Serum levels of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases-1 in human herpesvirus-6-infected infants with or without febrile seizures.

Human herpesvirus-6 (HHV-6) is a cause of exanthema subitum and, sometimes, of febrile seizures. However, the pathogenesis of febrile seizures associated with HHV-6 infection remains unclear. We investigated serum matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) levels in infants with HHV-6 infection. Serum levels of both MMP-9 and TIMP-1 were significantly higher in infants with HHV-6 infection than in controls. Serum TIMP-1 levels were significantly higher in infants with febrile seizures than in infants without febrile seizures. Serum MMP-9/TIMP-1 ratios were significantly lower in infants with febrile seizures than in infants without febrile seizures. In infants with HHV-6 infection, positive correlations were found between serum MMP-9 concentrations and the white blood cells (WBC) count, and between serum TIMP-1 concentrations and the WBC count. Positive correlations were also found between the amounts of HHV-6 DNA and the ratios of MMP-9/TIMP-1 in infants with HHV-6 infection. In conclusion, we suggest that high serum levels of MMP-9 and TIMP-1 in infants with HHV-6 infection may induce dysfunction of the blood-brain barrier, eventually causing febrile seizures.

Development of a human herpesvirus 6 species-specific immunoblotting assay.

In order to assess the full spectrum of human herpesvirus 6A (HHV-6A)- and HHV-6B-associated diseases, we sought to develop an HHV-6 species-specific serological assay based on immunoblot analysis. The immunodominant proteins encoded by open reading frame U11, p100 for HHV-6A (strain U1102) and 101K for HHV-6B (strain Z29), were selected to generate virus species-specific antigens. Recombinant p100 and 101K were produced in a prokaryotic expression system. The expression of these proteins was confirmed by using anti-His tag and 101K-specific monoclonal antibodies. HHV-6 species-specific antibodies were detected by immunoblotting in patient sera. Eighty-seven serum samples obtained from various subjects were utilized to determine the reliability of the method for clinical use. Ten of twelve exanthem subitum convalescent-phase sera reacted exclusively with 101K, whereas none of twelve acute-phase sera reacted with either protein. Two of three sera collected from HHV-6A-infected patients reacted with p100 and 101K. Although all five acute and convalescent-phase sera obtained from transplant recipients reacted exclusively with 101K, two of six convalescent-phase sera obtained from patients with drug-induced hypersensitivity syndrome reacted with both p100 and 101K. Of 38 sera obtained from healthy adults, 31 were positive for 101K antibody, while 4 reacted with both proteins. However, PCR analysis of peripheral blood mononuclear cells and saliva from these subjects did not detect HHV-6A DNA. In conclusion, this novel serological assay based on immunoblot analysis using recombinant HHV-6A p100 and HHV-6B 101K allowed us to discriminate between HHV-6A- and HHV-6B-specific antibodies.

Human herpes virus type 6 can cause skin lesions at the BCG inoculation site similar to Kawasaki Disease.

Kawasaki Disease (KD) is acute, febrile, multisystem vasculitis of early childhood, the detailed mechanism of which is still unclear. Skin symptoms occur in KD, such as edema of the hands and feet with subsequent desquamation and redness at the inoculation site of bacillus Calmette-Guerin (BCG). The change at the BCG inoculation site has been considered as a specific feature of KD, although its mechanism is not fully understood. We present an 11-month-old boy who developed fever with redness of the BCG site due to infection with human herpes virus type 6 (HHV6). At the age of 3 months, the patient received BCG. His fever remitted 7 days after the onset of skin redness, with sequential desquamation at the BCG site and extremities, which is not a common feature of HHV6 infection that typically lasts for 3 days. The final diagnosis was exanthema subitum. Characteristically, the HHV6 infection in our patient appeared to be associated with the invigoration of the T cell system, as represented by the elevated serum levels of soluble interleukin-2 receptor (3,490 U/ml vs. normal range 145-519 U/ml). This patient clearly showed redness and crusting at the BCG inoculation site, suggesting that HHV6 infection might cause skin changes similar to those of KD via an unknown mechanism. In addition, we suggest that the activation of the T cell system may account for the skin lesions in KD, characterized by redness and subsequent crusting of the BCG inoculation site and desquamation of the extremities.

Direct detection of human herpesvirus 6 DNA in serum by the loop-mediated isothermal amplification method.

BACKGROUND: A more rapid and easier method is needed for monitoring human herpesvirus 6 (HHV-6) infections. The loop-mediated isothermal amplification method (LAMP) can detect viral DNA with high specificity, efficiency, and speed under isothermal conditions. LAMP requires only simple equipment that is available in hospital laboratories. OBJECTIVES: We evaluated LAMP as a means of detecting HHV-6 DNA directly from patients' sera. RESULTS: The sensitivity of the HHV-6 LAMP protocol without heat denaturation was 1000 copies/tube; with heat denaturation 10 copies/tube were detected. Three hundred serum samples from children with fever were analyzed. Using HHV-6 isolation as a definition of HHV-6 infection, the sensitivity, specificity, positive predictive value, and negative predictive value of the HHV-6 LAMP method without DNA extraction were 95.5%, 95.2%, 94.0%, and 96.4%, respectively. CONCLUSION: Direct detection of HHV-6 DNA in serum with a modified HHV-6 LAMP could be used for rapid diagnosis of exanthem subitum (ES).

Prokaryotic expression of an immediate-early gene of human herpesvirus 6 and analysis of its viral antigen expression in human cells.

Segments of an immediate-early (1E) protein (1E03; 958 amino acids (aa)), encoded by clone pSTY03, of human herpesvirus 6 (HHV-6) variant B strain HST were expressed as beta-galactosidase fusion proteins in Escherichia coli. Using Western blot analysis, and the serum of a patient having high titer anti-HHV-6 antibodies, an antigenic region of the IE03 protein was mapped between residues 340 and 505 (pUE03IE-M). The fusion protein expressed in E. coli harboring plasmid pUE03IE-M was purified after electrophoresis in SDS-PAGE, and then immunized in mice to obtain a monospecific antibody. Monospecific antibody raised against the fusion protein reacted with IE03 protein species with apparent molecular weights of 155 and 170 kDa, and was detected as granular fluorescence in nuclei of infected cells by an immunofluorescence antibody test. Furthermore, this antibody reacted only with HHV-6 variant B, but did not react with HHV-6 variant A. The IE03 protein was confirmed to be an IE protein, since the synthesis of this protein was observed in infected cells that were first treated with cycloheximide, which was then replaced with actinomycin D. Further, it was also detected as early as 4 h after infection.

Transfer of human herpesvirus 6 and 7 antibodies from mothers to their offspring.

Placental transfer of maternal human herpesvirus (HHV) 6 and HHV 7 antibodies to infants was examined simultaneously in 69 paired plasma samples by an indirect immunofluorescence assay. All the mothers had antibodies to both viruses. The mean HHV 6 and HHV 7 antibody titers of infants were significantly higher than those of the mothers. The mean ratio of cord blood antibody titer to the maternal titer for both viruses was 1.89, suggesting active transport by placenta.

Frequent seizures with elevated interleukin-6 at the eruptive stage of exanthema subitum.

A 15-month-old girl developed frequent seizures at the eruptive stage of exanthema subitum. The eruption persisted for 2 weeks. Serum immunoglobulin G antibody to human herpes virus type 6 (HHV-6) increased markedly. Interleukin-6 was elevated whereas HHV-6 deoxyribonucleic acid was not detected in cerebrospinal fluid. These findings suggest that immune-mediated reactions after HHV-6 infection rather than direct action of active HHV-6 are responsible for frequent seizures in this case.

Isolation and identification of human herpesvirus 7 from an infant with exanthem subitum.

Exanthem subitum (ES) is a common childhood exanthematous disease. In a recent study of ES due to human herpesvirus 6 (HHV 6), we isolated human herpesvirus 7 (HHV 7) from the peripheral blood mononuclear cells (PBMC) of a seven month-old infant with typical symptoms of ES. The identity of the virus was confirmed by indirect immunofluorescence using HHV 7 specific monoclonal antibody and by amplification of the HHV 7 specific genomic sequences using the polymerase chain reaction (PCR). Paired serum samples from the infant showed serological conversion to the isolated virus. The clinical manifestations of ES in this infant appeared to be milder than the classical ES due to HHV 6.

Uvulo-palatoglossal junctional ulcers--an early clinical sign of exanthem subitum due to human herpesvirus 6.

A provisional clinical diagnosis of exanthem subitum was made in six febrile infants seen in the Paediatric Unit of Assunta Hospital, Petaling Jaya, Malaysia with uvulo-palatoglossal junctional ulcers prior to the eruption of maculopapular rash. On follow-up, all six infants developed maculopapular rash with the subsidence of fever at the end of the fourth febrile day. Human herpesvirus 6 was isolated from the peripheral blood mononuclear cells during the acute phase of the illness and HHV 6 specific genome was also detected in these cells by nested polymerase chain reaction. All the six infants showed seroconversion for both specific IgG and IgM to the isolated virus. This study suggests that the presence of uvulo-palatoglossal junctional ulcers could be a useful early clinical sign of exanthem subitum due to human herpesvirus 6.

[Leucocyte number, differential count and sedimentation rate in 9 "classic" childhood infections. (author's transl)]. / Leukozytenzahl, Differentialblutbild und Blutsenkung bei 9 "klassischen" Infektionskrankheiten.

A retrospective analysis of absolute numbers in 802 white blood counts and 396 sedimentation rates of 407 children, admitted between 1973-78, with 9 "classic" infections was done and evaluated for diagnostic usefulness. As diagnostic meaningful it was found: Lymphocytosis in pertussis; lymphocytopenia and slight increased sedimentation rate in measles; nothing particular in mumps; slight increased sedimentation rate in chicken pox; increase in mononuclear cells, particularly atypical lymphocytes and sedimentation rate in infectious mononucleosis; leucocytopenia caused by neutrocytopenia and lymphocytopenia in exanthema subitum (roseola infantum); increased sedimentation rate in scarlet fever; lymphocytopenia and a high sedimentation rate in mycoplasma-pneumonia; leucocytopenia with lymphocytopenia in rubella.

Detection by polymerase chain reaction amplification of human herpesvirus 6 DNA in peripheral blood of patients with exanthem subitum.

The polymerase chain reaction was used to detect human herpesvirus 6 (HHV-6) DNA in peripheral blood mononuclear cells from patients with exanthem subitum. Amplified products were detected by agarose gel electrophoresis and dot blot hybridization with a cloned DNA probe. No cross-hybridization with DNAs of five other human herpesviruses was observed in this system, and all HHV-6 strains gave positive reactions when the primer pairs were used. Immunoglobulin M antibody appeared about 5 days after the onset of disease, reaching a maximum after about 2 to 3 weeks and then decreasing to less than 1:10 1 month after the onset of disease in almost all patients. Mononuclear cells from seven patients with exanthem subitum all gave positive reactions in this system, and viral DNA was found in samples even during the convalescent phase of the disease. We conclude that this test, using polymerase chain reaction amplification, which takes only 1 to 2 days, is useful for the diagnosis of HHV-6 infection.

Human herpesvirus 6 (HHV-6) infection and exanthem subitum in Thailand.

Of 50 patients in Thailand suspected clinically of having exanthem, subitum, 31 (62%) were serodiagnosed as HHV-6 infection. Sixteen strains of HHV-6 from 31 patients (52%) whose antibody titers had converted during convalescence were isolated during the acute phase. The disease occurred in infants from 3 months to 1 year of age and most frequently at age 4-6 months. Antibody only to HHV-6 converted in 23 of 50 patients (46%), and seroconversion to HHV-6 and dengue virus was observed in 7 patients (14%), and to HHV-6 and Coxsackie B virus in 1 case (2%). In the 23 patients in whom seroconversion only to HHV-6 was observed, all had fever and rash which appeared after subsidence of the fever. Lymphadenopathy and relative lymphocytosis were recognized, associated with diarrhea, vomiting, running nose, cough and hepatomegaly. Febrile convulsions were seen in some cases. All patients recovered completely within a week.

[The blood picture in exanthema subitum (Zahorsky)/ critical 3-day fever-exanthema in young children]. / Das Blutbild beim Exanthema subitum (Zahorsky)/kritisches Dreitagefieber-Exanthem der Kleinkinder.

Exanthema subitum was described in 1910 by John Zahorsky/USA; in 1986 and 1988 the human herpesvirus 6 (HHV 6) was discovered as the causative agent of the disease and serologic tests were established for diagnostics (specific IgM and IgG antibodies). Up to this time the diagnosis was based on the typical clinical course: the prodromal febrile stage (3 days) followed by the onset of a (more or less characteristic) rash closely connected with the normalisation of the body temperature. Usually a typical white blood cell count was described for diagnostics on the first day of exanthema: leukocytopenia with eosino- and granulocytopenia associated with consequent lymphocytosis. We analysed the hematologic data for children with a serologically documented HHV6 infection including exanthema (group 1: n = 9), without exanthema (group 2: n = 11) or with a serologically unexplained febrile rash (group 3: n = 13). In children with exanthematous HHV6 infection (exanthema subitum) granulocytopenia and a decreased thrombocyte count (mean values) is the rule. But the total white blood cell count and the mean values for eosinophils did not differ between the groups studied.

Detection of human herpesvirus 6 DNAs in samples from several body sites of patients with exanthem subitum and their mothers by polymerase chain reaction assay.

Polymerase chain reaction amplification was used to detect human herpesvirus 6 (HHV-6) DNAs in peripheral blood mononuclear cells (MNCs), plasma, saliva, stool, and urine from three patients with exanthem subitum and in peripheral blood MNCs, plasma, and saliva from their mothers. HHV-6 DNAs were detected in MNCs during and after the disease and were found in plasma only in the acute phase. The virus DNAs were also detected in saliva after recovery from the illness and were found persistently or intermittently in stool but not in urine samples after the onset of the disease. In contrast, one of the three mothers excreted HHV-6 DNAs persistently in saliva. None of the mothers had the virus DNAs in peripheral blood MNCs and plasma nor a significant increase in antibody titers to HHV-6 after possible exposure from their children. These findings suggest systemic replication of HHV-6 during the acute phase in patients with exanthem subitum and persistent infection of the virus in several organs after recovery from the disease.

Seroconversion to human herpesvirus 6 and human herpesvirus 7 among Brazilian children with clinical diagnoses of measles or rubella.

We collected acute-phase and convalescent-phase serum samples from Brazilian patients who presented with exanthem of unknown origin and evaluated these samples by means of an immunoblot assay for seroconversion to human herpesvirus 6 (HIV-6) or human herpesvirus 7 (HIV-7). Measles or rubella had been clinically diagnosed in all these patients, but their sera were negative for antibodies to both measles virus and rubella virus. Twenty percent of the patients clearly seroconverted to HHV-6 after manifestation of the exanthem, and 8% seroconverted to HHV-7. All seroconversions to HHV-6 occurred in children aged < or = 5 years; a 41% frequency of seroconversion to HHV-6 was noted among children between 3 months and 23 months of age, whereas seroconversions to HHV-7 were detected during infancy and through adulthood. Our data indicate that primary infections due to HHV-6 or HHV-7 can be misdiagnosed as measles or rubella.

[Comparison of serological procedures used for the diagnosis of viral exanthema in laboratories participating in the measles elimination plan]. / Comparación de los procedimientos serológicos de los laboratorios del Plan para la Eliminación del Sarampión en el diagnóstico de exantemas víricos.

OBJECTIVE: The comparison of serological methods used by the laboratories participating in the Network for the Elimination of Measles to diagnose measles virus infection as well as differential diagnosis with other exanthematic diseases are compared. MATERIALS AND METHODS: One panel of 20 serum samples including measles (12), rubella (4), parvovirus B19 (2) and dengue (2) infections was established. All cases were diagnosed by detection of specific IgM. The panel was sent to the laboratories of the Network. The results were compared with those obtained at the reference laboratory. RESULTS AND DISCUSSION: Regarding measles, IgM response from 20 laboratories (19 by ELISA and 1 by indirect immunofluorescence) was obtained, with an agreement of 91.5%. Related to rubella IgM, replay from 6 laboratories, using ELISA, was received, with an agreement of 98.7%. With respect to parvovirus B19 IgM, response from 10 laboratories (8 by ELISA and 2 by indirect immunofluorescence) was obtained, with an agreement of 94.6%. Results about dengue virus were not reported by any laboratory. CONCLUSION: Some laboratories from the network should review the methods used for the diagnosis of measles and other exanthematic diseases. The results reassert the need for a reference laboratory to support confirmation of the results.