False-Negative Results of PCR Assay with Plasma of Patients with Severe Viral Hemorrhagic Fever

Drosten, Christian; Panning, Marcus; Guenther, S.; Schmitz, Herbert

doi:10.1128/jcm.40.11.4394-4395.2002

Cited by 36 publications

(30 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The use of inhibition controls is critical to prevent false-negative results (22 ). Use of an internal control that is copurified with the test samples and amplified with the same primers as the virus has been shown to be particularly efficient (17,18,23 ).…”

Section: Discussionmentioning

confidence: 99%

Rapid Detection and Differentiation of Human Pathogenic Orthopox Viruses by a Fluorescence Resonance Energy Transfer Real-Time PCR Assay

et al. 2004

Self Cite

View full text Add to dashboard Cite

Background:The orthopox viruses that are pathogenic for humans include variola major virus (VAR), monkeypox virus (MPV), cowpox virus (CPV), and to a lesser extent, camelpox virus (CML) and vaccinia virus (VAC). PCR is a powerful tool to detect and differentiate orthopox viruses, and real-time PCR has the further advantages of rapid turnaround time, low risk of contamination, capability of strain differentiation, and use of multiplexed probes. Methods: We used real-time PCR with fluorescence resonance energy transfer technology to simultaneously detect and differentiate VAR, MPV, CPV/VAC, and CML. An internal control generated by cloning and mutating the PCR target gene facilitated monitoring of PCR inhibition in each individual test reaction. Results: Strain differentiation results showed little interassay variability (CV, 0.4 -0.6%), and the test was 100-fold more sensitive than virus culture on Vero cells. Low copy numbers of DNA could be detected with >95% probability (235-849 genome copies/mL of plasma). Conclusions: The real-time PCR assay can detect and differentiate human pathogenic orthopox viruses. The use of an internal control qualifies the assay for high sample throughput, as is likely to be needed in situations of suspected acts of biological terrorism, e.g., use of VAR.

show abstract

Section: Discussionmentioning

confidence: 99%

Rapid Detection and Differentiation of Human Pathogenic Orthopox Viruses by a Fluorescence Resonance Energy Transfer Real-Time PCR Assay

et al. 2004

Self Cite

View full text Add to dashboard Cite

show abstract

“…Algunos autores han mostrado un especial interés en la posibilidad de falsos negativos a partir de muestras de suero, debido a la inhibición de la reacción de RT-PCR por componentes celulares derivados del daño tisular generado durante una fiebre hemorrágica viral grave, particularmente en infecciones causadas por el virus de la fiebre amarilla o el virus Ebola (29); en el caso de las muestras evaluadas en nuestro estudio, no se observó inhibición de la reacción en ninguno de los sueros evaluados; por el contrario, las bandas de amplificación fueron muy evidentes lo que demuestra una alta eficiencia del método de extracción del ARN cuando los posibles inhibidores pueden haber sido eliminados; sin embargo, la posibilidad de observar falsos negativos debe ser evaluada mediante el análisis de un mayor número de sueros y debe tenerse siempre en cuenta en el momento de aplicar la técnica para el diagnóstico de rutina en el laboratorio. Así, para detectar inhibidores se recomienda utilizar siempre un ARN control en un duplicado de la muestra y, además, probar en paralelo diluciones del suero original (29).…”

Section: Discussionunclassified

“…Así, para detectar inhibidores se recomienda utilizar siempre un ARN control en un duplicado de la muestra y, además, probar en paralelo diluciones del suero original (29).…”

Section: Discussionunclassified

Detección molecular del virus de la fiebre amarilla en muestras de suero de casos fatales humanos y en cerebros de ratón.

Méndez¹,

Rodríguez

Bernal³

et al. 2003

biomedica

View full text Add to dashboard Cite

Palabras clave: virus de fiebre amarilla, RT-PCR, herramientas diagnósticas. Molecular detection of yellow fever virus in human sera and mice brainsA molecular method for the diagnosis of yellow fever virus infection was developed based on reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification. Examinations were conducted on lyophilized sera from 3 fatal yellow fever cases and 4 fresh sera from 3 fatal cases and one from a symptomatic patient (positive IgM against yellow fever virus). Sera were extracted with TRIZOL-LS® to isolate viral RNA for RT treatment and the PCR reaction included 2 primers sets designed specifically for yellow fever virus: sense, JM2104 (5´-CGTTGGGAGAGGAGATTC-3´) y JM2249 (5´-TTCTTCACTTCGGTTGGG-3´), and antisense, JM2673 (5´-TCATCTGCCCTGCTTCTC-3´) y JM2751 (5´-CCTCTCTGGTAAACATTCT-3´). The technique for demonstrating the yellow fever virus in tissue samples was used in infected mice brains treated with lysis buffer before RNA extraction. PCR reactions were evaluated in agarose gels where single bands of the expected size for each primers pair (569bp and 502bp) were observed for all serum samples. In addition, the results for 2 fresh positive sera were supported by histopathologic finding of yellow fever virus. The RT-PCR method permits a rapid and specific demonstration of the presence of yellow fever virus.

show abstract

“…False-negative results in patients with severe hemorrhagic fever have been noted before (30). Therefore, analytical results obtained by this and any other test should be interpreted by trained and experienced diagnosticians together with all available laboratory results and clinical, pathological, and epidemiological data to ensure an accurate diagnosis.…”

Section: Discussionmentioning

confidence: 99%

Comparative Evaluation of the Diagnostic Performance of the Prototype Cepheid GeneXpert Ebola Assay

et al. 2016

View full text Add to dashboard Cite

The diagnostic sensitivity of the three platforms was 100% in 91 patient samples that were confirmed to be infectious by virus isolation. All three molecular platforms detected viral EBOV RNA in additional samples that did not contain viable EBOV. The analytical sensitivity of the GeneXpert Ebola POC for the detection of NP was higher, and comparable to that of polymerase gene detection, than that for the detection of GP when using a titrated laboratory stock of EBOV. There was no detectable cross-reactivity with other hemorrhagic fever viruses or arboviruses. The GeneXpert Ebola POC offers an easy to operate and sensitive diagnostic tool that can be used for the rapid screening of suspected EVD cases in treatment or in holding centers during EVD outbreaks.

show abstract

False-Negative Results of PCR Assay with Plasma of Patients with Severe Viral Hemorrhagic Fever

Cited by 36 publications

References 9 publications

Rapid Detection and Differentiation of Human Pathogenic Orthopox Viruses by a Fluorescence Resonance Energy Transfer Real-Time PCR Assay

Rapid Detection and Differentiation of Human Pathogenic Orthopox Viruses by a Fluorescence Resonance Energy Transfer Real-Time PCR Assay

Detección molecular del virus de la fiebre amarilla en muestras de suero de casos fatales humanos y en cerebros de ratón.

Comparative Evaluation of the Diagnostic Performance of the Prototype Cepheid GeneXpert Ebola Assay

Contact Info

Product

Resources

About