Immune response to inactivated bacterial vector carrying the recombinant K39 antigen of Leishmania infantum in mice

Objective:To evaluate the immunological response elicited by an inactivated bacterial vector carrying the K39 antigen of Leishmania infantum,and a purified antigen.Methods:Mice were subjected to the following treatments:(1)Purified recombinant K39(rK39)protein at a 20μg dose with complete Freund’s adjuvant;(2)Inactivated Escherichia coli(BL21 DE3)carrying the K39 protein at an equivalent total protein content of 200μg;(3)Inactivated bacteria lacking the K39 protein;(4)Non-immunized control animals.Serological monitoring was performed.All groups were challenged by intraperitoneal injection of 10^(7) Leishmania infantum promastigotes.After euthanasia,the liver and spleen were collected to analyze the levels of TNF,IFN-γ,IL-12,IL-4,and IL-10.Results:Mice immunized with purified rK39 or the inactivated bacterial vector carrying the K39 antigen of Leishmania infantum showed a long-lasting immune response with high levels of polyclonal antibodies specifically recognizing the recombinant proteins.The IgG1 subclass was the predominant immunoglobulin;however,the induction of IgG2a and the profile of cytokines produced were indicative of the induction of a mixed-type response.Conclusions:The inactivated bacterial vector carrying the K39 antigen,as well as the purified antigen can induce a long-lasting immune response in immunized mice,predominantly favouring a Th2 profile response.

Anti-leishmanial,immunomodulatory and anti-oxidative activity of quercetin against cutaneous leishmaniasis caused by Leishmania major

Objective:To evaluate the in vitro and in vivo efficacy of quercetin and its immunomodulatory and anti-oxidative activity against Leishmania major(L.major).Methods:L.major promastigotes and amastigotes were incubated with different concentrations of quercetin to estimate EC_(50).For in vivo study,the base of tails of mice was infected with L.major.After developing ulcers in the inoculation site,mice were treated with 50 mg/kg quercetin orally for 28 consecutive days.The wound-healing potential of quercetin was evaluated by histopathological analysis of tissue sections stained by hematoxylin and eosin as well as Masson's trichrome.In addition,the levels of tumor necrosis factor-α,interleukin-6,malondialdehyde,and adiponectin,the ferric reducing ability of plasma,as well as superoxide dismutase and glutathione peroxidase activities were measured.Results:The EC_(50)values of quercetin against L.major promastigotes and intracellular amastigotes were 0.27 and 0.85μM,respectively.Histopathological analysis showed that fewer inflammatory cells,more fibroblasts,and more collagen deposition were observed in tissue sections of quercetin-treated mice.In addition,treatment with quercetin markedly increased glutathione peroxidase activity,the ferric reducing ability of plasma and adiponectin levels while decreasing malondialdehyde,interleukin-6,and tumor necrosis factor-αlevels.Conclusions:Quercetin shows anti-leishmanial activity,immunomodulatory,anti-oxidative,and anti-inflammatory effects.Therefore,it may be further explored as an effective drug in treating leishmaniasis.

Antileishmanial Potential of <i>Piper</i><i>nigrum</i>Seed Extracts against <i>Leishmania</i><i>donovani</i>

Visceral leishmaniasis (VL) is a pestilent form of leishmaniasis that chiefly impinges the poorest sections of the society. The prototypical therapeutic interventions in vogue are handicapped due to toxicity and alarming increase in drug resistance. In the absence of vaccines, progressive emergence of HIV-VL co-infection and relapse in the form of post kala-azar dermal leishmaniasis, have fuelled the quest for alternative therapies. Herein, we report antileishmanial activity of Piper nigrum, which is endowed with multifarous medicinal properties. Hexane (PNH) and ethanolic (PNE) extracts of P. nigrum substantially inhibited the growth of Leishmania donovani promastigotes with 50% inhibitory concentration (IC50) of 31.6 and 37.8 μg&#183ml-1, respectively. Growth reversibility analysis revealed the leishmanicidal effect of PNH which caused cell shrinkage and flagellar disruption. In contrast, PNE treated promastigotes showed partial effect. PNH and PNE also abrogated the growth of intra-macrophagic Leishmania amastigotes with IC50 of 14.6 and 18.3 μg&#183ml-1, respectively. Anti-amastigote efficacy of PNH was accompanied by higher selectivity over host macrophages than PNE. Gas-Chromatography-Mass Spectrometry showed the presence of several secondary metabolites such as trans-β-caryophyllene, piperine, β-bisabolene and other sesquiterpenes in PNH and piperine, δ-(sup 9)-cis oleic acid and piperyline in PNE. Conclusively, our work revealed discernible antileishmanial activity of P. nigrum extracts.

Cutaneous leishmaniasis in Louisiana-one-year follow-up:A case report

Reports of leishmaniasis are scarce in North America.It is considered to be one of the neglected tropical diseases.It is seen in immigrants from endemic areas to United States.Treatments are not readily available in the United States.Untreated or inadequately treated cutaneous leishmaniasis not only causes localized disfigurement but can advance to more permanent and devastating mucosal disfigurement and perforation,if caused by a species that can also cause mucocutaneous leishmaniasis.CASE SUMMARY A 42-year-old human immunodeficiency virus negative male immigrant from Honduras presented to the emergency department of our facility in Louisiana with a 2-mo history of a left lower extremity ulcer.It started as a painless blister that progressed in size and developed into other smaller lesions tracking up the thigh and became tender and erythematous.Clinically looked nontoxic and healthy.He was afebrile.Blood tests,except inflammatory markers,were within normal limits.The cellulitis of the leg was treated with 6 d of vancomycin that also relieved the pain.Skin biopsy was obtained,and histopathology was suspicious for leishmania.Polymerase chain reaction/deoxyribonucleic acid sequencing done by centers for disease control and prevention confirmed the diagnosis as Leishmania panamensis.There was no involvement of nasooropharyngeal mucosa,confirmed by otolaryngology.The patient was treated with miltefosine for 28 d.Clinic follow-up after approximately 11 mo revealed a healed skin ulcer.CONCLUSION Cutaneous leishmaniasis should be in the differential diagnosis of skin ulcers of travelers from endemic areas.Awareness regarding diagnosis and treatment of leishmaniasis needs to be enhanced.

Cutaneous leishmaniasis in Iran: Results from an epidemiological study in urban and rural provinces

Objective: To examine the prevalence and clinical manifestations of cutaneous leishmaniasis(CL) in Iran.Methods: This study was conducted in Iran between 2011 and 2013. Sampling, preparing, developing, and fixing of suspicious skin lesions were completed in healthcare centers in 31 Iranian provinces as well as in the Academic Reference Laboratory and the National Reference Laboratory. The information was then analyzed at the Ministry of Health's Information Management Center of Contagious Diseases.Results: Over a three-year period, the number of people identified with CL was 56 546.The highest incidence was reported in 2011(27.5 per 100 000). Wet CL accounted for 43.7% of cases while 43.3% resulted from sporotrichoid leishmaniasis. The results showed that there was a higher incidence of CL due to Leishmania major(50.2%) than to Leishmania tropica. The results of this study found that the highest incidence of CL had happened respectively in Ilam, Fars and, Khorasan Razavi Provinces between 2011 and 2013.Conclusions: Although the incidence of the disease is declining, CL is still a public health concern and disease control protocols need to be established. Therefore, further studies are needed to identify the vectors, reservoirs, and disease species as well as to develop appropriate disease control strategies.

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.

Development ofLeishmania vaccines:predicting the future from past and present experience

Leishmaniasis is a disease that ranges in severity infection. Resistance to infection is associated with from skin lesions to serious disfigurement and fatal systemic a T-helper-1 immune response that activates macrophages to kill the intracellular parasite in a nitric oxide-dependent manner. Conversely, disease progression is generally associated with a T-helper-2 response that activates humoral immunity. Current control is based on chemothera- peutic treatments which are expensive, toxic and associated with high relapse and resistance rates. Vaccination remains the best hope for control of all forms of the disease, and the development of a safe, effective and affordable antileishmanial vaccine is a critical global public-health priority. Extensive evidence from studies in animal models indicates that solid protection can be achieved by immunization with defined subunit vaccines or live-at- tenuated strains of Leishmania. However, to date, no vaccine is available despite substantial efforts by many laboratories. Major impediments in Leishmania vaccine development include： lack of adequate funding from national and international agencies, problems related to the translation of data from animal models to human disease, and the transition from the laboratory to the field. Furthermore, a thorough understanding of protective immune responses and generation and maintenance of the immunological memory, an important but least-studied aspect of antiparasitic vaccine development, during Leishmania infection is needed. This review focuses on the progress of the search for an effective vaccine against human and canine leishmaniasis.

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.

Antileishmanial activities of caffeic acid phenethyl ester loaded PLGA nanoparticles against Leishmania infantum promastigotes and amastigotes in vitro

Objective: To investigate and compare the antileishmanial effects of CAPE and(CAPE)PLGA NPs on Leishmania infantum(L.infantum) promastigotes and amastigotes in vitro,Methods: Efficacies of CAPE,(CAPE)PLGA NPs and free PLGA nanoparticles(NPs) on promastigotes were evaluated using MTT and promastigote count assays,and their anti-amastigote effects were determined via infection index analysis,Griess reaction was also performed to calculate nitric oxide production of macrophages exposed to investigated molecules,Results: It was determined that CAPE and(CAPE)PLGA NPs demonstrated significant inhibitory effects on L.infantum promastigotes and amastigotes,while free NPs did not exhibit any meaningful antileishmanial effectiveness,The IC50 values of CAPE for L.infantum promastigotes and amastigotes were assessed as(51.0±0.8) and(19.0±1.4) μg/m L,respectively(P<0.05),On the other side,it was revealed that(CAPE)PLGA NPs had superior antileishmanial activity on both forms of parasites since its IC50 values for L.infantum promastigotes and amastigotes were(32.0±1.3) and(8.0±0.9) μg/m L,respectively(P<0.05),It was also determined that both agents strongly stimulated nitric oxide production of macrophages,Conclusions: The obtained results show that(CAPE)PLGA NPs have a great potential to be especially used in treatment of visceral leishmaniasis; however,in vivo antileishmanial screening of these molecules should be performed in the near future.

First report on molecular characterization of Leishmania species from cutaneous leishmaniasis patients in southern Khyber Pakhtunkhwa province of Pakistan

Objective: To report presence of Leishmania major in Khyber Pakhtunkhwa of Pakistan, where cutaneous leishmaniasis(CL) is endemic and was thought to be caused by Leishmania tropica only. Methods: Biopsy samples from 432 CL suspected patients were collected from 3 southern districts of Khyber Pakhtunkhwa during years 2011–2016. Microscopy on Giemsa stained slides were done followed by amplification of the ribosomal internal transcribed spacer 1 gene. Results: Leishmania amastigotes were detected by microscopy in 308 of 432 samples(71.3%) while 374 out of 432 samples(86.6%) were positive by ribosomal internal transcribed spacer 1 PCR. Subsequent restriction fragment length polymorphism confirmed Leishmania tropica in 351 and Leishmania major in 6 biopsy samples. Conclusions: This study is the first molecular characterization of Leishmania species in southern Khyber Pakhtunkhwa. It confirmed the previous assumptions that anthroponotic CL is the major CL form present in Khyber Pakhtunkhwa province. Furthermore, this is the first report of Leishmania major from a classical anthroponotic CL endemic focus identified in rural areas of Kohat district in southern Khyber Pakhtunkhwa.

A review of adjuvants forLeishmania vaccine candidates

Over the last decade, there has been a flurry of research on adjuvants for vaccines, and several novel adjuvants are now licensed products or in late stage clinical development. The success of adjuvants in enhancing the immune response to antigens has led many researchers to re-focus their vaccine development programs. Although several vaccine candidates have been tested against leishmaniasis, there is yet no effective vaccine against this parasitic disease. Recent research has documented that efforts to develop effective Leishmania vaccine have been limited due to lack of an appropriate adjuvant. In view of this, this review paper outlines some of the adjuvants that have been used in Leishmania vaccine candidates and cites a few of the responses obtained from these studies. The aim of the present review is to consolidate these findings to facilitate the application of these adjuvants in general and experimental vaccinology.

Disseminated cutaneous leishmaniasis due to Leishmania(Leishmania) amazonensis in human immunodeficiency virus(HIV)-infected patients: A report of two cases

Rationale: Co-infection of human immunodeficiency virus(HIV) and Leishmania spp. has impact on clinical and therapeutic outcomes of leishmaniases. Most studies do not present the identification of Leishmania species causing American tegumentary leishmaniasis in co-infections. In the Americas, Leishmania(L.) Viannia(V.) braziliensis and L.(V.) guyanensis have been identified. Patient concerns: In this study, two cases of American tegumentary leishmaniasis in patients infected with HIV are described. Patients presented several lesions with rapid dissemination and mucosal involvement. Diagnosis: Disseminated cutaneous leishmaniasis caused by L. amazonensis was identified by molecular test. Interventions: The patients were treated with conventional therapies for HIV infection and American tegumentary leishmaniasis. Outcomes: In co-infection, the clinical manifestations are atypical and the treatment response can be impaired. Lessons: These cases show that HIV infection impacts L. amazonensis infection and point to the relevance of identifying Leishmania species, which can lead to a better patient management.

Treatment of Acute Cutaneous Leishmaniasis by Oral Zinc Sulfate and Oral Ketocanazole Singly and in Combination

Background: Cutaneous Leishmaniasis (CL) is an endemic disease in many countries and caused by different species of Leishmania parasite. It results in a deformed scar after a relatively long period. Many therapies have been tried in treatment of this disease. Objective: To compare the effect of oral zinc sulfate and oral ketoconazole singly and in combination in the treatment of acute cutaneous leishmaniasis. Patients and Methods: This single, blinded, therapeutic, controlled study was conducted in the Department of Dermatology, Baghdad Teaching Hospital, Baghdad, Iraq, during the period, January 2015 to July 2015. Seventy-five patients with acute CL were enrolled in this study. The total numbers of lesions were 327, and the duration of lesions ranged from 4 to 12 (6.9 ± 0.7) weeks. The diagnosis was confirmed by smear and histopathology. Patients were divided into three groups: 24 patients in Group A were treated with oral zinc sulfate capsules 10 mg/kg/day for 6 weeks;24 patients in Group B were treated with ketoconazole tablets 200 mg twice daily for 6 weeks and 27 patients in Group C were treated orally with a combination of zinc sulfate and ketoconazole for 6 weeks. All patients were seen regularly every 2 weeks for 6 weeks of treatment period, then monthly for the next three months as follow up period. Healing of the lesions was assessed by using Sharquie’s modified Leishmania score to assess the objective response to the topical or systemic therapy. Results:After six weeks, 75 patients have completed the treatment, 24patients received zinc sulfate capsule, 24 patients received oral ketoconazole and 27 patients received a combination of both treatments. The cure rate was (60%) in the group receiving oral zinc sulfate capsuleand (50%) in the one receiving oral ketoconazole tablet (P = 0.146) and (96%) in the combination group (P ? 0.04). Conclusion: The combination therapy using oral zinc sulfate and oral ketoconazole gave a high cure rate. The combination therapy is a new mode of therapy as both drugs act in a synergistic way.

First molecular identification of Leishmania species in a new endemic area of cutaneous leishmaniasis in Lorestan,Iran

Objective:To identify Leishman{u using PCR.Methods:This studs was conducted from April2009 to March 2011 in order to identify Leishmania species in a new endemic area of CL in Lorestan.Iran.Samples were taken from 62 patients that referred to the health centers in different cities of Lorestan province,the presence of Leishmcania was confirmed using direct smear and then grown in NNN media and mass cultured in RPM!1640 medium supplemented with 10%heat-inactivated fetal bovine serum.DNA was extracted from cultured promastigotes and used in P15-PCR.Results:45(72.6%)samples out of 62 showed a hand in the range of 485 hp and 17(27.4%)with a hand in the range of 626 hp which were similar to standard strains of Leichmania tropica(L.tropical and Leishnrania major(L.major),respectively.50(65.80%)of samples were collected from people with no history of travel in at least a year prior to the onset which shows that indigenous source of infection.Conclusions:Since the vector and reservoir of the two species are different.so precise and extensive control and prevention methods should be designed and earned out.

Molecular detection of Leishmania isolated from Cutaneous leishmaniasis patients in Jask County,Hormozgan Province,Southern Iran,2008

Objective:To investigate on patients leishmanial infections in Jask County.Methods: Impression smears were prepared from patients in 2008,all,were chequed for leishmanial infection by microscopy and molecular assays.Whole D.NA was extracted using Proteinase K and Phenol/Chloroform/Isoamyl alcohol method.The variable segment on minicircles of kineloplasl DIVA was amplified via a Nested-PCR technique using species-specific primers(LIN R4-LIN 17 -Lin 19).Results:A total of 40 smears were prepared from 20 patients,from which,eight samples (40%) were positive for leishman body by microscopic method,while,18 samples(90%) were positive,molecularly.The parasite was identified as Leishmania major(L major).Conclusions: Zoonotic or Rural cutaneous leishmaniasis is endemic in Jask County whose pathogen is L major.Molecular assays using specific primers are very accurate and more sensitive and specific than microscopy which is time consuming and needs master mieroscopists.

Molecular characterization of sandflies and Leishmania detection in main vector of zoonotic cutaneous leishmaniasis in Abarkouh district of Yazd province,Iran

Ohjective:To assess molecular characterization,distribution,seasonal activities of sandfly species and Leishmania parasites infecting them for this zoonotic cutaneous leishmaniasis focus.Methods:The collections were carried out in 2009-2011 using CDC traps,Sticky Papers and manual aspirator in and around the villages in Abarkouh district.Individual sandflies were characterized by PCR amplification and sequencing of fragments of their mitochondrial cytochrome b gene.Leishmania parasite infections within sandflies were performed by targeting Cyt b,ITS-rDNA,k-DNA and microsatellite genes.Results:The PCR assays detected only Leishmania major(L.major).All infections(30)were found in the abundant and widespread vector Phlebolomus papalasi(P.papatasi).Small numbers of other sandfly species were also screened for infections,but none was found.Sergentomyia sinloni and P.papalasi were the predominant members in all locations of this district and in all habitats throughout the trapping season.Only five other sandfly species were found,namely Phlebolomus ansari,Phlebotomus caucasicus,Phlebotomus sergenti,Sergentomyia dentata and Sergentomyia merviney.Conclusions:In the current survey,the only infections detected are of L.major in females of P.papatasi(30 out of190).The rates of infection of P.papalasi by L.major are not significantly different in compare with other locations in Iran with no diversity of parasite strains.Zoonotic cutaneous leishmaniasis may have emerged only recently in Abarkouh district,and the reason may well be the instability of the transmission cycles there.

Unusual presentation of disseminated cutaneous leishmaniasis due to Leishmania major.Case reports of four Iranian patients

We report four disseminated cutaneous leishmaniasis(DCL) cases referred to leishmaniasis laboratory at the School of Public Health,Tehran University of Medical Sciences with multiple nodular,ulcerative and crusted lesions extended on the face,trunk,and extremities.None of the patients had any complication and historical involvement in their immunological system conditions that suggest as the criteria for DCL.Direct smears of ulcers were positive for Leishmania parasite.The parasite was isolated from the active lesions and identified as Leishmania major(L major) using PCR-RFLP assay and sequencing analysis.

Leishmaniosis phytotherapy: Review of plants used in Iranian traditional medicine on leishmaniasis

Many native plants in traditional medicine have been used for the treatment of cutaneous leishmaniasis and the recent clinical trials have proven the efficacy of some of them. Researches conducted on these plants have shown that garlic, shallots, wormwood, yarrow, walnuts, thyme, henna plant, mimosa, aloe, wood betony, medlar, periwinkle, yeah, savory, black beans, etc. are ef ective on cutaneous leishmania. Synthetic agents in Iranian market have some disadvantages such as high cost and side ef ects and are painful in injections. Given the ef ectiveness of these plants, they can be a source of natural and safe compounds for the treatment of Leishmania. Therefore, more clinical researches should be done to determine the ef ectiveness and safety of these medicinal plants, their active ingredients and their possible toxic substances which can lead to the production of ef ective and safe drugs for leishmaniasis. It also might be an ef ective way to prepare herbal ointment on wound healing.

Leishmaniasis in the knee area

Leishmaniasis is a parasitic infection caused by species leishmaniae,which can produce two types of manifestations:visceral and cutaneous.In south America cutaneous leishmaniasis is more common than visceral leishmaniasis.A case of primary cutaneous leishmaniasis from Bolivia is presented for its rarity.The patient of our case showed an ulcerated lesion of the knee. Montenegro’s intradermal test was positive.Giemsa-stained touch preparation of the skin biopsy revealed amastigotes inside macrophages,consistent with leishmaniasis.The patient was treated with meglumine antimoniate intramuscular(20 mg of Sb+/kg/day) three weeks,with complete cicatrization of the lesion.

Molecular detection of Leishmania species in human and animals from cutaneous leishmaniasis endemic areas of Waziristan, Khyber Pakhtunkhwa, Pakistan

Objectives: To detect Leishmania species in human patients, animal reservoirs and Phlebotomus sandflies in Waziristan, Pakistan. Methods: Tissue smears and aspirates from 448 cutaneous leishmaniasis(CL) suspected patients were analyzed. To sort out role of the reservoir hosts, skin scrapings, spleen and liver samples from 104 rodents were collected. Furthermore, buffy coat samples were obtained from 60 domestic animals. Sandflies were also trapped. All human, animals and sandfly samples were tested by microscopy, kinetoplastic PCR and internal transcribed spacer 1(ITS1) PCR followed by restriction fragment length polymorphism for detection of Leishmania species. Results: An overall prevalence of 3.83% and 5.21% through microscopy and ITS1 PCR respectively was found. However, the statistically non-significant correlation was found between area, gender, and number of lesions. The presence of rodents, sandflies, domestic animals and internally displaced people increased the risk of CL. Using ITS1-PCR-RFLP, Leishmania tropica(L. tropica) was confirmed in 106 samples while 25 of the isolates were diagnosed as Leishmania major(L. major). Similarly, 3/104 rodents were positive for L. major and 14 pools of DNA samples containing Phlebotomus sergenti sandflies were positive for L. tropica. None of samples from domestic animals were positive for leishmaniasis. Conclusions: In the present study, L. tropica and L. major are found to be the main causative agents of CL in study area. Movement of internally displaced people from CL endemic areas presents a risk for nearby CL free areas. To the best of our knowledge, we report for the first time L. major infection in rodents(Rattus rattus) and L. tropica in Phlebotomus sergenti sandflies trapped in Waziristan, Pakistan.