检测脊髓灰质炎IgM抗体捕捉法ELISA的建立及其在早期快速诊断中的应用

首次建立了用于脊髓灰质炎快速、分型诊断的血清IgM抗体捕捉法ELISA。检测了52例疑似病人,IgM阳性率为76.9％(40/52),而粪便病毒分离率仅为44.2％(23/52),前者的阳性检出率明显高于后者。做病毒分型诊断结果,与分离病毒中和试验定型的总符合率为92.86％(39/42)。检测601例来自全国各省的脊灰疑似病人血清,其阳性率波动于13％～92.3％,平均为61.1％。阳性率与收集血清的病日相关,发病0～3天收集者,阳性检出率为69.5％,4～25天者为64％,26～54天者为52％,55天以上者则未能检出。本检测方法需时1.5天,简便、敏感、特异,重复性良好,适用于脊灰的早期快速诊断。对其实用性进行了讨论。     

Rapid Detection of Oculopathogenic Adenovirus in Conjunctivitis

Ocular adenovirus (Ad) infections occur throughout the world in both sporadic and epidemic forms. Accurate laboratory diagnosis of Ad in conjunctival samples is always valuable. The present study was carried out to explore the presence of Ad as a causative agent in clinically suspected viral conjunctivitis and to compare the performance of conventional virus isolation on cell cultures, direct detection of Ad antigens in conjunctival cells by a direct fluorescence assay, Ad DNA detection by polymerase chain reaction (PCR), and specific IgM measurement by ELISA. Samples included scrapes from conjunctiva. Scrapes were subjected to study by direct immunofluorescence stain, culture on the Hep-2 cell line, and PCR for Ad detection. Blood samples were also taken and subjected to study for specific anti-Ad IgM determination. The culture for Ad was positive in 77.8%, direct antigen detection by fluorescent stain was positive in 72.2%, PCR was positive in 83.3%, and serology was positive in 88.9% of patients. Both determination of antibody IgM and PCR correctly identified a larger group of patients compared to cell culture. The most sensitive and specific method for diagnosis of Ad compared to culture was PCR (100%), followed by IgM detection (92.9%) then direct antigen detection by fluorescent stain (85.8%). From this study, we conclude that Ad is a common pathogen in sporadic cases of conjunctivitis. Screening of adenoviral conjunctivitis is possible by using specific IgM due to its high sensitivity. A confirmatory test can be done by PCR for diagnosis of Ad, as it is a rapid, specific, and accurate method.     

Experimental infection of Atlantic salmon Salmo salar pre-smolts by i.p. injection with new Irish and Norwegian salmonid alphavirus (SAV) isolates: a comparative study

Atlantic salmon Salmo salar L. pre-smolts were experimentally infected with 2 different isolates of salmonid alphavirus (SAV): a Subtype 1 isolate from Ireland and a Subtype 3 isolate from Norway. Sequential samples of tissue and blood were collected during a period of 20 wk post injection and subjected to virus isolation from kidney tissue and serum, detection of viral nucleic acid in heart tissue and serum by real-time RT-PCR, detection of specific antibodies by virus neutralisation assay, and histopathological examination. Successful reproduction of pancreas disease (PD) was obtained by intraperitoneal (i.p.) injection of both isolates. No mortality was observed post infection in either group, but typical PD histopathological lesions in heart and pancreas tissue were observed with both isolates. The prevalence and severity of lesions in the pancreas, heart, skeletal muscle and brain were similar in both groups with only subtle differences recorded. Re-isolation of virus from kidney tissue was performed at 7 and 14 d post infection (d p.i.) only and was positive for both test groups at both sampling points. Isolation of virus from sera from both groups was positive at 4 to 14 d p.i., but was negative at later sampling points when antibody production had begun. Virus may be detected only during the acute phase using both methods. Specific neutralising antibodies could be detected for both test groups from Day 21 p.i. until the end of the experiment at 140 d p.i. Peak antibody titres were seen 70 d p.i. Using real-time RT-PCR, pancreas disease virus (PDV)-specific RNA was detected frequently in serum samples up to 14 d p.i. and occasionally thereafter. In contrast, viral RNA could still be detected in the heart tissue of fish from both groups for at least 140 d p.i.     

Further Evaluation of Immunofluorescence Techniques for Detection of Shigellae in Fecal Specimens

The fluorescent antibody (FA) tests for group B and group D Shigella were reevaluated. Duplicate swab specimens from patients suspected of having shigellosis were cultured shortly after collection and after transport in a soft-agar holding medium. Smears for FA examination were made at the same time. Results obtained for the group D Shigella agree closely with those obtained in previous studies. The percentage of isolations of group B Shigella from transported specimens which were positive by FA was 59.6% as compared to 39.3 and 53.3% in previous studies. S. flexneri was isolated from 76.7% of the FA-positive specimens when they were examined shortly after collection. More isolates of Shigella were obtained when specimens were examined by both methods than when either method was used alone. The results indicated that FA tests for both group B and group D Shigella are practical and may be useful to laboratories engaged in Shigella isolation.     

Sensitive enzyme immunoassay for the rapid diagnosis of influenza a virus infections in clinical specimens

Samples of nasopharyngeal secretion (NPS) from 100 infants and small children admitted for acute respiratory disease during the period from January to March 1989 were examined for the presence of influenza A virus. All samples were tested by enzyme immunoassay (EIA), fluorescent antibody (FA) technique and by isolation in cell culture 3–6 h after they were obtained from the patients. Of 24 influenza strains found by isolation, 21 were detected by EIA and 19 were FA⁺. In comparison with virus isolation, EIA gave the following values: sensitivity 88 %, specificity 100 %, positive prognostic value (PPV) 100 %, and negative prognostic value (NPV) 96 %. A rabbit anti-influenza-A serum (A-13) was used as catching antibody and a monoclonal anti-influenza-A pool against NP protein was used as detector antibody in EIA. A-13 gave bands corresponding to influenza A core proteins (NP and M1) in Western blot (WB) studies when different H3N2 strains were employed as antigens. A-13 gave only a band corresponding to the NP protein when H1N1 strains were examined by WB. The detection level by EIA for both H3N2 and H1N1 strains precipitated by polyethylene glycol from tissue culture maintenance medium was 1–2 ng.     

Comparison of techniques for the detection of avian infectious bronchitis virus as a contaminant of vaccines

Techniques for detecting various levels of both field and vaccine strains of infectious bronchitis virus in a deliberately contaminated Newcastle disease vaccine were compared using chick embryos, chick kidney cells, chick tracheal organ cultures and chickens with a view to determining the most appropriate method for screening vaccines for freedom from IBV contamination. Techniques examined included detection of abnormalities and deaths in embryos, cytopathic effect in chick kidney cells, ciliostasis in chick tracheal organ cultures and clinical signs and virus isolation in chickens as well as the fluorescent antibody test, negative contrast electron microscopy and serology where appropriate. Results showed that the techniques capable of detecting both strains of infectious bronchitis virus were, in order of sensitivity, the fluorescent antibody test on allantoic cells from infected embryos, ciliostasis and direct electron microscopy of allantoic fluid. One surprising feature was the poor results obtained using chickens. Some detection was achieved with tracheal virus isolation and tracheal organ cultures prepared from inoculated birds and to a lesser extent with histology and clinical signs, but no technique detected the field strain.     

Measles virus replication in lungs of hispid cotton rats after intranasal inoculation.

Hispid cotton rats were inoculated intranasally with either measles virus (MV) Edmonston, a multipassaged, tissue culture-adapted strain of MV, or with one of three clinical MV isolates that had limited passages (three to five times) in tissue culture cells. MV Edmonston was recovered from the lungs of every (n = 37) hispid cotton rat inoculated with this virus for at least 7 days after virus inoculation. Peak pulmonary titers occurred on Day +4 (3.3-4.4 log10/g lung). Scattered areas of inflammation were observed interstitially in lung sections from infected animals stained with hematoxylin and eosin, and a similar pattern of diffuse fluorescence was seen in cryostat sections stained with an indirect fluorescent antibody procedure specific for virus antigens. Fluorescent antibody and virus isolation studies on lung lavage cells both suggested that lung leukocytes were a primary target of the virus. In contrast to these findings, virus was isolated only sporadically from hispid cotton rats inoculated with any of the clinical measles virus isolates. Despite the restricted growth of MV in these animals, cotton rats may be useful for studying certain aspects of measles virus pathogenesis and for screening potential antiviral compounds in vivo.     

Les anticorps fluorescents dans le diagnostic bactériologique des Escherichia coli entéro-pathogènes: Comparaison avec l'isolement de ces germes par culture classique

Enteropathogenic E. coli were sought routinely by the fluorescent antibody technique, using monovalent and polyvalent conjugates (rhodamine sulfonyl· chloride, fluorescein and rhodamine isothiocyanate).In 2061 stool specimens examined with monovalent antibody to E. coli 0127:B8, there were 61 false positives, 14 of which were from previously known cases of E. coli 0127:B8 infection, and 33 specimens that were negative by fluorescence but positive on culture. In 457 stool specimens examined with polyvalent antiserum, there were 15 false positives, five of which came from cases previously infected by the corresponding serotypes, and there were 20 specimens negative by fluorescence but positive on culture. The disagreement amounted, therefore, to 4.6% in the former instance and 7.6% in the latter. This fluorescent technique permits rapid sufficiently precise detection of enteropathogenic E. coli in stools.     

Detection and analysis of tumor fluorescence using a two-photon optical fiber probe

The utility of a two-photon optical fiber fluorescence probe (TPOFF) for sensing and quantifying tumor fluorescent signals was tested in vivo. Xenograft tumors were developed in athymic mice using MCA207 cells expressing green fluorescent protein (GFP). The TPOFF probe was able to detect ex vivo fluorescence from excised tumors containing as little as 0.3% GFP-expressing cells. TPOFF results were similar to both flow-cytometric analysis of tumor cells after isolation and suspension, and fluorescence determined by microscope images of cryosectioned tumors. TPOFF was then used to measure GFP fluorescence from tumors in live mice. The fiber probe detected fluorescently-labeled Herceptin antibody targeted to HER2-expressing tumors in severe combined immunodeficient mice. Dendrimer nanoparticles targeted by folic acid and having 6-TAMRA as a fluorescent probe were also used to label KB cell tumors in vivo. The fiber probe documented a fourfold increase in tumor fluorescence in animals that received the targeted dendrimer. These results suggest TPOFF can be used as a minimally invasive system for identifying tumor markers and monitoring drug therapy.     

Indirect immunohistochemistry on skin biopsy for the detection of persistently infected cattle with bovine viral diarrhoea virus in Italian dairy herds

Indirect immunohistochemistry (IHC) on skin biopsies for identification of persistently infected (PI) animals has been used as a parallel test to antigen and antibody ELISAs in a bovine viral diarrhoea (BVD) voluntary control program. The aim was to evaluate the reliability and feasibility of IHC on ear skin tissues to detect PI animals in field conditions, including both adult and calves under 6 months of age. In animals over 6 months of age skin biopsy and blood sample were collected at the same time, whereas in young calves blood sampling was performed when animals reached 6 months of age. One hundred and sixty-five animals were tested and immunohistochemical results were compared with those of antigen ELISA. In case of inconclusive results virus isolation and virus neutralization assays were performed. Agreement K value was 0.96. Immunohistochemical staining in positive animals was clearly detectable in the keratinocytes of the epidermis and adnexa.     

A five-year immunological follow-up study of the institutionalized handicapped children vaccinated with live varicella vaccine or infected with natural varicella

Twenty-two institutionalized handicapped children who were susceptible to varicella were vaccinated with live varicella vaccine of the Oka strain and their immune status was followed for 5 years under conditions without exposure to natural varicella. Simultaneously, 7 children infected with natural varicella were followed. Of the 22 vaccinees, 16 showed sero-positive conversion by the fluorescent antibody to membrane antigen (FAMA) test, the other 6 remaining seronegative during 5 years of observation period. All the 16 cases showing seroconversion had detectable antibody for 5 years after vaccination, and 14 of them gave a positive reaction in the varicella skin test. All the 7 cases after natural varicella gave positive reactions in both the FAMA and skin test. These results suggest that immunity conferred by the vaccination would persist long even in the absence of exposure to natural varicella, though further follow-up studies are needed.     

Application of Immunofluorescent Antibody Technique in Rapid Diagnosis of Respiratory Syncytial Virus Infection

Seventy-eight unselected children under the age of 2 years suffering from acute respiratory infections were investigated by the fluorescent antibody technique and a comparison was made with conventional isolation and serological methods. Sixty-nine per cent. of children with bronchiolitis were diagnosed as suffering from respiratory syncytial virus infection on the day of admission by examination of nasopharyngeal secretions. There were 44 children with respiratory syncytial virus infection diagnosed by conventional methods over a month, but by using fluorescent antibody techniques on tissue culture 53% were diagnosed by the second day, 71% by the fourth day, and 82% by the seventh day. The method of choice for a rapid diagnosis of respiratory syncytial virus infection is by examining nasopharyngeal secretions, when 90% of those with this infection can be diagnosed on the day of admission to hospital.     

Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.     

Direct Immunofluorescence Test for the Diagnosis of Genital Herpesvirus Infections

Herpetic lesions of the genitalia may be confused clinically with other ulcerative, genital lesions. Direct immunofluorescence (FA) provides a rapid method of diagnosis, and the utility of this method for the diagnosis of genital ulcers was examined. One hundred and ten patients with genital lesions were examined by darkfield for syphilis and by FA and culture for herpes simplex virus (HSV) infections. Satisfactory samples were obtained from 102 patients, of which 81 were clinically suspected cases of HSV. Acetone-fixed slides of scrapings of ulcerative lesions were stained with conjugated antiserum prepared in rabbits against HSV type 2. HSV was isolated from 73% of specimens of suspected herpetic lesions, and 77% of these specimens were positive by FA. Nine percent were positive by FA only and these were not thought to represent false positives. Five percent were positive by culture only. A comparison of clinical diagnoses with laboratory findings revealed that 4% of the cases were misdiagnosed when only the clinical evaluation was considered. The data suggest that the inclusion of a diagnostic FA test for HSV along with the darkfield examination may be useful for differentiating the etiological agents of ulcerative, genital lesions.     

Comparative sensitivities of diagnostic procedures used to detect bacterial kidney disease in salmonid fishes

Kidney and spleen homogenates from each of 60 coho salmon (Oncorhynchus kisutch) and steelhead trout (Salmo gairdneri) were examined for detection of Renibacterium salmoninarum. The proportions of positives differed widely with the detection procedures used: in coho salmon, 5% were positive by the Gram-stain procedure, 10% by the direct fluorescent antibody test, 48% by bacteriological isolation, 65% by staphylococcal coagglutination, and 73% by counterimmunoelectrophoresis; in steelhead trout, 3% were positive by Gram-stain, 8.3% by fluorescent antibody, 17% by bacteriological isolation, and 67% by counterimmunoelectrophoresis. Renibacterium salmoninarum was not detected in either coho salmon or steelhead trout by immunodiffusion analysis.     

The india-ink immunoreaction: a method for the rapid diagnosis of encephalitozoonosis.

Sera from 37 rabbits were assayed for antibodies against Encephalitozoon cuniculi (Nosema cuniculi) by the india-ink immunoreaction and the indirect fluorescent antibody tests: all animals seropositive to the former were also positive to the latter test. 27 of the rabbits were also tested for skin hypersensitivity and then autopsied. Animals positive to the skin test were also positive to the serological tests. At autopsy 18 of 22 rabbits positive in the immunological tests showed lesions typical of encephalitozoonosis. Sera from 200 rabbits originating from 6 institutes were assayed by the india-ink test: seropositive rabbits were found from all institutes (9.1 to 81.9% incidence), with serum titres ranging from 1:125 to 1:5000. The india-ink test appears to be a rapid and convenient method for diagnosis of encephalitozoonosis in rabbits.     

Rift valley fever on the east coast of Madagascar

In March 1990, a Rift Valley fever virus (RVFV) outbreak was suspected in the district of Fenerive on the east coast of Madagascar after an abnormally high incidence of abortions and disease in livestock. Sera from humans and cattle were tested for RVFV antibodies by immunofluorescence assay (IFA) and ELISA-IgM capture. Sera and mosquitoes collected in the same area were tested for virus isolation by tissue culture and suckling mouse intracerebral inoculation, and for antigen detection by an ELISA antigen capture assay. Among cattle from the area, RVFV antibody prevalence was 58.6 % by IFA and 29.6 % by ELISA-IgM. In contrast, human populations in the same area had a lower RVFV antibody prevalence, with 8.01 % IFA and 5.4 % IgM-positive sera. No RVFV antigen was detected and virus isolation was unsuccessful from the sera and mosquito pools tested. Different hypotheses concerning the emergence and diffusion of RVFV in this area and the occurrence of the outbreak are discussed.     

Detection of typhoid fever by Widal and indirect fluorescent antibody (IFA) tests. A comparative study

Widal test is a conventional method for the detection of typhoid fever. However, it takes 18-24 hours to complete the test. In the present study indirect fluorescent antibody test has been compared with the Widal test using single serum specimens and was found to be rapid, sensitive and specific. Serum specimens from 41 culture proven cases of typhoid fever, 14 clinically suspected cases and 22 normal individuals were collected. Whereas Widal test detected 63.41% positive cases, IFA test detected 87.80% from among culturally proven typhoid cases. Among the clinically suspected cases of typhoid fever, IFA test detected 85.71% (28.57 + 57.14%) while Widal test detected only 57.13% (35.71 + 21.42%) positive cases out of above 14 cases.     

Visualization of Mouse Neuronal Ganglia Infected by Herpes Simplex Virus 1 (HSV-1) Using Multimodal Non-Linear Optical Microscopy

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that causes skin lesions and goes on to enter a latent state in neurons of the trigeminal ganglia. Following stress, the virus may reactivate from latency leading to recurrent lesions. The in situ study of neuronal infections by HSV-1 is critical to understanding the mechanisms involved in the biology of this virus and how it causes disease; however, this normally requires fixation and sectioning of the target tissues followed by treatment with contrast agents to visualize key structures, which can lead to artifacts. To further our ability to study HSV-1 neuropathogenesis, we have generated a recombinant virus expressing a second generation red fluorescent protein (mCherry), which behaves like the parental virus in vivo. By optimizing the application of a multimodal non-linear optical microscopy platform, we have successfully visualized in unsectioned trigeminal ganglia of mice both infected cells by two-photon fluorescence microscopy, and myelinated axons of uninfected surrounding cells by coherent anti-Stokes Raman scattering (CARS) microscopy. These results represent the first report of CARS microscopy being combined with 2-photon fluorescence microscopy to visualize virus-infected cells deep within unsectioned explanted tissue, and demonstrate the application of multimodal non-linear optical microscopy for high spatial resolution biological imaging of tissues without the use of stains or fixatives.     

Isolation of dermatophytes from clinically normal sites in patients with tinea cruris

Sixty patients clinically suspected of tinea cruris were studied by collecting skin scrapings from the site of their lesions and six clinically normal sites including the thighs, scrotum, crural clefts, natal cleft and the web between their 4th and 5th toes. Dermatophytes were detected in scrapings in 46 (77%) and by culture in 36 (60%) patients from lesions. Trichophyton rubrum was isolated from 32 and Epidermophyton floccosum from 4 patients. Dermatophytes were also isolated with maximum isolation from the scrotum, crural clefts and the natal cleft in that order. Thus, when tinea cruris is treated with topical antifungal agents they should be applied also to the potential carriage sites to prevent recurrence.