Clinical Infectious Diseases Advance Access published May 28, 2015
1
Deep sequencing of HIV-1 in cerebrospinal fluid
Marta2,5, Ranjababu Kulasegaram6, Simon Rackstraw7,8 1
Department of Infection, Barts Health NHS Trust, London, UK
2
rip t
C.Y. William Tong1,2, Sinead Costelloe3, Jonathan Hubb1, Jane Mullen4, Siobhan O’Shea4, Monica
Blizard Institute, Barts & The London School of Medicine and Dentistry, Queen Mary University of
3
Directorate of Infection, Guy’s and St Thomas’ NHS Foundation Trust, London, UK
4
Infection Sciences, Viapath, Guy’s and St Thomas’ NHS Foundation Trust, London, UK
5
Department of Neurosciences, Barts Health NHS Trust, London, UK
6Department
and Immunology, Barts Health NHS Trust, London, UK
8Department
of Medicine, Mildmay Hospital UK, London, UK
M
7Infection
of Genitourinary Medicine, Guy’s and St Thomas’ NHS Foundation Trust, London, UK
ed
Correspondence: Dr C. Y. William Tong, Department of Infection (Virology), Barts Health NHS Trust
Ac
ce
pt
3rd Floor Pathology and Pharmacy Building, 80 Newark Street, London E1 2ES, [email protected]
© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected].
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
an us c
London, London, UK
2
Abstract Using deep sequencing, HIV resistance-associated mutations (RAM) were detected as minority
rip t
species in the CSF of 4 patients with higher CSF HIV-1 viral load than plasma, but not in 2 patients with higher plasma viral load. Deep sequencing could help our understanding of viral escape in the
M ed pt ce Ac
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
an us c
central nervous system.
3
Combination antiretroviral therapy (ART) is highly effective in the suppression of HIV-1 replication. However, recent reports have highlighted the possibility of viral escape in the central nervous
rip t
system (CNS) [1-3], which is believed to be due to compartmentalisation [4]. Other studies have found evidence of a greater diversity of viral strains in CSF [5], which may harbour resistance-
associated mutations (RAM) [1, 2]. The lack of penetration of some ART agents into the CNS was
an us c
postulated to account for this phenomenon [6], though this hypothesis is controversial [7-10]. The cause and significance of CNS viral escape remains poorly understood [6].
Minority viral species representing 1.0 log10) CSF HIV-1 viral load compared to plasma (mean: 1.87;
an us c
range: 1.28 – 2.79 log10 higher), whereas the CSF viral loads in patients 5 and 6 were lower than
plasma. All 6 patients had Magnetic Resonance Imaging (MRI) of the brain. The presence of white
patient 6 showed encephalomalacia and volume loss but no signal changes.
The plasma viral load of patient 4 was too low (47 copies/ml) to allow successful sequencing by both Sanger and deep sequencing. Sequencing was successful for all other samples by both
M
methods. Apart from one minor mutation (Q58E in patient 4) detected only by Sanger sequencing and a discrepancy in the mutation M184V or M184I in a plasma sample (patient 2), all the mutations
ed
identified by the Sanger method in plasma or CSF were also detected by deep sequencing. Deep sequencing did, however, identify a number of additional mutations not detected by the Sanger
pt
method, mainly in the CSF (patients 1 – 4) but also in plasma (patients 2 and 3). In the two patients (5 and 6) where the CSF viral load was lower than plasma, no additional mutations were detected by
ce
deep sequencing in either plasma or CSF.
The additional mutations detected by deep sequencing were mostly present as minority
Ac
species, which ranged from 3 to 40.1%. In patient 1, N88S was detected in CSF at a level of 8.8%. This is likely to be selected as a result of the previous use of nelfinavir. In patient 2, both M184V and M184I were detected in CSF by Sanger sequencing. However, deep sequencing was more precise, showing that 5.9% were M184I and 94.1% M184V. The result in plasma was significantly different in that Sanger sequencing only detected M184I, whereas deep sequencing only detected M184V. In addition, minority species of mutations in PR (V82I – 40.1%) and RT (D67G, K70S, two species of
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
matter signal changes suggestive of HIV encephalopathy was reported in patients 1 – 5. The MRI of
6 K70N) were only detected in the CSF of this patient using deep sequencing. In patient 3, the presence of K65R (CSF 2.4%) and K65N (plasma 11.7%, CSF 7.8%) were most likely selected by tenofovir; in patient 4, detection of I47V in CSF (3%) could be related to the previous and current use
rip t
of protease inhibitors including lopinavir and darunavir. A regimen change was made in patients 1 – 4 (Table 1). The choice of antiretroviral agents in the new regimen was largely based on a perceived improvement in CNS penetration. All patients
an us c
showed clinical improvement and one (patient 1) had radiological improvement. Patients 5 and 6 did not have regimen change. Patient 5 was empirically treated for tuberculosis and clinically improved.
DISCUSSION
To our knowledge, this is the first study using deep sequencing to characterise HIV-1 in the CSF of
M
patients with neurological symptoms suggestive of HIV encephalopathy. The mutations detected by deep sequencing showed good correlation with the standard Sanger method, and additional
ed
minority species in plasma and CSF were identified. The discrepancy in the M184V/I mutations and failure of the Sanger method to identify V82I at 40% in patient 2 could be due to amplification bias
pt
introduced at the PCR stage and inaccuracy of the current sequencing technology. Improved deep sequencing methods are now available and could help to address such problems.
ce
The presence of minority species in CSF was observed only in cases where the CSF viral load was significantly higher than that of plasma. This is consistent with the compartmental theory and
Ac
differential evolution of HIV-1 in the CNS. The RAMs detected in CSF were likely to be present as a result of previous or current exposure to specific ART agents; their absence in plasma could be related to a difference in drug pressure in the two compartments. The presence of RAMs as minority species in CSF could be due to a relative lack of CNS penetration of certain ART regimens or a result of poor adherence. A lack of drug selection pressure in CSF allows independent evolution of a full
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
Patient 6 died of an unrelated cause.
7 repertoire of viruses containing minority species, while such resistant viruses in plasma are suppressed. In the two patients (5 and 6) where the viral loads in CSF were lower than in plasma, viral
rip t
loads in both compartments were probably sustained by poor adherence as no relevant RAMs were present. Although patient 5 had some radiological features suggestive of HIV encephalopathy, this may not be the predominant pathology as she responded to empirical treatment for tuberculosis.
an us c
While the regimen change in patients 1 - 4 was aimed at improving antiviral penetration to
the CNS [8], better engagement and improved adherence may have been the reason for the clinical
study is limited by the small number of patients and the retrospective nature of the analysis with testing carried out at two different laboratories. Two of the six paired samples were more than 2 weeks apart which may have introduced bias to the sequence analysis. Also, not all patients had
M
radiological follow-up to confirm improvement. In addition, there were no follow-up samples to confirm the improvement in CSF viral load after the regimen change. Nevertheless, this study
ed
demonstrates the value of deep sequencing in improving our understanding of viral escape in the CNS. With improvements in sequencing technology and reduction in costs of deep sequencing, this
Notes
pt
could transform our approach to the analysis of HIV-1 sequences in different body compartments.
Ac
ce
The authors have no reported conflicts related to the content of this manuscript.
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
improvement. Re-treatment using susceptible ART with good compliance should be considered. This
8 REFERENCES 1. Canestri A, Lescure FX, Jaureguiberry S, et al. Discordance between cerebral spinal fluid and plasma HIV replication in patients with neurological symptoms who are receiving suppressive
rip t
antiretroviral therapy. Clin Infect Dis. 2010;50(5):773-8.
2. Peluso MJ, Ferretti F, Peterson J, et al. Cerebrospinal fluid HIV escape associated with progressive
AIDS. 2012;26(14):1765-74.
3. Béguelin C, Vázquez M, Bertschi M, et al. Viral escape in the CNS with multidrug-resistant HIV-1. J Int AIDS Soc. 2014;17(4 Suppl 3):19745.
M
4. Schnell G, Price RW, Swanstrom R, Spudich S. Compartmentalization and clonal amplification of
ed
HIV-1 variants in the cerebrospinal fluid during primary infection. J Virol. 2010;84(5):2395-407.
5. Soulié C, Fourati S, Lambert-Niclot S, et al. HIV genetic diversity between plasma and
pt
cerebrospinal fluid in patients with HIV encephalitis. AIDS. 2010;24(15):2412-4.
ce
6. Nightingale S, Winston A, Letendre S, et al. Controversies in HIV-associated neurocognitive
Ac
disorders. Lancet Neurol. 2014 Nov;13(11):1139-51.
7. Rawson T, Muir D, Mackie NE, Garvey LJ, Everitt A, Winston A. Factors associated with cerebrospinal fluid HIV RNA in HIV infected subjects undergoing lumbar puncture examination in a clinical setting. J Infect. 2012;65(3):239-45.
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
an us c
neurologic dysfunction in patients on antiretroviral therapy with well controlled plasma viral load.
9 8. Ciccarelli N, Fabbiani M, Colafigli M, et al. Revised central nervous system neuropenetrationeffectiveness score is associated with cognitive disorders in HIV-infected patients with controlled
rip t
plasma viraemia. Antivir Ther. 2013;18(2):153-60.
9. Cusini A, Vernazza PL, Yerly S, et al. Higher CNS penetration-effectiveness of long-term
combination antiretroviral therapy is associated with better HIV-1 viral suppression in cerebrospinal
10. Libertone R, Lorenzini P, Balestra P, et al. Central nervous system penetration-effectiveness rank does not reliably predict neurocognitive impairment in HIV-infected individuals. J Int AIDS Soc. 2014;17(4 Suppl 3):19655.
M
11. Ram D, Leshkowitz D, Gonzalez D, et al. Evaluation of GS Junior and MiSeq next-generation sequencing technologies as an alternative to Trugene population sequencing in the clinical HIV
ed
laboratory. J Virol Methods. 2014;212C:12-16.
pt
12. Mohamed S, Penaranda G, Gonzalez D, et al. Comparison of ultra-deep versus Sanger sequencing detection of minority mutations on the HIV-1 drug resistance interpretations after virological failure.
Ac
ce
AIDS. 2014;28(9):1315-24.
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
an us c
fluid. J Acquir Immune Defic Syndr. 2013;62(1):28-35.
an us cr ipt
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
10
Table 1. Characteristics and comparative sequencing results of the six patients using standard Sanger and deep sequencing methods. Abbreviations of antiretroviral agents: ABC:abacavir; ATV:atazanavir; DDI:didanosine; DRV:darunavir; D4T:stavudine; EFV:efavirenz; FPV:foasamprenavir; FTC:emtricitabine; IDV:indinavir; LPV:lopinavir; MVC: maraviroc; NFV:nelfinavir; NVP:nevirapine; r:ritonavir boosted protease inhibitor; SQV:saquinavir; TDF:tenofovir; ZDV:zidovudine; 3TC:lamivudine. NA: Not applicable.
1 63 Male A
2 42 Female C
3 35 Female C
4 28 Female CRF06_cpx
ART previously exposed to
ABC, D4T, DDI, TDF, ZDV, 3TC EFV, NVP DRVr, IDVr, LPVr, NFV
TDF, ZDV, FTC, 3TC ATVr, LPVr
TDF, ZDV, FTC, 3TC NVP ATVr
TDF, FTC ATVr, DRVr, LPVr
DRVr
TDF, FTC, ATVr
5 months
18 months
confusion, poor executive function, impaired memory and psychomotor slowness
headache, tiredness and lethargy
CSF viral load (copies/mL)
522803
5205
Plasma viral load (copies/mL)
848
No. of days between plasma and CSF Log10 differences in viral load (CSF - plasma)
37 2.79
3TC, DRVr
ABC, 3TC, DRVr
3 years 4 months
Intermittent
seizure (previous treated cerebral toxoplasmosis)
Headache, photophobia
Chest symptoms, acute on chronic confusion
79478
894
42959
6536
213
778
47
135760
23505
-1
1
15
-8
-3
1.39
2.01
1.28
-0.53
-0.55
TDF, FTC, DRVr
5 years
2 years
ed
TDF, FTC, ATVr
recurrent headache
pt
ce
Clinical presentation
6 57 Male B
Ac
ART regimen at presentation Duration of last ART regimen
5 52 Female A ABC, D4T, DDI, TDF, ZDV, FTC, 3TC NVP ATVr, FPVr, IDVr, LPVr, SQVr
M
Patient Age Gender HIV-1 Clade
Not known (Poor adherence)
145
119
336
68
43
225
5
65%
80%
90%
0.85
1.11
2.32
0.47
Glucose (mmol/L)
3
3.3
1.9
2.7
MRI with white matter changes suggestive of HIV encephalopathy
Yes
Yes
Yes
% lymphocytes Protein (g/dL)
L10V, I13L, G16E, K20R, I89M
K20R, M36I, D60E, L63P, L89I, I93L
RT mutations detected by both methods in plasma and CSF
V179I
V90I, M184I (CSF only), M184V
None
None
None
M184I (100%)
890
481
222
42
NA
Yes
Sequence not available for plasma due to low viral load. CSF - I13V, G16E, K20I, M36I. Q58E (population sequencing only), L89M Sequence not available for plasma due to low viral load. CSF - K65R, M41L, M184V
100%
100%
2.03
0.38
3.2
3.4
Yes
No
L10F, I13V, K20R, V32I, M36I, I47V, L89M
L10V, A71T, V77I, I93L
V179I
None
None
Sequence not available for plasma due to low viral load.
None
None
K65N (11.7%)
Sequence not available for plasma due to low viral load.
None
None
pt
ce
None
Ac
Additional PR mutations detected by deep sequencing in plasma Additional RT mutations detected by deep sequencing in plasma Additional PR mutations detected by deep sequencing in CSF Additional RT
I93L, L89M
ed
PR mutations detected by both methods in plasma and CSF
M
CSF wbc (/L)
11
an us cr ipt
612
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
CD4 count (/L)
N88S (8.8%)
V82I (40.1%)
None
I47V (3%)
None
None
None
D67G (5.75%), K70N
K65N (7.8%), K65R
None
None
None
TDF, FTC, DRVr, MVC
No follow-up MRI. Clinically improved
No follow-up MRI. Clinically improved
No follow-up MRI. Clinically improved
ed pt
12
an us cr ipt
ABC, 3TC, DRVr
M
Significant MRI improvement after 1 year
ABC, 3TC, DRVr
ce
Follow-up
ABC, 3TC, DRVr, MVC
(2.4%)
Ac
New ART introduced after LP
(AAC; 31%), K70N (AAT; 1.5%), K70S (5.8%), M184I (5.9%), M184V (94.1%)
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
mutations detected by deep sequencing in CSF
None
None
Back to baseline after empiric TB treatment
Died (Carcinoma of lung with metastasis)
1
Deep sequencing of HIV-1 in cerebrospinal fluid
Marta2,5, Ranjababu Kulasegaram6, Simon Rackstraw7,8 1
Department of Infection, Barts Health NHS Trust, London, UK
2
rip t
C.Y. William Tong1,2, Sinead Costelloe3, Jonathan Hubb1, Jane Mullen4, Siobhan O’Shea4, Monica
Blizard Institute, Barts & The London School of Medicine and Dentistry, Queen Mary University of
3
Directorate of Infection, Guy’s and St Thomas’ NHS Foundation Trust, London, UK
4
Infection Sciences, Viapath, Guy’s and St Thomas’ NHS Foundation Trust, London, UK
5
Department of Neurosciences, Barts Health NHS Trust, London, UK
6Department
and Immunology, Barts Health NHS Trust, London, UK
8Department
of Medicine, Mildmay Hospital UK, London, UK
M
7Infection
of Genitourinary Medicine, Guy’s and St Thomas’ NHS Foundation Trust, London, UK
ed
Correspondence: Dr C. Y. William Tong, Department of Infection (Virology), Barts Health NHS Trust
Ac
ce
pt
3rd Floor Pathology and Pharmacy Building, 80 Newark Street, London E1 2ES, [email protected]
© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected].
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
an us c
London, London, UK
2
Abstract Using deep sequencing, HIV resistance-associated mutations (RAM) were detected as minority
rip t
species in the CSF of 4 patients with higher CSF HIV-1 viral load than plasma, but not in 2 patients with higher plasma viral load. Deep sequencing could help our understanding of viral escape in the
M ed pt ce Ac
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
an us c
central nervous system.
3
Combination antiretroviral therapy (ART) is highly effective in the suppression of HIV-1 replication. However, recent reports have highlighted the possibility of viral escape in the central nervous
rip t
system (CNS) [1-3], which is believed to be due to compartmentalisation [4]. Other studies have found evidence of a greater diversity of viral strains in CSF [5], which may harbour resistance-
associated mutations (RAM) [1, 2]. The lack of penetration of some ART agents into the CNS was
an us c
postulated to account for this phenomenon [6], though this hypothesis is controversial [7-10]. The cause and significance of CNS viral escape remains poorly understood [6].
Minority viral species representing 1.0 log10) CSF HIV-1 viral load compared to plasma (mean: 1.87;
an us c
range: 1.28 – 2.79 log10 higher), whereas the CSF viral loads in patients 5 and 6 were lower than
plasma. All 6 patients had Magnetic Resonance Imaging (MRI) of the brain. The presence of white
patient 6 showed encephalomalacia and volume loss but no signal changes.
The plasma viral load of patient 4 was too low (47 copies/ml) to allow successful sequencing by both Sanger and deep sequencing. Sequencing was successful for all other samples by both
M
methods. Apart from one minor mutation (Q58E in patient 4) detected only by Sanger sequencing and a discrepancy in the mutation M184V or M184I in a plasma sample (patient 2), all the mutations
ed
identified by the Sanger method in plasma or CSF were also detected by deep sequencing. Deep sequencing did, however, identify a number of additional mutations not detected by the Sanger
pt
method, mainly in the CSF (patients 1 – 4) but also in plasma (patients 2 and 3). In the two patients (5 and 6) where the CSF viral load was lower than plasma, no additional mutations were detected by
ce
deep sequencing in either plasma or CSF.
The additional mutations detected by deep sequencing were mostly present as minority
Ac
species, which ranged from 3 to 40.1%. In patient 1, N88S was detected in CSF at a level of 8.8%. This is likely to be selected as a result of the previous use of nelfinavir. In patient 2, both M184V and M184I were detected in CSF by Sanger sequencing. However, deep sequencing was more precise, showing that 5.9% were M184I and 94.1% M184V. The result in plasma was significantly different in that Sanger sequencing only detected M184I, whereas deep sequencing only detected M184V. In addition, minority species of mutations in PR (V82I – 40.1%) and RT (D67G, K70S, two species of
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
matter signal changes suggestive of HIV encephalopathy was reported in patients 1 – 5. The MRI of
6 K70N) were only detected in the CSF of this patient using deep sequencing. In patient 3, the presence of K65R (CSF 2.4%) and K65N (plasma 11.7%, CSF 7.8%) were most likely selected by tenofovir; in patient 4, detection of I47V in CSF (3%) could be related to the previous and current use
rip t
of protease inhibitors including lopinavir and darunavir. A regimen change was made in patients 1 – 4 (Table 1). The choice of antiretroviral agents in the new regimen was largely based on a perceived improvement in CNS penetration. All patients
an us c
showed clinical improvement and one (patient 1) had radiological improvement. Patients 5 and 6 did not have regimen change. Patient 5 was empirically treated for tuberculosis and clinically improved.
DISCUSSION
To our knowledge, this is the first study using deep sequencing to characterise HIV-1 in the CSF of
M
patients with neurological symptoms suggestive of HIV encephalopathy. The mutations detected by deep sequencing showed good correlation with the standard Sanger method, and additional
ed
minority species in plasma and CSF were identified. The discrepancy in the M184V/I mutations and failure of the Sanger method to identify V82I at 40% in patient 2 could be due to amplification bias
pt
introduced at the PCR stage and inaccuracy of the current sequencing technology. Improved deep sequencing methods are now available and could help to address such problems.
ce
The presence of minority species in CSF was observed only in cases where the CSF viral load was significantly higher than that of plasma. This is consistent with the compartmental theory and
Ac
differential evolution of HIV-1 in the CNS. The RAMs detected in CSF were likely to be present as a result of previous or current exposure to specific ART agents; their absence in plasma could be related to a difference in drug pressure in the two compartments. The presence of RAMs as minority species in CSF could be due to a relative lack of CNS penetration of certain ART regimens or a result of poor adherence. A lack of drug selection pressure in CSF allows independent evolution of a full
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
Patient 6 died of an unrelated cause.
7 repertoire of viruses containing minority species, while such resistant viruses in plasma are suppressed. In the two patients (5 and 6) where the viral loads in CSF were lower than in plasma, viral
rip t
loads in both compartments were probably sustained by poor adherence as no relevant RAMs were present. Although patient 5 had some radiological features suggestive of HIV encephalopathy, this may not be the predominant pathology as she responded to empirical treatment for tuberculosis.
an us c
While the regimen change in patients 1 - 4 was aimed at improving antiviral penetration to
the CNS [8], better engagement and improved adherence may have been the reason for the clinical
study is limited by the small number of patients and the retrospective nature of the analysis with testing carried out at two different laboratories. Two of the six paired samples were more than 2 weeks apart which may have introduced bias to the sequence analysis. Also, not all patients had
M
radiological follow-up to confirm improvement. In addition, there were no follow-up samples to confirm the improvement in CSF viral load after the regimen change. Nevertheless, this study
ed
demonstrates the value of deep sequencing in improving our understanding of viral escape in the CNS. With improvements in sequencing technology and reduction in costs of deep sequencing, this
Notes
pt
could transform our approach to the analysis of HIV-1 sequences in different body compartments.
Ac
ce
The authors have no reported conflicts related to the content of this manuscript.
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
improvement. Re-treatment using susceptible ART with good compliance should be considered. This
8 REFERENCES 1. Canestri A, Lescure FX, Jaureguiberry S, et al. Discordance between cerebral spinal fluid and plasma HIV replication in patients with neurological symptoms who are receiving suppressive
rip t
antiretroviral therapy. Clin Infect Dis. 2010;50(5):773-8.
2. Peluso MJ, Ferretti F, Peterson J, et al. Cerebrospinal fluid HIV escape associated with progressive
AIDS. 2012;26(14):1765-74.
3. Béguelin C, Vázquez M, Bertschi M, et al. Viral escape in the CNS with multidrug-resistant HIV-1. J Int AIDS Soc. 2014;17(4 Suppl 3):19745.
M
4. Schnell G, Price RW, Swanstrom R, Spudich S. Compartmentalization and clonal amplification of
ed
HIV-1 variants in the cerebrospinal fluid during primary infection. J Virol. 2010;84(5):2395-407.
5. Soulié C, Fourati S, Lambert-Niclot S, et al. HIV genetic diversity between plasma and
pt
cerebrospinal fluid in patients with HIV encephalitis. AIDS. 2010;24(15):2412-4.
ce
6. Nightingale S, Winston A, Letendre S, et al. Controversies in HIV-associated neurocognitive
Ac
disorders. Lancet Neurol. 2014 Nov;13(11):1139-51.
7. Rawson T, Muir D, Mackie NE, Garvey LJ, Everitt A, Winston A. Factors associated with cerebrospinal fluid HIV RNA in HIV infected subjects undergoing lumbar puncture examination in a clinical setting. J Infect. 2012;65(3):239-45.
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
an us c
neurologic dysfunction in patients on antiretroviral therapy with well controlled plasma viral load.
9 8. Ciccarelli N, Fabbiani M, Colafigli M, et al. Revised central nervous system neuropenetrationeffectiveness score is associated with cognitive disorders in HIV-infected patients with controlled
rip t
plasma viraemia. Antivir Ther. 2013;18(2):153-60.
9. Cusini A, Vernazza PL, Yerly S, et al. Higher CNS penetration-effectiveness of long-term
combination antiretroviral therapy is associated with better HIV-1 viral suppression in cerebrospinal
10. Libertone R, Lorenzini P, Balestra P, et al. Central nervous system penetration-effectiveness rank does not reliably predict neurocognitive impairment in HIV-infected individuals. J Int AIDS Soc. 2014;17(4 Suppl 3):19655.
M
11. Ram D, Leshkowitz D, Gonzalez D, et al. Evaluation of GS Junior and MiSeq next-generation sequencing technologies as an alternative to Trugene population sequencing in the clinical HIV
ed
laboratory. J Virol Methods. 2014;212C:12-16.
pt
12. Mohamed S, Penaranda G, Gonzalez D, et al. Comparison of ultra-deep versus Sanger sequencing detection of minority mutations on the HIV-1 drug resistance interpretations after virological failure.
Ac
ce
AIDS. 2014;28(9):1315-24.
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
an us c
fluid. J Acquir Immune Defic Syndr. 2013;62(1):28-35.
an us cr ipt
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
10
Table 1. Characteristics and comparative sequencing results of the six patients using standard Sanger and deep sequencing methods. Abbreviations of antiretroviral agents: ABC:abacavir; ATV:atazanavir; DDI:didanosine; DRV:darunavir; D4T:stavudine; EFV:efavirenz; FPV:foasamprenavir; FTC:emtricitabine; IDV:indinavir; LPV:lopinavir; MVC: maraviroc; NFV:nelfinavir; NVP:nevirapine; r:ritonavir boosted protease inhibitor; SQV:saquinavir; TDF:tenofovir; ZDV:zidovudine; 3TC:lamivudine. NA: Not applicable.
1 63 Male A
2 42 Female C
3 35 Female C
4 28 Female CRF06_cpx
ART previously exposed to
ABC, D4T, DDI, TDF, ZDV, 3TC EFV, NVP DRVr, IDVr, LPVr, NFV
TDF, ZDV, FTC, 3TC ATVr, LPVr
TDF, ZDV, FTC, 3TC NVP ATVr
TDF, FTC ATVr, DRVr, LPVr
DRVr
TDF, FTC, ATVr
5 months
18 months
confusion, poor executive function, impaired memory and psychomotor slowness
headache, tiredness and lethargy
CSF viral load (copies/mL)
522803
5205
Plasma viral load (copies/mL)
848
No. of days between plasma and CSF Log10 differences in viral load (CSF - plasma)
37 2.79
3TC, DRVr
ABC, 3TC, DRVr
3 years 4 months
Intermittent
seizure (previous treated cerebral toxoplasmosis)
Headache, photophobia
Chest symptoms, acute on chronic confusion
79478
894
42959
6536
213
778
47
135760
23505
-1
1
15
-8
-3
1.39
2.01
1.28
-0.53
-0.55
TDF, FTC, DRVr
5 years
2 years
ed
TDF, FTC, ATVr
recurrent headache
pt
ce
Clinical presentation
6 57 Male B
Ac
ART regimen at presentation Duration of last ART regimen
5 52 Female A ABC, D4T, DDI, TDF, ZDV, FTC, 3TC NVP ATVr, FPVr, IDVr, LPVr, SQVr
M
Patient Age Gender HIV-1 Clade
Not known (Poor adherence)
145
119
336
68
43
225
5
65%
80%
90%
0.85
1.11
2.32
0.47
Glucose (mmol/L)
3
3.3
1.9
2.7
MRI with white matter changes suggestive of HIV encephalopathy
Yes
Yes
Yes
% lymphocytes Protein (g/dL)
L10V, I13L, G16E, K20R, I89M
K20R, M36I, D60E, L63P, L89I, I93L
RT mutations detected by both methods in plasma and CSF
V179I
V90I, M184I (CSF only), M184V
None
None
None
M184I (100%)
890
481
222
42
NA
Yes
Sequence not available for plasma due to low viral load. CSF - I13V, G16E, K20I, M36I. Q58E (population sequencing only), L89M Sequence not available for plasma due to low viral load. CSF - K65R, M41L, M184V
100%
100%
2.03
0.38
3.2
3.4
Yes
No
L10F, I13V, K20R, V32I, M36I, I47V, L89M
L10V, A71T, V77I, I93L
V179I
None
None
Sequence not available for plasma due to low viral load.
None
None
K65N (11.7%)
Sequence not available for plasma due to low viral load.
None
None
pt
ce
None
Ac
Additional PR mutations detected by deep sequencing in plasma Additional RT mutations detected by deep sequencing in plasma Additional PR mutations detected by deep sequencing in CSF Additional RT
I93L, L89M
ed
PR mutations detected by both methods in plasma and CSF
M
CSF wbc (/L)
11
an us cr ipt
612
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
CD4 count (/L)
N88S (8.8%)
V82I (40.1%)
None
I47V (3%)
None
None
None
D67G (5.75%), K70N
K65N (7.8%), K65R
None
None
None
TDF, FTC, DRVr, MVC
No follow-up MRI. Clinically improved
No follow-up MRI. Clinically improved
No follow-up MRI. Clinically improved
ed pt
12
an us cr ipt
ABC, 3TC, DRVr
M
Significant MRI improvement after 1 year
ABC, 3TC, DRVr
ce
Follow-up
ABC, 3TC, DRVr, MVC
(2.4%)
Ac
New ART introduced after LP
(AAC; 31%), K70N (AAT; 1.5%), K70S (5.8%), M184I (5.9%), M184V (94.1%)
Downloaded from http://cid.oxfordjournals.org/ at New York University on June 7, 2015
mutations detected by deep sequencing in CSF
None
None
Back to baseline after empiric TB treatment
Died (Carcinoma of lung with metastasis)
