Detection and Typing of Human Herpesvirus 6 by Molecular Methods in Specimens from Patients Diagnosed with Encephalitis or Meningitis

Detection and Typing of Human Herpesvirus 6 by Molecular Methods in Specimens from Patients Diagnosed with Encephalitis or Meningitis

Two siblings and their mother developed afebrile generalized lymphadenopathy. Atul Humar, Department of Medicine, Division of Infectious Diseases and Multi-Organ Transplantation, Toronto General Hospital-University Health Network, 200 Elizabeth St., NUW 9-132, Toronto, Ontario M5G 2C4, Canada (Atul.Humar{at}uhn.on.ca). It is usually a mild infection that causes no long-term problems. The primary HHV-6 infection, which occurred throughout the year, was observed in 94 boys and 82 girls (mean age, 7.3 months). According to our knowledge this well-documented case is probably the first report from Poland. The disease is common in children aged 3 months to 3 years and most common in those aged 6 months to 2 years. It is usually caused by a virus called human herpesvirus type 6 (HHV-6).

Treatment with antiviral agents was effective, with total resolution of her symptoms and the DNA of this virus disappeared from the CSF after 23 days of treatment. Immune system also seems to play a role in the etiology, mainly immunocompromised states. The article offers information on several medical researches including voriconazole, Clostridium difficile infection and human herpesvirus (HHV)-6 and HHV-7. Fifty percent of patients of 3 years of age or younger suffered from seizures. The detection of HHV-6 in specimens from patients diagnosed with encephalitis or meningitis, in the absence of a positive PCR result for other agents, strongly suggests a role for HHV-6 in the pathogenesis of these central nervous system diseases. Human herpesvirus—6 (HHV-6) is increasingly being recognized as an important pathogen of concern in patients who have undergone solid-organ and/or bone-marrow transplantation. The child can be flushed, irritable, and unwell with the fever.

There are two variants, HHV-6A and HHV-6B, which have an overall genetic identity of 90% (17). Clinical disease caused by HHV-6A is not well understood. A rash usually appears when the fever subsides, when the child is getting better. Small pink spots appear. By the age of three, almost all children have been infected by HHV-6 (25, 39), resulting in the majority of adults being seropositive (31). After primary infection, HHV-6 persists in the salivary glands and remains latent in monocytes and macrophages. This specific type of error is estimated to account for over 90 % of live births with DS, with maternal age being the best known risk factor for…

HHV-6 has been associated with delayed engraftment, high-grade graft-versus-host disease, and lymphoproliferative disorders (29, 32, 37). There are several reports of HHV-6 involvement in immunocompetent patients with acute encephalitis (1, 27, 36, 41) and meningitis (24). The CMV D+/R− subgroup received ganciclovir until 12 weeks posttransplant and also had blood samples collected for an 8-week period after the ganciclovir was discontinued. At first the high fever may cause concern to parents and doctors if it is not clear what is causing it. Once the virus is detected, it is then typed using a previously published conventional PCR assay (28). The target for the real-time PCR assay is a portion of the U6 gene. It indicates that the fever has been caused by the roseola virus and nothing more serious.

A fever can make a child feel uncomfortable and irritable. One of two primer sets was used for typing, targeting either the U86 or the U95 gene (28); both are immediate early/regulatory genes. Once amplified, HHV-6A and -6B produce fragments of different sizes, thereby allowing the type to be determined. In addition, the sequence of the amplified product was determined in order to verify the subtype. A total of 1,482 specimens from hospitalized patients in New York State who had been diagnosed with encephalitis or meningitis were tested for HHV-6 by real-time PCR. The majority of specimens (>95%) were derived from cerebrospinal fluid (CSF), but other matrices, including brain tissue, serum, plasma, and liver tissue, were also tested. Specimens were collected between the years 2003 and 2007.

They merely help to ease discomfort caused by the fever. HHV-6A (strain 350) was obtained from the NIH AIDS Research and Reference Reagent Program, and HHV-6B was obtained from the ATCC (Manassas, VA). The plasmid control for HHV-6 (pNT10) was constructed via conventional PCR performed with the real-time PCR primers for the HHV-6 assay (Table ) and HHV-6B as the template, and the product was then cloned into the Topo II plasmid (Invitrogen, Carlsbad, CA). If one of these medicines on its own is not enough to keep the temperature down, you can use both. For example, if the effect of paracetamol is wearing off but it is too soon to give another dose, you could then use ibuprofen. Genomic HHV-6 DNA was quantified by performing real-time PCR on serial dilutions of plasmid and genomic DNAs and by constructing a standard curve using serial dilutions of quantified plasmid DNA as standards. Nucleic acid was extracted from patient specimens by using the NucliSENS miniMAG or easyMAG system (bioMérieux, Durham, NC).

Two hundred and fifty microliters of each specimen was added to 2 ml of lysis buffer. Five microliters of plasmid pTU65 (1,900 gene copies) was spiked into the lysed sample. Following its extraction with miniMAG or easyMAG, the nucleic acid was eluted in 50 or 55 μl of elution buffer, respectively. Real-time PCR for the detection of HHV-6 was performed using primers and probe developed in-house (Table ). Do not cold-sponge a child who has a fever. Amplification was carried out in a 25-μl volume reaction mixture by using universal buffer (ABI, Foster City, CA), 1,100 nM each primer, and 200 nM probe. The reaction mixtures were incubated at 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min.

This reduces heat loss, and can trap heat in deeper parts of the body. The child may then get worse. The reaction mixture consisted of universal buffer, 900 nM each forward and reverse primer, and 250 nM probe. The reaction conditions were as described above. PCRs were performed using an ABI 7900 or ABI 7500 instrument. A previously reported PCR method for the typing (28) of HHV-6 was used essentially as described previously, with primer sets targeting the U86 or U95 gene. PCR products were analyzed by agarose gel electrophoresis and stained by ethidium bromide.

Detection and Typing of Human Herpesvirus 6 by Molecular Methods in Specimens from Patients Diagnosed with Encephalitis or Meningitis
The expected product sizes were 311 and 209 bp for HHV-6B and HHV-6A, respectively, when the U86 gene was targeted, and 342 and 264 bp, respectively, when the U95 gene was targeted. A fever caused by any illness may contribute to dehydration. Sequencing reactions of the PCR products were performed using the same primers that were used for the PCRs. The primer concentrations were 3.2 pmol, and sequencing reactions were performed at the Wadsworth Center Molecular Genetics Core facility on an automated DNA sequencer model 3100 (Applied Biosystems, Foster City, CA). Signs of dehydration include: a dry mouth, no tears, sunken eyes, drowsiness and generally becoming more unwell. Seek medical help if you suspect that your child is becoming dehydrated. The real-time assay for the detection of HHV-6 was determined to have a linear range from 5 × 106 gene copies/reaction (gc/rx) to 5 gc/rx, as determined from performance of real-time PCR on serial dilutions of quantified HHV-6 DNA extracted from culture.

The efficiency of the reaction was 100%, with an R2 value of 0.99 and an m value (slope of standard curve) of −3.32. The limit of detection of the assay was 5 gc/rx, equating to 200 gc/ml of patient specimen. A specificity assay was performed, in which the reactivity of the HHV-6 assay was determined against genomic nucleic acid from the following viruses and bacteria: influenza A-H1, influenza A-H3, influenza B, rhinovirus, echovirus 9, echovirus 11, echovirus 30, Coxsackie A and B viruses, human coronavirus 229E, respiratory syncytial virus, severe acute respiratory syndrome (SARS) coronavirus, human metapneumovirus, adenovirus, varicella-zoster virus, herpes simplex viruses 1 and 2, cytomegalovirus, Epstein-Barr virus (EBV), group A streptococcus, Haemophilus influenzae, Haemophilus parainfluenzae, Neisseria subflava, Streptococcus sanguis, and Corynebacterium xerosis. No cross-reactivity was observed between the primer and probe set and the organisms selected in the specificity panel, indicating that the assay is specific. We performed a retrospective screen of archived specimens from patients who had been hospitalized with a diagnosis of encephalitis or meningitis. Fourteen CSF specimens were from those collected in 2003, 86 from those collected in 2004, and 277 from those collected in 2005 selected randomly. We also tested a total of 1,105 specimens that were received from 1 January 2006 to 31 July 2007.

Twenty-six specimens (from 24 patients) of the total 1,482 specimens were positive for HHV-6 (Table ). Of these specimens, 20 could be typed and all 20 were found to be HHV-6B. The remaining specimens could not be typed, either because of insufficient specimen for testing or because the typing assay was not as sensitive as our real-time PCR detection assay was. All positive specimens were from CSF, except for a sample of liver tissue and a serum specimen (Table ). Dual infections were detected in three cases. These included two patients with dual infections of EBV and HHV-6 and one autopsy specimen in which both adenovirus and HHV-6 were detected. Apart from the dual infections, varicella-zoster virus, herpes simplex viruses 1 and 2, EBV, cytomegalovirus, adenovirus, and enterovirus were ruled out by PCR or real-time PCR for all specimens.

In addition, arboviruses, including West Nile virus, Saint Louis encephalitis virus, California serogroup viruses, Cache Valley virus, and eastern equine encephalitis virus were ruled out for specimens that were received during the arbovirus season (1 May to 30 November). Bacterial culture results were negative for seven specimens and not reported for the remaining specimens. Immunosuppression was not reported for any of the patients found to be positive for HHV-6. However, patient 9 had undergone a blood transfusion 4 weeks prior to the onset of his symptoms, and therefore may have been immunocompromised. Three specimens were received from one patient (patient 23 [Table ]) during the course of her illness. Two CSF specimens and one serum specimen were submitted for this patient. The first CSF specimen was collected 16 days after onset of illness; by performing real-time PCR, we were able to determine that the specimen had a viral count of approximately 13,000 gc/ml.

The serum was collected 27 days after onset and had a viral count of approximately 22,000 gc/ml. The second CSF was collected 34 days after onset, as the patient symptoms were resolving, and had a viral count of 6,600 gc/ml. The time between the onset of disease and specimen collection was reported for 20 of the 24 patients who tested positive for HHV-6 (Table ). For these patients, the time range was 0 to 34 days, with a mean of 8.5 days. Three of the positive specimens were autopsy specimens (patients 12, 20, and 21 [Table ]), and no onset date was available. The ages of the 24 patients who were positive for HHV-6 ranged from 1 year to 81 years (Table ). Ten patients were 3 years of age or younger, six were between 4 and 20 years old, and eight were between 21 and 81 years old.

Forty-two percent were therefore infants or very young children. Thirteen of 24 patients were male. Fifteen patients had been diagnosed with encephalitis, three with meningitis, one with encephalitis or meningitis, and one with meningoencephalitis (Table ). The diagnoses for the remaining four patients were unknown. Three of the CSF specimens were from autopsy cases, and the CSF profiles were not reported. An additional seven specimens did not have CSF profiles available. The CSF profiles were reported for 16 cases; of these, 12 profiles were abnormal (Table ).

An abnormal CSF profile is defined as having a higher than normal protein range (>45 mg/dl) and/or a higher than normal white blood cell (WBC) count (>5 WBC/mm3). Eight patients had proteins in the higher than normal range (49 to 483 mg/dl), with a mean of 166 mg/dl. Nine patients had higher than normal WBC counts, ranging from 10 to 228 WBC/mm3, with a mean of 65. Six patients had both abnormal protein levels and abnormal WBC counts, and of these patients, all except one had predominantly lymphocyte increases. Two patients had abnormal protein levels but normal WBC counts, and two patients had abnormal WBC counts (predominantly lymphocytes) but normal protein levels. For one patient, the WBC count was abnormal, but the protein level was not reported. Fever was reported for 17 (71%) patients, altered mental status was reported for 16 (67%) patients, headache was reported for 7 (29%) patients, and seizure was reported for 8 (33%) patients (Table ).

Other symptoms that were reported included muscle weakness, muscle pain, rash, and stiff neck. These are general symptoms seen in patients suffering from encephalitis or meningitis. Altered mental status is often also reported for patients with encephalitis. Following real-time PCR, the cycle threshold (CT) value was recorded for each specimen extract (Table ). The CT is defined as the number of cycles required for the fluorescent signal to cross the threshold (exceed background level). The CT values for the specimen extracts ranged from 25.03 to 39.92, indicating a substantial variation among the viral loads of the patients (Table ). Although the detection assay was not performed as a quantitative assay, the CT value from real-time PCR can be inversely correlated with the viral titer, with a low CT value indicating a high viral titer.

The various methods used for the diagnosis of HHV-6 include PCR, serology, viral culture, in situ hybridization, and immunohistochemistry. Since HHV-6 has the ability to persist and establish latency, the problem arises that diagnostic tests must be able to distinguish active viral replication from latent infection. Reverse transcription-PCR assays have been developed to detect the presence of viral mRNA, which constitutes a marker for active infection (44). The detection of immunoglobulin M or immunoglobulin G antibodies to HHV-6 in serum or CSF does not discriminate between active infection and latent/chronic persistent infection. The same is true for the detection of HHV-6 DNA by PCR in peripheral blood mononuclear cells (PBMCs), the site where the virus establishes latency. In these cases, HHV-6 DNA can be detected by PCR in PBMCs at low levels (9). Viral culture is a difficult and time-consuming process and is not routinely used in clinical laboratories to detect virus in CSF specimens.

Immunohistochemistry, although a valuable tool, requires brain specimens that are highly invasive and not generally available. In recent years, the use of quantitative PCR for absolute quantitation of viral load has come into wider usage (2, 19, 26) since viral load is a preferred indicator of active infection.

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