Identification of a new polymorphism on the wild-type canine distemper virus genome: could this contribute to vaccine failures?

The canine distemper virus (CDV) is responsible for a multisystem infectious disease with high prevalence in dogs and wild carnivores and has vaccination as the main control measure. However, recent studies show an increase in cases including vaccinated dogs in different parts of the world. There are several reasons for vaccine failures, including differences between vaccine strains and wild-type strains. In this study, a phylogenetic analysis of CDV strains from samples of naturally infected, vaccinated, and symptomatic dogs in Goiânia, Goiás, Brazil was performed with partial sequencing of the hemagglutinin (H) gene of CDV. Different sites of amino acid substitutions were found, and one strain had the Y549H mutation, typically present in samples from wild animals. Substitutions in epitopes (residues 367, 376, 379, 381, 386, and 388) that may interfere with the vaccine's ability to provide adequate protection against infection for CDV were observed. The identified strains were grouped in the South America 1/Europe lineage, with a significant difference from other lineages and vaccine strains. Twelve subgenotypes were characterized, considering a nucleotide identity of at least 98% among the strains. These findings highlight the relevance of canine distemper infection and support the need better monitoring of the circulating strains that contribute to elucidate if there is a need for vaccine update.

Performance of the procedure for ultra-rapid extraction and loop-mediated isothermal amplification (PURE-LAMP) method to detect malaria in Haiti.

BACKGROUND: Malaria continues to cause burden in various parts of the world. Haiti, a Caribbean country, is among those aiming to eliminate malaria within a few years. Two surveys were conducted in Haiti during which we aimed to evaluate the performance of the simple and rapid procedure for ultra-rapid extraction-loop-mediated isothermal amplification (PURE-LAMP) method with dried blood spots as an alternative diagnostic method for malaria in the context of low to very low rates of transmission. METHODS: Febrile and afebrile people were recruited from three administrative divisions within Haiti: Nippes, Sud and Grand'Anse, during the summers of 2017 (early August to early September) and 2018 (late July to late August). Their blood samples were tested by microscopy, rapid diagnostic tests (RDT), PURE-LAMP and nested PCR to detect Plasmodium infection. Sensitivity, specificity, positive and negative predictive values and kappa statistics were estimated with the nested PCR results as the gold standard. RESULTS: Among 1074 samples analyzed, a positive rate of 8.3% was calculated based on the nested PCR results. Among febrile participants, the rates in 2017 and 2018 were 14.6% and 1.4%, respectively. Three positives were detected among 172 afebrile participants in 2018 by PURE-LAMP and nested PCR, and all three were from the same locality. There was no afebrile participants recruited in 2017. The PURE-LAMP, RDT and microscopy had respective sensitivities of 100%, 85.4% and 49.4%. All of the testing methods had specificities over 99%. CONCLUSIONS: This study confirmed the high performance of the PURE-LAMP method to detect Plasmodium infection with dried blood spots and recommends its use in targeted mass screening and treatment activities in low endemic areas of malaria.

Molecular Detection of Toxoplasma gondii in Blood Samples of Domestic Livestock in the Republic of Korea.

Toxoplasma gondii, a major zoonotic pathogen distributed worldwide, causes severe infections in humans, animals, and birds. However, limited information is available regarding T. gondii infection in livestock in the Republic of Korea (ROK). Herein, we determined the prevalence of T. gondii infection in livestock in the ROK and identified animal species that can potentially transmit T. gondii to humans. B1 gene-targeting nested polymerase chain reaction detected T. gondii DNA in 3.3% (2/61), 2.9% (3/105), 14.1% (11/78), and 15.4% (14/91) of dairy cattle, beef cattle, Boer goats, and Korean native goats, respectively. The prevalence of T. gondii was significantly higher (p = 0.002) in goats than in cattle. The risk of contracting T. gondii infection was significantly higher by 6.18-fold in Korean native goats (95% confidence interval [CI]: 1.72-22.27%, p = 0.005) and by 5.58-fold in Boer goats (95% CI: 1.50-20.76%, p = 0.010) than in beef cattle. Our T. gondii DNA sequences exhibited 97.1-100% homology with those obtained from various hosts in other countries. To the best of our knowledge, this is the first study to report T. gondii infection using the blood samples of domestic ruminants in the ROK. The results revealed that the prevalence of T. gondii infection is higher in goats than in cattle as determined by molecular detection. Thus, these findings suggest that T. gondii can be transmitted from ruminants to humans via meat consumption.

PCR and peptide based PCMV detection in pig - development and application of a combined testing procedure differentiating newly from latent infected pigs.

Porcine cytomegalovirus (PCMV) is widely distributed in pigs and difficult to detect due to latency. PCMV infection of source pigs was associated with early graft failure after cardiac and renal xenotransplantation into nonhuman primates. Importantly, PCMV infection of the first genetically modified pig heart into a human may have contributed to the reduced survival of the patient. Sensitive and reliable assays for detection of latent PCMV infection are thus indispensable. Here, we report the development of five peptide-induced rabbit antisera specific for PCMV glycoprotein B (gB) and their validation for detection of PCMV in infected pig fallopian tube (PFT) cells by immunofluorescence and electron microscopy (EM). The anti-gB antibodies were also used for detection by Western blot analysis of PCMV purified from the supernatant of infected PFT cells. Sera of infected versus non-infected pigs have been compared. In parallel, PCMV viral load in blood samples of the animals was quantified by a novel highly sensitive nested-PCR and qPCR assay. A combination of four partly overlapping peptides from the gB C-terminus was used to establish a diagnostic ELISA for PCMV gB specific pig antibodies which is able to differentiate infected from non-infected animals and to quantify maternal antibodies in neonates. The combination of a highly sensitive nested PCR for direct virus detection with a sensitive peptide-based ELISA detecting anti-PCMV gB-antibodies, supplemented by Western blot analysis and/or immunohistochemistry for virus detection will reliably differentiate pigs with active infection, latently infected pigs, and non-infected pigs. It may significantly improve the virologic safety of xenotransplantation.

In vitro Culture and Multilocus Genotyping of Giardia duodenalis Trophozoites Obtained from Human Fecal Samples in Southwest Iran.

INTRODUCTION: The enteric protozoa, Giardia duodenalis (G. duodenalis), consists of eight distinct assemblages (A-H) with identical morphological characteristics and a direct life cycle. Successful axenic cultivation of this parasite is an important preliminary step for biological, drug resistance and phylogenetic studies. Moreover, G. duodenalis exhibits great genetic and biotypic diversity. The current study aimed to evaluate In vitro culture and multilocus genotyping of G. duodenalis trophozoites obtained from human fecal samples in southwest Iran. METHODS: Thirty human fecal specimens containing G. duodenalis cysts were collected from Ahvaz city (southwest of Iran). The purification of cysts was carried out by the sucrose flotation technique. The cysts were inoculated in a modified TYI-S-33 medium and was daily monitored for the development and viability of trophozoites. After extracting DNA, gdh, bg and tpi genes were evaluated using molecular techniques (the semi-nested PCR for gdh gene and the nested PCR for tpi and bg genes). Eventually, the amplified fragments were sequenced and then, the phylogenetic tree was drawn. RESULTS: Of 30, the trophozoites were encysted from five samples. All three genes were detected in two cases of five samples using molecular techniques. The multilocus phylogenetic analysis demonstrated that all the two samples belonged to assemblage A and sub-assemblage AІІ. CONCLUSION: Our findings indicated the presence of different numbers of trophozoites with variable development and survival rates in modified TYI-S-33 medium. Furthermore, the multilocus genotyping showed that these trophozoites belonged to assemblage A and sub-assemblage AІІ.

Development and evaluation of a semi-nested PCR technique for amplification and determination of different surface gene variation patterns in patients with chronic HBV infection: development of an amplification method for HBV S gene.

Aim: Development of an amplification method for further investigation of HBV S gene variation patterns. Background: Pre-S/S variants in patients with chronic HBV infection may contribute to the progression of liver damage and Hepatocellular carcinoma (HCC). Methods: This study was performed on ten patients with chronic HBV infection. Viral DNA was extracted from patient's plasma, primer design was performed, and a semi-nested PCR method was set up to amplify the pre-S/S region of HBV genome. Subsequently, sequencing was performed to analyze the variants of this region. Results: In the current study, the semi-nested PCR method was successfully set up, and types of variation in the studied samples were investigated. Conclusion: Pre-S/S variants should be routinely determined in HBV carriers to help identify individuals who may be at a high risk of less favorable liver disease progression. This study showed that the technique could accurately amplify the pre-S/S region, and the product can be successfully used for variation detection by direct sequencing.

Molecular and microscopy detection of Pneumocystis jirovecii in hospitalized patients during the COVID-19 pandemic.

Introduction: Early detection of Pneumocystis jirovecii as an opportunistic pathogen that may endanger predisposed persons, including COVID-19 patients, may help to choose the optimal management. Methods: In this study, 585, including 530 COVID-19 patients, with clinical and radiological evidence of respiratory diseases, were investigated for P. jirovecii screening. Clinical specimens were examined by direct microscopy and PCR, and randomly selected positive PCR products were confirmed through DNA sequence analysis. Results: Thirty-one (5.3%) samples were positive in P. jirovecii-specific nested-PCR, while by direct microscopic tests, Pneumocystis was observed in 22 (3.76%) samples. Males (61.7%) and patients over 50 years old (75.6%) were more commonly affected than others, and malaise and fatigue (84%), and wheezing (75%) were the most common symptoms, followed by fever (40.48%) and dyspnea (39.51%). Among the Pneumocystis-positive patients, three cases had coinfection with Aspergillus fumigatus, A. flavus, and A. niger (each n = 1), as documented by direct microscopy, culture, and species identification by PCR-sequencing. Conclusion: Pneumocystis pneumonia is still a diagnostic challenge; therefore, additional large-scale studies are needed to clarify the epidemiology of the disease in immunocompromised or COVID-19 patients.

Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp.

Leishmaniasis is an endemic parasitic disease in at least 98 countries. In Spain, it is considered a zoonosis caused by Leishmania infantum, with an annual incidence of 0.62 cases/100,000 inhabitants. The predominant clinical manifestations are the cutaneous (CL) and visceral forms (VL), and the diagnosis is performed by parasitological, serological, and molecular tests. At the WHO Collaborating Center for Leishmaniasis (WHOCCLeish), routine diagnostic tests are based on a nested PCR (Ln-PCR), culture, and serological tests. To simplify our PCR protocol, we aimed to develop and validate a ready-to-use nested gel-form PCR (LeishGelPCR) and a duplex real-time PCR (qPCR) that allowed simultaneous detection of Leishmania and mammalian DNA as an internal control (Leish-qPCR). Clinical validation was performed in 200 samples from the WHOCCLeish collection; 92 and 85 out of 94 and 87 samples were positive by LeishGelPCR and Leish-qPCR, respectively, showing a sensitivity of 98% in both approaches. The specificity was 100% for LeishGelPCR and 98% for Leish-qPCR. The limits of detection of both protocols were similar (0.5 and 0.2 parasites/reaction). Parasite loads in VL and CL forms were similar, although high loads were observed when invasive samples were tested. In conclusion, LeishGelPCR and Leish-qPCR showed excellent performance in the diagnosis of leishmaniasis. These new forms of 18S rRNA gene PCR are equivalent to Ln-PCR and can be introduced in the algorithm for CL and VL diagnosis. IMPORTANCE Although the gold standard for diagnosis of leishmaniasis is the microscopic observation of amastigotes, molecular techniques are becoming a cost-efficient alternative. Currently, PCR is a routine resource that is used in many reference microbiology laboratories. In this article, we have described two ways to improve the reproducibility and usability of the molecular detection of Leishmania spp. These new approaches could be introduced even in middle- and low-resource laboratories; one is a ready-to-use gel-form system of a nested PCR and the other is a real-time PCR. We show why molecular diagnosis is the best methodology to confirm a clinical suspicion of leishmaniasis with higher sensitivity than traditional methods, thus facilitating early diagnosis and timely treatment of human leishmaniasis.

Nested PCR Approach for petB Gene Metabarcoding of Marine Synechococcus Populations.

The molecular diversity of marine picocyanobacterial populations, an important component of phytoplankton communities, is better characterized using high-resolution marker genes than the 16S rRNA gene as they have greater sequence divergence to differentiate between closely related picocyanobacteria groups. Although specific ribosomal primers have been developed, another general disadvantage of bacterial ribosome-based diversity analyses is the variable number of rRNA gene copies. To overcome these issues, the single-copy petB gene, encoding the cytochrome b6 subunit of the cytochrome b6f complex, has been used as a high-resolution marker gene to characterize Synechococcus diversity. We have designed new primers targeting the petB gene and proposed a nested PCR method (termed Ong_2022) for metabarcoding of marine Synechococcus populations obtained by flow cytometry cell sorting. We evaluated the specificity and sensitivity of Ong_2022 against the standard amplification protocol (termed Mazard_2012) using filtered seawater samples. The Ong_2022 approach was also tested on flow cytometry-sorted Synechococcus populations. Samples (filtered and sorted) were obtained in the Southwest Pacific Ocean, from subtropical (ST) and subantarctic (SA) water masses. The two PCR approaches using filtered samples recovered the same dominant subclades, Ia, Ib, IVa, and IVb, with small differences in relative abundance across the distinct samples. For example, subclade IVa was dominant in ST samples with the Mazard_2012 approach, while the same samples processed with Ong_2022 showed similar contributions of subclades IVa and Ib to the total community. The Ong_2022 approach generally captured a higher genetic diversity of Synechococcus subcluster 5.1 than the Mazard_2012 approach while having a lower proportion of incorrectly assigned amplicon sequence variants (ASVs). All flow cytometry-sorted Synechococcus samples could be amplified only by our nested approach. The taxonomic diversity obtained with our primers on both sample types was in agreement with the clade distribution observed by previous studies that applied other marker genes or PCR-free metagenomic approaches under similar environmental conditions. IMPORTANCE The petB gene has been proposed as a high-resolution marker gene to access the diversity of marine Synechococcus populations. A systematic metabarcoding approach based on the petB gene would improve the characterization/assessment of the Synechococcus community structure in marine planktonic ecosystems. We have designed and tested specific primers to be applied in a nested PCR protocol (Ong_2022) for metabarcoding the petB gene. The Ong_2022 protocol can be applied to samples with low DNA content, such as those obtained by flow cytometry cell sorting, allowing the simultaneous assessment of the genetic diversity of Synechococcus populations and cellular properties and activities (e.g., nutrient cell ratios or carbon uptake rates). Our approach will allow future studies using flow cytometry to investigate the link between ecological traits and taxonomic diversity of marine Synechococcus.

Detection of Toxoplasma gondii DNA in milk of dairy cows from southern Brazil.

Consumption of unpasteurized cow's milk may be a transmission route for some pathogenic microorganisms, but there is little information about the risk of Toxoplasma gondii infection. Blood and milk samples were collected in a paired and random fashion from 106 dairy cows and bulk-tank milk samples were also collected from each of the six farms, in southern Brazil. Serum anti-T.gondii antibodies (IgG) were detected by an indirect fluorescent antibody test (IFAT) with a cutoff point of 1:64. Nested PCR targeting the ITS1 was performed on milk samples to detect the Sarcocystidae family, confirmed to be T.gondii by Sanger sequencing. The occurrence of anti-T.gondii antibodies in the herds was 14.1%, (15/106) with seropositive cows in all herds. Antibody titers in positive samples ranged from 64 to 128. T.gondii DNA was detected in 2.8% (03/106) of the milk samples. The ITS1 sequences generated in this study were ON809793 - ON809794 and the sequencing revealed 98-100% identity with T. gondii DNA sequences deposited in GenBank. All cows PCR positive for T.gondii in milk were negative for IgG antibodies in serum, suggesting that naturally infected cows may shed T. gondii in milk in the acute phase of infection. The results of this study demonstrate that T. gondii DNA may be detected in raw cow's milk, so the potential risks of lactogenic infection should be considered. The presence of T. gondii DNA in milk does not confirm that the protozoa are viable and infective, and further investigations into the role of cow's milk in the epidemiology of toxoplasmosis are needed.

Comparative Evaluation of LAMP and Nested PCR for the Rapid Diagnosis of Mycobacterium marinum Infection.

Purpose: Culture of Mycobacterium marinum is very time-consuming, taking several weeks to produce positive results. Seeking rapid and sensitive diagnostic methods for diagnosis can greatly improve patient treatment. Our study aimed to compare the rapid diagnostic abilities of polymerase chain reaction (PCR), nested PCR and loop mediated isothermal amplification (LAMP) of detecting M. marinum in skin samples from patients with M. marinum infection. Methods: A total of 6 M. marinum strains and 6 skin samples with definite diagnosis of M. marinum infection were included in the study. We optimized LAMP performance for detection of M. marinum genomic DNA and confirmed the specificity of the primers. Then, the sensitivity of the LAMP and nested PCR assays were assessed by M. marinum strains and clinical samples. Results: Nested PCR was 10-fold more sensitive than the LAMP assay by serial dilution of M. marinum DNA. PCR positive samples were all positive by LAMP detection of 6 clinical M. marinum strains. Out of 6 clinical skin specimens confirmed as M. marinum infection, 0 (0%), 3 (50%), 3 (50%), and 4 (66.6%) were positive by PCR, nested PCR, LAMP and culture. The LAMP shared the same sensitivity than nested PCR in M. marinum strains and clinical samples, but it was easy to perform and faster than nested PCR assay. Conclusion: Compared with conventional PCR, LAMP and nested PCR are more sensitive and have a higher detection rate of M. marinum in clinical skin specimens. The LAMP assay proved to be more suitable for rapid diagnosis of M. marinum infection in a shorter time, especially in resource-limited settings.

Morphological and Molecular Characterization of Eimeria spp. Infecting Domestic Poultry Gallus gallus in Riyadh City, Saudi Arabia.

Coccidiosis in chickens is one of the major problems in the poultry industry, caused by protozoan parasites of the genus Eimeria. The current study used morphological and molecular characteristics to identify Eimeria spp. infecting domestic chickens (Gallus gallus) in the Riyadh region of Saudi Arabia. In this study, 120 domestic poultry were examined and 30 were found to be infected with oocysts of Eimeria spp. (25%). According to the morphology of the recorded oocysts, five species were found. Eimeria necatrix was the first species discovered, and it was distinguished by oblong, ovoid-shaped oocysts with double-layered walls that measured 20 (23-23) and 17 (16-20) µm. The second species was Eimeria maxima, which had oval- to egg-shaped oocysts with double-layered walls and measurements of 28 (26-29) and 23 (20-24) µm. The third species was Eimeria tenella, characterized by oval-shaped oocysts with double-layered walls and measurements of 21 (20-24) × 17 (16-20) µm. Eimeria praecox was the fourth species that was characterized by spherical-shaped oocysts with single-layered walls and measurements of 21 (19-23) × 20 (19-20) µm. Eimeria acervulina was the last species to have oval-shaped oocysts with double-layered walls and measurements of 20 (18-25) and 17 (14-20) µm. The percentages of infection with Eimeria species were as follows: E. tenella, 10.84%; E. necatrix, 5.84%; E. acervulina, 4.16%; E. maxima, 2.5%; and E. praecox, 1.66%. Nested PCR based on the amplification of internal transcribed spacer I (ITS-I) regions confirmed the presence of the five Eimeria species in the examined fecal samples with their specific amplicon sizes: E. necatrix (383 bp), E. maxima (145 bp), E. tenella (278 bp), E. praecopx (116 bp), and E. acervulina (321 bp).

Pneumocystis jirovecii Colonization in Mexican Patients with Chronic Obstructive Pulmonary Disease.

The prevalence of colonization by Pneumocystis jirovecii (P. jirovecii) has not been studied in Mexico. We aimed to determine the prevalence of colonization by P. jirovecii using molecular detection in a population of Mexican patients with chronic obstructive pulmonary disease (COPD) and describe their clinical and sociodemographic profiles. We enrolled patients discharged from our hospital diagnosed with COPD and without pneumonia (n = 15). The primary outcome of this study was P. jirovecii colonization at the time of discharge, as detected by nested polymerase chain reaction (PCR) of oropharyngeal wash samples. The calculated prevalence of colonization for our study group was 26.66%. There were no statistically significant differences between COPD patients with and without colonization in our groups. Colonization of P. jirovecii in patients with COPD is frequent in the Mexican population; the clinical significance, if any, remains to be determined. Oropharyngeal wash and nested PCR are excellent cost-effective options to simplify sample collection and detection in developing countries and can be used for further studies.

PET-PCR reveals low parasitaemia and submicroscopic malarial infections in Honduran Moskitia.

BACKGROUND: Malaria remains a main parasitic disease of humans. Although the largest number of cases is reported in the African region, there are still endemic foci in the Americas. Central America reported 36,000 malaria cases in 2020, which represents 5.5% of cases in the Americas and 0.015% of cases globally. Most malaria infections in Central America are reported in La Moskitia, shared by Honduras and Nicaragua. In the Honduran Moskitia, less than 800 cases were registered in 2020, considering it an area of low endemicity. In low endemicity settings, the number of submicroscopic and asymptomatic infections tends to increase, leaving many cases undetected and untreated. These reservoirs challenge national malaria elimination programmes. This study aimed to assess the diagnostic performance of Light Microscopy (LM), a nested PCR test and a photoinduced electron transfer polymerase chain reaction (PET-PCR) in a population of febrile patients from La Moskitia. METHODS: A total of 309 febrile participants were recruited using a passive surveillance approach at the Puerto Lempira hospital. Blood samples were analysed by LM, nested PCR, and PET-PCR. Diagnostic performance including sensitivity, specificity, negative and positive predictive values, kappa index, accuracy, and ROC analysis was evaluated. The parasitaemia of the positive samples was quantified by both LM and PET-PCR. RESULTS: The overall prevalence of malaria was 19.1% by LM, 27.8% by nPCR, and 31.1% by PET-PCR. The sensitivity of LM was 67.4% compared to nPCR, and the sensitivity of LM and nPCR was 59.6% and 80.8%, respectively, compared to PET-PCR. LM showed a kappa index of 0.67, with a moderate level of agreement. Forty positive cases by PET-PCR were not detected by LM. CONCLUSIONS: This study demonstrated that LM is unable to detect parasitaemia at low levels and that there is a high degree of submicroscopic infections in the Honduran Moskitia.

Retrospective analysis of Porphyromonas gingivalis in patients with nasopharyngeal carcinoma in central China.

Little is known about the presence and possible role of Porphyromonas gingivalis (P. gingivalis) in nasopharyngeal carcinoma (NPC), its co-infection with Epstein-Barr virus (EBV), or their association with clinical characteristics of patients with NPC in Central China, where NPC is non-endemic. A total of 45 NPC formalin-fixed paraffin-embedded (FFPE) tissues were retrospectively analyzed using immunohistochemistry (IHC) and a nested PCR combined with DNA sequencing to detect the presence of P. gingivalis, and using reverse transcription-quantitative PCR to detect the presence of EBV. Clinical data including EBV and P. gingivalis status were associated with overall survival (OS). All tumors were undifferentiated, non-keratinizing carcinomas, of which 40/45 (88.9%) were positive for EBV (EBV+), 26/45 (57.8%) were positive for P. gingivalis (by IHC), and 7/45 (15.6%) were positive for P. gingivalis DNA (P. gingivalis +). All seven P. gingivalis DNA-positive NPCs were co-infected with EBV. The 5-year survival rates of the patients with EBV-/P. gingivalis -, EBV+/P. gingivalis -, and EBV+/P. gingivalis + tumors were 60.0% (3/5), 39.4% (13/33) and 42.9% (3/7), respectively. No significant difference was found between the OS of NPC patients among the different infection groups (P=0.793). In conclusion, to the best of our knowledge, this is the first study to describe and confirm the presence of P. gingivalis in FFPE tissues from patients with NPC. P. gingivalis was found to co-exist with EBV in NPC tumor tissues, but is not etiologically relevant to NPC in non-endemic areas, such as Central China.

Molecular investigation of ready-to-eat salads for Giardia duodenalis and Cryptosporidium spp. in Portugal.

Foodborne outbreaks are often associated with the consumption of salads. However, published studies on the detection of foodborne pathogens in ready-to-eat salads are scarce. The aim of this study was to detect Giardia duodenalis and Cryptosporidium DNA in ready-to-eat salads, by applying techniques of molecular biology to study the frequency of contamination in salads. A total of 100 packages of ready-to-eat salads containing assorted leafy green vegetables were randomly purchased from hypermarkets located in central regions of Portugal (Coimbra and Viseu). Nested-PCR and qPCR methods were used to detect G. duodenalis and Cryptosporidium DNA. Species and assemblages of the parasites were identified by sequence analysis and PCR. Eighteen of the 100 samples (18%) were positive for G. duodenalis and twelve were sequenced and identified as assemblage A. Cryptosporidium spp. were not detected in any salads. Overall, pre-harvest and post-harvest preventive measures may be need for G. duodenalis control throughout the food production industry, from the field to consumers.

Identification of Leishmania species and frequency distribution of LRV1 and LRV2 viruses on cutaneous leishmaniasis patients in Isfahan Province, Iran.

PURPOSE: Leishmaniasis is one of the most serious health problems in developing countries. Iran is one of the endemic regions of cutaneous leishmaniasis. Leishmania RNA virus (LRV) is a dsRNA virus member of the Totiviridae family, which was first detected in the promastigotes of Leishmania braziliensis guyanensis. Our study aimed to investigate possible changes in the predominant and causative strains of CL and screening the LRV1 and LRV2 species genome from Leishmania species isolated from the lesions of patients. MATERIALS AND METHODS: Direct smear samples obtained from 62 patients with leishmaniasis referring to the Skin Diseases and Leishmaniasis Research Center in Isfahan province during 2021-2022 were examined. Total DNA extraction procedures and conservation of site-specific multiplex PCR and nested PCR were performed for detecting Leishmania species. The molecular identification of LRV1 and LRV2 viruses, samples were used for total RNA extraction and real-time (RT)-PCR analysis, followed by conducting a restriction enzyme assay to confirm the PCR products. RESULTS: Of the total Leishmania isolates, 54 and 8 isolates were identified as L. major and L. tropica, respectively. LRV2 was identified in 18 samples affected by L. major, while LRV1 was only detected in one of the samples with L. tropica. No LRV2 was found in any samples with L. tropica. The results showed that there was a significant relationship between LRV1 and the type of leishmaniasis (Sig. â€‹= â€‹0.009, P â€‹≤ â€‹0.05), while this relationship was not observed between LRV2 and the type of leishmaniasis. CONCLUSIONS: The presence of a significant number of LRV2 in isolated samples, as well as the recognition of LRV1 in one of the Old World leishmaniasis species, which is a new result, could pave the way for investigating further aspects of this disease and successful treatment strategies in future studies.

Molecular detection of Babesia microti in dromedary camels in Egypt.

Babesia microti (Apicomplexa: Piroplasmida) causes a medically important tick-borne zoonotic protozoan disease. Egyptian camels are susceptible to Babesia infection; however, just a few cases have been documented. This study aimed to identify Babesia species, specifically Babesia microti, and their genetic diversity in dromedary camels in Egypt and associated hard ticks. Blood and hard tick samples were taken from 133 infested dromedary camels slaughtered in Cairo and Giza abattoirs. The study was conducted from February to November 2021. The 18S rRNA gene was amplified by polymerase chain reaction (PCR) to identify Babesia species. Nested PCR targeting the ß-tubulin gene was used to identify B. microti. The PCR results were confirmed by DNA sequencing. Phylogenetic analysis based on the ß-tubulin gene was used to detect and genotype B. microti. Three tick genera were identified in infested camels (Hyalomma, Rhipicephalus, and Amblyomma). Babesia species were detected in 3 out of 133 blood samples (2.3%), while Babesia spp. were not detected in hard ticks by using the 18S rRNA gene. B. microti was identified in 9 out of 133 blood samples (6.8%) and isolated from Rhipicephalus annulatus and Amblyomma cohaerens by the ß-tubulin gene. The phylogenetic analysis of the ß-tubulin gene revealed that USA-type B. microti was prevalent in Egyptian camels. The results of this study suggested that the Egyptian camels may be infected with Babesia spp. and the zoonotic B. microti strains, which pose a potential risk to public health.

Nested PCR-Based Rapid Detection of Phytoplasma Leaf Wilt Disease of Coconut in Sri Lanka and Systemic Movement of the Pathogen.

Phytoplasmas are associated with many plant diseases. In palms, lethal bronzing disease, Texas Phoenix palm decline, and coconut lethal yellowing decline are some of them. In Sri Lanka, coconut leaf wilt decline has been reported in the Weligama area of the Southern province, and the disease is called Weligama coconut leaf wilt disease (WCLWD). Unlike other phytoplasma diseases of palms, WCLWD shows slow disease progress. Pathogen detection entirely relies on nested polymerase chain reaction (PCR). However, inconsistencies in pathogen detection have been experienced, i.e., symptomatic plants often produce negative results. The objectives of this study were to reconsider the choice of primers and to determine the best sampling tissue types for consistent detection of the pathogen. Among the six universal primer combinations tested, P1/Tint nested with fU5/rU3 produced consistent results. BLASTn searches of the sequences showed 99-100% similarity to sugarcane white leaf disease (SWL) or grassy shoot (SGS) disease-causing phytoplasma. The optimized nested PCR protocol was successful, with the minimum success rating of 88% and 100% specificity. Midribs of milky white bud leaf samples were the best tissue type for rapid detection. Systemic movement of the pathogen and a tentative latent period were also reported. The findings are helpful in the early detection of the disease.

Development and evaluation of a simple PCR assay and nested PCR for rapid detection of clarithromycin-resistant Helicobacter pylori from culture and directly from the biopsy samples in India.

BACKGROUND: Eradication of Helicobacter pylori provides the most effective treatment for gastroduodenal diseases caused by H. pylori infection. Clarithromycin, a member of the macrolide family, still remains the most important antibiotic used in H. pylori eradication treatment. But the increasing prevalence of clarithromycin resistant H. pylori strains due to point mutations in the V region of the 23S rRNA, poses a great threat in treating the ailing patients. So, we aimed for PCR-mediated rapid detection of the point mutation at 2143 position of 23S rRNA gene in H. pylori that is relevant to clarithromycin resistance from culture and simultaneously from biopsy specimens to avoid the empirical treatment. RESULTS: Newly developed PCR assay using DNA of pure culture detected point mutation in 23S rRNA gene in 21 (8.04%) of 261 clinical strains tested. The agar dilution method showed that all these 21 strains were resistant to clarithromycin indicating the perfect match of the PCR based results. Additionally, the sequencing study also identified the A to G mutation at 2143 position in 23S rRNA gene of the resistant strains only. Consequently, the newly developed Nested-ASP-PCR dealing directly with 50 biopsy specimens demonstrated 100% sensitivity and specificity with the findings of agar dilution method taken as Gold standard. Bioinformatics based analysis such as accessibility analysis and dot plot clearly stated that the base pairing probability has increased due to mutation. Computational studies revealed that the point mutation confers more stability in secondary structure due to conversion of loop to stem. Furthermore, interaction studies showed binding affinity of the CLR to the mutant type is weaker than that to the wild type. CONCLUSION: This assay outlines a rapid, sensitive and simple approach to identify point mutation that confers clarithromycin resistance as well as clarithromycin sensitive strains, providing rapid initiation of effective antibiotic treatment. Additionally, it is simple to adopt for hospital based diagnostic laboratories to evaluate the degree of regional clarithromycin resistance from biopsy specimens itself. Furthermore, in silico studies provide evidence or a signal that the prevalence of clarithromycin resistance may rise in the near future as a result of this point mutation.