[Acyclovir-resistant perineal HSV infection revealing chronic lymphoid leukaemia]. / Herpès chronique périnéal résistant à l'aciclovir révélateur d'une leucémie lymphoïde chronique.

BACKGROUND: Chronic HSV infection is a cause of chronic perineal ulcerations. We report a case of a chronic and refractory HSV infection revealing chronic lymphoid leukaemia. PATIENTS AND METHODS: An 85-year-old woman with an 8-month history of chronic perineal ulcerations was referred to our dermatology department. She had no previous medical history of herpes infection. Skin biopsies ruled out carcinoma but were consistent with HSV infection. A local swab was positive for HSV2. Treatment with valaciclovir and intravenous acyclovir (ACV) at the recommended doses was ineffective. Laboratory tests revealed type-B chronic lymphoid leukaemia. Molecular biology studies confirmed the presence of ACV-resistant HSV via decreased thymidine kinase activity (stop codon: M183stop). Foscarnet was administered for a period of 3 weeks with almost complete healing of the ulcerations. Treatment was stopped prematurely due to acute renal insufficiency and the remaining lesions were treated using imiquimod cream. Valaciclovir was prescribed to prevent further episodes. The condition recurred a mere 11 months later. DISCUSSION: The prevalence of ACV-resistant HSV is 0.32 % in immunocompetent patients and 3.5 % in immunocompromised patients. Insufficient dosing regimens or prolonged treatment with TK inhibitors result in the local selection of pre-existing mutant HSV viruses. Foscarnet, a DNA polymerase inhibitor, is the treatment of choice in HSV-resistant infections. ACV-resistant HSV is less virulent and replicates less, with reactivations being mainly due to wild-type HSV latent in the neural ganglia. Valaciclovir can be used as a preventive treatment. To our knowledge, this is the first case of ACV-resistant HSV infection revealing chronic lymphoid leukaemia. CONCLUSION: Chronic perineal ulcerations can be the first manifestation of immunodeficiency seen for example with haematological diseases. In the event of clinical resistance of an HSV infection to recommended thymidine kinase inhibitor regimens, the use of foscarnet should be considered.

The use of a multiplex real-time PCR assay for diagnosing acute respiratory viral infections in children attending an emergency unit.

BACKGROUND: The use of a multiplex molecular technique to identify the etiological pathogen of respiratory viral infections might be a support as clinical signs are not characteristic. OBJECTIVES: The aim of the study was to evaluate a multiplex molecular real-time assay for the routine diagnosis of respiratory viruses, to analyze the symptoms associated with the pathogens detected and to determine the spread of virus during the period. STUDY DESIGN: Respiratory samples were collected from children presenting with respiratory symptoms and attending the emergency unit during the 2010-2011 winter seasons. Samples were tested with the multiplex RespiFinder(®) 15 assay (PathoFinder™) which potentially detects 15 viruses. RESULTS: 857 (88.7%) of the 966 samples collected from 914 children were positive for one (683 samples) or multiple viruses (174 samples). The most prevalent were the respiratory syncytial virus (39.5%) and the rhinovirus (24.4%). Influenza viruses were detected in 139 (14.4%) samples. Adenovirus was detected in 93 (9.6%) samples, coronaviruses in 88 (9.1%), metapneumovirus in 51 (5.3%) and parainfluenzae in 47 (4.9%). Rhinovirus (40%) was the most prevalent pathogen in upper respiratory tract infections while respiratory syncytial virus (49.9%) was the most prevalent in lower respiratory tract infections. Co-infections were associated with severe respiratory symptoms. CONCLUSION: The multiplex assay detected clinically important viruses in a single genomic test and thus will be useful for detecting several viruses causing respiratory tract disorders.

Maribavir use in practice for cytomegalovirus infection in French transplantation centers.

Maribavir (MBV), a UL97 inhibitor, shows good oral bioavailability, low host cell toxicity, and theoretical benefits to inhibit cross-resistant viruses. We herein examined clinical and virological outcomes of 12 patients, including 3 bone marrow recipients and 9 organ recipients infected with resistant cytomegalovirus (CMV) and treated with MBV during 2011-2012. All received at least 800-mg daily doses. They had developed clinical (12/12) and/or virological (11/12) resistance to CMV infection. Based on a decrease of viral load in blood >1.5 log copies/mL half of them responded to MBV treatment. The individual changes varied from a rapid decrease in viral load (n = 4) to no response (n = 3) with some late response slowly decreasing viremia (n = 3). In 2 cases MBV was used as secondary prophylaxis. No clear parameter emerged as a clinical surrogate for nonresponse to MBV. These results contrast with the lack of efficacy in phase III trials of MBV prophylaxis among stem cell recipients, which were possibly due to low doses or inadequate timing of drug initiation in the study. Additional clinical and surrogate laboratory markers are needed to determine antiviral responses to guide MBV use. Dosage ranging studies might benefit future MBV use.

Prospective evaluation of a new automated nucleic acid extraction system using routine clinical respiratory specimens.

The aim of the study was to evaluate the MagNA Pure 96™ nucleic acid extraction system using clinical respiratory specimens for identifying viruses by qualitative real-time PCR assays. Three extraction methods were tested, that is, the MagNA Pure LC™, the COBAS Ampliprep™, and the MagNA Pure 96™ with 10-fold dilutions of an influenza A(H1N1)pdm09 sample. Two hundred thirty-nine respiratory specimens, 35 throat swabs, 164 nasopharyngeal specimens, and 40 broncho-alveolar fluids, were extracted with the MagNA Pure 96™ and the COBAS Ampliprep™ instruments. Forty COBAS Ampliprep™ positive samples were also tested. Real-time PCRs were used to identify influenza A and influenza A(H1N1)pdm09, rhinovirus, enterovirus, adenovirus, varicella zoster virus, cytomegalovirus, and herpes simplex virus. Similar results were obtained on RNA extracted from dilutions of influenza A(H1N1)pdm09 with the three systems: the MagNA Pure LC™, the COBAS Ampliprep™, and the MagNA Pure 96™. Data from clinical respiratory specimens extracted with the MagNA Pure 96™ and COBAS Ampliprep™ instruments were in 98.5% in agreement (P < 0.0001) for influenza A and influenza A(H1N1)pdm09. Data for rhinovirus were in 97.3% agreement (P < 0.0001) and in 96.8% agreement for enterovirus. They were in 100% agreement for adenovirus. Data for cytomegalovirus and HSV1-2 were in 95.2% agreement (P < 0.0001). The MagNA Pure 96™ instrument is easy-to-use, reliable, and has a high throughput for extracting total nucleic acid from respiratory specimens. These extracts are suitable for molecular diagnosis with any type of real-time PCR assay.

Viral infections after kidney transplantation.

Chronic immunosuppression, required to maintain allograft function postorgan transplant, predisposes transplant patients to a variety of viral infections. These can occur at every stage of post-transplantation. Some infections, however, such as cytomegalovirus (CMV), Epstein Barr virus (EBV), or BK virus (BKV), tend to occur within months after transplantation. CMV infections can be easily prevented by prophylaxis therapy whereas EVB or BKV infections can be prevented by lowering (when possible) immunosuppression. Some viral infections can result in posttransplant lymphoproliferative disorders (EBV), Kaposi sarcoma (human herpes simplex virus type 8), or skin and/or cervical cancers (papillomavirus). Other viral infections, such as those due to influenza or para influenzae viruses, respiratory syncytial virus, or West nile fever virus, are mostly acquired through environmental spread. Thanks to modern laboratory technique, and a formidable antiviral armamentarium, viral infections in organ transplant patients i) can be easily detected at early stages, and ii) can be efficiently treated.

JC virus DNA in the peripheral blood of renal transplant patients: a 1-year prospective follow-up in France.

There is little information on JC virus (JCV) infection in renal transplant patients. A long-term prospective follow-up study was conducted to assess the incidence of JCV DNA in the blood of 103 adult renal transplant patients enrolled prospectively between 1 January and 31 December 2006. Patients were monitored until April 2008. JCV DNA was quantified by a real-time polymerase chain reaction in whole blood samples collected regularly for at least 1 year post-transplant. JCV was detected in seven patients (6.8%) (31/1,487 whole blood samples) at a median time of 139 days post-transplant. The median JC virus load of the first positive DNA blood sample was 3.4 log(10) copies/ml (1.9-5.7 log(10) copies/ml). Induction therapy were either anti-CD25 monoclonal antibodies (n = 5) or antithymocyte globulins (n = 2). Post-transplant immunosuppressive treatment included steroids with tacrolimus/mycophenolate mofetil (MMF) (n = 2), or ciclosporin/MMF (n = 1), or belatacept/MMF (n = 4). Two patients were also treated with rituximab. All seven patients infected with JCV had other viral infections(s): BK virus (3), Epstein-Barr virus (2), Cytomegalovirus (1) or both BK virus and Epstein-Barr virus (1). Three patients had BKV-associated nephropathy and decoy cells shedding. JCV infection was not associated with acute rejection episodes or nephropathy, regardless of the virus load. No patient developed progressive multifocal leukoencephalopathy during follow-up. Thus the incidence of JCV infection in renal transplant patients was low and not associated with any specific clinical manifestations. JCV replication must still be diagnosed and differentiated from BK virus infection because of its non-aggressive course.

Direct genotyping of cytomegalovirus envelope glycoproteins from toddler's saliva samples.

BACKGROUND: The polymorphism of genes encoding CMV envelope protein is used for strain classification and may influence pathogenesis and/or infectivity. CMV genotyping is usually based on sequencing or acrylamide gel-RFLP, but these methods are not suited to rapid screening of large populations. OBJECTIVES: We developed a high-throughput method to analyze CMV strains diversity and to detect multiple-strain infection in a large population of toddlers (six daycare centers (DCC) and an emergency unit (EU)). METHODS: We developed a new PCR-RFLP method coupled with capillary electrophoresis fragment detection for UL55-gB, UL75-gH and UL73-gN genotyping. To detect gB recombinants, gpUL55 typing was applied to two variable zones (NTerminal and central). We applied this method to 212 CMV-positive saliva samples and controlled the results by direct sequencing of PCR products. RESULTS: We identified 112 strains, that fell into eight groups in UL55-gB, two groups in UL75-gH, and seven groups in UL73-gN. The 79 samples from the emergency unit contained 30 strains, 28 children harboring 2 strains. The samples (n=133) from the six daycare centers contained respectively 4, 1, 6, 1 and 11 strains. Fifteen percent of strains were UL55-gB recombinants. CONCLUSION: Our new method can simultaneously determine gB, gH and gN genotypes and offers more precise classification of CMV strains than previous RFLP-based methods. This could constitute the basis for a new classification, particularly in UL55-gB. Easy direct identification of multiple strains and recombinants in pathological samples could facilitate large epidemiologic studies.

Comparison of two highly automated DNA extraction systems for quantifying Epstein-Barr virus in whole blood.

BACKGROUND: Optimal automated molecular methods are needed to monitor Epstein-Barr virus (EBV) infections in transplant recipients. OBJECTIVES: To compare the extraction of EBV DNA from whole blood using the COBAS Ampliprep and the MagNA Pure instruments (Roche) for quantifying EBV DNA by real-time PCR. STUDY DESIGN: EBV DNA content was determined on clinical samples extracted by both systems. RESULTS: The detection limit was 2.16log(10)copies/mL using the COBAS Ampliprep extraction system. Specificity was 100% and we saw no cross-contamination. Extraction was linear from 2.60 to 5.60log(10)copies/mL. The intra-assay variation was 1.91% for 3.60, 2% for 4.60 and 4.51% for 5.60log(10)copies/mL; inter-assay variation was 4.88%. Sixty-six samples were tested: 26 were positive and 28 were negative by both methods. One sample was MagNA Pure positive/COBAS Ampliprep negative (virus load 3.15log(10)copies/mL) and 10 samples were MagNA Pure negative/COBAS Ampliprep positive (virus loads from 1.59 to 3.51log(10)copies/mL) (P<0.0001). Both methods gave similar quantitative results (average difference 0.07log(10)copies/mL) which were well correlated (r=0.73, P<0.001). CONCLUSIONS: The COBAS Ampliprep extraction system is comparable to the MagNA Pure and offers a high reliability for extracting EBV DNA from whole blood.

Prospective evaluation of BK virus DNAemia in renal transplant patients and their transplant outcome.

BACKGROUND: After renal transplantation, the prevalence of BK virus (BKV) viruria, viremia, and nephritis (BKVAN) has been estimated at 30%, 13%, and 8%, respectively. PATIENTS AND METHODS: The aim of this prospective study was to assess the occurrence of BKV DNAemia during the first year after renal transplantation and to determine the prevalence of BKVAN, in the absence of immunosuppression alteration, following positive BKV DNA. BKV DNAemia was assessed systematically in 104 renal transplant patients on postoperative days 60, 90, 135, 180, 270, and 360. RESULTS: Of the 104 patients, 7 (6.7%) presented with at least 1 episode of BKV DNAemia. Those with positive BKV DNAemia had a cumulative steroid dose administered from days 0 to 7 which was higher than those without BKV DNAemia (2.13 +/- 0.6 vs 1.6 +/- 0.4; P = .024). The first BKV DNAemia occurred at 170 (30-460) days posttransplantation. Of the 7 patients who experienced at least 1 BKV DNAemia, 3 had 1 occurrence, but the other 4 had repeated occurrences. These 4 patients developed overt BKVAN at 1 (2 cases) to 2 weeks (2 cases) after the first occurrence of BKV DNAemia. These 4 patients were withdrawn from mycophenolate mofetil, which was in all cases replaced by leflunomide. With a follow-up ranging from 14 to 24 months after the first episode of BKV DNAemia, patient and graft survivals were both 100%. Current serum creatinine ranges from 97 to 173 micro mol/L for those who had only 1 episode of BKV DNAemia, and from 144 to 240 micro mol/L for those who had overt BKVAN. CONCLUSION: Although BKV DNAemia is a rare event after renal transplantation, it is often associated with BKVAN, which may be treated successfully by the alleviation of immunosuppression and leflunomide therapies.

Detection of ganciclovir resistance after valacyclovir-prophylaxis in renal transplant recipients with active cytomegalovirus infection.

Whether valaciclovir (VCV) prophylaxis could be responsible for ganciclovir (GCV)-resistance of Human cytomegalovirus (HCMV) in transplantation has never been documented. A multicentric retrospective pilot study was undertaken to detect GCV-resistance through mutations within the UL97 gene in renal transplant recipients who experienced active HCMV infection and received valacyclovir prophylaxis. Twenty-three patients who experienced HCMV antigenaemia or DNAemia during or at the end of prophylaxis were included. UL97 genotyping was carried out on peripheral blood samples, using a nested in-house PCR, which amplified the full-length UL97 gene. One patient has a resistance-related mutation (M460I); the major risk factor for emergence of resistance in this patient was the presence of early and persistent antigenaemia. GCV-resistance during VCV-prophylaxis was rare after renal transplantation. However, special attention must be paid to patients developing early active HCMV infection under prophylaxis.

Surveillance network for herpes simplex virus resistance to antiviral drugs: 3-year follow-up.

Herpes simplex virus (HSV) infections are very common in the general population and among immunocompromised patients. Acyclovir (ACV) is an effective treatment which is widely used. We deemed it essential to conduct a wide and coordinated survey of the emergence of ACV-resistant HSV strains. We have formed a network of 15 virology laboratories which have isolated and identified, between May 1999 and April 2002, HSV type 1 (HSV-1) and HSV-2 strains among hospitalized subjects. The sensitivity of each isolate to ACV was evaluated by a colorimetric test (C. Danve, F. Morfin, D. Thouvenot, and M. Aymard, J. Virol. Methods 105:207-217, 2002). During this study, 3900 isolated strains among 3357 patients were collected; 55% of the patients were immunocompetent. Only six immunocompetent patients excreted ACV-resistant HSV strains (0.32%), including one female patient not treated with ACV who was infected primary by an ACV-resistant strain. Among the 54 immunocompromised patients from whom ACV-resistant HSV strains were isolated (3.5%), the bone marrow transplantation patients showed the highest prevalence of resistance (10.9%), whereas among patients infected by human immunodeficiency virus, the prevalence was 4.2%. In 38% of the cases, the patients who excreted the ACV-resistant strains were treated with foscarnet (PFA), and 61% of them developed resistance to PFA. The collection of a large number of isolates enabled an evaluation of the prevalence of resistance of HSV strains to antiviral drugs to be made. This prevalence has remained stable over the last 10 years, as much among immunocompetent patients as among immunocompromised patients.

Automated extraction and quantification of human cytomegalovirus DNA in whole blood by real-time PCR assay.

The measurement of human cytomegalovirus (HCMV) DNA in blood is becoming the standard method for monitoring HCMV infection in immune-suppressed and unsuppressed patients. As various blood compartments can be used, we have compared the HCMV DNA measured in whole blood (WB), peripheral blood leukocytes (PBL), and plasma by real-time PCR. We tested 286 samples: HCMV DNA was extracted automatically from WB and PBL with the MagNA Pure instrument (Roche Molecular Biochemicals) and manually from plasma samples. The HCMV DNA from WB, PBL, and plasma was measured by real-time Light Cycler PCR. Primers and probe were located in the UL 83 region. HCMV DNA was detected more frequently in WB (88.5%) than in the PBL (65.7%) (P < 0.0001) or the plasma (55.2%) (P < 0.0001). There was a good correlation between the positive results in WB and in PBL (r = 0.68; P < 0.0001), and 3.15 log(10) genome copies in 200000 PBL, equivalent to the threshold value of 50 pp65-positive polymorphonuclear cells per 200000 leukocytes, was equivalent to 3.4 log(10) genome copies in 200 microl of WB. WB was shown to be suitable for automated extraction and the quantitation of HCMV DNA by real-time Light Cycler PCR by analysis of serial samples from representative patients of various populations. This system may be very useful for monitoring of immune-suppressed and unsuppressed patients.

Seroprevalence of retroviral infections among pregnant women in Martinique (French West Indies).

A seroepidemiologic study of human T cell lymphotrophic virus type-1 (HTLV-1) and human immunodeficiency virus (HIV) infections was carried out in Martinique among 467 pregnant women receiving prenatal care at the Martinique Department for the Protection of Motherhood and Childhood. A seroprevalence rate of 1.93% was found for HTLV-1 infection. No HIV serum marker was observed. Given the epidemiology of these viral diseases, it is suggested that serologic status should be determined for all pregnant women on this island. A further, large-scale, prospective survey of HIV seroprevalence in Martinique should be performed to confirm the results of the present study.

Risk factors for cytomegalovirus infection in BMT recipients transfused exclusively with seronegative blood products.

Risk factors for developing cytomegalovirus (CMV) infection and pneumonitis were analysed in 100 unselected consecutive patients undergoing allogenic BMT. This series is homogeneous because of the same diagnostic procedures, BMT technique and supportive care (exclusive seronegative blood products, no CMV immunoglobulin, no prophylactic antiviral). The incidence of CMV infection and CMV interstitial pneumonitis (CMV-IP) were 44% and 13% respectively, of whom five patients died. Variables such as age, sex, underlying disease, conditioning regimen and occurrence of GVHD were not found as significant risk factors. We confirm that the only major factor was recipient's serology as CMV infection and IP occurred in 4% and 0% respectively among negative recipients (R-) compared with 79% and 25% among positive R. In contrast to some studies among R+, neither donor's immunity nor recipient's CMV humoral response improved the clinical outcome. This study validates the good predictive value of viremia and urinary virus excretion for the occurrence of CMV-IP (respectively positive in 11 and 13 patients out of 13 with IP), always preceding IP by a median of 37 and 27 days. The highest risk patients for lethal CMV-IP were older recipients (> 30 years). Thus, prospective prophylactic trials with antiviral agents are suggested in such viremic or viruric patients. Furthermore, the use of seronegative blood products is highly effective and sufficient to prevent CMV infection in R-patients.

[Infectious complications of the treatment of acute lymphoblastic leukemia in children]. / Complications infectieuses du traitement des leucémies aiguës lymphoblastiques de l'enfant.

The authors report the infectious complications observed during the treatment of acute lymphoblastic leukemia in 70 children, followed by a same team, with the same protocole, for a period of 6 years (mean follow-up: 42.3 months). The complications were mainly bacterial during induction phase, mainly staphylococcic the microbiological follow-up and a rapid empiric antibiotic therapy allowed to control more than 80% of the febrile episodes. There was one death from fulminant pyocyanic infection.