Download HTLV in plasma

HTLV-1 viral RNA is detected rarely in plasma of
HTLV-1 infected subjects.
Maria Antonietta Demontis, Maaz Tahir Sadiq, Simon
Golz and Graham P Taylor
Section of Infectious Diseases, Department of Medicine
Imperial College
Norfolk Place
London W2 1PG
Running head: Detection of HTLV-1 in plasma samples
Keywords: HTLV-1, detection, plasma, diagnosis, nested PCR, quantitative PCR,
HTLV-1 RNA.
Abstract Word Count 223
Word Count – Body of Text 1893
Corresponding author:
Graham P. Taylor, Professor of Human Retrovirology, Infectious Diseases, Department of
Medicine, St Mary's Campus, Imperial College Norfolk Place London W2 1PG
Telephone:
020 7594 3910
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Fax Number: 020 7594 3910
E-Mail: [email protected]
Abstract
Background: Plasma of patients infected with HTLV-1 is considered non-infectious but
detection of HTLV-1 genomic RNA in plasma has been recently reported. The aim of
this project was to detect and quantify HTLV-1 RNA in plasma and assess its potential
value in diagnosis and prognosis.
Methods: RNA from one milliliter of plasma from 65 subjects infected with HTLV-1 (27
asymptomatic
carriers
(AC),
17
patients
with
HTLV-1-associated
myelopathy
(HAM/TSP), 14 with Adult T-cell Leukaemia/Lymphoma (ATLL), two co-infected with
HIV and five with other HTLV-1-associated disease, was extracted and reverse
transcribed. HTLV-1 specific nested PCR was performed using primers to amplify the
conserved Tax region. All samples were run in quadruplicate, nested PCR products
were detected by gel electrophoresis.
Results: HTLV-1 RNA was detected in plasma from 18 (28%) patients, always at a very
low copy number (3-13 copies viral cDNA per milliliter of plasma). Mean values of
HTLV-1 proviral load did not differ between patients in whom HTLV-1 RNA was
detected and patients in whom it was not possible to detect HTLV-1 RNA in plasma.
Conclusions: HTLV-1 genomic RNA can be detected in the plasma of a minority of
patients but not at a level or frequency to be useful clinically or diagnostically. Lack of
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transmission of HTLV-1 by plasma is due to the rare presence of HTLV-1 virions,
regardless of any other factor.
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Introduction
Human T-cell lymphotrophic virus (HTLV) was the first retrovirus to be associated with
human disease [Poiesz et al.1980].
HTLV Type 1 (HTLV-1) is accepted to be the
aetiological agent of adult T-cell leukemia/lymphoma (ATLL) [Yoshida et al. 1984;
Takatsuki
et
al.
1985]
and
HTLV-1-associated
myelopathy/Tropical
spastic
paraparesis(HAM/TSP) [Gessain et al. 1985] and is associated with a number of other
inflammatory conditions [Vernant et al. 1987; Morgan et al 1989; Nishioka et al 1989].
HTLV-1 exists predominantly as cell-bound provirus. HTLV-1 virions are considered to
be poorly infectious, requiring prolonged cell-cell contact for trasmission [Derse et al.
2001]. Additionally, already infected cells produce a very small number of free particles,
of which only 1 in 105 are infectious [Fan et al 1992].
HTLV-1 RNA transfers from an infected to an uninfected cell by means of a virological
synapse [Igakura et al. 2003]. This structure, similar to immunological synapses and
allowing for direct transmission of HTLV-1 viral particles across small intercellular
spaces, has been observed ex vivo [Majorovits et al. 2008]. It is believed that most cellto-cell transmission occurs via this mechanism [Bangham, 2003]. Whilst some degree of
such ‘infectious spread’ must occur in vivo, HTLV-1 Tax-induced mitotic clonal
expansion is considered to be a major contributor to the HTLV-1 infected cell burden
[Wattel et al. 1996; Tanaka et al. 2005;].
It has long been observed that plasma from HTLV-1 infected patients is not infectious.
Whether this is due to the absence of HTLV-1 virions from plasma or because they are
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non-infectious is not clear. Recently, Cabral et al [Cabral et al. 2012] reported the
detection of HTLV-1 RNA in 8% of plasma samples from asymptomatic carriers (AC)
and patients with HAM/TSP. Given the ease of handling of plasma compared with
peripheral blood mononuclear cells (PBMCs) the aims of this study are: 1) to determine
whether HTLV-1 RNA can be detected in plasma; 2) if detectable, whether this has
comparable diagnostic and prognostic value to the current assays, which quantify
HTLV-1 proviral DNA in PBMCs; finally, 3) to investigate whether viral load was
quantifiable also in plasma, as in PBMC. .
Methods
Plasma samples from 65 patients, whose HTLV-1 infection had been confirmed by
Western Blot, were analysed. HTLV-1 proviral load in PBMCs for each sample time
point was measured routinely as described previously [Demontis et al. 2013]. Samples
were categorized according to the donor’s HTLV-1-related clinical state: 27
asymptomatic carriers, 17 patients with HAM/TSP, 14 patients with ATLL, two patients
co-infected with HIV, two with polymyositis, two with neurological symptoms not HAM
and one with strongyloidiasis (Table I). Plasma samples from 13 HTLV-1 uninfected
subjects were used as negative controls. Seventeen samples (four from AC, seven from
patients with HAM/TSP and six from patients with ATLL) were repeated to assess the
reproducibility of the method. From six patients (two AC, two patients with HAM/TSP
and two with ATLL) a second time point, at least two years late, was analysed (sample
pair had a proviral load within the range of intra-patient variability)
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RNA Extraction and Reverse Transcription
Samples were thawed at room temperature and 1ml plasma was aliquoted. Plasma was
centrifuged at 20,000g, 4°C, for one hour. The upper 860µl of plasma were removed
and the remaining 140µl were processed using spin column protocol of the QIAMP Viral
RNA MiniKit (Qiagen, Germany) according to the manufacturer’s instructions. The
resulting RNA was eluted in 60µl of Elution Buffer and stored at -80°C until used. RNA
was reverse transcribed (RT) using the QuantiTect RNA Reverse Transcription Kit
(Qiagen, Germany) according to the manufacturer’s instructions. All apparatus and work
surfaces were treated with Ambion DNA zap and RNase zap prior to conducting reverse
transcription. A Geneamp 9700 PCR system was used for incubation at 42°C for 15
minutes followed by 95°C for 3min. The protocol also includes addition of 2µl gDNA
wipeout per 12µl RNA to prevent carryover of DNA contaminants. To exclude the
presence of genomic DNA from RNA preparation, a minus reverse-transcriptase control
was included in RT-PCR experiments. All resulting 20µl cDNA samples were purified
with the QIAquick PCR Purification Kit (Qiagen, Germany) and suspended in 50µl
elution buffer for storage at -80°C.
HTLV-1 viral RNA detection.
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All the cDNA samples were subjected to HTLV-1 detection by quantitative PCR in
duplicate [Demontis et al 2013] and by highly sensitive semi-quantitative nested PCR in
quadruplicate [Tosswill et al. 1998]. Primers published previously, SK43IF (5’CGGATACCCAGTCTACGTGT-3’) and SK44IR (5’-GAGCCGATAACGCGTCCATCG3’) [Kwok et al. 1988], were used to amplify a 159-bp fragment of DNA from the tax
region of HTLV-1 cDNA. The lower detection limit was three copies of HTLV cDNA per
milliliter of plasma.
To confirm the nature of the PCR products four of these were sequenced by cycle
sequencing using an ABI 3100 GeneticAnalyser according to the manufacturer’s
instruction. The sequences were then edited and analysed and compared to the HTLV1 tax reference sequence, using the programme Sequencher (Sequencer, GeneCodes
Corporation (URL: http://www.genecodes.com).
Validation of RT efficiency
To ensure the reliability of the RNA extraction and cDNA generation, serial dilutions of
cDNA obtained from a plasma sample of a patient with untreated HTLV/HIV co-infection
and with a known HIV viral load with a commercially used assay (1300 copies/ml,
COBAS TaqMan HIV-1 assay) was amplified by semi-nested PCR using HIV integrase
specific
primers
(SID1:
5’-AAGACAGCAGTACAAATGGCAGT-3”,
and
TACTGCCCCTTCACCTTTCCA-3’
SID3:
SID2:
5’5’-
CAATTTTAAAAGAAAAGGGGGGATT-3) to generate a 198bp product. In the first
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round the reaction volume of 25µl (composed of 2.5µl 10x buffer, 1.5µl MgCl2 (25mM),
0.5µl Primers, SID1 and SID2, 0.5µl dNTPs (10mM), 0.1µl Taq Polymerase (5U/µl),
14.9µl Nuclease free water and 5µl of sample cDNA) was subjected to the following
thermo cycling conditions: 94°C for 10mins, 25 cycles of 94°C for 30s, 55°C for 30s,
72°C for 30s and a single cycle of 72°C for 7mins.
The second round of PCR was conducted immediately after the first round using a
reaction mix of 25µl composed of 2.5µl 10x buffer, 1.5µl MgCl2 (25mM), 0.5µl Primers,
SID2 and SID3, 0.5µl dNTPs (10mM), 0.1µl Taq Polymerase (5U/µl), 18.9µl Nuclease
free water and 1µl of first round product. Amplification conditions were: 94°C for 10mins,
39 cycles of 94°C for 30s, 55°C for 30s, 72°C for 30s and a single cycle of 72°C for
7mins. Amplification products were separated on a 2% agarose gel at 100V for
40minutes employing SYBR Green (Invitrogen).
Results
Using the HTLV-1 specific nested PCR, products of the correct size (159bp) were
detected in the first plasma sample of 18/65 (28%) patients.
The frequency of detection of HTLV-1 viral RNA did not differ by clinical status.
The selected amplicons were confirmed to be HTLV-1 by sequencing. Using Poisson
distribution the HTLV-1 cDNA copy was 3-13 cDNA copies generated from 1 ml of
plasma.
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All no template controls, minus reverse-transcriptase controls and HTLV-1 uninfected
plasma controls were negative by quantitative PCR and nested PCR. HTLV RNA was
not detected in any sample from the patients by quantitative PCR. HIV integrase RNA
was detected in the stored plasma of the HIV-1/HTLV-1 co-infected patient at a
concentration of 560 RNA copies/ml compared to the 1300 RNA copies/ml obtained
with the commercial assay.
Due to the very low concentration of cDNA, to test the variability of the method and how
consistently results could be reproduced, the same samples of 16 patients (12 initially
negative and four initially positive) were assayed again. A concordant result was
obtained in 12/16 (75%) whilst 4/16 (25%) were discordant: two initially negative were
positive and two initially positive were negative. This degree of discordancy is consistent
with the target sequence being present at the limit of detection. A plasma sample from a
different time point was also assayed for six patients. Two were repeatedly negative and
four were discordant: negative to positive at the Lowest Detection Limit (three cDNAs
copies/ml) for two patients and positive to negative for two patients.
Plasma HTLV RNA was detectable in similar proportions of symptomatic patients
(grouped together) and asymptomatic carriers (p=0.41 by Fisher’s exacrt test).
The proviral load values in PBMC were lower in subjects who were positive for HTLV in
the plasma (10.8±21.9, mean±SD) compared to those who were negative (14.6±21.4);
however this difference was not significant (p=0.46 by analysis of variance, figure I).
Discussion
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Although whole blood, cellular blood products and tissue can transmit HTLV-1 it has
long been recognized that plasma from patients infected with HTLV-1 is not infectious.
Retrospective studies have never conclusively reported HTLV-1 transmission to
recipients of cell free blood products from infected donors [Sullivan et al. 1991; Manns
et al. 1992; Kleinman et al. 1993; Blejer et al. 1995; Namen-Lopes et al. 2009].
The apparent inability of plasma to transmit HTLV-1 could be explained by one, or a
combination of two theories. Either the plasma of patients infected with HTLV-1 does
not contain HTLV-1 virions, or only in quantities insufficient for transmission, or such
plasma contains virions but they are not infectious. In support of the second theory Fan
et al. demonstrated that only a very small minority of cell-free virions produced by
HTLV-1 infected cells or continuous producer cell lines such as MT-2, are infectious (in
vitro) [Fan et al. 1992]. However, the first hypothesis is widely believed, but data are
sparse with the single paper addressing this reporting the detection of HTLV-1 RNA in
the plasma of 8% of HTLV-1 infected persons, who were either asymptomatic carriers
or patients with HAM/TSP [Cabral et al. 2012].
In this study the frequency at which HTLV-1 RNA can be detected in patient plasma as
well as the relationship of plasma HTLV-1 RNA to disease including patients with ATLL,
who have the highest HTLV-1 proviral burden, has been explored. HTLV-1 viral RNA
was detected by a highly sensitive and specific nested RT-PCR in 28% of the subjects,
being AC, patients with HAM/TSP, or ATLL but only at or just above the threshold of
detection by nPCR and below that of qPCR. Using this semi-quantitative method 3 – 13
HTLV-1 cDNA copies were found per 1ml plasma which, assuming 25% RT efficiency,
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equates to 12 – 56 HTLV-1 RNA copies per ml plasma. This low level of HTLV-1 RNA
was confirmed by repeated testing of the same or subsequent samples indicating that at
such a low copy number sampling contributes significantly to the frequency of detection.
The presence of such a small number of HTLV-1 RNA copies is likely to be the prime
reason why plasma from HTLV-1 infected patients does not transmit this infection.
High proviral load is known to be a risk factor for the development of ATLL [Okayama et
al. 2004] but this did not correlate with detectability of HTLV-1 in plasma as patients with
and without malignant disease manifestations had comparable HTLV-1 plasma
viraemia. The potential of HTLV-1 plasma viraemia as a marker of disease progression
is thus extremely limited. Molecular detection of HTLV-1 RNA in plasma cannot be used
to screen for HTLV-1 infection and does not correlate with the presence of HTLV-1
associated disease. Since all patients in the study had chronic infection the possibility of
HTLV-1 RNA in plasma during acute infection cannot be discounted.
These data support the importance of the virological synapse and the role of cell-to-cell
transmission in infectious spread of HTLV-1 in vivo. Although not examined these data
also suggest that cell-to-cell spread is important in both mother-to-child transmission
and sexual transmission of HTLV-1 but this needs to be confirmed.
Sources of Funding
Internal. GPT is supported by the Imperial Comprehensive Biomedical Research Centre
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Competing interests
The authors declare that they have no competing interests.
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