A quantitative polymerase chain reaction-enzyme immunoassay for accurate measurements of human papillomavirus type 16 DNA levels in cervical scrapings

A quantitative polymerase chain reaction-enzyme immunoassay (Q-PCR-EIA) was developed to measure the amount of human papillomavirus (HPV) 16 DNA per genome equivalent in cervical scrapings. The quantitative approach was based on a combined competitive PCR for both HPV 16, using the general primer GP5+/6+ PCR, and β-globin DNA. The two competitive PCRs involve co-amplification of target sequences and exogenously added DNA constructs carrying a rearranged 30 bp sequence in the probe-binding region. The accuracy of quantification by combining the two competitive PCR assays was validated on mixtures of HPV 16 containing cervical cancer cells of CaSki and SiHa cell lines. Comparison of this fully quantitative PCR assay with two semi-quantitative HPV PCR assays on a series of crude cell suspensions from HPV 16 containing cervical scrapings revealed remarkable differences in the calculated relative HPV load between samples. We found evidence that correction for both intertube variations in PCR efficiency and number of input cells/integrity of DNA significantly influence the outcome of studies on viral DNA load in crude cell suspensions of cervical scrapings. Therefore, accurate measurements on viral DNA load in cervical scrapings require corrections for these phenomena, which can be achieved by application of this fully quantitative approach. © 1999 Cancer Research Campaig

Comparison of ELISA protocols measuring HPV16 IgG antibodies and evaluation of the association between HPV16 seropositivity and HPV DNA detection

Bien que les infections cervicales au VPH soient très courantes, la séroconversion ne se produit pas toujours. Nous avons comparé deux protocoles basés sur deux dilutions sériques pour mesurer la séroréactivité du papillomavirus humain (VPH) de type 16 et avons étudié si la présence de l'ADN du VPH était associé à la séropositivité au VPH16. Nous avons également évalué si l'association était influencée par la co-infection avec d’autres types de VPH et par la charge virale. Les données utilisées proviennent de femmes brésiliennes qui ont participées à l'étude de cohorte Ludwig-McGill portant sur l'histoire naturelle de l'infection du col de l’utérus par le VPH. Les protocoles de sérologie étaient basés sur des particules pseudo-virales (VLP) composés par les protéines L1 ou L1 et L2 qui sont, respectivement, les protéines principale et secondaire de la capside virale. Deux dilutions sériques ont aussi été utilisées, soient : 1:10 et 1:50. La séroréactivité au VPH16 a été exprimée en rapports d'absorbance normalisé (NAR). Le génotypage de l'ADN du VPH et la charge virale ont été évalués par des méthodes basées sur la PCR. La corrélation et la concordance entre les dilutions de chaque protocole (VLP L1 et L1+L2) ont été évaluées par la corrélation de Pearson (r) et la méthode de Bland-Altman, respectivement. La performance des différents protocoles a été comparée à l’aide de courbe ROC (receiver operating characteristic) en utilisant la présence de l'ADN de VPH16 comme étalon-or. La régression linéaire a été utilisée pour analyser l'association entre la séropositivité au VPH16 et la détection de l’ADN du VPH avec les deux protocoles. La présence de l’ADN du VPH a été analysée en fonction (1) des types spécifiques de VPH plus ou moins apparentés au VPH16 et (2) l’infection VPH16 détectée seule ou en co-infection avec d’autres types de VPH. Les modèles de régression linéaire présentés ci-haut ont aussi été utilisées sur l’ensemble de la cohorte testée avec le protocole VLP L1+L2 et dilution sérique 1:10. L'impact de l'âge en tant que facteur de confusion potentiel ou modificateur d'effet a été analysé dans ce modèle. Finalement, l’association entre la charge virale de VPH16 et la séroréactivité a été analysée à l’aide de la corrélation de Pearson. L’ampleur des différences de la moyenne des log10-NAR et les écart-types entre les dilutions sériques observées pour chaque protocole (VLP L1 et L1+L2) étaient, respectivement, -0,081 (0,123) et -0,026 (0,150) unités logarithmiques. Les NARs obtenus par les dilutions sériques utilisées (1:10 et 1:50) pour chaque protocole étaient fortement corrélés (r = 0,87 vs. 0,94, respectivement). Cependant, l'utilisation de VLP L1+L2 a augmenté la performance du test à détecter les anticorps IgG anti-VPH16 en particulier avec la dilution sérique 1:10 [l’aire sous la courbe ROC la plus élevée (IC 95%) = 0,7330 (0,6465 – 0,8495)]. Les modèles de régression ont montré que la séroréactivité au VPH16 n’étaient qu’associée à la présence de l’ADN du VPH16 et non pas aux autres types. Par exemple, les analyses avec le protocole VLP L1+L2 et la dilution sérique 1:10 ont montré que la séroréactivité au VPH16 était associée à la présence de l'ADN du VPH16, β (IC 95%) = 0,24 (0,14 – 0,34), et non pas aux VPH31 ou 35, β (IC 95%) = 0.03 (-0,19 – 0,25), ou VPH52, 67, 33 ou 58, β (IC 95%) = 0,15 (-0,04 – 0,34), comparativement aux femmes infectées par tout autre type de VPH ou négative. Les analyses sur la cohorte entière avec le même protocole ont aussi montré que l’association entre la séroréactivité et l’ADN du VPH16 était similaire quand l’infection était présente seule ou en co-infection, β (IC 95%) = 0,14 (0,07 – 0,21) et β (IC 95%) = 0,11 (0,01 – 0,21), respectivement, comparativement à celles infectées par tout autre type de VPH ou négative. L’âge n’a pas été un facteur de confusion important et n’a pas été un modificateur d’effet dans l'analyse de l'ensemble de la cohorte. La charge virale du VPH16 n’a pas été corrélée avec la séroréactivité du VPH16, r (95% IC) = -0,04 (-0,34 – 0,27); β (IC 95%) = -0,01 (-0,08 – 0,06). En conclusion, le protocole le plus fortement corrélé avec l’ADN du VPH-16 a été celui avec le VLP L1+L2 et la dilution sérique 1:10. Seule la présence de l'ADN du HPV16 a été associée à la séropositivité au HPV16 (pas d’autre type de HPV), et elle n'a pas été influencée par la co-infection ou la charge virale.Although cervical HPV infections are very common, seroconversion does not always occur. We compared two protocols based on two serum dilutions to measure human papillomavirus (HPV) type 16 seroreactivity and investigated if HPV DNA positivity was associated with HPV16 seropositivity. We also assessed if the association was influenced by co-infection with other HPV types and viral load. The data used are from Brazilian women participating in the Ludwig-McGill cohort study on the natural history of cervical HPV infection. The serology protocols were based on virus-like particles (VLPs) composed by the L1 or L1 and L2 proteins which are, respectively, the major and minor viral capsid proteins. Two serum dilutions were used: 1:10 and 1:50. HPV16 seroreactivity was expressed as normalized absorbance ratio (NAR). HPV DNA genotyping and viral load were evaluated by PCR-based methods. Correlation and agreement between serum dilutions of each protocol (L1 and L1+L2 VLP) were assessed by Pearson’s correlation (r) and Bland-Altman method, respectively. The performance of the different protocols was compared using the receiver operating characteristic (ROC) curve using the presence of HPV16 DNA as the gold standard. Linear regression was used to analyze the association between HPV16 seropositivity and the detection of HPV DNA infection with both protocols. The presence of HPV DNA was analyzed based on (1) specific HPV types more or less related to HPV16 and (2) HPV16 infection detected alone or in co-infection with other HPV types. The linear regression models presented above were also used on the entire cohort tested with VLP L1+L2 and serum dilution 1:10. The impact of age as a potential confounding factor or effect modifier was analyzed in this model. Finally, the association between HPV16 viral load and seroreactivity was analyzed using Pearson correlation. The magnitude of log10-NARs mean differences between serum dilutions and their standard deviations for each protocol (L1 and L1+L2 VLP) were -0,081 (0.123) and -0.026 (0.150) log units, respectively. The NARs obtained by the serum dilutions used (1:10 and 1:50) for each protocol were strongly correlated (r = 0.87 vs. 0.94, respectively). However, the use of L1+L2 VLPs increased the performance of the test to detect HPV16 IgG antibodies, especially with the 1:10 serum dilution [the highest ROC area (95% CI) = 0.7330 (0.6465 – 0.8495)]. The regression models showed that HPV16 seroreactivity was uniquely associated with the presence of HPV16 DNA and not with other HPV types. For example, the analyses with the protocol L1+L2 VLP and serum dilution 1:10 showed that HPV16 seroreactivity was associated with the presence of HPV16 DNA, β (95% CI) = 0.24 (0.14 - 0.34), and not to HPV31 or 35, β (95% CI) = 0.03 (-0.19 - 0.25), or HPV52, 67, 33 or 58, β (95% CI) = 0.15 (-0.04 - 0.34), compared to women infected with any other HPV type or negative. The analysis of the entire cohort shows that the association between HPV16 seroreactivity and HPV16 DNA infection was similar when the infection was present alone or in co-infection, β (95% CI) = 0.14 (0.07 - 0.21) and β (95% CI) = 0.11 (0.01 - 0.21), respectively, compared to those infected with any other HPV type or negative. Age was not a significant confounder nor an effect modifier in the analysis of the entire cohort. The HPV16 viral load was not correlated with HPV16 seroreactivity, r (95% CI) = -0.04 (-0.34 – 0.27); β (95% CI) = -0.01 (-0.08 – 0.06). In conclusion, the protocol with the higher correlation with HPV 16 positivity was that with the L1+L2 VLP and serum dilution 1:10. Only the presence of HPV16 DNA was associated with HPV16 seropositivity (no other HPV type), and it was not influenced by co-infection or viral load

Clinical Evaluation of a GP5+/6+-Based Luminex Assay Having Full High-Risk Human Papillomavirus Genotyping Capability and an Internal Control

The LMNX genotyping kit HPV GP (LMNX) is based on the clinically validated GP5+/6+ PCR, with a genotyping readout as an alternative for the more established enzyme immunoassay (EIA) detection of 14 targeted high-risk human papillomavirus (HPV) types. LMNX is additionally provided with an internal control probe. Here, we present an analysis of the clinical performance of the LMNX using a sample panel and infrastructure provided by the international VALGENT (Validation of Genotyping Tests) project. This panel consisted of cervical specimens from approximately 1,000 women attending routine screening, “enriched” with 300 women with abnormal cytology. Cases were defined as women classified with cervical intraepithelial neoplasia (CIN) grade 2+ (CIN2+) (n = 102) or CIN3+ (n = 55) within the previous 18 months. Controls were women who had normal cytology results over two subsequent screening rounds at a 3-year interval (n = 746). The GP5+/6+-PCR EIA (EIA) was used as a comparator assay and showed sensitivities of 94.1% and 98.2% for CIN2+ and CIN3+, respectively, with a clinical specificity of 92.4% among women aged ≥30 years. The LMNX demonstrated clinical sensitivities of 96.1% for CIN2+ and of 98.2% for CIN3+ and a clinical specificity of 92.6% for women aged ≥30 years. The LMNX and EIA were in high agreement (Cohen's kappa = 0.969) for the detection of 14 hrHPVs in aggregate, and no significant difference was observed (McNemar's P = 0.629). The LMNX internal control detected 0.6% inadequate specimens. Based on our study results, we consider the LMNX, similarly to the EIA, useful for HPV-based cervical cancer screening

Caractérisation par analyse moléculaire de variants de l'infection persistance par le virus du papillome humain de type 16

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Advancing Microbial Risk Assessments of Subsurface Water Sources

Source water microbial quality evaluations are essential for the selection, design, and management of drinking water supplies. As biological sciences and technology continue to advance, how these assessments are performed will continue to evolve. In this age of “big data”, more data than can be meaningfully interpreted are being generated. Data generators and data analysts take on increasingly specialized roles to handle data from progressively complex tools. Drinking water industry decision makers are ultimately left with a frequently overwhelming task of choosing and deploying the best combination of tools from an ever-expanding repertoire. This dissertation serves to inform a prudent level of microbial water quality monitoring, especially in subsurface water sources. The potential and limitations of existing and emerging microbial tools to support groundwater vulnerability assessments to fecal pathogen intrusion were explored. Approaches for handling microbial non-detects were critically reviewed; the value of increased sampling and method analytical recovery characterization efforts for improving the precision of mean microbial concentrations (and associated risk estimates) was also examined. A novel approach for elucidating patterns from biochemical data acquired during well purging was demonstrated, which maximizes the use of sequentially collected adenosine triphosphate measurements to gain insights about purging sufficiency for microbial water quality evaluation. Finally, the increasingly common use of a biomolecular method (16S rRNA gene amplicon sequencing) that may provide complementary, contextual lines of evidence in groundwater vulnerability assessments to fecal pathogen intrusion was evaluated. Overall, this dissertation emphasizes the need for consideration of the “fit-for-purpose” ability and the complementary use of existing and emerging microbial tools in these evaluations; no single tool can fully capture the elusive, multi-faceted nature of microbial water quality

Entwicklung einer PCR basierten Methode zur Quantifizierung der Erreger des Echten Gurkenmehltaus, Podosphaera xanthii und Golovinomyces orontii, in Mischinfektionen

[no abstract

An investigation of the natural history of early cervical human papillomavirus infection and its relationship to the acquisition of epithelial abnormalities of the cervix

Cervical human papillomavirus (HPV) infection is a very common sexually transmitted disease which is now considered to be a necessary, but not sufficient, cause of cervical cancer. It has been suggested that the association between HPV infection and cervical neoplasia can be exploited to improve the efficiency and effectiveness of primary- and secondary-prevention programmes for cervical cancer. However, whether this aspiration can be realized in practice requires a greater understanding of the natural history of early cervical HPV infection and its role in the acquisition of epithelial abnormalities of the cervix. In this thesis, a longitudinal study of young women who had recently embarked on sexual activity has provided sequential observations on the natural history of cervical HPV infection. This thesis addresses four aspects of this natural history: the association between HPV infection and the proximity of first sexual intercourse to menarche; the association between smoking, cervical HPV infection and high-grade cervical disease; the validation of a neutralising antibody assay and its use in defining the kinetics of the humoral immune response to cervical HPV16 and HPV18 infections; and the analysis of measurements of the viral load of HPV16 and HPV18, and their association with epithelial abnormalities of the cervi