Evaluation of Hydatid Cyst Antigen for Serological Diagnosis.

Background: Hydatidosis, a chronic zoonotic disease, has a distribution worldwide and is caused by the larval stage of the Echinococcus helminth. The Dot-ELISA test can diagnose hydatidosis quickly and accurately. Additionally, unlike other hydatid disease tests now used, this quick and affordable enzyme immunoassay is very serum-conservative and antigen-conservative, needing just nanogram levels of parasite antigen. Methods: In the present cross-sectional study, crude and B antigens of hydatid cyst fluid were obtained to diagnose human hydatidosis using CIEP (Counter Immunoelectrophoresis), ELISA (Enzyme-linked Immuno Sorbent assay), and Dot- ELISA (Dot Enzyme linked Immuno Sorbent Assay) methods. Infected liver with a hydatid cyst was collected from Tehran's slaughterhouses to prepare cyst fluid in different stages. After extracting and purifying the Cyst fluid, it is centrifuged at 4ºc, then prepared to concentrate. The study also included sera from hydatidosis (n=60), samples of helminth parasites (n=55), fascioliasis (n=35), toxocariasis (n=20) and negative control (n=35) were tested by CIEP (Counter Immunoelectrophoresis), ELISA (Enzyme-linked Immune Sorbent assay), and Dot- ELISA (Dot Enzyme linked Immuno Sorbent Assay) methods. All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) for Windows release 25.0 (SPSS Inc., Chicago, IL, USA). Results: Crude antigen of hydatid cyst showed a specificity of 76.7%, a sensitivity of 93.3% using the ELISA method, and B antigen showed a specificity of 96.7% and sensitivity of 88.3% using the same method. The crude antigen of the hydatid cyst exhibited a specificity of 68.9% and a sensitivity of 86.7% using CIEP. The B antigen showed a specificity of 87.8% and sensitivity of 83.3% using the same method.The crude antigen of hydatid cyst having serum dilution at 1:800 exhibited a specificity of 83.3% and sensitivity of 100% using the Dot-ELISA method and B antigen having serum dilution at 1:800 serum showed a specificity of 100% and sensitivity of 98.3% using the same method. The results of this finding showed that B antigen has the maximum specificity to diagnose hydatid test using the Dot- ELISA method. Conclusion: Hydatid cysts present with varied symptomatology. History of exposure to infected animals may not be present. A high degree of clinical suspicion combined with meticulous history and clinical examination supported by laboratory investigations are required for its diagnosis. The Dot-ELISA system with native antigen B is a viable approach for the immunodiagnosis of human hydatidosis that is preferred to infection.

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.

Serological Evaluation of Anti-Toxoplasma gondii Antibodies in Patients with Acute Leukemia and Lymphoma through Chemotherapy.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that belongs to the family Coccidae. We aimed to evaluate IgG avidity and the changes of anti-Toxoplasma immunoglobulins M (IgM) and G (IgG) in patients with acute leukemia and lymphoma. METHODS: Ninety eight patients with Acute myeloid leukemia (AML), Acute Lymphoblastic Leukemia (ALL) and lymphoma, selected from patients referring to Imam Reza Hospital of Tabriz (38°04'N 46°18'E), in terms of the presence of anti-Toxoplasma IgM, IgG, IgG avidity antibodies and the major risk factors were evaluated. RESULTS: The results of pre-chemotherapy evaluation showed that of the examined patients, only two cases, one patient with ALL and another patient with lymphoma, had a positive IgM titer. Overall, 46 cases had positive IgG titers, including 20 patients with AML, 15 patients with ALL and 11 patients with lymphoma. Three (3.06%) patients were positive for anti-T. gondii IgM and one of them was with new infection of toxoplasmosis in lymphoma patients. The post-chemotherapy IgG titer evaluation showed 46 [46.9% (95% CI 37.4-56.7)] positive IgG cases that this result was similar to the result of pre-treatment phase. One [1% (95% CI 0.2-5.6)] positive IgG avidity case was detected using ELISA method, in a patient with lymphoma whose IgM was also positive. There was no significant difference between the type of leukemia and the history of contact with cat. CONCLUSION: Performing specialized tests to diagnose toxoplasma infection before starting treatment, in immunodeficiency patients who undergo chemotherapy, is necessary; therefore, these tests should be considered in therapeutic protocols.

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.

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.

Prevalence and Genotyping of Trichomonas vaginalis Infected to dsRNA Virus by PCR-Restriction Fragment Length Polymorphism (RFLP).

BACKGROUND: Trichomonas vaginalis is a prevalent sexually transmitted infection cause trichomoniasis. In this study prevalence and genotype of Iranian isolates of T. vaginalis infected (dsRNA) viruses were evaluated by PCR-RFLP and obtained patterns were then confirmed by sequence analysis and genotype of these Iranian isolates confirmed again. METHODS: Ten strains of T.vaginalis were collected from 1700 vaginal samples of women referred to hospitals associated with Iran University of Medical Sciences in Tehran, Iran during Feb 2016 to Jul 2017, evaluated in points of infection to T. vaginalis Virus (TVV-1) were used in a PCR-RFLP. All of ten isolates of T. vaginalis were examined by designed nested PCR for actin gene and then digestion patterns of three endonuclease enzymes of HindII, MseI and RsaI were evaluated and genotype of these isolates was defined. RESULTS: By combination of fragments pattern of three enzymes of HindII, RsaI and MseI, three genotypes were found; six genotypes E, two genotypes G and two genotypes I. The most dominant genotypes were genotype E. Among four TVV infected isolates two genotype E, one genotype G and one genotype I were found, however among six uninfected T. vaginalis isolates to TVV-1, all of three genotypes were also found. CONCLUSION: Three genotypes E, G and I in T. vaginalis infected with dsRNA isolates were found, however, these three genotypes in T. vaginalis without virus were also observed. Further study is needed to evaluate genotypes of T. vaginalis, which infected virus in more great T. vaginalis population.

Fatty acid and retinol-binding protein: A novel antigen for immunodiagnosis of human strongyloidiasis.

The tenacious human parasitic helminth Strongyloides stercoralis is a significant health problem worldwide. The current lack of a definitive diagnostic laboratory test to rule out this infection necessitates designing more specific diagnostic methods. Fatty acid and retinol-binding protein (FAR) plays a crucial role in the development and reproduction of nematodes. We generated a recombinant form of this protein and determined its applicability for immunodiagnosis of S. stercoralis. The L3 form of S. stercoralis was harvested and used for RNA extraction and cDNA synthesis. The coding sequence of S. stercoralis FAR (SsFAR) was cloned into pET28a(+) vector, expressed in E. coli BL21 and purified. ELISA and immunoblotting were employed to determine the specificity and sensitivity of rSsFAR using a set of defined sera. In addition, we analyzed the phylogenetic relationship of SsFAR with different FAR sequences from other nematodes. The cloned SsFAR had an open reading frame of 447 bp encoding 147 amino acids, with a deduced molecular mass of 19 kD. The SsFAR amino acid sequence was 93% identical to FAR of S. ratti. For differential immunodiagnosis of strongyloidiasis, rSsFAR exhibited 100% sensitivity and 97% specificity. However, cross-reactivity with FAR proteins of other parasites, namely Toxocara canis and Echinococcus granulosus, was noted. Our results provide a novel approach for immunodiagnosis of S. stercoralis infections using rSsFAR with reliable sensitivity and specificity.

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.

Production of recombinant 14-3-3 protein and determination of its immunogenicity for application in serodiagnosis of strongyloidiasis.

BACKGROUND: Strongyloides stercoralis is the fourth most important intestinal nematode worldwide. The parasite load and larvae count are often low, thus conventional methods are not sufficiently sensitive to detect the infection. In this study we developed an immunoglobulin G-based enzyme-linked immunosorbent assay (ELISA) method to detect antibodies against S. stercoralis 14-3-3 protein in patients' sera. METHODS: S. stercoralis RNA was extracted and following complementary DNA synthesis, the 708-bp fragment of 14-3-3 protein was amplified by polymerase chain reaction and cloned into the pET28a+ expression vector. The 30-kDa recombinant 14-3-3 protein was expressed in Escherichia coli BL21 (DE3) cells and purified by affinity chromatography. Finally, its immunoreactivity was assessed by indirect ELISA and western blotting. RESULTS: The S. stercoralis 14-3-3 gene was successfully amplified and cloned into an expression vector. The 30-kDa recombinant protein was purified by affinity chromatography. An ELISA developed in-house detected infected patients' sera with 96% sensitivity. CONCLUSIONS: We concluded that the recombinant 14-3-3 protein has enough sensitivity and specificity for detection of strongyloidiasis in human sera and could be applied for serodiagnosis.

Sero-molecular detection, multi-locus genotyping, and clinical manifestations of ocular toxoplasmosis in patients in northwest Iran.

BACKGROUND: Our goal was to use molecular techniques to verify and characterise clinical diagnoses of ocular toxoplasmosis. Clinical cases were evaluated against IgM and IgG Toxoplasma antibodies, and IgG avidity was tested. B1 gene was assessed for molecular detection, and multi-locus genotyping were conducted to type Toxoplasma infections. METHODS: A cross-sectional study was conducted in 33 patients with suspected active ocular toxoplasmosis. Patients were examined by an ophthalmologist and clinical manifestations were recorded. Toxoplasma gondii IgG and IgM from serum samples were analysed by chemiluminescence immunoassay and ELISA. Acute vs chronic infection was evaluated by IgG avidity testing. Molecular diagnosis of T. gondii infection targeted the B1 gene, and the T. gondii genotype was determined by amplification of the GRA6, SAG2, SAG3, BTUB and APICO loci. The correlation of age, gender, occupation, education, contact with cats or soil, and the consumption of undercooked meat with the incidence of ocular toxoplasmosis was evaluated. RESULTS: Twenty-eight patients (84.8%) were seropositive, two (6%) were both IgG and IgM positive, while one (3%) showed IgG avidity <40%. Molecular testing confirmed toxoplasmosis in 27 patients (81.8%). Chorioretinal scarring (p=0.014) and posterior uveitis (p=0.004) was significantly associated with ocular toxoplasmosis patients. Multi-locus genotyping showed genotype I. Ocular toxoplasmosis showed no significant correlation with gender, age, behaviours, occupation or education. CONCLUSION: Clinical manifestations, serological and molecular detection of Toxoplasma were highly correlated in the diagnosis of ocular toxoplasmosis. Genotype I was predominant in ocular toxoplasmosis in northwest Iran. A larger comparative study should be conducted to provide a broader view of the molecular epidemiology of T. gondii genotypes and its role in toxoplasmosis.

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.

A 53 KDa Glycan Antigen of Hydatid Cyst Wall May Involve in Evasion from Host Immune System.

BACKGROUND: Recent studies have shown that similar host glycan antigens are expressed by helminths such as Echinococcus granulosus hydatid cysts to evade from host immune system. In this work to investigate these antigens further, immunological cross-reactivity between human sera and hydatid cyst wall antigens has been investigated. MATERIALS AND METHODS: Hydatid cyst wall antigens were used in enzyme-linked immunosorbent assay and Western immunoblotting and probed with pooled sera of hydatidosis patients and healthy controls. Sodium metaperiodate treatment was used to investigate glycan antigens. RESULTS: A band with molecular weight about 53 KDa reacted with both hydatid patients' sera and also normal human sera. It has been shown that this band was a glycan antigen. CONCLUSIONS: A 53 KDa glycan antigen of hydatid cyst wall that reacted with all human sera may have an important role for evasion from host immune system.

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.

Antibody response to glycan antigens of hydatid cyst fluid, laminated layer and protoscolex of Echinococcus granulosus.

Background: Hydatid disease is characterized by long-term growth of hydatid cysts in the human. The glycan antigens have an important role in the immunology of hydatid cyst. In this study immunological reaction of host sera to different glycan antigens of the cyst, has been investigated. Methods: The antibody responses were tested to glycoprotein and glycolipid of the laminated layer (LL), cyst fluid (CF) and protoscolex (PS) antigens of E. Granulosus using ELISA and western immunoblotting tests. Thin-layer chromatography and ß-elimination were used for glycan purification. Results: Both hydatid cyst and normal human sera reacted with hydatid cyst fluid, protoscolices, laminated layer, glycoprotein and glycolipid antigens. The most antigen-antibody reaction was related to CF and PS antigens, and LL antigens had the minimal reaction with the sera. Thin layer chromatography (TLC) of the antigens showed presence of many glycan bands in the laminated layer. Conclusion: The parasite may elaborate different glycan antigens in LL to evade host immune response.

Isolation and identification of cutaneous leishmaniasis species by PCR-RFLP in Ilam province, the west of Iran.

Cutaneous leishmaniasis (CL) is one of the most common parasitic diseases and public health problems in Iran. CL is endemic in most parts of Ilam province, in the west of Iran. The distance from the center of country, the great number of divers rural areas, and lack of specialists and laboratory facilities have been the major causes of Leishmania species remaining unknown in this region. Polymerase chain reaction followed by restriction fragment length polymorphism was performed to identify the Leishmania species in 61 patients with cutaneous lesions. Eventually L. major was confirmed as the cause of cutaneous leishmaniasis in Ilam province, the west of Iran.

Seroepidemiology of Toxoplasma gondii infection in pregnant women in west Iran: determined by ELISA and PCR analysis.

Congenital toxoplasmosis can lead to severe damage for the fetus and newborn. Considering that the seroepidemiology of Toxoplasma infection in the pregnant women is poorly studied in west of Iran, the main objective of this study was to estimate the seroprevalence and potential risk factors for congenital toxoplasmosis in Delfan, Iran. In this cross-sectional study, the serum samples obtained from pregnant women who were referred to health centers for routine monitoring of the pregnancy. Totally, 264 sera were screened for IgG and IgM anti-T. gondii antibodies by enzyme linked immunosorbent assay method. All women with IgM anti-T. gondii positive checked by RT-PCR and confirmed. In addition, structured questionnaires were used to obtain information on risk factors for T. gondii infection. Anti-Toxoplasma IgG and IgM were positive in 66 (25 %) and 15 (5.7 %) respectively. Seropositive subjects were more frequently seen in those with >30 years old compared to younger women (<25 years old) (p < 0.001). No significant relationship was found between the seroprevalence of T. gondii infection and level of education, and gestational age (p > 0.05), while there was statistical difference between the infection with cat exposure, consumption of raw/undercooked meat, eating raw or uncooked eggs, consumption of unwashed vegetables and drinking unpasteurized milk (p < 0.001). In the present study, it was found that T. gondii infection was present among pregnant women in west of Iran. Therefore, it is suggested to provide health education for preventing primary infection during pregnancy and subsequently congenital toxoplasmosis in the pregnant women.

Molecular identification and antifungal susceptibility profile of Candida species isolated from patients with vulvovaginitis in Tehran, Iran.

BACKGROUND: Rapid and accurate identification and evaluation of antifungal susceptibility pattern of Candida isolates are crucial to determine suitable antifungal drugs for the treatment of patients with vulvovaginitis candidiasis. MATERIALS AND METHODS: Vaginal samples were collected from 150 women with suspicious vaginal candidiasis, and then cultured on Sabouraoud's Dextrose Agar with chloramphenicol to isolate Candida species. After identification of Candida isolates using polymerase chain reaction-restriction fragment length polymorphism technique, antifungal susceptibility testing of four azolic antifungal drugs was carried out using broth microdilution method according to the CLSI M27-A3. RESULTS: Candida species were isolated from eighty suspected patients (61.79%). The most common pathogen was Candida albicans (63.75%). Resistance to fluconazole and ketoconazole was observed in 27.5% and 23.75% of Candida isolates, respectively, and only 2% of Candida isolates were resistant to miconazole. Interestingly, resistance to fluconazole in C. albicans was more than other Candida species. CONCLUSION: The results indicated that therapy should be selected according to the antifungal susceptibility tests for the prevention of treatment failure and miconazole therapy can be considered as the best therapeutic choice in the management of vulvovaginitis.

Evaluation of anti-Cathepsin L1: a more reliable method for serodiagnosis of human fasciolosis.

BACKGROUND: Coprological examinations are commonly used for diagnosis of fasciolosis. However, these methods are not useful during the acute phase of the infection and also show poor sensitivity during its chronic phase. In this study we compared the immunoreactivity of the native and recombinant forms of Fasciola hepatica excretory/secretory antigens and determined the most appropriate one for development of F. hepatica-specific immunoassays. METHODS: The coding sequences of previously-determined immunogenic proteins including cathepsin L1 (CL1), fatty acid binding protein (FABP) and glutathione-S-transferase (GST) were cloned and expressed in E. coli BL-21 cells. Native forms of FABP and GST were also purified. We evaluated the immunoreactivity of the native and recombinant proteins by ELISA using sera from 40 healthy individuals, 15 fasciolosis patients, and 57 patients with other infectious diseases. RESULTS: All of the studied proteins showed high sensitivity and specificity for F. hepatica serodiagnosis. However, CL1 was more sensitive and specific (100%) than the others for the detection of F. hepatica-specific antibodies. Notably, both FABP and GST showed significant cross-reactivity with hydatidosis patients' sera while CL1 did not. CONCLUSIONS: Cathepsin L1 has acceptable sensitivity and specificity for serodiagnosis of F. hepatica and its application could be advantageous in immunoassay development.

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.

SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples.

Parasitological methods for the diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures. The aim of this study was to detect Leishmania infantum (L. infantum) DNA by real time-PCR method in peripheral blood of symptomatic VL patient and compared its performance with nested PCR, an established molecular method with very high diagnostic indices. 47 parasitologically confirmed VL patients diagnosed by direct agglutination test (DAT > 3200), bone marrow aspiration and presented characteristic clinical features (fever, hepatosplenomegaly, and anemia) and 40 controls (non-endemic healthy control-30, Malaria-2, Toxoplasma gondii-2, Mycobacterium tuberculosis-2, HBV-1, HCV-1, HSV-1 and CMV-1) were enrolled in this study. SYBR-green based real time-PCR and nested PCR was performed to amplify the Kinetoplast DNA minicircle gene using the DNA extracted from Buffy coat. From among 47 patients, 45 (95.7 %) were positive by both nested-PCR and real time-PCR. These results indicate that real time-PCR was not only as sensitive as a nested-PCR assay for detection of Leishmania kDNA in clinical sample, but also more rapid. The advantage of real time-PCR based methods over nested-PCR is simple to perform, more faster in which nested-PCR requires post-PCR processing and reducing contamination risk.