Nasopharyngeal Swab Samples Research Articles

Improved heat shock method for extracting total RNA from nasopharyngeal swab samples even with low viral load

This study aimed to improve the heat shock method as a cost-effective and time-efficient for total RNA extraction. We compared the effectiveness of two total RNA extraction methods by using Real-Time PCR for nasopharynx swabs. Include: I; use of a commercial total RNA extraction kit as a standard. II; utilized a modified heat shock method (MHS). Time, centrifuge speed and duration, proteinase K, and RNA carrier were optimized. The optimized parameters included treating the sample with 5 μg/μL at 56°C for 5 minutes, heating at 95°C for 5 minutes followed by thermal shock in ice for 3 minutes, adding 4 μg/μL RNA carrier at room temperature for 3 minutes, and centrifuging at 7000 rpm for 10 minutes. This optimization demonstrated a sensitivity and specificity of 100% (CI: 95%) even in samples with low viral load. Our in-house method presents a rapid, and cost-effective alternative for total RNA extraction.

COVID-19 in patients presenting with malaria-like symptoms at a primary healthcare facility in Accra, Ghana.

Malaria is a common and severe public health problem in Ghana and largely responsible for febrile symptoms presented at health facilities in the country. Other infectious diseases, including COVID-19, may mimic malaria due to their shared non-specific symptoms such as fever and headache thus leading to misdiagnosis. This study therefore investigated COVID-19 among patients presenting with malaria-like symptoms at Korle-Bu Polyclinic, Accra, Ghana. This study enrolled 300 patients presenting with malaria-like symptoms aged ≥18yrs. After consent was obtained from study patients, two to three millilitres of whole blood, nasopharyngeal and oropharyngeal swab samples, were collected for screening of Plasmodium falciparum using malaria rapid diagnostic test, microscopy and nested PCR, and SARS-CoV-2 using SARS-CoV-2 antigen test and Real-time PCR, respectively. The plasma and whole blood were also used for COVID-19 antibody testing and full blood counts using hematological analyser. SARS-CoV-2 whole genome sequencing was performed using MinIon sequencing. The prevalence of malaria by microscopy, RDT and nested PCR were 2.3%, 2.3% and 2.7% respectively. The detection of SARS-CoV-2 by COVID-19 Rapid Antigen Test and Real-time PCR were 8.7% and 20% respectively. The Delta variant was reported in 23 of 25 SARS-CoV-2 positives with CT values below 30. Headache was the most common symptom presented by study participants (95%). Comorbidities reported were hypertension, asthma and diabetes. One hundred and thirteen (37.8%) of the study participants had prior exposure to SARS CoV-2 and (34/51) 66.7% of Astrazeneca vaccinated patients had no IgG antibody. It may be difficult to use clinical characteristics to distinguish between patients with COVID-19 having malaria-like symptoms. Detection of IgM using RDTs may be useful in predicting CT values for SARS-CoV-2 real-time PCR and therefore transmission.

Cycle threshold values of SARS-CoV-2 RNA in conjunctival swabs and nasopharyngeal secretions: a comparative study from a tertiary care center in India.

To detect the viral RNA load of SARS-CoV-2 in conjunctival swabs of COVID-19 patients, and compare with nasopharyngeal swabs. Conjunctival swabs of COVID-19 patients (with PCR positive nasopharyngeal swabs) were subjected to quantitative reverse transcription-polymerase chain reaction (RT-PCR) for detection of SARS-CoV-2 RNA. The cycle threshold (Ct) values of Open Reading Frame 1 (ORF 1 Ab gene) and nucleoprotein (N gene) PCRs were used to assess the viral RNA load, and compare them with the baseline values of nasopharyngeal swabs. Of 93 patients, 17 (18.27%) demonstrated SARS-CoV-2 RNA in conjunctival swabs. Baseline nasopharyngeal swabs were collected at a median of 2days; while, the conjunctival swabs were collected at median 7days, from onset of illness (p < 0.001). Despite a significant delay in conjunctival swab collection than nasopharyngeal swabs, the Ct values (ORF or N gene PCRs) were comparable between nasopharyngeal swab and conjunctival swab samples. Subsequently, during the recovery period, in four of these 17 patients (with conjunctival swab positivity), when the second nasopharyngeal swab was 'negative', the conjunctival swab was 'positive'. The conjunctival swabs demonstrated SARS-CoV-2 RNA in 17 (18.27%) of 93 COVID-19 patients. Our results may suggest a delayed or a prolonged shedding of the virus/viral RNA on the ocular surface than in nasopharyngeal mucosa.

Comparing Polymerase Chain Reaction Testing of Nasopharyngeal Swab and Lower Respiratory Tract Specimens for the Diagnosis of Pneumocystis jirovecii Pneumonia.

Using nasopharyngeal (NP) swab samples instead of lower respiratory tract specimens for polymerase chain reaction (PCR) to diagnose Pneumocystis jirovecii pneumonia (PJP) may be better tolerated and improve diagnostic accessibility. In this 2-year Australian retrospective cohort study of patients with clinically suspected PJP, P jirovecii PCR on NP swab samples had perfect specificity but low sensitivity (0.66).

Clinical and molecular epidemiology of enterovirus D68 from 2013 to 2020 in Shanghai

Enterovirus D68 (EV-D68) is an emerging pathogen that has caused outbreaks of severe respiratory disease worldwide, especially in children. We aim to investigate the prevalence and genetic characteristics of EV-D68 in children from Shanghai. Nasopharyngeal swab or bronchoalveolar lavage fluid samples collected from children hospitalized with community-acquired pneumonia were screened for EV-D68. Nine of 3997 samples were EV-D68-positive. Seven of nine positive samples were sequenced and submitted to GenBank. Based on partial polyprotein gene (3D) or complete sequence analysis, we found the seven strains belong to different clades and subclades, including three D1 (detected in 2013 and 2014), one D2 (2013), one D3 (2019), and two B3 (2014 and 2018). Overall, we show different clades and subclades of EV-D68 spread with low positive rates (0.2%) among children in Shanghai between 2013 and 2020. Amino acid mutations were found in the epitopes of the VP1 BC and DE loops and C-terminus; similarity analysis provided evidence for recombination as an important mechanism of genomic diversification. Both single nucleotide mutations and recombination play a role in evolution of EV-D68. Genetic instability within these clinical strains may indicate large outbreaks could occur following cumulative mutations.

Molecular Detection of Equine Herpesviruses from Field Outbreaks in Donkeys in Northwest Amhara Region, Ethiopia.

Equine herpesviruses pose a threat to equine health and potentially cause substantial economic losses to the global equine industry. EHV outbreaks have been reported in various parts of Ethiopia and the Amhara region specifically. This study aimed to detect EHVs from suspected outbreak cases in selected districts of the Northwest Amhara region. A cross-sectional study was performed from January 2022 to July 2022 to detect EHVs from suspected outbreak cases. Clinical observation was conducted for the presumptive identification of equine herpesvirus infection, and nasopharyngeal swab samples were collected for molecular detection of the viruses for confirmation. Out of 463 donkeys observed, 23 donkeys showed clinical signs suggestive of equine herpesvirus infection. Samples from 10 suspected donkeys were further subjected to polymerase chain reaction (PCR) test, amplifying ORF30 for EHV-1 and gB for EHV-2 and EHV-5. Among the 10 donkeys tested, seven (n = 7) were positive for EHV-5. All ten (n = 10) tested donkeys were negative for EHV-1 and EHV-2. EHV-5 was detected in animals with nervous signs, respiratory signs, a combination of nervous and respiratory signs, and a combination of abortion, respiratory, and nervous signs. Generally, only EHV-5 was identified from the outbreak, and more detailed epidemiological/molecular studies should be performed to better understand its dynamics and inform preventive measures.

Direct immunoassay on a polyester microwell plate for colorimetric detection of the spike protein in swab and saliva samples.

This study presents the development of a polyester microplate for detecting the S-protein of the SARS-CoV-2 virus in saliva and nasopharyngeal swab samples using direct enzyme-linked immunosorbent assay (ELISA) technology. The polyester microplate was designed to contain 96 zones with a 3 mm diameter each, and a volume of 2-3 μL. The experimental conditions including reagent concentration and reaction time were optimized. The microplate image was digitized and analyzed using graphical software. The linear range obtained between protein S concentrations and pixel intensity was 0-10 μg mL-1, with a correlation coefficient of 0.99 and a limit of detection of 0.44 μg mL-1. The developed methodology showed satisfactory intraplate and interplate repeatability with RSD values lower than 7.8%. The results achieved through immunoassay performed on polyester microplates were consistent with those of the RT-PCR method and showed a sensitivity of 100% and 90% and specificity of 85.71% and 100% for saliva and nasopharyngeal samples, respectively. The proposed direct immunoassay on polyester microplates emerges as an alternative to conventional immunoassays performed on commercial polystyrene plates, given the low cost of the device, low consumption of samples and reagents, lower waste generation, and shorter analysis time. Moreover, the immunoassay has shown great potential for diagnosing COVID-19 with precision and accuracy.

Cross comparison of alternative diagnostic protocols including substitution to the clinical sample, RNA extraction method and nucleic acid amplification technology for COVID-19 diagnosis.

the gold-standard diagnostic protocol (GSDP) for COVID-19 consists of a nasopharyngeal swab (NPS) sample processed through traditional RNA extraction (TRE) and amplified with retrotranscription quantitative polymerase chain reaction (RT-qPCR). Multiple alternatives were developed to decrease time/cost of GSDP, including alternative clinical samples, RNA extraction methods and nucleic acid amplification. Thus, we carried out a cross comparison of various alternatives methods against GSDP and each other. we tested alternative diagnostic methods using saliva, heat-induced RNA release (HIRR) and a colorimetric retrotranscription loop-mediated isothermal amplification (RT-LAMP) as substitutions to the GSDP. RT-LAMP using NPS processed by TRE showed high sensitivity (96%) and specificity (97%), closely matching GSDP. When saliva was processed by TRE and amplified with both RT-LAMP and RT-qPCR, RT-LAMP yielded high diagnostic parameters (88%-96% sensitivity and 95%-100% specificity) compared to RT-qPCR. Nonetheless, when saliva processed by TRE and detected by RT-LAMP was compared against the GSDP, the resulting diagnostic values for sensitivity (78%) and specificity (87%) were somewhat high but still short of those of the GSDP. Finally, saliva processed with HIRR and detected via RT-LAMP was the simplest and fastest method, but its sensitivity against GSDP was too low (56%) for any clinical application. Also, in this last method, the acidity of a large percentage of saliva samples (9%-22%) affected the pH-sensitive colorimetric indicator used in the test, requiring the exclusion of these acidic samples or an extra step for pH correction. our comparison shows that RT-LAMP technology has diagnostic performance on par with RT-qPCR; likewise, saliva offers the same diagnostic functionality as NPS when subjected to a TRE method. Nonetheless, use of direct saliva after a HIRR and detected with RT-LAMP does not produce an acceptable diagnostic performance.

Rapid detection of monkeypox virus and differentiation of West African and Congo Basin strains using endonuclease restriction-mediated real-time PCR-based testing.

The ongoing multi-country outbreak of monkeypox virus (MPXV) has continuously attracted global attention, highlighting the critical need for timely and accurate methods to detect MPXV and differentiate its clades. Herein, we devised a novel multiplex ET-PCR (endonuclease restriction-mediated real-time PCR) assay that integrates PCR amplification, restriction endonuclease cleavage and real-time fluorescence detection to diagnose MPXV infection and distinguish the Congo Basin and West African MPXV strains. In the MPXV ET-PCR system, three sets of specific primers were designed for MPXV, Congo Basin and West African strains. A short sequence, which could be recognized by restriction endonuclease enzyme BstUI, was added to the 5'end of amplification primers. Then, the modified primers were assigned different reporter dyes and corresponding quenching dyes to each of the three targets, enabling real-time fluorescence reporting of the results and multiplex detection. The designed assay enabled the detection of single or three targets in a single tube, with excellent specificity and analytical sensitivity in terms of plasmid and pseudotyped virus. Moreover, the clinical feasibility of our assay was validated using artificially simulated plasma, nasopharyngeal swab and skin swab samples. In conclusion, the multiplex ET-PCR assay devised here had the advantages of simple primer design, cost-effectiveness, low contamination risk, excellent sensitivity, high specificity and multiplex detection, making it a valuable and dependable tool for curbing the extensive spread of MPXV.

Pediatric Respiratory Pathogens Circulate in Children and Adults in Communities Near Susceptible Wild Great Ape Populations in Uganda

Respiratory infections are a leading cause of death in developing countries. Infants and young children are especially susceptible to disease because they lack immunity, whereas adults who have acquired immunity can be infected asymptomatically. Great ape species, all of which are endangered, are similarly susceptible to respiratory illnesses caused by human respiratory pathogens. We obtained 432 nasopharyngeal swab samples (127 from adults and 305 from children) in a cross‐sectional study that took place between February and October 2022 at four sites in Western Uganda (Budongo Central Forest Reserve, Bulindi Town Council, Bwindi Impenetrable National Park, and Kibale National Park) where the participants live in communities where interaction with apes is frequent. Prior research at Kibale has shown that locally circulating human respiratory pathogens have led to multiple lethal outbreaks in wild eastern chimpanzees (Pan troglodytes schweinfurthii). We used a multiplex PCR panel to characterize respiratory pathogens, with the goal of assessing whether respiratory illnesses in the chimpanzees of Budongo and Bulindi and the mountain gorillas (Gorilla beringei beringei) of Bwindi might have originated in local children and been introduced to the apes via asymptomatic adult carriers. The prevalence of respiratory pathogens was twice as high in Bwindi (44.0%) as it was in Budongo (24.0%) and Bulindi (20.8%), while the prevalence was intermediate at Kibale (34.4%). Rates of respiratory pathogen detection were higher but statistically indistinguishable in children compared to adults at Budongo and Bulindi, and children were 15.0 times more likely than adults to have positive detections at Kibale. At Bwindi, however, the pattern was reversed, with adults 2.6 times more likely than children to have positive detections. Rhinovirus, metapneumovirus, human parainfluenza virus 3, respiratory syncytial virus, and coronavirus OC43, all of which have been identified as causative agents of respiratory disease outbreaks in great ape populations across sub‐Saharan Africa, accounted for three quarters (73.6%) of detected pathogens. Our data support the idea that human respiratory pathogens that can infect great apes occur at high frequencies in human populations in Western Uganda that live close to and interact with wild apes that have suffered from lethal outbreaks caused by these same pathogens. Reducing respiratory infections in local children, thereby reducing both carriage of those infections into the forest by people and ape exposure to these pathogens when they enter human spaces, should decrease the risk of respiratory disease outbreaks in apes.

Evaluation of Two Commercial Kits for Qualitative Detection of SARS-CoV-2 Antigens in Nasopharyngeal Specimens.

Two rapid antigen tests (RATs) for COVID-19 targeting the nucleocapsid protein of SARS-CoV-2 were compared with real-time RT-PCR as the reference method. Ninety-six nasopharyngeal swab samples, comprising 56 positive and 40 negative samples confirmed through rRT-PCR were collected and retested to determine the reliability of the two nasopharyngeal RATs. The overall sensitivity and specificity of both RATs were 64.3% (95% confidence interval 50.4 - 76.6%) and 100% (95% confidence interval 91.2 - 100%), respectively. Cohen's kappa coefficient of agreement of both RATs to rRT-PCR was 0.600 (95% confidence interval 0.457 - 0.743) (p < 0.001), showing almost perfect agreement when the Ct values were less than 25 in rRT-PCR. A significant difference in Ct values between true positives and false negatives was observed (Mann-Whitney-Wilcoxon test; p < 0.001). Compared to rRT-PCR, RATs have fewer false negatives. In suspected COVID-19 cases, negative RAT results should be retested using either RAT or rRT-PCR.

Investigation of Urine Organic Acid Profile in Coronavirus Disease (COVID-19) Patients.

Coronavirus disease 2019 (COVID-19) is a viral infection caused by severe acute respiratory syn-drome coronavirus 2 (SARS-CoV-2). The high mortality and morbidity rate and its course with a wide variety of clinical symptoms indicate that the disease affects many metabolic pathways. The aim of our study was to investi-gate the metabolic effects of SARS-CoV-2 by evaluating the urinary organic acid profile in patients with SARS-CoV-2 infection. Can metabolites in urine guide the diagnosis, follow-up, treatment, and prognosis of COVID-19? Forty-two patients, including 30 SARS-CoV-2 RT-PCR positive patients and 12 SARS-CoV-2 RT-PCR negative controls, were studied. SARS-COV-2 RT-PCR of nasopharyngeal swab samples was studied. Urines were evaluated in the GCMS-QP2010 SE Gas Chromatograph Mass Spectrometer (SHIMADZU) device for organic acid profile in the metabolism laboratory. Urine organic acid profile was evaluated by studying 117 organic acids in each patient. Tiglylglycine, 2-hydroxybutyric acid, 4-hydroxyphenylpyruvic acid, 3-hydroxypropionic acid, erythro-4,5-dihydroxyhexanoic acid lactone, 2-hydroxyphenylacetic acid, N-acetyltyrosine, 3-phenyllactic acid, 5-hydroxyindolacetic acid, 3-hydroxysebacic acid, palmitic acid, 3-methylglutaconic acid, 3-methylglutaric acid, lactic acid, pyruvic acid-oxime, 3-hydroxysobutyric acid, and organic acids were found to be increased, compared to the control group. Tiglylglycine has been specifically identified as a potential biomarker of respiratory chain disorders. The deterioration in lipid metabolism and pyruvate pathway in COVID-19 patients was evaluated as remarkable.

The Performance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Virus Using the Colorimetric Reverse-Transcription Loop Mediated Isothermal Amplification (RT-LAMP) Method in Saliva Specimens of Suspected COVID-19 Patients.

Corona Virus Disease-19 (COVID-19) is a disease caused by Severe-Acute-Respiratory-Syndrome-Coronavirus-2 (SARS-CoV-2). The most reliable and widely accepted method for diagnosing this infection, despite facing various challenges, is the Reverse Transcription Polymerase Chain Reaction (RT-PCR) method, which utilizes nasopharyngeal swab sample. Reverse-transcription loop mediated isothermal amplification (RT-LAMP) is a simpler nucleic acid amplification method compared to the RT-PCR method. This method has several advantages, including: of amplification at constant temperature, faster results, and potentially greater examination capacity. This study aimed to compare the validity of the RT-LAMP method using saliva specimens with that of the RT-PCR method using nasopharyngeal smears. This was an analytical observational study with a cross-sectional design. The participants were inpatients in the COVID-19 special isolation building of Hasan Sadikin General Hospital, Indonesia with a probable (clinical symptoms of covid, but not confirm NAAT examination) or confirmed diagnosis of COVID-19 from September 2021 to February 2022. The inclusion criteria are COVID-19 patients with symptoms, adult subjects, and composite mentions. Patients who were unable to secrete saliva were also excluded. In total, 118 specimens were collected. The validity test results of the saliva specimens using the RT-LAMP method showed sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), of 65.5%, 100%, 100%, and 75%, respectively. The results increased in subjects treated between 3 and 7 days after symptom onset ie 73.2%, 100%, 100%, and 82.3%, respectively. The very strong specificity accompanied by good sensitivity and NPV in the group of subjects treated 3-7 days after the onset of symptoms indicates that the RT-LAMP method using saliva specimens can be an efficient and reliable alternative tool in detecting the SARS-CoV-2 virus.

Prevalence of Parainfluenza Viruses in Seoul, 2021 - 2022.

Parainfluenza virus (PIV) is a significant etiological agent of acute lower respiratory tract infections (ALRIs) in infants and young children. The present study has been conducted to investigate the prevalence of recently identified respiratory viruses. In total, 543 oropharyngeal or nasopharyngeal swab samples collected from hospitalized patients with acute respiratory symptoms (ARS) between January and December 2021 (5,653 females and 4,950 males) were tested for respiratory viruses using RT-PCR. At least one respiratory virus was detected by RT-PCR in 119 out of 175 samples (68%). The most frequently detected virus was human rhinovirus (HRV) (34, 6.5%), followed by human parainfluenza viruses (HPIVs) (19, 3.6%), human bocavirus (HBoV) (8, 1.5%), human adenovirus (HAdV) (7, 1.3%), and human respiratory syncytial virus (HRSV) (4, 0.8%). HPIV-3 accounted for 3.6% (19/175) of all viral pathogens and was the second most frequently detected viral pathogen in our study. HPIV-3 infections peaked in the fall (November) of 2021. Phylogenetic analysis of the coding region of the viral protein HA revealed that all 35 (100%) of 35 HPIV-infected patients were infected with HPIV-3. HPIV was an important causative pathogen associated with ALRI in children hospitalized in Korea in the late fall of 2021, as the social distancing rules for COVID-19 were relaxed. These findings highlight the im-portance of HPIV as a cause of ALRI.

Detection of human bocavirus (HBoV) in children with acute respiratory infection (ARI) during the covid-19 transition period

Acute respiratory infections (ARIs) are the highest cause of death in children in the world. Based on the 2018 Riset Kesehatan Dasar Nasional, ARI cases in Indonesia showed a prevalence of 4.4%, with the highest cases occurring in children. One of the new viruses first identified in 2005 in human nasopharyngeal samples is the human bocavirus (HBoV). HBoV is a single-strand DNA virus belonging to the Parvoviridae family. This study aimed to assess the prevalence of HBoV in children presenting with ARI during the transitional period of the Covid-19 era. HBoV detection was conducted using multiplex PCR and reverse hybridization methods on nasopharyngeal and oropharyngeal swab samples collected from symptomatic children. This study reported a prevalence rate of 4.94% for HBoV in 2022 and 5.04% in 2023. Furthermore, the study identified favorable detection rates for HBoV in children with ARIs as 14.81% in 2022 and 8.45% in 2023. These rates ranked 2nd and 5th highest among other pathogens detected in ARIs. Additionally, there was an increase in positive HBoV samples from 4 samples in 2022 to 6 samples in 2023, which was attributed to the relaxation of nonpharmaceutical Covid-19 interventions by mid-2022. HBoV was identified at a significant rate among children with ARI in Jakarta during the transitional phase of the Covid-19 era (2022-2023). Given its potential to induce severe ARI, HBoV necessitates heightened attention as an etiological agent.

Evaluation of Hospitalized Patients with Community-Acquired Influenza-Like Illness During Two Influenza Seasons.

Influenza is among the most important respiratory infections affecting all age groups and can lead to hospitalizations. We aimed to determine the frequency of influenza infections among acute admissions with influenza-like illness (ILI) and evaluate the demographic, clinical findings, and outcomes of patients with influenza. This prospective, active surveillance study was conducted in a university hospital between 2015 and 2017. Patients hospitalized for at least 24 hours in the selected units with community-acquired ILI were screened according to certain influenza-predicting ICD-10 codes. Nasopharyngeal and pharyngeal swab samples were taken from patients who were eligible for the study. Patients tested for influenza with real-time polymerase chain reaction. Univariate and multivariate analyses were performed for data. Among 440 patients screened according to influenza-related ICD-10 codes, 112 were included. Influenza positivity was detected in 37 of the 112 patients. Clinical findings were similar between influenza positive and negative groups and also between influenza subtypes, excluding sore throat, which was more common in the H1N1 group. Alanine transaminase (ALT), aspartate transaminase (AST), and creatine kinase (CK) elevations were found to be significantly higher in the influenza-positive group. When influenza-positive patients with and without pneumonia were compared, the rate of vaccination in the same season was higher in patients without pneumonia than in patients with pneumonia (38.8% and 10.5%, respectively; p=0.04). Integrating molecular tests detecting both influenza and other respiratory viruses into influenza surveillance programs can increase the efficacy and quality of these programs. The elevation of AST, ALT, and CK in influenza cases can be considered in distinguishing influenza from other ILI cases. Vaccination in the same season can reduce the risk of pneumonia in influenza-positive patients.

Sensitivity and Specificity of Rapid Antigen Antigen Examination for COVID-19 Immunochromatography Method at the Dadi Regional Special Hospitals

Currently COVID-19 is still in pandemic status so the transmission of this disease needs to be controlled. One of the government's efforts is to screen people suspected of COVID-19 with laboratory examinations using RT-PCR, but this is hampered by long test times. For this reason, the Rapid Antigen COVID-19 (RA COVID-19) immunochromatography (IT) method is used which is short in time and uses simple equipment. This research is a laboratory observational research which aims to determine the sensitivity and specificity of the RA COVID-19 method at RSKD Dadi, South Sulawesi Province. The research sample was 50 suspected COVID-19 patients being treated at RSKD Dadi, South Sulawesi Province and then nasopharyngeal swab samples were taken from these patients. The 50 swab samples were examined using the RA COVID-19 method and the Real Time Polymerase Chain Reaction (RT-PCR) method. Location The research was carried out at the RSKD Dani laboratory, South Sulawesi Province for 6 months (July-December 2022). The research results showed that the RA COVID-19 used at RSKD Dadi, South Sulawesi Province as a COVID-19 screening method for suspected COVID-19 patients had a sensitivity of 87% and a specificity of 97%. Conclusion: RA COVID-19 used at RSKD Dadi, South Sulawesi Province can be used as a screening method, but the results still need to be confirmed using the RT-PCR method

Study of the resistome of human microbial communities using a targeted panel of antibiotic resistance genes in COVID-19 patients

To study overall drug resistance genes (resistome) in the human gut microbiome and the changes in these genes during COVID-19 in-hospital therapy. A single-center retrospective cohort study was conducted. Only cases with laboratory-confirmed SARS-CoV-2 RNA using polymerase chain reaction in oro-/nasopharyngeal swab samples were subject to analysis. The patients with a documented history of or current comorbidities of the hepatobiliary system, malignant neoplasms of any localization, systemic and autoimmune diseases, as well as pregnant women were excluded. Feces were collected from all study subjects for subsequent metagenomic sequencing. The final cohort was divided into two groups depending on the disease severity: mild (group 1) and severe (group 2). Within group 2, five subgroups were formed, depending on the use of antibacterial drugs (ABD): group 2A (receiving ABD), group 2AC (receiving ABD before hospitalization), group 2AD (receiving ABD during hospitalization), group 2AE (receiving ABD during and before hospitalization), group 2B (not receiving ABD). The median number of antibiotic resistance (ABR) genes (cumulative at all time points) was significantly higher in the group of patients treated with ABD: 81.0 (95% CI 73.8-84.5) vs. 51.0 (95% CI 31.1-68.4). In the group of patients treated with ABD (2A), the average number of multidrug resistance genes (efflux systems) was significantly higher than in controls (group 2B): 47.0 (95% CI 46.0-51.2) vs. 21.5 (95% CI 7.0-43.9). Patients with severe coronavirus infection tended to have a higher median number of ABR genes but without statistical significance. Patients in the severe COVID-19 group who did not receive ABD before and during hospitalization also had more resistance genes than the patients in the comparison group. This study demonstrated that fewer ABR genes were identified in the group with a milder disease than in the group with a more severe disease associated with more ABR genes, with the following five being the most common: SULI, MSRC, ACRE, EFMA, SAT.

Plasmon-Enhanced Digital Fluoroimmunoassay for Subfemtomolar Detection of Protein Biomarkers.

Rapid and accurate quantification of low-abundance protein biomarkers in biofluids can transform the diagnosis of a range of pathologies, including infectious diseases. Here, we harness ultrabright plasmonic fluors as "digital nanolabels" and demonstrate the detection and quantification of subfemtomolar concentrations of human IL-6 and SARS-CoV-2 alpha and variant proteins in clinical nasopharyngeal swab and saliva samples from COVID-19 patients. The resulting digital plasmonic fluor-linked immunosorbent assay (digital p-FLISA) enables detection of SARS-CoV-2 nucleocapsid protein, both in solution and in live virions. Digital p-FLISA outperforms the "gold standard" enzyme-linked immunosorbent assay (ELISA), having a nearly 7000-fold lower limit-of-detection, and outperforms a commercial antigen test, having over 5000-fold improvement in analytical sensitivity. Detection and quantification of very low concentrations of target proteins holds potential for early detection of pathological conditions, treatment monitoring, and personalized medicine.

Effect of age and season on respiratory mucosal immune marker profiles

BackgroundThe upper respiratory tract is continuously exposed to microorganisms and noxious elements, leading to local immune responses and the secretion of immune markers. While several studies describe immune marker profiles in respiratory mucosal samples in defined patient cohorts, mucosal immune profiles from the general population during the different seasons are lacking. Such baseline profiles are essential to understand the impact of various exposures to the mucosal immune system throughout life. ObjectivesTo establish baseline, local upper respiratory mucosal immune profiles in the general population, and assess these profiles in regard to age, sex, seasonality, and basic health and lifestyle factors. MethodsWe measured the concentrations of 35 immune markers involved in a broad range of immunological processes at the mucosa in nasopharyngeal swab samples from 951 individuals from a nationwide study, aged 0-86 years. ResultsClustering analysis shows that the immune marker profiles clearly reflect immunological functions, such as tissue regeneration and antiviral responses. The concentrations of the immune markers changed strongly with seasonality and age -with the most profound changes occurring in the first 25 years of life- and also associated with sex, BMI, smoking, mild symptoms of airway infection, and chronic asthma and hay fever. ConclusionWe show that immunological analyses of non-invasive mucosal samples provide insight into mucosal immune responses to microbial and noxious element exposure in the general population. These data provide a baseline for future studies on respiratory mucosal immune responses and for the development of mucosal immune-based diagnostics.