A total of 323 patients with lymphadenopathy were selected in Belem, Brazil, between January 1996 and December 2001, and screened for the presence of human herpesvirus-6 (HHV-6) IgM- and- IgG antibodies by enzyme-linked immunosorbent assay (ELISA). When seroprevalence is analyzed by gender, similar rates arc found for female (60.6%) and male (55.7%) individuals. Seventy-seven (23.8%) patients were HHV-6-IgM-and- IgG-positive (IgM+ subgroup), with positivity rates of 29.7% and 17.7% (p = 0.0007) for female- and male individuals, respectively. Sera from a subgroup (n = 120) of these subjects, with high HHV-6 antibody levels (either IgM+ or IgG+ reactivities), were subsequently processed for the presence of HHV-6 DNA by polymerase chain reaction (PCR)/nested PCR. Active infections (IgM+ and/or IgG+ high levels specific antibodies plus detection of viral DNA) were diagnosed in 20/77 (20.0%) and 8/43 (18.6%); subgroup of the 120 individuals suspected of having HHV-6 suggestive recent infection. All (n = 28) cases of active infection were found to be associated with HHV-6 variant-A (HHV-6A), as detectable by PCR/nested PCR, using variant-specific primer that amplify regions of 195 base pairs (bp) (HHV-6A) and 423 bp (HHV-6B). Rates of HHV-6 DNA detection between female and male patients were similar (p > 0.05) in the IgM+ and IgG+ groups: 20.4% versus 35.7% and 25.0% versus 13.0%, respectively. HHV-6 DNA was detected across 50 age-groups at rates that ranged from 15.4% (male,
KEYWORDS: Herpesvirus-6; Variant-A; Lymphadenopathy.
Human herpesvirus=6 (HHV-6) is largely recognised as an ubiquitous virus that infects most persons up to the age of 2 years46. It was first isolated in 1986 by SALAHUDIN et al.50, from patients with lymphoproliferative disorders or acquired immunodeficiency syndrome. HHV-6 composes the Roseolovirus genus of the betaherpesvirus subfamily19,49. Two distinct variants (HHV-6A and HHV-6B) are currently recognised, with different genetic, biological and immunological properties1,19. The HHV-6B appears to be isolated more frequently than HHV-6A from blood, except in patients with AIDS2.
While HHV-6A has been obtained primarily from adults with chronic illnesses1,5, HHV-6B strains have mostly been isolated from infants with exanthem subitum (ES) (roseola infantum, sixth disease) and other paediatric febrile illnesses, including lymphadenopathy18,31,48,52. Since virtually all children become infected early life, primary HHV-6 infections among adults are rare events. Whenever these latter conditions occur, however, clinical findings such as lymphadenopathy38,44,53, heterophile-negative mononucleosis3,45 and hepatitis21 are likely to result. In most of the primary infections by HHV-6 the clinical symptoms are high fever and a rough skin rash, that is, the typical exanthema subitum; convulsions may also be present in those more severe cases. Primary infection in adults is not yet fully characterised, but a variety of clinical conditions have been described, including lymphoproliferative disorders6,7. On the other hand, reactivation may cause severe disease in immunocompromised patients and has also been reported to occur without symptoms in immunocompetent individuals26. The HHV-6A variant was found to be the predominant variant present in the lymph node biopsies from the HIV-infected patients15,34.
In vivo HHV-6 replication has been demonstrated in a variety of cells including lymphocytes, macrophages, histiocytes, and endothelial/ epithelial cells; CD4+ lymphocytes appear to be the main target cells for HHV-69,10,13,43,44. The frequent detection of HHV-6 antigen/DNA in salivary glands22,36, lymph node tissues38, neurons and glial cells of the brain12 suggests that HHV-6 can either persistently infect cells or establish latency with potential subsequent reactivation. It has been reported that saliva is likely to play a major role in horizontal transmission of HHV-6, leading to the widespread of viral infection30. Consequently, serological studies have in general shown that nearly 100% of infants become infected before the age of 3 years8,46. In addition to this, large serosurveys conducted elsewhere, reported HHV-6 seroprevalcnce rates of 80 to 95% in healthy adolescents and adults40,51,59. In Northeastern Brazil, HHV-6 seroprevalence rates of 76.5% - 77.2% were reported by LINHARES et al.41 among Brazilian and Japanese immigrants. Similar surveys were subsequently conducted in the Amazon region of Brazil, yielding prevalence rates that ranged from 5.4% to 14.9% among Amerindians25, and from 75.0% to 100.0% in urban communities23.
This study focussed on cases of lymphadenopathy occurring in Belcm, Para, Brazil for which a diagnosis could not be achieved following a routine screening for those potentially most common causative infectious agents of this clinical condition. Further assays were then extended to HHV-6 and specific results are discussed herein.
MATERIALS AND METHODS
This was a retrospective study in which scrum samples obtained from 323 patients with lymphoproliferative syndromes were examined for HHV-6 infection. These subjects had been referred by local physicians to the Virology section of Institulo Evandro Chagas, Belem, Brazil, during the period of January 1996 to December 2001 for serological diagnosis. Patients enrolled in this study (n = 323) were selected from a total of 4,154 subjects with fever and lymphadenopathies who tested negative for the following agents: rubella, cytomegalovirus, Epstein-Barr virus, Toxoplasma gondii, and Brucella spp. All patients presented with an acute disease of moderate severity, mainly characterised by fever and lymphadenopathy. We did not include those patients with skin rash, as well as those lymphoproliferative disorders affecting immuno-suppressed individuals. Study subjects (of whom 165 were female) were aged between 6 months and 78 years (mean, 19 years) at the time of blood sampling. Acute-phase blood samples were taken at presentation to the Institute, and sera were kept frozen at -20 [degrees]C until they were processed.
The detection of IgM and IgG antibodies to HHV-6 was performed using a commercial enzyme-linked immunosorbent assay (ELISA) developed by PANBIO(TM) (East Brisbane, Australia). This is a system that includes solid-phase wells coated with HHV-6-infected cells, essentially as described elsewhere4,16,47. For the determination of HHV-6 immunoglobulin M (IgM) sera were tested at single 1:100 (v/v) dilutions, with previous removal of IgG. This eliminates the interference phenomenon if rheumatoid factor is present in serum. All samples yielding optical density (OD) values which are at least twice the mean absorbance of the "cut off were regarded as suggestive of recent HHV-6 infection; this corresponds to > 20 IgM PANBIO units. A subgroup (n = 120) of subjects among study patients with HHV-6 IgM+ (> 20 PANBIO units for IgM) or IgG+ (> or = 50 PANBIO units for IgG) results were selected for HHV-6 DNA detection using a two-step polymerase chain reaction (PCR), as reported before35,53. First amplification was carried out using a mixture of external oligonucleotides primers designated as EX1 and EX2, followed by a second amplification cycle (the nested PCR) that includes a mixture of internal primers IN3 and IN4. A mixture including 1.5 mM MgCl^sub 2,^ 0.5 [mu]M of each primer, 2.5 units of Taq DNA polymerase (GIBCO-BRL) in the final concentration. Temperatures were used as follows: one initial cycle at 94 [degrees]C for 5 min, 40 cycles of denaturalization at 94 [degrees]C for one min, annealing at 55 [degrees]C for one min, and extension at 72 [degrees]C for 1.5 min, with a single final extension step at 72 [degrees]C for five min. Active HHV-6 infections were defined as the presence of IgM or/and high IgG antibody levels (> 50 PANBIO units) plus DNA detection.
Variant-specific primers were used for PCR/nested PCR amplification, targeting two distinct regions of the HHV-6 genome of 195 bp and 423 bp for variants A and B, respectively57.
Conventional ELISA and immunofluorescence indirect assays were used for the detection of both IgM and IgG to rubella, Epstein-Barr virus, cytomegalovirus and Toxoplasma gondii, as described before11,14,17,37. Only 28 samples were tested by standard seroagglutination to detect Bnicella spp antibodies.
Statistical analysis was done with EPI-INFO software, version 6.0 (Atlanta, GA, USA). Comparison of rates between groups was made with the Mantel-Haenszel chi-square test of association or, if assumptions required for the chi-square test were not met, with Fisher's exact test. Siqnificance was defined as p
Overall, 188 (58.2%) of the 323 patients had HHV-6-specific IgM and/or IgG antibodies in their sera, whereas 135 (41.8%) subjects were found to be IgM- and- IgG-seronegative (Table 1 ). Serological status as defined by IgG+ was identified in 111 patients. Similar rates of HHV-6 antibodies (IgM and/or IgG) were noted for female and male subjects: 60.6% versus 55.7%, respectively. Seventy-seven (23.8%) patients were HHV-6 IgM- and- IgG-positivc (IgM+ subgroup), with positivity rates of 29.7% and 17.7% (p = 0.0007) for female- and- male individuals, respectively. No significant differences (p > 0.05) across 50 years; a significant difference (p = 0.04) was observed between female and male patients belonging to the 6-10-year age-group. Sera from 120 patients with presumptive recent infections with IgM+ (n = 77) or IgG+ (n = 43) specific antibodies, were assessed for the presence of viral DNA by PCR and nested-PCR, as demonstrated in Table 2. HHV-6 DNA was amplified from the serum of 20/77 (25.9%) IgM+ patients and from 8/43 (18.6%) subjects whose serum samples reacted IgG+. There was no statistically significant difference (p > 0.05). Rates of HHV-6 DNA detection between female and male patients were similar (p > 0.05) in the IgM+ and IgG+ groups: 20.4% versus 35.7% and 25.0% versus 13.0%, respectively. HHV-6 DNA was detected across
Among patients whose serological status was IgG+, HHV-6 DNA was detected in 50 age-groups at rates that ranged from 15.4% (male,
Figure 1 shows the anatomical sites of enlarged lymph nodes observed in 28 patients with active HHV-6 infection, according to sex and duration of these clinical signs in days. Overall, lymph node swelling was noted at similar rates in female (15, 53.6%) - and - male (13, 46.4%) patients with active HHV-6 infection caused by variant A. Reactive cervical lymph nodes were predominant, affecting 9 (32.0%) patients of each gender group. In smaller proportions, occipital, retroauricular, axillary and inguinal enlarged lymph nodes were observed. As for the duration, symptoms lasted 1-5 days in 35.7% of the individuals, whereas in 64.3% of them the disease lasted 6-20 days. Figure 2 illustrates the nestcd-PCR products with molecular weights matching those for HHV6A variant are present for 3 of the 28 scrum samples from patients with active infections. No positive results were obtained from testing sera for potential pathogens other than HHV-6 that might be involved in aetiology of the lymphadenopathies.
The present analysis of the HHV-6 scrological status of n = 323 patients with lymphadcnopathy of previously unknown aetiology provided evidence that this herpcsvirus may play a role as a cause for this clinical condition, as already reported elsewhere3. This assumption is based on the findings showing that about one-third of these patients had serological suggestive evidence of recent HHV-6 infection, that is, their sera reacted either IgM+ or IgG+ (high IgG levels) when tested for the presence of HHV-6 antibodies by ELISA. The proposed association between lymphadenopathy and HHV-6 infection in the present study is strengthened by the detection of HHV-6 DNA, by PCR30,53 in 28 subjects among 120 patients whose sera were indicative of active HHV-6 infection. Although focussing on a specific group of patients suffering from lymphoprolifcralive disorders, the present study allows for a reappraisal of HHV-6 scroprevalcnce in Belem, Para, Brazil, an issue that has been assessed in previous local surveys conducted by FREITAS & LINHARES23. The overall prevalence of HHV-6 antibody in the present study (58.2%) was found to be lower than that yielded before for the "normal" population of Belem, Brazil, 90.0%23. Although this may have occurred by chance, it would be worth considering two other explanations for such a difference. First, in the previous, "normal" population-based survey23, an indirect immunofiuorescence assay was used that might be more sensitive than the ELISA employed to assess the scrological status of patients with lymphadcnopathy. We do not believe that false-positive results were obtained when using the IFI, because of the high specificity of the test used8,51. Second, it could be postulated that a proportion of selected patients who developed lymphadenopathy had indeed primary HHV-6 infection. An evidence that might possibly support the "primary infection" hypothesis is that more than 40% of patients with lymphadenopathy had no delectable HHV-6 antibodies of cither IgM or IgG classes, in contrast with the only 10% antibody-negative proportion in the normal population23. Our study showed an overall higher HHV-6 antibody (IgM and/or IgG) rate in female than in male patients with lymphadcnopathy in Belem (29.7% versus 17.7%, p = 0.007), in contrast with similar prevalence rates achieved when comparing female (90.5%) and male (89.3%) subjects of a previous "normal population" study in the same setting. It is likely that women are more frequently exposed to HHV-6, as a result of their close contact with infants who might develop ES and, therefore, act as a source of viral transmission to mothers and care-givers. Excretion of HHV-6 in saliva of infants with ES and their parents suggests this to be a source for transmission of HHV-6 infection55.
A major finding in our study was the detection of HHV-6A DNA in sera from patients with lymphadenopathy, a viral variant that has been associated mainly with active infections among immunocompromised individuals, namely HIV-positive patients2,7,18,34,42,57. Contrasting with these findings, our investigation has shown HHV-6A variant to be associated with apparently immunocompetent individuals. Of note, it has been reported that immunocompromised and immunocompetent subjects are usually associated with reactivation28,29 and primary infections32,45,54, respectively, whatever variant considered. Of note, latency and later reactivation are both biological properties attributable to HHV-6A and HHV-6B variants in cells and body tissues6,7,20,38,39. It seems plausible to point out that the detection of HHV-6 DNA in sera from our patients support the hypothesis of primary infection53,56. Several studies2,3,5,45 record that active HHV-6A infection occurs later in life when compared wilh HHV-6B infection. Indeed, the latter variant appears to infect mainly infants younger than 3 years24,33,58. It is noticeable, however, that this has not invariably happened among our study subjects. On the other hand, some authors2,18,50 have demonstrated that a peak incidence of the HHV-6A infection occurs during adolescence and adulthood a pattern which appears more compatible with that recorded in our investigation. In this regard, it should be pointed out that HHV-6A infection appeared to be less frequent in our children aged less than five years with lymphadenopathy.
Of interest, no cases of neurological disorders - even seizures among infants - were identified among our patients who were infected with HHV-6A, in spite of previous reports indicating that such A variant has a possible greater ncurotropism than HHV-6B27. In our investigation, the cervical lymph nodes were more affected in both sexes, as recorded in studies conducted elsewhere38,45. Although preliminary, our data suggest that it would be worth seeking for HHV-6 infection, whenever a patient (infant or adult) presents with lymphadenopathy as a prominent symptom in the course of an acute febrile illness.
We thank Dr. Elisabete O. Santos, Talita A.F. Monteiro and Marinete Povoa for carrying out the serological tests for rubella, cytomegalovirus, Epstein-Barr virus and Toxoplasma gondii. We are grateful to Dr. Naimes O. Paiva for his help in performing the Brucella spp.
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Received: 18 June 2003
Accepted: 23 September 2003
Secao de Virologia, Instituto Evandro Chagas, Secretaria de Vigilancia em Saude, Ministerio da Saude, Belem, Para, Brasil.
Correspondence to: Dr. Ronaldo B. Freitas, Instituo Evandro Chagas, Av. Almirante Barroso 492, 66.090-000 Belem, Para, Brasil; e-mail: firstname.lastname@example.org
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