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1.
Int J Food Microbiol ; 322: 108587, 2020 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-32203767

RESUMEN

Hepatitis E virus (HEV) is a zoonotic pathogen spreading worldwide. Pig was known as its first and main animal reservoir. In China, pork consumption is very large and the risk of potential HEV contamination should not be underestimated. The present study aims to develop a quantitative real-time reverse transcription combining recombinase polymerase amplification assay (RT-qRPA) for the rapid detection of HEV RNA presence in raw pork liver on the Jinzhou markets in China. Methods: the specific primers and probes for RT-qRPA assay were designed targeting the ORF2/3 conserved region in genotype 4 swine HEV isolate (accession no. DQ279091.2) according to the TwistDx manual instructions. The specificity, sensitivity and reproducibility evaluations of the RT-qRPA method were subsequently conducted in assessing agreement with the standard RT-qPCR method. Results: the qRPA method step exhibited the obvious time-saving advantage which worked under the isothermal condition at 39 °C within about 30 min to complete the run while the compared standard qPCR method in the same cycle took almost 60 min to do. Both methods could exclusively detect the HEV genome equivalents from the quantified HEV-VLPs spiked samples. And both methods shared the same limit of detection (LOD) that was estimated at 1.25 × 103 genome equivalents copies/g spiked sample by the probit analysis. The recovery rate of HEV-VLPs reached a range of 9.56-14.65% by the RT-qRPA method which was higher than that of 1.34-2.34% by the standard RT-qPCR method. The detected HEV RNA positive rate in the field was 1.8% (1 out of 55) by both methods under Cohen's kappa statistic accessing with perfect agreement (κ = 1.00, p < 0.0005). The viral load in positive sample detected by the RT-qRPA method was estimated at 2.2125 × 105 genome copies/g pork liver sample. Conclusions, the present reported RT-qRPA method mainly targeting genotype 4 HEV is a rapid and reliable method. Its time-saving quality offers a promising for the development of a portable tool used in the routine monitoring of HEV contamination in the field.


Asunto(s)
Microbiología de Alimentos/métodos , Virus de la Hepatitis E/aislamiento & purificación , Hígado/virología , Carne de Cerdo/virología , Carga Viral/métodos , Animales , China , Microbiología de Alimentos/normas , Genotipo , Virus de la Hepatitis E/genética , Límite de Detección , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Reproducibilidad de los Resultados , Porcinos , Factores de Tiempo , Carga Viral/normas
2.
Mol Immunol ; 116: 180-190, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31704501

RESUMEN

Infectious pancreatic necrosis virus (IPNV) and infectious hematopoietic necrosis virus (IHNV) are two common viral pathogens that cause severe economic losses in all salmonid species in culture, but especially in rainbow trout. Although vaccines against both diseases have been commercialized in some countries, no such vaccines are available for them in China. In this study, a recombinant virus was constructed using the IHNV U genogroup Blk94 virus as a backbone vector to express the antigenic gene, VP2, from IPNV via the reverse genetics system. The resulting recombinant virus (rBlk94-VP2) showed stable biological characteristics as confirmed by virus growth kinetic analyses, pathogenicity analyses, indirect immunofluorescence assays and western blotting. Rainbow trout were immunized with rBlk94-VP2 and then challenged with the IPNV ChRtm213 strain and the IHNV Sn1203 strain on day 45 post-vaccination. A significantly higher survival rate against IHNV was obtained in the rBlk94-VP2 group on day 45 post-vaccination (86%) compared with the PBS mock immunized group (2%). Additionally, IPNV loads decreased significantly in the rBlk94-VP2 immunized group in the liver (28.6-fold to 36.5-fold), anterior kidney (21.7-fold to 44.2-fold), and spleen (14.9-fold to 22.7-fold), as compared with the PBS mock control group. The mRNA transcripts for several innate and adaptive immune-related proteins (IFN-γ, IFN-1, Mx-1, CD4, CD8, IgM, and IgT) were also significantly upregulated after rBlk94-VP2 vaccination, and neutralizing antibodies against both IHNV and IPNV were induced on day 45 post-vaccination. Collectively, our results suggest that this recombinant virus could be developed as a vaccine vector to protect rainbow trout against two or more diseases, and our approach lays the foundations for developing live vaccines for rainbow trout.


Asunto(s)
Enfermedades de los Peces/inmunología , Virus de la Necrosis Hematopoyética Infecciosa/inmunología , Oncorhynchus mykiss/inmunología , Oncorhynchus mykiss/virología , Animales , Anticuerpos Antivirales/inmunología , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/virología , China , Riñón Cefálico/inmunología , Riñón Cefálico/virología , Virus de la Necrosis Pancreática Infecciosa/inmunología , Pancreatitis Aguda Necrotizante/inmunología , Pancreatitis Aguda Necrotizante/virología , Infecciones por Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/virología , Bazo/inmunología , Bazo/virología , Vacunación/métodos , Vacunas de ADN/inmunología , Carga Viral/métodos , Vacunas Virales/inmunología
3.
Microbiol Immunol ; 63(11): 449-457, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31373399

RESUMEN

Hepatitis C virus (HCV) infection is a major public health problem with about 1.75 million new HCV cases and 71 million chronic HCV infections worldwide. The study aimed to evaluate clinical, serological, molecular, and liver markers to develop a mathematical predictive model for the quantification of the HCV viral load in chronic HCV infected patients. In this cross-sectional study, blood samples were taken from 249 recently diagnosed HCV-infected subjects and were tested for liver condition, viral genotype, and HCV RNA load. Receiver operating characteristics (ROC) curves and multiple linear regression analysis were used to predict the HCV-RNA load. Genotype 3 followed by genotype 1 were the most prevalent genotypes in Mashhad, Northeastern Iran. The maximum levels of viral load were detected in the mixed genotype group, and the lowest levels in the undetectable genotype group. The log of the HCV viral load was significantly associated with thrombocytopenia and higher serum levels of alanine transaminase (ALT). In addition, the log HCV RNA was significantly higher in patients with arthralgia, fatigue, fever, vomiting, or dizziness. Moreover, genotype 3 was significantly associated with icterus. A ROC curve analysis revealed that the best cut-off points for serum levels of aspartate aminotransferase (AST), ALT, and alkaline phosphatase (ALP) were >31, >34, and ≤246 IU/L, respectively. Sensitivity, specificity, and positive predictive values for AST were 87.7%, 84.36%, and 44.6%, for ALT they were 83.51%, 81.11%, and 36%, and for ALP were 72.06%, 42.81%, and 8.3%, respectively. A mathematical regression model was developed that could estimate the HCV-RNA load. Regression model: log viral load = 7.69 - 1.01 × G3 - 0.7 × G1 + 0.002 × ALT - 0.86 × fatigue.


Asunto(s)
Hepacivirus/genética , Hepatitis C Crónica/virología , Hígado/patología , ARN Viral/sangre , Viremia/diagnóstico , Adulto , Biomarcadores/sangre , Estudios Transversales , Femenino , Genotipo , Humanos , Irán/epidemiología , Masculino , Persona de Mediana Edad , Modelos Teóricos , Pruebas Serológicas/métodos , Carga Viral/métodos
4.
Appl Microbiol Biotechnol ; 103(19): 8115-8125, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31435714

RESUMEN

Sensitive and rapid methods for determining viral contamination of water are critical, since illness can be caused by low numbers of viruses and bacterial indicators do not adequately predict viral loads. We developed novel rapid assays for detecting the viral water quality indicator human adenovirus (HAdV). A simple 15-min recombinase polymerase amplification step followed by a 5-min lateral flow detection is used. Species-specific assays were developed to discriminate HAdV A, B, C and F, and combined into a multiplex test (Ad-FAC). Species-specific assays enabled detection of 10-50 copies of the HAdV plasmid. Sample testing using methods optimised for wastewater analysis indicated the Ad-FAC assay showed 100% sensitivity and 100% specificity when compared with HAdV qPCR, with a detection limit as low as 50 gene copies. This is the first study to demonstrate the use of RPA for detecting enteric viruses in water samples, to assess virological water quality. The ability to rapidly detect enteric virus contamination of water could assist in more effective management of water safety and better protection of public health.


Asunto(s)
Adenovirus Humanos/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Carga Viral/métodos , Microbiología del Agua , Calidad del Agua , Adenovirus Humanos/genética , Sensibilidad y Especificidad , Factores de Tiempo
5.
BMJ Case Rep ; 12(8)2019 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-31451457

RESUMEN

Kaposi sarcoma (KS) is an angioproliferative disorder that is commonly associated with human herpes virus 8 as well as the HIV. In fact, KS is one of the most common AIDS-defining illnesses. KS typically presents with diffuse, violaceous cutaneous nodules, and may have concomitant visceral involvement. However, visceral involvement rarely occurs without skin manifestations. A rare case of localised bronchopulmonary KS without skin involvement is described in a patient with previously undiagnosed HIV. This atypical presentation represents a challenge for modern-day physicians in developed countries where the prevalence of AIDS-related diseases is decreasing.


Asunto(s)
Síndrome de Inmunodeficiencia Adquirida , Infecciones por VIH , Neoplasias Pulmonares , Oseltamivir/administración & dosificación , Sarcoma de Kaposi , Síndrome de Inmunodeficiencia Adquirida/diagnóstico , Síndrome de Inmunodeficiencia Adquirida/etiología , Antivirales/administración & dosificación , Lavado Broncoalveolar/métodos , Recuento de Linfocito CD4/métodos , Deterioro Clínico , Confusión/diagnóstico , Confusión/etiología , Deshidratación/complicaciones , Deshidratación/diagnóstico , Deshidratación/terapia , Diagnóstico Diferencial , Resultado Fatal , Fluidoterapia/métodos , Infecciones por VIH/complicaciones , Infecciones por VIH/diagnóstico , Infecciones por VIH/fisiopatología , Infecciones por VIH/terapia , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/fisiopatología , Masculino , Sarcoma de Kaposi/patología , Sarcoma de Kaposi/fisiopatología , Tomografía Computarizada por Rayos X/métodos , Carga Viral/métodos
6.
Diagn Microbiol Infect Dis ; 95(3): 114859, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31320237

RESUMEN

OBJECTIVE: To assess the predictive value of JC virus (JCV) PCR in cerebrospinal fluid (CSF) in the diagnosis of progressive multifocal leukoencephalopathy (PML). METHODS: We conducted a retrospective database query to identify patients with positive CSF JCV PCR. Clinical features, final diagnosis and quantitative PCR results were obtained. RESULTS: A positive CSF JCV PCR had a PPV of 10.4% for the diagnosis of PML. A weakly positive PCR had a PPV of 1.6%, whereas a moderately to highly positive PCR had a PPV of 92.3%. A PPV of 0.0% was observed in immunocompetent patients and in patients without compatible clinical or radiological features. CONCLUSIONS: A false-positive CSF JCV PCR is highly prevalent in our clinical practice. This test should be reserved for patients with a clinical suspicion of PML and the quantitative result of the PCR should be taken into account when making the diagnosis of PML.


Asunto(s)
Virus JC/aislamiento & purificación , Leucoencefalopatía Multifocal Progresiva/líquido cefalorraquídeo , Leucoencefalopatía Multifocal Progresiva/diagnóstico , Reacción en Cadena de la Polimerasa , Carga Viral/métodos , ADN Viral/sangre , ADN Viral/líquido cefalorraquídeo , ADN Viral/orina , Reacciones Falso Positivas , Humanos , Virus JC/genética , Leucoencefalopatía Multifocal Progresiva/sangre , Leucoencefalopatía Multifocal Progresiva/orina , Infecciones por Polyomavirus/líquido cefalorraquídeo , Infecciones por Polyomavirus/diagnóstico , Valor Predictivo de las Pruebas , Estudios Retrospectivos
7.
Medicine (Baltimore) ; 98(29): e16222, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-31335672

RESUMEN

RATIONALE: HIV-related lymphoma, especially non-Hodgkin lymphoma, is one of the most common malignant tumors in HIV/acquired immune deficiency syndrome (AIDS) patients. Autologous hematopoietic stem cell transplantation (AHSCT) for the patients with Burkitt lymphoma (BL) is needed to be further explored. PATIENT CONCERNS: A 57-year-old man was hospitalized with intermittent pain on upper abdomen and melena for >1 month. DIAGNOSIS: HIV antibody testing was positive. The upper gastrointestinal endoscopy was performed and histopathology and immunohistochemistry revealed BL. INTERVENTIONS: Highly effective antiretroviral therapy and sixth cycles of chemotherapy were administered, followed by autologous hematopoietic stem cell transplantation. OUTCOMES: The patient has had tumor-free survival for >6 years with normal CD4+ T cell counts and HIV viral load below the lowest detection LESSONS:: The patient was treated with AHSCT followed complete remission after chemotherapy and achieved long-term disease-free survival. AHSCT may be a promising way for clinical cure of HIV-related BL.


Asunto(s)
Terapia Antirretroviral Altamente Activa/métodos , Linfoma de Burkitt , Infecciones por VIH , Trasplante de Células Madre Hematopoyéticas/métodos , Acondicionamiento Pretrasplante/métodos , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Linfoma de Burkitt/complicaciones , Linfoma de Burkitt/patología , Linfoma de Burkitt/cirugía , Supervivencia sin Enfermedad , Endoscopía Gastrointestinal/métodos , Infecciones por VIH/complicaciones , Infecciones por VIH/diagnóstico , Infecciones por VIH/tratamiento farmacológico , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Trasplante Autólogo/métodos , Resultado del Tratamiento , Carga Viral/métodos
8.
Diagn Microbiol Infect Dis ; 95(2): 152-158, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31204110

RESUMEN

The present multicentric (n = 11 laboratories) study aimed to identify conversion factors from copies/mL to international units (IU)/mL for the normalization of HCMV DNA load using the first WHO International Standard for HCMV nucleic acid amplification techniques and to enhance interlaboratory agreement of HCMV DNA quantification methods. Study protocols for whole blood and plasma (extraction and amplification) were performed to calculate conversion factors from HCMV DNA copy number to IU. The greatest variability was observed in samples with lower HCMV concentrations (3.0 Log10) in both biological matrices. Overall, 73.1% (206/282) of whole blood and 82.2% (324/394) of plasma samples analyzed fell within an acceptable variation range (±0.5 Log10 difference). An average of 0.64 (range 0.21-1.17) was the conversion factor calculated for the HCMV whole blood panel and 0.82 (range 0.39-2.2) for the HCMV plasma panel.


Asunto(s)
Infecciones por Citomegalovirus/diagnóstico , Citomegalovirus/aislamiento & purificación , ADN Viral/sangre , Carga Viral/métodos , Carga Viral/normas , Citomegalovirus/genética , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/virología , ADN Viral/genética , Humanos , Técnicas de Amplificación de Ácido Nucleico/normas , Estándares de Referencia , Reproducibilidad de los Resultados , Organización Mundial de la Salud
9.
PLoS One ; 14(6): e0218369, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31246963

RESUMEN

BACKGROUND: Point of-care (POC) HIV-1 RNA tests which are accurate and easy to use with limited infrastructure are needed in resource-limited settings (RLS). We systematically reviewed evidence of POC test performance compared to laboratory-based HIV-1 RNA assays and the potential utility of these tests for diagnosis and care in RLS. METHODS: Studies published up to July 2018 were identified by a search of PUBMED, EMBASE, Web of Science, CINAHL and Cochrane Central Register of Controlled Trials. Studies evaluating the use of POC HIV-1 RNA testing for early infant diagnosis (EID), acute HIV infection (AHI) diagnosis, or viral load monitoring (VL), compared to centralized testing, were included. Separate search strategies were used for each testing objective. RESULTS: 197 abstracts were screened and 34 full-text articles were assessed, of which 32 met inclusion criteria. Thirty studies evaluated performance and diagnostic accuracy of POC tests compared to standard reference tests. Two of the thirty and two additional studies with no comparative testing reported on clinical utility of POC results. Five different POC tests (Cepheid GeneXpert HIV-1 Quantitative and Qualitative assays, Alere q HIV-1/2 Detect, SAMBA, Liat HIV Quant and Aptima HIV-1 Quant) were used in 21 studies of VL, 11 of EID and 2 of AHI. POC tests were easy to use, had rapid turnaround times, and comparable accuracy and precision to reference technologies. Sensitivity and specificity were high for EID and AHI but lower for VL. For VL, lower sensitivity was reported for whole blood and dried blood spots compared to plasma samples. Reported error rates for Cepheid GeneXpert Qual (2.0%-5.0%), GeneXpert Quant (2.5%-17.0%) and Alere q HIV-1/2 Detect (3.1%-11.0%) were higher than in WHO prequalification reports. Most errors resolved with retesting; however, inadequate sample volumes often precluded repeat testing. Only two studies used POC results for clinical management, one for EID and another for VL. POC EID resulted in shorter time-to-result, rapid ART initiation, and better retention in care compared to centralised testing. CONCLUSIONS: Performance of POC HIV-1 RNA tests is comparable to reference assays, and have potential to improve patient outcomes. Additional studies on implementation in limited-resources settings are needed.


Asunto(s)
Infecciones por VIH/diagnóstico , Infecciones por VIH/virología , VIH-1/genética , Pruebas en el Punto de Atención , ARN Viral , Factores de Edad , Infecciones por VIH/tratamiento farmacológico , VIH-1/clasificación , Humanos , Sistemas de Atención de Punto/normas , Pruebas en el Punto de Atención/normas , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Carga Viral/métodos
10.
Pediatrics ; 143(6)2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31101703

RESUMEN

BACKGROUND: Early HIV testing is needed for treatment success in young infants, but universal testing is expensive. In this study, we examined the feasibility of early infant HIV risk scores for targeted polymerase chain reaction (PCR) testing and early HIV diagnosis. METHODS: A cross-sectional cohort of newborns exposed to HIV was enrolled and PCR tested within 72 hours. We quantified associations between HIV infection and clinical and laboratory maternal-infant parameters by logistic regression models and determined sensitivity and specificity for derived risk scores. RESULTS: From August 2014 to December 2016, 1759 participants were enrolled. Mothers without antenatal care (5.7% [97 of 1688]) were more likely to deliver newborns who are PCR-positive (P = .0005). A total of 1 in 5 mothers (217 of 990; 21.9%) had HIV viral load (VL) >1000 copies per µL. A total of 432 of 1655 (26.1%) infants were preterm. Low birth weight was documented in 398 of 1598 (24.55%) and 13 of 31 (40.63%) newborns who are PCR-negative and -positive, respectively (P = .0329). A total of 204 of 1689 (12.08%) were growth restricted or small for gestational age, and 6 of 37 (16.22%) were PCR-positive. Symptomatic newborns frequently tested positive (P = .0042). The HIV PCR positivity rate was 2.2% (37 of 1703). Two-risk (combined 3-drug antiretroviral therapy [cART] duration, VL), 3-risk (cART duration, VL, symptomatic newborn), and 4-risk (cART duration, VL, symptomatic, small for gestational age newborn) models for HIV acquisition had predictive probability of 0.28, 0.498, and 0.57, respectively; this could guide targeted birth testing. However, using the 3- and 4-risk scores (probability 0.02 and 0.04), 20% and 24% will be missed compared with universal testing. CONCLUSIONS: Targeted newborn testing requires access to maternal VL. Even if risk models include parameters such as maternal cART history, birth weight, weeks' gestation, and symptoms, 1 in 5 newborns who are infected will not be targeted. At present, we support universal PCR testing at birth within the South African prevention of mother-to-child transmission of HIV context.


Asunto(s)
Infecciones por VIH/diagnóstico , Infecciones por VIH/genética , Transmisión Vertical de Enfermedad Infecciosa , Tamizaje Neonatal/métodos , Complicaciones Infecciosas del Embarazo/diagnóstico , Complicaciones Infecciosas del Embarazo/genética , Adulto , Estudios de Cohortes , Estudios Transversales , Diagnóstico Precoz , Femenino , Infecciones por VIH/epidemiología , Humanos , Recién Nacido , Embarazo , Complicaciones Infecciosas del Embarazo/epidemiología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Factores de Riesgo , Sudáfrica/epidemiología , Carga Viral/genética , Carga Viral/métodos
11.
Arch Virol ; 164(8): 2083-2090, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31134354

RESUMEN

Although a few studies have been done on transmissible blood-borne viral infections in high-risk groups, little attention has been given to assessing the infection status of the general population in Afghanistan. To investigate the epidemiological status in the general population, we tested the serological markers of hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis delta virus (HDV), human immunodeficiency virus 1 (HIV-1) and human T-cell leukemia virus (HTLV) infections. In total, 492 samples were selected randomly from Nangarhar, Herat, Mazar-e Sharif, Kandahar, and Kabul from subjects between 25 and 70 years old. The samples were tested for the presence of HBsAg, anti-HBs, anti-HBc, anti-HDV, anti-HCV, anti-HIV-1 and anti-HTLV I/II antibodies using chemiluminescent immunoassays on Abbott Architect automated platforms. In addition, 220 HBsAg-positive samples identified among 5897 samples from the general population of the same regions of Afghanistan were included in the study and tested for both HBsAg and anti-HDV to investigate HDV prevalence in the country. Viral loads of HBV, HCV and HDV were determined in all seropositive samples using Ampliprep/Cobas TaqMan HBV, HCV, Test Roche (CA, USA), and an in-house method, respectively. Out of 492 samples, 31 (6.3%), 136 (27.6%) and 149 (30.3%) were found to be positive for HBsAg, anti-HBs and anti-HBc, respectively. Anti-HDV positivity was detected in five (2.1%) out of 234 HBsAg-positive samples (including 14 of the randomly selected samples that were not among the 220 previously identified as HBsAg positive). Only eight out of 492 (1.6%) subjects were positive for anti-HCV antibodies. Seven out of 489 (1.4%) were positive for anti-HIV-1 antibodies, and three out of 466 cases (0.6%) were positive for anti-HTLV I/II antibodies. These results suggest that Afghanistan is an intermediate endemic region for HBV, HDV and HCV infection. The prevalence of HIV-1 seems to be significantly higher than the global prevalence and that of the eastern Mediterranean region. In addition, the HTLV I/II screening results suggest that these viruses should be monitored in Afghanistan to confirm the trend observed in the current study.


Asunto(s)
Virosis/epidemiología , Adulto , Afganistán/epidemiología , ADN Viral/genética , Femenino , Infecciones por VIH/epidemiología , Infecciones por VIH/inmunología , VIH-1/inmunología , Hepacivirus/inmunología , Anticuerpos Antihepatitis/inmunología , Hepatitis B/epidemiología , Hepatitis B/inmunología , Anticuerpos contra la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis C/epidemiología , Hepatitis C/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Hepatitis D/epidemiología , Hepatitis D/inmunología , Virus de la Hepatitis Delta/inmunología , Humanos , Masculino , Persona de Mediana Edad , Carga Viral/métodos , Virosis/inmunología
12.
PLoS Comput Biol ; 15(4): e1006849, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30978183

RESUMEN

Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4+ T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a "gold standard" for reservoir measurement, little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates-as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir-rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95% probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre- and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points.


Asunto(s)
Infecciones por VIH/virología , VIH-1 , Carga Viral/métodos , Latencia del Virus , Fármacos Anti-VIH/uso terapéutico , Teorema de Bayes , Linfocitos T CD4-Positivos/virología , Biología Computacional , Simulación por Computador , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Humanos , Leucocitos Mononucleares/virología , Funciones de Verosimilitud , Cadenas de Markov , Método de Montecarlo , Reproducibilidad de los Resultados , Carga Viral/estadística & datos numéricos , Replicación Viral
13.
BMC Womens Health ; 19(1): 47, 2019 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-30909894

RESUMEN

BACKGROUND: European guidelines for cervical cancer screening now recommend the use of clinically validated assays for high-risk HPV-DNA sequences as primary test in women older than 30 years, performed in centralized laboratories, and run on systems providing automated solutions for all steps. METHODS: We conducted a comparison study, according to the international guidelines, nested within the organized population-based cervical screening program, between the cobas 4800 and 6800 systems (Roche Diagnostics), to evaluate accuracy and reproducibility of HPV test results and laboratory workflow. In Italy implementation of HPV cervical screening is under way on a regional basis; in Veneto it started in June 2015, following a piloting phase; the assay in use in the three centralized laboratories is the cobas 4800 HPV test, run on the cobas 4800 system. Comparison of HPV results with a new version of the assay (cobas 6800/8800 HPV) run on the cobas 6800 system, and intra- and inter-reproducibility analyses have been conducted in samples collected in PreservCyt medium (Hologic) from women without and with a subsequent diagnosis of high-grade lesion. RESULTS: Samples from women older than 30 years attending organized cervical cancer screening were used. Clinical sensitivity and specificity were evaluated on 60 cases and 925 controls, respectively; intra-laboratory reproducibility and inter-laboratory agreement by the 6800 system were evaluated on 593 and 460 specimens, respectively. Our results showed a very high agreement (> 98%) for overall qualitative results between the two systems; clinical sensitivity and specificity of the HPV assay run on 6800 were non-inferior to those of the HPV assay run on 4800 (p = 0,0157 and p = 0,0056, respectively, at the recommended thresholds of 90 and 98%); kappa values of 0.967 and 0.969 were obtained for intra- and inter-laboratory reproducibility analyses in the 6800 system. The 6800 platform displayed several technological improvements over the 4800 system, with higher throughput and laboratory productivity, and lower operator's hands-on time. CONCLUSIONS: The new cobas 6800/8800 HPV assay run on the 6800 instrument is suitable for use in large centralized laboratories included within population-based cervical cancer screening programs.


Asunto(s)
Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Neoplasias del Cuello Uterino/diagnóstico , Carga Viral/métodos , Adulto , Técnicas Citológicas/normas , Detección Precoz del Cáncer/métodos , Femenino , Humanos , Italia , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular/instrumentación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
14.
Anal Chim Acta ; 1059: 86-93, 2019 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-30876636

RESUMEN

A novel, simple, and label-free amplification electrochemical impedance method for quantitative detection of Human T-lymphotropic virus types II(HTLV-II) via click chemistry-mediated of hairpin DNA probes (hairpins) with polymers was developed. The hairpins were firstly attached to the gold electrode surface by an S-Au bond, the azido terminals of hairpins were close to the electrode surface, which make it difficult to be approached. After hybridizing with HTLV-II, the hairpins were unfolded and experienced a big configuration change, which made the azido terminals of the hairpins available to conjugate with alkynyl-containing polymer, called P(DEB-DSDA), formed by 1,4-diacetylenebenzene (DEB) and 4,4'-Diazido-2,2'-stilbenedisulfonic acid disodium salt tetrahydrate (DSDA) via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). With amount of P(DEB-DSDA) conjugated with the hairpin probes via click polymerization, its electrochemical signal can have a great amplification. Under optimized experimental conditions, this new probe showed a low detection limit of 0.171 pM with a good liner in the range of 1 pM-1 nM. Meanwhile, the biosensor also exhibited good selectivity and reliability in detection of real serum samples, indicating that it has great application potential in clinical DNA diagnosis and detection.


Asunto(s)
Técnicas Biosensibles/métodos , Sondas de ADN/química , Técnicas Electroquímicas/métodos , Virus Linfotrópico T Tipo 2 Humano/aislamiento & purificación , Carga Viral/métodos , Azidas/síntesis química , Azidas/química , Sangre/virología , Química Clic , Reacción de Cicloadición , Sondas de ADN/genética , ADN Viral/genética , Secuencias Invertidas Repetidas , Límite de Detección , Hibridación de Ácido Nucleico , Polímeros/síntesis química , Polímeros/química , Estilbenos/síntesis química , Estilbenos/química
15.
Retrovirology ; 16(1): 4, 2019 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-30770748

RESUMEN

Latently infected CD4 lymphocytes preclude cure of HIV infection, even with the most effective antiretroviral therapy. The replication competent latent HIV reservoir has been quantified with the terminal dilution quantitative viral outgrowth assay, which induces virus propagation in CD4+ T cell culture supernatants following cellular activation. Efforts to improve the sensitivity of this inefficient assay have introduced more sensitive p24 ELISA and RNA PCR based endpoints, but these more sensitive endpoints have raised the question whether they are measuring induced replication competent or defective virions. Here we performed parallel terminal dilution assays with CD4 lymphocytes from subjects effectively treated with antiretroviral therapy. An HIV integrase inhibitor was incorporated into one set of parallel cultures to compare the frequency of cells that can be induced to produce virions to those that produce virus that can propagate and amplify with co-culture in permissive cells. The majority of cells that can be induced to generate virus particles are producing replication competent virus, thus justifying more sensitive and faster assays of this reservoir.


Asunto(s)
Linfocitos T CD4-Positivos/virología , Infecciones por VIH/virología , VIH/fisiología , Carga Viral/métodos , Activación Viral , Latencia del Virus , Replicación Viral , Antirretrovirales/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Humanos
16.
Diagn Microbiol Infect Dis ; 94(2): 135-139, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30777343

RESUMEN

The accurate measurement of the Epstein-Barr virus (EBV) DNA level in the blood is required for managing EBV-associated diseases. A new commercial Abbott Real-Time EBV assay, which targets the BLLF1 gene, was evaluated on 120 clinical whole blood samples and the Qnostics EBV analytical panel. The limit of detection of the assay was 48.9 IU/mL (95% confidence interval, 48.1-49.8 IU/mL). The assay was linear from 2 to 5 log10 IU/mL (R2 = 0.997). The within-run coefficients of variation (CVs) ranged from 1.68% to 4.75% and the between-run CVs ranged from 1.73% to 12.83% for samples with high, medium, and low viral loads. EBV DNA loads measured by Abbott EBV assay strongly correlated with the results quantified by another commercial Nanogen EBV Real-Time Alert Q-PCR assay (r = 0.879, P < 0.0001). The fully automated Abbott Real-Time EBV assay targeting BLLF1 reduced both hands-on time and turnaround time and demonstrated a reliable performance for EBV DNA quantification in whole blood.


Asunto(s)
Automatización de Laboratorios/métodos , Sangre/virología , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Carga Viral/métodos , ADN Viral/sangre , Herpesvirus Humano 4/genética , Humanos , Reproducibilidad de los Resultados , Proteínas Virales/genética
17.
Medicine (Baltimore) ; 98(3): e12735, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30653086

RESUMEN

BACKGROUND: Micronutrient deficiencies are common during pregnancy, especially in pregnant women from economically disadvantaged settings where diets with low content of minerals and vitamins are consumed. Selenium is a non-metallic chemical element of great importance to human health. This study will assess the effect of selenium supplementation on major pregnancy outcomes and disease progression among HIV-infected pregnant women in Lagos, Nigeria. METHODS: A randomized, double-blind, placebo-controlled trial involving confirmed HIV-positive pregnant women at the Lagos University Teaching Hospital (LUTH) between September 2018 and February 2019. Eligible participants are HIV-infected pregnant women aged 15 to 49 years and have a singleton gestation at 14 to 27 weeks' gestation. At enrolment, 90 women will be randomly assigned into each intervention arm to receive either a daily tablet of 200 µg elemental selenium or placebo. Relevant participants' data will be collected at enrolment and at delivery. Statistical analyses will be carried out using SPSS version 23.0 for Windows. The associations between any 2 groups of continuous variables will be tested using the t test or the Mann-Whitney U test and that of 2 groups of categorical variables with chi-square or Fishers exact test where appropriate. A series of multivariable analyses will also be carried out to identify and control for several possible confounders of the major pregnancy outcomes and HIV disease progression. Statistical significance will be defined as P < .05. Ethical approval for the study was obtained from the LUTH's Health Research and Ethics Committee (Approval number: ADM/DCST/HREC/APP/2438; 30th August 2018). DISCUSSION: This trial will assess the effect of selenium supplementation on pregnancy outcome and HIV disease progression among HIV-infected pregnant women in Lagos. This will help to determine if routine selenium supplementation in HIV-infected pregnant women will contribute to the improvement in the major adverse pregnancy outcomes such as preterm birth and low birth weight and the HIV disease surrogate markers such as CD4+ cells count and viral load. TRIAL REGISTRATION: PACTR, PACTR201809756724274. Registered on 3rd September 2018, https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=3571.


Asunto(s)
Complicaciones Infecciosas del Embarazo/tratamiento farmacológico , Resultado del Embarazo/epidemiología , Selenio/uso terapéutico , Oligoelementos/uso terapéutico , Adolescente , Adulto , Recuento de Linfocito CD4/métodos , Progresión de la Enfermedad , Método Doble Ciego , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , VIH-1/efectos de los fármacos , Humanos , Persona de Mediana Edad , Nigeria/epidemiología , Placebos , Embarazo , Complicaciones Infecciosas del Embarazo/epidemiología , Complicaciones Infecciosas del Embarazo/virología , Estudios Prospectivos , Selenio/administración & dosificación , Oligoelementos/administración & dosificación , Carga Viral/efectos de los fármacos , Carga Viral/métodos , Adulto Joven
18.
J Infect Dis ; 219(12): 1934-1939, 2019 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-30668796

RESUMEN

BACKGROUND: Drug-induced immunosuppression in kidney transplant recipients is crucial to prevent allograft rejection, but increases risk for infectious disease. Immunologic monitoring to tailor immunosuppressive drugs might prevent alloreactivity and adverse effects simultaneously. The apathogenic torque teno virus (TTV) reflects the immunocompetence of its host and might act as a potential candidate for a holistic monitoring. METHODS: We screened all 1010 consecutive patients from the prospective Vienna Kidney Transplant Cohort Study for availability of allograft biopsies and adequately stored sera for TTV quantification by polymerase chain reaction. RESULTS: Patients with acute biopsy-proven alloreactivity according to the Banff classification (n = 33) showed lower levels of TTV in the peripheral blood compared to patients without rejection (n = 80) at a median of 43 days before the biopsy. The risk for alloreactivity decreased by 10% per log level of TTV copies/mL (risk ratio, .90 [95% confidence interval, .84-.97]; P = .005). TTV levels >1 × 106 copies/mL exclude rejection with a sensitivity of 94%. Multivariable generalized linear modeling suggests an independent association between TTV level and alloreactivity. CONCLUSIONS: TTV is a prospective biomarker for risk stratification of acute biopsy-proven alloreactivity in kidney transplant recipients and might be a potential tool to tailor immunosuppressive drug therapy.


Asunto(s)
Infecciones por Virus ADN/etiología , Inmunosupresión/efectos adversos , Trasplante de Riñón/efectos adversos , Torque teno virus/patogenicidad , Adulto , Anciano , Biopsia , Infecciones por Virus ADN/virología , ADN Viral/genética , Femenino , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/virología , Humanos , Inmunosupresores/efectos adversos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Riesgo , Medición de Riesgo , Carga Viral/métodos
19.
Diagn Microbiol Infect Dis ; 94(2): 129-134, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30651176

RESUMEN

BK virus (BKV) nephropathy is a serious complication in renal transplant recipients due to the need for immunosuppression. Nearly 50% of renal transplant patients with BKV nephropathy experience a significant loss of function of the transplanted kidney. It is routine practice to screen renal transplant recipients regularly for BK viremia. In this study, we compared the performance of BKV quantitative polymerase chain reaction analyte specific reagents by ELITech and Luminex for measuring BKV viral load in plasma using the Roche Cobas® z480 instrument and the Luminex ARIES® platform, respectively. A total of 34 patients previously tested on the z480, with results spanning the test's linear range, were analyzed on the ARIES®. The BKV DNA copy number correlation between the 2 methods was very good, with an R2 value of 0.96. The average difference in log copy number between the 2 methods was -0.3, indicating that the ARIES® method may have slightly greater analytical sensitivity. BKV quantification results were closely matched between the 2 different methods. The workflow with the ARIES® System is greatly simplified by elimination of DNA extraction and most hands-on steps. The high degree of automation allows samples to be tested as they arrive into the laboratory, resulting in enhanced patient care due to more rapid turnaround time for results back to the ordering physician.


Asunto(s)
Virus BK/aislamiento & purificación , ADN Viral/sangre , Trasplante de Riñón/efectos adversos , Técnicas de Diagnóstico Molecular/métodos , Plasma/virología , Infecciones por Polyomavirus/diagnóstico , Carga Viral/métodos , Humanos , Huésped Inmunocomprometido , Infecciones por Polyomavirus/virología , Sensibilidad y Especificidad , Receptores de Trasplantes
20.
Int J Cancer ; 144(9): 2313-2319, 2019 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-30485420

RESUMEN

Detectable post-therapy plasma Epstein-Barr virus (EBV) DNA predicts poor survival in non-metastatic nasopharyngeal carcinoma (NPC). However, some patients subsequently experience spontaneous remission of residual EBV DNA during follow-up and it was unclear whether these patients were still at high risk of disease failure. Using the NPC database from an established big-data intelligence platform, 3269 NPC patients who had the plasma EBV DNA load measured at the end of therapy (± 1 week) were identified. In total, 93.0% (3031/3269) and 7.0% (238/3269) of patients had undetectable and detectable (> 0 copy/ml) plasma EBV DNA at the end of therapy (EBV DNAend ), respectively. Detectable EBV DNAend was a prognostic factor for poorer 3-year disease-free survival (DFS), overall survival (OS), distant metastasis-free survival (DMFS), and loco-regional recurrence-free survival (LRRFS) in both univariate and multivariate analyses. Of 238 patients with residual EBV DNAend , 192 underwent EBV DNA assay 3 months after and spontaneous remission occurred in 72.4% (139/192). However, these patients still had poorer 3-year DFS (55.1% vs. 89.8%), OS (79.1% vs. 96.2%), DMFS (68.4% vs. 94.1%) and LRRFS (84.5% vs. 95.0%) than patients with undetectable EBV DNAend (all p < 0.001). And patients with persistent detectable post-therapy EBV DNA had the worst outcomes. These results were confirmed in multivariate analysis. In conclusion, residual EBV DNA post therapy was a robust biomarker for NPC prognosis. Although residual post-therapy EBV DNA could spontaneous remit during follow-up, these patients were still at high risk of disease failure and such patients may benefit from adjuvant therapy.


Asunto(s)
ADN Viral/sangre , Infecciones por Virus de Epstein-Barr/terapia , Herpesvirus Humano 4/genética , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/terapia , Remisión Espontánea , Adulto , ADN Viral/genética , Supervivencia sin Enfermedad , Infecciones por Virus de Epstein-Barr/sangre , Femenino , Herpesvirus Humano 4/crecimiento & desarrollo , Humanos , Masculino , Persona de Mediana Edad , Carcinoma Nasofaríngeo/patología , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/virología , Carga Viral/métodos
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