NNN Medium Research Articles

Detection and isolation of Leishmania infantum from natural infected dog in Türkiye

Some clinical signs such as cachexia, alopecia, exfoliative dermatitis, hair loss, and swollen lymph nodes were observed in a one-year-old crossbred male dog living in a rural area of Kirikkale province, Türkiye, was presented to the veterinary clinic by its owner. Anaemia, leucopenia, hyperglobulinemia, and bilirubinemia were detected. Seropositivity was detected using Leishmania IgG/IgM Rapid Test. The amastigote forms of the parasite were observed in the lymph node aspirates. Viable promastigotes were observed in the samples obtained from the Novy-MacNeal-Nicolle medium (NNN) tubes. The parasite DNA was extracted from the promastigotes produced in the NNN medium using a DNA extraction kit according to the manufacturer's instructions. The specific bands indicating the gene regions of L. infantum (350 bp and 730 bp for HSP20 and HSP70, respectively) were observed. Additionally, BLASTn analysis revealed 100 % similarity with GenBank-deposited L. infantum sequences. The sequences were submitted to GenBank (Accession numbers: OR806945 and OR806946). According to the author's knowledge, it is the first dog isolate deposited in the collection of the Microbiology Reference Laboratories and Biological Products Department, General Directorate of Public Health, Ministry of Health, Ankara, Türkiye. The isolate (TR_L. infantum CanL-1_damla) has been preserved by cryopreservation at −150 °C for further studies.

In Vitro new experimentally culture media for Leishmania species cultivation

A fifty four different biphasic media were used to cultivate promastigote parasite of Leishmania tropica and compared with ordinary NNN medium, to find a cow milk agar medium can provide a good cultivation to the parasite as well as the ordinary medium, where milk is a cheap and simple, and a good source of lipids, protein, carbohydrate, and calcium. Also, using a 20% of human urine, can enhance the growth rate of parasite, this reduced the time that needed to cultivate the parasite in the ordinary NNN medium. So, using of a modified NNN medium (medium that used the solid phase of ordinary NNN and 20% of human urine as liquid phase) in the field of laboratory can provide a short duration to cultivate Leishmania parasite. The count of Leishmiania tropica, promastigotes ,taken from NNN medium reached, 3 × 106/ml at the end of the 6th day in our new medium, while in NNN medium the number of organisms reached only 1× 106 / ml. also 9.7×107 promastigotes found after the fourteen days of cultivation in media supplemented with urine and 22×106promaastigotes in the tenth days in media with milk. After several passages, the cultured medium ,prepared was .evaluated as, being quite simple, inexpensive, and successful ,compared with, other commercially ,available culture media.

Preparation of Thymol Nanoliposome and Solid Lipid Nanoparticle and Evaluation of their Inhibitory Effects on Leishmania Major Promastigotes

Introduction: Thymol has an antiprotozoal effect. Nanoparticulate systems are useful carriers for both small and large drug molecules, which can protect them from some chemical and biological damages as well as target drug delivery to specific organs or receptors. In this work, the nano-liposomal system and solid lipid nanoparticles loaded by thymol were prepared and the effectiveness of them were evaluated on Leishmania major promastigotes.
 Methods: Several formulations of nano-liposomes and solid lipid nanoparticles were prepared, and the amount of thymol loading, in-vitro release profile, particle size, and zeta potential were evaluated. Finally, the best formulations were serially diluted and incubated for 24, 48, and 72 hours on Leishmania major promastigotes, which cultured on Novy–MacNeal–Nicolle medium, and the results were analyzed.
 Results: The highest loading of thymol in nano-liposomes (92%) was seen in the formulations made with phosphatidylcholine (Called L3), and among the solid lipid nanoparticles, the formulation prepared with glycerol monostearate (S1) had the most entrapment efficiency of thymol (87%). These formulations were selected to evaluate the release rate of thymol. The results showed that S1 has a slower release rate than L3; this may be due to the presence of Glycerol monostearate in solid lipid nanoparticles structure. The best formulations, L3 and S1, were chosen for anti-Leishmaniosis assessment; which showed that all three forms of free thymol, nanoliposomes, and solid lipid nanoparticles inhibited Leishmania major. The half maximal inhibitory concentration (IC50) of free thymol, nanoliposome, and solid lipid nanoparticles for a 24-hour incubation are 7.8, 62.5, and 125, respectively, which decrease to 7.8, 7.8, and 15.6 for 48 hours and 7.8, 0.49, and 0 for 72 hours of incubation.
 Conclusion: Thymol has a significant effect on the inhibition of Leishmania major promastigotes and usage of thymol in the form of liposomes or solid lipid nanoparticles can sustain the drug release and have a lower IC50 during the longer incubation time.

Could RPMI-1640 Medium be Used in the Diagnosis and Isolation of Cutaneous Leishmaniasis Lacking NNN Medium?

Laboratory diagnosis of leishmaniasis is based on culture, microscopic examination, serological and molecular methods. The gold standard method is to see amastigotes in microscopic examination and to grow promastigotes in Novy, MacNeal, Nicolle (NNN) medium. NNN medium is frequently used for culture all over the world. In our study, it was aimed to investigate whether the use of RPMI-1640 medium is an appropriate method in cases where the gold standard NNN medium is not available for the diagnosis of cutaneous leishmaniasis (CL). Smears were prepared from the needle aspiration fluid sample from the patient who applied to Manisa Celal Bayar University Faculty of Medicine and had lesions suspicious of CL, and were stained with Giemsa for the presence of amastigotes. The samples taken were directly inoculated into RPMI-1640 broth and incubated at 26 °C for the presence of promastigotes. On consecutive days after incubation, it was checked for promastigote growth. Genotyping of the grown isolate was performed with primers and probes specific to the internal transcribed spacer-1 (ITS-1) gene region with the help of real-time polymerase chain reaction. The amastigote form was observed in the microscopic examination of the needle aspiration fluid sample smear preparations taken from the patient. On the other hand, promastigote growth was observed in RPMI-1640 broth from the 3rd day. In addition, the isolate obtained from the CL patient was determined to be Leishmania tropica as a result of the species determination made by genotyping. It is thought that this study is important in terms of suggesting an alternative medium for the diagnosis of leishmaniasis in laboratories where the gold standard NNN medium is easily accessible. RPMI-1640 medium, which is easily obtained and prepared in parasitology laboratories, can help in the diagnosis of the disease and treatment follow-up, Leishmania spp. isolation and drug resistance studies.

Screening and Characterization of Bioactive Molecules Derived From Medicinal Plants for Antileishmanial Activities for Leishmania donovani

Abstract- Leishmaniasis is a vector-borne disease caused by genus Leishmania. It causes significant morbidity and mortality in the endemic areas of several developing countries. Due to multidrug resistance in Leishmania sp and unavailability of an effective vaccine, discovery of new drugs is urgently needed. The aim of the present study was screening of medicinal plants used as Indian traditional medicine for leishmanicidal activity. Promastigote forms of Leishmania parasite were cultured in-vitro in NNN medium and further sub-cultured and maintained in RPMI-1640 medium containing 10% Fetal Bovine Serum for the screening of medicinal plants. A total of 26 medicinal plants were collected and screened for leishmanicidal activity. The methanolic extracts showing antileishmanial activity were subjected to LC-MS analysis to identify the major phyto-constituents in the crude methanolic extracts. The compounds were detected in the LC-MS of active extracts. The plant extract showing maximum antileishmanial activity was further fractionated to isolate the major compound(s).The compound isolated was characterised by IR, Mass spectrometry and subjected to in vitro antileishmanial activity against L. donovani promastigotes. In vitro antileishmanial assay revealed that crude methanolic extracts of 2/10 plants were active against L. donovani promastigotes. Methanolic root extracts of Inula racemosa were found active (54.83%) against the parasite while T. Terrestris were found least active with such as toxicity, percent inhibition 43.10% at concentration of 500 μg/ml. Inula racemosa methanolic extract further fractionated subjected for LC-MS analysis. The compound isolated from the methanolic root extract of I. racemosa was isoalantolactone, which did not show any antileishmanial activity against L. donovani. The study suggested that crude extract of Inula racemosa and T. Terrestris have shown potent antileishmanial activity while extracted bioactive molecules does not show efficacy against Leishmania parasite. Hence the antileishmanial activity could be due to any other compound which could not be detected, so further study is undertaken.

Characterisation of the <i>Leishmania donovani/L. infantum</i> Hybrid Isolated from an Autochothonous Kala-Azar Patient: Preliminary Results of an In Vivo Model

In the present study, preliminary outcomes of the in vivo assessment of a Leishmania donovani/L. infantum hybrid isolated from a hospitalised patient with visceral leishmaniasis in Manisa and identified through analysis of the Leishmania-specific ITS-1, hsp70 and cpb gene regions are presented in comparison with reference strains of L. donovani and L. infantum. Three different study groups [(SG); n=16 mice each] and a control group (n=8 mice) were established with female Balb/C mice weighing 25-30 g. Reference L. donovani (MHOM/IN/1980/DD8), reference L. infantum (MHOM/TN/1980/IP1) and a L. donovani/L. infantum hybrid (MHOM/TR/2014/CBVL-LI/ LD), stored in liquid nitrogen, were thawed, cultured and incubated at 25 °C. A 15-μL dose of 1x108/mL promastigotes of three strains was applied to the tail veins of mice in the SG. After the mice were sacrificed, the liver and spleen tissues were removed and stored for immunological, immunohistochemical and pathological analyses. The presence of infection in the liver and spleen tissues of mice was detected both by a specific enzyme-linked immunosorbent assay test and from the recovery of Leishmania promastigotes from liver and spleen tissues in NNN medium. However, Leishmania amastigotes were not observed in the touch biopsy smears of livers or spleens in either of the SGs. In addition, no evidence of tissue damage was identified in the SGs after immunohistochemical staining (with antibodies against IL-9, CD-117, MBP, CD163, CD4, CD8 and CD31). The obtained results show that hybrid Leishmania and reference L. donovani and L. infantum strains reached the liver and spleens of Balb/C mice in SGs but were of no pathological consequence. Yet, these three Leishmania isolates caused skin lesions when applied subcutaneously in Balb/C mice in another study. The findings presented in this study will be reassessed upon completion of the project, once the final results are obtained.

First Presentation of the Complete Infection Cycle of Visceralizing Leishmania infantum/ donovani Hybrid Strain in Turkey: The Host, Reservoir and Vector

Objective: Visceral Leishmaniasis (VL) is a widespread infectious disease of the reticuloendothelial system which is mostly caused by Leishmania infantum and L.donovani. VL is a zoonotic infection and dogs act as the main reservoirs of the disease while the vectors are the female Phlebotomus, that is to say, sandflies. Although Leishmania spp. are attributed to reproduce asexually, the genetic exchange between different strains has recently been demonstrated and causes interspecific hybrids. We aimed to present the complete infection cycle of a hybrid strain of Leishmania infantum/donovani with the host, reservoir, and vector. Materials and Methods: A patient presenting with VL proved to have a dog with canine leishmaniasis. Bone marrow aspirate from the patient and lymph node aspirates from the dog were inspected for Leishmania spp. amastigotes under light microscopy. Samples were cultured in enriched NNN medium. Serological examinations were made using IFAT for the patient serum and rK39 test for both human and dog samples. Light traps were placed to collect Phlebotomus. Leishmania molecular identification was executed by primers and probes specific to ITS-1 and cytochrome B gene region of Leishmania parasites. Results: Leishmania spp. amastigotes were detected in the slides while promastigotes were present in the enriched NNN medium. For patient serum, IFAT IgG was 1/256 positive, and rK39 was positive for both dog and human samples. Fifty-two Phlebotomus were caught (24 male, 28 female): 35 of them were P. similis, ten were P. neglectus/syriacus, six were P .papatasi, and one was P. tobbi. Molecular analysis showed that the Leishmania spp. isolates from the patient, dog, and P.tobbi were identical. Gel image revealed two bands in all three samples, thus leading to the conclusion that the infecting strain was a hybrid of L. infantum and L. donovani. Conclusion: We found identical isolates that are putative hybrids of L. donovani and L. infantum in human, dog reservoir and sandfly.. Keywords: Leishmaniasis, Phlebotomus, Hybrid, Epidemiology, Migration, Turkey

Köpeklerde Kanin Leishmaniasis Etkeni Leishmania tropica’da İlaç Dirençlerinin Araştırılması

Objective: Leishmaniasis is endemic in 98 countries, and poses risk to 1 billion people in the world. The infection caused by obligatory intracellular parasite Leishmania spp. in dogs is called Canine Leishmaniasis (CanL). In this study, it was aimed to detect the resistance status of Leishmania tropica strains isolated from dogs in our country against amphotericin B, meglumine antimoniate and sodium stibogluconate applied in leishmaniasis treatment. Method: Leishmania spp. promastigotes, taken out from the liquid nitrogen were first cultured in NNN media, then the growing isolates were transferred to RPMI-1640 medium and abundant amount of promastigotes were obtained. Isolates were genotyped using real-time polymerase chain reaction method with primers and probes specific to the ITS-1 region of Leishmania spp. and in five isolates that were found to be L. tropica causing CanL, resistance status against amphotericin B, meglumine antimoniate and sodium stibogluconate was investigated by hemocytometer and XTT methods. Results: The mean IC50 values were determined as 10.60 mg/ml for meglumine antimoniate, 0.1471 mg/ml for sodium stibogluconate, 0.0328 μM/ml for amphotericin B by hemocytometer method. Average IC50 values determined by XTT method were 10.48 mg/ml for meglumine antimoniate, 0.1470 mg/ml for sodium stibogluconate, 0.0326 μM/ml for amphotericin B. Conclusion: According to our data, while L. tropica strains which were isolated from CanL cases which are very rarely found causative agents in dogs, were not found to be resistant to amphoreticin B and sodium stibogluconate, parasite developed drug resistance against meglumine antimoniate. In future, this situation is thought to be a problem during treatment in human cutaneous leishmaniasis cases.

First report on feline leishmaniasis caused by Leishmania (Leishmania) amazonensis in Amazonian Brazil

In the present study, we reported the natural infection by Leishmania sp. in a domestic cat, in which the amastigote forms of the parasite were observed within a lesion on its ear-tip. Fragment of the lesion was obtained and cultured in NNN medium, and PCR-RFLP analysis of the isolated sample was performed, which revealed that the profile was compatible with Leishmania (L.) amazonensis. This is the first proven case of a cat infected by L. (L.) amazonensis reported in Belém city, Pará state, northern Brazil.

Observation on growth and reproduction of various Leishmania isolates in different culture media

To compare the growth and reproduction of the promastigotes of Leishmania isolates from various endemic areas of visceral leishmaniasis in China in various culture media, so as to provide experimental evidence for selecting an appropriate medium for the culture of Leishmania. A total of 3 × 105 promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates were inoculated into 1 mL NNN medium, 1 mL M199 medium supplemented with 20% fetal bovine serum medium, 1 mL M199 medium supplemented with 20% horse serum medium, and 1 mL brain heart infusion medium containing heme, respectively. All media were placed at 22 ℃ under a sterile condition, and the number of promastigotes was counted continuously for 8 days under a microscope. The growth curve was plotted for the three Leishmania isolates. The promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates all grew and reproduced in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium. The number of promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates was all significantly higher in the NNN medium than in the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05), and the number of promastigotes of the KS-2 isolate was all significantly greater than that of the Cy and JIASHI-5 isolates in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05). In ad dition, the promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates failed to grow and reproduce in the brain heart infusion medium. The growth and reproduction of the promastigotes of various Leishmania isolates from various endemic areas of visceral leishmaniasis in China vary in the same culture medium, and the growth and reproduction of a Leishmania isolate vary in different culture media. The NNN medium best fits for the culture of Leishmania isolates in the endemic areas of visceral leishmaniasis in China.

Isolation and Molecular Diagnosis of Leishmania major and Study Activity of Aqueous Extract of Plant Nigella sativa Against the Parasite In Vitro

The current study included isolating and diagnosing the cutaneous leishmaniasis parasite from patients with Baghdad boil and the reviewers of Al Hussein Teaching Hospital in Thi-Qar province for the period from November to December 2016 and then studying the effect of aqueous extract of the plant Nigella sativa in the vitality of isolated parasite in vitro. Through the results, the isolated parasite was diagnosed depend on lesions appearance and characterization then confirm the diagnosis by KDNA- PCR which revealed that leishmania spp. was Leishmania major . In this study three different concentrations from aqueous extract of the Nigella sativa plant were used (50, 100 and 150 mg / ml), the parasite growing in NNN medium (3 replicates used)It was observed that the percentage of parasite destruction increased with prolong exposure time of the extract and increased concentration of the extract, results also exhibited that the percentage of the destruction was 100% after four hours of exposure to the concentration of 150 mg / ml which was the highly killed percentage compared to control, which was 5. 98%.

Ocular manifestations of leishmaniasis in a cat: first case report from Brazil

ABSTRACT An 8-year-old domestic short hair female cat initially presented with bilateral uveitis with pseudotumoral appearance. The patient tested negative for feline immunodeficiency virus (FIV), feline leukemia virus (FeLV) and Toxoplasma gondii. Histopathology of a granulomatous lesion on the upper left conjunctiva revealed amastigotes compatible with Leishmania spp. Aqueous humor was aspired and the diagnosis was confirmed after isolation of promastigotes cultivated in biphasic NNN medium and by positive polymerase chain reaction (PCR) for Leishmania infantum. Treatment with allopurinol (10mg/kg/ BID/PO) was commenced and a natural insect repellent was prescribed. Six months of treatment with allopurinol associated with the initial topical medications helped to improve ocular signs. Leishmaniasis should be considered as a differential diagnosis in cats presenting uveitis with pseudotumoral appearance. To our knowledge, this is the first report of feline leishmaniasis with ocular manifestation in Brazil, in which diagnosis was confirmed by aqueous humor analysis.

Imported cutaneous leishmaniasis cases detected in truck drivers in Hatay

Leishmaniasis, seen in tropical and subtropical regions, is an infectious disease caused by the protozoan parasite Leishmania species. There are three main forms of leishmaniasis: cutaneous, mucocutaneous and visceral leishmaniasis. Cutaneous leishmaniasis (CL) has become an increasing problem as the number of travels around the world increases and people go to work in endemic areas. Turkey has received great numbers of immigrants in recent years, from its neighboring countries like Iraq, Islamic Republic of Iran, Afghanistan, Turkmenistan and the Syrian Arab Republic because of the political instabilities in these countries as well as the job opportunities caused by large-scale development projects undertaken by Turkey. In this report, imported CL cases detected in five truck drivers transporting from Hatay to Turkmenistan, Syria, Saudi Arabia, Iran and Georgia, Uzbekistan and Azarbaijan countries were presented. The patients admitted to Mustafa Kemal University, Faculty of Medicine Dermatology Policlinic, with wound complaints on their bodies were directed to the Department of Parasitology to obtain smear samples from their wounds. The age range of the patients were 38 to 43 years. Patients with wound trail for a period ranging from one month to one year had a number of lesions varying between 2-7 and in all cases, a smear preparation was prepared from the lesions for diagnostic purposes. Clinical material obtained from five patients with pre-diagnosis of CL was firstly examined with Giemsa stain. Samples taken from the patients were inoculated into modified NNN (Novy-MacNeal-Nicolle) medium for the evaluation of the presence of the promastigotes. Promastigotes obtained from the inoculated medium were also genotyped using the ITS1 region. In all of the slides prepared from the clinical material taken from the patients amastigotes were determined. The growth of promastigotes were observed in only three of the clinical specimen inoculated media. The genotyped three species were Leishmania tropica, Leishmania infantum/donovani and Leishmania major. In this study, the importance of support for the diagnosis of different microbiological methods used in the diagnosis of leishmaniasis infection which occurred during the outbreaks of the disease has been put forward. In addition, it was aimed to draw attention to the importance of imported CL cases in our country diagnosed in five truck drivers making transportation from Hatay to Turkmenistan, Syria, Saudi Arabia, Iran, Georgia, Uzbekistan and Azerbaijan.

Do the rodents have a role in transmission of cutaneous leishmaniasis in Turkey?

Leishmaniasis is a zoonotic/anthroponotic vector borne parasitic infection which is caused by Leishmania species and transmitted by sand flies (Phlebotomus spp.) The reservoirs of Leishmania species in nature are various wild and domestic carnivores, rodents and human. The aim of this study was to investigate whether the rodents in genera Meriones, Mesocricetus, Rattus and Mus which inhabit in the natural habitat of our country could be natural reservoirs of Leishmania tropica, Leishmania infantum, Leishmania major and Leishmania donovani for cutaneous Leishmaniasis (CL)., The rodents Mus musculus (Balb/C mouse), Mesocricetus auratus (hamster), Meriones unguiculatus (gerbil) and Rattus norvegicus (rat) which are part of the natural habitat in Turkey were used in the study. L.tropica, L.infantum, L.major and L.donovani promastigote isolates obtained from CL patients and cultured in enriched media were injected in the footpads of the animals intradermally using the density of 108 promastigote/ml. The scale of the lesions on the footpads of the animals were measured for 12 weeks. At the end of the experiment, the animals were sacrificed and "touch preparations" were prepared using footpad, liver, spleen and testicles of the sacrified animals and were examined using Giemsa stained slides following culturing in enriched NNN medium. Leishmania amastigotes were seen in the slides prepared from the footpads of the all experimental animals and all cultures were positive for promastigotes prepared from the same clinical material. But not all the experiment groups were positive for the liver, spleen and testicle preparations. According to these results it was concluded that while all rodents in the experiment groups were positive for CL, only a part of the experiment groups were positive for internal organ involvement. Accordingly, (a) All Leishmania strains caused both CL and internal organ involvement in M.unguiculatus and M.musculus, (b) only L.tropica caused CL and internal organ involvement in R.norvegicus, while other Leishmania strains only caused CL in this group, (c) in M.auratus only L.donovani caused CL while other strains caused both CL and internal organ involvement. In our study, it was determined that the rodents Meriones, Mesocricetus, Rattus and Mus genera which are part of our country's natural habitat could serve as natural reservoirs of L.tropica, L.infantum, L.major and L.donovani, thus having the potential for the spreading of Leishmaniasis in our country and important information were gathered concerning the clinical aspects of the infection caused by Leishmania species in their potential reservoir hosts.

Isolation of naturally infecting Leishmania infantum from canine samples in Novy-MacNeal-Nicolle medium prepared with defibrinated blood from different animal species

The most commonly used culture medium for the in vitro isolation of Leishmania spp. from canine biological samples is biphasic Novy-MacNeal-Nicolle (NNN) medium, whose solid phase is prepared using rabbit blood. Leishmania infantum parasites from natural infections are highly sensitive and demanding for growth in axenic conditions when firstly obtained from the dog’s body. The objective of this study was to evaluate whether NNN medium (NNN-test) prepared with chicken blood (NNN-C), ox blood (NNN-O), horse blood (NNN-H) or sheep blood (NNN-S) was viable for the isolation of parasites from naturally infected dogs, in an endemic area for visceral leishmaniasis caused by L. infantum. Spleen aspirates from six dogs previously diagnosed as infected by parasitological methods were simultaneously inoculated in each NNN-test medium, including the conventional medium prepared with rabbit blood (NNN-R), and the cultures were examined for three weeks under optic microscopy. Spleen samples were also analyzed for parasite loads by quantitative PCR (qPCR). Cultures from three of the six dogs (50%) were positive in at least one of the NNN-test media: one sample presented the highest spleen parasite load by qPCR (1.19 × 104 parasites/mL) and was positive in all test media; the second sample presented parasitic isolation in the first week of culture in all inoculated media, of which the NNN-C medium had the highest mean parasite count (NNN-C = 23.5 × 104/mL vs. NNN-R = 3.25 × 104/mL); the third sample was positive only in the NNN-S medium besides the conventional control NNN-R. Cultures from the three remaining dogs were negative in all NNN media, including the control and test media; of those three dogs, two presented the lowest spleen parasitic loads according to qPCR. Blood from chicken, ox, horse and sheep shown to be viable for the preparation of NNN culture medium for the primary isolation of L. infantum from samples of naturally infected dogs and can be considered as an alternative to rabbit blood when necessary.

EPIDEMIOLOGICAL STUDY OF CUTANEOUS LEISHMANIASIS IN SOME IRAQI PROVINCES

&#x0D; &#x0D; Background&#x0D; Cutaneous leishmaniasis (CL) remains a serious public health concern in some parts of Iraq. The aims of this study to report of CL in some parts of Iraq, by different parasitological, cultural, and molecular methods and evaluate sex differences among infected patients. This is the first study conducted to characterize Leishmania species causing CL among Iraqi patients using the sequence analysis of Internal Transcribed Spacer1(ITS1) at Wasit Province.&#x0D; Methods&#x0D; A total of 700 cases of suspected CL were referred to the Iraqi clinics and health centres and they checked for Leishmania amastigote using a Giemsa-stain; however, the Novy Macneal Nicolle (NNN) culture led to the growth of promastigotes in all samples, then the samples were examined using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP)-PCR methods.&#x0D; Results&#x0D; The present study indicated that the prevalence of CL as follow: AL-Diwaniyah 88(15.1%), Wasit 85 (14.5%), Najaf 79 (13.6%), Thi-Qar76 (13.1%), Basrah 67 (11.5%), Baghdad 65 (11.2%), Diyala 63(10.8%), and Salah-Edin province which recorded lower infection 60 (10.3%), and it appeared in 83.3% by using Giemsa- smeared stain. The highest infection rate (100%) appeared using PCR while the lowest infection (68%) appeared by culture on NNN media. The present study was revealed that the highest infection (60%) was caused by L.major rather than L.tropica (40%). Our results showed that 368 (52.6%) of CL patients were had single lesion and 215 (30.7%) had multiple lesions, and the ulcerative wet type lesions were present in 49.6%, while the nodule dry type lesions were present in 33.7%. The overall prevalence of cutaneous leishmaniasis in the study area was very high (83.3%) having a statistically significant association with sex; males are more prone (56.4%) to CL as compared to females (43.6%)&#x0D; &#x0D; Conclusions&#x0D; A clear and reliable bias toward males exists in some tropical diseases, such as leishmaniasis. CL is a major health problem in Iraq and CL caused by many countries including Iraq. Health authorities should be aware of the fact that war and terrorist activities induce expansion of the disease and increase the incidence rate in the situation that access to medical treatment is not easy especially in poor conditions in leishmaniasis endemic areas.&#x0D; &#x0D; &#x0D;

COMPARATIVE STUDY BETWEEN CLASSICAL AND MOLECULAR METHODS IN DIAGNOSIS OF LEISHMANIA SPECIES IN IRAQ

Background and Objective&#x0D; Cutaneous leishmaniasis (CL) remains a serious public health concern in some parts of Iraq. The aims of this study to diagnose the Leishmania sp. causative agent of CL in some parts of Iraq, by different parasitological, cultural, and molecular methods. It was carried during the period October 2014 to February 2015.&#x0D; Materials and Methods&#x0D; One hundred-sixty one of skin samples were examined by direct Giemsa-smeared, culture on NNN medium and Nested – PCR methods in different age groups.&#x0D; Results&#x0D; The results of our study showed that 110 (68.3%) gave positives by Giemsa-smeared, 104 (64.6%) by culture and 67 (100%) by Nested – PCR. Our results appeared that there was slight gender predilection ; CL cases were more frequent in males (62.1%) than females (37.9%). Also, the type of infection showed that disease was in wet type 133(82.6%) more than in dry type 28 (17.4%). It was found that 98 (60.9%) of CL cases were in contact with rodents while 63(39.1%) of cases were not. &#x0D; Conclusions&#x0D; CL disease is endemic in many parts of Iraq with high incidence and expanding to new foci that is considered a public health threat which needs special attention. Women appeared to be better equipped than men to contain the infection and its clinical consequences, but the sex factor tended to lessen at higher levels of exposure.

Cryopreservation of Leishmania Species in Manisa Province

It was aimed to assess the success of the cryopreservation process which is carried out in order to preserve the genetic material and the virulence of the Leishmania species that are an important health problem in our region. Leishmania tropica, L. infantum, L. major, and L. donovani strains in Novy-MacNeal-Nicolle (NNN) medium in MCBU were used. Promastigotes cultured in the NNN medium were transferred to RPMI 1640 medium; promastigotes in the logarithmic phase were washed three times with PBS, and 15% dimethylsulfoxide (DMSO) was added. Leishmania species were transferred to 12 separate tubes. The tubes were stored at -86°C for one night by placing them in Coolcell boxes. The tubes were transferred into a liquid nitrogen tank. One cryotube per Leishmania strain is thawed monthly and cultured in NNN medium. For the duration of study it was observed that each Leishmania isolate preserved 60-65% of their viability and entered the logarithmic phase on the 7th day following the inoculation in the NNN medium. Abnormalities in the structures and movements of the promastigotes were not observed in microscopic examinations. The following conclusions were made: cryopreservation is important for studies planned related to leishmaniasis and cryopreservation with DMSO is successful.

Pathological Study of Cutaneous Leishmaniasis in Al-Diwaniya province

In the present study, 42 patients were observed, as cutaneous leishmaniasis, from 1 September 2012 until the end of August 2013. They divided to 25 (595. %) males and 17 (40.5%) females, and the diagnosis was depend on clinical examination, pathological lesions and parasitic isolation. Lesions were classified depending on number, so the patients who have multiple lesions were (64.3%), while (35.7%) have single lesion, the number of lesions per patient ranged from one to 12 lesions. Most of the lesions were located on face (40.5%), but some lesions were on the face &amp; hand (21.4%), while few were found in hand and back (2.4%). Histopathologicaly, the cutaneous lesions were observed; keratinized layer and epidermal cells were necrosed or not present over the ulcer, Skin biopsies were taken, fixed with formalin, sectioned, and stained with hematoxyline and eosin stain (H &amp; E stain). Microscopic examination, showed accumulation of amastigote, inflammatory cells such as neutrophils and lymphocytes with macrophages engulf amastigotes. Eleven samples of aspirated blood were collected by fine needle, for making direct Giemsa-stained smears before the cultivation, 72.7% of the cases were positive to the presence of leishmania tropica. There are three culture media were used in this study, biphasic medium, NNN medium, and lock’s solution; 63.4% of cases were positive in NNN medium, and 72.7 % were positive in both of biphasic media and Lock’s solution.

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

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. Rodents were collected from selected study sites in kala-azar endemic areas based on information for localities of kala-azar cases for screening of Leishmania 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 2mg/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 used for DNA extractions and PCR amplification of Leishmania infection. 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 nilothicus compared to 17.6% (3/17) and 12.5% (2/16) for Acomys cahirinus and Tarera (G) 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. Leishmania donovani might circulate among different species of rodents in kala-azar endemic lowlands and valleys of Ethiopia by Phlebotomus orientalis and Phlebotomus martini. Detailed studies to substantiate the preliminary data on the possible role of these rodents are urgently needed.