Clinical Presentations of HHV-6 Infection in Infants and Children in Kuwait: A Retrospective Study

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.


Introduction
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 [1]. Like other herpesviruses, it has the ability to remain latent and can be reactivated with immunosuppression [2]. Human herpesvirus infects approximately 70% of infants by the age of two years [3]. Primary infection causes undifferentiated febrile illnesses, where approximately 30% of children exhibit the classical clinical manifestations of exanthema subitum (also known as roseola infantum or sixth disease) [4] [5]. In addition, immunocompetant children may present with acute febrile illnesses with or without a rash [6]. Seizures are the most common complication of primary HHV-6 infection noted in approximately 14% of patients [7]. However, nonspecific symptoms without fever, such as fussiness and diarrhea, can occur after infection with the virus [8]. Other clinical manifestations include hepatitis, myocarditis, and encephalitis may also occur. This is with or without co-infection with other viruses [9] [10]. Following immunosuppression, HHV-6 can be reactivated and may result in severe neurological complications, such as multiple sclerosis, mesial temporal sclerosis and encephalitis [11]- [13].
From a study by Hussain et al. [14], where they investigated the infectious etiologies of neutropenia among children in Kuwait, there has been no published data on the spectrum of signs and symptoms developed as a result of HHV-6 infection in infants and children in Kuwait. Therefore, this study describes the clinical manifestations and syndromes associated with HHV-6 infection among infants and children in Kuwait.

Patients
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.
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 [15]. Thus, active HHV-6 infection was defined as a positive plasma HHV-6 DNA PCR.

Detection of Other Viruses
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 [16]. CMV DNA was detected by ArtusCMV PCR Kit (Qiagen, Hilden, Germany) using ABI PRISM 7500 Fast Real-Time PCR System. HSV DNA detection was performed using LightCycler HSV 1/2 Detection Kit (Roche Molecular Systems Inc., Branchburg, USA).  Table 1). Only one patient was positive in both CSF and plasma samples. However, one patient was CSF positive and plasma negative, which suggest that this patient had latent infection.

HHV
The clinical manifestations and syndromes of the 42 HHV-6 positive infants and children are presented in Table 2. Fever was the most prominent finding, presented in 50% (n = 21) of the HHV-6 cases. Roseolainfantum presented in 19% (n = 8) of the cases. The rash was maculopapular and most frequently generalized, involving face, trunk, and limbs. Seizure and vomiting were seen in 7.1% (n = 3) and 4.8% (n = 2) of the cases, respectively. In addition, Diarrhea, cough, otitis media, (2.4% each) were less frequently seen in the HHV-6 cases. Fifteen children (35.8%) had neutropenia at the time of hospitalization. Another three children (7.1%) had meningitis. Encephalitis, thrombocytopenia, urinary tract infection (UTI), Bicytopenia, neonatal sepsis with shock, were seen in 2.4%. Of the patients, one child had bone marrow transplantation with severe combined immunodeficiency syndrome (SCID) with MHC class II deficiency and had graft versus host disease. This child presented with severe rash.
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 ( Table 3). Three children were positive for Enterovirus RNA, 2 children were positive for Parvovirus B19 positive, and two children were positive for EBV. In addition, one child was positive for Adenovirus and another one was positive for CMV.

Discussion
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 [14] [17].
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 [4]. Our results showed that fever presented in 31% (n = 2) of infants and children. Other studies have also shown that acute HHV-6 can present as a febrile illness without a rash. Fever was most commonly accompanied by irritability, otitis media, upper and lower respiratory symptoms, or diarrhea [7]. Another report showed outbreaks of HHV-6 infection that presented as a febrile illness with or without rash in a number of day-care centers in Brazil [18]. Roseola, a clinical syndrome that is relatively specific for HHV-6 infection, occurred in 19% (n = 8) of patients in this study. However, a study by Laina et al. (2010) showed a higher percentage; 60% of HHV-6 positive infants and children with primary infection had typical roseola [3]. On the other hand, others reported roseola in a minority of cases [8].
In recent years, attention has focused on febrile seizures due to HHV-6 infection. Febrile seizures are age-dependent condition, occurring in patients aged from 6 months to 5 years old [3]. Recently, it is considered that HHV-6 plays a causative role in febrile seizures, based on the neurotropic properties of this virus and the similarity of the groups in which febrile seizures and HHV-6 infection occur [19]. Our study showed that only 7.1% (n = 3) HHV-6 positive patients had febrile seizure at the time of admission to the hospital. Another study showed that among children with primary HHV-6 infection, 8% had febrile seizures, a percentage similar to the present findings [20]. However, Zerr et al. (2005) did not report any case with seizures [8]. In contrast, other studies showed higher percentage of seizures. Barone et al. (1995), for example, showed that 26% of HHV-6 positive young children experienced febrile seizures [21]. Other symptoms, diarrhea, cough and otitis media, were also reported in HHV-6 positive infants and children, but in a lesser percentage (2.4%; n = 1). Zerr et al. showed similar results [8].
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 [14]. A study by Karavanaki et al. (2006) showed that HHV-6 was associated with neutropenia in 2 of 24 pre-viously healthy children [22].
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. [23]. Interestingly, we had reported three children with HHV-6 DNA in CSF and with meningitis. One of the 3 children with meningitis was positive for both HHV-6 and Enterovirus RNA in CSF sample; however, the other 2 children were positive in CSF for HHV-6 DNA, suggesting that HHV-6 was the etiologic cause of meningitis in these children. We also reported a one-year-old child and who had febrile convulsion with left side weakness and with encephalitis. This child was only positive for HHV-6 in CSF. Our result is one of few that supports the assumption that primary infection with HHV-6 in infants and children may have an important role in CNS diseases. Our data are in agreement with another study by McCullers et al. (1995), who showed that 9 out of 129 patients with focal encephalitis were infected with HHV-6 [24]. In addition, three cases of HHV-6 meningoencephalitis were reported in previously healthy children by Bozzola et al. [11]. Another study found that out of 983 cases with acute encephalopathy in Japan, 17% were caused by HHV-6. They also found that 64% of the severe cases with biphasic seizures in the study were attributed to HHV-6 and that only half of which survived without permanent neurological sequelae [25].
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 transplantationrelated morbidity and mortality [26]. Out of 365 patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT) tested for HHV-6, 100 experienced HHV-6 reactivation. Furthermore, 69 of these infected patients developed GVHD. The authors confirmed the association between HHV-6 and GVHD and suggested that HHV-6 might play a role in triggering severe manifestations of GVHD [27].
Six children experienced co-infection with other viruses ( Table 3). One patient had triple infection with Enterovirus, Adenoviruse and EBV and another patient had dual infection with Parovirus B19 and EBV. In addition, four patients had single infection with Parvovirus B19, Enterovirus, CMV and Enterovirus, respectively. Our results are in agreement with the study of Hussain et al. (2012), which showed that neutropenia in previously healthy children in Kuwait was caused by demonstrable infection in 55% of cases: HHV-6, 30%; Enterovirus, 23%; Influenza A H1N1, 13%; Parvovirus, 10%; EBV, 10%; UTI by Eshcherichia coli, 7%; Adenovirus, 7% by [14].
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.