In vitro method for detecting and quantifying HIV-2 DNA

Abstract

The present invention relates to a method for detecting or quantifying deoxyribonucleic acid (DNA) of human immunodeficiency virus 2 (HIV-2) in a sample containing DNA comprising: a) performing a real-time polymerase chain reaction (PCR) on the sample, or a fraction thereof comprising DNA, with at least two sets of primers and probe each respectively comprising two primers and a labeled probe for the detection or quantification of HIV-2 DNA, at least one of the sets is selected from the group consisting of: a set comprising a primer comprising or consisting of a sequence SEQ ID NO: 1 or a sequence having al least 90% identity to SEQ ID NO: 1, a primer comprising or consisting of a sequence SEQ ID NO: 2 or a sequence having 90% identity to SEQ ID NO: 2 or the complement of these sequences, and a labeled probe comprising or consisting of a sequence SEQ ID NO: 3, or a sequence having at least 90% identify to SEQ ID NO: 3 or the complement of these sequences, and a set comprising a primer comprising or consisting of a sequence SEQ ID NO: 4 or a sequence having at least 90% identity to SEQ ID NO: 4, a primer comprising or consisting of a sequence SEQ ID NO: 5 or a sequence having 90% identity to SEQ ID NO: 5 or the complement 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 to SEQ ID NO: 6 or the complement of these sequences, and b) determining therefrom the presence or absence and/or the quantity of HIV-2 DNA in the biological sample.

Claims

1. A method for detecting or quantifying deoxyribonucleic acid (DNA) of human immunodeficiency virus 2 (HIV-2) in a sample containing DNA comprising: a) performing a real-time polymerase chain reaction (PCR) on the sample, or a fraction thereof comprising DNA, with at least two sets of primers and probe each respectively comprising two primers and a labeled probe for the detection or quantification of HIV-2 DNA, at least one of the sets being selected from the group consisting of: a set comprising a primer comprising or consisting of a sequence SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1, a primer comprising or consisting of a sequence SEQ ID NO: 2 or a sequence having 90% identity to SEQ ID NO: 2 or the complement of these sequences, and a labeled probe comprising or consisting of a sequence SEQ ID NO: 3, or a sequence having at least 90% identity to SEQ ID NO: 3 or the complement of these sequences, and a set comprising a primer comprising or consisting of a sequence SEQ ID NO: 4 or a sequence having at least 90% identity to SEQ ID NO: 4, a primer comprising or consisting of a sequence SEQ ID NO: 5 or a sequence having 90% identity to SEQ ID NO: 5 or the complement 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 to SEQ ID NO: 6 or the complement of these sequences, and b) determining therefrom the presence or absence and/or the quantity of HIV-2 DNA in the biological sample, wherein the primers and probes are each present at a concentration between 300 and 500 nM.

2. The method according to claim 1, wherein in step a) of performing a real-time polymerase chain reaction (PCR) on the sample, or a fraction thereof comprising DNA, is performed with: (i) at least 4 primers respectively comprising or consisting of: a sequence SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1 or the complement of these sequences, and a sequence SEQ ID NO: 2 or a sequence having 90% identity to SEQ ID NO: 2 or the complement of these sequences, and a sequence SEQ ID NO: 4 or a sequence having 90% identity to SEQ ID NO: 4 or the complement of these sequences, and a sequence SEQ ID NO: 5 or a sequence having 90% identity to SEQ ID NO: 5 or the complement of these sequences, and (ii) at least 2 labelled probes respectively comprising or consisting of: a sequence SEQ ID NO: 3, a sequence complement to SEQ ID NO: 3, or a sequence having at least 90% identity to SEQ ID NO: 3 or the complement thereof, and a sequence SEQ ID NO: 6, a sequence complement to SEQ ID NO: 6, or a sequence having at least 90% identity to SEQ ID NO: 6 or the complement thereof.

3. The method according to claim 1, wherein the sample containing DNA is a biological sample or a sample comprising cells infected in vitro by HIV-2.

4. The method according to claim 1, wherein the primers respectively consist of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5, and the labeled probes respectively consist of sequences SEQ ID NO: 3 and SEQ ID NO: 6.

5. The method according to claim 1, wherein the labelled probes are labelled with 6-carboxyfluorescein (FAM) at their 5′end and with Black Hole Quencher-1 (BHQ1) at their 3′ end.

6. The method according to claim 1, wherein at least one DNA virus or at least one DNA molecule having essentially no sequence similarity with the genomic sequence of HIV-2 is added to the sample or the fraction thereof, as well as primers and probe necessary for its amplification and detection by real-time PCR, as an internal control of extraction and/or inhibition.

7. The method according to claim 1, wherein the PCR comprises the following thermocycling conditions: 2 min at 50° C., followed by 10 min at 95° C., followed by 50 cycles of 95° C. for 15 s and 60° C. for 1 min.

8. The method according to claim 1, further comprising determining or quantifying HIV-1 nucleic acids in the sample or a fraction thereof.

9. A kit or a mix for detecting or quantifying HIV-2 DNA, comprising: a) at least two sets of primers and probe each respectively comprising two primers and one probe for the detection or quantification of HIV-2 DNA, the primers and probe being each present at a concentration between 300 and 500 nM, and at least one of the sets being selected from the group consisting of: a set comprising a primer comprising or consisting of a sequence SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1, a primer comprising or consisting of a sequence SEQ ID NO: 2 or the complement of these sequences, and a labelled probe comprising or consisting of a sequence SEQ ID NO: 3, or a sequence having at least 90% identity to SEQ ID NO: 3 or the complement of these sequences, and a set 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 to SEQ ID NO: 5 or the complement of these primers, and a labelled probe comprising or consisting of a sequence SEQ ID NO: 6, or a sequence having at least 90% identity to SEQ ID NO: 6 or the complement of these sequences, and b) at least one DNA virus or at least one DNA molecule having essentially no sequence similarity with the genomic sequence of HIV-2, as well as primers and probe necessary for its amplification and detection by real-time PCR, as an internal control of extraction and/or inhibition, and c) optionally additional reagents for performing a PCR.

10. The kit or the mix according to claim 9, comprising: a1) at least 4 primers, at a concentration between 300 and 500 nm, respectively comprising or consisting of: a sequence SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1 or the complement of these sequences, and a sequence SEQ ID NO: 2 or a sequence having 90% identity to SEQ ID NO: 2 or the complement of these sequences, and a sequence SEQ ID NO: 4 or a sequence having 90% identity to SEQ ID NO: 4 or the complement of these sequences, and a sequence SEQ ID NO: 5 or a sequence having 90% identity to SEQ ID NO: 5 or the complement of these sequences, and a2) at least 2 labelled probes at a concentration between 300 and 500 nM, respectively comprising or consisting of: a sequence SEQ ID NO: 3, a sequence complement to SEQ ID NO: 3, or a sequence having at least 90% identity to SEQ ID NO: 3 or the complement thereof, and a sequence SEQ ID NO: 6, a sequence complement to SEQ ID NO: 6, or a sequence having at least 90% identity to SEQ ID NO: 6 or the complement thereof.

11. The kit or the mix according to claim 9, further comprising primers and at least one labelled probe for detecting or quantifying HIV-1 nucleic acids.

12. An in vitro method for determining HIV-2 viral load, in an individual, comprising the steps of: (c) performing the method for detecting or quantifying HIV-2 DNA in a biological sample taken from the individual as defined in claim 1; and (d) determining therefrom the HIV-2 viral load of the individual.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1

(2) FIG. 1 represents the standard curve of the threshold cycle (C.sub.T) (vertical axis) determined by performing a real-time PCR according to the invention as a function of the number of HIV-2 DNA copies introduced (horizontal axis, logarithmic scale). Median values and interquartiles ranges at 25% and 75% of C.sub.T are shown.
FIGS. 2 and 3
FIGS. 2A-2B represent log.sub.10 of the number of HIV-2 DNA copies obtained by automatic extraction from 10.sup.6 PBMCs (FIG. 2A) or leukocytes (FIG. 2B) (vertical axis) as a function of log.sub.10 of the number of HIV-2 DNA copies obtained by manual extraction from 10.sup.6 PBMCs (FIG. 2A) or leukocytes (FIG. 2B) (horizontal axis). PBMCs being from cellular pellets (FIG. 2A) and leukocytes from whole blood (FIG. 2B). The correlation coefficient (r) and the significance threshold (p) are represented.
FIGS. 4 and 5
FIGS. 4 and 5 represent the quantity of total HIV-2 DNA (vertical axis, log.sub.10 of the number of copies for 10.sup.6 PBMCs) as a function of the quantity of HIV-2 RNA quantity (horizontal axis, in log.sub.10 of the number of copies per mL (FIG. 3) or of strata of the number of copies per mL (FIG. 4)) for treated group A HIV-2-positive patients (black triangles), non-treated group A HIV-2-positive patients (white triangles), treated group B HIV-2-positive patients (black circles) and non-treated group B HIV-2-positive patients des patients (white circles). An arbitrary value of 1.60 log.sub.10 copies/mL was attributed to samples with an undetectable plasma viral load.

EXAMPLE

(3) Materials and Methods

(4) Biological Samples According to the Invention

(5) Frozen aliquots of peripheral blood mononuclear cells (PBMCs) from 63 HIV-2 infected patients included in the French National HIV-2 Cohort (ANRS CO5) were selected according to viral genotype and plasma HIV-2 RNA load, determined as described in Avettand-Fenoel et al. (2014) J. Clin. Microbiol. 52: 3017-3022. Written informed consent was obtained from all patients at the time of inclusion in the cohort. Among the 63 patients, 71% (n=45) were treated by antiretroviral therapy, and concerning the viral group, determined as described in Charpentier et al. (2014) AIDS Lond Engl 28: 1161-1169, 35 of them presented a group A virus (71% treated) and 28 presented a group B virus (71% treated). The selected samples had the following characteristics: HIV-2 RNA load<40 copies/ml (n=28:15 group A and 13 group B), 40 to 500 copies/ml (n=18; 10 group A and 8 group B), 500 to 5.000 copies/ml (n=9; 4 group A and 5 group B) and 5,000 to 50,000 copies/ml (n=8; 6 group A and 2 group B). These samples were used to evaluate the clinical performance of the assay. Eleven whole blood samples from HIV-2 infected patients (6 group A and 5 group B) were also used to evaluate performance of the method on whole blood.

(6) Blood samples from 30 HIV-negative subjects, 40 HIV-1 group M-positive patients and 10 HIV-1 group O patients were tested to evaluate the specificity of the method according to the invention.

(7) Detection and Quantification of HIV-2 DNA According to the Invention

(8) Several extraction methods were used for whole blood and cell pellets, depending on the laboratory out of three (A, B and C) involved in the study. For whole blood, total DNA was extracted from 200 μl using the NucleoSpin® Blood Kit (Macherey-Nagel, Düren, Germany) in labs A and B and the QIAsymphony DSP DNA mini kit (Qiagen, Courtaboeuf, France) in lab C. For cell pellets, the QIAamp DNA mini kit and the QIAsymphony DSP DNA mini kit were used for total DNA extraction from 3 to 5 million cells in labs A and C, respectively.

(9) To normalize the HIV-2 DNA quantification, the amount of total DNA in extracts was determined by spectrophotometry (Nanodrop, Thermo Scientific, Wilmington. N.C. USA) (labs A and B) or quantification of the albumin gene (lab C) using the LightCycler® FastStart DNA Master Hydroprobe kit (Roche. Mannheim. Germany) and serial dilutions of Human Genomic DNA (Roche) as standard (Laurendeau et al. (1999) Clin. Chem. 45: 982-986: Dehee (2001) J. Med. Viol. 65: 543-552).

(10) The quantification method according to the invention is based in particular on a triplex TaqMan PCR approach targeting the conserved consensus regions in the long terminal repeat (LTR) and the gag gene. It includes an internal control (yellow dye universal DNA extraction and inhibition control. Diagenode, Liege, Belgium) added before extraction.

(11) The forward and reverse primers for the LTR region were 5′-AGCAGGTAGAGCCTGGGTGTT-3′ (SEQ ID NO: 1) and 5′-TCTTTAAGCAAGCAAGCGTGG-3′ (SEQ ID NO: 2) respectively (Rouet et al. J. Clin. Microbiol. 42: 4147-53), with an internal probe 5′-FAM-CTTGGCCGGYRCTGGGCAGA-BHQ1-3′. (SEQ ID NO: 3) where FAM means carboxyfluorescein, and BHQ1 means black hole quencher 1.

(12) The forward and reverse primers for the gag region were 5′-GCGCGAGAAACTCCGTCTTG-3′ (SEQ ID NO: 4) and 5′-TTCGCTGCCCACACAATATGTT-3′ (SEQ ID NO: 5), respectively, with an internal probe 5′-FAM-TAGGTTACGGCCCGGCGGAAAGA-BHQ1-3′ (SEQ ID NO: 6) (Damond et al. (2005) J. Clin. Microbiol. 43: 4234-6).

(13) TABLE-US-00002 TABLE 1 Primers and probes used for PCR analysis LTR Gag Forward 5′-AGCAGGTAGAGCCTGGG 5′-GCGCGAGAAACTCCGTC primers TGTT-3′ (SEQ ID NO: TTG-3′ (SEQ ID NO: 4) 1) Reverse 5′-TCTTTAAGCAAGCAAGC 5′-TTCGCTGCCCACACAAT primers GTGG-3′ (SEQ ID NO: ATGTT-3′ (SEQ ID NO: 2) 5) Probe 5′-CTTGGCCGGYRCTGGGC 5′-TAGGTTACGGCCCGGCG AGA-3′ (SEQ ID NO: GAAAGA-3′ (SEQ ID NO: 3) 6)

(14) The reaction mix consists of a 50-μl volume containing the DNA extract (20 μl), primers and probes for HIV-2 (400 nM each), primers and probe for the internal control (1 μl), and IX PCR buffer (2× qPCR MasterMix Plus; Eurogentec, Seraing. Belgium).

(15) The thermocycling conditions are as follows: 2 min at 50° C. and 10 min at 95° C., followed by 50 cycles of: 95° C. for 15 s and 60° C. for 1 min. Amplification and data acquisition were carried out with the CFX96 system (Biorad, Hercules, Calif. USA) (Lab A) and TaqMan ABI 7900 and 7500 real-time PCR system (Applied Biosystems, Courtaboeuf, France) (Labs B and C, respectively). The log.sub.10 number of targets initially present is proportional to the cycle threshold (C.sub.T) and was determined from the external standard curve.

(16) DNA from HIV-2 (NIH-Z strain) infected cells (Advanced Biotechnologies Inc, Eldersburg, Md. USA) is used as the external standard. This standard, evaluated at 131.300 copies/μl using a previously described assay (Damond et al. (2005) J. Clin. Microbiol. 43: 4234-4236), was first diluted in 200 ng/μl human genomic DNA (Promega, Madison, Wis., USA) to a theoretical concentration of 60,000 copies/20 μl, followed by serial 1/10 dilutions in 25 ng/μl of human genomic DNA to concentrations from 6,000 copies/20 μl down to a final dilution of 2 copies/20 μl.

(17) HIV-2 DNA levels were first reported as HIV-2 DNA copies/PCR. The number of copies of HIV-2 DNA/μg total DNA was then calculated using the extract concentration and the final results were reported as the number of copies/10.sup.6 cells. The formula used to convert these results is: HIV-2 DNA (copies/μg total DNA)×1,000,000/150,000=HIV-2 DNA (copies/10.sup.6 cells) (Avettand-Fénoël et al. (2009) J. Med. Virol. 81: 217-223: Dib et al. (1996) Nature 380: 152-154).

(18) Determination of the Analytical Performance of the HIV-2 DNA Detection and Quantification Method According to the Invention

(19) Specificity was determined by testing blood samples from HIV-negative subjects (10 per lab), HIV-1 group M-positive patients (lab A) and HIV-1 group O patients (lab B).

(20) Linearity was assessed in the three labs using serial dilutions of the external standard at 6,000, 600, 60, and 6 copies/20 μl (22 PCRs).

(21) Analytical sensitivity was determined by testing dilutions of the external standard to 10, 6, 4, 3, and 2 copies/20 μl (20 replicates each) (lab A and lab B).

(22) Within-PCR reproducibility was determined by testing the external standard at dilutions of 6000, 600, 60, and 6 copies/20 μl (10 replicates for each dilution) (lab B).

(23) To determine between-PCR reproducibility, an HIV-2-positive control was prepared by dilution of a DNA extract from cell cultures of an HIV-2 group A isolate (GenBank accession number M15390, SEQ ID NO: 13) and quantified at 2.13 log.sub.10 copies/20 μl by a previously described assay (Damond et al. (2005) J. Clin. Microbiol. 43: 4234-4236). This solution was tested in the three laboratories in separate PCRs (n=24).

(24) Manual and automated extractions were compared using blood cell pellets and whole blood: 15 PBMCs pellets (8 group A and 7 group B) and 11 whole blood samples (6 group A and 5 group B) were extracted and quantified in parallel in labs A (manual) and C (automated).

(25) Statistical Analysis

(26) Comparisons between groups were performed with the Mann-Whitney test. Pearson's correlation coefficients were calculated to estimate the relationship between the C.sub.T values and log.sub.10 of the number of HIV-2 DNA copies/PCR, the relationship between the HIV-2 DNA and HIV-2 RNA loads, and to compare manual versus automated extractions. ANOVA was performed to evaluate the HIV-2 DNA quantification values according to HIV-2 RNA quantification values.

(27) Results

(28) Analytical Performance of the Detection and Quantification Method According to the Invention

(29) The 50 HIV-1 positive and 30 HIV-negative DNA samples were negative in the assay, giving a specificity of 100%.

(30) The standard curve shows strong linearity between the C.sub.T values and log.sub.10 of the number of HIV-2 DNA copies/PCR, with a limit of quantification of six copies/PCR (FIG. 1). The median correlation coefficient is 0.997 (range, 0.982 to 1), and the median slope is −3.45 (range, −3.11 to −3.64).

(31) The analytical sensitivity of the assay is 100% at 4 copies/PCR, 95% at 3 copies/PCR, and 85% at 2 copies/PCR.

(32) Within-PCR reproducibility was evaluated using the external standard with theoretical concentrations of 6.000, 600, 60, and 6 copies/PCR (log.sub.10 of the number of copies/PCR respectively 3.78, 2.78, 1.78, and 0.78). The inventors obtained a mean of 3.80 in log.sub.10 copies/PCR for the expected value of 3.78 in log.sub.10 copies/PCR with a within-PCR coefficient of variation (CV) of 1.03% and mean values of 2.79, 1.83, and 0.85 in log.sub.10 copies/PCR respectively for the expected concentrations of 2.78, 1.78, and 0.78 in log.sub.10 copies/PCR, and within-PCR CVs of 1.60%, 3.43%, and 27.02%, respectively.

(33) The positive control was determined to be 2.19 in log.sub.10 copies/PCR for the between-PCR assays performed in the three laboratories, with a CV of 5.10%.

(34) Manual and automated extractions were compared using samples extracted and quantified in parallel in labs A and C, respectively. As such, the median values of HIV-2 DNA obtained from the 15 cell pellets were 2.34 in log.sub.10 copies/10.sup.6 PBMC with manual extraction and 2.29 in log.sub.10 copies/10.sup.6 PBMC with automated extraction, with a median of differences of 0.22 in log.sub.10 and a correlation coefficient of 0.97 (IC 95%=[0.92; 0.99], p<0.0001). Median values obtained from the 11 whole blood samples were 2.05 in log.sub.10 copies/10.sup.6 leukocytes with manual extraction and 1.73 in log.sub.10 copies/10.sup.6 leukocytes with automated extraction, with a median difference of 0.3 in log.sub.10 and a correlation coefficient of 0.96 (IC95%=[0.85:0.99], p<0.0001) (FIGS. 2 and 3).

(35) Clinical Performance

(36) Clinical performance was evaluated in lab C. All samples from HIV-2-infected patients were validated according to the internal control manufacturer's instructions.

(37) 122 to 1,000 ng (median 548 ng) of total DNA per PCR well was analyzed, depending on the total DNA concentration in extracts. HIV-2 DNA was detectable for all 63 patients. HIV-2 DNA was detectable but not quantifiable (<6 copies/PCR) for 20 patients (32%) and quantifiable (≤6 copies/PCR) for 43 patients (68%) with a median HIV-2 DNA load of 2.45 in log.sub.10 copies/10.sup.6 PBMCs (InterQuartile range [IQR]=2.15-3.00 log.sub.10). For the 20 patients with detectable, but not quantifiable, HIV-2 DNA, the same DNA extracts were retested using 2-6 PCR replicates. Eighteen samples gave positive results in all replicates, one sample had two positive results out of three and one had three positive results out of four, at a level lower than six copies per PCR.

(38) Among the 35 group A samples. HIV-2 DNA was quantifiable in 21 samples (60%), with a median load of 2.56 in log.sub.10 copies/10.sup.6 PBMCs (IQR=2.29-3.03 log.sub.10). Among the 28 group B samples, HIV-2 DNA was quantifiable in 20 samples (71%), with a median load of 2.27 log.sub.10 copies/10.sup.6 PBMCs (IQR=1.97-2.81 log.sub.10). There was no difference between groups A and B in the proportion of patients displaying an HIV-2 DNA load below the limit of quantification (p=0.79), nor in the median load (2.56 vs. 2.27 log.sub.10 copies/10.sup.6 PBMCs respectively, p=0.17) when this load was quantifiable.

(39) Among the 18 patients having never received antiretroviral therapy, HIV-2 DNA was quantifiable for 10 of them (56%), with a median load of 2.08 in log.sub.10 copies/10.sup.6 PBMCs (IQR=1.88-2.28 log.sub.10). Among the 45 antiretroviral-treated patients. HIV-2 DNA was quantifiable in 33 of them (73%), with a median load of 2.60 in log.sub.10 copies/10.sup.6 PBMCs (IQR=2.26-3.09 log.sub.10). There was no difference in the proportion of patients displaying an HIV-2 DNA load below the limit of quantification between patients having never received antiretroviral therapy and treated patients (p=0.23 for all patients, p=0.74 for group A, and 0.65 for group B). The median HIV-2 DNA load was significantly higher in antiretroviral-treated than in patients having never been treated (p=0.003 for all patients, p=0.068 for group A, and 0.03 for group B), when quantifiable.

(40) When quantifiable, the HIV-2 DNA load correlated with the HIV-2 RNA load (r=0.68, 95% CI [0.4-0.8], p<0.0001 for the whole group and r=0.73, 95% CI [0.4-0.9], p<0.0002 for treated patients) (FIG. 4). The proportion of patients displaying an HIV-2 DNA load below the limit of quantification was respectively 46%, 39%, 0%, and 0% for an HIV-2 RNA load<40 copies/ml, 40 to 500 copies/ml, 500 to 5,000 copies/ml, and 5,000 to 50,000 copies/ml respectively. When quantifiable, the median (IQR) HIV-2 DNA loads were 2.26 in log.sub.10 (2.02-2.56 log.sub.10), 2.37 in log.sub.10 (2.01-2.50 log.sub.10), 2.74 in log.sub.10 (2.17-3.24 log.sub.10), and 3.42 in log.sub.10 copies/10.sup.6 PBMCs (3.06-3.58 log.sub.10), respectively (FIG. 5). HIV-2 DNA loads were significantly different depending on the HIV-2 RNA load strata (ANOVA test, p<0.0001). A comparison between subgroups (group A/group B, antiretroviral-treated patients/patients having never been treated) according to HIV-2 RNA load was not relevant because of the small number of patients in each subgroup.

CONCLUSION

(41) Infection by HIV-2 is different from that by HIV-1, especially with respect to its slower progression, therapeutic management, and the level of HIV-2 genetic diversity. Specific molecular methods are therefore necessary for the diagnosis and monitoring of HIV-2 infection (either alone or in the context of a co-infection with HIV-1), the diagnosis of infants born to seropositive HIV-2 mothers, and studying the HIV-2 reservoir. Plasma viremia is undetectable in many HIV-2 infected patients in the absence of antiretroviral therapy, particularly in those with high CD4.sup.+ T cell count. HIV-2 DNA may be the only detectable marker in these patients.

(42) The aim of the present invention is thus in particular to develop a highly sensitive quantitative assay for HIV-2 DNA that is easy to implement in developing countries where HIV-2 is endemic.

(43) The detection and quantification method according to the invention exhibits 100% specificity which means the HIV-2 primers did not hybridize to HIV-1 genome. This method also displays good linearity (6 to 6,000 copies/PCR) and good within-PCR reproducibility. The inventors have also shown a good inter-laboratory reproducibility. In addition, the detection and quantification method which may include an external standard for quantification improves reliability and comparisons between different studies. The excellent sensitivity (95% limit of detection: 3 copies/PCR) is useful both for clinical diagnosis and pathophysiological studies.

(44) Both manual and automated extraction methods, as well as two real-time PCR instruments, were validated for compatibility with local practices in resource-limited countries. Eventually, the inventors also showed that the method according to the invention can be performed both on blood cell pellets and on whole blood samples.

(45) 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 with success in many resource-limited countries. This thus facilitates the use of the test according to the invention either for HIV-2 alone or jointly with HIV-1 in the same PCR. This allows for a reduction of analytical costs by increasing the number of samples per PCR and facilitates molecular diagnosis of dual HIV-1/HIV-2 infection within the same sample or diagnosis of HIV infection in babies.

(46) HIV-2 DNA was detectable in blood cells of all patients (infected either with HIV-2 group A or HIV-2 group B): the sensitivity of the method according to the invention has thus been improved relative to previous assays which reported undetectable HIV-2 DNA in 17% (5/29) of samples from HIV-2-infected patients (Damond et al. (2001) J. Clin. Microbiol. 39: 4264-4268).

(47) The addition of an internal control, which was absent in the previous assays (Damond et al. (2001) J. Clin. Microbiol. 39: 4264-4268: Gueudin et al. (2005) Meth. Mol. Biol. 304: 215-220), allows validating the analytical process.

(48) The inventors also showed that HIV-2-infected patients receiving antiretroviral therapy had higher HIV-2 DNA loads than those of patients having never received antiretroviral treatment, probably because patients who had been treated had more advanced disease, which necessitated the initiation of antiretroviral treatment. Among treated patients, a higher HIV-2 DNA viral load correlated with higher HIV-2 RNA loads, similar to what has been reported for HIV-1 infection (Visseaux et al. (2016) 23th Conference on Retroviruses and Opportunistic Infections Abstract 214. Boston, Mass., USA).

(49) Quantification of HIV-2 DNA levels could also provide information about the HIV-2 reservoir, as HIV-1 DNA load has been reported to be a relevant marker of the HIV-1 reservoir. Indeed, the HIV-1 DNA load is predictive of immunological and clinical progression, regardless of CD4.sup.+ cell counts and RNA viral load. HIV-1 DNA levels vary depending on the stage of HIV infection, with the highest levels found during primary HIV infection and AIDS. In addition, HIV-1 DNA quantification has allowed reservoir studies in various types of patient cohorts: long-term non-progressors, elite controllers, chronically infected untreated patients, and post-treatment controllers. It has offered a better understanding of the natural history of HIV-1 infection.

(50) In conclusion, the inventors have developed a detection or quantification method (or assay), HIV-2 DNA which has good analytical performance and good clinical sensitivity. This method is particularly successful for both HIV-2 groups A and B, the most prevalent, in comparison with previously described assays (Damond et al. (2001) J. Clin. Microbiol. 39: 4264-4268; Gueudin et al. (2008) AIDS Lond. Engl. 22: 211-215). The method according to the invention has been validated on a large, well-characterized panel of patient samples. This method is easy to perform assay and is appropriate for use in resource-limited countries in which multiple HIV-2 variants circulate. It can also be particularly useful for HIV-2 diagnosis in babies born to seropositive mothers, for diagnosis of mono- or co-infections with HIV-1, which is important because of monitoring and therapeutic consequences. Eventually, this method is also useful in pathogenesis studies on HIV-2 reservoirs, exploring new insights into the natural history of HIV-2 infection at different stages, and improving opportunities for clinical studies in treated patients.