Entamoeba histolytica and Probable Effect on Production Microsatellite Instability in Colorectal Cancer.

The mortality rate of Entamoeba histolytica is still high and approximately 100,000 per year. Environmental factors and different pathogens can cause microsatellite instability (MSI) positive, which may be one reason for colorectal cancer. MSI status can play an essential role in treatment. Moreover, E. histolytica might be one of the pathogens which raise the incidence of colorectal cancer. Therefore, the probable relationship of E. histolytica with MSI production was evaluated. Four hundred samples of colorectal biopsies based on pathological reports were divided into four groups: colitis, polyps, hyperplasia or dysplasia, and adenocarcinoma. The prevalence of E. histolytica was examined with PCR and immunohistochemical staining (IHC) for the light chain lectin HK-9. The adenocarcinoma formalin-fixed paraffin-embedded colorectal tumours sections were tested for MSI genes. We detected E. histolytica in 6% and 4% of colitis samples by PCR and IHC technique, respectively. However, it did not identify in polyp and hyperplasia samples. The MSI test was examined in the colorectal cancer group, which became positive in 19%. Entamoeba histolytica was detected in 26.3% (5/19) of MSI-positive and 2.5% (2/81) of MSI-negative cases by IHC technique however was not identified by PCR assay in this group. It is concluded PCR and IHC assay is recommended as complementary tests in colitis biopsies. Simultaneous PCR and IHC negative results could confirm the non-existence of the parasite with more confidence. Consequently, E. histolytica might be one of the biotic  factors which raise the incidence of colorectal cancer because of the coincidence of the IHC positive results in MSI-positive adenocarcinoma.

Application of Dirofilaria immitis immunoreactive proteins in serodiagnosis.

Dirofilariasis is a zoonotic global vector-borne disease caused by Dirofilaria immitis. The present study focuses on the somatic and excretory/secretory (E/S) proteins released from adult D. immitis. We aimed to fractionate and identify adult D. immitis immunoreactive proteins. Somatic and E/S extracts were immunoblotted to identify the immunoreactive proteins. In the current study, we used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF/MS) to characterize the immunogenic proteins. Additionally, we used fast protein liquid chromatography (FPLC) to fractionate and evaluate the immunogenicity of the D. immitis secretome. The most immunoreactive proteins were between 10 and 48 kDa. Six proteins including polyprotein antigen, P22u, pepsin inhibitor Dit33, neutrophil chemotactic factor (DiNCF) precursor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat-shock protein 70 (HSP70) were found in both somatic and E/S extracts. Eluting the FPLC column with NaCl resolved two peaks in which the immunoreactivities of the purified proteins were conserved. Characterization of these proteins could provide a novel perspective for understanding the pathogenesis and diagnosing of this disease.

Multiple zoonotic helminth infections in domestic dogs in a rural area of Khuzestan Province in Iran.

BACKGROUND: Echinococcosis and toxocarosis caused by the genus of Echinococcus and Toxocara spp. are among important helminthic diseases worldwide. Limited data on the prevalence of these parasites persuaded us to determine the prevalence of E. granulosus, E. multilocularis, and T. canis infections in domestic dogs in rural areas of Ahvaz, southwestern Iran. Fecal samples from 167 domestic dogs were examined using both microscopy and PCR techniques. Multiplex PCR was performed for the presence of Echinococcus, and Taenia spp. and single PCR for detection of T. canis and Toxascaris leonina. RESULTS: The total occurrence of identified parasites was 65 (38.9%). The microscopic examinations showed that 40 (24%), 18 (10.8%), and four (2.4%) of dogs were infected with taeniid-like, ascarid, and both genera eggs, respectively. Echinococcus granulosus was identified in seven (4.2%), Taenia spp. in 29 (17.4%), and mixed infection with both in 11 (6.6%) samples. Sequencing of PCR-positive samples identified E. granulosus s.s. (G1), 18 T. hydatigena (10.8%), five T. multiceps (3%), three T. serialis (1.8%), one T. ovis (0.6%), one Spirometra erinaceieuropaei voucher (0.6%), and two Mesocestoides corti (1.2%). This is the first report of S. erinaceieuropaei voucher and M. corti in dogs in Iran. Nine (5.4%) and 16 (9.6%) dogs showed infection with T. canis and T. leonina, respectively. Two samples showed coinfection with both ascarids. CONCLUSIONS: Several studies have reported echinococcosis and toxocarosis in intermediate hosts from the southwest of Iran; however, this study is the first molecular research on E. granulosus and T. canis in domestic dogs in a rural area of southwestern Iran. Furthermore, issues of soil contamination with dogs' feces and recent dust storms in Khuzestan may have a role in the spreading of these zoonotic infections to other provinces close to it, and neighboring countries such as Iraq.

Comparative assessment of recombinant and native immunogenic forms of Fasciola hepatica proteins for serodiagnosis of sheep fasciolosis.

Laboratory diagnosis of sheep fasciolosis is commonly performed by coprological examinations; however, this method may lead to false negative results during the acute phase of the infection. Furthermore, the poor sensitivity of coprological methods is considered to be a paradox in the chronic phase of the infection. In this study, we compared the immunoreactivity of native and recombinant forms of Fasciola hepatica excretory/secretory antigens and determined their capabilities for the development of F. hepatica-specific immunoassays. Immunoreactivity and specificity of recombinant and native forms of F. hepatica antigens, including fatty acid binding protein (FABP), glutathione-S-transferase (GST), and cathepsin L-1 (CL1), in parallel with native forms of FABP and GST, were studied for serodiagnosis of the chronic form of sheep fasciolosis, individually or in combination with each other by enzyme-linked immunosorbent assays (ELISA). The correlation of the findings was assessed by receiver-operator characteristic (ROC); furthermore, the specificity and sensitivity were assessed by Youden's J. Serologic cross-reactivity was evaluated using samples from healthy sheep (n = 40), Fasciola-infected sheep (n = 30), and sheep with other parasitic infections (n = 43). The FABPs were determined to be greater than 95% sensitive for F. hepatica serodiagnosis. The most desirable diagnostic recombinant antigen was rCL1, which showed 100% sensitivity and 97% specificity in ELISA and was capable of discriminating the positive and negative samples by maximum Youden's J results. We conclude that rCL1 can be used for routine serodiagnosis of chronic fasciolosis. Thus, it could be advantageous in development of immunoassays for screening of ovine herds in fasciolosis-endemic areas and as a reliable agent for detection of fasciolosis in non-endemic regions.

Serodiagnosis of fasciolosis by fast protein liquid chromatography-fractionated excretory/secretory antigens.

In several studies, different antigenic preparations and diverse immunological tests were applied for serodiagnosis of Fasciola hepatica infections. Most of these preparations showed cross-reactivity with proteins of other parasites. Application of purified antigens might reduce these cross-reactivities. Here, we used fast protein liquid chromatography (FPLC)-fractionated extracts of F. hepatica excretory/secretory antigens (E/S Ags) for serodiagnosis of human and sheep fasciolosis. To develop an improved diagnostic method, we fractionated F. hepatica E/S Ags by anion exchange chromatography on a Sepharose CL-6B column and then tested the serodiagnostic values of the fractions. We used sera from F. hepatica-infected human and sheep as positive controls. Sera from patients with hydatidosis and strongyloidiasis were used for cross-reactivity studies. Enzyme-linked immunosorbent assays (ELISA) of the second FPLC peak, containing 20, 25, and 70 kDa proteins, discriminated between F. hepatica-infected and uninfected human and sheep samples. Fractionation of F. hepatica E/S Ags by FPLC is a fast and reproducible way of obtaining antigens useful for serodiagnosis of human and sheep fasciolosis with acceptable sensitivity and specificity. Graphical abstract ᅟ.

Expression of Heat Shock Protein 70 (HSP70) and Astacin Genes of Strongyloides stercoralis as well as HSP70 and HSP17.1 Genes of S. ratti in Adult and Larval Stages of S. stercoralis.

Background: Uncovering the roles and characteristics of pathogenesis-related molecules can help us develop novel management methods in parasitology. In this study, we studied the expression levels of Strongyloides stercoralis heat shock protein70 (HSP70) (Sst-hsp-70) and astacin (Sst-ast) as pathogenesis-related genes as well as the expression of S. ratti HSP70 and HSP17.1 (Sra-hsp-70, Sra-hsp-17.1) in the larvae and adult stages of S. stercoralis. Methods: A hyperinfection isolate of S. stercoralis from Gilan Province, northern Iran was cultivated on nutrient agar. After a couple of days, parasites in different stages of life were collected, and total RNA was extracted. The expression levels of astacin and HSP genes were compared by real-time PCR. Results: Statistically higher expression levels of Sst-ast, Sst-hsp-70, and Sra-hsp-70 genes in L3 larvae than in adults were observed. However, the expression level of Sra-hsp-17.1 was non-significantly lower in the larval stage than in adult worms. Conclusion: Higher expression levels of Sst-ast, Sst-hsp-70, and Sra-hsp-70 genes in the larval stages of S. stercoralis suggest the potential role of these enzymes in parasite cutaneous invasion and pathogenesis. However, higher expression of Srahsp-17.1 in adult forms is probably involved in resistance and survival mechanisms. The similarity in gene expression between S. stercoralis and S. ratti can provide helpful hints to better understand strongyloidiasis from various perspectives, including pathogenesis, proper diagnosis, and targeted treatment.

Molecular identification of Cryptosporidium, Giardia, and Blastocystis from stray and household cats and cat owners in Tehran, Iran.

Cryptosporidiosis, giardiasis, and blastocystosis are among the most important parasitic diseases common between humans and cats. In addition, there are concerns about the possible transmission of zoonotic parasites from infected cats to humans. Hence, we investigated the molecular epidemiology of Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. in stray and household cats and cat owners. Our study was performed on 132, 33, and 33 fecal samples of stray and household cats, as well as cat owners in Tehran, Iran. Cryptosporidium spp. was identified using a nested PCR targeting the small subunit ribosomal RNA gene (SSU rRNA) and sequencing the internal amplified fragments. Furthermore, to perform multilocus genotyping of G. duodenalis, the ß-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes were amplified to assess the DNA of G. duodenalis in the fecal samples of cats and cat owners. In addition, Blastocystis was detected by targeting the SSU rRNA gene, and the subtypes of Blastocystis were determined via the sequencing of amplicons. Cryptosporidium felis and Cryptosporidium canis were detected in seven stray cats (5.3%) and one household cat (3%). The bg gene of G. duodenalis was amplified and successfully sequenced in two (1.5%) stray cats and revealed assemblages F and B of G. duodenalis. Sequencing and phylogenic analysis of SSU rRNA gene nucleotide sequences of Blastocystis detected ST5 and ST10 in stray cats (1.5%), ST1 in household cats (9.1%), and ST1, ST2, ST3, and ST7 in owners (30.3%). The low prevalence of Cryptosporidium, Giardia and Blastocystis in cats and the presence of species/assemblages/subtypes with limited zoonotic potential indicate that cats had a minor role in their owners' infection in the investigated population. However, the presence of zoonotic protozoa in cats suggests the necessity of special attention to high-risk individuals during close contact with cats. Therefore, it is recommended that veterinarians, physicians, and urban managers plan to prevent, control, or treat these parasites to help the urban community live healthily alongside cats.

A shotgun metagenomic analysis of the fecal microbiome in humans infected with Giardia duodenalis.

BACKGROUND: The mechanisms underlying the clinical outcome disparity during human infection with Giardia duodenalis are still unclear. In recent years, evidence has pointed to the roles of host factors as well as parasite's genetic heterogeneity as major contributing factors in the development of symptomatic human giardiasis. However, it remains contested as to how only a small fraction of individuals infected with G. duodenalis develop clinical gastrointestinal manifestations, whereas the majority of infected individuals remain asymptomatic. Here, we demonstrate that diversity in the fecal microbiome correlates with the clinical outcome of human giardiasis. METHODS: The genetic heterogeneity of G. duodenalis clinical isolates from human subjects with asymptomatic and symptomatic giardiasis was determined using a multilocus analysis approach. We also assessed the genetic proximity of G. duodenalis isolates by constructing phylogenetic trees using the maximum likelihood. Total genomic DNA (gDNA) from fecal specimens was utilized to construct DNA libraries, followed by performing paired-end sequencing using the HiSeq X platform. The Kraken2-generated, filtered FASTQ files were assigned to microbial metabolic pathways and functions using HUMAnN 3.04 and the UniRef90 diamond annotated full reference database (version 201901b). Results from HUMAnN for each sample were evaluated for differences among the biological groups using the Kruskal-Wallis non-parametric test with a post hoc Dunn test. RESULTS: We found that a total of 8/11 (72.73%) human subjects were infected with assemblage A (sub-assemblage AII) of G. duodenalis, whereas 3/11 (27.27%) human subjects in the current study were infected with assemblage B of the parasite. We also found that the parasite's genetic diversity was not associated with the clinical outcome of the infection. Further phylogenetic analysis based on the tpi and gdh loci indicated that those clinical isolates belonging to assemblage A of G. duodenalis subjects clustered compactly together in a monophyletic clade despite being isolated from human subjects with asymptomatic and symptomatic human giardiasis. Using a metagenomic shotgun sequencing approach, we observed that infected individuals with asymptomatic and symptomatic giardiasis represented distinctive microbial diversity profiles, and that both were distinguishable from the profiles of healthy volunteers. CONCLUSIONS: These findings identify a potential association between host microbiome disparity with the development of clinical disease during human giardiasis, and may provide insights into the mechanisms by which the parasite induces pathological changes in the gut. These observations may also lead to the development of novel selective therapeutic targets for preventing human enteric microbial infections.

Multilocus sequence typing of Giardia duodenalis genotypes circulating in humans in a major metropolitan area.

Giardia duodenalis is an intestinal protozoan parasite of humans and animal hosts and comprises eight microscopically indistinguishable molecularly-diverse lineages designated as assemblages A-H. Assemblages A and B are the primary sources of infections in humans and a wide range of mammals. Here, we identified assemblages, and inter-/intra-assemblage genetic diversity of human G. duodenalis isolates based on the multilocus sequence typing of the triosephosphate isomerase (tpi), ß -giardin (bg), and glutamate dehydrogenase (gdh) loci. Multilocus sequence analysis of 62 microscopically-positive G. duodenalis fecal samples identified 26 (41.9%), 27 (43.5%), and nine (14.5%) isolates belonging to assemblages A, B, and discordant assemblages, respectively. The tpi locus assemblage-specific primers identified dual infections with A and B assemblages (45.2%). The sequence analysis of multiple alignments and phylogenetic analysis showed low genetic polymorphism in assemblage A isolates, classified as sub-assemblage AII at three loci, subtype A2 at tpi and gdh loci, and subtype A2 or A3 at bg locus. High genetic variations were found in assemblage B isolates with 14, 15, and 23 nucleotide patterns at tpi, bg, and gdh loci, respectively. Further concatenated sequence analysis revealed four multilocus genotypes (MLG) in 24 assemblages A isolates, two previously-identified (AII-1 and AII-5), with one novel multilocus genotype. However, the high genetic variations observed in assemblage B isolates among and within the three genetic loci prevented the definitive designation of specific MLGs for these isolates. Multilocus sequence typing may provide new insight into the genetic diversity of G. duodenalis isolates in Tehran, suggesting that humans are likely a potential source of G. duodenalis infection. Further host-specific experimental transmission studies are warranted to elucidate the modes of transmission within multiple host populations.

Prevalence and Molecular Characterization of Toxoplasma gondii and Toxocara cati Among Stray and Household Cats and Cat Owners in Tehran, Iran.

Toxoplasma gondii and Toxocara spp. are the most critical parasites common between humans and cats. The close association of cats with humans in urban areas persuaded us to investigate the prevalence of these parasites in stray and household cats and their possible role in the owners' infection. Herein, 132 and 33 fecal samples of stray and household cats, respectively, and 33 blood samples of their owners were collected in Tehran, Iran. The prevalence of T. gondii was determined by targeting the B1 gene in the feces of stray and household cats and the blood of cat owners. Furthermore, genotypes of T. gondii were identified based on the multilocus genotyping of BTUB, GRA6, SAG3, and APICO loci. Toxocara spp. were detected by targeting the second internal transcribed spacer (ITS-2) of the ribosomal DNA of these parasites in the cats' feces and the humans' blood. Also, Toxocara IgG was assessed in the human serum samples. The B1 gene amplification showed that 15.2% of stray cats, 18.2% of household cats, and 51.5% of cat owners were infected with T. gondii. The multilocus sequence analysis revealed the predominance of genotype I of T. gondii in stray cats and genotype II of T. gondii in household cats and cat owners. The amplifying of ITS-2 revealed a high prevalence of T. cati infection (47.0%) in stray cats, whereas no infection was found in the feces of household cats or the serum of cat owners. Likewise, Toxocara IgG was not detected in the serum of humans. The lower prevalence of T. gondii in stray/household cats than in the cat owners indicates the limited impact of close contact with infected cats in human toxoplasmosis. However, the high prevalence of T. cati infection in stray cats can cause contamination of the environment by excreting eggs that may lead to infecting humans through soil or water. Therefore, public health education in urban management planning is necessary for routine urban cat deworming programs and for training the healthcare workers to prevent, control, and treat these infections.

Giardia intestinalis: DNA extraction approaches to improve PCR results.

Difficulty in disrupting cysts of Giardia intestinalis, a cosmopolitan protozoan parasite, decreases the yield of DNA extracted and reduces the effectiveness of the polymerase chain reaction (PCR). To improve the detection of the Giardia Glutamate Dehydrogenase (gdh) gene, we re-evaluated the effects of deoxyribonucleic acid (DNA) extraction methods. Purified and concentrated cysts from 33 fecal samples were disrupted using conventional methods, and DNA extraction was conducted using two protocols: the QIAamp Stool Mini Kit and phenol/chloroform/isoamyl alcohol (PCI). PCR amplification was successful for 12 extracted DNA samples (36%) using PCI following a glass bead and freeze/thaw pretreatment and for all 33 samples (100%) using the QIAamp Stool Mini Kit following the aforementioned pretreatment. Consequently, the pretreatment of cysts with glass beads and freeze/thaw cycles followed by extraction of DNA with the QIAamp Stool Mini kit was the more effective protocol.

Molecular Identification and Genotyping of Babesia canis in Dogs from Meshkin Shahr County, Northwestern Iran.

BACKGROUND: Canine babesiosis is one of the mainly worldwide-distributed tick-borne haemoprotozoan parasitic diseases in dogs. METHODS: A total of 43 blood samples were randomly collected from naturally infected dogs in seven villages from different geographical areas of Meshkin Shahr, Ardabil Province, Iran. The presence of Babesia species detected with standard methods including parasitological and gene sequencing techniques targeting the 18S rRNA gene. RESULTS: Our results revealed that four dogs 9.3% (4/43) including one female and three male dogs were infected with Babesia. All four Babesia-infected dogs were confirmed B. canis by the molecular-based method. Sequence alignments comparison of the B. canis genotypes A and B, it was revealed that all B. canis isolates belonged to genotype B. CONCLUSION: This study provides essential data for subsequently define the critical importance of the molecular studies in management and prevention of the canine babesiosis in Iran.

Restricted genetic heterogeneity of the Plasmodium vivax transmission-blocking vaccine (TBV) candidate Pvs48/45 in a low transmission setting: Implications for the Plasmodium vivax malaria vaccine development.

Plasmodium vivax is the most widespread malaria species parasitizing humans outside Africa, with approximately 100 million cases reported per year. Most human cases of P. vivax are asymptomatic with low parasitemia, making active case detection-based elimination programme challenging and less effective. Despite the widespread distribution of P. vivax, no effective vaccines are currently available. Transmission blocking vaccines have recently emerged as potential vaccine candidates to reduce transmission rates to below the essential levels required for the maintenance of the parasite life cycle. Here, we demonstrated that P. vivax was the predominant species found in a malaria-endemic area, although P. vivax/P. falciparum co-infections were also common. Through genomic sequence analysis and neighbor-joining algorithms, we demonstrated limited genetic heterogeneity in the P. vivax transmission-blocking vaccine candidate Pvs48/45 among clinical isolates of P. vivax. Restricted genetic polymorphism occurred at both nucleotide and amino acid levels. The most frequent mutation was A â†’ G at nucleotide position 77 (46.7%), whereas the least frequent was C â†’ T at nucleotide position 1230 (3.3%). The occurrence of single nucleotide polymorphisms (SNPs) distribution at 6/8 positions (75%) led to changes in amino acid sequences in the Pvs48/45 loci, whereas 2/8 (25%) of SNPs resulted in no amino acid sequence variations. Consistently, the nucleotide diversity in the Pvs48/45 locus among the P. vivax population studied was extremely low (π = 0.000525). Changes in amino acid sequences in the Pvs48/45 protein did not result in substantial conformational modifications in the tertiary structures of these proteins. Unveiling the population genetic structure and genetic heterogeneity of vaccine target antigens are necessary for rational design of transmission-blocking antibody vaccines and to monitor the vaccine efficacy in clinical trials in endemic areas for malaria.

Photodynamic Therapy Using Toluidine Blue O (TBO) Dye as a Photosensitizer against Leishmania major.

BACKGROUND: Photodynamic therapy (PDT) is alternative treatment of cutaneous leishmaniasis (CL), and phenolthiazine dyes such as Toluidine Blue O (TBO) have the potential role in PDT and notably affect parasites inactivation. This study aimed to evaluate the effectiveness of PDT by using TBO and a light-emitting diode (LED) in the treatment of zoonotic CL (ZCL). METHODS: The study was conducted in Iran University of Medical Sciences, Tehran, Iran in 2018-2020. Different concentration (7.8 µg/mL up to 500 µg/mL) of TBO as a photosensitizer and a 630 nm LED light as a source of light were used for antileishmanial activity against both forms of Leishmania major promastigotes and intracellular amastigotes. Effective concentration (EC50) and cell cytotoxicity (CC50) were calculated in both infected and non-infected J774.A1 macrophages, respectively. As well as inhibitory concentration (IC50) was quantified in L. major promastigotes for 2 h, 24 h, and 48 h after incubation using a MTT colorimetric assay. RESULTS: TBO dye in combination with the PDT significantly decreases the L. major promastigotes and intra-cellular amastigotes viability when compared with TBO alone. Both TBO dye in combination with the PDT and TBO alone had no toxic effects on the mice macrophages; however, it significantly killed the entered parasites inside the cells. Our results in the current study established satisfactory findings in clearing intracellular L. major parasites in in-vitro conditions. CONCLUSION: TBO dye in combination with the PDT can be considered as a harmless, effective and importantly perfect treatment against L. major, causative agent of ZCL, in an in-vitro situation without any negative toxicity to the mice macrophages.

The fatty acid-binding protein (FABP) decreases the clinical signs and modulates immune responses in a mouse model of experimental autoimmune encephalomyelitis (EAE).

BACKGROUND: An increasing body of studies has shown that Fasciola hepatica can affect immune responses. This study explored whether the fatty acid-binding protein (FABP) of F. hepatica can modulate the immune system in a mouse model of experimental autoimmune encephalomyelitis (EAE). METHODS: EAE-induced C57BL/6 mice were treated with vehicle, F. hepatica total extract (TE) or FABP. The clinical signs, body weights, and the expression of IFN-γ, T-bet, IL-4, GATA3, IL-17, RORγ, TGF-ß, FOXP3, IL-10, TNF-α genes and proteins were determined in the isolated CD4+ splenocytes. Besides, the percentage of Treg cells and degree of demyelination were evaluated. RESULTS: We found that TE and FABP treatments decreased the clinical scores, lymphocyte infiltration rate, and demyelinated plaques in EAE mice. The expressions of IL-4 and GATA3 were increased, whereas IL-17 and TNF-α were down-regulated. FABP did not affect the expression of IFN-γ, RORγ, IL-10, and TGF-ß genes or proteins but reduced the expression of T-bet. TE administration did not affect the expression of IL-10 and the Tbet genes, and increased the expression levels of IFN-γ and FOXP3 in CD4+ lymphocytes. Both FABP and TE treatment did not affect the Treg cell percentage. CONCLUSION: This study indicates that F. hepatica FABP and TE can suppress the inflammatory responses in EAE-induced mice and shift the immune system toward Th2 responses. However, FABP exerts stronger anti-inflammatory effects and seems to be more effective than TE for EAE treatment.

Morphological Description, Phylogenetic and Molecular Analysis of Dirofilaria immitis Isolated from Dogs in the Northwest of Iran.

BACKGROUND: Dirofilariasis is a globally distributed arthropod-borne parasitic disease of mainly canids and felids. We evaluated to extend the knowledge of morpho-molecular characteristics and outer ultrastructure of Dirofilaria immitis isolated from Northwest of Iran. METHODS: Overall, 67 filarial worms including 41 females and 26 males parasites were collected from the cardiovascular system of the 43 stray dogs in Meshkinshar, Ardebil Province, Northwest of Iran in 2017, and subjected to light and scanning electron microscopy (SEM) as well as carmine alum staining for morpho-molecular and identification. Molecular methods were used for confirmation of morphological findings by sequencing of Cyto-chrome c oxidase subunit I (cox1) gene. RESULTS: The partial DNA sequencing of cox1 gene of adult parasites showed considerable homology and close proximity to the previously isolated from Kerman and Meshkinshahr, Iran. The lowest genetic variation and the highest intra-species variability was found in D. immitis and Dirofilaria repens, respectively. No similarity was identified between D. immitis nucleotide sequence and Wolbachia species as its endosymbiont bacteria. CONCLUSION: The SEM technique is an excellent tool for differential recognition of the parasite surface morphology and molecular techniques could differentiate and identify Dirofilaria spp.

Low prevalence of antifungal resistant Candida africana, in the C. albicans complex causing vulvovaginal candidiasis.

The Candida (C.) albicans complex includes C. albicans, C. dubliniensis, C. stellatoidea, and C. africana, with the last mentioned as an important emerging agent of vulvovaginal candidiasis (VVC). The aim of the study was to identify C. africana and C. dubliniensis and assess their drug susceptibility in vaginitis. One-hundred Candida isolates of the C. albicans complex from women diagnosed with vaginitis and from vaginal samples in the culture collection of a medical mycology laboratory were examined. Species of the C. albicans complex were identified with conventional and molecular methods using polymerase chain reaction (PCR) for amplification and sequencing of the internal transcribed spacer (ITS) region, PCR for partial amplification of hyphal wall protein 1 (HWP1) gene and duplex PCR. The effects of antifungal drugs were evaluated according to standard broth microdilution protocols. Ninety-seven C. albicans (97%) and three C. africana (3%) isolates were identified. Results of susceptibility testing revealed one isolate of C. africana to be resistant to both clotrimazole and fluconazole, and one showed reduced susceptibility to itraconazole. Identification of Candida species especially C. africana in vaginitis is crucial, there are varying levels of resistance to antifungal drugs.

A retrospective study of hydatid cysts in patients undergoing liver and lung surgery in Tehran, Iran.

Hydatid cyst, caused by larval stages of Echinococcus granulosus, is a zoonotic parasitic disease with public health importance. The disease is cosmopolitan and endemic in Iran. We conducted a retrospective study of the records of Milad Hospital, Tehran, Iran to establish the proportion of lung and liver surgical procedures that were performed for removal of hydatid cyst and to investigate the demography of the population undergoing lung and liver hydatid cyst surgery in this hospital. A retrospective cross-sectional study was conducted of records of 682 patients who underwent liver (n = 404) or lung (n = 278) surgery from April 2009 to March 2013. In 404 liver surgeries, 111 (27.5%) diagnoses of hydatid cyst were verified. Liver hydatid infection demonstrated a significant age-related difference (p < 0.05). Cysts were found in 64 of 217 females (29.5%) and 47 of 187 males (25.1%). While in both sexes, more cysts were found in liver, the liver/lung ratio in females was significantly higher than in males (p < 0.001). Hydatid cyst was verified in 59 (21.2%) of 278 lung surgeries: 27 of 105 females (25.7%) and 32 of 173 males (18.5%). There was a significant relationship between sex and organ site (p < 0.001) with the proportion of hydatid cysts in males occurring in lung higher than seen in females. In the five investigated years, approximately 25% of liver and lung surgeries conducted at Milad Hospital were related to hydatidosis. Increasing public awareness of principles of avoiding infection could reduce the risk of nearly a quarter of liver and lung surgeries and costs associated with the treatment of hydatid cysts.

Molecular Detection and Phylogenetic Analysis of Endosymbiont Wolbachia pipientis (Rickettsiales: Anaplasmataceae) Isolated from Dirofilaria immitis in Northwest of Iran.

BACKGROUND: The purpose of this study was molecular detection and phylogenetic analysis of Wolbachia species of Dirofilaria immitis. METHODS: Adult filarial nematodes were collected from the cardiovascular and pulmonary arterial systems of naturally infected dogs, which caught in different geographical areas of Meshkin Shahr in Ardabil Province, Iran, during 2017. Dirofilaria immitis genomic DNA were extracted. Phylogenetic analysis for proofing of D. immitis was carried out using cytochrome oxidase I (COI) gene. Afterward, the purified DNA was used to determine the molecular pattern of the Wolbachia surface protein (WSP) gene sequence by PCR. RESULTS: Phylogeny and homology studies showed high consistency of the COI gene with the previously-registered sequences for D. immitis. Comparison of DNA sequences revealed no nucleotide variation between them. PCR showed that all of the collected parasites were infected with W. pipientis. The sequence of the WSP gene in Wolbachia species from D. immitis was significantly different from other species of Dirofilaria as well as other filarial species. The maximum homology was observed with the Wolbachia isolated from D. immitis. The greatest distance between WSP nucleotides of Wolbachia species found between D. immitis and those isolated from Onchocerca lupi. CONCLUSION: PCR could be a simple but suitable method for detection of Wolbachia species. There is a pattern of host specificity between Wolbachia and Dirofilaria that can be related to ancestral evolutions. The results of this phylogenetic analysis and molecular characterization may help us for better identification of Wolbachia species and understanding of their coevolution.

Detection and genetic characterization of Echinococcus granulosus mitochondrial DNA in serum and formalin-fixed paraffin embedded cyst tissue samples of cystic echinococcosis patients.

Cystic echinococcosis (CE) is a worldwide zoonotic disease caused by the larval stage of Echinococcus granulosus. We investigated the presence of E. granulosus-specific DNA in the serum of CE patients by detecting the cytochrome c oxidase I (cox1) and NADH dehydrogenase subunit I (nad1) mitochondrial genes. Serum and formalin-fixed paraffin embedded (FFPE) cyst tissue samples of 80 CE patients were analyzed. The extracted DNA of samples was submitted to PCR amplification of cox1 and nad1 genes, and products were sequenced and genotyped. Nineteen (23.8%; 95% CI 15.8-34.1) serum and 78 (97.5%; 95% CI 91.3-99.3) FFPE cyst tissue samples were successfully amplified with at least one gene. Echinococcus DNA was detected in the sera of 15.0% (95% CI: 8.8-24.4) and 10.0% (95% CI: 5.2-18.5) and in cyst tissue of 91.3% (95% CI: 83.0-95.7) and 83.8% (95% CI: 74.2-90.3) of 80 patients by cox1 and nad1 gene, respectively. Four genotypes of E. granulosus were distinguished in the CE patients, with predominance of genotype G1, followed by G3, G2, and G6. The finding of E. granulosus DNA in 23.8% of serum samples from CE patients confirmed that E. granulosus releases cell-free DNA into the circulatory system, but quantities may be inadequate for the diagnosis of CE. Genotype G1 predominance suggests the sheep-dog cycle as the primary route of human infection.