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Evaluation of herpes simplex detection in corneal scrapings by three molecular methods.

Herpes simplex virus (HSV)-1 keratitis (HSK) is a sight-threatening ocular infection with worldwide occurrence. A prompt laboratory diagnosis is often very useful. The purpose of this study was to evaluate molecular methods as rapid diagnostic tools compared with cell culture of HSK. Corneal scrapings from patients with clinically suspected HSK were tested by direct immunofluorescence assay (IFA) for HSV-1 antigen and by polymerase chain reaction (PCR) for HSV-1 DNA, and an attempt for viral isolation was performed on Vero cell line culture. Positive samples by cell culture were 20.8%, whereas PCR was positive in 29.2%, and IFA was positive in 33.3%. IFA had better sensitivity (80%) and negative predictive value (81.8%) than PCR (70% and 76.9%, respectively); however, PCR had better specificity (71.4%) and positive predictive value (63.6%). This indicates that a combination of cell culture, IFA and PCR constitutes the best set of tools for diagnosis of clinically suspected cases of HSK. Documented infection can be further assessed by cell-culture technique or PCR depending laboratory availability.

Animals↗

Prevalence and incidence of Lyme borreliosis among Slovene forestry workers during the period of tick activity.

OBJECTIVES: To establish the prevalence and incidence of symptomatic and asymptomatic infection with Borrelia burgdorferi sensu lato during the period of tick activity, to compare the risk of infection with B. burgdorferi s.l. for forestry workers and indoor workers in Slovenia, and to compare the outcome of an in-house immunofluorescent assay (IFA) and a commercially available enzyme-linked immunosorbent assay (ELISA). METHODS: The study included 122 forestry workers; the control group consisted of 93 indoor workers. All participants were examined twice in 2002: before the beginning of tick activity (March) and at the end of tick activity (November). At each examination, principal demographic and epidemiological data were collected and a blood sample taken for serological analysis. Specific IgM and IgG antibodies against B. burgdorferi s.l. in the paired sera were determined with an in-house IFA and a commercially available ELISA flagellin test (DAKO). RESULTS: 9.8% of the forestry workers and 4.3% of the indoor workers tested positive for IgG with the IFA (p = 0.26); 23.8% of the forestry workers and 9.7% of the indoor workers tested positive for IgG with the ELISA (p = 0.02). During the study period the incidence of symptomatic Lyme borreliosis was 2.3% and the rate of IgG and/or IgM seroconversion of 10.2% was the same with both tests. CONCLUSIONS: The seroprevalence of antibodies against B. burgdorferi s.l. among the Slovene forestry workers was greater than among the indoor workers, but the difference between the two groups was not significant when the IFA was used. The incidence of Lyme borreliosis during the period of tick activity was lower than we expected, with a large proportion of seroconversions being asymptomatic.

Adult↗

Effect of guinea pig thyroglobulin in incomplete Freund's adjuvant on experimental autoimmune thyroiditis induced by in vitro-activated lymph node cells.

Guinea pigs injected with guinea pig thyroglobulin (GPTG) in incomplete Freund's adjuvant (IFA) have been shown to be unresponsive to challenge with GPTG in complete Freund's adjuvant (CFA). However, effector cells which transfer experimental autoimmune thyroiditis (EAT) can be demonstrated in cultured lymph node cells (LNC) of unresponsive animals, indicating that GPTG in IFA does not suppress the initial sensitization of EAT effector cells. LNC from unresponsive animals were unable to suppress the in vitro activation of effector LNC or to suppress EAT when cotransferred with effector cells. When GPTG in IFA was given to animals which were used as recipients of effector cells, the production of EAT was markedly suppressed. These results suggest that GPTG in IFA can suppress EAT either by preventing effector cells from interacting with the thyroid or by interfering with the function of a cell in the normal recipient which may interact with effector cells to result in the lesions of EAT.

Animals↗

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax and its use in a seroepidemiological survey of the Eastern Caribbean Basin.

An enzyme-linked immunosorbent assay (ELISA) for IgG antibodies against a South American (New World) strain of Trypanosoma vivax was developed and used for mass screening of cattle from 20 islands in the Eastern Caribbean Basin. The sensitivity and specificity of antigens prepared from a bovine-derived field strain and a murine-adapted laboratory strain of T. vivax, both of New World origin, were compared using an indirect fluorescent antibody (IFA) test, and an antigen prepared from the murine-adapted strain was subsequently used to develop an ELISA test. The results of the ELISA test were then compared with the results of a concurrently run IFA test. There was no cross-reactivity with either test using serum from a Trypanosoma theileri-infected cow. Both tests were weakly cross-reactive with sera from a T. brucei-infected steer, and the IFA test was moderately cross-reactive with several serum samples from a T. evansi-infected steer. For bovine sera collected from herds on islands in the Eastern Caribbean region, only five of 640 tested positive with the ELISA test. Thirty five of 653 sera tested were positive by IFA although the fluorescence elicited was weak as compared to that elicited by sera from known infected animals. Sera collected from 27 cattle in a region known to be free of T. vivax (OH, U.S.A) were negative with the ELISA test, whereas seven of 30 sera from a herd in French Guiana known to be infected with T. vivax were positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Endemic Lassa fever in Liberia. VI. Village serological surveys for evidence of Lassa virus activity in Lofa County, Liberia.

Six villages in Lofa County, north-west Liberia, and one near the coast were surveyed for the presence of indirect fluorescent antibodies (IFA) to Lassa virus (LV). Prevalences were similar among males and females, and among various age groups. The prevalence of IFA positive sera, 6.4%, in two roadside villages was significantly higher than in two matched villages "in the bush", 1.9%. It was also higher in Gbanwei, a roadside village which did not maintain traditional sanitary measures, than in Zuwulo, similarly located but with maintenance of clean-swept areas without shrubbery or rubble between the houses. In another pair of villages, the one adjacent to a Mission Clinic with a very high prevalence of IFA positive staff members had significantly higher prevalence, 14.1%, than did the other, a roadside village with 5.1% seropositives. LV antibodies were also found in 4.3% of the inhabitants of a small coastal village near Robertsfield International Airport. Though LV infections are more common in villages in which traditional practices have been modified, they are present even in villages which are relatively unchanged. In the former they appear to be continuous while sporadic in the latter. The prevalences of IFA in the villages with the highest rates are about one third of what is found in personnel of hospitals near them, suggesting that hospital staff members acquire infections from patients as well as from the communities in which they live.

Adolescent↗

Endemic Lassa fever in Liberia. IV. Selection of optimally effective plasma for treatment by passive immunization.

The efficacy of passive immunization for treatment of Lassa Fever (LF) is believed to depend on the titre of the neutralizing antibody infused. For the purpose of identifying optimal donors of LV-immune plasma, a population of LF-convalescent patients in Liberia was tested for prevalence of neutralizing antibody. Minimally protective titres, expressed as a log10 neutralization index, (LNI), were established in animal models as LNI greater than 2. LNI titres for 26 donors, tested eight or more months after illness, were modest: 16 titred 1 less than LNI less than 2, 4 titred 2 greater than LNI less than 3, and only 4 titred LNI greater than 3. Sequentially obtained plasma from six donors indicated that the LNI response was delayed relative to the indirect fluorescent antibody (IFA) response, that high titres (LNI greater than 3) occurred only after seven months and in only two of six patients. Most of the unselected LV-immune plasma will require concentration to therapeutically useful LNI titres. In a passive immunization experiment, guinea-pigs were protected by a late convalescent plasma (LNI = 4.8, IFA = 320) but not by an early plasma, (LNI = 0.6, IFA = 640), thus supporting the selection of immune plasma on the basis of the LNI. Cross serological testing with LV strains and convalescent plasma from patients in Sierra Leone, Liberia and Nigeria suggested that these LV strains were indistinguishable by cross-IFA, but were readily distinguishable by cross neutralization tests. Geographical matching of LV and plasma origins may thus be a factor in selection of optimal plasma for passive immunization of Lassa fever.

Animals↗

Imported fire ant immunotherapy: effectiveness of whole body extracts.

The purpose of this study was to determine if whole body extract (WBE) immunotherapy for imported fire ant (IFA) hypersensitivity is effective. This evaluation was carried out by retrospectively interviewing 76 patients with a history of generalized allergic reactions to IFA stings and positive skin tests to IFA-WBE. The study groups consisted of 65 patients on immunotherapy and 11 similar patients who were not treated for various reasons. In addition, an IFA sting challenge was performed in 30 volunteers of the 65 patients on immunotherapy. The retrospective review showed that of the 65 patients on immunotherapy there had been 112 subsequent field-sting episodes in 47 patients. Only one sting episode in this group (2.1%) produced an anaphylactic reaction. Six of the 11 patients not on immunotherapy have had subsequent field re-sting episodes, and each has had a systemic reaction. Repeat skin testing on 31 of the 65 patients in the immunotherapy group showed persistent positive responses in five (16%), but each was at a lower dilution than initially. Responses of the other 26 of the 31 patients who had skin testing had become negative. The four untreated patients who were available for skin testing continued to have positive responses at comparable dilutions on skin testing. Sting challenges carried out on 30 volunteers from the 65 patients (all from the 31 who had repeat skin tests) on immunotherapy resulted in only local reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaphylaxis↗

Participation of encephalitogen in incomplete Freund's adjuvant in the induction of experimental allergic encephalomyelitis in Hartley guinea pigs.

The addition of complete Freund's adjuvant (CFA) to encephalitogen is required for the induction of experimental allergic encephalomyelitis (EAE). On the other hand, administration of encephalitogen in incomplete Freund's adjuvant (IFA) protects the animal from the development of EAE. It is shown in this work that injection of homologous central nervous system (CNS) tissue or myelin basic protein (BP) in IFA, before challenge with CNS tissue in CFA, accelerated the onset of the disease in Hartley guinea pigs. It also appeared to protect the animals, however, because 22% of the group did not develop EAE at all, and in those which did, the disease was not as lethal as in controls. To produce this accelerated form of EAE with encephalitogen in IFA required (1) a time interval shorter than 9 days between the first injection and challenge and (2) that the first injection and the challenge be done in the same site, which could be hind or front foot pads but not the nuchal area. The results indicated that 'priming' by encephalitogen in IFA occurred when this two-step induction procedure was used. The experimental conditions may have bypassed suppressive mechanisms.

Animals↗

Studies of V beta 8 T cell receptor peptide treatment in experimental autoimmune encephalomyelitis.

Lewis rats immunized with T cell receptor (TCR) variable region peptide V beta 8 in complete Freund's adjuvant (CFA) were protected against experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein in CFA, although variable protection was also observed in rats injected with control peptide in CFA, or CFA alone. However, this adjuvant-mediated protection could be avoided by immunizing with TCR peptide in incomplete adjuvant (IFA). Clinical, but not histologic EAE was suppressed in rats given V beta 8 peptide in IFA, whereas control animals injected with V beta 14 peptide in IFA, or IFA alone developed severe clinical EAE. Anti-V beta 8 antibodies were present in the sera of all V beta 8-treated rats. These findings lend support to the hypothesis that autoimmune disease can be suppressed by inducing an immune response against the TCR-idiotope of autoreactive T cells.

Amino Acid Sequence↗

Comparison of immunofluorescence and enzyme-linked immunosorbent assays for the serology of Hantaan virus infections.

Three enzyme-linked immunosorbent assay (ELISA) systems based upon different principles were developed for the serology of Hantaan virus infections and compared with an indirect immunofluorescence assay (IFA). The indirect IFA was carried out with gamma-irradiated Hantaan virus-infected and uninfected Vero E6 cells fixed with ethanol (-70 degrees C) or acetone (20 degrees C) on drop slides and a FITC-coupled sheep anti-human Ig preparation. Atypical staining in the IFA was avoided by using ethanol (-70 degrees C) instead of acetone (20 degrees C) fixation. In the first ELISA ('cell-assay'), Hantaan virus-infected or uninfected Vero E6 cells were used as antigens, which after gamma-irradiation were seeded into microtiter ELISA strips. Serial dilutions of human sera were incubated and specific antibodies were demonstrated with a horseradish peroxidase (HRPO)-conjugated sheep anti-human Ig preparation. In the second ELISA ('competition-assay') an affinity-purified human Ig preparation was used as a capture antibody for Hantaan virus antigen. After incubation of serial dilutions of human sera with this coat, the reactivity of the affinity purified anti-Hantaan virus Ig coupled to HRPO was determined. In the third ELISA ('complex trapping blocking [CTB]-assay') the same capture antibody was used to react with a mixture of the antigen and serial dilutions of human sera. The reactivity with the same HRPO conjugate was then determined. The results obtained in the respective assay systems with sera from people at risk or suspected of Hantaan virus infection coincided well. The CTB-ELISA proved to be faster and more sensitive than both the other ELISA systems, without giving more non-specific reactions: it detected almost all the IFA positive samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Application of peroxidase labelled antibody assays for detection of porcine IgG antibodies to hog cholera and bovine viral diarrhea viruses.

Rapid, sensitive peroxidase labelled antibody (PLA) assays using microtiter systems, were developed for detection of hog cholera virus (HCV) and cross-reacting bovine viral diarrhea virus (BVDV) antibodies in pig sera. HCV-infected pig kidney cell line (PK 15) prepared in microtiter plates were fixed and used in PLA assays. After inoculation with test serum, bound antibodies (HCV/BVDV) were reacted with either horseradish peroxidase (HRP) conjugated anti-porcine immunoglobulin (H & L) or biotinylated protein A (BPA) and subsequent HRP labelled avidin (A). Positive reactions were easily visualized under an inverted light microscope as foci of brown colored cells after enzyme degradation of hydrogen peroxidase in the presence of amino-ethylcarbazole (AEC). The PLA assays were superior to the indirect fluorescent antibody (IFA) test in detecting anti-HCV antibodies in porcine sera collected early after inoculation of pigs with a lapinized HCV vaccine. The performances of the PLA, IFA and FA neutralization (FAN) tests in measuring the immune response in the vaccinated pigs were comparable. Cross-reacting anti-BVDV antibody, as measured by a microtiter serum neutralization (MTSN) test, was not demonstrable in vaccinated pigs until they were challenged with a virulent HCV, 13 weeks later. The PLA assays relative to the IFA test detected more reactive samples among porcine field sera collected from HC-free pigs in Canada. Of 795 samples, 24 (3.01%) were reactive in the PLA employing HRP anti-porcine IgG, and 21 (2.6%) in the PLA, using BPA-HRP-A. When 324 of these sera were screened by the IFA test (using HC antigen), only one sample (0.30%) was found reactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Recovery of human parainfluenza virus types one and two.

The ability to recover human parainfluenza virus types 1 and 2 (HPIV-1, 2) from infected individuals has been highly variable. During the autumn of 1991, 158 nasal wash specimens collected from children with lower respiratory symptoms were split and cultured independently at two laboratories using different tissue culture techniques. Immunofluorescent antibody (IFA) and hemadsorption (HAd) assays were compared for their speed and efficiency in viral detection. 45 isolates [HPIV-1 (17) and HPIV-2 (28)] were recovered by one laboratory and only one (HPIV-2) by the other. IFA was the most sensitive assay detecting 87% of HPIV-1 and 70% of HPIV-2 by the fourth day of culture. HAd assay detected 87% of HPIV-1 isolates by the time they were positive by IFA, but only 35% of the HPIV-2 isolates. Significant methodologic differences between laboratories were then compared simultaneously for effect on virus recovery from culture positive frozen clinical specimens. Recovery of 100% of the isolates was achieved. Factors that contributed to differences in recovery of HPIV-1 and 2 were: (1) primary African green monkey (AGMK) cells were inferior to cynomolgus monkey kidney or LLC-MK2 cells, (2) addition of trypsin to culture medium for AGMK and LLC-MK2 cells enhanced recovery, (3) use of IFA was essential for rapid detection of HPIV-2, and (4) use of microtiter plate culture without specimen dilution enhanced virus recovery. A survey of clinical virology laboratories demonstrated considerable variability in the use of these techniques for routine respiratory virus culture.

Animals↗

Prevalence of Anaplasma marginale, Babesia bigemina and B. bovis in Nigerian cattle using serological methods.

Serum samples collected randomly from 500 cattle from the 10 northern states of Nigeria were tested for antibodies against Anaplasma marginale by indirect fluorescent antibody (IFA), card agglutination (CT) and capillary tube-agglutination (CA) tests. The serum samples were also examined for antibodies to Babesia bigemina and B. bovis by the IFA test only. Of the serum samples tested, 79.4% had antibodies against A. marginale by the IFA test, 40 and 25% in the CT and CA tests, respectively. The IFA test results for B. bigemina and B. bovis were 29.4 and 14.1%, respectively.

Agglutination Tests↗

Comparison between an enzyme-linked immunosorbent assay using a detergent-soluble Leishmania infantum antigen and indirect immunofluorescence for the diagnosis of canine leishmaniosis.

Serum samples collected from 290 dogs--186 Leishmania-infected and 104 control animals--were screened to detect the presence of specific antibodies to Leishmania infantum antigens in Tuscany, Italy. Two different techniques were compared: an indirect immunofluorescence assay (IFA) and an indirect enzyme-linked immunosorbent assay (ELISA). The ELISA was developed using a detergent-soluble antigen of L. infantum promastigotes. Triton X-100 and protease inhibitors were used as detergent and to increase reproducibility of the assay, respectively. A strong correlation between the anti-Leishmania antibody levels obtained by ELISA and those obtained using IFA was observed. The ELISA appeared to be somewhat more sensitive than IFA (99.5% vs. 98.4%), while its specificity was lower (97.1% vs. 100%), even if not significantly different. Immunoblotting analysis, using the detergent-soluble L. infantum antigen, demonstrated that proteins of M(r) 30 and 73 kDa were recognized by all positive sera, regardless of the serum titre. Furthermore, antigens of M(r) 16, 18, 26, 33, 50 and 117 kDa were also frequently reactive with a large proportion of sera from infected dogs. This ELISA demonstrated a high sensitivity and specificity as well as the IFA, and it seems to be a suitable assay for large scale epidemiological studies.

Animals↗

Serological titers of equine monocytic ehrlichiosis associated with gastro-intestinal disorders and serological follow-up on two endemic farms.

The purpose of this work was to study the association of positive serological titers to Ehrlichia risticii, the causative agent of equine monocytic ehrlichiosis (EME) with gastro-intestinal disorders in hospitalized horses referred to The Ohio State University College of Veterinary Medicine Teaching Hospital (OSU VMTH). In addition, serological titers for E. risticii were monitored in two horse populations with endemic EME for one season to monitor temporal changes in titers. A statistically significant difference was found between the proportion of the total hospitalized horse population presented with a gastro-intestinal disorder during the study period, and study horses with IFA titers > or = 1:80 with these signs (P < 0.05). No such difference was found between the proportion of the total hospital horse population presented with signs of gastro-intestinal disorder, and the study horses with IFA titers of 1:20-1:40 with these signs, suggesting that these titers may not have any clinical significance for EME (P > 0.05). Thirty-eight horses on two farms endemic for EME were tested approximately every 3 weeks, 33 of which were tested serially at least two times. Five of the 38 horses (13.2%) had IFA titers > or = 1:80--two that were positive initially and three that seroconverted during the study; 15 horses' titers fluctuated between negative (IFA titers < 1:20) and exposed titers (1:20 through 1:40); and 18 horses remained negative throughout the study.

Animals↗

Comparative seroreactivity to Bartonella henselae and Bartonella quintana among cats from Israel and North Carolina.

Bartonella henselae, the predominant cause of cat scratch disease, and Bartonella quintana, the cause of trench fever, are closely related Bartonella species that induce cross-reactivity when cat or human sera are tested using an indirect immunofluorescence antibody (IFA) test. Cats are the natural reservoir for B. henselae, whereas a mammalian reservoir host for B. quintana has not been identified. Serum samples from 114 cats from Israel and 114 cats from North Carolina were tested by IFA for seroreactivity to B. henselae and B. quintana antigens. Similar numbers of cats from Israel [45 (39.5%)] and from North Carolina [46(40.4%)] were seroreactive to both antigens, however, as compared to cats from North Carolina [8 (7%)], a significantly (P = 0.001) larger number of cats from Israel were seroreactive to B. quintana antigen only [23 (20.2%)]. In addition, mean antibody titers were lower to B. henselae than to B. quintana (P = 0.0001) in the cats from Israel, whereas similar mean titers to both antigens were identified in cats from North Carolina. Absorption of serum using whole B. henselae organisms resulted in a significantly greater (P = 0.0001) decrease in antibody titer to B. henselae between absorbed and non-absorbed sera, as compared to the decrease in antibody titer following absorption with whole B. quintana organisms. There was a similar decrease in antibody titer in sera from cats experimentally infected with B. henselae and in cats naturally exposed to Bartonella species from Israel and North Carolina. Our results indicate that absorption of serum will, in most instances, distinguish species-specific reactivity by IFA to B. henselae from cross-reactivity to B. quintana in cats experimentally infected with B. henselae. The data support the conclusion that B. henselae is the principal Bartonella species responsible for seroreactivity against B. henselae and B. quintana in naturally exposed cats from Israel or North Carolina. It also suggests that in Israel, cats are exposed to one or more antigenically different Bartonella species, sub-species or strains, that seroreact by IFA more intensely with B. quintana antigen.

Animals↗

A modified indirect fluorescent antibody test for the diagnosis of brucellosis.

We adapted the conventional indirect fluorescent antibody (IFA) test to assay IgM and IgG Brucella-specific antibodies to differentiate acute from chronic infections rather than measure total antihuman globulin specific antibodies. The results were compared with the slide agglutination test (SAT) used for screening and the quantitative microagglutination test (MAT). Of a total of 118 randomly selected samples sent for anti-Brucella antibodies received at a general hospital laboratory, 58 (47.9%) were found to be positive for IFA-IgG test but not necessarily by other tests. Eleven of these cases were positive for Brucella melitensis by culture. Sixty serum samples found negative for Brucella antibodies by IFA and other tests were of patients with medical conditions other than brucellosis. Fifty serum samples from healthy blood donors were negative for Brucella spp. antibodies by all the three tests. The IFA test was found to be a more sensitive test than MAT and distinguished an acute infection from chronic disease.

Acute Disease↗

Cytomegalovirus antibody detection in blood donors and mothers of very low birth weight neonates by using three serologic methods.

We compared three serologic methods for cytomegalovirus (CMV) antibody detection and determined the CMV antibody seroprevalence of blood donors and mothers of very low birth weight (less than 1250 g) neonates in the Greater Hartford region. CMV serology was determined for 577 healthy blood donors as well as for 147 mothers of premature infants. Plasma from blood donors and sera from mothers were tested by either latex agglutination (LA) or by an immunofluorescent antibody assay (IFA), and results were compared with those from an enzyme-linked immunosorbent assay (ELISA). Sensitivity and specificity for LA to ELISA were significantly better than for IFA to ELISA [sensitivity 79/81 (97%) vs 171/202 (85%), and specificity 90/94 (96%) vs 257/347 (74%), p less than 0.01]. These differences remained whether plasma or sera were tested. Borderline values explained only two (33%) of six LA-ELISA as well as only 70 (58%) of 121 IFA-ELISA discordance. CMV seroprevalence rate for the donor blood population was 38%, and for the mothers was 53%. The LA assay is superior to the IFA assay for CMV screening of blood donors and maternal populations.

Antibodies, Viral↗