Molecular epidemiology and multilocus genotyping of Giardia duodenalis in individuals attending major public hospitals in Shiraz, southwestern Iran: A public health concern.

Giardia duodenalis is one of the most common causes of waterborne disease worldwide, and is often associated with outbreaks of diarrhea in areas with poor sanitation and hygiene. This study aimed to assess the prevalence and genetic diversity of G. duodenalis assemblages in individuals attending major public hospitals in Shiraz, southwestern Iran. From August 2022 to May 2023, a total of 614 stool samples from individuals were collected and initially examined for G. duodenalis cysts using parasitological techniques, sucrose flotation, and microscopy. Microscopy-positive samples were validated by SSU-PCR amplification of the parasite DNA. A multilocus genotyping (MLG) scheme, which focused on the triose phosphate isomerase (tpi) and the glutamate dehydrogenase (gdh) genes, was employed for genotyping purposes. G. duodenalis cysts were found in 7.5% (46/614) and 8.5% (52/614) of samples through microscopy and SSU-PCR, respectively. Successful amplification and sequencing results were obtained for 77.3% (17/22) and 45.5% (10/22) of the infected samples at the tpi and gdh loci, respectively. MLG data for the two loci were available for only five samples. Out of the 22 samples genotyped at any loci, 54.5% (12/22) were identified as assemblage A, while 45.5% (10/22) were identified as assemblage B. AII was the most predominant sub-assemblage identified [54.5% (12/22)], followed by BIII [27% (6/22)], discordant BIII/BIV [13.6% (3/22)], and BIV [4.5% (1/22)]. In the present study, no assemblages suited for non-human animal hosts (e.g., C-F) were detected. This suggests that the transmission of human giardiasis in Shiraz is primarily anthroponotic. Further molecular-based analyses are necessary to confirm and expand upon these findings. These analyses will also help determine the presence and public health importance of the parasite in environmental samples, such as drinking water.

Preparation and characterization of artemether-loaded niosomes in Leishmania major-induced cutaneous leishmaniasis.

Cutaneous leishmaniasis is the most prevalent form of leishmaniasis worldwide. Although various anti-leishmanial regimens have been considered, due to the lack of efficacy or occurrence of adverse reactions, design and development of novel topical delivery systems would be essential. This study aimed to prepare artemether (ART)-loaded niosomes and evaluate their anti-leishmanial effects against Leishmania major. ART-loaded niosomes were prepared through the thin-film hydration technique and characterized in terms of particle size, zeta potential, morphology, differential scanning calorimetry, drug loading, and drug release. Furthermore, anti-leishmanial effect of the preparation was assessed in vitro and in vivo. The prepared ART-loaded niosomes were spherical with an average diameter of about 100 and 300 nm with high encapsulation efficiencies of > 99%. The results of in vitro cytotoxicity revealed that ART-loaded niosomes had significantly higher anti-leishmanial activity, lower general toxicity, and higher selectivity index (SI). Half-maximal inhibitory concentration (IC50) values of ART, ART-loaded niosomes, and liposomal amphotericin B were 39.09, 15.12, and 20 µg/mL, respectively. Also, according to the in vivo study results, ART-loaded niosomes with an average size of 300 nm showed the highest anti-leishmanial effects in animal studies. ART-loaded niosomes would be promising topical drug delivery system for the management of cutaneous leishmaniasis.

Development of New PCR Protocols to Detect Genetic Diversity in the Metronidazole Metabolism Genes in Susceptible and Refractory Clinical Samples of Giardia duodenalis.

PURPOSE: Investigating the genetic variation in thioredoxin reductase (TrxR) and nitroreductase (NR) genes in both treatment-resistant and -sensitive Giardia duodenalis isolates can provide valuable information in identifying potential markers of resistance to metronidazole. The rapid increase in metronidazole treatment failures suggests the presence of genetic resistance mechanisms. By analyzing these genes, researchers can gain insights into the efficacy of metronidazole against G. duodenalis and potentially develop alternative treatment strategies. In this regard, four G. duodenalis isolates (two clinically sensitive and two clinically resistant to metronidazole) were collected from various hospitals of Shiraz, southwestern Iran. METHODS: Parasitological methods including sucrose flotation and microscopy were employed for the primary confirmation of G. duodenalis cysts in stool samples. Microscopy-positive samples were approved by SSU-PCR amplification of the parasite DNA. All four positive G. duodenalis specimens at SSU-PCR were afterward analyzed utilizing designed primers based on important metronidazole metabolism genes including TrxR, NR1, and NR2. RESULTS: Unlike TrxR gene, the results of NR1 and NR2 genes showed that there are non-synonymous variations between sequences of treatment-sensitive and -resistant samples compared to reference sequences. Furthermore, the outcomes of molecular docking revealed that there is an interaction between the protein sequence and spatial shape of treatment-resistant samples and metronidazole in the position of serine amino acid based on the NR1 gene. CONCLUSION: This issue can be one of the possible factors involved in the resistance of Giardia parasites to metronidazole. To reach more accurate results, a large sample size along with simulation and advanced molecular dynamics investigations are needed.

The in vitro and in vivo effects of Lucilia sericata larval secretions on Leishmania major.

The emerging of drug resistant against Leishmania parasites prompts scientists to seek for novel therapeutic strategies against theses infectious protozoan parasites. Among different strategies, the use of larvae secretions could be suggested as a possible therapy with low side effects. Accordingly, the current study evaluated the in vitro and in vivo effects of Lucilia sericata larval secretions on Leishmania major, the causative agent of cutaneous leishmaniasis (CL). After preparation of L. sericata larval stages (L2 and L3) secretions, the potential effects of secretions were evaluated against L. major promastigotes and amastigotes (in vitro) using MTT assay. The cytotoxicity effects of secretions were also checked on uninfected macrophages. In addition, in vivo experiments were also conducted to investigate the effects of larvae's secretions on the CL lesions induced in the BALB/c mice. Although the increased concentration of larvae secretions exhibited a direct effect on the promastigotes proliferation (viability), contrarily, L2 secretions at a concentration of 96 µg/ml represented the highest inhibitory effect on parasite (amastigotes) burden in infected macrophages. Interestingly, L3 secretions > 60 µg/ml induced inhibitory effects on amastigotes. The results relevant to the cytotoxicity effects of L2 and L3 secretions on uninfected-macrophages showed a dose dependent correlation. In vivo results were also significant, compared to the positive control group. This study suggested the plausible inhibitory effects of L. sericata larvae's secretions on the L. major amastigotes and CL lesions progression. It seems that the characterization of all effective components/proteins in the larvae secretions and their specific targets in parasite structure or in cell (macrophage) responses could further reveal more details regarding the anti-leishmanial properties of these compounds.

Molecular diagnosis of Trichomonas vaginalis in liquid-based Papanicolaou samples in Shiraz, southern Iran.

BACKGROUND: Trichomoniasis is a parasitic infection of the urinary and genital tract, caused by Trichomonas vaginalis. This study aimed to investigate the molecular diagnosis of T. vaginalis infection in liquid-based Papanicolaou samples in Shiraz, southern Iran. MATERIALS AND METHODS: In this cross-sectional study, 534 liquid-based Papanicolaou samples were collected from women referring to the laboratory of Motahari Clinic of Shiraz University of Medical Sciences in 2021. Genomic DNA were extracted from the samples and examined for evidence of T. vaginalis using polymerase chain reaction (PCR) using TVK3 and TVK7 specific primers. RESULTS: The mean age of participants was 39.28 ± 9.89 with a maximum age of 65 and a minimum age of 19 years. T. vaginalis DNA fragments were detected in 4.86% (26/534) of the cases. There was significantly higher prevalence in the age groups of 21 to 30 and 41 to 50 years (46.15%, p = 0.001 and 38.46%, p = 0.015, respectively). Furthermore, the results showed an association between a history of foamy discharge and Trichomonas positivity (p = 0.001). CONCLUSION: T. vaginalis infection is common in liquid-based Papanicolaou samples of women who attended regular health check-ups in the study area. Screening for trichomoniasis in populations, particularly if using highly sensitive methods such as PCR, may lead to increased detection and treatment.

Use of Recombinant CP2 and CP23 Antigens of Cryptosporidium parvum for Serodiagnosis of Human Cryptosporidiosis

Background: Cryptosporidium parvum is an important coccidian parasite infecting many mammals, including human. This parasite can manifest as chronic severe diarrhea in immunocompromised individuals, especially those with AIDS. The present study reports the recombinant production of recombinant (r)P2 and rP23 antigens of C. parvum as antigens for detecting human cryptosporidiosis using indirect ELISA tests. Methods: The coding sequences of rP2 and rP23 proteins were codon-optimized, commercially synthesized and sub-cloned in the pET28a expression vector. The expressed proteins were purified by Ni-NTA column chromatography and confirmed by Western blotting. The efficacy of rP2/rP23 proteins for serodiagnosis was evaluated by positive (n = 20) and negative (n = 20) human sera, confirmed by the Ziehl-Neelsen staining as the gold standard test. Results: In ELISA test, the sera from C. parvum-infected patients reacted strongly to rP2/rP23. The sensitivity and specificity related to the diagnostic potential of rP2/rP23 in the ELISA assay were 100%. Conclusion: Our results showed that combination of rP23 and rP2 antigens in ELISA significantly increases the performance of C. parvum serodiagnosis in human cryptosporidiosis.

The level of interleukin-17, 23, and gamma interferon in cutaneous leishmaniasis patients before and after intra lesion treatment.

Leishmaniasis is a zoonotic vector-borne disease that is endemic in tropical and sub-tropical districts. The immune system response is one of the most important factors that has affected parasitic treatment. In this research, the production of IL-17 (Interleukin 17), IL-23 (Interleukin 23), and IFN-ɤ (Interferon-gamma) in peripheral blood mononuclear cells (PBMCs) isolated from patients with cutaneous leishmaniasis caused by L. major before and after treatment were compared to evaluate their roles in the recovery process. For this experimental study, we recruited 23 patients in Iran. Ten milliliters of peripheral blood samples were collected before and after one month of treatment, and PBMCs were isolated. Production of IL-17, IL-23, and IFN-ɤ was assessed by sandwich ELISA technique. The production of IFN-ɤ and IL-17 in patients (before treatment sensitive leishmaniasis and resistance leishmaniasis) was more than the healthy controls (P < 0.05). Moreover, both of the cytokines productions in sensitive leishmaniasis cases were more than the resistance leishmaniasis patients. In this study, we observed lower levels of IL-23 in patients compared to healthy controls. And among the patients, IL-23 production was lower in sensitive leishmaniasis cases (P < 0.05). Conclusion: It appears that the production of IFN-ɤ is necessary for the treatment of leishmaniasis, but further studies are required to address the role of IL-17 and IL-23 in this disease.

Prevalence and molecular characterization of Giardia duodenalis in small ruminants of Shiraz, southwestern Iran: A zoonotic concern.

Livestock are commonly affected by gastrointestinal protozoan parasites, including Giardia duodenalis. In small ruminants, G. duodenalis infection may decrease carcass weight and dressing percentage. Current study was done to determine the prevalence, assemblage distribution, and zoonotic significance of Giardia infection in sheep and goats in Shiraz, southwestern Iran. In total, 200 fecal samples were randomly collected from 100 sheep and 100 goats in 10 farms in Shiraz, southwest of Iran (June 2021-February 2022) and directly examined for G. duodenalis cyst by saline/iodine wet mount examination using a light microscope (400 ×). Positive samples were further genotyped using a nested-PCR and sequencing methods. A mean prevalence of 5.5% (11/200) was estimated for G. duodenalis infection among small ruminants, with 7% and 4% for sheep and goats, respectively. Next, 9 out of 11 positive samples were amplified and only 5 were successfully sequenced at the tpi locus. Our results showed that 80% (4/5) isolates belonged to assemblage E, while only 20% (1/5) were associated with the assemblage A (subtype AI). Of note, 2 E assemblages were isolated from goats and 2 E assemblages plus 1 assemblage A were isolated from sheep. The major finding in the present study was the isolation of assemblage A from sheep in Shiraz, highlighting the zoonotic transmission of Giardia infection in the study area. In general, the information mentioned in the present study is very limited and more extensive studies in this field are needed to achieve more conclusive results.

Molecular Epidemiology, Species Distribution, and Zoonotic Importance of the Neglected Meat-Borne Pathogen Sarcocystis spp. in Cattle (Bos taurus): A Global Systematic Review and Meta-analysis.

BACKGROUND: Sarcocystis species are diverse apicomplexan parasites, though only two zoonotic species (S. hominis and S. heydorni) circulate between cattle and humans. Due to the importance of cattle in the human food chain and to prevent the consequences of parasitosis in humans, the first global systematic review and meta-analysis on molecular epidemiology, species distribution, and zoonotic significance of Sarcocystis infection in cattle was performed. METHODS: For this aim, four international English databases (PubMed, Scopus, Google Scholar, and Web of Science) were systematically searched till 20th September 2021, and random-effect models were drawn to calculate total estimates and their 95% confidence intervals (CIs). RESULTS: Finally, 44 papers from 21 countries were qualified for this review which examined 8526 cattle regarding Sarcocystis infection, rendering a total prevalence of 62.7% (95% CI 53-71.5%). Globally, 12 Sarcocystis spp. have been reported from cattle, including S. cruzi, S. hominis, S. hirsuta, S. rommeli, S. heydorni, S. bovifelis, S. bovini, S. sinensis, S. gigantea, S. fusiformis, S. hjorti and S. tenella. Among them, S. cruzi (37 studies), S. hominis (22 studies) and S. hirsuta (19 studies) were the 3 most common species, with 76.4% (95% CI 64.8-85%), 30.2% (95% CI 19.3-44%) and 8.7% (95% CI 3.8-18.6%), respectively. However, molecular identification was not performed in 48.4% (95% CI 27.3-70.1%) of the positive samples. CONCLUSION: Despite the zoonotic significance of Sarcocystis spp., particularly S. hominis, the epidemiology and distribution of Sarcocystis infection in cattle remains unclear and demands more extensive researches around the world.

Occurrence, genetic characterization, and zoonotic importance of Giardia duodenalis in various species of rodents (Mus musculus, Rattus norvegicus, and Rattus rattus).

Giardia duodenalis is a well-known flagellated parasite and the causative agent of protozoal diarrhea in animals and humans worldwide. Current study was aimed at determination of G. duodenalis prevalence, genetic variation and zoonotic significance in various species of rodents in Shiraz, southwestern Iran. In brief, 120 fecal specimens were collected from rodents (Rattus rattus, Rattus norvegicus, and Mus musculus) during May up to November 2021 and microscopically examined for Giardia cysts. Further molecular characterization of positive samples was done by nested-PCR, followed by nucleotide sequencing of the triose phosphate isomerase (tpi) gene. A total prevalence of 3.3% (4/120) was observed in rodents, with highest rate in black rats [5% (2/40)]. Regarding brown rats and house mice, only one sample was found to be positive, showing 2.5% and 2.5% prevalence, respectively. It is noteworthy that Giardia B and G assemblages were found in black rats (one case/genotype), whereas the only positive samples from brown rats and house mice were characterized as assemblage G. The major findings of the present study were the presence of both zoonotic and non-zoonotic Giardia assemblages in examined rats in Shiraz and the potential of black rats to harbor Giardia infection to humans. These concerns should be taken seriously in terms of public health. Nevertheless, the true epidemiology and assemblage distribution of Giardia is still open to question.

Artemether-loaded nanostructured lipid carriers: preparation, characterization, and evaluation of in vitro effect on Leishmania major.

BACKGROUND AND PURPOSE: Cutaneous leishmaniasis is a global health problem. The discovery of new and highly efficient anti-leishmanial treatments with lower toxicity is globally needed. The current study was carried out to evaluate the anti-leishmanial effects of artemether (ART) and ART-loaded nanostructured lipid carriers (ART-NLCs) against promastigotes and amastigotes of Leishmania major. EXPERIMENTAL APPROACH: Solvent diffusion evaporation technique was applied to prepare ART-NLCs. These nanoparticles were characterized using a particle size analyzer (PSA), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The antiparasitic activity on amastigote was assessed in J774 cell culture. The drug cytotoxicity on promastigote and macrophage was assessed using the MTT technique after 24 and 48 h and compared with NLCs, ART, and amphotericin B, as the control agents. The selectivity index was calculated for the agents. FINDINGS/RESULTS: The DLS and PSA techniques confirmed that ART-NLCs were homogenous in size with an average diameter of 101 ± 2.0 nm and span index of 0.9. The ART-NLCs significantly heighten the anti-leishmanial activity of ART (P < 0.001). The IC50 values of ART and ART-NLCs on promastigotes after 24 and 48 h were 76.08, 36.71 and 35.14, 14.81 µg/mL, respectively while they were calculated 53.97, 25.43 and 20.13, 11.92 for amastigotes. Also, ART-NLCs had the lowest cytotoxicity against macrophages. Furthermore, among the agents tested, ART-NLCs had the highest selectivity index. CONCLUSION AND IMPLICATIONS: ART-NLCs had lower cytotoxic effects than ART and amphotericin B, also its selectivity index was significantly higher. Based on the findings of the study, this formulation could be a promising candidate for further research into leishmaniasis treatment.

In vitro effect of artemether-loaded nanostructured lipid carrier (NLC) on Leishmania infantum.

Visceral leishmaniasis (VL) is an acute and deadly form of leishmaniasis, caused by Leishmania infantum parasite. Due to the toxicity and side effects of conventional treatment options, such as glucantime and other pentavalent drugs, finding novel drugs with fewer adverse effects is required. Artemether (ART), is one of the derivatives of artemisinin, which was shown to be effective in treating malaria and more recently, leishmaniasis. In this fundamental-applied research, we compared the effect of ART and nanostructure loaded with artemether (NLC-ART) on Leishmania infantum promastigotes and amastigotes, at different concentrations (2.5-5-10-25-50-100 µg/ml) using the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay method after 24 and 48 h of treatment. Inhibitory concentration (IC50) values (µg/ml) of promastigote and amastigote of L. infantum to ART/ NLC-ART, after 48 h of treatment, were found to be 37.12 / 32.1 and 16.43 / 15.42, respectively. Moreover, we found that (NLC-ART), had the lowest cytotoxicity against the J774 macrophage cell line. Conclusion: The NLC-ART can be a good candidate for the treatment of visceral leishmaniasis.

Expression of Hexokinase in the Proteome Profile of Leishmania major and Crithidia

Objective: The relationship between drug resistance and the expression of hexokinase (HK) has been indicated in leishmaniasis. According to the prolonged treatment period in cutaneous leishmaniasis (CL) patients co-infected with Crithidia in Iran, this study aims to investigate the expression of HK in the proteome of Leishmania major and Crithidia using a proteomic approach. Methods: A total of 205 samples were removed from the lesions of patients in Fars province, Iran, for the characterization of L. major and Crithidia using polymerase chain reaction (PCR). After protein extraction, two-dimensional gel electrophoresis was employed for protein separation. Several spots were isolated for HK determination in the proteomes of L. major and Crithidia using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS). Results: The PCR results showed 5 positive cases for Crithidia and 96 positive cases for L. major. MALDI TOF/TOF MS indicated HK as a common protein in the proteome of L. major and Crithidia. HK was up-regulated in the Crithidia proteome in comparison with the L. major proteome. Conclusion: Since a relationship between HK expression and drug resistance has been indicated in leishmaniasis, the overexpression of HK in Crithidia might be related to the increased duration of the treatment period in CL patients co-infected with Crithidia.

High Prevalence of Toxoplasma gondii Infection in Type I Diabetic Patients.

BACKGROUND: Type I diabetes (TΙDM) is a genetic or autoimmune disorder, which may be stimulated by induced immune system components due to the underlying infectious diseases. This study was undertaken to find out any possible association between Toxoplasma gondii infection and TIDM. MATERIALS AND METHODS: One hundred and eighty-two blood samples were taken from individuals who were referred to outpatient clinics in Shiraz city, Southern Iran, during a 6-month period. The age of type I diabetic subjects (n = 91) and the control group (n = 91) was identical, which were less than 30 years. The sera were examined for IgG and IgM antibodies by ELISA and correlated with epidemiological factors such as age, sex, and family history of diabetes. RESULTS: Out of 91 diabetic patients, 54 (59.3%) were female and 37 (40.7%) were male. The highest frequency of diabetes belonged to 6-10- and 11-15-year groups (P = 0.17). Toxoplasma infection prevalence in diabetic and control groups was 28.6% and 7.7%, respectively (P = 0.001). A significantly positive family history of diabetes was observed between diabetic patients (31 cases, 34.1%) and the control group (3 cases, 3.3%) (P = 0.01). Interestingly, IgG positivity was seen in 13 cases (41.9%) of patients with positive family history of type I diabetes and 13 cases (21.7%) of subjects with no positive family history of type I diabetes (P = 0.04). CONCLUSION: Our study showed a higher prevalence of Toxoplasma infection in type I diabetes patients. It is likely that the prevalence of TIDM decreases by increasing hygiene and preventing toxoplasmosis.

First molecular subtyping and phylogeny of Blastocystis sp. isolated from domestic and synanthropic animals (dogs, cats and brown rats) in southern Iran.

BACKGROUND: Blastocystis sp. is a common intestinal protist that infects humans and many animals globally. Thus far, 22 subtypes (STs) have been identified in mammalian and avian hosts. Since various STs are common to humans and animals, it was suggested that some human infections might arise from zoonotic transmission. Therefore, the aim of this study was to assess the presence of Blastocystis sp. in domestic (dogs and cats) and synanthropic animals (rats) of Fars Province, Iran, and to genetically characterize the samples. METHODS: A total of 400 fresh faecal samples from 154 dogs, 119 cats, and 127 rats were inspected by direct microscopy, Wheatley's trichrome staining, in vitro culture, and 18S rRNA gene nested-PCR. Finally, sequencing and phylogenetic analyses were performed. RESULTS: Out of 400 samples, 47 (11.8%) and 61 (15.3%) samples were detected as positive by direct wet mount and culture, respectively. Molecular analysis detected a larger number of positive samples (n = 70, 17.5%): nested-PCR showed that 29 (18.8%) dogs, 21 (17.7%) cats, and 20 (15.8%) rats were infected by Blastocystis sp. Sequence analysis of positive samples indicated the presence of zoonotic STs in all investigated host species. Specifically, ST2 (allele 9), ST3 (allele 34), ST4 (allele 94), ST7 (allele 99), ST8 (allele 21), and ST10 (allele 152) were detected in dogs; ST1 (allele 2), ST3 (allele 34), ST4 (allele 94), ST10 (allele 152), and ST14 (allele 159) were detected in cats; and ST1 (allele 2), ST3 (allele 34), and ST4 (allele 92) were detected in rats. CONCLUSIONS: Our data suggest that domestic dogs and cats can serve as possible reservoirs for in-contact humans, especially those who handle shelter-resident and client-owned animals. Moreover, rats as synanthropic animals can function as a potential source of human infections. Conversely, humans can act as a source of infections to animals. These results should be reinforced in future molecular epidemiological studies.

Alarming: high prevalence of Leishmania infantum infection in cats from southern Iran based on molecular and serological methods.

Recently, Leishmania infantum has increasingly been detected in stray cats in endemic regions of the world. Cats have been considered playing a role in the epidemiology of visceral leishmaniosis, an endemic zoonosis in Iran. The studies concerning feline leishmaniosis (FeL) allow the hypothesis that cats can be considered as potential reservoirs. The investigations on Leishmania infection in cats are very few in Iran and therefore we aimed to assess the L. infantum infection in stray cats and its possible role in transmission of the disease to human by direct agglutination test (DAT), ELISA, nested-PCR and confirmation via sequencing and phylogenetic analysis in Fars province, Iran. Whole blood samples were obtained from 174 stray cats. Anti-Leishmania antibodies were detected in the sera using DAT and ELISA. DNA was extracted from the buffy coat of each subject and PCR amplified, targeting Leishmania kDNA gene. PCR results were confirmed by sequence analysis. Prevalence of clinical signs in positive cats was 19.0%. Anti-Leishmania antibodies with different titers were detected in 48 (27.59%) and leishmanial DNA in 36 (20.69%) of the cats. The sequencing of PCR-positive cats revealed the parasite as L. infantum. A high seroprevalence of L. infantum was revealed, with higher levels in males, adult cats, and those living in rural districts and southern zones. Despite the reservoir task of cats in nature is still ambiguous, the high serological and molecular detection of L. infantum in stray cats indicates that cats are regularly bitten by infected sand flies in Fars province, southern Iran, and may have a potential reservoir role in the maintenance of L. infantum in the endemic areas of zoonotic visceral leishmaniosis in Iran. Anyway, Leishmania infection must be appraised in the differential diagnosis of cutaneous or systemic clinical signs in cats.

DNA-based detection of Leishmania and Crithidia species isolated from humans in cutaneous and post-kala-azar dermal leishmaniasis from Shiraz and Kharameh, southern Iran.

BACKGROUND & OBJECTIVES: Leishmania major and L. tropica are the main pathogens of cutaneous leishmaniasis (CL) in several rural and some urban regions of Iran, respectively. The aim of this study was to detect Leishmania species, and update the distribution data of these species in humans suspected to CL in two endemic foci in southern Iran. METHODS: From March 2016 to March 2017, 276 positive samples from of 350 suspected cases were diagnosed and compared by different diagnostic methods, viz. microscopy, culture, and PCR. In PCR assay, four different gene identifications were performed including minicircle kDNA, and cysteine protease B genes for Leishmania detection, and glyceraldehyde-3-phosphate dehydrogenase, and internal transcribed spacer 1 genes for Crithidia detection. RESULTS: In total, 68% (235/350) and 65.3% (177/271) of patients suspected of leishmaniasis were positive by microscopy and cultivation methods. In PCR assay, L. major, and L. tropica were detected in 86.2% (238/276), and 13.1% (36/276) of CL cases, respectively. Also, dermal L. infantum strain was isolated from 0.7% (2/276) of post-kala-azar dermal leishmaniasis patients. In addition, Crithidia fasciculata was detected in two CL patients chronically infected with L. major. INTERPRETATION & CONCLUSION: It appears that the epidemiology of CL has changed during the last decades and can complicate the control strategy aspects of CL in southern Iran. Therefore, more epidemiological, ecological, and gene polymorphism studies are needed to understand the pathogenic role of these species in human, as a main host of leishmaniasis in Iran.

Molecular Identification of Species Caused Cutaneous Leishmaniasis in Southern Zone of Iran.

BACKGROUND: Leishmania major and Leishmania tropica are two main species causing cutaneous leishmaniasis (CL) in Iran. Recently, Crithidia spp. has also been reported in the wound of patients with CL. In this study, we determined the species causing CL in the southern of Iran and the role of Crithidia spp. in creating skin ulcers. METHODS: In this cross-sectional study from Apr to Sep 2016, 66 patients with CL referred to Diagnostic Lab of Leishmaniasis, Valfajr Health Center, Shiraz, Iran, were selected. After DNA extraction from the Giemsa stained smears, all samples were amplified in two separate steps using specific primers, firstly, to differentiate Leishmania species and then to identify Crithidia spp. RESULTS: Two species L. major and L. tropica were responsible for 60 and 6 cases, respectively. Moreover, in two patients, mixed infection with Crithidia was confirmed. In mix infection cases, the morphology of the cutaneous ulcers was not different from the wounds of other patients. CONCLUSION: Leishmania major is responsible for the most common CL in southern Iran. In addition, in two patients with L. major and L. tropica, mix infection with Crithidia was confirmed. The potential role of Crithidia as the main factor for CL and the probability of this parasite to have synergistic effects on Leishmania, as a hypothesis, requires more comprehensive researches on the ambiguity of this protozoon.

Thermotolerant Acanthamoeba spp. isolated from recreational water in Gorgan City, north of Iran.

Acanthamoeba as free-living parasites are scattered ubiquitously, throughout the world. This study was aimed to evaluate the presence of Acanthamoeba spp. genotypes in the recreational water sources in Gorgan County, the capital of Golestan Province using both morphological and molecular approaches. Thirty water samples were collected from different recreational waters in Gorgan, the capital of Golestan Province, northern Iran during 2015-2016. Samples were filtered and followed by culture in non-nutrient agar. Acanthamoeba were identified both by morphological and molecular analysis. The pathogenical potential of positive cloned samples were also determined using tolerance test. Twenty-six percent of recreational water were identified as Acanthamoeba spp. based on the morphological analysis and from these positive samples, five samples were successfully sequenced after molecular studies. Phylogenetic analysis showed the clustering of four samples in T4 genotype group and only one sample as T15 genotype. Thermotolerance test revealed that all cloned samples were highly positive. Since the attractiveness of recreational places for people is increasing, the potential risk of this water should be monitored routinely in each region. More studies are needed to better evaluate the risk of this ubiquitous parasite for the human.

Leishmania cytochrome b gene sequence polymorphisms in southern Iran: relationships with different cutaneous clinical manifestations.

BACKGROUND: Cutaneous leishmaniasis (CL) caused by Leishmania species, is a geographically extensive disease that infects humans and animals. CL is endemic in half of the 31 provinces of Iran, with 29,201 incidence cases reported in Fars province from 2010 to 2015. CL is polymorphic and may result in lesions characterized by different clinical features. Parasite genetic diversity is proposed to be one of the factors affecting the clinical outcome and lesion characteristics in CL patients. However, there is still very limited data regarding the genetic variation of Leishmania spp. based on the sequencing of Cytochrome b (Cyt b) gene. METHODS: All patients originated from endemic regions in Fars province. The amplification of the Cyt b gene from isolates of 100 patients with disparate clinical forms of CL was accomplished using Nested-PCR. Sequence analysis of the amplified Cyt b was used to scrutinize the genetic variations among Leishmania isolates and connect the results with clinical pictures. The clinical demonstrations were basically of two types, typical and atypical lesions. Molecular phylogenetic tree was constructed using the Neighbor-Joining method, with species/strains from this study compared to species/strains from other geographical regions. RESULTS: Leishmania major was identified as the predominant infecting Leishmania spp. (86% of cases), with the remainder of cases being infected by Leishmania tropica. Clinical examination of patients revealed 12 different clinical CL forms. Among Leishmania samples analyzed, five distinct haplotypes were recognized: three in L. major and two in L. tropica. We found a correlation between clinical outcomes and Cyt b sequence variation of Leishmania spp. involved. Moreover, we observed a higher presence of polymorphisms in L. major compared with L. tropica. This difference may be due to the different eco-epidemiologies of both species, with L. tropica being an anthroponosis compared to L. major, which is a zoonosis. CONCLUSIONS: The sequence analysis of Cyt b gene from 25 L. major and L. tropica strains demonstrated genetic variability of L. major and L. tropica causing CL in southern Iran, and a feasible connection amid the genetic heterogeneity of the parasite, geographical source and clinical appearance of the disease in human was detected.