- Email: [email protected]
Human polyomavirus 6 DNA in the cerebrospinal fluid of an HIV-positive patient with leukoencephalopathy
Journal of Clinical Virology 68 (2015) 24–27
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Human polyomavirus 6 DNA in the cerebrospinal fluid of an HIV-positive patient with leukoencephalopathy Serena Delbue a,∗ , Francesca Elia a , Lucia Signorini a , Ramona Bella a , Sonia Villani a , Enrico Marchioni b , Pasquale Ferrante a , Tung Gia Phan c,d , Eric Delwart c,d a
Laboratory of Translational Medicine, Department of Biomedical, Surgical and Dental Sciences, University of Milano, Italy Department of General Neurology, IRCCS National Neurological Institute C. Mondino Foundation, Pavia, Italy c Blood Systems Research Institute, San Francisco, CA 94118, USA d Department of Laboratory Medicine, University of California at San Francisco, San Francisco, CA 94118, USA b
a r t i c l e
i n f o
Article history: Received 11 March 2015 Received in revised form 17 April 2015 Accepted 21 April 2015 Keywords: Polyomaviruses Leukoencephalopathy Cerebrospinal fluid Deep sequencing
a b s t r a c t Background: Leukoencephalopathies in HAART-treated, HIV-positive patients include progressive multifocal leukoencephalopathy (PML), a result of lytic infection oligodendrocytes by JC polyomavirus (JCV), and another form characterized by the absence of JCV genome in cerebrospinal fluid (CSF). Objectives: To test the potential viral etiology of JCV-negative leukoencephalopathy. Study design: CSF was collected from 43 HIV-positive patients with MRI suggestive of leukoencephalopathies. DNA was isolated and real-time PCR assays for neurotropic viruses (Herpes Simplex Viruses 1/2, Varicella Zoster Virus, Epstein Barr Virus, Human Cytomegalovirus, Human Herpesvirus 6, JCV and HIV) were conducted. CSF from 14 non-reactive cases were subjected to random nucleic acid amplification, deep sequencing, and in silico search for viral sequences. Results: JCV genome was detected in the CSF of 19/43 PML patients, HIV genome in the CSF of 5 PML patients including 2 JCV negative patients, and no viruses were detected in 22 patients. Human Polyomavirus 6 (HPyV6) DNA was detected by deep sequencing in one JCV-negative leukoencephalopathy CSF sample. Conclusions: HPyV6 DNA was detected in CSF of a case of demyelinating disease. HPyV6 has not been previously reported in CSF or associated with any disease. Demonstrating a causative role will require further studies. © 2015 Elsevier B.V. All rights reserved.
1. Background Progressive multifocal leukoencephalopathy (PML) is a severe, often fatal, demyelinating disease of the white matter of the central nervous system (CNS), due to polyomavirus JC (JCV) lytic infection of oligodendrocytes [1]. PML occurs in severely immunocompromised patients, such as in the context of HIV infection, hematological malignancy, exposure to chemotherapy or immunomodulatory antibodies, transplantation and various causes of lymphocyte depletion [2,3]. Although the introduction of highly active antiretroviral therapy (HAART) significantly reduced PML mortality, it remains the third most common neurological infectious disease in HIV/AIDS, following Toxoplasma
∗ Corresponding author at: Department of Biomedical, Surgical and Dental Sciences, University of Milano, Via Pascal, 36 -20133 Milano, Italy. Tel.: +39 0250315070. E-mail address: [email protected] (S. Delbue). http://dx.doi.org/10.1016/j.jcv.2015.04.016 1386-6532/© 2015 Elsevier B.V. All rights reserved.
encephalitis and HIV encephalopathy [4]. Additionally, atypical forms with an uncommon clinical presentation were described. These pathologies were characterized by the presence of demyelinating lesions in magnetic resonance imaging (MRI) and mild symptoms resembling those of PML but in the absence of the JCV in the cerebrospinal fluid (CSF) [5,6]. 2. Objectives To gather new insights on the potential viral etiology of the JCVnegative forms of leukoencephalopathies. 3. Study design 3.1. Patients From 2007 to 2010, 43 HIV-positive patients, showing MRI lesions indicative of demyelinating leukoencephalopathy, were
S. Delbue et al. / Journal of Clinical Virology 68 (2015) 24–27
25
Fig. 1. Sequence alignment of the HPyV6 gene fragment found in the CSF of a JCV-negative patient by deep sequencing analysis, with the HM011561 HPyV6 sequence.
identified from those patients who underwent lumbar puncture at the Neurological Department of the Mondino Institute, Pavia. CSF was collected for diagnosis purpose and part of the clinical specimen was stored at −80 ◦ C for research purpose. The patients’ demographic and immune-virological characteristics were acquired from clinical records. The study protocol was approved by the local ethical review boards (IRB numbers 1835 and 2319), and all patients provided written informed consent.
Table 1 Clinical and demographic features of the patients. Sex
31 Male; 12 Female
Age ± sd (years) CD4 (cells/mmc ± sd) HIV viremia (copies/ml ± sd) Risk factor (sex/drug abuse/maternal) HIV duration (years ± sd) Therapy duration (years ± sd) Presence of neurological symptoms (yes/no)
42.6 ± 8.6 266.8 ± 173.2 155450.8 ± 8000 11/31/1 10.5 ± 5.3 4.6 ± 7.9 31/12
4. Virological assays 4.1. Nucleic acids isolations and real-time PCRs (Q-PCRs) assays JCV and Herpesviruses loads were quantified after the viral DNA was extracted from 0.15 ml CSF, which was done using the Nucleospin RNA virus kit (MachereyNagels, Germany) according to the manufacturers’ instructions. The HIV loads were quantified after the viral RNA was extracted from 0.14 mL CSF using the Qiamp viral RNA mini kit (QIAGEN, USA), according to the manufacturer’s instructions. The Q-PCR protocol for the detection of JCV DNA, particularly for targeting the LT region, has been described elsewhere [7]. To detect the presence of the Herpes Simplex Virus 1 and 2 (HSV 1/2), Varicella Zoster Virus (VZV), Epstein Barr Virus (EBV), Human CytomegaloVirus (HCMV), Human Herpes Virus 6 (HHV 6) DNA, different Q-PCR were performed using primers and probes whose sequences are proprietary (Applied Biosystem, UK). To detect the presence of the HIV genome, a Q-PCR targeting the gag gene was performed, as described before [8]. Each sample was analyzed in triplicate, and each run contained a negative control consisting of the reaction mixture without the DNA template. Standard curves for quantification of viral genome were constructed using serial dilutions of a plasmid containing the whole viral genome (range: 10–106 plasmid copies/mL). Data were expressed as log [copies/CSF mL]. As the Q-PCR is highly sensitive, strict precautionary measures were taken to avoid cross-contamination [9]: separate rooms were used to extract the nucleic acids, to prepare the amplification mixes, and to run the Q-PCR; multiple negative controls (containing water instead of DNA templates) were included with each Q-PCR batch. 4.1.1. Deep sequencing and bioinformatic analysis A total of 14 CSF samples non-reactive for the tested viruses were analyzed in pools of three to four samples using a
metagenomics approach. Samples were pooled to reduce cost. Total nucleic acids were extracted using a Maxwell® 16 automated extractor (Promega) from supernatant of CSF spun 15 min in a microfuge. A DNA library was constructed using ScriptSeqTM v2 RNA-Seq Library Preparation Kit (Epicentre) which amplifies both RNA and DNA, and sequenced using the Illumina MiSeq platform. Viral sequences were identified through translated protein sequence similarity search (BLASTx) to annotated viral proteins available in GenBank’s viral RefSeq database. 4.1.2. Human Polyomavirus 6 (HPyV6) PCR amplification The 14 CSF samples analyzed by deep sequencing were subjected to PCR targeting the HPyV6 VP1 region to identify the positive individual sample. The primers employed have been previously published [10]; the annealing temperature of the first PCR (30 cycle) was 60 ◦ C, and 5 l of the PCR products were than subjected to further 25 cycle of amplification, with the annealing temperature of 62 ◦ C. 5. Results 5.1. Patients Clinical and demographic characteristics of the 43 enrolled patients are summarized in Table 1. 5.2. Q-PCRs assays JCV genome was detected in the CSF of 19/43 patients (44.2%), diagnosed as PML patients, with a mean viral load of log 4.7 copies/mL (range: log 3.23–6.9 copies/mL); HIV genome was detected in the CSF of 5/19 PML patients (26.3%), with a mean
Table 2 Virological findings in CSF collected from HIV patients affected with leukoencephalopathies.
CSF+/total (by Q- PCRs) CSF+/total (by deep sequencing) a b
Herpesviruses DNA
JCV DNA
HIV RNA
HPyV6
0/43 0/14
19a /43 (44.2%) 0/14
7a /43 (16.3%) 0/14
n.a. 1/14b (7.1%)
5 CSF contained both JCV and HIV genomic sequences. Deep sequencing was performed on 14 not reactive CSF and the finding was confirmed by PCR n.a. not available.
26
S. Delbue et al. / Journal of Clinical Virology 68 (2015) 24–27
viral load of log 2.8 copies/ml (range: log 1.7–6.0 copies/mL) and in 2/22 (9.1%) JCV negative patients, with a mean viral load of log 1.9 copies/ml (log 1.7–4.07 copies/mL); Human Herpesvirus genomes was not detected in any of the analyzed CSF (Table 2). Consequently, 22 cases were diagnosed as JCV-negative leukoencephalopathy patients.
previously reported in CSF [23], supports the possibility of contamination with skin-associated viruses. Whether HPyV6 detection in CSF is associated with this patient’s neurological disease, is indicative of contamination with skin microbiota, or reflects increased blood-brain permeability is unknown. The involvement of HPyV6 in JCV-negative leukoencephalopathy therefore remains theoretical pending further investigations.
5.3. Deep sequencing and bioinformatic analysis Translated sequence reads showing similarity to viral sequences were identified using BLASTx. Using a BLASTx cutoff of E scores <10−5 one pool of four CSF samples containing 3.3 × 106 sequence reads showed the presence of 6 anellovirus reads, 70 papillomavirus reads (data not shown), and 4 HPyV6 reads (Fig. 1). 5.4. HPyV6 PCR
Funding This work was partially supported by an Italian Minister of the University Grant (PRIN 2010- 2011) to Pasquale Ferrante and NHLBI grant R01HL105770 to E.L.D. Conflict of interest
PCR specific for HPyV6 DNA was performed on the 14 notreactive CSF and confirmed the presence of the HPyV6 genome in one of the CSF in the positive pool, collected from a 39 years old male patient with CD4 cell count 320/mm2 , HIV-positive for at least 24 months before the CSF collection, with MRI lesions resembling those of PML. The PCR product was sequenced and the result confirmed the presence of HPyV6 genome (Fig. 1).
Ethical approval
6. Discussion
References
Virological analysis of CSF from HIV-positive patients with leukoencephalopathy was conducted, and the rate of JCV findings was within the expectation. A recently published study observed that JCV was detected in the CSF of 44% HIV-positive patients presenting MRI lesions accounting for leukoencephalopathies, and these patients were classified as PML patients, whereas the others (JCV negative) were classified as possible PML, since no viral genome was detected in the CSF [11]. The concomitant presence of both JCV and HIV in the CSF of 26.3% PML patients was also not surprising [8], since it is well known the synergistic effect of HIV and JCV [12]. Indeed, evidence for a direct role of HIV in JCV activation comes from several studies showing up-regulation of the JCV late promoter by the HIV-encoded regulatory protein, Tat [13]. The HPyV6 genome was first described in 2010, by Buck et al., who detected it in 5/35 skin swab specimens collected from healthy subjects. Additionally, that group showed that antibody reactivity against HPyV6 was very common, with 69% healthy subjects scoring seropositive [14]. Other studies also detected persistent infections with HPyV6 and seropositivity rates of 35–90% by adulthood [15,16]. Detection of HPyV6 DNA on skin swabs was common in both immunocompetent and immunocompromised subjects [17,18]. The presence of HPyV6 has not yet been associated with a specific disease [19]. To date there is only one publication that tested for HPyV6 DNA in several body fluids, detecting it in 2/1232 respiratory secretions and in 1/185 fecal samples, but not in 161 blood, 171 CSF and 189 urine from either healthy or symptomatic subjects [20]. Here, HPyV6 genome was detected in the CSF from an HIVpositive patient, affected with JCV-negative leukoencephalopathy. JCV being a recognized cause of PML in immunodeficient subjects provides a precedent for polyomavirus causing neurological symptoms but alternative explanations should also be considered. Since HPyV6 has been commonly detected in skin swabs, precautions (disposable plastic, gloves) were taken both during the CSF collection, and during sample preparation, to prevent contamination. It is nonetheless possible that the HPyV6 sequences originated from the skin during CSF collection as happen with bacterial contamination of blood [21,22] after venipuncture. Our detection of papillomavirus sequences in the CSF, an observation
[1] B.R. Brooks, D.L. Walker, Progressive multifocal leukoencephalopathy, Neurol. Clin. 2 (1984) 299–313. [2] J.R. Berger, Progressive multifocal leukoencephalopathy and newer biological agents, Drug Saf. 33 (2010) 969–983. [3] M.S. Dworkin, A review of progressive multifocal leukoencephalopathy in persons with and without AIDS, Curr. Clin. Top. Infect. Dis. 22 (2002) 181–195. [4] M.A. Lima, F. Bernal-Cano, D.B. Clifford, R.T. Gandhi, I.J. Koralnik, Clinical outcome of long-term survivors of progressive multifocal leukoencephalopathy, J. Neurol. Neurosurg. Psychiatry 81 (2010) 1288–1291. [5] T.D. Langford, S.L. Letendre, G.J. Larrea, E. Masliah, Changing patterns in the neuropathogenesis of HIV during the HAART era, Brain Pathol. 13 (2003) e195–e210. [6] F.R. Guerini, S. Delbue, M. Zanzottera, C. Agliardi, M. Saresella, R. Mancuso, R. Maserati, E. Marchioni, A. Gori, P. Ferrante, Analysis of CCR5CCR2, SDF1 and RANTES gene polymorphisms in subjects with HIV-related PML and not determined leukoencephalopathy, Biomed. Pharmacother. 62 (2008) 26–30. [7] S. Delbue, G. Sotgiu, D. Fumagalli, M. Valli, E. Borghi, R. Mancuso, E. Marchioni, R. Maserati, P. Ferrante, A case of a progressive multifocal leukoencephalopathy patient with four different JC virus transcriptional control region rearrangements in cerebrospinal fluid, blood, serum, and urine, J. Neurovirol. 11 (2005) 51–57. [8] S. Delbue, F. Elia, C. Carloni, E. Tavazzi, E. Marchioni, S. Carluccio, L. Signorini, S. Novati, R. Maserati, P. Ferrante, JC virus load in cerebrospinal fluid and transcriptional control region rearrangements may predict the clinical course of progressive multifocal leukoencephalopathy, J. Cell Physiol. 227 (2012) 3511–3517. [9] S. Kwok, R. Higuchi, Avoiding false positives with PCR, Nature 339 (1989) 237–238. [10] E.A. Siebrasse, I. Bauer, L.R. Holtz, B.-M. Le, S. Lassa-Claxton, et al., Human polyomaviruses in children undergoing transplantation, United States, 2008–2010, Emerging Infect. Dis. 18 (2012) 1676–1679. [11] R.M. Schowalter, D.V. Pastrana, K.A. Pumphrey, A.L. Moyer, C.B. Buck, Merkel cell polyomavirus and two previously unknown polyomaviruses are chronically shed from human skin, Cell Host Microbe 7 (2010) 509–515. [12] J.L. Casado, I. Corral, J. García, J. Martinez-San Millán, E. Navas, A. Moreno, S. Moreno, Continued declining incidence and improved survival of progressive multifocal leukoencephalopathy in HIV/AIDS patients in the current era, Eur. J. Clin. Microbiol. Infect. Dis. 33 (2014) 179–187. [13] A. Tretiakova, B. Krynska, J. Gordon, K. Khalili, Human neurotropic JC virus early protein deregulates glial cell cycle pathway and impairs cell differentiation, J. Neurosci. Res. 55 (1999) 588–599. [14] D. Kaniowska, R. Kaminski, S. Amini, S. Radhakrishnan, J. Rappaport, E. Johnson, K. Khalili, L. Del Valle, A. Darbinyan, Cross-interaction between JC virus agnoprotein and human immunodeficiency virus type 1 (HIV-1) Tat modulates transcription of the HIV-1 long terminal repeat in glial cells, J. Virol. 80 (2006) 9288–9299. [15] J.T. Nicol, R. Robinot, A. Carpentier, G. Carandina, E. Mazzoni, M. Tognon, A. Touzé, P. Coursaget, Age-specific seroprevalences of merkel cell polyomavirus, human polyomaviruses 6, 7, and 9: and trichodysplasia spinulosa-associatedpolyomavirus, Clin. Vaccine Immunol. 20 (2013) 363–368.
I approved the final manuscript.
The study protocol was approved by the local ethical review boards (IRB numbers 1835 and 2319).
S. Delbue et al. / Journal of Clinical Virology 68 (2015) 24–27 [16] E. van der Meijden, S. Bialasiewicz, R.J. Rockett, S.J. Tozer, T.P. Sloots, M.C. Feltkamp, Different serologic behavior of MCPyV, TSPyV, HPyV6, HPyV7 and HPyV9 polyomaviruses found on the skin, PLoS ONE 8 (2013) e81078. [17] U. Wieland, S. Silling, M. Hellmich, A. Potthoff, H. Pfister, A. Kreuter, Human polyomaviruses 6, 7, 9, 10 and Trichodysplasia spinulosa-associated Polyomavirus in HIV-infected men, J. Gen. Virol. 95 (2014) 928–932. [18] D. Schrama, L. Groesser, S. Ugurel, C. Hafner, D.V. Pastrana, C.B. Buck, L. Cerroni, A. Theiler, J.C. Becker, Presence of human polyomavirus 6 in mutation-specific BRAF inhibitor-induced epithelial proliferations, JAMA Dermatol. 150 (2014) 1180–1186. [19] E.J. Duncavage, J.D. Pfeifer, Human polyomaviruses 6 and 7 are not detectable in Merkel cell polyomavirus-negative Merkel cell carcinoma, J. Cutan. Pathol. 38 (2011) 790–796. [20] R.J. Rockett, T.P. Sloots, S. Bowes, N. O’Neill, S. Ye, J. Robson, D.M. Whiley, S.B. Lambert, D. Wang, M.D. Nissen, S. Bialasiewicz, Detection of novel polyomaviruses, TSPyV, HPyV6, HPyV7, HPyV9 and MWPyV in feces, urine, blood, respiratory swabs and cerebrospinal fluid, PLoS ONE 8 (2013) e62764.
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[21] C.P. McDonald, A. Roy, P. Mahajan, R. Smith, A. Charlett, J.A. Barbara, Relative values of the interventions of diversion and improved donor-arm disinfection to reduce the bacterial risk from blood transfusion, Vox Sang. 86 (2004) 178–182. [22] C. Bruneau, P. Perez, M. Chassaigne, P. Allouch, A. Audurier, C. Gulian, G. Janus, G. Boulard, P. De Micco, L.R. Salmi, L. Noel, Efficacy of a new collection procedure for preventing bacterial contamination of whole-blood donations, Transfusion 41 (2001) 74–81. [23] A.L. Greninger, S.N. Naccache, K. Messacar, A. Clayton, G. Yu, S. Somasekar, S. Federman, D. Stryke, C. Anderson, S. Yagi, S. Messenger, D. Wadford, D. Xia, J.P. Watt, K. Van Haren, S.R. Dominguez, C. Glaser, G. Aldrovandi, C.Y. Chiu, A novel outbreak enterovirus D68 strain associated with acute flaccid myelitis cases in the USA (2012–14): a retrospective cohort study, Lancet. Infect. Dis. (Suppl. 1473–3099) (2015) 70093–70099.
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Human polyomavirus 6 DNA in the cerebrospinal fluid of an HIV-positive patient with leukoencephalopathy Serena Delbue a,∗ , Francesca Elia a , Lucia Signorini a , Ramona Bella a , Sonia Villani a , Enrico Marchioni b , Pasquale Ferrante a , Tung Gia Phan c,d , Eric Delwart c,d a
Laboratory of Translational Medicine, Department of Biomedical, Surgical and Dental Sciences, University of Milano, Italy Department of General Neurology, IRCCS National Neurological Institute C. Mondino Foundation, Pavia, Italy c Blood Systems Research Institute, San Francisco, CA 94118, USA d Department of Laboratory Medicine, University of California at San Francisco, San Francisco, CA 94118, USA b
a r t i c l e
i n f o
Article history: Received 11 March 2015 Received in revised form 17 April 2015 Accepted 21 April 2015 Keywords: Polyomaviruses Leukoencephalopathy Cerebrospinal fluid Deep sequencing
a b s t r a c t Background: Leukoencephalopathies in HAART-treated, HIV-positive patients include progressive multifocal leukoencephalopathy (PML), a result of lytic infection oligodendrocytes by JC polyomavirus (JCV), and another form characterized by the absence of JCV genome in cerebrospinal fluid (CSF). Objectives: To test the potential viral etiology of JCV-negative leukoencephalopathy. Study design: CSF was collected from 43 HIV-positive patients with MRI suggestive of leukoencephalopathies. DNA was isolated and real-time PCR assays for neurotropic viruses (Herpes Simplex Viruses 1/2, Varicella Zoster Virus, Epstein Barr Virus, Human Cytomegalovirus, Human Herpesvirus 6, JCV and HIV) were conducted. CSF from 14 non-reactive cases were subjected to random nucleic acid amplification, deep sequencing, and in silico search for viral sequences. Results: JCV genome was detected in the CSF of 19/43 PML patients, HIV genome in the CSF of 5 PML patients including 2 JCV negative patients, and no viruses were detected in 22 patients. Human Polyomavirus 6 (HPyV6) DNA was detected by deep sequencing in one JCV-negative leukoencephalopathy CSF sample. Conclusions: HPyV6 DNA was detected in CSF of a case of demyelinating disease. HPyV6 has not been previously reported in CSF or associated with any disease. Demonstrating a causative role will require further studies. © 2015 Elsevier B.V. All rights reserved.
1. Background Progressive multifocal leukoencephalopathy (PML) is a severe, often fatal, demyelinating disease of the white matter of the central nervous system (CNS), due to polyomavirus JC (JCV) lytic infection of oligodendrocytes [1]. PML occurs in severely immunocompromised patients, such as in the context of HIV infection, hematological malignancy, exposure to chemotherapy or immunomodulatory antibodies, transplantation and various causes of lymphocyte depletion [2,3]. Although the introduction of highly active antiretroviral therapy (HAART) significantly reduced PML mortality, it remains the third most common neurological infectious disease in HIV/AIDS, following Toxoplasma
∗ Corresponding author at: Department of Biomedical, Surgical and Dental Sciences, University of Milano, Via Pascal, 36 -20133 Milano, Italy. Tel.: +39 0250315070. E-mail address: [email protected] (S. Delbue). http://dx.doi.org/10.1016/j.jcv.2015.04.016 1386-6532/© 2015 Elsevier B.V. All rights reserved.
encephalitis and HIV encephalopathy [4]. Additionally, atypical forms with an uncommon clinical presentation were described. These pathologies were characterized by the presence of demyelinating lesions in magnetic resonance imaging (MRI) and mild symptoms resembling those of PML but in the absence of the JCV in the cerebrospinal fluid (CSF) [5,6]. 2. Objectives To gather new insights on the potential viral etiology of the JCVnegative forms of leukoencephalopathies. 3. Study design 3.1. Patients From 2007 to 2010, 43 HIV-positive patients, showing MRI lesions indicative of demyelinating leukoencephalopathy, were
S. Delbue et al. / Journal of Clinical Virology 68 (2015) 24–27
25
Fig. 1. Sequence alignment of the HPyV6 gene fragment found in the CSF of a JCV-negative patient by deep sequencing analysis, with the HM011561 HPyV6 sequence.
identified from those patients who underwent lumbar puncture at the Neurological Department of the Mondino Institute, Pavia. CSF was collected for diagnosis purpose and part of the clinical specimen was stored at −80 ◦ C for research purpose. The patients’ demographic and immune-virological characteristics were acquired from clinical records. The study protocol was approved by the local ethical review boards (IRB numbers 1835 and 2319), and all patients provided written informed consent.
Table 1 Clinical and demographic features of the patients. Sex
31 Male; 12 Female
Age ± sd (years) CD4 (cells/mmc ± sd) HIV viremia (copies/ml ± sd) Risk factor (sex/drug abuse/maternal) HIV duration (years ± sd) Therapy duration (years ± sd) Presence of neurological symptoms (yes/no)
42.6 ± 8.6 266.8 ± 173.2 155450.8 ± 8000 11/31/1 10.5 ± 5.3 4.6 ± 7.9 31/12
4. Virological assays 4.1. Nucleic acids isolations and real-time PCRs (Q-PCRs) assays JCV and Herpesviruses loads were quantified after the viral DNA was extracted from 0.15 ml CSF, which was done using the Nucleospin RNA virus kit (MachereyNagels, Germany) according to the manufacturers’ instructions. The HIV loads were quantified after the viral RNA was extracted from 0.14 mL CSF using the Qiamp viral RNA mini kit (QIAGEN, USA), according to the manufacturer’s instructions. The Q-PCR protocol for the detection of JCV DNA, particularly for targeting the LT region, has been described elsewhere [7]. To detect the presence of the Herpes Simplex Virus 1 and 2 (HSV 1/2), Varicella Zoster Virus (VZV), Epstein Barr Virus (EBV), Human CytomegaloVirus (HCMV), Human Herpes Virus 6 (HHV 6) DNA, different Q-PCR were performed using primers and probes whose sequences are proprietary (Applied Biosystem, UK). To detect the presence of the HIV genome, a Q-PCR targeting the gag gene was performed, as described before [8]. Each sample was analyzed in triplicate, and each run contained a negative control consisting of the reaction mixture without the DNA template. Standard curves for quantification of viral genome were constructed using serial dilutions of a plasmid containing the whole viral genome (range: 10–106 plasmid copies/mL). Data were expressed as log [copies/CSF mL]. As the Q-PCR is highly sensitive, strict precautionary measures were taken to avoid cross-contamination [9]: separate rooms were used to extract the nucleic acids, to prepare the amplification mixes, and to run the Q-PCR; multiple negative controls (containing water instead of DNA templates) were included with each Q-PCR batch. 4.1.1. Deep sequencing and bioinformatic analysis A total of 14 CSF samples non-reactive for the tested viruses were analyzed in pools of three to four samples using a
metagenomics approach. Samples were pooled to reduce cost. Total nucleic acids were extracted using a Maxwell® 16 automated extractor (Promega) from supernatant of CSF spun 15 min in a microfuge. A DNA library was constructed using ScriptSeqTM v2 RNA-Seq Library Preparation Kit (Epicentre) which amplifies both RNA and DNA, and sequenced using the Illumina MiSeq platform. Viral sequences were identified through translated protein sequence similarity search (BLASTx) to annotated viral proteins available in GenBank’s viral RefSeq database. 4.1.2. Human Polyomavirus 6 (HPyV6) PCR amplification The 14 CSF samples analyzed by deep sequencing were subjected to PCR targeting the HPyV6 VP1 region to identify the positive individual sample. The primers employed have been previously published [10]; the annealing temperature of the first PCR (30 cycle) was 60 ◦ C, and 5 l of the PCR products were than subjected to further 25 cycle of amplification, with the annealing temperature of 62 ◦ C. 5. Results 5.1. Patients Clinical and demographic characteristics of the 43 enrolled patients are summarized in Table 1. 5.2. Q-PCRs assays JCV genome was detected in the CSF of 19/43 patients (44.2%), diagnosed as PML patients, with a mean viral load of log 4.7 copies/mL (range: log 3.23–6.9 copies/mL); HIV genome was detected in the CSF of 5/19 PML patients (26.3%), with a mean
Table 2 Virological findings in CSF collected from HIV patients affected with leukoencephalopathies.
CSF+/total (by Q- PCRs) CSF+/total (by deep sequencing) a b
Herpesviruses DNA
JCV DNA
HIV RNA
HPyV6
0/43 0/14
19a /43 (44.2%) 0/14
7a /43 (16.3%) 0/14
n.a. 1/14b (7.1%)
5 CSF contained both JCV and HIV genomic sequences. Deep sequencing was performed on 14 not reactive CSF and the finding was confirmed by PCR n.a. not available.
26
S. Delbue et al. / Journal of Clinical Virology 68 (2015) 24–27
viral load of log 2.8 copies/ml (range: log 1.7–6.0 copies/mL) and in 2/22 (9.1%) JCV negative patients, with a mean viral load of log 1.9 copies/ml (log 1.7–4.07 copies/mL); Human Herpesvirus genomes was not detected in any of the analyzed CSF (Table 2). Consequently, 22 cases were diagnosed as JCV-negative leukoencephalopathy patients.
previously reported in CSF [23], supports the possibility of contamination with skin-associated viruses. Whether HPyV6 detection in CSF is associated with this patient’s neurological disease, is indicative of contamination with skin microbiota, or reflects increased blood-brain permeability is unknown. The involvement of HPyV6 in JCV-negative leukoencephalopathy therefore remains theoretical pending further investigations.
5.3. Deep sequencing and bioinformatic analysis Translated sequence reads showing similarity to viral sequences were identified using BLASTx. Using a BLASTx cutoff of E scores <10−5 one pool of four CSF samples containing 3.3 × 106 sequence reads showed the presence of 6 anellovirus reads, 70 papillomavirus reads (data not shown), and 4 HPyV6 reads (Fig. 1). 5.4. HPyV6 PCR
Funding This work was partially supported by an Italian Minister of the University Grant (PRIN 2010- 2011) to Pasquale Ferrante and NHLBI grant R01HL105770 to E.L.D. Conflict of interest
PCR specific for HPyV6 DNA was performed on the 14 notreactive CSF and confirmed the presence of the HPyV6 genome in one of the CSF in the positive pool, collected from a 39 years old male patient with CD4 cell count 320/mm2 , HIV-positive for at least 24 months before the CSF collection, with MRI lesions resembling those of PML. The PCR product was sequenced and the result confirmed the presence of HPyV6 genome (Fig. 1).
Ethical approval
6. Discussion
References
Virological analysis of CSF from HIV-positive patients with leukoencephalopathy was conducted, and the rate of JCV findings was within the expectation. A recently published study observed that JCV was detected in the CSF of 44% HIV-positive patients presenting MRI lesions accounting for leukoencephalopathies, and these patients were classified as PML patients, whereas the others (JCV negative) were classified as possible PML, since no viral genome was detected in the CSF [11]. The concomitant presence of both JCV and HIV in the CSF of 26.3% PML patients was also not surprising [8], since it is well known the synergistic effect of HIV and JCV [12]. Indeed, evidence for a direct role of HIV in JCV activation comes from several studies showing up-regulation of the JCV late promoter by the HIV-encoded regulatory protein, Tat [13]. The HPyV6 genome was first described in 2010, by Buck et al., who detected it in 5/35 skin swab specimens collected from healthy subjects. Additionally, that group showed that antibody reactivity against HPyV6 was very common, with 69% healthy subjects scoring seropositive [14]. Other studies also detected persistent infections with HPyV6 and seropositivity rates of 35–90% by adulthood [15,16]. Detection of HPyV6 DNA on skin swabs was common in both immunocompetent and immunocompromised subjects [17,18]. The presence of HPyV6 has not yet been associated with a specific disease [19]. To date there is only one publication that tested for HPyV6 DNA in several body fluids, detecting it in 2/1232 respiratory secretions and in 1/185 fecal samples, but not in 161 blood, 171 CSF and 189 urine from either healthy or symptomatic subjects [20]. Here, HPyV6 genome was detected in the CSF from an HIVpositive patient, affected with JCV-negative leukoencephalopathy. JCV being a recognized cause of PML in immunodeficient subjects provides a precedent for polyomavirus causing neurological symptoms but alternative explanations should also be considered. Since HPyV6 has been commonly detected in skin swabs, precautions (disposable plastic, gloves) were taken both during the CSF collection, and during sample preparation, to prevent contamination. It is nonetheless possible that the HPyV6 sequences originated from the skin during CSF collection as happen with bacterial contamination of blood [21,22] after venipuncture. Our detection of papillomavirus sequences in the CSF, an observation
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I approved the final manuscript.
The study protocol was approved by the local ethical review boards (IRB numbers 1835 and 2319).
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