Assessment of Clinical and Laboratory Use of the Cutaneous Direct Immunofluorescence Assay.

IMPORTANCE: Dermatologists submit direct immunofluorescence (DIF) biopsies on a daily basis, using an assay detecting immunoreactant deposition with a panel that has traditionally comprised immunoglobulin (Ig) G, IgA, IgM, C3, and fibrin, with or without albumin antibodies. OBJECTIVES: To evaluate and compare the frequency of immunoreactants in DIF biopsies submitted over an 8-year period and assess use by dermatologists based on clinical impression. DESIGN, SETTING, AND PARTICIPANTS: A quality improvement study was conducted in a community outreach reference laboratory associated with a large academic medical center. Results of 2050 consecutive DIF skin biopsies submitted to the laboratory between April 1, 2012, and June 12, 2020, were analyzed by final pathologic diagnosis and antibody subtype positivity, in comparison with clinical impression. Biopsies in which the submitting physician had not performed the biopsy were excluded. MAIN OUTCOMES AND MEASURES: Histopathologic findings and the results of DIF biopsies using the standard 6-antibody panel were evaluated in correlation with the submitted clinical diagnosis to assess immunoreactivity of the assay. RESULTS: Of 2050 DIF biopsies submitted, 367 (17.9%) were positive; IgG, IgA, and C3 alone identified all primary immunobullous disease cases (pemphigoid, pemphigus, linear IgA, and dermatitis herpetiformis), and IgA, C3, and fibrin antibodies alone identified all vasculitis cases. A panel of IgG, IgA, IgM, and fibrin identified all cases of lupus erythematosus. DIF results were positive in less than half of cases of hematoxylin and eosin biopsy-confirmed lupus erythematosus (23 of 47 [49%]). A total of 247 biopsies were submitted for clinical diagnoses not optimally supported on DIF: lichen planus, porphyria, and connective tissue disease. CONCLUSIONS AND RELEVANCE: The findings of this study suggest that there is a knowledge gap among dermatologists relating to the opportunity for high-value, cost-conscious use of DIF. The practice of reflexive antibody testing using a 6-antibody panel for all DIF biopsies is likely unnecessary. DIF protocols tailored to the clinical diagnosis may enhance cost-effectiveness without loss of test sensitivity or specificity.

New-generation diagnostics in inflammatory skin diseases: Immunofluorescence and histopathological assessment using ex vivo confocal laser scanning microscopy in cutaneous lupus erythematosus.

Ex vivo confocal laser scanning microscopy (CLSM) offers real-time examination of excised tissue in reflectance, fluorescence and digital haematoxylin-eosin (H&E)-like staining modes enabling application of fluorescent-labelled antibodies. We aimed to assess the diagnostic performance of ex vivo CLSM in identifying histopathological features and lupus band test in cutaneous lupus erythematosus (CLE) with comparison to conventional histopathology and direct immunofluorescence (DIF). A total of 72 sections of 18 CLE patients were stained with acridine orange (AO), anti-IgG, anti-IgM and anti-IgA; 21 control samples were stained with AO. Subsequently, ex vivo CLSM examination of all samples was performed in reflectance, fluorescence and digital H&E-like staining modes. Superficial and deep perivascular inflammatory infiltration (94.4%), interface dermatitis (88.9%), spongiosis (83.3%) and vacuolar degeneration (77.7%) were the most common features detected with ex vivo CLSM. Kappa test revealed a level of agreement ranging within "perfect" to "good" between ex vivo CLSM and conventional histopathology. ROC analysis showed that the combination of perivascular infiltration, interface dermatitis and spongiosis detected by ex vivo CLSM has the potential to distinguish between CLE and controls. Basement membrane immunoreactivity with IgG, IgM and IgA was identified in 88.8% (n = 15), 55.5% (n = 10) and 55.5% (n = 10) of the CLE samples using ex vivo CLSM, respectively, whereas DIF showed IgG, IgM and IgA positivity in 94.4% (n = 17), 100% (n = 18) and 88.9% (n = 16) of patients, respectively. In conclusion, ex vivo CLSM enables simultaneous histopathological and immunofluorescence examination in CLE showing a high agreement with conventional histopathology, albeit with a lower performance than conventional DIF.

Immunofluorescence and histopathological assessment using ex vivo confocal laser scanning microscopy in lichen planus.

Ex vivo confocal laser scanning microscopy (CLSM) provides rapid, high-resolution imaging, fluorescence detection and digital haematoxylin-eosin (H&E)-like staining. We aimed to assess the performance of ex vivo CLSM in identifying histomorphology and immunoreactivity in lichen planus (LP) and comparing its accuracy with conventional histopathology and direct immunofluorescence (DIF). Thirty-three sections of 17 LP patients stained with acridine orange (AO) and FITC-labelled anti-fibrinogen antibody and 21 control samples stained with AO were examined using ex vivo CLSM. Ex vivo CLSM was in perfect agreement with conventional histopathology in identifying interface dermatitis, vacuolar degeneration and band-like infiltration. ROC analysis showed that the presence of vacuolar degeneration, interface dermatitis and band-like infiltration was useful to distinguish LP sections from controls (p < .0001). The detection rates of fibrinogen deposition using DIF and in conclusion ex vivo CLSM were 93.8% and 62.5%, respectively. ex vivo CLSM enables histopathological and immunofluorescence examination in LP with the advantage of digital H&E-like staining.

Economic and feasibility comparison of the dRIT and DFA for decentralized rabies diagnosis in resource-limited settings: The use of Nigerian dog meat markets as a case study.

BACKGROUND: Rabies lyssavirus (RABV) is the aetiologic agent of rabies, a disease that is severely underreported in Nigeria as well as elsewhere in Africa and Asia. Despite the role that rabies diagnosis plays towards elucidating the true burden of the disease, Nigeria-a country of 180 million inhabitants-has a limited number of diagnostic facilities. In this study, we sought to investigate two of the World Organization for Animal Health (OIE)-recommended diagnostic assays for rabies-viz; the direct fluorescent antibody test (DFA) and the direct rapid immunohistochemical test (dRIT) in terms of their relative suitability in resource-limited settings. Our primary considerations were (1) the financial feasibility for implementation and (2) the diagnostic efficacy. As a case study, we used suspect rabies samples from dog meat markets in Nigeria. METHODS/PRINCIPAL FINDINGS: By developing a simple simulation framework, we suggested that the assay with the lowest cost to implement and routinely use was the dRIT assay. The costs associated with the dRIT were lower in all simulated scenarios, irrespective of the number of samples tested per year. In addition to the cost analysis, the diagnostic efficacies of the two assays were evaluated. To do this, a cohort of DFA-positive and -negative samples collected from dog meat markets in Nigeria were initially diagnosed using the DFA in Nigeria and subsequently sent to South Africa for diagnostic confirmation. In South Africa, all the specimens were re-tested with the DFA, the dRIT and a quantitative real-time polymerase chain reaction (qRT-PCR). In our investigation, discrepancies were observed between the three diagnostic assays; with the incongruent results being resolved by means of confirmatory testing using the heminested reverse transcription polymerase reaction and sequencing to confirm that they were not contamination. CONCLUSIONS/SIGNIFICANCE: The data obtained from this study suggested that the dRIT was not only an effective diagnostic assay that could be used to routinely diagnose rabies, but that the assay was also the most cost-effective option among all of the OIE recommended methods. In addition, the results of our investigation confirmed that some of the dogs slaughtered in dog markets were rabies-positive and that the markets posed a potential public health threat. Lastly, our data showed that the DFA, although regarded as the gold standard test for rabies, has some limitations-particularly at low antigen levels. Based on the results reported here and the current challenges faced in Nigeria, we believe that the dRIT assay would be the most suitable laboratory test for decentralized or confirmatory rabies diagnosis in Nigeria, given its relative speed, accuracy, cost and ease of use.

Clinical evaluation and cost analysis of a Trioplex real-time PCR assay for the detection and differentiation of herpes simplex virus 1 and 2 in cutaneous and mucocutaneous lesions.

PURPOSE: Herpes simplex virus (HSV) is a common lifelong sexually transmitted infection. HSV-1 typically manifests as oral cold sores, while HSV-2 is more traditionally associated with sexual transmission and infection. We have developed a real-time PCR (Trioplex) for the simultaneous detection of HSV-1 and -2 and the bacterial phage internal control (IC) MS2. METHODOLOGY: To determine the performance of the Trioplex method and resolve discrepancies, 178 clinical specimens from cutaneous and mucocutaneous sources were tested using 3 different methods; virus culture with direct fluorescent antibody (DFA) immunostaining, Trioplex and a commercially available HSV analyte-specific reagent (ASR). RESULTS: HSV Trioplex was significantly more sensitive than virus culture (89 and 67 % HSV 1/2, respectively) and comparable to the commercial assay (P<0.001). Cost analysis revealed that the Trioplex reduced cost by 80  % compared to cell culture. CONCLUSIONS: The implementation of the HSV Trioplex improved the detection turnaround time from 3-10 days to 2.5 h, thus streamlining Herpes detection, improving sensitivity and reducing laboratory costs.

Comparative cost-effectiveness of immunoassays and FLOTAC for diagnosing Giardia spp. infection in dogs.

BACKGROUND: Giardia spp. is a protozoan pathogen and is the most common enteric parasite of domestic animals and humans. Assays for detecting infection in fecal samples using direct or indirect examinations are important tools for diagnosing the disease. The objective of the present study was to compare the cost-effectiveness of immunoassays and FLOTAC technique for diagnosing Giardia spp. infection in dogs. RESULTS: Fecal samples from 80 positive stray dogs were tested for the presence of copro-antigens of Giardia spp. using the direct immunofluorescence assay (IFA), a rapid enzyme-linked immunosorbent assay (ELISA) and the FLOTAC double technique. All methods were performed in accordance with the instructions reported in the original description for each technique. The results showed that ELISA can be run in less time than IFA and almost at the same time of the FLOTAC technique. Among the tests used in this study, FLOTAC had the lowest cost per correct diagnosis, compared with immunoassays. CONCLUSIONS: The results from this cost-effectiveness analysis, in combination with the sensitivity and specificity of the FLOTAC technique, suggest that the FLOTAC technique can be use in the routine diagnosis of Giardia spp. infection in dogs.

Performance evaluation of direct fluorescent antibody, Focus Diagnostics Simplexa™ Flu A/B & RSV and multi-parameter customized respiratory Taqman® array card in immunocompromised patients.

BACKGROUND: Molecular assays for diagnosis of Flu A, Flu B, and RSV with short turn-around-time (TAT) are of considerable clinical importance. In addition, rapid and accurate diagnosis of a large panel of viral and atypical pathogens can be crucial in immunocompromised patients. OBJECTIVES: First, to evaluate the performance of the Simplexa™ Direct assay system in comparison with direct fluorescent antibody (DFA) and customized Taqman® Array Card (TAC) testing for RSV, Flu A, and Flu B in immunocompromised patients. Second, to evaluate different algorithms for the detection of respiratory pathogens in terms of cost, turn-around-time (TAT) and diagnostic yield. STUDY DESIGN: We collected 125 nasopharyngeal swabs (NTS) and 25 BAL samples from symptomatic immunocompromised patients. Samples for which Simplexa™ and TAC results were discordant underwent verification testing. The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. RESULTS: The overall sensitivity was significantly lower for DFA testing than for the two molecular methods (p<0.05). Performance characteristics of Simplexa™ testing were not significantly different compared to TAC testing (p>0.1). For BAL samples only, the sensitivity and specificity of the Simplexa™ assay was 100%. In total, 6.7, 16 and 18% of samples were positive for Flu A, Flu B or RSV by DFA, Simplexa™ and TAC testing, respectively. When considering not only these pathogens but also all results for TAC, the method identified 93 samples with one or more respiratory pathogens (62%). A co-infection rate of 15.3% was found by TAC. The estimated costs and TAT were 8.2€ and 2h for DFA, 31.8€ and 1.5h for Simplexa™ and 55€ and 3h for TAC testing. CONCLUSIONS: Performing the Simplexa™ test 24h a day/7 days a week instead of DFA would considerably improve the overall sensitivity and time-to-result, albeit at a higher cost generated in the laboratory. Performing the TAC would increase the diagnostic yield and detection of co-infections significantly.

Comparative assessment of seller's staining test (SST) and direct fluorescent antibody test for rapid and accurate laboratory diagnosis of rabies.

BACKGROUND: Rabies causes 55, 000 annual human deaths globally and about 10,000 people are exposed annually in Nigeria. Diagnosis of animal rabies in most African countries has been by direct microscopic examination. In Nigeria, the Seller's stain test (SST) was employed until 2009. Before then, both SST and dFAT were used concurrently until the dFAT became the only standard method. OBJECTIVE: This study was designed to assess the sensitivity and specificity of the SST in relation to the 'gold standard' dFAT in diagnosis of rabies in Nigeria. METHODS: A total of 88 animal specimens submitted to the Rabies National Reference Laboratory, Nigeria were routinely tested for rabies by SST and dFAT. RESULTS: Overall, 65.9% of the specimens were positive for rabies by SST, while 81.8% were positive by dFAT. The sensitivity of SST in relation to the gold standard dFAT was 81.0% (95% CIs; 69.7% - 88.6%), while the specificity was 100% (95% CIs; 76% - 100%). CONCLUSION: The relatively low sensitivity of the SST observed in this study calls for its replacement with the dFAT for accurate diagnosis of rabies and timely decisions on administration of PEP to prevent untimely deaths of exposed humans.

Valoración de exposición y endemicidad de Toxoplasma gondii en donantes de sangre en la provincia de Holguín, Cuba / Assessment of exposure and endemicity of Toxoplasma gondii among blood donors from the province of Holguín, Cuba

INTRODUCCIÓN: la toxoplasmosis es la enfermedad parasitaria más difundida en el mundo que afecta al hombre, descrita hace poco más de 100 años y producida por Toxoplasma gondii. Una de las formas que el hombre adquiere la enfermedad es a través de la placenta, de órganos trasplantados y por transfusiones de sangre; la forma infectante del parásito denominada "taquizoitos" es la responsable de este tipo de infección y ocurre durante la fase hematógena del mismo en un individuo seropositivo a T gondii. En Cuba al igual que en otras partes del mundo se ha demostrado la presencia de este parásito; en la provincia de Holguín su circulación se ha confirmado en receptores de trasplante renal. OBJETIVOS: conocer la seroprevalencia en donantes de sangre que motivó el interés para la realización de este trabajo. MÉTODOS: se evaluaron 892 muestras de sueros de donantes de los 14 municipios de la provincia de Holguín, el comportamiento serológico se determinó por la Técnica de Inmunofluorescencia Indirecta. RESULTADOS: la seropositividad para Inmunoglobulina G, anti Toxoplasma gondii de un 38,2 %, CONCLUSIONES: los donantes de la provincia de Holguín, están expuesto al Toxoplasma gondii, donde existe endemicidad del parásito en todo sus municipios y queda demostrado que los individuos procedentes de áreas rurales, tienen mayor incidencia de seropositivos al Toxoplasma gondii que los de áreas urbanas.

INTRODUCTION: Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii. First described a little over 100 years ago, it is the most widely distributed parasitic disease affecting humans. The disease may be acquired from the placenta, from transplanted organs or from blood transfusions. The infecting form of the parasite, known as "tachyzoite", is responsible for this type of infection, which occurs during the hematogenous stage in a T. gondii-seropositive individual. The presence of this parasite has been demonstrated both in Cuba and in other regions of the world. In the province of Holguín its circulation has been confirmed in renal transplant recipients. OBJECTIVES: The purpose of the present study was to determine the seroprevalence of toxoplasmosis among blood donors. METHODS: An evaluation was conducted of 892 serum samples from donors from the 14 municipalities in the province of Holguín. Serological behavior was determined by indirect immunofluorescence technique. RESULTS: Seropositivity for anti-Toxoplasma gondii Immunoglobulin G was found to be 38.2 %. CONCLUSIONS: Blood donors from the province of Holguín are exposed to Toxoplasma gondii. The parasite is endemic in all municipalities, and it has been shown that Toxoplasma gondii seropositivity is higher in rural areas than in urban areas.

A Survey of Oral Cancer Screening Insurance Coverage in New York City.

Clinical studies show that fewer than 25% of people who visit a dentist regularly are screened for oral cancer, and that the majority of oral cancers present at an advanced stage, when cure rates are already abysmal. This study explores the current status of oral cancer screening coverage among a variety of insurance providers in New York City. The study focuses on determining the coverage and frequency of the cluster of salient CDT (dental) codes surrounding oral cancer screenings.

Diagnostic evaluation of rapid tests for scrub typhus in the Indian population is needed.

BACKGROUND: Owing to frequent outbreaks witnessed in different parts of the country in the recent past, scrub typhus is being described as a re-emerging infectious disease in India. Differentiating scrub typhus from other endemic diseases like malaria, leptospirosis, dengue fever, typhoid, etc. is difficult due to overlapping clinical features and a lower positivity for eschars in Asian populations. Hence, the diagnosis heavily relies on laboratory tests. DISCUSSION: Costs and the need of technical expertise limit the wide use of indirect immunoperoxidase or immunofluorescence assays, ELISA and PCR. The Weil-Felix test is the most commonly used and least expensive serological test, but lacks both sensitivity and specificity. Hence, the diagnosis of scrub typhus is often delayed or overlooked. With due consideration of the cost, rapidity, single test result and simplicity of interpretation, rapid diagnostic tests have come into vogue. However, evaluation of rapid diagnostic tests for scrub typhus in the Indian population is needed to justify or discourage their use. CONCLUSION: Research studies are needed to find the most suitable test in terms of the rapidity of the result, simplicity of the procedure, ease of interpretation and cost to be used in the Indian populace.

Detección de anticuerpos circulantes en biopsia de tejido para el diagnóstico de dermatitis herpetiforme / Detection of circulating antibodies in tissue biopsy for the diagnosis of dermatitis herpetiformis

Introducción: la dermatitis herpertiforme (DH), es una enfermedad crónica inflamatoria de la piel. Se caracteriza por la presencia de lesiones pruriginosas de diversas formas y de hallazgos histopatológicos típicos. Se ha considerado como la expresión cutánea de la enteropatía sensible al gluten (ESG), indistinguible de la enfermedad celiaca. Es una entidad con baja prevalencia y se presenta con más frecuencia en la tercera década de la vida. Afecta principalmente a individuos caucásicos. El diagnóstico de realiza a través de la detección de un depósito granular de Inmunoglobulina A (IgA) en la unión dermoepidérmica, a través de un estudio de inmunofluorescencia directa (IFD) de una muestra de piel sana perilesional. Estos depósitos con frecuencia se localizan en las puntas de las papilas dérmicas. La negatividad del estudio de inmunofluorescencia directa debe hacer dudar del diagnóstico. El tratamiento consiste en mantener una dieta estricta libre de gluten, el manejo de las lesiones y la prescripción de dapsona para el manejo de las erupciones cutáneas. Objetivo: evaluar la utilidad de la detección de anticuerpos circulantes en biopsia de tejido para el diagnóstico de la dermatitis herpetiforme. \r\nResultados: se identificaron 174 publicaciones. Con los resultados obtenidos, no fue posible identificar revisiones sistemáticas de la literatura ni estudios de validez diagnóstica de la IFD. Se hizo una preselección de 18 estudios observacionales descriptivos. Fueron incluidos nueve series de casos. Se presentan los datos descriptivos sobre la positividad de la IFD para el diagnóstico de la DH. Conclusiones: \r\nactualmente se considera que la IFD es el patrón de oro para el diagnóstico de la DH.(AU)

An assessment of zoonotic and production limiting pathogens in rusa deer (Cervus timorensis rusa) from Mauritius.

A population of approximately 70,000 rusa deer (Cervus timorensis russa) represents the most important mammal species reared for food on the island of Mauritius, being the main source of red meat for the local population. However, very limited information is available on the circulation of pathogens affecting the productivity and health of this species. To produce baseline data on the circulation of infectious pathogens in rusa deer under production, a serological survey and/or direct pathogen detection for six selected infectious diseases was undertaken in 2007 in a sample of 53% of the herds reared in semi-free-ranging conditions in hunting estates. Seropositive results were recorded for Johne's disease with an indirect ELISA test (1.7%, n = 351), heartwater with an immunofluorescence antibody test (IFAT) (95.5%, n = 178) and leptospirosis with a Microscopic Agglutination Test (MAT) (25.9%, n = 363). Significant associations were found between seroprevalence to some of the leptospiral serogroups detected (Tarassovi, Pomona, Sejroe and Mini) and age of the animals, animal density or location of the estates (being more prevalent in hotter and more humid areas). In addition, Mycobacterium bovis and M. avium subspecies paratuberculosis were confirmed in two deer carcasses by culture and PCR, respectively. No antibodies against Brucella spp. nor Rift Valley Fever virus were detected with the use of respective indirect ELISA's. The results obtained suggest that the population of rusa deer from Mauritius is exposed to a wide range of pathogens which may affect their productivity. In addition, the results highlight the potential public health risks incurred by deer industry workers and consumers. This survey fills an important gap in knowledge regarding the health of tropical deer meat in Mauritius and justifies the need to implement more regular surveys of selected pathogens in the deer population.

Assessment of risk of possible exposure to rabies among processors and consumers of dog meat in Zaria and Kafanchan, Kaduna state, Nigeria.

Canine rabies is endemic in Nigeria. Some of the dogs slaughtered for human consumption may be infected with rabies virus, thus exposing handlers of raw dog meat to the disease since the virus may be present in the nerves in the meat. A cross-sectional study was designed and a structured questionnaire was designed and administered to a convenience sample of 160 processors and consumers (100 from Zaria and 60 from Kafanchan), by face to face interview at the slaughter sites or dog meat sale points. The questionnaire sought information on demographic characteristics of the respondents, rabies knowledge, attitude and actions the respondents would take if exposure occurs. Associations between demographic variables and categorized knowledge, attitude or practice scores were assessed using x2 analysis. The relationship between non-categorized scores was assessed using multiple regression analysis. Also, 154 brain samples from slaughtered dogs (74 from Zaria and 80 from Kafanchan) were checked for rabies antigen using direct fluorescent antibody test. Of the 160 respondents, 49 (30.6%) were involved in the slaughtering and sale of dog meat while 111(69.4%) were involved in handling and consumption of processed dog meat. Only 123(76.9%) knew that dogs are common source of rabies in Nigeria and 105(65.6%) knew that rabies affect humans. Also 110(68.8%) did not have adequate knowledge of the clinical signs of rabies. The level of knowledge, having positive attitudes and knowing acceptable practices were directly proportional to the level of education. Respondents from Kafanchan had higher level of knowledge and more positive attitudes towards rabies than those from Zaria. There were significant correlations between knowledge and attitude scores (r=0.49) and between knowledge and practice scores (r=0.43) at p<0.001. Rabies antigen was detected in the brain of 6 (3.9%) of the slaughtered dogs. The findings indicate that processors and consumers of dog meat are deficient in the knowledge of rabies. There is therefore a need for educational programmes targeted at this high risk group to increase their level of knowledge and reduce the risk of exposure.

A survey strategy for human respiratory syncytial virus detection among haematopoietic stem cell transplant patients: epidemiological and methodological analysis.

Human respiratory syncytial virus (HRSV) causes severe infections among children and immunocompromised patients. We compared HRSV infections among Haematopoietic Stem Cell Transplant program (HSCT) patients and children using direct immunofluorescence (DFA), point-of-care RSV Bio Easy® and a polymerase chain reaction (PCR) assay. Overall, 102 samples from HSCT patients and 128 from children obtained positivity rate of 18.6% and 14.1% respectively. PCR sensitivity was highest mainly on samples collected after five days of symptoms onset. A combination of both DFA and reverse transcriptase-PCR methods for HSCT high-risk patients is the best diagnostic flow for HRSV diagnosis among these patients.

A survey strategy for human respiratory syncytial virus detection among haematopoietic stem cell transplant patients: epidemiological and methodological analysis

Human respiratory syncytial virus (HRSV) causes severe infections among children and immunocompromised patients. We compared HRSV infections among Haematopoietic Stem Cell Transplant program (HSCT) patients and children using direct immunofluorescence (DFA), point-of-care RSV Bio Easy® and a polymerase chain reaction (PCR) assay. Overall, 102 samples from HSCT patients and 128 from children obtained positivity rate of 18.6% and 14.1% respectively. PCR sensitivity was highest mainly on samples collected after five days of symptoms onset. A combination of both DFA and reverse transcriptase-PCR methods for HSCT high-risk patients is the best diagnostic flow for HRSV diagnosis among these patients.

Accuracy of rapid influenza diagnostic test and immunofluorescence assay compared to real time RT-PCR in children with influenza A(H1N1)pdm09 infection.

INTRODUCTION: The influenza burden among children is underestimated. The aim of our study was to estimate the accuracy of the rapid influenza detection test (RIDT) BD Directigen™ EZ Flu A+B® and direct immunofluorescence assay (DFA) used among children with influenza-like illness (ILI) consulted in the ambulatory care clinic. MATERIAL/METHODS: A total of 150 patients were enrolled in the study. Inclusion criteria were: age less than 59 months, presentation of ILI according to the CDC (Centers for Disease Control and Prevention) definition (fever >37.8°C, cough and/or sore throat in the absence of another known cause of illness), duration of symptoms shorter than 96 hours. Two nasal swabs and one pharyngeal swab were obtained from patients and tested by RIDT, DFA and real time RT-PCR as the reference method. RESULTS: For influenza A (H1N1)pdm09 virus sensitivity of RIDT was 62.2% (95% CI 46.5-76.2%), specificity 97.1% (95% CI 91.8-99.4%), PPV 90.3% (95% CI 74.3-98%), NPV 85.7% (95% CI 78.1-91.5%), for DFA sensitivity was 60% (95% CI 51.9-63.2%), specificity 96% (95% CI 88.7-98.8%), PPV 93.1% (95% CI 80.5-98%), NPV 72.7% (95% CI 67.2-74.9%). Analysis of logistic regression revealed that the chance of receiving a true positive result of RIDT was twice as high when the test was conducted during the first 48 hours of symptoms (OR 0.40 vs OR 0.22). CONCLUSIONS: The accuracy of RIDT is comparable with DFA and both methods are very specific but moderately sensitive in diagnosis of influenza in young children. Both methods may be recommended for screening for influenza among children.

Re-assessment of direct fluorescent antibody negative brain tissues with a real-time PCR assay to detect the presence of raccoon rabies virus RNA.

The first report of the raccoon variant of rabies virus was in Ontario, Canada in 1999. As part of the control of this outbreak a Point Infection Control (PIC) strategy of trapping and euthanizing vector species was implemented. To evaluate whether this strategy was indeed removing diseased animals, rabies diagnosis was performed on these specimens. During a PIC program conducted in 2003, 721 animals (raccoons, striped skunks and red foxes) were collected and euthanized and brain material from each specimen was divided into two halves; one half was submitted for rabies diagnosis by a direct fluorescent antibody (DFA) test while the other was tested using a sensitive real-time reverse-transcriptase polymerase chain reaction (RT-qPCR), to detect raccoon rabies virus (RRV) RNA. This latter assay can detect less than ten viral copies in 200ng of total cellular RNA. All 721 PIC brain samples were negative by the DFA test but ten of them (5 raccoons, 5 skunks) tested positive for raccoon rabies virus by the RT-qPCR assay albeit at low levels. Three of these samples were confirmed by sequencing of the PCR products. Little correlation was observed between clinical rabies DFA positive scoring categories and viral copy number as determined by RT-qPCR.

Accurate estimation of vascular endothelial growth factor levels in microsamples with a low-cost bead-based assay.

PURPOSE: The purpose of this study was to evaluate the accuracy of measurement of a known concentration of vascular endothelial growth factor (VEGF) using cytometric bead-based assay in microsamples (50 microL) and compare it with that obtained by enzyme-linked immunosorbent assay. METHODS: A known concentration of VEGF samples was prepared with addition of VEGF (100 pg/mL) to 1 mL of Hank balanced salt solution. This preparation was further diluted serially to 50, 25, and 12.5 pg/mL; and VEGF estimation was done on each of these samples using Luminex, a commercially available cytometric bead-based assay and enzyme-linked immunosorbent assay. The assay was replicated three times at each dilution. RESULTS: The mean concentrations of VEGF measured using Luminex were 97.7 +/- 6.2, 47.3 +/- 8.1, 24.9 +/- 2.2, and 14.2 +/- 1.8 pg/mL at 100, 50, 25, and 12.5 pg/mL dilutions, respectively. Similarly, the mean concentrations of VEGF measured using enzyme-linked immunosorbent assay for equivalent dilutions were 100.6 +/- 24.4, 53.8 +/- 16.8, 44.8 +/- 35.5, and 14.19 +/- 0.7 pg/mL, respectively. CONCLUSION: Bead-based assay accurately measures VEGF concentration in a microsample and has better validity and reliability as compared with enzyme-linked immunosorbent assay.

The burden of respiratory syncytial virus bronchiolitis on a pediatric inpatient service.

BACKGROUND: Respiratory syncytial virus bronchiolitis is the single leading cause of pediatric admissions for infants in the first year of life, presenting regularly in epidemic proportions in the winter months and impacting in major way on pediatric inpatient services. OBJECTIVES: To quantitate the burden of RSV disease on a pediatric service with the purpose of providing a database for proper health planning and resource allocation. METHODS: We conducted a prospective 5 year study of documented RSV infections in a single pediatric service. RSV disease was confirmed by direct immunofluorescence testing of nasal swabs from all hospitalized cases of bronchiolitis. RESULTS: On average, 147 17 cases of RSV bronchiolitis were admitted annually in the November-March RSV season, representing 7%-9% of admissions and 10%-14% of hospital days. There was a consistent male preponderance of admissions (55-64%) and 15-23% of admissions were patients less than 1 month old. In peak months RSS ses accounted for as many of 40% of the hospitalized infants and was the leading cause of over-occupancy (up to 126%) in the pediatric ward during the winter. CONCLUSIONS: RSV infection is a major burden for pediatric inpatient services during the winter season. This recurrent and predictable "epidemic," which regularly leads to over-occupancy, requires increased manpower (nursing) and resources (beds, pulse oximeters) to facilitate proper care. Since this annual event is not a surprise nor an unexpected peak, but rather a cyclical predictable epidemiological phenomeon, proper planning and allocation of services are crucial.