Isolation and Molecular Identification of Vahlkampfiidae and Vermamoeba Vermiformis from Fresh Vegetables: A Neglected Source of Infections

Background: Naegleria spp., Tetramitus spp., and Vermamoeba vermiformis are potential pathogenic free-living amoebae (FLA) causing diseases such as keratitis, meningoencephalitis, and lung infections. We aimed to investigate the presence of Vahlkampfiidae and V. vermiformis in raw vegetables commonly consumed in Iran. Methods: Totally, 70 samples of vegetables samples including watercress (22), leeks (12), parsley (10), basil (13) and mint (13) were collected from municipal markets of Tehran, the capital of Iran during June to October 2021. After washing vegetables, samples were cultivated onto 2% non-nutrient agar (NNA) medium. After morphological confirmations, DNA was extracted and identical fragments of the FLA were amplified and sequenced. Results: Out of 70 cultured samples, 11 samples (15.71 %) were morphologically positive, of which four and seven were V. vermiformis and Vahlkampfiidae isolates, respectively. According to the PCR/sequencing results two, one, one, and one strains belonged to N. australiensis, N. americana, Vahlkampfia sp., V. inornata, and T. aberdonicus, respectively. All Vermamoeba genus were characterized as V. vermiformis. Conclusion: The results of current study revealed the contamination of fresh raw vegetables with Vahlkampfiidae and V. vermiformis. In addition, to our knowledge this is the first report of T. aberdonicus in raw vegetables. Our findings highlight the public health importance of vegetables in transmission of FLA, as well as the potential role of FLA in transmission of potential pathogenic microorganisms via consuming of fresh raw vegetables

Genetic Diversity and Phylogenetic Study of Leishmania Species in Iran by Multilocus Sequence Typing

Background: Leishmaniasis is an important public health parasitic infection, which is endemic in many parts of the world, including Iran. We aimed to investigate genetic diversity and phylogenetic relationship among different Leishmania isolates using multilocus sequence typing (MLST). Methods: Totally, 41 isolates collected either from patients referred to Leishmaniasis Diagnostics and Treatment Center at Tehran University of Medical Sciences, Tehran, Iran or from animals during 2019-2021, were subjected to the study. They included L. major and L. tropica from human, L. infantum from canine, and L. turanica from rodents from different endemic foci of Iran analyzed using MLST including gp63, g6pdh, lack, nagt, and hsp70 genes. Results: A total of 5010 bps was analyzed from each isolate. The three targets, nagt, lack, and g6pdh, generated better topology comparing to the other genes. In the 44 isolates, 22 haplotypes (STs) were identified. Leishmania tropica contained the highest number of haplotypes (n=12) comparing to L. major (n=8), L. infantum (n=1) and L. turanica (n=1). All five genomic loci caused separation of Iranian Leishmania species at the species level, indicating conservation of these genes in the Leishmania parasite. Conclusion: The highest number of haplotypes belonged to L. tropica, indicating that the genetic diversity of this species is higher than that of L. major. It was further confirmed that the MLST is a suitable method to examine genetic variation of Leishmania parasites with respect to evolutionary and epidemiological studies

Can Giardia Infection Impair the Diagnostic Level of Fecal Calprotectin in Patients with Inflammatory Bowel Disease? A Case Report

Inflammatory bowel disease (IBD) is attributed to complex conditions of gastrointestinal tract that is frequently reported all over the world. Fecal calprotectin evaluation is described as a primary tool to screen IBD patients. There are reports showing the confounding role of some microbial agents in diagnostic levels of calprotectin. A 32-yr-old woman with symptoms like IBD/IBS (irritable bowel syndrome); admitted to IBD Clinic of Behbood Research Center for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran, Jan 2017 for evaluation of the level of fecal calprotectin. In spite of high level of calprotectin, trophozoite of Giardia intestinalis was observed in direct examination of stool sample. Microbial pathogens can lead to false elevation of fecal calprotectin and misdiagnosis of gastrointestinal patients suspected to IBD

Molecular Detection and Genotyping of Intestinal Microspor-idia from Stray Dogs in Iran

Background: Microsporidia as one of the most important pathogens in veterinary and agricultural settings, have emerged in immunocompromised patients in Iran. To date, different Enterocytozoon bieneusi genotypes have been identified in humans and animals, supporting the possibility of zoonotic zoonosis transmission potential. The aim of this study was to evaluate the distribution of E. bieneusi genotypes among overpopulated stray dogs in vicinity of Tehran, the capital city of Iran. Methods: Totally, 75 stool and 75 urine samples were obtained from 75 stray dogs during the time period from Mar 2015 to Oct 2015. DNA extraction was performed on all the samples and specific fragment of small subunit ribosomal RNA gene of E. bieneusi and Encephalitozoon spp. was amplified. Furthermore, specific primers targeting the internal transcribed spacer region of E. bieneusi were applied to determine the genotype of the microorganism. Results: Microsporidia was detected in 5.3% of stool samples, while none of the urine samples was positive for microsporidia species. Overall, 440 bp fragment of E. bieneusi was amplified in all the samples and there was no amplification for Encephalitozoon spp. The results of sequencing of 410 bp fragment of internal transcribed spacer region showed that all the E. bieneusi were genotype D. Conclusion: E. bieneusi was the most prevalent microsporidian species in the stray dogs and all the positive isolates were characterized as genotype D

Gastrointestinal Parasites of Domestic Mammalian Hosts in Southeastern Iran

Gastrointestinal parasites (GIP) are a major cause of disease and production loss in livestock. Some have zoonotic potential, so production animals can be a source of human infections. We describe the prevalence of GIP in domestic mammals in Southeastern Iran. Fresh fecal samples (n = 200) collected from cattle (n = 88), sheep (n = 50), goats (n = 23), camels (n = 30), donkeys (n = 5), horse (n = 1), and dogs (n = 3) were subjected to conventional coprological examination for the detection of protozoan (oo)cysts and helminth ova. Overall, 83% (166/200) of the samples were positive for one or more GIP. Helminths were found in dogs, donkeys, sheep (42%), camels (37%), goats (30%), and cattle (19%), but not in the horse. Protozoa were found in cattle (82%), goats (78%), sheep (60%), and camels (13%), but not in donkeys, dogs, or the horse. Lambs were 3.5 times more likely to be infected by protozoa than sheep (OR = 3.5, 95% CI: 1.05-11.66), whereas sheep were at higher odds of being infected by helminths than lambs (OR = 4.09, 95% CI: 1.06-16.59). This is the first study assessing the prevalence of GIP in domestic mammals in Southeastern Iran.This research was funded by the Office of Vice-chancellor for Research of Iranshahr University of Medical Sciences (Grant No. 9900039) and within the scope of the project CICECOAveiro Institute of Materials, UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020, financed by national funds through the FCT/MEC (PIDDAC).S

Mapping 123 million neonatal, infant and child deaths between 2000 and 2017

Since 2000, many countries have achieved considerable success in improving child survival, but localized progress remains unclear. To inform efforts towards United Nations Sustainable Development Goal 3.2—to end preventable child deaths by 2030—we need consistently estimated data at the subnational level regarding child mortality rates and trends. Here we quantified, for the period 2000–2017, the subnational variation in mortality rates and number of deaths of neonates, infants and children under 5 years of age within 99 low- and middle-income countries using a geostatistical survival model. We estimated that 32% of children under 5 in these countries lived in districts that had attained rates of 25 or fewer child deaths per 1,000 live births by 2017, and that 58% of child deaths between 2000 and 2017 in these countries could have been averted in the absence of geographical inequality. This study enables the identification of high-mortality clusters, patterns of progress and geographical inequalities to inform appropriate investments and implementations that will help to improve the health of all populations

Mapping local patterns of childhood overweight and wasting in low- and middle-income countries between 2000 and 2017

A double burden of malnutrition occurs when individuals, household members or communities experience both undernutrition and overweight. Here, we show geospatial estimates of overweight and wasting prevalence among children under 5 years of age in 105 low- and middle-income countries (LMICs) from 2000 to 2017 and aggregate these to policy-relevant administrative units. Wasting decreased overall across LMICs between 2000 and 2017, from 8.4% (62.3 (55.1–70.8) million) to 6.4% (58.3 (47.6–70.7) million), but is predicted to remain above the World Health Organization’s Global Nutrition Target of <5% in over half of LMICs by 2025. Prevalence of overweight increased from 5.2% (30 (22.8–38.5) million) in 2000 to 6.0% (55.5 (44.8–67.9) million) children aged under 5 years in 2017. Areas most affected by double burden of malnutrition were located in Indonesia, Thailand, southeastern China, Botswana, Cameroon and central Nigeria. Our estimates provide a new perspective to researchers, policy makers and public health agencies in their efforts to address this global childhood syndemic

Prevalence of Intestinal Coccidial Infections among Different Groups of Immunocompromised Patients

Background: Cryptosporidium and Isospora are known as one of the main cause of diarrhea in both immunocompetent and immunocompromised subjects, all over the world. Incidence of enteropathogens such as Cryptosporidium spp. and Isospora belli considerably has increased, since immunodeficiency virus (HIV) rapidly disseminated. In addition, cancer patients are highly susceptible to opportunistic infections. This study aimed to estimate the prevalence of cryptosporidiosis and isosporiasis in immunocompromised patients in Tehran. Methods: This study carried out on patients admitted to Imam Khomeini hospital during 2013-2014. Stool samples collected from 350 immunocompromised patients.Formol-ether concentration was performed for all stool samples. Zeil- Neelsen technique was applied to stain the prepared smears and finally, all slides were examined by light microscope. Results: Out of 350 patients, 195 (55.7%) and 155 (44.3%) were male and female,respectively. Cryptosporidium oocysts were detected in 3 (0.9%) samples including one sample from HIV+/AIDS patients and 2 samples from organ transplant recipients.Isospora oocysts were detected in 4 (1.1%) samples consisting 2 HIV+/AIDS patients, one patients suffering from malignancy and one patients with other immunodeficiency diseases. Conclusion: Cryptosporidium sp, and I. belli are the most prevalent gastrointestinal parasitic protozoans that infect a broad range of individuals, particularly those patients who have a suppressed or deficient immunity system

Comparison of Three Staining Methods for the Detection of Intestinal Microspora Spp.

 Background: This study aimed to compare three staining methods including: Calcofluor white, Chromotrope and Quick Hot Gram chromotrope used in diagnosis of intestinal microsporidial spores. Methods: One hundred and seventy five stool specimens were collected from patients referred to Laboratory of Intestinal Protozoology at the School of Public Health, Tehran university of Medical Sciences during 2012-2013. All of specimens were evaluated by nested PCR. The formalin–fixed stool samples were prepared from each specimen and dried at room temperature for 10 min, followed by 10 min methanol fixation. All the collected stoolsamples were evaluated blindly by calcofluor white, Chromotrope and Quick Hot Gram chromotrope staining methods separately. Results : Microsporidial spores were recognized using Chromotrope, Quick Hot Gram chromotrope and Calcofluor white, in16 of 18 (88.8%), 17 of 18 (94.4%) and 18 of18(100%) samples that were positive by nested PCR respectively. Regarding 14 stool samples that were negative by nested PCR, 14 cases were negative bychromotrope and Quick hot Gram chromotrope and 13 samples were negative by Calcofluor white. One discordant sample interpreted as false positive. Conclusion: Calcofluor white staining had the best performance for the detection of intestinal Microsprora spores and can be used as initial screen test for the detection of intestinal Microspora spp

Multigene typing of Giardia Duodenalis isolated from tuberculosis and non-tuberculosis subjects.

Giardia duodenalis is a cryptic protozoan, which has eight assemblages (A-H). Assemblages A and B are the main genotypes reported from humans with probable anthroponotic and zoonotic transmission. The current study aimed to characterize G. duodenalis assemblages in tuberculosis (TB) patients and healthy subjects using multilocus genotyping (MLG). Thirty Giardia-positive stool samples, which were obtained from TB patients and healthy subjects were included in the study. After total DNA extraction, three β-giardin (bg), triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh) genes were amplified and sequenced. Obtained sequences were compared to the GenBank database to characterize assemblages. Phylogenetic analysis using Maximum Likelihood (ML) and Tamura 3-parameter was performed for each gene. From 30 Giardia-positive subjects, 17 (57%) and 13 (43%) were from healthy and TB-infected subjects, respectively. There was no significant co-existence of Giardia and tuberculosis (P-value = 0.051). In addition, 14 (46.7%) and 16 (53.3%) of Giardia isolates were from asymptomatic and symptomatic subjects, respectively. PCR amplification was successful in 25 single samples (83.3%) consisted of 20 for tpi, 15 for bg, and 13 for gdh genes. Accordingly, 13/25 (52%) and 8/25 (32%) belonged to assemblage A and assemblages B, respectively, whereas 4/25 (16%) were either assemblage A or B with different genes at the same time. Significant correlation between assemblages and TB, age, and symptoms was not seen. The phylogenetic analyses represented no separation based on TB and gastrointestinal symptoms. Assemblage A was the predominant genotype in samples. The high frequency of assemblage AII indicated importance of anthroponotic transmission of Giardia in both healthy and TB patients. In addition, considering the exclusive reports of sub-assemblage AIII in wild ruminants, the presence of AIII in the current study have to be carefully interpreted. The inconsistency between the assemblage results of either bg or gdh loci with tpi gene signifies the insufficiency of single gene analysis and the necessity for MLG in molecular epidemiology of G. duodenalis