Human Herpes Virus Type 6 is Associated with CNS Infections in Children in Sudan

Objective HHV-6 is increasingly recognized as febrile agent in children, however, less is known in sub-Saharan African countries. In here, we aimed to investigate the involvement of HHV-6 in pediatric CNS infections in Khartoum, Sudan. This report is part of a larger study on the microbial aetiologies of CNS infections in this population. Out of 503, 13 (2.6%) CSF specimens were positive for HHV-6 DNA which constituted 33% (13/40) of cases with proven infectious meningitis. Median Ct for all HHV-6 positive specimens was 38 with range of 31.9 to 40.8. Median virus copy was 281.3/PCR run (1x10 5 virus copies/ml CSF) with range of 30 to 44x10 3 copies/PCR run (12x10 3 and 18x10 6 virus copies/ml CSF). All positive patients presented with fever, vomiting and 86% with seizures. Male to female ratio was 1:1; 50% were toddlers, 42% infants and 8% teenagers. Most (83%) were admitted in the dry season and 17% in the rainy season. CSF leukocytosis was seen in 33%. Normal and low CSF glucose levels were seen in 86% and 14%, respectively. CSF proteins levels were low in 14% and high in 43%. In conclusion, HHV-6 is common in CNS infections in children in Sudan.

Introduction HHV-6 is major cause of acute febrile illnesses in young children [1]; most are infected by age of three [2].
After primary infection, HHV-6 persists in salivary glands and remains latent in leukocytes [3]. Immunosuppression can lead to virus reactivation and various complications can occur, including CNS disease [3]. CNS disease can also occur in primary infections, especially in young immunocompetent children with fever and seizures [4,5]. In this matter, several authors described the major role the virus could play as a cause of pediatric neuroinfections [6][7][8][9][10][11].
In the absence of primary infection, the key nding linking HHV-6 to CNS infections is detection of viral DNA in CSF by PCR; indicating active replication. This interpretation has been questioned in view of the phenomenon of HHV-6 chromosomal integration [12,13]. HHV-6 is the only human herpes virus found integrated into human genome and can be transmitted vertically from parent to child [14]. Such individual is easily identi able, since every leukocyte contains viral sequences and there are thus characteristically persistent high levels of HHV-6 DNA in both serum and whole blood [12,13]. In contrast, integrated HHV-6 DNA is highly unexpected in normal cell-free body compartments, including CSF [11,13,15]. Ward [16] stated that viral load should be high to identify a condition as chromosomal integration and low virus copies would indicate an infection.
The outcome for HHV-6 infection is generally favorable, with most patients recovering fully. Nevertheless, mild to moderate neurological impairment as well as death due to complications has been reported [22,23]. Little is known on HHV-6 infections in sub-Saharan Africa, and the infection has never been investigated in Sudan. We, therefore, intended to identify the involvement of HHV-6 (among other microbial aetiologies reported elsewhere) in CNS infections in large group of children in Khartoum, Sudan.

Study Materials
Total of 503 CSF specimens were obtained from febrile ( 37 o C) meningitis-suspected attendee of Omdurman Hospital for Children in Khartoum, Sudan. Patients ages 0 to 15 years who were admitted in all seasons of 2010 were included. Clinical and demographic data were obtained from hospital records.
Routine CSF analyses were performed at the Microbiology Laboratory of Omdurman Hospital. An aliquot of CSF was frozen in -80 o C for further analysis for HHV-6 DNA at the department of Clinical Microbiology, Umeå University, Sweden. Permission to collect data and specimens was granted from hospital authorities. Ethical clearance was obtained from the Ethical Committee Board of Al-Neelain University.

HHV-6 DNA Analysis
QIAampUltraSensVirusTechnology (Qiagen®) was used to extract viral DNA in 1 mL CSF following the manufacturer recommendations. Pure viral nucleic acids were eluted in 30 µl low-salt buffer AVE twice.
Each elute (60 µl) was divided into two aliquots and preserved at -80 o C. Viral gDNA ampli cation and detection was performed by real-time analysis using Applied Biosystems®7900HT Fast Real-Time PCR system and TaqMan® probe (reporter dye FAM™ on 5´ end and quencher dye TAMRA™ on 3´ end). Forward and reverse primers (Table S1) and probes (DNATechnology®) were diluted to reach nal working concentration of 25 µM (standardized concentration by Umeå University Hospital). Commercially provided oligonucleotide products were diluted to the suitable working solution and the recommendation of QuantiTect®QPCR Protocol was followed.
Another study in New York [11] revealed signi cantly higher prevalence of 40% (14/35) in patients with CNS infections who tested negative for other CNS pathogens. Among well-de ned groups in our study, the prevalence was also high accounting for 33% out of 40 cases with proven infectious meningitis (Table 3). Unlike Yao [11] approach, we did not exclude cases with other detected CNS pathogens because of possible co-infections, as frequently reported [21,[25][26][27]. In fact, we were able to identify mixed microbial infections in three cases.
Primary HHV-6 infection almost invariably occurs in the rst 2 years of life [5,28,29], but rare cases of CNS infections, presumed due to HHV-6 reactivation, have been reported in immunocompetent older children and adults [22]. Among 24 HHV-6 positive patients in Tavakoli study [21], 42% were infants ≤ 3 years and 12.5% were teenagers ≤ 17 years. Out of our 13 HHV-6 positive cases; 92% were infants ≤ 2.3 years and 8% was a 15 years old. The ratio of males to females was 1:1 which agrees with Tavakoli's ndings of 1.3:1.1.
Clinical signs and symptoms in HHV-6 meningitis are not speci c [30]. Tavakoli [21] reported fever in 71%, altered mental status in 67%, headache in 29% and seizures in 33%. Other reported symptoms were muscle weakness, muscle pain and stiff neck; which are general symptoms for meningitis or encephalitis. We report fever and vomiting in 100% of HHV-6 positive cases, seizures in 86%, chills in 14.3% and stiff neck in 14.3%. None of our patients developed skin rash being the only speci c -but rare-symptom in HHV-6 meningitis [30]; other studies [21,24] concur.
Normal CSF glucose with normal or elevated proteins is the usual nding in viral CNS infections [17], as found in this study. Unfortunately, chemical analysis of the CSF is ineffective in case of viral infections; however, its signi cance is in distinguishing bacterial from aseptic aetiologies which is crucial preliminary step in deciding adequate treatment. CSF leukocytosis was seen in 33%. Increased numbers of CSF leukocytes ( 5cell/mm 3 ) indicates in ammation, however, normal cellular counts do not rule out viral aetiologies. In fact, normal CSF cellular counts in patients with proven CNS infections were frequently reported [17][18][19][20]. Normal CSF pro le was reported in 25% of HHV-6 positive cases by Tavakoli [21], accordingly, HHV-6 testing should not be limited to patients with abnormal CSF pro les.
Thermal cycle (Ct) is de ned as the number of cycles required for the uorescent signal to cross the threshold (exceed background level). Median Ct for our positive cases was 38 (range: 31.9-40.8).
Tavakoli reported Ct values range 25.03 to 39.92. In quantitative real-time PCR, Ct values inversely correlate with viral loads, therefore, low Ct value indicates high viral load and vice versa. Our Ct values and viral loads were found to be signi cantly (p = 0.029) inversely correlated (-0.6, 95% CI:-0.9 to -0.1) indicating signi cant variation of viral loads among our patients. Substantial variation among viral loads in patients was also reported [21].
The phenomenon of HHV-6 chromosomal integration is in debate; while some [11][12][13] consider it an easily identi able condition based on the presence or absence of nucleus containing blood cells in different body compartments, Ward [16] believes the few leukocytes that are usually present in normal CSF can reveal positive viral DNA in case of HHV-6 integration. In here, CSF was clear with no observed cells in most cases (86%), while single case (14%) showed increased CSF leukocytes. Ward [16] elaborated that in order to identify a condition as chromosomal integration; viral load should be high while low virus copies would indicate infection. The average concentration of CSF HHV-6 DNA in 9 children with primary infection (2.4 log 10 copies/ml) was signi cantly lower than that of 21 patients with viral chromosomal integration (4.0 log 10 copies/ml) in Ward study. In ours, the median CSF virus concentration was 1 × 10 5 copies/ml with minimum and maximum virus concentrations of 12 × 10 3 and 18 × 10 6 copies/ml. While Ward [16] recommended identifying low virus copies (≤ 10 3 ) as an acute HHV-6 infection and high virus copies (≥ 10 4 ) as chromosomal integration, Collot [31] identi ed viral integration in approximate concentrations of 10 3 to 10 6 copies/ml. We identi ed viral concentrations as low as 10 4 and as high as 10 7 . Others [11,21] reported high CSF viral loads in patients with HHV-6 CNS infections. Some authors [12,13] insist that chromosomal integration is a rare condition. Accordingly, we assume the detected HHV-6 DNA in our mostly cell-free CSF specimens is more likely to be from free replicating virus than from chromosomally integrated virus.
Febrile seizures appear to be caused by primary HHV-6 infection in infants with incidence of 13% in the United States [4]. Knowing that febrile seizures and vomiting were dominant symptoms among our population and the most frequent age group was children ≤ 2.3 years, therefore further supporting our assumption. Despite this, and for the sake of scienti c relevance, we are not ruling out the possibility of integration among our identi ed cases. For this reason, studies to identify the prevalence of HHV-6 integration among healthy Sudanese population are warranted.

Conclusion And Limitation
We conclude that HHV-6 CNS infection is frequent in this population (i.e. identi ed in one third of cases with proven infectious meningitis). A major limitation in this study is that we were unable to further genotype our identi ed HHV-6 DNA copies because of limited CSF volume (i.e. all available CSF was consumed in repeated testing and identi cations).

Declarations
Ethics approval and consent to participate The ethical clearance for conducting this study was obtained from the Ethical Committee Board of Al-Neelain University. Permission to collect data and specimens was granted from hospital authorities. Patients were not contacted directly; data were obtained from hospital les and were kept anonymous at all stages of the study. Specimens were obtained from the hospital main laboratory after all requested tests were applied.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study will be available from the corresponding author on reasonable request once all related articles are published.

Competing interests
We, the authors, declare that we have no competing interests with respect to the research, authorship and/or publication of this article. Authors' contributions NA developed research questions and design, collected and managed all data, performed all laboratory work, statistical analysis and interpretation, wrote and edited the text. NM advised on the approach and methodology and guided and supervised the molecular laboratory work. ME and CA contributed on the approach and methodology, supervised the molecular laboratory work, edited and proofread the manuscript and were major contributors in the overall study. IF supervised the research process throughout; contributed in the development of research questions, design and methodology, managed all logistics and clinic based activities. All authors have read and approved the nal manuscript.