Diagnostic value of quantitative PCR for adenovirus detection in stool samples as compared with antigen detection and cell culture in haematopoietic stem cell transplant recipients.

Adenovirus (AdV) infections constitute a significant cause of morbidity and mortality during haematopoietic stem cell transplantation. Recent guidelines recommend repeated screening for AdV in whole blood (WB), with quantitative PCR (qPCR) as the reference standard. Despite pre-emptive antiviral treatment based on qPCR in WB, the mortality rate after disseminated AdV infection remains very high. The aim of our study was to advance early screening for AdV, using a standardized method, so as to enable the earlier initiation of antiviral treatment or adoptive immunotherapy. The diagnostic value of AdV DNA quantification in stool samples was investigated retrospectively and compared with antigen detection and cell culture in 21 patients with AdV infection, from 182 patients followed in the Transplant Unit of Nancy University Hospital Centre, including 18 patients with systemic infection. In 16/18 patients with positive AdV viraemia, AdV DNA was present in stool samples earlier than in WB (median, 42 days; range, 3-199 days), whereas both antigen detection and cell culture were still negative for 11/18 patients with systemic AdV infection. The course of AdV viral loads in stool samples was predictive of adenoviraemia (sensitivity, 89%). Very late and lethal AdV infections were observed in cord blood transplant recipients, and would have been detected much earlier with the use of qPCR on stool samples. This study confirmed that quantification of AdV in stool samples by qPCR is beneficial for the management of transplant recipients, with or without antigen detection.

Occult hepatitis B infection in patients infected with HIV: report of two cases of hepatitis B reactivation and prevalence in a hospital cohort.

Patients co-infected with human immunodeficiency virus (HIV) and hepatitis B virus (HBV) are particularly at risk of hepatitis B reactivation. Two cases of patients infected with HIV with isolated anti-HBc antibodies who had experienced an HBV reactivation are described. In the two cases HBV reactivation occurred after withdrawal of anti-retroviral treatment with anti-HBV activity from the patients' highly active antiretroviral therapy (HAART), in accordance with HIV genotypic resistance profiles. Consequently, plasma samples from 383 patients infected with HIV were tested to assess the prevalence of occult HBV infection in the Infectious Diseases Department Unit of Nancy Hospital by investigating serological patterns and HBV replication. Forty-five percent (172/383) of patients had had previous contact with HBV. Isolated anti-HBc antibodies were observed in 48 patients (48/383, 12%) and, among these, 2 were HBV-DNA positive. Since 75% (288/383) of the patients were treated with HAART, including at least one drug active against HBV, occult HBV infection was perhaps unrecognized. In cases of HIV infection, all patients should be screened for HBV infection and the knowledge of HBV status as well as the monitoring of HBV viral load are essential in preventing HBV reactivation. Consideration should be given to the continuation of drugs with anti-HBV activity in co-infected patients receiving HAART, as cessation of therapy is associated with a risk of HBV reactivation. At least, close monitoring of the HBV viral load is warranted in such situations.

[Neutropenia and/or thrombocytopenia due to acute parvovirus B19 infection]. / Neutropénie et/ou thrombopénie associée(s) à une infection aiguë par le parvovirus B19.

Erythema infectiosum (fifth disease) is the most common clinical presentation of acute parvovirus B19 infection in infancy. In healthy adults, most cases of infection are asymptomatic or accompanied by a flu-like syndrome like headaches and myalgia. Haematological manifestations are dominated by transient aplasia of erythroid progenitor cells which remains asymptomatic in most of non immunocompromised patients. Patients with sickle cell disease, thalassemia or other disorders associated with shortened red blood cell survival are at particular risk for marked anemia or red blood cell aplasia. In immunosuppressed patients, anemia may be chronic because of persistent viral load. Neutropenia, lymphopenia or thrombocytopenia have also been reported in acute parvovirus B19 infection. Mechanisms of these cytopenias are not yet elucidated. We present two patients with thrombopenia and/or neutropenia but without anemia due to acute parvovirus B19 infection.

Effective ribavirin concentration in mice brain using cyclodextrin as a drug carrier: evaluation in a measles encephalitis model.

Ribavirin (RBV) is a water-soluble synthetic nucleoside with broad spectrum antiviral properties, but it is ineffective against major viral encephalitis because of a failure to cross the blood-brain barrier (BBB). The antiviral activity of the complex ribavirin/alpha-cyclodextrin was previously demonstrated to be stronger than free ribavirin, in an in vivo model of measles virus (MV) encephalitis in mice. The role of cyclodextrin (CD) on ribavirin uptake into the brain needs to be defined. Ribavirin specific extraction from brain tissue was developed, based on a solid phase extraction. It was quantified by high performance liquid chromatography at different time points after intraperitoneal injection of single or multiple doses of free ribavirin or of the complex ribavirin/alpha-cyclodextrin. Whatever the tested dose (40 or 100mg/kg), the amount of ribavirin in the brain was significantly higher (p<0.001) when the drug was injected as a complex with alpha-cyclodextrin, in healthy or measles virus-infected mice.

[What future for ribavirin beyond hepatitis C therapy?] / Quel avenir pour la ribavirine en dehors de l'hépatite C ?

Most of emerging and re-emerging diseases are due to RNA viruses and available drugs are insufficient. Currently the ribavirin is only licensed for the treatment of chronic hepatitis C infection, whereas this guanosin analogue has a broad-spectrum in vitro activity against many DNA and RNA viruses. It was consequently used as a last resort, in emergency state like avian influenza or in front of new viruses (SARS). The strategies for development of new antiviral drugs are usually based on virus structure and properties. In regard to recent development of chemical vector designed for improving drug bioavailability, use of former drug, like ribavirin, could be reconsidered. For example, complexation of ribavirin with cyclodextrins, cyclic oligosaccharide vectors, can improve its bioavailability in central nervous system and improve encephalitis treatment.

[Characterization of hepatitis B virus strains from the Central African Republic: preliminary results]. / Caractérisation des souches de virus de l'hépatite B circulant en République centrafricaine: résultats préliminaires.

OBJECTIVES: Genotyping of Hepatitis B virus (HBV) strains from patients in Central African Republic and comparison with results obtained in other African countries. PATIENTS AND METHODS: Sera were collected from patients admitted with symptoms of acute or chronic hepatitis to the "Hôpital de l'Amitié de Bangui", Central African Republic (CAR). The complete sequence of preS2/S gene has been defined for determining genotypes. RESULTS: Hundred and ninety-six sera were collected from 112 men and 84 women. Ninety-two percent of patients had contact with HBV (anti-HBc postitive) and the HBsAg prevalence was about 62%. HBV DNA was detected in 66% of HBsAg positive sera. No HBV-DNA was evidenced among patients with negative HBsAg. Ninety-three percent of the HBV strains belonged to genotype E; one (3.4%) belonged to genotype A1, and one (3.4%) belonged to genotype D. CONCLUSIONS: The high prevalence of HBV infection in the studied population is due to their recruitment. The genotype E is predominant in CAR and the intragroup variability of HBV genotype E reached only 1.8%. Genotypes A and D were less common in CAR their presence may be explained by importation.

In vivo antiviral activity of ribavirin/alpha-cyclodextrin complex: evaluation on experimental measles virus encephalitis in mice.

Intracranial injection of the rodent adapted CAM/RB strain of measles virus (MV) induces encephalitis in CBA/ca mice. It has already been shown that cyclodextrins can be used as carriers to increase the antiviral activity of ribavirin (RBV) against MV in cellular model. In this study, the antiviral activity of a RBV/alpha-cyclodextrin complex has been evaluated in vivo using the above model. CBA/ca mice were treated by intraperitoneal injection of free ribavirin (40 mg/kg) or a RBV/alpha-cyclodextrin complex (molar ratio 1:3). After 21 days, intracerebral injection of CAM/RB resulted in 100% mortality in the mock group. In contrast, mortality rates of 80% and 40%, respectively, were observed in RBV and RBV/alpha-CD-treated mice (p<0.05 and p=0.06 for distilled water and RBV, respectively). The viral load of MV in the mouse brain was monitored daily by real-time PCR until day 6 after infection, to compare virus production in treated and non-treated mice. This data shows that RBV complexation with alpha-cyclodextrin can increase the antiviral activity of ribavirin in a measles virus encephalitis model in mice.

Evaluation by Q-RTPCR of the efficacy of ribavirin complexed with beta-cyclodextrin against measles virus in a mouse encephalitis model.

The objective of this work was to study the antiviral activity of ribavirin on measles encephalitis infection when using cyclodextrins as carriers. The use of cyclic oligosaccharides can promote the activity of many drugs and the benefit of the association of ribavirin with beta-cyclodextrin has already been demonstrated in vitro. Intracranial inoculation of the rodent adapted neurovirulent CAM/RB strain of measles virus induces encephalitis in CBA/ca mice. The antiviral activity of the complex ribavirin/beta-cyclodextrin at molar ratio 1:1 has been evaluated in vivo in the above encephalitis model. CBA/ca mice were treated with daily intraperitoneal injection of ribavirin (40 mg/kg) with or without beta-cyclodextrin. The viral load in the brain of mice was quantified by real-time Reverse transcription-Polymerase chain reaction. Treatment of mice by the complex ribavirin/beta-cyclodextrin (1:1) by intraperitoneal route decreases the viral load in the brain of 1.1 and 0.7 log(10) Eq copies x mL(-1) compared to distillated water and ribavirin treatment, respectively. At the same time, free ribavirin injection shows a negligible difference compared to treatment by distillated water.

Infection due to acyclovir resistant herpes simplex virus in patients undergoing allogeneic hematopoietic stem cell transplantation.

Over an eight-month period from October 1997 to May 1998, four patients who had received bone marrow transplant (BMT) from unrelated donor presented with severe mucosal cutaneous infections involving acyclovir resistant herpes simplex virus 1 (HSV-1). The four isolates were acyclovir (ACV) resistant, three of which were also foscarnet resistant as determined by the dye uptake method. The sequencing of the thymidine kinase (TK) gene did not permit to establish a relation between mutations and resistance to ACV. Three patients were considered as clinically cured of their HSV infection by replacement of ACV or foscarnet with either valacyclovir (one case) or cidofovir (two cases) but eventually two of them died of graft vs host disease. One patient died of extensive HSV infection despite administration of cidofovir. This study emphasizes the importance of monitoring the herpes virus resistance to antiviral drugs in bone marrow transplant recipients and the usefulness of the evaluation of novel antiviral drug for treatment of infections due to strains of HSV resistant to ACV and foscarnet that occur in about 5% of immunocompromised patients.

Treatment of adenovirus infections in patients undergoing allogeneic hematopoietic stem cell transplantation.

Retrospective analysis of 303 patients who underwent allogeneic hematopoietic stem cell transplantation identified 35 (11.5%) with adenovirus infection. Among them, 22 received specific therapy. As first-line therapy, 18 were treated with intravenous ribavirin, 3 with cidofovir, and 1 with vidarabine. Moreover, 2 received donor leukocyte infusion in combination with ribavirin, and 1 received it after failing to respond to other therapies. Seven survived (31.8%; 3 of 13 who received ribavirin alone and 2 of 3 who received cidofovir). Among the 5 patients treated with combined strategies, 2 who received donor leukocyte infusions showed clearance of all symptoms. Acute graft-versus-host disease grade > or = 3 (P = .01) and a long delay between infection and treatment (P = .05) correlated with a greater risk of treatment failure. In conclusion, ribavirin and vidarabine are ineffective options, particularly for patients at who are high risk of acquiring disseminated adenovirus disease. Conversely, cidofovir or donor leukocyte infusions seem to be encouraging approaches if initiated early.

Investigation of aciclovir-resistant herpes simplex virus I infection in a bone marrow transplantation unit: genotyping shows that different strains are involved.

Over an eight-month period from October 1997 to May 1998, four patients who had received a bone marrow transplant (BMT) from an unrelated donor presented with severe mucosal cutaneous infections involving aciclovir resistant herpes simplex virus 1 (HSV-1). The emergence within a short period of resistant HSV-1 strains in the bone marrow transplantation unit raised fears of hospital-acquired infections. The hypothesis was investigated by restriction fragment length polymorphism (RFLP), sequencing of the thymidine kinase (TK) gene and genotyping of hypervariable regions of these four strains. Restriction fragment length polymorphism proved to be poorly discriminant and the TK sequence did not rule out transmission between these patients. Amplification of reiterating hypervariable genomic HSV-1 regions designated Re IV and Re VII clearly differentiated patients' strains. Thus, in this study, there was no evidence of nosocomial transmission of HSV-1 strains between the four patients.

A convenient semi-quantitative method for the diagnosis of Epstein-Barr virus reactivation.

A semi-quantitative determination of Epstein-Barr virus (EBV) viremia has been devised. Peripheral blood mononuclear cells are recovered by Ficoll gradient and numerated. Five microl aliquots of recovered cell suspension and 5 microl of two standard dilutions (containing 500 and 100 cells, respectively) are subjected to a nested polymerase chain reaction (PCR). This technique has been evaluated over 3 years for the follow-up of 45 patients attending the Bone Marrow Transplantation Unit of the "Centre Hospitalier et Universitaire de Nancy". EBV reactivation was diagnosed in 13 patients (28%). Positivity of PCR for 100 cells was found in 9 patients of whom 6 developed lymphoma or lymphoproliferative disorder. This technique is easy to perform and doesn't necessitate any specific material besides the one necessary for routine genic amplification.

Hepatitis B virus X gene variability in French-born patients with chronic hepatitis and hepatocellular carcinoma.

The polymorphism of the hepatitis B virus (HBV) X gene from patients born in Lorraine has been studied in serum samples from 22 HBV infected patients, 14 presenting with chronic hepatitis and 8 with hepatocellular carcinoma (HCC). Subtypes adw and ayw represented 21 of the 22 sequenced isolates. The sequence of the X gene of HBV strains from these patients differed from the ones of Far East origin by A to T(1678) and G to A(1759) changes for subtype ayw and C to T(1792) for adw. The expression of the preC region, as indicated by the detection of HBe antigen (HBeAg), was not observed in 11 patients. In 6 patients (3 HCC and 3 non HCC), the absence of HBeAg could be related to a stop codon at position 28. For the 5 remaining patients, the precore stop mutation at codon 28 was not evidenced but 3 out these 5 patients had mutations at nt 1764 and nt 1766 in the promoter of the preC/C gene. These two mutations were also observed in 2 patients with HBeAg, indicating that they are not implicated in the suppression of expression of this gene. Independently of the serotype, two main differences were noted between aminoacid (aa) sequences of chronic hepatitis and HCC related strains: first, twice as many aa changes were found in HCC patients than in chronic hepatitis B carriers (mean of aa changes per patient 4.1 vs. 2.0) and second, we found apparition of polar aa in HCC patients. Most mutations already described in patients from the Far East with HCC have been found in strains of patients from Lorraine. The changes K130M and V131I, considered as "hot spot mutations," were found in strains of HCC patients carrying an ayw subtype of the HBV genome but not in the ones of chronically infected patients. In contrast, strains of the adw subtype had these two changes in the two groups of patients. However when considering the 22 sequenced genes, these hot spot mutations were associated with HCC (P = 0.034).

Genotyping of adenoviruses isolated in an outbreak in a bone marrow transplant unit shows that diverse strains are involved.

In the Bone Marrow Transplant Unit of the "Centre Hospitalier Universitaire" in Nancy, from October 1995 to May 1996, 13 patients of 65 (20%) had a positive adenovirus (Ad) culture after bone marrow transplant. This unusually high rate raised fears of nosocomial spread and so isolates were serotyped. Fourteen Ad strains were isolated from the 13 patients, nine were of serotype 1, 2 or 3, and 5 were non-typable. These five latter strains were responsible for four cases of severe infection with fatal outcome within a two-month period. They were further submitted to restriction fragment length polymorphism analysis of their DNA which showed the isolates differed by a percentage similarity of 8-79%. In this outbreak, different strains were involved, and there was no evidence of a nosocomial origin of Ad infection.

Multiplex PCR for rapid and differential diagnosis of Mycoplasma pneumoniae and Chlamydia pneumoniae in respiratory infections.

A duplex polymerase chain reaction (PCR) was developed for the simultaneous detection of Chlamydia pneumoniae and Mycoplasma pneumoniae. A study of 163 respiratory specimens from in-patients of the "Centre Hospitalier et Universitaire de Nancy" showed the good sensitivity of this duplex PCR allowing the detection of C. pneumoniae and M. pneumoniae from 8 and 13 patients, respectively, whereas the culture was negative for C. pneumoniae for all the samples and positive for M. pneumoniae only in 9 cases. The value of these results has been confirmed by running on the same samples specific nested PCRs for these two microorganisms that gave the same results. Thus, the proposed duplex amplification technique may facilitate the diagnosis of infection by these two agents that are difficult to isolate.

Molecular epidemiology of ocular isolates of adenovirus 8 obtained over nine years.

Twenty nine strains of adenovirus 8 have been isolated over nine years in Strasbourg, France, 22 of which were from one private ophthalmologist. To assess a possible relation between these strains, the DNA of adenovirus was analysed by restriction fragment length polymorphism using eight different enzymes. Among these, three proved discriminant (Xba I, Bgl II, Eco RI) and made it possible to define 13 genotypes differing from each other by one to three DNA bands. Seven genotypes were unique isolates, while three, representing 16 strains, were isolated over five to eight years. All the genotypes but one were closely related, with 87% homology. All 13 differed from an adenovirus 8 strain from Lyon (homology 68-76%). This study confirmed the stability of adenovirus 8 in a given population.