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.

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.

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.

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.

Predicting the number of visceral leishmaniasis cases in Kashgar, Xinjiang, China using the ARIMA-EGARCH model

Objective:To forecast the visceral leishmaniasis cases using autoregress integrated moving average(ARIMA)and hybrid ARIMAEGARCH model,which offers a scientific basis to control visceral leishmaniasis spread in Kashgar Prefecture of Xinjiang,China.Methods:The data used in this paper are monthly visceral leishmaniasis cases in the Kashgar Prefecture of Xinjiang from 2004 to 2016.The sample data between 2004 and 2015 were used for the estimation to choose the best model and the sample data in 2016 were used for the forecast.Time series of visceral leishmaniasis started on 1 January 2004 and ended on 31 December 2016,consisting of 1790 observations reported in Kashgar Prefecture.Results:For Xinjiang,the total number of reported cases were 2187,the male-to-female ratio of cases was 1:1.42.Patients aged between 0 and 10 years accounted for 82.72%of all reported cases and the largest percentage of visceral leishmaniasis cases was detected among scattered children who accounted for 68.82%.The monthly incidences fitted by ARIMA(2,1,2)(1,1,1)12 model were consistent with the real data collected from 2004 to 2015.However,the predicted cases failed to comply with the observed case number;we then attempted to establish a hybrid ARIMA-EGARCH model to fit visceral leishmaniasis.Finally,the ARIMA(2,1,2)(1,1,1)12-EGARCH(1,1)model showed a good estimation when dealing with volatility clustering in the data series.Conclusions:The combined model has been determined as the best prediction model with the root-mean-square error(RMSE)of 7.23%in the validation phase,which means that this model has high validity and rationality and can be used for short-term prediction of visceral leishmaniasis and could be applied to the prevention and control of the disease.

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.

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.

Visceral leishmaniasis among children in an endemic area of northwestern Iran between 2016 and 2017: An epidemiological study

Objective: To clarify the epidemiological aspects of visceral leishmaniasis in Kaleybar and Khoda-Afarin districts, north-west of Iran.Methods: A total of 1 420 human(children under 12 years) samples, 101 domestic dogs samples(Canis familiaris), and 577 female sand fly samples were collected. Sera of human and dogs were tested using the direct agglutination test, and sand flies were identified at species level using the microscopic method. Furthermore, a structured questionnaire was applied to evaluate the correlation between the potential risk factors and the related clinical signs/symptoms with the human and dogs’ seropositivity.Results: Totally, 2.18% of human samples were positive at titers≥1: 800;among them, 13 cases(41.94%) were above 1:3 200, and clinical symptoms were observed in all of them except for an 11-year old girl. Anti-Leishmania infantum antibodies were found at titer≥1: 320 in 9.90% of dogs’ samples, half of them had at least one sign of canine visceral leishmaniasis. Moreover, 10 Phlebotomus species were identified in the study areas, and Phlebotomus(Larroussius) major group was the predominant species. There are significant correlations between the presence of anti-Leishmania infantum antibodies and the fever(P<0.001), anemia(P=0.001) and weight loss(P=0.016) in children. On the other hand, significant correlations were revealed between the Leishmania infection and the shelter(P=0.039), cutaneous lesion(P=0.005), lymphadenopathy(P=0.001) and weight loss(P<0.001) in the infected dogs.Conclusions: Visceral Leishmania infection is prevalent in rural areas of Kaleybar and KhodaAfar districts located in East-Azerbaijan province, therefore active detection and treatment of visceral leishmaniasis cases should not be neglected.

Evaluation of in vitro and in vivo immunostimulatory activities of poly(lactic-co-glycolic acid) nanoparticles loaded with soluble and autoclaved Leishmania infantum antigens: A novel vaccine candidate against visceral leishmaniasis

Objective: To prepare and characterize poly lactic-co-glycolic acid(PLGA) nanoparticles loaded with soluble leishmanial antigen or autoclaved leishmanial antigen and explore in vitro and in vivo immunogenicity of antigen encapsulated nanoparticles. Methods: Water/oil/water double emulsion technique was employed to synthesize PLGA nanoparticles, and scanning electron microscopy, Fourier transform infrared spectroscopy and Zeta-potential measurements were used to identify the characteristics of nanoparticles. Cytotoxicity of synthetized nanoparticles on J774 macrophage were investigated by MTT assays. To determine the in vitro immunostimulatory efficacies of nanoparticles, griess reaction and ELISA was used to measure the amounts of NO and cytokines. During the in vivo analysis, Balb/c mice were immunized with vaccine formulations, and protective properties of nanoparticles were measured by Leishman Donovan unit in the liver following the infection. Cytokine levels in spleens of mice were determined by ELISA. Results: MTT assay showed that neither soluble leishmanial antigen nor autoclaved leishmanial antigen encapsulated nanoparticles showed cytotoxicity against J774 macrophage cells. Contrary to free antigens, both autoclaved leishmanial antigen-nanoparticle and soluble leishmanial antigen-nanoparticle formulations led to a 10 and 16-fold increase in NO amounts by macrophages, respectively. Leishman Donovan unit calculations revealed that soluble leishmanial antigen-nanoparticles and autoclaved leishmanial antigen-nanoparticles yielded 52% and 64% protection against visceral leishmaniasis in mouse models. Besides, in vitro and in vivo tests demonstrated that by increasing IFN-γ and IL-12 levels and inhibiting IL-4 and IL-10 secretions, autoclaved leishmanial antigen-nanoparticles and soluble leishmanial antigennanoparticles triggered Th1 immune response. Conclusions: Both autoclaved leishmanial antigen-nanoparticles and soluble leishmanial antigen-nanoparticles formulations provide exceptional in vitro and in vivo immunostimulatory activities. Hence, PLGA-based antigen delivery systems are recommended as potential vaccine candidates against visceral leishmaniasis.

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.

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.

Phenology and population dynamics of sand flies in a new focus of visceral leishmaniasis in Eastern Azarbaijan Province,North western of Iran

Objective:To investigate species composition,density,accumulated degree-day and diversity of sand flies during April to October 2010 in Azarshahr district,a new focus of visceral leishmaniasis in north western Iran.Methods:Sand flies were collected using sticky traps biweekly and were stored in 96%ethanol.All specimens were mounted in Puri’s medium for species identification using valid keys of sandflies.The density was calculated by the formula: number of specimens/m of sticky traps and number of specimens/ number of traps.Degree-day was calculated as follows:(Maximum temperature + Minimum temperature)/2—Minimum threshold.Diversity indices of the collected sand flies within different villages were estimated by the Shannon- weaver formula(H’=sum form(i=1) to s Pi log_e Pi ).Results:Totally 5 557 specimens comprising 16 Species(14 Phlebotomus,and 2 Sergentomyia) were indentified.The activity of the species extended from April to October.Common sand-flies in resting places were Phlebotomus papatasi, Phlebotomus sergenti and Phlebotomus mongolensis.The monthly average density was 37.6.41.1, 40.23,30.38 and 30.67 for Almalodash,Jaragil,Segaiesh,Amirdizaj and Germezgol villages, respectively.Accumulated degree-day from early January to late May was approximately 289 degree days.The minimum threshold temperature for calculating of accumulated degree-day was 17.32℃.According on the Shannon-weaver(H’),diversity of sand flies within area study were estimated as 0.917,1.867,1.339,1.673,and 1.562 in Almalodash,Jaragil,Segaiesh,Amirdizaj and Germezgol villages,respectively.Conclusions:This study is the first detailed research in terms of species composition,density,accumulated degree-day and diversity of sand flies in an endemic focus of visceral leishamaniasis in Azarshahr district.The population dynamics of sand flies in Azarshahr district were greatly affected by climatic factors.According to this study the highest activity of the collected sand fly species occurs at the teritary week of August.It could help health authorities to predicate period of maximum risk of visceral leishamaniasis transmission and implement control program.

Sandfly fauna and ecological analysis of Phlebotomus orientalis and Phlebotomus martini in the lowland foci of visceral leishmaniasis in Somali Regional State, southeast Ethiopia

Objective:To identify the sandfly fauna and analyze ecology of sandfly vector(s)of visceral leishmaniasis in three districts of Somali Regional State,southeast Ethiopia.Methods:Sandflies were collected from four sampling habitats,including indoor,peri-domestic,farm field and mixed forest using light and sticky traps in July 2016,and February and April 2017 in Liben and Dawa zones in the Somali Regional State,southeastern Ethiopia.Results:In total,4367 sandfly specimens,belonging to 12 species(three Phlebotomus spp.and nine Sergentomyia spp.)were identified.Phlebotomus(P.)heischi,P.orientalis,and P.martini constituted 45.7%,31.1%,and 23.1%of the sandfly collection,respectively.There were significant differences in the median number of P.orientalis,and P.martini captured per CDC trap/night between the three sampling districts(P<0.05).In light trap capture,collection habitats had significant effects on the abundance of P.orientalis,and P.martini(P<0.05).More median numbers of P.orientalis,and P.martini species were collected in agricultural fields followed by mixed forest and peri-domestic habitats.P.orientalis and P.martini were caught more in outdoor than indoor habitats,suggesting exophilic behaviour.Conclusions:The study demonstrated that the presence of P.orientalis and P.martini are probable vectors of visceral leishmaniasis in this new focus.The findings of our study will improve the understanding of the dynamics of visceral leishmaniasis transmission and will facilitate the implementation of integrated disease control measures based on ecological knowledge of visceral leishmaniasis vector in Liben and Dawa zones and its surrounding regions.

Immunogenicity and efficacy of recombinant 78 kDa antigen of Leishmania donovani formulated in various adjuvants against murine visceral leishmaniasis

Objective: To analyze the protective efficacy of recombinant 78 kDa antigen of Leishmania donovani in combination with two adjuvants, that is, cationic liposomes or MPL-A against visceral leishmaniasis in BALB/c mice. Methods: The genomic DNA of promastigotes was isolated and 583 bp of T cell epitopes of gene encoding 78 k Da was amplified using specific primers. The amplified gene was cloned into p ET28 c, transformed into Escherichia coli BL21(DE3) and got expressed after IPTG induction. The recombinant protein was then purified using Ni-NTA and named r78. Three groups of mice were immunized with 10 μg of r78 plus MPL-A, r78 encapsulated in positively charged liposomes and control animals immunized with PBS. Two booster doses were given with the respective vaccine at an interval of 2 weeks each. Mice were challenged with 1×107 Leishmania promastigotes and sacrificed on different post infection/challenge days. Results: Immunization with r78 along with MPL-A and liposomeencapsulated r78 brought a significant reduction in parasite load. In comparison to the infected controls, the parasite load declined by 96.2% in mice immunized with r78 plus MPL-A and 97.23% in animals immunized with liposome-encapsulated r78. The immunized animals also exhibited profound DTH response. The serum antibody responses increased from 15 to 90 days post infection/challenge. Immunized animals showed greater IgG2 a levels and lesser Ig G1 levels in comparison to the infected controls. The splenocytes from immunized mice were cultured, stimulated with r78 and analyzed for cytokine profile. The levels of IL-2 and IFN-γ were greater in immunized animals as compared to control mice. Conclusions: The study proves that r78 in combination with suitable adjuvants is a potential vaccine candidate and may be instrumental in control of visceral leishmaniasis.

Oral mucosal involvement in visceral leishmaniasis

Leishmaniasis affects both the visceral and cutaneous tissues in body.Oral Mucosal involvement in leishmaniasis is rare and is often overlooked.We present a case 17 vear old boy from the north east region of Bihar who has a hislorv of visceral leishmaniasis one year back,came to the department of oral surgery for treatment of persistent oral ulcers.Oral examination did not give any diagnostic information while systemic examination revealed enlarged spleen and low grade fever.Patient was screened for leishmaniasis by rK39 based immunochromatographic strip test which came to be positive.Biopsy of the ulcer as well as splenic and bone marrow aspirate confirmed the presence of leishmaniasis.Patient was administered Amphotericin B for 20 days following which significant clinical and haematological improvement followed.

Characteristics of bacterial sepsis among patients with visceral leishmaniasis

Sepsis is one of the major causes and predictors of death in patients with visceral leishmaniasis(VL).Globally,incidence rate of sepsis ranged from 56-91 cases per 100000 people,with a mortality rate of 30%.Incidence of sepsis has been raised due to aging of the population and the higher incidence of immunosuppressive conditions such as HIV.VL and others.The prevalence of sepsis was reported from 4.2%to 32.3%and 14.1%in VL and VL-HIV coinfectcd patients,respectively.The mortality rate of VL patients with sepsis is greater than50%.Factors associated with sepsis in VL patients are immune suppression,pancytopenia.HIV co-infection,age <l year old and >40 years old,indwelling of central venous lines and hospitalization.Although antimicrobial susceptibility patterns were not well reported,both Gram-positive and Gram-negative bacteria were isolated from patients with VL.So far.limited information is available on sepsis in VL.especially in VL-HIV coinfected patients.Therefore,further studies about sepsis prevalence,causative agents and their antibiotic patterns,and associated factors among VL and VL-HIV coinfectcd patients arc necessary.This review provides information about bacterial sepsis in patients with VL.

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.

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.