Use Of RT-PCR Research Articles

Association of Oncotype-DX HER2 Single Gene Score With HER2 Expression Assessed by Immunohistochemistry in HER2-low Breast Cancer.

"HER2-low" is an emerging subtype of breast cancer, with a documented role in predicting response to treatment with novel antibody-drug conjugates. It is defined based on immunohistochemistry, but increasing evidence is challenging this approach as appropriate for identifying the HER2-low subgroup, due to both interobserver variability and limitations of the method itself. We retrospectively analyzed data from 430 patients from our departmental databases who had been subjected to an Oncotype-DX score and assessed the correlation of the Oncotype-DX HER2 single-gene score with the HER2 expression on immunohistochemistry. The Oncotype-DX Recurrence Score was also evaluated in the HER2-0 versus HER2-low subgroups. The HER2 single-gene score was found to accurately correlate with the HER2 result on immunohistochemistry, with a statistically significant difference both between HER2-0 and HER2 +1 tumors (p<0.0001), as well as between HER2 +1 and +2 tumors (p<0.0001). There was no statistically significant difference in the recurrence score between the HER2-0 and the HER2-low subgroups. Oncotype-DX single-gene scores for HER2 are a potential surrogate marker for assessing the precise HER2 status, with better reproducibility and less interobserver variance compared to immunohistochemistry. The use of rt-PCR emerges as an alternative method of assessment of the HER2-low subgroup.

Use of RT-PCR in conjunction with a respiratory pathogen assay to concurrently determine the prevalence of bacteria and SARS-CoV-2 from the nasopharynx of outpatients.

COVID-19 has emerged as a highly contagious and debilitating disease caused by the SARS-CoV-2 virus and has claimed the lives of over 7.7 million people worldwide. Bacterial co-infections are one of many co-morbidities that have been suggested to impact the outcome of COVID-19 in patients. The goals of this study are to elucidate the presence of bacteria in the nasopharynx of SARS-CoV-2 positive and negative patients and to describe demographic categories that may be associated with the detection of these organisms during one of the initial waves of the COVID-19 pandemic. To this end, we investigated SARS-CoV-2 and bacterial co-detection from outpatient RT-PCR testing in Texas. The results indicate that Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae were the most frequently detected bacteria in both SARS-CoV-2 positive and SARS-CoV-2 negative patients and that these bacteria were present in these two patient populations at similar proportions. We also detected Staphylococcus aureus in a significantly larger proportion of males relative to females and people under 65 years of age relative to those 65 and over. Finally, we observed that SARS-CoV-2 was more commonly detected in Hispanics compared to non-Hispanics; however, low disclosure rates make volunteer bias a concern when interpreting the effects of demographic variables. This study describes the bacteria present in the nasopharynx of SARS-CoV-2 positive and negative patients, highlights associations between patient demographics and SARS-CoV-2 as well as bacterial co-detection. In addition, this study highlights RT-PCR based molecular testing as a tool to detect bacteria simultaneously when SARS-CoV-2 tests are performed.

Sequential infection of human norovirus and Salmonella enterica resulted in higher mortality and ACOD1/IRG1 upregulation in zebrafish larvae

Human norovirus (HNoVs) and Salmonella are both very important foodborne pathogens with mixed infection of HNoV and Salmonella reported clinically. With the use of model organism zebrafish (Danio rerio), it was observed that the sequential infection of HNoVs and Salmonella caused lower survival rates (12.5 ± 4.2%) than the single-pathogen infection by Salmonella (31.6 ± 7.3%, P < 0.05) or HNoVs (no mortality observed). Gene expression study with the use of RT-PCR and global transcriptomic analysis revealed that the mortality of zebrafish larvae was very likely due to the harmful inflammatory responses. Specifically, it was noted that the genes encoding aconitate decarboxylase 1 (ACOD1), also known as immunoresponsive gene 1 (IRG1), were significantly upregulated in the sequentially infected zebrafish larvae. The expression of acod1 could lead to mitochondrial reactive oxygen species (ROS) production. The ROS levels were indeed higher in sequentially infected zebrafish larvae than the single-pathogen infected ones (P < 0.05). An immersion treatment of glutathione or citraconate did not affect the microbial loads of HNoVs and Salmonella but significantly reduced the ROS levels and protected the zebrafish larvae by inducing higher survival rates in the sequentially infected zebrafish larvae (P < 0.05). Taken together, this study accumulated new knowledge over the function of ACOD1/IRG1 pathway in infectious diseases, and proposed possible treatment strategies accordingly.

Cost-effectiveness analysis of COVID-19 tests in the unified health system

BackgroundTo evaluate the cost-effectiveness ratio and economic impact of the Rapid Antigen Test (TR-Ag) to replace RT-PCR for the detection of the new Coronavirus in the Unified Health System (SUS).MethodsThis is a cost-effectiveness analysis. Clinical protocols were used for the diagnosis of COVID-19 at the São José Municipal Hospital, located in the city of Itaberá-SP. The Incremental Cost-Effectiveness Ratio (ICER) was divided into two scenarios. In the first, the accuracy reported by the test manufacturers was included, and in the second, the cost resulting from a systematic review. Both were compared with the performance of the RT-PCR test. The increase in diagnoses was chosen as a health outcome and absenteeism was used as a criterion for assessing the economic impact.ResultsThe analysis resulted in incremental cost-effectiveness ratios of R$ 42,136.67 and R$ 68,329.73 for every thousand tests, according to the accuracy of the manufacturers’ TR-Ag tests and what is reported in the literature in relation to RT-PCR, respectively. The average value found for the RT-PCR test (R$ 202.87) represents an increase of 165.32% in cost in relation to the value found for the TR-Ag. 4,305 tests were performed between April 2020 and December 2021 at the referral hospital. Also, maintaining the use of RT-PCR as the first choice for diagnosing COVID-19 and regulating absenteeism in the economically active population could have an impact of up to R$ 1,022,779.68 on municipal management.ConclusionIt is concluded that the TR-Ag are configured as a cost-effective alternative for the SUS in the detection of the new Coronavirus. The strategy becomes economically favorable for the expansion of testing, combating the COVID-19 pandemic and reducing the impact on the local economy. However, studies are needed to validate the accuracy of the tests so that economic evaluations on the subject are more assertive.

The use of RT-PCR in the diagnosis and differentiation of vaccine strains of chicken infectious bronchitis and Newcastle disease.

Infectious diseases of young and adult birds with respiratory syndrome are a significant deterrent to the development of industrial poultry farming due to decreased productivity and significant mortality. The only effective method of combating viral diseases is timely and targeted vaccination, which largely depends on laboratory diagnostic results. This article aims to study the real-time reverse transcription polymerase chain reaction, (RT-PCR) which has the prospect of more effective diagnosis of vaccine strains of chicken infectious bronchitis and Newcastle disease. The fastest and most accurate method for the differential diagnosis of pathogens in an associative viral infection is RT-PCR. The method proposed in the article for selecting primers for amplification made it possible to use this method for the simultaneous interspecies differential diagnosis of two or more viral agents, significantly accelerating their diagnosis. The correlation of the nucleotide sequence obtained from sequencing to a specific virus strain is complicated by the lack of a single nomenclature mechanism for separating genetic groups. The results of this study will allow easy and fast typing of sequences into known and databased virus strains and avoid further confusion in the nomenclature of genetic groups in the future.

Newcastle Disease Genotype VII Prevalence in Poultry and Wild Birds in Egypt.

Newcastle Disease Virus (NDV) genotype VII is a highly pathogenic Orthoavulavirus that has caused multiple outbreaks among poultry in Egypt since 2011. This study aimed to observe the prevalence and genetic diversity of NDV prevailing in domestic and wild birds in Egyptian governorates. A total of 37 oropharyngeal swabs from wild birds and 101 swabs from domestic bird flocks including chickens, ducks, turkeys, and pelicans, were collected from different geographic regions within 13 governorates during 2019–2020. Virus isolation and propagation via embryonated eggs revealed 91 swab samples produced allantoic fluid containing haemagglutination activity, suggestive of virus presence. The use of RT-PCR targeted to the F gene successfully detected NDV in 85 samples. The geographical prevalence of NDV was isolated in 12 governorates in domestic birds, migratory, and non-migratory wild birds. Following whole genome sequencing, we assembled six NDV genome sequences (70–99% of genome coverage), including five full F gene sequences. All NDV strains carried high virulence, with phylogenetic analysis revealing that the strains belonged to class II within genotype VII.1.1. The genetically similar yet geographically distinct virulent NDV isolates in poultry and a wild bird may allude to an external role contributing to the dissemination of NDV in poultry populations across Egypt. One such contribution may be the migratory behaviour of wild birds; however further investigation must be implemented to support the findings of this study. Additionally, continued genomic surveillance in both wild birds and poultry would be necessary for monitoring NDV dissemination and genetic diversification across Egypt, with the aim of controlling the disease and protecting poultry production.

Sex-based differences in conjunctival goblet cell responses to pro-inflammatory and pro-resolving mediators

Many conjunctival inflammatory diseases differ between the sexes and altered conjunctival goblet cells (CGCs) response is often involved. Inflammation is initiated by the release of pro-inflammatory mediators and terminated by the biosynthesis of specialized pro-resolution mediators (SPMs). Herein, we determined the sex-based difference in the responses of CGCs to inflammatory stimuli or pro-resolving lipid SPMs and their interaction with sex hormones. GCs were cultured from pieces of human conjunctiva in RPMI media. CGCs were transferred 24 h before the start of experiments to phenol red-free and FBS-free media to minimize exogenous hormones. RT-PCR, immunofluorescence microscopy (IF), and Western Blot (WB) were performed to determine the presence of sex hormone receptors. Cellular response to pro-inflammatory stimuli or SPMs was studied by measuring the increase in intracellular [Ca2+] ([Ca2+]i) using fura 2/AM microscopy. Use of RT-PCR demonstrated estrogen receptor (ER) α in 4/5 males and 3/3 females; ERβ in 2/4 males and 2/3 females; and androgen receptors (AR) in 3/3 male and 3/3 female CGCs. Positive immunoreactivity by IF and protein expression by WB was detected using antibodies for the ERα and ERβ in 3/3 males and 3/3 females, while AR were only present in males. Significantly different Ca2+ responses between sexes were found with carbachol only at 10–3 M, but not with histamine or leukotriene (LT) B4 at any concentration used. Incubation with dihydrotestosterone (DHT), estrone (E1), or estradiol (E2) at 10–7 M for 30 min significantly inhibited the LTB4-stimulated [Ca2+]i increase in male and female CGCs. Incubation with DHT, E1, and E2 overnight significantly inhibited the LTB4 response in females, while DHT and E2 significantly inhibited the LTB4 response in males. The SPM lipoxin A4 (LXA4) (10–9–10−8 M), but not the resolvins D1 or D2, induced an [Ca2+]i increase that was significantly higher in males compared to females. We conclude that male and female CGCs showed differences in the expression of sex hormone receptors. Treatment with sex hormones altered pro-inflammatory mediator LTB4-induced response. Males compared to females have a higher response to the ω-6-fatty acid derived SPM LXA4, indicating males may terminate inflammation in conjunctival goblet cells faster than females.

Investigation of foot and mouth disease virus and other animal pathogens in cattle, buffaloes and goats at the interface with Akagera National Park 2017 – 2020

BackgroundFoot-and-Mouth Disease Virus (FMDV) is a positive-sense RNA virus of the family of the picornaviridæ that is responsible for one of the livestock diseases with the highest economic impact, the Foot-and-Mouth Disease (FMD). FMD is endemic in Rwanda but there are gaps in knowing its seroprevalence and molecular epidemiology. This study reports the FMD seroprevalence and molecular characterization of FMDV in Eastern Rwanda.ResultsThe overall seroprevalence of FMD in the study area is at 9.36% in cattle and 2.65% in goats. We detected FMDV using molecular diagnostic tools such as RT-PCR and RT-LAMP and the phylogenetic analysis of the obtained sequences revealed the presence of FMDV serotype SAT 2, lineage II. Sequencing of the oropharyngeal fluid samples collected from African buffaloes revealed the presence of Prevotela ruminicola, Spathidium amphoriforme, Moraxella bovoculi Onchocerca flexuosa, Eudiplodinium moggii, Metadinium medium and Verrucomicrobia bacterium among other pathogens but no FMDV was detected in African buffaloes.ConclusionsWe recommend further studies to focus on sampling more African buffaloes since the number sampled was statistically insignificant to conclusively exclude the presence or absence of FMDV in Eastern Rwanda buffaloes. The use of RT-PCR alongside RT-LAMP demonstrates that the latter can be adopted in endemic areas such as Rwanda to fill in the gaps in terms of molecular diagnostics. The identification of lineage II of SAT 2 in Rwanda for the first time shows that the categorised FMDV pools as previously established are not static over time.

CKS2 and S100A12: Two Novel Diagnostic Biomarkers for Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systematicness autoimmunity disease with joint inflammation. RA etiology is still unknown. Early and exact diagnosing is still hard to reach. In the paper, we purposed to discover novel diagnosis biological marker for RA. Two open, usable gene expression profiles of human RA as well as controlled specimens (dataset GSE17755 as well as GSE93272) were downloaded from the GEO database. Differentially expressed genes (DEGs) were screened between 331 RA and 88 control samples. Functional enrichment analysis was applied to explore the possible function of DEGs. Expression levels as well as diagnosis values of biological marker in RA were further verified in our cohort by the use of RT-PCR and ROC assays. We identified 13 DEGs between RA samples and control samples. 13 DEGs were remarkably abundant in NF-kappa B signal pathway. Among the 13 DEGs, CKS2, S100A12, LY96, and ANXA3 exhibited a strong diagnostic ability in screening RA specimens from normal specimens using all AUC > 0.8. Moreover, we confirmed that the expression of CKS2 and S100A12 was distinctly upregulated in RA specimens contrasted to normal specimens. Overall, serum CKS2 and S100A12 could be used as novel diagnosis biological markers for RA patients.

Identification of Biomarkers Associated with Diagnosis of Osteoarthritis Patients Based on Bioinformatics and Machine Learning.

Osteoarthritis (OA) is thought to be the most prevalent chronic joint disease. The incidence of OA is rising because of the ageing population and the epidemic of obesity. This research was designed for the identification of novel diagnostic biomarkers for OA and analyzing the possible association between critical genes and infiltrated immune cells. 10 OA samples from patients with spinal OA and 10 normal samples were collected. GSE55235 and GSE55457 datasets including human OA and normal samples were downloaded from the GEO datasets. Differentially expressed genes (DEGs) were identified between 20 OA and 20 controls. SVM-RFE analysis and LASSO regression model were carried out to screen possible markers. The compositional patterns of the 22 types of immune cell fraction in OA were determined by the use of CIBERSORT. The expression level of the biomarkers in OA was examined by the use of RT-PCR. In this study, an overall 44 DEGs were identified: 18 genes were remarkably upregulated and 26 genes were distinctly downregulated. KEGG pathway analyses revealed that pathways were significantly enriched including IL-17 signal path, rheumatoid arthritis, TNF signal path, and lipid and atherosclerosis. Based on the results of machine learning, we identified APOLD1 and EPYC as critical diagnostic genes in OA, which were further confirmed using ROC assays. Immune cell infiltration analysis revealed that APOLD1 was correlated with mastocytes stimulated, NK cells resting, T cells CD4 memory resting, DCs stimulated, T cells gamma delta, macrophages M0, NK cells stimulated, and mastocytes resting. Moreover, we found that EPYC was correlated with mastocytes stimulated, NK cells resting, T cells CD4 memory resting, DCs stimulated, T cells gamma delta, macrophages M0, NK cells stimulated, and mastocytes resting. Overall, our findings might provide some novel clue for the exploration of novel markers for OA diagnosis. The critical genes and their associations with immune infiltration may offer new insight into understanding OA developments.

FCGBP Is a Promising Prognostic Biomarker and Correlates with Immunotherapy Efficacy in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies of the head and neck. In OSCC patients, the prognosis was dramatically different. In this research, we aimed to study the expressions and prognostic values of IgG Fc binding protein (FCGBP) in OSCC patients. The expression of FCGBP was analyzed using TCGA datasets and GEO datasets. FCGBP was evaluated for its predictive significance in OSCC patients by the use of a Kaplan-Meier and Cox regression model. Enrichment analysis for the GO and KEGG databases were conducted. CIBERSORT used TCGA datasets to show immune cell infiltration. In addition, researchers looked into the relationships between FCGBP and immune cells. The levels of FCGBP in OSCC cells was examined through the use of RT-PCR. FCGBP overexpression was tested for its effects on OSCC cell proliferation and invasion using CCK-8 and Transwell assays. We observed that FCGBP expressions were distinctly downregulated in OSCC specimens compared with nontumor tissues in both TCGA and GEO datasets, which was further confirmed by RT-PCR. OSCC patients with advanced clinical stages and poor prognoses had lower levels of FCGBP expression. Many immune-related biological activities and signaling pathways were found to be considerably abundant in KEGG tests and GO analysis results. The correlation analysis indicated that FCGBP was associated with a number of immune cells in a positive way. We found that FCGBP expressions were strongly and distinctly linked to the expressions of known immunological checkpoints, and FCGBP expression had significant positive connections with tumor mutational burden. FCGBP upregulation distinctly slowed the growth and invasion of OSCC cells in functional experiments. FCGBP has the potential to be a therapeutic target for OSCC and a biomarker for OSCC patients' prognosis.

Phylogenetic analysis of RT-PCR detected infectious bronchitis virus locally infected broiler farms in Diyala Governorate-IRAQ

Background and Aim: One of the primary pathogenic causes linked to economic losses in the chicken business is the infectious bronchitis virus. This virus exploited its S (spike) gene-encoded protein to hook into the host receptor. The biological diversity of the viral S gene may be linked to the chicken industry's vaccination status. Recently, the virus was detected in broilers of six farms located in different districts of Diyala Governorate by the use of RT-PCR. The current study aimed to explore the pattern of biological diversity of infectious bronchitis viruses based on the possibility of genetic variations of the S gene. Materials and Methods: Ten tissue samples were collected from broilers of six farms located in different districts of Diyala Governorate and named R1 to R10. The chickens were recently vaccinated, but naturally infected with infectious bronchitis virus; a particular RT-PCR fragment partially encompassing the coding domains of the S gene was recently obtained. The amplified fragments were immediately submitted to direct sequencing tests to examine the genetic variation pattern in the samples acquired from various chicken sources. Then unique comprehensive trees were generated to validate the correct genotyping of the identified variations and their phylogenetic distribution.

Microglial Cytokines Induce Invasiveness and Proliferation of Human Glioblastoma through Pyk2 and FAK Activation

Simple SummaryMicroglia infiltrate most gliomas and have been demonstrated to promote tumor growth, invasion, and treatment resistance. To develop improved treatment methods, that take into consideration the supporting role of microglia in tumor progression, the functional and mechanistic pathways of glioma–microglia interactions need to be identified and experimentally dissected. Our recent studies and literature reports revealed the overexpression of Pyk2 and FAK in glioblastomas. Pyk2 and FAK signaling pathways have been shown to regulate migration and proliferation in glioma cells, including microglia-promoted glioma cell migration. However, the specific factors released by microglia that modulate Pyk2 and FAK to promote glioma invasiveness and proliferation are poorly understood. The aim of this study was to identify key microglia-derived signaling molecules that induce the activation of Pyk2- and FAK-dependent glioma cell proliferation and invasiveness.Glioblastoma is the most aggressive brain tumor in adults. Multiple lines of evidence suggest that microglia create a microenvironment favoring glioma invasion and proliferation. Our previous studies and literature reports indicated the involvement of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) in glioma cell proliferation and invasion, stimulated by tumor-infiltrating microglia. However, the specific microglia-released factors that modulate Pyk2 and FAK signaling in glioma cells are unknown. In this study, 20 human glioblastoma specimens were evaluated with the use of RT-PCR and western blotting. A Pierson correlation test demonstrated a correlation (0.6–1.0) between the gene expression levels for platelet-derived growth factor β(PDGFβ), stromal-derived factor 1α (SDF-1α), IL-6, IL-8, and epidermal growth factor (EGF) in tumor-purified microglia and levels of p-Pyk2 (Y579/Y580) and p-FAK(Y925) in glioma cells. siRNA knockdown against Pyk2 or FAK in three primary glioblastoma cell lines, developed from the investigated specimens, in combination with the cytokine receptor inhibitors gefitinib (1 μM), DMPQ (200 nM), and burixafor (1 μM) identified EGF, PDGFβ, and SDF-1α as key extracellular factors in the Pyk2- and FAK-dependent activation of invadopodia formation and the migration of glioma cells. EGF and IL-6 were identified as regulators of the Pyk2- and FAK-dependent activation of cell viability and mitosis.

Detection of dengue virus serotype 3 in Cajamarca, Peru

Objective: To describe and molecularly characterize an outbreak of dengue virus (DENV) infection in Cajamarca, an Andean region in Peru. Methods: A total of 359 serum samples from patients with acute febrile illness were assessed for the presence of DENV via RT-PCR, ELISA NS1, IgM and IgG in Cajamarca, Peru from January 2017 to June 2017. The evaluation of the different diagnostic tests and their applicability was performed. Results: Dengue virus was detected in 24.7% of samples by RT-PCR. Meanwhile, serological analysis detected 30.3% positive cases via ELISA NS1 antigen, 16.7% via ELISA IgG and 9.7% via ELISA IgM. Most of the cases corresponded to DENV-3 (77.5%). The use of RT-PCR performed better in primary infections (P&lt;0.01), while detection of ELISA IgM performed better in secondary infections (P&lt;0.01). The combination of NS1 and IgM performed better than the other assays in detecting primary (92.5%) and secondary infections (96.6%). The most frequent symptoms associated with fever were headaches, myalgias, and arthralgias across all groups. Conclusions: We report an important outbreak of dengue infection caused by DENV-3 in Cajamarca, Peru. Our findings encourage the use of NS1 antigen and IgM co-detection. These findings demonstrate an increasing expansion of DENV-3 in Peru and highlight the importance of molecular diagnosis and serotype characterization among the clinically defined dengue cases to strengthen the Peruvian epidemiological surveillance.

Laboratory Diagnosis of Rabies. Current State and Trends in Development

The review considers the relevant aspects of laboratory diagnosis of rabies. The methods of laboratory diagnostics of rabies infection, standardized by WHO in 2018, and their use in the Russian Federation are presented. The scheme of laboratory diagnostics of rabies, applied by specialists of the “48th CRI” of the Ministry of Defense of Russia, for the study of biological samples from deceased people is outlined. Between 2002 and 2018, the study of biomaterial was carried out using molecular-biological, virological methods of diagnosis and in some cases electron microscopy, which allowed to detect and identify the pathogen in 257 samples from 71 people, to certify and deposit new isolates of the rabies virus. Accumulation and analysis of the lessons learned in the application of molecular-biological method of rabies diagnosis allows us to recommend the use of RT-PCR, real-time RT-PCR sets (having a certificate of state registration) in healthcare and veterinary medicine practice to identify the causative agent of rabies infection. The use of molecular-biological methods is promising in terms of the development of rabies diagnosis to improve epidemiological surveillance and raise the efficiency of the biological protection of the population of the Russian Federation.

Comparison of droplet digital PCR vs real-time PCR for Yersinia enterocolitica detection in vegetables

Yersiniosis - the 4th most commonly reported zoonosis in the European Union - is caused by the consumption of food contaminated with the bacterium Yersinia enterocolitica. The number of human cases and contaminated food samples is probably underestimated since conventional molecular methods currently proposed for Yersinia enterocolitica detection proved to have several limitations. Critical issues associated with the detection of Yersinia enterocolitica in meat and/or meat product has already been investigated, whereas data on the possible limits of the molecular methods for Yersinia enterocolitica detection in vegetables are still lacking. According to ISO method (ISO 18867:2015), real-time polymerase chain reaction (rtPCR) should be adopted for Yersinia enterocolitica detection, even if it proved to be affected by some biases. Recently, Droplet Digital PCR (ddPCR) has been introduced as a useful tool to detect and quantify different pathogenic bacteria in complex food matrices. However, its potential application for Yersinia enterocolitica detection in vegetables has never been investigated before.In the present study two molecular platforms (rtPCR and ddPCR) were used to evaluate the pathogen's behaviour in experimentally contaminated leafy greens (Lactuca sativa L.) and to assess the rate of detection achievable after the incubation for eleven days at different temperatures. By comparing, noticeable differences emerged between the two technical approaches: only ddPCR allowed the detection of the pathogen in leafy greens when contaminated at low levels. Moreover, results of the present work highlighted the importance of length and temperature of incubation on the survival and/or the growth of Yersinia enterocolitica in vegetables: at 18 and 25 °C the concentration of the pathogen considerably decreases along incubation. Based on data, the use of rtPCR leads to an underestimation of the true prevalence of pathogenic Y. enterocolitica in vegetables, while temperature and time currently proposed for Y. enterocolitica (25 °C for 24 h), allow optimizing detection. To conclude, ddPCR may be undoubtedly proposed as a reliable alternative strategy for the quick detection of the pathogen in food samples.

Improving of the Nested PCR for Detection of Bovine Leukemia Virus

Enzootic bovine leukosis caused by a bovine leukemia virus has a significant economic impact and is reported in World Organization for Animal Health(OIE). Aim. The purpose of our work was to improve the nested polymerase chain reaction (PCR) recommended by the OIE conducting it second-stage in real-time (RT) PCR. Such modification does not require the stage of gel electrophoresis and consequently reduces contamination risks and prevents false positive results. Methods. Primers that are recommended by the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (OIE) were used for the first amplification stage. For the second stage of the proposed modification of nested PCR, the primers and probe were designed based on the alignment of the sequences envelope gene of different isolates of bovine leukemia virus including Ukrainian isolates. Amplification of the internal control was carried out for the second stage to prevent false negative results. Results. Comparative studies of 48 blood samples for bovine leukemia virus identification by a proposed nested RT-PCR, nested PCR recommended by the protocol of the OIE, and RT-PCR were conducted. The sample panel included both positive and negative samples. A 100% match of the results of the bovine leukemia virus presence in nested PCR proposed by the OIE and in our proposed nested RT-PCR was obtained. Comparative analysis of results that were obtained using the RT-PCR and the proposed nested RT-PCR showed that false-negative results in 5 samples and 3 doubtful results that require retesting were obtained by use of RT-PCR. The interpretation of the results using nested RT-PCR is more efficient than RT-PCR since the cycle threshold value of positive samples obtained using RT-PCR was in the range of 24–40 cycles, whereas in the case of nested RT-PCR using, the value of Ct was in the range of 4–20 cycles. Conclusions. Proposed nested PCR modification includes the combination of the OIE recommendation about nested PCR and the reduction of the risk of contamination by conducting the second stage in RT-PCR. Results of approbation of proposed nested RT-PCR give a reason to recommend it for the identification of bovine leukemia virus.

Molecular detection of Sarcocystis neurona in cerebrospinal fluid from 210 horses with suspected neurologic disease

An ante-mortem diagnosis of equine protozoal myeloencephalitis (EPM) is presently based on clinical presentation, immunodiagnostics performed on serum and cerebrospinal fluid (CSF), and ruling out other neurological disorders. Molecular techniques introduce a novel and promising approach for the detection of protozoal agents in CSF. Hypothesizing that real-time PCR (rtPCR) can be a useful complement to EPM diagnostics, 210 CSF samples from horses suspected of neurological disease with EPM included as a differential diagnosis were tested using rtPCR to detect Sarcocystis neurona DNA and immunodiagnostics targeting antibodies against the same pathogen, performed on serum and CSF samples. Molecular and immunological results were compared with respect to origin of the horse, time of the year, signalment, clinical signs and treatment history. Twenty-five horses tested positive in CSF for S. neurona by rtPCR only, while 30 horses had intrathecally-derived antibodies to S. neurona only (serum to CSF ratio ≤ 64 by indirect fluorescent antibody test - IFAT), and 13 horses tested rtPCR-positive in CSF with evidence of intrathecally-derived antibodies to S. neurona. Previous treatment for EPM was the only variable presenting statistical difference between the two testing modalities, highlighting that animals with history of anti-protozoal treatment were more likely to test positive solely in IFAT, while horses without treatment were more likely to test positive by rtPCR only. The results support the use of molecular diagnosis for EPM caused by S. neurona as a complement to immunodiagnostics. The use of rtPCR in CSF for the detection of S. neurona may improve the diagnostic work-up of neurologic disease suspected horses, especially in animals without previous anti-protozoal treatment.

Algorithms for testing COVID-19 focused on use of RT-PCR and high-affinity serological testing: A consensus statement from a panel of Latin American experts

The COVID-19 pandemic has caused an unprecedented public health, social, and economic crisis. Improving understanding on available tests for detecting COVID-19 is critical for effective management of the pandemic. We proposed that a multidisciplinary expert panel can establish recommendations on ideal use of diagnostic tools, with a focus on RT-PCR and serological high-affinity antibodies (both IgM and IgG) tests for the Latin America region. Study designA collaborative multidisciplinary panel of 5 recognized experts in Latin America (an infectious disease specialist, three pathologists, and an immunologist) was convened and supported by Roche Diagnostics to develop standard guidelines and an evidence-based document of best practices on the use of diagnostic tools for COVID-19. ResultsThe authors reached consensus on the applicability of diagnostic tools to provide testing algorithms for the use of RT-PCR and serological high-affinity antibodies (both IgM and IgG) tests in three settings: 1) For asymptomatic subjects exposed to a SARS-CoV-2 infected person; 2) For epidemiological purposes and; 3) For symptomatic subjects. ConclusionThe serological high-affinity SARS-CoV-2 antibodies (both IgM and IgG) tests play a key role in COVID-19 diagnosis. These tests can be applied for suspected false-negative RT-PCR results and for individual determination of response. The use of these tests can also contribute greatly to public health strategies, such as population screening and supporting vaccination planning. Serological status for high-affinity antibodies (both IgM and IgG) should be performed ideally 21 days after potential infectious contact, given that the majority of exposed individuals will have seroconverted.

Added value of anti-SARS-CoV-2 antibody testing in a Flemish nursing home during an acute COVID-19 outbreak in April 2020

ABSTRACT Objectives To examine the added value of anti-SARS-CoV-2 antibody testing in a nursing home during an acute COVID-19 outbreak. RT-PCR is the gold standard, but can be false-negative. Methods 119 residents and 93 staff members were tested with RT-PCR test and/or a rapid IgM/IgG test. Of these participants, 176 had both tests, 24 only RT-PCR, and 12 only IgM/IgG in the period April 14 to 16 April 2020. Results 40 (34%) residents and 11 (13%) staff were PCR-positive. Using a rapid IgM/IgG test, 17 (17%) residents and 18 (20%) staff were positive for IgM and/or IgG (IgM/IgG). Thirty-two PCR-positive residents had an IgM/IgG test: 9 (28%), 11 (34%), and 13 (41%) were positive for IgM, IgG, and IgM/IgG. Ten PCR-positive staff had an IgM/IgG test: 3 (30%), 6 (60%), and 6 (60%) were positive for IgM, IgG, and IgM/IgG. Additional IgM/IgG tests were performed in 9 residents 11 to 14 days after the positive RT-PCR test. Of those, 7 (78%) tested positive for IgM/IgG. When retested 3 weeks later, the 2 remaining residents also tested positive. Of the 134 PCR-negative participants who had an IgM/IgG test, 15 were positive for IgM/IgG (8% of the 200 participants tested with RT-PCR). Conclusions During an acute outbreak in a nursing home, 26% of residents and staff were PCR-positive. An additional 8% was diagnosed using IgM/IgG antibody testing. The use of RT-PCR alone as the sole diagnostic method for surveillance during an acute outbreak is insufficient to grab the full extent of the outbreak.