Molecular Identification and Phylogenetic Analysis of Leishmania major Isolated from Zoonotic Cutaneous Leishmaniasis Foci in Northeast Iran.

Background: Epidemiological studies, classification and genetic studies of Leishmania species are effective in treatment, control and prevention in endemic areas. We aimed to investigate the genetic diversity and phylogeny of Leishmania in Zoonotic foci located in northeastern Iran using nagt gene for the first time. Methods: DNA of 100 confirmed positive slides collected from the health centers of Sarkhes, Darghez, Fariman, Esfarayen, and Sabzevar were extracted during 2020-2021. The partial sequence of kDNA was amplified to identify the species. Twenty-five DNA samples were randomly subjected to amplify by nagt gene primes and were sequenced. The sequences were aligned with reference sequences in National Center for Biotechnology Information (NCBI). Then, the genetic similarities of the sequences were checked using Clustalx2.1 software and the phylogenetic tree was drawn by Mega 7 software. Results: All the positive samples were diagnosed as L. major. Approximately, half of the sequences of species were similar to two reference genes JX103550.1:404-712 L. major Esfahan and KX759012.1:568-807 L. Major Ilam (more than 90% similarity). According to the results of the phylogeny tree, the closest genotype to our study samples was JX103550.1:404-712 L. major Esfahan. Conclusion: The most causative agent CL in these areas was L. major. The genetic diversity of L. major was high such as other zoonotic foci in Iran. Due to the high similarity of the strains in the study areas with the strains of Isfahan and Ilam, similar control and prevention methods is suggested in these areas.

Toxoplasmosis Frequency Rate in Rheumatoid Arthritis Patients in Northeastern Iran.

Background: Toxoplasmosis is a zoonotic disease caused by the parasite Toxo-plasma gondii, a cosmopolitan intracellular parasite. It can be a risk factor for auto-immune diseases, including rheumatoid arthritis (RA). This study was designed to investigate the possible association between serological history of T. gondii infection and defined clinical manifestation of RA in Northeast of Iran. Methods: Overall, serum samples were collected from 50 RA patients and 40 healthy controls, from Qaem Hospital in Mashhad City, northeastern Iran in 2018. Seroprevalence of T. gondii infection was determined by ELISA. Results: The prevalence of anti -T. gondii IgG in RA patients 48% (24.50) was significantly higher than the control group 10% (4.40) (P <0.001). Erythrocyte sedimentation rate (ESR), anti-cyclic citrullinated peptide (anti-CCP) and (rheumatoid factor) RF levels between the RA and control groups (P <0.01). Control group were matched with patients for age, gender and living area. Conclusion: Given that a high correlation has been demonstrated between positivity rate of anti-T. gondii IgG and RA in Northeastern Iran, further studies will be necessary to clarify the pathogenesis of T. gondii among these patients.

Impact of a health educational interventional program on reducing the head lice infestation among pupils in an elementary school of a sub-tropical region: a quasi-experimental study.

BACKGROUND: Pediculosis is an important social challenge that can be caused by human head louse, Pediculus humanus capitis. This infestation is cosmopolitan, especially in countries with low hygiene and sanitation. Regular health education classes for students and their parents are required to reduce this infestation in schools and families. This study aimed to evaluate the impact of a health education intervention program on reducing head lice among pupils of an elementary school. METHODS: In a quasi-experimental study, a total of 880 elementary school girls were screened for pediculosis in (2017-2018). The prevalence of pediculosis follow-up continued monthly until the end of the school year after a two months educational intervention course. Visual inspection was applied for initial diagnosis of infection. The suspected cases were confirmed by wood lamp and potassium hydroxide (KOH) microscopic examination. Also eight questions about diagnostic, epidemiology, treatment and prevention were answered in a questionnaire by 50 volunteer parents before and after the training intervention course, to evaluate their general knowledge and measure the effectiveness of learning. All data were statistically analyzed using SPSS software (version 20.0, SPSS, Inc., Chicago, IL, USA). McNemar's test was also applied to investigate the prevalence rate before and after the intervention. A p -value of less than 0.05 was considered as statistically significant. RESULTS: At the beginning of study, pre-intervention prevalence of pediculosis among pupils was 8.4% (49/594). The mean age in all pupils was 9.86 ± 1.83 years old and the most infestation was shown in fourth-grade students with 10 years old. Analysis of statistics demonstrated a significant difference between having infestation and the number of members in the families. Post-intervention phase led to a decreased prevalence of pediculosis in pupils to 3% (8/594) (p-value < 0.05). Based on statistical analysis of questions before and after the training in the questionnaire, a significant increase of parental knowledge was observed on the prevention program of pediculosis (p-value < 0.001). CONCLUSION: The prevalence of pediculosis was significantly reduced following the educational interventions in the school. The applied interventions may be implemented in other residual centers to get rid of this important infestation.

Validation of a mixture of rK26 and rK39 antigens from Iranian strain of Leishmania infantum to detect anti-Leishmania antibodies in human and reservoir hosts.

Mediterranean type of visceral leishmaniasis (VL) is a zoonotic parasitic infection. Some provinces of Iran are endemic for VL while other parts are considered as sporadic areas. This study aimed to assess a combination of recombinant K26 and rK39 antigens as well as crude antigen (CA), derived from an Iranian strain of L. infantum, compared to direct agglutination test (DAT) for the detection of VL in humans and domestic dogs as animal reservoir hosts of the disease. A combination of rK26 and rK39 antigens and also CA was evaluated using indirect ELISA on serum samples of 171 VL confirmed humans (n = 84) and domestic dogs (n = 87) as well as 176 healthy humans (n = 86) and domestic dogs (n = 90). Moreover, 36 serum samples of humans (n = 20) and canines (n = 16) with other potentially infectious diseases were collected and tested for finding cross- reactivity. The results of ELISA were compared to DAT, currently considered as gold standard for the serodiagnosis of VL. The sensitivity and specificity, positive predictive and negative predictive values were calculated compared to DAT. The positive sera had previously shown a positive DAT titer ≥ 1:800 for humans and ≥ 1:80 for dogs. Analysis was done by MedCalc and SPSS softwares. Using the combination of rK26 and rK39 in ELISA, a sensitivity of 95.2% and a specificity of 93.0% % were found in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 98.9% and specificity of 96.7%% were found at a 1:80 (cut-off) titer compared with DAT. A good degree of agreement was found between the combined rK39 and rK26-ELISA with DAT in human (0.882) and dog serum samples (0.955) by kappa analysis (p < 0.05). The ELISA using the CA test showed 75% sensitivity in human and 93.1% in dog serum samples as well as 53.5% specificity in human and 83.3% in dog,s sera, respectively. The combination of rK26 and rK39 recombinant antigen prepared from Iranian strain of Leishmania infantum showed high accuracy for the serodiagnosis of VL in human and domestic dogs. Further extended field trial with a larger sample size is recommended.

Evaluation of zoonotic platyhelminthe infections identified in slaughtered livestock in Iran, 2015-2019.

BACKGROUND: Platyhelminth infections of livestock can result in considerable economic losses. This study aims to evaluate the spatial frequency of cystic echinococcosis (CE), dicrocoeliasis, and fascioliasis in livestock slaughtered in Iran during the years 2015-2019 and estimate direct costs associated with organ condemnation due to these parasites. METHODS: Abattoir data from 413 abattoirs representing all 31 Iranian provinces were collected from the Iran Veterinary Organization. Infection prevalence was calculated per year at the province level. The Local Moran's I statistic was performed to evaluate spatial autocorrelation of animals positive at slaughter for the years 2015-2019. Direct costs associated with condemned livers were calculated for each parasitic condition, with costs associated with condemned lungs also included for CE. RESULTS: Overall prevalence values for the study timeframe were as follows: sheep and goat fascioliasis 1.56% (95% CI: 1.56-1.56%), cattle fascioliasis 3.86% (95% CI: 3.85-3.88%), sheep and goat dicrocoeliasis 4.63% (95% CI: 4.62-4.63%), cattle dicrocoeliasis 3.08% (95% CI: 3.07-3.09%), sheep and goat CE 5.32% (95% CI: 5.32-5.33%), and cattle CE 7.26% (95% CI: 7.24-7.28%). Northwest Iran had the highest prevalence of CE and fascioliasis. High infection areas for Dicrocoelium spp. included the provinces of Zanjan, Gilan, Qazvin, and Tehran, which are located in northern Iran. Direct economic losses for sheep and goat fascioliasis, dicrocoeliasis, and CE for the study period were US$13,842,759, US$41,771,377, and US$22,801,054, respectively. Direct economic losses for cattle fascioliasis, dicrocoeliasis, and CE for the study period were US$1,989,200, US$1,668,986, and US$2,656,568, respectively. CONCLUSION: Our findings provide valuable data for future monitoring of these important parasitic diseases in Iranian livestock. Disease control strategies are required to reduce the economic and public health impact of these platyhelminths.

Diagnosis of Acanthamoeba keratitis in Mashhad, Northeastern Iran: A Gene-Based PCR Assay.

BACKGROUND: The genus Acanthamoeba is a free-living opportunistic protozoan parasite, which widely distributed in soil and fresh water. Acanthamoeba keratitis, which causes a sight-threating infection of the cornea, is going to rise in Iran and worldwide. The aim of this study was to compare direct microscopy, culture and PCR for detection of Acanthamoeba spp. in clinical samples and to determine the genotypes of Acanthamoeba spp. by sequencing 18SrRNA gene. METHODS: Among patients clinically suspected to AK referred to a tertiary ophthalmology center at Mashhad, northeastern Iran. During 2017-18, twenty corneal scrapes specimens obtained. The samples were divided into three parts, subjected to direct microscopic examination, culture onto non-nutrient agar and PCR technique. Sensitivity, specificity, accuracy and likelihood ratio were evaluated. RESULTS: Among 20 persons clinically suspected to amoebic keratitis, 13(69.2%) patients definitely diagnosed as Acanthamoeba keratitis. Wearing contact lens, eye trauma due to foreign particle and swimming in fresh water were the main predisposing factors. Most of patients suffered from pain and photophobia. Corneal ring infiltration and epithelial defect were common clinical sings. Direct examination had the lowest sensitivity and sensitivity of both Nelson-PCR and JDP-PCR methods were equal and highest. In addition, the results of sequencing identified that all strains belonged to T4 genotype. CONCLUSION: Amoebic keratitis is a sporadic parasitic keratitis, which is mainly seen in contact lens user in Mashhad. PCR based on 18S ribosomal DNA with JDP primers is a reliable and highly sensitive method for diagnosis of Acanthamoeba keratitis in clinically suspected cases.

Study of Lophomonas blattarum Infection in Kidney Transplant Patients in Mashhad City, Iran.

BACKGROUND: Lophomonas blattarum is a flagellate protozoan which is known as an emerging parasite in the human respiratory system. Organ transplant recipients are considered as immunocompromised patients due to prescription of immunosuppressive drugs. This group of patients is susceptible to opportunistic infection as well as lophomoniasis. This study aims to investigate the prevalence and clinical manifestation of pulmonary infections caused by L. blattarum in kidney transplant recipients. METHODS: This is a case-control study including 50 kidney transplant recipients and 50 controls. The sputum samples were collected from 50 kidney transplant recipients with bronchopulmonary infection signs suspected to lophomoniasis admitted in Montaserieh and Imam Reza hospitals, Mashhad, Iran. 50 healthy individuals as the control group were matched for sex and age with case ones. The consent form, checklist, and required information were provided for each patient. All samples were microscopically examined for the flagellated protozoan, L. blattarum, using direct smear. RESULTS: Among 50 kidney transplant recipients suspected to lophomoniasis, L. blattarum was identified in sputum samples of 4 (8%) participants of the case group including one female and three males. None of the samples were positive among the control group. Symptoms in patients of this study were high fever (4 out of 4 patients), cough (3 out of 4 patients), and dyspnea (2 out of 4 patients). Three patients showed a positive response to metronidazole treatment. CONCLUSION: The results of this study suggest that L. blattarum should be considered as a pathogenic agent in kidney transplant recipients. It is necessary to examine sputum samples in posttransplant pneumonia patients, especially in those resistant to antibacterial therapy.

The rK39 Antigen from an Iranian Strain of Leishmania infantum: Detection of Anti-Leishmania Antibodies in Humans and Dogs.

BACKGROUND: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis in Iran with high mortality rates in the case of inaccurate diagnosis and treatment. This study aimed to prepare and evaluate a new rk39 recombinant antigen from an Iranian strain of Leishmania infantum for diagnosis of VL in humans and dogs. METHODS: rK39-based enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for the detection of anti L. infantum antibodies. We screened 84 human sera and 87 dog sera from clinical cases in the endemic area of Meshkin-Shahr, Iran along with 176 sera from healthy controls (collected from 86 humans and 90 dogs) during 2013-2016. RESULTS: Using the rK39 ELISA, a sensitivity of 85.7% (95% CI, 95-99%) and a specificity of 86.0% (95% CI, 95%-99%) were detected in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 96.6% (95% CI, 95%-99%) and specificity of 94.4% (95% CI, 95%-99%) were found at a 1:80 (cut-off) titer compared with DAT. Kappa analysis indicated agreement between the rK39 ELISA and DAT (0.718) when using human sera at a 1:800 (cut-off) titer as well as (0.910) at a 1:80 (cut-off) titer when using dog sera (P<0.05). CONCLUSION: New rk39 recombinant antigen from an Iranian strain of Leishmania infantum seems to be used for diagnosis of VL in humans and dogs. Further extended field studies are recommended.

Fish tank granuloma: An emerging skin disease in Iran mimicking Cutaneous Leishmaniasis.

OBJECTIVE: Mycobacterium marinum causes a rare cutaneous disease known as fish tank granuloma (FTG). The disease manifestations resemble those associated with Cutaneous Leishmaniasis (CL). The aim of this study was to determine whether FTG was the cause of cutaneous lesions in patients who were referred to the Parasitology laboratory of Imam Reza Hospital in Mashhad to be investigated for CL. MATERIALS/METHODS: One hundered patients, clinically diagnosed with CL between April 2014 and March 2015, were included in this study. Ziehl-Neelsen staining was performed to identify acid-fast Mycobacterium in addition to bacterial cultures using Löwenstein-Jensen medium. Skin lesion samples were also collected and kept on DNA banking cards for PCR testing. RESULTS: Twenty-nine of the 100 individuals with skin lesions, and therefore suspected of suffering from CL, tested positive for Mycobacterium marinum by PCR. Of these, 21 (72.4%) were male and 8(27.6%) were female. In 97% of these cases the lesions were located on hands and fingers. These patients had a history of manipulating fish and had been in contact with aquarium water. A sporotrichoid appearance was observed in 58.6% of the patients with mycobacterial lesions; 67% of patients had multiple head appearance. CONCLUSION: Patients suspected to have CL and who test negative for CL could be affected by FTG. Therefore, after obtaining an accurate case history, molecular diagnosis is recommended for cases that give a negative result by conventional methods.

Diagnosis of acute toxoplasmosis by IgG avidity method in pregnant women referred to health centers in south-eastern Iran.

Toxoplasmosis, one of the most common parasitic infections, can cause abortions in human. The purpose of this study was to determine seroprevalence of toxoplasmosis and acute form of toxoplasmosis in pregnant women. 208 pregnant women who referred to health centers in south-eastern Iran were taken under study after signing the informed consent forms and filling out the checklists. For those with high levels of IgG or IgM antibody titers, IgG Avidity test was performed to determine the acute infection. High level of IgG anti-body were found in sera of 81 pregnant women and 7 persons had borderline titer. IgM antibody results were positive in 33 and borderline in 4 cases. Then, the samples with positive and borderline results for IgM and IgG were evaluated by IgG avidity test. In this study, approximately 60% of pregnant women were not immune to risks posed by Toxoplasma gondii. Therefore, this group are at risk of acquiring primary infection of toxoplasmosis during gestation.

Urinary Canthariasis Due to Tenebrio molitor Larva in a Ten-Year-Old Boy.

Canthariasis is a human disease caused by infestation of beetle larvae. We report here an unusual cause of urogenital infection due to Tenebrio molitor in a 10-year-old boy suffering from severe and intermittent suprapubic pain from Nehbandan City, Northeastern Iran in 2018. After 9 months, three larvae were excreted. Keratinization of bladder wall was observed in histopathology. All laboratory evaluations were normal except for presence of microscopic hematuria. This report implicated that T. molitor could infest bladder accidentally and cause canthariasis and clinical symptoms that may lead to severe pain and bladder inflammation and hyperemia.

Diagnosis of Acute Toxoplasmosis by IgG and IgM Antibodies and IgG Avidity in Pregnant Women from Mashhad, Eastern Iran.

BACKGROUND: We aimed to evaluate the diagnosis of acute toxoplasmosis by IgG avidity test in pregnant women. METHODS: In this cross-sectional study, 250 blood samples were collected from pregnant women with the first month of their pregnancy referring to health centers of University in Mashhad during 2016. Samples were centrifuged at 3000 rpm for 5 min for separation of serum and were kept in the -20 until use. To detection of acute and chronic toxoplasmosis, anti-Toxoplasma antibodies (IgG and IgM, and IgG avidity tests were performed using ELISA. Then, data analyzed using SPSS software by Frequency, Pearson Chi-Square, Likelihood Ratio, and Exact tests. And P<0.05 was statistically considered as significant. RESULTS: Total prevalence of IgG and IgM was 23.2% and 7.2%, respectively. A significant correlation was observed between the mean age and IgG level (P<0.05). It was not found any correlation between the history of raw meat consumption, cats keeping, education, and residency site. Moreover, 16 people (6.4%) had IgM antibody, of which, 10 cases (62.5%) with low avidity for IgG and 1 people (6.2%) with moderate avidity and 5 cases (31.3%) with high avidity for IgG. Moreover, 76% of pregnant women were seronegative. CONCLUSION: More than half of the women (62.5%) with positive IgM antibody in their serum had a low avidity for IgG which revealed an acute infection among pregnant women. Toxoplasma infection should be considered as an important factor that affects the pregnancy and IgG avidity as an important test for screening the women who need the treatment.

Naegleria species population found in pond water of parks in Mashhad city, Can the physicochemical factors affect it?

Naegleria species are the ubiquitous free-living amoebas that are found worldwide in soil and water. Among Naegleria spp., N. fowleri can cause primary amebic meningoencephalitis (PAM). Ninety water samples were collected from the pond of parks. Also, the water quality parameters were measured at the sampling site (such as temperature, pH, total dissolved solids (TDS), electrical conductivity (EC) and Turbidity). After filtering, the samples were cultured on Bacto-agar enriched with Escherichia coli. A PCR assay was conducted on the culture-positive samples in the ITS1, 5.8SrDNA and ITS2 regions, and then the PCR products were sequenced. The pond water of parks was contaminated with some Naegleria spp. (except N. fowleri) and a Vahlkampfia avara. There was no significant relationship between water quality parameters and the presence of Naegleria (p > 0.05). Our protocol investigates to detect Naegleria spp. from ponds water of parks in Mashhad city and the relations between the water quality parameters and its presence.

Identification of Hymenolepis diminuta Cysticercoid Larvae in Tribolium castaneum (Coleoptera: Tenebrionidae) Beetles from Iran.

BACKGROUND: Hymenolepis diminuta is a cestod of rodents and rarely infects humans. Infection in humans is via ingestion of infected insects. This study was aimed to detect H. diminuta cysticercoids in red flour beetles, Tribolium castaneum, and cockroaches originated from different regions of Iran. METHODS: The red flour beetles and cockroaches were collected from local bakeries in five cities including Tehran, Ahvaz, Kazerun, and Sabzevar during 2010-2011. Some beetles and cockroaches were colonized in insectary and adults from F1 generation were fed on H. diminuta eggs. Both laboratory-infected and field-collected samples were dissected and examined for cysticercoids. Detection of H. diminuta DNA in T. castaneum beetles was performed by targeting a partial sequence of Ribosomal gene. RESULTS: Except the beetles from Ahvaz, all specimens were negative for cysticercoid by microscopy. Of the four dissected beetles from Ahvaz, one harbored 12 cysticercoids. Also, 110 (52%) of laboratory-infected beetles showed infection with an average of 12-14 larvae. None of the cockroaches was infected. Two beetles from Ahvaz, including the remainder of the microscopic positive specimen, yielded the expected amplicon in PCR assay. The H. diminuta DNA sequences generated in this study were identical and matched 97-100% with similar sequences from GenBank database. CONCLUSION: Lack of infection in the majority of beetles may reflect a low rat infestation rate in those areas, alternatively, the examined specimens might not have been the representative samples of the T. castaneum populations.

Cloning of K26 Hydrophilic Antigen from Iranian Strain of Leishmania infantum.

BACKGROUND: Visceral leishmaniasis (VL) caused by Leishmania infantum is the most severe form of leishmaniasis in Iran, which causes a high mortality rate in the case of inaccurate diagnosis and treatment. This study aimed to clone of K26 gene from Iranian strain of L. infantum and register the sequencing results in Genbank to facilitate the preparation a new K26 antigen for the detection of L. infantum infection. METHODS: L. infantum was obtained from an infected domestic dog in Meshkin-Shahr area from northwestern Iran in 2015. Canine visceral leishmaniasis was confirmed by direct agglutination test (DAT), rK39 dipstick and parasitological methods. L. infantum was confirmed by N-acetyl glucosamine -1-phosphate transferase (nagt)-PCR and its sequencing. The band of interest for k26 form Iranian strain of L. infantum was purified by gel extraction kit after PCR amplification and then ligated into pBluescript II SK (+) and pET-32a (+), respectively. The sequences of recombinant plasmids were analyzed and submitted to Genbank. RESULTS: The submission of rk26 nucleotide sequence was performed to the GeneBank/NCBI Data Base under accession number KY212883. The related gene was showed a homology about 99% to L. chagasi and L. infantum k26 gene, while the level of homology in comparison with different strains of L. donovani ranged from 84-94%. CONCLUSION: The successful rk26 cloning into an expression vector performed in this study could help to produce a new recombinant antigen for serodiagnosis of VL especially in areas where L. infantum is the main causative agent.

A Case of Secondary Ophthalmomyiasis Caused by Chrysomya bezziana (Diptera: Calliphoridae).

Myiasis is the invasion of vertebrates' tissue by the larvae of a fly of the order Diptera. The objective of this paper is to present a rare case of secondary myiasis of ocular infestation by Chrysomya bezziana. A 55-year-old female from Sar village of Mazandaran Province, northern Iran), referred to Khatam Al-Anbia Hospital of Mashhad with extensive destruction of left orbital cavity. Existence of larvae was the major complaint in recent months. Some live larvae were removed from her destructed left eye. Primary diagnosis was myiasis of left upper lid (LUL) and suspected recurrent Basal cell carcinoma (BCC). The laboratory diagnosis was done in parasitology lab of Imam Reza Hospital of Mashhad and collected larvae were identified and confirmed to be larvae of the C. bezziana (Diptera: Calliphoridae). It is a case report of secondary ophtalmiomyiasis due to C. bezziana of a patient lives in Mazandaran Province.