Clinical manifestations of human herpesvirus 6 (HHV-6) have not been clearly defined, and the role of HHV-6 in human disease among infants and children in Kuwait remains to be fully elucidated. A retrospective study covering the period between 2008 and 2014 was conducted on infants and children aged from 1 month to 5 years. Blood and CSF samples from infants and children who presented with symptoms suggestive HHV-6 infection were subjected to PCR test for HHV-6. Results showed that 9.3% (n = 42) of infants and children were positive for HHV-6. Fever was the most noticeable symptoms, presenting in 50% (n = 21) of the patients. Also, neutropenia was highly associated with HHV-6 infection, where it presented in 35.8% (n = 15) of infants and children. Our results provided important information about the clinical outcome of HHV-6 infection among infants and children in Kuwait.
Human herpesvirus 6 (HHV-6) is a ubiquitous virus, which is widespread thought the world. It belongs to the β-herpes group family to which cytomegalovirus (CMV) and HHV-7 are members [
From a study by Hussain et al. [
This is a retrospective study covering the period between January 2008 and May 2014 on patients in different Hospitals in Kuwait including Mubarak, Amiri, Sabah, Adan, Farwania Hospital. The Organ Transplant Center (OTC) and Kuwait Cancer Control Center (KCCC) were also included in this study. Patients suspected of possible HHV-6 infection were investigated by PCR test, which was performed in the Virology Unit, Faculty of Medicine, Kuwait University. The samples were taken from 449 infants and children aged from 1 month to 5 years who presented with symptoms suggestive HHV-6 infection such as fever, roseola, and seizures and were hospitalized during the study period. Of these infants and children, 251 were males and 198 were females, with a mean age of 1.3 months. Among these patients, 212 were Kuwaiti and 237 were non-Kuwaiti. Clinical data for infants and children with positive results were retrospectively registered. This retrospective study based on patient referral with no disclosure of names and this study was approved by the ethical committee at the Faculty of Medicine, Kuwait University.
HHV-6 DNA was extracted from clinical samples (blood, CSF) using Roche ®MagNA Pure LC system (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s instructions. HHV-6 DNA in the clinical samples was detected by amplifying the conserved region in HHV-6A (U1102) and HHV-6B (Z29) by nested PCR. The first PCR reaction was performed using primers HHV (6-1) (F): 5’AAGCTTGCACAATGCCAAAAAACAG-3’ and HHV (6-2) (R): 5’CTCGAGTATGCCGAGACCCCTAATC-3’. Ten ml of the extracted DNA was mixed with 15 ml PCR mix that consisted of 12.5 ml AmpliTaq Gold PCR Master Mix (Applied Biosystem, Branchburg, New Jersey, USA), 0.5 ml of each primer (10 pmol) (Roche, Applied Biosystem, USA), and 1.5 ml H2O. The positive control was ATCC (American Type Culture Collection) strain of HHV-6. A total of 30 amplification cycles (each cycle consisted of 94˚C for 1 min, 55˚C for 1 min, and 72˚C for 1 min) were carried out to amplify the target DNA. Second PCR reaction was performed using primers HHV (6-3) (F): 5’-TCCATTATTTTGGCC- GCATTCGT-3’ and HHV (6-4) (R): 5’-TGTTAGGATATACCGATGTGCGT-3’. One ml of the amplified product was mixed with 24 ml PCR mix which consisted of 12.5 ml AmpliTaq Gold PCR Master Mix, 0.5 ml of each primer (10 pmol), and 10.5 ml of H2O. Thermal cycling condition was similar to the first PCR. The 173 bp amplification products were visualized by QIAxcel System (Qiagen, Hilden, Germany) according to manufacturer’s instructions.
DNAemia was defined as the presence of a positive HHV-6 DNA in blood or plasma. In addition, it has been previously determined that the detection of HHV-6 DNA in plasma is consistent with active infection [
Detection of IgM antibodies to Epstein Bar virus (EBV) in blood was performed using Trinty Biotech Captia Qualitative ELISA Kit (Trinty Biotech Plc, Bray, Co., Wicklow, Ireland) according to the manufacturer’s instructions. Also, detection of IgM antibodies to Parvovirus B19 in blood was performed using Biotrin ELISA Kit (Biotrin Blackrock Co. Dublin, Ireland) according to the manufacturer’s instructions. IgM antibodies to Adenovirus were detected in blood using NovaLisa Qualitative ELISA Kit (Nova Tech Immundiagnostica GmbH-Technologie & Wald Park, Dietzenbach, Germany) according to the manufacturer’s instructions. Enteroviruse RNA was detected by conventional PCR using QIAGEN One step RT-PCR Kit (Qiagen, Hilden, Germany) as described previously [
HHV-6 infection was verified in 42/449 (9.3%) infants and children. Of the positive infants and children, there were 24 male and 18 female with a male to female ratio of 1.3:1.
Among the 42 cases, the breakdown of positive HHV-6 DNA PCR results according to the sources of samples was as follows: 35 (83.3%) were positive in plasma and 8 (19%) were positive in CSF (
The clinical manifestations and syndromes of the 42 HHV-6 positive infants and children are presented in
The relationship between the presence of HHV-6 DNA and other viruses was also examined. Six children showed HHV-6 co-infection with other viruses (
Infants and children aged from 1 month to 5 years were admitted to different hospitals in Kuwait based on clinical presentations suggestive of HHV-6 infection. These clinical presentations included high fever, seizures and rash. Blood samples were taken from these children in addition to CSF sample in cases of febrile convulsion or meningitis. The clinical samples were investigated for HHV-6 by PCR test in addition to other viruses. Results showed that HHV-6 infection was found in 42 (9.3%) of infants and children. Male to female ratio was 1.3:1. This result was consistent with other studies reporting a predominance of male infants and children with HHV-6 infection [
The results showed that 83.3% (n = 35) of children had active primary infection as indicated by positive plasma results. However, 19% (n = 8) of children were positive for HHV-6 in CSF since it was the only type of sample collected from them. Active infection in these children could be confirmed if plasma sample were available and tested for HHV-6 infection. It should be noted that the patients were considered to have primary infection because of their age and that they presented with symptoms of HHV-6 infection (fever-induced seizures and rash) plus virus DNA positive by PCR.
The clinical manifestations presented in our patients with positive HHV-6 DNA were varied. The classic manifestation of HHV-6 infection was acute febrile illnesses with or without rash [
Type of Sample | Number of HHV-6 PCR Positive (%) |
---|---|
Plasma | 35 (83.3%) |
CSF | 8 (19%) |
Total | 43 |
Clinical Manifestations | n (%) | Syndrome | n (%) |
---|---|---|---|
Fever | 21 (50) | Neutropenia | 15 (35.8) |
Roseolainfantum | 8 (19) | Meningitis | 3 (7.1) |
Seizure | 3 (7.1) | Encephalitis | 1 (2.4) |
Vomiting | 2 (4.8) | Thrombocytosis | 1 (2.4) |
Diarrhea | 1 (2.4) | ·UTI | 1 (2.4) |
Cough | 1 (2.4) | Bicytopenia | 1 (2.4) |
Otitis media | 1 (2.4) | Neonatal sepsis with shock | 1 (2.4) |
ØSCID | 1 (2.4) |
·Urinary Tract Infection; ØSevere Combined Immunodeficiency.
Patient No. | Virus | Associated Condition | |||||||
---|---|---|---|---|---|---|---|---|---|
Enterovirus RNA | Parvovirus B19 DNA | Parvovirus B19 IgM | CMV DNA | Adenovirus IgM | EBV IgM | Neutropenia | Febrile Neutropenia | Meningitis | |
1 | − | + | ØND | − | − | + | Yes | No | No |
2 | + | − | − | − | + | + | Yes | No | No |
3 | − | ND | + | − | − | − | No | Yes | No |
4 | + | − | − | − | − | − | Yes | No | No |
5 | − | − | − | + | − | − | No | Yes | No |
6 | + | − | − | − | − | − | No | No | Yes |
ØND: Not Done.
children with primary infection had typical roseola [
In recent years, attention has focused on febrile seizures due to HHV-6 infection. Febrile seizures are age-de- pendent condition, occurring in patients aged from 6 months to 5 years old [
In this study, the most frequent condition associated with HHV-6 was neutropenia, which was detected in 35.8% (n = 15) children. This result is in agreement with another study by Hussain et al. (2012), where they investigated the infectious causes of isolated transient neutropenia in previously healthy children. They showed that HHV-6 infection was the leading cause of transient neutropenia in previously healthy children in Kuwait [
The role and frequency of HHV-6 in central nervous system (CNS) diseases of children are unclear and it is an area of ongoing investigation. A study by Ansari et al. (2004) showed that HHV-6 DNA was found in 2 of 245 CSF samples from pediatric patients with meningitis who lacked evidence of another microbiologic cause. [
We found one child aged two years and 6 months who had undergone bone marrow transplant had SCID with MHC class II deficiency and graft-versus-host disease (GVHD). This child had only severe rash and was positive for HHV-6 in plasma. Unfortunately, it was difficult to define clearly whether this infection was primary or reactivation of latent infection since information on antibody seroconversion test was not available. HHV-6 reactivation is very common after bone marrow transplantation and is associated with serious transplantation- related morbidity and mortality [
Six children experienced co-infection with other viruses (
In conclusion, the study in Kuwait provides important information about the clinical outcome of HHV-6 infection among infants and children. Fever is the main clinical manifestation of HHV-6 infection. In addition, neutropenia is highly associated with HHV-6 infection among infants and children. Furthermore, encephalitis and meningitis are linked to HHV-6 infection in immunocompetant children, which indicate that the virus can result in different neurological complications. Finally, our results also suggest that HHV-6 may trigger the manifestations of GVHD.
The laboratory investigations were conducted at the virology unit at Faculty of Medicine, Kuwait University. The authors especially thank Dina A. Khalik and the laboratory technicians.