Short Communication:A Rapid and Sensitive Real-Time PCR Assay for the K65R Drug Resistance Mutation in SIV Reverse Transcriptase

Introduction: Treatment for All recommendations have allowed access to antiretroviral (ARV) treatment for an increasing number of patients. This minimizes the transmission of infection but can potentiate the risk of transmitted (TDR) and acquired drug resistance (ADR). Objective: To study the trends of TDR and ADR in patients followed up in Portuguese hospitals between 2001 and 2017. Methods: In total, 11,911 patients of the Portuguese REGA database were included. TDR was defined as the presence of one or more surveillance drug resistance mutation according to the WHO surveillance list. Genotypic resistance to ARV was evaluated with Stanford HIVdb v7.0. Patterns of TDR, ADR and the prevalence of mutations over time were analyzed using logistic regression. Results and Discussion: The prevalence of TDR increased from 7.9% in 2003 to 13.1% in 2017 (p < 0.001). This was due to a significant increase in both resistance to nucleotide reverse transcriptase inhibitors (NRTIs) and non-nucleotide reverse transcriptase inhibitors (NNRTIs), from 5.6% to 6.7% (p = 0.002) and 2.9% to 8.9% (p < 0.001), respectively. TDR was associated with infection with subtype B, and with lower viral load levels (p < 0.05). The prevalence of ADR declined from 86.6% in 2001 to 51.0% in 2017 (p < 0.001), caused by decreasing drug resistance to all antiretroviral (ARV) classes (p < 0.001). Conclusions: While ADR has been decreasing since 2001, TDR has been increasing, reaching a value of 13.1% by the end of 2017. It is urgently necessary to develop public health programs to monitor the levels and patterns of TDR in newly diagnosed patients.

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Development of a Highly Sensitive Genus-Specific Quantitative Reverse Transcriptase Real-Time PCR Assay for Detection and Quantitation of Plasmodium by Amplifying RNA and DNA of the 18S rRNA Genes

Journal of Clinical Microbiology ◽

10.1128/jcm.00276-11 ◽

2011 ◽

Vol 49 (8) ◽

pp. 2946-2953 ◽

Cited By ~ 102

Author(s):

E. Kamau ◽

L. S. Tolbert ◽

L. Kortepeter ◽

M. Pratt ◽

N. Nyakoe ◽

...

Keyword(s):

Reverse Transcriptase ◽

Real Time ◽

Real Time Pcr ◽

18S Rrna ◽

Rrna Genes ◽

Pcr Assay ◽

Highly Sensitive ◽

18S Rrna Genes ◽

Rna And Dna

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P132 A novel PCR-CRISPR based diagnosis for Treponema pallidum detection, genotyping, and drug-resistance mutation identification in real-time

10.1136/sextrans-2021-sti.246 ◽

2021 ◽

Author(s):

W Chen ◽

H Luo ◽

Z Mai ◽

Y Jiang ◽

Z Hu ◽

...

Keyword(s):

Drug Resistance ◽

Real Time ◽

Treponema Pallidum ◽

Resistance Mutation ◽

Drug Resistance Mutation ◽

Mutation Identification

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Human Immunodeficiency Virus-1 Sequence Changes and Drug Resistance Mutation Among Virologic Failures of Lopinavir/Ritonavir Monotherapy: AIDS Clinical Trials Group Protocol A5230

Open Forum Infectious Diseases ◽

10.1093/ofid/ofw154 ◽

2016 ◽

Vol 3 (3) ◽

Cited By ~ 1

Author(s):

Saran Vardhanabhuti ◽

David Katzenstein ◽

John Bartlett ◽

Nagalingeswaran Kumarasamy ◽

Carole L. Wallis

Keyword(s):

Human Immunodeficiency Virus ◽

Drug Resistance ◽

Reverse Transcriptase ◽

Genetic Distance ◽

Hamming Distance ◽

Virologic Failure ◽

Resistance Mutation ◽

Drug Resistance Mutation ◽

Immunodeficiency Virus ◽

Human Immunodeficiency Virus 1

Abstract Background. The mechanism of virologic failure (VF) of lopinavir/ritonavir (LPV/r) monotherapy is not well understood. We assessed sequence changes in human immunodeficiency virus-1 reverse-transcriptase (RT) and protease (PR) regions. Methods. Human immunodeficiency virus-1 pol sequences from 34 participants who failed second-line LPV/r monotherapy were obtained at study entry (SE) and VF. Sequence changes were evaluated using phylogenetic analysis and hamming distance. Results. Human immunodeficiency virus-1 sequence change was higher over drug resistance mutation (DRM) sites (median genetic distance, 2.2%; Q1 to Q3, 2.1%–2.5%) from SE to VF compared with non-DRM sites (median genetic distance, 1.3%; Q1 to Q3, 1.0%–1.4%; P < .0001). Evolution over DRM sites was mainly driven by changes in the RT (median genetic distance, 2.7%; Q1 to Q3, 2.2%–3.2%) compared with PR (median genetic distance, 1.1%; Q1 to Q3, 0.0%–1.1%; P < .0001). Most RT DRMs present at SE were lost at VF. At VF, 19 (56%) and 26 (76%) were susceptible to efavirenz/nevirapine and etravirine (ETV)/rilpivirine (RPV), respectively, compared with 1 (3%) and 12 (35%) at SE. Participants who retained nonnucleoside reverse-transcriptase inhibitor (NNRTI) DRMs and those without evolution of LPV/r DRMs had significantly shorter time to VF. Conclusions. The selection of LPV/r DRMs in participants with longer time to VF suggests better adherence and more selective pressure. Fading NNRTI mutations and an increase in genotypic susceptibility to ETV and RPV could allow for the reuse of NNRTI. Further studies are warranted to understand mechanisms of PR failure.

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Establishment and Application of a Taqman Reverse Transcriptase Quantitative Real Time Pcr Assay for Feline Calicivirus

10.21203/rs.3.rs-267291/v1 ◽

2021 ◽

Author(s):

jingjie zhao ◽

Lin Liang ◽

Guangzhi Zhang ◽

Wenhui Li ◽

Shaohan Li ◽

...

Keyword(s):

Reverse Transcriptase ◽

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Clinical Samples ◽

Feline Calicivirus ◽

Quantitative Real Time Pcr ◽

Pcr Assay ◽

Virus Content ◽

Nasal Swabs

Abstract Feline calicivirus (FCV) is an infectious pathogen that causes disease in cats. With the current emergence of FCV-associated virulent systemic disease (FCV VSD) worldwide, the establishment of a rapid, sensitive, and reproducible diagnostic assay for its detection is important to inform prevention and control strategies. In this study, specific primers and TaqMan-FAM probes were designed based on the conserved regions of the FCV genome sequence, and a TaqMan reverse transcriptase quantitative real time PCR assay was established. This assay could specifically detected the FCV genome. The assay had a wide dynamic range, with linear detection in the range of 9.6×109 copies/μL to 9.6×100 copies/μL, with a limit of detection of 9.6×100 copies/μL, showing high sensitivity and repeatability. In addition, we used this assay to evaluated clinical samples (n=100) taken from cats from across China for the presence/absence of FCV genetic material For samples with low virus content, the positive detection rate of TaqMan reverse transcriptase quantitative real time PCR assay (RT-qPCR) was much higher than that of conventional reverse transcriptase PCR assay (cRT-PCR). And The qRT-PCR assay was used to detect the viral load of cat swabs within 17 days after FCV infection. From days 1-9, the oral and nasal swabs generally had higher viral loads than the anal swabs. While from days 10-17, the levels in the oral and nasal swabs being generally lower than those in the anal swabs. Overall, this FCV TaqMan RT-qPCR assay assay represents a rapid and accurate.

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Reverse Transcriptase Real-Time PCR Assay for the Rapid Enumeration of Live Candida auris from the Healthcare Environment

10.1101/2021.03.18.436063 ◽

2021 ◽

Author(s):

Bryanna Lexus Freitas ◽

Lynn Leach ◽

Vishnu Chaturvedi ◽

Sudha Chaturvedi

Keyword(s):

Reverse Transcriptase ◽

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Multidrug Resistant ◽

Molecular Diagnostic ◽

Pcr Assay ◽

Candida Auris ◽

Healthcare Environments ◽

Rapid Enumeration

Ongoing healthcare-associated outbreaks of multidrug-resistant yeast Candida auris have prompted development of several rapid DNA-based molecular diagnostic tests. These tests do not distinguish between live and dead C. auris cells, limiting their use for environmental surveillance and containment efforts. We addressed this critical gap by developing a reverse transcriptase (RT) real-time PCR assay for rapid detection of live C. auris in healthcare environments. The assay targets the internal transcribed spacer 2 (ITS2) cDNA of C. auris, amplified by obtaining pure RNA followed by reverse transcription and real-time PCR assay. The assay was highly sensitive, with the detection limit of ten colony-forming units (CFU) per RT real-time PCR reaction. In C. auris viability studies, ITS2 cDNA was detectable in heat- and ethanol-killed C. auris cells, but not bleach-killed cells. Validation studies showed no cycle threshold (Ct) values were obtained from samples on a sponge matrix spiked with either dead C. auris (105/ml) or other Candida species (105/ml), while most environmental samples spiked with 102 to 105 CFU of live C. auris yielded positive Ct values. Finally, 33 environmental samples positive for C. auris DNA but negative by culture were all negative by RT real-time PCR assay, confirming concordance between culture and the assay. The RT real-time PCR assay appears highly reproducible, robust, and specific for detecting live C. auris from environmental samples, and could be an invaluable tool in surveillance efforts to control the spread of live C. auris in healthcare environments.

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