RAPID IDENTIFICATION OF ETIOLOGICAL AGENTS OF RESPIRATORY TRACT INFECTIONS DURING THE COVID-19 PANDEMIC USING THE MULTIPLEX-PCR METHOD.

Objective. Most respiratory infections have similar symptoms, so it is clinically difficult to determine their etiology. This study aimed to show the importance of molecular diagnostics in identifying the etiological agent of respiratory infections, especially during the coronavirus disease 2019 (COVID-19) pandemic. Methods. A total of 849 samples from patients hospitalized at the University Clinical Center Kragujevac (from January 1 to August 1, 2022) were examined using automated multiplex-polymerase chain reaction (PCR) tests. The BioFire-FilmArray-Respiratory Panel 2.1 test was used for 742 nasopharyngeal swabs [identification of 19 viruses (including SARS-CoV-2) and four bacteria], while the BioFire-FilmArray-Pneumonia Panel was used [identification of 18 bacteria and nine viruses] (BioMerieux, Marcy l'Etoile, France) for 107 tracheal aspirates. The tests were performed according to the manufacturer's instructions, and the results were available within an hour. Results. In 582 (78.4%) samples, the BioFire-FilmArray-Respiratory Panel 2.1 plus test identified at least one pathogen. The rhinovirus (20.6%), SARS-CoV-2 (17.7%), influenza A (17.5%), respiratory syncytial virus (12.4%), and parainfluenza 3 (10.1%) were the most common. Other viruses were found less frequently, and Bordetella parapertussis was detected in one sample. In 85 (79.4%) samples, the BioFire-FilmArray-Pneumonia Panel test identified at least one bacterium or virus. The most prevalent bacteria were Staphylococcus aureus (42.4%), Haemophilus influenzae (41.2%), Streptococcus pneumoniae (36.5%), Moraxella catarrhalis (22.3%), and Legionella pneumophila (2.4%). Among viruses, rhinovirus (36.5%), adenovirus (23.5%), influenza A (11.8%), and the genus Coronavirus (4.7%), were detected. Conclusion. Multiplex-PCR tests improved the implementation of therapeutic and epidemiological measures, preventing the spread of the COVID-19 infection and Legionnaires' disease.Copyright © 2022, Serbian Medical Society. All rights reserved.

Prevalence of multidrug-resistant Gram-negative bacteria from blood cultures and rapid detection of beta-lactamase-encoding genes by multiplex PCR assay

Introduction This study aimed to determine the prevalence of multidrug-resistant Gram-negative bacteria (GNB) from blood cultures in a tertiary-care hospital and the multiplex PCR assay's ability to detect resistance genes. Methods A total of 388 GNB isolates obtained from hospitalized patients between November 2019 and November 2021 were included in the study. Antimicrobial susceptibility testing was done by VITEK 2 system and broth microdilution method. Beta-lactamase-encoding genes were detected by multiplex PCR assays, BioFire-Blood Culture Identification 2 (BCID2) panel (bioMerieux, France). Extended-spectrum beta-lactamases (ESBLs) were detected phenotypically with VITEK AST-GN71 card (bioMerieux, France). The isolates of GNB were classified into multidrug-resistant, extensively-drug-resistant, and pandrug-resistant categories, and their prevalence and distribution in different wards, including coronavirus diseases 2019 (COVID-19) intensive care units (ICU), were calculated. Results Results revealed that all isolates of Acinetobacter baumannii and Pseudomonas aeruginosa were multidrug-resistant as well as 91.6% of Enterobacter cloacae, 80.6% of Proteus mirabilis, and 76.1% of Klebsiella pneumoniae, respectively. In fermentative bacteria, blaOXA-48-like (58.1%), blaNDM (16.1%), blaKPC (9.7%) and blaVIM (6.5%) genes were detected. More than half of Enterobacter cloacae (58.3%) and Klebsiella pneumoniae (53.7%) produced ESBLs. Among non-fermenters, the blaNDM gene was carried by 55% of Pseudomonas aeruginosa and 19.5% of Acinetobacter baumannii. In the COVID-19 ICU, Acinetobacter baumannii was the most common isolate (86.1%). Conclusions This study revealed high proportions of multidrug-resistant blood isolates and various underlying resistance genes in Gram-negative strains. The BCID2 panel seems to be helpful for the detection of the most prevalent resistance genes of fermentative bacteria.Copyright © GERMS 2022.

Single-step multiplex reverse transcription-polymerase chain reaction for the detection and differentiation of QX-like infectious bronchitis virus from the Thai variant and vaccine strains H120, Ma5, and 4/91

Background and Aim: QX-like infectious bronchitis virus (IBV) is a highly infectious avian coronavirus that causes respiratory and kidney disease. It is linked to increased mortality and loss of performance in infected chickens worldwide, including Thailand. Thus, a simple and rapid diagnostic method for the diagnosis of QX-like IBV is needed. This study aimed to develop a single-step multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay to detect and differentiate QX-like IBV from Thai IBV and vaccine strains used in the poultry industry (H120, Ma5, and 4/91). Materials and Methods: Primer sets specific for QX-like and Thai IBV were designed to target the S1 gene. The specificity of the technique was verified using nine isolates of QX-like IBV, four isolates of Thai IBV, and other avian viral respiratory pathogens. The detection limit was evaluated using a serial ten-fold dilution of QX-like and Thai IBV. Results: The results showed that single-step mRT-PCR could detect QX-like IBV and differentiate it from Thai IBV and the vaccine strains H120, Ma5, and 4/91. The limit of detection of the developed assay was 102.2 embryo infectious dose (EID)50/mL for QX-like IBV and 101.8 EID50/mL for Thai IBV. Interestingly, the developed assay could identify mixed infection by both IBVs in a single sample. Conclusion: The single-step mRT-PCR assay developed in this study can potentially discriminate QX-like IBV from Thai IBV and the vaccine strains H120, Ma5, and 4/91 in a single reaction. It is also suitable for use in all laboratories with access to conventional PCR equipment. [ FROM AUTHOR] Copyright of Veterinary World is the property of Veterinary World and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Molecular epidemiology and genetic diversity of canine coronavirus from domestic dogs in Chengdu, China from 2020 to 2021 using a multiplex RT-PCR.

Recent reports on identification of canine coronavirus (CCoV) in humans have emphasized the urgency to strengthen surveillance of animal CoVs. The fact that recombinations between CCoV with feline, porcine CoVs brought about new types of CoVs indicated that more attention should be paid to domestic animals like dogs, cats and pigs, and the CoVs they carried. However, there are about ten kinds of CoVs that infect above animals, and thus representative CoVs with zoonotic potentials were considered in this study. Multiplex RT-PCR against CCoV, Feline coronavirus (FCoV), porcine deltacoronavirus and porcine acute diarrhea syndrome coronavirus was developed to investigate the prevalence of CoVs from domestic dogs in Chengdu, Southwest China. Samples from a total of 117 dogs were collected from a veterinary hospital, and only CCoV (34.2%, 40/117) was detected. Therefore, this study focused on CCoV and its characteristics of S, E, M, N and ORF3abc genes. Compared with CoVs that are capable of infecting humans, CCoV strains showed highest nucleotide identity with the novel canine-feline recombinant detected from humans (CCoV-Hupn-2018). Phylogenetic analysis based on S gene, CCoV strains were not only clustered with CCoV-II strains, but also closely related to FCoV-II strains ZJU1617 and SMU-CD59/2018. As for assembled ORF3abc, E, M, N sequences, CCoV strains had the closest relationship with CCoV-II (B203_GZ_2019, B135_JS_2018 and JS2103). What's more, specific amino acid variations were found, especially in S and N proteins, and some mutations were consistent with FCoV, TGEV strains. Altogether, this study provided a novel insight into the identification, diversification and evolution of CoVs from domestic dogs. It is of top priority to recognize zoonotic potential of CoVs, and continued comprehensive surveillance will help better understand the emergence, spreading, and ecology of animal CoVs.

A rapid and highly sensitive multiple detection of human adenovirus type 3, type 7 and respiratory syncytial virus by recombinase-aided reverse transcription PCR.

BACKGROUND: Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase-aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology. METHODS: In this study, we developed and validated the multiplex reverse transcription recombinase-aided PCR (multiplex RT-RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process. RESULTS: Using recombinant plasmids, the sensitivity of multiplex RT-RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT-RAP showed no cross-reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT-RAP and the results were found to be consistent with those of corresponding RT-qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT-RAP was two to eightfold higher than that of corresponding RT-qPCR. CONCLUSION: We conclude the multiplex RT-RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load.

FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity.

Recombinase polymerase amplification (RPA) running at 37-42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics.

Evaluation of BioFire® FilmArray® Pneumonia Panel in Bronchoalveolar Lavage Samples From Immunocompromised Patients With Suspected Pneumonia.

Objectives Immunocompromised patients, specifically those with solid organ transplants or cancer on chemotherapy, are at particularly high risk of severe pneumonia and opportunistic infections. In select patients, bronchoalveolar lavage (BAL) is performed to provide high-quality samples for analysis. We compare BioFire® FilmArray® Pneumonia Panel (BioFire Diagnostics, Salt Lake City, Utah, United States), a multiplex polymerase chain reaction (PCR) assay, with standard of care diagnostics in BAL samples from immunocompromised patients to identify opportunities for this test to affect clinical decision making. Methods Patients hospitalized with pneumonia based on clinical and radiographic findings who underwent evaluation with bronchoscopy between May 2019 to January 2020 were reviewed. Among those patients undergoing bronchoscopy, those who were immunocompromised were selected for inclusion in the study. BAL specimens submitted to the microbiology laboratory were chosen based on as part of the internal validation of the panel in comparison with sputum culture at our hospitals. We compared the outcomes of the multiplex PCR assay with traditional culture methods and evaluated the role of PCR assay in de-escalating antimicrobial therapy. Results Twenty-four patients were identified for testing with the multiplex PCR assay. Of the 24 patients, 16 were immunocompromised, all with solid or hematological malignancy or a history of organ transplant. Seventeen individual BAL samples from the 16 patients were reviewed. BAL culture results and the multiplex PCR assay were in agreement in 13 samples (76.5%). In four cases, the multiplex PCR assay identified a possible causative pathogen not detected by standard workup. The median time to de-escalation of antimicrobials was three days (interquartile range (IQR) 2-4) from the day of collection of the BAL samples. Conclusions Studies have established the additive role of multiplex PCR testing in addition to traditional diagnostic tools like sputum culture in diagnosing the etiology of pneumonia. Limited data exist specifically looking at immunocompromised patients, in whom a timely and accurate diagnosis is particularly important. There is a potential benefit for performing multiplex PCR assays as an additive diagnostic tool in BAL samples for these patients.

Clinical influence of multiplex polymerase chain reaction routine uses in urgent pediatric admissions.

BACKGROUND: The coronavirus disease 2019 outbreak has prompted some hospitals to implement screening tests upon admission since 2020. FilmArray® Respiratory 2.1 Panel (FilmArray) is a multiplex polymerase chain reaction (PCR) test with high sensitivity and specificity for detecting respiratory pathogens. We aimed to assess the clinical influence of the routine use of FilmArray for pediatric patients, including those without symptoms suggestive of an infection. METHODS: We conducted a single-center retrospective observational study, which investigated patients aged ≤15 years who underwent FilmArray on admission in 2021. We collected the patients' epidemiological information, symptoms, and FilmArray results from their electronic health records. RESULTS: A positive result was observed in 58.6% of patients admitted to the general ward or intensive care unit (ICU) but only in 1.5% of patients in the neonatal ward. Among the patients admitted to the general ward or ICU who tested positive, 93.3% had symptoms suggestive of infections, 44.6% had a sick contact before admission, and 70.5% had siblings. However, 62 (28.2%) out of 220 patients without the four (fever, respiratory, gastrointestinal, and dermal) symptoms also had positive results. Among them, 18 patients with adenovirus and three with respiratory syncytial virus were isolated to private rooms. However, 12 (57.1%) patients were discharged without symptoms suggestive of viral infection. CONCLUSION: Multiplex PCR routine use for all inpatients may lead to excessive management of positive cases because FilmArray cannot quantify microorganisms. Thus, targets for testing should be considered carefully based on patients' symptoms and histories of sick contacts.

["When it rains, it pours" - de novo diagnosed multiple myeloma with invasive Streptococcus pneumoniae infection in severe COVID-19 complicated with cytokine storm]. / "A baj nem jár egyedül" ­ de novo diagnosztizált myeloma multiplex invazív Streptococcus pneumoniae fertozéssel citokinviharral szövodött COVID­19-ben.

With the appearance of SARS-CoV-2, the range of infections, considered the most common cause of death for people with multiple myeloma, has expanded. Although the omicron variant (PANGO B.1.1.529) of SARS-CoV-2, that dominates the world at the time of manuscript writing, is less likely to cause fatal infection in immunocompetent patients compared to the delta variant (PANGO B.1.617.2), its transmissibility did not decrease. The likelihood of a severe or critical course of COVID-19 in patients with multiple myeloma is increased by the humoral and cellular immunosuppression caused by the malignancy itself, its targeted hematological treatment, and other comorbidities associated with the disease (e.g., chronic kidney failure). Antiviral therapies, monoclonal antibody preparations used as pre- or post-exposure prophylaxis, and possibly convalescent plasma therapy, started as early as possible might prevent the clinical progression of COVID-19. While the incidence of community-acquired co-infections accompanying COVID-19 in the average population is not exceptionally high, in people with multiple myeloma, Streptococcus pneumoniae infection that follows respiratory viral diseases is approximately 150 times more likely to cause invasive disease. As a result of modern oncohematological treatment, multiple myeloma has now become a chronic disease accompanied by relapses, and those affected should be immunized against the above two pathogens. In our manuscript, we describe the case of an adult patient with severe COVID-19 complicated by cytokine storm and invasive Streptococcus pneumoniae infection who was diagnosed with de novo multiple myeloma during hospital care, and, finally, we briefly review the related literature data. Orv Hetil. 2023; 164(20): 763-769.

MultiSero: An Open-Source Multiplex-ELISA Platform for Measuring Antibody Responses to Infection.

A multiplexed enzyme-linked immunosorbent assay (ELISA) that simultaneously measures antibody binding to multiple antigens can extend the impact of serosurveillance studies, particularly if the assay approaches the simplicity, robustness, and accuracy of a conventional single-antigen ELISA. Here, we report on the development of multiSero, an open-source multiplex ELISA platform for measuring antibody responses to viral infection. Our assay consists of three parts: (1) an ELISA against an array of proteins in a 96-well format; (2) automated imaging of each well of the ELISA array using an open-source plate reader; and (3) automated measurement of optical densities for each protein within the array using an open-source analysis pipeline. We validated the platform by comparing antibody binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigens in 217 human sera samples, showing high sensitivity (0.978), specificity (0.977), positive predictive value (0.978), and negative predictive value (0.977) for classifying seropositivity, a high correlation of multiSero determined antibody titers with commercially available SARS-CoV-2 antibody tests, and antigen-specific changes in antibody titer dynamics upon vaccination. The open-source format and accessibility of our multiSero platform can contribute to the adoption of multiplexed ELISA arrays for serosurveillance studies, for SARS-CoV-2 and other pathogens of significance.

Antibiotic therapy of Acinetobacter baumannii bloodstream infection in COVID-19 patients

Secondary bacterial infection is one of the important risk factors for the development of severe course and death in COVID-19. The rational choice of antibacterial therapy is based on the data of microbiological monitoring of pathogens of healthcare-associated infections. The aim of the study is to determine the main options for antibiotic therapy of Acinetobacter baumannii bloodstream infection in COVID-19 patients. Material and methods. A retrospective, single-centre, uncontrolled study of the incidence of A. baumannii bacteremia in COVID-19 patients treated at the City Clinical Hospital No. 52 in Moscow from October 2020 to September 2021 was performed. For each strain of A. baumannii sensitivity to the main antibacterial agents was determined. Genetic determinants of antibiotic resistance were studied by real-time multiplex polymerase chain reaction. The main therapeutic options for A. baumannii bloodstream infection were analyzed. Results and discussion. Bloodstream infections were diagnosed in 4.7% of hospitalized patients with COVID-19 (758/16 047). Gram-negative bacteria were the causative agents of bloodstream infections in 76% of cases. A. baumannii were isolated from the blood of 143 patients (0.89%). Detection of the pathogen in the blood of COVID-19 patients was associated with severe and extremely severe course of the disease. Most of the strains (93%) were isolated in the intensive care unit. The A. baumannii strains studied were carbapenem-resistant (CRAb) and phenotypically belonged to the XDR class. According to a PCR study, A. baumannii strains were producers of oxacillinases OXA-23, OXA-40, and OXA-51. Conclusion. The circulation of A. baumannii CRAb in intensive care units makes empiric therapy based on carbapenems irrational and ineffective. For the etiotropic therapy of A. baumannii bloodstream infection it is recommended to use combined antibiotic therapy regimens with the inclusion of polymyxin B and sulbactam.Copyright © Eco-Vector, 2022.

Investigation of Microbial Etiology in Endotracheal Aspirate Samples of Severe Pneumonia Patients by Multiplex PCR and Culture Method

Objectives: The probability of detecting viral and atypical agents in pneumonia patients has increased with the molecular methods used in recent years. We aimed to investigate pneumonia pathogens in endotracheal aspiration samples (ETA) of patients with severe community-acquired (CAP), hospital-acquired pneumonia (HAP), and ventilator-associated pneumonia (VAP) by multiplex polymerase chain reaction (m-PCR) and culture method. Methods: A prospective study was performed between December 2019 and October 2020. Patients 18 years and older with pneumonia followed in ICU on the mechanical ventilator were included. COVID-19 patients were excluded. Patients were grouped as CAP, HAP, and VAP. Two ETA samples were obtained from patients within 48 hours of the pneumonia diagnosis. Respiratory pathogens were investigated in samples by viral-bacterial m-PCR and bacterial culture methods. Results: 74 patients were included in the study. m-PCR of ETA samples achieved pathogen detection in 87.8% of patients compared with 58.1% with culture methods. The most common pathogen detected by m-PCR was Streptococcus pneumoniae in both CAP and HAP patients and Klebsiella spp. in VAP patients. The most common pathogen isolated by culture was Staphylococcus aureus in both CAP and HAP patients and Klebsiella spp. in VAP patients. Atypical pneumonia pathogens were positive for 14.9% of the patients. Atypical pathogens were recovered from 28.5% of CAP patients and 23.1% of HAP patients. Viruses constituted all of the atypical pathogens recovered from HAP patients. No atypical pathogen was found in VAP patients. Conclusion: In this study, S. pneumoniae was the most common pathogen detected with m-PCR, and S. aureus and Klebsiella spp. were the most common pathogens detected with culture. Determination of microbial etiology of lower respiratory tract samples by molecular methods for diagnosing severe CAP and HAP may be beneficial in terms of treatment.

RAPID IDENTIFICATION OF ETIOLOGICAL AGENTS OF RESPIRATORY TRACT INFECTIONS DURING THE COVID-19 PANDEMIC USING THE MULTIPLEX-PCR METHOD

Objective. Most respiratory infections have similar symptoms, so it is clinically difficult to determine their etiology. This study aimed to show the importance of molecular diagnostics in identifying the etiological agent of respiratory infections, especially during the coronavirus disease 2019 (COVID-19) pandemic. Methods. A total of 849 samples from patients hospitalized at the University Clinical Center Kragujevac (from January 1 to August 1, 2022) were examined using automated multiplex-polymerase chain reaction (PCR) tests. The BioFire-FilmArray-Respiratory Panel 2.1 test was used for 742 nasopharyngeal swabs [identification of 19 viruses (including SARS-CoV-2) and four bacteria], while the BioFire-FilmArray-Pneumonia Panel was used [identification of 18 bacteria and nine viruses] (BioMérieux, Marcy l'Etoile, France) for 107 tracheal aspirates. The tests were performed according to the manufacturer's instructions, and the results were available within an hour. Results. In 582 (78.4%) samples, the BioFire-FilmArray-Respiratory Panel 2.1 plus test identified at least one pathogen. The rhinovirus (20.6%), SARS-CoV-2 (17.7%), influenza A (17.5%), respiratory syncytial virus (12.4%), and parainfluenza 3 (10.1%) were the most common. Other viruses were found less frequently, and Bordetella parapertussis was detected in one sample. In 85 (79.4%) samples, the BioFire-FilmArray-Pneumonia Panel test identified at least one bacterium or virus. The most prevalent bacteria were Staphylococcus aureus (42.4%), Haemophilus influenzae (41.2%), Streptococcus pneumoniae (36.5%), Moraxella catarrhalis (22.3%), and Legionella pneumophila (2.4%). Among viruses, rhinovirus (36.5%), adenovirus (23.5%), influenza A (11.8%), and the genus Coronavirus (4.7%), were detected. Conclusion. Multiplex-PCR tests improved the implementation of therapeutic and epidemiological measures, preventing the spread of the COVID-19 infection and Legionnaires' disease. © 2022, Serbian Medical Society. All rights reserved.

A Case of Ocular Myasthenia Gravis Seen by Rheumatology as Suspected Mononeuritis Multiplex in an Adult Male Shortly after Pfizerbiontech Sars-Cov-2 Vaccination

Background/Aims Myasthenia gravis (MG) is an antibody-mediated autoimmune disease targeting proteins at the postsynaptic membrane of the neuromuscular junction. MG is thought to occur in genetically susceptible individuals following an environmental trigger. SARS-CoV-2 infection has been associated with new-onset autoimmune disease, new-onset MG, and exacerbations of pre-existing MG, with molecular mimicry between SARS-CoV-2 epitopes and autoantigen-induced autoreactivity thought to be part of the underlying mechanism. We report a case of newonset ocular MG following first dose Pfizer-BioNTech BNT162b2 SARS-COV2 vaccination which was referred to rheumatology as suspected mononeuritis multiplex. Methods A 53-year-old man of East Asian ethnicity presented to the emergency department (ED) with sudden onset diplopia and left lateral gaze restriction 7 days after receiving his first dose of the Pfizer-BioNTech BNT162b2 SARS-COV2 vaccination. He had longstanding myopia and dry eyes but no other medical history, no regular medications or significant family history. He was a current smoker, with a 50-pack year history. He did not drink alcohol or use any recreational drugs. He was found to have an isolated left VI cranial nerve (CN) palsy with an otherwise normal ocular and physical examination. Blood tests were unremarkable apart from raised cholesterol, and he was discharged with a suspected self-limiting microvascular CN lesion. Three weeks later he presented to ED with worsening diplopia, increasingly restricted eye movements, headache, nausea, vomiting and blurred vision. Ophthalmology assessment noted new right sided CN III and VI palsy, persistent left CN VI palsy, and vertical diplopia in all fields of gaze. Neurological and physical examination were normal. Bloods including an autoimmune screen were unremarkable. SARS-CoV-2 Spike antibodies were positive consistent with SARS-CoV-2 vaccination but not infection. Intracranial and thoracic imaging were unremarkable. He was referred to and seen by both rheumatology and neurology as a case of suspected mononeuritis multiplex. Results A diagnosis of ocular MG was confirmed with positive serum acetylcholine receptor antibodies, and he was started on prednisolone, and pyridostigmine to good effect. Daily forced vital capacity (FVC) showed no respiratory muscle involvement, and nerve conduction studies and electromyography were normal, excluding secondary generalisation. Conclusion A review of the literature found 14 reported cases of new-onset MG all within 4 weeks following SARS-CoV-2 vaccine. Whilst these cases provide interesting insights into the pathogenesis of autoimmune conditions such as MG, they are not epidemiological studies to inform vaccine safety. Ultimately, current evidence suggests that the risks of SARS-COV-2 infection outweigh the risk of vaccine-related adverse events, therefore we suggest clinicians should be aware of potential new-onset autoimmune conditions, but support the safety of SARSCOV2 vaccination. Further, research into possible immunological mechanisms behind this phenomenon, including identifying potential epitopes inducing molecular mimicry, could help establish the likelihood of a causative link.

Long Term Circulation of Sars-Cov-2 Related Lineages in Bats in Cambodia

Intro: Recent evidence shows the Greater Mekong Subregion to be a hotspot for Sarbecoviruses in bats, especially insectivorous Horseshoe bats (genus Rhinolophus). However, prevalence, maintenance, and evolution of these viruses in Rhinolophids is still poorly understood. Sampling efforts are still limited and generally only cover cross-sectional surveillance at single points in time. Following the detection of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2)-related viruses in Rhinolophus shameli from 2010 in Steung Treng, Cambodia, further active longitudinal surveillance in the same area between 2020-2021 continued the detection of these viruses. Method(s): Live bat capture and sampling has been implemented in several sites located in Stung Treng province. All rectal swabs of bats were tested for the detection of SARS-CoV-2 or Sarbecoviruses by real time RT-PCR. RNA samples from positive RT-PCR bats were then sequenced using a highly multiplexed PCR amplicon approach using new designed primers set guided by the ARTIC Network multiplex PCR primers set (https://artic.network/ncov-2019), on Oxford Nanopore technology. Finding(s): The sarbecoviruses were detected in four Rhinolophus shameli bats, a percentage of similarity ranging at the nucleotide level between 98.8% - 99.1% when compared to two other Cambodian bat sarbecoviruses from 2010 and between 92.4% - 94.5% when compared to human SARS-CoV-2 across the whole genome. Discussion(s): The bat SARS-CoV-2 related virus recently detected in four positive bats in 2020-2021 are genetically homologous with the virus detected in 2010, indicating a geographically/host limited population that is stable over time in the past ten years. Conclusion(s): Overall, our findings indicate further complexity in the diversity and evolution of sarbecoviruses and add intricacy to the search for the origins of the Coronavirus Disease 2019 (COVID-19) pandemic.Copyright © 2023

Medical diagnostic value of digital PCR (dPCR): A systematic review

Digital polymerase chain reaction (dPCR) is an emerging technique for the absolute quantification of target nucleic acids. dPCR got attention as a precise quantification tool in preclinical research, particularly when used to detect genetic mutations and result in highly precise measurements. In dPCR, the statistic of Poisson distribution was followed for the random distribution of molecules in different partitions, which is essential for dPCR quantification. Amplified target sequences in different partitions are identified by fluorescence and each partition functions as a separate PCR microreactor. Without the need for calibration, the percentage of PCR-positive partitions is sufficient to estimate the concentration of the target sequence. The present revolution in digital quantification was made possible by advancements in microfluidics, which provided effective partitioning techniques. In this paper, the contrast of the underlying ideas of quantitative real-time PCR with dPCR for the measurement of nucleic acids quantity Polymerase chain reaction (q-PCR). This review study briefly introduced the background of dPCR and compared different types of PCR, particularly the quantity of real-time qPCR and digital PCR. The fundamental concept of dPCR is also explained and also briefly compares the advantages of dPCR over qPCR and analyzes the applications of dPCR as a diagnostic tool for cancer and different types of viral species.

RAPID IDENTIFICATION OF ETIOLOGICAL AGENTS OF RESPIRATORY TRACT INFECTIONS DURING THE COVID-19 PANDEMIC USING THE MULTIPLEX-PCR METHOD.

Objective. Most respiratory infections have similar symptoms, so it is clinically difficult to determine their etiology. This study aimed to show the importance of molecular diagnostics in identifying the etiological agent of respiratory infections, especially during the coronavirus disease 2019 (COVID-19) pandemic. Methods. A total of 849 samples from patients hospitalized at the University Clinical Center Kragujevac (from January 1 to August 1, 2022) were examined using automated multiplex-polymerase chain reaction (PCR) tests. The BioFire-FilmArray-Respiratory Panel 2.1 test was used for 742 nasopharyngeal swabs [identification of 19 viruses (including SARS-CoV-2) and four bacteria], while the BioFire-FilmArray-Pneumonia Panel was used [identification of 18 bacteria and nine viruses] (BioMerieux, Marcy l'Etoile, France) for 107 tracheal aspirates. The tests were performed according to the manufacturer's instructions, and the results were available within an hour. Results. In 582 (78.4%) samples, the BioFire-FilmArray-Respiratory Panel 2.1 plus test identified at least one pathogen. The rhinovirus (20.6%), SARS-CoV-2 (17.7%), influenza A (17.5%), respiratory syncytial virus (12.4%), and parainfluenza 3 (10.1%) were the most common. Other viruses were found less frequently, and Bordetella parapertussis was detected in one sample. In 85 (79.4%) samples, the BioFire-FilmArray-Pneumonia Panel test identified at least one bacterium or virus. The most prevalent bacteria were Staphylococcus aureus (42.4%), Haemophilus influenzae (41.2%), Streptococcus pneumoniae (36.5%), Moraxella catarrhalis (22.3%), and Legionella pneumophila (2.4%). Among viruses, rhinovirus (36.5%), adenovirus (23.5%), influenza A (11.8%), and the genus Coronavirus (4.7%), were detected. Conclusion. Multiplex-PCR tests improved the implementation of therapeutic and epidemiological measures, preventing the spread of the COVID-19 infection and Legionnaires' disease.Copyright © 2022, Serbian Medical Society. All rights reserved.

Role of antiviral therapy in respiratory infections in children: Analysis of clinical and laboratory observations.

Objective. To evaluate the efficacy of therapy for acute respiratory viral infections (ARVIs) in children with antiviral medications: inosine pranobex (Groprinosin, Gedeon Richter) and Kagocel (Kagocel, Niarmedic Pharma LLC) in comparison with symptomatic treatment without etiotropic agents based on clinical and laboratory parameters. Patients and methods. The clinical and laboratory observation was conducted in an outpatient setting in the pre-COVID-19 period between 2018 and 2020. Acute respiratory infections were diagnosed using licensed testing systems by multiplex polymerase chain reaction (PCR) with detection of nucleic acid viral genomes: influenza, rhinovirus, respiratory syncytial virus, metapneumovirus, parainfluenza, seasonal coronaviruses, adenoviruses, and bocavirus). A total of 151 children aged 3 to 15 years were examined and monitored in dynamics, with 78.7% of positive and 21.3% of negative results detected by PCR in the nasopharyngeal and oropharyngeal swabs. The patients were randomized into three groups depending on the antiviral medication prescribed: group 1 (53 children) received Groprinosin;group 2 (52 children) received Kagocel;group 3 (control, 46 children) received only symptomatic therapy without antiviral agents. Results. The study demonstrated a significant positive effect in patients in group 1 treated with Groprinosin (n = 53). At the end of therapy for both mono- and mixed infections, there were 95.8% of negative results (according to PCR diagnosis, that is, the absence of viral genome). In children in group 2 (n = 52) treated with Kagocel, the absence of viral nucleic acids (NAs) was observed less frequently (in 77.3% of cases). In children in group 3 (n = 46) who did not receive etiotropic antiviral therapy, there were only 40.3% of negative results after the end of treatment, and viral NAs were detected in 59.7% of patients. In this case, a 5-day course of Groprinosin was prescribed, after which the PCR results became negative in all patients. Therefore, children with recurrent respiratory infections, mixed infections, and herpesvirus infections require longer therapy. Additionally, a high frequency of ARVI complications was noted in group 3 (5 (10.9%) patients, where otitis was observed in 1 case, sinusitis - in 2 cases, bronchitis - in 2 cases), whereas 1 (1.8%) patient taking Groprinosin had otitis, and 1 (1.9%) patient taking Kagocel had pneumonia. Conclusion. This study was the first to investigate antibody titers to respiratory viruses in dynamics at 3, 6 and 12 months after the onset of ARVI. It showed that the development of antibodies to respiratory viruses is very unstable and does not occur in all patients. Antibodies almost disappeared by the third month after ARVI and were no longer detectable by the sixth month. After 12 months, patients suffered a new ARVI and developed the corresponding antibodies. This information will be especially relevant in conditions of the rise in the incidence of ARVIs, as well as the COVID-19 pandemic observed in recent years.Copyright © 2022, Voprosy Prakticheskoi Pediatrii. All rights reserved.

Multiplex Assays Enable Simultaneous Detection and Identification of SARS-CoV-2 Variants of Concern in Clinical and Wastewater Samples

The targeted screening and sequencing approaches for COVID-19 surveillance need to be adjusted to fit the evolving surveillance objectives which necessarily change over time. We present the development of variant screening assays that can be applied to new targets in a timely manner and enable multiplexing of targets for efficient implementation in the laboratory. By targeting the HV69/70 deletion for Alpha, K417N for Beta, K417T for Gamma, and HV69/70 deletion plus K417N for sub-variants BA.1, BA.3, BA.4, and BA.5 of Omicron, we achieved simultaneous detection and differentiation of Alpha, Beta, Gamma, and Omicron in a single assay. Targeting both T478K and P681R mutations enabled specific detection of the Delta variant. The multiplex assays used in combination, targeting K417N and T478K, specifically detected the Omicron sub-variant BA.2. The limits of detection for the five variants of concern were 4-16 copies of the viral RNA per reaction. Both assays achieved 100% clinical sensitivity and 100% specificity. Analyses of 377 clinical samples and 24 wastewater samples revealed the Delta variant in 100 clinical samples (nasopharyngeal and throat swab) collected in November 2021. Omicron BA.1 was detected in 79 nasopharyngeal swab samples collected in January 2022. Alpha, Beta, and Gamma variants were detected in 24 wastewater samples collected in May-June 2021 from two major cities of Alberta (Canada), and the results were consistent with the clinical cases of multiple variants reported in the community. © 2023 The Authors. Published by American Chemical Society.