Nérina Profizi

Effects of a Combination of Zidovudine, Didanosine, and Lamivudine on Primary Human Immunodeficiency Virus Type 1 Infection

A combination of zidovudine, didanosine, and lamivudine was used to treat 10 patients with primary human immunodeficiency virus type 1 (HIV-1) infection 5-28 days after the onset of symptoms. When therapy began, the mean plasma HIV-1 RNA level was 5.31 +/- 0.33 log10 copies/mL and the mean CD4 T cell count was 630 +/- 112 x 10(6)/L. The plasma HIV-1 RNA level decreased rapidly, and levels dropped below the cutoff in each case after 108 +/- 32 days. Lymph nodes from 5 patients were biopsied before therapy and during follow-up. Infectious HIV-1 could not be cultivated from any lymph node mononuclear cells taken on day 90, and HIV-1 RNA was at very low levels in lymph nodes after 1 year. In some cases, waning of the antibody response to HIV-1 was shown by Western blot after several months of undetectable plasma RNA. These data demonstrate that triple-drug therapy has a potent antiviral effect during primary HIV-1 infection.

Residual human immunodeficiency virus type 1 RNA in lymphoid tissue of patients with sustained plasma RNA of <200 copies/mL.

Human immunodeficiency virus type 1 (HIV-1) RNA was measured in lymph node (LN) mononuclear cells of 50 patients with sustained plasma RNA of <200 copies/mL with therapy. Six patients had received a combination of three reverse transcriptase inhibitors (RTIs) since primary infection, 11 received this same combination during chronic disease, 21 received a combination of two RTIs plus a protease inhibitor (PI), and 12 received three RTIs plus a PI. The mean overall duration of therapy was 8.9 +/- 0.5 months (range, 5-24), with no significant difference between groups. LN HIV-1 RNA levels varied from undetectable to 1.7 million copies/10(6) cells according to cases. The mean LN HIV-1 RNA level was 2.99 +/- 0.42 log10 copies/10(6) cells in the 17 patients receiving three RTIs compared with 1.93 +/- 0.25 log10 copies/10(6) cells in the 33 patients receiving a PI (t test, P = .02). These data demonstrate that highly active antiretroviral regimens have unequivalent effects on LNs and invite redefinition of suboptimal therapy at this level.

Correlation between plasma levels of cytokines and HIV-1 RNA copy number in HIV-infected patients

SummaryThe plasma levels of HIV-1 RNA, tumor necrosis factor alpha (TNF-α), soluble receptors type II of TNF-α (sTNF-α RII), soluble receptors of interleukin-4 (sR IL-4), interleukin-6 (IL-6), soluble receptors of interleukin-6 (sR IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), soluble receptors of GM-CSF (sR GM-CSF), RANTES, MIP-1α and MIP-1β were measured in 80 HIV-infected patients. All patients had not been treated previously with antiretroviral drugs and did not present a recent history of opportunistic infection. A statistically significant correlation was found between HIV-1 RNA and TNF-α (p=0.005) or sTNF-α RII levels (p<0.001). RANTES and MIP-1α levels did not correlate with HIV-1 RNA. MIP-1β levels were correlated with plasma RNA titers in patients with CD4+ T cells <200 × 106/l (p=0.03). MIP-1α and sR IL-4 levels were significantly different according to the CD4+ T cell range (p=0.003 and p=0.0002, respectively). GM-CSF and sR GM-CSF were undetectable in each case. These data confirm that HIV-1 replication in the plasma is correlated with TNF-α levels, but do not show a clear correlation with levels of the chemokines studied.

Multicenter Quality Control for the Detection of Hepatitis C Virus RNA in Seminal Plasma Specimens

ABSTRACT The discrepant results available in the literature about the presence of hepatitis C virus (HCV) RNA in seminal plasma of men chronically infected by this agent are related, at least in part, to the molecular techniques used and particularly to the wide range of protocols dedicated to RNA extraction. In order to evaluate these protocols and to standardize the method of detection of HCV RNA in this fluid, a panel of coded specimens was tested blindly in 12 French laboratories; it included 14 seminal plasma specimens and four water controls spiked with HCV RNA ranging from 10 to 20,000 IU/ml and two HCV-negative seminal plasma specimens. The extraction step was performed according to methods using either silica beads (NucliSens [Organon Teknika S.A., Fresnes, France]; RNA viral kit [Qiagen, Courtaboeuf, France]) or guanidinium thiocyanate (Amplicor HCV assay; Roche Diagnostics, Meylan, France), preceded or not by a centrifugation of the seminal plasma. For the amplification step, all the laboratories performed the same reverse transcription-PCR technique (Amplicor HCV Cobas assay). The percentage of correct results ranged from 53.3 to 100, the poorest results being obtained when no centrifugation step preceded the Amplicor extraction protocol. The rate of correct results was significantly higher in laboratories using a preliminary centrifugation of the specimen (P = 0.034 by chi-square test). By contrast, the overall number of correct results was not correlated to the initial volume of sample used for the test. These results allowed us to validate standardized techniques adapted to the performance of this test on a routine basis, especially in men infected with HCV and involved in programs of medically assisted reproduction.

Long-term evaluation of triple nucleoside therapy administered from primary HIV-1 infection

OBJECTIVE To study the long-term effect of triple-drug therapy initiated at the time of primary HIV-1 Infection and to evaluate the persistance of replication-competent virus in responding patients. METHODS Prospective open-label pilot study. Patients received a combination of zidovudine, didanosine and lamivudine. Viral sequencing of the reverse transcriptase gene was performed before therapy and during follow-up. HIV-1 RNA and DNA as well as CD4 and CD8 T lymphocyte subsets were measured in blood and in lymph node biopsies during therapy. Isolated blood CD4 T cells were cultured in conditions that improved HIV isolation. Three patients received in vivo interleukin-2 and gamma-interferon in order to try to identify intracellular pools of replication-competent virus. SETTING A tertiary care general hospital. PATIENTS Fifteen patients observed within 28 days following the acute retroviral syndrome. RESULTS After a mean follow-up of 27.5+/-2.9 months, plasma RNA remained < 20 copies/ml (four patients), fluctuated between 20 and 120 copies/ml (six patients) or rebounded (five patients). M184V and/or T215Y mutations were demonstrated in two of these last five patients. Proviral DNA in peripheral blood mononuclear cells (PBMC) decreased by an average of -1 log after 16+/-3 months, reaching undetectable levels in three patients. The culture of isolated CD4 T cells yielded virus in all but two patients. These last were characterized by a waning antibody reactivity on the Western blot, undetectable proviral DNA in PBMC and undetectable RNA in lymph nodes. Cytokine administration in vivo had no effect in one patient and unmasked plasma RNA in the other. Stopping therapy in the first patient led to a rebound in plasma RNA. CONCLUSION Despite a lack of detectable plasma viral activity in some patients after 3 years of triple nucleoside therapy administered since the acute retroviral syndrome, replication-competent virus can still be demonstrated.

Human Immunodeficiency Virus Type 1 Dynamics in Different Lymphoid Tissue Compartments

Human immunodeficiency virus type 1 (HIV-1) RNA was measured in total lymph node (LN) tissue and isolated LN mononuclear cells (LNMC) in sequential LN biopsy samples from 1 patient with primary HIV-1 infection and from 5 previously untreated patients with chronic disease. HIV-1 RNA levels were an average of 210-fold higher in total LN tissue compared with levels in LNMC, even during primary infection, when circulating antibodies were absent. After the patients were treated with a three- or four-drug regimen, total HIV-1 RNA decreased exponentially in total LN tissue and in LNMC (mean half-lives of 8.5 +/- 1.8 and 7.9 +/- 2.2 days, respectively). In addition, the evolution of the infectious virus in LNMC was analyzed for the 5 patients with chronic disease: Titers decreased, with a mean half-life of 7.5 +/- 2.3 days. Extracellular virions are the most important virus compartments in LNs; however, they exhibit the same dynamics as virions situated in LNMC, with a mean virus decay half-life of approximately 1 week.

Role of autoimmunity in protein S deficiency during HIV-1 infection

SummaryThe objective of the study was to evaluate the role of autoimmune mechanisms in the pathophysiology of protein S deficiency during HIV-1 infection. In a prospective study the correlation between protein S activity and the presence of anti-protein S autoantibodies or anti-cardiolipin antibodies in HIV-1-positive patients and in a population of patients without HIV infection was investigated. Fifty-five HIV-1-infected patients and 15 hospitalized patients without HIV infection were analysed for protein S activity (functional assay), complement system activation, presence of autoantibodies against protein S (Dot Immunobinding) and levels of anti-cardiolipin IgG antibodies (ELISA). The presence of anti-protein S antibodies was detected in 31 (56.36%) out of the 55 HIV-1-positive patients and in three (20%) of the 15 control patients (Fishers exact test, p=0.012). The average value (± standard deviation) of protein S activity was 100.93 (14.73)% in the control group. For the HIV-1-infected patients it was 73.70 (20.67)% in those with anti-protein S antibodies compared to 88.08 (25.48)% in those without antibodies (Mann-Whitney U Test, p=0.01). In the HIV-1-positive group protein S activity was correlated with concentrations of circulating immune complexes (Spearman rank sum test, r=−0.41, p=0.018) and in the control group with concentrations of anti-cardiolipin antibodies (Spearman rank sum test, r=0.709, p=0.032). In conclusion, HIV-1 infection is associated with a high prevalence of antibodies against protein S. These antibodies are associated with a significantly low protein S activity. The exact correlation with plasma levels of these antibodies remains to be investigated.ZusammenfassungIn der vorliegenden Studie wurden Untersuchungen zur Bedeutung von Autoimmunmechanismen in der Pathophysiologie des Protein-S-Mangels bei HIV-1-Infektion durchgeführt. Die Beziehung zwischen Protein-S-Aktivität und Antikörpern gegen Protein S oder Kardiolipin wurde bei Patienten mit HIV-1-Infektion und ohne HIV-Infektion prospektiv untersucht. In einem funktionalen Assay wurde bei 55 HIV-1-infizierten Patienten und 15 stationären Patienten ohne HIV-Infektion Protein S bestimmt sowie die Aktivierung des Komplementsystems, Vorliegen von Autoantikörpern gegen Protein S (Dot Immunbindung) und IgG-Antikörperspiegel gegen Kardiolipin (ELISA) gemessen. Bei 31/55 HIV-1-positiven Patienten (56,36%) und bei 3/15 (20%) Kontrollpatienten waren Antikörper gegen Protein S nachzuweisen (Fishers exact test p=0,012). Die Protein-S-Aktivität betrug im Durchschnitt (± Standardabweichung) in der Kontrollgruppe 100,93 (14,73)%, bei HIV-Infizierten 73,70 (20,67)% bei Fällen mit Anti-Protein-S-Antikörpern und 88,08 (25,48)% bei Patienten ohne Anti-Protein-S-Antikörper (Mann-Whitney-U-Test p=0,01). In der HIV-1-positiven Gruppe korrelierte die Protein-S-Aktivität mit zirkulierenden Immunkomplexen (Spearman rank sum test r=− 0,41; p=0,018) und in der Kontrollgruppe mit Anti-Kardiolipin-Antikörpern (Spearman rank sum test r=0,709; p=0,032). Die HIV-Infektion ist folglich mit einer hohen Prävalenz von Antikörpern gegen Protein S und niedriger Protein-S-Aktivität assoziiert. Die genaue Korrelation dieser Antikörper mit Plasmaspiegeln muß noch bestimmt werden.

HIV-1 replication in the plasma and cerebrospinal fluid

SummaryThe HIV-1 RNA in plasma and CSF samples from 40 HIV-1 infected patients was measured by a polymerase chain reaction (PCR) technique. The possible implication of cytokines in HIV-1 replication was investigated by measuring the concentrations of tumor necrosis factor α (TNF-α), macrophage colony stimulation factor (M-CSF) and interleukin-6 (IL-6) in these fluids. HIV-1 RNA was quantified in all plasma samples and in 87.5% of the CSF samples. CSF HIV-1 RNA titers did not correlate with the stage of disease or the CD4+ T cell counts, unlike the plasma HIV-1 RNA titers. These results were confirmed when patients with a blood brain barrier damage, as assessed by the CSF/plasma albumin ratio, were excluded from the analysis. TNF-α levels were statistically correlated with the HIV-1 RNA in plasma and CSF. These data demonstrate that HIV-1 replication in the CSF at each clinical stage can be accurately measured with PCR and, although the titers of HIV-1 RNA copies in the CSF are correlated with those in the plasma, the magnitude of HIV-1 replication in CSF is not directly linked to the stage of disease, or to the CD4+ T cell count. The significance of early high levels of HIV-1 RNA in CSF is now being studied prospectively.ZusammenfassungHIV-1-RNA wurde mittels Polymerasekettenreaktion (PCR) in Plasma- und Liquorproben von 40 HIV-1-infizierten Patienten quantifiziert. Um mögliche Einflüsse durch Zytokine auf die HIV-1-Replikation zu erfassen, wurden Tumornekrosefaktor-α (TNF-α), Makrophagen-Kolonie-stimulierender Faktor (M-CSF) und Interleukin-6 (IL-6) in diesen Flüssigkeiten ebenfalls bestimmt. Eine Quantifizierung von HIV-1 RNA war in allen Plasmaproben und in 87,5% der Liquorproben möglich. Im Gegensatz zu den HIV-1-RNA-Titern im Plasma fand sich zwischen HIV-1-RNA-Titern im Liquor und dem Krankheitsstadium oder den CD4+-T-Zell-Zahlen keine Korrelation. Diese Ergebnisse bestätigten sich auch bei Patienten, bei denen gemessen am Liquor-Ausschluß von Plasma-Albumin-Quotienten eine Blut-Liquor-Schrankenstörung bestand. Die HIV-1-Replikation kann in allen klinischen Stadien mittels PCR exakt quantifiziert werden. Obwohl die Liquor-Titer an HIV-1-RNA-Kopien mit den Plasmatitern korrelieren, besteht dennoch keine direkte Beziehung zum Krankheitsstadium oder zur CD4+-Zellzahl. In einer prospektiven Studie wird derzeit die Bedeutung frühzeitig auftretender HIV-1-RNA-Spiegel im Liquor untersucht.The HIV-1 RNA in plasma and CSF samples from 40 HIV-1 infected patients was measured by a polymerase chain reaction (PCR) technique. The possible implication of cytokines in HIV-1 replication was investigated by measuring the concentrations of tumor necrosis factor α (TNF-α), macrophage colony stimulation factor (M-CSF) and interleukin-6 (IL-6) in these fluids. HIV-1 RNA was quantified in all plasma samples and in 87.5% of the CSF samples. CSF HIV-1 RNA titers did not correlate with the stage of disease or the CD4+ T cell counts, unlike the plasma HIV-1 RNA titers. These results were confirmed when patients with a blood brain barrier damage, as assessed by the CSF/plasma albumin ratio, were excluded from the analysis. TNF-α levels were statistically correlated with the HIV-1 RNA in plasma and CSF. These data demonstrate that HIV-1 replication in the CSF at each clinical stage can be accurately measured with PCR and, although the titers of HIV-1 RNA copies in the CSF are correlated with those in the plasma, the magnitude of HIV-1 replication in CSF is not directly linked to the stage of disease, or to the CD4+ T cell count. The significance of early high levels of HIV-1 RNA in CSF is now being studied prospectively. HIV-1-RNA wurde mittels Polymerasekettenreaktion (PCR) in Plasma- und Liquorproben von 40 HIV-1-infizierten Patienten quantifiziert. Um mögliche Einflüsse durch Zytokine auf die HIV-1-Replikation zu erfassen, wurden Tumornekrosefaktor-α (TNF-α), Makrophagen-Kolonie-stimulierender Faktor (M-CSF) und Interleukin-6 (IL-6) in diesen Flüssigkeiten ebenfalls bestimmt. Eine Quantifizierung von HIV-1 RNA war in allen Plasmaproben und in 87,5% der Liquorproben möglich. Im Gegensatz zu den HIV-1-RNA-Titern im Plasma fand sich zwischen HIV-1-RNA-Titern im Liquor und dem Krankheitsstadium oder den CD4+-T-Zell-Zahlen keine Korrelation. Diese Ergebnisse bestätigten sich auch bei Patienten, bei denen gemessen am Liquor-Ausschluß von Plasma-Albumin-Quotienten eine Blut-Liquor-Schrankenstörung bestand. Die HIV-1-Replikation kann in allen klinischen Stadien mittels PCR exakt quantifiziert werden. Obwohl die Liquor-Titer an HIV-1-RNA-Kopien mit den Plasmatitern korrelieren, besteht dennoch keine direkte Beziehung zum Krankheitsstadium oder zur CD4+-Zellzahl. In einer prospektiven Studie wird derzeit die Bedeutung frühzeitig auftretender HIV-1-RNA-Spiegel im Liquor untersucht.

HHV-8 IN PBMC AND KAPOSI'S SARCOMA ACTIVITY

Human herpes virus 8 (HHV-8) has recently been linked to Kaposi s sarcoma (KS) [1] and detected in several tissue specimens [2] comprising peripheral blood mononuclear cells (PBMC) [3, 4]. However, though HHV-8 viremia seems to be correlated with the presence of disseminated clinical lesions [5], the frequency of detection in patients who have achieved a complete tumour remission is unknown. To address this issue we have analysed PBMC from 26 HIV-infected patients, 16 showing clinical activity of KS (skin lesions only: five cases, skin and organ involvement: 11 cases) and ten with complete clinical remission (mean duration: 4.7 _+ 2.3 years). Peripheral blood was mixed with lymphocyte separating medium (Eurobio, Les Ulis, France) and centrifuged to isolate 2 million PBMC that were lysed with the whole blood specimen preparation kit (Roche Diagnostic Systems, Neuilly/Seine, France). D N A was extracted by standard methods. HHV-8 D N A was detected by nested-PCR using the primers described by Chang et al. [1] which amplify a 233-bp D N A fragment (KS330Bam), and a 150-bp internal primer. PCR was done for 35 cycles (30 sec at 95°C, 30 sec at 55°C and 1 min at 72°C) in a thermocycler (Perkin-Elmer Cetus, Norwalk, CT, USA). KS had been proven by biopsy in each case at the time of diagnosis. HHV-8 detection in PBMC was positive in 13 patients (81.25%) with clinically active KS and in two (20%) with complete remission of KS (Fishers exact test, p = 0.004). These last two patients were initially treated with alpha-interferon and were in complete remission for 2 and 5 years. The other patients with a past history of KS had been treated with chemotherapy. Cryopreserved PBMC were available at the time of KS diagnosis in seven cases and all tested positive for KSHV. One of these patients has at present clinically inactive skin lesions but receives monthly low-dose chemotherapy because relapse occurs each time when this regimen is stopped. Biopsied lesions that had been stable for 4 years showed only fibrosis and a negative PCR for HHV-8. On the other hand, lesions clinically stable for 2 years and apparently inactive showed some histological aspects evocative of KS cells and a strong positivity for HHV-8 by PCR. This patient repeatedly tested negative for HHV-8 in PBMC. To further analyse the relation between HHV-8 cell-associated viremia and the presence of the virus in organ tissues, we performed PCR on surgical lymph node biopsies from ten HIV-infected patients without a history of KS. HHV-8 D N A was detected in lymph nodes in five cases, two of which were repeatedly negative in PBMC at the time of the biopsy and during the 12 months following. These results demonstrate that HHV-8 detection in PBMC is correlated with the clinical extension of KS. However, the presence of HHV-8 in circulating cells, that are mainly B lymphocytes [3], cannot be explained only by the infection of these cells when trafficking through the infected tissues. This is reinforced by the possibility to observe HHV-8 infection in lymph nodes without detectable viremia. Consequently, the infection of circulating cells by HHV-8 could play a major role in KS pathogenesis. C. Poggi, A. Lafeuillade, N. Profizi, I. Poizot-Martin

Serum HIV-1 RNA load to predict CD4+ T-cell depletion in asymptomatic patients.

SummaryThe temporal association between the increase in viral replication and the depletion in CD4+ T cells in HIV-1 infection is not yet clear. To investigate this phenomenon HIV-1 RNA was quantified in several frozen sera from 20 asymptomatic HIV-1 infected patients in the 2 years preceding CD4+ T cell depletion of 50% or more, and compared with 20 HIV-1 infected paired patients who were stable in the same period. In each group, no statistically significant variation in the mean HIV-1 RNA titre was found between the last checkup and the one 24 months earlier. The mean HIV-1 RNA titre was 103.86 copies/ml in the non-progressor group and 105.12 copies/ml in the progressor group. These data support the view that the quantity of circulating HIV-1 RNA is an early predictor of disease progression that is relatively constant during the asymptomatic period of HIV-1 infection.ZusammenfassungDie zeitliche Beziehung zwischen der zunehmenden Virusreplikation und Depletion der CD4+ T-Zellen bei HIV-1 Infektion ist noch nicht geklärt. Um dieses Phänomen zu untersuchen, haben wir eine Quantifizierung der HIV-1 RNA in mehreren Seren von 20 asymptomatischen HIV-1 infizierten Patienten durchgeführt, die in den Jahren vor einer Verminderung der CD4+ T-Zellen um 50% und mehr eingefroren worden waren. Die Werte wurden mit denjenigen von 20 HIV-1 infizierten Patientenpaaren verglichen, die während desselben Zeitraums stabil geblieben waren. In keiner der Gruppen fanden sich statistisch signifikante Unterschiede im mittleren HIV-1 RNA-Titer zwischen der Untersuchung zum Zeitpunkt 24 Monate und der ersten Untersuchung. In der nicht progredienten Gruppe fand sich ein mittlerer HIV-1 RNA-Titer von 103,86/ml Kopien, in der progredienten Gruppe 105,12/ml. Diese Ergebnisse sprechen dafür, daß die Menge an zirkulierender HIV-1 RNA ein früher Prädiktor der Krankheitsprogression ist, der während der asymptomatischen Phase der HIV-1 Infektion relativ konstant bleibt.The temporal association between the increase in viral replication and the depletion in CD4+ T cells in HIV-1 infection is not yet clear. To investigate this phenomenon HIV-1 RNA was quantified in several frozen sera from 20 asymptomatic HIV-1 infected patients in the 2 years preceding CD4+ T cell depletion of 50% or more, and compared with 20 HIV-1 infected paired patients who were stable in the same period. In each group, no statistically significant variation in the mean HIV-1 RNA titre was found between the last checkup and the one 24 months earlier. The mean HIV-1 RNA titre was 103.86 copies/ml in the non-progressor group and 105.12 copies/ml in the progressor group. These data support the view that the quantity of circulating HIV-1 RNA is an early predictor of disease progression that is relatively constant during the asymptomatic period of HIV-1 infection. Die zeitliche Beziehung zwischen der zunehmenden Virusreplikation und Depletion der CD4+ T-Zellen bei HIV-1 Infektion ist noch nicht geklärt. Um dieses Phänomen zu untersuchen, haben wir eine Quantifizierung der HIV-1 RNA in mehreren Seren von 20 asymptomatischen HIV-1 infizierten Patienten durchgeführt, die in den Jahren vor einer Verminderung der CD4+ T-Zellen um 50% und mehr eingefroren worden waren. Die Werte wurden mit denjenigen von 20 HIV-1 infizierten Patientenpaaren verglichen, die während desselben Zeitraums stabil geblieben waren. In keiner der Gruppen fanden sich statistisch signifikante Unterschiede im mittleren HIV-1 RNA-Titer zwischen der Untersuchung zum Zeitpunkt 24 Monate und der ersten Untersuchung. In der nicht progredienten Gruppe fand sich ein mittlerer HIV-1 RNA-Titer von 103,86/ml Kopien, in der progredienten Gruppe 105,12/ml. Diese Ergebnisse sprechen dafür, daß die Menge an zirkulierender HIV-1 RNA ein früher Prädiktor der Krankheitsprogression ist, der während der asymptomatischen Phase der HIV-1 Infektion relativ konstant bleibt.