scholarly journals Evaluation of the relationship between quantitative PCR results and cell culturing of SARS2-CoV with respect to symptoms onset and Viral load – a systematic review

2021 ◽  
Author(s):  
gilad rozenberg ◽  
Oran Erster ◽  
Itai Ghersin ◽  
Michal Mandelboim ◽  
Ami neuberger ◽  
...  
Keyword(s):  
Viral Load ◽  
Viral Genome ◽  
Positive Culture ◽  
Accurate Method ◽  
Positive Sample ◽  
Immunocompromised Patients ◽  
Ct Value ◽  
Asymptomatic Patients ◽  
Pcr Assays ◽  
Culture Positive

Background: Viral culture is currently the most accurate method to demonstrate viability and infectivity of Severe acute respiratory syndrome Coronavirus (SARS–2 CoV). Routine clinical diagnosis, however, is mostly performed by PCR – based assays that do not discriminate between infectious and non–virus. Herein, we aimed to determine the correlation between positive viral cultures and either PCR positivity, the Cycle Threshold (Ct) or the number of viral copies. Methods: A systematic electronic literature search was performed and studies that reported both viral SARS–CoV–2 culture and PCR–based assays were included. A separate search for samples from blood, urine, stool, breast milk and tears were performed. To convert Ct values reported in the reviewed studies were to viral genomic copies, calibration experiments with four different reaction performed, using quantified RNA molecules. Results: A total 540 articles were reviewed, and 38 studies were included in this review. Out of 276 positive–culture of non-severe patients, 272 (98.55%) were negative ten days after symptoms onset, while PCR assays remained positive for up to 67 days. In severely ill or immunocompromised patients positive-culture was obtained up to 32 days and out of 168 cultures, 31 (18.45%) stayed positive after day 10. In non-severe patients, in Ct value greater than 30 only 10.8% were still culture–positive while in Ct >35 it was nearly universally negative. The minimal calculated number of viral genome copies in culture- positive sample was 2.5 X 103 copies / mL. These findings were similar in immunocompromised patients. Recovering positive culture from non–respiratory samples was sporadically obtained in stool or urine samples. Conversion of Ct values to viral genome copies showed variability between different PCR assays and highlighted the need to standardize reports to correctly compare results obtained in different laboratories. Conclusion: During the pandemic phase, non-severe COVID-19 patients who are recovering and are not immuno-suppressed, can be regarded as non–infectious, within 10 days from symptom onset, or with Ct value greater than 35 (or a calculated viral load lower than 1.2X103 copies / mL). These findings have important implications for recovering patients and asymptomatic patients, with respect to isolation criteria. The conversion of Cq values to viral genome copies described herein may be useful in future work, enabling a more standardized comparison between results reported in different studies from different laboratories.

scholarly journals Direct Detection of Shigella in Stool Specimens by Use of a Metagenomic Approach

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
Jie Liu ◽  
Mathieu Almeida ◽  
Furqan Kabir ◽  
Sadia Shakoor ◽  
Shahida Qureshi ◽  
...  
Keyword(s):  
Direct Detection ◽  
Positive Culture ◽  
Accurate Method ◽  
Diagnostic Methods ◽  
Metagenomic Sequencing ◽  
Illumina Hiseq ◽  
Content Type ◽  
Sequence Composition ◽  
Culture Positive ◽  
Culture Negative

ABSTRACTThe underestimation ofShigellaspecies as a cause of childhood diarrhea disease has become increasingly apparent with quantitative PCR (qPCR)-based diagnostic methods versus culture. We sought to confirm qPCR-based detection ofShigellavia a metagenomics approach. Three groups of samples were selected from diarrheal cases from the Global Enteric Multicenter Study: nineShigellaculture-positive and qPCR-positive (culture+qPCR+) samples, nine culture-negative but qPCR-positive (culture−qPCR+) samples, and nine culture-negative and qPCR-negative (culture−qPCR−) samples. Fecal DNA was sequenced using paired-end Illumina HiSeq, whereby 3.26 × 108± 5.6 × 107high-quality reads were generated for each sample. We used Kraken software to compare the read counts specific to “Shigella” among the three groups. The proportions ofShigella-specific nonhuman sequence reads between culture+qPCR+(0.65 ± 0.42%) and culture−qPCR+(0.55 ± 0.31%) samples were similar (Mann-Whitney U test,P= 0.627) and distinct from the culture−qPCR−group (0.17 ± 0.15%,P< 0.05). The read counts of sequences previously targeted byShigella/enteroinvasiveEscherichia coli(EIEC) qPCR assays, namely,ipaH,virA,virG,ial,ShET2, andipaH3, were also similar between the culture+qPCR+and culture−qPCR+groups and distinct from the culture−qPCR−groups (P< 0.001). Kraken performed well versus other methods: its precision and recall ofShigellawere excellent at the genus level but variable at the species level. In summary, metagenomic sequencing indicates thatShigella/EIEC qPCR-positive samples are similar to those ofShigellaculture-positive samples inShigellasequence composition, thus supporting qPCR as an accurate method for detectingShigella.

scholarly journals Secondary bacterial infections and antimicrobial resistance in COVID-19: comparative evaluation of pre-pandemic and pandemic-era, a retrospective single center study

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Mustafa Karataş ◽  
Melike Yaşar-Duman ◽  
Alper Tünger ◽  
Feriha Çilli ◽  
Şöhret Aydemir ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Bacterial Infections ◽  
Respiratory Infections ◽  
Low Frequency ◽  
Positive Culture ◽  
Positive Sample ◽  
University Hospital ◽  
Control Groups ◽  
Extended Spectrum Beta Lactamase ◽  
Culture Positive

Abstract Purpose In this study, we aimed to evaluate the epidemiology and antimicrobial resistance (AMR) patterns of bacterial pathogens in COVID-19 patients and to compare the results with control groups from the pre-pandemic and pandemic era. Methods Microbiological database records of all the COVID-19 diagnosed patients in the Ege University Hospital between March 15, 2020, and June 15, 2020, evaluated retrospectively. Patients who acquired secondary bacterial infections (SBIs) and bacterial co-infections were analyzed. Etiology and AMR data of the bacterial infections were collected. Results were also compared to control groups from pre-pandemic and pandemic era data. Results In total, 4859 positive culture results from 3532 patients were analyzed. Fifty-two (3.59%) patients had 78 SBIs and 38 (2.62%) patients had 45 bacterial co-infections among 1447 COVID-19 patients. 22/85 (25.88%) patients died who had bacterial infections. The respiratory culture-positive sample rate was 39.02% among all culture-positive samples in the COVID-19 group. There was a significant decrease in extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales (8.94%) compared to samples from the pre-pandemic (20.76%) and pandemic era (20.74%) (p = 0.001 for both comparisons). Interestingly, Acinetobacter baumannii was the main pathogen in the respiratory infections of COVID-19 patients (9.76%) and the rate was significantly higher than pre-pandemic (3.49%, p < 0.002) and pandemic era control groups (3.11%, p < 0.001). Conclusion Due to the low frequency of SBIs reported during the ongoing pandemic, a more careful and targeted antimicrobial prescription should be taken. While patients with COVID-19 had lower levels of ESBL-producing Enterobacterales, the frequency of multidrug-resistant (MDR) A. baumannii is higher.

scholarly journals Secondary Bacterial Infections and Antimicrobial Resistance in COVID-19: Comparative Evaluation of Pre-Pandemic and Pandemic-Era, A Retrospective Single Center Study

2021 ◽  
Author(s):  
Mustafa Karataş ◽  
Melike Yaşar Duman ◽  
Alper Tünger ◽  
Feriha Çilli ◽  
Şöhret Aydemir ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Bacterial Infections ◽  
Respiratory Infections ◽  
Low Frequency ◽  
Positive Culture ◽  
Positive Sample ◽  
University Hospital ◽  
Control Groups ◽  
Extended Spectrum Beta Lactamase ◽  
Culture Positive

Abstract Purpose: In this study, we aimed to evaluate the epidemiology and antimicrobial resistance (AMR) patterns of bacterial pathogens in COVID-19 patients and to compare the results with control groups from the pre-pandemic and pandemic era.Methods: Microbiological database records of all the COVID-19 diagnosed patients in the Ege University Hospital between March 15, 2020, and June 15, 2020, evaluated retrospectively. Patients who acquired secondary bacterial infections (SBIs) and bacterial co-infections were analyzed. Etiology and AMR data of the bacterial infections were collected. Results were also compared to control groups from pre-pandemic and pandemic era data.Results: In total, 4,859 positive culture results from 3,532 patients were analyzed. Fifty-two (3.59%) patients had 78 SBIs and 38 (2.62%) patients had 45 bacterial co-infections among 1,447 COVID-19 patients. 22/85 (25.88%) patients died who had bacterial infections. The respiratory culture-positive sample rate was 39.02% among all culture-positive samples in the COVID-19 group. There was a significant decrease in extended-spectrum beta-lactamase-producing Enterobacterales (8.94%) compared to samples from the pre-pandemic (20.76%) and pandemic era (20.74%) (p=0.001 for both comparisons). Interestingly, Acinetobacter baumannii was the main pathogen in the respiratory infections of COVID-19 patients (%9.76) and the rate was significantly higher than pre-pandemic (3.49%, p<0.002) and pandemic era control groups (3.11%, p<0.001).Conclusion: Due to the low frequency of SBIs reported during the ongoing pandemic, a more careful and targeted antimicrobial prescription should be taken. While patients with COVID-19 had lower levels of ESBL producing Enterobacterales, the frequency of MDR A. baumannii is higher.

scholarly journals Viral Dynamics and Real-Time RT-PCR Ct Values Correlation with Disease Severity in COVID-19

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1091
Author(s):  
Ali A. Rabaan ◽  
Raghavendra Tirupathi ◽  
Anupam A Sule ◽  
Jehad Aldali ◽  
Abbas Al Mutair ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Disease Severity ◽  
False Negative ◽  
Influential Factors ◽  
Confirmatory Test ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Viral Kinetics ◽  
Ct Value

Real-time RT-PCR is considered the gold standard confirmatory test for coronavirus disease 2019 (COVID-19). However, many scientists disagree, and it is essential to understand that several factors and variables can cause a false-negative test. In this context, cycle threshold (Ct) values are being utilized to diagnose or predict SARS-CoV-2 infection. This practice has a significant clinical utility as Ct values can be correlated with the viral load. In addition, Ct values have a strong correlation with multiple haematological and biochemical markers. However, it is essential to consider that Ct values might be affected by pre-analytic, analytic, and post-analytical variables such as collection technique, specimen type, sampling time, viral kinetics, transport and storage conditions, nucleic acid extraction, viral RNA load, primer designing, real-time PCR efficiency, and Ct value determination method. Therefore, understanding the interpretation of Ct values and other influential factors could play a crucial role in interpreting viral load and disease severity. In several clinical studies consisting of small or large sample sizes, several discrepancies exist regarding a significant positive correlation between the Ct value and disease severity in COVID-19. In this context, a revised review of the literature has been conducted to fill the knowledge gaps regarding the correlations between Ct values and severity/fatality rates of patients with COVID-19. Various databases such as PubMed, Science Direct, Medline, Scopus, and Google Scholar were searched up to April 2021 by using keywords including “RT-PCR or viral load”, “SARS-CoV-2 and RT-PCR”, “Ct value and viral load”, “Ct value or COVID-19”. Research articles were extracted and selected independently by the authors and included in the present review based on their relevance to the study. The current narrative review explores the correlation of Ct values with mortality, disease progression, severity, and infectivity. We also discuss the factors that can affect these values, such as collection technique, type of swab, sampling method, etc.

scholarly journals Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽  
2021 ◽  
pp. 1-6
Author(s):  
Salman Khan ◽  
Syed Asad Ali Shah ◽  
Syed Muhammad Jamal
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Kappa Value ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Level Of Agreement

<b><i>Background:</i></b> Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. <b><i>Methods:</i></b> A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. <b><i>Results:</i></b> S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (<i>p</i> = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. <b><i>Conclusions:</i></b> The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

scholarly journals 520. Longitudinal Analysis of SARS-CoV-2 Viruses in Hospitalized Adults

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S325-S326
Author(s):  
Lacy Simons ◽  
Ramon Lorenzo-Redondo ◽  
Hannah Nam ◽  
Scott C Roberts ◽  
Michael G Ison ◽  
...  
Keyword(s):  
Viral Load ◽  
Genome Sequencing ◽  
Viral Genome ◽  
Temporal Dynamics ◽  
Population Level ◽  
Hospital Staff ◽  
Therapeutic Interventions ◽  
Viral Loads ◽  
Qrt Pcr ◽  
Over Time

Abstract Background The rapid spread of SARS-CoV-2, the causative agent of Coronavirus disease 2019 (COVID-19), has been accompanied by the emergence of viral mutations, some of which may have distinct virological and clinical consequences. While whole genome sequencing efforts have worked to map this viral diversity at the population level, little is known about how SARS-CoV-2 may diversify within a host over time. This is particularly important for understanding the emergence of viral resistance to therapeutic interventions and immune pressure. The goal of this study was to assess the change in viral load and viral genome sequence within patients over time and determine if these changes correlate with clinical and/or demographic parameters. Methods Hospitalized patients admitted to Northwestern Memorial Hospital with a positive SARS-CoV-2 test were enrolled in a longitudinal study for the serial collection of nasopharyngeal specimens. Swabs were administered to patients by hospital staff every 4 ± 1 days for up to 32 days or until the patients were discharged. RNA was extracted from each specimen and viral loads were calculated by quantitative reverse transcriptase PCR (qRT-PCR). Specimens with qRT-PCR cycle threshold values less than or equal to 30 were subject to whole viral genome sequencing by reverse transcription, multiplex PCR, and deep sequencing. Variant populations sizes were estimated and subject to phylogenetic analysis relative to publicly available SARS-CoV-2 sequences. Sequence and viral load data were subsequently correlated to available demographic and clinical data. Results 60 patients were enrolled from March 26th to June 20th, 2020. We observed an overall decrease in nasopharyngeal viral load over time across all patients. However, the temporal dynamics of viral load differed on a patient-by-patient basis. Several mutations were also observed to have emerged within patients over time. Distribution of SARS-CoV-2 viral loads in serially collected nasopharyngeal swabs in hospitalized adults as determined by qRT-PCR. Samples were collected every 4 ± 1 days (T#1–8) and viral load is displayed by log(copy number). Conclusion These data indicate that SARS-CoV-2 viral loads in the nasopharynx decrease over time and that the virus can accumulate mutations during replication within individual patients. Future studies will examine if some of these mutations may provide fitness advantages in the presence of therapeutic and/or immune selective pressures. Disclosures Michael G. Ison, MD MS, AlloVir (Consultant)

scholarly journals Percutaneous Bone Biopsy for Diabetic Foot Osteomyelitis: A Systematic Review and Meta-Analysis

2020 ◽  
Vol 7 (10) ◽  
Author(s):  
Marcos C Schechter ◽  
Mohammed K Ali ◽  
Benjamin B Risk ◽  
Adam D Singer ◽  
Gabriel Santamarina ◽  
...  
Keyword(s):  
Adverse Events ◽  
Diabetic Foot ◽  
Bone Biopsy ◽  
Meta Analysis ◽  
Positive Culture ◽  
High Yield ◽  
Analysis Model ◽  
Antibiotic Management ◽  
Culture Positive ◽  
Diabetic Foot Osteomyelitis

Abstract Background Diabetes is the leading cause of lower extremity nontraumatic amputation globally, and diabetic foot osteomyelitis (DFO) is usually the terminal event before limb loss. Although guidelines recommend percutaneous bone biopsy (PBB) for microbiological diagnosis of DFO in several common scenarios, it is unclear how frequently PBBs yield positive cultures and whether they cause harm or improve outcomes. Methods We searched the PubMed, EMBASE, and Cochrane Trials databases for articles in any language published up to December 31, 2019, reporting the frequency of culture-positive PBBs. We calculated the pooled proportion of culture-positive PBBs using a random-effects meta-analysis model and reported on PBB-related adverse events, DFO outcomes, and antibiotic adjustment based on PBB culture results where available. Results Among 861 articles, 11 studies met inclusion criteria and included 780 patients with 837 PBBs. Mean age ranged between 56.6 and 71.0 years old. The proportion of males ranged from 62% to 86%. All studies were longitudinal observational cohorts, and 10 were from Europe. The range of culture-positive PBBs was 56%–99%, and the pooled proportion of PBBs with a positive culture was 84% (95% confidence interval, 73%–91%). There was heterogeneity between studies and no consistency in definitions used to define adverse events. Impact of PBB on DFO outcomes or antibiotic management were seldom reported. Conclusions This meta-analysis suggests PBBs have a high yield of culture-positive results. However, this is an understudied topic, especially in low- and middle-income countries, and the current literature provides very limited data regarding procedure safety and impact on clinical outcomes or antibiotic management.

scholarly journals Differential Kinetics of Cycle Threshold Values during Admission by Symptoms among Patients with Mild COVID-19: A Prospective Cohort Study

2021 ◽  
Vol 18 (15) ◽  
pp. 8181
Author(s):  
Teppei Sakano ◽  
Mitsuyoshi Urashima ◽  
Hiroyuki Takao ◽  
Kohei Takeshita ◽  
Hiroe Kobashi ◽  
...  
Keyword(s):  
Cohort Study ◽  
Viral Load ◽  
Prospective Cohort Study ◽  
Prospective Cohort ◽  
Polymerase Chain Reaction Test ◽  
Cycle Threshold ◽  
Viral Loads ◽  
Asymptomatic Patients ◽  
Asymptomatic Individuals ◽  
Chain Reaction Test

In the coronavirus disease 2019 (COVID-19) pandemic, more than half of the cases of transmission may occur via asymptomatic individuals, which makes it difficult to contain. However, whether viral load in the throat during admission is different between asymptomatic and symptomatic patients is not well known. By conducting a prospective cohort study of patients with asymptomatic or mild COVID-19, cycle threshold (Ct) values of the polymerase chain reaction test for COVID-19 were examined every other day during admission. The Ct values during admission increased more steadily in symptomatic patients and febrile patients than in asymptomatic patients, with significance (p = 0.01 and p = 0.004, respectively), although the Ct values as a whole were not significantly different between the two groups. Moreover, the Ct values as a whole were higher in patients with dysosmia/dysgeusia than in those without it (p = 0.02), whereas they were lower in patients with a headache than those without (p = 0.01). Patients who were IgG-positive at discharge maintained higher Ct values, e.g., more than 35, during admission than those with IgG-negative (p = 0.03). Assuming that viral load and Ct values are negatively associated, the viral loads as a whole and their changes by time may be different by symptoms and immune reaction, i.e., IgG-positive at discharge.

scholarly journals 1484. Microbial Etiology of Community-acquired Pneumonia in Immunocompromised Patients

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S743-S744
Author(s):  
Abhishek Deshpande ◽  
Pei-Chun Yu ◽  
Michael Rothberg
Keyword(s):  
Panel Member ◽  
Positive Culture ◽  
Empiric Therapy ◽  
Community Acquired Pneumonia ◽  
Immunocompromised Patients ◽  
Viral Pathogens ◽  
Chest X Ray ◽  
Microbial Etiology ◽  
Almost All ◽  
Immunocompetent Patients

Abstract Background Community-acquired pneumonia (CAP) is a leading cause of infection related mortality. Few studies have specifically evaluated the microbial etiology of CAP in immunocompromised patients. Using a large national inpatient database, we compared the microbial etiology of CAP in immunocompromised patients compared to immunocompetent patients. Methods We included adult patients admitted with pneumonia from 2010-2015 to 176 US hospitals participating in Premier. Patients were identified as having CAP if they had a chest X-ray and were on antimicrobials on the first day. Immunocompromised was defined by the receipt of immunosuppressive medications or ICD-9 codes for neutropenia/ hematological malignancy/ organ transplantation or comorbidities with AIDS. For microbial etiology, patients were included if they had a positive culture or test collected by hospital day 0 through 3. Patients with identical bacteria in blood and urine were excluded. Results A total of 168,159 patients had a diagnosis of CAP with a culture/test performed on first 3 days. A pathogen was detected in 18.8% of patients. Among pathogen positive patients, 4,851 patients were identified as immunocompromised and 26,752 as immunocompetent. Almost all patients (99%) had at least one culture, blood (96%) and respiratory (51%). Among patients who were immunocompromised, the most common bacterial pathogens (compared to immunocompetent patients) were, S. pneumoniae (17.7% vs 19.0%), MRSA (13.1% vs 14.4%), MSSA (12.0% vs 11.8%), P. aeruginosa (12.0% vs 9.9%), E. coli (7.4% vs 6.4%), K. pneumoniae (5.8% vs 4.9%), H. influenzae (5.5% vs 5.5%), M. pneumoniae (3.0% vs 3.0%) and L. pneumophila (0.93% vs 1.2%). Among viral pathogens, while the most common were influenza virus (12.9% vs 14.1%) followed by rhinovirus (1.5% vs 0.89%), immunocompromised patients has a higher prevalence of noninfluennza viruses (3.42% vs 2.43%). Conclusion In a large US inpatient sample, the causative organisms in immunocompromised patients did not differ much from those in immunocompetent patients. CAP pathogens in immunocompromised patients were more likely to involve gram-negative bacilli such as P.aeruginosa and E.coli, than gram-positive cocci. These findings may have implications when deciding on empiric therapy in these patients. Disclosures Abhishek Deshpande, MD, PhD, Ferring Pharmaceuticals (Advisor or Review Panel member)Merck (Consultant)

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