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...

The antemortem diagnosis of rabies in humans employs techniques that require accuracy, speed, and sensitivity. A combination of histochemical analysis, in vitro virus isolation, immunological methods, and molecular amplification procedures are utilized in efforts to diagnose the disease. Modern medicine now offers potentially life-saving treatment for a disease that was considered invariably fatal once clinical signs develop. However, medical intervention efforts require a rapid and accurate diagnosis as early in the course of clinical disease as possible. Indirect fluorescent-antibody (IFA) testing on cerebrospinal fluid and serum specimens provides rapid results, but the specificity of the assay has not been well studied. Because false-positive IFA results could significantly affect patient treatment and outcomes, it is critical to understand the specificity of this assay. In this study, IFA testing was performed on 135 cerebrospinal fluid and serum specimens taken from patients with viral encephalitis or a presumed viral infection involving an agent other than rabies virus. Results indicate that false-positive results can occur in interpreting the rabies IFA test. Staining patterns morphologically similar to antirabies staining were observed in 7 of the 135 cerebrospinal fluid specimens examined. In addition, a majority of the cerebrospinal fluid specimens tested from patients with encephalitis presented immunoglobulin that bound to antigens present in the cell culture substrate. Of marked concern was the frequent presence of cross-reactive antibodies in encephalitis cases associated with West Nile and Powassan flaviviruses. Because IFA testing for rabies on human specimens may result in false-positive results, it should not be used as the sole basis for initiating antirabies treatment. Rapid accurate antemortem rabies diagnosis in humans has been imperative for palliative patient care and for treatment of individuals potentially exposed to the patient. The Milwaukee protocol (1) was introduced as a potentially life-saving treatment for human rabies, and the sooner the protocol is initiated the greater the chances of success. This paradigm demands speed and accuracy from the rabies diagnostician. The test most likely to provide a quick rabies diagnosis is the direct fluorescent-antibody (DFA) test (see Protocol for Postmortem Diagnosis of Rabies in Animals by Direct Fluorescent Antibody Testing [www.cdc.gov /rabies/pdf/RabiesDFASPv2.pdf]) performed on a nuchal skin biopsy specimen from the patient. However, since the results of this test may be negative in earlier stages of the disease, other procedures are relied upon and are carried out concurrently with the DFA test. The indirect fluorescent-antibody (IFA) test performed with cerebrospinal fluid (CSF) and serum specimens from rabiessuspect patients can yield results within a few hours. To perform an IFA test, serial dilutions of serum or CSF samples are placed on fixed, rabies virus-infected, cultured cells. If the serum or CSF contains antibodies to rabie...

Geomyces destructans is the etiologic agent of bat geomycosis, commonly referred to as white nose syndrome (WNS). This infection has caused severe morbidity and mortality in little brown bats (Myotis lucifugus) and has also spread to other bat species with significant decline in the populations. Currently, G. destructans infection is identified by culture, ITS-PCR, and histopathology. We hypothesized that a real-time PCR assay would considerably improve detection of G. destructans in bats. The 100 bp sequence of the Alpha-L-Rhamnosidase gene was validated as a target for real-time PCR. The assay sensitivity was determined from serial dilution of DNA extracted from G. destructans conidia (5 × 10(-1)-5 × 10(7)), and the specificity was tested using DNA from 30 closely and distantly related fungi and 5 common bacterial pathogens. The real-time PCR assay was highly sensitive with detection limit of two G. destructans conidia per reaction at 40 PCR cycles. The assay was also highly specific as none of the other fungal or bacterial DNA cross-reacted in the real-time PCR assay. One hundred and forty-seven bat tissue samples, suspected of infection with G. destructans, were used to compare the real-time PCR assay to other methods employed for the detection of G. destructans. Real-time PCR was highly sensitive with 80 of 147 (55%) samples testing positive for G. destructans DNA. In comparison, histopathology examination revealed 64/147 (44%) positive samples. The internal transcribed spacer (ITS)-PCR yielded positive amplicon for G. destructans from 37 tissue samples (25%). The least sensitive assay was the fungal culture with only 17 tissue samples (12%) yielding G. destructans in culture. The data suggested that the real-time PCR assay is highly promising for rapid, sensitive, and specific identification of G. destructans. Further trials and inter-laboratory comparisons of this novel assay are recommended to improve the diagnosis of bat geomycosis.