Studies on human polyomavirus infection in immunosuppressed patients with polyoma related tumors

Polyomaviruses are potentially oncogenic viruses, found in humans, in other mammals and in birds all over the world. The polyomaviruses that have been observed in humans are BK virus (BKV) and JC virus (JCV) as well as the primate polyomavirus Simian Virus 40 (SV40). BKV and JCV persistent latent in humans, but are not believed do not cause any disease or symptoms in immunocompetent individuals. However, in immunosuppressed individuals, e.g in bone marrow transplanted (BMT) patients BKV has been reported to be associated with hemorrhagic cystitis (HC), and in HIV positive patients JCV has been shown to cause progressive multifocal leukoencephalopathy (PML). SV40 is believed to have been transferred to humans via contaminated polio vaccine 195561, and recent studies have detected fragments of SV40 DNA in malignant mesotheliomas (MM), brain tumors and osteosarcomas. However, the reported frequency varies widely between different studies and countries. 'Me aim of the present study was to further investigate human polyomavirus infections in immunosuppressed patients and in patients with polyoma virus related tumors. In BMT patients with late onset HC, BKV is excreted in the urine, but all BMT patients with BK viruria do not develop HC. To investigate if primary BKV infection transmitted from donor to recipient during BMT could cause HC we investigated anti BKV serum titers in BMT recipients and their donors. We also sequenced the non coding control region (NCCR) and the VP1 region of BKV from BMT patients to search for mutations correlated to the development of HC. Primary BKV infection was not the major cause of HC in BMT patients since all patients had anti BKV antibody titers before BMT. However, after BMT significantly more patients with HC had serological changes compared to patients without HC. BKV with C to G mutations in the NCCR Sp1 binding site was significantly over represented in BMT patients with HC. However, when we by Real Time PCR investigated if these mutations resulted in a higher BKV load in the urine of affected patients, we found that this was not the case. To study if human polyomavirus reactivation was related to graft cold ischemia time and if this could cause graft rejections in renal transplant (RTX) patients, we analyzed urine samples by PCR from RTX patients for presence of BKV or JCV and in parallel we examined their files for graft rejection. Reactivation of BKV or JCV was not correlated to the length of the cold ischemia time or to complications after RTX, such as rejection. JCV can be detected by PCR in the cerebrospinal fluid (CSF) of patients with PML. To investigate if JCV is present in CSF during other central nervous system (CNS) infections or in Multiple Sclerosis (MS) patients, we analyzed CSF samples by PCR from patients with other vital CNS infections and MS. JCV was not detected by PCR in CSF from patients with HSV-1 encephalitis, enteroviral meningitis, nonenteroviral meningitis or during MS. Thus, detection of JCV by PCR in CSF is indicative for PML. In order to investigate if SV40 or human polyomaviruses were present in Swedish malignant mesotheliomas (MM), and possibly related to the distribution of early batches of SV40 contaminated polio vaccine, we analyzed Swedish MM samples for SV40 by PCR. SV40 was present in only 10% of the Swedish MM (from three men hom 1905-1953), which was less than that reported, 40-69% in Italy and the USA. This indicates that SV40 contaminated polio vaccine not was distributed widely in Sweden even before 1958

A related donor and reduced intensity conditioning reduces the risk of development of BK virus-positive haemorrhagic cystitis in allogeneic haematopoetic stem cell-transplanted patients

The significance of the BK virus (BKV) and possible co-factors for the development of late onset haemorrhagic cystitis (HC) in allogeneic haematopoetic stem cell (HSCT)-transplanted patients is reviewed. BKV-associated HC causes significant morbidity and mortality in HSCT patients, however, BK-viruria cannot distinguish patients at risk of HC, since it is observed in patients with and without HC. Several studies have therefore attempted to identify co-factors for the development of HC. Acute graft versus host disease was in the past, though less so recently, reported to correlate to the incidence of HC. However, patients who had received grafts from unrelated donors (URD) and had had full conditioning prior to HSCT were shown to have an increased risk of HC, compared to patients who had received HSCT from a related donor (RD) or patients who had received reduced intensity conditioning. In conclusion, HSCT patients with BK-viruria, an URD and receiving full conditioning have an increased risk of developing HC.</p

Human parvovirus B19 DNA is not detected in Guthrie cards from children who have developed acute lymphoblastic leukemia.

Udgivelsesdato: 2004-AprBACKGROUND: There has been much speculation about the cause of childhood acute lymphoblastic leukemia (ALL). It has been suggested, on the basis of findings in epidemiological studies, that ALL may be initiated by an in utero infection of the fetus. The human parvovirus B19 (B19) is etiologically related to human diseases, including erythema infectiosum and aplastic crisis, but it has not yet been considered to be involved in the development of ALL. Therefore, the aim of this study was to investigate, whether prenatal B19 infection could still be indirectly correlated with the development of childhood ALL. PROCEDURES: Fifty-four Guthrie cards, collected at 3-5 days of age, from Swedish children who subsequently developed ALL, as well as from 50 healthy controls, were investigated by nested PCR for the presence of B19 DNA. RESULTS: B19 DNA was not detected in any of the Guthrie cards from ALL patients or from healthy controls, although all tested samples had amplifiable cellular DNA as confirmed by an HLA DQ specific PCR. CONCLUSION: B19 DNA was not found in any of the Guthrie cards from children who later developed ALL or in the healthy controls. These findings suggest that it is less likely that childhood ALL is associated with an in utero in fection with B19

DNA from Potentally Oncogenic Viruses Is Not Detected in Guthrie Cards from Chlidren Who Later Developed Acute Lymphoblastic Leukaemia.

Abstract INTRODUCTION Acute lymphoblastic leukemia (ALL) is the most frequent childhood malignancy in the western countries. Epidemiological reports and animal observations suggest a possible association between maternal viral infections during pregnancy and subsequent development of childhood ALL. A maternal viral infection during pregnancy, which is transferred to the foetus in a critical time during foetal hematopoesis, could initiate a preleukemic clone. A postnatal molecular event, during maximum stress on lymfocyte precursor proliferation, could then expand the preleukemic clone. A possible infectious agent should be able to cross the placenta and induce genomic instability in the foetal lymphocytes, without causing severe foetal abnormalities. Candidate viruses could be the Human Polyoma viruses (JC Virus and BK virus), Human Parvovirus B 19, Human Herpes virus 6 (HHV 6), Epstein-Barr virus (EBV) or Cytomegalo virus (CMV). In order to investigate if children who later develop ALL were prenatally infected with these viruses, Guthrie cards taken at birth were analysed by PCR. PATIENTS, MATERIAL AND METHODS Capillary blood routinely collected at 3–5 days of age and stored on Guthrie cards, were retrieved from 54 patients who later developed ALL. A total of 50 of these children had been diagnosed as pre-B ALL (either CD10+, CD20+, FAB LI or L2) and four were diagnosed as T-ALL (CD3+ or CD8+). The median age of diagnosis was 5 years (range from 9 months to 17 years, mean 5,9 years). These children had been admitted for treatment at four different Paediatric Oncology Swedish Centres from 1980–2001. The control group were 47 healthy controls matched for age and birth place. DNA was extracted from the original Guthrie cards, using Minimal Essential Medium (MEM) to eluate the blood. Presence of BKV and JCV DNA were analysed by a nested PCR, amplifying a 176 and 173 bp segment respectively. Presence of Parvovirus B19 DNA was analysed by the NS1-PCR, amplifying a 284 bp segment. Presence of HHV 6 DNA was analysed by a nested PCR, amplifying a 173 bp segment. Presence of EBV DNA was analysed by a nested PCR, amplifying a 208 bp segment. Presence of CMV DNA was analysed by a Real Time PCR. To exclude the possibility of negative results due to failure to extract DNA, all samples were tested by a HLA-DQ PCR. RESULTS AND DISCUSSION In order to detect if a viral in utero infection could initiate the development of ALL we analysed presence of BKV, JCV, HHV 6, Parvorirus B19 and CMV by PCR in from Guthrie cards at birth, in 54 patents that later developed ALL and 47 matched healthy control patients. No viral DNA was detected in the samples from ALL patients or in the healthy controls. All samples contained amplifiable DNA when tested by HLA-DQ PCR. Thus, it is less likely that childhood ALL is associated to an in utero infection of BKV, JCV, HHV 6, Parvorirus B19, EBVor CMV. However, it is not possible to exclude an association with a latent utero infection with very low levels of circulating virus at birth. In view of the epidemiological evidence between childhood ALL and infection, the search for a viral etiology must continue.</jats:p

Human Polyomaviruses Were Not Detected in Cerebrospinal Fluid of Patients with Neurological Complications After Hematopoetic Stem Cell Transplantation

Abstract Abstract 4503 Neurological complications after allogenetic hematopoetic stem cell transplantation (HSCT) are associated with increased mortality. Reactivation of JC-virus (JCV), a well-known human polyomavirus (HPyV) can be associated with progressive multifocal leukoencephalopathy (PML) after HSCT. Knowledge of whether reactivation of the newly discovered HPyVs KIPyV, WUPyV, Merkel cell PyV (MCV), HPyV 6, HPyV7, trichodysplasia spinulosa PyV (TSV) and HPyV9 is associated to neurological complications after HSCT is however limited. Cerebrospinal fluid (CSF) from 32 HSCT patients with neurological symptoms was therefore analyzed for presence of the above HPyVs including BK-virus (BKV), JCV, and primate PyVs lymphotropic PyV (LPV) and Simian Virus 40 (SV40), but found negative by PCR for all these PyVs. Introduction The incidence of neurological complications after HSCT is up to 15% and related to increased mortality. Risk factors are e.g. busulfan (Bu), electrolyte disturbances, low platelet count, high blood pressure, ciklosporin toxicity and viral reactivation, of which HPyV JCV is one. 2007–2008, three new HPyVs were described: KIPyV, WUPyV, both found in nasopharyngeal aspirates and Merkel cell PyV (MCV), detected in Merkel Cell Carcinoma, a skin cancer detected in elderly persons or in immunocompromized persons. We earlier examined for these three HPyVs in CSFs of 20 patients with neurological complications after HSCT, but they were all negative by PCR. From 2009 and on, another five HPyVs (HPV6, HPV7, TSV and HPyV9) were detected, and to possibly find whether these PyVs were responsible for neurological conditions in patients having undergone HSCT, we have extended our study and analyzed CSF from 32 patients who presented with neurological complications after HSCT for all known HPyVs. Patients, Material and Methods Patients From January 1st 2000 to April 20th 2011 843 patients underwent HSCT at the Centre for Allogenic Stemcell Transplantation, Karolinska University Hospital Huddinge, Sweden. Of these patients, 65 suffered from neurological symptoms and 32 had sufficient amounts of CSF for further analysis. Mean age was 38 years (1–63). The female/male ratio was 13/19. 15 patients received stem cell from a matched unrelated donor, 3 from cord blood and 6 from siblings. 20/32 patients had Bu in the conditioning therapy, 7 received TBI and 5 patients got other conditioning therapy. Multiplex PCR analysis A PCR detecting the presently known 9 HPyVs, including, BKV, JCV, KIPyV, WUPyV, MCV, HPyV6, 7, TSV and HPyV9 as well as the primate LPV and SV40 was developed and used for the analysis. The PCR was initiated with denaturation of CSFs (10 μl) for 9 min at 94°. DNA was extracted from 5 μl CSF/sample using 2×Qiagen Multiplex PCR Master Mix and a PCR protocol containing 15 min in 94°C for denaturation 94°C 15 min followed by 40 cycles with 94°C 20 sec, 50°C, 1 min 30 sec, 71°C 1 min 20 sec and ended with 71°C for 4 min. PyVs amplicons were identified using bead based multiplex analysis with a MagPix system from Luminex. The assay could detect all 11 viruses with a sensitivity of &lt;10 copies/sample. Results and Discussion Among the 32 HSCT patients with neurological symptoms, the most frequent were headache and fever, (41%), followed by seizures (25%) and confusions/hallucinations (16%). At the end of the study period 16/32 patients with a mean follow up time of 3.5 years had survived. Because HyPV reactivation is increased in immunocompromised patients, neurological signs due to reactivation of these viruses are more likely to occur. Despite neurological symptoms, no DNA from the known 9 HPyVs or from LPV or SV40 was detected by the Luminex multiplex based assay in any of the 32 CSF samples. The detection assay used, with detection limits of between 5–10 copies of the respective viruses, should be sensitive enough to detect viral DNA if the corresponding virus was responsible for neurological disease. Many episodes of neurological complications after HSCT are still not given a diagnosis. Our negative finding emphasizes the need for a broader search for causes, especially when new treatment options are becoming available. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Successful Hematopoietic Stem Cell Transplantation in a Patient with LPS-Responsive Beige-Like Anchor (LRBA) Gene Mutation

Purpose: Autosomal recessive mutations in LRBA, encoding for LPS-responsive beige-like anchor protein, were described in patients with a common variable immunodeficiency (CVID)-like disease characterized by hypogammaglobulinemia, autoimmune cytopenias, and enteropathy. Here, we detail the clinical, immunological, and genetic features of a patient with severe autoimmune manifestations. Methods: Whole exome sequencing was performed to establish a molecular diagnosis. Evaluation of lymphocyte subsets was performed for immunological characterization. Medical files were reviewed to collect clinical and immunological data. Results: A 7-year-old boy, born to consanguineous parents, presented with autoimmune hemolytic anemia, hepatosplenomegaly, autoimmune thyroiditis, and severe autoimmune gastrointestinal manifestations. Immunological investigations revealed low immunoglobulin levels and low numbers of B and NK cells. Treatment included immunoglobulin replacement and immunosuppressive therapy. Seven years after disease onset, the patient developed severe neurological symptoms resembling acute disseminated encephalomyelitis, prompting allogeneic hematopoietic stem cell transplantation (HSCT) with the HLA-identical mother as donor. Whole exome sequencing of the patient uncovered a homozygous 1 bp deletion in LRBA (c.7162delA:p.T2388Pfs*7). Importantly, during 2 years of follow-up post-HSCT, marked clinical improvement and recovery of immune function was observed. Conclusions: Our data suggest a beneficial effect of HSCT in patients with LRBA deficiency