Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “IFA”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 775 records · Page 43Linked to original sources
PubMed1990

Comparison of four methods for rapid detection of Pneumocystis carinii in respiratory specimens.

Four stains for the detection of Pneumocystis carinii in respiratory specimens were compared for sensitivity, specificity, preparation time, and ease of interpretation. One hundred specimens were collected. Of these, 50 were induced sputum specimens and 50 were bronchoalveolar lavage fluid. All specimens were stained with Diff-Quik (DQ) (a modified Giemsa stain), a quick silver stain, and direct and indirect immunofluorescence stains. A positive specimen was defined as any smear positive by two or more of the methods. Fifty-eight percent of specimens were positive. Seventy-four percent of the sputum specimens and 42% of the bronchoalveolar lavages were positive. The sensitivities for detection of P. carinii in sputum were 92% with silver stain, 97% with direct immunofluorescence assay (DFA), 97% with indirect immunofluorescence assay (IFA), and 92% with DQ. The sensitivities for detection in bronchoalveolar lavage were 86% with silver stain, 90% with DFA, 86% with IFA, and 81% with DQ. Preparation times varied from 90 min for the silver stain and IFA to 3 min for DQ. Costs of the tests varied from $1.50 per slide for DQ to $10.00 per slide for the silver stain and DFA. Reading times varied from 10 to 30 min for the silver stain and DQ to less than 5 min for the immunofluorescence assays. We conclude that all of these tests are viable options for the clinical laboratory, and the choice will be influenced by factors such as clinical volume, ability to batch specimens, and expertise of technological support. A reasonable option may be to use the quick and inexpensive DQ as a screening test and to confirm negative smears with a more sensitive assay.

Bacteriological Techniques↗
PubMed1992

Comparison of indirect immunofluorescence and membrane fluorescence assays for the differentiation of antibodies to human immunodeficiency virus types 1 and 2.

Serum samples from 20 human immunodeficiency virus type 1 (HIV-1)- and 30 HIV-2-infected and 7 dually infected individuals were reacted by using the indirect immunofluorescence assay (IFA) and membrane fluorescence assay in order to determine whether these methods were useful for typing HIV-1 and HIV-2 antibodies. Although 41 of 50 (82%) of the HIV-1- and HIV-2-positive specimens cross-reacted to some extent with the heterologous antigen in the IFA, the antigen with the higher titer correlated completely with the infecting type. The IFA could not distinguish single from dual infections, however. In contrast, only 4 of the 50 (8%) serum samples cross-reacted in the membrane fluorescence test. All seven of the specimens from patients with mixed infections reacted with both antigens. The membrane fluorescence test appears to be reliable for serodifferentiation of HIV-1 and HIV-2 infections and may be useful for laboratories with low-volume typing requirements.

Antibody Specificity↗
PubMed1994

Evaluation of specificity of indirect enzyme-linked immunosorbent assay for diagnosis of human Q fever.

Ninety-five acute- and convalescent-phase serum specimens from 48 patients suspected of having rickettsial or Legionella infections were assayed for antibodies to Coxiella burnetii, the causative agent of Q fever. To evaluate the specificity of the indirect enzyme-linked immunosorbent assay (ELISA) for human Q fever, we compared the ELISA results with those of the indirect immunofluorescence antibody (IFA) test. The ELISA data were analyzed by two different criteria for a positive test. The first criterion for positive results by ELISA was based upon diagnostic titers established in a study of 150 subjects who had no demonstrable cellular or humoral immune responses to C. burnetii phase I or phase II whole cells or phase I lipopolysaccharide. The second criterion was based upon diagnostic antibody titers in a study of 51 subjects who had been diagnosed as having clinical Q fever and had fourfold or greater rises in humoral immune responses to C. burnetii phase I and phase II whole-cell antigens. A comparison of the ELISA and IFA test results of the 95 serum specimens indicated excellent agreement between the tests (Kappa = 92.9%; P < 0.05). None of the 38 patients whose etiologies were confirmed serologically as Legionnaires' disease or rickettsial diseases other than Q fever were classified as positive for C. burnetii by the ELISA. Only one patient identified by the IFA test as having Q fever was not scored positive by the ELISA. These results suggest that the ELISA is useful for epidemiologic screening and as a diagnostic test for human Q fever.

Acute Disease↗
PubMed1994

Comparison of two methods for detecting intrathecal synthesis of Borrelia burgdorferi-specific antibodies and PCR for diagnosis of Lyme neuroborreliosis.

An indirect immunofluorescence assay (IFA), a commercial capture enzyme-linked immunosorbent assay (ELISA), and PCR were compared for the diagnosis of Lyme neuroborreliosis in 16 individuals. Borrelia burgdorferi-specific intrathecal immunoglobulin G was found in 10 of 16 patients by IFA and in 8 of 12 patients by capture ELISA. PCRs performed on cerebrospinal fluid samples stored at -70 degrees C were positive for one of eight children and one of four adults with intrathecal antibody production and negative for all four adults without intrathecal antibody production. For two children with facial palsy following erythema chronicum migrans, neither IFA nor capture ELISA detected specific intrathecal antibodies and PCR was also negative. We conclude that both clinical manifestations and laboratory parameters are important for the diagnosis of Lyme neuroborreliosis.

Adolescent↗
PubMed1994

Comparison of four immunoserologic assays for detection of antibodies to Borrelia burgdorferi in patients with culture-positive erythema migrans.

In view of the significant sequelae associated with Lyme borreliosis, there is a need for timely and accurate diagnosis of erythema migrans (EM). Although Borrelia burgdorferi can be cultured from biopsies of EM lesions, immunodiagnostic testing is more widely available. Four immunoserologic methods were studied by using the sera of 51 patients with EM lesions that were culture positive for B. burgdorferi. Nineteen patients had single primary lesions, and thirty-two had multiple secondary lesions. At the time of biopsy, 40 patients, 8 with primary lesions and all patients with secondary lesions, were seropositive by an immunoglobulin M (IgM) indirect fluorescent-antibody (IgM IFA) test (Bion Enterprises). Twenty-three patients were seropositive by a whole-cell fluorescence enzyme immunoassay (EIA) (BioWhittaker, Inc.), twenty-two were positive by immunoblotting (ViroStat, Inc.), and one was positive by a P39 recombinant EIA (P39 EIA) (General Biometrics, Inc.). Sera from various patient control groups were tested: rheumatoid arthritis (n = 19), infectious mononucleosis (n = 20), systemic lupus (n = 22), syphilis (n = 13), streptococcal sequelae (n = 20), and healthy subjects (n = 16). None of these sera reacted with the IgM IFA test or P39 EIA. Fifteen reacted with the fluorescence EIA. We conclude that the IgM IFA test is an effective and reliable assay for the diagnosis of EM, particularly for patients with secondary lesions. Immunoblot, fluorescence EIA, and P39 EIA lack the sensitivity to reliably diagnose EM.

Adolescent↗
PubMed1995

Detection of respiratory syncytial virus by reverse transcription-PCR and hybridization with a DNA enzyme immunoassay.

Nasal aspirates from 238 infants hospitalized with acute respiratory infections during the winter of 1994 and 1995 were tested for respiratory syncytial virus (RSV) by immunofluorescence assay (IFA) and the viral isolation technique (VIT) and by two PCR and hybridization methods: reverse transcription PCR 1 (RT-PCR1), which amplifies the RNAs of all RSV strains, and RT-PCR-2, which allows subgroup classification of RSV. RT-PCR-1 and RT-PCR-2 detected viral sequences in 56.7% (135 of 238) and 48.3% (115 of 238) of the samples, respectively, while only 80 (33.6%) samples were found to be positive by IFA and VIT. Of the PCR-positive specimens, 57 were missed by these routine techniques in RT-PCR-1 and 45 were missed in RT-PCR-2. Although the RSV-PCR-1 and RSV-PCR-2 techniques amplified two different sequences of the RSV genome, they gave similar results for 218 (91.6%) nasal aspirates. Compared with conventional methods, the sensitivity, specificity, and agreement were 97.5, 63.9, and 75.2%, respectively, for RT-PCR-1 and 89.7, 71.9, and 77.7%, respectively, for RT-PCR-2, and for these two RT-PCR assays, the positive predictive value (PPV) and the index of agreement (kappa) were comparable and moderate, respectively: PPV was 57.8% and kappa was 0.52 in RT-PCR-1, and PPV was 60.9% and kappa was 0.54 in RT-PCR-2. However, there was a perfect correlation between the two RT-PCRs, with a PPV of 100% and an excellent index of agreement (kappa = 0.88). Therefore, most RT-PCR results were really true positive, and VIT and IFA, which missed some of them, appeared to be less sensitive.

Base Sequence↗
PubMed1995

Development of specific fluorescent-antibody test for tissue form of Penicillium marneffei.

The diagnosis of penicilliosis marneffei can be difficult because the clinical manifestations mimic those of tuberculosis, histoplasmosis, and other mycotic infections. Furthermore, the tissue form of Penicillium marneffei can be confused with those of Histoplasma capsulatum and Cryptococcus neoformans. To facilitate the rapid detection and identification of P. marneffei in clinical materials, we sought to develop a specific indirect fluorescent-antibody (IFA) reagent for this dimorphic pathogen. Preliminary IFA studies with yeast-like cells (fission arthroconidia) of P. marneffei indicated that these cellular elements stained with antiglobulins against culture filtrate antigens and whole yeast-like cellular antigens. Both types of antiglobulins reacted with the yeast-like cells of P. marneffei and with H. capsulatum, but not with their respective mycelial forms. The antiglobulins also failed to react with the yeast and hyphal forms of a variety of other heterologous fungi. Specific antiglobulins useful in an IFA test for identifying P. marneffei yeast-like cells in culture or in clinical materials were produced by adsorptions with yeast-form cells of H. capsulatum. The yeast-like culture filtrate antigens of P. marneffei are preferred for use in the production of the specific antiglobulins because they stained P. marneffei yeast-like elements more intensely than antiglobulins produced against intact yeast-like cells.

Animals↗
PubMed1996

Evaluation of a commercial enzyme-linked immunosorbent assay for detection of serum immunoglobulin G response to human herpesvirus 6.

A rapid (60-min) commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G (IgG) class antibodies to human herpesvirus 6 (HHV-6) was evaluated. The specificity of the ELISA for HHV-6 was confirmed by absorption studies, with the reactivities of HHV-6-positive sera being unaffected by other herpesviruses (cytomegalovirus, herpes simplex virus, and varicella-zoster virus) or the HSB2 cell line used to culture HHV-6. HHV-6 IgG antibody levels in a panel of 502 serum samples were determined by ELISA and an indirect immunofluorescence assay (IFA). Results obtained by the two methods were in close agreement, suggesting that the ELISA provides a suitable test method for the determination of HHV-6 IgG antibodies in a routine clinical laboratory. Both tests were positive in 398 cases (79%), and both were negative in 71 cases (14%), with a different result obtained by IFA and ELISA in only 33 cases (7%). Furthermore, absorption of sera with HHV-6 prior to assay revealed that the majority of these results were false positive (n = 8) or false negative (n = 23) in the IFA (true positives or negatives in the ELISA). Subsequently, the ELISA showed a sensitivity of 99.76% and a specificity of 98.75%. HHV-6-specific IgG levels were also determined in paired serum samples collected from 49 donors--14 with exanthem subitum (ES), 15 with ES which was complicated with central nervous system involvement, and 20 undergoing bone marrow transplantation--in whom HHV-6 infection had been demonstrated by virus isolation and/or PCR. All patients with ES or central nervous system complications showed an increase in HHV-6-specific IgG, indicating that this ELISA may be a useful aid in the diagnosis of these conditions. Furthermore, 14 of 20 patients undergoing bone marrow transplantation showed an increase in HHV-6-specific IgG levels, possibly reflecting a reactivation of HHV-6 in these patients.

Adolescent↗
PubMed1996

Serological diagnosis of ovine enzootic abortion by comparative inclusion immunofluorescence assay, recombinant lipopolysaccharide enzyme-linked immunosorbent assay, and complement fixation test.

Since the 1950s, serological diagnosis of ovine enzootic abortion (OEA), caused by strains of Chlamydia psittaci, has been based mainly on the complement fixation test (CFT), which is neither particularly sensitive nor specific since antibodies to other chlamydial and enterobacterial pathogens may be detected. In this study. a recombinant enzyme-linked immunosorbent assay (rELISA) (medac, Hamburg, Germany), based on a unique chlamydial genus-specific epitope of Chlamydia trachomatis L2 lipopolysaccharide, was evaluated for sensitivity and specificity as a primary screening assay for OEA by comparison with the CFT. A comparative inclusion immunofluorescence assay (IFA), in which antibody titers to C. psittaci and Chlamydia pecorum were examined, was used as the reference test for 573 serum samples from four flocks. Reactivity to C. pecorum was measured since inapparent intestinal infections by C. pecorum are believed to be common in British flocks. In detecting positive sera from an abortion-affected flock, in which a C. pecorum infection was also suggested by IFA, the rELISA outperformed the CFT with significant evidence for increased sensitivity (P = 0.003). In two flocks in which C. pecorum infections alone were suggested by IFA, the rELISA and CFT were prone to detect low levels of false-positive results, but the values were not significant. The rELISA provided results in one flock in which sera that were anticomplementary could not be resolved by the CFT. In another flock in which abortion had not occurred but infection by both chlamydial species was suspected, no significant difference was found between the sensitivities of the rELISA and CFT. The rELISA could not differentiate ovine C. psittaci and C. pecorum infections but was shown to be a more sensitive primary screening test for OEA than was the CFT, particularly where abortion had occurred and even when antibodies due to additional inapparent infection(s) by C. pecorum were present.

Abortion, Veterinary↗
PubMed1997

Comparison of major antigenic proteins of six strains of the human granulocytic ehrlichiosis agent by Western immunoblot analysis.

The etiologic agent of human granulocytic ehrlichiosis (HGE) is an obligate intracellular bacterium. In 1996, blood specimens from 53 patients suspected of having HGE were examined by indirect fluorescent antibody (IFA) testing with the HGE agent no. 13 isolate as the antigen, by nested PCR, and by culture. All patients resided in Westchester County, N.Y. Twelve patient specimens were positive for IFA (titer > or = 1:40). Seven of these were also positive by PCR. Of the seven specimens positive by both IFA testing and PCR, the HGE agent was isolated from four (no. 2, 3, 6, and 11) and continuously cultured in HL-60 cells. These were confirmed as the HGE agent by sequencing of 16S rDNA. Both purified whole-cell organisms and the outer membrane fractions of the new isolates were compared with no. 13 isolate and a tick (USG) isolate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analysis. No. 11 and 13 isolates had identical SDS-PAGE patterns with respect to 49- and 47-kDa proteins. No. 3 and USG isolates lacked the 47-kDa protein, and no. 6 isolate lacked the 49-kDa protein. Both 49- and 47-kDa bands were absent in no. 2 isolate. Western blot results with seven different sera, including five convalescent-phase sera from these patients, one dog anti-USG isolate, and one horse anti-BDS isolate, showed that all major antigens in six isolates were recognized by all sera. However, the molecular sizes and the numbers of major antigens recognized varied among the six isolates. Overall, HGE agent no. 3, 6, 11, and 13, and USG isolates had similar patterns, with 1 or 2 major antigens with molecular masses of around 49 and 47 kDa. No. 2 isolate was quite distinct in having a major antigen of 43 kDa. This indicates that although these antigenic epitopes are all cross-reactive among strains, the HGE agent has a strain pleomorphism in its major antigenic proteins. The major antigen profiles of the outer membrane protein fractions and of whole organisms of six HGE agent isolates were similar, suggesting that 49- and 47-kDa major antigens are the outer membrane proteins of the HGE agent.

Animals↗
PubMed1997

Prevalence of granulocytic Ehrlichia infection among white-tailed deer in Wisconsin.

Human granulocytic ehrlichiosis (HGE) is caused by an agent that is nearly indistinguishable from the veterinary pathogens Ehrlichia equi and Ehrlichia phagocytophila. The deer tick, Ixodes scapularis, is a vector of the HGE agent, and the white-tailed deer is the primary host for adult Ixodes ticks. We assessed the distribution of granulocytic Ehrlichia infection among deer living within (Wisconsin) and outside (western and southern Iowa) the geographic range of L. scapularis. Whole-blood samples were tested for HGE 16S ribosomal DNA (rDNA) by PCR, and E. equi antibody was detected by indirect immunofluorescence assay (IFA). Antibody titers of > or = 1:64 were defined as positive, and all positive samples were retested with a second lot of substrate antigen. E. equi antibody was present in 14 (8%) of 187 Wisconsin deer and 0 of 60 Iowa specimens (rate ratio undefined; P = 0.025). An additional 30 serum samples from Wisconsin deer were excluded because IFA results were discrepant between substrate lots. The reciprocal antibody titers ranged from 64 to 512 (geometric mean, 141) for positive samples. PCR results were positive for 27 (15%) of 181 Wisconsin deer. The prevalence of infection in northwestern Wisconsin deer was not significantly different from that in central Wisconsin deer, as determined by IFA and PCR. In two samples that were sequenced, the 16S rDNA was nearly identical to that of the granulocytic Ehrlichia species but distinct from that of Anaplasma marginale. The DNA sequences of the samples differed from the published sequences for E. equi, E. phagocytophila, and the HGE agent by 1 or 2 nucleotides (> or = 99.1% homology) at phylogenetically informative sites. Granulocytic Ehrlichia organisms in deer are widely distributed within the geographic range of L. scapularis in Wisconsin. Deer may serve as useful sentinels for areas where HGE transmission to humans may occur.

Animals↗
PubMed1997

Comparison of PCR and culture to the indirect fluorescent-antibody test for diagnosis of Potomac horse fever.

Potomac horse fever is an acute systemic equine disease caused by Ehrlichia risticii. Currently, serologic methods are widely used to diagnose this disease. However, serologic methods cannot determine whether the horse is presently infected or has been exposed to ehrlichial antigens in the past. The purpose of the present study was to compare the sensitivities of the nested PCR and cell culture with that of the indirect fluorescent-antibody (IFA) test for the diagnosis of Potomac horse fever. Blood and fecal specimens serially collected from a pony experimentally infected with E. risticii Maryland, blood specimens serially collected from mice inoculated with E. risticii Ohio 380, and blood and/or fecal specimens collected from 27 horses which had clinical signs compatible with Potomac horse fever were examined. These horses resided in Kentucky, Indiana, Pennsylvania, and Vermont. The IFA test titer became positive after 6 days postinoculation (p.i.) for the pony. A culture of the blood of the pony was positive for E. risticii starting on day 1 and was positive through day 28 p.i. By the nested PCR, E. risticii was detectable in the blood and feces of the pony starting on day 1 and was detectable through day 32 p.i. E. risticii was detected in the blood of subclinically infected mice by the nested PCR. Twenty-two clinical specimens were seropositive for E. risticii by the IFA test, with titers ranging from 1:20 to 1:1,280. E. risticii was cultured from 95% (20 of 21) of seropositive clinical blood specimens. E. risticii was detected in the blood by PCR in 81% (17 of 20) of the culture-positive clinical specimens. The study indicated that the nested PCR is as sensitive as culture for detecting infection with E. risticii.

Animals↗
PubMed1998

Association of deficiency in antibody response to vaccine and heterogeneity of Ehrlichia risticii strains with Potomac horse fever vaccine failure in horses.

Ehrlichia risticii is the causative agent of Potomac horse fever (PHF), which continues to be an important disease of horses. Commercial inactivated whole-cell vaccines are regularly used for immunization of horses against the disease. However, PHF is occurring in large numbers of horses in spite of vaccination. In a limited study, 43 confirmed cases of PHF occurred between the 1994 and 1996 seasons; of these, 38 (89%) were in horses that had been vaccinated for the respective season, thereby clearly indicating vaccine failure. A field study of horses vaccinated with two PHF vaccines indicated a poor antibody response, as determined by immunofluorescence assay (IFA) titers. In a majority of horses, the final antibody titer ranged between 40 and 1,280, in spite of repeated vaccinations. None of the vaccinated horses developed in vitro neutralizing antibody in their sera. Similarly, one horse experimentally vaccinated three times with one of the vaccines showed a poor antibody response, with final IFA titers between 80 and 160. The horse did not develop in vitro neutralizing antibody or antibody against the 50/85-kDa strain-specific antigen (SSA), which is the protective antigen of the original strain, 25-D, and the variant strain of our laboratory, strain 90-12. Upon challenge infection with the 90-12 strain, the horse showed clinical signs of the disease. The horse developed neutralizing antibody and antibody to the 50/85-kDa SSA following the infection. Studies of the new E. risticii isolates from the field cases indicated that they were heterogeneous among themselves and showed differences from the 25-D and 90-12 strains as determined by IFA reactivity pattern, DNA amplification finger printing profile, and in vitro neutralization activity. Most importantly, the molecular sizes of the SSA of these isolates varied, ranging from 48 to 85 kDa. These studies suggest that the deficiency in the antibody response to the PHF vaccines and the heterogeneity of E. risticii isolates may be associated with the vaccine failure.

Animals↗
PubMed1998

Use of fluorescent-antibody staining of cytocentrifuge-prepared smears in combination with cell culture for direct detection of respiratory viruses.

Over a 3-year period, 1,003 respiratory samples were collected and examined for selected respiratory viruses with cytocentrifuged prepared smears stained with fluorescently labeled antibodies (IFA) in conjunction with cell culture. IFA results were compared with results obtained by cell culture. Viruses were isolated or detected by direct means in 401 samples. Agreement between culture and IFA was 90%.

Cells, Cultured↗
PubMed1999

Comparison of PCR, culture, and serological tests for diagnosis of Mycoplasma pneumoniae respiratory tract infection in children.

For diagnosis of Mycoplasma pneumoniae infection we compared two rapid tests, PCR and the immunoglobulin M immunofluorescence assay (IgM IFA), with culture and the complement fixation test (CFT), in a prospective study among 92 children with respiratory tract infection and 74 controls. Based on positivity of culture and/or CFT as the diagnostic criterion, nine patients (10%) were diagnosed with M. pneumoniae infection. All patients positive by culture were also positive by PCR. In all controls cultures, PCRs, and serological assays were negative, except in one with a positive IgM IFA. The IgM IFA had a low positive predictive value of 50%. Only a combination of PCR (seven patients) and CFT (seven patients) allowed diagnosis of all cases.

Adolescent↗
PubMed1999

Comparison of Ehrlichia chaffeensis recombinant proteins for serologic diagnosis of human monocytotropic ehrlichiosis.

Diagnosis of human monocytotropic ehrlichiosis (HME) generally depends on serology that detects the antibody response to immunodominant proteins of Ehrlichia chaffeensis. Protein immunoblotting was used to evaluate the reaction of the antibodies in patients' sera with the recombinant E. chaffeensis 120- and 28-kDa proteins as well as the 106- and the 37-kDa proteins. The cloning of the genes encoding the latter two proteins is described in this report. Immunoelectron microscopy demonstrated that the 106-kDa protein is located at the surfaces of ehrlichiae and on the intramorular fibrillar structures associated with E. chaffeensis. The 37-kDa protein is homologous to the iron-binding protein of gram-negative bacteria. Forty-two serum samples from patients who were suspected to have HME were tested by immunofluorescence (IFA) using E. chaffeensis antigen and by protein immunoblotting using recombinant E. chaffeensis proteins expressed in Escherichia coli. Thirty-two serum samples contained IFA antibodies at a titer of 1:64 or greater. The correlation of IFA and recombinant protein immunoblotting was 100% for the 120-kDa protein, 41% for the 28-kDa protein, 9.4% for the 106-kDa protein, and 0% for the 37-kDa protein. None of the recombinant antigens yielded false-positive results. All the sera reactive with the recombinant 28- or the 106-kDa proteins also reacted with the recombinant 120-kDa protein.

Amino Acid Sequence↗
PubMed2000

Improved sensitivity of PCR for diagnosis of human granulocytic ehrlichiosis using epank1 genes of Ehrlichia phagocytophila-group ehrlichiae.

The agent of human granulocytic ehrlichiosis (HGE), Ehrlichia phagocytophila, and Ehrlichia equi probably comprise variants of a single Ehrlichia species now called the Ehrlichia phagocytophila genogroup. These variants share a unique 153-kDa protein antigen with ankyrin repeat motifs encoded by the epank1 gene. The epank1 gene was investigated as an improved target for PCR diagnosis of HGE compared with the currently used 16S rRNA gene target. Primers for epank1 flanking a region that spans part of the 5' ankyrin repeat coding region and part of the unique 3' region were synthesized. Blood samples from 31 patients with suspected HGE who were previously tested by 16S rRNA gene (16S) PCR and indirect immunofluorescent antibody test (IFA) were retrospectively tested with the epank1 primers. Eleven patients were 16S PCR positive and had a seroconversion detected by IFA (group A), 10 patients were 16S PCR negative but had a seroconversion detected by IFA (group B), and 10 patients were 16S PCR negative and seronegative (group C). Ten of the 11 group A patients were epank1 PCR positive, all 10 of the group B patients were epank1 PCR positive, and all of the PCR-negative and seronegative patients (group C) were epank1 PCR negative. The epank1 primers are more sensitive than the previously used 16S rRNA gene primers and therefore may be more useful in diagnostic testing for HGE.

Ankyrin Repeat↗
PubMed2000

Serodiagnosis of Louse-Borne relapsing fever with glycerophosphodiester phosphodiesterase (GlpQ) from Borrelia recurrentis.

Human louse-borne relapsing fever occurs in sporadic outbreaks in central and eastern Africa that are characterized by significant morbidity and mortality. Isolates of the causative agent, Borrelia recurrentis, were obtained from the blood of four patients during a recent epidemic of the disease in southern Sudan. The glpQ gene, encoding glycerophosphodiester phosphodiesterase, from these isolates was sequenced and compared with the glpQ sequences obtained from other relapsing-fever spirochetes. Previously we showed that GlpQ of Borrelia hermsii is an immunogenic protein with utility as a serological test antigen for discriminating tick-borne relapsing fever from Lyme disease. In the present work, we cloned and expressed the glpQ gene from B. recurrentis and used recombinant GlpQ in serological tests. Acute- and convalescent-phase serum samples obtained from 42 patients with louse-borne relapsing fever were tested with an indirect immunofluorescence assay (IFA) and an enzyme-linked immunosorbent assay (ELISA) that used whole cells of B. recurrentis and with immunoblotting to whole-cell lysates of the spirochete and Escherichia coli producing recombinant GlpQ. The geometric mean titers of the acute- and convalescent-phase serum samples measured by IFA were 1:83 and 1:575, respectively. The immunoblot analysis identified a high level of reactivity and seroconversion to GlpQ, and the assay was more sensitive than the whole-cell IFA and ELISA using purified, recombinant histidine-tagged GlpQ. Serum antibodies to GlpQ and other antigens persisted for 27 years in one patient. We conclude that assessment of anti-GlpQ antibodies will allow serological confirmation of louse-borne relapsing fever and determination of disease prevalence.

Amino Acid Sequence↗