Persistent detection of varicella-zoster virus DNA in a previously healthy child after severe chickenpox.

In immunocompetent children with primary varicella-zoster virus (VZV) infection, peak viral loads are detected in peripheral blood near the onset of the vesicular rash. VZV DNA concentrations normally diminish and become undetectable within 3 weeks after the appearance of the exanthem. Here, we present a previously healthy, human immunodeficiency virus-negative, 4-year-old boy admitted with severe varicella. High viral loads (>340,000 copies/ml) were found in his blood, and the viral loads remained high for at least 1.5 years. Clinical recovery preceded complete clearance of the virus. General and VZV-specific immune reactivity were intact. NK cells and CD8(+) T cells were activated during acute infection, and VZV-specific CD4(+) T cells were detected at high frequencies. VZV DNA was initially detected in B cells, NK cells, and both CD4(+) and CD8(+) T cells. In contrast, during the persistent phase of VZV DNA detection, the viral DNA was primarily located in CD8(+) T cells. For the first time, we describe the persistent detection of VZV DNA in a previously healthy child.

Cytomegalovirus rather than HIV triggers the outgrowth of effector CD8+CD45RA+CD27- T cells in HIV-1-infected children.

OBJECTIVE: To analyse the effect of viral coinfections on immune reconstitution in HIV-1-infected children (< 18 years) taking highly active antiretroviral therapy (HAART). METHODS: Absolute lymphocyte numbers of various subsets of CD8 T cells were measured. RESULTS: Prior cytomegalovirus (CMV) infection correlated with an increased number of CD8 effector T cells (i.e., CD45RA+CD27-) at baseline (CMV-seropositive versus CMV-seronegative patients; P = 0.009), as well as an increased state of T cell activation as defined by HLA-DR and CD38 expression. The expansion of effector CD8 T cells persisted over time, independent of the HIV response to HAART. Numbers of CD8 effector T cells were significantly higher in patients with CMV replication as reflected by persistent urinary CMV shedding and periodic CMV DNAaemia (P = 0.02). These patients also showed an increase in CMV-specific antibodies compared with those without CMV shedding (P = 0.007). The number of CMV-specific interferon-gamma (IFN-gamma)-producing CD8 T cells was lower in children who persistently shed CMV compared with those who did not (P = 0.02). In contrast, CMV-specific CD4 T cell responses were detected at similar levels in both groups. CONCLUSIONS: In HIV-1-infected children, CMV infection correlated with the outgrowth of CD8+CD45RA+CD27- effector T cells. Activation of the immune system by persistent CMV secretion resulted in increasing CMV-specific IgG and higher numbers of CD8 effector T cells. Despite these increases, the CMV-specific IFN-gamma-producing CD8 T cell response was diminished, which could explain the inability to suppress CMV completely in 41% of HIV-1-infected children.

Survey of acyclovir-resistant herpes simplex virus in the Netherlands: prevalence and characterization.

BACKGROUND: Widespread and frequent use of acyclovir (ACV) for treatment, suppressive therapy and prophylaxis of herpes simplex virus (HSV) infections and its over the counter availability may be associated with emergence of HSV resistance. OBJECTIVES: To determine the prevalence of ACV-resistant HSV isolates in different patient groups between 1999 and 2002 in the Netherlands. STUDY DESIGN: A total of 542 isolates, 410 HSV-1 and 132 HSV-2, from 496 patients were screened for reduced susceptibility to ACV. A newly developed ELVIRA HSV screening assay was used that allowed a high throughput screening. The genotypic analysis of the HSV thymidine kinase gene was performed to identify resistance-associated mutations. RESULTS: Thirteen isolates, 8 HSV-1 and 5 HSV-2, from 10 patients (2%) were found resistant to ACV. A single ACV-resistant strain was identified among isolates from 368 immunocompetent patients (0.27%; 95% confidence interval [CI], 0.007%-1.5%), whereas in nine isolates from 128 immunocompromised patients resistant HSV was identified (7%; 95% CI, 3.26%-12.93%). The highest frequency of ACV-resistant HSV was associated with bone marrow transplantation: four patients out of 28 (14.3%) shed resistant virus. In addition, resistant virus was obtained from two HIV-positive patients, one patient with a hematological malignancy and two patients on immunosuppressive drugs. Further testing showed that none of the isolates was resistant to foscarnet. Several new mutations were identified in the thymidine kinase gene of these resistant isolates, and their effect on ACV-resistance is discussed. CONCLUSIONS: Our study shows that the prevalence of ACV resistance is low in immunocompetent patients (0.27%), whereas ACV-resistant HSV infections occur relatively frequently in immunocompromised patients (7%; P < 0.0001). This emphasizes the need for drug susceptibility monitoring of HSV infections in immunocompromised patients with persisting infections despite antiviral therapy.

Emergence of a CD4+CD28- granzyme B+, cytomegalovirus-specific T cell subset after recovery of primary cytomegalovirus infection.

Cytotoxic CD4(+)CD28(-) T cells form a rare subset in human peripheral blood. The presence of CD4(+)CD28(-) cells has been associated with chronic viral infections, but how these particular cells are generated is unknown. In this study, we show that in primary CMV infections, CD4(+)CD28(-) T cells emerge just after cessation of the viral load, indicating that infection with CMV triggers the formation of CD4(+)CD28(-) T cells. In line with this, we found these cells only in CMV-infected persons. CD4(+)CD28(-) cells had an Ag-primed phenotype and expressed the cytolytic molecules granzyme B and perforin. Importantly, CD4(+)CD28(-) cells were to a large extent CMV-specific because proliferation was only induced by CMV-Ag, but not by recall Ags such as purified protein derivative or tetanus toxoid. CD4(+)CD28(-) cells only produced IFN-gamma after stimulation with CMV-Ag, whereas CD4(+)CD28(+) cells also produced IFN-gamma in response to varicella-zoster virus and purified protein derivative. Thus, CD4(+)CD28(-) T cells emerge as a consequence of CMV infection.

Cross-reactivity of cytomegalovirus-specific CD8+ T cells to allo-major histocompatibility complex class I molecules.

BACKGROUND: In transplantation settings, cytomegalovirus (CMV) infection is a common complication. CMV infection is associated with a higher incidence of graft rejection in solid organ transplantation and graft-versus-host disease in bone marrow transplantation. The underlying mechanism of this association could be the generation of CMV-specific CD8 T cells capable of cross-reacting with alloantigens present on graft and host, respectively. METHODS: Whereas as to date, no direct ex vivo analysis can be performed of the CD8 T-cell repertoire directed at allo-major histocompatibility complex (MHC) class I molecules, virus-specific cells can be readily enumerated by use of MHC-peptide tetrameric complexes. In this study, the authors used this technique to analyze potential overlapping CD8 T-cell repertoires between self-MHC-viral peptide and allo-MHC complexes by stimulating CMV-specific CD8 T cells with alloantigens. RESULTS.: The authors found that CMV-specific CD8 T cells are activated and proliferate on stimulation with alloantigens. CONCLUSIONS: Although these cells are cytotoxic against CMV-peptide pulsed target cells, no cytotoxicity of CMV-specific cells to alloantigens could be detected, inferring that there are other mechanisms of graft damage by alloantigen-stimulated virus-specific CTL.

The size and phenotype of virus-specific T cell populations is determined by repetitive antigenic stimulation and environmental cytokines.

Based on the expression of the TNFR SFP CD27, two Ag-primed CD8(+) T cell subsets can be discerned in the circulation of healthy individuals: CD27(+) T cells that produce a variety of cytokines but do not display immediate cytolytic activity; and cytotoxic CD27(-) T cells, which secrete only IFN-gamma and TNF-alpha. The mechanism that controls the generation of these different phenotypes is unknown. We show that CMV reactivation not only increases the number of virus-specific T cells but also induces their transition from a CD27(+) to a CD27(-) phenotype. In support of a relation between pool size and phenotype in a cohort of latently infected individuals, the number of Ag-specific CD27(-) CD8(+) T cells was found to be linearly related to the total number of CMV-specific CD8(+) T cells. In vitro studies revealed that the acquisition of the CD27(-) phenotype on CMV-specific T cells depended on the interaction of CD27 with its cellular ligand, CD70. Expression of CD70 was proportional to the amount of antigenic stimulation and blocked by the CD4(+) T cell-derived cytokine IL-21. Thus, induction of CD70, which may vary in distinct viral infections, appears to be a key factor in determining the size and phenotype of the CMV-specific T cell population in latently infected individuals.

Identification of a new human coronavirus.

Three human coronaviruses are known to exist: human coronavirus 229E (HCoV-229E), HCoV-OC43 and severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV). Here we report the identification of a fourth human coronavirus, HCoV-NL63, using a new method of virus discovery. The virus was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis. The complete genome sequence indicates that this virus is not a recombinant, but rather a new group 1 coronavirus. The in vitro host cell range of HCoV-NL63 is notable because it replicates on tertiary monkey kidney cells and the monkey kidney LLC-MK2 cell line. The viral genome contains distinctive features, including a unique N-terminal fragment within the spike protein. Screening of clinical specimens from individuals suffering from respiratory illness identified seven additional HCoV-NL63-infected individuals, indicating that the virus was widely spread within the human population.

Epidemiology of hepatitis B infection among expatriates in Nigeria.

Adult expatriates in countries where hepatitis B virus (HBV) is highly endemic have an increased risk of HBV infection, but little is known about risks to their children or about patterns of spread. The epidemiology of HBV infection was studied among 124 unvaccinated Dutch missionaries and family members who lived in a rural area of Nigeria. Antibodies to hepatitis B core antigen were found in 5 (9.8%) of 51 adults (incidence rate, 1.7 per 1000 person-months at risk [PMAR]) and 9 (12.3%) of 73 children (incidence rate, 2.8 per 1000 PMAR). Vertical transmission of HBV was a likely source of infection in 1 child and was a possible source of infection in 2 others. The prevalence of HBV infection showed strong family clustering (P<.0001), was associated with a history of temporary adoption of Nigerian children (P=.004), and increased with both the number of adoptive children (P=.009) and the total time that these children had stayed in the family (P=.036). Horizontal transmission from adoptive Nigerian children probably played an important role in the spread of HBV infection in this expatriate community.

Spontaneous outgrowth of EBV-transformed B-cells reflects EBV-specific immunity in vivo; a useful tool in the follow-up of EBV-driven immunoproliferative disorders in allograft recipients.

During immunosuppressive medication, Epstein-Barr virus (EBV) infection is associated with a risk of developing posttransplant lymphoproliferative disease (PTLD). The appropriateness of a spontaneous EBV B-cell transformation (SET) assay as a monitor of EBV-specific immunity was evaluated to investigate if it safely allows reducing immunosuppressive medication, thereby decreasing the risk of developing PTLD. PBMC were isolated longitudinally from 20 pediatric renal allograft recipients treated with prednisone and cyclosporine combined with either azathioprine or mycophenolate mofetil. Most significantly, EBV-peptide-specific CD8+ T cells were detectable in the blood of patients with negative SET assays, coinciding with significantly lower EBV loads, whereas these cells were less frequent in the blood of patients with positive SET assays. Reducing the levels of immunosuppression resulted in normalization of the SET assays. Therefore, the SET assay is a reflection of the interaction between viral replication, transformation of B cells, and EBV-specific immunity in vivo and hence a valuable screening test for EBV-driven lymphoproliferative phenomena in allograft recipients.

A woman with a lobar infiltrate due to psittacosis detected by polymerase chain reaction.

This report presents a case of community-acquired pneumonia due to Chlamydia psittaci presenting with a lobar infiltrate and diagnosed by a newly developed ompA gene-based polymerase chain reaction (PCR). This gene encodes a specific C. psittaci major outer membrane protein. This kind of PCR could reduce antibiotic consumption and expedite outbreak management.

Frequencies of circulating cytolytic, CD45RA+CD27-, CD8+ T lymphocytes depend on infection with CMV.

Viral infections may cause serious disease unless the adaptive immune system is able to clear the viral agents through its effector arms. Recent identification and functional characterization of subpopulations of human CD8(+) T cells has set the stage to study the correlation between the appearance of particular subsets and common viral infections during childhood, i.e., EBV, CMV, varicella-zoster virus (VZV), and the attenuated measles-mumps-rubella (MMR) vaccine strains. In a cohort of 220 healthy children we analyzed lymphocytes and subpopulations of CD4(+) and CD8(+) T cells. The presence of the cytolytic CD45RA(+)CD27(-) subset of CD8(+) T cells correlated with prior CMV infection as defined by seroconversion (p < 0.0001). The number of this CD8(+) T cell subset remained stable during follow-up over 3 years in 40 children. The CD45RA(+)CD27(-) subset of CD8(+) T cells first appeared during acute CMV infection and subsequently stabilized at an individual set-point defined by age and immunocompetence. The functional importance of these cells in CMV surveillance was reflected by their increased numbers in immunosuppressed pediatric kidney transplant patients. Preferential expansion of CD8(+)CD45RA(+)CD27(-) cytolytic T cells seems unique for CMV.

Human cytomegalovirus DNA in plasma and serum specimens of renal transplant recipients is highly fragmented.

Quantitation of cytomegalovirus (CMV) DNA in plasma and serum by PCR is increasingly used to identify patients at risk for developing CMV disease and to monitor the efficacy of antiviral therapy. Although CMV DNA levels are generally interpreted as viral loads, the exact nature of the viral DNA in these specimens is unknown. We studied the state of CMV DNA in plasma and serum specimens obtained from three renal transplant recipients at peak viral DNA levels during primary CMV infection. For this purpose, DNA isolated from these specimens was fractionated by size, and CMV DNA levels in the resulting DNA fractions were measured by quantitative PCR targeted at large (578-bp) and small (134-bp) amplicons. These experiments showed that the molecular sizes of DNA fragments from which CMV DNA is amplified were small (<2,000 bp), indicating that CMV DNA in plasma and serum is highly fragmented. Furthermore, CMV DNA levels were consistently higher when targeted at the smaller amplicon, providing additional evidence for the fragmentation of viral DNA. In conclusion, the first results with three patients have shown that CMV DNA in plasma and serum is highly fragmented and does not necessarily reflect the amount of infectious virus. These observations have potential consequences for understanding CMV pathogenesis and interpreting CMV DNA levels in individual patient management.

Incidence and risk factors of probable dengue virus infection among Dutch travellers to Asia.

We studied the incidence of dengue virus (DEN) infections in a cohort of Dutch short-term travellers to endemic areas in Asia during 1991-92. Sera were collected before and after travel. All post-travel sera were tested for DEN immunoglobulin M (IgM) [IgM capture (MAC)-enzyme-linked immunosorbent assay (ELISA)] and IgG (indirect ELISA). Probable DEN infection was defined as IgM seroconversion or a fourfold rise in IgG ratio in the absence of cross-reaction with antibody to Japanese encephalitis virus (JEV). Infections were considered clinically apparent in case of febrile illness (> 24 H) with headache, myalgia, arthralgia or rash. Probable DEN infection was found in 13 of 447 travellers (incidence rate 30/1000 person-months, 95% CI 17.4-51.6). One infection was considered secondary; no haemorrhagic fever occurred. The clinical-to-subclinical infection rate was 1 : 3.3. The risk of infection showed marked seasonal variation. DEN infections are frequent in travellers to endemic areas in Asia; most remain subclinical.