Liposomal dual delivery systems in visceral leishmaniasis enhance the synergistic effects of combination therapy:A promise for the future

Visceral leishmaniasis(VL)is a neglected tropical disease,and this review has summarized the current treatment scenario and its prospects.It also highlights alternative approaches used by research groups in India and around the world to develop cutting-edge and potent anti-leishmanial treatments.Even though numerous medications could be utilized to treat VL,the limitations of current treatments including their toxicity,cost,route of administration,and duration of doses,have contributed to the emergence of resistance.Combination therapy might be a better option due to its shorter duration,easier route of administration,and ability to extend the lifespan of individual drugs.However,there is a risk of not delivering both the drugs to the target site together,which can be overcome by the liposomal entrapment of those drugs and at a time knock an opportunity to reduce the dosage of amphotericin B if the combination drug provides a synergistic effect with it.Therefore,this review presents a novel strategy to fight against VL by introducing dual drug-loaded liposomes.

Hematological picture of pediatric Sudanese patients with visceral leishmaniasis and prediction of leishmania donovani parasite load

BACKGROUND Visceral leishmaniasis(VL)is a systemic protozoan infection caused by Leishmania donovani(L.donovani)and transmitted by sand flies,causing macrophage invasion in the liver,spleen,and bone marrow.Diagnosis of VL is currently based on clinical signs,symptoms,and specific in-vitro markers and bone marrow investigations.However,VL's specific hematological and bone marrow manifestation in Sudanese pediatric patients is not well studied.AIM To examine the blood and bone marrow characteristics in pediatric patients from Sudan who have VL.METHODS This is a retrospective hospital-based study with a sample of 107 consecutive Sudanese pediatric patients.The data focused on hematological and bone marrow results.We included only the completed records of the pediatric patients with VL in the Tropical Disease Teaching Hospital in Khartoum,Sudan from the period of 2016 to 2020.RESULTS The majority of pediatric patients included in this study are below 5-years-old(n=59,55.2%).Moreover,anemia,thrombocytopenia,and leukopenia were among the prevalent characteristics in the population under study.To further analyze the data,we developed a machine learning model using boosted forest algorithms to predict L.donovani parasites load,with a mean accuracy of 0.88 for the training dataset and an accuracy of 0.46,0.50,and 0.74 for mild,moderate,and severe L.donovani parasite load in the validation dataset.CONCLUSION This study shows that the most common bone marrow change among Sudanese VL children was increased chronic inflammatory cells(n=88,82.2%)with present macrophage hemophagocytes(n=103,96.3%).While anemia and thrombocytopenia were the most common hematological changes.These results will hopefully lead to an early diagnosis and hence better management for Sudanese pediatric patients with suspected VL.

Visceral Leishmaniasis at the National Reference University Hospital Center of N’Djamena (Chad): Epidemiological, Clinical, Diagnostic, Therapeutic and Prognostic Aspects

Introduction: leishmaniasis is a group of parasitic diseases caused by a parasite of the genus Leishmania transmitted by the bite of an infected vector called a sandfly. There are four forms. Visceral leishmaniasis is the most severe form. The aim of our work is to study the epidemiological, clinical, diagnostic, therapeutic and prognostic aspects of the disease in the Infectious Diseases Department of the Centre Hospitalier Universitaire de Référence Nationale in N'Djaména. Methodology: Patients were recruited on the basis of clinical signs suggestive of visceral leishmaniasis, i.e. prolonged fever, splenomegaly and altered general condition. Biological confirmation was performed with a rapid diagnostic test using recombinant K39 parasite antigen, which is known to have good specificity and sensitivity. Epidata version 3.1 software was used to process patient data. Results: From 05/04/21 to 15/12/23, 153 positive cases were managed. The mean age of patients was 18 years, with a sex ratio of 9.2. Of these patients, 103 (67.3%) had recently stayed at gold mining sites. Patients testing positive were treated with sodium stibogluconate combined with paromomycin for 17 days. The mortality rate was 13.2%. Conclusion: Leishmaniasis is a serious and little-known disease in Chad. In order to respond to the disease, it is necessary to reinforce the capacities of health structures and to carry out appropriate actions in the outbreaks.

Next-generation sequencing technology for diagnosis and efficacy evaluation of a patient with visceral leishmaniasis: A case report

BACKGROUND Visceral leishmaniasis(VL)is a parasitic disease caused by Leishmania and transmitted by infected sand flies.VL has a low incidence in China,and its clinical presentation is complex and atypical.This disease is easily misdiagnosed and can become life-threatening within a short period of time.Therefore,early,rapid and accurate diagnosis and treatment of the disease are essential.CASE SUMMARY A 25-year-old male patient presented with the clinical manifestations of irregular fever,hepatosplenomegaly,increased polyclonal globulin,and pancytopenia.The first bone marrow puncture biopsy did not provide a clear diagnosis.In order to relieve the pressure and discomfort of the organs caused by the enlarged spleen and to confirm the diagnosis,splenectomy was performed,and hemophagocytic syndrome was diagnosed by pathological examination of the spleen biopsy.Following bone marrow and spleen pathological re-diagnosis and metagenomic next-generation sequencing(mNGS)technology detection,the patient was finally diagnosed with VL.After treatment with liposomal amphotericin B,the body temperature quickly returned to normal and the hemocytes recovered gradually.Post-treatment re-examination of the bone marrow puncture and mNGS data showed that Leishmania was not detected.CONCLUSION As a fast and accurate detection method,mNGS can diagnose and evaluate the efficacy of treatment in suspicious cases of leishmaniasis.

In vitro activity and in vivo efficacy of a combination therapy of diminazene and chloroquine against murine visceral leishmaniasis

The present study evaluated the in vitro activity and in vivo efficacy of diminazene combined with chloroquine as a potential drug against Leishmania donovani. Amphotericin B was used as a positive control drug. In vitro activity involved incubation of various drug concentrations with promastigotes or vero cells in culture before determination of parasite growth inhibition or cell death while in vivo evaluations involved infection of various mice groups with virulent L. donovani parasites and treatment with test drug compounds following disease establishment. Weight changes in experimental mice were also evaluated before infection and throughout the experiment. The results indicated that the diminazene-chloroquine combination was at least nine times more efficacious than individual drugs in killing promastigotes in culture. The diminazene-chloroquine combination was safer （Ld50=0.03±0.04） than Amphotericin B （Ld50=0.02±0.01）. Body weight in infected mice increased significantly （P=0.0007） from day 7 to day 37 following infection （P=0.026）. However, body weight remained comparable in all mice groups during treatment （P=0.16）. The diminazene-chloroquine combination significantly reduced splenic parasite numbers as compared to individual drug therapies （P=0.0001） although Amphotericin B was still more efficacious than any other treatment （P=0.0001）. Amongst the test compounds, the diminazene-chloroquine combination showed the lowest level of IgG antibody responses with results indicating significant negative correlation between antileishmanial antibody responses and protection against disease. These findings demonstrate the positive advantage and the potential use of a combined therapy of diminazene-chloroquine over the constituent drugs. Further evaluation is recommended to determine the most efficacious combination ratio of the two compounds.

Conventional treatments and non-PEGylated liposome encapsulated doxorubicin for visceral leishmaniasis:A scoping review

Visceral leishmaniasis(VL),also known as Kala-azar,is caused by Leishmania(L.)donovani complex,which includes L.donovani and L.infantum and is associated with a high death rate as compared to the cutaneous and subcutaneous form.Treatment of VL includes chemotherapeutic agents which are associated with some major hurdles like toxicities,parenteral administration,high cost,parasite resistance and stability.Hence,there is an urgent requirement to develop novel chemotherapeutic agents or repurposing of existing drugs against VL.Developing formulation of new chemical entity for the treatment of VL is laborious,time consuming and associated with huge financial burden.However,screening of existing chemotherapeutic agents is a good alternative to avail cost-effective treatment option for VL.Non-PEGylated liposome encapsulated doxorubicin(Myocet®)is proposed as an alternative treatment option for VL in this review article.Here,we covered the fundamental aspects of VL,loophole associated with available current treatment strategies and non-PEGylated liposome encapsulated doxorubicin as a novel alternative formulation for treating VL,as this liposomal delivery system of doxorubicin might passively target the intra-cellular regions of macrophage.

Leishmania donovani whole cell antigen delivered with adjuvants protects against visceral leishmaniasis in vervet monkeys (Chlorocebus aethiops)

In a previous immunogenicity and efficacy study in mice, montanide ISA 720 （MISA） was indicated to be a better adjuvant than bacillus calmette guerin vaccine （BCG） for a Leishmania vaccine. In the present study, we report the safety, immunogenicity and efficacy of Leishmania donovani （L. donovani） sonicated antigen delivered with alum-BCG （A1BCG）, MISA or monophosphoryl lipid A （MPLA） in vervet monkeys following intradermal inoculums. Vaccinated and control animals were challenged with virulent L. donovani parasites and the parasitic burden was determined. Only animals vaccinated with alum-BCG adversely reacted to the inoculum by produc- ing ulcerative erythematous skin indurations. Non-parametric ANOVA followed by a post test showed signifi- cantly higher IgG antibodies, and revealed the presence of lymphoproliferative and interferon gamma responses in both AIBCG＋Ag and MISA＋Ag as compared to the MPLA＋Ag or other groups （P 〈 0.001）. We conclude that L. donovani sonicated antigen containing MISA is safe and is associated with protective immune response against Leishmania donovani infection in the vervet monkey model.

Diagnostic value of bone marrow cell morphology in visceral leishmaniasis-associated hemophagocytic syndrome:Two case reports

BACKGROUND Visceral leishmaniasis related-hemophagocytic lymphohistiocytosis(VL-HLH)is a hemophagocytic syndrome caused by Leishmania infection.VL-HLH is rare,especially in nonendemic areas where the disease is severe,and mortality rates are high.The key to diagnosing VL-HLH is to find the pathogen;therefore,the Leishmania must be accurately identified for timely clinical treatment.CASE SUMMARY We retrospectively analyzed the clinical data,laboratory examination results,and bone marrow cell morphology of two children with VL-HLH diagnosed via bone marrow cell morphology at Kunming Children’s Hospital of Yunnan,China.Both cases suspected of having malignant tumors at other hospitals and who were unresponsive to treatment were transferred to Kunming Children’s Hospital.They are Han Chinese girls,one was 2 years old and the other one is 9 mo old.They had repeated fevers,pancytopenia,hepatosplenomegaly,hypertriglyceridemia,and hypofibrinogenemia over a long period and met the HLH-2004 criteria.Their HLH genetic test results were negative.Both children underwent chemotherapy as per the HLH-2004 chemotherapy regimen,but it was ineffective and accompanied by serious infections.We found Leishmania amastigotes in their bone marrow via morphological examination of their bone marrow cells,which showed hemophagocytic cells;thus,the children were diagnosed with VL-HLH.After being transferred to a specialty hospital for treatment,the condition was well-controlled.CONCLUSION Morphological examination of bone marrow cells plays an important role in diagnosing VL-HLH.When clinically diagnosing secondary HLH,VL-HLH should be considered in addition to common pathogens,especially in patients for whom HLH-2004 chemotherapy regimens are ineffective.For infants and young children,bone marrow cytology examinations should be performed several times and as early as possible to find the pathogens to reduce potential misdiagnoses.

Visceral leishmaniasis: An immunological viewpoint on asymptomatic infections and post kala azar dermal leishmaniasis

Elimination of visceral leishmaniasis is a priority programme in Indian subcontinent.The World Health Organization has set a new target to eliminate kala-azar by the year 2020 as previous target elimination year(2015) has passed.The elimination programme has successfully curbed the rate of infection in endemic regions; however, there are still few challenges in its route.The current drug control regime is extremely limited and comprises only two(amphotericin B and miltefosine) drugs, which are also susceptible for parasites resistance.Moreover, these drugs do not produce sterile cure, and cured patients may develop post kala-azar dermal leishmaniasis even after a decade of cure leaving behind a potent source of parasitic reservoirs for further disease transmission.A significant proportion of endemic population remain seropositive but aymptomatic for many years without any clinical symptom that serve as latent parasitic reservoirs.The lack of tools to identify live parasites in asymptomatic infections and there association in disease transmission, parameters of sterile cure along with post kala-azar dermal leishmaniasis progression remain a major threat in its elimination.In this review, we discuss the potential of host immune inhibitory mechanisms to identify immune correlates of protective immunity to understand the mystery of asymptomatic infections, sterile cure and post kala azar dermal leishmaniasis.

Preliminary study on investigation of zoonotic visceral leishmaniasis in endemic foci of Ethiopia by detecting Leishmania infections in rodents

Objective:To investigate the zoonotic visceral leishmaniasis(ZVL) by identification of the most probable reservoir hosts using parasite isolation and analysis of a possible transmission dynamics of the disease in extra-domestic agricultural fields and rural villages.Methods:Rodents were collected from selected study sites in kala-azar endemic areas based on information for localities of kala-azar cases for screening of Leshmania infections using parasitological,serological and polymerase chain reaction(PCR) from March.2013 to January,2014.Ketamine(Clorketam Veterinary) was used to anaesthesize the rodents according the prescribed dosage(average 2 mg/kg for intra-venous route).The blood obtained using sterile needle was dropped into sterile filter paper and allowed to air dry before sealing in plastic bags.The tissues from liver,spleen and skin were macerated in Locke's solution before transferring them into NNN medium.Blood and touch smears of liver,spleen,skin and bone marrow were prepared for fixing using methanol and staining by Giemsa stain for microscopy.These tissues were also ascd for DNA extractions and PCR amplification of Leishmania infection.Results:A total of 335 rodents(13 species) were analyzed by sampling internal organs.The infection rate by PCR was 11.1%(6/54) for Arvicanthis nilothicas compared to 17.6%(3/17) and 12.5%(2/16) for Acomys cahirinus and Tarera(C) robustus respectively.Almost all the infections were found from bone marrow samples(8/48 or 16.7%) compared with 1/91(1.1%) liver,2/87(2.2%) spleen and 0/87(0%) skin.In all study sites with past human VL cases,rodents and proved vectors shared similar habitats.Conclusions:Leishmania donovani might circulate among different species of rodents in kala-azar endemic lowlands and valleys of Ethiopia by Phlebotamus orientalis and Phlebotomus martini.Detailed studies to substantiate the preliminary data on the possible role of these rodents arc urgently needed.

Novel PCR primers to diagnose visceral Leishmaniasis using peripheral blood, spleen or bone marrow aspirates

Objective: To establish a suitable method of diagnosis of visceral Leishmania sis(VL) using peripheral blood, spleen or bone marrow aspirates. Methods: Peripheral blood, bone marrow and spleen aspirate samples were collected from clinically suspected VL patients(n=26). A new PCR primer pair(MK1F/R) was designed targeting kinetoplast mini circle DNA sequences of Leishmania donovani, and Leishmania infantum, and was used to diagnose VL along with some other established primers for VL in polymerase chain reactions. Test was validated by comparing with several other diagnostic methods. Results: The designed primer set showed 100% specificity and 98% sensitivity in detecting VL using blood samples, when compared with more invasive samples: bone marrow or spleen aspirates. Conclusions: The newly designed primer MK1F/R could be a better alternative for PCR based diagnosis of VL using less invasive sample, peripheral blood instead of bone marrow or spleen aspirates.

A PCR and RFLP-based molecular diagnostic algorithm for visceral leishmaniasis

Objective:To determine an algorithm for molecular diagnosis of visceral leishmaniasis(VL)by kinetoplast DNA(kDNA)(RV1/RV2)and internal transcriber spacer(ITS1)(LITSR/L5.8 S)polymerase chain reaction(PCR),complemented by ITS 1 PCR restriction fragment length polymorphism(RFLP),using peripheral blood or bone marrow aspirate from patients with suspected VL.Methods:Biological samples were submitted to the gold standard for the diagnosis of VL and molecular diagnosis represented by ITS 1 PCR,kDNA PCR,and ITS 1 PCR RFLP.The samples were obtained from seven groups:groupⅠ,82 samples from patients with confirmed VL;groupⅡ,16 samples from patients under treatment for VL;groupⅢ,14 samples from dogs with canine visceral leishmaniasis(CVL);groupⅣ,a pool of six experimentally infected sandflies(Lutzomya longipalpis);group V,18 samples from patients with confirmed tegumentary leishmaniasis(TL)and groupsⅥandⅦwere from control groups without VL.Results:The following gold standard and molecular examination results were obtained for each of the seven groups:groupⅠ:parasitologic and immunochromatographic tests showed a sensitivity of 76.3%(61 of 80)and 68.8%(55 of 80),respectively,and a sensitivity of 97.6%(80 of 82)and 92.7%(76 of 82)by ITS1 and kDNA PCR,respectively.After ITS1 PCR RFLP(HaeⅢ)analysis of the 80 positive samples,52.5%(42 of 80)generated three fragments of 180,70,and 50 bp,corresponding to the pattern of Leishmania infantum infantum;groupⅡ:negative for the parasitologic methods and positive for IrK39(100%,16 of 16),presented 12.5%(2 of 16)of positivity by ITS 1 PCR and 25.0%(4 of 16)by kDNA PCR;groupⅢ:positive in the parasitologic and serologic tests(100%,14 of 14),presented 85.7%(12 of 14)of positivity by ITS1 PCR and kDNA PCR.ITS1 PCR RFLP showed that 83.3%(10 of 12)of the canine samples contained parasites with profiles similar to L.infantum;groupⅣpresented amplifications by ITS1 PCR and kDNA PCR.ITS1 PCR products were analyzed by RFLP,generating a profile similar to that of L.infantum;groupⅤ:positive in the parasitologic examination(100%,18 of 18),presented 72.2%(13 of 18)of the samples by ITS1 PCR positive.A total of 69.2%(9 of 13)showed profiles corresponding to a Viannia complex by ITS1 PCR RFLP;and groupⅥand groupⅥwere negative by ITS 1 and kDNA molecular tests.Comparing the molecular results with the parasitologic and serologic diagnosis from groupⅠ,almost perfect agreement was found(κboth>0.80,P<0.001).ITS1 and RV1/RV2 PCR detected 90.2%(74 of 82)of the samples.Two samples positive by RV1/RV2 were negative by LITSR/L5.8 S,and six samples positive by LITSR/L5.8 S were negative by RV1/RV2.Therefore,these two systems complemented each other;they diagnosed 100%of the samples as belonging to the Leishmania genus.Conclusions:We suggest an algorithm for the molecular diagnosis of VL,which must consider previous parasitologic and serologic(immunochromatographic)diagnoses,and should combine kDNA and ITS1 to determine the Leishmania subgenus using RFLP as a complement method to define the L.infantum species.

Efficacy and safety of pharmacotherapeutic interventions used in visceral leishmaniasis clinical trials:A systematic review and network meta-analysis

Objective:To compare the efficacy and safety outcomes of different antileishmanial agents used in visceral leishmaniasis clinical trials.Methods:A systematic literature search in PubMed/MEDLINE,EMBASE,Cochrane,and Google Scholar was done using keywords“randomized controlled trials”,“antileishmanial”and“visceral leishmaniasis”.The outcomes included were cure rate,overall withdrawals,relapse rate,and treatment-emergent adverse events.Effect estimates through the frequentist network meta-analysis approach were presented as OR with 95%CI.Rankogram plots were used for identifying the“best intervention”based on p-scores obtained using the surface under the cumulative ranking.The risk of bias was evaluated by using Pedro Scale.Results:Seventeen randomized controlled trials with 5143 visceral leishmaniasis patients who received different antileishmanial agents(amphotericin B,miltefosine,paromomycin,meglumine antimoniate,sodium stibogluconate,sitamaquine,and pentavalent antimonials)and met the inclusion criteria were included.For efficacy outcomes of the treatments,the rankogram of the network meta-analysis revealed that paromomycin(p-score=0.8148)has the highest probability of being best in the pool,followed by sodium stibogluconate(OR 0.82,95%CI 0.24-2.79,p-score=0.7580),amphotericin B+miltefosine(OR 0.66,95%CI 0.02-19.04,p-score=0.7329)as compared to the remaining treatments;however,the most of the treatment-emergent adverse events were reported with sitamaquine.Conclusions:Paromomycin reported the highest cure rates,while the maximum treatment-emergent adverse events were seen with sitamaquine.

Immunogenicity and protective efficacy of DNA vaccine against visceral leishmaniasis in BALB/c mice

The current study was designed to examine the protective efficacy of DNA vaccines based on gp63 and Hsp70 against murine visceral leishmaniasis. Inbred BALB/c mice were immunized subcutaneously twice at an interval of three weeks with pcDNA3.1 （＋） encoding T cell epitopes of gp63 and Hsp70 individually and in combination. Animals were challenged intracardially with 107 promastigotes ofLeishmania donovani 10 days post immunization and sacrificed 1,2 and 3 months post challenge. The immunized animals revealed a significant reduction （P 〈 0.05） in splenic and hepatic parasite burden as compared to the infected controls. Maximum reduction in parasite load （P 〈 0.05） was observed in animals treated with a combination ofpcDNA/gp63 and pcDNA/Hsp70. These animals also showed heightened DTH response, increased IgG2a, elevated Thl cytokines （IFN-γ and IL-2） and reduced IgG 1 and IL-10 levels. Thus, mice immunized with the cocktail vaccine exhibited significantly greater protection in comparison to those immunized with individual antigens.

Evaluation of interleukin 8 +2767 A/T polymorphism in visceral leishmaniasis

Objective:To evaluated the relationship between the genetic variations at IL-8 +2767 position with VL pathogenesis among Iranian patients.Methods:Three groups including patients with VL clinical presentation and leishmania seropositive(n=124).patients seropositive but without clinical presentation(n=82) and healthy controls(n=63) were selected to conduct this cross-sectional study.Polymorphism at +2767 position of IL-8 was investigated using PCR-RPLP techniques.Anti-leishmania antibody titration was evaluated by the immunoflorescence technique.Results:We observed higher significant frequencies +2767 A/A and A/T genotypes in groups I compared to group 2 and healthy controls(P=0.001).Also,patients in Group 1 carriyng A/A genotype showed higher liter of antileshmania antibody than patients with A/T and T/T genotypes(P=0.05).The validity of the data was analyzed using Hardy-Weinberg equilibrium and one way analysis of variance(ANOVA),as well as x^2tests.Conclusions:Our findings indicate that the IL-8 +2767 polymorphism is significantly involved in impaired immune responses against VL and it could be considered as a risk factor for the VL progress.

A New Vaccine Strategy of Dendritic Cell Presented Kinetoplastid Membrane(KMP-11)as Immunogen for Control against Experimental Visceral Leishmaniasis

Available reports suggest that, Leishmania donovani antigen KMP-11 may be significant in the modulation of immune responses in visceral leishmaniasis (VL). This study evaluated vaccine prospect of presentation of KMP-11 antigen through murine dendritic cells against VL in infected BALB/c mice. We report here that immunization with KMP-11 delivered through bone marrow derived dendritic cells can lead to killing of L. donovani in infected BALB/c mice. Furthermore, the strategy to use KMP-11 as vaccine delivered through DCs can stimulate the production of IFN-g, IL-12, IL-2R and TNF-α with concomitant down-regulation of IL-10 and IL-4. Furthermore, anti-leishmanial defence function (ROS) of splenocytes was observed increased in the presence of DC-delivered KMP-11 vaccination accompanied with an increased p38-MAPK signalling in vaccinated splenocytes. We summarized from our data that KMP-11 delivered through DCs has potential for eliciting protective immunity through pro-inflammatory cytokines (IFN-γ, IL-12, IL-2, TNF-α) following an up-regulation in signalling event of p38-MAPK. Therefore the study suggests a new control strategy against VL in future.

Association of Nutritional Supplements with Nutritional Status and Treatment Outcomes of Visceral Leishmaniasis among Children Aged 5 - 12 Years in Baringo and West Pokot Counties, Kenya

Introduction: Visceral leishmaniasis is a severe form of leishmaniasis that affects about 90,000 people annually worldwide. It is mainly transmitted by Leishmania donovani and infantum, which can cause damage to internal organs, such as the spleen, liver, and bone marrow. If left untreated, severe cases can be fatal, as the disease can lead to severe secondary diseases, mycological and bacterial infections, and hemorrhages. Nutritional deficiencies and concurrent infections increase the incidence of visceral leishmaniasis and the likelihood of lethality. There is limited information about the relationship between the disease and nutrition in endemic areas in Kenya. Objective: The study was to analyze the association of nutritional supplements with the nutritional status and treatment outcomes of visceral leishmaniasis among children aged 5 - 12 years in Baringo and West Counties in Kenya. Methods: A quasi-experimental study design adopting quantitative data collection method was used in this study. A total of 204 children aged 5 - 12 years were included in the study. Data on nutritional status and treatment outcomes for VL was collected using a questionnaire and consent form. Pre- and post-intervention assessments were conducted to compare BMI, fever, spleen size, splenic aspirate after treatment, and the presence of PKDL in a 3-month follow-up. The data was analyzed using R statistical software with descriptive and inferential statistics, including chi-square tests and t-tests. The impact of treatment was estimated using the difference-in-difference method to compare changes in outcomes over time between the intervention and comparison groups. Results: The baseline characteristics assessed in this study were socio-demographics (age, gender, marital status, education and religion), vitamins (A, B, C, D) and minerals (zinc, Iron and Iodine). The results showed that the mean age was 8.72, children aged 5 - 9 years were 64.7%, and those aged 10 - 12 years were 35.3% in the intervention and comparison groups. There were more males than females in the study (53.9% in the intervention and 52.9% in the comparison group respectively). All the children in the study were from a Christian background, were underweight, had enlarged spleen, and were positive for VL by Splenic Aspirate. Those who presented with fever were 88.2% (88% in both intervention and comparison groups). Most children had lower levels of zinc, iron, vitamins A, B12, and D at baseline (54.9%, 91.2%, 54.4%, 57.8%, and 58.8% respectively). The majority (93.1%) were deficient in vitamin C (90.2% in the intervention and 96.1% in the comparison group). Conclusion: According to the study findings, the effect of administering micronutrients is significant at 5% significance level with the intervention having a positive effect. The administration of the nutritional supplement led on average to an increase of the minerals, vitamins and BMI levels in the body.

Difference in DNA sequences in SSU rDNA variable regions among pathogens isolated from different epidemic foci of visceral leishmaniasis in China

To confirm the existence of point mutations in the SSU rDNA variable regions of 5 Leishmania donovani (L d ) isolates from different epidemic foci in China Methods Specific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEM R T Easy Vectors After that, the specific fragments were sequenced by an automated DNA sequencer Results Sequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length All 5 point mutations were located in two unique sequence blocks (UQ Ⅰ and UQ Ⅱ), and no insertions or deletions were found The identities of comparison of Leishmania in GeneBank were more than 98% Conclusion Five point mutations exist in the SSU rDNA variable region of 5 L d isolates from different epidemic foci of visceral leishmaniasis (VL) in China Sequence differences of the SSU rDNA variable region exist among L d isolates from different foci

Historical experience in the elimination of visceral leishmaniasis in the plain region of Eastern and Central China

Visceral leishmaniasis(VL)(kala-azar)was most seriously prevalent in the plain regions of eight provinces/municipalities in the eastern and central parts of China.In the early 1950s,the number of counties/cities endemic for VL and the number of cases in the plain regions accounted for 60%and 80%,respectively,of the total numbers in the entire country.By implementing comprehensive control measures,including treatment of patients for eliminating the source of infection and spraying insecticide in endemic villages to kill sandflies,VL transmission has been brought under control in this region by the early 1960s,and no new infected cases have been found since 1983,achieving the goal of eliminating VL.

Phlebotomine sand flies(Diptera:Psychodidae)transmitting visceral leishmaniasis and their geographical distribution in China:a review

After the existence of phlebotomine sand flies was first reported in China in 1910,the distribution of different species and their role in the transmission of visceral leishmaniasis(VL)have been extensively studied.Up until 2008,four species have been verified as vectors of VL,namely,Phlebotomus chinensis(Ph.sichuanensis),Ph.longiductus(Ph.chinensis longiductus),Ph.wui(Ph.major wui),and Ph.alexandri.The sand fly species vary greatly depending on the natural environments in the different geographic areas where they are endemic.Ph.chinensis is euryecious and adaptable to different ecologies,and is thus distributed widely in the plain,mountainous,and Loess Plateau regions north of the Yangtze River.Ph.longiductus is mainly distributed in ancient oasis areas south of Mt.Tianshan in the Xinjiang Uygur autonomous region.Ph.wui is the predominant species in deserts with Populus diversifolia and Tamarix vegetation in Xinjiang and the western part of the Inner Mongolia autonomous region.Finally,Ph.alexandri is steroecious and found only in stony desert areas,such as at the foot of the mountains in Xinjiang and the western Hexi Corridor,in Gansu province.This review summarized the relationship between the geographic distribution pattern of the four sand fly species and their geographical landscape in order to foster research on disease distribution and sand fly control planning.Furthermore,some problems that remained to be solved about vectors of VL in China were discussed.