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PubMed2006

Evaluation of sampling technique and transport media for the diagnostics of adenoviral eye infections. Adenovirus sampling and transport.

BACKGROUND: Human adenoviruses (HAdV) may cause pharyngoconjunctival fever, follicular conjunctivitis or epidemic keratoconjunctivitis (EKC). Especially, outbreaks of the latter may lead to severe economic losses when preventive measures are implemented too late. Thus, a safe sampling method, proper specimen transport conditions and a fast and sensitive diagnostic technique is mandatory. METHODS: Two commercially available virus transport systems (VTS) were compared with two NaCl-moisturised sampling devices, one of which comprises Dacron-tipped plastic-shafted swabs and the other a cotton-tipped wood-shafted swab, available in most ophthalmologists' offices. Downstream methods for specific detection of HAdV included direct immunofluorescence assay (IFA) of conjunctival swabs, virus isolation by cell culture and quantitative real-time polymerase chain reaction (qPCR). Furthermore, the influence of application of local anaesthetics prior to swabbing on subsequent detection of HAdV was investigated. RESULTS: Application of local anaesthetics had a positive influence on the amount of swabbed cells, thus increasing the chance of obtaining positive results by IFA. Neither isolation of HAdV by cell culture nor by qPCR was negatively influenced by this pretreatment. Surprisingly, both commercially available VTS performed significantly worse than the NaCl-moisturised swabs. This was shown with regard to virus recovery rates in cell culture as well as viral genome copy numbers in the qPCR. CONCLUSIONS: Based on our results, the following recommendations are provided to improve sampling, transport and diagnostic techniques regarding conjunctival swabs for diagnosis of human adenovirus infection: (1) application of local anaesthetics, (2) NaCl-moisturised VTS for shipment of specimens, and (3) detection of HAdV by qPCR. The latter method proved to be superior to virus isolation by cell culture, including subsequent identification by IFA, because it is faster, more sensitive and allows simultaneous handling of a number of samples. Hence, countermeasures to prevent further virus spread in an outbreak situation can be implemented earlier, thus reducing the number of subsequent adenoviral infections.

Adenovirus Infections, Human↗
PubMed2005

Prevalence and genetic diversity of Entamoeba histolytica in an institution for the mentally retarded in the Philippines.

A total of 113 mentally retarded patients residing in a mental institution in Metropolitan Manila, Philippines, were screened for the presence of Entamoeba histolytica based on microscopy and polymerase chain reaction (PCR). Anti-E. histolytica antibodies were also screened in 97 serum samples collected using immunofluorescence antibody (IFA) test. Parasitological examination showed E. histolytica/Entamoeba dispar in 43 cases (38.05%), while PCR detected 74 cases (65.48%) positive for E. histolytica and 6 cases (5.30%) positive for E. dispar. Interestingly, these 6 samples were coinfected with E. histolytica. IFA test revealed that 80.41% (78/97) of the respondents possessed significant antibody titers for intestinal infection of E. histolytica. Of this number, there were 5 patients negative in IFA test but positive in PCR. The genetic diversity of E. histolytica isolates was also investigated by analyzing polymorphism in the serine-rich gene by nested PCR on DNA directly extracted from stool specimens. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded six distinct DNA banding patterns among the 74 stool isolates. An apparent clustering of E. histolytica strains was observed in patients living in different residential cottages of the institution. These results indicate the high prevalence of E. histolytica in an institution for the mentally retarded in the Philippines.

Animals↗
PubMed2006

Quantitative assessment of viable Cryptosporidium parvum load in commercial oysters (Crassostrea virginica) in the Chesapeake Bay.

The epidemiological importance of increasing reports worldwide on Cryptosporidium contamination of oysters remains unknown in relation to foodborne cryptosporidiosis. Thirty market-size oysters (Crassostrea virginica), collected from each of 53 commercial harvesting sites in Chesapeake Bay, MD, were quantitatively tested in groups of six for Cryptosporidium sp. oocysts by immunofluorescent antibody (IFA). After IFA analysis, the samples were retrospectively retested for viable Cryptosporidium parvum oocysts by combined fluorescent in situ hybridization (FISH) and IFA. The mean cumulative numbers of Cryptosporidium sp. oocysts in six oysters (overall, 42.1+/-4.1) were significantly higher than in the numbers of viable C. parvum oocysts (overall, 28.0+/-2.9). Of 265 oyster groups, 221 (83.4%) contained viable C. parvum oocysts, and overall, from 10-32% (mean, 23%) of the total viable oocysts were identified in the hemolymph as distinct from gill washings. The amount of viable C. parvum oocysts was not related to oyster size or to the level of fecal coliforms at the sampling site. This study demonstrated that, although oysters are frequently contaminated with oocysts, the levels of viable oocysts may be too low to cause infection in healthy individuals. FISH assay for identification can be retrospectively applied to properly stored samples.

Animals↗
PubMed2002

Regioselectivity of CYP2B6: homology modeling, molecular dynamics simulation, docking.

Human cytochrome P450 (CYP) 2B6 activates the anticancer prodrug cyclophosphamide (CPA) by 4-hydroxylation. In contrast, the same enzyme catalyzes N-deethylation of a structural isomer, the prodrug ifosfamide (IFA), thus causing severe adverse drug effects. To model the molecular interactions leading to a switch in regioselectivity, the structure of CYP2B6 was modeled based on the structure of rabbit CYP2C5. We modeled the missing 22-residue loop in CYP2C5 between helices F and G (the F-G loop), which is not resolved in the X-ray structure, by molecular dynamics (MD) simulations using a simulated annealing protocol. The modeled conformation of the loop was validated by unconstrained MD simulations of the complete enzymes (CYP2C5 and CYP2B6) in water for 70 and 120 ps, respectively. The simulations were stable and led to a backbone r.m.s. deviation of 1.7 A between the two CYPs. The shape of the substrate binding site of CYP2B6 was further analyzed. It consists of three well-defined hydrophobic binding pockets adjacent to the catalytic heme. Size, shape and hydrophobicity of these pockets were compared to the shapes of the two structurally isomeric substrates. In their preferred orientation in the binding site, both substrates fill all three binding pockets without repulsive interactions. The distance to the heme iron is short enough for 4-hydroxylation and N-deethylation to occur for CPA and IFA, respectively. However, if the substrates are docked in the non-preferred orientation (such that 4-hydroxylation and N-deethylation would occur for IFA and CPA, respectively), one pocket is left empty, and clashes were observed between the substrates and the enzyme.

Amino Acid Sequence↗
PubMed2005

Controlled, randomized trial of island flap anoplasty for treatment of trans-sphincteric fistula-in-ano: early results.

Treatment of trans-sphincteric fistula is usually a compromise between recurrence and incontinence. Dermal island flap anoplasty has been found to be useful in the treatment of these fistulas. We performed a randomized trial to compare dermal island flap anoplasty with conventional treatment for trans-sphincteric fistula-in-ano. Seventy nine patients with fistula-in-ano were recruited; twenty patients with trans-sphincteric fistula confirmed by endoanal ultrasound were prospectively randomized to receive either dermal island flap anoplasty (IFA) or conventional treatment (CVN) for trans-sphincteric fistula-inano. Conventional treatment consisted of lay open fistulotomy or seton insertion if deemed unsuitable for fistulotomy. Dermal island flap anoplasty involved a cutaneous advancement flap into the rectum. Pain scores, fecal incontinence scores, operative complications, wound healing and recurrence rates were charted. Two patients in the CVN group required seton insertions, which were still intact at the 9-month follow-up. Two patients with similar high trans-sphincteric fistula in the IFA group avoided having a long-term seton. There were no differences in the postoperative pain score, incontinence score, complications, wound healing and recurrence rates between the two groups. IFA is a safe and useful method for treating transsphincteric fistula. It can be considered when a suprasphincteric extension is suspected, thus avoiding risk of incontinence or the discomfort of a long-term seton.

Adult↗
PubMed1992

Intermediate filament antigens of 60 and 65 kDa in the nuclear matrix of plants: their detection and localization.

Although the presence of a matrix in plant nuclei has been reported, major questions remain about its structural and biochemical features. We have used an intermediate filament antibody of broad specificity to explore whether Daucus carota (carrot) nuclei and nuclear matrices contain intermediate filament/lamin antigens and, if so, where specifically they are localized. SDS-PAGE and Western blotting revealed two bands, at 60 and 65 kDa, that were highly immunoreactive with the intermediate filament antibody (IFA) of Pruss et al. (1981, Cell 27, 419-428). This pattern was observed consistently, not only with carrot cell-free nuclei and nuclear matrices, but also with nuclear preparations from Vicia faba (broad bean) and Pisum sativum (pea). Immunofluorescence studies with whole carrot nuclei localized the IFA antigens to the nucleoplasm and disclosed no accentuated peripheral labeling. Agarose-embedded nuclear matrices showed not only fluorescence throughout the nucleoplasm but also heavy labeling surrounding the nucleoli and suggestions of peripheral labeling. At the ultrastructural level, immunogold results from pre- and postembedment treatments supported the conclusion that IFA antigens occur throughout the nucleoplasm, with possibly a slight concentration at the periphery. These combined results provide substantial evidence that plant nuclei and their matrices possess at least two major intermediate filament antigens with molecular weights characteristic of animal lamins. Whether or not these antigens represent plant lamins, their nonperipheral localization hints at significant differences among the eukaryotic kingdoms in nuclear organization.

Antigens↗
PubMed1993

Intrathecal synthesis of specific antibodies in neuroborreliosis. Comparison of different ELISA techniques and calculation methods.

The sensitivity of six different ELISA techniques and calculation methods for the determination of intrathecal synthesis of IgG antibodies specific to Borrelia burgdorferi was investigated in paired CSF and serum specimens from 33 patients with neuroborreliosis. The diagnostic value of the Antibody Index (AI) was compared with the meaningfulness of serum antibodies to B. burgdorferi (Bb), established by immunofluorescence assay (IFA). The AI, as a standard for intrathecal antibody synthesis was determined from specific antibody ratios (QBb) in the CSF and serum and the CSF/serum ratio of IgG (QIgG) or albumin (QAlb). Using Western blotting with identical concentrations of IgG in the CSF and serum all patients displayed intrathecal synthesis of specific antibodies to at least two B. burgdorferi proteins. The different ELISA methods and calculation procedures were almost equivalent in demonstrating intrathecal synthesis of specific antibodies (32 and 33/33). Calculation of AI from IFA titers was somewhat less sensitive (30/33). In 5 patients titers of serum IgG- and IgM-antibodies to B. burgdorferi determined by IFA were within the normal range or borderline, while elevated AIBb values indicated an autochthonous immune response to B. burgdorferi in the CSF. In uncertain cases of neuroborreliosis calculation of AI from ELISA titers will be useful in clarifying the diagnosis.

Antibodies, Bacterial↗
PubMed1983

Development of antibody to Rickettsia tsutsugamushi in soldiers in Malaysia.

Malaysian, British and New Zealand soldiers were tested for evidence of infection with Rickettsia tsutsugamushi after several weeks' exposure to the infection during field exercises in Malaysia. 39 (5.0%) of 787 British and New Zealand soldiers developed immunofluorescent antibody (IFA) to R. tsutsugamushi to a titre of 1:50 and two (0.3%) to a titre of 1:100. 11 (1.5%) of 751 Malaysian soldiers also developed low titres less than or equal to 1:100. These low antibody levels were not correlated with clinical disease, and their significance is unknown. Seven (0.9%) of the Malaysians showed an IFA rise to greater than or equal to 1:200, and three of these experienced febrile illnesses, one lasting two weeks. An additional eight Malaysian soldiers had an IFA titre of greater than or equal to 1:400 when first tested and six of these also had a Proteus OXK agglutinin titre of greater than or equal to 1:160, indicating infection shortly before the study.

Antibodies, Bacterial↗
PubMed1984

Endemic Lassa fever in Liberia. V. Distribution of Lassa virus activity in Liberia: hospital staff surveys.

Serological testing of hospital personnel by the indirect fluorescent antibody (IFA) technique was used to indicate the distribution of Lassa virus (LV) activity in Liberia. Determination of the places of origin of the staff members as well as the sites of the hospitals indicated that LV is active in throughout Liberia. Prevalences of IFA varied from 3.8% at the J. J. Dossen Hospital on the coast in the south-east to 22.3, 23.5 and 40.4% in Lofa County hospitals inland in the north-west. Rises in LV antibody prevalences, high prevalences and relatively high IFA titres in hospital personnel suggest the LV activity is particularly high in Lofa, Grand Cape Mount and Nimba Counties.

Antigens, Viral↗
PubMedDate not supplied

Seroepidemiological survey of rickettsial infections among blood donors in central Tunisia.

In this report we attempt to evaluate the prevalence of antibodies against Rickettsia conorii, R. typhi, Coxiella burnettii, and Ehrlichia chaffeensis in central Tunisia. Five hundred sera from blood donors, collected between March and June 1993, were tested for these 4 antibodies using an indirect immunofluorescence antibody assay (IFA). Nine percent of the sera had antibodies against R. conorii (IgG > 1:32) by IFA, and 8% by Western blotting; with IFA, 3.6% had antibodies to R. typhi, 26% to C. burnetii (> 1:50), and none to E. chaffeensis. Infection rates with R. conorii and R. typhi did not differ significantly between the sexes, but fewer young people had antibodies to R. typhi. A significantly higher prevalence of antibodies to C. burnetii was noted for males with no difference between age classes. No significant difference was detected between urban and rural areas. This study confirmed the endemicity of rickettsioses, and revealed a high seroprevalence of Q fever, in central Tunisia.

Adolescent↗
PubMed1991

Homotypic antibody responses to fresh clinical isolates of human immunodeficiency virus.

Human immunodeficiency virus type 1 (HIV-1) exhibits extensive genomic and antigenic diversity, which is thought to contribute to the failure of the host's immune response to control infection and prevent clinical progression. Part of this failure may be due to utilization by the virus of antigenic variation as a means to escape protective immune responses. Antibody-escape variants of HIV-1 were studied here using fresh clinical isolates and autologous plasmas. HIV-1 was isolated from the plasma of seven people who were all seropositive for at least 2 years, and symptomatic sometime during that period. Isolated viruses were confirmed as HIV-1 by the presence of reverse transcriptase activity in infected culture supernatants, and by positive immunofluorescence using human monoclonal antibody to HIV-1 core protein. Plasma from these people were positive by Western immunoblot (DuPont) for most major HIV-1 (strain IIIB) antigens. These plasmas neutralized three laboratory strains of HIV-1 (i.e., IIIB, RF, and MN) but did not neutralize the homotypic strain in five cases, and had greatly reduced neutralizing titers against the homotypic strain in two cases. Homotypic neutralizing antibodies were absent in autologous plasma obtained 3 months later. When antibody titers were measured by fixed-cell indirect immunofluorescence assays (IFAs), high titers of IgG (1:6400 to 1:25,600) were detected against HIV-1 IIIB, while low titers of only 1:20 to 1:160 were detected against homotypic viral antigens at the time of virus isolation, and remained low 12 and 16 weeks later. No class IgA, IgD, IgE, or IgM antibodies to homotypic viral antigens, as possible IgG-blocking antibodies, were detected by fixed-cell IFAs. Cross-reactions with heterologous donor's plasmas were observed in some cases, and in these cases the cross-reactions were unidirectional. Live-cell IFAs detected IgG in patient's plasma to HIV-1 IIIB-infected cells but not to cells infected with homotypic isolates. These results suggest that it is common for neutralization-resistant HIV-1 variants to appear during the course of infection, and that all or most antigens of these variants are capable of escaping antibody recognition.

AIDS-Related Complex↗
PubMed1988

Standardization and kinetics of in vitro bovine blood lymphocyte stimulation with bovine rotavirus.

Two groups of 3-month old calves were immunized intramuscularly with attenuated bovine rotavirus and boosted 21 and 42 days later. The first group of three calves were vaccinated with live virus emulsified with incomplete Freund's adjuvant (IFA) and the second group was immunized with live virus suspended in phosphate buffered saline (PBS). Three other calves, serving as controls, were inoculated with PBS emulsified with IFA. The specific cell-mediated and antibody responses of the animals were studied. Preliminary analysis of in vitro peripheral blood lymphocyte transformation to bovine rotavirus determined optimal conditions as: 96 h culture period, 5 X 10(5) cells per culture in RPMI 1640 medium containing 10% heat-inactivated bovine fetal serum and the use of inactivated virus in the cell culture at a concentration of 5 X 10(6) median tissue culture infective dose before inactivation. Specific blastic stimulation was observed on calves immunized with the rotavirus emulsified with IFA after the second and third vaccine inoculation with stimulation index values varying from 2.00 to 5.73. Serum neutralizing antibody titers of 1/25,600 were also induced in the same calves. Calves immunized with rotavirus-PBS suspension developed a mean antibody titer of 1/1,600, but showed no specific lymphocyte stimulation. No increase in specific immune responses was detected in the control animals.

Animals↗
PubMed1983

Application of an indirect carbon immunoassay (CIA) for the rapid diagnosis of antibody to Toxoplasma gondii in sheep.

Carbon immunoassay (CIA), a novel indirect rapid test for Toxoplasma antibody in sheep, was compared with indirect fluorescent antibody assay (IFA). CIA relies on the adherence of carbon particles of India-ink to rabbit immunoglobulin G.l Carbon labelled anti-sheep rabbit IgG was used for the detection of sheep antibody when attached to tachyzoites of Toxoplasma gondii. The result was read in an ordinary light microscope and there was a clearcut difference between negative and positive reactions. Out of a total of 97 sheep sera tested, 15 sera were negative in both tests and 12 were negative in CIA but showed low positive titres in IFA. The remaining 70 sera were positive in both tests but the titres were usually about 3 dilution steps lower when investigated with CIA as compared to IFA.

Animals↗
PubMed1995

Effective immune protection of pigs against cysticercosis.

A scolex protein antigen (SPA) was prepared from cysticerci of Taenia solium obtained from naturally infected pigs. Yorkshire pigs were vaccinated with SPA plus incomplete Freund's adjuvant (IFA) or with SPA plus Corynebacterium parvum (CP). Controls were given IFA plus phosphate-buffered saline (PBS) or CP plus PBS. All animals were given three subcutaneous injections at 20-day intervals. Ten days after the third injection, the pigs were fed with 10(4) viable eggs of T. solium. All pigs developed a delayed type hypersensitivity, and a transient eosinophilia after the first dose of vaccine. High titers of specific antibodies were detected in the sera of vaccinated animals and in infected controls. A protection level of 71.43% was recorded in animals vaccinated with SPA plus IFA and of 75.00% in those vaccinated with SPA plus CP.

Animals↗
PubMed1990

An enzyme-linked immunosorbent assay for IgG antibodies to human herpes virus 6.

An indirect enzyme-linked immunosorbent assay (ELISA) with human herpes virus 6 (HHV6) membrane antigen was compared with indirect immunofluorescence assay (IFA) for measurement of HHV6 IgG antibodies. Five hundred serum samples from 403 Swedish patients with suspected symptomatic Epstein-Barr virus (EBV) infections were examined. The specificity of the ELISA compared with IFA was 98.7% and the sensitivity was 98.4%. In 90% of the patients, IgG antibodies to HHV6 were detected with both assays. The highest HHV6 IgG titers were found mainly in patients with EBV or CMV infections, but HHV6 mononucleosis was not diagnosed. The same HHV6 antigen was assessed for IgM ELISA but was found to be of limited value due to high IgM reactivity with the control antigen. The HHV6 IgM ELISA requires further investigation. The IgG ELISA described is a reliable alternative to IFA for measurement of HHV6 IgG antibodies and for large scale epidemiological studies.

Antibodies, Viral↗
PubMed1993

Detection of human parvovirus B19-specific IgM and IgG antibodies using a recombinant viral VP1 antigen expressed in insect cells and estimation of time of infection by testing for antibody avidity.

Sera from patients with symptoms of recent human parvovirus B19 (B19) infection were tested for B19-specific IgM in an immunofluorescence assay (IFA) using insect cells expressing B19 recombinant VP1 coat protein as an antigen. A highly significant correlation (P < 0.001) was found between titres obtained in the IgM IFA and the units obtained in an IgM antibody-capture RIA using plasma derived native B19 antigen. An IgG IFA using the recombinant antigen was performed on 57 sera and the antibody avidity determined. There was a highly significant correlation (P < 0.001) between the relative amounts of low avidity B19-specific IgG antibodies and time after onset of illness. This finding allows the detection of IgG to be used for diagnosing acute infection.

Animals↗
PubMed1996

Competitive ELISA for detection of HHV-6 antibody: seroprevalence in a danish population.

A competitive HHV-6 enzyme-linked immunosorbent assay (ELISA) was established, utilizing HHV-6 rabbit polyclonal antibodies and HHV-6 cellular antigen. In total, 285 sera from Danish children aged 1-6 years were screened for HHV-6 antibodies. The lowest seropositive rate, 78% was found in the youngest age group from 1 year to 15 months. All 120 (100%) children aged 3 years and above were seropositive. The ELISA was compared with indirect immunofluorescence assay (IFA) using a group of 83 serum samples from 65 Danish patients (aged 4 weeks to 72 years). All but one of the sera (100% of patients) were positive in the ELISA, but only 71% of sera (65% of patients) were positive with both assays. The ELISA detected one seroconversion in a baby, which was not detected by IFA. The ELISA was found to be more sensitive than IFA.

Adolescent↗
PubMed1988

A newly developed immunofluorescent assay for determining the Pichinde virus-inhibitory effects of selected nucleoside analogues.

An immunofluorescent assay (IFA) for Pichinde virus (PCV), a member of the family Arenaviridae, was developed for antiviral drug assays against the virus. The assay was performed by adding fluorescein-labeled anti-PCV monoclonal antibody to virus-infected cells at 24 h after the initial infection and counting the infected cells with an epifluorescence microscope. The average 50% effective dose (ED50) for a series of nucleoside analogues tested against PCV using this IFA was: 2-beta-D-ribofuranosylselenazole-4-carboxamide (selenazofurin), less than 1.0 microgram/ml; 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin), 6.0 micrograms/ml; ammonium 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide- 5'-phosphate hydrate (ribavirin-5'-monophosphate), 15.8 micrograms/ml; ammonium 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5'-hemisuccinate (ribavirin-5'-hemisuccinate), 14.7 micrograms/ml; ammonium 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5'-(2,3- dimethyl)hemisuccinate [ribavirin-5'-(2,3-dimethyl)hemisuccinate], 213.5 micrograms/ml; 4-hydroxy-1-beta-D-ribofuranosyl-2-pyridone (3-deazauridine), 5.2 micrograms/ml; and (S)-9-(2,3-dihydroxypropyl)adenine, ([S]-DHPA), 471.0 micrograms/ml. In comparison, the ED50 of ribavirin using inhibition of marginal PCV-induced cytopathogenic effect after 12 days was 6.0 micrograms/ml and using plaque reduction after 5 days was 2.5 micrograms/ml, indicating that this IFA was of comparable sensitivity to these other tests.

Animals↗