Plasmodium falciparum Allelic Diversity: A Comparison of DNA Extraction from Isolates Collected on Rapid Diagnostic Tests (Rdts) and Filter Paper

Background: To perform molecular epidemiologic studies based on large cohorts, material such as RDTs or filter papers are useful for biological sample collection and extraction of RNA or DNA of good quality. Thus, we aimed to assess the quality of DNA extracted from malaria rapid diagnostic tests (RDTs) stored at various temperatures for the analysis of Plasmodium falciparum genetic diversity. Methods: Febrile patients benefitted from free malaria diagnosis using microscopy in a malaria sentinel site, at the Regional Hospital Estuaire-Melen, in Gabon, in 2015. P. falciparum isolates were collected onto one filter paper and 2 similar RDTs devices (Acon®) per patient. Nucleic acids were extracted with QiAmp Qiagen kit from paper and RDTs and the quality of the DNA was analyzed by msp1 gene amplification. Results: Msp1gene amplification was achieved in nucleic acids extracted from all filter papers and RDTs devices (n = 45, 100%). K1 alleles were detected in 93.3% (n = 42/45) of the samples and Mad20 alleles in 73.3% (n = 33/45). The number and the intensity of K1 and/or Mad20 fragments were comparable according to the sample collection material and the storage conditions (room temperature vs -20°C) of the samples. The size of the fragments indicating allelic diversity was comparable in 80% (n=36) of the samples. Conclusion: These data show that RDTs are a valuable source of DNA for malaria parasite genetic polymorphism analysis. Storage conditions of the devices did not influence the quality of DNA extracted from RDTs device, although some alleles may be missed.

Toxoplasma gondii in Milk of Human and Goat from the Desert Area in Central Iran

Background: Toxoplasma gondii, an obligate intracellular protozoan, causes toxoplasmosis. The aim of this study was molecular detection of T. gondii in breast and goat milk samples by the molecular method in the central Iran. Methods: Totally, 300 human' and 200 goats' milk samples were collected randomly from different regions of central Iran in 2018. DNA extraction was performed by the salting-out method. Molecular detection of the parasite was done by nested-PCR using the specific primer pairs. Statistical analysis was performed by SPSS 23 using descriptive and Chi-square tests. Results: Out of 300 human milk samples, 1 sample (0.3%) was infected with T. gondii. Out of 200 samples of goat milk, 11 samples (5.5%) showed infection with T. gondii. The frequency of infection in goat's milk samples was 4.36% in the south and west, 1.9% in northern regions, and 2% in eastern regions of the province. The statistical analysis showed no significant difference between toxoplasmosis and different geographical regions in the province. Conclusion: Because of the popularity of the goat milk and the transfection probability with the milk to humans, it is recommended to boil milk prior to use. Furthermore, case contamination of T. gondii in the human milk sample showed one of the important paths for infection transmission, which requires further studies.

Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples

Background: Toxoplasmosis is a worldwide-distributed infection that can cause serious diseases, mainly in congenitally infected and immunodeficient individuals. PCR assays play an indispensable role in the detection of Toxoplasma gondii in different biological samples. Methods: This study was conducted in the Parasitology Department at Pasteur Institute of Iran (Tehran) during 2016-2018. We designed a highly sensitive quantitative real-time PCR (RT-qPCR) targeted REP-529, a noncoding repetitive DNA. We cloned the amplicon in a plasmid (pTZREP-529) and used it to generate the standard curve. The Toxoplasma RT-qPCR characteristics, i.e., detection limit, specificity, linear dynamic range, linearity, intra-, and inter-assay precisions, were determined. The detection limit of the assay was one plasmid copy number (PCN) per reaction (about 0.004 T. gondii genome), and the linear dynamic range was equal to 6 logs (1× 101 to 1× 107 PCN per reaction). Results: The assay showed no signal when genomic DNA of Plasmodium falciparum, Leishmania major, and Trichomonas vaginallis were used. The standard curve was drawn using dilutions of pTZREP-529 plasmid spiked with genomic DNA from a mouse brain, and test characteristics were shown unaffected. Applying the Toxoplasma RT-qPCR, we showed brain cysts were significantly decreased in mice vaccinated with GRA2 antigen of Toxoplasma formulated in Monophosphoryl Lipid A (MPL) adjuvant. Conclusion: We have developed a quantitative, specific, and highly sensitive PCR for detecting T. gondii in biological samples.

Predisposing Factors to Lymphatic Filariasis among Residents in Igbo-Eze North; an Endemic Area in Nigeria

Background: The study on lymphatic filariasis (LF) in Igbo-Eze North was conducted to determine the prevalence and predisposing factors to LF among its residents between May and October 2018. Methods: A total of 201 residents who have lived in the area for at least one year were recruited. They were stratified according to age, gender and occupation, and were clinically examined firstly by rapid assessment method for any lymphoedema and hydrocele. At recruitment, blood samples were collected from all volunteered participants for LF test. In addition, demographic information and risk factors of the respondents were captured using a structured questionnaire by oral interview. Results: The overall prevalence for LF was 84 (41.8%). Furthermore, LF prevalence was significant (P < 0.05) in all the studied communities: 61.5% in Umuogbuagu, 48.1% in Aguibege, 32.7% in Umuagama and 21.7% in Umuopu. The sex-related prevalence of LF was higher among females than males, with slight significant difference (P = 0.046). In relation to age and occupation, higher prevalences (P < 0.0001) were recorded among older (≥ 50 years) subjects (49, 61.2%) and traders (55, 57.9%) respectively. The risk associated with LF implicated lack of knowledge, non-use of mosquito nets, as well as visit and proximity to water bodies as major predispositions (P < 0.05). Conclusion: The prevalence of LF in this study was high. Higher prevalence was among females, older people and traders. Notable risks to the disease outcome are environmental, attitudinal and occupational with chances of scaled up prevalence and burden overtime.

Wound Myiasis in Diabetic Foot Ulcer: Calliphoridae and Sar-cophagidae Family

Background: Myiasis is a parasitic infestation of tissues or body cavities of mammals with dipterous larvae. The patients with diabetic foot ulcers are more vulnerable to acquiring infestation; however, the infestation may be neglected and mistreated in some cases. Methods: Data were collected of twelve myiasis cases with diabetic foot ulcers in Nazli-Selim Eren Chronic Wound and Infections Care Unit, Aydin, Turkey between 2017 and 2019. Demographic, clinical characteristics of the patients and clinical examination of the wound were recorded. To morphology-based identification method of the agents, the developmental stages of the maggots were examined. Results: The cases aged between 46 and 81 years (10 males, two females). Eight of the larvae collected from wounds had Calliphoridae and four had Sarcophagidae family. The larvae were infested right/left foot sole, thumb, ankle, and mostly left toes. The number of larvae collected from the cases ranged from 2 to 48. Third-stage larvae (L3) were mostly detected. Mixed (L1-L2, L2-L3) larvae were detected in a patient. The infestations were more common in July and August. According to the score of Infectious Diseases Society of America (IDSA), ten (83%) cases had moderate and two (17%) cases were mild diabetic foot infections (DFIs). Conclusion: Diabetic foot ulcers should be evaluated in terms of myiasis. This was the first study in our province indicating that myiasis should not be neglected and different species of flies were responsible for myiasis cases.

Laboratory Evidence of the Presence of Demodex in Urine La-boratory: A Case Report

Folliculorum mites (Demodex folliculorum, and D. brevis) are part of the common external parasites in humans as the exclusive host of them. The highest focus of these mites is on those parts of the body that have fat glands and fatty products in the skin. This is proven by the dermal – epidermal separation method. In the present study, the presence of Demodex is reported in a urine sample containing hematuria, which has not been observed so far according to the previous investigations. The case was related to a 44-yr-old woman with symptoms of headache, chills, and joint pain referring to the medical diagnostic laboratory of Sanandaj, Kurdistan Province, northwest of Iran. After historiography and collecting the urine sample as middle, the live parasite of Demodex was observed. The presence or migration of mite in the atypical areas of the body (genital, urinary, eye, etc.), which are close to hairy tissues (especially in women), may be one of the causes of allergic reactions and clinical symptoms in people.

Chronic Toxoplasma gondii Infection Potentiates Parkinson’s Disease Course in Mice Model

Background: Toxoplasma gondii is a neuroinvasive protozoa pathogen that could manipulate its intermediate host's behavior. However, the possible link between T. gondii infection and the development of neurodegenerative disorders such as Parkinson’s disease (PD) has been proposed, we tested the hypothesis that in chronic toxoplasmosis neuroinflammation, and molecular mediators potentiate behavioral-cognitive impairments in BALB/c mice with PD. Methods: To establish chronic toxoplasmosis by Tehran strain, cysts of T. gondii were injected intraperitoneally into BALB/c mice in Kerman, Iran in 2019. To induce the PD model, mice (BALB/c) were treated with Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The behavioral experiments such as anxiety and motor coordination were performed using the Open field and Rotarod tests. Additionally, we investigated the contribution of Toxoplasma-induced neuroinflammation, and behavioral-cognitive impairments in the PD mice model. Results: Chronic toxoplasmosis caused PD-like symptoms and induced various behavioral changes in infected BALB/c mice. In T. gondii infected+MPTP treated group, T. gondii infection could potentiate PD in infected mice receiving MPTP and caused remarkable dysfunction in motor coordination and change in anxiety and depression-like behaviors similar or more severe than PD group. Conclusion: Chronic T. gondii infection exacerbates pathological progression of PD in BALB/c mice brain by promoting neuroinflammation, and behavioral changes establishing.

Phylogenetic Analysis and Genetics Polymorphisms Evaluation of ROP8 and B1 Genes of Toxoplasma gondii in Livestock and Poul-try Hosts of Yazd, Qom and Golestan Provinces of Iran

Background: A high correlation is observed between specific clonal lineages and host types in toxoplasmosis. The main objectives of this study were comparing polymorphism and evolutionary analysis of the B1 and ROP8 genes, as well as the evaluation of phylogenic and Toxoplasma gondii isolates obtained from different hosts and regions. Methods: Overall 96 brain/ diaphragm tissue samples of livestock and poultry from three provinces of Iran (cows: 9 from Yazd, 9 from Qom; sheep: 19 from Yazd, 7 from Qom; goats: 7 from Yazd, 4 from Qom; one camel from Yazd and 37 chickens, 2 roosters and one duck from Golestan) were tested during 2018-19. A nested PCR and PCR-PCR methods were developed with the B1 and ROP8 genes. Evaluation of genetic proximity, genetic diversity and evolutionary analysis were done using MEGA-X and DnaSP5 software. Thirty samples of both genes were sequenced (18 B1 and 12 ROP8 genes), and submitted to the GenBank (MN275903-MN275932). Results: Tajima's D index analyses showed that both genes were in the negative direction of evolution. The B1 gene was more sensitive than the ROP8 gene. The ROP8 gene showed better and more acceptable results in terms of the relationship between the host and the genotyping of the samples. Conclusion: The B1 gene was only an attractive target for rapid detection of T. gondii parasites, whereas the ROP8 gene due to a high level of polymorphism was able to isolate the three clonal lineages (type I, II and III), inter-types and even atypical strains from different isolates of T. gondii.

Seasonal Prevalence and Novel Multilocus Genotypes of Giardia duodenalis in Yaks (Bos grunniens) in Qinghai Province, Western China

Background: Giardia duodenalis is an important opportunistic zoonotic intestinal protozoon, which could parasitize yaks. However, a few studies have been conducted on the seasonal infection of G. duodenalis in yaks in China. Methods: Overall, 1,027 fecal samples were collected from yaks of two age groups in seven cities of Qinghai Province, China at four seasons between May 2016 and Sep 2017. The prevalence and assemblages were analyzed by nested PCR and multilocus sequence typing (MLST). Results: The overall prevalence of G. duodenalis was 2.04% (21/1027) based on triose phosphate isomease (tpi) locus. No significant differences in prevalence of the organism in yaks were found among different sampling areas. Additionally, same result was also presented in different seasons. However, there was statistically significant difference between young yaks within 6 months (8.33%, 4/48) and adult yaks over 6 months (1.73%, 17/979). The assemblage A recognized as a zoonotic assemblage (n=3) was found in yaks (>6 months) from Xining, while assemblage E (n=18) was detected from yaks in six cities. There were 5, 2 and 3 G. duodenalis subtypes detected positive at the tpi, the β-giardin (bg), and the glutamate dehydrogenase (gdh) loci, with 2, 2 and 3 novel subtypes, respectively. Three samples were successfully sequenced at all three loci, forming 1 assemblages A multilocus genotype (MLG) and 2 assemblages E MLGs, not reported. Conclusion: This study indicated a zoonotic potential of G. duodenalis in yaks from Qinghai Province and provides basic information about the epidemiology of G. duodenalis.

Seroprevalence of Toxocara Infection among Asthmatic Children in Shiraz City, Southern Iran

Background: Human toxocariasis is caused by Toxocara canis and T. cati, the nematodes in the intestine of dogs and cats, respectively. Since the association between asthma and toxocariasis is controversial, the aim of the present study was to investigate the seroprevalence of Toxocara infection among asthmatic children in comparison with healthy children. Methods: This case-control study was conducted on 92 asthmatic and 91 healthy children aged 1-16 years old in Shiraz City, Southern Iran in 2019-2020. The serum samples were tested for IgG anti-Toxocara antibodies by ELISA method using the T. canis larval excretory-secretory (E/S) antigens. The collected data were analyzed using SPSS software. Results: The seroprevalence of toxocariasis in asthmatic patients was higher than the healthy children with no significant difference in Toxocara seropositivity between two groups (9.8% vs 8.8%, P = 0.817). The association between Toxocara infection and variables such as gender and age were not statistically significant. Conclusion: There was no significant association between toxocariasis and childhood asthma. Further study on different regions such as urban and rural areas with a large sample size and using questionnaire for considering risk factors of asthma and toxocariasis is recommended.