RFLP Assay Research Articles

Molecular investigation of Anaplasma phagocytophilum and related strains among sheep flocks from different parts of Türkiye; with a note of phylogenetic analyses of Anaplasma phagocytophilum- like 1

Anaplasma phagocytophilum is a vector-borne zoonotic pathogen and can infect various vertebrate hosts, especially cattle, sheep, goats, horses, and dogs. Molecular-based studies have revealed that the agent has a high genetic diversity and closely related strains circulate in hosts. In this study, 618 sheep blood samples obtained from different geographic regions of Türkiye were researched for A.phagocytophilum and related strains with PCR, RFLP, and DNA sequence analyses. The DNA of these pathogens was detected in 110 (17.79%) samples. RFLP assay showed that all positive samples were infected with A.phagocytophilum-like 1, whereas A.phagocytophilum–like 2 and A.phagocytophilum were not detected. Partial parts of 16 S rRNA gene of seven randomly selected positive samples were sequenced. The phylogenetic analyses of these isolates revealed that at least two A.phagocytophilum-like 1 isolates circulate among hosts in Türkiye and around the world. A.phagocytophilum-related strains have been reported in molecular-based studies over the last few years, but there is a lack of data on the vector competence, epidemiology, clinical symptoms, and genetic diversity of these pathogens. Therefore, large-scale molecular studies are still needed to obtain detailed data on the above-mentioned topics.

MEFV gene pathogenic variants: a risk factor for dysmenorrhea in the Turkish population

Background/Aim The Mediterranean fever (MEFV) gene codes for protein pyrin, which is among the modulators of inflammasome activity in innate immune cells. It was suggested that there is a relation between MEFV variations and inflammatory diseases. The aim of this study was to investigate MEFV gene variations in the patients with primary dysmenorrhea. Methods The prevalence of common MEFV gene variations (M694V, M680I, V726A, E148Q and R202Q) was investigated in 145 young women with primary dysmenorrhea and 135 unrelated healthy controls. MEFV gene variations were genotyped using PCR-based RFLP assay. Results Number of childbirth and marriage were significantly lower in the study group than the controls, respectvely (p < 0.001, p = 0.001). Family history was statistically higher in the patient group (p < 0.001). In total, MEFV genotype and allele frequencies were significantly higher in patients than controls, respectively (p = 0.008 and p = 0.005, respectively). It was found that MEFV gene E148Q allele was more common in patient group (p = 0.039). MEFV R202Q A allele was higher in the patients than the controls (p = 0.045). A significant association was observed when the patients were compared with the controls according to R202Q variant AA versus GG+GA genotypes (p=0.020). Conclusion Our findings suggest that MEFV variations may be a risk factor for patients with dysmenorrhea in a Turkish cohort. Highlights There are very few studies in the literature regarding the relationship between pathological variants of MEFV and dysmenorrhea disease. The common MEFV mutations/variants were evaluated in primary dysmenorrhea patients. Family history was statistically higher in the patient group (p <.001). MEFV gene variations were found 52 (35.9%) in patients and 29 (21.5%2) in controls. MEFV gene allele frequency was significantly higher in-patient group than control (p =.005).

Species composition, infection rate and detection of resistant alleles in Anopheles funestus (Diptera: Culicidae) from Lare, a malaria hotspot district of Ethiopia

BackgroundAnopheles funestus, which is considered as secondary vector of malaria in Ethiopia, is known to have several morphologically indistinguishable (sibling) species. Accurate identification of sibling species is crucial to understand their biology, behaviour and vector competence. In this study, molecular identification was conducted on the Ethiopian An. funestus populations. Moreover, insecticide resistance mechanism markers were detected, including ace N485I, kdr L1014F, L1014S, and CYP6P9a TaqMan qPCR was used to detect the infective stage of the parasite from field collected adult female An. funestus populations.MethodsAdult female mosquito collection was conducted from Lare, Gambella Regional State of Ethiopia between June 2018 to July 2020 using CDC light traps and HLC. Sub-samples of the morphologically identified An. funestus mosquitoes were molecularly identified using species-specific PCR, and the possible presence of insecticide resistance alleles was investigated using TaqMan qPCR (N485I-Ace-1), PCR-Sanger sequencing (L1014F-kdr), and PCR–RFLP (CYP6P9a resistance allele). Following head/thorax dissection, the TaqMan qPCR assay was used to investigate the presence of the infective stage Plasmodium parasite species.ResultsA total of 1086 adult female An. funestus mosquitoes were collected during the study period. All sub-samples (N = 20) that were morphologically identified as An. funestus sensu lato (s.l.) were identified as An. funestus sensu stricto (s.s.) using species- specific PCR assay. The PCR–RFLP assay that detects the CYP6P9a resistance allele that confers pyrethroid resistance in An. funestus was applied in N = 30 randomly selected An. funestus s.l. specimens. None of the specimens showed a digestion pattern consistent with the presence of the CYP6P9a resistance allele in contrast to what was observed in the positive control. Consequently, all samples were characterized as wild type. The qPCR TaqMan assay that detects the N485I acetylcholinesterase-1 mutation conferring resistance to organophosphates/carbamates in An. funestus was used in (N = 144) samples. All samples were characterized as wild type. The kdr L1014F and L1014S mutations in the VGSC gene that confer resistance to pyrethroids and DDT were analysed with direct Sanger sequencing after PCR and clean-up of the PCR products were also characterized as wild type. None of the samples (N = 169) were found positive for Plasmodium (P. falciparum/ovale/malariae/vivax) detection.ConclusionAll An. funestus s.l. samples from Lare were molecularly identified as An. funestus s.s. No CYP6P9, N485I acetylcholinesterase 1, kdr L1014F or L1014S mutations were detected in the An. funestus samples. None of the An. funestus samples were positive for Plasmodium. Although the current study did not detect any insecticide resistant mechanism, it provides a reference for future vector monitoring programmes. Regular monitoring of resistance mechanisms covering wider geographical areas of Ethiopia where this vector is distributed is important for improving the efficacy of vector control programs.

Genetic association of vitamin D receptor gene with female infertility

Background Infertility is defined as failure to achieve a clinical pregnancy after 12 months of unprotected intercourse. It affects 15% of couples globally and 22% of couples within Pakistan. Female infertility can be caused by numerous genetic or environmental factors including hormone imbalances and exposure to chemicals or radiation. The prevalence of vitamin D deficiency among the adult population was reported to be 14–59% with a higher prevalence in Asian countries. Furthermore, the expression of Vitamin D receptor (VDR) can play a vital role in the reproductive organs of females. Hence, the aim of our present study was to check the association of VDR polymorphisms with infertile females. For this purpose, blood samples were collected for genotyping of four known VDR mutations [FokI (rs2228570), TaqI (rs731236), ApaI (rs7975232), and BsmI (rs1544410)] via PCR-based RFLP assay. Results Genotyping indicated that FokI, TaqI, and ApaI are associated with infertility (p = 0.004*, p = 0.013*, and p = 0.033*, respectively). However, BsmI did not show any significance. Multinomial regression analysis indicated that FokI heterozygous genotypes increase the risk of infertility by 2.5 times (hetero: OR = 2.5, 95%, p = 0.001*) as compared to wild type. Heterozygous genotypes of TaqI and ApaI were found to play a protective role and reduce the risk of infertility by 58 and 52%, respectively [TaqI: OR = 0.42, 95%, p = 0.004*, ApaI: OR = 0.48, 95%, p = 0.01*, respectively] as compared to wild type. Multinomial logistic regression analysis was also performed for allelic data as well. Conclusion Thus, it could be summarized that among the studied polymorphisms of VDR, FokI SNP greatly increased the risk of infertility, while TaqI and ApaI genotypes protect from infertility. However, BsmI does not influence the risk of infertility in Pakistani females.

Vaccinia virus induces endoplasmic reticulum stress and activates unfolded protein responses through the ATF6α transcription factor

BackgroundCell responses to different stress inducers are efficient mechanisms that prevent and fight the accumulation of harmful macromolecules in the cells and also reinforce the defenses of the host against pathogens. Vaccinia virus (VACV) is an enveloped, DNA virus, belonging to the Poxviridae family. Members of this family have evolved numerous strategies to manipulate host responses to stress controlling cell survival and enhancing their replicative success. In this study, we investigated the activation of the response signaling to malformed proteins (UPR) by the VACV virulent strain—Western Reserve (WR)—or the non-virulent strain—Modified Vaccinia Ankara (MVA).MethodsThrough RT-PCR RFLP and qPCR assays, we detected negative regulation of XBP1 mRNA processing in VACV-infected cells. On the other hand, through assays of reporter genes for the ATF6 component, we observed its translocation to the nucleus of infected cells and a robust increase in its transcriptional activity, which seems to be important for virus replication. WR strain single-cycle viral multiplication curves in ATF6α-knockout MEFs showed reduced viral yield.ResultsWe observed that VACV WR and MVA strains modulate the UPR pathway, triggering the expression of endoplasmic reticulum chaperones through ATF6α signaling while preventing IRE1α-XBP1 activation.ConclusionsThe ATF6α sensor is robustly activated during infection while the IRE1α-XBP1 branch is down-regulated.

PCR–RFLP assays for the identification of Anopheles (Diptera: Culicidae) species circulating in Honduras

BackgroundVector populations are a key target for malaria control and elimination. In Honduras, there are at least 12 reported anopheline species, however, the definitive number of species remains uncertain. Due to the inherent limitations of morphological identification of Anopheles species, molecular approaches have been developed to provide accurate identification and robust surveillance of local malaria vectors. The aim of this study was to design and assess three PCR–RFLP assays to identify anopheline species known to presently occur in Honduras.MethodsMosquitoes captured between 2018 and 2022 in seven malaria-endemic and non-endemic departments in Honduras were analysed. The ITS2 ribosomal region and three restriction enzyme-based assays were evaluated in silico and experimentally.ResultsA total of 132 sequences from 12 anopheline species were analysed. The ITS2 marker showed length polymorphisms that generated products between 388 and 592 bp and no relevant intraspecies polymorphisms were found. Furthermore, the three PCR–RFLP assays were able to differentiate 11 species with sufficient precision and resolution.ConclusionThe ITS2 region was shown to be a useful molecular marker for identifying local Anopheles species. In addition, the PCR–RFLP assays evaluated here proved to be capable of discriminating most of the anopheline species present in Honduras. These methods provide alternatives to improve entomological surveillance of Anopheles in Honduras and other Mesoamerican countries.

The Association of Angiotensin-Converting Enzyme 2 Polymorphism and SARS-CoV-2 Infection in the Iraqi Population.

The quick and advancing prevalence of the new coronavirus SARS-CoV-2 produced a global crisis surge with a profound impact on human health and worldwide economic constancy. The virus is known as one strain of coronavirus, which causes the respiratory infection responsible for the current pandemic of COVID-19. The virus spike protein has a high binding affinity to human ACE2, depending on crystallization analysis and biochemical interaction studies. Studies consistently reveal that rs2285666, a polymorphism found in ACE2, diverse significantly between Europeans and Asians, changing ACE2 expression. The alternating allele TT of rs2285666 SNP increased gene expression to 50%; thus, it may have a role in SARS-COV-2 infection vulnerability. This study aimed to investigate rs2285666 SNP association with SARS-CoV2 infection as a first report in the Iraqi population. Fifty (20 Male/30 Female) Covid-19 patients with severe symptoms with mean age (of 41.5±10.7) and 50 (20 Male/30 Female) healthy people as a control group with mean age (of 41.5±10.7) were included in this study. Sample of a patient tested as a mutant genotype (TT) by RFLP assay. The results reveal a MAF value of 0.3 for this gene in Iraqi samples, more than Europeans (0.2) and less than East Asians (0.55). The codominant model had significant OR of both alleles CT and TT (OR=4.26 & 6.7; P-value=0.012 & 0.023 respectively). In conclusion, there is an association between increased severity of SARS-Cov-2 infection and rs2285666 polymorphism of the codominant genotype model of the Iraqi population. However, several other factors may affect disease severity, such as ethnic group differences, sex, comorbidity, virus strain, and others.

Detection of Leishmania donovani using ITS1-RFLP from positive and negative smear samples among clinically reported patients visiting University of Gondar Comprehensive Specialized Hospital

BackgroundVisceral leishmaniasis is caused by the Leishmania donovani species complex that can spread to internal organs and leading to death if not treated on time. Diagnosis of leishmaniasis is based on clinical signs and symptoms, microscopy, serological and molecular techniques. Because of a broad spectrum of diverse clinical manifestations and similarities of the responses to different species, identification to the species level is often difficult for the proper patient treatment and management. Therefore, the objective of this study was to evaluate the PCR- RFLP assay of the ITS1 region for identification of L. donovani species from clinical smear slide patient samples.MethodDNA extraction was performed on a total of 90 smear slide samples using phenol—chloroform method. The PCR detection limit was determined by L. donovani reference strain DNA. The ITS1 region was amplified at 320 bp using LITSR/L5.8S genus specific primers and then the ITS1-PCR products were subjected to RFLP assay for confirmation of L. donovani species using HaeIII restriction enzyme.ResultsOf the total samples ITS1-PCR revealed the true positive, false positive, true negative, and false negative results of 42 (46.7%), 6 (6.7%), 37 (41.1%) and 5 (5.6%), respectively. Considering microscopy as the gold standard, the sensitivity, specificity, positive predictive values, and negative predictive values of the ITS1- PCR technique was 89.4%, 86.0%, 87.5%, and 88.1% respectively. All ITS1-PCR positive clinical samples were confirmed as L. donovani species by PCR–RFLP patterns.ConclusionIn conclusion, the ITS1- RFLP method is highly sensitive and more specific for identification of L. donovani species in the smear negative clinical samples of visceral leishmaniasis patients. There is also significant association and degree of agreement between the two methods. For direct identification of L. donovani species from clinical samples, irrespective of genus and species level, PCR–RFLP is more recommendable than a microscope.

Allelic Variation in Protein Tyrosine Phosphatase Receptor Type-C in Cattle Influences Erythrocyte, Leukocyte and Humoral Responses to Infestation With the Cattle Tick Rhipicephalus australis.

The protein tyrosine phosphatase receptor type-C (PTPRC) gene encodes the common leukocyte antigen (CD45) receptor. CD45 affects cell adhesion, migration, cytokine signalling, cell development, and activation state. Four families of the gene have been identified in cattle: a taurine group (Family 1), two indicine groups (Families 2 and 4) and an African “taurindicine” group (Family 3). Host resistance in cattle to infestation with ticks is moderately heritable and primarily manifests as prevention of attachment and feeding by larvae. This study was conducted to describe the effects of PTPRC genotype on immune-response phenotypes in cattle that display a variable immune responsiveness to ticks. Thirty tick-naïve Santa-Gertrudis cattle (a stabilized composite of 5/8 taurine and 3/8 indicine) were artificially infested with ticks weekly for 13 weeks and ranked according to their tick counts. Blood samples were taken from control and tick-challenged cattle immediately before, then at 21 d after infestation and each subsequent week for 9 weeks. Assays included erythrocyte profiles, white blood cell counts, the percentage of cellular subsets comprising the peripheral blood mononuclear cell (PBMC) population, and the ability of PBMC to recognize and proliferate in response to stimulation with tick antigens in vitro. The cattle were PTPRC genotyped using a RFLP assay that differentiated Family 1 and 3 together (220 bp), from Family 2 (462 bp), and from Family 4 (486 bp). The PTPRC allele frequencies were Family 1/3 = 0.34; Family 2 = 0.47; Family 4 = 0.19. There was no significant association between PTPRC genotype and tick count. Each copy of the Family 1/3 allele significantly decreased total leucocyte count (WCC) and CD8+ cells. Increasing dosage of Family 2 alleles significantly increased red blood cell count (RCC), haematocrit (PCV), and haemoglobin (Hb) concentration in blood. Increasing dosage of the Family 4 allele was associated with increased WCC, reduced RCC, reduced PCV and reduced Hb. Homozygote Family 1/3 animals had consistently lower IgG1 in response to tick Ag than homozygote Family 2 animals. The PTPRC genotype influences the bovine immune response to ticks but was not associated with the observed variation in resistance to tick infestation in this study.

PCR–RFLP and species-specific PCR efficiency for the identification of adulteries in meat and meat products

The traceability of meat origin has become a necessity to safeguard consumer's confidence in commercial meat products. Our study recommends a meat species detection using qualitative approach based on PCR–RFLP and species-specific primers PCR. Here, we targeted a 359 bp fragment of the mitochondrial cytochrome b gene amplified by PCR using universal primers followed by three enzymatic digestions. Seven animal species including dromedary, rabbit, goat, turkey, rat, donkey and pork, have been efficiently detected in pure and mixed samples. The combination of PCR–RFLP and triplex PCR assays offers, in addition, the identification of chicken, dog and cat species in meat. In conclusion, by the mean of PCR-based techniques using universal primers followed by enzymatic digestion and multiplex primer-specific approach, we developed an extensible protocol by which we identified 10 animal species that could be integrated in meat analysis daily routine.

Human β-Defensin 2 Mutations Are Associated With Asthma and Atopy in Children and Its Application Prevents Atopic Asthma in a Mouse Model.

Asthma and allergies are complex, chronic inflammatory diseases in which genetic and environmental factors are crucial. Protection against asthma and allergy development in the context of farming environment is established by early animal contact, unpasteurized milk consumption and gut microbiota maturation. The human β-defensin 2 (hBD-2) is a host defense peptide present almost exclusively in epithelial tissues, with pronounced immunomodulatory properties, which has recently been shown to ameliorate asthma and IBD in animal models. We hypothesized that adequate hBD-2 secretion plays a role in the protection against asthma and allergy development and that genetic variations in the complex gene locus coding for hBD-2 may be a risk factor for developing these diseases, if as a consequence, hBD-2 is insufficiently produced. We used MALDI-TOF MS genotyping, sequencing and a RFLP assay to study the genetic variation including mutations, polymorphisms and copy number variations in the locus harboring both genes coding for hBD-2 (DEFB4A and DEFB4B). We administered hBD-2 orally in a mouse model of house dust mite (HDM)-asthma before allergy challenge to explore its prophylactic potential, thereby mimicking a protective farm effect. Despite the high complexity of the region harboring DEFB4A and DEFB4B we identified numerous genetic variants to be associated with asthma and allergy in the GABRIELA Ulm population of 1,238 children living in rural areas, including rare mutations, polymorphisms and a lack of the DEFB4A. Furthermore, we found that prophylactic oral administration of hBD-2 significantly curbed lung resistance and pulmonary inflammation in our HDM mouse model. These data indicate that inadequate genetic capacity for hBD-2 is associated with increased asthma and allergy risk while adequate and early hBD-2 administration (in a mouse model) prevents atopic asthma. This suggests that hBD-2 could be involved in the protective farm effect and may be an excellent candidate to confer protection against asthma development.

Loop-mediated isothermal amplification (LAMP) assay for detection of sesame phyllody phytoplasmas in Vietnam.

Phloem-limiting phytoplasmas are known to be causal agents of phyllody, which is recognized by the abnormal development of floral structures resulting in serious yield losses in sesame plants. Currently, identification of the various groups of phytoplasmas that cause sesame phyllody (SP) is conducted by nested PCR, RFLP, and multiplex real-time qPCR assays. However, these methods require intensive labor and are costly and time-consuming so can only be undertaken in well-equipped labs. Here, diagnostic loop-mediated isothermal amplification (LAMP)-based assays allowing rapid detection of specific groups of phytoplasmas within 30min were developed based on detection of the 16S rRNA sequence of phytoplasmas. Universal 16S rRNA phytoplasma primers and seven primer sets of different 16Sr group phytoplasmas (16SrI, 16SrII, 16SrIII, 16SrIV, 16SrV, 16SrX, 16SrXI) and universal plant cytochrome oxidase (cox) gene primers were used to detect 16S rRNA group phytoplasma sequences and the cox gene in sesame plants. The LAMP assays were carried out using a real-time fluorometer with amplification plots and annealing curves visualized directly. Results demonstrated that the 16SrI and 16SrII group phytoplasmas were causal agents of sesame phyllody in Vietnam. LAMP-based assays for in-field detection of sesame phyllody-causing phytoplasmas revealed advantages and potential applicability in comparison with conventional approaches. To the best of our knowledge, this is the first assessment of multiple phytoplasma infection associated with sesame phyllody disease in Vietnam using LAMP-based assays.

Rapid Genetic Identification of the Blue Crab Callinectes sapidus and Other Callinectes spp. Using Restriction Enzyme Digestion and High Resolution Melt (HRM) Assays

The blue crab Callinectes sapidus is one of the most widely studied marine crustaceans due to its high economic value and ecological significance. Despite extensive research on the blue crab in North America, many questions remain about the distribution and abundance of the species in the subtropics and tropics. In many places, C. sapidus is sympatric with morphologically similar Callinectes spp., which has implications for seafood mislabeling. To enable rapid identification of the species, we designed and tested two PCR-based assays targeting the 12S rRNA mitochondrial gene. The first assay discriminates C. sapidus from other Callinectes spp. via post-PCR restriction digestion (PCR-RFLP) and the second assay discriminates among multiple Callinectes spp. through High Resolution Melting (HRM) analysis and supervised machine learning analyses. A total of 58 DNA samples from five Callinectes spp. (validated via 12S gene sequencing) were used for assay testing. The PCR RFLP assay was 100% accurate identifying C. sapidus from other Callinectes spp. HRM analysis of amplicons showed good discrimination among species, with distinct clusters formed between species with higher sequence homology. Linear discriminant analysis (LDA) classification of HRM curves was quite successful given the small dataset available, producing ~90-91% mean accuracy in classification over all species with 100-fold cross validation. Much of the error came from misclassifications between C. similis and C. danae, which are ~99% similar in sequence for the amplicon; collapsing them into a single class increased overall classification success to 94%. Error also arose from C. bocourti classifications, which had a reference set containing only three samples. Classification accuracy of C. sapidus alone via HRM was 97.5%. Overall, these assays show great promise as rapid and inexpensive methods to identify Callinectes spp. and have application for both ecological research and seafood identification or labeling.

Molecular identification of Leishmania in free-ranging black and gold howler monkeys (Alouatta caraya) in northeastern Argentina

Parasitological surveys of non-human primates provides an important opportunity to better understand the epidemiology, transmission dynamics and emergence risk of anthropozoonoses such as leishmaniasis, which affect human populations in several regions accross South America. Our study area, in northeastern Argentina, can be considered a southern marginal region for the presence of leishmaniases and includes the habitat of black and gold howler monkeys, Alouatta caraya. To evaluate if A. caraya serve as potential hosts in the Leishmania cycle, we used molecular methods to examine infection by Leishmania spp. in 109 howler monkeys of different ages captured between July and August 2010. External ear tissue samples were subjected to PCR amplification for the Leishmania ribosomal internal transcribed spacer (ITS-1) and a RFLP assay with the Hae III restriction enzyme, and finally confirmed by sequencing. Nine howler monkeys (8.3%) were infected with Le. braziliensis (2.8%), Le. amazonensis (2.8%) and/or Le. infantum (3.7%). The results also suggest a case of co-infection between Le. braziliensis and Le. amazonensis. Further, we report the first observation of Le. amazonensis in the northeastern region of Argentina. The detection of Leishmania spp. in free-ranging howler monkeys gives rise to questions about the actual prevalence of the parasite in the wild, as well as if the number of infected wild monkeys detected may present a risk of leishmaniasis emergence in surronding human populations. Anyway, the presence of Leishmania spp. in A. caraya suggests the possible importance of these monkeys in the sylvatic and periurban transmission.

Sex identification PCR–RFLP assay tested in eight species of Sebastes rockfish

The phenotypic identification of sex in Sebastes rockfish is difficult and often impractical from a management perspective, and the genetic basis of sex determination in the genus is currently uncertain. We tested a previously developed sex identification polymerase chain reaction restriction fragment length polymorphism (PCR–RFLP) assay in eight species of Sebastes rockfish. Results indicated that the association of this restriction site with sex is species-dependent.

Molecular epidemiological survey of cutaneous leishmaniasis from Azad Jammu and Kashmir, Pakistan.

Cutaneous leishmaniasis (CL) is an emerging neglected tropical disease in Azad Jammu and Kashmir which is an underdeveloped area. Prevalence and parasite species identification are the key factors to control disease in a particular population, which were the objectives of the present study. Due to a lack of previous data, we performed a district-based active CL surveillance in 2018. The data of CL, suspected (n = 20,000) cases were analyzed statistically and identified the parasite species in microscopic positive cases by ITS1-PCR RFLP and also obtained accession numbers MN891719-28 from gene Bank. The phylogenetic tree was constructed using MEGA6 software. Out of 20,000 CL, suspected cases the highest rate of 4.02% (135/3360) of CL in Mirpur and the lowest 1.58% (8/505) in Neelum was reported. The slide positivity rate, annual parasite incidence rate and annual blood examination rate were 2.27 per 1000 population, 0.08 and 0.34%. The males were more infected 58.12% (297/511) than females 41.88% (214/511) and the age group of 1–20 years were found highly infected 82.78% (423/511) than 21–40 years 13.89% (71/511) and 41–60 years 3.33% (17/511) in the studied population. The patients 56.36% (288/511) had a single lesion whereas 29.35% (150/511) had two, only 10.76% (31/288) and 8% (12/150) were using bed nets. The patients 14.29% (73/511) had three or more lesions were not using bed nets. Only 27.98% (143/511) patients had received treatment, while 72.02% (368/511) didn't. Microscopically positive cases were found to be 2.56% (511/20,000) and ITS1-PCR positive cases were found to be 91.39% (467/511). The RFLP assay confirmed the presence of Leishmania tropica in 467 samples.

First detection of “Candidatus Phytoplasma asteris” ‐ and “Candidatus Phytoplasma solani”‐related strains in fig trees

AbstractIn July 2017, a survey was conducted in a fig collection plot at Locorotondo (south of Italy) to investigate the possible presence of phytoplasmas in plants showing yellowing, deformed leaves, short internodes, mottling and mosaic. Samples were collected from symptomatic plants and tested by nested PCR assays using universal and specific primers to amplify the 16S rDNA of these prokaryotes. PCR results detected the presence of phytoplasma sequences in twenty plant samples that resulted clustering two phylogenetically distinct phytoplasmas, i.e., “Candidatus Phytoplasma asteris” and “Candidatus Phytoplasma solani” affiliated to 16SrI and 16SrXII ribosomal groups, respectively. The presence of phytoplasmas belonging to both ribosomal groups was confirmed with group specific quantitative PCR and RFLP assays on 16S ribosomal amplicons. Results of this study indicate for the first time the occurrence of phytoplasmas in fig; however, more work should be carried out to verify their association with the symptoms observed on diseased fig plants.

Association of HSPA1B Polymorphisms with Paranoid Schizophrenia in a Polish Population

This study aimed to find the potential association between HSPA1B polymorphisms and risk of paranoid schizophrenia, clinical variables of the disease, and suicidal behavior. A total of 901 unrelated Polish subjects of Caucasian origin (377 schizophrenia patients and 524 controls) were recruited. Four single-nucleotide polymorphisms (SNP) were genotyped using PCR–RFLP (rs539689, rs9281590) and TaqMan assays (rs263979, rs6547452). A strong tendency towards statistical significance (p = 0.051) was observed in rs539689 allele distribution between patients and controls in overall study subjects. After stratification according to gender, we found that rs539689 was significantly associated with schizophrenia in males, but not in females. The minor allele C had a protective effect in males [OR 0.73 (95% CI 0.61–0.88, p < 0.05)]. In addition, two SNPs (rs539689, rs9281590) were significantly associated with PANSS scores. Another important finding was a strong significant association between the HSPA1B rs539689 polymorphism and attempted suicide in schizophrenic patients. The C/C genotype and C allele were protective against suicidal behavior in entire sample (p < 0.001), in males (p < 001), and in females (p < 0.05), although associations were weaker than in males. Our findings support that HSPA1B gene may be involved in susceptibility to schizophrenia and clinical presentation of the disease in a sex-dependent manner, and may play a role in suicidal behavior in the Polish population of schizophrenic patients. Further independent analyses in different populations should be performed to clarify the role of HSPA1B in the pathogenesis of schizophrenia.

Single nucleotide polymorphism rs10889677 in miRNAs Let-7e and Let-7f binding site of IL23R gene is a strong colorectal cancer determinant: Report and meta-analysis

Single nucleotide polymorphisms (SNPs) in the recognition sites of microRNAs (miRNAs), located at 3′ untranslated region (UTR) of mRNAs, interfere with posttranslational gene regulation. Deregulation of genes may contribute to some disease susceptibility including colorectal cancer (CRC). In the present study, in a case-control setup, 167 CRC patients and 161 control subjects were studied for allele and genotype frequency of rs10889677 polymorphism in miRNAs Let-7e and Let-7f binding sites at 3′ UTR of IL23R gene using PCR–RFLP assay. Also, related articles were retrieved from MEDLINE, Cochrane review, Google Scholar and Scopus databases for meta-analysis study. According to our results, AA genotype of SNP rs10889677 was significantly correlated with increased risk of CRC (OR = 3.10; 95% CI [1.86–5.18]; P: < 0.001). In a meta-analysis on 10 risk estimates for the CC versus AA genotype, we found an inverse association between CC SNPs and risk of all cancer (OR = 0.59; 95% CI [0.49–0.71]; P < 0.001). In conclusion, our results demonstrate that rs10889677 polymorphism is significantly associated with CRC risk.

Improving specific detection and updating phylogenetic data related to Anaplasma platys-like strains infecting camels (Camelus dromedarius) and their ticks

In camels and their infesting ectoparasites, specific detection of pathogenic Anaplasma platys and genetically related strains (A. platys-like strains) remains problematic. This requires sequencing of the hemi-nested PCR products specific to A. platys and related strains. In this study, a PCR/RFLP method, earlier developed for specific detection of A. platys-like strains in animal species other than camels, was adapted in order to subtype A. platys-like strains isolated from camels and their ticks and to differentiate them from pathogenic A. platys without going through a sequencing step. This approach was used for investigating the infections with A. platys and related strains in 412 Camelus dromedarius camels and 334 feeding ticks from five Tunisian governorates. Microscopic examination using Giemsa-stained blood smears was performed in order to specify which types of cells were infected. Ticks were identified as Hyalomma dromedarii (n = 164, 49%), H. impeltatum (n = 161, 48.3%) and H. excavatum (n = 9, 2.7%). A. platys was not detected in any of the tested camels or ticks. The overall prevalence of A. platys-like strains was 5.6% (23/412) in camels and microscopic examination of infected cells showed a tropism for neutrophil granulocytes. One tick identified as H. dromedarii out of 327 analyzed ticks was found to be infected with A. platys-like strains (0.3%). Alignment, identity comparison and phylogenetic analysis of the 16S rRNA partial sequences obtained in this study suggest that Tunisian dromedaries and feeding ticks are infected with different Anaplasma strains genetically related to A. platys. Sequence analysis and phylogenetic study based on the groEL gene confirm the RFLP results and show that camel strains formed a separate sub-cluster relatively close to A. platys-like strains infecting Tunisian cattle. This adapted RFLP assay allows fast and specific detection of pathogenic A. platys and A. platys-like strains in camels and infesting ticks and has the intrinsic potential of revealing co-infections with these two types of bacteria in the same sample, reducing the time and costs associated with cloning and sequencing during molecular diagnosis.