Significance of herpesvirus 6 in BAL fluid of hematology patients with acute respiratory failure.

PURPOSE: Human herpesvirus 6 (HHV6) is an emerging cause of interstitial pneumonia in immunocompromised hosts. However, the clinical significance of a positive PCR test for HHV6 in respiratory samples from patients with hematological malignancies remains unclear. METHODS: We retrospectively studied the features and outcomes of 29 critically ill hematology patients with acute respiratory failure and lung pulmonary infiltrates visible on a chest radiograph, who tested positive for a qualitative PCR for HHV6 in bronchoalveolar lavage fluid. RESULTS: Of the 29 patients, 18 (62%) were stem cell transplant recipients and 11 (38%) had received chemotherapy. All patients had a fever. Clinical manifestations consistent with extra-pulmonary HHV6 disease were noted in 17 (59%) patients. One or more co-pathogens were found in 25 (86%) patients. The four remaining patients diagnosed with HHV6 pneumonia and subsequently recovered with foscarnet therapy. Antiviral therapy was also given to seven patients with co-infections, of whom two ultimately died. CONCLUSIONS: In most cases, HHV6 recovered from BAL fluid is a co-pathogen whose clinical relevance remains undetermined. However, in some cases, HHV6 is the only pathogen, along with disseminated systemic viral disease, and the patient is likely to benefit from foscarnet therapy.

Detection of human herpesviruses HHV-6, HHV-7 and HHV-8 in whole blood by real-time PCR using the new CMV, HHV-6, 7, 8 R-gene kit.

Human herpesviruses 6 (HHV-6), 7 (HHV-7) and 8 (HHV-8) are lymphotropic herpesviruses that may cause opportunistic diseases in immunosuppressed patients such as transplant or AIDS patients. The new commercial CMV HHV-6, 7, 8 R-gene kit (Argene, Varilhes, France) for the simultaneous quantitation of HHV-6 and qualitative detection of HHV-7 and HHV-8 was evaluated using whole blood samples (respectively, n=175, 100 and 161) and using different extraction and real-time PCR platforms in two Centers A and B. In comparison with HHV-6 in-house real-time PCR the commercial kit showed agreements of 96% (n=75) and 85% (n=100) in A and B, respectively, with significant Spearman's correlation between both techniques (in A: r=0.97 [p<0.001]; in B: r=0.70 [p<0.001]). The Bland-Altman test results and prospective monitoring of patients confirmed the accuracy of these HHV-6 real-time PCR techniques. The agreement between the in-house HHV-7 PCR and commercial kit was of 86% (n=100). In comparison with in-house HHV-8 real-time PCRs, the commercial kit showed agreements of 100% (n=61) and 93.7% (n=96) in A and B, respectively. These results demonstrate that the new commercial CMV HHV-6, 7, 8 R-gene kit was an efficient and reliable tool for the diagnosis of herpesvirus 6, 7, 8 infections.

Multicentric evaluation of a new commercial cytomegalovirus real-time PCR quantitation assay.

Automated real-time PCR systems have become the most common method in the quantitation of viral load during cytomegalovirus (CMV) infection in immuno-compromised patients. In order to evaluate a new commercially available CMV real-time PCR assay (CMV R-gene, Argene, France), a pp65 antigenemia assay and four different "in-house" real-time PCR assays were compared to the CMV R-gene for the detection and the quantitation of CMV load in 506 specimens of whole blood from transplant patients in four French hospital laboratories. The CMV R-gene was more sensitive than the pp65 antigenemia: there were 18% antigenemia-negative versus CMV R-gene-positive samples. A significant correlation was found between DNA quantitation by CMV R-gene and the number of positive cells detected by the pp65 antigenemia test (Spearman's rank test, r=0.63, p<0.0001). A CMV DNA load equivalent to 50 pp65-positive cells/200000 polymorphonuclear leukocytes was 5.26log(10)copies/mL of whole blood. When the CMV R-gene kit was compared to the four other "in-house" real-time PCR assays, there were few discordant results (6.7% total for the four laboratories), all detected with a weak positive CMV DNA viral load. Spearman's coefficients showed a good (r=0.82 for laboratory 1, r=0.66 for laboratory 3) to excellent (r=0.99 for laboratory 2, r=0.94 for laboratory 4) correlation between CMV R-gene and the four real-time "in-house" PCR assays. However, the results of CMV DNA viral load generated by CMV R-gene test were constantly higher than those generated by three out of four "in-house" PCR assays. This mean variation in CMV DNA viral load measured by CMV R-gene and "in-house" PCRs was of 0.77log(10), 0.04log(10), 0.77log(10) and 0.97log(10), for laboratories 1, 2, 3 and 4, respectively. We concluded that there was variability between results of different real-time PCR assays for CMV DNA quantitation. This observation emphasized the need of a standardised commercial assay to allow an "inter-laboratory" comparison of results. Our study showed that CMV R-gene is an accurate, efficient, reliable and versatile tool for rapid diagnosis and monitoring of CMV disease in transplantation recipients.

Virological, serological and epidemiological study of 255 consecutive cases of genital herpes in a sexually transmitted disease clinic of Paris (France): a prospective study.

Some studies (mostly retrospective) have pointed to an increasing frequency (up to 60%) of herpes simplex virus type 1 (HSV-1) in genital herpes (GH), but they were biased towards severe or atypical cases. We wished to evaluate the frequency of HSV-1 in patients attending our clinic for both first and recurrent episodes of GH. All patients (men and women) with genital lesions compatible with GH were included in a prospective study between May 1999 and April 2002. For all patients a standardized questionnaire, clinical examination, MRC5 culture (Dade Behring), polymerase chain reaction (PCR)-herpes consensus (Argène Biosoft) in case of negative culture and type-specific herpes serology HSV-1 and HSV-2 (Elisa Eurobio) were obtained. Predictive factors associated with HSV-1 and HSV-2 GH were studied by uni- and multivariable analyses. In all, 255 patients had a positive culture (n = 216) or PCR (n = 39). A total of 248 patients had typable herpes (148 men and 100 women). Median age was 33 (27-43); 20% had anal herpes; 48% had clinically recurrent lesions; 21% were HIV +; 20% of men were homosexual; 77% practised oral sex. In all, 36 were HSV-1 (14.5%): more in women, 25/100 (25%), than in men, 11/148 (7.5%) (odds ratio [OR]: 4 [1.8-9.1], P = 0.008). HSV-1 accounted for 23% of cases of first clinical episodes (women: 31.5%; men: 14.7%) (P = 0.02) and 6% of clinically recurrent episodes (women: 15%; men: 1.2%) (OR: 3.8 [1.6-9.1], P = 0.0033). Serological study was done in 239: primary infection was disclosed in 33 (HSV-1: 61%), HSV-2 non-primary first episode in 22 and recurrence in 184 (HSV-1: 8%). In all, 37% of recurrent episodes presented as a first clinical episode. HSV-1 was linked in men with homosexuality (P<0.01) and anilingus (P<0.01), in women with younger age (P<0.01), more sexual intercourses (P<0.0001) and more oral sex (P<0.001). Although HSV-1 is frequent in first clinical (23%) and primary (61%) episodes of GH, recurrent GH remains mostly due to HSV-2 (94%).

Rectal lymphogranuloma venereum surveillance in France 2004-2005.

Lymphogranuloma venereum (LGV) is a sexually transmitted infection (STI) caused by Chlamydia trachomatis strains belonging to the L1, L2 or L3 genotype. An alert about an outbreak of LGV among MSM in the Netherlands was published in January 2004. The first cases of rectal LGV in France were retrospectively diagnosed in March 2004 and sentinel surveillance for LGV was implemented in April 2004. Most of the participating centres were located in the cities of Paris and Bordeaux. Only confirmed rectal LGV cases were included in the surveillance. Rectal specimens from men that were found to be positive for C trachomatis by PCR were sent to the National Reference Centre for Chlamydia infection for genotyping. Simple epidemiological data provided by clinicians and genotyping results were sent to the Institut de Veille Sanitaire (InVS) where data were anonymously recorded. A total of 328 C. trachomatis rectal strains isolated in men were genotyped by the end of December 2005. Of these, 244 (74%) were LGV strains belonging to the L2 genotype. No L1 or L3 C. trachomatis genotype was found. Diagnosis was made retrospectively for 46 cases. The median age of patients with LGV was 39 years. HIV status was known for 96 patients: 82/96 (85%) were HIV-infected. Most LGV cases were diagnosed in the Paris area (92%). Among the remaining 26% C. trachomatis strains, genotypes Da and G were the most frequent. As with syphilis in recent years, the emergence of LGV in Europe is mainly affecting HIV-infected MSM. The screening and treatment of STIs should be included in the clinical follow-up of all HIV-infected MSM.

Rectal lymphogranuloma venereum surveillance in France 2004-2005.

Lymphogranuloma venereum (LGV) is a sexually transmitted infection (STI) caused by Chlamydia trachomatis strains belonging to the L1, L2 or L3 genotype. An alert about an outbreak of LGV among MSM in the Netherlands was published in January 2004. The first cases of rectal LGV in France were retrospectively diagnosed in March 2004 and sentinel surveillance for LGV was implemented in April 2004. Most of the participating centres were located in the cities of Paris and Bordeaux. Only confirmed rectal LGV cases were included in the surveillance. Rectal specimens from men that were found to be positive for C trachomatis by PCR were sent to the National Reference Centre for Chlamydia infection for genotyping. Simple epidemiological data provided by clinicians and genotyping results were sent to the Institut de Veille Sanitaire (InVS) where data were anonymously recorded. A total of 328 C. trachomatis rectal strains isolated in men were genotyped by the end of December 2005. Of these, 244 (74%) were LGV strains belonging to the L2 genotype. No L1 or L3 C. trachomatis genotype was found. Diagnosis was made retrospectively for 46 cases. The median age of patients with LGV was 39 years. HIV status was known for 96 patients: 82/96 (85%) were HIV-infected. Most LGV cases were diagnosed in the Paris area (92%). Among the remaining 26% C. trachomatis strains, genotypes Da and G were the most frequent. As with syphilis in recent years, the emergence of LGV in Europe is mainly affecting HIV-infected MSM. The screening and treatment of STIs should be included in the clinical follow-up of all HIV-infected MSM.

Response to antiviral therapy in haematopoietic stem cell transplant recipients with cytomegalovirus (CMV) reactivation according to the donor CMV serological status.

Pre-emptive antiviral treatment efficiently prevents occurrence of cytomegalovirus (CMV) disease in allogeneic stem cell transplant recipients. However, successive treatment courses can be necessary. The current study was aimed at determining factors that could influence the response to antiviral treatment, in particular the donor CMV serostatus. A total of 147 consecutive CMV-seropositive recipients (R+) were included and prospectively monitored for 6 months after transplantation. Reactivation of CMV occurred in 111 patients, 61 of 78 with a CMV-positive donor (D+) and in 50 of 69 with a CMV-negative donor (D-) (p 0.45). Baseline viral loads and initial viral doubling times did not differ between D+/R+ and D-/R+. Fifteen D+/R+ and four D-/R+ had self-resolving CMV infections. A total of 92 patients received antiviral treatment and 81 (88%) had a significant decrease in CMV load under therapy. Repeated CMV episodes were observed in 67% of those and were significantly more frequent in D-/R+ than in D+/R+ (p <0001). Half-life of CMV under treatment was significantly longer in D-/R+ than in D+/R+. Treatment failure observed in eight recipients was associated with low leucocyte count at reactivation onset, and was significantly more frequent in D-/R+ (six patients) than in D+/R+ (two patients) (p <0.0001). CMV strains resistant to antivirals were found in two D-/R+. Donor CMV serostatus influenced neither CMV reactivation occurrence nor the kinetics of CMV DNA load in the early phase of CMV replication but had a significant impact on response to antiviral therapy. Virological drug-resistance remained rare.

Treatment of chronic hepatitis C virus in allogeneic bone marrow transplant recipients.

We recently reported an increased incidence of cirrhosis in hepatitis C virus (HCV)-infected stem cell transplant (SCT) recipients. Here, we describe our experience in the treatment of these patients, which has been, to date, poorly reported in the literature. Among 99 HCV-infected HCT recipients, 36 had HCV-related liver lesions on biopsy requiring therapy. Owing to HCV treatment contraindications, only 61% of patients (22/36) could be treated. In all, 12 patients received more than one course of anti-HCV treatment if they had HCV RNA still detectable after the first course of treatment and no treatment contraindications. Combined therapy (pegylated interferon (IFN): n=9, or standard IFN: n=9, in combination with ribavirin) led to sustained virological response in 4/18 (20%) patients as compared to 2/20 (10%) in patients who received IFN alone. Hematological toxicity was more frequent with combined therapy. While anemia responded to erythropoietin and/or dose modification, thrombocytopenia usually led to treatment interruption (n=3). This study thus highlights the efficacy of combined therapy and emphasizes the fact that the undue safety concerns are not a problem when treating this particular population.

Ninety-six-week efficacy of combination therapy with lamivudine and tenofovir in patients coinfected with HIV-1 and wild-type hepatitis B virus.

We describe 6 patients who were coinfected with human immunodeficiency virus (HIV) type 1 and wild-type hepatitis B virus (HBV), in whom complete and sustained antiviral activity against wild-type HBV strains was attained during 96 weeks of combination therapy with lamivudine and tenofovir. The use of combination therapy with lamivudine and tenofovir for the treatment of HBV infection is very promising in the treatment of HIV/HBV coinfection.

Development of cytomegalovirus resistance to ganciclovir after oral maintenance treatment in a renal transplant recipient.

The emergence of a resistant strain is a theoretical threat after extensive use of antiviral drugs. We report the emergence of a ganciclovir-resistant cytomegalovirus (CMV) strain in a kidney transplant recipient during oral ganciclovir maintenance treatment. The patient was treated by oral ganciclovir for 2 months after successful treatment of CMV primary infection by intravenous ganciclovir. He developed a new episode of CMV infection with no clinical response to intravenous ganciclovir. The CMV isolate exhibited both phenotypic and genotypic resistance to ganciclovir. The CMV isolate was constituted of a mixture of strains, with and without a mutation at codon 460 of the UL97 gene. The clinical condition improved when mycophenolate mofetil (MMF) was discontinued, and a short course of intravenous globulin was added to ganciclovir. The emergence of the CMV strain could be secondary to more potent immunosuppression provide by MMF or subtherapeutic level obtained during oral ganciclovir treatment. We believe that ganciclovir resistance must be part of the differential diagnosis when a patient relapses or fails to respond to ganciclovir treatment.

Squamous cell carcinomas after allogeneic bone marrow transplantation for aplastic anemia: further evidence of a multistep process.

BACKGROUND: Secondary solid tumors are rare events occurring in patients who underwent allogeneic marrow transplantation for aplastic anemia and Fanconi's anemia. Human herpes virus 8 (HHV8), Epstein-Barr virus (EBV), and human papillomaviruses (HPV) sequences have been found in squamous cell carcinoma (SCC) occurring in organ transplant recipients. The tumor suppressor gene p53 has been strongly linked to the occurrence of SCC in the nonimmunocompromised population. PATIENTS AND METHODS: In eight patients with SCC, we searched for HHV8, EBV, varicella zoster virus, adenovirus, and HPV sequences from DNA extracted from selected areas of SCC. We also looked for p53 expression in those specimens as well as the presence of anti-p53 antibodies in the serum of these patients at the onset of SCC. RESULTS: In one patient, we found the presence of both HHV8 and EBV sequences, and in another patient we found HPV16 sequences. All five tumors that could be studied disclosed evidence of p53 accumulation, but none of the eight patients had anti-p53 antibodies in the sera. CONCLUSION: SCC developing in marrow transplant recipients seems to occur via a multistep process. Genetic predisposition may be present, as in patients with Fanconi's anemia. Transplantation-related factors, such as irradiation and chronic graft-versus-host disease, also have a role. In this article, we add two more potent risk factors: p53 alteration(s) and in some cases the presence of oncogenic viruses.

DNA fingerprinting of Chlamydia trachomatis by use of ribosomal RNA, oligonucleotide and randomly cloned DNA probes.

DNA fingerprinting of 15 reference strains and 24 clinical isolates of Chlamydia trachomatis, 2 strains of C. psittaci and one strain of C. pneumoniae was studied by use of universal 16 + 23S RNA from Escherichia coli, 16S rDNA-directed oligonucleotide and randomly cloned chlamydial DNA probes. The rRNA-gene restriction patterns (ribotypes) enabled the differentiation of chlamydial species. Following DNA cleavage by restriction endonuclease PvuII, lymphogranuloma venereum and trachoma biovars of C. trachomatis could be differentiated. An oligonucleotide, designed to hybridize the C. trachomatis 16S rDNA, also allowed for both species-specific identification and biovar typing of C. trachomatis human strains. Molecular typing system using 3 lambda clones containing C. trachomatis serotype E random DNA inserts, combined to ribotyping, revealed 12 groups of variable banding patterns within C. trachomatis, and could provide an alternative epidemiological tool.

Molecular typing of Chlamydia trachomatis by random amplification of polymorphic DNA.

The random amplification of polymorphic DNA (RAPD) was used for epidemiological typing of Chlamydia trachomatis strains. DNA samples from 39 C. trachomatis, 1 C. pneumoniae and 2 C. psittaci strains were screened by the use of 4 single 10-mer primers. Different and reproducible banding profiles were observed on agarose gel electrophoresis. No common profiles were recorded for strains from different Chlamydia species. All C. trachomatis strains of trachoma biovar were distinguished from lymphogranuloma venereum biovar. Moreover, serotypes A to C were separated from serotypes D to K, and some groups of strains sharing the same serotype D to K were further subdivided by RAPD. Conversely, strains of different serotypes could produce identical patterns of amplification, indicating that RAPD did not reflect serotyping. The patterns of amplified products were compared to the restriction fragment length polymorphism of the omp1 gene after amplification and to DNA fingerprinting by use of ribosomal RNA or randomly cloned DNA probes. RAPD seemed to be an alternative molecular typing procedure for epidemiological study and strain identification in urogenital infections due to serotypes D to K.

Inhibition of human immunodeficiency type 1 virus replication in monocytic U-937 cells by superinfection with Chlamydia trachomatis.

The aim of this study was to investigate the effects of infection of monocytic cells with both the human immunodeficiency type 1 virus (HIV-1) and Chlamydia trachomatis on the replication of each pathogen. U-937 cells, chronically infected with HIV-1 (strain LaVLai), either induced to differentiate into immature macrophage-like cells by 32 pM 12-O-tetradecanol phorbol-13-acetate or uninduced, were superinfected with C. trachomatis serovar L2. Both HIV-1 infection and differentiation rendered the U-937 cells highly susceptible to C. trachomatis lytic infection. Differentiation and superinfection of HIV-1-infected cells with C. trachomatis both affected cell viability and reduced viral production in vitro. RT activity was one tenth the original value after differentiation of HIV-1-infected cells, one twentieth the original value after superinfection with C. trachomatis, and one hundredth the original value after differentiation and superinfection with C. trachomatis.

[Chlamydia trachomatis: risk factors in women in the Parisian region. Importance of smoking and cervical ectropion]. / Chlamydia trachomatis: facteurs de risque chez les femmes de la région parisienne. Importance du tabagisme et de l'ectropion cervical.

A study of chlamydial infection and its clinical correlates was undertaken collaboratively among french women attending sexually transmitted disease (STD, prenatal, and teen clinics (n = 148). A complete sexual and gynecologic history and pelvic exam was performed on all women. Endocervical and urethral cultures were obtained for C. trachomatis and N. gonorrhoeae. Reason for visit included suspected STD in 97% of STD, 5% of prenatal and 17% of teen women. N. gonorrhoeae was isolated from STD clinic patients only (17%). C. trachomatis was found in 22% of teen, 17% of STD and 2% of prenatal clinic women. C. trachomatis was significantly associated with smoking, a history of urethral discharge in the male partner, and endocervical ectopy > 50% of total cervical surface.

[Diagnostic value of the study of anti-Chlamidia antibodies in salpingitis. 379 cases]. / Intérêt diagnostique de la recherche des anticorps anti-chlamydiens au cours des salpingites. 379 observations.

Antibodies to Chlamydia were assayed by complement fixation (CF) and inclusion indirect immunofluorescence (IFI) in sera collected from 379 patients with salpingitis: 30.3% of the patients had total and IgM antibodies at IFI and CF antibodies (profile I); 26.6% of the patients had total and IgM antibodies at IFI without CF antibodies (profile II); 31.6% of the patients had only total antibodies at IFI without specific IgM and without CF antibodies (profile III); 11.3% of the patients were Chlamydia antibody negative (profile IV). In the control group of 50 pregnant women apparently non infected, the profile distribution was 2% profile I, 8% profile II, 38% profile III, and 54% profile IV. Detection of IgM antibodies to Chlamydia trachomatis in 57% of patients with salpingitis, taking only one specimen, suggested recent or active chlamydial infection. CF antibodies indicated diffuse infection. Total antibodies correlated well with IgG antibodies detected by ELISA. Their finding was by no means diagnosis for Chlamydia being the cause of tubal infection, although titers observed in salpingitis patients were higher than in controls.

[Non-gonococcal urethritis and cervicitis. Isolation of Chlamydia trachomatis and detection of anti-Chlamydia antibodies by indirect immunofluorescence]. / Urétrites et cervicites non gonococciques. Isolement de Chlamydia trachomatis et détection des anticorps anti-chlamydiens par immunofluorescence indirecte.

The responsibility of Chlamydia trachomatis in non-gonococcal urethritis and cervicitis was investigated in 267 patients of both sexes. It was confirmed in 36.3% of patients with urethritis and 20.9% of patients with cervicitis by isolating C. trachomatis on Hela 229 cells in the presence of cytochalasin B. No clinical feature specific of C. trachomatis infection could be elicited. The patients were tested for total IgM-type serum anti-chlamydia antibodies by indirect immunofluorescence (IF), using as antigen the inclusions formed in Hela 229 cells by an L2 serotype of C. trachomatis. The serological study was also performed in 86 blood-donors used as controls. The diagnostic value of IF serology is limited in lower genito-urinary infections; the presence of specific IgM's correlates well with the isolation of C. trachomatis, but these IgM's are not detected in protracted urethritis or cervicitis. In such cases, the aetiological diagnosis can only be made by isolation of C. trachomatis from the focus of infection.

[Asymptomatic carriage of Chlamydia trachomatis in men consulting for condylomata acuminata]. / Portage asymptomatique de Chlamydia trachomatis chez des hommes consultant pour condylomes acuminés.

A search for Chlamydia trachomatis by cell culture was carried out in the urethra of 82 male patients consulting for condyloma acuminata at the Clinical and Biological Centre for Sexually Transmissible Diseases of the Saint-Louis hospital, Paris. Three patients had discreet urethral signs, but none had urethral discharge. Cell culture was positive for C. trachomatis in 36 of the 82 patients (44 percent). This high prevalence suggests that C. trachomatis should systematically be looked for in the urethra of male patients consulting for condylomata acuminata. If this cannot be done, then a systematic treatment with tetracyclines should be instituted.