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1.
Cochrane Database Syst Rev ; 8: CD013207, 2021 08 12.
Article in English | MEDLINE | ID: covidwho-1813441

ABSTRACT

BACKGROUND: The standard method of diagnosing HIV in infants and children less than 18 months is with a nucleic acid amplification test reverse transcriptase polymerase chain reaction test (NAT RT-PCR) detecting viral ribonucleic acid (RNA). Laboratory testing using the RT-PCR platform for HIV infection is limited by poor access, logistical support, and delays in relaying test results and initiating therapy in low-resource settings. The use of rapid diagnostic tests at or near the point-of-care (POC) can increase access to early diagnosis of HIV infection in infants and children less than 18 months of age and timely initiation of antiretroviral therapy (ART). OBJECTIVES: To summarize the diagnostic accuracy of point-of-care nucleic acid-based testing (POC NAT) to detect HIV-1/HIV-2 infection in infants and children aged 18 months or less exposed to HIV infection. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (until 2 February 2021), MEDLINE and Embase (until 1 February 2021), and LILACS and Web of Science (until 2 February 2021) with no language or publication status restriction. We also searched conference websites and clinical trial registries, tracked reference lists of included studies and relevant systematic reviews, and consulted experts for potentially eligible studies. SELECTION CRITERIA: We defined POC tests as rapid diagnostic tests conducted at or near the patient site. We included any primary study that compared the results of a POC NAT to a reference standard of laboratory NAT RT-PCR or total nucleic acid testing to detect the presence or absence of HIV infection denoted by HIV viral nucleic acids in infants and children aged 18 months or less who were exposed to HIV-1/HIV-2 infection. We included cross-sectional, prospective, and retrospective study designs and those that provided sufficient data to create the 2 × 2 table to calculate sensitivity and specificity. We excluded diagnostic case control studies with healthy controls. DATA COLLECTION AND ANALYSIS: We extracted information on study characteristics using a pretested standardized data extraction form. We used the QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies) tool to assess the risk of bias and applicability concerns of the included studies. Two review authors independently selected and assessed the included studies, resolving any disagreements by consensus. The unit of analysis was the participant. We first conducted preliminary exploratory analyses by plotting estimates of sensitivity and specificity from each study on forest plots and in receiver operating characteristic (ROC) space. For the overall meta-analyses, we pooled estimates of sensitivity and specificity using the bivariate meta-analysis model at a common threshold (presence or absence of infection). MAIN RESULTS: We identified a total of 12 studies (15 evaluations, 15,120 participants). All studies were conducted in sub-Saharan Africa. The ages of included infants and children in the evaluations were as follows: at birth (n = 6), ≤ 12 months (n = 3), ≤ 18 months (n = 5), and ≤ 24 months (n = 1). Ten evaluations were field evaluations of the POC NAT test at the point of care, and five were laboratory evaluations of the POC NAT tests.The POC NAT tests evaluated included Alere q HIV-1/2 Detect qualitative test (recently renamed m-PIMA q HIV-1/2 Detect qualitative test) (n = 6), Xpert HIV-1 qualitative test (n = 6), and SAMBA HIV-1 qualitative test (n = 3). POC NAT pooled sensitivity and specificity (95% confidence interval (CI)) against laboratory reference standard tests were 98.6% (96.1 to 99.5) (15 evaluations, 1728 participants) and 99.9% (99.7 to 99.9) (15 evaluations, 13,392 participants) in infants and children ≤ 18 months. Risk of bias in the included studies was mostly low or unclear due to poor reporting. Five evaluations had some concerns for applicability for the index test, as they were POC tests evaluated in a laboratory setting, but there was no difference detected between settings in sensitivity (-1.3% (95% CI -4.1 to 1.5)); and specificity results were similar. AUTHORS' CONCLUSIONS: For the diagnosis of HIV-1/HIV-2 infection, we found the sensitivity and specificity of POC NAT tests to be high in infants and children aged 18 months or less who were exposed to HIV infection.


Subject(s)
HIV Infections/diagnosis , HIV-1/genetics , HIV-2/genetics , Point-of-Care Testing , Polymerase Chain Reaction/methods , Cross-Sectional Studies , Female , HIV-1/isolation & purification , HIV-2/isolation & purification , Humans , Infant , Infant, Newborn , Male , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity
2.
Virol J ; 18(1): 189, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1779657

ABSTRACT

BACKGROUND: The importance of clinicolaboratory characteristics of COVID-19 made us report our findings in the Alborz province according to the latest National Guideline for the diagnosis and treatment of COVID-19 in outpatients and inpatients (trial five versions, 25 March 2020) of Iran by emphasizing rRT-PCR results, clinical features, comorbidities, and other laboratory findings in patients according to the severity of the disease. METHODS: In this study, 202 patients were included, primarily of whom 164 had fulfilled the inclusion criteria. This cross-sectional, two-center study that involved 164 symptomatic adults hospitalized with the diagnosis of COVID-19 between March 5 and April 5, 2020, was performed to analyze the frequency of rRT-PCR results, distribution of comorbidities, and initial clinicolaboratory data in severe and non-severe cases, comparing the compatibility of two methods for categorizing the severity of the disease. RESULTS: According to our findings, 111 patients were rRT-PCR positive (67.6%), and 53 were rRT-PCR negative (32.4%), indicating no significant difference between severity groups that were not related to the date of symptoms' onset before admission. Based on the National Guideline, among vital signs and symptoms, mean oxygen saturation and frequency of nausea showed a significant difference between the two groups (P < 0.05); however, no significant difference was observed in comorbidities. In CURB-65 groups, among vital signs and comorbidities, mean oxygen saturation, diabetes, hypertension (HTN), hyperlipidemia, chronic heart disease (CHD), and asthma showed a significant difference between the two groups (P < 0.05), but no significant difference was seen in symptoms. CONCLUSION: In this study, rRT-PCR results of hospitalized patients with COVID-19 were not related to severity categories. From initial clinical characteristics, decreased oxygen saturation appears to be a more common abnormality in severe and non-severe categories. National Guideline indices seem to be more comprehensive to categorize patients in severity groups than CURB-65, and there was compatibility just in non-severe groups of National Guideline and CURB-65 categories.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , Adult , Aged , COVID-19/physiopathology , Comorbidity , Cross-Sectional Studies , Female , Hospitalization , Humans , Iran , Male , Middle Aged , SARS-CoV-2/genetics , Severity of Illness Index , World Health Organization
3.
PLoS One ; 17(2): e0264201, 2022.
Article in English | MEDLINE | ID: covidwho-1714776

ABSTRACT

Activating mutations in EGFR predict benefit from tyrosine kinase inhibitor therapy for patients with advanced non-small cell lung cancer. Directing patients to appropriate therapy depends on accurate and timely EGFR assessment in the molecular pathology laboratory. This article describes the analytical design, performance characteristics, and clinical implementation of an assay for the rapid detection of EGFR L858R and exon 19 deletion mutations. A droplet digital polymerase chain reaction (ddPCR) assay was implemented with probe hydrolysis-dependent signal detection. A mutation-specific probe was used to detect EGFR L858R. A loss of signal design was used to detect EGFR exon 19 deletion mutations. Analytical sensitivity was dependent on DNA input and was as low as 0.01% variant allele fraction for the EGFR L858R assay and 0.1% variant allele fraction for the EGFR exon 19 deletion assay. Correlation of 20 clinical specimens tested by ddPCR and next generation sequencing showed 100% concordance. ddPCR showed 53% clinical sensitivity in the detection of EGFR mutations in plasma cell-free DNA from patients with lung cancer. The median clinical turnaround time was 5 days for ddPCR compared to 13 days for next generation sequencing. The findings show that ddPCR is an accurate and rapid method for detecting EGFR mutations in patients with non-small cell lung cancer.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , DNA Mutational Analysis/methods , Lung Neoplasms/genetics , Polymerase Chain Reaction/methods , ErbB Receptors/genetics , Humans , Mutation , Sensitivity and Specificity
4.
Indoor Air ; 32(2): e13002, 2022 02.
Article in English | MEDLINE | ID: covidwho-1700268

ABSTRACT

The airborne route is the dominant form of COVID-19 transmission, and therefore, the development of methodologies to quantify SARS-CoV-2 in bioaerosols is needed. We aimed to identify SARS-CoV-2 in bioaerosols by using a highly efficient sampler for the collection of 1-3 µm particles, followed by a highly sensitive detection method. 65 bioaerosol samples were collected in hospital rooms in the presence of a COVID-19 patient using a liquid impinger sampler. The SARS-CoV-2 genome was detected by ddPCR using different primer/probe sets. 44.6% of the samples resulted positive for SARS-CoV-2 following this protocol. By increasing the sampled air volume from 339 to 650 L, the percentage of positive samples went from 41% to 50%. We detected five times less positives with a commercial one-step RT-PCR assay. However, the selection of primer/probe sets might be one of the most determining factor for bioaerosol SARS-CoV-2 detection since with the ORF1ab set more than 40% of the samples were positive, compared to <10% with other sets. In conclusion, the use of a liquid impinger collector and ddPCR is an adequate strategy to detect SARS-CoV-2 in bioaerosols. However, there are still some methodological aspects that must be adjusted to optimize and standardize a definitive protocol.


Subject(s)
Air Pollution, Indoor , COVID-19 , SARS-CoV-2/isolation & purification , COVID-19/diagnosis , Hospitals , Humans , Polymerase Chain Reaction/methods , RNA, Viral/analysis
5.
Sci Rep ; 12(1): 1650, 2022 01 31.
Article in English | MEDLINE | ID: covidwho-1661981

ABSTRACT

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the coronavirus strain causing the respiratory pandemic COVID-19 (coronavirus disease 2019). To understand the pathobiology of SARS-CoV-2 in humans it is necessary to unravel the metabolic changes that are produced in the individuals once the infection has taken place. The goal of this work is to provide new information about the altered biomolecule profile and with that the altered biological pathways of patients in different clinical situations due to SARS-CoV-2 infection. This is done via metabolomics using HPLC-QTOF-MS analysis of plasma samples at COVID-diagnose from a total of 145 adult patients, divided into different clinical stages based on their subsequent clinical outcome (25 negative controls (non-COVID); 28 positive patients with asymptomatic disease not requiring hospitalization; 27 positive patients with mild disease defined by a total time in hospital lower than 10 days; 36 positive patients with severe disease defined by a total time in hospital over 20 days and/or admission at the ICU; and 29 positive patients with fatal outcome or deceased). Moreover, follow up samples between 2 and 3 months after hospital discharge were also obtained from the hospitalized patients with mild prognosis. The final goal of this work is to provide biomarkers that can help to better understand how the COVID-19 illness evolves and to predict how a patient could progress based on the metabolites profile of plasma obtained at an early stage of the infection. In the present work, several metabolites were found as potential biomarkers to distinguish between the end-stage and the early-stage (or non-COVID) disease groups. These metabolites are mainly involved in the metabolism of carnitines, ketone bodies, fatty acids, lysophosphatidylcholines/phosphatidylcholines, tryptophan, bile acids and purines, but also omeprazole. In addition, the levels of several of these metabolites decreased to "normal" values at hospital discharge, suggesting some of them as early prognosis biomarkers in COVID-19 at diagnose.


Subject(s)
Asymptomatic Infections/epidemiology , COVID-19/blood , COVID-19/diagnosis , Metabolome , Metabolomics/methods , Pandemics , SARS-CoV-2/genetics , Severity of Illness Index , Adult , Aged , Aged, 80 and over , Biomarkers/blood , COVID-19/epidemiology , COVID-19/physiopathology , Case-Control Studies , Cohort Studies , Female , Follow-Up Studies , Humans , Male , Middle Aged , Patient Admission , Polymerase Chain Reaction/methods , Spain/epidemiology
6.
Anal Biochem ; 641: 114565, 2022 03 15.
Article in English | MEDLINE | ID: covidwho-1632512

ABSTRACT

Polymerase chain reaction (PCR) is the most widely used method for nucleic acids amplification. To date, a huge number of versatile PCR techniques have been developed. One of the relevant goals is to shorten PCR duration, which can be achieved in several ways. Here, we report on the results regarding nucleic acids amplification by convective PCR (cPCR) in standard 0.2 ml polypropylene microtubes. The following conditions were found to be optimal for such amplification: 1) 70 µl reaction volume, 2) the supply of external temperature 145°Ð¡ for the denaturation zone and 0°Ð¡ for the annealing zone, 3) ∼30° inclination of the microtube main axis, 4) the use of nearby primers, and 5) duration of the reaction 15-20 min. At these conditions, the amplification products are accumulated in an amount sufficient to be registered by gel electrophoresis, and high sensitivity of the reaction comparable to that of conventional PCR is achieved. cPCR provided the reliable detection of SARS-CoV-2 coronavirus RNA isolated from nasopharyngeal swabs of COVID-19 patients.


Subject(s)
COVID-19 Nucleic Acid Testing/instrumentation , COVID-19/diagnosis , Polymerase Chain Reaction/instrumentation , SARS-CoV-2/isolation & purification , COVID-19/virology , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/methods , Convection , Humans , Polymerase Chain Reaction/economics , Polymerase Chain Reaction/methods , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Temperature , Time Factors
7.
JAMA Netw Open ; 5(1): e2142796, 2022 01 04.
Article in English | MEDLINE | ID: covidwho-1615909

ABSTRACT

Importance: The SARS-CoV-2 viral trajectory has not been well characterized in incident infections. These data are needed to inform natural history, prevention practices, and therapeutic development. Objective: To characterize early SARS-CoV-2 viral RNA load (hereafter referred to as viral load) in individuals with incident infections in association with COVID-19 symptom onset and severity. Design, Setting, and Participants: This prospective cohort study was a secondary data analysis of a remotely conducted study that enrolled 829 asymptomatic community-based participants recently exposed (<96 hours) to persons with SARS-CoV-2 from 41 US states from March 31 to August 21, 2020. Two cohorts were studied: (1) participants who were SARS-CoV-2 negative at baseline and tested positive during study follow-up, and (2) participants who had 2 or more positive swabs during follow-up, regardless of the initial (baseline) swab result. Participants collected daily midturbinate swab samples for SARS-CoV-2 RNA detection and maintained symptom diaries for 14 days. Exposure: Laboratory-confirmed SARS-CoV-2 infection. Main Outcomes and Measures: The observed SARS-CoV-2 viral load among incident infections was summarized, and piecewise linear mixed-effects models were used to estimate the characteristics of viral trajectories in association with COVID-19 symptom onset and severity. Results: A total of 97 participants (55 women [57%]; median age, 37 years [IQR, 27-52 years]) developed incident infections during follow-up. Forty-two participants (43%) had viral shedding for 1 day (median peak viral load cycle threshold [Ct] value, 38.5 [95% CI, 38.3-39.0]), 18 (19%) for 2 to 6 days (median Ct value, 36.7 [95% CI, 30.2-38.1]), and 31 (32%) for 7 days or more (median Ct value, 18.3 [95% CI, 17.4-22.0]). The cycle threshold value has an inverse association with viral load. Six participants (6%) had 1 to 6 days of viral shedding with censored duration. The peak mean (SD) viral load was observed on day 3 of shedding (Ct value, 33.8 [95% CI, 31.9-35.6]). Based on the statistical models fitted to 129 participants (60 men [47%]; median age, 38 years [IQR, 25-54 years]) with 2 or more SARS-CoV-2-positive swab samples, persons reporting moderate or severe symptoms tended to have a higher peak mean viral load than those who were asymptomatic (Ct value, 23.3 [95% CI, 22.6-24.0] vs 30.7 [95% CI, 29.8-31.4]). Mild symptoms generally started within 1 day of peak viral load, and moderate or severe symptoms 2 days after peak viral load. All 535 sequenced samples detected the G614 variant (Wuhan strain). Conclusions and Relevance: This cohort study suggests that having incident SARS-CoV-2 G614 infection was associated with a rapid viral load peak followed by slower decay. COVID-19 symptom onset generally coincided with peak viral load, which correlated positively with symptom severity. This longitudinal evaluation of the SARS-CoV-2 G614 with frequent molecular testing serves as a reference for comparing emergent viral lineages to inform clinical trial designs and public health strategies to contain the spread of the virus.


Subject(s)
COVID-19/virology , RNA, Viral , SARS-CoV-2 , Severity of Illness Index , Viral Load , Virus Shedding , Adult , COVID-19/complications , Female , Humans , Incidence , Longitudinal Studies , Male , Middle Aged , Molecular Diagnostic Techniques/methods , Polymerase Chain Reaction/methods , Prospective Studies , Serologic Tests
8.
J Clin Lab Anal ; 36(1): e24152, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1565196

ABSTRACT

The ongoing COVID-19 pandemic constitutes a new challenge for public health. Prevention and control of infection have become urgent and serious issues. To meet the clinical demand for higher accuracy of COVID-19 detection, the development of fast and efficient methods represents an important step. The most common methods of COVID-19 diagnosis, relying on real-time fluorescent quantitative PCR(RT-qPCR), computed tomography, and new-generation sequencing technologies, have a series of advantages, especially for early diagnosis and screening. In addition, joint efforts of researchers all over the world have led to the development of other rapid detection methods with high sensitivity, ease of use, cost-effectiveness, or allowing multiplex analysis based on technologies such as dPCR, ELISA, fluorescence immunochromatography assay, and the microfluidic detection chip method. The main goal of this review was to provide a critical discussion on the development and application of these different analytical methods, which based on etiology, serology, and molecular biology, as well as to compare their respective advantages and disadvantages. In addition to these methods, hematology and biochemistry, as well as auxiliary analysis based on pathological anatomy, ultrasonography, and cytokine detection, will help understand COVID-19 pathogenesis. Together, these technologies may promote and open new windows to unravel issues surrounding symptomatic and asymptomatic COVID-19 infections and improve clinical strategies toward reducing mortality.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnostic imaging , Polymerase Chain Reaction/methods , COVID-19/pathology , Chromatography, Affinity/methods , Cytokines/blood , Enzyme-Linked Immunosorbent Assay , Four-Dimensional Computed Tomography , Gold Colloid , Humans , Mass Spectrometry/methods , Nasopharynx/virology , SARS-CoV-2/genetics
9.
PLoS One ; 16(11): e0260298, 2021.
Article in English | MEDLINE | ID: covidwho-1554516

ABSTRACT

BACKGROUND: Some studies revealed that despite having sufficient sun exposure and dietary supply, the level of serum 25(OH)D in Bangladeshi adults is lower than its normal range. Genetic pattern of an individual is also an essential factor that regulates the level of serum 25(OH)D. However, the genetic variations of CYP2R1 (rs10741657) and their association with low serum 25(OH)D level in Bangladeshi adults are yet to be explored. OBJECTIVE: This study was conducted to determine the frequency of variants of rs10741657 of CYP2R1 gene and its association with low serum 25(OH)D level among Bangladeshi adults. METHOD: This pilot study was conducted among thirty individuals with low serum 25(OH)D level as the study population and ten subjects with sufficient serum 25(OH)D level as controls based on the inclusion and exclusion criteria. Genetic analysis of rs10741657 of CYP2R1 including primer designing, DNA extraction, PCR of target region with purification and Sanger sequencing of the PCR products were done accordingly. For statistical analysis, One-way ANOVA followed by LSD test, Freeman-Halton extension of Fisher's exact test, Chi-square test (χ2) test and unpaired student t-test were performed. RESULTS: In this study, genetic variants of CYP2R1 (rs10741657) among the study population were genotype GG (63.30%), GA (30%) and AA (6.7%). Minor allele frequency of the study population was 0.217. The association between GG and GA genotypes of CYP2R1 (rs10741657) with low serum 25(OH)D level among the study population was found and it was statistically significant. Statistically significant differences were also observed between the genotypes and alleles of the study population and controls. CONCLUSIONS: The presence of 'GG' and 'GA' genotypes of rs1041657 in CYP2R1 gene is associated with low serum 25(OH)D level among Bangladeshi adults in this pilot study.


Subject(s)
/genetics , Cholestanetriol 26-Monooxygenase/genetics , Cytochrome P450 Family 2/genetics , Genetic Variation/genetics , Vitamin D/analogs & derivatives , Adult , Alleles , Chi-Square Distribution , Female , Gene Frequency/genetics , Genetic Testing/methods , Genotype , Humans , Male , Pilot Projects , Polymerase Chain Reaction/methods , Vitamin D/blood
10.
Pan Afr Med J ; 39: 228, 2021.
Article in French | MEDLINE | ID: covidwho-1551883

ABSTRACT

INTRODUCTION: the COVID-19 pandemic causes biological diagnostic problems that remain relevant in low-income countries in general and in Cameroon in particular. Rapids tests that reliably detect SARS-CoV-2 virus antigen present themselves as an important alternative in several contexts. The objective of our study was to evaluate the diagnostic performance of two rapid diagnostic tests BIOSYNEX® COVID-19 Ag BSS and BIOSYNEX® COVID-19 Ag + BSS, compared to each other and to the AmpliQuick® SARS-CoV-2 PCR test. METHODS: a cross-sectional and comparative study was carried out from April 27 to May 29, 2021 in the city of Douala in Cameroon. The samples consisted of nasopharyngeal swabs received at the molecular biology laboratory of the Douala Gyneco-obstetric and pediatric hospital, whatever their origin. The socio-demographic parameters (age, profession, football players, travelers, others), marital status, nationality), comorbidity and known status of COVID-19, were recorded on the collection sites. The main collection sites were the Deïdo Health District and the Douala Gyneco-Obstetric and Pediatric Hospital. We performed the diagnosis of COVID-19 using the rapid diagnostic test (RDT) BIOSYNEX® COVID-19 Ag BSS and RDT BIOSYNEX® COVID-19 Ag + BSS compared to each other and to the AmpliQuick® SARS-CoV-2 polymerase chain reaction (PCR) test on each sample. Statistical analysis of the data was performed using Microsoft Excel and SPSS version 17 software. To determine the sensitivity of the two RDTs, the Bayesian latent class model was performed on the median with a 95% confidence interval with p<0.05 as the significant level. An ethical clearance was sought and obtained from the University of Douala Institutional Ethics Committee. RESULTS: a total of 1813 participants were included in our study, with a predominance of men (1226, 68.68 %) and the most represented age group was that of 31 to 40 years (568, 31.33 %). Most of the participants were married (888, 53.46%) and only a few had a known COVID-19 status (75, 5.47%). The two rapid tests on our study population show much closed COVID-19 prevalence values, respectively 2.03 for BIOSYNEX® COVID-19 Ag BSS and 2.17 for BIOSYNEX® COVID-19 Ag + BSS. RDT BIOSYNEX® COVID-19 Ag + BSS showed higher sensitivity 94.1% vs. 87.5% for RDT BIOSYNEX® COVID-19 Ag BSS with almost identical specificity 98.9% for RDT BIOSYNEX® COVID-19 Ag + BSS vs. 98.7% for RDT BIOSYNEX® COVID-19 Ag BSS compared to AmpliQuick® SARS-CoV-2. BIOSYNEX® COVID-19 Ag + BSS RDT showed a negative predictive value of 99.9% compared to BIOSYNEX® COVID-19 Ag BSS RDT. There is a 99.9% agreement between the RDT BIOSYNEX® COVID-19 Ag BSS and the RDT BIOSYNEX® COVID-19 Ag + BSS. Conclusion: the RDT BIOSYNEX®COVID-19 Ag + BSS and RDT BIOSYNEX® COVID-19 Ag BSS can be used for the diagnosis of SARS-CoV-2 and can have an important contribution in the context of mass screenings and screening in remote areas.


Subject(s)
Antigens, Viral/analysis , COVID-19 Testing/methods , COVID-19/diagnosis , Polymerase Chain Reaction/methods , Adolescent , Adult , Aged , Cameroon , Child , Child, Preschool , Cross-Sectional Studies , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , Predictive Value of Tests , SARS-CoV-2 , Sensitivity and Specificity , Young Adult
11.
Res Microbiol ; 173(3): 103915, 2022.
Article in English | MEDLINE | ID: covidwho-1540936

ABSTRACT

Despite the scientific advances observed in the recent decades and the emergence of new methodologies, the diagnosis of systemic fungal infections persists as a problematic issue. Fungal cultivation, the standard method that allows a proven diagnosis, has numerous disadvantages, as low sensitivity (only 50% of the patients present positive fungal cultures), and long growth time. These are factors that delay the patient's treatment and, consequently, lead to higher hospital costs. To improve the accuracy and quickness of fungal infections diagnosis, several new methodologies attempt to be implemented in clinical microbiology laboratories. Most of these innovative methods are independent of pathogen isolation, which means that the diagnosis goes from being considered proven to probable. In spite of the advantage of being culture-independent, the majority of the methods lack standardization. PCR-based methods are becoming more and more commonly used, which has earned them an important place in hospital laboratories. This can be perceived now, as PCR-based methodologies have proved to be an essential tool fighting against the COVID-19 pandemic. This review aims to go through the main steps of the diagnosis for systemic fungal infection, from diagnostic classifications, through methodologies considered as "gold standard", to the molecular methods currently used, and finally mentioning some of the more futuristic approaches.


Subject(s)
COVID-19 , Mycoses , COVID-19/diagnosis , Humans , Mycoses/diagnosis , Pandemics , Polymerase Chain Reaction/methods
12.
PLoS One ; 16(11): e0259398, 2021.
Article in English | MEDLINE | ID: covidwho-1502074

ABSTRACT

The first case of COVID-19 in Nigeria was recorded on February 27, 2020, being an imported case by an Italian expatriate, to the country. Since then, there has been steady increase in the number of cases. However, the number of cases in Nigeria is low in comparison to cases reported by other countries with similar large populations, despite the poor health system prevailing in the country. This has been mainly attributed to the low testing capacity in Nigeria among other factors. Therefore, there is a need for innovative ways to increase the number of persons testing for COVID-19. The aim of the study was to pilot a nasopharyngeal swab self-sample collection model that would help increase COVID-19 testing while ensuring minimal person-to-person contact being experienced at the testing center. 216 participants took part in this study which was carried out at the Nigerian Institute of Medical Research between June and July 2020. Amongst the 216 participants, 174 tested negatives for both self-collected samples and samples collected by Professionals, 30 tested positive for both arms, with discrepancies occurring in 6 samples where the self-collected samples were positive while the ones collected by the professionals were negative. The same occurred in another set of 6 samples with the self-collected samples being negative and the professional-collected sample coming out positive, with a sensitivity of 83.3% and a specificity of 96.7%. The results of the interrater analysis are Kappa = 0.800 (95% CI, 0.690 to 0.910) which implies an outstanding agreement between the two COVID-19 sampling methods. Furthermore, since p< 0.001 Kappa (k) coefficient is statistically different from zero, our findings have shown that self-collected samples can be reliable in the diagnosis of COVID-19.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/prevention & control , Polymerase Chain Reaction/methods , Telemedicine/methods , Adolescent , Adult , Aged , COVID-19 Testing/statistics & numerical data , Diagnostic Tests, Routine , Female , Humans , Male , Middle Aged , Nigeria/epidemiology , Remote Consultation/methods , Reproducibility of Results , SARS-CoV-2 , Sensitivity and Specificity , Specimen Handling/methods , Young Adult
13.
Bioanalysis ; 13(23): 1723-1729, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1497587

ABSTRACT

Polymerase chain reaction (PCR) is widely used in various fields of laboratory testing, ranging from forensic, molecular biology, medical and diagnostic applications to a wide array of basic research purposes. COVID-19 infection testing has brought the three-letter PCR abbreviation into the vocabulary of billions of people, making it likely the most well-known laboratory test worldwide. With new modalities and translational medicine gaining importance in pharmaceutical research and development, PCR or more specifically, quantitative PCR (qPCR) is now becoming a standard tool in the (regulated) bioanalytical laboratory, driving the bioanalytical community to define best practices for method development, characterization and validation. In absence of specific guidance from health authorities, qPCR may be vulnerable to scope creep from pharmacokinetics (PK) assay validation as defined in bioanalytical method validation guidance/guidelines. In this manuscript, the European Bioanalysis Forum builds a rationale for applying context of use principles when defining requirements for qPCR assay performance and validation criteria.


Subject(s)
Biological Assay/methods , Polymerase Chain Reaction/methods , Europe , Humans , Research Design
14.
Commun Biol ; 4(1): 1215, 2021 10 22.
Article in English | MEDLINE | ID: covidwho-1479821

ABSTRACT

SARS-CoV-2 replication requires the synthesis of a set of structural proteins expressed through discontinuous transcription of ten subgenomic mRNAs (sgmRNAs). Here, we have fine-tuned droplet digital PCR (ddPCR) assays to accurately detect and quantify SARS-CoV-2 genomic ORF1ab and sgmRNAs for the nucleocapsid (N) and spike (S) proteins. We analyzed 166 RNA samples from anonymized SARS-CoV-2 positive subjects and we observed a recurrent and characteristic pattern of sgmRNAs expression in relation to the total viral RNA content. Additionally, expression profiles of sgmRNAs, as determined by meta-transcriptomics sequencing of a subset of 110 RNA samples, were highly correlated with those obtained by ddPCR. By providing a comprehensive and dynamic snapshot of the levels of SARS-CoV-2 sgmRNAs in infected individuals, our results may contribute a better understanding of the dynamics of transcription and expression of the genome of SARS-CoV-2 and facilitate the development of more accurate molecular diagnostic tools for the stratification of COVID-19 patients.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/genetics , COVID-19/metabolism , Coronavirus Nucleocapsid Proteins , Polymerase Chain Reaction/methods , RNA, Viral/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Transcriptome , Computational Biology , Humans , Limit of Detection , Open Reading Frames , Phosphoproteins , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Reproducibility of Results
15.
S Afr Med J ; 111(10): 957-960, 2021 10 05.
Article in English | MEDLINE | ID: covidwho-1478408

ABSTRACT

BACKGROUND: The onset of the COVID-19 pandemic in South Africa (SA) created numerous supply challenges. Demand for diagnostic testing overwhelmed the capacity to deliver. We describe the utility and outcomes of a mobile laboratory staffed by non-laboratory healthcare workers and established to perform polymerase chain reaction (PCR) testing for the rapid diagnosis of COVID-19 at a large hospital in SA. OBJECTIVES: To describe the performance of the mobile PCR COVID-19 laboratory. The secondary objective was to determine the prevalence of COVID-19 infections in the non-COVID intensive care unit (ICU). METHODS: This was a retrospective descriptive study of data from the newly established mobile COVID-19 PCR laboratory database and the non-COVID ICU database during the first peak of the COVID-19 pandemic (20 May - 8 August 2020) at a tertiary hospital in SA. RESULTS: The mobile laboratory received 1 113 emergency COVID-19 PCR test requests for patients with non-COVID clinical presentations. The median (interquartile range) turnaround time was 152 (123 - 184) minutes (n=36). Primary outcome (20 May - 19 June, n=315): The sensitivity and specificity were 95% and 97%, respectively, and the positive and negative predictive values 82.4% and 99.2%, respectively. Secondary outcomes (9 June - 8 August): The prevalence of COVID-19 infections among patients admitted to the multidisciplinary adult and paediatric non-COVID ICU was 2.4% (n=4/168). The mean (standard deviation) COVID-19 positive rate for the mobile laboratory during this period was 18.1% (6%). The prevalence of COVID-19 infections among medical staff in the non-COVID ICU was 3.1% (n=1/32). CONCLUSIONS: The establishment of a mobile PCR laboratory staffed by non-laboratory healthcare workers during the COVID-19 pandemic provided a rapid, accurate and clinically effective solution for emergency hospital admissions with non-COVID-19 presentations.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Hospitalization/statistics & numerical data , Mobile Health Units , Adolescent , Adult , COVID-19/epidemiology , Child , Cross-Sectional Studies , Female , Humans , Intensive Care Units/statistics & numerical data , Laboratories , Male , Middle Aged , Polymerase Chain Reaction/methods , Predictive Value of Tests , Retrospective Studies , Sensitivity and Specificity , South Africa , Tertiary Care Centers , Time Factors , Young Adult
16.
Mol Biotechnol ; 64(4): 339-354, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1469770

ABSTRACT

The outbreak of COVID-19 pandemic and its consequences have inflicted a substantial damage on the world. In this study, it was attempted to review the recent coronaviruses appeared among the human being and their epidemic/pandemic spread throughout the world. Currently, there is an inevitable need for the establishment of a quick and easily available biosensor for tracing COVID-19 in all countries. It has been known that the incubation time of COVID-19 lasts about 14 days and 25% of the infected individuals are asymptomatic. To improve the ability to determine SARS-CoV-2 precisely and reduce the risk of eliciting false-negative results produced by mutating nature of coronaviruses, many researchers have established a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using mismatch-tolerant molecular beacons as multiplex real-time RT-PCR to distinguish between pathogenic and non-pathogenic strains of coronaviruses. The possible mechanisms and pathways for the detection of coronaviruses by biosensors have been reviewed in this study.


Subject(s)
COVID-19 Testing/methods , Biosensing Techniques/methods , COVID-19 Testing/instrumentation , CRISPR-Cas Systems , Electrochemical Techniques , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique/methods , Humans , Molecular Diagnostic Techniques/instrumentation , Molecular Diagnostic Techniques/methods , Neutralization Tests , Nucleic Acid Amplification Techniques/instrumentation , Nucleic Acid Amplification Techniques/methods , Polymerase Chain Reaction/instrumentation , Polymerase Chain Reaction/methods , SARS-CoV-2/pathogenicity , Surface Plasmon Resonance
17.
Curr Issues Mol Biol ; 43(3): 1212-1225, 2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1438531

ABSTRACT

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Most SARS-CoV-2 infections are mild or even asymptomatic. However, a small fraction of infected individuals develops severe, life-threatening disease, which is caused by an uncontrolled immune response resulting in hyperinflammation. However, the factors predisposing individuals to severe disease remain poorly understood. Here, we show that levels of CD47, which is known to mediate immune escape in cancer and virus-infected cells, are elevated in SARS-CoV-2-infected Caco-2 cells, Calu-3 cells, and air-liquid interface cultures of primary human bronchial epithelial cells. Moreover, SARS-CoV-2 infection increases SIRPalpha levels, the binding partner of CD47, on primary human monocytes. Systematic literature searches further indicated that known risk factors such as older age and diabetes are associated with increased CD47 levels. High CD47 levels contribute to vascular disease, vasoconstriction, and hypertension, conditions that may predispose SARS-CoV-2-infected individuals to COVID-19-related complications such as pulmonary hypertension, lung fibrosis, myocardial injury, stroke, and acute kidney injury. Hence, age-related and virus-induced CD47 expression is a candidate mechanism potentially contributing to severe COVID-19, as well as a therapeutic target, which may be addressed by antibodies and small molecules. Further research will be needed to investigate the potential involvement of CD47 and SIRPalpha in COVID-19 pathology. Our data should encourage other research groups to consider the potential relevance of the CD47/ SIRPalpha axis in their COVID-19 research.


Subject(s)
Antigens, Differentiation/metabolism , CD47 Antigen/metabolism , COVID-19/epidemiology , COVID-19/metabolism , Pandemics , Receptors, Immunologic/metabolism , SARS-CoV-2/metabolism , Severity of Illness Index , Signal Transduction/immunology , Blood Donors , Blotting, Western/methods , Bronchi/cytology , COVID-19/pathology , COVID-19/virology , Caco-2 Cells , Epithelial Cells/metabolism , Epithelial Cells/virology , Healthy Volunteers , Humans , Monocytes/metabolism , Monocytes/virology , Polymerase Chain Reaction/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification
18.
J Mol Diagn ; 23(10): 1207-1217, 2021 10.
Article in English | MEDLINE | ID: covidwho-1428187

ABSTRACT

The coronavirus disease 2019 (COVID-19) response necessitated innovations and a series of regulatory deviations that also affected laboratory-developed tests (LDTs). To examine real-world consequences and specify regulatory paradigm shifts, legislative proposals were aligned on a common timeline with Emergency Use Authorization (EUA) of LDTs and the US Food and Drug Administration (FDA)-orchestrated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) labeling update study. The initial EUA adoption by LDT developers shows that the FDA can have oversight over LDTs. We used efficiency-corrected microcosting of our EUA PCR assay to estimate the national cost of the labeling update study to $0.3 to $1.4 million US dollars. Labeling update study performance data showed lower average detection limits in commercial in vitro diagnostic (IVD) assays versus LDTs (32,000 ± 75,000 versus 71,000 ± 147,000 nucleic acid amplification tests/mL; P = 0.04); however, comparison also shows that FDA review of IVD assays and LDTs did not prevent differences between initial and labeling update performance (IVD assay, P < 0.0001; LDT, P = 0.003). The regulatory shifts re-emphasized that both commercial tests and LDTs rely heavily on laboratory competence and procedures; however, lack of performance data on authorized tests, when clinically implemented, precludes assessment of the benefit related to regulatory review. Temporary regulatory deviations during the pandemic and regulatory science tools (ie, reference material) have generated valuable real-world evidence to inform pending legislation regarding LDT regulation.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Polymerase Chain Reaction/methods , United States Food and Drug Administration/legislation & jurisprudence , COVID-19 Nucleic Acid Testing/economics , Humans , Laboratories/statistics & numerical data , Limit of Detection , Polymerase Chain Reaction/economics , Time Factors , United States
19.
Biotechniques ; 71(4): 510-515, 2021 10.
Article in English | MEDLINE | ID: covidwho-1411518

ABSTRACT

Purity and integrity are two important criteria for any RNA extraction process to qualify the RNA for meaningful gene expression analysis. This study compares four commercially available RNA extraction kits using silica membrane and magnetic bead separation methods. The performance was evaluated in terms of both quantity (total RNA amount in µg/µl) and purity (260/280 ratio). The concentration and purity of each kit was significantly different from those of the others (p < 0.001). Although quantity obtained from Mag MAX is comparatively lower than QIAGEN, the quality is comparable as evident from real-time PCR performance. This study suggests that there are practical differences between these RNA extraction kits that should be taken into account while isolating RNA required for gene expression analysis.


Subject(s)
Magnets/chemistry , Membranes, Artificial , RNA, Viral/isolation & purification , Reagent Kits, Diagnostic , Silicon Dioxide/chemistry , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing/methods , Gene Expression Profiling/methods , Humans , Polymerase Chain Reaction/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification
20.
Genes Genomics ; 43(11): 1277-1288, 2021 11.
Article in English | MEDLINE | ID: covidwho-1409152

ABSTRACT

BACKGROUND: Coronavirus disease of 2019 (COVID-19) is well known as a fatal disease, first discovered at Wuhan in China, ranging from mild to death, such as shortness of breath and fever. Early diagnosis of COVID-19 is a crucial point in preventing global prevalence. OBJECTIVE: We aimed to evaluate the diagnostic competency and efficiency with the Allplex™ 2019-nCoV Assay kit and the Dr. PCR 20 K COVID-19 Detection kit, designed based on the qRT-PCR and dPCR technologies, respectively. METHODS: A total of 30 negative and 20 COVID-19 positive specimens were assigned to the diagnostic test by using different COVID-19 diagnosis kits. Diagnostic accuracy was measured by statistical testing with sensitivity, specificity, and co-efficiency calculations. RESULTS: Comparing both diagnostic kits, we confirmed that the diagnostic results of 30 negative and 20 positive cases were the same pre-diagnostic results. The diagnostic statistics test results were perfectly matched with value (1). Cohen's Kappa coefficient was demonstrated that the given kits in two different ways were "almost perfect" with value (1). In evaluating the detection capability, the dilutional linearity experiments substantiate that the Dr. PCR 20 K COVID-19 Detection kit could detect SARS-CoV-2 viral load at a concentration ten times lower than that of the Allplex™ 2019-nCoV Assay kit. CONCLUSIONS: In this study, we propose that the dPCR diagnosis using LOAA dPCR could be a powerful method for COVID-19 point-of-care tests requiring immediate diagnosis in a limited time and space through the advantages of relatively low sample concentration and small equipment size compared to conventional qRT-PCR.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , COVID-19 , Genes, Viral/genetics , Humans , Republic of Korea , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral Load
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