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Fofana DB, Coulibaly TA, Maiga M, Nguyen T, Gozlan J, Diarra Z, Koné A, Cissoko Y, Maiga AI, Hawkins CA, Murphy RL, Morand-Joubert L, Diakité M, Holl JL, McFall SM. A multiplexed real-time PCR assay for simultaneous quantification of human immunodeficiency virus and Hepatitis B virus for low-and-middle- income countries. J Virol Methods 2024; 330:115026. [PMID: 39233060 DOI: 10.1016/j.jviromet.2024.115026] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2024] [Revised: 08/21/2024] [Accepted: 08/29/2024] [Indexed: 09/06/2024]
Abstract
Due to shared routes of transmission, including sexual contact and vertical transmission, HIV-HBV co-infection is common, particularly in sub-Saharan Africa. Measurement of viral load (VL), for both HIV and HBV, plays a critical role for determining their infectious phase and monitoring response to antiviral therapy. Implementation of viral load testing in clinical settings is a significant challenge in resource-limited countries, notably because of cost and availability issues. We designed HIV and HBV primers for conserved regions of the HIV and HBV genomes that were specifically adapted to viral strains circulating in West Africa that are HIV-1 subtype CRF02AG and HBV genotype E. We first validated two monoplex qPCR assays for individual quantification and, then developed a multiplex qPCR for simultaneous quantification of both viruses. HIV RNA and HBV DNA amplification was performed in a single tube using a one-step reverse transcription-PCR reaction with primers and probes targeting both viruses. Performance characteristics such as the quantification range, sensitivity, and specificity of this multiplex qPCR assay were compared to reference qPCR tests for both HIV and HBV viral load quantification. The multiplex assay was validated using clinical samples from co- or mono-infected patients and gave comparable viral load quantification to the HIV and HBV reference test respectively. The multiplex qPCR demonstrated an overall sensitivity of 71.25 % [68.16-74.3] for HBV and 82 % [78.09-85.90] for HIV and an overall specificity of 100 % [94.95-100] for both viruses. Although the overall sensitivities of the HIV and HBV assays were lower than the commercial comparator assays, the sensitivity in the clinical decision range of >1000 copies/mL for HIV was 80 % [71.26-88.73] and >1000 IU/mL for HBV was 100 % [95.51-100] which indicates the test results can be used to guide treatment decisions. This in-house developed multiplex qPCR assay represents a useful diagnostic tool as it can be performed on affordable "open" real-time PCR platforms currently used for HIV or SARS-Cov-2 infection surveillance in Mali.
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Affiliation(s)
- Djeneba Bocar Fofana
- Faculty of Medicine, University of Sciences, Techniques and Technologies of Bamako (USTTB), Bamako BP 1805, Mali.
| | - Tenin Aminatou Coulibaly
- University Clinical Research Center, International Centers for Excellence in Research (UCRC), University of Sciences, Techniques and Technologies of Bamako, Bamako, Mali
| | - Mamoudou Maiga
- Institute for Global Health, Northwestern University, Chicago, IL 60208, USA
| | - Thuy Nguyen
- Clinical Retrovirology Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD, USA
| | - Joël Gozlan
- Sorbonne Université, INSERM, Institut Pierre Louis d'Epidémiologie et de Santé Publique (iPLESP), for Department of Virology, Assistance Publique-Hôpitaux de Paris (AP-HP), Saint-Antoine Hospital, Paris F-75012, France
| | - Zoumana Diarra
- Faculty of Medicine, University of Sciences, Techniques and Technologies of Bamako (USTTB), Bamako BP 1805, Mali
| | - Amadou Koné
- University Clinical Research Center, International Centers for Excellence in Research (UCRC), University of Sciences, Techniques and Technologies of Bamako, Bamako, Mali
| | - Yacouba Cissoko
- Faculty of Medicine, University of Sciences, Techniques and Technologies of Bamako (USTTB), Bamako BP 1805, Mali
| | - Almoustapha Issiaka Maiga
- Faculty of Medicine, University of Sciences, Techniques and Technologies of Bamako (USTTB), Bamako BP 1805, Mali
| | - Claudia A Hawkins
- Institute for Global Health, Northwestern University, Chicago, IL 60208, USA
| | - Robert L Murphy
- Institute for Global Health, Northwestern University, Chicago, IL 60208, USA
| | - Laurence Morand-Joubert
- Sorbonne Université, INSERM, Institut Pierre Louis d'Epidémiologie et de Santé Publique (iPLESP), for Department of Virology, Assistance Publique-Hôpitaux de Paris (AP-HP), Saint-Antoine Hospital, Paris F-75012, France
| | - Mahamadou Diakité
- Faculty of Medicine, University of Sciences, Techniques and Technologies of Bamako (USTTB), Bamako BP 1805, Mali
| | - Jane L Holl
- Biological Sciences Division University of Chicago, IL 60637, USA
| | - Sally M McFall
- Institute for Global Health, Northwestern University, Chicago, IL 60208, USA
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2
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Nemr WA, Nashwa RK. Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1. World J Virol 2024; 13:88164. [PMID: 38616859 PMCID: PMC11008401 DOI: 10.5501/wjv.v13.i1.88164] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/16/2023] [Revised: 12/07/2023] [Accepted: 01/10/2024] [Indexed: 03/11/2024] Open
Abstract
BACKGROUND Hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus 1 (HIV-1) are the most epidemic blood-borne viruses, posing threats to human health and causing economic losses to nations for combating the infection transmission. The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive, but they are more accurate than serological testing. AIM To develop a rapid, cost-effective, and accurate diagnostic multiplex polymerase chain reaction (PCR) assay for simultaneous detection of HCV, HBV, and HIV-1. METHODS The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electrophoretic molecular weight inside each viral genome. Therefore, this diagnostic method will be appropriate for application in both conventional (combined with electrophoresis) and real-time PCR facilities. Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus. Then, Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay. RESULTS The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay. The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis. Compared to related published assays, the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays. CONCLUSION This study provides a simple, cost-effective, and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses; this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.
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Affiliation(s)
- Waleed Abdelgaber Nemr
- Department of Radiation Microbiology, National Center for Radiation Research and Technology, Egyptian Atomic Energy Authority, Cairo 11371, Egypt
| | - Radwan K Nashwa
- Department of Health Radiation Research, National Center for Radiation Research and Technology, Egyptian Atomic Energy Authority, Cairo 11371, Egypt
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Yi HW, Wang XM, Tan X, Ding CZ, Zhang CL, Wu JH, Li Q, Xin CQ, Fan W. Simultaneous detection of human norovirus GI, GII and SARS-CoV-2 by a quantitative one-step triplex RT-qPCR. Front Microbiol 2024; 14:1269275. [PMID: 38260899 PMCID: PMC10800780 DOI: 10.3389/fmicb.2023.1269275] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2023] [Accepted: 12/20/2023] [Indexed: 01/24/2024] Open
Abstract
Background There are many similarities in the clinical manifestations of human norovirus and SARS-CoV-2 infections, and nucleic acid detection is the gold standard for diagnosing both diseases. In order to expedite the identification of norovirus and SARS-CoV-2, a quantitative one-step triplex reverse transcription PCR (RT-qPCR) method was designed in this paper. Methods A one-step triplex RT-qPCR assay was developed for simultaneous detection and differentiation of human norovirus GI (NoV-GI), GII (NoV-GII) and SARS-CoV-2 from fecal specimens. Results The triplex RT-qPCR assay had high detection reproducibility (CV < 1%) and sensitivity. The lower limits of detection (LLOD95) of the triplex RT-qPCR assay for each target site were 128.5-172.8 copies/mL, and LLOD95 of the singleplex RT-qPCR assay were 110.3-142.0 copies/mL. Meanwhile, among the detection of clinical oropharyngeal swabs and fecal specimens, the results of the singleplex and triplex RT-qPCR assay showed high agreement. Conclusion The triplex RT-qPCR assay for simultaneous detection of NoV-GI, NoV-GII and SARS-CoV-2 from fecal specimens has high clinical application value.
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Affiliation(s)
- Hua-Wei Yi
- The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China
- The First People's Hospital of Jingzhou, Jingzhou, Hubei, China
| | - Xian-Mo Wang
- The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China
- The First People's Hospital of Jingzhou, Jingzhou, Hubei, China
| | - Xin Tan
- Health Science Center of Yangtze University, Jingzhou, Hubei, China
| | - Cai-Zhi Ding
- The People's Hospital of Songzi, Jingzhou, Hubei, China
| | - Chang-Li Zhang
- The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China
- The First People's Hospital of Jingzhou, Jingzhou, Hubei, China
| | - Jia-Hao Wu
- The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China
- The First People's Hospital of Jingzhou, Jingzhou, Hubei, China
| | - Qi Li
- The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China
- The First People's Hospital of Jingzhou, Jingzhou, Hubei, China
| | - Chen-Qi Xin
- The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China
- The First People's Hospital of Jingzhou, Jingzhou, Hubei, China
| | - Wen Fan
- The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China
- The First People's Hospital of Jingzhou, Jingzhou, Hubei, China
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Alex-Sanders N, Woodhall N, Farkas K, Scott G, Jones DL, Walker DI. Development and validation of a duplex RT-qPCR assay for norovirus quantification in wastewater samples. J Virol Methods 2023; 321:114804. [PMID: 37643662 DOI: 10.1016/j.jviromet.2023.114804] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2023] [Revised: 08/21/2023] [Accepted: 08/26/2023] [Indexed: 08/31/2023]
Abstract
Norovirus (NoV) is a highly contagious enteric virus that causes widespread outbreaks and a substantial number of deaths across communities. As clinical surveillance is often insufficient, wastewater-based epidemiology (WBE) may provide novel pathways of tracking outbreaks. To utilise WBE, it is important to use accurate and sensitive methods for viral quantification. In this study, we developed a one-step duplex RT-qPCR assay to simultaneously test the two main human pathogenic NoV genogroups, GI and GII, in wastewater samples. The assay had low limits of detection (LOD), namely 0.52 genome copies (gc)/µl for NoVGI and 1.37 gc/µl for NoVGII. No significant concentration-dependent interactions were noted for both NoVGI and for NoVGII when the two targets were mixed at different concentrations in the samples. When tested on wastewater-derived RNA eluents, no significant difference between duplex and singleplex concentrations were found for either target. Low levels of inhibition (up to 32 %) were noted due to organic matter present in the wastewater extracts. From these results we argue that the duplex RT-qPCR assay developed enables the sensitive detection of both NoVGI and NoVGII in wastewater-derived RNA eluents, in a time and cost-effective way and may be used for surveillance to monitor public and environmental health.
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Affiliation(s)
| | - Nick Woodhall
- School of Natural Sciences, Bangor University, Bangor, Gwynedd LL57 2UW, UK
| | - Kata Farkas
- School of Natural Sciences, Bangor University, Bangor, Gwynedd LL57 2UW, UK
| | - George Scott
- Centre for Environment, Fisheries and Aquaculture Science, Weymouth, Dorset, UK
| | - Davey L Jones
- School of Natural Sciences, Bangor University, Bangor, Gwynedd LL57 2UW, UK; Food Futures Institute, Murdoch University, 90 South Street, Murdoch, WA 6150, Australia
| | - David I Walker
- Centre for Environment, Fisheries and Aquaculture Science, Weymouth, Dorset, UK
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Nishiya A, Salles N, de Almeida-Neto C, Ferreira S, Nogueira F, Rocha V, Mendrone-Júnior A. Detection of unreported usage of the antiretroviral drug lamivudine in two blood donors. Transfusion 2023; 63:2106-2113. [PMID: 37702479 DOI: 10.1111/trf.17544] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2023] [Revised: 08/25/2023] [Accepted: 08/27/2023] [Indexed: 09/14/2023]
Abstract
BACKGROUND Unreported HIV antiretroviral (ARV) drug usage by blood donors compromises the ability to detect evidence of HIV infection in blood screening tests and represents a risk for blood transfusion safety. Our objective was to determine the frequency of undeclared ARV drug use by blood donors with altered HIV markers. STUDY DESIGN AND METHODS This was a retrospective cross-sectional analysis of donations that were tested for HIV antibody (ab), antigen (ag), and RNA by chemiluminescent immunoassay and nucleic acid screening tests. Positive samples were retested and were subjected to ARV drug testing by high-performance liquid chromatography-tandem mass spectrometry. RESULTS Of 345,252 blood donations, 361 (0.1%) were positive on initial testing. Samples from 296 (81.9%) of these donations were available for further analysis. The presence of HIV ab/ag and/or RNA was confirmed in 83 (28.0%) of these samples. All 296 bloods were subjected to ARV testing. The ARV drug lamivudine, at 11.3 and 6.7 ng/mL, was detected in 2 of 83 (2.4%) donations that were HIV positive. Other drugs were not detected. CONCLUSION Unreported ARV usage was identified in two candidates for blood donation. More intensive efforts to educate donors about disclosure and to investigate the extent of this phenomenon in Brazil are needed.
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Affiliation(s)
- Anna Nishiya
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil
- Laboratory of Medical Investigation in Pathogenesis and targeted therapy in Oncoimmunohematology (LIM-31), Department of Hematology, Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil
| | - Nanci Salles
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil
| | - Cesar de Almeida-Neto
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil
- Disciplina de Ciências Médicas, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil
| | - Suzete Ferreira
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil
- Laboratory of Medical Investigation in Pathogenesis and targeted therapy in Oncoimmunohematology (LIM-31), Department of Hematology, Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil
| | - Fátima Nogueira
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil
| | - Vanderson Rocha
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil
- Laboratory of Medical Investigation in Pathogenesis and targeted therapy in Oncoimmunohematology (LIM-31), Department of Hematology, Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil
- Disciplina de Ciências Médicas, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil
- Churchill Hospital, Oxford University, Oxford, UK
| | - Alfredo Mendrone-Júnior
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil
- Laboratory of Medical Investigation in Pathogenesis and targeted therapy in Oncoimmunohematology (LIM-31), Department of Hematology, Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil
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Floridia V, Sfulcini M, D’Alessandro E, Cattaneo L, Mezzetti M, Liotta L, Trevisi E, Lopreiato V, Minuti A. Effect of Different Anticoagulant Agents on Immune-Related Genes in Leukocytes Isolated from the Whole-Blood of Holstein Cows. Genes (Basel) 2023; 14:406. [PMID: 36833333 PMCID: PMC9957540 DOI: 10.3390/genes14020406] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2023] [Revised: 01/30/2023] [Accepted: 02/02/2023] [Indexed: 02/08/2023] Open
Abstract
Anticoagulants, such as ethylenediaminetetraacetic acid (EDTA), sodium citrate (Na-citrate), or heparin are normally used in hematological clinical tests to prevent coagulation. Although anticoagulants are fundamental for the correct application of clinical tests, they produce adverse effects in different fields, such as those involving specific molecular techniques; for instance, quantitative real time polymerase chain reactions (qPCR) and gene expression evaluation. For this reason, the aim of this study was to evaluate the expression of 14 genes in leukocytes that were isolated from the blood of Holstein cows, and which were collected in Li-heparin, K-EDTA, or Na-citrate tubes; then, they were analyzed using qPCR. Only the SDHA gene showed a significant dependence (p ≤ 0.05) on the anticoagulant that was used with the lowest expression; this was observed in Na-Citrate after being compared with Li-heparin and K-EDTA (p < 0.05). Although a variation in transcript abundance with the three anticoagulants was observed in almost all the investigated genes, the relative abundance levels were not statistically significant. In conclusion, the qPCR results were not influenced by the presence of the anticoagulant; thus, we had the opportunity to choose the test tube that was used in the experiment without interfering effects impacting the gene expression levels caused by the anticoagulant.
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Affiliation(s)
- Viviana Floridia
- Department of Veterinary Sciences, Università di Messina, Viale Palatucci, 13, 98168 Messina, Italy
| | - Marta Sfulcini
- Department of Animal Sciences, Food and Nutrition, Faculty of Agriculture, Food and Environmental Science, Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy
| | - Enrico D’Alessandro
- Department of Veterinary Sciences, Università di Messina, Viale Palatucci, 13, 98168 Messina, Italy
| | - Luca Cattaneo
- Department of Animal Sciences, Food and Nutrition, Faculty of Agriculture, Food and Environmental Science, Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy
| | - Matteo Mezzetti
- Department of Animal Sciences, Food and Nutrition, Faculty of Agriculture, Food and Environmental Science, Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy
| | - Luigi Liotta
- Department of Veterinary Sciences, Università di Messina, Viale Palatucci, 13, 98168 Messina, Italy
| | - Erminio Trevisi
- Department of Animal Sciences, Food and Nutrition, Faculty of Agriculture, Food and Environmental Science, Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy
| | - Vincenzo Lopreiato
- Department of Veterinary Sciences, Università di Messina, Viale Palatucci, 13, 98168 Messina, Italy
| | - Andrea Minuti
- Department of Animal Sciences, Food and Nutrition, Faculty of Agriculture, Food and Environmental Science, Università Cattolica del Sacro Cuore, 29122 Piacenza, Italy
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Chia CT, Bender AT, Lillis L, Sullivan BP, Martin CD, Burke W, Landis C, Boyle DS, Posner JD. Rapid detection of hepatitis C virus using recombinase polymerase amplification. PLoS One 2022; 17:e0276582. [PMID: 36282844 PMCID: PMC9595512 DOI: 10.1371/journal.pone.0276582] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2022] [Accepted: 10/11/2022] [Indexed: 11/06/2022] Open
Abstract
Over 71 million people are infected with hepatitis C virus (HCV) worldwide, and approximately 400,000 global deaths result from complications of untreated chronic HCV. Pan-genomic direct-acting antivirals (DAAs) have recently become widely available and feature high cure rates in less than 12 weeks of treatment. The rollout of DAAs is reliant on diagnostic tests for HCV RNA to identify eligible patients with viremic HCV infections. Current PCR-based HCV RNA assays are restricted to well-resourced central laboratories, and there remains a prevailing clinical need for expanded access to decentralized HCV RNA testing to provide rapid chronic HCV diagnosis and linkage to DAAs in outpatient clinics. This paper reports a rapid, highly accurate, and minimally instrumented assay for HCV RNA detection using reverse transcription recombinase polymerase amplification (RT-RPA). The assay detects all HCV genotypes with a limit of detection of 25 copies per reaction for genotype 1, the most prevalent in the United States and worldwide. The clinical sensitivity and specificity of the RT-RPA assay were both 100% when evaluated using 78 diverse clinical serum specimens. The accuracy, short runtime, and low heating demands of RT-RPA may enable implementation in a point-of-care HCV test to expand global access to effective treatment via rapid chronic HCV diagnosis.
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Affiliation(s)
- Catherine T. Chia
- Department of Biochemistry, University of Washington, Seattle, Washington, United States of America
| | - Andrew T. Bender
- Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America
| | | | - Benjamin P. Sullivan
- Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America
| | - Coleman D. Martin
- Department of Chemical Engineering, University of Washington, Seattle, Washington, United States of America
| | - Wynn Burke
- Department of Medicine, Division of Gastroenterology, University of Washington, Seattle, Washington, United States of America
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, United States of America
| | - Charles Landis
- Department of Medicine, Division of Gastroenterology, University of Washington, Seattle, Washington, United States of America
| | | | - Jonathan D. Posner
- Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America
- Department of Chemical Engineering, University of Washington, Seattle, Washington, United States of America
- Family Medicine, School of Medicine, University of Washington, Seattle, Washington, United States of America
- * E-mail:
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Mousavi SM, Hashemi SA, Kalashgrani MY, Gholami A, Omidifar N, Babapoor A, Vijayakameswara Rao N, Chiang WH. Recent Advances in Plasma-Engineered Polymers for Biomarker-Based Viral Detection and Highly Multiplexed Analysis. BIOSENSORS 2022; 12:286. [PMID: 35624587 PMCID: PMC9138656 DOI: 10.3390/bios12050286] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/30/2022] [Revised: 04/22/2022] [Accepted: 04/27/2022] [Indexed: 05/07/2023]
Abstract
Infectious diseases remain a pervasive threat to global and public health, especially in many countries and rural urban areas. The main causes of such severe diseases are the lack of appropriate analytical methods and subsequent treatment strategies due to limited access to centralized and equipped medical centers for detection. Rapid and accurate diagnosis in biomedicine and healthcare is essential for the effective treatment of pathogenic viruses as well as early detection. Plasma-engineered polymers are used worldwide for viral infections in conjunction with molecular detection of biomarkers. Plasma-engineered polymers for biomarker-based viral detection are generally inexpensive and offer great potential. For biomarker-based virus detection, plasma-based polymers appear to be potential biological probes and have been used directly with physiological components to perform highly multiplexed analyses simultaneously. The simultaneous measurement of multiple clinical parameters from the same sample volume is possible using highly multiplexed analysis to detect human viral infections, thereby reducing the time and cost required to collect each data point. This article reviews recent studies on the efficacy of plasma-engineered polymers as a detection method against human pandemic viruses. In this review study, we examine polymer biomarkers, plasma-engineered polymers, highly multiplexed analyses for viral infections, and recent applications of polymer-based biomarkers for virus detection. Finally, we provide an outlook on recent advances in the field of plasma-engineered polymers for biomarker-based virus detection and highly multiplexed analysis.
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Affiliation(s)
- Seyyed Mojtaba Mousavi
- Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei City 106335, Taiwan;
| | - Seyyed Alireza Hashemi
- Nanomaterials and Polymer Nanocomposites Laboratory, School of Engineering, University of British Columbia, Kelowna, BC V1V 1V7, Canada;
| | - Masoomeh Yari Kalashgrani
- Biotechnology Research Center, Shiraz University of Medical Sciences, Shiraz 71468-64685, Iran; (M.Y.K.); (A.G.)
| | - Ahmad Gholami
- Biotechnology Research Center, Shiraz University of Medical Sciences, Shiraz 71468-64685, Iran; (M.Y.K.); (A.G.)
| | - Navid Omidifar
- Department of Pathology, Shiraz University of Medical Sciences, Shiraz 71468-64685, Iran;
| | - Aziz Babapoor
- Department of Chemical Engineering, University of Mohaghegh Ardabil, Ardabil 56199-11367, Iran;
| | - Neralla Vijayakameswara Rao
- Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei City 106335, Taiwan;
| | - Wei-Hung Chiang
- Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei City 106335, Taiwan;
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9
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Nishiya AS, Salles NA, de Almeida-Neto C, Witkin SS, Ferreira SC, Nogueira FAH, Facincani T, Rocha V, Mendrone-Jr A. Influence of unreported HIV prophylaxis on the kinetics of post-blood donation HIV seroconversion. Transfusion 2021; 61:3488-3492. [PMID: 34617611 DOI: 10.1111/trf.16698] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2021] [Revised: 09/21/2021] [Accepted: 09/23/2021] [Indexed: 02/06/2023]
Abstract
BACKGROUND In 2020, of 110,000 blood donors screened for HIV exposure two individuals were identified who were viral RNA-positive but seronegative. One of the donors, borderline negative in a pooled screening test for HIV RNA, utilized antiretroviral drugs as post-exposure, pre-donation prophylaxis. The kinetics of subsequent HIV seropositivity in both donors are described. STUDY DESIGN AND METHODS Both donors were recalled and interviewed, and blood was obtained at intervals for HIV antibodies and RNA testing. RESULTS One donor used antiretroviral prophylaxis for 30 days due to a relationship with an HIV-positive partner. In follow-up samples, seroconversion was noted at 70 days, and viral RNA was detected at 105 days, after blood donation. In contrast, the other donor seroconverted in <25 days and the appearance and titer of HIV RNA was in accordance with the typical pre-seroconversion window. CONCLUSION The use of anti-viral prophylaxis by blood donors in the acute phase of HIV infection delays seroconversion. A 6-month deferral in blood donation after HIV prophylaxis, as currently recommended in Brazil, would have been sufficient in this case to mitigate the risk of transfusion-transmitted HIV. Ultimately, improvement in donor compliance with selection procedures for blood donation is needed to optimize blood safety.
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Affiliation(s)
- Anna S Nishiya
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil.,Laboratory of Medical Investigation in Pathogenesis and targeted therapy in Oncoimmunohematology (LIM-31), Department of Hematology, Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil
| | - Nanci A Salles
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil
| | - Cesar de Almeida-Neto
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil.,Disciplina de Ciências Médicas, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil
| | - Steven S Witkin
- Instituto de Medicina Tropical, Universidade de São Paulo, São Paulo, Brazil.,Department of Obstetrics and Gynecology, Weill Cornell Medicine, New York, New York, USA
| | - Suzete C Ferreira
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil.,Laboratory of Medical Investigation in Pathogenesis and targeted therapy in Oncoimmunohematology (LIM-31), Department of Hematology, Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil
| | | | - Tila Facincani
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil
| | - Vanderson Rocha
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil.,Laboratory of Medical Investigation in Pathogenesis and targeted therapy in Oncoimmunohematology (LIM-31), Department of Hematology, Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.,Disciplina de Ciências Médicas, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil.,Churchill Hospital, Oxford University, Oxford, UK
| | - Alfredo Mendrone-Jr
- Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil.,Laboratory of Medical Investigation in Pathogenesis and targeted therapy in Oncoimmunohematology (LIM-31), Department of Hematology, Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil
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10
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Jarchi M, Bokharaei-Salim F, Esghaei M, Kiani SJ, Jahanbakhsh F, Monavari SH, Ataei-Pirkooh A, Marjani A, Keyvani H. The Frequency of HIV-1 Infection in Iranian Children and Determination of the Transmitted Drug Resistance in Treatment-Naïve Children. Curr HIV Res 2021; 17:397-407. [PMID: 31702525 DOI: 10.2174/1570162x17666191106111211] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Revised: 10/23/2019] [Accepted: 10/31/2019] [Indexed: 12/23/2022]
Abstract
BACKGROUND The advent of resistance-associated mutations in HIV-1 is a barrier to the success of the ARTs. OBJECTIVE In this study, the abundance of HIV-1 infection in Iranian children, and also detection of the TDR in naïve HIV-1 infected pediatric (under 12 years old) were evaluated. MATERIALS From June 2014 to January 2019, a total of 544 consecutive treatment-naïve HIV-1- infected individuals enrolled in this study. After RNA extraction, amplification, and sequencing of the HIV-1 pol gene, the DRM and phylogenetic analysis were successfully performed on the plasma specimens of the ART-naïve HIV-1-infected-children under 12 years old. The DRMs were recognized using the Stanford HIV Drug Resistance Database. RESULTS Out of the 544 evaluated treatment-naïve HIV-1-infected individuals, 15 (2.8%) cases were children under 12 years old. The phylogenetic analyses of the amplified region of pol gene indicated that all of the 15 HIV-1-infected pediatric patients were infected by CRF35_AD, and a total of 13.3% (2/15) of these children were infected with HIV-1 variants with SDRMs (one child harbored two related SDRMs [D67N, V179F], and another child had three related SDRMs [M184V, T215F, and K103N]), according to the last algorithm of the WHO. No PIs-related SDRMs were observed in HIV-1-infected children. CONCLUSION The current study demonstrated that a total of 13.3% of treatment-naïve HIV-1-infected Iranian pediatrics (under 12 years old) were infected with HIV-1 variants with SDRMs. Therefore, it seems that screening to recognize resistance-associated mutations before the initiation of ARTs among Iranian children is essential for favorable medication efficacy and dependable prognosis.
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Affiliation(s)
- Maryam Jarchi
- Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Farah Bokharaei-Salim
- Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Maryam Esghaei
- Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Seyed Jalal Kiani
- Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | | | | | - Angila Ataei-Pirkooh
- Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Arezoo Marjani
- Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Hossein Keyvani
- Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
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11
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Salles NA, Nishiya AS, Ferreira SC, Rocha VG, Mendrone-Junior A. Detection of HIV-1 infections in blood donors during the pre-seroconversion window period in São Paulo, Brazil. Rev Soc Bras Med Trop 2019; 52:e20180432. [DOI: 10.1590/0037-8682-0432-2018] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2018] [Accepted: 03/27/2019] [Indexed: 11/22/2022] Open
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12
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Lee H, Lee D, Park JH, Song SH, Jeong IG, Kim CS, Searson PC, Lee KH. High throughput differential identification of TMPRSS2-ERG fusion genes in prostate cancer patient urine. Biomaterials 2017; 135:23-29. [DOI: 10.1016/j.biomaterials.2017.04.049] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2017] [Revised: 04/26/2017] [Accepted: 04/27/2017] [Indexed: 12/18/2022]
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13
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Assih M, Feteke L, Bisseye C, Ouermi D, Djigma F, Karou SD, Simpore J. Molecular diagnosis of the human immunodeficiency, Hepatitis B and C viruses among blood donors in Lomé (Togo) by multiplex real time PCR. Pan Afr Med J 2016; 25:242. [PMID: 28293358 PMCID: PMC5337291 DOI: 10.11604/pamj.2016.25.242.7096] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2015] [Accepted: 09/21/2016] [Indexed: 11/30/2022] Open
Abstract
This study aimed to compare the sensitivity of multiplex PCR to ELISA technique in the instantaneous detection of HBV, HCV and HIVin blood samples from donors of the National blood Transfusion Centre in Togo. A total of 440 blood samplesfrom volunteer were collected and tested by ELISA and multiplex PCR for HBV, HCV and HIV detection. Among the 440 volunteer blood donors, 83% were female and 17% were male. Age range of 20-29 years was more represented (73%). Whereas, multiplex PCR detected more cases of HBV than ELISA (50% vs 33%, P=0.0155);ELISA more detected HCV than PCR (34% vs 3%, P<0.0001) and HIV (26% vs 7%, P<0.0001). Confirming these observations our data showed that multiplex PCR was more sensitive in the detection of HBV. The sensitivity of ELISA for the detection of HCV and HIV was elevated compared to multiplex PCR. Multiplex PCR was more specific that ELISA for the detection of HCV and HIV.Interestingly, our data showed that the gender do not influenced the sensitivity of either ELISA or multiplex PCR to detect these viruses. This study showed the limit of both ELISA and multiplex PCR in the detection of HBV, HCV and HIV.
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Affiliation(s)
- Maléki Assih
- Center for Biomolecular Research Pietro Annigoni, CERBA/LABIOGENE, University of Ouagadougou, Burkina Faso
| | - Lochina Feteke
- National Center for Blood Transfusion (CNTS), Lomé, Togo
| | - Cyrille Bisseye
- Center for Biomolecular Research Pietro Annigoni, CERBA/LABIOGENE, University of Ouagadougou, Burkina Faso; Laboratory of Molecular and Cellular Biology, University of Sciences and Techniques of Masuku (USTM), Franceville, Gabon
| | - Djeneba Ouermi
- Center for Biomolecular Research Pietro Annigoni, CERBA/LABIOGENE, University of Ouagadougou, Burkina Faso
| | - Florencia Djigma
- Center for Biomolecular Research Pietro Annigoni, CERBA/LABIOGENE, University of Ouagadougou, Burkina Faso
| | - Simplice Damintoti Karou
- Center for Biomolecular Research Pietro Annigoni, CERBA/LABIOGENE, University of Ouagadougou, Burkina Faso; High School of Food and Biological Techniques (ESTBA-UL), University of Lomé, Togo
| | - Jacques Simpore
- Center for Biomolecular Research Pietro Annigoni, CERBA/LABIOGENE, University of Ouagadougou, Burkina Faso
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14
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Irshad M, Gupta P, Mankotia DS, Ansari MA. Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review. World J Gastroenterol 2016; 22:4824-4834. [PMID: 27239109 PMCID: PMC4873875 DOI: 10.3748/wjg.v22.i20.4824] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/09/2016] [Revised: 03/09/2016] [Accepted: 03/30/2016] [Indexed: 02/06/2023] Open
Abstract
The present review describes the current status of multiplex quantitative real time polymerase chain reaction (qPCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex qPCR for the detection of hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). In addition, multiplex qPCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex qPCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex qPCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process.
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MESH Headings
- DNA, Viral/blood
- DNA, Viral/genetics
- Hepatitis Viruses/classification
- Hepatitis Viruses/genetics
- Hepatitis Viruses/immunology
- Hepatitis, Viral, Human/blood
- Hepatitis, Viral, Human/diagnosis
- Hepatitis, Viral, Human/genetics
- Hepatitis, Viral, Human/immunology
- Humans
- Multiplex Polymerase Chain Reaction
- Predictive Value of Tests
- Reproducibility of Results
- Serogroup
- Serologic Tests/methods
- Serotyping
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15
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Mao X, Liu S, Yang C, Liu F, Wang K, Chen G. Colorimetric detection of hepatitis B virus (HBV) DNA based on DNA-templated copper nanoclusters. Anal Chim Acta 2016; 909:101-8. [PMID: 26851090 DOI: 10.1016/j.aca.2016.01.009] [Citation(s) in RCA: 55] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2015] [Revised: 12/31/2015] [Accepted: 01/03/2016] [Indexed: 12/18/2022]
Abstract
DNA detection plays an important role in early diagnosis of genetic disease. The conventional detection methods of DNA are based on expensive equipment, which do not meet the demands of developing countries. Thus, we developed a colorimetric method, which could be observed with naked eye and used copper nanoclusters for cost-effective. Moreover, the target of this method is the DNA in Hepatitis B virus that is one of the most popular chronic viral infections in developing countries over the past years. Our method was sensitive and the limit of detection was 12 × 10(9) molecules. Three-base-pair mismatches target DNA was detected easily. These results revealed the favorable sensitivity and selectivity of this approach. Most importantly, our method may have potential applications in correct diagnosis of genetic disease and monitoring of gene therapy in the poverty-stricken areas.
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Affiliation(s)
- Xiaoxia Mao
- Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, China; Department of Life Science, Anqing Normal University, Anqing, Anhui 246011, China
| | - Siyu Liu
- Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, China
| | - Chao Yang
- Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, China
| | - Fengzhen Liu
- Department of Oncology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China
| | - Keming Wang
- Department of Oncology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China.
| | - Guifang Chen
- Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, China.
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16
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Zhou L, Gong R, Lu X, Zhang Y, Tang J. Development of a Multiplex Real-Time PCR Assay for the Detection of Treponema pallidum, HCV, HIV-1, and HBV. Jpn J Infect Dis 2015; 68:481-7. [PMID: 25866106 DOI: 10.7883/yoken.jjid.2014.416] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Treponema pallidum, hepatitis C virus (HCV), human immunodeficiency virus (HIV)-1, and hepatitis B virus (HBV) are major causes of sexually transmitted diseases passed through blood contact. The development of a sensitive and efficient method for detection is critical for early diagnosis and for large-scale screening of blood specimens in China. This study aims to establish an assay to detect these pathogens in clinical serum specimens. We established a TaqMan-locked nucleic acid (LNA) real-time polymerase chain reaction (PCR) assay for rapid, sensitive, specific, quantitative, and simultaneous detection and identification. The copy numbers of standards of these 4 pathogens were quantified. Standard curves were generated by determining the mean cycle threshold values versus 10-fold serial dilutions of standards over a range of 10(6) to 10(1) copies/μL, with the lowest detection limit of the assay being 10(1) copies/μL. The assay was applied to 328 clinical specimens and compared with enzyme-linked immunosorbent assay (ELISA) and commercial nucleic acid testing (NAT) methods. The assay identified 39 T. pallidum-, 96 HCV-, 13 HIV-1-, 123 HBV-, 5 HBV/HCV-, 1 T. pallidum/HBV-, 1 HIV-1/HCV-, and 1 HIV-1/T. pallidum-positive specimens. The high sensitivity of the assay confers strong potential for its use as a highly reliable, cost-effective, and useful molecular diagnostic tool for large-scale screening of clinical specimens. This assay will assist in the study of the pathogenesis and epidemiology of sexually transmitted blood diseases.
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Affiliation(s)
- Li Zhou
- ABSL-III Laboratory at Center for Animal Experiment, State Key Laboratory of Virology, Wuhan University School of Medicine
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17
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Titchmarsh L, Zeh C, Verpoort T, Allain JP, Lee H. Leukodepletion as a point-of-care method for monitoring HIV-1 viral load in whole blood. J Clin Microbiol 2015; 53:1080-6. [PMID: 25428162 PMCID: PMC4365239 DOI: 10.1128/jcm.02853-14] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2014] [Accepted: 11/14/2014] [Indexed: 11/20/2022] Open
Abstract
In order to limit the interference of HIV-1 cellular nucleic acids in estimating viral load (VL), the feasibility of leukodepletion of a small whole-blood (WB) volume to eliminate only leukocyte cell content was investigated, using a selection of filters. The efficacy of leukocyte filtration was evaluated by counting, CD45 quantitative PCR, and HIV-1 DNA quantification. Plasma HIV-1 was tested by real-time reverse transcription (RT)-PCR. A specific, miniaturized filter was developed and tested for leukocyte and plasma virus retention, WB sample dilution, and filtration parameters in HIV-1-spiked WB samples. This device proved effective to retain >99.9% of white blood cells in 100 μl of WB without affecting plasma VL. The Samba sample preparation chemistry was adapted to use a leukodepleted WB sample for VL monitoring using the point-of-care Samba-1 semiautomated system. The clinical performance of the assay was evaluated by testing 207 consecutive venous EDTA WB samples from HIV-1-infected patients attending a CD4 testing clinic. Most patients were on antiretroviral treatment (ART), but their VL status was unknown. Compared to the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test, the new Samba assay had a concordance of 96.5%. The use of the Samba system with a VL test for WB might contribute to HIV-1 ART management and reduce loss-to-follow-up rates in resource-limited settings.
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Affiliation(s)
- Logan Titchmarsh
- Diagnostic Development Unit, Department of Haematology, University of Cambridge, Cambridge, United Kingdom
| | - Clement Zeh
- U.S. Centers for Disease Control and Prevention, HIV-Research Branch, Kisumu, Kenya, and Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
| | - Thierry Verpoort
- Macopharma, Department of Research and Development, Tourcoing, France
| | - Jean-Pierre Allain
- Division of Transfusion Medicine, Department of Haematology, University of Cambridge, Cambridge, United Kingdom
| | - Helen Lee
- Diagnostic Development Unit, Department of Haematology, University of Cambridge, Cambridge, United Kingdom
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18
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Wu H, Rao P, Jiang Y, Opriessnig T, Yang Z. A sensitive multiplex real-time PCR panel for rapid diagnosis of viruses associated with porcine respiratory and reproductive disorders. Mol Cell Probes 2014; 28:264-70. [DOI: 10.1016/j.mcp.2014.07.001] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2014] [Revised: 06/19/2014] [Accepted: 07/11/2014] [Indexed: 10/25/2022]
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19
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Opare-Sem O, Bedu-Addo G, Karikari P, Boateng P, Sarkodie F, Rahman R, Asenso-Mensah K, Awuah B, Osei Akoto A, Munin SA, Mensah-Acheampong F, Allain JP, Owusu-Ofori S. Fourteen-year experience of a tertiary hospital transfusion committee in West Africa. Transfusion 2014; 54:2852-62. [DOI: 10.1111/trf.12690] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2014] [Revised: 03/19/2014] [Accepted: 03/19/2014] [Indexed: 12/23/2022]
Affiliation(s)
- Ohene Opare-Sem
- Department of Medicine; Komfo Anokye Teaching Hospital; Kumasi Ghana UK
| | - George Bedu-Addo
- Department of Medicine; Komfo Anokye Teaching Hospital; Kumasi Ghana UK
| | - Patrick Karikari
- Department of Oral Health; Komfo Anokye Teaching Hospital; Kumasi Ghana UK
| | - Peter Boateng
- Transfusion Medicine Unit; Komfo Anokye Teaching Hospital; Kumasi Ghana UK
| | - Francis Sarkodie
- Transfusion Medicine Unit; Komfo Anokye Teaching Hospital; Kumasi Ghana UK
| | - Rabiniatu Rahman
- Transfusion Medicine Unit; Komfo Anokye Teaching Hospital; Kumasi Ghana UK
| | | | - Baffour Awuah
- Department of Oncology; Komfo Anokye Teaching Hospital; Kumasi Ghana UK
| | - Alex Osei Akoto
- Department of Paediatrics; Komfo Anokye Teaching Hospital; Kumasi Ghana UK
| | - S.A. Abdul Munin
- Department of Obstetrics and Gynaecology; Komfo Anokye Teaching Hospital; Kumasi Ghana UK
| | - Fred Mensah-Acheampong
- Policy, Planning, Monitoring and Evaluation Unit; Komfo Anokye Teaching Hospital; Kumasi Ghana UK
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20
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Zhou H, Chen S, Gan N, Li T, Cao Y, Jiang Q. Design of Sensitive Biocompatible Quantum-Dots Embedded in Mesoporous Silica Microspheres for the Quantitative Immunoassay of Human Immunodeficiency Virus-1 Antibodies. ELECTROANAL 2013. [DOI: 10.1002/elan.201300307] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
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Takizawa K, Nakashima T, Mizukami T, Kuramitsu M, Endoh D, Kawauchi S, Sasaki K, Momose H, Kiba Y, Mizutani T, Furuta RA, Yamaguchi K, Hamaguchi I. Degenerate polymerase chain reaction strategy with DNA microarray for detection of multiple and various subtypes of virus during blood screening. Transfusion 2013; 53:2545-55. [PMID: 23590180 DOI: 10.1111/trf.12193] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2012] [Revised: 01/26/2013] [Accepted: 01/27/2013] [Indexed: 12/18/2022]
Abstract
BACKGROUND The risk of transferring blood-borne infections during transfusion is continually increasing because of newly emerging and reemerging viruses. Development of a rapid screening method for emerging viruses that might be transmitted by transfusion is required to eliminate such pathogens during blood donor screening. Owing to increased use of human materials in organ transplants and cell therapy, the risk of donor-transmitted viral infections is also increasing. Although nucleic acid amplification technology (NAT) is dedicated to blood screening, a small, convenient detection system is needed at the laboratory and hospital level. STUDY DESIGN AND METHODS We developed a new pathogen detection system that can detect multiple viruses simultaneously, using originally designed degenerate polymerase chain reaction primers to amplify a wide range of viral genotypes. Amplified samples were identified using a DNA microarray of pathogen-specific probes. RESULTS We detected very low copy numbers of multiple subtypes of viruses, such as human hepatitis C virus (HCV), human hepatitis B virus (HBV), human parvovirus B19 (PVB19), and West Nile virus (WNV), using a single plate. We also detected all genotypes of human immunodeficiency virus (HIV) but sensitivity was less than for the other viruses. CONCLUSION We developed a microarray assay using novel primers for detection of a wide range of multiple pathogens and subtypes. Our NAT system was accurate and reliable for detection of HIV, HBV, HCV, PVB19, and WNV, with respect to specificity, sensitivity, and genotype inclusivity. Our system could be customized and extended for emerging pathogens and is suitable as a future NAT system.
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Affiliation(s)
- Kazuya Takizawa
- Department of Safety Research on Blood and Biologics, Department of Virology, Medicine, National Institute of Infectious Diseases, Tokyo, Japan; Department of Pathology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan; Department of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan; Osaka Red Cross Blood Center, Osaka, Japan; Nihon Parkerizing Hiroshima Works Co. Ltd, Hiroshima, Japan
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Hu YL, Luo FL, Fu JL, Chen TL, Wu SM, Zhou YD, Zhang XL. Early increased ficolin-2 concentrations are associated with severity of liver inflammation and efficacy of anti-viral therapy in chronic hepatitis C patients. Scand J Immunol 2013; 77:144-50. [PMID: 23298162 DOI: 10.1111/sji.12014] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2012] [Accepted: 11/28/2012] [Indexed: 12/31/2022]
Abstract
Ficolin-2 is a kind of human serum complement lectin with a structure similar to mannan-binding lectin (MBL), and it has been implicated in innate immunity. Recent studies have shown that complement pathway activation may contribute to hepatitis. However, the relationship between ficolin-2 and viral hepatitis remains largely elusive. The aim of this study was to determine the dynamics of ficolin-2 in patients with chronic hepatitis C. Forty nine patients who had not yet received therapy [24 patients with abnormal alanine aminotransferase (ALT) levels (>40 U/L) and 25 patients with normal ALT levels (≤ 40 U/L)], 28 patients with hepatitis C who received therapy for 2 weeks and 16 patients received therapy for a full month or longer were included in the study. A sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure the ficolin-2 concentrations in all serum samples of patients and 42 healthy donors. We found the concentrations of ficolin-2 were significantly higher in chronic hepatitis C patients with abnormal ALT values than in chronic hepatitis C patients with normal ALT values and healthy controls. Ficolin-2 concentrations in chronic hepatitis C patients with abnormal ALT values were positively correlated with ALT levels (P < 0.05). After therapy, the concentrations of ficolin-2 decreased and accompany with ALT and Hepatitis C virus (HCV) RNA levels. Then, we found ficolin-2 concentrations in rapid viral response (RVR) group decreased significantly (P < 0.05), while in non-RVR group, ficolin-2 decreased slightly (P > 0.05). Our findings suggest that early increased ficolin-2 is highly correlated with hepatic inflammation and rapid viral response.
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Affiliation(s)
- Y-L Hu
- State Key Laboratory of Virology, Department of Immunology, Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University School of Medicine, Wuhan, China
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Zhang J, Wu X, Yang W, Chen J, Fu F. Ultra-sensitive electrochemical detection of single nucleotide polymorphisms based on an electrically controllable magnetic gold electrode. Chem Commun (Camb) 2013; 49:996-8. [DOI: 10.1039/c2cc37783g] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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Molecular virology in transfusion medicine laboratory. BLOOD TRANSFUSION = TRASFUSIONE DEL SANGUE 2012; 11:203-16. [PMID: 23356973 DOI: 10.2450/2012.0219-12] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
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A novel multiplex real-time PCR assay for the concurrent detection of hepatitis A, B and C viruses in patients with acute hepatitis. PLoS One 2012; 7:e49106. [PMID: 23145085 PMCID: PMC3493491 DOI: 10.1371/journal.pone.0049106] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2012] [Accepted: 10/04/2012] [Indexed: 12/16/2022] Open
Abstract
A novel multiplex real-time PCR assay for concurrent detection of hepatitis viruses was evaluated for its clinical performance in screening patients with acute hepatitis. A total of 648 serum samples were collected from patients with acute symptoms of hepatitis. Concurrent detection of nucleic acids of HAV, HBV and HCV was performed using the Magicplex™ HepaTrio Real-time Detection test. Serum nucleic acid levels of HBV and HCV were also quantified by the Cobas® AmpliPrep/Cobas® TaqMan® (CAP/CTM) HBV and HCV tests. Patients’ medical records were also reviewed. Concordance rates between the results from the HepaTrio and the CAP/CTM tests for the detection of HBV and HCV were 94.9% (k = 0.88) and 99.2% (k = 0.98), respectively. The cycle threshold values with the HepaTrio test were also correlated well with the levels of HBV DNA (r = −0.9230) and HCV RNA (r = −0.8458). The sensitivity and specificity of the HepaTrio test were 93.8% and 98.2%, respectively, for detecting HBV infection, and 99.1% and 100.0%, respectively, for HCV infection. For the HepaTrio test, 21 (3.2%) cases were positive for both HBV and HCV. Among the positive cases, 6 (0.9%) were true coinfections. This test also detected 18 (2.8%) HAV positives. The HepaTrio test demonstrated good clinical performance and produced results that agreed well with those of the CAP/CTM assays, especially for the detection of HCV. This assay was also able to detect HAV RNA from anti-HAV IgM-positive individuals. Therefore, this new multiplex PCR assay could be useful for the concurrent detection of the three hepatitis viruses.
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Low cost HIV-1 quantitative RT-PCR assay in resource-limited settings: Improvement and implementation. J Virol Methods 2012; 185:118-23. [DOI: 10.1016/j.jviromet.2012.06.015] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2011] [Revised: 06/06/2012] [Accepted: 06/12/2012] [Indexed: 11/18/2022]
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27
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Wang S, Sarenac D, Chen MH, Huang SH, Giguel FF, Kuritzkes DR, Demirci U. Simple filter microchip for rapid separation of plasma and viruses from whole blood. Int J Nanomedicine 2012; 7:5019-28. [PMID: 23055720 PMCID: PMC3457680 DOI: 10.2147/ijn.s32579] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
Sample preparation is a significant challenge for detection and sensing technologies, since the presence of blood cells can interfere with the accuracy and reliability of virus detection at the nanoscale for point-of-care testing. To the best of our knowledge, there is not an existing on-chip virus isolation technology that does not use complex fluidic pumps. Here, we presented a lab-on-a-chip filter device to isolate plasma and viruses from unprocessed whole blood based on size exclusion without using a micropump. We demonstrated that viruses (eg, HIV) can be separated on a filter-based chip (2-μm pore size) from HIV-spiked whole blood at high recovery efficiencies of 89.9% ± 5.0%, 80.5% ± 4.3%, and 78.2% ± 3.8%, for viral loads of 1000, 10,000 and 100,000 copies/mL, respectively. Meanwhile, 81.7% ± 6.7% of red blood cells and 89.5% ± 2.4% of white blood cells were retained on 2 μm pore–sized filter microchips. We also tested these filter microchips with seven HIV-infected patient samples and observed recovery efficiencies ranging from 73.1% ± 8.3% to 82.5% ± 4.1%. These results are first steps towards developing disposable point-of-care diagnostics and monitoring devices for resource-constrained settings, as well as hospital and primary care settings.
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Affiliation(s)
- ShuQi Wang
- Bio-acoustic MEMS in Medicine Laboratory, Department of Medicine, Division of Biomedical Engineering, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
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Design and development of an in-house multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV in plasma samples. IRANIAN JOURNAL OF MICROBIOLOGY 2012; 52:456-63. [PMID: 22783455 DOI: 10.1007/s12088-012-0271-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/25/2011] [Accepted: 04/26/2012] [Indexed: 01/12/2023]
Abstract
BACKGROUND AND OBJECTIVES HIV-1 and HCV infections are life threatening problems in patients who receive blood products. Serological methods have proven useful in detecting these infections, but there are setbacks that make it challenging to detect these infectious agents. By the advent of Nucleic Acid Testing (NAT) methods, especially in multiplex format, more precise detection is possible. MATERIALS AND METHODS We have developed a multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV. Primers were designed for highly conserved region of genome of each virus. Using these primers and standard plasmids, we determined the limit of detection, clinical and analytical specificity and sensitivity of the assay. Monoplex and multiplex RT-PCR were performed. RESULTS Analytical sensitivity was considered to be 100 and 200 copies/ml for HIV-1 and HCV, respectively. High concentration of one virus had no significant effect on the detection of the other one with low concentration. By analysis of 40 samples, clinical sensitivity of the assay was determined to be 97.5%. Using different viral and human genome samples, the specificity of the assay was evaluated to be 100%. CONCLUSIONS The aim of this study was to develop a reliable, rapid and cost effective method to detect HIV-1 and HCV simultaneously. Results showed that this simple and rapid method is perfectly capable of detecting two viruses in clinical samples.
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Wang S, Esfahani M, Gurkan UA, Inci F, Kuritzkes DR, Demirci U. Efficient on-chip isolation of HIV subtypes. LAB ON A CHIP 2012; 12:1508-15. [PMID: 22391989 PMCID: PMC3777392 DOI: 10.1039/c2lc20706k] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/05/2023]
Abstract
HIV has caused a global pandemic over the last three decades. There is an unmet need to develop point-of-care (POC) viral load diagnostics to initiate and monitor antiretroviral treatment in resource-constrained settings. Particularly, geographical distribution of HIV subtypes poses significant challenges for POC immunoassays. Here, we demonstrated a microfluidic device that can effectively capture various subtypes of HIV particles through anti-gp120 antibodies, which were immobilized on the microchannel surface. We first optimized an antibody immobilization process using fluorescent antibodies, quantum dot staining and AFM studies. The results showed that anti-gp120 antibodies were immobilized on the microchannel surface with an elevated antibody density and uniform antibody orientation using a Protein G-based surface chemistry. Further, RT-qPCR analysis showed that HIV particles of subtypes A, B and C were captured repeatably with high efficiencies of 77.2 ± 13.2%, 82.1 ± 18.8, and 80.9 ± 14.0% from culture supernatant, and 73.2 ± 13.6, 74.4 ± 14.6 and 78.3 ± 13.3% from spiked whole blood at a viral load of 1000 copies per mL, respectively. HIV particles of subtypes A, B and C were captured with high efficiencies of 81.8 ± 9.4%, 72.5 ± 18.7, and 87.8 ± 3.2% from culture supernatant, and 74.6 ± 12.9, 75.5 ± 6.7 and 69.7 ± 9.5% from spiked whole blood at a viral load of 10,000 copies per mL, respectively. The presented immuno-sensing device enables the development of POC on-chip technologies to monitor viral load and guide antiretroviral treatment (ART) in resource-constrained settings.
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Affiliation(s)
- ShuQi Wang
- Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Harvard-MIT Health Sciences and Technology, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 65 Landsdowne St., # 267, Cambridge, MA 02139, USA
| | - Matin Esfahani
- Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Harvard-MIT Health Sciences and Technology, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 65 Landsdowne St., # 267, Cambridge, MA 02139, USA
| | - Umut A. Gurkan
- Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Harvard-MIT Health Sciences and Technology, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 65 Landsdowne St., # 267, Cambridge, MA 02139, USA
| | - Fatih Inci
- Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Harvard-MIT Health Sciences and Technology, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 65 Landsdowne St., # 267, Cambridge, MA 02139, USA
| | - Daniel R. Kuritzkes
- Section of Retroviral Therapeutics, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
| | - Utkan Demirci
- Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Harvard-MIT Health Sciences and Technology, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 65 Landsdowne St., # 267, Cambridge, MA 02139, USA
- Harvard-MIT Health Sciences and Technology, Cambridge, MA 02139, USA
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Freimanis GL, Loua A, Allain JP. HIV-1 subtypes D and F are prevalent in Guinea Conakry. J Clin Virol 2012; 53:350-3. [PMID: 22269393 DOI: 10.1016/j.jcv.2011.12.026] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2011] [Revised: 12/15/2011] [Accepted: 12/22/2011] [Indexed: 11/17/2022]
Abstract
BACKGROUND Limited data is available upon the distribution of different HIV-1/2 genotypes in the blood donor population from Guinea Conakry. OBJECTIVES To investigate the prevalence of HIV-1/2 subtypes in asymptomatic blood donors in Guinea Conakry, in order to update knowledge of HIV-1/2 epidemiology within this country. STUDY DESIGN Samples from 104 blood donors seropositive for HIV-1/2 were tested for HIV-1 by real-time RT-PCR. Those negative for HIV-1 were tested with HIV-2 nested RT-PCR. Positive samples were further amplified in the HIV-1 gag and pol regions and sequenced. Subtypes were determined by phylogenetic analysis on amplicon sequences. RESULTS 61 samples were positive by HIV-1 real-time RT-PCR. Of the 43 negative, 2 (4.6%) were positive for HIV-2. 52/61 (85.3%) samples were positive by nested RT-PCR. Of the 52, 43 (70.5%) and 31(59.6%) sequences were obtained in the gag and pol regions, respectively; 23 for both regions. HIV-1 subtype distribution was 1 B (2.1%), 8 F (17%), 8 D (17%) and 28 CRF02_AG (59.6%) with 2 unclassified recombinants (4.3%). Unique clusters for subtype D and F distinguished Guinea from HIV-1 subtype distribution in neighboring countries. CONCLUSIONS Subtype F and subtype D strains, uncommon in West Africa, are a substantial part of HIV-1 epidemiology in Guinea.
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Affiliation(s)
- G L Freimanis
- Division of Transfusion Medicine, Dept of Haematology, University of Cambridge, Cambridge Blood Centre, Cambridge CB2 2PT, UK
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31
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Daly GM, Bexfield N, Heaney J, Stubbs S, Mayer AP, Palser A, Kellam P, Drou N, Caccamo M, Tiley L, Alexander GJM, Bernal W, Heeney JL. A viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing. PLoS One 2011; 6:e28879. [PMID: 22216131 PMCID: PMC3244418 DOI: 10.1371/journal.pone.0028879] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2011] [Accepted: 11/16/2011] [Indexed: 12/22/2022] Open
Abstract
Here we describe a virus discovery protocol for a range of different virus genera, that can be applied to biopsy-sized tissue samples. Our viral enrichment procedure, validated using canine and human liver samples, significantly improves viral read copy number and increases the length of viral contigs that can be generated by de novo assembly. This in turn enables the Illumina next generation sequencing (NGS) platform to be used as an effective tool for viral discovery from tissue samples.
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Affiliation(s)
- Gordon M. Daly
- Department of Veterinary Medicine, The University of Cambridge, Cambridge, United Kingdom
| | - Nick Bexfield
- Department of Veterinary Medicine, The University of Cambridge, Cambridge, United Kingdom
| | - Judith Heaney
- Department of Veterinary Medicine, The University of Cambridge, Cambridge, United Kingdom
| | - Sam Stubbs
- Department of Veterinary Medicine, The University of Cambridge, Cambridge, United Kingdom
| | - Antonia P. Mayer
- Department of Veterinary Medicine, The University of Cambridge, Cambridge, United Kingdom
| | - Anne Palser
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom
| | - Paul Kellam
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom
| | - Nizar Drou
- The Genome Analysis Centre, Norwich Research Park, Norwich, United Kingdom
| | - Mario Caccamo
- The Genome Analysis Centre, Norwich Research Park, Norwich, United Kingdom
| | - Laurence Tiley
- Department of Veterinary Medicine, The University of Cambridge, Cambridge, United Kingdom
| | - Graeme J. M. Alexander
- Department of Hepatology, Addenbrookes Hospital, Cambridge University Hospitals National Health Services Foundation Trust, Cambridge, United Kingdom
| | - William Bernal
- Institute of Liver Studies, King's College London School of Medicine, King's College Hospital, Denmark Hill, London, United Kingdom
| | - Jonathan L. Heeney
- Department of Veterinary Medicine, The University of Cambridge, Cambridge, United Kingdom
- * E-mail:
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Wang QQ, Zhang J, Hu JS, Chen HT, Du L, Wu LQ, Ding YZ, Xiong SH, Huang XC, Zhang YH, Liu YS. Rapid detection of hepatitis C virus RNA by a reverse transcription loop-mediated isothermal amplification assay. ACTA ACUST UNITED AC 2011; 63:144-7. [PMID: 21635570 DOI: 10.1111/j.1574-695x.2011.00828.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The usefulness of reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the rapid diagnosis of hepatitis C virus (HCV) RNA was evaluated. This assay showed higher sensitivities than that of nested RT-PCR, with a detection limit of 600 IU mL(-1) , and no cross-reactivity was observed with hepatitis A virus, hepatitis B virus and hepatitis E virus. Furthermore, 106 stored sera from recently diagnosed cases were retrospectively investigated with real-time RT-PCR, the nested RT-PCR, in parallel with this new assay. The general detection rates of HCV RT-LAMP, real-time PCR and the nested RT-PCR for 106 stored sera samples were 95%, 96% and 88%, respectively. This study provides the first data on the usefulness of HCV RT-LAMP in the diagnosis of HCV RNA, especially in the early clinical diagnosis of acute HCV infection.
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Affiliation(s)
- Qin-qin Wang
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China
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33
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Sabino EC, Salles NA, de Almeida-Neto C, Barreto AM, Basques F, Barros EA, Mendrone A, Busch MP, NHLBI Retrovirus Epidemiology Donor Study-II (REDS-II) and International Component. Performance of parallel screening of Brazilian blood donors with two human immunodeficiency virus immunoassays: implications for sequential immunoassay testing algorithms in other countries. Transfusion 2011; 51:175-83. [PMID: 20633245 PMCID: PMC3019293 DOI: 10.1111/j.1537-2995.2010.02773.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
BACKGROUND In Brazil it is mandatory to screen donors for human immunodeficiency virus (HIV) antibodies using two immunoassays (IAs) in parallel. Confirmatory testing is performed only on reactive donors who return for counseling. The goal of this analysis was to determine if concordant IA reactivity accurately predicts infection and can be used for HIV incidence and/or prevalence analyses. STUDY DESIGN AND METHODS We reviewed HIV screening and confirmatory results obtained for 307,407 donations in the first year of the REDS-II study in Brazil (2007) and for 2,304,755 donations collected from 1996 to 2006 in one of the REDS-II sites (São Paulo, Brazil). RESULTS In the São Paulo site, 11,410 (0.50%) HIV IA-reactive donations were discarded, but only 2095 (0.09%) were reactive to both IAs. Western blot was positive on 1002 (48%) dual-IA-reactive donors who returned for counseling. Only four HIV-infected donors were detected who had been missed at screening by one of the IAs; all occurred before 2002. The positive predictive value (PPV) of dual-IA reactivity varied from 45.8 to 100%, with 80% to 90% PPVs when using IAs from different manufacturers. If both assays yielded signal-to-cutoff (S/C) values of 3.0 or more, PPVs ranged from 91% to 99%, with approximately 99% sensitivity for true HIV seropositivity. CONCLUSION Parallel testing of all donations has limited efficacy when highly sensitive IAs are used. Reactivity by two sequential IAs is useful for prevalence studies if the assays are from different manufacturers and especially if high S/C values are considered.
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Affiliation(s)
- Ester C Sabino
- Fundação Pró-Sangue/Hemocentro de São Paulo, São Paulo, Brazil.
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Foglieni B, Candotti D, Guarnori I, Raffaele L, Berzuini A, Spreafico M, Orani A, Rossotti R, Rossi D, Allain JP, Prati D. A cluster of human immunodeficiency virus Type 1 recombinant form escaping detection by commercial genomic amplification assays. Transfusion 2010; 51:719-30. [PMID: 21087286 DOI: 10.1111/j.1537-2995.2010.02942.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
BACKGROUND Nucleic acid testing (NAT)-based methods for the detection and quantification of human immunodeficiency virus Type 1 (HIV-1) RNA are used to increase transfusion safety and to diagnose and manage HIV-1-infected patients. We describe a novel HIV-1 recombinant form associated with lack of reactivity or substantial underestimation of viral load by commercial NAT assays. STUDY DESIGN AND METHODS We observed a repeat blood donor seroconverting to anti-HIV in whom HIV RNA was initially undetectable with routine NAT was observed. During donor follow-up, HIV RNA became detectable, but the viral load was 2 to 3 log lower than measured with other NATs targeting different genome regions. Genome sequencing revealed a novel B/F recombinant with mutations affecting primers and probe annealing accounting for the poor performance of routine NAT. A total of 553 HIV-1-infected patients attending the hospital clinic were subsequently tested prospectively using the routine assay and an in-house assay specifically designed to detect the B/F strains. RESULTS The routine assay substantially underestimated viremia (1-5 log) in 19 cases (3.5%), 11 (58%) of which were infected with the same B/F strain observed in the index donor samples. Two other non-B circulating recombinant forms of HIV-1 (A/G, B/G subtypes) were identified as poorly detected. Newly introduced NATs targeting two HIV-1 regions improved assay performance. CONCLUSION HIV-1 increasing heterogeneity affects the efficiency of NATs and consequently the safety of the blood supply as well as diagnosis and patient management.
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Affiliation(s)
- Barbara Foglieni
- Department of Transfusion Medicine and Hematology and Infectious Diseases Unit, Ospedale A. Manzoni, Lecco, Italy.
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Lefrère JJ, Dahourouh H, Dokekias AE, Kouao MD, Diarra A, Diop S, Tapko JB, Murphy EL, Laperche S, Pillonel J. Estimate of the residual risk of transfusion-transmitted human immunodeficiency virus infection in sub-Saharan Africa: a multinational collaborative study. Transfusion 2010; 51:486-92. [DOI: 10.1111/j.1537-2995.2010.02886.x] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Abstract
The high prevalence of numerous endemic and epidemic diseases such as malaria, HIV infection and viral hepatitis in some areas of sub-Saharan Africa (SSA) affects the health status of blood donors. Considering the difficulties in ensuring sufficient and safe blood supply, analysing epidemiological factors that impact blood donors in this community may further bring light on issues of supply and safety, and help in planning for its rational use. This review does not aim to propose new strategies but describes the main characteristics of blood donors in SSA as collected from different reports. Data were mainly obtained from the reports of the World Health Organization and national blood transfusion programmes and also from relevant literature and conference reports. Several characteristics are common in blood donors, such as the predominance of young adult males, the high frequency of Transmission-transmitted Infections (TTIs) and some erythrocytic phenotypes. The data indicate variations in the level of improvement of blood collection and blood safety from one area to another, particularly in the field of donor motivation or screening strategies for TTIs. These data could be useful to supplement previous reports and to provide updates for governments and international organizations' programs involved in the improvement of blood safety in Africa.
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Affiliation(s)
- C T Tagny
- University Teaching Hospital of Yaoundé, Cameroon.
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Perandin F, Pollara PC, Gargiulo F, Bonfanti C, Manca N. Performance evaluation of the automated NucliSens easyMAG nucleic acid extraction platform in comparison with QIAamp Mini kit from clinical specimens. Diagn Microbiol Infect Dis 2009; 64:158-65. [PMID: 19500527 DOI: 10.1016/j.diagmicrobio.2009.02.013] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2008] [Revised: 02/16/2009] [Accepted: 02/19/2009] [Indexed: 11/30/2022]
Abstract
The performance of the NucliSens easyMAG platform for the extraction of nucleic acid from different clinical specimens was compared with a manual procedure. A total of 308 specimens were analyzed: 209 plasma samples collected for virus detection and quantification of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) (n = 70), and 29 for HIV genotyping for drug resistance. Linearity of extraction was tested on dilution series of CMV and EBV; the correlation coefficient (R(2)) for standard curves based on repeated extraction runs was 0.99 for CMV and EBV. Inter- and intrarun variability was in accordance with previous studies, and the correlation between automated and manual extraction was very high. The concordant results were 95.7% for CMV and 100% for EBV. The results of sequence analysis for HIV drug resistance showed a concordance in 24 of 29 specimens. The NucliSens easyMAG is extremely easy to perform, is automated, and resulted in a strong reduction of hands-on time compared with manual protocol.
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Affiliation(s)
- Francesca Perandin
- Department of Experimental and Applied Medicine, University of Brescia, Italy.
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Simultaneous detection of enterovirus 70 and coxsackievirus A24 variant by multiplex real-time RT-PCR using an internal control. J Virol Methods 2009; 159:23-8. [DOI: 10.1016/j.jviromet.2009.02.022] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2008] [Revised: 02/17/2009] [Accepted: 02/17/2009] [Indexed: 11/19/2022]
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40
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Stals A, Baert L, Botteldoorn N, Werbrouck H, Herman L, Uyttendaele M, Van Coillie E. Multiplex real-time RT-PCR for simultaneous detection of GI/GII noroviruses and murine norovirus 1. J Virol Methods 2009; 161:247-53. [PMID: 19563828 DOI: 10.1016/j.jviromet.2009.06.019] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2009] [Revised: 06/14/2009] [Accepted: 06/21/2009] [Indexed: 11/15/2022]
Abstract
A quantitative two-step multiplex real-time reverse transcriptase (RT-) PCR assay for the simultaneous detection of genogroup I (GI) and genogroup II (GII) noroviruses (NoVs) is described below. A murine norovirus 1 (MNV-1) real-time PCR detection assay described recently was integrated successfully into the multiplex assay, making it possible to detect GI and GII NoVs and MNV-1 in one reaction tube with MNV-1 plasmid DNA as real-time PCR internal amplification control (IAC). The results showed a nearly complete concordance between the multiplex assay and the corresponding single-target PCRs. Analysis of competition between the individual reactions within the multiplex real-time PCR assay showed that GI and GII NoV plasmid DNAs mixed at equimolar concentrations were detected reproducibly and quantitatively, while a 4 log excess between GI and GII plasmid DNAs hindered amplification of the target with the lowest concentration. High concentrations of the real-time PCR IAC (MNV-1 plasmid DNA) also interfered with the possibility of the developed multiplex real-time RT-PCR assay to detect quantitatively and simultaneously the presence of GI and GII NoVs within one sample. The specificity of the multiplex assay was evaluated by testing a NoV RNA reference panel containing nine GI, eight GII, and one GIV in vitro synthesized RNA fragment, plus 16 clinical samples found positive for GI and GII NoVs previously. In addition, a collection of bovine NoVs and other (non-NoV) enteric viruses were found to be negative, and no cross-amplification between genogroups was observed.
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Affiliation(s)
- Ambroos Stals
- Flemish Government, Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Sciences Unit, Oost-Vlaanderen, Belgium.
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Development of a Novel, Ultra-rapid Biosensor for the Qualitative Detection of Hepatitis B Virus-associated Antigens and Anti-HBV, Based on "Membrane-engineered" Fibroblast Cells with Virus-Specific Antibodies and Antigens. SENSORS 2009; 9:2176-86. [PMID: 22574007 PMCID: PMC3345828 DOI: 10.3390/s90302176] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/03/2009] [Revised: 03/16/2009] [Accepted: 03/25/2009] [Indexed: 11/30/2022]
Abstract
A novel miniature cell biosensor detection system for the detection of Hepatis B virus (HBV)-associated antigens and anti-HBV is described. The biosensor is based on “membrane-engineered” Vero fibroblast cells immobilized in an alginate matrix. The membrane-engineering process involved the electroinsertion of anti-HBV specific antibodies (anti-HBs, anti-HBe) or antigens (HBsAg) in the membranes of the Vero cells. The attachment of a homologous antigen to the electroinserted antibody (or, respectively, of the antibody to the electroinserted antigen) triggered specific changes to the cell membrane potential that were measured by appropriate microelectrodes, according to the principle of the Bioelectric Recognition Assay (BERA). The sensor was used for screening 133 clinical blood serum samples according to a double-blind protocol. Considerably higher sensor responses were observed against HBV-positive samples, compared with responses against negative samples or samples positive for heterologous hepatitis viruses such as Hepatitis C (HCV) virus. Detection of anti-HBs antibodies was made possible by using a biosensor based on immobilized Vero cells bearing the respective antigen (HBsAg). The observed response was rapid (45 sec) and quite reproducible. Fluorescence microscopy observations showed that attachment of HBV particles to cells membrane-engineered with anti-HBs was associated with a decrease of [Ca2+]cyt. The perspectives for using the novel biosensor as a qualitative, rapid screening, high throughput assay for HBV antigens and anti-HBs in clinical samples is discussed.
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Rouet F, Ménan H, Viljoen J, Ngo-Giang-Huong N, Mandaliya K, Valéa D, Lien TX, Danaviah S, Rousset D, Ganon A, Nerrienet E. In-house HIV-1 RNA real-time RT-PCR assays: principle, available tests and usefulness in developing countries. Expert Rev Mol Diagn 2009; 8:635-50. [PMID: 18785811 DOI: 10.1586/14737159.8.5.635] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The principle of currently available licensed HIV-1 RNA assays is based on real-time technologies that continuously monitor the fluorescence emitted by the amplification products. Besides these assays, in-house quantitative (q) real-time reverse transcription (RT)-PCR (RT-qPCR) tests have been developed and evaluated particularly in developing countries, for two main reasons. First, affordable and generalized access to HIV-1 RNA viral load is urgently needed in the context of expected universal access to prevention and antiretroviral treatment programs in these settings. Second, since many non-B subtypes, circulating recombinant forms and unique recombinant forms circulate in these areas, in-house HIV-1 RNA RT-qPCR assays are ideal academic tools to thoroughly evaluate the impact of HIV-1 genetic diversity on the accuracy of HIV-1 RNA quantification, as compared with licensed techniques. To date, at least 15 distinct in-house assays have been designed. They differ by their chemistry and the HIV-1 target sequence (located in gag, Pol-IN or LTR gene). Analytical performances of the tests that have been extensively evaluated appear at least as good as (or even better than) those of approved assays, with regard to HIV-1 strain diversity. Their clinical usefulness has been clearly demonstrated for early diagnosis of pediatric HIV-1 infection and monitoring of highly active antiretroviral therapy efficacy. The LTR-based HIV-1 RNA RT-qPCR assay has been evaluated by several groups under the auspices of the Agence Nationale de Recherches sur le SIDA et les hépatites virales B et C. It exists now as a complete standardized commercial test.
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Affiliation(s)
- François Rouet
- Laboratoire de Virologie, Centre Muraz, BP390 Bobo-Dioulasso 01, Burkina Faso.
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Davis C, Berry N, Heath A, Holmes H. An international collaborative study to establish a replacement World Health Organization International Standard for human immunodeficiency virus 1 RNA nucleic acid assays. Vox Sang 2008; 95:218-25. [PMID: 19121186 DOI: 10.1111/j.1423-0410.2008.01086.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
BACKGROUND AND OBJECTIVES An international collaborative study was undertaken to identify a replacement for the World Health Organization (WHO) 1st International Standard for human immunodeficiency virus 1 (HIV-1) RNA for use in nucleic acid-based techniques (NAT) (code 97/656). In the original study to establish the 1st International Standard, a second candidate material (code 97/650) had been shown to perform well and this was re-evaluated to establish whether it would be a suitable replacement. MATERIALS AND METHODS Eight laboratories from six different countries participated in the collaborative study to evaluate the candidate replacement standard. A total of eight different NATs were used, five in a quantitative format and three qualitative, of which five were commercially available. RESULTS The results showed that the estimates of RNA copies in the current study were generally in line with those of the original study and there was no evidence of any drift in overall levels expressed in International Units (IU) for the candidate standard between the two studies. Furthermore, it was shown to be stable over long-term storage at -20 degrees C. CONCLUSIONS The candidate material code 97/650 was established by the WHO as the 2nd International Standard for HIV-1 RNA for use in NAT and assigned a unitage of 5.56 log(10) (363 078) IU/vial.
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Affiliation(s)
- C Davis
- Divisions of Retrovirology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, UK
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Tagny CT, Mbanya D, Tapko JB, Lefrère JJ. Blood safety in Sub-Saharan Africa: a multi-factorial problem. Transfusion 2008; 48:1256-61. [PMID: 18713111 DOI: 10.1111/j.1537-2995.2008.01697.x] [Citation(s) in RCA: 83] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Although the World Health Organization (WHO) has set targets for safe blood by 2012, Sub-Saharan Africa remains confronted with multi-factorial issues that compromise blood safety in most countries of the region. Some of these include the development and implementation of national policies for transfusion, the recruitment of voluntary and unpaid donors, proper screening of collected blood as well as a strategy for its rational use in a setting already plagued by a high prevalence of blood-borne agents, poverty, and sometimes organizational deficits. Furthermore, the organization of hemovigilance, as well as quality systems that could monitor transfusion practices is lacking in these settings. There is no funding and global improvement of blood safety has to be cheap to be feasible. Specific solutions for the African continent need to be developed and implemented. This paper examines the current status and difficulties of blood safety in Africa and reviews available data on transfusion medicine in the region.
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Affiliation(s)
- Claude Tayou Tagny
- University Teaching Hospital and Faculty of Medicine and Biomedical Sciences, Yaoundé, Cameroon
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Dineva MA, MahiLum-Tapay L, Lee H. Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings. Analyst 2008; 132:1193-9. [PMID: 18318279 DOI: 10.1039/b705672a] [Citation(s) in RCA: 128] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Currently available nucleic acid testing (NAT)-based assays are complex and time-consuming, and they require expensive instrumentation and dedicated laboratory spaces for sample preparation as well as for amplification and detection of the nucleic acid target. Reagents required for these tests are also expensive and must be transported and stored refrigerated or frozen. These characteristics have limited the use of such assays for point-of-care (POC) testing, especially in resource-poor settings. Efforts to develop simple and rapid NAT-based assays have focused predominantly on the amplification and detection steps, with sample preparation and nucleic acid extraction remaining the bottleneck in the development of NAT systems suitable for POC applications or resource-limited settings. A review of NAT platforms and technologies currently under development and validation for rapid field testing revealed that, in addition to requiring expensive and complex instrumentation, many of these systems also require off-line sample preparation and reagent handling. In their current format, they are therefore not appropriate for POC testing in resource-limited settings. We evaluated several commercially available technologies and procedures for the isolation of nucleic acid with the extraction of HIV-1 RNA from human plasma as a model system. Our results indicate that solid-phase extraction with silica or glass in the presence of a chaotropic salt provides the highest extraction efficiency. However, none of the existing methods and technologies is readily adaptable to a POC system. The integration of sample preparation procedures well suited to NAT-based assays in resource-limited settings therefore remains a challenge.
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Cheng D, Zhao JJ, Li N, Sun Y, Zhou YJ, Zhu Y, Tian ZJ, Tu C, Tong GZ, Qiu HJ. Simultaneous detection of Classical swine fever virus and North American genotype Porcine reproductive and respiratory syndrome virus using a duplex real-time RT-PCR. J Virol Methods 2008; 151:194-199. [PMID: 18582953 DOI: 10.1016/j.jviromet.2008.05.011] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2008] [Revised: 04/16/2008] [Accepted: 05/12/2008] [Indexed: 11/29/2022]
Abstract
Classical swine fever and porcine reproductive and respiratory syndrome are both notifiable diseases of the World Organization for Animal Health (OIE). The two diseases exhibit indistinguishable clinical symptoms and sometimes co-exist in swine herds. In this study, a duplex real-time RT-PCR for simultaneous detection of Classical swine fever virus (CSFV) and North American (NA) genotype Porcine reproductive and respiratory syndrome virus (PRRSV) based on two differently labeled TaqMan probes was developed and evaluated. The detection limit of the assay was 3.2 TCID(50) or 13 RNA copies for CSFV and 1.8 TCID(50) or 10 RNA copies for PRRSV, about 50 times more sensitive than conventional RT-PCRs. The duplex real-time RT-PCR was capable of specifically detecting different subgroups of wild-type CSFV and different strains of NA-genotype PRRSV, whereas a number of non-CSFV/PRRSV porcine viruses and bovine pestivirus were tested negative. Out of 155 field samples, 16 were tested positive for CSFV, 73 were positive for PRRSV, and 13 were co-infected with the two viruses. These results were 99.4% in agreement with those using conventional RT-PCRs. Therefore, the assay provides sensitive and simultaneous detection and differentiation of CSFV and PRRSV.
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Affiliation(s)
- Dan Cheng
- Division of Swine Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, 427 Maduan Street, Harbin 150001, Heilongjiang, China
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Compston LI, Sarkobie F, Li C, Candotti D, Opare-Sem O, Allain JP. Multiplex real-time PCR for the detection and quantification of latent and persistent viral genomes in cellular or plasma blood fractions. J Virol Methods 2008; 151:47-54. [PMID: 18479760 DOI: 10.1016/j.jviromet.2008.03.023] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2008] [Revised: 03/11/2008] [Accepted: 03/17/2008] [Indexed: 01/12/2023]
Abstract
In common with latent viruses such as herpesviruses, parvovirus B19, HBV and GBV-C are contained successfully by the immune response and persist in the host. When immune control breaks down, reactivation of both latent and persistent viruses occurs. Two multiplex assays were developed (B19, HBV, HHV-8), (EBV, CMV, VZV) for blood screening, and tested on blood donor samples from Ghana to determine baseline prevalence of viraemia in immunocompetent persons. Single-virus real-time quantitative PCR (qPCR) assays were optimised for viral load determination of positive initial screening. The qPCR method utilised was absolute quantification with external standards. Multiplex and single-virus qPCR assays had similar sensitivity, except for the B19 assay in which sensitivity was 100-fold lower. Assays were optimised for reproducibility and repeatability, with R(2) of 0.9 being obtained for most assays. With the exception of B19 and CMV, assays had 100% detection limit ranging between 10(1) and 10(2) copies, IU or arbitrary units under single-virus and multiplex assay conditions. The prevalence of viraemia was 1.6% HBV (0.8% DNA+/HBsAg-, 0.8% DNA+/HBsAg+), 0.8% parvovirus B19, and 3.3% GBV-C viraemia in the plasma fraction. The prevalence of four herpesviruses was 1.0% HHV-8, 0.85% CMV, and 8.3% EBV, and no detectable VZV viraemia.
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Affiliation(s)
- Lara Isobel Compston
- Division of Transfusion Medicine, Department of Haematology, University of Cambridge, Cambridge, UK
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Liu Z, Teng Y, Liu H, Jiang Y, Xie X, Li H, Lv J, Gao L, He J, Shi X, Tian F, Yang J, Xie C. Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay. J Virol Methods 2008; 149:103-9. [DOI: 10.1016/j.jviromet.2007.12.017] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2007] [Revised: 12/01/2007] [Accepted: 12/20/2007] [Indexed: 11/25/2022]
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An oligonucleotide microarray for multiplex real-time PCR identification of HIV-1, HBV, and HCV. Biotechniques 2008; 44:241-6, 248. [PMID: 18330353 DOI: 10.2144/000112628] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
We describe a novel microarray-based approach for simultaneous identification and quantification of human immunodeficiency virus type 1 (HIV-1) and hepatitis B and C viruses (HBV and HCV) in donor plasma specimens. The method is based on multiplex real-time RT-PCR performed within the microarray hydrogel pads. Double-stranded amplification products are simultaneously detected using nonspecific SYBR Green I dye due to the reaction run in separate pads bearing 5'-immobilized specific primers. Both the sensitivity and specificity of the assay, based on 132 blood specimens analyzed, were 100% (56, 26, and 8 specimens were seropositive to HBV HCV and HIV-1, respectively; 22 were positive to both HIV-1 and HCV and 2 positive to all three viruses; 18 samples were pathogen-negative). The dynamic range of the quantitative analysis covered a six-order interval ranging from 100 to 106 genome equivalents per assay. The 95% detection limits were 14 gEq for HIV-1, 10 gEq (1.7 IU) for HBV, and 15 gEq (7.5 IU) for HCV per assay. The proposed approach is considered to be versatile and could be adapted for simultaneous identification and quantification of numerous genetic targets.
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Multiplex RT-PCR amplification of HIV genes to create a completely autologous DC-based immunotherapy for the treatment of HIV infection. PLoS One 2008; 3:e1489. [PMID: 18231576 PMCID: PMC2211536 DOI: 10.1371/journal.pone.0001489] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2007] [Accepted: 12/13/2007] [Indexed: 11/19/2022] Open
Abstract
BACKGROUND Effective therapy for HIV-infected individuals remains an unmet medical need. Promising clinical trials with dendritic cell (DC)-based immunotherapy consisting of autologous DC loaded with autologous virus have been reported, however, these approaches depend on large numbers of HIV virions to generate sufficient doses for even limited treatment regimens. METHODOLOGY/PRINCIPAL FINDINGS The present study describes a novel approach for RT-PCR amplification of HIV antigens. Previously, RT-PCR amplification of autologous viral sequences has been confounded by the high mutation rate of the virus which results in unreliable primer-template binding. To resolve this problem we developed a multiplex RT-PCR strategy that allows reliable strain-independent amplification of highly polymorphic target antigens from any patient and requires neither viral sequence data nor custom-designed PCR primers for each individual. We demonstrate the application of our RT-PCR process to amplify translationally-competent RNA encoding regions of Gag, Vpr, Rev and Nef. The products amplified using this method represent a complex mixture of autologous antigens encoded by viral quasispecies. We further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro. CONCLUSION/SIGNIFICANCE This study describes a strategy to overcome patient to patient viral diversity enabling strain-independent RT-PCR amplification of RNAs encoding sequence divergent quasispecies of Gag, Vpr, Rev and Nef from small volumes of infectious plasma. The approach allows creation of a completely autologous therapy that does not require advance knowledge of the HIV genomic sequences, does not have yield limitations and has no intact virus in the final product. The simultaneous use of autologous viral antigens and DCs may provoke broad patient-specific immune responses that could potentially induce effective control of viral loads in the absence of conventional antiretroviral drug therapy.
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