Yann Gérard

Symptomatic hyperlactataemia: an emerging complication of antiretroviral therapy.

BackgroundFatal lactic acidosis is a serious complication of therapy with nucleoside analogues. ObjectiveTo examine symptomatic hyperlactataemia in HIV-infected adults treated with antiretroviral drugs. MethodsIn this prospective study, arterial blood lactate levels were measured in patients presenting with unexplained clinical symptoms. When these levels were high, functional respiratory tests (FRT) were carried out. Liver or muscle biopsies were further proposed. Incidences were calculated by comparison with the entire cohort of patients treated in the department. ResultsFourteen HIV-infected adults treated with antiretroviral drugs were identified with symptomatic hyperlactataemia during a 2-year period follow-up study. The incidence of hyperlactataemia was 0.8% per year but reached 1.2% if only patients treated with a regimen including stavudine were considered. Clinical symptoms included abnormal fatigue, tachycardia, abdominal pain, weight loss, peripheral neuropathy, and more specifically exercise-induced dyspnoea occurring despite effective antiretroviral treatment. FRT showed a metabolic deviation towards anaerobiosis with a high lactate/pyruvate ratio. Ultrastructural mitochondrial abnormalities were seen in all four patients for whom this was examined. There was a marked decrease in complex IV activity in muscle biopsies from four of five patients, consistent with a mitochondrial dysfunction. Evolution was favourable in 13 patients, probably because of an early diagnosis. ConclusionsPotentially fatal adverse events occurring during antiretroviral treatment may be avoided by close monitoring of clinical signs and blood lactate levels. If other studies confirm that the cumulative long-term toxicity of antiretroviral drugs results from mitochondrial dysfunction, the incidence of hyperlactataemia and its clinical consequences may become more important.

Simultaneous detection of 6 human herpesviruses in cerebrospinal fluid and aqueous fluid by a single PCR using stair primers

A Herpes Consensus allows the simultaneous detection of 6 human herpesviruses: herpes simplex virus type 1 (HSV‐1) and type 2 (HSV‐2), human cytomegalovirus (HCMV), varicella‐zoster virus (VZV), Epstein‐Barr virus (EBV), and human herpes virus 6 (HHV‐6). This technique was used first to examine retrospectively 100 DNA extracts from 95 CSF and 5 aqueous fluids, prepared by treatment by saturated NaCl followed by ethanol precipitation (n = 63) or by simple boiling (n = 37) and stored at −80°C, and secondly to test prospectively 38 CSF samples for which two DNA extracts were prepared with commercially available DNA extraction kits. In all cases, the results were compared with those of an “in‐house” PCR. Concordant results between both PCR and the Herpes Consensus techniques were obtained in 61 of 63 DNA extracts prepared by treatment by saturated NaCl (97%) and in only 31 of 37 boiled samples (84%). Both commercially available methods of DNA extraction examined appear to be suitable for Herpes Consensus PCR, although they cannot remove completely PCR inhibitors that must be sought in case of negative results. This preliminary study shows that the Herpes Consensus method should be of value for rapid diagnosis of herpesvirus infections on condition that it is performed on purified DNA extracts. J. Med. Virol. 62:349–353, 2000.

Tolerance, compliance and psychological consequences of post-exposure prophylaxis in health-care workers

Our objectives were to evaluate tolerance and compliance of postexposure triple therapy in health-care workers (HCWs) by retrospective observational study. Structured telephone interview of HCWs identified through data from antiretroviral prescribing centres. Twenty HCWs who received triple prophylaxis were identified over one year. Sixteen agreed to participate in the study. All but one source patient had documented HIV infection. Half HCWs were not aware of post-exposure therapy. Most HCWs received a zidovudine, lamivudine and indinavir combination. All completed at least 4 weeks of therapy. Only 50% received their first dosage less than 4 h after exposure. Nearly all experienced adverse events, mostly digestive (nausea and abdominal pain n =15) or psychological (anxiety and depression n =15), none resulting in therapy discontinuation. Most events occurred 2 to 7 days after therapy initiation. Most modified their sexual life with abstinence or condom use. Compliance was excellent. Half HCWs did not miss any tablet, 4 forgot one dosing a month and 4 one dosing a week. Follow up is over 6 months in all but one HCW. No HIV seroconversion has been observed to date. In France, post-exposure triple antiretroviral therapy is widely available 24 h a day in every emergency room but further training and development of HCWs is needed to decrease consulting time and increase referral to specialized physicians. Notable moderate adverse events, both physical and psychological are noted, however, compliance is excellent.

A decreased production of IL12 in vitro is associated with isolation of cytopathic HIV-1 strains in HIV-1-infected patients

The changes in type 1 (IL12, IFNγ, IL2) and type 2 (IL4, IL10) cytokine profiles may be associated with virological parameters of progression of the disease in HIV‐1‐infected patients. The production of cytokines was studied in LPS + PHA‐activated whole‐blood culture in HIV‐1‐infected individuals at different stages of the disease. The association was investigated between IL12p40 and IL12p70 profiles and other cytokines (IFNγ, IL4, IL10), as well as the isolation of cytopathogenic HIV‐1 strains. The phenotype of HIV strains was studied by a micromethod based on P4 cell line, allowing detection of cytopathic effects of HIV‐1 isolates (syncytium‐induction and cell‐killing without syncytium induction). The individual variations in IL12p40 and IL12p70 production were limited in the healthy controls. Low values were observed in HIV‐1‐infected patients. The production of IL12 (p40 and p70) and the IL12p70/IL4 ratio and the IFNγ/IL4 ratio were significantly lower in patients with cytopathic isolates compared with patients with noncytopathic isolates, and a correlation was obtained between the values of IL12 (IL12p40 and IL12p70) and those of IFNγ/IL4 ratio. There was no increase in the secretion of IL4 and IL10 in patients with cytopathic strains compared with other patients. The results indicate a decreased production of type 1 cytokines (IL12, IFNγ) in the presence of a relatively preserved production of type 2 cytokines (IL4, IL10) in HIV‐1‐infected patients. In conclusion, the defect of production of IL12 by whole blood is associated with virological correlates of progression of HIV‐1 disease. J. Med. Virol. 55:209–214, 1998.

Production of TNFα and IL-6 by Activated Whole Blood from HIV-1 Infected Patients Detected by a One-Stage Procedure: Relationship with the Phenotype of HIV-1 Isolates

Diluted whole blood (WB) culturing may be the most appropriate milieu in which to study cytokine production in vitro. We tested TNFα and IL‐6 production using small volumes of WB (25 μl) from HIV‐1 positive patients with a one‐step procedure that combines WB stimulation with LPS, PHA and cytokine measurement. We studied 49 patients without secondary infection or at distance of secondary infection staged according to the 1993 classification of the CDC and 12 healthy seronegative subjects. Heparinized blood from 5 control subjects had been collected sequentially during a period of 5 months. The individual variations of TNFα and IL‐6 production were limited for all these individuals. In 1 out of 20 CDC group A patients, 6 out of 17 CDC group B patients and 3 out of 12 CDC group C patients, we obtained higher values of TNFα than the mean + 2 S.D. of the control group. In 3 out of 20 CDC group A patients, 1 out of 17 CDC group B patients without AIDS and 5 out of 12 CDC group C patients, the TNFα values were lower than the mean −2 S.D. of the control group. Low IL‐6 values were obtained in 1 out of 20 CDC group A patients and 1 out of 17 CDC group B patients and 3 out of 12 CDC group C patients. There was no correlation between TNFα production in vitro and plasma level of TNFα. We found no correlation between the levels of cytokines and monocyte count or between the levels of cytokines and CD4 T‐cell count in peripheral blood. Our data point out a disarray in TNFα and IL‐6 production by WB from HIV‐1 infected patients. The relationship between the disarray of cytokine production and cytopathogenicity of HIV‐1 isolates in the P4 cell line was investigated in this study. We found a correlation between the high level of TNFα produced by WB and the phenotype of HIV‐1 isolates isolated from patients. The one‐stage procedure used in this work is of potential value to investigate the activation status of cells for monitoring HIV‐1 positive individuals and predicting HIV‐1 phenotype.

A Microassay for Determination of the Cytopathogenicity of Human Immunodeficiency Virus Type-1 Isolates

Correlations between the in vitro biological properties of HIV strains isolated from patients and the prognosis of their disease have been reported. We developed a technique to study the phenotype of HIV strains isolated from patients. We used the P4 cell line, derived from HeLa cells, which has been transfected with receptor CD4 gene. HIV laboratory strain (HIVLAI) and peripheral blood leukocytes (PBLs) from donors infected with HIVLAI induce syncytium in P4 cell cultures in vitro. The presence of reporter gene (LacZ gene) under the control of the HIV‐1 long terminal repeat (LTR) in these cells allows colorimetric visualization of syncytia in the cytoplasm using a β‐galactosidase (βgal) assay in the presence of X‐gal. We cocultivated 1 × 106 patient PBLs with 2 × 106 normal PHA‐activated normal PBLs for 4 days in the presence of IL‐2 in 24‐well plates. Half of the medium was replaced twice a week and PHA‐activated normal PBLs were added every 7 days. HIV‐1 was isolated from cocultured PBLs of 18 patients with advanced‐stage HIV infection as assessed by the production of HIV p24 detected with a commercially available HIV‐1 p24 ELISA. Supernatant and 105 cells were collected twice a week from cocultured PBLs and were added to P4 cells in 96‐well microtiter plates. The cultures were observed every day for 3 days and then the βgal assay was performed. We did not observe any effect with cells and supernatant from 8 patients, harvested from cultures incubated for as long as 28 days. The phenotype of these isolates was called NC (noncytopathic). In cells from 2 patients, we obtained blue multinucleated giant cells; the phenotype of these strains was called SI (syncytium inducing). In cultures from 8 other patients, we obtained the death of P4 cells without syncytium formation, and the phenotype of these strains was called CI (cell‐killing inducing). In every case, the cytopathic effect of HIV‐1 isolates could be detected with cocultured PBLs collected as early as day 4 of culture. Cocultured PBLs from 13 healthy controls did not alter the P4 cells. We displayed the replication of CI strains of HIV‐1, but not the one of NC strains in P4 cell line. Our micromethod allowed the detection of cytopathic effects of HIV isolates. Further investigations should define the clinical applications of this method.

Imbalance in cytokine production by whole blood related to presence of cytopathogenic HIV-1 strains in HIV-1-infected patients

SummaryThe possible association between the emergence of cytopathogenic HIV-1 variants and disturbance of the cytokine production in the course of HIV-1 infection was studied in 18 infected patients. The cytopathogenicity of the isolates was studied in a microassay based on the use of HIV-1-infectible Hela-CD4 cells carrying the bacterial LacZ gene under the control of the HIV-LTR (P4 cells). In addition, the production of cytokines by heparinized whole blood (HWB) obtained the same day from HIV-1(+) patients was measured. TNF-α was determined in a one-step procedure combining HWB culture in the presence of LPS+PHA for 24 h and detection of cytokines in the same wells. In separate experiments HWB was cultured in the presence of LPS+PHA for 48 h, then the supernatants were collected and stored until assayed by ELISA for IFN-γ and IL-4. Higher TNF-α levels were found in activated HWB of patients with cytopathic strains (n=9) than in patients with non-cytopathic strains (n=9, p=0.02) as assed with P4 cells. A defective production of type 1 cytokine (IFN-γ) and no increased secretion of type 2 cytokines (IL-4) was observed in patients with cytopathic strains. IFN-γ/IL-4 ratios were significantly lower in patients with cytopathic strains (n=9) than in other patients (n=9, p=0.009). The results show that the disarray of cytokine production, as assessed with whole blood culture, is associated with the cytopathogenicity of HIV-1 isolates in HIV-1-infected individuals.

A Study of Two Procedures of HIV-1 Isolation from Whole Blood Cultures

Culture techniques for isolation of HIV‐1 from small amounts of whole blood (WB) treated with anticoagulant have been reported and gave results identical to those of culture of separated peripheral blood mononuclear cells. Some authors obtained much higher isolation rates when EDTA was used instead of heparin. We compared two previously described techniques for cultivation of HIV‐1 from WB of adult HIV+ patients staged according to the CDC classification. In addition, we assessed the influence of the type of anticoagulant used for the collection of blood in viral replication in cell cultures from whole blood. Small volumes of WB treated with either heparin or EDTA were cocultivated with phytohemagglutinin (PHA)‐stimulated peripheral blood mononuclear cells (PHA‐PBMC) from healthy donors. We used two procedures for WB culture: procedure I, based on the culture of 250 μl of WB with 1 × 106 PHA‐PBMC from donors; and procedure II based on the culture of 500 μl of WB with 4× 106 PHA‐PBMC from donors. The cocultures were placed in 24‐well plates and incubated for as long as 28 days in medium containing interleukin 2 (IL‐2). Twice weekly half of the medium was replaced with fresh medium. In procedure II, one million fresh PHA‐PBMC from donors was added on the 7th day of culture. The culture supernatant was assayed for the presence of HIV‐1 p24 antigen in an enzyme immunoassay. The kinetics of HIV‐1 replication in cultures of WB from 7 AIDS patients were similar using procedures I and II. In 8 HIV + patients the isolation rate was higher with heparin‐ than with EDTA‐treated samples. The isolation rate was higher in AIDS patients (n = 8) than in others with both methods. In stage IV patients without AIDS (n = 8) we failed to isolate HIV‐1 in 1 patient with procedure I, whereas we succeeded with procedure II. In stage II, HIV‐I was isolated in 1 of 4 patients with both methods. HIV was isolated in cultures of WB from patients receiving zidovudine or related nucleoside analogues and in cultures of WB from untreated patients. HIV‐1 could not be isolated from WB of patients with more than 400 CD4+ T lymphocytes in their peripheral blood (n=4); however, it was isolated from 14 of 16 patients with less than 400 CD4+ T lymphocytes. Our results suggest that procedure II is more sensitive than procedure I and that heparin is better than EDTA for collecting WB. We showed that the rate of HIV‐1 isolation from WB increased in advanced‐stage patients. Further studies are needed to define the clinical applications of WB culture.

Combination of whole blood culture and a rapid and sensitive cell assay for the determination of the cytopathogenicity of human immunodeficiency virus type-1 isolates.

It has been reported that in vitro biological properties of human immunodeficiency virus type 1 (HIV-1) isolates from patients are correlated with the prognosis of HIV-1 infection. A rapid assay was developed to study the phenotype of HIV-1 isolates. The P4 cell line is a HIV-1 infectible Hela CD4 cell carrying the bacterial LacZ gene under the control of the HIV-1 LTR (long terminal repeat). Conventional peripheral blood mononuclear cells (PBMCs) co-culture and heparinized whole blood (HWB) co-culture with normal PBMCs were used for HIV-1 isolated strains from 17 HIV-1-infected patients. The sensitivity of P4 cells was higher than that of MT-2 cells for detecting syncytia induced by HIV-1LAI (lymphadenopathy-associated virus). Like MT-2 cells, P4 cells enable the detection of syncytium inducing strains isolated in peripheral blood mononuclear cells (PBMCs) and HWB cultures. HIV-1 isolates with both culture methods from certain patients induced cytolysis without syncytium in P4 cells but had no cytopathic effect on MT-2 cells. The experiments are in favour of the direct effect of HIV-1 isolates of these patients in the lysis of P4 cells but its mechanism has not been elucidated. It was shown that the combination of whole blood culture for HIV-1 isolation and phenotype study with P4 cell assay is rapid and sensitive and could be used to monitor HIV-1-infected patients.

Bacterial infections in hospitalized HIV patients

Summary A prospective screening of 473 hospitalized HIV infected patients gave 253 bacterial infections in 169 patients. Pneumonia was the most frequent infection (n=106). Infections were documented in 111/253 (44%) episodes. The most frequent isolated pathogens were Staphylococcus sp. (n=25), Pseudomonas aeruginosa (n=21), Escherichia coli (n=20), and Streptococcus pneumoniae (n=18). Factors associated with infections were: intravenous drug use (RR: 1.38; 95% CI: 1.07–1.77, p −3 ) was the only significant factor for community acquired infections while CD4 −3 ) and AIDS (RR 4.71, 95% CI 2.05–10.85, p −4 ) were significant only for nosocomial infections. We conclude that the main risk factors for community acquired and nosocomial infections are intravenous drug use and AIDS, respectively.