Indirect Immunofluorescent Antibody Technique Research Articles

Anti-nuclear antibodies: A practical approach to testing and interpretation

Anti-nuclear antibodies (ANAs) are a group of antibodies that are characteristically associated with connective tissue diseases (CTDs). Indirect immunofluorescence antibody technique, having a high sensitivity, is the most common technique used for detection, results of which are expressed in terms of the pattern of fluorescence, substrate used, and the titer of a positive test. Other methods include solid-phase assays. ANA test must be performed only when there is a clinical suspicion of an autoimmune CTD. ANA should not be used as a screening tool for asymptomatic individuals. It is essential in clinical practice to be aware of when to order ANA testing, and how to correctly interpret the test results.

Recent Approaches To Optimize Laboratory Assessment of Antinuclear Antibodies.

The presence of antinuclear antibodies (ANAs) is a hallmark of a number of systemic autoimmune rheumatic diseases, and testing is usually performed as part of the initial diagnostic workup when suspicion of an underlying autoimmune disorder is high. The indirect immunofluorescence antibody (IFA) technique is the preferred method for detecting ANAs, as it demonstrates binding to specific intracellular structures within the cells, resulting in a number of staining patterns that are usually categorized based on the cellular components recognized and the degree of binding, as reflected by the fluorescence intensity or titer. As a screening tool, the ANA patterns can guide confirmatory testing useful in elucidating a specific clinical diagnosis or prognosis. However, routine use of ANA IFA testing as a global screening test is hampered by its labor-intensiveness, subjectivity, and limited diagnostic specificity, among other factors. This review focuses on current efforts to standardize the nomenclature of ANA patterns and on alternative methods for ANA determination, as well as on recent advances in image-based computer algorithms to automate IFA testing in clinical laboratories.

Seropositivity for West Nile Virus Antibodies in Patients Affected by Myasthenia Gravis.

BackgroundMyasthenia gravis (MG) is an autoimmune neuromuscular disease characterized by varying degrees of weakness of the skeletal muscles. Specific auto-antibodies against acetylcholine receptor (AChR) are present in the majority of MG patients, although the main cause behind its development still remains unclear. Recently MG development following West Nile virus (WNV) infection has been described in patients without any earlier evidence of MG. It is known that infectious agents trigger immune response and occasionally initiate autoimmune disease. WNV, the causative agent of both benign illness and neuroinvasive disease, has become endemic in many countries in all continents.MethodsIn the present study, 29 patients (15 males and 14 females, 19 - 78 years old) with confirmed diagnosis of MG and elevated levels of AChR autoantibodies were screened for the presence of serum anti-WNV antibodies and compared to a similar population affected by different autoimmune diseases. Indirect immunofluorescent antibody technique was used to evaluate the reaction of patients’ sera on cells infected by WNV.ResultsPositive fluorescent signals for anti-WNV IgG were obtained in 17% of MG patients, although no clinical manifestations related to WNV infection were reported. These results are in agreement with previous data and appear of great interest in the understanding of the pathogenic autoimmune mechanisms at the bases of MG development.ConclusionAs already observed in other human autoimmune diseases, pathogen-triggered autoimmunity could be involved in MG by breaking immunological self-tolerance through possible mechanisms of molecular mimicry between virus proteins and AChR subunits. In predisposed individuals, WNV infection could also represent an additional risk factor to initiate MG.

Evaluation of Peripheral Blood Lymphocytes as Cell Line for the Propagation of Human Herpes Simplex 1

Objectives: This study was planned to evaluate the use of peripheral blood lymphocytes as cell line for propagation of human herpes simplex 1, by the using of modern diagnostic techniques. Methodology: Primarily, 40 samples were collected from dermal lesions, investigated by RT-PCR techniquedirected to certify human herpes simplex1 infections Bosphore® HSV 1-2 Genotyping Kit v1(Anatoliagene works, Turkey) was used for the detection protocol. Results: the results revealed that HSV1 was correlated with 23(57.5%) of the total cases investigated. Five of HSV1positive samples were selected and applied to the assay of in vitro studying of specific cytopathic effects(CPE) viacell culture technique. Peripheral blood lymphocytes were isolated and propagated as a cell line. The demonstrationof specific HSV1 cytopathic effect was demonstrated by indirect immunofluorescent antibody technique. Thisapproach was revealed different degrees of sensitivity for supporting the growth of human herpes simplex1 virus;these cells were sensitive enough to support the growth of HSV1 virus. Conclusions: We concluded that Bosphore® HSV 1 Genotyping Kit v1allows very rapid detection of HSV DNA indermal leisions. peripheral blood lymphocytes were efficient enough for the studying of CPE of HSV1, while PCRassay was more efficient and more précised as a diagnostic technique. Recommendations: Real-time polymerase chain reaction is complementary to cell culture technique in diagnosis ofHSV, and it`s preferred to use specific primers of viral virulence factors and monitoring their pathogenicity.

Prevalence of <i>Besnoitia besnoiti</i> antibodies in bovine sera and milk in Northern Nigeria

Besnoitia besnoiti , a re-emergent parasite of cattle in Europe, occurs in many countries of Africa and other parts of the world. Clinical observations and incidental findings of B. besnoiti in cattle have been reported in the Southern and Northern regions of Nigeria, but the prevalence of antibodies against this parasite is not yet known. This investigation was designed to determine the seroprevalence of bovine besnoitiosis in Northern Nigeria. A total of 400 cattle were selected at random through cluster sampling of herds from two Local Government Areas (LGA) each, of 5 States in the region (Kano, Kaduna, Katsina, Sokoto and Borno States), between May, 2008 and November, 2009. Sera samples obtained from cows, bulls and calves, and milk from lactating cows with suckling calves were screened with indirect immunofluorescent antibody technique (IFAT) for antibodies to B. besnoiti. Out of the 400 samples 321 (80.3%) were positive for antibodies to B. besnoiti . Cattle sampled in Borno had the highest (87.5%) prevalence of antibodies to B. besnoiti, while those sampled from Katsina State had the least prevalence (62.3%). Wamako LGA of Sokoto State had the highest prevalence of the antibodies (100.0%), while Dan Musa LGA in Katsina had the least prevalence (53.0%) among the ten LGA sampled, however, these differences were not statistically significant (p > 0.05). Similarly, the overall prevalence of antibodies to B. besnoiti did not vary significantly between bulls (84.0%) and cows (79.0%), or in the dry (83.6%) and wet (77.1%) seasons (p > 0.05). The high prevalence of antibodies to B. besnoiti in cattle in Northern Nigeria indicates an endemic state of the disease in this region. Keywords : Antibodies, Besnoitia , Cattle, Northern Nigeria, Prevalence

FRECUENCIA DE SERORREACTORES A Lawsonia intracellularis EN GRANJAS PORCINAS TECNIFICADAS

The objective of this study was to detect the presence of Lawsonia intracellularis antibodies in commercial swine herds in the Peruvian departments of Ica, Arequipa, La Libertad and Lambayeque. The antibodies were detected using the Indirect Immunofluorescence Antibody Technique (IFAT) in serum samples of 293 animals in five different age groups. La Libertad department was the most affected (14.6%) and female breeders were the most affected age group (23.1%). Positive results were found in 5.5 ± 0.6% of the samples. The results showed that L. intracellularis is an enteric ethiologic agent that affects the commercial swine production system of Peru.

A Study of the Distribution Patterns and Levels of Salmonella Enteritidis in the Immune Organs of Ducklings After Oral Challenge by Serovar-Specific Real-Time PCR

The objective of this study was to understand the distribution patterns and levels of Salmonella Enteritidis (SE) in the immune organs of ducklings after oral challenge. We conducted serovar-specific real-time polymerase chain reaction (PCR) for SE to detect the genomic DNA of SE in the blood and immune organs, including the bursa of Fabricius, thymus, spleen, and Harderian gland, from ducklings after oral challenge at different time points. The results showed that SE was consistently detected in all the samples. The Harderian gland and spleen tested positive at 8 hr postinoculation (PI). The organism was detected in the blood, bursa of Fabricius, and thymus at 10 hr PI. The copy number of SE DNA in each tissue reached a peak at 24-36 hr PI. The spleen, blood, and Harderian gland contained high concentrations of SE, whereas the thymus and bursa of Fabricius had low concentrations. SE populations began to decrease and were not detectable at 2 days PI, but they were still present up to 9 days PI in the spleen, without producing any apparent symptoms. To validate these results, the indirect immunofluorescent antibody (IFA) technique was used, and the IFA results were similar to those of the fluorescent quantitative-PCR. In conclusion, the results provided insight into the SE life cycle in the immune organs; furthermore, the Harderian gland and spleen were determined to be the primary sites of invasion among the immune organs of normal ducklings after oral SE challenge. This study will help in understanding the pathogenesis of SE infection in vivo and may help in the development of a live Salmonella vaccine in the future.

Emerging rickettsial infections in Sri Lanka: the pattern in the hilly Central Province.

To identify different rickettsial infections using a specific immunofluorescent technique in patients clinically diagnosed as 'typhus fever' in the Central Province of Sri Lanka, and to define the clinical picture, assess the severity of infection and to determine the pattern of geographical distribution of the infections of the hospital-based patients. A specific indirect immunofluorescent antibody technique was used on the sera of two groups of patients in laboratories in Japan and Thailand. We serodiagnosed infections with Orientia tsutsugamushi, Rickettsia typhi and spotted fever group in 56 of 118 clinically investigated patients. There were eight infections with O. tsutsugamushi, two with R. typhi and 10 spotted fever group patients with IgM antibodies suggestive of acute infection. Nineteen patients had antibodies against these three rickettsial species, suggestive of past exposure, co-infection or cross-reactivity of antigens. Discrete, erythematous maculopapular rash was common to all three types of infection except for five patients who had no rash. Five patients positive for spotted fever antibodies developed fern-leaf type skin necrosis with severe illness. Duration of the febrile period ranged from 4 to 23 days with defervescence occurring after specific antibiotic treatment. The study has shown the presence of different types of rickettsial infections in the Central Province of Sri Lanka. The characterization of the clinical picture and the severity of infection provide useful information for the proper management of the patients in the future.

Prevalence of porcine proliferative enteropathy and its control with tylosin in Korea

Porcine proliferative enteropathy(PPE) is an enteric disease been caused by Lawsonia intracellularis. It has become one of the critical problems in the pig industry. To investigate the prevalence of PPE in Korea, serum samples of 828 pigs from 65 herds were tested using indirect immunofluorescence antibody technique(IFA). The infection rate in individual pigs varied from 44 to 69%, whereas 100% in pig farms. The infection frequency was 57, 44.9, and 59.4% according to age respectively. Administration of tylosin in feed at a concentration of 110 ppm for 14 days reduced the infection rate of the farms. These data indicated that the high prevalence of PPE may be controlled by tylosin.

An indirect immunofluorescent antibody technique for detection and enumeration of Vibrio vulnificus serovar E (biotype 2): development and applications.

The applications of an indirect fluorescent antibody technique (IFAT), developed to detect and enumerate the pathogenic bacterium Vibrio vulnificus serovar E from water and clinical samples, are described. This technique proved accurate for detecting V. vulnificus, even under starvation conditions and in the non-culturable state, and could differentiate this species from other bacteria which share the same habitats. The IFAT was successfully used to diagnose vibriosis from naturally- and artificially-infected eels. The overall data suggest that applying this technique properly in environmental and epidemiological/epizootiological studies could significantly increase our knowledge of this bacterium.

Comparison of a Clinic-Based ELISA Test Kit with the Immunofluorescence Test for the Assay of Ehrlichia Canis Antibodies in Dogs

The "gold standard" for the detection of antibodies to Ehrlichia canis, the cause of canine monocytic ehrlichiosis (CME), is the indirect immunofluorescence antibody (IFA) test. The IFA test however is generally available only in selected laboratories and requires extensive equipment and trained personnel. A double-blind study was conducted to compare the ability of an in-clinic standardized enzyme-linked immunosorbent assay (ELISA) test kit to measure E. canis IgG antibodies in dogs compared with the standard IFA technique. A good correlation was found between the 2 techniques (r2 = 0.8793; P < 0.0001). Evidence for the sensitivity of the ELISA technique for the early detection of E. canis IgG antibodies was demonstrated by comparing the appearance of E. canis antibody titers by the IFA and ELISA techniques after artificial infection of 2 sets of dogs. In both experimental infections, both tests were equally sensitive for the early detection of IgG antibodies against E. canis, and the results correlated well with the appearance of fever and clinical signs. Proposed application of the in-clinic ELISA test is to aid in the diagnosis of CME.

PRESUMED PARVOVIRUS B19-ASSOCIATED RETINAL PIGMENT EPITHELIOPATHY

To describe a patient with a white dot syndrome associated with acute erythema infectiosum. A patient with a clinical history of erythema infectiosum and multifocal punctate white lesions at the level of the retinal pigment epithelium was followed up for an 8-month interval. Serum was tested for immunoglobulin M (IgM) and IgG antibodies to parvovirus B19 at the time of the initial evaluation and during convalescence using an indirect immunofluorescence antibody technique. Serial photographs and fluorescein angiograms were obtained. IgM and IgG antibodies to parvovirus were detected in the serum at the time of initial evaluation; IgM antibodies had disappeared but IgG antibodies persisted in serum obtained at 1-month follow-up. Fundus evaluation revealed clinical disappearance of some lesions, with increased pigmentation of others over the course of follow-up. The authors have identified an adult patient who presented with a white dot syndrome associated with acute erythema infectiosum documented by serologically proved parvovirus B19 infection.

Waterborne Transmission of Flavobacterium psychrophilum in Coho Salmon Eggs.

Fertilized coho salmonOncorhynchus kisutcheggs were immersed in diluted broth culture of Flavobacterium psychrophilum(1.0×108, 1.0t106, 1.0×104CFU/mL) for 30 min before water-hardening, after water-hardening and at eyed-egg stages. Just after exposure to the bacteria, the eggs were disinfected with povidone-iodine, and then discretely incubated in flowing water. After 7-28 days of incubation, F. psychrophilumwas isolated only from the egg group which had been exposed to 1.0 t 108CFU/mL before water-hardening. No bacteria were detected from the other egg groups exposed to 1.0 t 106or 1.0 t 104CFU/mL as well as the groups exposed to 1.0 t 108 CFU/mL after water-hardening or the group at the eyed-egg stage. The viable counts ofF. psychrophilumin the contents of the infected eggs ranged from 103to 107 CFU/g. Observations on the frozen sections stained by indirect immunofluorescent antibody technique revealed that manyF. psychrophilumcells were located within the infected eggs at the eyed stage. It was concluded that F.psychrophilumentered the eggs during the water-hardening stage.

Distribution of Immunocompetent Cells in Equine Lymphoid Tissues

Distribution of immunocompetent cells in lymphoid tissues of healthy nulliparous horses were revealed by the double-staining indirect immunofluorescence antibody technique with monoclonal antibodies. In the thymus, immature T cells (CD4+ 8+ T cells) aggregated in the cortex, while a few mature T cells (CD4+ 8- or CD4- 8+ T cells) were scattered in the medulla. MHC class II+ cells aggregated in the cortico medullary region were considered to be macrophages and dendritic cells, while the MHC class II+ cells in the cortex were thought to be macrophages, dendritic cells or cortical epithelial cells. The existence of T cell-dependent areas in the spleen, lymph nodes and ileum was confirmed by the presence of CD5+ T cells. The number of helper T cells (CD4+ 5+ T cells) was slightly greater than that of killer T cells (CD5+ 8+ T cells) in these areas. Within the follicles of the spleen, lymph nodes and ileum, aggregated MHC class II+ cells were considered to be B cells and dendritic cells. Almost all the IgM+ MHC class II+ B cells aggregated within the center of the follicles, whereas IgM- MHC class II+ B cells were found in the marginal zone of the follicles. These findings suggest that activated B cells are contained in the center of the follicle and inactivated B cells in the marginal zone of the follicle and that the follicles are adjoined to T cell-dependent areas.

Q fever in the Greek island of Crete: detection, isolation, and molecular identification of eight strains of Coxiella burnetii from clinical samples.

Over a period of 6 years (1989 to 1995), serum samples from 3,300 patients suspected to be infected by Coxiella burnetii were assayed for the presence of antibodies against antigen phase II of the microorganism by the indirect immunofluorescence antibody technique (IFAT). One hundred fifty-two cases were recorded, and blood samples from 17 patients were cultured for the isolation of the pathogen. By a centrifugation shell vial technique, eight strains were isolated from patients suffering from acute Q fever. The microorganism was detected in the cultures by IFAT, by Gimenez staining, and by the cytopathogenic effect on Vero and human embryonic lung (HEL) cells. PCR followed by restriction fragment length polymorphism analysis was used to confirm the diagnosis and identify the Coxiella burnetii strains within the cell cultures as well as to compare them with reference strains. In order to avoid time-consuming cultures, to achieve direct detection of Coxiella burnetii in clinical samples (blood, buffy coat, etc.), and to increase the specificity and sensitivity of the detection, nested PCR was performed. The first step of DNA extraction was performed with the QIAamp blood kit 250. For the second step of the PCR assays, the conditions of temperature and times of recycling were properly modified, and the microorganism was detected within 4 h. Our study demonstrates that Q fever is an endemic disease in Crete and that the diagnosis of Coxiella burnetii infection can be rapidly achieved by the detection of the microorganism in buffy coat samples by nested PCR. Although the presenting symptoms of the disease in this study differed from those in other studies, the Cretan strains do not differ genotypically from the reference strains (Nine Mile and Q212).

A maternal screening program for congenital toxoplasmosis in Quindio, Colombia and application of mathematical models to estimate incidences using age-stratified data.

We studied 937 pregnant women from Quindio, Colombia for the presence of specific anti-Toxoplasma gondii IgG antibodies using the indirect immunofluorescence antibody technique (IFAT-IgG). Specific anti-T. gondii IgM antibodies detected using the immunosorbent agglutination assay (ISAgA-IgM) were investigated in patients with high titers in the IFAT-IgG (dilutions > or = 1:1,024). We used mathematical models based on the age prevalence results of the IFAT-IgG to estimate the number of seroconversions and these were compared with the results predicted by the IgM based-incidence results. We found 15 positive cases by ISAgA-IgM and we were able to follow the children of six mothers from this group in which we found one case of congenital toxoplasmosis with the development of a retinal scar despite prenatal and postnatal treatment. The estimation of new cases for the annual total of pregnancies (approximately 8,000) in the Quindio region was 30-120 according to the ISAgA-IgM results and 57-85 using mathematical models. Thus, mathematical models based on age prevalence can give useful estimations of the magnitude of the problem.

Primary surveillance of spotted fever group antibodies on rats in the Kinmen area

The positive rate of rickettsial antibodies of 107 rats in the Kinmen area by indirect immunofluorescent antibody (IFA) technique was 0% (0/107) in typhus fever, 38.3% (41/107) in scrub typhus and 66.4% (71/107) in spotted fever group; the positive rate (42.9%) of spotted fever group of 21 rats in Taiwan island also higher than scrub typhus (19.0). It suggests that spotted fever group patients may be present in our country but have not been discovered.

Study on the Suitability of Sow Colostrum for the Serological Diagnosis of Porcine Reproductive and Respiratory Syndrome (PRRS)

Serum and colostrum from 73 sows were collected. The serum samples were tested by Immuno. Peroxidase Monolayer Assay (IPMA) and the corresponding colostrum samples with the indirect Immuno fluorescent Antibody (IFA) technique. All serum positive sows were colostrum positive and all colostrum negative were serum negative. Eight sows only reacted positively in the colostral testing. Compared to the serum standard test the specificity was 82.6% and the sensitivity 100%. The observed agreement between both tests was 89.2%. In addition all serum samples were also tested with the IF test (IFT). Of the eight sows which were negative in the IPMA serum test and positive in the IFA colostrum test, three were found positive when the serum was tested with IFA. Consequently, the observed agreement was higher at 93.2%. After the suitability of colostrum for porcine reproductive and respiratory syndrome (PRRS) diagnosis was demonstrated, 1915 colostrum samples collected from 135 different farms were tested in a comparative study with the IPMA and IFA techniques. Of the 1915 colostrum samples 139 were positive with both IPMA and IFA. With IPMA only, 43 samples were positive compared with 192 samples found positive with the IFA technique. A total of 1541 samples were negative in both tests. The observed agreement between both tests was 87.5%. The quotient of the observed agreement minus chance agreement and the maximum possible agreement beyond chance level (Kappa Quotient) was 0.49. In 90% of the farms that tested IFA positive there was a seroconversion of more than 50% of all colostrum tested. By comparison only 29% of the IPMA positive farms were positive with more than 50%. Based on the epidemiological findings on PRRS it was concluded that the IFA technique indicates a higher sensitivity for the detection of PRRS virus antibodies in sow colostrum. Finally the possible advantages and disadvantages of sow colostrum testing and serum testing are discussed.

Isolation of herpes simplex virus from sexually transmitted disease patients in Ibadan, Nigeria.

Despite the public health importance of herpes simplex virus infection in Nigeria, attempts have not been made to isolate and identify the virus. To isolate, identify, and type the virus from patients attending a sexually transmitted disease (STD) clinic in Ibadan, Nigeria. One hundred fifty-three clinical samples from 116 patients were taken and inoculated into tissue culture for virus isolation. The isolates were identified and typed using chloroform sensitivity test, histocytological study, Complement Fixation Test (CFT), and indirect immunofluorescent antibody technique (IFAT). Virus was isolated from seven of the 153 samples. Two of the seven were HSV-1, and five were HSV-2. This is the first documented isolation of the herpes simplex virus in Nigeria. The study has provided baseline data for future studies on genital herpes in Nigeria. These results may have interesting implications with respect of HIV transmission and other related sexually transmitted diseases in Nigeria.

A monoclonal antibody-based immunoperoxidase method for rapid detection of haemorrhagic enteritis virus of turkeys

An indirect immunoperoxidase ( ip) technique involving avidin-biotin peroxidase complex, using a monoclonal antibody was developed for the detection of haemorrhagic enteritis ( he) virus antigen in frozen and formalin-fixed paraffin-embedded tissue sections. This 1P procedure was compared with an indirect immunofluorescence antibody technique ( ifat) and an agar gel precipitation test ( agpt). Spleens from turkeys experimentally infected with HE virus were collected and examined for the presence of viral antigen. The IP staining procedure detected HE viral antigen as early as 48 hours after infection and continued to demonstrate the presence of viral antigen for up to 11 days after infection at which time the experiment was terminated. The antigen was detected from three to seven days and from two to nine days after infection by the agpt and ifat, respectively. The ifat and agpt had sensitivities of 74·19 and 48 per cent, respectively, compared with ip. Because of its high sensitivity and specificity, the ip technique could be useful for studying the pathogenesis and rapid laboratory detection of he virus.