Comparison of Automated Quantitative Reverse Transcription-PCR and Direct Fluorescent-Antibody Detection for Routine Rabies Diagnosis in the United States

Abstract

Rabies virus found worldwide and prevalent throughout the United States continues to be a public health concern. Direct-fluorescent antibody (DFA) detection remains the gold standard for rabies virus diagnostics. Assessing the utility of a high-throughput molecular platform such as the QIAsymphony SP/AS, in conjunction with quantitative reverse transcription-PCR (qRT-PCR), to augment or potentially replace the DFA test, was the focus of this project. Here we describe a triplex qRT-PCR assay, including assembly and evaluation for sensitivity, specificity, and ability to detect variants. Additionally, we compared the qRT-PCR assay to the gold standard direct fluorescent-antibody test. More than 1,000 specimens submitted for routine rabies diagnosis were tested to directly compare the two methods. All results were in agreement between the two methods, with one additional specimen detected by qRT-PCR below the limits of the DFA sensitivity. With the proper continued validation for variant detection, molecular methods have a place in routine rabies diagnostics within the United States. Rabies virus, in the Lyssavirus genus of the Rhabdoviridae family, is found worldwide and has been described since antiquity (1). The fatality rate of clinical rabies infections remains greater than 99%. There are at least 14 recognized species of lyssaviruses circulating in the world (2), yet in the Western Hemisphere, only classical rabies virus has been identified in terrestrial mammals and bats.The cost of rabies control in the United States exceeds 300 million dollars annually (http://www.cdc.gov/rabies/location/usa /cost.html). A great portion of this expense results from the cost of administering postexposure prophylaxis (PEP) to individuals meeting risk assessment guidelines for contracting rabies virus after a contact with a rabid or suspect rabid animal. The need for accurate and timely diagnostic tools to identify positive rabies cases will continue, as this disease is prevalent in the continental United States, Canada, and Mexico (3). The direct-fluorescent antibody test (DFA) (4) is a rapid and sensitive method for diagnosing rabies infection, and as a World Organisation for Animal Health (OIE)/World Health Organization (WHO)-prescribed rabies test, it is considered the worldwide gold standard for rabies diagnosis. The accuracy of the DFA relies upon the examination of fresh brain tissue, the experience of a trained microscopist with access to a quality fluorescence microscope, and the availability of high-quality antirabies diagnostic conjugates.The Rabies Laboratory of the New York State Department of Health (NYSDOH) performs diagnostic testing on approximately 7,000 animal specimens a year. During the summer months, the specimen submission rate can approach 150 to 200 samples per day, making a high-throughput method of detection a necessity. In most laboratories, the DFA can be completed within 3 h from receipt of specimen, and it has proven to be highly specific, sensitive, and reliable. Because a rabies laboratory can...

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses

“…TaqMan probe based real-time RT-PCR assays often have lower sensitivity relative to SYBR Green based assays due to the sensitivity of TaqMan probes to the diversity of target sequences in the genomes of different RABV and other lyssaviruses [18, 22–24]. SYBR Green technology based real-time RT-PCR assays may provide greater sensitivity but can carry significant risk of false positive results when performed without a confirmatory sequencing step [20]. Typically, a combination of two or more assays is used to improve assay sensitivity and account for the breadth of lyssavirus diversity [25].…”

Development and evaluation of the RT-PCR kit for the rabies virus diagnosis

“…По данным сравнительного анализа диагностическая чувствительность МФА и ОТ-ПЦР в реальном времени сравнимы только в случае проведения анализа ex tempore. В противном случае диагностическая чувствительность ОТ-ПЦР в реальном времени существенно выше и составляет от 10 2 до 10 4 копий в 1 мл для разных штаммов [19]. Исследования зарубежных ученых, направленные на оценку молекулярных методов для диагностики бешенства, неоднократно демонстрировали их перспективность, особенно в формате ОТ-ПЦР в реальном времени.…”

“…В частности, продемонстрирована способность выявлять РНК 67 изолятов RABV, циркулировавших на территории Северной Америки. При этом среднее пороговое значение флуоресценции Ct для тестируемых образцов составило 16,4 [19].…”

Susceptibility of neuroblastoma cells to rabies virus may be affected by passage number

“…Following this incubation, the plates were removed from the incubator and the supernatant was removed from each well retained for real time RT-PCR. Supernatant recovered from cells was processed for real-time RT-PCR as described elsewhere (Dupuis et al, 2015). …”