs in most patients [18]. While often subclinical, overt clinical manifestations with pronounced bleeding have been reported as well [13,19]. Liver dysfunction, vitamin K malabsorption and sepsis could potentially further worsen coagulation disturbances [13]. In our center, vitamin K supplements, fresh frozen plasma and/or platelets are routinely given (if available) in cases of severe laboratory abnormalities or overt bleeding. Although the evidence-base supporting such practices remains limited, particularly with ongoing disseminated intravascular coagulation, it might be prudent to refer severe VL cases with spontaneous bleeding to referral centers capable of administering blood products.
In line with other studies, liver disturbances were common, often pronounced and liver failure was considered the main cause of death in eight individuals [20,21]. While hepatic effects of VL have been consistently reported [13,20–23], other factors could further exacerbate this, including use of traditional medicine, sepsis and concurrent medication given during admission [13]. This warrants careful use of potentially hepatotoxic medication, with close laboratory monitoring, access to AmBisome for those with pronounced liver dysfunction, and early identification of sepsis. A similar approach could apply to renal dysfunction. While rare on admission, it required treatment change in eight individuals and contributed to death in several cases. As herbal medicine can be liver or nephrotoxic [24], more work is needed to understand the types of herbal medicine being used in VL endemic areas and how it affects the course and management of VL.
In summary, strategies to prevent clinical deterioration and reduce case fatality should focus on careful evaluation for bacterial infections and early antibiotic use, effective measures for infection prevention and control, judicious use of potentially toxic concurrent medication with close monitoring, cardiovascular support, and careful administration of blood products. With two deaths potentially related to fatal pulmonary embolism, the role of prophylactic anticoagulation should be further explored, despite the increased bleeding tendency commonly seen in VL [25,26].
Strengths of the study are that it was conducted in a highly experienced Leishmania treatment and research center, with a comprehensive laboratory assessment supported by high-quality laboratory facilities. Additionally, we included the full spectrum of VL cases, in contrast with clinical trials, and hence more representative of the overall VL population seeking care in an East-African setting. There are a number of important limitations to acknowledge. Because this was a descriptive cohort study and not a hypothesis-driven analytical study, differences observed between trial-eligible and trial-ineligible patients should be interpreted as descriptive patterns rather than statistically confirmed or causal associations. In addition, factors such as HIV co-infection, disease severity, and treatment regimen may confound observed differences between groups. Not all patients diagnosed with VL at the center could be included. Fifty-three patients were enrolled in other studies of which 21 are included in a clinical trial, which could over-represent the “trial-ineligible” patients in the present study. It also needs to be acknowledged that LRTC is a referral center receiving complicated cases, and it may not fully represent other treatment centers managing VL. Another important limitation of this study is that the study population consisted almost exclusively of young adult males, reflecting the epidemiology of VL in this setting, where the disease disproportionately affects seasonal migrant workers engaged in agricultural labor in endemic lowland areas. Consequently, women, young children, and elderly individuals were underrepresented in our cohort. This demographic pattern differs from that observed in several other East-African and global VL-endemic settings, where VL more commonly affects children and includes a broader representation of women and older patients. Therefore, the external validity of our findings is limited, and caution is required when generalizing these results to other endemic regions or to the broader VL population. Our findings should thus primarily be interpreted in the context of VL among migrant-worker populations in Northwest Ethiopia and other similar settings. Some tests such as additional coagulation tests or blood culture could have been of value. Due to the ongoing conflict and instability in the region during the study, long-term outcomes were not available for a substantial proportion of study participants. Loss to follow-up mainly affected the ascertainment of long-term outcomes after discharge, including relapse and late mortality, but did not affect the primary in-hospital outcomes reported in this study.
In conclusion, our study shows that at the main VL treatment center in Ethiopia, most VL patients present with severe disease, with multiple organs affected. Severe bacterial infections, severe bleeding and liver failure were commonly diagnosed and potentially leading to death. Most of our patients would typically not be included in clinical trials, and this group displayed high mortality. Besides strategies aiming at early VL diagnosis and treatment, enhanced VL care will be needed to decrease case fatality rates, the main indicator of success for the East-African VL elimination initiative.
Methods
Ethics statement
The study protocol was approved by the institutional review board of the Institute of Tropical Medicine Antwerp, Antwerp, Belgium (ref 1544/21) and the University of Gondar, Ethiopia (ref VP/RTT/05/965/2022), the ethical committee of the University of Antwerp, Belgium (ref 3276) and the Ethiopian national research ethics review committee (ref 17/152/147/23). Written informed consent was obtained from all participants
Study setting
The study was conducted at the Leishmania Research and Treatment center (LRTC) at the University of Gondar, North-West Ethiopia. The center, supported by the University of Gondar, the Drugs for Neglected Diseases Initiative (DNDi), and the Institute of Tropical Medicine (ITM) conducts clinical trials on VL and provides free health care to routine VL patients. LRTC serves as the key referral center for treatment of VL in the Amhara region since 2004 [6]. Most VL cases treated at the center are seasonal migrant workers, traveling from various parts of the country to the VL-endemic lowland cash crop farming areas of North-West Ethiopia. Additionally, the center treats residents of these lowland areas and patients referred from other centers for further diagnostic work-up or due to severe disease [7].
Study design and population
We conducted a prospective cohort study including all VL cases treated at LRTC between February 2023 and June 2024. We excluded patients below the age of 12 years, those already on VL treatment for three days or more, and those unable to give blood or come for follow-up. Over the planned recruitment period of 18 months, we expected to recruit 741 patients from LRTC and the Abdurafi health center supported by Médecins Sans Frontières (MSF), but due to insecurities in the area the study was only conducted at LRTC. The study was registered on ClinicalTrials.gov (identifier NCT05602610).
Routine VL diagnosis and treatment
Individuals presenting at the University of Gondar Comprehensive Specialized Hospital and meeting the VL clinical case definition (fever for two or more weeks and enlarged spleen or either loss of weight, anemia or leukopenia) with a travel history to or residents of a VL endemic area were considered as VL suspects and referred to LRTC for diagnostic assessment. In each primary VL case (no previous VL), rK39 rapid diagnostic test (RDT) (IT-LEISH, Bio-Rad, USA) and spleen aspiration was done. In case of contraindications to spleen aspiration (e.g., platelets<40,000 or signs of increased bleeding tendency), bone marrow aspiration was performed. For those with previous VL history (i.e., cases of relapse), only tissue aspiration was done. A VL diagnosis was made if Leishmania parasites were detected microscopically in the tissue aspirate. If no parasites were detected, a diagnosis of primary VL could (occasionally) still be made based on the combination of high clinical suspicion and a positive rK39 RDT result. All VL cases were also tested for HIV co-infection.
VL treatment was provided according to the national guideline [8]. Most primary VL was treated with a combination of sodium stibogluconate (SSG) (20 mg/kg intramuscular (IM) or intravenous (IV) with a maximum dose of 850 mg) and paromomycin (PM) (11 mg/kg IM) for 17 days. Liposomal amphotericin B (L-AmB; AmBisome) (30mg/kg IV over 12 days) was reserved for complicated VL cases, including those with VL relapse, or older age (> 45 years) and more severe VL symptoms (e.g., critical condition, severe laboratory abnormalities). In case of HIV co-infection, L-AmB 30 mg/kg was combined with miltefosine (MF; 2.5 mg/kg per os (PO) daily for 28 days) for 1 or 2 cycles. All patients were admitted for monitoring, with regular clinical and laboratory assessments during the treatment period. Treatment response was assessed at the end of treatment visit (day 17 for SSG&PM and AmB- and day 28 for L-AmB&MF-treated patients) and was based on clinical signs (such as absence of fever, improved appetite, weight gain, and reduced spleen size) and laboratory results (improvement in hematologic profile). If there was no significant clinical improvement and no change in hematologic profile, or in case of HIV coinfection, a parasitological test of cure was performed by microscopic evaluation of spleen or bone marrow tissue aspirates for the detection of parasites using Giemsa staining. If parasites remained detectable, the patient was defined to have treatment failure and additional treatment was given. If there was suspicion of relapse during the follow-up period after treatment, VL was confirmed by spleen or bone marrow tissue aspiration except for HIV coinfected patients with chronic, relapsing VL who received treatment based on clinical symptoms, as they did not manage to clear parasites with treatment anymore and it was decided to start treatment on clinical grounds.
Investigations, data recording and patient follow-up
After VL diagnosis, a comprehensive clinical evaluation was conducted and data were collected on VL-related symptoms, signs and clinical parameters. Blood, stool and urine was collected for further analysis, and electrocardiogram (ECG) (GE Healthcare, Mac 2000, India) and chest X-ray (DogX-ray, China) were performed. If there was a productive cough, sputum was collected and sent for tuberculosis (TB) testing using GeneXpert (Cepheid, USA). In case of clinical suspicion of disseminated TB, an abdominal ultrasound (Samsung Medison, Republic of Korea) was performed.
During treatment, clinical and laboratory assessments were performed on day 3, day 7, and at the end of treatment study visit. After discharge, follow-up visits were planned for three and six months after the end of treatment for clinical and laboratory evaluation. Phone calls were made on month 9 and month 12 after admission to document the long-term outcomes. Individuals missing planned study visits were also contacted by phone and invited for a clinical assessment at LRTC or, if not able to attend, to collect information on their VL-related health. Participants were defined as lost to follow-up if they were not seen at the follow-up visit or for those missing visits, if the participant could not be reached by phone. For these participants, we had no further information on their outcome. In case of a relapse and subsequent treatment, a post-relapse visit after 6 months was scheduled. The study closed at the end of June 2025.
We documented the anti-Leishmania treatment given, the occurrence of adverse events, reasons for treatment change and the response to treatment. Ancillary and supportive care were documented as well, including all concomitant treatments received with indications. The need of advanced supportive care (e.g., intensive care, dialysis and ionotropic use…) and transfusion of blood products (e.g., whole blood, fresh frozen plasma, platelets) was also recorded. Patients were given antibiotics on indication, based on the suspected focus. Prescriptions of antibiotics was not informed by CRP-levels, as this was a study-related procedure. For patients with severe neutropenia (an absolute neutrophil count <500 cells/ µL), antibiotics were given (ciprofloxacin PO or ceftriaxone IV) for prophylaxis or presumed sepsis treatment. In case an individual died, the main cause of death was defined by the treating clinician. To avoid directing the treating clinician towards certain terms or causes of death, the information was entered using free text without pre-defined categories for causes of death or contributing factors.
Laboratory assays and radiological investigations
The following laboratory tests were performed on blood at baseline: a complete blood count (SysmexXN350 and SX500, Japan), kidney and liver function tests, liver enzymes (Humastar 200, Germany), electrolytes (Humalyte Plus Human Germany), C-Reactive Protein (CRP) (Quick Read Go instrument, Finland), International Normalized Ratio (INR; Coagucheck XS, Germany) and HIV serology (One Step Anti-HIV (1 & 2), China). For Leishmania, besides the routine rK39 RDT and microscopy of Giemsa-stained spleen or bone marrow aspirates, real-time PCR targeting kinetoplast DNA (kDNA) on whole blood was done in batches as described before [9].
Additional tests included urine dipstick and microscopy, Giemsa-stained thick and thin blood films for malaria and stool wet mount microscopy for intestinal parasite eggs and trophozoites (Lugol’s iodine and normal saline wet mount). For chest X-ray and abdominal ultrasound analyses, we used the routine report from the radiologist. Based on the abdominal ultrasound report, the treating VL clinician – extensively experienced in VL care - defined whether the radiological findings were compatible with disseminated TB.
Data collection and statistical analysis
The study was conducted in compliance with Good Clinical Practice (GCP). Data were collected electronically using the GCP compliant REDCap software.
Treatment outcomes were described after the first course of treatment (at the end of treatment visit) and at discharge from hospital (end of the VL episode), also taking into consideration additional treatment for treatment failures. Outcomes at the end of treatment and at discharge were defined as cure, treatment failure, deceased, defaulted or referred. The number and proportion of patients with relapse after discharge was reported as well.
To define the group of patients that would typically be excluded from clinical trials (“trial-ineligible patients”), we applied the revised trial exclusion criteria from the phase III clinical trial comparing MF&PM and SSG&PM, conducted by DNDi at seven sites in Kenya, Uganda, Sudan, and Ethiopia, including at the LRTC in Gondar [10]. In this trial, the original (stricter) exclusion criteria were revised during the study to allow for recruitment of a more representative VL population. In the present paper, we will refer to this trial as the MF&PM trial [10]. The revised exclusion criteria we applied were age < 4yrs or >45yrs, relapse, HIV coinfection, concurrent TB, clinically significant ECG abnormalities, haemoglobin <5 g/dL and severe VL according to the investigator’s judgement, requiring AmBisome therapy (see S1 Table).
The analysis was primarily descriptive. Demographic, clinical, laboratory, treatment and outcome variables using medians with interquartile ranges (IQR) for continuous variables and frequencies with percentages for categorical variables. Where relevant, continuous variables were categorized using clinically relevant cut-offs. The analysis describing differences between trial-eligible and trial-ineligible patients was intended to provide an overall characterization of differences in case mix, supportive care needs, and outcomes in routine care. No formal hypothesis testing was performed, as the primary objective was to characterize the cohort and describe real-world patient profiles. Emphasis was placed on the magnitude and clinical relevance of observed differences. Analysis was done using Stata version 18 (StataCorp LP, College Station, TX, USA).
We summarized the information provided by the treating clinician on the main cause of death for each individual, grouped by the main underlying process or organ affected (e.g., severe infection or acute liver failure). The cause(s) of death for each individual can be found in S2 Table. Only deaths occurring during hospitalization for VL treatment were recorded. Hence individuals known to have died after discharge outside the hospital were not included in S2 Table.
Supporting information
S1 Text. Additional information on the diagnosis of visceral leishmaniasis.
https://doi.org/10.1371/journal.pntd.0013878.s001
(DOCX)
S1 Table. Overview of the main exclusion criteria from the miltefosine/paromomycin trial that were used to define the non-trial group within the study populationa.
https://doi.org/10.1371/journal.pntd.0013878.s002
(DOCX)
S2 Table. Line list of the main causes of death in VL cases in Gondar, Ethiopia (2023–2024).
https://doi.org/10.1371/journal.pntd.0013878.s003
(DOCX)
S3 Table. Prescription of systemic therapeutic antibiotics within 48 hours of VL diagnosis, stratified by CRP values at baseline in VL cases in Gondar, Ethiopia (2023–2024).
https://doi.org/10.1371/journal.pntd.0013878.s004
(DOCX)
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