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Blood transfusion remains an important aspect of patient management in visceral leishmaniasis (VL). However, transfusion triggers considered are poorly understood. This review summarises the transfusion practices adopted in VL efficacy studies using the Infectious Diseases Data Observatory VL clinical trials library. Of the 160 studies (1980-2021) indexed in the IDDO VL library, description of blood transfusion was presented in 16 (10.0%) (n=3459 patients) studies. Transfusion was initiated solely based on haemoglobin (Hb) measurement in nine studies, combining Hb measurement with an additional condition (epistaxis/poor health/clinical instability) in three studies and the criteria was not mentioned in four studies. The Hb threshold range for triggering transfusion was 3-8 g/dL. The number of patients receiving transfusion was explicitly reported in 10 studies (2421 patients enrolled, 217 underwent transfusion). The median proportion of patients who received transfusion in a study was 8.0% (Interquartile range: 4.7% to 47.2%; range: 0-100%; n=10 studies). Of the 217 patients requiring transfusion, 58 occurred before VL treatment initiation, 46 during the treatment/follow-up phase and the time was not mentioned in 113. This review describes the variation in clinical practice and is an important initial step in policy/guideline development, where both the patient's Hb concentration and clinical status must be considered., (© The Author(s) 2024. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Visceral leishmaniasis is a neglected tropical disease endemic in East Africa where improved patient-adapted treatments are needed. The Leishmaniasis East Africa Platform (LEAP) was created in 2003 to strengthen clinical research capacity, serve as a base for training, and evaluate and facilitate implementation of new treatments. Major infrastructure upgrades and personnel training have been carried out. A short course of Sodium Stibogluconate and Paramomycin (SSG&PM) was evaluated and is now first-line treatment in the region; alternative treatments have also been assessed. LEAP can serve as a successful model of collaboration between different partners and countries when conducting clinical research in endemic countries to international standards., (© The Author 2016. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

In established Leishmania donovani visceral infection in normal mice, anti-interleukin (IL)-10 receptor (IL-10R) monoclonal antibody (MAb) treatment induced intracellular parasite killing within liver macrophages. IL-10R blockade maintained IL-12 protein 40, markedly increased interferon (IFN)-gamma serum levels, and enhanced tissue inducible nitric oxide synthase (iNOS) expression and granuloma assembly. Optimal MAb-induced killing, including synergism with antimony chemotherapy, required endogenous IL-12 and/or IFN-gamma and at least one IFN-gamma-regulated macrophage mechanism-iNOS or phagocyte oxidase. However, in IFN-gamma knockout mice, anti-IL-10R also induced both granuloma formation and leishmanistatic activity. As judged by IL-10R blockade, endogenous IL-10 primarily regulates killing in L. donovani infection by suppressing production of and responses to the Th1 cell-type cytokines, IL-12, and IFN-gamma. However, because anti-IL-10R also released IFN-gamma-independent effects, IL-10 appears to act more broadly and suppresses multiple antileishmanial mechanisms.

The gene encoding type II DNA topoisomerase from the kinetoplastid hemoflagellated protozoan parasite Leishmania donovani (LdTOP2) was isolated from a genomic DNA library of this parasite. DNA sequence analysis revealed an ORF of 3711 bp encoding a putative protein of 1236 amino acids with no introns. The deduced amino acid sequence of LdTOP2 showed strong homologies to TOP2 sequences from other kinetoplastids, namely Crithidia and Trypanosoma spp. with estimated identities of 86 and 68%, respectively. LdTOP2 shares a much lower identity of 32% with its human homologue. LdTOP2 is located as a single copy on a chromosome in the 0.7 Mb region in the L.donovani genome and is expressed as a 5 kb transcript. 5'-Mapping studies indicate that the LdTOP2 gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon occurring at -639 from the predicted initiation site. Antiserum raised in rabbit against glutathione S-transferase fusion protein containing the major catalytic portion of the recombinant L.donovani topoisomerase II protein could detect a band on western blots at approximately 132 kDa, the expected size of the entire protein. Use of the same antiserum for immunolocalisation analysis led to the identification of nuclear, as well as kinetoplast, antigens for L.donovani topoisomerase II. The in vitro biochemical properties of the full-length recombinant LdTOP2 when overexpressed in E.coli were similar to the Mg(II) and ATP-dependent activity found in cell extracts of L.donovani.

From the early 1900s, visceral leishmaniasis (VL; kala-azar) has been among the most important health problems in Sudan, particularly in the main endemic area in the eastern and central regions. Several major epidemics have occurred, the most recent--in Western Upper Nile province in southern Sudan, detected in 1988--claiming over 100,000 lives. The disease spread to other areas that were previously not known to be endemic for VL. A major upsurge in the number of cases was noted in the endemic area. These events triggered renewed interest in the disease. Epidemiological and entomological studies confirmed Phlebotomus orientalis as the vector in several parts of the country, typically associated with Acacia seyal and Balanites aegyptiaca vegetation. Infection rates with Leishmania were high, but subject to seasonal variation, as were the numbers of sand flies. Parasites isolated from humans and sand flies belonged to three zymodemes (MON-18, MON-30 and MON-82), which all belong to the L. donovani sensu lato cluster. Transmission dynamics have not been elucidated fully; heavy transmission in relatively scarcely populated areas such as Dinder national park suggested zoonotic transmission whereas the large numbers of patients with post kala-azar dermal leishmaniasis (PKDL) in heavily affected villages may indicate a human reservoir and anthroponotic transmission. Clinical presentation in adults and in children did not differ significantly, except that children were more anaemic. Fever, weight loss, hepato-splenomegaly and lymphadenopathy were the most common findings. PKDL was much more common than expected (56% of patients with VL developed PKDL), but other post-VL manifestations were also found affecting the eyes (uveitis, conjunctivitis, blepharitis), nasal and/or oral mucosa. Evaluation of diagnostic methods showed that parasitological diagnosis should still be the mainstay in diagnosis, with sensitivities for lymph node, bone marrow and spleen aspirates of 58%, 70% and 96%, respectively. Simple, cheap serological tests are needed. The direct agglutination test (DAT) had a sensitivity of 72%, specificity of 94%, positive predictive value of 78% and negative predictive value of 92%. As with other serological tests, the DAT cannot distinguish between active disease, subclinical infection or past infection. The introduction of freeze-dried antigen and control sera greatly improved the practicality and accuracy of the DAT in the field. An enzyme-linked immunosorbent assay using recombinant K39 antigen had higher sensitivity than DAT (93%). The polymerase chain reaction using peripheral blood gave a sensitivity of 70-93% and was more sensitive than microscopy of lymph node or bone marrow aspirates in patients with suspected VL. The leishmanin skin test (LST) was typically negative during active VL and converted to positive in c. 80% of patients 6 months after treatment. Immunological studies showed that both Th1 and Th2 cell responses could be demonstrated in lymph nodes from VL patients as evidenced by the presence of messenger ribonucleic acid for interleukin (IL)-10, interferon gamma and IL-2. Treatment of peripheral blood mononuclear cells from VL patients with IL-12 was found to drive the immune response toward a Th1 type response with the production of interferon gamma, indicating a potential therapeutic role for IL-12. VL responded well to treatment with sodium stibogluconate, which is still the first line drug at a dose of 20 mg/kg intravenously or intramuscularly per day for 15-30 d. Side effects and resistance were rare. Liposomal amphotericin B was effective, with few side effects. Control measures have not been implemented. Based on observations that VL does not occur in individuals who have a positive LST, probably because of previous cutaneous leishmaniasis, a vaccine containing heat-killed L. major promastigotes is currently undergoing a phase III trial.

Active targeting of doxorubicin to macrophages was studied by incorporating it in mannose-coated liposomes by use of visceral leishmaniasis in BALB/c mice as the model macrophage disease. Mannosylated liposomal doxorubicin was more effective than liposomal doxorubicin or free doxorubicin. Because leishmaniasis is accompanied by immunosuppression, immunostimulation by interferon (IFN)-gamma was evaluated to act synergistically with mannosylated liposomal doxorubicin therapy. Combination chemotherapy with a suboptimal dose of IFN-gamma resulted in possibly complete elimination of spleen parasite burden. Analysis of mRNA levels of infected spleen cells suggested that targeted drug treatment together with IFN-gamma, in addition to greatly reducing parasite numbers, resulted in reduced levels of interleukin (IL)-4 but increased levels of IL-12 and inducible nitric oxide synthase. Such combination chemotherapy may provide a promising alternative for the cure of leishmaniasis, with a plausible conversion of antiparasitic T cell response from a Th2 to Th1 pattern indicative of long-term resistance.

Tests on a panel of 421 serum samples from Ethiopia and 11 from Europe with the direct agglutination test (DAT) and freeze-dried antigen (Leishmania donovani 1S) showed that a cut-off titre of 1:200 or 1:400 could be employed. Using a cut-off titre of 1:400, 181 (88.3%) of 205 serum samples from visceral leishmaniasis (VL) patients tested positive, as did 5 (13.9%) of 36 sera from patients with localized cutaneous leishmaniasis, 5 (71.4%) sera from 7 patients with diffuse cutaneous leishmaniasis and 8 (89.9%) sera from 9 HIV-positive VL patients. We also found that 19 (57.6%) sera from 33 treated VL patients were still positive in the DAT at 1.5 to 5 years after treatment.

Kenyan subjects with visceral leishmaniasis were examined for evidence of increased production of soluble interleukin-4 receptor (sIL-4R). Soluble IL-4R regulates the bioactivity of IL-4, a cytokine important in mediating progressive forms of leishmaniasis. Persons with visceral leishmaniasis sustained 8- to 10-fold more circulating sIL-4R compared with Papua New Guinea residents with documented filariasis or uninfected Kenyan and North American subjects. Soluble IL-2R concentrations were elevated nonspecifically in both visceral leishmaniasis and filariasis patients. These findings are significant given that IL-4 induces sIL-4R in mice, and treatment with recombinant sIL-4R cures progressive murine leishmaniasis dependent on IL-4 bioactivity. Further studies are indicated to determine whether the immunologic detection of IL-4 produced in human visceral leishmaniasis is obscured because of sequestration by soluble receptor and whether the production of sIL-4R is relevant to the pathogenesis of visceral leishmaniasis.

Interferon (IFN)-gamma, in both natural endogenous form and administered as exogenous protein, induces control over visceral Leishmania donovani in experimentally infected BALB/c mice. To further characterize the therapeutic role of IFN-gamma in host defense against intracellular L. donovani, the efficacy of IFN-gamma delivered by gene transfer was tested. One week after infection, normal and IFN-gamma gene-disrupted (GKO) BALB/c mice were injected with an IFN-gamma gene-bearing mammalian expression plasmid (pIFN). Plasmid-specific IFN-gamma transcripts were detected in liver and spleen. Whereas liver parasite burdens more than doubled in untreated and mock-treated normal and GKO mice during the subsequent 2 weeks, animals injected with pIFN had controlled visceral infection and reduced parasite burden. These results indicate that, in infected tissues, IFN-gamma delivered by gene transfer enhances control over disseminated intracellular infection.

Interferon-gamma (IFN-gamma) enhances the therapeutic response to pentavalent antimony in patients with visceral leishmaniasis. To determine the effect of cytokine immunotherapy alone, 9 patients with kala-azar were treated with IFN-gamma before receiving antimony. After 20 days of IFN-gamma therapy, 4 patients showed no parasitologic response; in the remaining 5 patients, however, splenic aspirate parasite scores declined from 4.2 +/- 0.2 to 1.2 +/- 0.5 (mean +/- SE). These results indicate that treatment with IFN-gamma alone can induce visceral antileishmanial activity. However, the limited efficacy in this uncontrolled pilot trial suggests that the therapeutic role of IFN-gamma in kala-azar is that of an adjunct to conventional antimony treatment.

To determine if interferon (IFN)-gamma can enhance intracellular antimicrobial defense in a T cell-deficient host, nude BALB/c mice were infected with Leishmania donovani and treated with IFN-gamma. IFN-gamma induced killing of L. donovani in livers of euthymic mice but had no effect in nude mice despite activating peritoneal macrophages in vivo. Transfer of CD4+ or CD8+ T cells permitted nude mice to respond to IFN-gamma; treatment with T cell-regulated antileishmanial cytokines (interleukin-2, granulocyte-macrophage colony-stimulating factor, or tumor necrosis factor-alpha) could not substitute for T cells. NK cells played no apparent role. In reconstituted nude mice, the antileishmanial effect of IFN-gamma correlated with markedly enhanced mononuclear cell recruitment to infected liver foci. Thus, although IFN-gamma activates macrophages in the absence of host T cells, a T cell mechanism is required for antileishmanial activity in tissue. Provided one T cell subset is adequately preserved, IFN-gamma may prove useful in intracellular infections in the T cell-deficient host.

Fifteen Indian patients with relapsing or drug-refractory visceral leishmaniasis were retreated for 30 days with antimony plus interferon-gamma (IFN-gamma). All 15 had failure of an initial course of antimony and at least one additional course of antimony or pentamidine; 7 patients had failure of three or four prior courses of therapy. During the study, treatment was discontinued in 2 patients because of anemia and congestive heart failure in 1 and intractable vomiting in the other; both subsequently died. In the remaining 13 patients, IFN-gamma plus antimony treatment was associated with daily fever but no other adverse reactions. After 30 days of therapy, 9 (69%) of the 13 patients were apparently cured. Six months after treatment, all 9 were healthy, had parasite-free bone marrow aspirate smears, and were considered cured. None have relapsed during a mean follow-up of 15.9 +/- 1.7 months. These results support the use of antimony plus IFN-gamma as an immunochemotherapeutic alternative for kala-azar patients who have repeated failures of conventional treatment.

The optimal therapy for visceral leishmaniasis in patients with AIDS is unknown. We describe herein three patients with advanced AIDS and disseminated leishmaniasis who were treated with meglumine antimoniate plus interferon-gamma. The conditions of the three patients rapidly improved, and the therapeutic combination drastically reduced the parasite burden; after administration of antiprotozoal therapy for 4 weeks, cultures of bone marrow were negative for two of the three patients. Interferon-gamma was well tolerated. Because the patients died of other AIDS-related diseases in the months following therapy, the efficacy of the combination of meglumine antimoniate and interferon-gamma for preventing relapses remains unknown.

Traditionally, the mainstay of visceral and diffuse cutaneous leishmaniasis therapy has been pentavalent antimony, with pentamidine and amphotericin B reserved for refractory cases. Interferon-gamma (IFN-gamma) in combination with pentavalent antimony has been used successfully in treating patients refractory to pentavalent antimony and in patients with previously untreated visceral leishmaniasis (39 total). In addition, 6 patients with diffuse cutaneous leishmaniasis have been treated with combination IFN-gamma and antimony therapy. Preliminary experience with these patients indicates that IFN-gamma is a useful adjunct therapy for severe or refractory cases of visceral leishmaniasis; however, the potential of IFN-gamma in treating diffuse cutaneous leishmaniasis cannot be assessed as current clinical experience is too limited.

Disturbance of T cell-mediated immunity has been reported in acute visceral leishmaniasis (AVL). In a study of 16 patients with AVL, defective production of interleukin-1 (IL-1) by peripheral blood mononuclear cells was demonstrated in response to leishmania antigens, heat-killed Listeria organisms, and lipopolysaccharide when compared to posttherapy values or controls. This global defect in IL-1 production was corrected after successful therapy. Twelve of 16 patients responded with a greater than or equal to 2.5-fold increase in IL-1 production that correlated with clinical cure, P less than .01. Depressed production of tumor necrosis factor (TNF) was leishmania antigen-specific and similarly recovered after therapy. In vitro TNF production during the follow-up period did not correlate with clinical status but high serum levels were associated with AVL. Since T cells are activated by processed antigens presented on class II major histocompatibility molecules and by newly synthesized IL-1, defective IL-1 production may contribute to the immunosuppression observed in AVL.

The epidemiology, clinicopathological features, and response to therapy of 63 Saudi patients with visceral leishmaniasis are described. The clinical features in our cases were similar to those described from Asir province, India, and Ethiopia, except for the presence of lymphadenopathy. Fever, hepatosplenomegaly, pancytopenia, and liver dysfunction were common findings. The unusual feature is the seasonal variation in the distribution of the disease. The response to sodium stibogluconate was excellent and the mortality rate was low (less than 1 per cent).

High tumor necrosis factor-alpha (TNF alpha) levels were present in the serum of 24 of 28 active visceral leishmaniasis (VL) patients (142.9 +/- 113.9 pg/ml, mean +/- SD), whereas levels were not elevated in 26 of 30 patients with cryptic leishmanial infection (16 asymptomatic, 4 with self-healing subclinical infection, and 10 posttreatment VL cases). Serum TNF alpha levels were also not elevated in 15 normal volunteers (11.3 +/- 15.6 pg/ml) and in 10 patients with tegumentary leishmaniasis (19.1 +/- 10.8 pg/ml). Leishmanial infection of human monocyte-derived macrophages enhanced the basal TNF alpha production by these cells, and this effect was further potentiated by treatment with recombinant interferon-gamma. After effective treatment of VL patients, serum TNF alpha levels dropped rapidly (129 +/- 112 vs. 9 +/- 13 pg/ml in 10 days), even before clinical parameters such as spleen size or parasitism, white blood cell count, or levels of hemoglobin returned to normal values. On the other hand, patients unresponsive to treatment remained with elevated levels (276 +/- 69 vs. 155 +/- 71 pg/ml in 10 days). Thus, serum TNF alpha levels in VL patients are a good parameter to monitor in determining host response to therapy.

In experimental visceral leishmaniasis, intermittently administered interferon-gamma (IFN-gamma) induces antileishmanial activity, which is primarily microbistatic. To determine if the efficacy of IFN-gamma immunotherapy could be enhanced by continuous delivery, Leishmania donovani-infected mice were treated using a subcutaneous osmotic pump. Once-daily intraperitoneal injections of 10(5) or 10(6) units of IFN-gamma inhibited the replication of L. donovani within liver macrophages but overall did not reduce liver parasite burdens. In contrast, a comparable dose of IFN-gamma (2.4 x 10(5) units/day) administered continuously induced an enhanced effect and reduced liver burdens by almost 50%. Although pump delivery did not similarly increase the efficacy of antimony chemotherapy in infected mice, continuous treatment with IFN-gamma plus antimony produced an additive antileishmanial effect. These results suggest that continuous infusions of macrophage-activating lymphokines such as IFN-gamma (used alone or in combination with chemotherapy) may be required to optimize in vivo antimicrobial effects.

To determine if the macrophage-activating T cell lymphokine gamma interferon (IFN-gamma) can enhance the effect of conventional chemotherapy against intracellular Leishmania donovani, we treated human macrophages in vitro with both recombinant (r) IFN-gamma and sodium stibogluconate (Pentostam). After pretreatment with a nonactivating dose of rIFN-gamma (10 U/mL), ineffective concentrations of Pentostam (1 and 5 micrograms/mL) were converted to leishmanistatic concentrations, and a leishmanistatic dose (10 micrograms/mL) was converted to uptake of Pentostam. In a model of visceral leishmaniasis, infected mice were treated with ineffective concentrations of rIFN-gamma (10(4) U) plus suboptimal doses of Pentostam (10 or 50 mg/kg). With combination therapy, the doses of Pentostam required to achieve 50% inhibition or killing of visceral L. donovani were reduced by 10-fold and fourfold, respectively. These results suggest that IFN-gamma therapy may be a useful adjunct in visceral leishmaniasis and illustrate one potential role for IFN-gamma in the treatment of systemic intracellular infections.

Antigens with molecular weight ranges of 94-67 kDa (LiF2), 30-20 kDa (LiF5), or below 20 kDa (LiF6), isolated from lysates of Leishmania infantum promastigotes by electroelution from polyacrylamide gels were injected into mice which were genetically either partially resistant (C57BL/6) or susceptible (BALB/c) to Leishmania infection. One month after the completion of the intravenous (C57BL/6) or subcutaneous (BALB/c) schedules, the mice were challenged with 1 x 10(3) L. major promastigotes. All mice immunized with LiF2, LiF5 and LiF6 were completely resistant. Furthermore, the C57BL/6 mice immunized with LiF2 resisted a second challenge with 1 x 10(4) L. major amastigotes. 5 months later, LiF2 antigen was used for immunotherapy of L. major leishmaniasis; parasites disappeared from the treated skin lesions, although ensuing systemic infection could not be averted.