Laboratory surveillance of arboviral infections in a southern France region colonized by Aedes albopictus.

The establishment of Aedes albopictus in southern France, a recognized competent vector for several arboviruses, represents a new threat for the local transmission and spread of what were until recently considered as tropical diseases. A preparedness and response plan, based on vigilance of both clinicians and laboratories, has introduced significant changes in guidelines and behaviour regarding patients' care specifically during the activity period of mosquitoes. In the present study, we report the results of a 1-year activity in arboviral infection diagnosis. A total of 141 patients were included in this retrospective study. The number of suspected imported and autochthonous cases was 69 and 72, respectively. A diagnosis of arboviral infection was confirmed for 15 (21·7%) suspected imported cases, with identification of 13 dengue viruses, one chikungunya virus and one Zika virus. No autochthonous cases were detected. This report illustrates the increase in requests for arboviral infection diagnosis and confirms the challenge with identifying autochthonous arboviral infection cases in many unspecific febrile syndromes.

Merkel cell polyomavirus DNA detection in lesional and nonlesional skin from patients with Merkel cell carcinoma or other skin diseases.

Background A novel polyomavirus, the Merkel cell polyomavirus (MCPyV), has recently been identified in Merkel cell carcinoma (MCC). Objectives To investigate the specificity of this association through the detection, quantification and analysis of MCPyV DNA in lesional and nonlesional skin biopsies from patients with MCC or with other cutaneous diseases, as well as in normal skin from clinically healthy individuals. Methods DNA was extracted from lesional and nonlesional skin samples of patients with MCC or with other cutaneous diseases and from normal-appearing skin of clinically healthy subjects. MCPyV DNA was detected by polymerase chain reaction (PCR) and quantified by real-time PCR. Additionally, the T antigen coding region was sequenced in eight samples from seven patients. Results MCPyV DNA was detected in 14 of 18 (78%) patients with MCC, five of 18 (28%) patients with other skin diseases (P = 0.007) and one of six (17%) clinically healthy subjects. In patients with MCC, viral DNA was detected in nine of 11 (82%) tumours and in 10 of 14 (71%) nontumoral skin samples (P = 0.66). MCPyV DNA levels were higher in MCC tumours than in nontumoral skin from patients with MCC, and than in lesional or nonlesional skin from patients with other cutaneous disorders. Signature mutations in the T antigen gene were not identified in the two MCC tumour specimens analysed. Conclusions High prevalence and higher levels of MCPyV DNA in MCC supports a role for MCPyV in tumorigenesis. However, the high prevalence of MCPyV in the nontumoral skin and in subjects without MCC suggests that MCPyV is a ubiquitous virus.

Effect of HIV-1 and antiretroviral therapy on herpes simplex virus type 2: a prospective study in African women.

OBJECTIVES: To document the natural history of herpes simplex virus type 2 (HSV-2) in relation to HIV and highly active antiretroviral therapy (HAART) in Africa, a longitudinal study was conducted of women in the placebo arms of two randomised controlled trials of HSV-suppressive therapy in Burkina Faso. METHODS: 22 HIV-uninfected women (group 1), 30 HIV-1-infected women taking HAART (group 2), and 68 HIV-1-infected women not eligible for HAART (group 3) were followed over 24 weeks. HSV-2 DNA was detected on alternate weeks using real-time PCR from cervicovaginal lavages. Plasma HIV-1 RNA was measured every month. CD4 cell counts were measured at enrollment. RESULTS: Ulcers occurred on 1.9%, 3.1% and 7.2% of visits in groups 1, 2 and 3 (p = 0.02). Cervicovaginal HSV-2 DNA was detected in 45.5%, 63.3% and 67.6% of women (p = 0.11), and on 4.3%, 9.7% and 15.5% of visits in the three groups (p<0.001). Among HIV-infected women, cervicovaginal HSV-2 DNA was detected more frequently during ulcer episodes (adjusted risk ratio (aRR) 2.79, 95% CI 2.01 to 3.86) and less frequently among women practising vaginal douching (aRR 0.60, 95% CI 0.40 to 0.91). Compared with women not taking HAART and with CD4 cell counts of 500 cells/microl or greater, women on HAART had a similar risk of HSV-2 shedding (aRR 0.95, 95% CI 0.52 to 1.73), whereas women with CD4 cell counts of 200-500 cells/microl were more likely to shed HSV-2 (aRR 1.71, 95% CI 1.02 to 2.86). CONCLUSIONS: HSV-2 reactivations occur more frequently among HIV-infected women, particularly those with low CD4 cell counts, and are only partly reduced by HAART. HSV therapy may benefit HIV-infected individuals during HAART.

Longitudinal effect following initiation of highly active antiretroviral therapy on plasma and cervico-vaginal HIV-1 RNA among women in Burkina Faso.

BACKGROUND: Highly active antiretroviral therapy (HAART) could decrease HIV-1 transmissibility by reducing genital and plasma HIV-1 RNA. METHODS: We evaluated the effect of HAART on genital and plasma HIV-1 RNA in a cohort of 39 antiretroviral-naïve women in Burkina Faso. Cervico-vaginal lavages were collected before HAART initiation and at six visits over 28 weeks while on HAART. Blood samples were collected at baseline and at three and four visits for CD4 and plasma HIV-1 RNA measurements, respectively. RESULTS: Before HAART, 72% of women had detectable genital HIV-1 RNA. After 18 weeks on HAART, only one woman (2.5%) had detectable plasma HIV-1 RNA and two women (5.1%) had detectable genital HIV-1 RNA. Similar results were observed at each follow-up visit. However, 16/34 (47%) women with consistently undetectable plasma HIV-1 RNA shed HIV-1 at least once between weeks 18 and 28. In samples with detectable genital HIV-1, the mean quantity of HIV-1 RNA decreased from 3.87 prior to HAART to 3.04 log(10) copies/mL at last visit (median 29 weeks; a 6.8-fold decrease in absolute number of copies/mL) (p = 0.04). A significant median CD4 lymphocyte cell gain of 121 cells/muL (interquartile range 59 to 204) was measured between pre-HAART and last visit. CONCLUSION: These findings suggest that HAART could play a role in reducing HIV transmission in Africa; however, they underscore the need to emphasise safe sex practices with patients taking HAART.

[Bocavirus: a new human parvovirus]. / Le bocavirus : un nouveau parvovirus humain.

A new virus was recently discovered by molecular techniques in respiratory samples collected from young children with respiratory disease. This virus, which represents a new member of the Parvoviridae family is genetically related to the bovine parvovirus and the canine minute virus that belong to the Bocavirus genus. It has been classified in the Bocavirus genus and named human bocavirus (HBoV). Recent studies conducted in different countries have shown that HBoV is found in 3 to 18 %of children with respiratory disease worldwide. Genetic analysis indicate that this virus shows low genetic variability. The clinical signs observed in patients infected with HBoV are not different from those caused by other respiratory viruses.A frequent association of HBoV with other respiratory pathogens may be observed. Therefore, the exact role played by this virus in human diseases still remains unclear. Further studies including control populations are needed to ascertain the pathogenic potential of this virus.

Characteristics of HPV infection in women at risk in Western Algeria.

OBJECTIVES: We aimed to characterize HPV infections and cervical lesions in Western Algeria. PATIENTS AND METHODS: A total of 96 cervical samples obtained from women at risk of HPV infection (HIV-1-infected or presenting with a gynecological disease) were analyzed to characterize this infection and search for cytological abnormalities. RESULTS: A total of 60% of women at risk had an HPV infection. The rate of high-risk HPV (HR-HPV) infection among these women was 84.5% and that of intraepithelial lesions was 29.3%. The frequency of HPV infection was significantly higher among HIV-1-infected patients. An association between the presence of HR-HPV and the polygamy of the partner was observed. An association between cytological abnormalities and the use of oral contraceptives was observed among HIV-1-infected women. CONCLUSION: Given the high frequency of HPV infection in this at risk population, close monitoring and regular gynecological screening are essential.

Multicenter evaluation of the NucliSens EasyQ HIV-1 v1.1 assay for the quantitative detection of HIV-1 RNA in plasma.

The Nuclisens EasyQ HIV-1 v1.1 assay (Biomerieux) is a real-time detection method combined with NASBA technology designed to measure plasma HIV-RNA. Its performance was assessed in 1008 clinical specimens collected from individuals infected with clade B (774) and non-B (234) HIV-1 variants at four European laboratories. The results were compared with those obtained using three other commercial viral load assays: Cobas Amplicor Monitor HIV-1 v1.5 (Roche), Versant HIV-1 RNA assay (Bayer) and Nuclisens HIV-1 QT (Biomerieux). Overall, the linearity, specificity and reproducibility of the EasyQ assay was comparable with that from the other tests. The correlation coefficient (R) between methodologies was 0.85 for Amplicor; 0.87 for Versant; and 0.91 for Nuclisens. The specificity of the assay was 99.4%. Of note, Versant missed 17% of specimens with non-B subtypes which could be detected by EasyQ, while Amplicor provided similar results than EasyQ. HIV-1 group O specimens were only detected by the EasyQ assay. In conclusion, the performance of the EasyQ assay seems to be similar to that of other HIV-1 viral load tests currently on the market, but it is more sensitive than Versant for HIV-1 non-B subtypes and shows a wider dynamic range than Amplicor. Moreover, as it incorporates the advantage of real-time detection procedures, it facilitates high throughput and short turnaround time.

Impairment of B-lymphocyte differentiation induced by dual triggering of the B-cell antigen receptor and CD40 in advanced HIV-1-disease.

OBJECTIVE: This study was performed to investigate the hyporeactivity of purified B lymphocytes from HIV-1-infected patients. DESIGN: Given the importance of the B-cell Ag receptor (BCR) and CD40 in B-lymphocyte activation, we assessed the capacity of purified peripheral blood B lymphocytes from HIV-1-infected patients to differentiate into Ig-secreting cells in a T-cell- and accessory-cell-independent system of BCR and CD40 costimulation. METHODS: B lymphocytes from 21 HIV-1-infected patients were purified by immunomagnetic cell separation and costimulated with immobilized anti-CD40 monoclonal antibodies and Staphylococcus aureus Cowan I particles in the presence of interleukin (IL)-2 and IL-10. Homotypic aggregate formation, apoptosis, cell cycle entrance, proliferation and Ig secretion of B cells were analysed. RESULTS: Costimulation by the BCR and CD40 induced proliferation and differentiation of B lymphocytes into Ig-secreting cells in 13 patients (group I) but not in eight patients (group II). For three patients in group II, the dual triggering induced apoptosis of B cells. The unexpected inability of these cells to differentiate was associated with a high CD38 expression and a weak spontaneous production of Ig or anti-HIV-1 antibodies in patients with a high viral load and a low CD4+ lymphocyte count. Despite this anomaly, the B cells from group II were able to progress through the cell cycle after stimulation with a combination of phorbol ester and ionomycin in complete medium, suggesting an impairment in BCR and CD40 early signal transduction. CONCLUSION: Intrinsic in vitro hyporeactivity of B lymphocytes to dual triggering of BCR and CD40 was observed in advanced HIV-1 disease and appeared to be related to in vivo hyperactivation of B cells.

Emergence of zidovudine and multidrug-resistance mutations in the HIV-1 reverse transcriptase gene in therapy-naive patients receiving stavudine plus didanosine combination therapy. STADI Group.

OBJECTIVE: Assessment of genotypic changes in the reverse transcriptase gene of HIV-1 occurring in antiretroviral naive patients treated by stavudine plus didanosine combination therapy. METHODS: Sequence analysis (codons 1-230) was performed after amplification of the reverse transcriptase gene from plasma samples collected at baseline and at the end of treatment from 39 previously treatment-naive patients treated for 24-48 weeks. RESULTS: At baseline, mutations associated with zidovudine resistance were detected in plasma from two patients: Asp67Asn/Lys219Gln and Leu210Trp. Among the 39 subjects, 18 (46%) developed mutations: one developed the Val75Thr/Ala mutation, four (10%) developed a Gln151Met multidrug-resistance mutation (MDR), associated in one of them with the Phe77Leu and the Phe116Tyr MDR mutations and 14 (36%) developed one or more zidovudine-specific mutations (Met41Leu, Asp67Asn, Lys70Arg, Leu210Trp, Thr215Tyr/Phe). The development of a Met41Leu zidovudine-specific mutation was associated with the development of a Gln151Met mutation in one patient. Other reverse transcriptase mutations known to confer resistance to nucleoside analogues were not detected. At inclusion, there was no statistical difference in HIV-1 load between patients who developed resistance mutations and those who did not. RNA HIV-1 load decrease was higher (P = 0.05) in patients who maintained a wild-type reverse transcriptase genotype (-2.22 log10 copies/ml) than in patients who developed resistance mutations (-1.14 log10 copies/ml). CONCLUSION: Stavudine/didanosine combination therapy is associated with emergence of zidovudine-related resistance or MDR mutations in naive patients. These findings should be considered when optimizing salvage therapy for patients who have received a treatment including stavudine/didanosine combination.

CD4 T cell surface CCR5 density as a host factor in HIV-1 disease progression.

OBJECTIVE AND DESIGN: We have recently shown that the number of CCR5 molecules at the surface of peripheral blood CD4 T cells (CCR5 density) correlates with the viral RNA plasma level in HIV-1-infected individuals. As viral load is a strong predictor of outcome in HIV infection, the present study examines the correlation between CCR5 density and HIV-1 disease progression. METHODS: Using a quantitative flow cytometry assay, we measured CCR5 density in HIV-1-infected adults and control healthy volunteers. The CCR5 genotype (presence of a Delta 32 allele) was also determined. RESULTS: CCR5 density was stable over time on non-activated, HLA-DR(-)CD4 T cells of infected individuals. In a study cohort of 25 patients, asymptomatic and non-treated, we observed a correlation between CCR5 density on HLA-DR(-)CD4 T cells and the CD4 T cell slope (P = 0.026), which was independent of the presence or absence of the Delta 32CCR5 deletion. In particular, slow progressors expressed lower CCR5 densities than non-slow progressors (P = 0.004) and non-infected control subjects (P = 0.002). CONCLUSION: These results are compatible with the hypothesis that CCR5 density, which is a key factor of HIV-1 infectability, determines in-vivo HIV production, and thereby the rate of CD4 cell decline. Consequently, CCR5 density quantitation could be a new valuable prognostic tool in HIV-1 infection. Moreover, these data emphasize the therapeutic potential of treatments that reduce functional CCR5 density.

Ganciclovir resistance and UL97 gene mutations in cytomegalovirus blood isolates from patients with AIDS treated with ganciclovir.

BACKGROUND: Treatment of cytomegalovirus (CMV) diseases with protracted administration of ganciclovir can promote the development of resistant CMV that is associated with a poor response to therapy. It has been shown that the majority of ganciclovir-resistant CMV isolates carry mutations in the UL97 phosphotransferase gene. OBJECTIVES: To evaluate the frequency of CMV resistance to ganciclovir in patients with AIDS treated with ganciclovir and to identify the UL97 gene mutations associated with ganciclovir resistance. STUDY DESIGN: Analysis of CMV blood isolates obtained over 1 year from patients treated with ganciclovir. CMV susceptibility to ganciclovir was determined by an immediate early antigen plaque reduction assay; UL97 gene mutations were identified by restriction enzyme digest analysis and sequencing. RESULTS: Twenty-nine patients were followed-up; 17 CMV blood isolates were obtained from 10 ganciclovir-experienced patients. Thirteen (76%) of these isolates, obtained from seven (24%) patients after a median treatment duration of 5.5 months, were resistant to ganciclovir. Five of the seven patients who had a ganciclovir-resistant CMV in blood showed retinitis progression. UL97 gene mutations were identified in nine CMV isolates at codons 460 (M --> V), 594 (A --> V and A --> T), and 595(L --> S). Three patients developed a ganciclovir-resistant virus after a ganciclovir treatment shorter than 60 days (28-58 days). In another patient, we observed that ganciclovir resistance persisted 4 months after discontinuation of ganciclovir therapy. CONCLUSION: Our results indicate that ganciclovir resistance due to UL97 gene mutations is common in subjects with AIDS-related CMV diseases treated with ganciclovir. Detection of these mutations represents a tool for the management of patients with ganciclovir therapy.

Virus isolation, polymerase chain reaction and in vitro antibody production for the diagnosis of pediatric human immunodeficiency virus infection.

Viral culture (VC), polymerase chain reaction (PCR) and in vitro antibody production (IVAP) by peripheral blood mononuclear cells were compared for the early diagnosis of HIV-1 infection in 46 infants born to HIV-1 seropositive mothers. The ten children considered infected on the basis of clinical signs and persistence of anti-HIV-1 antibodies had at least one positive viral culture and seven were always positive in both PCR and IVAP tests. PCR and IVAP tests were occasionally negative in three infected children. Among 30 healthy children who became seronegative and were always negative for viral culture, 22 (73.3%) were also repeatedly negative in PCR and IVAP. We report 6 cases of children classified as P2A at the term of this study but who had lost anti-HIV-1 antibodies. They presented at least one positive viral culture and occasional positive PCR and/or IVAP results. The results indicate that the combination of viral culture, PCR and IVAP tests improves the early diagnosis of pediatric HIV infection.

Unlikely role of Epstein-Barr virus in the pathogenesis of primary cutaneous CD30+ anaplastic large cell lymphoma.

BACKGROUND: Primary cutaneous CD30+ anaplastic large cell lymphoma (ALCL) is a rare subset of cutaneous lymphoma, with a much better prognosis than its nodal counterpart. The pathogenesis of both nodal and primary cutaneous CD30+ ALCL is largely unknown but experimental data support the hypothesis that the Epstein-Barr virus could play a role in the nodal subset. OBJECTIVE: To evaluate the involvement of Epstein-Barr Virus (EBV) in primary cutaneous CD30+ ALCL by searching for both nucleic acids and EBV proteins in cutaneous lesions. SETTING: Two University Hospitals in Southern France (secondary referral hospitals). PATIENTS: Eight consecutive patients with typical primary cutaneous CD30+ anaplastic large cell lymphoma were studied. METHODS: Search for the presence of DNA, RNA and EBV proteins in cutaneous lesions by PCR, in situ hybridization and immunohistochemistry. RESULTS: EBV DNA and RNA was identified in only one lesion of primary cutaneous CD30+ ALCL and in none of the normal adjacent skin samples. In situ hybridization and immunohistological studies were consistently negative in all samples. CONCLUSION: These results do not support an early role of EBV in the oncogenetic pathogenesis of primary cutaneous CD30+ ALCL.

Detection of rubella virus in amniotic fluid by electron microscopy.

We report in this paper the observation of rubella virus by electron microscopy in an amniotic fluid sample, collected from a pregnant woman with rubella infection. Virological investigations by inoculation of cell cultures with amniotic fluid and fetal blood remained negative, due probably to the presence of neutralizing antibodies in the samples. Electron microscopy is a rapid but weakly sensitive method to detect viruses in clinical specimens. However, this unusual observation would indicate that in some cases electron microscopy could be a useful technique to evidence a fetal rubella infection.

Ganciclovir resistance mutations in UL97 and UL54 genes of Human cytomegalovirus isolates resistant to ganciclovir.

Human cytomegalovirus (HCMV) resistance to ganciclovir results from mutations in viral phosphotransferase (UL97) and/or DNA polymerase (UL54) genes. The HCMV isolates from the blood of immunocompromised patients with persisting presence of the pp65 antigen in the blood in spite of ganciclovir therapy were tested for ganciclovir susceptibility by an immediate-early antigen plaque reduction assay, and the UL54 and UL97 genes were sequenced. Nine isolates from eight patients (six patients with acquired immune deficiency syndrome (AIDS), one liver transplant recipient and one renal transplant recipient) showed phenotypic resistance to ganciclovir. All these ganciclovir-resistant HCMV isolates harbored one or more of the following UL97 mutations: M460V, A594V, A594T, L595S, C603W, and M615V. Two isolates harbored the P522S mutation in the UL54 gene. The M615V mutation in the UL97 gene has not been reported earlier and its role in ganciclovir resistance remains to be elucidated. In ganciclovir-resistant HCMV isolates the UL54 gene was less frequently mutated than the UL97 gene. The P522S mutation was relatively frequent in UL54-mutated HCMV isolates.

[Facial palsy and central nervous system infection with varicella virus following adult chickenpox]. / Paralysie faciale et infection du système nerveux par le virus VZV après une varicelle de l'adulte.

INTRODUCTION: VZV virus-related peripheral neuropathies usually occur after shingles in adults and more rarely after chickenpox in childhood. CASE REPORT: A 54-year-old patient presented with a right VIIth nerve palsy following a chickenpox rash and recovered after antiviral treatment. CSF analysis revealed lymphocytic meningitis and the virus was identified by PCR. CONCLUSIONS: Although previous chickenpox was not found in the patient's past history, the probability of reinfection is likely. The virus can be assumed to affect the nervous system directly; the axonal or demyelinating mechanism of the neuropathy may be discussed.

[RT-PCR in the prenatal diagnosis of rubella. Report of 2 cases]. / Apport de la RT-PCR pour le diagnostic prénatal de la rubéole. A propos de 2 observations.

OBJECTIVE: To confirm the interest of the reverse transcription-coupled polymerase chain reaction (RT-PCR) in prenatal diagnosis of rubella. PATIENTS AND METHODS: Maternal blood, amniotic fluid and foetal blood specimens collected at 21 to 24 weeks of amenorrhea from two pregnant women with serologically proven rubella infection were tested by RT-PCR and cell culture for detection of rubella virus. Rubella virus-specific IgM antibodies were also assayed in foetal blood. RESULTS: In both cases, rubella virus was detected in foetal blood by RT-PCR and cell culture, whereas it was not detected in maternal blood. Rubella virus-specific IgM were also detected in foetal blood. In amniotic fluid specimens, rubella virus was detected by RT-PCR in one case and by cell culture in the other one. A termination of pregnancy was carried out in both cases. COMMENTS: RT-PCR is a rapid test. It could be useful to confirm foetal infection and could represent a criterion for considering a pregnancy termination. However, this technique which is not yet standardized for detection of rubella virus, needs further validation by additional studies.

[Acquired cytomegalovirus infections in immunocompetent adults: 116 cases]. / Cytomégalovirose acquise de l'adulte immunocompétent. 116 observations.

OBJECTIVE: To analyse retrospectively the clinical, biological and epidemiological features of cytomegalovirus (CMV) infection in the immunocompetent host. PATIENTS AND METHODS: A retrospective study was conducted in 116 cases of CMV infection (74 inpatients, 42 out-patients) collected from 1981 to 1997 in a university hospital. Diagnostic was established on serological criteria in all cases. RESULTS: Fever was observed in all cases but one (mean duration: 21 days). The most frequent symptoms were headache (51%) and myalgia (46%). Splenomegaly was the most frequent sign (36%). Pulmonary interstitial opacities on chest x-ray were found in 8.5% of patients. Pulmonary (1 case) and neurological (2 cases) complications occurred. Concomitant HIV primary infection was observed in 2 patients. Mononucleosis and ALAT and LDH elevations were observed in 95%, 85% and 95% of cases respectively. Viremia was positive in 79% (30/38). pp65 antigenemia was useful to establish the diagnosis in 9 patients. Antibiotics were prescribed in 46% before diagnosis was established. CONCLUSION: CMV infection in the immunocompetent host is a frequent and rarely complicated disease. pp65 antigenemia should be evaluated more in this setting. Most hospitalizations due to this affection could be avoided.