Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
  • C12Q1/702—Specific hybridization probes for retroviruses
  • C12Q1/703—Viruses associated with AIDS
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/16—Primer sets for multiplex assays

Definitions

• the present invention relates to an in vitro method for the detection and quantification of human immunodeficiency virus 2 (VI H-2).
• HIV H - 2 Infection with HIV H - 2, mainly limited to West Africa, is characterized by a slow progression of the disease associated with a slow decrease in the number of CD4 + T lymphocytes, a low rate of transmission by pathway. sexually or vertically and has a lower viral replication than HIV-1.
• NRTIs non-nucleoside reverse transcriptase inhibitors
• fusion inhibitors eg, Enfuvirtide
• protease inhibitors PIs
• HIV-2 DNA is currently considered to be the only detectable alternative marker in individuals whose H-2 VI RNA is undetectable.
• This marker is particularly useful for confirming the diagnosis of mono or co-infection with HIV-1 in cases of high serological cross reactivity.
• the detection of HIV-2 DNA is essential for early diagnosis in children born to HIV-2 infected mothers.
• this marker may be useful for physiopathological studies of VI H-2 reservoirs.
• Several techniques for quantifying HIV-2 DNA are part of the state of the art, such as that described by Damond et al. (2001) J. Clin. Microbiol. 39: 4264-4268, in which the authors perform a real-time polymerase chain reaction (PCR) to amplify the gag region of the viral genome.
• HIV-2 DNA has difficulty quantifying HIV-2 DNA in group B. In addition, they lack sensitivity and are not always able to detect DNA. HIV-2 in some samples from infected individuals.
• the object of the invention is to provide a method for the detection and quantification of HIV-2 DNA to overcome these difficulties, in particular by improving the reproducibility, sensitivity and / or specificity of the methods of the invention. state of the art, particularly for the two endemic groups A and B of HIV-2.
• the present invention results from the unexpected discovery, by the inventors, that a real-time PCR carried out with a set of primers of SEQ ID NO: 1 and 2 sequences and a probe of sequence SEQ ID NO: 3 , and a set of primers of SEQ ID NO: 4 and 5 and of a probe of sequence SEQ ID NO: 6, made it possible to reproducibly detect and quantify HIV-2 DNA of types A and B with a specificity of 100%, a detection limit of 3 copies / PCR and a limit of quantitation at 6 copies / PCR.
• the present invention thus relates to a method for detecting or quantifying deoxyribonucleic acid (DNA) of human immunodeficiency virus 2 (HIV-2) in a sample comprising DNA comprising:
• PCR real-time polymerase chain reaction
• an assembly comprising a primer comprising or consisting of a sequence SEQ ID NO: 4 or a sequence having at least 90% identity with SEQ ID NO: 4, a primer comprising or consisting of a sequence SEQ ID NO: 5 or a sequence having 90% identity with SEQ ID NO: 5 or the complementary of these sequences, and a labeled probe comprising or consisting of a sequence SEQ ID NO: 6, or a sequence having at least 90% identity with SEQ ID NO: 6 or the complement of these sequences;
• step a) of carrying out a polymerase chain reaction (PCR) in real time on the sample, or a fraction thereof comprising DNA is carried out with
• sequence SEQ ID NO: 3 a sequence complementary to SEQ ID NO: 3, or a sequence having at least 90% identity with SEQ ID NO: 3 or the complement thereof
• sequence SEQ ID NO: 6 a sequence complementary to SEQ ID NO: 6, or a sequence having at least 90% identity with SEQ ID NO: 6 or the complement thereof.
• At least one DNA virus or at least one DNA molecule having essentially no similarity in the sample or fraction thereof is added to the sample or fraction thereof.
• the method for detecting or quantifying HIV-2 DNA as defined above further comprises determining or quantifying HIV-1 nucleic acids in a sample or fraction of it.
• the present invention also relates to a kit or mixture for detecting or quantifying HIV-2 DNA, comprising:
• an assembly comprising a primer comprising or consisting of a sequence SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO: 1, a primer comprising or consisting of a sequence SEQ ID NO: 2 or a sequence having 90% identity with SEQ ID NO: 2 or the complementary of these sequences, and a labeled probe, in particular with 6-carboxyfluorescein (FAM) at its 5 'end and Black Hole Quencher-1 ( BHQ1) at its 3 'end, comprising or consisting of a sequence SEQ ID NO: 3, or a sequence having at least 90% identity with SEQ ID NO: 3 or the complement of these sequences, and
• FAM 6-carboxyfluorescein
• BHQ1 Black Hole Quencher-1
• an assembly comprising a primer comprising or consisting of a sequence SEQ ID NO: 4 or a sequence having at least 90% identity with SEQ ID NO: 4, a primer comprising or consisting of a sequence SEQ ID NO: 5 or a sequence having 90% identity with SEQ ID NO: 5 or complementary to these primers, and a labeled probe, in particular with 6-carboxyfluorescein (FAM) at its 5 'end and Black Hole Quencher-1 (BHQ1) at its 3' end, comprising or consisting of a sequence SEQ ID NO : 6, or a sequence having at least 90% identity with SEQ ID NO: 6 or the complement of these sequences, and
• FAM 6-carboxyfluorescein
• BHQ1 Black Hole Quencher-1
• the kit or mixture as defined above comprises:
• At least 4 primers preferably at a concentration of between 300 and 500 nM, in particular 400 nM, comprising or consisting respectively of:
• sequence SEQ ID NO 3 a sequence complementary to SEQ ID NO: 3, or a sequence having at least 90% identity with SEQ ID NI: 3 or the complement thereof, and
• the kit or mixture as defined above further comprises primers and at least one labeled probe for detecting or quantifying HIV-1 nucleic acids.
• the present invention also relates to the use of the kit or mixture as defined above, for detecting or quantifying the HIV-2 nucleic acids of a sample containing DNA.
• the present invention also relates to a method, particularly an in vitro method, for diagnosing an HIV-2 infection, or determining an HIV-2 viral load, in an individual, comprising the steps of:
• the present invention also relates to a method, particularly an in vitro method, for determining whether an individual is likely to benefit from treatment of an HIV-2 infection or a treatment adjustment, comprising performing the method for diagnose an HIV-2 infection, or determine a viral load of HIV-2, as defined above.
• the present invention also relates to a method, particularly an in vitro method, for monitoring the HIV-2 viral load of an individual treated for HIV-2 infection, comprising performing the method for diagnosing HIV infection. -2, or determine a viral load of HIV-2, as defined above.
• the present invention also provides nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (PIs) and / or integrase inhibitors for use in the prevention or treatment of HIV-2 infection in a human subject. individual, wherein the individual has been determined to be susceptible to treatment of HIV-2 infection by the method as defined above.
• NRTIs nucleoside reverse transcriptase inhibitors
• PIs protease inhibitors
• integrase inhibitors for use in the prevention or treatment of HIV-2 infection in a human subject. individual, wherein the individual has been determined to be susceptible to treatment of HIV-2 infection by the method as defined above.
• HIV-2 The human immunodeficiency virus 2 (HIV-2), the infection of which can lead to Acquired Immunodeficiency Syndrome (AIDS), is well known to those skilled in the art.
• HIV-2 is a member of the genus Lentivirus, which belongs to the family Retroviridae. Its replication cycle involves the entry of HIV-2 single-stranded RNA into a cell, reverse transcription to give single-stranded DNA, and then double-stranded DNA that is integrated into the genome of the host cell.
• the HIV-2 according to the invention may be of any group, in particular of group A, B or of non-endemic groups, in particular C, D, E or H.
• the method according to the invention detects or quantifies the Group A or B HIV-2 DNA in a sample with a specificity of the order of 100%.
• HIV-2 DNA DNA that encodes the entire HIV-2 genome in whole or in part.
• the HIV-2 DNA to be detected or quantified according to the invention can thus be in particular a single-stranded DNA, especially a sense or antisense single-stranded DNA, or a double-stranded DNA.
• PCR Real-time polymerase chain reaction
• qPCR quantitative PCR
• a signal usually a fluorescent signal, the intensity of which is a consequence of amplified DNA accumulation, is measured by the thermal cycler that performs the PCR.
• the intensity of the measured signal in particular the fluorescent signal, is greater than a signal threshold, generally the intensity of the background signal, it is deduced that the amplification has taken place, .e. that the HIV-2 DNA is present in the biological sample.
• CT threshold cycle
• PCR techniques can be used according to the invention, such as Taqman or Molecular Beacons technologies (molecular beacons).
• the PCR according to the invention is of Taqman type.
• Real-time Taqman PCR is well known to those skilled in the art and was originally described in 1965 by Holland et al. (1991) Proc. Natl. Acad. Sci. USA 88: 7276-7280.
• the Taqman probes consist of a fluorophore covalently attached to the 5 'end of an oligonucleotide probe and another type of fluorophore called "quencher" located at the 3' end.
• the probe is such that the quencher turns off the fluorescence emitted by the fluorophore when it is excited by a thermocycler light source, this phenomenon being known as fluorescence resonance energy transfer or FRET (Fluorescence Resonance Energy Transfer).
• FRET Fluorescence Resonance Energy Transfer
• Taqman probes are designed so that they hybridize to a target within an amplified DNA region.
• the thermostable polymerase used to perform the PCR extends the primer and synthesizes the nascent strand, its 5'-3 'exonuclease activity degrades the probe that hybridized to the target.
• the degradation of the probe causes the release of the fluorophore and breaks the proximity to the quencher, dissipating the quenching effect and thus triggering the fluorescence of the fluorophore.
• the fluorescence detected in the quantitative PCR thermocycler is directly proportional to the released fluorophore and the amount of DNA template present in the PCR.
• the real-time PCR according to the invention comprises the following thermocycling conditions:
• thermocycling conditions defined above are compatible with or identical to those used for the detection and / or quantification of HIV-1 DNA, in particular using the GENERIC HIV DNA Cell test. Biocentric company (Grasse, France). Consequently, the method for detecting and quantifying HIV-2 DNA in a sample containing DNA according to the invention can be used in the same thermocycler, with the same computer program and even, if necessary, in the same amplification plate as samples intended for the detection or quantification of HIV - 1 DNA, or even in the same samples.
• a "primer” is an oligonucleotide, preferably a DNA oligonucleotide, useful for initiating replication by a DNA polymerase, particularly a thermostable DNA polymerase.
• the primers according to the invention do not comprise more than 50 nucleotides, more preferably not more than 40 nucleotides, and more preferably not more than 30 nucleotides.
• a "probe” is an oligonucleotide, preferably a DNA oligonucleotide, that can hybridize to amplified DNA molecules, i.e., amplimers, as part of PCR. in real time according to the invention.
• the probes according to the invention do not comprise more than 50 nucleotides, more preferably no more than 40 nucleotides, and more preferably no more than 30 nucleotides.
• the labeled probe is such that a detectable signal, preferably fluorescence, is emitted and increases in intensity as a consequence of the accumulation of the amplified DNA molecules.
• the probes according to the invention are labeled, in particular covalently labeled, with a first fluorophore and with a second fluorophore of type ⁇ (quencher)), the fluorophore-quencher pair being such that the quencher extinguishes the emission fluorescence emitted by the fluorophore.
• the fluorophore be attached at or near the 5 'end of the probe and that the quencher be attached at or near the 3' end of the probe.
• Many fluorophore-quencher pairs adapted according to the invention can be designed by those skilled in the art.
• the pairs fluorophore-quencher 6-carboxyfluorescein (FAM) -carboxytetramethylrhodamine (TAMRA) e 6 6-carboxyfluorescein (FAM) -Black Hole Quencher-1 (BHQ1) are suitable for labeled probes according to the invention.
• a particularly preferred fluorophore-quencher pair according to the invention is the 6-carboxyfluorescein (FAM) -Black Hole Quencher-1 (BHQ1) pair.
• the two labeled probes according to the invention be labeled with the same fluorophore-quencher pair.
• the two labeled probes are labeled with a 6-carboxyfluorescein (FAM) at their 5 'ends and with a Black Hole Quencher-1 (BHQ1) at their 3' ends.
• FAM 6-carboxyfluorescein
• BHQ1 Black Hole Quencher-1
• the primer or probe when a primer or probe according to the invention is said to "understand" a particular sequence, the primer or probe may also include additional sequences extending from the 5 'end and / or or the 3 'end of the particular sequence. In contrast, when a primer or probe according to the invention "consists of” or “consists of” a particular sequence, the primer or probe does not include additional sequences in addition to the particular sequence.
• the at least 4 primers according to the invention consist respectively of the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5, and the two probes labeled according to the invention consist of respectively in the sequences SEQ ID NO: 3 and SEQ ID NO: 6.
• a "sequence having at least 90% identity with SEQ ID NO: X” differs from SEQ ID NO: X by inserting, deleting or substituting at least one nucleotide.
• the percentage identity between two nucleotide sequences is defined here as the number of positions for which the bases are identical when the two sequences are optimally aligned, divided by the total number of bases of the longest of the two. sequences. Two sequences are said to be optimally aligned when the percentage of identity is maximum.
• a sequence according to the invention having at least 90% identity with SEQ ID NO: 1, 2, 3, 4, 5 or 6 respectively has at least 95%, more preferably at least 98% identity with SEQ ID NO: 1, 2, 3, 4, 5 or 6.
• primers according to the invention comprising the sequences SEQ ID NO: 1 and SEQ ID NO: 2 or the sequences having at least 90% identity with SEQ ID NO: 1 and SEQ ID NO: 2 and the probe comprising or consisting of SEQ ID NO: 3, a sequence complementary to SEQ ID NO: 3, or a sequence having at least 90% identity with SEQ ID NO: 3 or the complement thereof, are used to detect a portion of the LTR region of the HIV-2 genome.
• primers according to the invention comprising the sequences SEQ ID NO: 4 and
• SEQ ID NO: 5 or sequences having at least 90% identity with SEQ ID NO: 4 and SEQ ID NO: 5 and the probe comprising or consisting of the sequence SEQ ID NO: 6, a sequence complementary to the SEQ ID NO: 6, or a sequence having at least 90% identity with SEQ ID NO: 6 or the complement thereof, are useful for detecting a portion of the gag region of the HIV-2 genome.
• 5'-AGCAGGTAGAGCCTGGGTGTT-3 ' (SEQ ID NO: 1) is 5'-AACACCCAGGCTCTACCTGCT-3' (SEQ ID NO: 7);
• 5'-CTTGGCCGGYRCTGGGCAGA-3 ' (SEQ ID NO: 3) is 5'-TCTGCCCAGYRCCGGCCAAG-3' (SEQ ID NO: 9);
• 5'-GCGCGAGAAACTCCGTCTTG-3 ' (SEQ ID NO: 4) is 5'-CAAGACGGAGTTTCTCGCGC-3' (SEQ ID NO: 10);
• 5'-TTCGCTGCCCACACAATATGTT-3 ' (SEQ ID NO: 5) is 5'-AACATATTGTGTGGGCAGCGAA-3' (SEQ ID NO: 1 1);
• NO: 3 (CTTGGCCGGYRCTGGGCAGA) is a so-called degenerate sequence wherein Y is C and T, and R is A and G.
• the primers and probes according to the invention are present in a concentration of between 300 and 500 nM each. Preferably also primers and probes are all present at the same concentration. Most preferably, the primers and probes are present at a concentration of 400 nM.
• primers and probes according to the invention in a real-time PCR allows the specific detection and quantification of HIV-2 DNA relative to the nucleic acids of HIV-1.
• iii) allows better detection and quantification of Group B HIV - 2 DNA.
• the internal control of extraction and / or inhibition makes it possible in particular to validate the entire analytical process by real - time PCR, from extraction to amplification, without competition between HIV DNA. -2 and internal control, including for weak values.
• the internal extraction and / or inhibition control is meant here an exogenous DNA or nucleic acid molecule, having essentially no sequence similarity with the human genome or with pathogens affecting the humans.
• the internal extraction and / or inhibition control according to the invention is a DNA molecule.
• the internal control according to the invention as well as the primers and probe necessary for its amplification and detection by real-time PCR are added before the extraction.
• the internal extraction and / or inhibition control according to the invention is labeled with a fluorochrome. More preferably, the internal control of extraction and / or inhibition according to the invention is marked by the fluorochrome Yellow Dye ref. DICD-YD-L1 00.
• sample containing DNA relates to any sample, in particular solid, fluid or liquid, likely to contain HIV-2 DNA.
• the sample containing the DNA according to the invention may in particular be a biological sample or a sample, in particular of experimental type, containing cells or fractions of cells infected in vitro by HIV-2.
• the biological sample according to the invention is derived from a sample taken from an individual, in particular a human individual, and may especially be a whole blood sample, a plasma sample, a serum sample, a seminal sample. , a sample of peripheral blood mononuclear cells (PBMC), or fractions thereof, or a sample of leukocytes, or fractions thereof.
• PBMC peripheral blood mononuclear cells
• a biological sample according to the invention may be a DNA solution of a sample, such as a whole blood sample, taken from a human individual.
• kit it is understood that one or more of the kit components may be packaged or compartmentalized separately from the rest of the components.
• the mixture it is understood that all components of the mixture are in a single compartment.
• the mixture according to the invention is a PCR mixture.
• Additional reagents with respect to the primers and probes according to the invention can be easily designed by those skilled in the art and can include, in particular, a thermostable DNA polymerase, a reverse transcriptase, dNTPs, salts, in particular Mn 2+ and Mg 2+ , and buffers.
• treatment of HIV-2 infection refers to any treatment that prevents or treats an HIV-2 infection, including reducing the HIV-2 viral load or rendering it undetectable. .
• the treatment of an HIV-2 infection according to the invention is selected from the group consisting of antiretroviral therapy (ART), in particular using a nucleoside reverse transcriptase inhibitor (NRTI). , a protease inhibitor (PI) and / or an integrase inhibitor, an immunomodulator, a molecule capable of acting at the level of the immune control points, a monoclonal antibody, a molecule anti -Late with an epigenetic mechanism of action and a vaccine.
• ART antiretroviral therapy
• NRTI nucleoside reverse transcriptase inhibitor
• PI protease inhibitor
• integrase inhibitor an immunomodulator
• a molecule capable of acting at the level of the immune control points a monoclonal antibody
• a molecule anti -Late with an epigenetic mechanism of action and a vaccine.
• FIG. 1 represents the standard curve of the threshold cycle (Cr) (vertical axis) determined by the implementation of a real-time PCR according to the invention as a function of the number of HIV-2 DNA copies introduced (axis horizontal, logarithmic scale). Median values and 25% and 75% interquartile ranges of Cr are indicated.
• Cr threshold cycle
• FIGS. 2A-2B show the logio of the copy number of HIV-2 DNA obtained by automatic extraction from 10 6 PBMC (FIG. 2A) or leukocytes (FIG. 2B) (vertical axis) as a function of log 10 of the number copies of HIV-2 DNA obtained by manual extraction from 10 6 PBMC ( Figure 2A) or leukocytes ( Figure 2B) (horizontal axis), the PBMCs being derived from cell pellets ( Figure 2A) and leukocytes from whole blood ( Figure 2B).
• the correlation coefficient (r) and significance threshold (p) are represented.
• Figures 4 and 5 represent the amount of total HIV-2 DNA (vertical axis, log 10 copy number for 10 6 PBMC) as a function of the amount of HIV-2 RNA (horizontal axis, in log 10 of the number of copies per mL ( Figure 3) or in copy-number strata per mL ( Figure 4)) for HIV-2 group A-treated patients (black triangles), group A non-HIV-2 positive patients treated (white triangles), group B HIV-2 positive patients treated (black circles) and group B HIV-2 positive patients (white circles).
• An arbitrary value of 1, 60 logio copies / mL was attributed to samples with undetectable plasma viral load.
• PBMCs peripheral blood mononuclear cells
• the quantification method according to the invention is based in particular on a Taqman type triplex PCR approach targeting the consensus regions conserved in the long repetitive terminal sequence (LTR) and in the gag gene. It includes an internal control (Ye // ow Dye universal DNA extraction and inhibition control, Diagenode, Liège, Belgium) added before extraction.
• the sense and antisense primers for the LTR region are 5'-
• the sense and antisense primers for the gag region are 5'-GCGCGAGAAACTCCGTCTTG-3 '(SEQ ID NO: 4) and 5'-TTCGCTGCCC AC AC AATATGTT-3', respectively (SEQ ID NO: 5), with an internal probe.
• 5'-FAM-TAGGTTACGGCCCGGCGGAAAGA-BHQ 1 -3 '(SEQ ID NO: 6) (Damond et al (2005) J. Clin Microbiol 43: 4234-6).
• the reaction mixture consists of a volume of 50 ⁇ l containing the DNA extract (20 ⁇ l), primers and probes for HIV-2 (400 nM each), the primers and the probe for internal control (1). ⁇ ) and PCR buffer IX (2X qPCR MasterMixPlus, Eurogentec, Seraing, Belgium).
• thermocycling conditions are as follows: 2 minutes at 50 ° C. and 10 minutes at 95 ° C., followed by 50 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute.
• the amplification and data acquisition were carried out using the CFX96 system (Biorad, Hercules, CA, USA) (Laboratory A) and the real-time PCR system TaqMan ABI 7900 and 7500 (Applied Biosystems, Courtaboeuf, France). France) (Laboratories B and C respectively).
• the logio of the number of targets initially present is proportional to the threshold cycle (Cr) and has been determined from the external standard curve.
• DNA from HIV-2 infected cells (NIH-Z strain) (Advanced Biotechnologies Inc, Eldersburg, MD, United States of America) is used as an external standard. This standard, evaluated at 131,300 copies / ⁇ l using a previously described test (Damond et al (2005) J Clin Microbiol 43: 4234-4236), was first diluted in 200 ng.
• Analytical sensitivity was determined by testing dilutions of the external standard at 10, 6, 4, 3, and 2 copies / 20Ml (replicated 20-fold) (laboratory A and laboratory B).
• Intra-PCR reproducibility was determined by testing the external standard at 6000, 600, 60, and 6 copies / 20Ml dilutions (replicated 10 times for each dilution) (laboratory B).
• the 50 positive or HIV-1 DNA samples and 30 HIV-negative DNA samples are negative in the assay, giving a specificity of 100%.
• the standard curve shows a strong linearity between the CT values and the logio of the HIV-2 DNA copy number / PCR, with a limit of 6 copies / PCR quantification ( Figure 1).
• the median correlation coefficient is 0.997 (in a range 0.982 to 1), and the median slope is -3.45 (in a range of -3.1 to -3.64).
• the analytical sensitivity of the assay is 100% at 4 copies / PCR, 95% at 3 copies / PCR and 85% at 2 copies / PCR.
• Intra-PCR reproducibility was evaluated using the external standard with theoretical concentrations of 6000, 600, 60 and 6 copies / PCR (log 10 copies / PCR respectively 3.78, 2.78, 1, 78 and 0.78).
• the inventors obtained an average of 3.80 log10 copies / PCR for the expected value of 3.78 log10 copies / PCR with an intra-PCR coefficient of variation (CV) of 1.03% as well as average values of 2.79, 1, 83 and 0.85 log10 copies / PCR respectively for expected concentrations of 2.78, 1, 78, and 0.78 log10 copies / PCR, and intra- PCR resumes of 1.60 % 3.43% and 27.02%, respectively.
• CV intra-PCR coefficient of variation
• the positive control was evaluated at 2.19 log10 copies / PCR for inter-PCR assays performed in the three laboratories, with a CV of 5.10%.
• PIQ 2.29-3, 03 logio
• p 0.79
• PIQs 2.08 log 10 copies / 10 6 PBMCs
• the proportions of patients with HIV-2 DNA load below the limit of quantification were 46%, 39%, 0%, and 0%, respectively, for HIV-2 RNA load ⁇ 40 copies / ml, from 40 to 500 copies / ml, from 500 to 5000 copies / ml, and from 5,000 to 50,000 copies / ml.
• the median HIV-2 DNA (PIQ) loads were 2.26 in log10 (2.02-2.56 log 10), 2.37 in log 10 (2.01 -2, 50 log 10), 2.74 log 10 (2.7-3.24 log 10) and 3.42 log 10 copies / 10 6 PBMC (3.06-3.58 log 10) respectively (FIG. 5).
• HIV-2 DNA loads were significantly different depending on the stratification of the HIV-2 RNA loads (Anova Test, p ⁇ 0.0001).
• a comparison between subgroups (group A / group B, antiretroviral / untreated patients) versus HIV-2 RNA load was not relevant because of low numbers of patients in each subgroup.
• HIV-2 infection is very different from that of HIV-1, particularly with respect to its slower progression, its therapeutic management and the level of genetic diversity of HIV-2.
• Specific molecular methods are therefore needed for the diagnosis and monitoring of HIV-2 infection (either alone or in the context of HIV-1 co-infection), for the diagnosis of infants born to mothers positive for HIV-2, as well as for the study of the HIV-2 reservoir.
• Plasma viraemia is undetectable in a large number of patients infected with HIV-2 in the absence of antiretroviral therapy, particularly in those with high CD4 + T cell counts. HIV-2 DNA may be the only detectable marker in these patients.
• the object of the present invention is therefore in particular to develop a quantitative assay of HIV-2 DNA highly sensitive and easy to implement in developing countries where HIV-2 is endemic.
• the method of detection and quantification according to the invention has a specificity of 100% which means that the primers of HIV-2 have not hybridized with the genome of HIV-1.
• This method also has good linearity (6 to 6000 copies / PCR) as well as good intra-PCR reproducibility.
• the inventors have elsewhere shown good inter-laboratory reproducibility.
• the detection and quantification method according to the invention which may include an external standard for quantification makes it possible to improve reliability and comparisons between different studies.
• the excellent sensitivity (95% detection limit: 3 copies / PCR) is both useful for clinical diagnosis and for physiopathological studies.
• the inventors used the same protocol as the Generic HIV DNA cell kit (Biocentric, Bandol, France) used to detect and quantify HIV-1 DNA. This test is currently being used successfully in several resource-limited countries. This thus makes it easier to use the test according to the invention for either HIV-2 alone or together with HIV-1, in the same PCR. This reduces analytical costs by increasing the number of PCR samples and facilitates the molecular diagnosis of HIV-1 and HIV-2 dual infection in the same sample or the diagnosis of HIV infection in patients with HIV. infants.
• HIV-2 DNA could be detected in the blood cells of all patients (infected with either HIV-2 group A or group B HIV-2); the sensitivity of the method according to the invention was thus improved over previous assays which reported that HIV-2 DNA was undetectable in 17% (5/29) of HIV-2 infected patient specimens ( Damond et al (2001) J. Clin Microbiol 39: 4264-4268).
• HIV-1 (Visseaux et al (201 6) 23th Conference on Retroviruses and Opportunism Infections Abstract 2 14. Boston, Massachusetts, USA).
• HIV-2 DNA levels could also provide information about the HIV-2 reservoir, just as the HIV-1 DNA load has been described as a relevant marker of the HIV-1 reservoir .
• the DNA load of HIV-1 is predictive of immunological and clinical progression, regardless of the amount of CD4 + lymphocytes and the viral RNA load.
• HIV-1 DNA levels vary according to the stage of HIV infection, with the highest levels found during primary HIV infection and during AIDS.
• quantification of HIV-1 DNA has allowed for reservoir studies in different types of cohorts of patients: long-term non-progressors, elite controllers, untreated chronic infected patients, and controllers. after treatment. This paves the way for a better understanding of the natural history of HIV - 1 infection.
• the inventors have developed a method for the detection and quantification (or assay) of HIV-2 DNA which has good analytical performance as well as good clinical sensitivity.
• This method is particularly effective for the more frequent HIV-2 groups A and B compared to the previously described tests (Damond et al (2001) J. Clin Microbiol 39: 4264-4268, Gueudin et al. (2008) AIDS Lond, Engl 22: 21-215).
• the method according to the invention has been validated over a wide and well characterized range of patient samples. This method is easy to implement and is suitable for use in resource-limited countries in which multiple variants of HIV-2 circulate.

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  • 2018-05-04 WO PCT/EP2018/061624 patent/WO2018202904A1/fr unknown
  • 2018-05-04 EP EP18726743.0A patent/EP3619327A1/de active Pending

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