Analysis of serum proteomic profiles of endangered Siamese and Burmese Eld's deer infected with subclinical Babesia bovis in Thailand.

The endangered Eld's deer is a conserved species in Thailand, where tropical parasitic infections are endemic. Although Eld's deer with babesiosis are generally asymptomatic, they can still harbor the parasite and serve as reservoirs for ticks, spreading the infection to healthy animals within the herd. The present study aimed to investigate potential serum proteome biomarkers of Eld's deer with subclinical Babesia bovis infection. A total of 67 blood samples were collected from captive Siamese and Burmese Eld's deer showing no signs of parasitic infection. The nested polymerase chain reaction (nPCR) of a conserved spherical body protein 2 (sbp-2) gene of B. bovis was utilized to classify Eld's deer groups, with 25.37 % (17/67) testing positive for B. bovis. Additionally, the application of proteomic studies showed that six B. bovis proteins, such as Obg-like ATPase 1 (OLA1) and heat shock protein 90 (HSP90), were significantly upregulated by more than a two-fold change compared with the PCR-negative samples. Of the 55 overexpressed serum proteins in the PCR-positives, alpha 2-HS glycoprotein (AHSG) and immunoglobulin lambda variable 2-8 (IGLV2-8) were notably among the top 10 proteins with the highest area under curve (AUC) values. Hence, they were proposed as potential biomarkers for subclinical B. bovis infection in Eld's deer. Analysis of the protein interaction network revealed interactions between Eld's deer AHSG and B. bovis OLA1 and HSP90, alongside associations with other proteins such as erb-b2 receptor tyrosine kinase 2 (ERBB2) and epidermal growth factor receptor (EGFR). These interactions were involved in the immune system pathway and inflammatory responses. Our findings shed light on subclinical infection of B. bovis in Eld's deer and identify potential biomarkers, contributing to the further effective detection and monitoring of B. bovis infection in this endangered species.

Novel Semi-Nested Real-Time PCR Assay Leveraging Extendable Blocking Probes for Improved SHOX2 Methylation Analysis in Lung Cancer.

Lung cancer is the leading cause of cancer deaths globally, necessitating effective early detection methods. Traditional diagnostics like low-dose computed tomography (LDCT) often yield high false positive rates. SHOX2 gene methylation has emerged as a promising biomarker. This study aimed to develop and validate a novel semi-nested real-time PCR assay enhancing sensitivity and specificity for detecting SHOX2 methylation using extendable blocking probes (ExBPs). The assay integrates a semi-nested PCR approach with ExBPs, enhancing the detection of low-abundance methylated SHOX2 DNA amidst unmethylated sequences. It was tested on spiked samples with varied methylation levels and on clinical samples from lung cancer patients and individuals with benign lung conditions. The assay detected methylated SHOX2 DNA down to 0.01%. Clinical evaluations confirmed its ability to effectively differentiate between lung cancer patients and those with benign conditions, demonstrating enhanced sensitivity and specificity. The use of ExBPs minimized non-target sequence amplification, crucial for reducing false positives. The novel semi-nested real-time PCR assay offers a cost-effective, highly sensitive, and specific method for detecting SHOX2 methylation, enhancing early lung cancer detection and monitoring, particularly valuable in resource-limited settings.

Establishment and evaluation of a qPCR method for the detection of pfmdr1 mutations in Plasmodium falciparum, the causal agent of fatal malaria.

Drug resistance surveillance is a major integral part of malaria control programs. Molecular methods play a pivotal role in drug resistance detection and related molecular research. This study aimed to develop a rapid and accurate detection method for drug resistance of Plasmodium falciparum (P. falciparum). A quantitative real-time PCR (qPCR) assay has been developed that identifies the mutation at locus A256T in the P.falciparum multi-drug resistance(pfmdr1) gene producing amino acid change at position 86. The results of 198 samples detected by qPCR were consistent with nested PCR and sequencing, giving an accuracy of 94.3%. The sensitivity, specificity, positive and negative predictive value of qPCR were 85.7%, 97.6%, 90.0% and 96.4%, respectively. The results of qPCR are basically consistent with the nested PCR, which is expected to replace the nested PCR as a new molecular biological method for drug resistance detection, providing reliable technical support for global malaria prevention and control.

Rapid Molecular Diagnosis of Sporotrichosis Directly from Biological Samples from a Reference Center in Brazil.

The gold standard diagnosis of sporotrichosis is the isolation of Sporothrix sp. in culture media, but this is a time-consuming test that is susceptible to contamination and can be affected by the fungal load. Molecular methods such as nested PCR are gaining more ground in the management of several infections as they are tools for the rapid and accurate identification of microorganisms from pure cultures or directly from biological samples. This study aimed to apply a nested PCR molecular protocol for the rapid detection of Sporothrix spp. directly from clinical samples. Thirteen samples-six from skin biopsies, five from skin exudates, and two from conjunctival secretions-were obtained from patients diagnosed with sporotrichosis due to S. brasiliensis. Calmodulin gene sequencing identified all the isolates as S. brasiliensis. Nested PCR was able to detect all the Sporothrix sensu lato directly from clinical samples as well as the CBS 120339 reference strain. The nested PCR protocol stands out as a diagnostic alternative, as it allows the identification of Sporothrix spp. directly from clinical samples without the need for fungal isolation.

Impact of whole grain highland hull-less barley on the denaturing gradient gel electrophoresis profiles of gut microbial communities in rats fed high-fat diets.

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) is a traditional non-culture technique that can provide a fingerprint of the microbial community. In the field of gut microbiota analysis, PCR-DGGE still holds potential for development. In the present study, we utilized an improved nested PCR-DGGE approach targeting the V3 region of 16S ribosomal DNA to investigate the impact of whole grain highland hull-less barley (WHLB), a cereal known for its significant hypocholesterolemic effect, on the gut microbiota profiles of high-fat diet rats. Seventy-two male Sprague-Dawley rats were divided into four groups and fed a normal control diet, a high-fat diet, or a high-fat diet supplemented with a low or high dose of WHLB for 4 or 8 weeks. The results revealed that the dominant bands varied among different dose groups and further changed with different treatment times. The compositions of bacterial communities in feces and cecal content were similar, but the dominant bacterial bands differed. After performing double DGGE, extracting the bands, sequencing the DNA, and aligning the sequences, a total of 19 bands were classified under the Firmicutes and Bacteroidetes phyla, while two bands were identified as unclassified uncultured bacteria. The relative abundance of Lactobacillus gasseri, Uncultured Prevotella sp., and Clostridium sp. increased following the administration of WHLB. Illumina-based sequencing was employed to assess the reliability of DGGE, demonstrating its reliability in analyzing the dominant taxonomic composition, although it may have limitations in accurately detecting the alpha diversity of bacterial species. IMPORTANCE: While next-generation sequencing has overshadowed polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), the latter still holds promise for advancing gut microbiota analysis due to its unique advantages. In this study, we used optimized nested PCR-DGGE to investigate the gut microbiota profile of high-fat diet rats after administering whole grain highland hull-less barley. High-throughput sequencing was employed to validate the DGGE results. Our results proved the reliability of PCR-DGGE for analyzing the dominant taxonomic composition while also providing visual evidence of a notable relationship between the composition of cecal and fecal microbial communities, highlighting substantial differences in both richness and abundance.

Detection of small ruminant Lentivirus proviral DNA in red deer from Poland.

Small ruminant lentiviruses (SRLVs) are widespread and infect goats and sheep. Several reports also suggest that SRLVs can infect wild ruminants. The presence of specific antibodies against SRLVs has been identified in wild ruminants from Poland, but no studies have been conducted to detect proviral DNA of SRLVs in these animals. Therefore, the purpose of this study was to examine samples from Polish wild ruminants to determine whether these animals can serve as reservoirs of SRLVs under natural conditions. A total of 314 samples were tested from red deer (n = 255), roe deer (n = 52) and fallow deer (n = 7) using nested real-time PCR. DNA from positive real-time PCR samples was subsequently used to amplify a CA fragment (625 bp) of the gag gene, a 1.2 kb fragment of the pol gene and an LTR-gag fragment. Three samples (0.95%) were positive according to nested real-time PCR using primers and probe specific for CAEV (SRLV group B). All the samples were negative for the primers and probe specific for MVV (SRLV A group). Only SRLV LTR-gag sequences were obtained from two red deer. Phylogenetic analysis revealed that these sequences were more closely related to CAEV than to MVV. Our results revealed that deer can carry SRLV proviral sequences and therefore may play a role in the epidemiology of SRLVs. To our knowledge, this is the first study describing SRLV sequences from red deer.

Molecular detection of Histoplasma capsulatum in organ samples from bats randomly captured in urban areas of Araraquara, São Paulo state, Brazil.

The mycosis histoplasmosis is also considered a zoonosis that affects humans and other mammalian species worldwide. Among the wild mammals predisposed to be infected with the etiologic agent of histoplasmosis, bats are relevant because they are reservoir of Histoplasma species, and they play a fundamental role in maintaining and spreading fungal propagules in the environments since the infective mycelial phase of Histoplasma grows in their accumulated guano. In this study, we detected the fungal presence in organ samples of bats randomly captured in urban areas of Araraquara City, São Paulo, Brazil. Fungal detection was performed using a nested polymerase chain reaction to amplify a molecular marker (Hcp100) unique to H. capsulatum, which revealed the pathogen presence in organ samples from 15 out of 37 captured bats, indicating 40.5% of infection. Out of 22 Hcp100-amplicons generated, 41% corresponded to lung and trachea samples and 59% to spleen, liver, and kidney samples. Data from these last three organs suggest that bats develop disseminated infections. Considering that infected bats create environments with a high risk of infection, it is important to register the percentage of infected bats living in urban areas to avoid risks of infection to humans, domestic animals, and wildlife.

Molecular detection and phylogenetic analysis of Borrelia Spp. In blood samples of cats and dogs by the nested-PCR method in West Azerbaijan Province, Iran.

The objective of this study was to investigate the presence and genetic attributes of Borrelia spp. in cats and dogs from the West Azerbaijan Province, located in the northwest of Iran. A total of 250 blood samples from cats and 300 blood samples from dogs were collected, and information regarding their age, sex, breed, ownership status, sampling time and region was recorded. The identification of positive samples was accomplished through nested-PCR and sequencing, with subsequent analysis of the gene sequences conducted using BioEdit software. The gene sequences for Borrelia spp. in this study showed 100% similarity to reference sequences in the GenBank® database. Phylogenetic trees were built using MEGA11. The outcomes indicated that among 250 blood samples from cats, 48 (19.2%) tested positive for Borrelia spp. gene, with a CI from 14.8 to 24.53% for cats. Similarly, out of 300 blood samples from dogs, 45 (15%) tested positive for the Borrelia spp. gene, with a CI from 11.4 to 19.48% for dogs.

Establishment of Nested PCR for the Detection of Pseudomonas plecoglossicida and Epidemiological Survey of Larimichthys crocea in the Southeast Coastal Region.

The visceral white nodules disease in the internal organs of Larimichthys crocea has caused significant harm in the aquaculture of this species, with Pseudomonas plecoglossicida considered one of the core pathogens causing this disease. In this study, we designed three pairs of specific nested PCR primers targeting the sctU gene of P. plecoglossicida, a crucial component of the Type III secretion system (T3SS), which is instrumental in bacterial pathogenesis and virulence. Through the optimization of PCR reaction conditions, specificity testing, and sensitivity determination, a method was established for the accurate detection of P. plecoglossicida. This method yielded single amplification products, exhibited a false positive rate of zero for reference bacteria, and achieved a detection sensitivity of a minimum of 2.62 copies/reaction for the target sequence. Using the detection method, we conducted analyses on the diseased populations of L. crocea, involving a total of 64 screened fishes along the southeast coast of China from 2021 to 2023. The results revealed that the infection rate of P. plecoglossicida in diseased L. crocea exceeded over 90% in March and April, while in other months, the maximum recorded infection rate was merely 10%. The detection method developed in this study shows potential for early warning and routine monitoring of visceral white nodules disease in the internal organs of species such as L. crocea.

Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples.

The aim of the present study was to compare the performance of a nested polymerase chain reaction (nPCR) and a real-time PCR based on the amplification of the HlyA gene from Listeria monocytogenes using a plasmid DNA standard. Nested PCR was developed with an internal amplification control (IAC). Both techniques were validated in soft cheese samples by comparing their results with the results of the microbiological reference method ISO 11290-1:2017. Cheese samples artificially contaminated with 3.5 to 3,500 UFC/25 g were processed by ISO 11290-1:2017 and, at several times of culture, DNA samples were extracted. All cheeses contaminated with L. monocytogenes were positive for the microbiological method 96 h post contamination and for nPCR and real-time PCR 48 h post contamination. At this time, the HlyA gene was amplified in all contaminated samples. Both molecular techniques showed the same sensitivity, 30 copies/reaction or 3.5 UFC/25 g, when plasmid DNA standard or artificially contaminated cheese samples were used. Finally, eighty soft cheese samples obtained from local retail stores and tested by three methods were negative, indicating a 100% concordance in results. The development of an nPCR with IAC reinforces the reliability of the negative results without increasing the costs of the reaction. Besides, nPCR showed less sensitivity to the presence of inhibitory substances in the reaction. The use of one of these molecular techniques could be easily coupled to the microbiological method, serving as a screening method in the food industry for hygiene monitoring and early identification of contaminated foods.

Development of a novel sensitive single-tube nested PCR assay for the detection of African swine fever virus.

African swine fever (ASF) is a highly fatal and contagious viral disease caused by African swine fever virus (ASFV). It has caused significant economic losses to the swine industry and poses a serious threat to food security worldwide. Diagnostic tests with high sensitivity are essential for the effective management of ASF. Here, we describe a single-tube nested PCR (STN-PCR) assay for the detection of ASFV in which two consecutive amplification steps are carried out within a single tube. Two pairs of primers (outer and inner) were designed to target the p72 gene of ASFV. The primer concentrations, annealing temperatures, and number of amplification cycles were optimized to ensure the consecutive utilization of outer and inner primer pairs during amplification while minimizing the likelihood of amplicon contamination. In comparison with two conventional endpoint PCR assays (one of which is recommended by the World Organization for Animal Health), the newly developed STN-PCR assay demonstrated a 100-fold improvement in the limit of detection (LOD), detecting 100 copies of ASFV genomic DNA, whereas the endpoint PCR assays could detect no fewer than 10,000 copies. The clinical performance of the STN-PCR assay was validated using 95 tissue samples suspected of being positive for ASFV, and the assay showed 100% specificity. A Cohen's kappa value of 0.91 indicated perfect agreement between the assays. This new STN-PCR assay is a potentially valuable tool that will facilitate the control of ASF.

Prevalence of Epstein Barr Virus and Herpes Simplex Virus Among Human Papillomavirus Negative Oral Cancer Patients: A Cross-Sectional Study from South India.

The high incidence of oral carcinomas is due to its multifactorial etiology and the presence of various risk factors. Human Papillomavirus (HPV) has a proven role in the pathogenesis of oral carcinomas, but in the recent times there has been an increasing incidence of oral cancers who are negative for HPV infection. Also, these patients are non-smokers and non-drinkers so it could be speculated that these oral cancers are due to some other etiological factor probably of other viral infections. Therefore, this study examined the prevalence of Epstein Barr Virus (EBV) and Herpes Simplex Virus (HSV) among oral cancer patients. This cross-sectional study was conducted from January 2019 to June 2020. Biopsy samples from 47 newly diagnosed untreated patients with oral malignancies were collected along with their demographic and clinicopathological information. DNA extracted from the biopsies was processed for nested PCR for the detection of EBV and HSV. All the samples tested negative for HPV and HSV infection. Nested PCR detected 29 cases (70.7%) to be positive for EBV. The non-cancerous adjacent tissues also were negative for HPV, EBV and HSV. The prevalence of EBV was found to be more in males (62.1%) and the highest number of cases was of the left buccal mucosa compromising 34% of the total cases. From the present study it can be concluded that EBV but not HSV infection is associated with an increased risk of developing oral cancers. Although, 70.7% of the patients were found to be positive for EBV whether the viral infection played any role in the driving the malignancy needs to be further elucidated.

Molecular and Serological Investigation of Infectious Bronchitis Virus in the East Shewa, Central Ethiopia.

Background: Infectious bronchitis (IB) is an economically important disease in poultry with worldwide distribution. The occurrence of IB has been reported both in commercial and backyard poultry in Ethiopia, although comprehensive information lacks available prevalence of the disease and the circulating serotypes. Methods: A cross-sectional study was conducted from November 2021 to June 2022 in seven commercial farms found in East Shewa, Central Ethiopia. Serological assay using indirect ELISA, virus isolation techniques in embryonated eggs, and molecular techniques such as one-step reverse transcriptase polymerase chain reaction (RT-PCR) and nested polymerase chain reaction (PCR) targeting a 466 bp S1 gene were employed. Results: A total of 196 blood samples, 7 pools (35) of swab samples, and 5 pools of tracheal samples were investigated. The results of serological analysis revealed that 97.96% (192/196; 95% CI: 94.86-99.44) of the sera samples were found to be positive for antibodies against IBV. Out of the 7 pools of swab and 5 pools of tracheal tissue samples analyzed using RT-PCR 33.3% (4/12) of them gave positive results all from swab samples. The RT-PCR-positive samples were subjected to a nested PCR yielding 295bp and 154bp indicating the circulation of Mass and 793/B (4/91) strains of IBV, respectively. The 12 pools of samples inoculated into embryonated egg showed cytopathic changes such as congestion, bleeding, and deformation only after three passages. Conclusion: Two serotypes of IBV are circulating in Ethiopian chickens, and molecular identification of the Massachusetts serotype is the first report in Ethiopia. Further epidemiological investigation is needed in order to devise effective control measures.

Genetic Characterization of the RAP-1A and SBP-4 Genes of Babesia Species Infecting Cattle from Selangor, Malaysia, and Ribah, Nigeria.

Bovine babesiosis has substantial economic implications in the cattle industry, emphasizing the need for a thorough understanding of the genetic diversity of the causative apicomplexan pathogen. Although babesiosis has been extensively studied globally, the genetic diversity of Babesia species in Malaysian and Nigerian cattle remains unreported. This study aims to bridge this gap by detecting and characterizing Babesia species in selected cattle herds. Our investigation explores the genetic diversity of Babesia species in cattle from Selangor, Malaysia, and Ribah, Nigeria. Blood samples revealed a 32.9% infection rate via PCR analysis. Further genetic analysis detected variations in Malaysian Babesia bigemina isolates but genetic similarity among Nigerian isolates. Conversely, all Babesia bovis isolates displayed genetic homogeneity. In summary, this research identifies genetic diversity in Babesia species affecting Malaysian and Nigerian cattle, highlighting regional disparities.

Single primer site-specific nested PCR for accurate and rapid genome-walking.

Genome-walking is a molecular tool used to unveil uncharacterized DNA regions flanking a known DNA, which has been widely used in bioscience and related areas. This study developed a reliable and efficient PCR-based genome-walking approach, named as single primer site-specific nested PCR (SPN-PCR). A SPN-PCR set sequentially consists of three single-primer nested PCR amplifications. The primary relaxed thermal cycle promotes outmost nested site-specific primer (NSSP) to partially combine with numerous places on DNA template, synthesizing many single-stranded DNAs (ssDNA). Among them, the target ssDNA is exponentially amplified in the subsequent stringent cycles, as its 3' part possesses the outmost NSSP complement; but a non-target ssDNA cannot be amplified, because it does not possess such a complement. Stringent secondary and tertiary PCRs also exclusively enrich this target DNA. Finally, the target DNA product becomes predominant. The feasibility of SPN-PCR was validated by genome-walking several selected genes from two divergent species.

Epidemiological investigation of Cryptosporidium in children with diarrhea in middle Inner Mongolia, China.

Cryptosporidia (Cryptosporidium) is a protozoan that is widely parasitic in the intestinal cells of humans and animals, and it is also an important zoonotic parasite. However, there is no epidemiological investigation on Cryptosporidium spp. infection in infants with diarrhea of Inner Mongolia, the largest livestock region in China. To investigate the prevalence of Cryptosporidium, 2435 fresh fecal samples were collected from children with diarrhea in Inner Mongolia Maternal and Child Health Care Hospital. Molecular characterization of Cryptosporidium was carried out based on its 18S rRNA and gp60 gene sequences. The overall prevalence was 12.85% (313/2435), and in Hohhot (12.15%), it was lower than that in the surrounding city (14.87%) (P < 0.05). Moreover, Cryptosporidium was detected in different seasons and sexes. Concerning the age of children with diarrhea, the prevalence of those age groups between 0 and 1 was obviously lower than others, and there were significant differences in the prevalence at different ages (P < 0.001). Analysis of the 18S rRNA gene sequence revealed that all the positive samples were Cryptosporidium parvum, and there were 5 subtypes (IIdA23G3, IIdA24G3, IIdA24G4, IIdA25G3, and IIdA25G4). To the best of our knowledge, the above subtypes have not been reported. Our results provide a relevant basis for control and education on food safety and foodborne illness prevention.

Confirmatory Assay for Laboratory Diagnosis of Malaria Using Molecular Approach.

BACKGROUND: Prompt malarial treatment and surveillance is crucial for accurate diagnosis of Plasmodium Sp. Gold standard microscopic examination has been widely applied for diagnosis of malaria in most part of the endemic areas. But in case of submicroscopic and asymptomatic microscopic diagnosis is questioned. The study aims to develop a simple, cost effective & robust nucleic acid amplification technique for the detection of malaria parasite. METHODS: Study population included 50 clinically diagnosed positive malaria patient samples from various pathological laboratories. Microscopy by preparing thick film was carried out of every sample for primary screening in the available facility of Surat Raktadan Kendra & Research Centre- Blood Bank. The conventional PCR (Polymerase Chain Reaction) was applied for genus-specific amplification targeting the 18 S rRNA gene of Plasmodium. Agarose gel electrophoresis was used to separate and analyze the amplified PCR product using 2% Agarose gel. RESULTS AND CONCLUSION: The study shows that nested PCR not only detected all microscopic positive samples, but also detected submicroscopic infections that were missed or misread by microscopy. Hence, the sensitivity of molecular based detection technique is proved to be more compared to microscopic examination.

Agreement between Clinical Assessment and Laboratory Diagnosis of Ringworm in Calves at Auction Markets.

To limit the spread of bovine ringworm, control measures such as movement restrictions are highly recommended. In this context, calves at auction markets in Styria, Austria, displaying skin lesions characteristic for bovine ringworm, are excluded from the auctions. To investigate whether these clinical assessments correspond to laboratory diagnosis, a total of 166 samples taken from skin lesions assigned to the three clinical categories 'ringworm very likely (v), likely (l) or unlikely (u)' were mycologically examined using microscopy, culture, and nested PCR followed by amplicon sequencing. Further, the relationships of isolated dermatophytes were determined through multi-locus sequence typing (MLST). Overall, a high agreement between clinical assessment and laboratory results were observed with microscopy and nested PCR, providing more consistent results and molecular detection possessing an analytical sensitivity superior to that of cultural isolation (culture 21.7% vs. nested PCR 48.2%). Phylogenetic analyses revealed that most of the isolated dermatophytes belong to a unique Trichophyton verrucosum MLST genotype. In conclusion, clinical assessments were largely confirmed through laboratory diagnosis with nested PCR and sequencing, providing rapid, sensitive, and species-specific detection of dermatophytes in calves at auction markets displaying skin lesions typical for ringworm; this seems to be predominantly caused by a single Trichophyton verrucosum strain.

Toxoplasma gondii molecular detection and phylogenetic analysis in hemodialysis patients from Khuzestan, Southwest Iran.

BACKGROUND: The diagnosis and genetic characterization of Toxoplasma gondii (T. gondii) infection can make a significant influence to the prevention of the dangerous consequences of toxoplasmosis, particularly in immunocompromised people. OBJECTIVE: The aim of this investigation was to assess the frequency and genotyping of T. gondii in blood samples of patients with hemodialysis. MATERIALS AND METHODS: In the current investigation, a total of 379 blood samples were taken from subjects with hemodialysis who were referred to teaching hospital of Ahvaz in the southwest of Iran. The samples were evaluated using the Nested PCR by targeting the B1 gene, and then, sequencing and phylogenetic tree were constructed. RESULTS: T. gondii DNA was found in 112 (29.55%) of the blood samples by Nested PCR. Amplicons from T. gondii revealed high identity with GenBank sequences. The phylogenetic analysis revealed that all sequences were closely related to Type I of T. gondii. CONCLUSION: Because of the high incidence of toxoplasmosis with type I prevalent in hemodialysis patients, we recommend a systematic screening for toxoplasmosis to carry out for monitoring the possible dissemination of toxoplasmosis during hemodialysis.

A Comparative Analysis of Molecular Biological Methods for the Detection of SARS-CoV-2 and Testing the In Vitro Infectivity of the Virus.

The virus discovered in 2019 in the city of Wuhan, China, which was later identified as SARS-CoV-2 and which spread to the level of a pandemic, put diagnostic methods to the test. Early in the pandemic, we developed a nested PCR assay for the detection of SARS-CoV-2, which we validated and applied to detect the virus in feline samples. The present study describes the application of the nested PCR test in parallel with LAMP for the detection of the virus in 427 nasopharyngeal and oropharyngeal human samples taken between October 2020 and January 2022. Of the swabs tested, there were 43 positives, accounting for 10.1% of all samples tested, with the negatives numbering 382, i.e., 89.5%, and there were 2 (0.4%) invalid ones. The nPCR results confirmed those obtained by using LAMP, with results concordant in both methods. Nasal swabs tested using nPCR confirmed the results of oropharyngeal and nasopharyngeal swab samples tested using LAMP and nPCR. The focus of the discussion is on the two techniques: the actual practical application of the laboratory-developed assays and the diagnostic value of nasal samples. The nPCR used is a reliable and sensitive technique for the detection of SARS-CoV-2 in nasopharyngeal, oropharyngeal, and nasal swab samples. However, it has some disadvantages related to the duration of the entire process, as well as a risk of contamination. Experiments were performed to demonstrate the infectivity of the virus from the positive isolates in vitro. A discrepancy was reported between direct and indirect methods of testing the virus and accounting for its ability to cause infection in vitro.