Identification of 3 novel herpesviruses in prosimians with lymphoproliferative disease.

Although many studies have characterized catarrhine and platyrrhine primate herpesviruses, little is known about herpesviruses in prosimians. We aimed to identify and characterize herpesviruses in prosimians with proliferative lymphocytic disease. DNA was extracted from tissues of 9 gray mouse lemurs (Microcebus murinus) and 3 pygmy slow lorises (Nycticebus pygmaeus) with lymphoproliferative lesions, and we performed nested PCR and sequencing for detection of herpesviruses and polyomaviruses. We identified 3 novel herpesviruses and performed phylogenetic analyses to characterize their relationship with other herpesviruses. A gray mouse lemur herpesvirus clustered with other primate herpesviruses within the subfamily Betaherpesvirinae, just basal to the genus Cytomegalovirus. The other gray mouse lemur herpesvirus and the pygmy slow loris herpesvirus clustered within the subfamily Gammaherpesvirinae, although the relationships within the subfamily were less resolved. Quantitative PCR assays were developed for the 2 new gray mouse lemur viruses, providing specific, faster, less expensive, and quantitative detection tools. Further studies are needed to elucidate the relationship between the presence of these viruses and the severity or presence of lymphoproliferative lesions in prosimians.

DEVELOPMENT AND VALIDATION OF A NOVEL DUPLEX PROBE-HYBRIDIZATION QUANTITATIVE PCR FOR LYMPHOMA-ASSOCIATED MIROUNGINE GAMMAHERPESVIRUS 3 IN NORTHERN ELEPHANT SEALS (MIROUNGA ANGUSTIROSTRIS).

Recently, a novel gammaherpesvirus, miroungine gammaherpesvirus 3 (MirGHV3), was described in two juvenile elephant seals (Mirounga angustirostris) with diffuse large B-cell lymphoma. We developed and validated a quantitative (q)PCR for rapid detection of MirGHV3 and investigated its potential association with lymphoma. We developed a duplex probe-hybridization qPCR with MirGHV3 DNA polymerase (pol) as the target gene. Each primer-probe combination was cross-validated against the others. Interference was not seen when they were run in the same well as a duplex assay. Twenty-three samples from seven northern elephant seals were tested using the duplex assay. Viral DNA was detected by the assay in 9 of 9 (100%) tissues affected by lymphoma and in 6 of 14 (43%) samples from tissues unaffected by lymphoma. There was a strong correlation between viral copies detected with each of the assays (P=0.0002). Viral load was significantly higher in tissues affected by lymphoma than in those unaffected (P<0.0001). Excluding the virus-negative samples, viral load was still significantly higher in tissues affected by lymphoma than in those unaffected (P=0.0004). This is consistent with a potential role of MirGHV3 in oncogenesis in northern elephant seals, although more studies are needed to determine this definitively. The qPCR developed has utility for further investigations of MirGHV3.

Prevalence of mecA, mecC and Panton-Valentine-Leukocidin Genes in Clinical Isolates of Coagulase Positive Staphylococci from Dermatological Canine Patients.

Coagulase positive Staphylococci (CoPS) are the leading cause of canine cutaneous and otic infections. Virulence factors associated with Staphylococci include the expression of mec and panton-valentine leukocidin (pvl) genes. Methicillin-resistance (MR) is commonly associated with mecA gene expression, although a recently identified variant, mecC, has been reported. This study aims to evaluate the prevalence of mecA, mecC and pvl genes in 232 clinical isolates of CoPS collected from dogs with pyoderma. A multiplex PCR, and Kirby-Bauer disk diffusion susceptibility test for cefoxitin was performed for all isolates. PBP2a agglutination test was performed on 127 isolates. Standard MRSA isolates were used as positive controls. The mecA gene was identified in 149/232 isolates (64.2%): 116 S. pseudintermedius, 30 S. coagulans and three S. aureus. The pvl gene was present in only 1 isolate of S. pseudintermedius (0.4%), whereas no isolates carried the mecC gene. 34 isolates were resistant to cefoxitin (14.6%) and they were all mecA positive. The results of this study show an MR prevalence of 64.2% confirming concerns about antibiotic resistance in veterinary medicine. In conclusion, this is the first study analyzing the prevalence of mecC and pvl in comparison to mecA, in a large cohort of CoPS clinical isolates from dogs with pyoderma. A multimodal surveillance on the prevalence of mecC and pvl in veterinary medicine is essential to appropriate antimicrobial management.

Identification of a novel Neorickettsia species in a Kemp's ridley sea turtle with granulomatous nephritis and development of a quantitative PCR assay.

An adult male Kemp's ridley turtle was found dead on the coast of Kenedy County, Texas, in August 2019 with bilateral severe, diffuse granulomatous nephritis. Pan-bacterial 16S rRNA gene polymerase chain reaction (PCR) and amplicon sequencing of affected tissue indicated the presence of a Neorickettsia. Neorickettsia is a genus of obligate intracellular Alphaproteobacteria that are transmitted by digenean trematodes. For further characterization, primers were designed to amplify and sequence the groEL gene. Phylogenetic analysis found that the organism was distinct from other known species to a degree consistent with a novel species. Immunohistochemistry using an antibody directed against a Neorickettsia surface protein showed bacterial clusters within the renal granulomas. A species-specific quantitative PCR was designed and detected the organism within the liver and colon of the index case. A quantitative PCR survey of grossly normal kidneys opportunistically collected from additional stranded sea turtle kidneys detected this organism in five of 15 Kemp's ridley turtles, two of nine green turtles, and neither of two loggerhead turtles. Recognition of this novel organism in an endangered species is concerning; additional work is underway to further characterize the potential of this organism as a pathogen of sea turtles.

Diffuse large B cell lymphoma and a novel gammaherpesvirus in northern elephant seals Mirounga angustirostris.

Two emaciated male northern elephant seal (NES) Mirounga angustirostris pups were admitted to The Marine Mammal Center (Sausalito, California, USA) and treated for malnutrition. Complete blood counts showed a progressive moderate to marked leukocytosis characterized by a predominance of large monomorphic mononuclear cells of probable lymphoid origin, frequently with flower-shaped nuclei. Both seals were euthanized due to suspected lymphoid neoplasia. At necropsy, most lymph nodes in both pups were markedly enlarged, some with distinct white nodules, the spleens were diffusely enlarged, and the intestinal mucosae were thickened. Histopathologic features consistent with disseminated large cell lymphoma were identified to varying degrees of severity in lymph nodes, bone marrow, liver, tonsils, spleen, liver, intestines, kidneys, lower urinary tract, and several other organs. Immunohistochemical staining of neoplastic cells was most consistent with B lymphocyte origin, with most cells staining positively for Pax 5 and CD20 with admixed small CD3-positive T lymphocytes and CD204-positive macrophages. PCR and sequencing identified a novel gammaherpesvirus, herein called miroungine gammaherpesvirus 3, from affected tissues. This virus is in a clade outside of named genera that utilize hosts in the suborder Caniformia. The present study is the first description of diffuse large B cell lymphoma with leukemic manifestation and concomitant detection of a novel gammaherpesvirus in free-living NESs. Further research regarding the prevalence of this new gammaherpesvirus and its associated pathogenesis in this species is indicated.

Evaluation of cutaneous and circulating (serum and exosomes) levels of chemokines (CCL17, CCL22, CCL27 and CCL28) in atopic dogs and their correlation with severity of the disease.

BACKGROUND: Canine atopic dermatitis (AD) is a complex multifactorial disease characterised by an exaggerated immunological response. Little is known about the role that cutaneous and circulating chemokines play in disease severity. OBJECTIVE: To evaluate the messenger (m)RNA and protein levels of selected chemokines in skin and serum of healthy and atopic dogs, and in the atopic group to determine whether there is a correlation with disease severity. MATERIALS AND METHODS: Skin biopsies and blood samples were taken from 12 privately owned atopic [lesional (AD-L) and nonlesional (AD-NL) skin] and 12 privately owned healthy dogs. Circulating exosomes were extracted from the serum. Cutaneous and exosomal mRNA levels of CCL17, CCL22, CCL27 and CCL28 were quantified using quantitative real-time PCR. Protein levels were evaluated using canine-specific ELISA kits. The severity and extent of the clinical signs also were assessed in the atopic dogs using Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04) and a validated pruritus Visual Analog Scale (pVAS). RESULTS: The expression of CCL28 exosomes in skin was greater in AD-L when compared to healthy (P = 0.019) and AD-NL (P = 0.002) samples. However, serum expression was lower in dogs with AD compared to healthy dogs (P = 0.03). A higher expression of CCL17 and CCL22 was seen in AD-L when compared to healthy skin (P = 0.018 and P = 0.019, respectively). There also was a positive correlation between clinical scores and CCL22 (AD-NL; r = 0.6, P = 0.05) and between the pruritus score and CCL22 (AD-L; r = 0.6, P = 0.05). Differences in CCL27 concentrations were not observed. CONCLUSIONS AND CLINICAL IMPORTANCE: This study suggests that CCL17, CCL22 and CCL28 may play a role in the cutaneous inflammatory response in atopic dogs. They may be considered as markers of disease severity, although further studies are needed to validate these findings.

Efficacy of ethylene oxide-sterilized waterproof cases for handheld cameras as sterile barriers for intraoperative imaging and recording.

OBJECTIVE: To evaluate the efficacy of ethylene oxide (EtOH) sterilization of 4 different waterproof camera cases and the ability of those sterilized cases to maintain a sterile barrier for intraoperative camera use. SAMPLE: 3 action cameras, 1 smartphone, and associated waterproof cases. PROCEDURES: Cases were inoculated by immersion in medium containing Staphylococcus pseudintermedius, Escherichia coli, and Pseudomonas aeruginosa and then manually cleaned and subjected to EtOH sterilization. Cameras were disinfected, loaded into sterile cases, and sterilely operated for 2 hours. Samples were collected from cases after inoculation, EtOH sterilization, camera loading, and 1 and 2 hours of operation and from all cameras after 2 hours of operation. Procedures were repeated twice, followed by an additional challenge round wherein cameras were purposefully contaminated prior to loading. All samples underwent bacterial culture. RESULTS: All cases were successfully sterilized, and loading of nonsterile cameras into sterile cases caused no contamination when cameras had been disinfected beforehand. Nonpathogenic environmental contaminants were recovered from 6 of 64 culture samples and 2 of 4 room samples. During the challenge round, only the postload sample for 1 case yielded E coli, suggesting sterile glove contamination; however, postload, 1-hour, and 2-hour samples for the GoPro case yielded E coli and S pseudintermedius, suggesting major contamination. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that the evaluated cases can be safely sterilized with EtOH and used for image acquisition by aseptically prepared surgeons when cameras are disinfected prior to loading. Except for the GoPro camera, camera use did not jeopardize sterile integrity.

Identification of a novel mortality-associated Helicobacter species in gopher tortoises (Gopherus polyphemus), qPCR test development and validation, and correlation with mortality in a wildlife rehabilitation population.

The genus Helicobacter includes spiral-shaped bacteria in the phylum Proteobacteria, class Epsilonproteobacteria, order Campylobacteriales, that have been associated with disease in animals, including reptiles. Three wild gopher tortoise (Gopherus polyphemus) index cases presented between 2012 and 2019 with nasal discharge, lethargy, and weight loss. Cytological examination of nasal discharge from all 3 tortoises identified marked heterophilic and mild histiocytic rhinitis with abundant extracellular and phagocytized spiral shaped bacteria that stained positive with Warthin-Starry stain. Polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene revealed this to be a novel Helicobacter species. Two tortoises died despite treatment attempts, and the third was moribund and was euthanized. Histological examination of the nasal mucosa (n = 3) showed granulocytic to lymphocytic rhinitis with variable mucosal hyperplasia, erosion, and ulceration; Warthin-Starry staining highlighted the presence of spiral bacteria in the untreated tortoise. Genus-specific primers were designed, and the gyrA and groEL genes were amplified by PCR and sequenced. Phylogenetic analysis shows that this organism and other previously characterized Helicobacter from tortoises form a clade. Development and cross-validation of two qPCR diagnostic assays for the gyrA and groEL genes showed significant correlation of the results of two assays (P < 0.0001). These assays were used to survey nasal wash samples from 31 rehabilitating gopher tortoises. Mortality of tortoises significantly correlated with higher Helicobacter loads detected by qPCR (P = 0.028). Appropriate quarantine protocols for tortoises during rehabilitation should consider this organism. Upper respiratory disease in tortoises may involve complex microbial ecology; factors beyond Mycoplasmopsis (Mycoplasma) agassizii should be taken into account.

Intradermal immunotherapy with actinomycetales preparations as treatment for feline atopic syndrome: a randomized, placebo-controlled, double-blinded study.

BACKGROUND: Feline atopic syndrome (FAS) is a common disease. Single intradermal injections of heat-killed actinomycetales have shown beneficial effects in canine allergies. HYPOTHESIS/OBJECTIVE: To evaluate the clinical effects of heat-killed actinomycetales [Gordonia bronchialis (GB) and Rodococcus coprophilus (RC)], alone or in combination, in FAS. METHODS AND MATERIALS: Privately owned cats with a diagnosis of FAS were assigned randomly in three treatment groups (GB, RC and GB/RC combination) or placebo. Five intradermal injections were performed over a one year period. At each visit [Day (D)0, D20, D40, D60, D90, D180 and D365], clinical signs, global owner assessment score, use of rescue medications, clinical adverse effects, skin hydration and cutaneous pH were assessed. RESULTS: Seventeen cats were enrolled. When compared to the placebo group and improvement in treatment GB was sustained from D90. When compared with D0 significant improvement in the GB group was seen from D60. Over one year, a complete remission of the clinical signs was seen in 30-67% of cats in the treatment groups. A reduction in the pruritus score was seen for RC after 365 days of treatment (P = 0.04). Differences in the other variables were not seen. CONCLUSIONS AND CLINICAL IMPORTANCE: The use of multiple intradermal injections of heat-killed GB shows promise as effective and well-tolerated treatment for FAS. Because of the low cost and the lack of adverse effects, GB could be a beneficial treatment option for FAS. A larger study is needed to confirm these data and to evaluate the immunological changes occurring in the treated cats.

A defective release of host defense peptides is present in canine atopic skin.

The use of dogs as animal model for human atopic dermatitis (AD) is well known. Striking similarities in the pathogenesis of AD have been demonstrated. Similar alteration of host defense peptides (HDP) have been identified in both species. However, the ultrastructural/molecular alterations associated with HDPs secretion in AD have not been elucidated. We were able to use a multidisciplinary approach to investigate the secretion of HDP in canine skin. The contemporary use of indirect immunofluorescence, ELISA and scanning immune-electron microscopy gave fundamental insights in the pathomechanism of HDP alteration in AD. An increased intracellular expression and a reduced secretion of HDPs is present in atopic skin. An increased presence of HDPs was seen on the surface of atopic skin. These results suggested a defective secretion and an increased adhesion of HDPs to atopic corneocytes might be the reason of the reduced killing activity of HDPs in AD.

Pilot study using five methods to evaluate skin barrier function in healthy dogs and in dogs with atopic dermatitis.

BACKGROUND: Atopic dermatitis is associated with skin barrier defects. In people, noninvasive techniques are used to quantify the skin barrier functionality. In dogs, transepidermal water loss (TEWL), stratum corneum hydration and pH have been used to assess skin barrier function. However, few studies have determined their repeatability. OBJECTIVE: To assess the repeatability of measurements of skin hydration, TEWL, pH, skin absorbance and erythema in healthy and atopic dogs. ANIMALS: Fifteen healthy and 15 atopic privately owned dogs. METHODS AND MATERIALS: Three repeated measurements using Corneometer®, Skin-pH-Meter®, Colorimeter® and VapoMeter® were obtained from inguinal, axilla, pinna and interdigital space by three investigators. Intra- and interobserver variability (coefficient of variation, correlation coefficients and intraclass correlation coefficients) and difference between the two groups (t-test or Mann-Whitney U-test) were determined. RESULTS: High repeatability and low variation were observed both intra- and interobservers for all devices except the VapoMeter®. The most repeatable device was the Skin-pH-Meter®, whereas the VapoMeter® was the device with the highest intra- and interobserver variability. Atopic dogs had a significantly increased pH (inguinal P = 0.03; axilla P = 0.02) and erythema (inguinal P = 0.01; axilla P = 0.02) compared to healthy dogs. No differences between the two groups were detected using the Corneometer®, VapoMeter® or Colorimeter® (tartrazine absorption). CONCLUSION AND CLINICAL SIGNIFICANCE: The results of this pilot study support the use of Corneometer®, Skin-pH-Meter® and Colorimeter® in the assessment of skin barrier function in dogs; further investigations to optimize measurements and confirm these results are needed.

PREVALENCE OF PARELAPHOSTRONGYLUS ANDERSONI IN WHITE-TAILED DEER, OTHER CERVIDS, AND BOVIDS IN NORTHERN FLORIDA.

Parelaphostrongylus andersoni, the muscleworm, commonly infects white-tailed deer (Odocoileus virginianus) and also infects caribou (Rangifer tarandus [R.t.] groenlanicus, R.t. grantii, R.t. tarandus, and R.t. caribou). Heavy infection with P. andersoni leads to weakness in the hindquarters, abnormal gait, and pulmonary lesions. The geographical range and full host spectrum of this parasite are not fully known. This study aims to understand host specificity better, especially in nonnative cervids and bovids. This study involved the collection of 140 fecal samples from native and nonnative cervid and bovid species, and 34 snail specimens. With the use of real-time PCR, we found 4/47 (8.5%) O. virginianus fecal samples were positive for P. andersoni. No previously undocumented species of cervids or bovids were found to be infected. Further research is warranted to understand P. andersoni range, host distribution, and potential impact on host health.

Prevention of medical errors and malpractice: Is creating resilience in physicians part of the answer?

In this article, we present key concepts regarding physician and resident resilience and burnout, the legal and educational context for these distinctions, and the effects of improved physician resilience through self-care on a reduction in medical errors and malpractice. Resilience here indicates the mental processes and behaviors that enable an individual to overcome the potential negative effects of stressors. In order to explore the multiple factors that contribute to physician resilience, the authors approached the topic from a variety of perspectives, including the current ways of thinking about medical malpractice in the United States, physician resilience and medical errors, and building resilience during postgraduate medical education. The authors review steps taken and in process to mitigate physician burnout and enhance physician resilience.

Development and validation of a probe hybridization reverse-transcription quantitative PCR for detection of mamastrovirus 2 in domestic cats.

Astroviruses are viral pathogens that have been associated with enteric and neurologic disease in a variety of species. The domestic cat is a prominent host, with reports of astroviral infection being both highly prevalent and widely distributed in the feline population. Despite the potential for inducing significant disease, especially within shelter environments, there is currently only one reliable method of detection: standard reverse-transcription PCR using pan-astrovirus degenerate primers (consensus RT-PCR) with product sequencing. Unfortunately, this process is relatively slow and costly. Quantitative real-time PCR (qPCR) represents an efficient, economical alternative, with the added benefit of viral load quantification. We developed a RT-qPCR assay using probe hybridization technique to detect conserved regions of mamastrovirus 2 extracted from fecal samples of domestic cats. Known positive and negative samples were tested, and results were compared with gold standard consensus RT-PCR and sequencing. A standard curve was employed to determine limits of detection. In order to assess analytic specificity, we tested several additional samples that had been collected from non-felid species and were known to contain non-target astroviruses. Discrepant results between consensus RT-PCR and RT-qPCR testing were further analyzed with a validation RT-PCR assay, using mamastrovirus 2-specific primers. Our probe hybridization RT-qPCR assay is reliable and effective for the detection of mamastrovirus 2. This assay will allow rapid, affordable detection and facilitate further research on astroviral infection within domestic cats.

Managing emotionally charged conversations between faculty and residents: a skill-building workshop.

This article was migrated. The article was marked as recommended. Background: Discussions with residents regarding remediation, probation, or dismissal are challenging, but only limited practical guidance about constructive conversations is available. Aims: To provide useful assistance to those who must conduct such conversations. Methods: Literature review, surveys, workshop development. Results: The workshop offers guidance about conducting challenging conversations, including opportunities for role playing. Conclusions: Development of the workshop is ongoing, and initial feedback recognizes its importance and applicability to help faculty manage challenging or confrontational conversations.

PHYLOGENETIC ANALYSIS OF THE GENOME OF AN ENTERITIS-ASSOCIATED BOTTLENOSE DOLPHIN MASTADENOVIRUS SUPPORTS A CLADE INFECTING THE CETARTIODACTYLA.

: Adenoviruses are nonenveloped, double-stranded DNA viruses, known to infect members of all tetrapod classes, with a similarity between phylogenies of hosts and viruses observed. We characterized bottlenose dolphin adenovirus 2 (BdAdV-2) found in a bottlenose dolphin ( Tursiops truncatus) with enteritis. Virions were seen by negative staining electron microscopy of feces. Initial sequences obtained using conserved PCR primers were expanded using primer walking techniques, and the complete coding sequence was obtained. Phylogenetic analyses were consistent with coevolution of this virus and its bottlenose dolphin host, placing BdAdV-2 into a monophyletic group with other mastadenoviruses of Cetartiodactyla. When considering the low guanine/cytosine (G/C) content of BdAdV-2 with the phylogenetic data, this virus may represent a host-jumping event from another member of Cetartiodactyla. Analysis of partial polymerase indicated that bottlenose dolphin adenovirus 1, previously identified in Spain, and BdAdV-2 are sister taxa with harbor porpoise adenovirus 1, forming a cetacean clade. Bottlenose dolphin adenovirus 2 includes a highly divergent fiber gene. Two genes homologous to the dUTPase superfamily are also present which could play a role in enabling viral replication in nondividing cells. We used sequence data to develop a probe hybridization quantitative PCR assay specific to BdAdV-2 with a limit of detection of 10 copies.

Determination of the diversity of astroviruses in feces from cats in Florida.

Astroviruses are small, nonenveloped RNA viruses that have been linked to numerous diseases in a variety of species, including enteric disease in humans and cheetahs. Species Mamastrovirus 2, previously known as feline astrovirus, has been isolated from the feces of domestic cats and cheetahs. A total of 122 cat fecal samples from Alachua County, FL Animal Services and the Veterinary Community Outreach Program at the University of Florida were analyzed, and 35 contained astroviral RNA that was amplified and identified using consensus RT-PCR and sequence analysis. Using phylogenetic analysis, 19 of the astroviral sequences were identified as Mamastrovirus 2, making it the most prevalent astrovirus in this population. Three samples were identified as an astrovirus similar to viruses previously identified in foxes in The Netherlands and a cat in California, and one was similar to a bat astrovirus. One astroviral sequence was identified as an Avastrovirus. Although a causative relationship between mamastroviruses and enteric disease in cats has yet to be established, it is clear that mamastroviruses are prevalent, and an understanding of prevalence of astroviral types may help direct future test development.

Development of a quantitative PCR assay for measurement of trichechid herpesvirus 1 load in the Florida manatee ( Trichechus manatus latirostris).

Trichechid herpesvirus 1 (TrHV-1) is currently the only known herpesvirus in any sirenian. We hypothesized that stress may lead to recrudescence of TrHV-1 in manatees, thus making TrHV-1 a potential biomarker of stress. We optimized and validated a TrHV-1 real-time quantitative probe hybridization PCR (qPCR) assay that was used to quantify TrHV-1 in manatee peripheral blood mononuclear cells (PBMCs). Average baseline TrHV-1 loads in a clinically healthy wild Florida manatee ( Trichechus manatus latirostris) population ( n = 42) were 40.9 ± SD 21.2 copies/100 ng DNA; 19 of 42 manatees were positive. TrHV-1 loads were significantly different between the 2 field seasons ( p < 0.025). This optimized and validated qPCR assay may be used as a tool for further research into TrHV-1 in Florida manatees.

Development and validation of a quantitative PCR for rapid and specific detection of California sea lion adenovirus 1 and prevalence in wild and managed populations.

California sea lion adenovirus 1 (CSLAdV-1) has been associated with hepatitis and enteritis in several wild and captive populations of diverse pinniped species. Currently available tests have been limited to pan-adenoviral polymerase chain reaction (PCR) followed by sequencing. We present the development of a quantitative probe-hybridization PCR (qPCR) assay for rapid, sensitive, and specific detection of this virus in California sea lions ( Zalophus californianus) and other pinnipeds. This assay did not amplify other mammalian adenoviruses and is able to detect consistently down to 10 viral copies per well. Compared with the gold standard conventional pan-adenovirus PCR/sequencing assay, diagnostic sensitivity and specificity of 100% and 88.2% were found, respectively. The lower diagnostic specificity of this qPCR assay may be the result of the lower limit of detection of this assay compared with the gold standard rather than the result of detection of true false-positives.