Modern diagnosis of Epstein-Barr virus infections and post-transplant lymphoproliferative disease

Infection by Epstein-Barr virus (EBV) occurs in approximately 95% of the world s population. EBV was the first human virus implicated in oncogenesis. Characteristic for EBV primary infection are detectable IgM and IgG antibodies against viral capsid antigen (VCA). During convalescence the VCA IgM disappears while the VCA IgG persists for life. Reactivations of EBV occur both among immunocompromised and immunocompetent individuals. In serological diagnosis, measurement of avidity of VCA IgG separates primary from secondary infections. However, in serodiagnosis of mononucleosis it is quite common to encounter, paradoxically, VCA IgM together with high-avidity VCA IgG, indicating past immunity. We determined the etiology of this phenomenon and found that, among patients with cytomegalovirus (CMV) primary infection a large proportion (23%) showed antibody profiles of EBV reactivation. In contrast, EBV primary infection did not appear to induce immunoreactivation of CMV. EBV-associated post-transplant lymphoproliferative disease (PTLD) is a life threatening complication of allogeneic stem cell or solid organ transplantation. PTLD may present with a diverse spectrum of clinical symptoms and signs. Due to rapidity of PTLD progression especially after stem cell transplantation, the diagnosis must be obtained quickly. Pending timely detection, the evolution of the fatal disease may be halted by reduction of immunosuppression. A promising new PTLD treatment (also in Finland) is based on anti-CD-20 monoclonal antibodies. Diagnosis of PTLD has been demanding because of immunosuppression, blood transfusions and the latent nature of the virus. We set up in 1999 to our knowledge first in Finland for any microbial pathogen a real-time quantitative PCR (qPCR) for detection of EBV DNA in blood serum/plasma. In addition, we set up an in situ hybridisation assay for EBV RNA in tissue sections. In collaboration with a group of haematologists at Helsinki University Central Hospital we retrospectively determined the incidence of PTLD among 257 allogenic stem cell transplantations (SCT) performed during 1994-1999. Post-mortem analysis revealed 18 cases of PTLD. From a subset of PTLD cases (12/18) and a series of corresponding controls (36), consecutive samples of serum were studied by the new EBV-qPCR. All the PTLD patients were positive for EBV-DNA with progressively rising copy numbers. In most PTLD patients EBV DNA became detectable within 70 days of SCT. Of note, the appearance of EBV DNA preceded the PTLD symptoms (fever, lymphadenopathy, atypical lymphocytes). Among the SCT controls, EBV DNA occurred only sporadically, and the EBV-DNA levels remained relatively low. We concluded that EBV qPCR is a highly sensitive (100%) and specific (96%) new diagnostic approach. We also looked for and found risk factors for the development of PTLD. Together with a liver transplantation group at the Transplantation and Liver Surgery Clinic we wanted to clarify how often and how severely do EBV infections occur after liver transplantation. We studied by the EBV qPCR 1284 plasma samples obtained from 105 adult liver transplant recipients. EBV DNA was detected in 14 patients (13%) during the first 12 months. The peak viral loads of 13 asymptomatic patients were relatively low (<6600/ml), and EBV DNA subsided quickly from circulation. Fatal PTLD was diagnosed in one patient. Finally, we wanted to determine the number and clinical significance of EBV infections of various types occurring among a large, retrospective, nonselected cohort of allogenic SCT recipients. We analysed by EBV qPCR 5479 serum samples of 406 SCT recipients obtained during 1988-1999. EBV DNA was seen in 57 (14%) patients, of whom 22 (5%) showed progressively rising and ultimately high levels of EBV DNA (median 54 million /ml). Among the SCT survivors, EBV DNA was transiently detectable in 19 (5%) asymptomatic patients. Thereby, low-level EBV-DNA positivity in serum occurs relatively often after SCT and may subside without specific treatment. However, high molecular copy numbers (>50 000) are diagnostic for life-threatening EBV infection. We furthermore developed a mathematical algorithm for the prediction of development of life-threatening EBV infection.

New aspects of the diagnosis of Helicobacter pylori infection

Aims: Helicobacter pylori infection, although the prevalence is declining in Western world, is still responsible for several clinically important diseases. None of the diagnostic tests is perfect and in this study, the performance of three stool antigen tests was assessed. In areas of high H. pylori prevalence, the definition of patients with the greatest benefit from eradication therapy may be a problem; the role of duodenal gastric metaplasia in categorizing patients at risk for duodenal ulcer was evaluated in this respect. Whether persistent chronic inflammation and elevated H. pylori antibodies after successful eradication are associated with each other or with atrophic gastritis, a long term sequelae of H. pylori infection, were also studied. Patients and methods: The three stool antigen tests were assessed in pre- and post-eradication settings among 364 subjects in two studies as compared to the rapid urease test (RUT), histology, culture, the 13C-urea breath test (UBT) and enzyme immunoassay (EIA) based H. pylori serology. The association between duodenal gastric metaplasia with duodenal ulcer was evaluated in a retrospective study including 1054 patients gastroscopied due to clinical indications and 154 patients previously operated for duodenal ulcer. The extent of duodenal gastric metaplasia was assessed from histological specimens in different patient groups formed on the basis of gastroscopy findings and H. pylori infection. Chronic gastric inflammation (108 patients) and H. pylori antibodies and serum markers for atrophy (77 patients) were assessed in patients earlier treated for H. pylori. Results: Of the stool antigen tests studied, the monoclonal antibody-based EIA-test showed the highest sensitivity and specificity both in the pre-treatment setting (96.9% and 95.9%) and after therapy (96.9% and 97.8%). The polyclonal stool antigen test and the in-office test had at baseline a sensitivity of 91% and 94%, and a specificity of 96% and 89%, respectively and in a post-treatment setting, a sensitivity of 78% and 91%, and a specificity of 97%, respectively. Duodenal gastric metaplasia was strongly associated with H. pylori positive duodenal ulcer (odds ratio 42). Although common still five years after eradication, persistent chronic gastric inflammation (21%) and elevated H. pylori antibodies (33%) were neither associated with each other nor with atrophic gastritis. Conclusions: Current H. pylori infection can feasibly be diagnosed by a monoclonal antibody-based EIA test with the accuracy comparable to that of reference methods. The performance of the polyclonal test as compared to the monoclonal test was inferior especially in the post-treatment setting. The in-office test had a low specificity for primary diagnosis and hence positive test results should probably be confirmed with another test before eradication therapy is prescribed. The presence of widespread duodenal gastric metaplasia showed promising results in detecting patients who should be treated for H. pylori due to an increased risk of duodenal ulcer. If serology is used later on in patients with earlier successfully treated for H. pylori, it should be taken into account that H. pylori antibodies may persist elevated for years for unknown reason. However, this phenomenon was not found to be associated with persistent chronic inflammation or atrophic changes.

Toksoplasman esiintyvyys suomalaisissa kissoissa

Toxoplasma gondii on kokkideihin kuuluva alkueläinloinen. Sen pääisäntiä ovat kissaeläimet, joissa tapahtuvan suvullisen lisääntymisen tuloksena tuotetaan ympäristöön ookystia. Väli-isäntiä voivat olla kaikki tasalämpöiset eläimet. Toksoplasma muodostaa isäntien kudoksiin infektiivisiä kudoskystia. Ihminen voi saada tartunnan syömällä kudoskystia tai ookystia, tai sikiö voi infektoitua jo kohdussa istukan kautta. Toxoplasma gondii voi aiheuttaa isäntänsä vakavan sairastumisen ja on siksi maailmanlaajuisesti merkittävä zoonoottinen loinen. Toksoplasman torjunnassa on oleellista tuntea alueen kissojen toksoplasmaseroprevalenssi ja arvioida ympäristön ookystakuormitusta: seropositiivisten kissojen voidaan olettaa joskus erittäneen ookystia. Tässä tutkimuksessa selvitettiin toksoplasman esiintymistä suomalaisissa kissoissa: suoralla agglutinaatiotestillä (ToxoScreen-DA) määritettiin IgG-vasta-aineiden esiintymistä seerumissa sekä vasta-ainetasoja (tiitteri). Flotac® - flotaatiomenetelmällä tutkittiin ookystien esiintymistä ulostenäytteissä. Tutkimuksessa selvitettiin kissojen toksoplasma-vasta-aineiden esiintymiseen vaikuttavia tekijöitä: serologisessa tutkimuksessa oli mukana sekä löytöettä rotukissoja, ja lisäksi tutkittiin kissojen sukupuolen, iän, sekä rotukissoilla lihansyönnin vaikutusta vastaaineiden esiintymiseen seerumissa. Serologisessa tutkimuksessa 398 kissan aineistossa seroprevalenssiksi saatiin 48,2 %. Tutkituista 369 rotukissasta 49,9 % oli seropositiivisia, kun taas 27 tutkitusta löytökissasta seropositiivisia oli 25,9 % (P<0,05). Sukupuolella ei todettu olevan merkitystä kissan seropositiivisuuteen. Aikuiset kissat olivat nuoria kissoja useammin seropositiivisia (53,7 % vs. 23,5 %) (P<0,001), koska kerran tartunnan saaneen kissan seerumista voidaan todeta vasta-aineita, ja vanhemmat kissat ovat nuoria todennäköisemmin ehtineet törmätä loiseen elämänsä aikana ja saada tartunnan. Ruokavalio oli tiedossa 347 rotukissalta, ja näistä 270 (77,8 %) oli joskus saanut raakaa lihaa. Raakaa lihaa saaneista kissoista 151 (55,9 %) oli seropositiivisia; 77 kissasta, jotka eivät olleet saaneet raakaa lihaa sitä vastoin ainoastaan 26 (33,8 %) oli seropositiivisia (P<0,001). Ulostenäytteistä 131 kissan aineistosta 1,5 % eritti toksoplasman kaltaisia ookystia ulosteessaan tutkimushetkellä. Suomessa kissojen ravinnon ja toksoplasmaseropositiivisuuden välistä yhteyttä ei ole aikaisemmin tutkittu. Näiden uusien tulosten valossa olisi toksoplasman torjunnassa erityisen tärkeää kiinnittää huomiota loisen tartuntareitteihin kissan osalta. Kissoja ei tulisi ruokkia raa’alla lihalla: kissalle annettava liha olisi hyvä kuumentaa yli 67ºC:een tai pakastaa alle -12ºC:n lämpötilassa toksoplasmatartunnan ehkäisemiseksi.