Reliability at the Lower Limits of HIV-1 RNA Quantification in Clinical Samples: A Comparison of RT-PCR versus bDNA Assays

ABSTRACTThis study aimed to validate a comprehensive diagnostic protocol based on real-time PCR for the rapid detection and identification ofBordetella pertussis,Bordetella parapertussis, andBordetella holmesii, as well as its implementation in the diagnostic routine of a reference children’s hospital. The new algorithm included a triplex quantitative PCR (qPCR) targeting IS481gene (inB. pertussis,B. holmesii, and someBordetella bronchisepticastrains), pIS1001(B. parapertussis-specific) andrnaseP as the human internal control. Two confirmatory singleplex tests forB. pertussis(ptxA-Pr) andB. holmesii(hIS1001) were performed if IS481was positive. Analytical validation included determination of linear range, linearity, efficiency, precision, sensitivity, and a reference panel with clinical samples. Once validated, the new algorithm was prospectively implemented in children with clinical suspicion of whooping cough presenting to Hospital Sant Joan de Deu (Barcelona, Spain) over 12 months. Lower limits of detection obtained were 4.4, 13.9, and 27.3 genomic equivalents/ml of sample for IS481(onB. pertussis), pIS1001and hIS1001, and 777.9 forptxA-Pr. qPCR efficiencies ranged from 86.0% to 96.9%. Intra- and interassay variabilities were <3% and <5%, respectively. Among 566 samples analyzed,B. pertussis,B. holmesii, andB. parapertussiswere detected in 11.1%, 0.9% (only in females >4 years old), and 0.2% of samples, respectively. The new algorithm proved to be a useful microbiological diagnostic tool for whooping cough, demonstrating a low rate of other non-pertussisBordetellaspecies in our surveilled area.

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Quantitative measurement of infectious virus in SARS-CoV-2 Alpha, Delta and Epsilon variants reveals higher infectivity (viral titer:RNA ratio) in clinical samples containing the Delta and Epsilon variants.

10.1101/2021.09.07.21263229 ◽

2021 ◽

Author(s):

Hannah W Despres ◽

Margaret G Mills ◽

David J Shirley ◽

Madaline M Schmidt ◽

Meei-Li Huang ◽

...

Keyword(s):

Quantitative Measurement ◽

Infectious Virus ◽

Viral Rna ◽

Clinical Samples ◽

Rt Pcr ◽

Genome Copy ◽

Live Virus ◽

High Degree ◽

The Relationship ◽

Rna Levels

ABSTRACT Background Novel SARS-CoV-2 Variants of Concern (VoC) pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between viral RNA and infectious virus for individual variants is unknown. Methods We measured infectious viral titer (using a micro-focus forming assay) as well as total and subgenomic viral RNA levels (using RT-PCR) in a set of 165 clinical samples containing SARS-CoV-2 Alpha, Delta and Epsilon variants that were processed within two days of collection from the patient. Results We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite the variability we observed for individual samples the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (6 and 4 times as much, p=0.0002 and 0.009 respectively) or subgenomic E RNA (11 and 7 times as much, p<0.0001 and 0.006 respectively). Conclusion In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may also be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity of the Delta variant may further explain increased spread and suggests a need for increased measures to prevent viral transmission.

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Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

PLoS ONE ◽

10.1371/journal.pone.0252687 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0252687

Author(s):

Sukalyani Banik ◽

Kaheerman Saibire ◽

Shraddha Suryavanshi ◽

Glenn Johns ◽

Soumitesh Chakravorty ◽

...

Keyword(s):

Limit Of Detection ◽

Viral Rna ◽

Clinical Samples ◽

Sample Collection ◽

Rt Pcr ◽

Diagnostic Assays ◽

Guanidinium Thiocyanate ◽

Upper Respiratory ◽

Polymerase Chain ◽

The Stability

Background Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. Methods We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. Results SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. Conclusion eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

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Improved Genotyping Vaccine and Wild-Type Poliovirus Strains by Restriction Fragment Length Polymorphism Analysis: Clinical Diagnostic Implications

Journal of Clinical Microbiology ◽

10.1128/jcm.38.12.4337-4342.2000 ◽

2000 ◽

Vol 38 (12) ◽

pp. 4337-4342 ◽

Cited By ~ 25

Author(s):

Amalia Georgopoulou ◽

Panayotis Markoulatos ◽

Niki Spyrou ◽

Nicholas C. Vamvakopoulos

Keyword(s):

Restriction Fragment Length Polymorphism ◽

Restriction Fragment ◽

Length Polymorphism ◽

Fragment Length ◽

Fragment Length Polymorphism ◽

Rflp Analysis ◽

Clinical Samples ◽

Polymorphism Analysis ◽

Rt Pcr ◽

Restriction Fragment Length

The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5′ noncoding region of polioviruses was selected for RT-PCR amplification by the UC53-UG52 primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, andAvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, orNcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success.

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High speed large scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled with high content screening

10.1101/2020.05.14.097295 ◽

2020 ◽

Cited By ~ 2

Author(s):

Rania Francis ◽

Marion Le Bideau ◽

Priscilla Jardot ◽

Clio Grimaldier ◽

Didier Raoult ◽

...

Keyword(s):

High Speed ◽

Large Scale ◽

Vaccine Development ◽

Work Load ◽

Drug Susceptibility Testing ◽

High Content Screening ◽

Clinical Samples ◽

Rt Pcr ◽

Positive Elements

AbstractSARS-CoV-2, a novel coronavirus infecting humans, is responsible for the current COVID-19 global pandemic. If several strains could be isolated worldwide, especially for in-vitro drug susceptibility testing and vaccine development, few laboratories routinely isolate SARS-CoV-2. This is due to the fact that the current co-culture strategy is highly time consuming and requires working in a biosafety level 3 laboratory. In this work, we present a new strategy based on high content screening automated microscopy (HCS) allowing large scale isolation of SARS-CoV-2 from clinical samples in 1 week. A randomized panel of 104 samples, including 72 tested positive by RT-PCR and 32 tested negative, were processed with our HCS procedure and were compared to the classical isolation procedure. Isolation rate was 43 % with both strategies on RT-PCR positive samples, and was correlated with the initial RNA viral load in the samples, where we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with HCS strategy, where 80 % of the positive samples were recovered by the third day of co-culture, as compared to only 25 % with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive elements after 1 week of co-culture. This system allows rapid and automated screening of clinical samples with minimal operator work load, thus reducing the risks of contamination.

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The evolution of regulatory elements in the emerging promoter variant strains of HIV-1

10.1101/2021.04.28.441760 ◽

2021 ◽

Author(s):

Disha Bhange ◽

Nityanand Prasad ◽

Swati Singh ◽

Harshit Kumar Prajapati ◽

Shesh Prakash Maurya ◽

...

Keyword(s):

Viral Latency ◽

Regulatory Elements ◽

The Other ◽

Clinical Samples ◽

Latent Reservoir ◽

Viral Gene ◽

Viral Promoter ◽

Reservoir Characteristics ◽

Hiv 1

AbstractIn a multicentric, observational, investigator-blinded, and longitudinal clinical study of 764 ART-naïve subjects, we identified nine different promoter-variant strains of HIV-1 subtype C (HIV-1C) emerging in the Indian population, with some of these variants being reported for the first time. Unlike several previous studies, our work here focuses on the evolving viral regulatory elements, not coding sequences. The emerging viral strains contain additional copies of the existing transcription factor binding sites (TFBS), including TCF-1α/LEF-1, RBEIII, AP-1, and NF-κB, created by sequence duplication. The additional TFBS are genetically diverse and may blur the distinction between the modulatory region of the promoter and the viral enhancer. In a follow-up analysis, we found trends, but not significant associations between any specific variant promoter and prognostic markers, probably because the emerging viral strains might not have established mono infections yet. Illumina sequencing of four clinical samples containing a co-infection indicated the domination of one strain over the other and establishing a stable ratio with the second strain at the follow-up time-points. Since a single promoter regulates viral gene expression and constitutes the master regulatory circuit with Tat, the acquisition of additional and variant copies of the TFBS may significantly impact viral latency and latent reservoir characteristics. Further studies are urgently warranted to understand how the diverse TFBS profiles of the viral promoter may modulate the characteristics of the latent reservoir, especially following the initiation of antiretroviral therapy.Significance StatementA unique conglomeration of TFBS enables the HIV-1 promoter to accomplish two diametrically opposite functions – transcriptional activation and transcriptional silencing. The various phases of viral latency - establishment, maintenance, and reversal - collectively determine the replication fitness of individual viral strains. A profound variation in the TFBS composition of the viral promoter may significantly alter the viral latency properties and the latent reservoir characteristics. Although the duplication of certain TFBS remains a quality unique to HIV-1C, the high-level genetic recombination of HIV-1 may promote the transfer of such molecular properties to the other HIV-1 subtypes. The emergence of several promoter-variant viral strains may make the task of a ‘functional cure’ more challenging in HIV-1C.

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Detecting SARS-CoV-2 by Realtime RT-PCR in clinical samples collected in the North of Vietnam

Tạp chí Y học Dự phòng ◽

10.51403/0868-2836/2020/114 ◽

2021 ◽

Vol 30 (9) ◽

pp. 11-17

Author(s):

Hoang Vu Mai Phuong ◽

Ung Thi Hong Trang ◽

Nguyen Vu Son ◽

Le Thi Thanh ◽

Nguyen Le Khanh Hang ◽

...

Keyword(s):

Viral Load ◽

Average Rate ◽

Clinical Samples ◽

Viet Nam ◽

Rt Pcr ◽

Ct Value ◽

The North ◽

Viral Load Data ◽

The Mean ◽

High Level

From January to August 2020, Northern Viet Nam faced a COVID-19 outbreak, up to September 2020, there were 1122 confrmed cases of SARS-CoV-2, of which 465 cases were imported from Europe, America and Asia, 657 cases were identifed domestically. A total of 30,686 samples were collected during the SARS-CoV-2 outbreak in Northern Viet Nam and examined by Real-time RT-PCR using primers and probe from Charite - Berlin protocol. This study showed the initial results of SARS-CoV-2 detection and RNA quantitative in positive samples. The positive rate was 0.8%, ranging from 0.4 to 3.5% according to collection sites. Out of 251 positive samples, the mean Ct value was 28 (IQR: 22.3-32; range 14 - 38). The positive samples had a Ct value below 30 was 68.5%, there was no signifcant difference between the Ct value of the group ≤ 30 and > 30. The mean of the RNA copies/µl was 8.4.107, (IQR: 2.29.106 - 1.83.109 RNA copies/µl, range: 1.95.103 – 4.95.1011). In the group of imported COVID-19 cases, the rate of virus at low level was 29%, an average was 56% and at high level was 15%. In the community groups, the viral load data showed that the average rate at low, intermediate and high level were 20%, 63% and 17% respectively. The proportion of high-level viral load may raise an alert to start the quarantine process to reduce the transmission of SARS-CoV-2

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Comparison of the Abbott Alinity m and m2000 assays for the quantification of HIV-1, HCV and HBV in clinical samples

Journal of Clinical Virology ◽

10.1016/j.jcv.2020.104331 ◽

2020 ◽

Vol 126 ◽

pp. 104331

Author(s):

Lina Mouna ◽

Coralie Pallier ◽

Stéphanie Proust ◽

Corinne Prégermain ◽

Anne-Marie Roque-Afonso

Keyword(s):

Clinical Samples ◽

Hiv 1

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