Detection of EBV infection in different etiologic groups of patients

The ability of two diagnostic tests (ELISA and IF) to detect of EBV infection in etiologically different group of patients was compared: cases of chronic lymphoadenopatis, confirmed mononucleosis or suspected EBV infection, tumors like leukemia or lymphoma. The presence of specific IgM and IgG antibodies for different EBV antigens was studied. The results obtained indicated that as many as 17 out of 32 tested serum samples presented different interpretation of EBV infection in both tests used. High number of discordant results was observed in detection of EA-IgG. The highest number of discordant results was observed in group of patients with tumors, while the lowest in group of cases diagnosed or suspected for EBV infection.     

Serodiagnostics of chlamydial infections--significance of positivity in IgA and/or IgM antibody classes only

There was followed the development of serological findings in patients with proved positivity only in classes IgA and/or IgM of chlamydial antibodies (without IgG), which can be suspected of showing "false" positivity. 184 patients were repeatedly examined for chlamydial antibodies in their sera (interval between collections up to three months) using a genus specific rELISA. Sera were also tested for the evidence of IgM antibodies against capside antigen of Epstein-Barr virus (EBV) and against cytomegalovirus (CMV) using ELISA methods. In 75 (40.8%) of patients, IgA/IgM individual positivities were demonstrated even during the following sample test(s). In 28 (15.2%) of them, IgG evidence preceded and in 29 (15.7%) other patients positive seroconversion followed in this class. In 13 (7.1%) patients, IgG antibodies disappeared and subsequently reappeared. Only in 39 (21.2%) of these probands, antibodies IgA/IgM were not demonstrated at another examination. Active EBV, resp. CMV infection was proved in 24 (13.0%), resp. in 18 (9.8%) of patients. It is concluded that the evidence of positivities only in classes IgA and/or IgM mostly signal the onset of a primary infection (reinfection) or an active infection in patients with IgG production failures respectively. In these cases, a "false" positivity can be supposed to occur only in a minor extent.     

Problems and limitations of conventional and innovative methods for the diagnosis of Toxoplasmosis in humans and animals

Diagnosis of toxoplasmosis is useful for human and animal health. Several techniques are employed for the diagnosis in feline and canine population. Coprological tests for the detection of oocysts in cat faeces are of little significance owing to short patency (15 days). Histological examinations of biological samples show a lack of reliability when the animals are infected with few parasites; the mouse inoculation is the most reliable method even if the detection of cysts in mice brain require 40 days. However tachyzoites of virulent strains can be isolated from peritoneal exudate 3-4 days after inoculation. Samples inoculation in cell cultures (VERO, human fibroblasts) requires specialized laboratories and fails if non viable parasites are present due to tissutal autolysis. Serological tests are the most used diagnostic methods; Dye test and IFAT that require intact tachyzoites are more sensitive and specific compared to IHA, LA, ELISA because, during the infection, the first significant increase of IgM and IgG antibodies was observed against cuticolar antigens. A PCR to identify T. gondii DNA in canine and feline biological samples was developed. The B1 PCR performed on blood samples was less sensitive than when it was performed on other biological fluids requiring 100 tachyzoites, instead of 10. Aqueous humor PCR results could be negative if the infection is low grade or is restricted to the posterior segment or the animal was previously treated with anti-Toxoplasma drugs. SNC disease may be also difficult to diagnose because an high serum IgG titer may be associated with locally production or leakage from serum through a compromised blood-CSF barrier. AB1 PCR was successfully applied for the diagnosis of Toxoplasma abortion in ewes requiring only 10 parasites in placental cotyledon samples; the test compared with mouse inoculation showed similar sensitivity. Discrepancies may have been due to a low and focal distribution of parasites in the tissues or to the presence of non viable parasites if the tissues are autolysed. In regard to diagnostic methods adaptable to slaughter testing, several serological tests have been studied (IFAT, ELISA, IHA) for detection of IgG in sheep, pigs, cattle using also recombinant antigens (gene fragments H4 and H11) to lack the cross reactivity. The problem is the antibodies fall to near background levels as the infection became chronic (6-10 months p.-i.). A highly sensitive and specific method (Toxo Taq Man) has been developed to detect and quantitate T. gondii burden in animal tissue samples (0.1 pg of T. gondii genomic DNA, which is equivalent to 1 bradyzoite) using T. gondii ITS1-derived primers and a fluorogenic probe via Real-Time PCR. This assay is compatible with automation technology for potential slaughterhouse use. The diagnosis of acute infection in human pregnancy is difficult since IgM antibodies can be detected for a very long time after the acute phase; an IgA increase is of more diagnostic value because can be detected only for 6-7 months while the short kinetics of IgE can be useful only to date the infection precisely. In addition an IgG seroconversion is essential for the diagnosis. Among the most reliable tests, IgG avidity test is useful when a single serum sample, in the first months of gestation, is available, but low avidity results may persist for as long as 1 year. For this purpose a panel of serologic tests must be performed (ELISA, EIA, ISAGA, IgG avidity, IFAT, Dye test) for IgM, IgA, IgG and IgE. The serological diagnosis of prenatal infection is difficult since maternal IgG are passively transferred in utero to the foetus and caution must be exercised in interpretation of IgM or IgA results. A technique of Western blots of paired maternal and baby sera for evidencing different bands in the blots of two sera was developed for this purpose (specificity 97-100%, sensitivity 96-98%). The most reliable methods for prenatal diagnosis are PCR, mouse inoculation and cultural techniques performed on amniotic fluid, foetal blood and peripheral maternal blood in pregnants serologically positive. PCR (targets B1, SAG-1, rDNA) with amniotic fluid performed from 18 weeks of gestation is more sensitive and more rapid than conventional diagnostic procedures. PCR has been successfully used to diagnose Toxoplasma encephalitis in immunocompromised patients (cerebral biopsy is the only diagnostic method) and in ocular toxoplasmosis. In this evenience it is useful the study of IgG, IgM, IgA profile of paired serum and aqueous humor (Western blots).     

Polymerase chain reaction for detection ofToxoplasma gondii in human biological samples

Using the polymerase chain reaction (PCR), Toxoplasma gondii from gene TGR1E with primers TGR1E-1, TGR1E-2 (standard PCR), and from B1 gene with primers TM1, TM2, TM3 (hemi-nested PCR) was detected in biological samples from 347 individuals (441 biological materials). Of the total of 441 biological materials, T. gondii DNA was detected in 5.2 %; it was positive in the following samples: blood (n = 6), blood from newborns (2), biopsies (2) and samples of progenitor cells (2) (from candidates for bone marrow transplantation). DNA of T. gondii was also revealed in 11 samples (8.3 %) of 120 cases of pregnant women during prenatal examinations. A positive result in the blood was also found in two cases of newborn babies from mothers who were infected in later pregnancy. The positive PCR examination was confirmed by serological methods (ELISA and complement fixation test). Agreement of PCR results and the detection of antibodies against toxoplasma was found in 83.3 %. Rapid PCR examination for the confirmation of acute parasitemia T. gondii is particularly important for the patients in whom the infection may cause serious consequences (e.g., for fetus in pregnant women or for patients suffering from imunosuppression).     

Hepatitis C virus and Epstein-Barr virus: dual infection or atypical antibody response

Positive serological reactions against hepatitis C virus (HCV) appeared in the course of Epstein-Barr virus (EBV) infectious mononucleosis. In 429 consecutive patients with high levels of transaminases, 28 patients with EBV primary infection were found. The presence of anti-HCV antibodies and HCV RNA was studied in these subjects. In seven patients anti-HCV antibodies (C33 and C22c RIBA bands) were detected, but all were polymerase chain reaction (PCR) negative. These results may have been due solely to a HCV infection or were an atypical response to HCV in the course of infectious mononucleosis.     

The role of IgG avidity determination in diagnosis of Epstein-Barr virus infection in immunocompetent and immunocompromised patients

There is a high degree of variability in the serologic response to Epstein-Barr virus (EBV) infection, especially in viral capsid antigen (VCA)-IgM antibodies. Therefore, additional tests are needed to confirm primary infection. We evaluated the value of IgG avidity determination in diagnosis of EBV infection in immunocompetent and immunocompromised patients. A total of 236 serum samples from immunocompetent patients with symptoms suggestive of EBV infection were tested for the presence of VCA-IgM/IgG antibodies and IgG avidity. Using IgG avidity, acute primary infection was confirmed in 56.7% of the immunocompetent patients with positive and in 1.8% of patients with negative VCA-IgM. Recent primary infection was documented in 8.9% of the IgM positive and 3.5% of the IgM negative patients. In patients with indeterminate serology (equivocal IgM), 6.7% were classified by avidity index (AI) as acute primary infection, 10.0% as post-acute and 83.3% as past infection cases. Concerning the 32 immunocompromised patients, recent primary infection was documented in 3 of the 14 IgM positive patients. High AI was detected in 11 of these patients, indicating an IgM response due to reactivation. Determination of IgG avidity in combination with classical serologic markers seems to be a reliable method to confirm primary infection both in immunocompetent and immunocompromised patients. It may be especially useful to differentiate cases of primary infection in patients with undetectable VCA-IgM antibodies or indeterminate routine EBV serology.     

The value of ultrasonography alone in screening surveys of cystic echinococcosis in children in Turkey

A total of 1205 primary school children were examined for cystic echinococcosis in five villages of Manisa, Turkey, to evaluate the efficacy of diagnostic methods of this infection in community-based screening surveys. Six hundred and thirty children from three villages, examined by a portable ultrasound scanner, chest microfilm and serological methods (ELISA, indirect hemagglutination) in our previous study, were designated as Study Group 1; and 575 children, from two adjacent villages, examined by ultrasonography alone in the present study, were designated as Study Group 2. In Study Group 1, hepatic cystic echinococcosis was detected in two cases (0.3%) by ultrasonography, while 43 (8.9%) and 49 (10.1%) cases were found to be positive for cystic echinococcosis by ELISA and indirect hemagglutination, respectively. Three of 575 children (0.5%) were diagnosed with cystic echinococcosis (two hepatic and one renal involvement) by ultrasonography alone in Study Group 2; and lung lesions were later detected in both cases with liver involvement by chest radiography.

Our results suggested that serological tests may be beneficial in suspected cases for confirmation and differential diagnosis, but have some drawbacks, such as discrepancy in results and high false seropositivity rates. Chest microfilm is not easy in field studies and exposure to X-ray is undesirable. As a reliable, simple, inexpensive and rapid technique, ultrasonography alone is recommended to be used in community-based screening surveys for cystic echinococcosis with confirmatory tests for suspected cases found during the screening program.     

Routine diagnosis of herpes simplex virus (HSV) encephalitis by an internal DNA controlled HSV PCR and an IgG-capture assay for intrathecal synthesis of HSV antibodies

Background: The development of antiviral therapy increases the need for rapid, sensitive and reliable methods or combination of methods for diagnosis and monitoring herpes simplex encephalitis, HSE.Objectives: Evaluation of diagnostic performance of three successively developed HSV PCR assays when combined with a new capture ELISA for HSV intrathecal antibody production (ITT).Study design: During a 3.6 year period a total of 4.206 CSF and serum samples from about 4.140 hospitalized patients with a tentative diagnosis of HSE were analyzed by a new ELISA for ITT. 1.962 CSF samples were examined also by PCR. Clinical signs and symptoms and additional tests were obtained on all ITT and/or PCR positive patients. In 1993 the PCR was a double PCR. In 1994 the PCR was a single PCR with internal inhibition control. Positive samples were confirmed by a different confirmative PCR to increase the specificity. From 1995 the PCR was as in 1994 but samples were no longer divided in the serology routine laboratory.Results: A total of 33 HSE cases was found (incidence 1.8 HSE per million people). All patients were treated with aciclovir. Three patients died, 9 patients had primary infection, 2 patients had HSE previously, and 2 patients relapsed. Only 11 patients recovered satisfactory. Of all 37 positive ITT 7 were unlikely positive. False positive PCR was seen in 1993 and 1994, due to sample-to-sample contamination during division of samples, but was not seen since 1995 when this procedure was changed. The test results depended on the state of the disease. Thus, the sensitivity, specificity, PPV and NPV for ITT were highest when performed more than 1 week after debut of symptoms whereas these values were highest using PCR within the first week.Conclusion: Routine PCR diagnosis of HSE type 1 and 2 is a highly sensitive and specific method that should be performed together with serological ITT to cover the whole time span from debut of symptoms to several weeks after hospitalization.     

Agar gel immunodiffusion test (AGID) evaluation for detection of bovine paratuberculosis in Rio de Janeiro,Brazil

AIMS: The aim of this study is to evaluate the AGID serological test for detection of antibodies anti-Mycobacterium paratuberculosis and its possible adoption as diagnostic method in our field conditions. METHODS: Bovine serum samples from dairy herds in Rio de Janeiro State, Brazil, were screened for the presence of antibodies against Myco. paratuberculosis using three different ELISA tests. A panel of 48 randomly selected sera were evaluated by an Agarose Gel Immunodiffusion (AGID) test using Protoplasmatic Paratuberculosis Antigen (PPA). AGID results were compared to the standards--the results of the three ELISA tests, and the specificity and sensitivity were calculated. RESULTS: From 48 sera tested for AGID, 14 (29.17%) were positive and 34 (70.83%) were negative. AGID sensitivity was 57% with two false-positive reactions, and specificity was 92.5% with nine false-negative results. The positive predictive value was calculated in 85.7% for a confidence interval of 95%. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to its low sensitivity and specificity rates, AGID test has shown to be unsatisfactory as a screening diagnostic method for subclinical herd infection, but it can be useful as a confirmatory test for clinical suspect animals.     

Laboratory diagnosis of leptospirosis

Percentage of serological positivity examined in 4205 blood sera by serological method microscopic agglutination test (MAT) on the hinterland territory of our laboratory (East Bohemia; 1999-2003) was 0.38-4.7 %. By the PCR method for detection of DNA of pathogenic leptospires (L. interrogans, L. borgpetersenii and L. kirschneri) from 57 samples of different biological materials from patients with fever of unknown etiology positive results were obtained in 4 specimens (7 %; 3 samples of urine and 1 sample of blood). This method was shown to distinguish between pathogenic and nonpathogenic strains and can detect 2.5-10 cells per mL of biological material. As an important presumption of successful detection of pathogenic leptospires a correct collecting of blood, urine samples or liquor is required before starting antibody therapy. The PCR method possesses a clear advantage over other methods, such as MAT, which relies on the detection of antibodies the presence of which cannot be detected until days after infection.     

Second International Diagnostic Accuracy Study for the Serological Detection of West Nile Virus Infection

Background

In recent decades, sporadic cases and outbreaks in humans of West Nile virus (WNV) infection have increased. Serological diagnosis of WNV infection can be performed by enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA) neutralization test (NT) and by hemagglutination-inhibition assay. The aim of this study is to collect updated information regarding the performance accuracy of WNV serological diagnostics.

Methodology/Principal findings

In 2011, the European Network for the Diagnostics of Imported Viral Diseases-Collaborative Laboratory Response Network (ENIVD-CLRN) organized the second external quality assurance (EQA) study for the serological diagnosis of WNV infection. A serum panel of 13 samples (included sera reactive against WNV, plus specificity and negative controls) was sent to 48 laboratories involved in WNV diagnostics. Forty-seven of 48 laboratories from 30 countries participated in the study. Eight laboratories achieved 100% of concurrent and correct results. The main obstacle in other laboratories to achieving similar performances was the cross-reactivity of antibodies amongst heterologous flaviviruses. No differences were observed in performances of in-house and commercial test used by the laboratories. IFA was significantly more specific compared to ELISA in detecting IgG antibodies. The overall analytical sensitivity and specificity of diagnostic tests for IgM detection were 50% and 95%, respectively. In comparison, the overall sensitivity and specificity of diagnostic tests for IgG detection were 86% and 69%, respectively.

Conclusions/Significance

This EQA study demonstrates that there is still need to improve serological tests for WNV diagnosis. The low sensitivity of IgM detection suggests that there is a risk of overlooking WNV acute infections, whereas the low specificity for IgG detection demonstrates a high level of cross-reactivity with heterologous flaviviruses.     

Hepatitis C prevalence and risk factors in hemodialysis patients in Central Brazil: a survey by polymerase chain reaction and serological methods

An hemodialysis population in Central Brazil was screened by polymerase chain reaction (PCR) and serological methods to assess the prevalence of hepatitis C virus (HCV) infection and to investigate associated risk factors. All hemodialysis patients (n=428) were interviewed in eight dialysis units in Goiania city. Blood samples were collected and serum samples screened for anti-HCV antibodies by an enzyme-linked immunosorbent assay (ELISA). Positive samples were retested for confirmation with a line immunoassay (LIA). All samples were also tested for HCV RNA by the PCR. An overall prevalence of 46.7% (CI 95%: 42-51.5) was found, ranging from 20.7% (CI 95%: 8.8-38.1) to 90.4% (CI 95%: 79.9-96.4) depending on the dialysis unit. Of the 428 patients, 185 were found to be seropositive by ELISA, and 167 were confirmed positive by LIA, resulting in an anti-HCV prevalence of 39%. A total of 131 patients were HCV RNA-positive. HCV viremia was present in 63.5% of the anti-HCV-positive patients and in 10.3% of the anti-HCV-negative patients. Univariate analysis of risk factors showed that the number of previous blood transfusions, transfusion of blood before mandatory screening for anti-HCV, length of time on hemodialysis, and treatment in multiple units were associated with HCV positivity. However, multivariate analysis revealed that blood transfusion before screening for anti-HCV and length of time on hemodialysis were significantly associated with HCV infection in this population. These data suggest that nosocomial transmission may play a role in the spread of HCV in the dialysis units studied. In addition to anti-HCV screening, HCV RNA detection is necessary for the diagnosis of HCV infection in hemodialysis patients.     

False positive diagnosis of meningococcal infection by the IS1106 PCR ELISA

At a time when optimal case ascertainment for meningococcal infection is a high priority, the need for non-culture case confirmation, in particular by DNA amplification, is seen as being of vital importance to assist contact management and cluster recognition. A solution hybridisation assay with colorimetric microtitre plate detection (polymerase chain reaction-enzyme-linked immunosorbent assay (PCR ELISA)) has been developed using the multicopy insertion sequence IS1106 which had reportedly achieved a specificity of 100% and was described as being meningococcal specific. This PCR ELISA assay was evaluated on specimens from over 5000 patients at the national Meningococcal Reference Unit (MRU) between late 1995 and early 1997 and was found to be highly sensitive. Insertion sequences, however, are genetically mobile with the ability to spread between species and even genera. During the evaluation period of the IS1106 PCR ELISA a number of false positives proved to be caused by organisms other than N. meningitidis were recorded resulting in the withdrawal of this assay as a front line screening assay for routine confirmation of meningococcal infection.     

Specific antibodies in children excreting cytomegalovirus

Acute-and convalescent-phase sera from 22 children were examined by ELISA in comparison with a routine complement fixation (CF) test for detection of anti-CMV antibodies. All these subjects were excreting CMV from urine and/or saliva. The results showed that ELISA is more sensitive than CF test. Particularly ten children showed, by ELISA, anti-CMV antibody titers more agreeing with clinical-virological features. Generally, in other subjects the results of the two serological tests were similar. Three cases showed discordances both between the two methods and between serological data and clinical virological findings.     

Detection of Epstein Barr virus in formalin-fixed paraffin tissues by fluorescent direct in situ PCR

Specific viral laboratory diagnosis of primary Epstein-Barr Virus (EBV) infection is usually based on antibody-detection assays. However, molecular detection is also considered the reference standard assay for diagnosis of central nervous system infections and of most cases of nasopharyngeal carcinoma (NPC). One-step or nested polymerase chain reaction (PCR) has rapidly replaced immunological assays based on virus-specific Ig antibodies for the laboratory diagnosis of Herpesvirus infections, even if serological methods are considered an additional tool for defining clinical diagnosis. In this article, we will present a rapid, sensitive and robust molecular tool for the viral detection of EBV (EBNA-1) within tissue specimens by making use of in situ PCR (IS-PCR).     

Evaluation of detection of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> DNA in animal blood samples by quantitative PCR

Aim of the study: The purpose of this study was to find out the relationship between the phase of infection (acute or persistent) and the ability of quantitative PCR to detect DNA of Toxoplasma gondii in circulating leukocytes in blood. Methodology: Animal serum samples were examined (50 sheep, 47 dogs, 32 dairy cows, 91 wild boars and 36 rabbits) for the occurrence of IgM and IgG antibodies to T. gondii by ELISA. Uncoagulated blood samples from the same animals were examined for the detection of T. gondii DNA in circulating leukocytes by real-time PCR. Results: Only IgM antibodies, characteristic for acute infection, were detected in 45 of the 256 serum samples (17.6%). Only IgG antibodies, corresponding with chronic infection, were detected in 120 of the 256 samples (46.8%). In 91 of the 256 samples (35.5%) neither IgM or IgG were detected by ELISA. For real-time PCR, animals were divided into three groups based on the serological results: (group I — acute infection, group II — chronic infection, and group III — no infection). In group I, the presence of T. gondii DNA was detected in 9 out of 45 samples (20%), whereas in group II only 1 of 120 samples was positive for T. gondii DNA (0.8%). In group III, no DNA of T. gondii (0/91 samples) was detected by real-time PCR. Significance: The proof of DNA by real-time PCR in IgM positive samples was statistically significant in comparison to IgG positive samples (P<0.0001).     

Serological evidence of infectious salmon anaemia virus (ISAV) infection in farmed fishes,using an indirect enzyme-linked immunosorbent assay (ELISA)

Antibody detection tests are rarely used for diagnostic purposes in fish diseases. Infectious salmon anaemia (ISA) caused by ISA virus (ISAV) is an emerging disease of Atlantic salmon Salmo salar L. The virus has also been isolated from diseased coho salmon Oncorhynchus kisutch in Chile. An indirect enzyme-linked immunosorbent assay (ELISA) that should facilitate serodiagnosis of ISAV infection, the study of epidemiology, and the control of ISA in farmed fishes has been developed using purified ISAV as the coating antigen, and monoclonal antibodies that detect fish immunoglobulins bound to the antigen on the plate. Application of the test to a random sample of farmed Atlantic salmon from the Bay of Fundy, New Brunswick, Canada, positively identified 5 of the 7 ISAV RT-PCR-positive fish, and all 10 RT-PCR-negative fish were also negative in the ELISA. Some RT-PCR-negative fish had an elevated non-specific antibody reactivity suggestive of chronic infection or resistance to ISAV. This test was also able to detect 11 of the 14 coho salmon pooled serum samples from a clinically affected farm in Chile that were positive by the virus neutralization (VN) test, and 2 of the 4 VN-negative samples. We conclude that this ELISA would be suitable as a routine test for ISAV infection or for assessing ISAV vaccine efficacy before placing smolts in sea cages, and for testing fishes in sea cages to detect level of resistance to ISA. The assay enables vaccination in combination with depopulation control methods.     

Evaluation of the sensitivity of PCR methods for the detection of extraneous agents and comparison with in vivo testing

In this study the sensitivity of polymerase chain reaction (PCR) methods for the detection of Newcastle disease virus (NDV), avian reovirus (ARV), avian influenza virus (AIV) and avian infectious bronchitis virus (IBV) was compared to the sensitivity of the corresponding serological tests described in the European Pharmacopoeia (Ph. Eur.). For this purpose, serial 10-fold dilutions of the respective inactivated vaccines were prepared and groups of SPF chickens were vaccinated with a double dose of the vaccine dilutions. After a period of 21 days, the animals were revaccinated with a single dose. Two weeks later, serum samples from each animal were tested for antibodies using an Idexx enzyme linked immunosorbent assay (ELISA). In parallel, samples of the diluted vaccines were tested by PCR. It was found that the sensitivity of the four PCR tests is comparable to or even slightly better than that of the corresponding serological tests. Thus these PCR tests fulfil the sensitivity requirements of the Ph. Eur. and could be used as alternative tests for the detection of extraneous agents in final batches of inactivated vaccines.     

Waveguide model of the hearing aid earmold system

Background

Epstein-Barr virus (EBV) is associated to the etio-pathogenesis of an increasing number of tumors. Detection of EBV in pathology samples is relevant since its high prevalence in some cancers makes the virus a promising target of specific therapies. RNA in situ hybridization (RISH) is the standard diagnostic procedure, while polymerase chain reaction (PCR)-based methods are used for strain (EBV type-1 or 2) distinction. We performed a systematic comparison between RISH and PCR for EBV detection, in a group of childhood B-cell Non-Hodgkin lymphomas (NHL), aiming to validate PCR as a first, rapid method for the diagnosis of EBV-associated B-cell NHL.

Methods

EBV infection was investigated in formalin fixed paraffin-embedded tumor samples of 41 children with B-cell NHL, including 35 Burkitt's lymphoma (BL), from Rio de Janeiro, Brazil, by in situ hybridization of EBV-encoded small RNA (EBER-RISH) and PCR assays based on EBNA2 amplification.

Results

EBV genomes were detected in 68% of all NHL. Type 1 and 2 accounted for 80% and 20% of EBV infection, respectively. PCR and RISH were highly concordant (95%), as well as single- and nested-PCR results, allowing the use of a single PCR round for diagnostic purposes. PCR assays showed a sensitivity and specificity of 96% and 100%, respectively, with a detection level of 1 EBV genome in 5,000–10,000 EBV-negative cells, excluding the possibility of detecting low-number EBV-bearing memory cells.

Conclusion

We describe adequate PCR conditions with similar sensitivity and reliability to RISH, to be used for EBV diagnostic screening in high grade B-NHL, in "at risk" geographic regions.     

The presence of anti-sheep red blood cell heterophile antibodies and their characteristics in murine schistosomiasis japonica

Sensitive methods of an enzyme-linked immunosorbent assay (ELISA) using red blood cells (RBC) have been developed and were applied to the detection of anti-sheep red blood cell (SRBC) heterophile antibodies (Ab) present in sera of Schistosoma japonicum (SJ)-infected mice. The indirect hemagglutination test (IHA) was used for the purpose as well. By these methods a significant increase in the heterophile Ab levels was demonstrated in the mice particularly after 6-10 weeks of infection. The heterophile Ab in SJ-infected mice were predominantly immunoglobulins resistant to 2-mercaptoethanol treatment and had temperature-independent reactivity. In an attempt to investigate the immunological specificity of the heterophile Ab, various absorption tests were performed; Davidsohn's differential absorption test revealed that the heterophile Ab were distinct from Forssman antibody, Paul-Bunnell antibody and heterophile agglutinins known to appear in serum sickness. The heterophile Ab were absorbed only with SRBC and goat red blood cells, not with other species of RBC such as human O Rh-, O Rh+, A Rh+, B Rh+, mouse, ox, chicken, horse, rabbit, guinea pig, and rat RBC, or syngeneic tissue homogenates including brain, lung, liver, spleen, kidney, muscle, and thymus. This heterophile antibody response is not a consequence of a specific immune response directed to the antigens of SJ parasites, since absorption of the heterophile Ab with SJ adult worms or an egg preparation did not reduce the heterophile Ab level.