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PubMed2005

[Coxiella burnetii infections as the public-health problem].

THE AIM OF WORK: To evaluate the incidence of human Q-fever in individual regions of Bosnia and Herzegovina, to determine the prevalence and areas at risk of Q-fever incidence, to assess Q-fever seroprevalence and to demonstrate seroprevalence ratings in regard to relevant criteria (sex, age, location of residence). METHODS: The research has involved 708 individuals from different cantons, whose sera were tested in Laboratory for Specific Diagnosis of Human Q-fever on the Microbiology Desk of Medical Faculty of University in Sarajevo, during the period from July 2000-December 2003. Sera were tested by Indirect Immunofluorescence Assay (IFA), using commercial test kit, reagents for Q-fever IFA IgM and IFA IgG for in vitro diagnostic use (Focus technologies). THE RESULTS: Specific anti-C. burnetii antibodies were detected in 249 (35.2%) individuals, whereof 75 (30.12%) female individuals, and 174 (69.9%) male individuals. Individuals suffering from Q-fever were mostly included in 31-40 aged group (26.8%). Most seropositive individuals were from Zenica-Doboj Canton (35.4%), then from Herzegovinian-Neretvian Canton (22.9%), from Sarajevo Canton (14.5%), from Una-Sana Canton (11.6%), and from Middle-Bosnian Canton (11.6%). CONCLUSIONS: In our four-year study, it is serologically demonstrated that Q-fever is imminent in Bosnia and Herzegovina and that it is going to be the actual concern of public health. The number and distribution of seropositive individuals suggest that Q-fever is endemic phenomenon in Bosnia and Herzegovina.

Adolescent↗
PubMed2005

Development and evaluation of a latex agglutination test for the rapid diagnosis of scrub typhus.

The purpose of this research was to develop a simple and rapid diagnostic test for scrub typhus using a latex agglutination test (LAT) to detect antibodies against Orientia tsutsugamushi. Five strains of O. tsutsugamushi were propagated in L929 cells. The rickettsiae were purified and concentrated with percoll density gradient centrifugation. A suitable concentration of O. tsutsugamushi soluble antigen was used to sensitize latex to prepare the latex antigen. The specificity, sensitivity, and accuracy of the latex antigen were assessed. The LAT, indirect immunofluorescent antibody test (IFA), and Weil-Felix agglutination test (WF) were compared by testing 109 acute febrile illness cases and 100 confirmed non-scrub typhus cases (50 other febrile disease cases and 50 healthy controls). By using the IFA as the standard reference method, the overall sensitivity, specificity, and accuracy of the LAT were 89.1, 98.2, and 93.6%, respectively. By contrast, the sensitivity of the WF, compared with the IFA, was only 47.3%, while the specificity and accuracy were 92.6 and 69.7%, respectively. Thus, the LAT described here is another important alternative test for the diagnosis of scrub typhus.

Antibodies, Bacterial↗
PubMed2005

[An immunofluorescence assay for the detection of SARS associated coronavirus antibody based on recombinant nucleocapsid antigen and its application].

OBJECTIVE: To establish a new technique for SARS-CoV antibody test to detect infection of severer acute respiratory syndrome (SARS). METHODS: Nucleocapsid gene was obtained by reverse transcription and polymerase chain reaction from a SARS patient and inserted into the vector pFastBacHTa expressing baculovirus. Insect Sf9 cells were transfected with the recombinant baculovirus expressing SARS nucleocapsid antigen and then cultured, fixed by acetone so as to make SARS-specific antigen. Immunofluorescence assay (IFA) technique and plaque reduction neutralization test (PRNT) were used to detect 7 samples of sera of 4 newly diagnosed SARS patients collected in different days, 48 samples of convalescent sera of SARS patients, 24 serum samples of healthy person undergoing physical examination, and 40 serum samples from non-SARS patients with fever by double blind test. RESULTS: The recombinant SARS-specific antigen reacted only with SARS positive sera but not with normal sera. Double blind test showed that 45 of the 46 PRNT positive sera were IFA positive with an accordance rate of 97.8%. 7 samples of sera from 4 SARS patients in acute progressive stage in Guangdong province were all IFA positive. SARS antibody could be detected since the sixth day after onset, and the titer increased from 1:40 to 1:600 on the ninth day. CONCLUSION: Immunofluorescence assay is highly specific and sensitive in detection of SARS. This reagent is safe and easy to prepare.

Adult↗
PubMed1992

Localization of a low molecular weight antigen of Eimeria tenella by use of hybridoma antibodies.

Three monoclonal antibodies (Mabs), found by western blot analysis to recognize 10-kDa bands of Eimeria tenella sporozoite preparations, were used with immunoelectron (IE) microscopy, immunogold-silver staining (IGSS), and indirect immunofluorescent antibody (IFA) light microscopy to determine the location and distribution of the antigens in or on extra- and intracellular parasites. All 3 of the Mabs (designated C3, E5, and 1231) were found by IE microscopy to label amylopectin granules of extracellular sporozoites. Additionally, these Mabs extensively gold-labeled the sporocyst wall. In cultured primary chicken kidney cells inoculated with sporozoites of E. tenella, IGSS showed surface labeling of the parasite and intense labeling of the infected host cells by 6 hr postinoculation (PI). At 24 hr PI, host cell vacuoles in infected and uninfected cells were labeled by the 3 Mabs by IFA. The E5 and C3 Mabs also were seen to label the host cell membrane of newly infected cells. The C3 and 1231 Mabs showed little label of the host cells by 48 hr PI, but the parasites still were labeled up to 96 hr PI. The E5 Mab had intense IFA labeling of infected host cells at 48 hr PI. The results of this study indicate that parasites apparently release antigenic material during the early stages of parasite development and that this material is found internally and/or on the surface of the infected host cells.

Animals↗
PubMed2005

A pilot study for serological evidence of hantavirus infection in human population in south India.

BACKGROUND AND OBJECTIVES: Hantaviruses are rodent-borne viruses of the family Bunyaviridae that have been identified as aetiological agents of two human diseases, haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). There are no reports of hantavirus infections in humans from India, hence this pilot study was undertaken to provide the serological evidence of hantavirus infections in humans in south India. METHODS: Serum samples were obtained from individuals with acute febrile illness and from voluntary blood donors, majority of whom were from south India. Serum samples were tested for anti-hantavirus IgM using a commercial enzyme immunoassay (EIA). Samples found positive by the EIA were tested by an indirect immunofluorescence assay (IFA) using slides coated with Seoul virus (SEOV) infected cells as substrate. RESULTS: Of the 152 serum samples from individuals with pyrexic illness, 23 (14.7%) were positive for anti-hantavirus IgM by EIA. In contrast, only 5.7 per cent of healthy blood donors were positive by this assay. Eighteen of the 22 (82%) EIA-positive samples from patients were positive by the IFA assay. In contrast, only 2 of the 5 (40%) blood donor EIA positive samples were positive in the IFA assay. INTERPRETATION AND CONCLUSION: The finding of this study indicated the possible presence of hantavirus infections in the human population of India presenting both as asymptomatic and symptomatic infections. Further studies need to be done to confirm the findings on a larger sample using molecular techniques.

Fluorescent Antibody Technique, Indirect↗
PubMed1991

[The role of phagocytic cells in the pathogenesis of idiopathic fibrosing alveolitis].

The paper deals with the examination of the role of phagocytes--alveolar macrophages and neutrophils--and peripheral monocytes, in the pathogenesis of idiopathic fibrosing alveolitis (IFA). As the disease aggravates, activation of the absorbing capacity of monocyte-macrophagal cells corresponds to a sharp rise in the level of circulating immune complexes in the blood of IFA patients. Higher activities of elastase and collagenase are observed in the IFA patients' bronchial lavage fluid.

Adult↗
PubMed1991

[Idiopathic fibrosing alveolitis and bronchioloalveolar cancer].

A case of bronchiolo-alveolar carcinoma (BAC) developing in the presence of idiopathic fibrosing alveolitis (IFA) in a man of 67 with a long history of pulmonary lesion is described. A honeycomb bung was found at the autopsy. Histologically, multiple foci of BAC of mixed-cell structure and those of mucus-producing cells against the background of fibrosing alveolitis at the stage of honeycomb lung were observed. An increased content of collagen type III, IV and V was found immunohistochemically. The role of sclerotic changes and defected immunological surveillance over the epithelial regeneration in the IFA-affected lung in relevant carcinoma genesis is suggested. The origin of the lung carcinoma in IFA is considered to be the regenerating epithelium of the low respiratory tract.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed1991

Development of the immunofluorescent antibody test for detection of feline leukemia virus infection in cats.

Studies of the immunodetection of various microorganisms by various assay systems indicated that the most specific and sensitive assays are immunofluorescence, radioimmunoassay, and immunoblot analysis (western blot), followed by sensitive but less specific ELISA and agglutination assays and, finally, by even less sensitive but very specific virus isolation and double immunodiffusion techniques. The first test for the clinical detection of FeLV infection in pet cats was the immunofluorescent antibody (IFA) test, which was introduced in 1972. The FeLV test is used for detection for FeLV infection and not as a test for leukemia or any other feline disease. The IFA test was compared with an immunodiffusion (ID) test and with tissue culture isolation (TCI) of the virus in 26 cats to establish a standard for FeLV tests. Excellent correlation was observed between the IFA and the ID tests (100%).

Animals↗
PubMed1991

Evaluation of feline leukemia virus diagnostic tests available for in-office use by veterinarians.

Blood samples from cats were tested for FeLV group-specific antigen by use of an indirect immunofluorescent antibody (IFA) test, ELISA, or both. Several ELISA kits were evaluated as they would be used in a veterinary clinic, and some ELISA kits were compared with an IFA test for agreement of results. Good agreement of results was evident among the commercially available ELISA kits; results for most blood samples were clearly negative or decidely positive. Occasionally, test results were equivocal and retesting was necessary to obtain clear interpretation. Negative results are highly reliable, which makes ELISA valuable screening tests for FeLV in healthy cats. Results of tests on blood from healthy cats in Alabama indicated positivity rate of 4.0% in 1978 with an IFA test and 4.8% with ELISA in 1988 through 1989. Infection rate in sick cats ranged from 11.6 to 35.8%. The frequency of positive test results for FeLV in cats tested at Auburn University decreased from a 5-year average of 27.7% during 1980 through 1984 to a 5-year average of 14.9% during 1986 through 1990; the rate in 1990 was 11.6%.

Alabama↗
PubMed2006

Diagnostic methods for detection & isolation of dengue viruses from vector mosquitoes.

Dengue is a deadly mosquito-borne infection warranting urgent attention for its containment particularly in the tropical and subtropical countries. In the absence of a vaccine or any specific drug for its treatment, an early diagnosis is considered indispensable to prevent any casualty. Detection of viruses in human sera particularly in endemic areas is cumbersome, difficult and also not desirable. Therefore, as an alternative approach, detection of the dengue virus antigen in mosquitoes has provided a reliable tool to (i) comprehend the types of viruses circulating in nature; and (ii) help in designing vector-specific control strategies. A mélange of diagnostic techniques are currently available with some advantages or disadvantages. Traditionally, cell cultures and suckling mice have been employed for virus isolations. While the virus isolation method in baby mice is time consuming, slow and expensive, the mosquito cell cultures offer a good degree of specificity. Mosquito inoculation techniques have been reported for detection and propagation of flaviviruses. Though this technique is sensitive for routine virological confirmation of dengue fever, it requires large number of infected mosquitoes, besides being time consuming. Insect bioassays (Toxo-IFA) are generally cumbersome requiring special facilities and are not suitable for large-scale epidemiological surveillance. ELISA has been shown to be a rapid and sensitive alternative to insect bioassays for monitoring arboviruses in wild populations. Reverse transcriptase polymerase chain reaction (RT-PCR) is a recent molecular diagnostic technology used for detecting virus infections in mosquitoes, which gives rapid results but is expensive and prone to contamination. This review describes the development of various techniques involved in detection and isolation of dengue viruses in mosquitoes. Definite diagnosis of the impending dengue epidemic can be made using ELISA for virological surveillance system on dengue virus antigen in the mosquito vectors. Therefore, ELISA offers a potential tool and a convenient system for quickly screening large number of samples up to the serotype level which can be employed effectively and efficiently for large scale dengue surveillance programmes on wild caught mosquito vectors. ELISA positive samples can be screened further by Toxo-IFA system for virus isolation. On the other hand, techniques like mosquitoes cell culture, mosquito inoculation (Toxo-IFA) and RT-PCR techniques can be employed for dengue virus amplification.

Aedes↗
PubMed1991

Comparative study of respiratory syncytial virus in nasopharyngeal aspirates using conventional cell culture, shell viral centrifugation culture, immunofluorescence and biotin-avidin enzyme linked immunosorbent assays.

133 nasopharyngeal aspirates (NPA) were simultaneously tested for the presence of respiratory syncytial virus (RSV) by conventional cell culture (CCC), shell vial centrifugation culture (SVC), immunofluorescence assay (IFA) and biotin-avidin enzyme linked immunosorbent assay (B-A ELISA). These yielded positive results in 32(24%), 45(33.8%), 36(27%) and 40(30%) of specimens, respectively. Specimens positive by IFA and B-A ELISA were all also positive by SVC. The sensitivity of CCC, IFA, and B-A ELISA comparing to SVC was 71%, 80%, and 88.9%, respectively. For rapid detection of RSV, we recommend the SVC method where a cell culture laboratory is available and the B-A ELISA method where a cell culture laboratory is not available.

Centrifugation↗
PubMed1991

Onchocerca volvulus: characterization of monoclonal antibodies reactive with surface components of third-stage larvae.

Five murine monoclonal antibodies (Mab's) have been produced which are reactive with the surface of the third-stage larvae (L3) of Onchocerca volvulus. These were produced from a fusion performed after intrasplenic injection of 10 live O. volvulus L3. Hybridomas were first screened by Elisa using a PBS extract of O. volvulus female adult worms. Elisa negative wells were screened by IFA on whole formalin-fixed L3. Five Mab's were isolated which were reactive with the surface of L3, all were found to be of the IgM isotype. All five Mab's were cross-reactive with the surface of O. lienalis L3, but not with the L3 of Brugia malayi, B. pahangi, Dirofilaria immitis, or Acanthocheilonema viteae. One of the Mab's, OV3-9, reacted with an antigen common to many species of helminths. The other four were genus specific reacting only with O. volvulus and O. lienalis. All 5 Mab's reacted with L2 of O. volvulus, but not with the L4 by IFA on whole fixed larvae. Only Mab's OV3-8 and OV3-9 reacted with cryosections of adult O. volvulus by IFA indicating that the other 3 Mab's are specific for the surface of L2/L3 of the genus Onchocerca. None of the Mab's reacted with detergent dissociated L3 proteins in Western Blot analysis. The ability of L3 to bind the Mab's was abolished by incubating larvae in either proteinase K or trypsin but was unaffected by incubation of larvae in detergents, or by treatment with periodate, indicating the proteinaceous nature of the Mab specific epitopes. Anti-phosphorylcholine Mab (Gib-13) did not react with the surface of L3.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

[Ophthalmological application of HSV-monoclonal antibodies].

Monoclonal antibodies against HSV (HSV-McAb) were used in the diagnosis of 127 cases of keratitis by immunofluorescence assay (IFA). The positive rate in 43 cases of epithelial and 50 cases of stromal HSK were 100% and 92% respectively. The IFA was negative in 20 cases of keratitis of non-HSV etiology and 38 cases of normal cornea (31 cases of epithelial smears and 7 cases of cornea paraffin sections). Among 14 cases of clinically doubtful HSK, 4 cases were positive. The IFA was completed within 2-3 hours after sampling. The procedure proved rapid, sensitive, and highly specific for etiological diagnosis of HSK.

Antibodies, Monoclonal↗
PubMed1991

[Studies on RESA in Plasmodium falciparum culture medium].

P. falciparum grown in the complete RPMI 1640 medium (containing about 10% normal rabbit serum) for 24 h was collected at about 5% parasitemia. The medium was centrifuged at 500g for 20 min and the supernatant was then centrifuged once more at 40,000g for 30 min. Ten serum samples from falciparum malaria patients with anti-RESA antibodies collected from Yunnan Province were mixed with different dilutions of the culture supernatant, incubated at 37 degrees C for 2h and then at 4 degrees C overnight, and used for conducting RESA-IFA test. The titers of RESA-IFA test of all samples were reduced as compared with controls. When heated supernatant was used in the test, the inhibitory activity remained unchanged. The results indicate that RESA in the supernatants was soluble and heat-stable. To establish the origin of the reacting antigens in the inhibition experiments, the soluble fraction of sonicated merozoite-enriched collection and sonicates of ghost made from normal O phenotype erythrocytes were used in the inhibition RESA-IFA test with sera containing anti-RESA antibodies. The results showed that merozoite-enriched preparations were fully inhibitory at the concentration of 3.1 micrograms/ml, while no inhibition was observed at 0.2 micrograms/ml. In contrast, similar extracts made from normal erythrocyte ghosts were not inhibitory until concentrations about 30 times higher than that of merozoite antigen.

Adolescent↗
PubMedDate not supplied

Detection of infectious bronchitis virus antigen from experimentally infected chickens by indirect immunofluorescent assay with monoclonal antibody.

Infectious bronchitis virus (IBV) was detected by indirect immunofluorescent assay with a monoclonal antibody (MAb-IFA). The monoclonal antibody was specific for the nucleocapsid protein of IBV strain M41. The MAb-IFA clearly detected IBV with high specificity in infected chicken kidney cells. The assay furthermore detected IBV in tracheal smears and sliced tracheas from experimentally infected chickens. The positive reaction was found to be longer than that in the virus recovery test. These results indicate that MAb-IFA is a useful method for the detection of IBV from chickens suspected to have infectious bronchitis.

Animals↗
PubMed1990

An economical procedure for screening of hybridoma supernatants for surface reactive antibodies to filarial larvae.

Third- and fourth-stage larvae of Onchocerca volvulus can be attached to the lids of 96-well microtiter plates to facilitate the screening of hybridoma supernatants reactive with filarial larvae. Plates are coated with a solution consisting of 0.1% gelatin, and 0.01% chrome alum. Following coating of plate lids one larva per depression is placed on the lid. An immunofluorescence assay (IFA) was conducted in which there were two 45 minute incubations and eight separate washes by immersion in PBS. At the completion of the assay over 90% of the larvae remain attached. The background in IFA is minimal and the results are consistent with those obtained using a suspension IFA. This technique permits rapid identification of antibody producing hybridomas with small numbers of larvae.

Animals↗
PubMed1990

A limited role of IL-1 in immune-enhancement by adjuvants.

Generation of interleukin-1 (IL-1) in the draining lymph nodes after injection of complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum was studied in line with IL-1 mRNA expression (cytoplasmic slot blot analysis) and IL-1 beta antigen detection (ELISA and immunohistochemistry) in rabbits. The expression of IL-1 beta mRNA was marked from 6 to 96 hr, with a maximum at around 24 hr post-injection of CFA, while injection of the other two adjuvants elicited only a moderate or negligible response. On the other hand, IL-1 alpha mRNA expression was almost negligible during the entire 8-week observation period after injection of the above three adjuvants. Generation of IL-1 beta antigen in the draining lymph nodes after CFA injection paralleled the expression of IL-1 beta mRNA. Immunohistochemistry revealed that cells containing IL-1 beta resided in the medullary sinuses, marginal sinuses and para-cortical area, but not in the follicles. Despite marked generation of IL-1 beta in CFA-treated draining lymph nodes, the primary antibody response (IgG) to ovalbumin differed only slightly between the three animal groups that were immunized with the antigen incorporated in CFA, IFA and alum. Further, rIL-1 beta did not significantly enhance the immune response when it was entrapped together with the antigen in IFA and alum. IL-1 beta enhanced the immune response only when it was injected with antigen without adjuvant. Thus, IL-1 seemed to play only a limited role, if any, in the augmentation of the primary immune response by the above-mentioned adjuvants.

Adjuvants, Immunologic↗
PubMed1979

Detection of antibodies to cytomegalovirus, herpes simplex virus and rubellavirus with the micro-enzyme immunoassay.

Serum samples of pregnant women (200), non-pregnant women (100) and children (36) were tested for the presence of antibodies to cytomegalovirus (CMV), herpes simplex virus (HSV) and rubellavirus with the micro-enzyme immunoassay (micro-EAI), the indirect immunofluorescence antibody (IFA) technique and the haemagglutination-inhibition (HI) test. Micro-EIA gave the highest positivity rate: 78% for CMV, 86% for HSV and 85% for rubellavirus compared to 67% (CMV) and 84% (HSV) of the IFA technique and 79% (rubellavirus) of the HI test, respectively. Among IFA and HI positive sera the occurrence of micro-EIA negative ones was 6% for CMV, 4% for HSV and 9% for rubellavirus. It is concluded that the introduction of micro-EIA will improve the diagnostic and screening work of virus laboratories.

Antibodies, Viral↗