Molecular prevalence, subtype distribution, and zoonotic potential of Blastocystis sp. in wild rodents and shrews inhabiting Zhejiang province of China.

Introduction: Globally, rodents and shrew populations constitute crucial elements of diverse environments and animal communities. It is imperative to study their population dynamics to mitigate any potential negative impact on humans, as they can be involved in the transmission of critical zoonotic agents, such as Blastocystis. Therefore, this study aimed to identify the prevalence and genetic composition of Blastocystis in wild rodents and shrews residing in the Zhejiang provinces of China. Methods: A total of 652 wild rodents and and shrews were captured from three different regions in Zhejiang Province from April 1st to October 31, 2023. The DNA was isolated by collecting fresh feces from the intestines of each rodent or and shrew. Rodent and shrew species were examined by vertebrate cytochrome b (cytb) analysis and PCR amplification. Blastocystis was also found in all fecal samples using PCR analysis and sequencing of the partial small subunit of ribosomal RNA (SSU rRNA) gene. Results: Among all the samples, 6.6% (43/652) showed a positive result for Blastocystis. In the results, 6 species of rodent and shrew were identified with Blastocystis, including Apodemus agrarius (n = 36) (2.8%), Niviventer confucianus (n = 75) (17.3%), Rattus losea (n = 18) (5.6%), R. norvegicus (n = 155) (2.6%), R. tanezumi (n = 86) (3.5%), and Suncus murinus (n = 282) (7.4%). The existence of 6 Blastocystis subtypes, ST4 (n = 33), ST1 (4), ST7 (n = 3), ST2 (n = 1), ST3 (n = 1), and ST5 (n = 1), were confirmed by sequence analysis. Discussion: Based on the molecular data obtained, the wild rodents and shrews under investigation were found to be concurrently infected with zoonotic subtypes of Blastocystis, including ST1 to ST5 and ST7. This suggests that these animals could potentially pose a zoonotic threat to humans and other animals susceptible to Blastocystis infection.

An affordable detection system based on RT-LAMP and DNA-nanoprobes for avian metapneumovirus.

Airborne animal viral pathogens can rapidly spread and become a global threat, resulting in substantial socioeconomic and health consequences. To prevent and control potential epidemic outbreaks, accurate, fast, and affordable point-of-care (POC) tests are essential. As a proof-of-concept, we have developed a molecular system based on the loop-mediated isothermal amplification (LAMP) technique for avian metapneumovirus (aMPV) detection, an airborne communicable agent mainly infecting turkeys and chickens. For this purpose, a colorimetric system was obtained by coupling the LAMP technique with specific DNA-functionalized AuNPs (gold nanoparticles). The system was validated using 50 different samples (pharyngeal swabs and tracheal tissue) collected from aMPV-infected and non-infected chickens and turkeys. Viral detection can be achieved in about 60 min with the naked eye, with 100% specificity and 87.88% sensitivity for aMPV. In summary, this novel molecular detection system allows suitable virus testing in the field, with accuracy and limit of detection (LOD) values highly close to qRT-PCR-based diagnosis. Furthermore, this system can be easily scalable to a platform for the detection of other viruses, addressing the current gap in the availability of POC tests for viral detection in poultry farming. KEY POINTS: •aMPV diagnosis using RT-LAMP is achieved with high sensitivity and specificity. •Fifty field samples have been visualized using DNA-nanoprobe validation. •The developed system is a reliable, fast, and cost-effective option for POCT.

Evaluation of the FPMC respiratory panel for detection of respiratory tract pathogens in nasopharyngeal swab and sputum specimens.

OBJECTIVES: The performance of the new Respiratory Pathogen panel (fluorescent probe melting curve, FPMC) for the qualitative detection of 12 organisms (chlamydia pneumoniae, mycoplasma pneumoniae, adenovirus, influenza A virus, influenza B virus, parainfluenza virus, rhinovirus, etc.) was assessed. METHODS: Prospectively collected nasopharyngeal swab (NPS) and sputum specimens (n = 635) were detected by using the FPMC panel, with the Sanger sequencing method as the comparative method. RESULTS: The overall percent concordance between the FPMC analysis method and the Sanger sequencing method was 100% and 99.66% for NPS and sputum specimens, respectively. The FPMC testified an overall positive percent concordance of 100% for both NPS and sputum specimens. The FPMC analysis method also testified an overall negative percent concordance of 100% and 99.38% for NPS and sputum specimens, respectively. CONCLUSIONS: The FPMC analysis method is a stable and accurate assay for rapid, comprehensive detecting for respiratory pathogens.

Cross-species transmission of Cryptosporidium in wild rodents from the southern region of Zhejiang Province of China and its possible impact on public health.

Wild rodents serve as reservoirs for Cryptosporidium and are overpopulated globally. However, genetic data regarding Cryptosporidium in these animals from China are limited. Here, we have determined the prevalence and genetic characteristics of Cryptosporidium among 370 wild rodents captured from three distinct locations in the southern region of Zhejiang Province, China. Fresh feces were collected from the rectum of each rodent, and DNA was extracted from them. The rodent species was identified by PCR amplifying the vertebrate cytochrome b gene. Cryptosporidium was detected by PCR amplification and amplicon sequencing the small subunit of ribosomal RNA gene. Positive samples of C. viatorum and C. parvum were further subtyped by analyzing the 60-kDa glycoprotein gene. A positive Cryptosporidium result was found in 7% (26/370) of samples, involving five rodent species: Apodemus agrarius (36), Niviventer niviventer (75), Rattus losea (18), R. norvegicus (155), and R. tanezumi (86). Their respective Cryptosporidium positive rates were 8.3%, 5.3%, 11.1%, 7.1%, and 7.0%. Sequence analysis confirmed the presence of three Cryptosporidium species: C. parvum (4), C. viatorum (1), and C. muris (1), and two genotypes: Cryptosporidium rat genotype IV (16) and C. mortiferum-like (4). Additionally, two subtypes of C. parvum (IIdA15G1 and IIpA19) and one subtype of C. viatorum (XVdA3) were detected. These results demonstrate that various wild rodent species in Zhejiang were concurrently infected with rodent-adapted and zoonotic species/genotypes of Cryptosporidium, indicating that these rodents can play a role in maintaining and dispersing this parasite into the environment and other hosts, including humans.

Title: Transmission interspécifique de Cryptosporidium chez les rongeurs sauvages de la région sud de la province chinoise du Zhejiang et son impact possible sur la santé publique. Abstract: Les rongeurs sauvages servent de réservoirs à Cryptosporidium et ont des grandes populations à l'échelle mondiale. Cependant, les données génétiques concernant Cryptosporidium chez ces animaux en Chine sont limitées. Ici, nous avons déterminé la prévalence et les caractéristiques génétiques de Cryptosporidium parmi 370 rongeurs sauvages capturés dans trois endroits distincts de la région sud de la province du Zhejiang, en Chine. Des excréments frais ont été collectés dans le rectum de chaque rongeur et l'ADN en a été extrait. L'espèce de rongeur a été identifiée par amplification par PCR du gène du cytochrome b des vertébrés. Cryptosporidium a été détecté par amplification PCR et séquençage d'amplicons de la petite sous-unité du gène de l'ARN ribosomal. Les échantillons positifs de C. viatorum et C. parvum ont ensuite été sous-typés en analysant le gène de la glycoprotéine de 60 kDa. Un résultat positif pour Cryptosporidium a été trouvé dans 7 % (26/370) des échantillons, impliquant cinq espèces de rongeurs : Apodemus agrarius (36), Niviventer niviventer (75), Rattus losea (18), R. norvegicus (155) et R. tanezumi (86). Leurs taux respectifs de positivité pour Cryptosporidium étaient de 8,3 %, 5,3 %, 11,1 %, 7,1 % et 7,0 %. L'analyse des séquences a confirmé la présence de trois espèces de Cryptosporidium : C. parvum (4), C. viatorum (1) et C. muris (1), et de deux génotypes : Cryptosporidium génotype IV de rat (16) et C. mortiferum-like (4). De plus, deux sous-types de C. parvum (IIdA15G1 et IIpA19) et un sous-type de C. viatorum (XVdA3) ont été détectés. Ces résultats démontrent que diverses espèces de rongeurs sauvages du Zhejiang sont simultanément infectées par des espèces/génotypes de Cryptosporidium zoonotiques et adaptés aux rongeurs, ce qui indique que ces rongeurs peuvent jouer un rôle dans le maintien et la dispersion de ce parasite dans l'environnement et d'autres hôtes, y compris les humains.

Rapid detection of pathogenic E. coli based on CRISPR Cas system.

Access to safe and nutritious food is critical for maintaining life and supporting good health. Eating food that is contaminated with pathogens leads to serious diseases ranging from diarrhea to cancer. Many foodborne infections can cause long-term impairment or even death. Hence, early detection of foodborne pathogens such as pathogenic Escherichia coli strains is essential for public safety. Conventional methods for detecting these bacteria are based on culturing on selective media and following standard biochemical identification. Despite their accuracy, these methods are time-consuming. PCR-based detection of pathogens relies on sophisticated equipment and specialized technicians which are difficult to find in areas with limited resources. Whereas CRISPR technology is more specific and sensitive for identifying pathogenic bacteria because it employs programmable CRISPR-Cas systems that target particular DNA sequences, minimizing non-specific binding and cross-reactivity. In this project, a robust detection method based on CRISPR-Cas12a sensing was developed, which is rapid, sensitive and specific for detection of pathogenic E. coli isolates that were collected from the fecal samples from adult goats from 17 farms in Tennessee. Detection reaction contained amplified PCR products for the pathogenic regions, reporter probe, Cas12a enzyme, and crRNA specific to three pathogenic genes-stx1, stx2, and hlyA. The CRISPR reaction with the pathogenic bacteria emitted fluorescence when excited under UV light. To evaluate the detection sensitivity and specificity of this assay, its results were compared with PCR based detection assay. Both methods resulted in similar results for the same samples. This technique is very precise, highly sensitive, quick, cost effective, and easy to use, and can easily overcome the limitations of the present detection methods. This project can result in a versatile detection method that is easily adaptable for rapid response in the detection and surveillance of diseases that pose large-scale biosecurity threats to human health, and plant and animal production.

Role of miRNA-21 in cancer and its application in electrochemical bioanalysis.

GRAPHICAL ABSTRACT[Formula: see text].

Molecular detection and genetic variability of Cryptosporidium spp. in wild Asian house shrews (Suncus murinus) from southern Zhejiang province, China.

Shrews play a crucial role as repositories for diverse pathogens linked to zoonotic infectious diseases. However, the genetic information regarding Cryptosporidium in Chinese shrews remains unexplored. The objectives of this study were twofold: to determine the occurrence rate of Cryptosporidium spp. in wild shrews residing in the southern part of Zhejiang Province, China, and to investigate their genetic characteristics. A total of 282 wild shrews were captured between April and October of 2023. The detection of Cryptosporidium in fecal samples, collected from each animal's rectum, was performed using PCR and sequencing of the partial small subunit of ribosomal RNA (SSU rRNA) gene. The 60-kDa glycoprotein (gp60) gene was utilized to further subtype the positive samples of C. viatorum and C. parvum. All animals were identified as Suncus murinus, and a positive result for Cryptosporidium was obtained in 14.2 % (40/282) of the samples. The following species and genotypes were identified: C. ratti (n = 19), C. parvum (n = 2), C. viatorum (n = 1), Cryptosporidium rat genotype IV (n = 13), and Cryptosporidium skunk genotype (n = 5). Furthermore, the subtypes IIdA15G1 and XVdA3 were detected within C. parvum and C. viatorum, respectively. Molecular evidence indicates that S. murinus is concurrently infected with rodent-adapted and zoonotic species/genotypes, actively contributing to the dissemination of cryptosporidiosis.

Molecular detection and phylogenetic analysis of Orf viruses from goats in Jiangxi province, China.

Orf is a zoonosis caused by the Orf virus (ORFV), which is endemic in goats, sheep, and wild ruminants worldwide. Orf infection is prevalent in China, with outbreaks reported in several provinces. Currently, there is limited information available regarding the characterization of ORFV strains in Jiangxi province. This study investigated an acute outbreak of Orf that occurred in 2021 in a goat herd in the Jiangxi province of China. Clinical signs in this case included lesions on the lips, nose, and inside the mouth. The presence of ORFV was confirmed from tissue samples by polymerase chain reaction (PCR). The nucleotide sequences of the B2L and F1L genes were fully sequenced and used to construct phylogenetic trees. The results of this investigation identified the ORFV JXxy2021 as the cause of the outbreak. The phylogenetic analysis revealed that the ORFV strain JXxy2021 had the highest similarity to the ORFV strains GO and FJ-SL from the neighboring province of Fujian. This suggests that JXxy2021 was likely transmitted from Fujian province. The results have provided valuable information on the genetic characteristics of JXxy2021 and the endemic situations of Orf in China.

Borrelia theileri infections in Rhipicephalus annulatus ticks from the north of Iran.

Ticks serve as vectors and reservoirs of various Borrelia species, potentially causing diseases in humans and animals. Mazandaran, a fertile green land in northern Iran, provides ample grazing grounds for livestock and harbors at least 26 hard tick species. This study investigated Borrelia infection in hard ticks from forest areas in this region and compared their genetic identity with the species data in the GenBank database. A total of 2,049 ticks were collected manually from mammalian hosts or using dragging and flagging methods. These ticks were then grouped into 190 pools and 41 individuals based on host, species, developmental stage, and gender. A real-time PCR (qPCR) detected Borrelia DNA in 26 pools from female, male, and nymph of Rhipicephalus annulatus (n = 17) and Ixodes ricinus (n = 9) ticks and one individual female Haemaphysalis punctata tick. The generated partial flaB and glpQ sequences from qPCR-positive Rh. annulatus ticks exhibited the highest identities of 98.1-100% and 98.2% with Borrelia theileri and closely related undefined isolates. Additionally, in phylogenetic analysis, these sequences clustered within well-supported clades with B. theileri and the closely related undefined isolates from various geographic regions, confirming the presence of B. theileri in the north of Iran. Divergence in B. theileri flaB and glpQ sequences across various geographical areas suggests potential subspeciation driven by adaptations to different tick species. This divergence in our flaB sequences implies the possible introduction of B. theileri-infected ticks from different geographical origins into Iran.

Enhanced Molecular Interaction of 3D Plasmonic Nanoporous Gold Alloys by Electronic Modulation for Sensitive Molecular Detection.

Three-dimensional gold and its alloyed nanoporous structures possess high surface areas and strong local electric fields, rendering them ideal substrates for plasmonic molecular detection. Despite enhancing plasmonic properties and altering molecular interactions, the effect of alloy composition on molecular detection capability has not yet been explored. Here, we report molecular interactions between nanoporous gold alloys and charged molecules by controlling the alloy composition. We demonstrate enhanced adsorption of negatively charged molecules onto the alloy surface due to positively charged gold atoms and a shifted d-band center through charge transfer between gold and other metals. Despite similar EM field intensities, nanoporous gold with silver (Au/Ag) achieves SERS enhancement factors (EF) up to 6 orders of magnitude higher than those of other alloys for negatively charged molecules. Finally, nanoporous Au/Ag detects amyloid-beta at concentrations as low as approximately 1 fM, with SERS EF up to 10 orders of magnitude higher than that of a monolayer of Au nanoparticles.

Molecular Detection of SARS-CoV-2 From Throat Swabs Performed With or Without Specimen Collection From the Tonsils: Protocol for a Multicenter Randomized Controlled Trial.

BACKGROUND: Testing for SARS-CoV-2 is essential to provide early COVID-19 treatment for people at high risk of severe illness and to limit the spread of infection in society. Proper upper respiratory specimen collection is the most critical step in the diagnosis of the SARS-CoV-2 virus in public settings, and throat swabs were the preferred specimens used for mass testing in many countries during the COVID-19 pandemic. However, there is still a discussion about whether throat swabs have a high enough sensitivity for SARS-CoV-2 diagnostic testing, as previous studies have reported a large variability in the sensitivity from 52% to 100%. Many previous studies exploring the diagnostic accuracy of throat swabs lack a detailed description of the sampling technique, which makes it difficult to compare the different diagnostic accuracy results. Some studies perform a throat swab by only collecting specimens from the posterior oropharyngeal wall, while others also include a swab of the palatine tonsils for SARS-CoV-2 testing. However, studies suggest that the palatine tonsils could have a tissue tropism for SARS-CoV-2 that may improve the SARS-CoV-2 detection during sampling. This may explain the variation of sensitivity reported, but no clinical studies have yet explored the differences in sensitivity and patient discomfort whether the palatine tonsils are included during the throat swab or not. OBJECTIVE: The objective of this study is to examine the sensitivity and patient discomfort of a throat swab including the palatine tonsils compared to only swabbing the posterior oropharyngeal wall in molecular testing for SARS-CoV-2. METHODS: We will conduct a randomized controlled study to compare the molecular detection rate of SARS-CoV-2 by a throat swab performed from the posterior oropharyngeal wall and the palatine tonsils (intervention group) or the posterior oropharyngeal wall only (control group). Participants will be randomized in a 1:1 ratio. All participants fill out a baseline questionnaire upon enrollment in the trial, examining their reason for being tested, symptoms, and previous tonsillectomy. A follow-up questionnaire will be sent to participants to explore the development of symptoms after testing. RESULTS: A total of 2315 participants were enrolled in this study between November 10, 2022, and December 22, 2022. The results from the follow-up questionnaire are expected to be completed at the beginning of 2024. CONCLUSIONS: This randomized clinical trial will provide us with information about whether throat swabs including specimens from the palatine tonsils will improve the diagnostic sensitivity for SARS-CoV-2 molecular detection. These results can, therefore, be used to improve future testing recommendations and provide additional information about tissue tropism for SARS-CoV-2. TRIAL REGISTRATION: ClinicalTrials.gov NCT05611203; https://clinicaltrials.gov/study/NCT05611203. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/47446.

A comparative analysis of real-time quantitative PCR and metabarcoding methods for eDNA-based detection of the toxic dinophyte Alexandrium tamiyavanichii (Dinophyceae).

The marine dinophyte Alexandrium tamiyavanichii is a toxigenic species that produces a group of neurotoxins that is responsible for paralytic shellfish poisoning in humans. Early detection of the species is essential for efficient monitoring. Harmful microalgal monitoring systems have evolved over the years with the advent of environmental DNA (eDNA)-based species detection techniques. In this study, eDNA samples were collected from a large-scale sampling covering the southern South China Sea. The sensitivity and specificity of metabarcoding of the V4 and V9 18S ribosomal DNA barcodes by high-throughput sequencing (HTS) were compared to the species-specific real-time qPCR targeting the A. tamiyavanichii ITS2 region. Environmental samples were screened for A. tamiyavanichii by qPCR (n = 43) and analyzed with metabarcoding (n = 30). Our results revealed a high occupancy profile across samples for both methods; 88% by qPCR, and 80-83% by HTS. When comparing the consistency between the two approaches, only two samples out of 30 were discordant. The V4 and V9 molecular units detected in each sample were positively correlated with the qPCR ITS2 gene copies (V4, rs = 0.67, p < 0.0001; V9, rs = 0.65, p < 0.0001), indicating that metabarcoding could be used as a useful tool for early detection of the species. Our results also revealed that the estimation of A. tamiyavanichii cell abundances based on the HTS read abundances was comparable to that of the qPCR quantification. For long-term monitoring, metabarcoding could serve as a cost-effective screening of detecting not only single HAB species but also simultaneously detecting a multitude of potentially harmful species, which is valuable in informing the subsequent implementation of species-specific monitoring strategies.

Detection of Ochyromera ligustri (Coleoptera: Curculionidae: Curculioninae) in Ligustrum spp. (Oleaceae) Using Newly Developed PCR Primers.

Ligustrum spp. (Oleaceae) have become invasive species in the US and negatively affect native plant diversity and richness in forests. Ochyromera ligustri (Coleoptera: Curculionidae) is considered a potential biological control agent in the US because adults feed on the foliage and larvae are seed-feeders of Ligustrum spp. To discover the relationships between O. ligustri and Ligustrum spp., fruit dissections or rearing and field observations are required. In the current research project, novel PCR primers were developed to rapidly detect the DNA of O. ligustri in molecular analyses without rearing and observation. The developed PCR primers worked even with 0.01 ng of DNA and did not amplify the DNA of the other five curculionid species tested. When the novel primers were tested with three Ligustrum spp. species common in the southeastern US, the DNA of O. ligustri was detected from all three species. We expect that the novel primers will be utilized to find out the presence and impact of O. ligustri on Ligustrum spp rapidly and accurately.

Amphistome Infection and Species Diversity of Freshwater Snails Collected from Selected Wildlife Drinking Water Sources in Matebeleland Region of Zimbabwe.

This study aimed at determining the identity of freshwater snails collected from selected water habitats frequented by wildlife as source of drinking water in the Matebeleland region of Zimbabwe and further screening the identified snails for natural infections with amphistomes using PCR. A total of 487 freshwater snails were collected from six areas in the Matebeleland region of Zimbabwe for identification and screening of amphistome infection. Eight freshwater snail species were morphologically identified and Biomphalaria pfeifferi, Bul. tropicus, Bul. truncatus, Bul. globosus, and L. (R.) natalensis were confirmed using the COI gene. Bulinus tropicus and Phy. acuta were the most abundant species at 33.9% (165/487) and 31.2% (155/487), respectively. DNA of amphistome was detected in 11.9% (58/487) of the collected snails. The highest infection rate was detected in Bul. globosus (44.4%). West Nicholson recorded the highest infection rate (33.9%), and infection was not detected in L. (R.) natalensis, Phy. acuta, and Bellamya spp. Amphistome DNA from M. tuberculata was successfully sequenced and identified as Calicophoron microbothrium. An additional band was detected in M. tuberculata, Bul. tropicus, and Bul. trancatus, which showed a 96.42% similarity to Paragonimus sp. sequence in the GenBank.

Tailoring strategies of SERS tags-based sensors for cellular molecules detection and imaging.

As an emerging nanoprobe, surface enhanced Raman scattering (SERS) tags hold significant promise in sensing and bioimaging applications due to their attractive merits of anti-photobleaching ability, high sensitivity and specificity, multiplex, and low background capabilities. Recently, several reviews have proposed the application of SERS tags in different fields, however, the specific sensing strategies of SERS tags-based sensors for cellular molecules have not yet been systematically summarized. To provide beneficial and comprehensive insights into the advanced SERS tags technique at the cellular level, this review systematically elaborated on the latest advances in SERS tags-based sensors for cellular molecules detection and imaging. The general SERS tags-based sensing strategies for biomolecules and ions were first introduced according to molecular classes. Then, aiming at such molecules located in the extracellular, cellular membrane and intracellular regions, the tailored strategies by designing and manipulating SERS tags were summarized and explored through several key examples. Finally, the challenges and perspectives of developing high performance of advanced SERS tags were briefly discussed to provide effective guidance for further development and extended applications.

Resurgence of common respiratory viruses in patients with community-acquired pneumonia (CAP)-A prospective multicenter study.

BACKGROUND: Community-acquired pneumonia (CAP) is a major global cause of death and hospitalization. Bacteria or community-acquired viruses (CARVs) cause CAP. COVID-19 associated restrictions effectively reduced the circulation of CARVs. OBJECTIVES: The aim of this study was to analyze the proportion of CARVs in adult patients with CAP from mid-2020 to mid-2023. Specifically, we aimed to compare the rate of influenza virus, SARS-CoV-2, and RSV detections in patients aged 18-59 years and ≥60 years. STUDY DESIGN: We analyze the proportion of 21 community-acquired respiratory viruses (CARVs) and three atypical bacteria (Bordetella pertussis, Legionella pneumophila, and Mycoplasma pneumoniae) in nasopharyngeal swab samples using molecular multiplex methods within the prospective, multicentre, multinational study of the German study Group CAPNETZ. We used stringent inclusion criteria throughout the study. RESULTS: We identified CARVs in 364/1,388 (26.2 %) patients. In detail, we detected SARS-CoV-2 in 210/1,388 (15.1 %), rhino-/enterovirus in 64/1,388 (4.6 %), influenza virus in 23/1,388 (1.6 %) and RSV in 17/1,388 (1.2 %) of all patients. We detected RSV and influenza more frequently in patients ≥60 years, especially in 22/23 compared to the previous season. None of the atypical bacteria were detected. CONCLUSIONS: Beginning in 2023, we demonstrate a re-emergence of CARVs in CAP patients. Effective vaccines or specific antiviral therapies for more than two thirds of the detected viral infections are currently available. High detection rates of vaccine-preventable viruses in older age groups support targeted vaccination campaigns.

Development and validation of a multi-target TaqMan qPCR method for detection of Borrelia burgdorferi sensu lato.

Reliable detection of bacteria belonging to the Borrelia burgdorferi sensu lato species complex in vertebrate reservoirs, tick vectors, and patients is key to answer questions regarding Lyme borreliosis epidemiology. Nevertheless, the description of characteristics of qPCRs for the detection of B. burgdorferi s. l. are often limited. This study covers the development and validation of two duplex taqman qPCR assays used to target four markers on the chromosome of genospecies of B. burgdorferi s. l. Analytical specificity was determined with a panel of spirochete strains. qPCR characteristics were specified using water or tick DNA spiked with controlled quantities of the targeted DNA sequences of B. afzelii, B. burgdorferi sensu stricto or B. bavariensis. The effectiveness of detection results was finally evaluated using DNA extracted from ticks and biopsies from mammals whose infectious status had been determined by other detection assays. The developed qPCR assays allow exclusive detection of B. burgdorferi s. l. with the exception of the M16 marker which also detect relapsing fever Borreliae. The limit of detection is between 10 and 40 copies per qPCR reaction depending on the sample type, the B. burgdorferi genospecies and the targeted marker. Detection tests performed on various kind of samples illustrated the accuracy and robustness of our qPCR assays. Within the defined limits, this multi-target qPCR method allows a versatile detection of B. burgdorferi s. l., regardless of the genospecies and the sample material analyzed, with a sensitivity that would be compatible with most applications and a reproducibility of 100% under measurement conditions of limits of detection, thereby limiting result ambiguities.

Advancements in SARS-CoV-2 detection: Navigating the molecular landscape and diagnostic technologies.

According to information from the World Health Organization, the world has experienced about 430 million cases of COVID-19, a world-wide health crisis caused by the SARS-CoV-2 virus. This outbreak, originating from China in 2019, has led to nearly 6 million deaths worldwide. As the number of confirmed infections continues to rise, the need for cutting-edge techniques that can detect SARS-CoV-2 infections early and accurately has become more critical. To address this, the Federal Drug Administration (FDA) has issued emergency use authorizations (EUAs) for a wide range of diagnostic tools. These include tests based on detecting nucleic acids and antigen-antibody reactions. The quantitative real-time reverse transcription PCR (qRT-PCR) assay stands out as the gold standard for early virus detection. However, despite its accuracy, qRT-PCR has limitations, such as complex testing protocols and a risk of false negatives, which drive the continuous improvement in nucleic acid and serological testing approaches. The emergence of highly contagious variants of the coronavirus, such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529), has increased the need for tests that can specifically identify these mutations. This article explores both nucleic acid-based and antigen-antibody serological assays, assessing the performance of recently approved FDA tests and those documented in scientific research, especially in identifying new coronavirus strains.

Molecular Epidemiology and Phylogeny of Theileria ovis and Theileria lestoquardi in Sheep and Goats from Southern Punjab, Pakistan.

Background: Theileria spp. are responsible for ovine and caprine theileriosis, leading to significant morbidity and mortality in small ruminants. The present study aims to investigate Theileria spp. infections in small ruminants from Southern Punjab in Pakistan, and genetic characterize revealed Theileria spp. isolates. Methods: A total of 93 sheep and 107 goats were sampled between May and August 2022. Blood smears were examined microscopically, and PCR amplification targeting the 18S rRNA gene was performed to detect Theileria spp. Additionally, specific PCR assays targeting 18S rRNA and ms1 partial sequences were used to identify Theileria ovis and T. lestoquardi, respectively.  Results: The prevalence of Theileria spp. was significantly higher using PCR (13.5%) compared to microscopic screening (5%). Sheep showed a higher prevalence rate (19.4%) compared to goats (8.4%) (p = 0.024). Young sheep aged ≤ 1 year were more commonly infected with Theileria spp. (41%) compared to older sheep (p = 0.006). The prevalence of Theileria spp. was higher in sheep-only herds (37.3%) compared to goat-only herds (18%) or mixed-species herds (8.1%) (p = 0.015). The prevalence rates of T. ovis and T. lestoquardi were 9% and 2.5%, respectively, with four animals (2 goats and 2 sheep) showing co-infection. Phylogenetic analysis revealed that our T. ovis 18S rRNA sequence clustered with previously reported sequences from sheep in Turkey, China, Spain, and goats in Tanzania. The obtained T. lestoquardi ms1 partial sequence formed a distinct cluster from other T. lestoquardi isolates in Pakistan and neighboring countries.  Conclusion: Theileria spp. co-circulation in Pakistani small ruminants, particularly the presence of T. ovis and T. lestoquardi, highlights the need for attention from animal health decision-makers due to their financial and health impacts.

Rapid Direct Identification of Microbial Pathogens and Antimicrobial Resistance Genes in Positive Blood Cultures Using a Fully Automated Multiplex PCR Assay.

This study assessed the performance of the BioFire Blood Culture Identification 2 (BCID2) panel in identifying microorganisms and antimicrobial resistance (AMR) profiles in positive blood cultures (BCs) and its influence on turnaround time (TAT) compared with conventional culture methods. We obtained 117 positive BCs, of these, 102 (87.2%) were correctly identified using BCID2. The discordance was due to off-panel pathogens detected by culture (n = 13), and additional pathogens identified by BCID2 (n = 2). On-panel pathogen concordance between the conventional culture and BCID2 methods was 98.1% (102/104). The conventional method detected 19 carbapenemase-producing organisms, 14 extended-spectrum beta-lactamase-producing Enterobacterales, 18 methicillin-resistant Staphylococcus spp., and four vancomycin-resistant Enterococcus faecium. BCID2 correctly predicted 53 (96.4%) of 55 phenotypic resistance patterns by detecting AMR genes. The TAT for BCID2 was significantly lower than that for the conventional method. BCID2 rapidly identifies pathogens and AMR genes in positive BCs.