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Article: Accuracy of a RT-qPCR SARS-CoV-2 detection assay without prior RNA extraction

Beltrán-Pavez, Carolina / Alonso-Palomares, Luis A. / Valiente-Echeverría, Fernando / Gaggero, Aldo / Soto-Rifo, Ricardo / Barriga, Gonzalo P.

Journal of virological methods. 2021 Jan., v. 287

2021

Abstract: The current COVID‐19 pandemic constitutes a threat to the population worldwide with over 21 million infected people. There is an urgent need for the development of rapid and massive detection tools as well as the identification and isolation of infected ... ...

Abstract	The current COVID‐19 pandemic constitutes a threat to the population worldwide with over 21 million infected people. There is an urgent need for the development of rapid and massive detection tools as well as the identification and isolation of infected individuals. we sought to evaluate different RT-qPCR kits and protocols to evaluate the best approach to be used omitting an RNA extraction step. We have investigated the sensitivity and performance of different commercially available RT-qPCR kits in detecting SARS-CoV-2 using 80 extracted RNA and NSS from COVID-19 diagnosed patients. We evaluated the ability of each kit to detect viral RNA from both kit-extracted or directly from a pre-boiled NSS observing that direct RNA detection is possible when Ct values are lower than 30 with the three kits tested. Since SARS-CoV-2 testing in most locations occurs once COVID-19 symptoms are evident and, therefore, viral loads are expected to be high, our protocol will be useful in supporting SARS-CoV-2 diagnosis, especially in America where COVID-19 cases have exploded in the recent weeks as well as in low- and middle-income countries, which would not have massive access to kit-based diagnosis. The information provided in this work paves the way for the development of more efficient SARS-CoV-2 detection approaches avoiding an RNA extraction step.
Keywords	COVID-19 infection ; RNA ; Severe acute respiratory syndrome coronavirus 2
Language	English
Dates of publication	2021-01
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2020.113969
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Transmitted Drug Resistance and HIV Diversity Among Adolescents Newly Diagnosed With HIV in Spain.

Epalza, Cristina / Valadés-Alcaraz, Ana / González-Alba, José María / Beltrán-Pavez, Carolina / Gutiérrez-López, Miguel / Rubio-Garrido, Marina / Fortuny, Clàudia / Frick, Marie Antoinette / Muñoz Medina, Leopoldo / Moreno, Santiago / Sanz, José / Rojo, Pablo / Navarro, María Luisa / Holguín, África

The Pediatric infectious disease journal

2023 Volume 43, Issue 1, Page(s) 40–48

Abstract: Background: Virologic characterization of newly HIV-diagnosed adolescents could help to improve their specific needs. The objective was to describe the transmitted drug resistance mutations (TDR) and its transmission by clusters in this population in ... ...

Abstract	Background: Virologic characterization of newly HIV-diagnosed adolescents could help to improve their specific needs. The objective was to describe the transmitted drug resistance mutations (TDR) and its transmission by clusters in this population in Spain. Methods: TDR to retrotranscriptase and protease inhibitors included in the WHO TDR list 2009 implemented in the Calibrated Population Resistance tool v8.0 (Stanford) were studied in HIV pol sequences from all HIV-diagnosed adolescents (12-19-year-old) enrolled during 2004-2019 period in the Spanish pediatric and adult (CoRISpe-CoRIS) cohorts. The found TDR were compared with the provided by the Stanford algorithm v9.0 2021. HIV-1 variants and transmission clusters were also studied. Results: Among 410 HIV-1 adolescents diagnosed, 141 (34.4%) had available ART-naive sequences. They were mostly male (81.6%), Spanish (55.3%) and with behavioral risk (92.2%), mainly male-to-male sexual contact (63.1%). TDR prevalence was significantly higher by Stanford versus WHO list (18.4% vs. 7.1%; P = 0.004). The most prevalent TDR by the WHO list was K103N (3.6%) and by Stanford E138A (6.6%), both at retrotranscriptase. E138A, related to rilpivirine/etravirine resistance, was absent in the WHO list. One in 4 adolescents carried HIV-1 non-B variants. We described 5 transmission clusters, and 2 carried TDR mutations. Conclusions: Our data suggest a high TDR prevalence in adolescents with a new HIV diagnosis in Spain, similar to adults, 2 active TDR transmission clusters, and the need for the WHO TDR list update. These findings could have implications for the options of the recently available rilpivirine-related long-acting treatment and in first-line regimen election.
MeSH term(s)	Adult ; Humans ; Male ; Adolescent ; Child ; Young Adult ; Female ; HIV Infections/drug therapy ; HIV Infections/epidemiology ; Spain/epidemiology ; Drug Resistance, Viral/genetics ; HIV Seropositivity ; Mutation ; HIV-1/genetics ; Rilpivirine/therapeutic use ; Prevalence ; Genotype ; Anti-HIV Agents/pharmacology ; Anti-HIV Agents/therapeutic use
Chemical Substances	Rilpivirine (FI96A8X663) ; Anti-HIV Agents
Language	English
Publishing date	2023-11-16
Publishing country	United States
Document type	Journal Article
ZDB-ID	392481-6
ISSN	1532-0987 ; 0891-3668
ISSN (online)	1532-0987
ISSN	0891-3668
DOI	10.1097/INF.0000000000004138
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Article ; Online: Accuracy of a RT-qPCR SARS-CoV-2 detection assay without prior RNA extraction.

Beltrán-Pavez, Carolina / Alonso-Palomares, Luis A / Valiente-Echeverría, Fernando / Gaggero, Aldo / Soto-Rifo, Ricardo / Barriga, Gonzalo P

Journal of virological methods

2020 Volume 287, Page(s) 113969

Abstract: The current COVID-19 pandemic constitutes a threat to the population worldwide with over 21 million infected people. There is an urgent need for the development of rapid and massive detection tools as well as the identification and isolation of infected ... ...

Abstract	The current COVID-19 pandemic constitutes a threat to the population worldwide with over 21 million infected people. There is an urgent need for the development of rapid and massive detection tools as well as the identification and isolation of infected individuals. we sought to evaluate different RT-qPCR kits and protocols to evaluate the best approach to be used omitting an RNA extraction step. We have investigated the sensitivity and performance of different commercially available RT-qPCR kits in detecting SARS-CoV-2 using 80 extracted RNA and NSS from COVID-19 diagnosed patients. We evaluated the ability of each kit to detect viral RNA from both kit-extracted or directly from a pre-boiled NSS observing that direct RNA detection is possible when Ct values are lower than 30 with the three kits tested. Since SARS-CoV-2 testing in most locations occurs once COVID-19 symptoms are evident and, therefore, viral loads are expected to be high, our protocol will be useful in supporting SARS-CoV-2 diagnosis, especially in America where COVID-19 cases have exploded in the recent weeks as well as in low- and middle-income countries, which would not have massive access to kit-based diagnosis. The information provided in this work paves the way for the development of more efficient SARS-CoV-2 detection approaches avoiding an RNA extraction step.
MeSH term(s)	COVID-19/diagnosis ; COVID-19/virology ; COVID-19 Nucleic Acid Testing/methods ; Humans ; Nasopharynx/virology ; RNA, Viral/genetics ; RNA, Viral/isolation & purification ; Reagent Kits, Diagnostic ; SARS-CoV-2/genetics ; SARS-CoV-2/isolation & purification ; Sensitivity and Specificity ; Specimen Handling/methods ; Time Factors ; Viral Load
Chemical Substances	RNA, Viral ; Reagent Kits, Diagnostic
Keywords	covid19
Language	English
Publishing date	2020-09-09
Publishing country	Netherlands
Document type	Evaluation Study ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2020.113969
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Article ; Online: Virological outcome among HIV infected patients transferred from pediatric care to adult units in Madrid, Spain (1997-2017).

Beltrán-Pavez, Carolina / Gutiérrez-López, Miguel / Rubio-Garrido, Marina / Valadés-Alcaraz, Ana / Prieto, Luis / Ramos, José Tomás / Jiménez De Ory, Santiago / Navarro, Marisa / Díez-Romero, Cristina / Pulido, Federico / Valencia, Eulalia / Holguín, África

Scientific reports

2020 Volume 10, Issue 1, Page(s) 16891

Abstract: The aim of this transversal study was to describe the virological and immunological features of HIV-infected youths transferred from pediatric to adult care units since 1997 vs. the non-transferred patients from the Madrid Cohort of HIV-infected children ...

Abstract	The aim of this transversal study was to describe the virological and immunological features of HIV-infected youths transferred from pediatric to adult care units since 1997 vs. the non-transferred patients from the Madrid Cohort of HIV-infected children and adolescents in Spain. We included 106 non-transferred and 184 transferred patients under clinical follow-up in 17 public hospitals in Madrid by the end of December 2017. Virological and immunological outcomes were compared in transferred vs. non-transferred patients. ART drug resistance mutations and HIV-variants were analyzed in all subjects with available resistance pol genotypes and/or genotypic resistance profiles. Among the study cohort, 133 (72.3%) of 184 transferred and 75 (70.7%) of 106 non-transferred patients had available resistance genotypes. Most (88.9%) of transferred had ART experience at sampling. A third (33.3%) had had a triple-class experience. Acquired drug resistance (ADR) prevalence was significantly higher in pretreated transferred than non-transferred patients (71.8% vs. 44%; p = 0.0009), mainly to NRTI (72.8% vs. 31.1%; p < 0.0001) and PI (29.1% vs. 12%; p = 0.0262). HIV-1 non-B variants were less frequent in transferred vs. non-transferred (6.9% vs. 32%; p < 0.0001). In conclusion, the frequent resistant genotypes found in transferred youths justifies the reinforcement of HIV resistance monitoring after the transition to avoid future therapeutic failures.
MeSH term(s)	Adolescent ; Adult ; Anti-HIV Agents/therapeutic use ; Child ; Child, Preschool ; Cohort Studies ; Drug Resistance, Viral/genetics ; Female ; Genotype ; HIV Infections/drug therapy ; HIV Infections/genetics ; HIV Infections/virology ; HIV-1/drug effects ; HIV-1/genetics ; Humans ; Infant ; Male ; Mutation/genetics ; Pediatrics ; Spain ; Viral Load/genetics ; Young Adult
Chemical Substances	Anti-HIV Agents
Language	English
Publishing date	2020-10-09
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-020-70861-x
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: SARS-CoV-2 detection from nasopharyngeal swab samples without RNA extraction

Beltrán-Pavez, Carolina / Márquez, Chantal L. / Muñoz, Gabriela / Valiente-Echeverría, Fernando / Gaggero, Aldo / Soto-Rifo, Ricardo / Barriga, Gonzalo P.

bioRxiv

Abstract: The ongoing COVID-19 pandemic has reached more than 200 countries and territories worldwide. Given the large requirement of SARS-CoV-2 diagnosis and considering that RNA extraction kits are in short supply, we investigated whether two commercial RT-qPCR ... ...

Abstract	The ongoing COVID-19 pandemic has reached more than 200 countries and territories worldwide. Given the large requirement of SARS-CoV-2 diagnosis and considering that RNA extraction kits are in short supply, we investigated whether two commercial RT-qPCR kits were compatible with direct SARS-CoV-2 detection from nasopharyngeal swab samples. We show that one of the tested kits is fully compatible with direct SARS-CoV-2 detection suggesting that omission of RNA extraction step should be considered in SARS-CoV-2 diagnosis.
Keywords	covid19
Language	English
Publishing date	2020-03-30
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2020.03.28.013508
Database	COVID19

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Article ; Online: SARS-CoV-2 detection from nasopharyngeal swab samples without RNA extraction

Beltrán-Pavez, Carolina / Márquez, Chantal L. / Muñoz, Gabriela / Valiente-Echeverría, Fernando / Gaggero, Aldo / Soto-Rifo, Ricardo / Barriga, Gonzalo P.

bioRxiv

Abstract	The ongoing COVID-19 pandemic has reached more than 200 countries and territories worldwide. Given the large requirement of SARS-CoV-2 diagnosis and considering that RNA extraction kits are in short supply, we investigated whether two commercial RT-qPCR kits were compatible with direct SARS-CoV-2 detection from nasopharyngeal swab samples. We show that one of the tested kits is fully compatible with direct SARS-CoV-2 detection suggesting that omission of an RNA extraction step should be considered in SARS-CoV-2 diagnosis.
Keywords	covid19
Publisher	BioRxiv; WHO
Document type	Article ; Online
DOI	10.1101/2020.03.28.013508
Database	COVID19

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Article ; Online: Prevalence of M184V and K65R in proviral DNA from PBMCs in HIV-infected youths with lamivudine/emtricitabine exposure.

Jiménez de Ory, Santiago / Beltrán-Pavez, Carolina / Gutiérrez-López, Miguel / Santos, María Del Mar / Prieto, Luis / Sainz, Talía / Guillen, Sara / Aguilera-Alonso, David / Díez, Cristina / Bernardino, Jose Ignacio / Mellado, María José / Ramos, José Tomás / Holguín, África / Navarro, Marisa

The Journal of antimicrobial chemotherapy

2021 Volume 76, Issue 7, Page(s) 1886–1892

Abstract: Objectives: We analysed the prevalence of M184V/I and/or K65R/E/N mutations archived in proviral DNA (pDNA) in youths with perinatal HIV, virological control and who previously carried these resistance mutations in historic plasma samples.: Methods: ... ...

Abstract	Objectives: We analysed the prevalence of M184V/I and/or K65R/E/N mutations archived in proviral DNA (pDNA) in youths with perinatal HIV, virological control and who previously carried these resistance mutations in historic plasma samples. Methods: We included vertically HIV-infected youths/young adults aged ≥10 years in the Madrid Cohort of HIV-1 Infected Children and Adolescents, exposed to lamivudine and/or emtricitabine, with M184V/I and/or K65R/E/N in historic plasma samples, on antiretroviral therapy (ART), virologically suppressed (HIV-1 RNA <50 copies/mL), and with available PBMCs in the Spanish HIV BioBank. Genomic DNA was extracted from PBMCs and HIV-1 RT gene was amplified and sequenced for resistance testing by Stanford HIV Resistance tool. Results: Among the 225 patients under follow-up in the study cohort, 13 (5.8%) met selection criteria, and RT sequences were recovered in 12 (92.3%) of them. All but one were Spaniards, carrying subtype B, with a median age at PBMCs sampling of 21.3 years (IQR: 15.6-23.1) with 4 years (IQR 2.1-6.5) of suppressed viral load (VL). Nine (75%) youths did not present M184V/I in pDNA after at least 1 year of viral suppression. In December 2019, the remaining three subjects carrying M184V/I in pDNA maintained suppressed viraemia, and two still used emtricitabine in ART. Conclusions: The prevalence of resistance mutations to lamivudine and emtricitabine in pDNA in a cohort of youths perinatally infected with HIV who remain with undetectable VL, previously lamivudine and/or emtricitabine experienced, was infrequent. Our results indicate that ART including lamivudine or emtricitabine may also be safe and successful in youths with perinatal HIV with previous experience of and resistances to these drugs detected in plasma.
MeSH term(s)	Adolescent ; Anti-HIV Agents/pharmacology ; Anti-HIV Agents/therapeutic use ; Child ; DNA ; Drug Resistance, Viral ; Emtricitabine/therapeutic use ; HIV Infections/drug therapy ; HIV Infections/epidemiology ; Humans ; Lamivudine/therapeutic use ; Prevalence ; Proviruses/genetics ; Viral Load
Chemical Substances	Anti-HIV Agents ; Lamivudine (2T8Q726O95) ; DNA (9007-49-2) ; Emtricitabine (G70B4ETF4S)
Language	English
Publishing date	2021-03-18
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	191709-2
ISSN	1460-2091 ; 0305-7453
ISSN (online)	1460-2091
ISSN	0305-7453
DOI	10.1093/jac/dkab080
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Article: Accuracy of a RT-qPCR SARS-CoV-2 detection assay without prior RNA extraction

Beltrán-Pavez, Carolina / Alonso-Palomares, Luis A / Valiente-Echeverría, Fernando / Gaggero, Aldo / Soto-Rifo, Ricardo / Barriga, Gonzalo P

J Virol Methods

Abstract	The current COVID-19 pandemic constitutes a threat to the population worldwide with over 21 million infected people. There is an urgent need for the development of rapid and massive detection tools as well as the identification and isolation of infected individuals. we sought to evaluate different RT-qPCR kits and protocols to evaluate the best approach to be used omitting an RNA extraction step. We have investigated the sensitivity and performance of different commercially available RT-qPCR kits in detecting SARS-CoV-2 using 80 extracted RNA and NSS from COVID-19 diagnosed patients. We evaluated the ability of each kit to detect viral RNA from both kit-extracted or directly from a pre-boiled NSS observing that direct RNA detection is possible when Ct values are lower than 30 with the three kits tested. Since SARS-CoV-2 testing in most locations occurs once COVID-19 symptoms are evident and, therefore, viral loads are expected to be high, our protocol will be useful in supporting SARS-CoV-2 diagnosis, especially in America where COVID-19 cases have exploded in the recent weeks as well as in low- and middle-income countries, which would not have massive access to kit-based diagnosis. The information provided in this work paves the way for the development of more efficient SARS-CoV-2 detection approaches avoiding an RNA extraction step.
Keywords	covid19
Publisher	WHO
Document type	Article
Note	WHO #Covidence: #753173
Database	COVID19

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Article ; Online: Potent Induction of Envelope-Specific Antibody Responses by Virus-Like Particle Immunogens Based on HIV-1 Envelopes from Patients with Early Broadly Neutralizing Responses.

Beltran-Pavez, Carolina / Bontjer, Ilja / Gonzalez, Nuria / Pernas, Maria / Merino-Mansilla, Alberto / Olvera, Alex / Miro, Jose M / Brander, Christian / Alcami, Jose / Sanders, Rogier W / Sanchez-Merino, Victor / Yuste, Eloisa

Journal of virology

2021 Volume 96, Issue 1, Page(s) e0134321

Abstract: Longitudinal studies in HIV-1-infected individuals have indicated that 2 to 3 years of infection are required to develop broadly neutralizing antibodies. However, we have previously identified individuals with broadly neutralizing activity (bNA) in early ...

Abstract	Longitudinal studies in HIV-1-infected individuals have indicated that 2 to 3 years of infection are required to develop broadly neutralizing antibodies. However, we have previously identified individuals with broadly neutralizing activity (bNA) in early HIV-1 infection, indicating that a vaccine may be capable of bNA induction after short periods of antigen exposure. Here, we describe 5 HIV-1 envelope sequences from individuals who have developed bNA within the first 100 days of infection (early neutralizers) and selected two of them to design immunogens based on HIV-1-Gag virus-like particles (VLPs). These VLPs were homogeneous and incorporated the corresponding envelopes (7 to 9 μg of gp120 in 10
MeSH term(s)	AIDS Vaccines/immunology ; Adult ; Antibodies, Neutralizing/immunology ; Antibody Formation ; Broadly Neutralizing Antibodies/immunology ; CD4 Lymphocyte Count ; CD4-CD8 Ratio ; Epitope Mapping ; Epitopes/immunology ; Female ; HIV Antibodies/chemistry ; HIV Antibodies/immunology ; HIV Infections/immunology ; HIV Infections/virology ; HIV-1/immunology ; Host-Pathogen Interactions/immunology ; Humans ; Immunization ; Male ; Middle Aged ; Models, Molecular ; Protein Conformation ; Structure-Activity Relationship ; Vaccines, Virus-Like Particle/immunology ; env Gene Products, Human Immunodeficiency Virus/chemistry ; env Gene Products, Human Immunodeficiency Virus/immunology
Chemical Substances	AIDS Vaccines ; Antibodies, Neutralizing ; Broadly Neutralizing Antibodies ; Epitopes ; HIV Antibodies ; Vaccines, Virus-Like Particle ; env Gene Products, Human Immunodeficiency Virus
Language	English
Publishing date	2021-10-20
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	80174-4
ISSN	1098-5514 ; 0022-538X
ISSN (online)	1098-5514
ISSN	0022-538X
DOI	10.1128/JVI.01343-21
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Article ; Online: Insights into neutralizing antibody responses in individuals exposed to SARS-CoV-2 in Chile.

Beltrán-Pavez, Carolina / Riquelme-Barrios, Sebastián / Oyarzún-Arrau, Aarón / Gaete-Argel, Aracelly / González-Stegmaier, Roxana / Cereceda-Solis, Karina / Aguirre, Adam / Travisany, Dante / Palma-Vejares, Ricardo / Barriga, Gonzalo P / Gaggero, Aldo / Martínez-Valdebenito, Constanza / Corre, Nicole Le / Ferrés, Marcela / Balcells, María Elvira / Fernandez, Jorge / Ramírez, Eugenio / Villarroel, Franz / Valiente-Echeverría, Fernando /

Soto-Rifo, Ricardo

Science advances

2021 Volume 7, Issue 7

Abstract: Chile has one of the worst numbers worldwide in terms of SARS-CoV-2 positive cases and COVID-19-related deaths per million inhabitants; thus, characterization of neutralizing antibody (NAb) responses in the general population is critical to understanding ...

Abstract	Chile has one of the worst numbers worldwide in terms of SARS-CoV-2 positive cases and COVID-19-related deaths per million inhabitants; thus, characterization of neutralizing antibody (NAb) responses in the general population is critical to understanding of immunity at the local level. Given our inability to perform massive classical neutralization assays due to the scarce availability of BSL-3 facilities in the country, we developed and fully characterized an HIV-based SARS-CoV-2 pseudotype, which was used in a 96-well plate format to investigate NAb responses in samples from individuals exposed to SARS-CoV-2 or treated with convalescent plasma. We also identified samples with decreased or enhanced neutralization activity against the D614G spike variant compared with the wild type, indicating the relevance of this variant in host immunity. The data presented here represent the first insights into NAb responses in individuals from Chile, serving as a guide for future studies in the country.
MeSH term(s)	Amino Acid Substitution ; Animals ; Antibodies, Neutralizing/blood ; Antibodies, Viral/blood ; COVID-19/blood ; COVID-19/genetics ; COVID-19 Serological Testing ; Chile ; Chlorocebus aethiops ; Female ; HEK293 Cells ; Humans ; Male ; Mutation, Missense ; SARS-CoV-2/genetics ; SARS-CoV-2/metabolism ; Spike Glycoprotein, Coronavirus/blood ; Spike Glycoprotein, Coronavirus/genetics ; Vero Cells
Chemical Substances	Antibodies, Neutralizing ; Antibodies, Viral ; Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2
Language	English
Publishing date	2021-02-12
Publishing country	United States
Document type	Clinical Trial ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2810933-8
ISSN	2375-2548 ; 2375-2548
ISSN (online)	2375-2548
ISSN	2375-2548
DOI	10.1126/sciadv.abe6855
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