Expert consensus on organ donation from patients infected with novel coronavirus in China / 器官移植

The spread, prevention and control of novel coronavirus infection and the potential risks and uncertainties of novel coronavirus transmission from donor to recipient have brought serious impacts and great challenges to organ donation and transplantation. There is increasing evidence that the use of non-pulmonary organs (kidney, liver and heart) from novel coronavirus infected donors carries a low risk of transmission, regardless of whether they were symptomatic at the time of acquisition. Delaying organ donation after the death of those who are positive for novel coronavirus antigen or nucleic acid testing, and then waiting until turns negative, will result in the discarding of a significant number of organs that are medically suitable for transplantation. In order to maximally meet the demand for transplantation in patients with end-stage organ failure, Branch of Organ Transplantation of Chinese Medical Association organized relevant experts formulated the "Expert consensus on organ donation from patients infected with novel coronavirus in China" after citizen' s death by taking into account the epidemic situation of novel coronavirus infection in China and the clinical practice of organ donation and transplantation, and by referring to relevant research results and clinical research evidence at home and abroad. It aims to provide recommendations and references for the procurement and application of donor organs from patients infected with novel coronavirus.

Establishment and application of sandwich ELISA for detection of specific antigen of Seoul virus L99 strain / 中华微生物学和免疫学杂志

Objective:To establish a double antibody sandwich ELISA for detecting the specific antigen of Seoul virus (SEOV) L99 strain and to provide a means for antigen detection in the development, production and verification of vaccine against hemorrhagic fever with renal syndrome (HFRS).Methods:Monoclonal antibodies (McAbs) aganist L99 virus were induced in mice using four hybridoma cell lines and purified by Protein-A affinity chromatography. The purity, titer and specificity of McAbs were determined by SDS-PAGE, indirect ELISA and Western blot, respectively. Four McAbs were paired with each other and the additivity indices of paired McAbs were analyzed. After labeling McAbs with horseradish peroxidase (HRP), the concentrations of the coated and labeled antibodies were optimized by orthogonal test, and then a double antibody sandwich ELISA for virus antigen detection was established. Type Ⅱ HFRS inactivated vaccine standard was used as a quantitative standard to verify the sensitivity, linearity, specificity, accuracy and precision of the developed method. The applicability of the method was verified by testing three batches of vaccine stock solutions.Results:Four McAbs were at titers of greater than 1∶10 6 and their purity was all greater than 98%. The McAbs secreted by 1D5, 3A4 and 5B7 cells could specifically recognize the nucleocapsid protein of SEOV L99. There was cross-reaction between McAb secreted by 1D5 cells and Hantaan virus PS-6. The McAbs secreted by 3A4 and 1D5 were used as coating and labeling antibodies based on the results of antibody pairs. The working concentrations of the coating antibody and the horseradish peroxidase (HRP)-labeled antibody were 20 μg/ml and 1∶4 000, respectively. The minimum detection limit of the established method for the detection of SEOV L99 antigen was 0.078 1 μg/ml, and the linear range was 0.078 1-2.500 0 μg/ml with a R2 value of more than 0.99. There was no cross reaction with other HFRS vaccine. The virus antigen recovery rate was between 95.8% and 108.7%, and the coefficients of variation of precision was less than 10%. Three batches of Type II HFRS inactivated vaccine stocks were detected by this method and the results was dose-dependent. Conclusions:This study successfully established a double antibody sandwich ELISA method for specific detection of SEOV L99 strain antigen in the production of bivalent HFRS vaccines produced from hamster kidney cells.

Evaluation of the SD Bioline Strep A Ultra Test in Relation With Number of Colony Forming Units and Color Intensity

BACKGROUND: The SD Bioline Strep A Ultra (SD, Yongin, Korea) is a recently developed rapid antigen detection test (RADT) for diagnosing bacterial pharyngitis caused by Group A Streptococcus, We evaluated the performance of SD Bioline Strep A Ultra, using the number of colony forming units and color intensity. METHODS: Three throat swabs each were taken from 343 children with pharyngitis who visited pediatric clinics. We evaluated the performance of SD Bioline Strep A Ultra and compared its positive rate with the number of colony forming units, using the Fisher exact test. RESULTS: The sensitivity, specificity, positive predictive value, and negative predictive value (95% confidence interval) were 97.4% (94.0–99.1%), 90.8% (85.0–94.9%), 93.0% (88.5–96.1%), and 96.5% (92.0–98.9%), respectively. Positive rate significantly differed by number of colony forming units (P=0.021). ROC plot for color intensity showed 0.938 of AUC (area under curve). CONCLUSIONS: SD Bioline Strep A Ultra showed excellent performance, and its positive rate differed by the number of colony counts. This RADT could be used as a sensitive and semi-quantitative method detecting bacterial pharyngitis.

Research progress of detective techniques for influenza virus infections / 中华实用儿科临床杂志

Influenza is one of the most common and widespread respiratory diseases in humans.In addition to pose a serious threat to the susceptible and high-risk population,influenza has an important impact on public health.Rapid and accurate diagnosis of influenza virus infections is essential for administrating prompt antiviral treatments and undertaking effective prevention and control measures during seasonal epidemics and pandemics,which to reduce morbidity and mortality associated with influenza virus infections.Several different approaches are currently available for diagnosis of influenza infections in humans,including virus isolation,antigen detections,nucleic acid amplifications,etc.New diagnostic methods are being developed to overcome the limitations of some traditional detection methods.Here is an overview of diagnostic techniques for influenza virus infection.

Expanded diagnostic approach to hepatitis E virus detection in patients with acute-on-chronic liver failure: A pilot study

Introduction: Acute decompensation of pre-existing chronic liver disease (CLD), known as acute-on-chronic liver failure (ACLF), is associated with high mortality. Hepatitis E virus (HEV) as a potential cause was studied. Objectives: The objectives of this study are to evaluate the role of HEV in ACLF patients using an IgM anti-HEV antibody enzyme-linked immunosorbent assay (ELISA), HEV antigen ELISA, and a quantitative HEV polymerase chain reaction (PCR). Materials and Methods: In this prospective cross-sectional study, blood samples were collected from 50 ACLF (cases) as defined by the standard guidelines (APASL, 2014) and 50 patients with stable CLD (controls) from January 2015 to August 2016, after obtaining informed consent. Two IgM ELISAs (MP Diagnostics HEV IgM ELISA 3.0, Singapore and Wantai HEV IgM ELISA, Beijing, China) were compared using plasma from cases and controls. In addition, an HEV antigen detection by ELISA (Wantai, Beijing, China) and a real-time PCR for quantification of HEV RNA in plasma and stool were employed. Results: Ethanol was the leading cause of acute insult in ACLF (54%) cases. HEV infection accounted for 20% of cases. Ten ACLF patients (20%) had 1–3 markers of HEV versus two (4%) among controls (P = 0.0138). Among ACLF cases, one had HEV viraemia (403 IU/ml), faecal shedding (2790 IU/ml) and detectable HEV antigenaemia. Agreement between the two anti-HEV IgM ELISAs was 0.638 (kappa value). Conclusion: This study shows that alcohol is a major contributing factor for both underlying CLD and ACLF while HEV is the most common infectious cause for ACLF, suggesting a need for a vaccination in such patients, whenever made available.

Estudio Prospectivo Observacional: Determinar la Sensibilidad del Criterio Clínico usando la Escala de "Centor" versus el Test de Detección Rápida de Estreptococo para el diagnóstico de Faringitis Estreptocócica del Grupo A en el Centro de Salud Bárbara / Observational Prospective Study: Determine the Sensitivity of the Clinical Criteria using the "Centor" Scale versus the Rapid Streptococcal Screening Test for the diagnosis of Group A Streptococcal Pharyngitis at the Bárbara Health Center

Introducción: Las faringitis producidas por el estreptococo beta hemolítico del grupo A no se pueden distinguir clínicamente de las faringitis producidas por otros gérmenes, sin embargo la utilización de los criterios de Centor y el test de detección rápida de antígeno son de gran utilidad para determinar las probabilidades que estos sean causados por el estreptococo beta hemolítico del grupo A. En este estudio se comparó la sensibilidad entre ambos métodos. Objetivos: Se realizó un estudio para determinar la sensibilidad del criterio clínico en el diagnóstico de faringitis causada por Estreptococo en comparación a la sensibilidad del test de detección rápida de antígeno. Metodología: En el Centro de Salud Bárbara, se tomaron a los pacientes pediátricos que consultaron por dolor de garganta durante dos meses. Se puntuó según la escala de Centor y se tomó una muestra para el test de detección rápida de antígeno, luego, se comparó con el cultivo de orofaringe. Resultados: Se comparó la sensibilidad de ambos parámetros. Discusión: Un puntaje ≥ 3 puntos en la escala de Centor tuvo una sensibilidad de 81.8% y especificidad de 50%. Mientras que el RADT presentó una sensibilidad del 83.3% y especificidad de 84.2%.

Introduction: Pharyngitis caused by group A beta-hemolytic streptococci cannot be distinguished clinically from pharyngitis caused by other germs, however the use of the Centor criteria and the rapid antigen detection test are very useful to determine this pathogen. These are likely to be caused by group A beta hemolytic streptococcus. In this study the sensitivity between the two methods were compared. Objectives: A study was conducted to determine the sensitivity of clinical criteria in the diagnosis of pharyngitis caused by Streptococcus in comparison to the sensitivity of the rapid antigen detection test. Methodology: In the Barbara Health Center, pediatric patients who consulted for sore throat for two months were taken. It was scored according to the Centor scale and a sample was taken for the rapid antigen detection test, later these were compared with the oropharynx culture. Results: The sensitivity of both parameters were compared. Discussion: A score ≥ 3 points on the Centor scale had a sensitivity of 81.8% and specificity of 50%. While the RADT presented a sensitivity of 83.3% and specificity of 84.2%.

Foreigners To Pulp And Periodontium - Report Of 2 Cases

Presence of a foreign object embedded in and around a tooth is unusual. Such object may get lodged and become a source of pain and infection, causing the patient to present to the dentist. This paper present two such case reports of foreign body imbedded within or around the tooth. One of the reported cases is the first case in which staple pin is present in primary molar. Present article emphasizes upon maintenance of oral hygiene and regular dental check up can prevent such undesirable situation.

Rapid Antigen Testing For Strep Sore Throat

This was a cross sectional study to estimate the prevalence of Group AStreptococcal (GAS) infection among children with acute sore throat and to compare results of Rapid antigen detection test (RADT) with throat culture. Children aged 3-15 years who presented with acute sore throat (throat pain ± redness of pharynx, palate, tonsils), whose parents were willing to participate in the study by giving a written consent were included. Two sterile throat swabs were taken by vigorously rubbing the tonsils or posterior pharyngeal wall, one for RADT (cerTEST Strep Acard test) and 2nd for bacterial culture. The samples were sent to the in-hospital NABLcertified laboratory (SRLLtd). The results of RADT were obtained within 15 minutes while the culture report was available after 72 hours. During the 7 months study period we took 90 throat samples from 86 children. Of these 26 were RADT positive and 22 were culture positive. The prevalence of GAS by RADT was 28.88% and by culture was 25.56%. The sensitivity and specificity of RADTwas 95.65% and 94.02 % respectively. Since the RADThad high sensitivity and specificity and the results were available within 15 minutes, the need for throat swab culture (with additional cost and delay in results) could be avoided. Appropriate antibiotic may be started on the basis of RADT. If RADTis negative culture should be sent.

Usefulness of Centor Score to Diagnosis of Group a Streptococcal Pharyngitis and Decision Making of Antibiotics Use / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: This study aims to verify the usefulness of Centor scores to diagnose the Group A Streptococcal pharyngitis. SUBJECTS AND METHOD: The subjects of this study were 379 patients who had been examined by the rapid antigen detection test (RADT) for Group A Streptococcus. We analyzed their medical records and laboratory test results retrospectively and compared the results of Centor symptom scores with those of RADT. Then we analyzed the association of RADT, the Centor score and the laboratory test results statistically. RESULTS: There were no correlation between the RADT results and fever, cough, tonsillar enlargement, nasal symptoms, myalgia or chilling (p>0.05). In the RADT positive group, there were more patients with tonsillar exudate, neck lymph node enlargement, tenderness and pharyngeal abscess formation significantly (p<0.05). The Centor score and C-reactive protein were significantly higher in the RADT positive group (p<0.05). CONCLUSION: The results of this study suggest that Centor symptom scores can be used to determine which antibiotics to use. The Centor score system can help reduce medical costs and detect the problematic Group A Streptococcal pharyngitis.

In silico analysis of amino acid variation in human respiratory syncytial virus: insights into immunodiagnostics

BACKGROUND The highly contagious nature of human respiratory syncytial virus (HRSV) and the gravity of its infection in newborns and vulnerable adults pose a serious public health problem. Thus, a rapid and sensitive diagnostic test for viral detection that can be implemented upon the first appearance of symptoms is needed. The genetic variation of the virus must be considered for immunodiagnostic purposes. OBJECTIVES To analyse HRSV genetic variation and discuss the possible consequences for capture immunoassay development. METHODS We performed a wide analysis of N, F and G protein variation based on the HRSV sequences currently available in the GenBank database. We also evaluated their similarity with homologous proteins from other viruses. FINDINGS The mean amino acid divergences for the N, F, and G proteins between HRSV-A and HRSV-B were determined to be approximately 4%, 10% and 47%, respectively. Due to their high conservation, assays based on the full-length N and F proteins may not distinguish HRSV from human metapneumovirus and other Mononegavirales viruses, and the full-length G protein would most likely produce false negative results due to its high divergence. MAIN CONCLUSIONS We have identified specific regions in each of these three proteins that have higher potential to produce specific results, and their combined utilisation should be considered for immunoassay development.

Rapid antigen detection test for respiratory syncytial virus diagnosis as a diagnostic tool / Teste rápido de detecção de antígenos para o diagnóstico do Vírus Sincicial Respiratório como ferramenta de diagnóstico

Abstract Objective: The aim of this study was to evaluate the QuickVue® RSV Test Kit (QUIDEL Corp, CA, USA) as a screening tool for respiratory syncytial virus in children with acute respiratory disease in comparison with the indirect immunofluorescence assay as gold standard. In Brazil, rapid antigen detection tests for respiratory syncytial virus are not routinely utilized as a diagnostic tool, except for the diagnosis of dengue and influenza. Methods: The authors retrospectively analyzed 486 nasopharyngeal aspirate samples from children under age 5 with acute respiratory infection, between December 2013 and August 2014, the samples were analyzed by indirect immunofluorescence assay and QuickVue® RSV Test kit. Samples with discordant results were analyzed by real time PCR and nucleotide sequencing. Results: From 313 positive samples by immunofluorescence assays, 282 (90%) were also positive by the rapid antigen detection test, two were positive only by rapid antigen detection test, 33 were positive only by immunofluorescence assays, and 171 were positive by both methods. The 35 samples with discordant results were analyzed by real time PCR; the two samples positive only by rapid antigen detection test and the five positive only by immunofluorescence assays were also positive by real time PCR. There was no relation between the negativity by QuickVue® RSV Test and viral load or specific strain. The QuickVue® RSV Test showed sensitivity of 90%, specificity of 98.8%, predictive positive value of 99.3%, and negative predictive value of 94.6%, with accuracy of 93.2% and agreement κ index of 0.85 in comparison to immunofluorescence assay. Conclusions: This study demonstrated that the QuickVue® RSV Test Kit can be effective in early detection of Respiratory syncytial virus in nasopharyngeal aspirate and is reliable for use as a diagnostic tool in pediatrics.

Resumo Objetivo: Avaliar o teste QuickVue® RSV Test Kit (QUIDEL Corp, CA, EUA) para o diagnóstico rápido do vírus sincicial respiratório em crianças com doença respiratória aguda, comparandoo com a imunofluorescência indireta como padrão ouro. Visto que, no Brasil, testes rápidos para detecção de antígenos para vírus sincicial respiratório não são rotineiramente utilizados como ferramenta de diagnóstico, exceto para Dengue e Influenza. Métodos: Um total de 486 amostras de aspirado de nasofaringe de crianças menores de 5 anos com doença respiratória aguda, coletadas entre dezembro de 2013 e agosto de 2014, foram analisadas por imunofluorescência e pelo teste QuickVue®. Amostras com resultados discordantes entre os métodos foram submetidas a PCR em tempo real e sequenciamento. Resultados: Das 313 amostras positivas por IFI, 282 foram positivas no teste rápido (90%), 2 amostras foram positivas apenas no teste rápido (0.6%), 33 apenas na imunofluorescência (10.5%) e 171 foram negativas em ambos os métodos. As 35 amostras com resultados discordantes foram testadas por PCR em tempo real, sendo que duas que foram positivas apenas no teste rápido e 5 apenas na imunofluorescência confirmaram-se positivas. Não houve relação entre a ausência de positividade no teste QuickVue® com a carga ou com a cepa viral. O teste QuickVue® mostrou sensibilidade de 90.1%, especificidade 98.9%, valor preditivo positivo 99.3%, valor preditivo negativo de 94.6%, acurácia de 93.2% e índice de concordância de 0.85 em comparação à imunofluorescência. Conclusões: Nosso estudo demonstrou que o teste QuickVue® RSV pode ser efetivo na detecção precoce do vírus sincicial respiratório em amostras de aspirado de nasofaringe e é confiável como uma ferramenta de diagnósticos em pediatria.

Evaluación del desempeño de dos pruebas para la detección de antígeno de Helicobacter pylori en heces / Evaluation of the accuracy of two stool antigen tests for Helicobacter pylori infection

Introducción: la detección de antígeno en heces se ha considerado una prueba prometedora para el diagnóstico de la infección por Helicobacter pylori. Para su introducción en la práctica médica, es un requisito indispensable demostrar el desempeño adecuado del método en la población de estudio. Objetivo: evaluar la capacidad diagnóstica de los sistemas comerciales ELISA SD y SD BIOLINE, del fabricante Standard Diagnostics, Corea, en pacientes cubanos con síntomas gastroduodenales. Métodos: se evaluaron 101 muestras de heces de pacientes previamente clasificados como H. pylori positivos y negativos por las pruebas de referencia de histología y prueba rápida de la ureasa. Se calcularon los parámetros de desempeño de ambos sistemas diagnósticos por el programa EPIDAT 3.1. Resultados: la sensibilidad para los sistemas ELISA SD y SD BIOLINE fue de 85,25 por ciento y 75,41 por ciento, respectivamente. La especificidad para ambos fue de 92,50 por ciento. Los valores predictivos positivos y negativos, los índices de validez y de Youden y la confiabilidad diagnóstica de ambas pruebas fueron satisfactorios. Conclusiones: Los sistemas evaluados exhibieron un desempeño comparable con la histología y la prueba rápida de ureasa para la detección activa de la infección por H. pylori, lo que demuestra su utilidad para el diagnóstico y el manejo oportuno del paciente, sin la necesidad de emplear pruebas invasivas(AU)

Introduction: stool antigen tests have been considered to be promising for the diagnosis of Helicobacter pylori infection. For their incorporation into medical practice, it is indispensable to demonstrate their accuracy in a study population. Objective: evaluate the diagnostic capacity of the commercial systems ELISA SD and SD BIOLINE, Standards Diagnostics, Korea, in Cuban patients with gastroduodenal symptoms. Methods: an evaluation was conducted of 101 stool samples from patients previously classified as H. pylori positive and negative by reference histological tests and the rapid urease test. Estimation was made of performance parameters for both diagnostic systems using the software EPIDAT 3.1. Results: Sensitivity for the systems ELISA SD and SD BIOLINE was 85.25 percent and 75.41 percent, respectively. Specificity for both was 92.50 percent. Positive and negative predictive values, validity and Youden's indices, and diagnostic reliability were satisfactory for both tests. Conclusions: the systems evaluated were found to have a performance level comparable with histological tests and the rapid urease test for active detection of H. pylori infection. This confirms their usefulness for the diagnosis and timely management of patients without having to use invasive tests(AU)

Clinical value of a rapid respiratory syncytial virus antigen detection in point-of-care testing / 中华儿科杂志

Objective@#To evaluate the clinical value of a rapid respiratory syncytial virus (RSV) antigen detection in point-of-care testing (POCT).@*Method@#A total of 209 specimens, including 78 throat swabs (TS) and 131 nasopharyngeal aspirates (NPAs), were collected from inpatients who visited the Children′s Hospital Affiliated to the Capital Institute of Pediatrics and were diagnosed as acute respiratory infection from 5 January to 7 February, 2015. These specimens were tested for RSV by a rapid antigen detection kit which was compared with reverse transcription polymerase chain reaction (RT-PCR) and direct immunofluorescence assay (DFA) for RSV detection.@*Result@#Compared with DFA for NPAs, the sensitivity and specificity of rapid antigen detection were 83.9% and 97.3%, respectively, with Kappa value of 0.86; Compared with RT-PCR, the sensitivity (NPAs, 74.2%; TS, 77.8%) and specificity (NPAs, 100.0%; TS, 92.0%) of rapid antigen detection were high, too, with Kappa value of 0.74 in NPAs and 0.62 in TS. However, the RSV positive rate of rapid antigen detection in TS (21.7%) from pediatric patients with acute lower respiratory tract infection was lower than that in NPAs (78.3%), as well as that of RT-PCR (7.3% in TS verse 78% in NPAs). The RSV rapid antigen detection kit can be finished in about 10 minutes.@*Conclusion@#With characteristics of high specificity, high sensitivity, being rapid, efficient and easy to operate in comparison with DFA and RT-PCR, RSV rapid antigen detection in this study is suitable for POCT. For pediatric patients with acute respiratory tract infection, NPA was better than TS for RSV detection.

Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection

Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis.

Comparative evaluation of antigen detection ELISA and reverse transcriptase PCR in acute stage of Japanese encephalitis prevalent in endemic areas of North-Eastern part of Uttar Pradesh, India.

Background: Objective of current study was to compare and evaluate different diagnostic tests to establish a reliable and less time consuming diagnostic test for prompt diagnosis of acute Japanese encephalitis cases mainly amongst children and young adults from North-Eastern part of Uttar Pradesh. Methods: A total of 100 subjects, including 50 suspected cases and 50 diseased controls were investigated to establish the diagnosis of JE in acute encephalitis patients. All CSF samples were subjected to MAC-ELISA, virus cultivation and RT-PCR. Results: Out of 50 cases, 6 showed negative results by all the three tests. 50% (22 out of 44), 47.5% (21 out of 44), and 66% (23 out of 44) were found positive by MAC-ELISA, Virus cultivation and RT-PCR respectively. The rate of detection by Virus cultivation and RT-PCR was higher in case of MAC-ELISA negative cases (57.1% and 78.5%) than that of MAC-ELISA positive cases (22.7% and 27.2%) respectively. Mean age of the patients was 12.5 years, which ranged from 1-24 years with male to female ratio of 3:1. Conclusions: The RT-PCR was found most reliable, sensitive and specific method amongst the three chosen methods for detection of JEV in suspected encephalitis patients.

Comparison of Different Methods of Detection of Enteric Pathogenic Protozoa.

Purpose: The study was conducted to compare different methods of detection of pathogenic protozoan parasites in stool specimens of People Living with HIV/AIDS (PLHA). Materials and Methods: Stool specimens of 242 HIV sero-positive patients were examined using the wet mount technique, modified Ziehl-Neelsen's (ZN) staining, auto-fluorescence and auramine fluorescence staining. Patient specimens, 94 and 40 out of 242, were also subjected to Giardia antigen detection using an enzyme immunoassay and Cryptosporidium antigen detection by immuno-chromatography, respectively. For calculation of sensitivity, specificity, positive and negative predictive values, light microscopy of wet mounts and modified ZN stained smears for Giardia and Coccidia, respectively, were considered as gold standards. Results: Sensitivity of auto-fluorescence, auramine-O staining and antigen detection techniques was found to be 100% as compared to the routine standards. The specificity of auto-fluorescence was 90.6% and 100% for Cyclospora and Isospora, respectively; that of auramine-O staining was 98.9% for Cryptosporidium, 99.30% for Cyclospora and 100% for Isospora; and that of antigen detection was 90.6% and 97.7% for Cryptosporidium and Giardia, respectively. Conclusion: In laboratories requiring screening of large number of stool specimens for detection of protozoan parasites, fluorescence microscopy and antigen detection can be useful techniques. Confirmation of positive results, however, needs to be done with the standard techniques.

Ocular Chlamydia trachomatis infections in patients attending a tertiary eye care hospital in north India A twelve year study.

Background & objectives: Ocular infection with Chlamydia trachomatis is a major public health problem in densely populated countries like India. The true prevalence of such infections is uncertain due to insufficient data available from India. The aim of this study was to do a retrospective analysis of C. trachomatis eye infections in patients attending the outpatient department of Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, over a period of 12 years. Methods: From 1997 to 2008, the Chlamydia laboratory received conjunctival swabs from 1281 consecutive patients for C. trachomatis detection after thorough clinical examination. Specimens were subjected to direct fluorescent antigen detection assay using monoclonal antibody based commercial kit to detect the presence of C. trachomatis antigen. Results: Antigen positivity varied between 22-28 per cent. Children below 11 yr and people above the age of 60 yr showed comparatively higher antigen positivity (25.7 and 27.8%, respectively). As compared to males significantly (P<0.05) higher number of females in the age group of 31-60 yr were positive for C. trachomatis antigen. Patients with the clinical diagnosis of follicular/allergic conjunctivitis and trachoma showed higher rate of antigen positivity. Interpretation & conclusions: Northern India having dry and arid climatic conditions in most parts of the year was considered in the past as one of the trachoma hyper-endemic foci. The study indicated that laboratory proven C. trachomatis eye infection still persisted in this part of the country throughout the study period of 12 years.

Development of two potential diagnostic monoclonal antibodies against human cytomegalovirus glycoprotein B

Human cytomegalovirus glycoprotein B (gB) represents a target for diagnosis and treatment in view of the role it plays in virus entry and spread. Nevertheless, to our knowledge, rare detection of a gB antigen has been reported in transplant patients and limited information is available about diagnostic gB monoclonal antibodies (mAbs). Our aim was to develop gB mAbs with diagnostic potential. Hydrophilic gB peptides (ST: amino acids 27-40, SH: amino acids 81-94) of favorable immunogenicity were synthesized and used to immunize BALB/c mice. Two mAbs, named ZJU-FH6 and ZJU-FE6, were generated by the hybridoma technique and limited serial dilution and then characterized by indirect ELISA, Western blotting, immunoprecipitation, and immunohistochemical staining. The mAbs displayed high titers of specific binding affinities for the ST and SH synthetic peptides at an mAb dilution of 1:60,000 and 1:240,000, respectively. Western blotting and immunoprecipitation indicated that these mAbs recognized both denatured and native gB of the Towne and AD169 strains. The mAbs, when used as the primary antibody, showed positive staining in cells infected with both Towne and AD169 strains. The mAbs were then tested on patients submitted to allogeneic hematopoietic stem cell transplantation. The gB antigen positivity rates of the patients tested using ZJU-FH6 and ZJU-FE6 were 62.0 and 63.0%, respectively. The gB antigen showed a significant correlation with the level of pp65 antigen in peripheral blood leukocytes. In conclusion, two potential diagnostic gB mAbs were developed and were shown to be capable of recognizing gB in peripheral blood leukocytes in a reliable manner.

Aspectos clínicos, patológicos, imuno-histoquímicos e virológicos em cinco bezerros persistentemente infectados com o vírus da diarreia viral bovina em uma propriedade do Rio Grande do Sul / Clinical, pathological, immunohistochemical and viral aspects of five calves persistently infected with bovine viral diarrhea virus in a farm of Rio Grande do Sul, Brazil

O vírus da diarreia viral bovina (BVDV) é responsável por diferentes síndromes que afetam bovinos em todo o mundo, causando grandes perdas econômicas. O presente trabalho analisou as características clínicas, patológicas e imuno-histoquímicas e virais de cinco bovinos persistentemente infectados pelo BVDV de uma mesma propriedade, localizada no Município de Viamão, Rio Grande do Sul. Dentre os sinais clínicos verificados destacaram-se subdesenvolvimento, secreções nasais e oculares, além de catarata congênita unilateral em dois bovinos. As principais lesões observadas durante a necropsia consistiram de aumento dos linfonodos mesentéricos, evidenciação das placas de Peyer e pododermatite e lesões crostosas no plano nasal e na região periocular em um animal. Os achados microscópicos caracterizavam-se, principalmente, por infiltrado mononuclear na lâmina do intestino delgado e rarefação linfoide com infiltrado histiocitário nos centrofoliculares de linfonodos e nas placas de Peyer. Antígenos virais foram detectados por imuno-histoquímica principalmente em queratinócitos da epiderme, no epitélio de folículos pilosos e células mononucleares da derme de orelhas e pele; histiócitos e em linfócitos dos linfonodos; células foliculares da tireoide; no citoplasma de neurônios e, em menor escala, em células da micróglia no córtex cerebral e no hipocampo. O isolamento viral de amostras de sangue e órgãos dos animais confirmou a presença de BVDV não citopático. Também foi possível detectar a presença do genoma viral por RT-PCR no soro dos animais. A análise filogenética do fragmento parcial da região 5' não traduzida do genoma viral permitiu a classificação da amostra viral como BVDV tipo 2b. O presente estudo reforça a necessidade de investigar e caracterizar surtos de BVD e descrever suas diferentes for-mas de apresentação.

Bovine viral diarrhea virus (BVDV) is responsible for different syndromes that affect cattle worldwide causing important economic losses. This study analyzed the clinical, pathological, immunohistochemical and viral aspects of persistent infection by BVDV in five animals of a farm located in the county of Viamão, Rio Grande do Sul, southern Brazil. The clinical signs included growth impairment, nasal and ocular discharge and, in two animals, congenital cataract. The main gross lesions observed at the necropsy were enlargement of mesenteric lymph nodes and Peyer's patches, and in one case, pododermatitis and crusted lesions on nasal planum and periocular region. Microscopic findings were characterized mostly by mononuclear infiltrate in the lamina propria, primarily in the small intestine and lymphoid depletion with histiocytic infiltrate in follicular centers of lymph nodes and Peyer's patches. Viral antigens were more frequently demonstrated in epidermal keratinocytes, epithelium of hair follicles and dendritic cells of the dermis of the ears and skin, histiocytes and lymphocytes in lymph nodes, thyroid follicular cells, in the cytoplasm of neurons and to a lesser extent, in glial cells in the cerebral cortex and hippocampus. Viral isolation from blood samples and organs confirmed the presence of non-cytopathic BVDV. Moreover, viral RNA was detected by RT-PCR in serum samples. Phylogenetic analysis of a partial fragment of the5' non-translated region of the viral genome allowed the classification of the sample as BVDV type 2b. The present study strengthens the need to investigate and to characterize BVD outbreaks and to describe its different clinic-pathological presentations.

Electroimmunotransferblot assay for the detection of mycobacterial antigens in the cerebrospinal fluid for diagnosis of tubercular meningitis.

Background: The diagnosis of tubercular meningitis (TBM) is often delayed as it presents initially with non-specific signs and symptoms leading to delay in appropriate therapy. Conventional modes of diagnosis are time-taking and immunodiagnosis has its own pitfalls. Antigen detection assays have been found to be quite promising in this aspect. Aim: In the present study, attempts were made to evaluate the ElectroImmunoTransferBlot (EITB) test for detection of Mycobacterium tuberculosis antigens in CSF. Methods: A total of 46 CSF specimens were collected from 26 clinically suspected cases of TBM and 20 non-TBM cases. The mycobacterial antigens were concentrated by immunoprecipitation and separated based on their molecular weight by SDS-PAGE which were further transferred and immobilized onto a matrix and detected by EITB. Results: In TBM CSF specimens distinct bands of molecular weight 12kDa, 30-32kDa, 71kDa, 86kDa, 96kDa, 110kDa and 120kDa were seen in addition to 50kDa Immunoglobulin (Ig) heavy chain, 25kDa Ig light chain and an indistinct human albumin band at 69kDa. The control group CSF specimens also showed the Ig and albumin bands but showed no cross-reactive antigens. The following proteins 12kDa (7.7%), 30-32kDa (23%), 71kDa (19.2%), 86kDa (77%), 96kDa (57.5), 110kDa (23%) and 120kDa (15.4%) were identified as reactive bands. The results were compared to the reverse passive latex agglutination test. Conclusion: The likelihood of diagnosing TBM as evidenced by detecting at least a single mycobacterium specific band was 88.4% by our protocol for antigen detection in CSF. The specificity of EITB for diagnosing TBM was found to be 100% when the 86kDa antigen was excluded from the analysis. However, the method of diagnosis is labour/reagent intensive and needs substantial validation.