Identification of Leishmania Species Using PCR Assay on Giemsa-Stained Slides Prepared From Cutaneous Leishmaniasis Patients

Background

Leishmaniasis is a group of diseases that are created by intracellular parasites of Leishmania. Cutaneous leishmaniasis is considered as one of the health problems in some provinces of Iran.

Methods

In this study, a total of 178 Giemsa-stained slides from confirmed cases of cutaneous leishmaniasis were examined. The slides were prepared from the patients with cutaneous leishmaniasis that referred to health centers and infected during the epidemic of cutaneous leishmaniasis in Poldokhtar city, Lorestan Province, Iran in 2006.Genomic DNA from each slide was extracted. After DNA extraction, ITS-PCR was used.

Results

Out of 178 slides, 129 (72.47%) samples had a band in the range of 485 bp and 49 (27.53%) samples 626 bp that matched L. tropica and L. major standard samples, respectively.

Conclusion

This study showed that Leishmania DNA could be efficiently extracted and amplified even from old Giemsa-stained microscopic slides that were stored more than 6 yr. In this study was shown that both L. tropica and L. major species exist in Lorestan Province.     

Investigation of Double-Band Electrophoretic Pattern of ITS-rDNA Region in Iranian Isolates of Leishmania tropica

Background

Leishmania tropica is a genetically divergent species. Amplification of entire internal transcribed spacer (ITS) region of L. tropica isolates obtained from Bam district, one of the well known focus of anthroponotic cutaneous leishmaniasis (ACL) in Iran, revealed a double-band pattern in agarose gel electrophoresis. This study explains how this pattern occurs.

Methods

Twenty seven L. tropica smear preparations were collected from Bam district, south east Iran, and eight L. major and one L. infantum smear preparations were gathered from Shiraz, south west Iran. Furthermore one L. major and one L. infantum cultured standard strains were tested using entire ITS-PCR to survey their electrophoretic pattern. The ITS sequences of L. tropica, L. major, and L. infantum already deposited in GenBank were analyzed. Analysis of GenBank sequences of L. tropica revealed two groups of sequences based on length size, one group having a 100 bp gap. Therefore, a new reverse primer namely LITS-MG was designed to exclude this gap in PCR products.

Results

Whole ITS fragment amplification resulted in a double-band pattern in all L. tropica cases, while a sharp single band was observed for L. infantum and L. major isolates. This result was corresponding to the result obtained from in silico analysis of GenBank sequences. Use of LITS-MG primer was expectedly resulted in a single band including ITS1, 5.8s and partial ITS2 product for L. tropica which is appropriate for following molecular studies such as sequencing or restriction analysis.

Conclusion

Sequences analysis of GenBank L. tropica sequences and following practical laboratory tests revealed at least two alleles in L. tropica which were confirmed in Bam isolates. This especial double-band pattern is because of a 100 bp fragment difference within ITS-rDNA alleles.     

Epidemiology,Pathology and Treatment of Cutaneous Leishmaniasis in Taif Region of Saudi Arabia

Background

Cutaneous leishmaniasis is an annoying and disfiguring disease affecting around 1,500,000 individuals globally. There are endemic pockets of this disease in Taif region. In some patients, lesion often weeps and leads to scar formation. The study was conducted to see the efficacy of fluconazole and itraconazole in the patients of cutaneous leishmaniasis and the effect of these drugs on liver enzymes and kidney markers.

Methods

Positivity of Leishmania was recorded by microscopic examinations of smears. Specific diagnosis for Leishmania major and L. tropica was made with the help of nested polymerase chain reaction. Fluconazole was given at the rate of 200mg/day while itraconazole was given at 150mg/day for six weeks. AST, ALT, creatinine and urea were estimated during medication.

Results

Leishmania major and L. tropica were the species responsible for cutaneous leishmaniasis in Taif region. 81% patients had single lesions, mostly on face followed by hands and legs. 15% of the lesions had bacterial contamination. In terms of efficacy, fluconazole gave slightly better results compared to itraconazole. After 6 weeks of medications, slightly elevated values were recorded for liver enzymes and creatinine.

Conclusion

Transmission of leishmaniasis in Taif region is probably because of poor coverage of residual insecticides spraying at hiding places in pile-ups of rocks and abandoned houses from where sand flies visit nearby houses and cattle sheds during night. Fluconazole and itraconazole may be used for the treatment of cutaneous leishmaniasis with good recovery rate and fewer side effects.     

IL-23 and IL-27 Levels in Macrophages Collected from Peripheral Blood of Patients with Healing Vs Non-Healing Form of Cutaneous Leishmaniasis

Background

In this study the level of IL-23 and IL-27 produced by macrophages derived from peripheral blood mononuclear cell culture collected from patients with healing or non-healing form of cutaneous leishmaniasis lesion were compared before and after treatment with live Leishmania to explore whether IL-23 or IL-27 plays any role in healing process of cutaneous lesions induced by L. major.

Methods

Twenty patients resident in Isfahan Province, with healing or non-healing form of cutaneous leishmaniasis lesion caused by Leishmania major participated in this study. In vitro productions of IL-23 and IL-27 by peripheral blood derived macrophages, before and after stimulation with live L. major (MRHO/IR/75/ER) promastigotes were evaluated using ELISA method. Patient with healing form of lesion received no treatment and patient with non-healing form of lesion received at least 2 courses of glucantime.

Results

The mean production of IL-23 and IL-27 from macrophages of patients with healing form of lesion was significantly higher than patients with non-healing form of lesion. The levels of IL-23 and IL-27 in culture supernatants before and after stimulation in healing form of CL was significantly higher than non- healing form of CL (P < 0.001).

Conclusion

IL-23 and IL-27 might play a role in human leishmaniasis and further studies are needed to understand the role of IL-23 and IL-27 in leishmaniasis.     

Cutaneous Leishmaniasis in Suspected Patients Referred To the Center for Research and Training in Skin Diseases and Leprosy,Tehran, Iran from 2008 To 2011

Background

Cutaneous leishmaniasis (CL) is a major health problem in many parts of Iran, although diagnosis of CL especially in the endemic area is easy, but treatment and management of the disease is a global dilemma. Diagnosis of CL in non-endemic area is not as simple as in endemic foci. In this study, the status and the proportions of CL induced by Leishmania major and L. tropica among CL suspected patients referred to the Center for Research and Training in Skin Diseases and Leprosy, (CRTSDL) during 2008 to 2011 are described.

Methods

CL patients with suspected lesions were clinically examined. History of trip to zoonotic CL and/or anthroponotic CL endemic areas and the characteristics of their lesion(s) were recorded. Diagnosis of the lesion was done using direct smear microscopy, culture and conventional polymerase chain reaction (PCR).

Results

A total of 404 (M = 256, F = 148) patients with 776 lesions were recruited and parasitologically examined. The results showed that 255 of the patients with 613 lesions; patients with lesion(s) induced by L. major=147 (M = 63, 43%, F = 84, 57%) and lesion(s) induced by L. tropica=108 (M = 35, 32%, F = 73, 68%). History of travel to endemic area was not always correlated with isolated Leishmania species.

Conclusion

Although travel history to endemic area is an important factor to be considered for diagnosis, but parasitological confirmation is necessary initiation of treatment.     

Leishmaniasis in Turkey: Determination of Leishmania Species by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS)

Background

Cutaneous leishmaniasis (CL) is endemic in Southeastern Anatolia, mainly in Sanliurfa and Hatay provinces, and the causative agents are mostly Leishmania tropica and less frequently L. infantum. Here, we report the first MALDI-TOF analyses of Leishmania promastigotes obtained from the cultures of two CL cases from Osmaniye and Hatay provinces who were initially diagnosed by microscopy, culture and identified as L. infantum with Real-Time PCR (RT-PCR).

Methods

Samples obtained from the skin lesions of patients were initially stained with Giemsa and cultivated in NNN medium. Examination of the smears and cultures revealed Leishmania amastigotes and promastigotes, respectively. The promastigotes (MHOM/TR/2012/CBU15 and MHOM/TR/2012/MK05) obtained from the cultures of both patients were used for RT-PCR targeting the ITS-1 region in the SSU of rRNA. The reference strains of four Leishmania species (L. infantum, L. donovani, L. tropica and L. major) were initially assessed with MALDI-TOF and their data were added to MALDI-TOF Biotyper Library.

Results

Both RT-PCR and MALDI-TOF analyses indicated that the causative agent in both patient samples was L. infantum.

Conclusion

Despite disadvantages such as requirement of culture fluid with nothing but promastigotes and high cost, MALDI-TOF analysis may be a fast, sensitive and specific diagnostic tool in especially large-scale research studies, where the cost declines, relatively.     

Molecular Identification of Leishmania Species Using Samples Obtained from Negative Stained Smears

Background

Cutaneous Leishmaniasis (CL) is a parasitic skin disease. Diagnosis primarily is based on clinical signs and microscopic observation of parasite on direct stained smears or tissue sections. Sensitivity of direct smear is not as high as molecular methods. The aim of this study was to identify and characterize Leishmania species among the negative direct smears obtained from skin ulcers suspected to CL by PCR method.

Methods

Among 81 patients with suspicious skin lesions to CL referred to the Parasitology lab, negative Giemsa stained smears were collected. DNA extraction performed by scraping stained smears, then PCR was performed.

Results

Among the DNA extracted from smears, L. tropica was isolated from 9 (11.1%) of the smears and L.major was not isolated from any samples.

Conclusion

Direct microscopy on stained smears for diagnosis of leishmaniasis is not enough accurate. PCR is recommended for clinically suspected lesions with negative result of direct smear.     

The Problem of Mixing up of Leishmania Isolates in the Laboratory: Suggestion of ITS1 Gene Sequencing for Verification of Species

Background

Leishmaniasis is endemic in Iran. Different species of Leishmania (L.) parasites are causative agents of this disease. Correct identification of Leishmania species is important for clinical studies, prevention, and control of the diseases. Mix up of Leishmania isolates is possible in the laboratory, so there is need for verification of species for isolates of uncertain identity. Different methods may be used for this purpose including isoenzyme electrophoresis and molecular methods. The isoenzyme electrophoresis, due to its drawbacks, is feasible only in specialized laboratories while molecular methods may be more feasible. The aim of this research was to study the application of the internal transcribed spacer 1 (ITS1) sequencing method, in comparison to isoenzyme electrophoresis method, for verification of Leishmania species.

Methods

Six Leishmania isolates were received from different research institutions in Iran. The species of these isolates were known by donating institution according to their isoenzyme profile. The species of these isolates were re-identified in Pasteur Institute of Iran by PCR amplification of ITS1 followed by sequencing and comparison of these sequences with Leishmania sequences in GenBank. Isoenzyme electrophoresis was performed for confirmation of the results of ITS1.

Results

ITS1 sequence showed that some isolates were mixed up or contaminated with Crithidia. Isoenzyme electrophoresis confirmed the results of ITS1 sequences.

Conclusion

ITS1 sequencing is relatively more feasible than the traditional isoenzyme electrophoresis method and is suggested for verification of Leishmania species.     

Interleukin-10 and Transforming Growth Factor-β in Early and Late Lesions of Patients with Leishmania major induced Cutaneous Leishmaniasis

Background

Cutaneous leishmaniasis is a neglected parasitic disease, which imposes massive human distress and financial costs to the endemic countries. Better understanding of host immune response to the parasite leads to helpful strategies for disease control. Interleukin (IL)-10 and transforming growth factor (TGF)-β are important immune regulatory cytokines, which appear to develop non-healing forms of leishmaniasis. However, there is little information about the function of IL-10 and TGF-β in old world cutaneous leismaniasis. The aim of this study was to analyze the role of IL-10 and TGF-β in human cutaneous leishmaniasis due to Leishmania major infection.

Methods

Biopsies were obtained from lesions of twenty proven cases of L. major induced cutaneous leishmaniasis. IL-10 and TGF-β positive cells were detected by immunofluorescence staining of frozen sections and compared between two groups of patients with early and late lesions.

Results

The mean percentage of IL-10 positive cells were significantly (P= 0.035) higher in late lesions (0.51±0.24) than early ones (0.15±0.07). Similar results were obtained for TGF-β with mean percentages of 0.16±0.05 and 0.53±0.28 in early and late lesions respectively (P= 0.008).

Conclusion

IL-10 and TGF-β are present in lesions of L. major induced cutaneous leishmaniasis and contribute to the pathogenesis of long lasting disease forms.     

Interleukin-10 and Transforming Growth Factor-β in Early and Late Lesions of Patients with Leishmania major Induced Cutaneous Leishmaniasis

Background

Cutaneous leishmaniasis is a neglected parasitic disease, which imposes massive human distress and financial costs to the endemic countries. Better understanding of host immune response to the parasite leads to helpful strategies for disease control. Interleukin (IL)-10 and transforming growth factor (TGF)-β are important immune regulatory cytokines, which appear to develop non-healing forms of leishmaniasis. However, there is little information about the function of IL-10 and TGF-β in old world cutaneous leismaniasis. The aim of this study was to analyze the role of IL-10 and TGF-β in human cutaneous leishmaniasis due to Leishmania major infection.

Methods

Biopsies were obtained from lesions of twenty proven cases of L. major induced cutaneous leishmaniasis. IL-10 and TGF-β positive cells were detected by immunofluorescence staining of frozen sections and compared between two groups of patients with early and late lesions.

Results

The mean percentage of IL-10 positive cells were significantly (P= 0.035) higher in late lesions (0.51±0.24) than early ones (0.15±0.07). Similar results were obtained for TGF-β with mean percentages of 0.16±0.05 and 0.53±0.28 in early and late lesions respectively (P= 0.008).

Conclusion

IL-10 and TGF-β are present in lesions of L. major induced cutaneous leishmaniasis and contribute to the pathogenesis of long lasting disease forms.     

Comparison of Three Methods for Diagnosis of Cutaneous Leishmaniasis

Background

Leishmaniasis is one of the infectious parasitic diseases of highest incidence in the world. Cutaneous Leishmaniasis (CL) has long been reported in Shiraz, Southern Iran. There is a need to find a sensitive and specific method for treatment and control of the disease.

Methods

We have compared the sensitivity of the conventional methods microscopy and cultivation of lesion scrapes against PCR amplification of parasite kinetoplast DNA from these samples. The samples (n=219) were obtained from the patients clinically suspected of CL. The smears were stained with Giemsa for microscopy and cultured in Novy-Nicolle-McNeal (NNN) blood agar for promastigote growth. For PCR, the dry smears were scraped off the slides and DNA was extracted.

Results

The positive rates from 219 specimens were 76.71%, 50.68%, and 93.61% for microscopy, cultivation, and PCR, respectively. The highest correlation was found between PCR and microscopy method (P=0.014). In PCR assay, 95.61%, 3.9%, and 0.49% of the samples were identified as Leishmania major, L. tropica, and dermatropic L. infantum, respectively.

Conclusion

The PCR method appears to be the most sensitive for the diagnosis of CL and is valuable for identifying the other species of Leishmania with confusing dermatropic signs.     

Inhibition of Murine Systemic Leishmaniasis by Acetyl Salicylic Acid via Nitric Oxide Immunomodulation

Background

The purpose of this study was to evaluate antileishmanial effects of ASA via NO pathway in Leishmania major infected Balb/c mice. Moreover, toxicity and pathological consequences of ASA administration were investigated.

Methods

Balb/c mice were infected with L. major and ASA was inoculated orally after lesion appearance for its ability to modulate NO and to modify Leishmania infection in host, in order to evaluate the effects of NO production on size and lesion macroscopy, delay of lesion formation and proliferation of amastigotes inside macrophages. Liver, spleen, and lymph nodes were also studied as target organs to detect amastigotes. In addition, plasma was investigated for NO induction using Griess microassay.

Results

ASA increased NO production in plasma of both naïve and Leishmania test groups at the ultimate of the experimental period. A decline was observed in proliferation of amastigotes inside macrophages of test group when compared with control one. ASA reduced lesion size, inhibited Leishmania visceralisation in spleen, lymph node, and decreased hepato/splenomegaly in ASA treated animals.

Conclusions

Some antileishmanial effects of ASA by NO-modulation were indicated during systemic leishmaniasis in mice. Despite slight effects on lesion size, ASA decreased parasite visceralization in target organs and declined their proliferation inside macrophages. Therefore, ASA may be indicated to inhibit systemic leishmaniasis via NO pathway in mice model.     

Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct

Background

Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines especially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression.

Methods

L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 µg of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR–tansfected promastigotes. Western blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits.

Results

The PTR1 protein was not expressed in pcDNA-rPTR– tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR– tansfected promastigotes.

Conclusion

This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.     

Canine Visceral Leishmaniasis in Boyer Ahmad District,Kohgiluyeh & Boyer Ahmad Province,Southwest of Iran

Background

Mediterranean type of visceral leishmaniasis (VL) is present in different parts of Iran. Several studies have identified dogs as the main reservoirs of the VL caused by Leishmania infantum in Iran and other Mediterranean regions. This study aimed to determine the seroprevalence of canine visceral leishmaniasis as animal reservoir host for human visceral leishmaniasis in Boyer Ahmad district in southwest of Iran.

Methods

A seroepidemiological study was carried out to determine the seroprevalence of canine visceral leishmaniasis (CVL) among ownership dogs by using direct agglutination test (DAT) in 23 of 182 villages of Boyer Ahmad district, during August 2009 to August 2010. One hundred and seventy serum samples from ownership dogs were selected by multi-stage cluster sampling in villages of Boyer Ahmad district. All samples were tested by DAT and anti-Leishmania antibodies titers at ≥ 1:320 was considered as positive.

Results

Of the 170 serum samples, 10% were positive by DAT at titers of 1:320 and higher. No statistical significant difference was found between male (10.7%) and female (8.3%) seroprevalence. The highest seroprevalence rate (15.1%) was observed among the ownership dogs of four to seven years age. Altogether, seventeen (25.4%) of the seropositive dogs had clinical signs and symptoms.

Conclusion

It seems that Boyer Ahmad district is an endemic area for canine visceral leishmaniasis in Iran.     

Molecular Detection and Conventional Identification of Leishmania Species in Reservoir Hosts of Zoonotic Cutaneous Leishmaniasis in Fars Province,South of Iran

Background

The objectives of our research were to search for Leishmania species in rodents in Fars province, south of Iran, and to compare molecular with conventional methods for detecting these parasites.

Methods

Rodents were captured using live traps and screened for Leishmania species using molecular and conventional methods, including the taking of smears from each ear. Nested PCR was employed to detect Leishmania in rodents by amplifying a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA-ITS2) that is species-specific by DNA sequence.

Results

Totally, 122 rodents were captured. Leishmania parasites were detected using the nested PCR and three conventional methods (direct smear, NNN culture and Balb/C inoculation. 41 (33.6%) out of 122 rodents had Leishmania infections (34 Meriones lybicus and 7 M. persicus). All PCR products of the ITS-rDNA gene were sequenced. Sequence analysis revealed that 28 out of 41 positive samples were Leishmania major. Thirteen sequences were unreadable and therefore not identified.

Conclusion

At least two gerbil species common in Fars ZCL foci, M. lybicus and M. persicus, are acquiring infections of L. major and may be reservoir hosts of one predominant parasite haplotype. Most infections were detected molecularly not by conventional methods, because most rodents died in the traps.     

Therapeutic Effect of Sodium Selenite and Zinc Sulphate as Supplementary with Meglumine Antimoniate( Glucantime?) Against Cutaneous Leishmaniasis In BALB/C Mice

Background

Successful therapy of leishmaniasis depends on effective cellular immune response. We evaluated the effectiveness of sodium selenite and zinc sulphate as known immunomodulator materials, in combination with Glucantime® in treatment of cutaneous leishmaniasis lesions resulting from Leishmania major in susceptible animal model.

Methods

Thirty three female mice weighing 18–20 g at the age of 7–8 week infected with L. major were randomly divided into 3 groups: group1: treated by sodium selenite (0.35 mg/kg for 30 days), group2: treated by zinc sulphate (2 mg/kg for 30 days) and group3: treated by distilled water (0.01 ml/gr body weight for 30 days) as control. All groups received Glucantime® as a standard anti- leishmanial agent (60 mg/kg, ip) for 14 days. To assess the results of treatment measurement of lesions size and parasitological tests were done weekly.

Results

The lesion sizes increased continuously in sodium selenite group.Although, in zinc group did not increase compared to baseline But with considering the time- group interaction there was no significant difference between zinc and control group during this study. There was no difference between lesion sizes and Leishmanial loads in the interventional and control groups, respectively.

Conclusion

Sodium selenite and zinc sulphate at mentioned doses and duration of treatment did not show any treatment effect on cutaneous leishmaniasis caused by L. major in BALB/c mice. Increasing the dose of supplements and considering the follow up period after treatment can help more certain conclusion.     

The Role of Liposomal CpG ODN on the Course of L. major Infection in BALB/C Mice

Background

Historically, leishmanization is the most effective protective measure against Cutaneous Leishmaniasis (CL), CL lesion induced by leishmanization sometimes takes a long time to heal. Manipulation of leishmanization inoculums needed to induce a mild and acceptable CL lesion. The aim of this study was to explore if liposomal form of CpG ODN (Cytosin phosphate Guanin Oligodeoxynucleotides) mixed with Leishmania major would induce a milder lesion size in Balb/c mice.

Methods

This study was performed in Biotechnology Research Center, Mashhad, and Center for Research and Training in Skin Diseases and Leprosy, Tehran, Iran during 2008–2009. Different groups of BALB/c mice were subcutaneously (SC) inoculated with L. major mixed with liposomal form of CpG ODN, or L. major plus free CpG ODN, or L. major mixed with empty liposomes or L. major in PBS. The lesion onset and the size of lesion were recorded; the death rate was also monitored.

Result

Footpad thickness was significantly (P<0.01) smaller, death rate was also significantly (P<0.05) lower in the mice received L. major mixed with liposomal CpG ODN or free CpG ODN than control groups received L. major in PBS or L. major plus liposomes, also mice which received L. major mixed with CpG ODN in soluble form showed a significantly (P<0.001) smaller lesion size than control groups.

Conclusion

CpG ODN seems to be an appropriate immunopotentiator mixed with Leishmania stabilate in leishmanization.     

The Multiple Forms of Leishmania major in BALB/C Mice Lung in Iran

Cutaneous leishmaniasis is one of the most important parasitic diseases, which are endemic in different parts of Iran. Leishmania major and L. tropica are the primary causative agents of this disease. The aim of the present study was to detect the multiple forms of L. major in lung. Ppromastigotes of L. major at stationary phase were injected to BALB/c mice. After 60 days, the different forms of Leishmania parasites were checked in lung tissue. Promastigote and amastigote forms of Leishmania parasites were detected.