Indirect Fluorescent Antibody Titers Research Articles

Specificity and sensitivity of an indirect fluorescent antibody test to detect antibodies against bovine viral diarrhea virus.

Since being reported in 1979 and 2006, indirect fluorescent antibody (IFA) tests have not been reported to detect bovine viral diarrhea virus (BVDV) antibodies to our knowledge. Thus, we re-evaluated the efficacy and usefulness of IFA tests for BVDV serology. We tested 4 combinations of 2 antibody conjugates (fluorescein isothiocyanate [FITC]-conjugated rabbit IgG anti-bovine IgG; rabbit IgG F(ab')2 fragment anti-bovine IgG [F(ab')2 FITC-IgG]) and 2 washing solutions (PBS; carbonate-bicarbonate-buffered saline [CBBS]) to evaluate the specificity of an IFA test for BVDV. We compared the sensitivity of the optimal combination with virus neutralization (VN) tests and an ELISA, and compared IFA with VN titers against different genotype (subgenotype) strains. For the F(ab')2 FITC-IgG/CBBS combination, only 1 of the 156 (0.6%) 4-fold diluted cattle sera resulted in a nonspecific reaction; other combinations led to a much higher incidence (22.9-37.2%). For the F(ab')2 FITC-IgG/CBBS combination, IFA detection rates were identical (36 of 59) for BVDV1 and BVDV2 genotypes, and IFA titers against them were strongly correlated (r = 0.99). The antibody-detection rates of the IFA tests were almost identical to those of VN tests and the ELISA (κ: 0.96 and 0.89, respectively). The IFA titers against 4 strains (BVDV1a, BVDV1j, BVDV2a, and an unidentified strain) were similar, 1,024 to ≥4,096, although the VN titers were different. Thus, our IFA tests were specific and sensitive, and more useful than VN tests given that the IFA tests could evaluate the immune status of cattle using a representative strain, regardless of genotype (subgenotype).

Could Antinuclear Antibody Positivity Be a Factor Affecting Treatment Response in Immune Thrombocytopenia Patients on Eltrombopag?

Objective:Eltrombopag remains a prominent option in the treatment of steroid-dependent or steroid-refractory immune thrombocytopenia (ITP) patients. Unfortunately, not all patients respond to eltrombopag. Antinuclear antibody (ANA) positivity can be seen at rates of up to 30% in ITP patients. Despite being widely used, more markers to predict the response to eltrombopag are still needed. In the present study, we aimed to show the association between ANA positivity and eltrombopag response in ITP patients.Materials and Methods:Patients who were diagnosed with ITP in the Trakya University Faculty of Medicine’s Department of Hematology and who underwent eltrombopag treatment due to their resistance to steroids and other treatments were included in our study. ANA measurement was performed by indirect fluorescent antibody method and titers of 1:160 and above were considered positive. ANA measurements were made before starting eltrombopag.Results:Forty-five patients were included in our study, 33 being women and 12 men. The mean age of the patients was 45.73 years. There were 14 patients with ANA positivity and 31 patients were found to be ANA-negative. Response rates were higher in ANA-negative patients compared to ANA-positive patients in the 1st and 6th months of eltrombopag treatment (p<0.05).Conclusion:ANA positivity in ITP may indicate unresponsiveness to eltrombopag treatment, a finding that should be further supported by prospective studies involving more patients.

A walk through the tall grass: A case of transaminitis, thrombocytopenia, and leukopenia resulting from an emerging zoonotic infection in Nova Scotia

Tick born zoonotic infections spread by Ixodes scapularis are on the rise in Canada. Tick-borne illnesses have a large constellation of symptoms associated with them which can often overlap and cloud diagnostic certainty. Human granulocytic anaplasmosis (HGA) is an emerging infectious disease in Canada, with tick carriage rates as high as 15% in some parts of the country. The majority of cases are associated with fever, headache, myalgia and malaise, thrombocytopenia, and transaminitis. The diagnosis is made through indirect fluorescent antibody titres, examination of peripheral smear, or polymerase chain reaction. Here we present the case of an otherwise health 71-year-old male presenting with fevers, thrombocytopenia, leukopenia, and a mixed transaminitis following a tick bite. He was subsequently diagnosed as the first case of HGA in Nova Scotia confirmed by indirect fluorescent antibody titre.

Serological prevalence of toxoplasmosis and neosporosis in dogs diagnosed with suspected meningoencephalitis in the UK.

To assess the prevalence of antibodies to Toxoplasma gondii and Neospora caninum in a population of dogs with a diagnosis of suspected inflammatory meningoencephalitis. Medical records of three referral centres were reviewed from 2008 to 2016 to identify a cohort of dogs diagnosed and treated for suspected inflammatory meningoencephalitis after testing for evidence of exposure to these pathogens. In our sample of 400 dogs the prevalence for exposure (IgG>1:50) to Toxoplasma gondii was 8/201 (3∙98%). Active infection (IgG titre >1:400 or/and an IgM titre >1:64 and/or positive PCR in CSF) was suspected in 1/400 (0∙25%). The prevalence for exposure [Indirect fluorescent antibody (IFA) titre >1:50] and active infection (IFA titres ≥⃒1:400 and/or positive PCR in CSF) with Neospora caninum were 14/201 (6∙96%) and 9/400 (2∙25%), respectively. In view of the low prevalence of protozoan infections, the risk associated with starting immunosuppressive medication in dogs with evidence of inflammatory meningitis or encephalitis in the UK appears low.

Feasibility of Using Convalescent Plasma Immunotherapy for MERS-CoV Infection, Saudi Arabia.

We explored the feasibility of collecting convalescent plasma for passive immunotherapy of Middle East respiratory syndrome coronavirus (MERS-CoV) infection by using ELISA to screen serum samples from 443 potential plasma donors: 196 patients with suspected or laboratory-confirmed MERS-CoV infection, 230 healthcare workers, and 17 household contacts exposed to MERS-CoV. ELISA-reactive samples were further tested by indirect fluorescent antibody and microneutralization assays. Of the 443 tested samples, 12 (2.7%) had a reactive ELISA result, and 9 of the 12 had reactive indirect fluorescent antibody and microneutralization assay titers. Undertaking clinical trials of convalescent plasma for passive immunotherapy of MERS-CoV infection may be feasible, but such trials would be challenging because of the small pool of potential donors with sufficiently high antibody titers. Alternative strategies to identify convalescent plasma donors with adequate antibody titers should be explored, including the sampling of serum from patients with more severe disease and sampling at earlier points during illness.

Feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with Middle East respiratory syndrome coronavirus infection: a study protocol.

As of September 30, 2015, a total of 1589 laboratory-confirmed cases of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) have been reported to the World Health Organization (WHO). At present there is no effective specific therapy against MERS-CoV. The use of convalescent plasma (CP) has been suggested as a potential therapy based on existing evidence from other viral infections. We aim to study the feasibility of CP therapy as well as its safety and clinical and laboratory effects in critically ill patients with MERS-CoV infection. We will also examine the pharmacokinetics of the MERS-CoV antibody response and viral load over the course of MERS-CoV infection. This study will inform a future randomized controlled trial that will examine the efficacy of CP therapy for MERS-CoV infection. In the CP collection phase, potential donors will be tested by the enzyme linked immunosorbent assay (ELISA) and the indirect fluorescent antibody (IFA) techniques for the presence of anti-MERS-CoV antibodies. Subjects with anti-MERS-CoV IFA titer of ≥1:160 and no clinical or laboratory evidence of MERS-CoV infection will be screened for eligibility for plasma donation according to standard donation criteria. In the CP therapy phase, 20 consecutive critically ill patients admitted to intensive care unit with laboratory-confirmed MERS-CoV infection will be enrolled and each will receive 2 units of CP. Post enrollment, patients will be followed for clinical and laboratory outcomes that include anti-MERS-CoV antibodies and viral load. This protocol was developed collaboratively by King Abdullah International Medical Research Center (KAIMRC), Gulf Cooperation Council (GCC) Infection Control Center Group and the World Health Organization—International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC-WHO) MERS-CoV Working Group. It was approved in June 2014 by the Ministry of the National Guard Health Affairs Institutional Review Board (IRB). A data safety monitoring board (DSMB) was formulated. The study is registered at http://www.clinicaltrials.gov (NCT02190799).

A longitudinal study of Babesia microti infection in seropositive blood donors

Babesia infection is caused by intraerythrocytic tick-borne parasites. Cases of transfusion-transmitted babesiosis have been increasingly recognized. To date, no Babesia test has been licensed for screening US blood donors. We conducted a longitudinal study to assess the course and markers of Babesia infection among seropositive donors identified in a seroprevalence study. Eligible donors had B. microti indirect fluorescent antibody (IFA) titers of 64 or greater. Enrollees were monitored up to 3 years, by IFA and three methods for evidence of parasitemia: B. microti nested polymerase chain reaction (PCR) analysis (at two laboratories), hamster inoculation, and blood-smear examination. Among 115 eligible donors, 84 (73%) enrolled. Eighteen enrollees (21%) had evidence of parasitemia for 30 total specimens (17% of 181), which were collected in 9 different months and tested positive by various approaches: PCR (25 specimens/16 persons), hamster inoculation (13 specimens/8 persons), and blood smear (one specimen positive by all three approaches). Overall, 14 persons had one or more specimen with positive PCR results at both laboratories (12 persons) and/or had parasitologically confirmed infection (eight persons). Three of nine persons who had more than one specimen with evidence of parasitemia had nonconsecutive positives. Several enrollees likely had been infected at least 1 year when their last positive specimen was collected. The final three specimens for seven persons tested negative by all study methods, including IFA. Seropositive blood donors can have protracted low-level parasitemia that is variably and intermittently detected by parasitologic and molecular methods. Donor-screening algorithms should include serologic testing and not solely rely on molecular testing.

The third described case of transfusion‐transmitted Babesia duncani

Almost all of the reported US tick-borne and transfusion-associated Babesia cases have been caused by Babesia microti, which is endemic in the Northeast and upper Midwest. We investigated a case caused by B. duncani (formerly, the WA1-type parasite), in a 59-year-old California resident with sickle cell disease (HbSS) whose only risk factor for infection was receipt of red blood cell transfusions. The patient's case was diagnosed in September 2008: intraerythrocytic parasites were noted on a blood smear, after a several-month history of increasing transfusion requirements. Molecular and indirect fluorescent antibody (IFA) analyses were negative for B. microti but were positive for B. duncani (IFA titer, 1:1024). The complete 18S ribosomal RNA gene of the parasite was amplified from a blood specimen; the DNA sequence was identical to the sequence for the index WA1 parasite isolated in 1991. The patient's case prompted a transfusion investigation: 34 of 38 pertinent blood donors were evaluated, none of whom tested positive by B. microti IFA. The implicated donor-a 67-year-old California resident-had a B. duncani titer of 1:4096; B. duncani also was isolated by inoculating jirds (Mongolian gerbils) with a blood specimen from March 2009, more than 10 months after his index donation in April 2008. The patient's case was diagnosed more than 4 months after the implicated transfusion in May 2008. This patient had the third documented transfusion case caused by B. duncani. His case underscores the fact that babesiosis can be caused by agents not detected by molecular or serologic analyses for B. microti.

Leishmaniasis in a dog native to Colorado

A 1-year-old 32.5-kg (71.5-lb) sexually intact male foxhound-Treeing Walker Coonhound cross was evaluated because of a 2.5-month history of dermatologic lesions, weight loss, and diarrhea. Physical examination revealed muscle wasting, lymphadenopathy, and multifocal pruritic dermatologic lesions of alopecia, thickening, erythema, and follicular casting. Hematologic and serum biochemical analyses revealed nonregenerative anemia, mono-cytosis, hypercalcemia, hyperproteinemia, and hyperglobulinemia. Proteinuria was identified on urinalysis. Hepatomegaly, splenomegaly, and diffuse abdominal lymphadenomegaly were detected on abdominal ultrasonography. A diagnosis of leishmaniasis was confirmed by ELISA detection of serum antibodies against Leishmania spp, a high serum indirect fluorescent antibody titer (1:1,024) against Leishmania infantum, amplification of Leishmania DNA on PCR assay of a whole blood sample and a lymph node aspirate, and histologic identification of suspected Leishmania amastigotes in skin specimens. In addition, the dog had a low CD4+:CD8+ lymphocyte ratio of 1:1. The dog was euthanized because of the severity of leishmaniasis and poor prognosis. This dog was from a litter of 10 puppies that included 4 stillborn puppies, 2 puppies that died as neonates, and 1 littermate that was euthanized at 1 year of age because of a high serum antibody titer against Leishmania spp. Eventually the foxhound dam was euthanized because of a high serum antibody titer against Leishmania spp. The dog had been raised with an unaffected littermate, its sire, and an unrelated Treeing Walker Coonhound female that were seronegative for Leishmania infection. Although vertical disease transmission was suspected, it is possible that L infantum is now endemic in Colorado. Leishmaniasis should be considered in dogs with scaly dermatoses.

A Serological Study on Toxoplasma gondii Infection Among People in South of Tehran, Iran

Although Toxoplasma gondii infection generally shows subclinical courses in adults, it may have severe pathologic effects on the fetus. For a survey of toxoplasmosis in Shahr-e-Rey area, south of Tehran, Iran, we conducted a serological study on 1,187 general populations using indirect fluorescent antibody (IFA) test. In this cross sectional study, cases were selected randomly by a local primary health care (PHC) system. Blood samples were collected in microcapillary tubes and sera were examined by IFA after centrifugation. It has been shown that 68.3% of cases in all age groups had IFA titer of 1 : 10 and higher, which we consider as positive cases, and the rate for 1 : 400 or over was 30.2%. There was meaningful correlation between anti-Toxoplasma antibody titers and age, sex, and occupation of subjected people (P < 0.05%). The incidence was higher in females and highest in the children age group (10-14 years old). The present study demonstrates high prevalence of Toxoplasma infection among the people in south of Tehran, Iran.

White-tailed deer ( Odocoileus virginianus) develop spirochetemia following experimental infection with Borrelia lonestari

Borrelia lonestari is considered a putative agent of southern tick-associated rash illness (STARI) and is known to occur naturally only in lone star ticks ( Amblyomma americanum) and white-tailed deer ( Odocoileus virginianus). We used a low passage isolate of B. lonestari (LS-1) to inoculate white-tailed deer, C3H mice, Holstein cattle, and beagles. Animals were monitored via examination of Giemsa and acridine orange stained blood smears, polymerase chain reaction (PCR), indirect fluorescent antibody (IFA) test, and/or culture isolation. Spirochetes were visualized in blood smears of both deer on days post-inoculation (DPI) 6, 8, 12 and one deer on DPI 15. Whole blood collected from deer tested PCR positive starting on DPI 4 and remained positive as long as DPI 28. Both deer developed antibody titers of >64, with a maximum IFA titer of 1024. The organism was reisolated from the blood of both deer on DPI 6 and one deer on DPI 12. All isolation attempts from mice, calves, or dogs were negative, although one of seven mice was transiently PCR positive. Mice and dogs developed an IFA titer ≥64, while calves lacked a detectable antibody response. These preliminary experimental infection trials show that white-tailed deer are susceptible to infection with B. lonestari and develop a spirochetemia following needle-inoculation, while C3H mice, calves, and dogs do not. Results suggest that deer may serve as a vertebrate reservoir host. Tick transmission studies are needed to confirm that this organism can be maintained in a natural cycle involving deer and A. americanum.

Central Nervous System Dysfunction Associated With Rocky Mountain Spotted Fever Infection in Five Dogs

Five dogs from the northeastern United States were presented with clinical signs of neurological disease associated with Rocky Mountain spotted fever (RMSF) infection. Four of the five dogs had vestibular system dysfunction. Other neurological signs included paresis, tremors, and changes in mentation. All of the dogs had an elevated indirect fluorescent antibody titer or a positive semiquantitative enzyme screening immunoassay titer for Rickettsia rickettsii at the time of presentation. Although a higher mortality rate has been reported for dogs with neurological symptoms and RMSF infection, all of the dogs in this study improved with appropriate medical therapy and supportive care.

Seronegative conversion in four Neospora caninum-infected cows, with a low rate of transplacental transmission

Four Neospora-seropositive pregnant cows (prebreeding indirect fluorescent antibody (IFA) titers between 1:400 and 1:1600) were confined and observed until parturition. All cows gave birth to normal calves. Selected tissues were tested for NC by histopathology, immunohistochemical (IHC) and polymerase chain reaction (PCR). Parasite isolation was attempted in vero cell cultures. At parturition, all cows were seronegative at 1:200 and two of four cows had a titer of 1:100 when further tested. Three of four calves were not infected, as determined by negative results of precolostral serology (1:25 cut-off), histopathology, IHC and PCR. One calf was congenitally infected, as shown by the presence of a thick-walled cyst labelled by IHC in its brain, positive PCR of brain and a precolostral IFA titer of 1:100. It was concluded that NC antibody titers may drop or convert to seronegative status in chronically infected cows by the time of parturition and this finding in four of four cows indicates that this could be a common occurrence. Similarly, the finding of an infected calf with a low antibody titer indicates that precolostral serology may not be a fool-proof means of identifying calves with congenital Neospora caninum infections. These findings call into question conclusions of other studies that have estimated rates of congenital transmission of this parasite based on serological tests at calving. This study is the first confirmed report of congenital NC infection in a calf in Thailand.

Cross-sectional study of serum antibodies against Sarcocystis neurona in cats tested for antibodies against Toxoplasma gondii.

To determine apparent seroprevalence of antibodies against Sarcocystis neurona in a population of domestic cats previously tested for antibodies against Toxoplasma gondii. Cross-sectional study. Serum from 196 domestic cats. Banked serum samples submitted to the Michigan State University Animal Health Diagnostic Laboratory for T. gondii diagnostic testing were tested for antibodies against S. neurona by use of an indirect fluorescent antibody (IFA) test and a western blot test. Submission records were analyzed to determine descriptive statistics and test for associations between positive results of a test for S. neurona and other variables in the data set. 10 of 196 (5%) samples yielded positive results for antibodies against S. neurona by use of western blot analysis, whereas 27 samples yielded positive results by use of the IFA. No association was found between S. neurona western blot test results and T. gondii test results, age, sex, or the reason for T. gondii testing. The S. neurona IFA titer was positively and significantly associated with positive results of western blot analysis. Domestic cats are not likely to play a substantial role as intermediate hosts in the natural life cycle of S. neurona. Results indicate that natural infection of domestic cats may occur, and small animal practitioners should be aware of this fact when evaluating cats with neurologic disease. The S. neurona IFA test had lower specificity than western blot analysis.

Isolation of Neospora caninum from the brain of a naturally infected adult dairy cow

Neospora caninum was isolated from the brain of a 2-year-old dairy cow that had aborted confirmed N. caninum-infected fetuses on two occasions. The cow had an indirect fluorescent antibody titer of 1:1600 to N. caninum. The cow was killed 24 days after its second abortion and the brain was bioassayed for N. caninum in nude mice. Multifocal areas of perivascular cuffing and glial nodules were observed in the cerebrum and mesencephalon of the cow, but N. caninum was not identified in histological sections of the brain. All three nude mice inoculated with brain homogenate of the cow, developed emaciation and paralysis. Microscopical examination of the nude mice revealed systemic N. caninum infection with demonstrable tachyzoites in various organs. The parasites isolated from fresh mouse brain were transferred successfully into Vero cell cultures. PCR procedure on the purified tachyzoites obtained from the Vero cell cultures amplified the specific DNA sequence for N. caninum.

Cat-scratch disease encephalopathy: A cause of status epilepticus in school-aged children

We describe 6 school-aged patients who presented with status epilepticus (SE) secondary to cat-scratch disease (CSD) encephalopathy to alert clinicians to this distinctive clinical entity. The hospital database for admissions during 1 year was reviewed for patients presenting with SE; 4 of 5 previously healthy school-aged children with SE had CSD encephalopathy based on elevated indirect fluorescent antibody titers to Bartonella henselae . CSD encephalopathy should be included in the differential diagnosis of school-aged children presenting with SE. (J Pediatr 1999;134:635-8)

Application of Seroepidemiology in the Evaluation of a Community-Based Malaria Control Program in Parawan, the Philippines.

Seroepidemiology has several proven applications in malaria endemic areas. In this study, it was used to assess the effectiveness of a community-based malaria control program in the focus of malaria transmission. The first serological survey was done before the implementation of a community-based malaria control program (pre-intervention), and the second one was done after 5 years of intervention in the study area. Comparison of the distribution of the indirect fluorescent antibody (IFA) titers showed a reduction in the high titer-responses after the intervention. Moreover, there was a statistically significant reduction in the geometric mean reciprocal titer (GMRT) after the intervention. These findings were suggestive of a reduction in malaria transmission resulting from the intervention. Results of a parallel parasitologic study revealed the same findings. Therefore, seroepidemiology, when used to complement the parasitologic measurement, is valuable in monitoring the effectiveness of malaria control measures.

Cloning and expression of the 44-kilodalton major outer membrane protein gene of the human granulocytic ehrlichiosis agent and application of the recombinant protein to serodiagnosis.

A 44-kDa major outer membrane protein of the human granulocytic ehrlichiosis (HGE) agent is an immunodominant antigen in human infection. A gene encoding this protein was cloned and sequenced. Southern blot results revealed the existence of multigenes homologous to the P44 gene in the genome of the HGE agent. The recombinant 44-kDa protein (rP44) was expressed by using expression vector pET30a. The reactivity of the affinity-purified rP44 was evaluated by Western immunoblot analysis and dot blot immunoassay. Western immunoblot analysis showed that mouse anti-rP44 serum reacted with 44- to 42-kDa proteins in six different HGE agent strains tested except strain 2, in which three proteins of 42, 40, and 38 kDa were recognized. Eleven HGE patient serum samples, a horse anti-HGE serum, and a horse anti-Ehrlichia equi serum recognized the rP44 protein. This suggests that rP44 is an HGE-E. equi group-specific antigen. Neither human anti-Ehrlichia chaffeensis serum nor rabbit anti-Borrelia burgdorferi serum reacted with rP44. Sera from two patients coinfected with the HGE agent and B. burgdorferi reacted positively with rP44 and the HGE agent. Sera from 20 HGE patients with indirect fluorescent-antibody (IFA) titers ranging from 1:20 to 1:2,560 gave distinct positive reactions in a dot immunoblot assay. There was a positive correlation between the color densities of the dot reactions and the IFA titers when greater than 50 ng of recombinant antigen per dot was used. The use of the affinity-purified rP44 protein as antigen would provide a more specific, consistent, and simpler serodiagnosis for HGE than the use of whole infected cells or purified HGE agents.

Recurrent deep pyoderma in German Shepherd dogs with underlying ehrlichioses and hypergammaglobulinaemia.

Four German Shepherd dogs with recurrent deep pyoderma and associated ehrlichioses are described. All the dogs had already received appropriate long-term antibacterial therapy but the clinical signs had consistently returned within 3-4 weeks of cessation of the treatment. Haematological and serum biochemical findings, and indirect fluorescent antibody (IFA) tests were indicative of Ehrlichia spp. infection. Treatment with doxycycline monohydrate was associated with the return of blood values to within normal ranges and diminution of the IFA titres. No further episodes of pyoderma were observed during a 1-2-year follow-up period.

Serologic survey for antibodies to Borrelia burgdorferi in white-tailed deer in Ontario.

Serum samples collected from 623 white-tailed deer (Odocoileus virginianus) in southern Ontario (Canada) from 1985 to 1989 were tested for antibodies to Borrelia burgdorferi using an indirect fluorescent antibody (IFA) staining method. Samples from 150 of the deer were also tested using an enzyme-linked immunosorbent assay (ELISA). At IFA titers of 1:64 and 1:128 deer with antibodies to B. burgdorferi appeared to be widespread throughout southern Ontario, with an apparent prevalence ranging from 3 to 47%. At IFA titres > or = 1:256 and ELISA titres > or = 1:160 deer with antibodies to B. burgdorferi were only present on Long Point which is the only known endemic focus of Ixodes scapularis, the primary vector for B. burgdorferi, in southern Ontario. At these titres the apparent prevalence of antibodies to B. burgdorferi on Long Point was only 5 to 7%, even though the mean intensity of infestation of adult I. scapularis on deer was > 180, and 60% of the adult ticks are infected with B. burgdorferi. Based on these results, white-tailed deer do not appear to be a good sentinel species for the distribution of B. burgdorferi.