Indirect Fluorescent Antibody Method Research Articles

Development of an Indirect Fluorescent Antibody (IFA) Assay for the Detection of Leishmania RNA Virus 2 (LRV2) in Leishmania Parasites.

Detection of Leishmania RNA virus (LRV) in Old World Leishmania species and their possible role in the disease prognosis requires sensitive and specific methods, preferably independent of the viral genome. We aimed to develop an indirect immunofluorescence antibody (IFA) assay to detect LRV in the Old World Leishmania parasites. Clinical samples were collected from 86 cutaneous leishmaniasis (CL) patients in different endemic areas of CL in Iran, during 2017-2019. For antibody preparation, the viruses were obtained from sediment of an LRV-infected L. major culture-using freeze and thaw cycles followed by gradient cesium chloride centrifugation. The purified viruses were used to immunize a male 3-4 months rabbit. Various dilutions of the LRV-immunized rabbit's serum and a conjugated antibody were deployed to detect LRV in 48 isolates by IFA assay. LRV virus was detected in four of the 48 CL cases using IFA method. Amplification of a partial fragment of RNA-dependent RNA polymerase (RdRp) gene from the isolates confirmed the IFA results. In phylogeny, the generated RdRp sequences from four isolates were grouped with the other Old World LRVs, but separate from L. aethiopica LRVs, which appeared as a highly supported distinct clade. Further optimization of this approach to detect the LRV directly in lesion scrapings can make it a more reliable tool for field studies and disclosing the virus's possible role in disseminating and unusual clinical features.

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.

Investigation of Intestinal Protozoon Prevalence in Immunocompromised Patients at a University Hospital

Immunocompromised patients are at a greater risk of developing intestinal parasite infections. In this study, we examined the presence of Enterocytozoon bieneusi, Encaphalitozoon intestinalis and other intestinal protozoa in stool samples of immunosuppressed patients. A total of 100 stool samples were obtained from patients receiving chemotherapy because of solid organ tumour with haematological malignancies and those receiving immunosuppressive treatment because of rheumatic diseases, organ transplant patients and patients receiving treatment for HIV-related infections. Stool samples were examined by using the native-lugol method in which the stool concentration, modified Kinyoun acid-fast and trichrome staining methods and parasite presence were analysed. The stool samples were also examined for the presence of Enterocytozoon bieneusi and Encephalitozoon intestinalis using an indirect fluorescent antibody method. Intestinal parasites were detected in 12% of all patients. The distribution of intestinal parasites in patients were 7% Blastocystis spp., 2% Blastocystis spp. + Dientamoeba fragilis, 1% Blastocystis spp. + Entamoeba coli, 1% Blastocystis spp. + Giardia intestinalis and 1% G. intestinalis. Microsporidia spp. were detected in 4% of all patients by the IFAT method and in 8% of all patients by calcoflour staining method. In our study, the most prevalent parasite detected in the immunosuppressed patients was Blastocystis spp. The pathogenesis of Blastocystis spp. remains to be controversial, and their role in immunocompromised patients continues to remain unknown. Although these rates detected in our study are similar to the prevalence in the normal population, it is important to study these microorganisms in immunocompromised patients in terms of the associated decreasing morbidity and mortality rates.

Detection of autoantibodies by indirect immunofluorescence and related techniques: the pathologist´s view

The increasing importance of autoimmunity based diseases attracts the interest of pathologists, for whom the wide acceptance of indirect fluorescent antibody (IFA) technique at autoantibody screening is relevant diagnostic approach. The IFA method has gained perfection by introducing the so called diagnostic mosaics, consisting of various tissue sections (primate and/or mammalian in origin), of Hep-2 cells, of microbeads (biochips) coated with specific purified antigen(s) and/or of transfected HEK cells expressing the target autoantigens. As result of this, the IFA technique dominates not only in the detection of anti-nuclear antibodies (ANA) and of anti-neutrophil cytoplasmic antibodies (ANCA), but is useful also in the search for serological markers in many other autoimmune disorders. Among diseases in question, the inflammatory bowel disease (GAB, PAB, ASCA), celiac disease (ARA, AEmA, AGA) and/or autoimmune hepatitis (AMA, ASMA, ABCA, anti-LKM1, SLA/LP) have emerged as important. Furthermore, antibodies to gastric parietal cells (APCA), to skin epidermal structures (anti-BP180, anti-Dsg1, anti-Dsg3), antibodies against neurons (anti-Hu, anti -Yo) and glial cells (anti-AGNA) and to several endocrine gland cells (AICA, SCA, OAB, anti-TPO, anti-TG) have recently gained attention. In addition to ANA, the detection of autoantibodies against skeletal or cardiac striated muscles (ASMA/ACMA) is of prognostic value in systemic sclerosis, Sjoegren´s syndrome, dilated cardiomyopathy, myositis and dermatomyositis. At last but not least, detection of anti-sperm antibodies (ASA) has become important due to their association with infertility occurring in the absence of mechanical obstruction of female reproductive system. We bring here an overview of reasonable applications of IFA, which, in combination with additional immunological techniques such as immunoblot (IB), allows a precise identification of several autoantibody target proteins. Though this paper stresses the advantages of IB technique, the enzyme linked immunosorbent assay (ELISA) cannot be neglected, since it is inevitable for certain diagnostic decisions (Table 1).

Investigation of Pneumocystis jirovecii in Respiratory Samples of Immunocompromised Patients with PCR, IFA and Giemsa Staining Methods

Pneumocystis jirovecii is an important opportunistic agent leading to pneumonia in immunocompromised patients. In this study, the presence of P.jirovecii were investigated by using Giemsa stain, indirect fluorescent antibody (IFA) test and two different nested polymerase chain reaction (nPCR) assays in respiratory samples obtained from 50 immunocompromised patients presenting with respiratory symptoms. The target genes used for nested PCR were mitochondrial large subunit ribosomal RNA (MtLSUrRNA) and internal transcribed spacer (ITS) region. P.jirovecii was detected in 7 (14%) and 11 (22%) respiratory samples by IFA and PCR, respectively, although all samples were negative with Giemsa stain. As a result, IFA and PCR were found to be rapid and reliable tests for the diagnosis of P.jirovecii infections and they should better be used together for accurate diagnosis.

Survey on Tick-Borne Pathogens in Thoroughbred Horses in the Hidaka District, Hokkaido, Japan

A total of 87 Thoroughbred horses and 10 ixodid ticks from a ranch in Hidaka district, Hokkaido were tested for tick-borne diseases. Using the indirect fluorescent antibody (IFA) method, 3.4, 92.0 and 97.7% of the horses showed antibody titers of ≥ 80 against Anaplasma phagocytophilum, Rickettsia helvetica, and Borrelia garinii, respectively. This is the first report of infection with the 3 pathogens in horses in Japan. Using PCR, DNAs from the peripheral blood of all horses were found negative with any Anaplasma, Rickettsia and Borrelia spp., while those from Haemaphysalis megaspinosa ticks were found positive for Anaplasma sp. closely related to A. phagocytophilum in Japan, and A. bovis. B. japonica was also detected in an H. flava tick for the first time.

Seroprevalence of Anaplasma phagocytophilum in horses in the Czech Republic

Anaplasma phagocytophilum is the aetiological agent of equine granulocytic anaplasmosis (EGA). The aim of this survey was to assess the prevalence of anti- A. phagocytophilum antibodies in the horse population of the Czech Republic (CZ) and to investigate possible links between seropositivity and the geographic origin, age, and/or sex of the tested horses. Antibodies against A. phagocytophilum were screened using an indirect fluorescent antibody method (IFA). Serum samples from 96 healthy horses from 8 localities (7 within the CZ and one control upland/tick-free locality in Ukraine) were examined. While the controls tested negative, the seroprevalence in the Czech localities was estimated at 73%, which suggests that subclinical EGA is common among horses in the CZ. Significant differences between seropositivity rates in individual stables were demonstrated (explicable by the different environment and/or management styles of the horses stabled therein). Horses in stables from which previous manifest cases had been reported tended to have higher average titres. Mares displayed a somewhat higher seropositivity rate than did stallions; age had no demonstrable effect upon the serological status of the examined horses.

Microscopic polyangiitis with renal mass-like lesion : A case report

A 63-year-old man was admitted to our hospital because of persistent fever, weight loss, painful foot, and purpura on his extremities. He had lower extremity peripheral neuropathy, and skin biopsy of the purpura revealed vasculitis. Serum tests for myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA), proteinase 3-ANCA, and ANCA (indirect fluorescent antibody method) were all negative. Computed tomography revealed a 6-cm large, irregularly shaped lesion in the right kidney, while the nasal sinuses and lungs were intact. Based on these findings, a diagnosis of suspected systemic vasculitis associated with renal cell carcinoma was made. Thus, right nephrectomy was performed. However, the pathological findings showed a large infarct with necrotizing vasculitis of the arcuate, interlobular, and perinephric small arteries and a crescent formation in the glomerulus. Based on these findings, he was diagnosed with microscopic polyangiitis. Due to rapidly worsening symptoms of purpura and neuropathy, treatment with a high dose of corticosteroid was initiated on postoperative day 2, which led to improvement of his symptoms. Vasculitis accompanied with a mass-like lesion is occasionally confused with malignancy. The lesion in our patient was considered to have originated by asymptomatic renal infarction. This case suggests that a renal mass-like lesion with vasculitis should be diagnosed with care.

Effects of the collagenolytic cell wall component of Fusobacterium necrophorum subsp. necrophorum on bovine hepatocytes

The effects of the collagenolytic cell wall component (CCWC) of Fusobacterium necrophorum subsp. necrophorum on bovine hepatic cell and cytoskeletons were investigated. Scanning electron microscopy (SEM) demonstrated that CCWC damaged the cell surfaces, forming tiny holes on the cell membranes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles revealed that CCWC degraded bovine cytokeratin and vimentin and by indirect fluorescent antibody (IFA) method, it was shown that CCWC caused the deformation of hepatocellular vimentin. This suggested that CCWC contributes to bovine hepatic injury and it may be as important pathogenic factor in the development of bovine hepatic abscesses.

The emergence of another tickborne infection in the 12-town area around Lyme, Connecticut: human granulocytic ehrlichiosis.

Human granulocytic ehrlichiosis (HGE) is an emerging tickborne infection, increasingly recognized in areas in which Lyme disease is endemic, but there are few data on the incidence of HGE. Prospective population-based surveillance was conducted in the 12-town area around Lyme, Connecticut, by means of both active and passive methods, from April through November of 1997, 1998, and 1999. Five hundred thirty-seven residents presenting to their primary care provider with an acute febrile illness suggestive of HGE were identified. Of these, 137 (26%) had laboratory evidence (by indirect fluorescent antibody staining or polymerase chain reaction) of HGE; 89 were confirmed cases, and 48 were probable cases. The incidence of confirmed HGE was 31 cases/100,000 in 1997, 51 cases/100,000 in 1998, and 24 cases/100,000 in 1999. A subset of sera was tested by use of immunoblot assays, and results were in agreement with indirect fluorescent antibody methods for 86% of samples analyzed. Thus, HGE is an important cause of morbidity and is now the second most common tickborne infection in southeastern Connecticut.

Antibodies to granulocytic ehrlichiae in white-footed and cotton mice in eastern United States.

Serum samples, collected from Peromyscus leucopus (white-footed mouse) or Peromyscus gossypinus (cotton mouse) during 1987 through 1990 in Florida, Georgia, Maryland, Mississippi, and North Carolina (USA), and in 1997 in southern Connecticut were analyzed by indirect fluorescent antibody (IFA) staining methods or Western blot procedures for antibodies to granulocytic ehrlichiae. Of the 82 sera from white-footed mice in Connecticut tested by IFA methods with either the BDS or NCH-1 strain of the human granulocytic ehrlichiosis (HGE) agent, 45 (55%) and 42 (51%) of the samples contained antibodies to these strains, respectively, at concentrations ranging from 1:80 to 1:2560. One (2%) of 43 sera from P. leucopus captured in Assateague Island (Maryland) had a titer of 1:80, while three (20%) of 15 sera from P. gossypinus, captured in Sapelo Island (Georgia) and four (40%) of 10 sera from cotton mice caught in Amelia Island (Florida) had antibodies to the NCH-1 strain at titers of 1:160 to 1:1,280. Fifty-five sera from P. leucopus in Cape Hatteras (North Carolina) and 30 sera from P. gossypinus in Mississippi were negative. Western blot analyses confirmed seropositivity for 19 (95%) of 20 mouse sera positive by IFA staining methods, including samples from both mouse species captured in Connecticut, Maryland, or Florida. There were key banding patterns to proteins having molecular masses of about 44, 80, 105, 110, or 120 kDa. Both serologic assays can be used to determine if mice have been exposed to granulocytic ehrlichiae. These rodents also may be useful in surveillance programs to identify endemic sites for HGE and in performing laboratory studies on immune responses to the etiologic agent.

Present state and problems of antinuclear antibody test by indirect fluorescent antibody method.

Present state and problems of antinuclear antibody test by indirect fluorescent antibody method.

A rapid viability assay for Cryptosporidium oocysts and Giardia cysts for use in conjunction with indirect fluorescent antibody detection.

There is an urgent need to develop rapid methods to determine the viability of Cryptosporidium oocysts and Giardia cysts in environmental samples, especially water. The inclusion of the vital dye propidium iodide (PI) by oocysts and cysts has been previously shown to correlate well with nonviability. The ability of nonviable oocysts and cysts to include PI has been employed to develop a rapid viability determination method that could be used in conjunction with the current indirect fluorescent antibody (IFA) method for detecting oocysts and cysts. The efficacy of this PI-IFA method to detect and determine the viability status of oocysts/cysts has been tested using oocyst samples inactivated by three different approaches. The ability to incorporate PI staining with IFA detection provides the advantage of both detection and viability determination at the same time, using the same sample.

Indirect fluorescent IgM antibody response of pigs infected with porcine reproductive and respiratory syndrome syndrome virus

IgG and IgM antibody responses were examined by an indirect fluorescent antibody method in pigs following inoculation with different porcine reproductive and respiratory syndrome virus (PRRSV) isolates or a vaccine virus. Viremia was also examined in the pigs. The IgG antibody was first detected between 9 and 14 days post inoculation (PI) and maintained high titers for at least 7 weeks PI. No change in IgG antibody titers was observed when the pigs were reinoculated with PRRSV 35 days PI. IgM antibody was detected between 5 and 28 days PI in the pigs. Reinoculation at 35 days PI caused a short term rise of IgM antibody. Virus was isolated from sera collected between 2 and 21 days PI. The IgM antibody was detected regularly in sera collected during viremia and up to 1–2 weeks after the viremic periods. These results suggest that pigs with detectable IgM antibody are probably pigs with recent infection and that routine testing of IgM antibody in purchased breeding pigs from seropositive farms may be useful in identification of pigs with recent infection.

Relation between the polymerase chain reaction and the indirect fluorescent antibody method in the diagnosis of Legionella infection.

There were only 43 culture-proven cases of legionellosis in Japan between 1980 and 1995. As fewer cases were diagnosed in Japan than in Europe and the United States, we decided to examine the application of PCR as an alternative method for detecting

Screening for Antinuclear Antibodies by Enzyme Immunoassay

Indirect fluorescent antibody (IFA) ia most widely used method in clin clinical laboratories to screen for autoantibodies against a wide variety of nuclear antigens. Recently, a number of antinuclear antibody (ANA) enzyme immunoassay (EIA) screens have become commercially available and claim to be an alternative method to screen for ANAs. Given the subjectivity of technical interpretation of IFA and the high number of ANA negative samples, a suitable EIA method for ANA screening would be beneficial to clinical laboratories with large samples volumes. Five ANA EIA screens were compared (Elias, Helix, Sanofi, TheraTest and Zeus) to IFA using a human epithelial cell line (HEp-2). Sera from 601 patients submitted to our reference laboratory for autoimmune testing, and from 202 normal healthy blood donors, were included in this study. Samples with discordant results between IFA and EIA were further analyzed using single antigen EIAs for SSA, SSB, Sm, RNP, Scl-70, histones, dsDNA, and ssDNA. Analyses were based on clinically significant IFA titers of > or equal to 1:160 as positive and <1:40 as negative. When compared to IFA, agreement, sensitivity and specificity for each ANA EIA screen were as follows: Elias: 87.0%, 69.5% and 97.9%; Helix: 94.6%, 90.2%, and 97.3%; Sanofi: 95.0%, 93.7%, and 95.9%; TheraTest: 95.3%, 97.7%, and 93.5%; Zeus: 87.1%, 96.2%, and 81.4%, respectively. In conclusion, screening for ANAs by EIA using several commercial assays was both sensitive and specific when compared to IFA. Moreover, the EIA is objective and much less labor intensive when screening a large number of clinical specimens. None of the EIAs were 100% sensitive and, thus, may fail to detect a few of the nonspecific ANAs that demonstrate atypical as well as classical IFA patterns. The advantages of employing these nonsubjective assays to screen out the vast majority of ANA negative sera is clear. The authors still recommend confirming titers and patterns of sera with positive EIA screens using classical IFA methods employing HEp-2 cells.

Prevention of the spread of porcine reproductive and respiratory syndrome virus in endemically infected pig herds by nursery depopulation

An attempt was made to prevent the spread of the virus of porcine reproductive and respiratory syndrome (PRRS) on three Minnesota pig farms that had been experiencing chronic nursing pig problems, including poor growth rates and increased mortality of post weaning pigs. The PRRS virus and different bacterial pathogens were isolated from all three farms during the initial investigation, and all the farms had a high prevalence of PRRS virus-seronegative breeding animals. All the pigs tested within one week after weaning when they were 18 to 22 days old, were seronegative, whereas 80 to 100 per cent of the pigs tested at eight to nine weeks had antibody titres ranging from 1:64 to 1:1024 by an indirect fluorescent antibody method. The seroprevalence among the finishing pigs on the three farms ranged from 25 to 50 per cent. An eradication protocol was established on each farm, involving emptying the nurseries, followed by pumping out the slurry pits and cleaning, washing and disinfecting three times in 14 days. After the nurseries were repopulated there were improvements in nursery mortality and average daily weight gain, and no seropositive animals were detected in the nurseries on any of the farms; the seronegative status was maintained for the six-month testing period.

Serodiagnosis of Legionella pneumonia--data of our laboratory in the recent 3 years

We had examined antibody titers against Legionella spp. of patients' sera which were mainly sent from other hospitals, performed with the indirect fluorescent antibody (IFA) method. The clinical status of the cases diagnosed as Legionella pneumonia serologically, were also studied. Out of 105 cases with clinically suspected Legionella pneumonia, 15 cases (14.3%) were seropositive. In 9 out of the 15 cases (60.0%) were caused by Legionella pneumophila serogroup 1. Clinical outline of these 15 cases did not contradict those reported in the literature, and erythromycin was effective in many cases. Significant rises (more than four times) of the titer were observed 3 to 4 weeks after onset in most of cases. We would like to emphasize that this should be performed serodiagnosis of Legionella pneumonia.

Comparison of four enzyme immunoassays for the detection of cytomegalovirus IgG antibodies

Four commercial enzyme immunoassays (EIA), namely the Behring Enzygnost EIA (BE-EIA), Abbott IMx, Whittaker CMV STAT Test Kit and Diamedix assay, were evaluated for the detection of CMV IgG. The methods were compared as to sensitivity, specificity, positive and negative predictive values, global agreement, ease of performance and, for a small number of specimens, reproducibility. Discordant results were resolved by using the Gull CMV indirect fluorescent antibody (IFA) method. Our data suggest that all four assays were valuable screening tools for the detection of CMV IgG based on their high sensitivity and high negative predictive value. However, differences were noted in the reproducibility level and in the incidence of false-positive, equivocal and nonspecific results regarding certain tests in particular. In our hands, the Abbott IMx and the BE-EIA ranked high in the performance characteristics for a good screening test, yet the Abbott IMx offers the added advantages of being the easiest to perform and having the most rapid turnaround time.

Immunological detection of cytoskeletal proteins in the exoerythrocytic stages of malaria by fluorescence and confocal laser scanning microscopy.

Using monospecific antibodies, the presence and distribution of tubulin, actin, myosin, intermediate filaments, and lamins were examined in the exoerythrocytic liver schizont of Plasmodium berghei by conventional indirect fluorescent antibody methods and confocal laser scanning microscopy. The binding reactivity of the antibodies to parasite proteins was determined by Western blot analysis. The localisation of all antibodies in control host hepatocytes followed expected distributions in both uninfected and infected hepatocytes; by contrast, reactivity to the exoerythrocytic schizont was variable. The parasite reacted positively with selected anti-tubulin, -actin, and -myosin antibodies in both fluorescence and Western blot analysis. Anti-lamin antibodies were positive by confocal indirect fluorescent antibody labelling, but no labelling was detected with anti-intermediate filament antibody. Within the technical limits of resolution of the methods as applied to asynchronous parasite infections, not one of the antibodies reacting positively with the parasite by the indirect fluorescent antibody technique could be shown to identify unequivocally the classic architectural features associated with their respective target organelles, i.e. microtubules, stress-fibres or the nuclear envelope.