Comparison of genotypic and phenotypic resistance patterns of human immunodeficiency virus type 1 isolates from patients treated with stavudine and didanosine or zidovudine and lamivudine.

Sequencing of reverse-transcriptase genes and recombinant virus assays were performed on paired isolates from antiretroviral drug-naive patients randomized to stavudine and didanosine (group 1; n = 21) or zidovudine and lamivudine (group 2; n = 21) at baseline and after > or = 12 months of follow-up. The T215Y mutation emerged in 13 (61.9%) and 2 (9.5%) isolates in groups 1 and 2, respectively (P < .0001). Furthermore, in group 1, mutations associated with multidideoxynucleoside resistance were selected in 3 isolates. In group 2, all isolates carried the M184V mutation. The median fold changes in susceptibilities to zidovudine, stavudine, and lamivudine were 16.4 and 1, 2.2 and 0.6, and 4.5 and > 38 in groups 1 and 2, respectively (P < .0001, all comparisons). These results suggest that the combination of stavudine and didanosine is associated more frequently with the emergence of zidovudine resistance and a decrease in susceptibility to stavudine than the combination of zidovudine and lamivudine.

Laboratory guidelines for the practical use of HIV drug resistance tests in patient follow-up.

HIV drug resistance is one of the major limitations in the successful treatment of HIV-infected patients using currently available antiretroviral combination therapies. When appropriate, drug susceptibility profiles should be taken into consideration in the choice of a specific combination therapy. Guidelines recommending resistance testing in certain circumstances have been issued. Many clinicians have access to resistance testing and will increasingly use these results in their treatment decisions. In this document, we comment on the different methods available, and the relevant issues relating to the clinical application of these tests. Specifically, the following recommendations can be made: (i) genotypic and phenotypic HIV-1 drug resistance analyses can yield complementary information for the clinician. However, insufficient information currently exists as to which approach is preferable in any particular clinical setting; (ii) when HIV-1 drug resistance testing is required, it is recommended that testing be performed on plasma samples obtained before starting, stopping or changing therapy, on samples that have a viral load above the detection limit of the resistance test; (iii) the panel recommends that genotypic and phenotypic HIV-1 drug resistance testing for clinical purposes be performed in a certified laboratory under strict quality control and quality assurance standards; and (iv) the panel recommends that resistance testing laboratories provide clinicians with resistance reports that include a list of drug-related resistance mutations (genotype) and/or a list of drug-related fold resistance values (phenotype), with interpretations of each by an experienced virologist. The interpretation of genotypic and phenotypic analysis is a complex and developing science, and in order to understand HIV-1 drug resistance reports, communication between the requesting clinician and the expert that interpreted the resistance report is recommended.

Genotypic and phenotypic resistance patterns of human immunodeficiency virus type 1 variants with insertions or deletions in the reverse transcriptase (RT): multicenter study of patients treated with RT inhibitors.

Genomic rearrangements in the 5' part of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been involved in multidrug resistance to nucleoside RT inhibitors (NRTI). We carried out a retrospective, multicenter study to investigate the prevalence, variability, and phenotypic consequences of such rearrangements. Data concerning the HIV-1 RT genotype and the biological and clinical characteristics of NRTI-treated patients were collected from 10 virology laboratories. Sensitivities of the different HIV-1 variants to RT inhibitors were analyzed in a single-cycle recombinant virus assay. Fifty-two of 2,152 (2.4%) RT sequences had a rearrangement in the 5' part of the RT, with an extensive molecular variation. The number of codons inserted between positions 68 and 69 ranged from 1 (3 samples) or 2 (41 samples) to 5 and 11 in one case each. In four cases, codon 67 was deleted. High levels of phenotypic resistance to zidovudine (AZT), lamivudine (3TC), stavudine (d4T), abacavir (ABC), and didanosine (ddI) were found in 95, 92, 72, 62, and 15% of the 40 samples analyzed, respectively. Resistance to AZT, d4T, and ABC could be found in the absence of the T215Y/F mutations. Resistance to 3TC could develop in the absence of specific mutations. Low-level resistance to ddI was noticed in 40% of the patients. The deletions of codon 67 seemed to have little effect on NRTI sensitivity. Most of the rearrangements were shown to contribute to cross-resistance to NRTI. The results regarding susceptibility to ddI raise the question of the interpretation of the phenotypic data concerning this drug.

Attachment disruptions in seriously emotionally disturbed children: implications for treatment.

In this paper, we consider the effect of attachment disruptions on severe adjustment problems in school-age boys. Three groups of 9-11-year-old boys were sampled based on their degree of risk for adjustment difficulties: (1) boys in regular classrooms, (2) boys in regular classrooms who are at risk due to poverty, and (3) boys who have been placed in special education classrooms as a result of serious emotional disturbance (SED). Attachment disruptions were categorized according to the severity of major separations from the biological mother. SED children experienced significantly more severe disruptions of their relationships with their biological mothers and fathers than either the high-risk or comparison boys. Teachers' ratings indicated that both the high-risk and SED boys experienced more externalizing symptoms than comparison boys in regular classrooms. However, SED children were most clearly discriminated from their high-risk and comparison counterparts by higher levels of dissociative symptoms. Regression analyses indicated that children who had experienced maternal attachment disruptions were more likely to show dissociative symptomatology in the classroom setting and were more likely to develop dependent relationships with their teachers after risk group status, child age and family structure were controlled. Implications of these findings for the treatment of SED children are discussed.

Analysis of HIV cross-resistance to protease inhibitors using a rapid single-cycle recombinant virus assay for patients failing on combination therapies.

OBJECTIVE: To assess the patterns of HIV phenotypic cross-resistance to protease inhibitors (PI) in patients experiencing viral load rebound on combination therapy including a PI. METHODS: Phenotypic analysis of sensitivity to indinavir, nelfinavir, saquinavir, ritonavir and amprenavir was carried out using a single-cycle recombinant virus assay. Viral protease was sequenced by automated dideoxynucleotide chain termination. RESULTS: Of the 108 patients studied, 68 had received indinavir, 50 ritonavir, 25 saquinavir and eight nelfinavir. The majority (71%) had received only one PI. The incidence of cross-resistance between indinavir, nelfinavir, ritonavir and saquinavir was high (60-90%). Cross-resistance to amprenavir was less frequent (37-40%). However there was some correlation between levels of sensitivity to amprenavir and indinavir (r2 = 0.34; P < 0.01). Conversely, the correlation between levels of sensitivity to indinavir and saquinavir was poor (r2 = 0.25), particularly for patients who had not received saquinavir. The degree of cross-resistance correlated with the level of resistance and with the total number of mutations in the protease gene (P < 0.05, chi square test) but could not be significantly correlated to any one particular mutation or combination of mutations. Mutation 184V was significantly associated with cross-resistance to amprenavir, with no mutations at codon 50 observed, while mutations associated with cross-resistance to saquinavir differed according to the treatment received. CONCLUSIONS: These results suggest that, although the total number of protease mutations correlates with the degree of cross-resistance, the specific mechanisms accounting for primary resistance and for cross-resistance may be different.

Transmission of antiretroviral-drug-resistant HIV-1 variants.

BACKGROUND: Resistance of HIV-1 to antiretroviral drugs is the main cause of antiretroviral-treatment failure. We assessed the transmission of drug-resistant variants among individuals with primary HIV-1 infection. METHODS: Population-based sequencing of the viral reverse-transcriptase and protease genes derived from plasma viral RNA was done in 82 consecutive individuals with documented primary HIV-1 infection from January, 1996, to July, 1998. Phenotypic resistance to protease inhibitors was assessed by recombinant virus assay in individuals with two or more mutations associated with resistance to protease inhibitors. FINDINGS: Zidovudine-resistance mutations were detected in seven (9%) of 82 individuals. Mutations associated with resistance to other reverse-transcriptase inhibitors (RTIs) were detected in two individuals. Primary-resistance mutations associated with protease inhibitors (V82A, L90M) were detected in three (4%) of 70 individuals; two of these had also RTI-resistance mutations. Decreased sensitivity to three or four protease inhibitors was seen in three individuals, one of whom was infected with HIV-1 variants that harboured 12 mutations associated with resistance to multiple RTI and protease inhibitors. INTERPRETATION: To introduce the best antiretroviral treatment, resistance testing should be done in recently HIV-1-infected individuals.

Efficacy of a five-drug combination including ritonavir, saquinavir and efavirenz in patients who failed on a conventional triple-drug regimen: phenotypic resistance to protease inhibitors predicts outcome of therapy.

OBJECTIVE: to assess the safety and efficacy of a combination of ritonavir, efavirenz and two recycled nucleosides in patients who failed on a conventional triple-drug regimen including indinavir or ritonavir. METHODS: An open label study of ritonavir (100 mg twice daily), saquinavir (1000 mg twice daily), efavirenz (600 mg per day) and nucleoside analogues in 32 saquinavir- and efavirenz-naive protease inhibitor-experienced patients. Patients were included on the basis of plasma levels of HIV RNA above 5000 copies/ml while on conventional antiretroviral therapy. Phenotypic resistance and genotypic resistance mutations to saquinavir were assessed at baseline. Peak and trough plasma levels of saquinavir were monitored throughout the study. RESULTS: Median CD4 cell counts and median plasma HIV RNA at baseline were 258 x 10(6)/l and 4.31 log10 copies/ml, respectively. The plasma viral load decreased by a median of 1.20 log10 copies/ml and the CD4 cell count increased by a median 60 x 10(6) cells/l at week 24 of therapy. Seventy-one per cent of the patients achieved a plasma viral load < 500 copies/ml and 45% achieved a viral load < 50 copies/ml. Patients exhibiting phenotypic resistance to saquinavir at baseline experienced a median decrease in HIV RNA of 0.91 log10 copies/ml at week 24 of therapy, as compared with a decrease of 1.52 log10 copies/ml in those exhibiting sensitive viral strains (P = 0.03). Genotypic resistance to saquinavir was not predictive of virologic failure. CONCLUSION: Our results indicate that the combination of ritonavir, saquinavir and efavirenz is safe and effective at 24 weeks in over two-thirds of patients who previously failed on highly active antiretroviral therapy, and that the determination of phenotypic resistance may be of greater value than the detection of resistance mutations to predict the outcome of salvage therapy in this setting.

Zidovudine resensitization and dual HIV-1 resistance to zidovudine and lamivudine in the delta lamivudine roll-over study.

OBJECTIVE: To study zidovudine resensitization and dual resistance to zidovudine/lamivudine in HIV-1 isolates from nucleoside reverse transcriptase (RT) inhibitor-experienced patients during selective pressure exerted by zidovudine/lamivudine combination therapy. DESIGN AND METHODS: HIV-1 isolates from 29 patients receiving zidovudine/lamivudine combination therapy in the Delta roll-over study were analysed at entry and during a 1 year follow-up period for phenotypic susceptibility to zidovudine and lamivudine in the ANRS PBMC assay. The RT gene from codon 20 to 230 and at codon 333 was analysed by nucleotide sequencing of the corresponding isolates. RESULTS: HIV-1 isolates from 23 of the 29 patients were phenotypically resistant to zidovudine at baseline; 61% of these patients showed significant zidovudine resensitization during follow-up. The zidovudine IC50 value correlated positively with log10 plasma HIV-1 RNA (P = 0.02) and negatively with the CD4 cell count (P = 0.004). Zidovudine resensitization (related to acquisition of the M184V mutation) was transient, with evolution towards dual resistance to zidovudine and lamivudine in 20 of the 29 patients. The phenotype of certain dually resistant isolates coincided with the emergence of multiple mutations in the 5' part of the RT gene. CONCLUSIONS: M184V-mediated zidovudine resensitization of HIV-1 is transient in most patients who are given zidovudine/lamivudine combination therapy when zidovudine resistance has already emerged. The subsequent evolution towards dual phenotypic resistance to zidovudine/lamivudine corresponds to complex genotypic profiles.

HIV protease genotype and viral sensitivity to HIV protease inhibitors following saquinavir therapy.

OBJECTIVE: To examine the relationship between HIV protease genotype and altered protease inhibitor sensitivity of isolates from patients after therapy with saquinavir (SQV) in its hard gelatin formulation. DESIGN: Forty-one post-therapy isolates and corresponding baseline samples were obtained from 37 patients in four different clinical trials after therapy with SQV for 16-147 weeks. Post-therapy isolates were selected on the basis of preliminary sequence or drug sensitivity data. RESULTS: Fifteen out of 17 isolates without detectable Val-48 or Met-90 mutations retained sensitivity to SQV. (The remaining isolates showed only a marginal increase in median inhibitory concentration.) In addition, three out of 15 isolates with Met-90 retained sensitivity to all other protease inhibitors tested (indinavir, ritonavir, amprenavir, nelfinavir). Of the isolates showing reduced sensitivity to SQV, six out of 22 retained sensitivity to all other protease inhibitors, whereas only four out of 22 showed broad cross-resistance to all protease inhibitors tested. The reduction in sensitivity correlated closely with the presence of Val-48 or Met-90. Subsequent accessory substitutions were also linked to reduced sensitivity. However, significant linkage was observed only between mutations at residues 48 and 82 and between those at residues 82 and 74. CONCLUSIONS: Recruitment of Val-48/Met-90 mutations was not found to be synonymous with cross-resistance. Indeed, the majority of isolates with these mutations retained sensitivity to at least one protease inhibitor (Val-48, 86%; Met-90, 77%). The recruitment of accessory mutations may occur only after the selection of key resistance mutations. Furthermore, Met-90 was found to be a poor marker of cross-resistance in SQV-treated patients.

Correlation of response to treatment and HIV genotypic changes during phase III trials with saquinavir and reverse transcriptase inhibitor combination therapy.

OBJECTIVES: Assessment of genotypic change in HIV protease during treatment with saquinavir (SQV) in combination with zidovudine (ZDV) and/or zalcitabine (ddC), to determine the influence of such changes on viral phenotype and response to treatment. DESIGN: Virologic substudies of Phase III clinical trials NV14256 and SV14604. METHODS: Population sequencing of HIV protease genes amplified from pre- and post-treatment plasma. Phenotyping of peripheral blood mononuclear cell (PBMC)-derived virus isolates, and genotyping of proviral DNA clones amplified from PBMC used in the expansion of virus isolates. RESULTS: In both trials the incidence of Met90 remained at < or = 20% in subjects receiving SQV in combination with ddC (with or without ZDV) for 1 year. A Val48 substitution was observed in two out of 81 subjects after 24 weeks and in two out of 75 subjects after 48 weeks. In 12 out of 13 NV14256 subjects with viral load rebound during SQV monotherapy these substitutions were associated with the rebound. In subjects treated with SQV plus ddC, rebound was associated with SQV resistance in six out of 22 cases and ddC resistance in five out of 22 cases. The incidences of non-BRU residues at positions 10, 63 and 71 were increased significantly (P < 0.05, Fisher's exact test) after SQV treatment with or without ZDV. However, comparison of genotypic and phenotypic data showed that these changes were not associated with reduced sensitivity to SQV. CONCLUSIONS: Virological failure during combination therapy can be due to resistance to either treatment drug, emphasising the need to change both the reverse transcriptase inhibitor and the protease inhibitor. Only Val48 and Met90 correlated directly with the development of reduced drug sensitivity during treatment with SQV in vivo.

Focal mycobacterial lymphadenitis following initiation of protease-inhibitor therapy in patients with advanced HIV-1 disease.

BACKGROUND: Inhibitors of HIV-1 protease produce a rapid decrease in plasma HIV-1 RNA, with concomitant increases in CD4 T-helper lymphocyte counts. The main side-effects of the protease inhibitors currently in use include gastrointestinal disturbances, paraesthesias, hyperbilirubinaemia, and nephrolithiasis. The increasing use of these agents in patients with advanced HIV-1 infection and CD4 counts of less than 50 cells/microL may be associated with unforeseen adverse effects not observed in earlier studies of patients with higher CD4 counts. METHODS: Five HIV-infected patients with baseline CD4 lymphocyte counts of less than 50 cells/mL were admitted to the Beth Israel Deaconess Medical Center (Boston, MA, USA) with high fever (> 39 degrees C), leucocytosis, and evidence of lymph-node enlargement within 1-3 weeks of starting indinavir therapy. Informed consent was obtained for studies that entailed CD4 lymphocyte counts, immunophenotyping, isolator blood cultures, and radiological scans. Biopsy samples of cervical, paratracheal, or mesenteric lymph nodes were taken for culture and pathology in four patients. FINDINGS: Lymph-node biopsy samples showed that focal lymphadenitis after initiation of indinavir resulted from unsuspected local or disseminated Mycobacterium avium complex (MAC) infection. The prominent inflammatory response to previously subclinical MAC infection was associated with leucocytosis in all patients and with an increase in the absolute lymphocyte counts in four patients. Three patients with follow-up CD4 counts showed two-fold to 19-fold increases after 1-3 weeks of indinavir therapy. Immunophenotyping after therapy in two patients showed that more than 90% of the CD4 cells were of the memory phenotype. INTERPRETATION: The initiation of indinavir therapy in patients with CD4 counts of less than 50 cells/mL and subclinical MAC infection may be associated with a severe illness, consisting of fever (> 39 degrees C), leucocytosis, and lymphadenitis (cervical, thoracic, or abdominal). The intense inflammatory reactions that make admission to hospital necessary may be secondary to significant numbers of functionally competent immune cells becoming available to respond to a heavy mycobacterial burden. Prophylaxis or screening for subclinical MAC infection, or both, should therefore be done before the beginning of protease-inhibitor therapy in patients with advanced HIV infection.

The Family Atherosclerosis Risk Intervention Study (FARIS): risk factor profiles of patients and their relatives following an acute cardiac event.

BACKGROUND: Relatives of patients with coronary heart disease have a heightened risk of cardiovascular disease. Attendance at a family-based screening clinic after an acute cardiac event could motivate patients and relatives to modify their lifestyles. AIMS: The Family Atherosclerosis Risk Intervention Study (FARIS) aimed to determine (i) whether a high proportion of patients and relatives would attend a special screening and prevention programme; (ii) whether the risk factor profiles of relatives would be worse than those in the general community; and (iii) whether ongoing management of patients and families together in a special clinic would improve risk factor profiles. METHODS: Consecutive patients, together with spouse, siblings and offspring, aged 18 to 69 years, were randomly allocated three months after an acute cardiac event to attend a special outpatient clinic, a screening and advice group, or a control group. Risk factor measures were total cholesterol, HDL cholesterol (HDLC), systolic blood pressure (SBP), body mass index (BMI) and smoking behaviour. This paper presents the risk factor profiles of all FARIS attenders and compares those of family members, age adjusted, with risk factors measured in a multicentre urban cross-sectional survey conducted in the same period. Differences between groups were compared using t-tests for numerical variables and ANOVA and chi-square for categorical variables. RESULTS: Six hundred and twenty-eight patients and 1723 family members were enrolled, representing 85.9% and 82.7% of eligible patients and relatives respectively. Risk factors were significantly worse amongst family members than among those in the population survey.

A multistep procedure for the chemical inactivation of human immunodeficiency virus for use as an experimental vaccine.

The kinetics of inactivation of four different strains of HIV-1 (RF, MN, SF2 and IIIB) by beta-propiolactone (BPL) and binary ethylenimine (BEI) were studied under various conditions. The conditions that would be required for the reduction of virus infectivity by at least 10(20) TCID50 ml-1 were estimated on the basis of the experimental rates of inactivation obtained. A multiple step procedure including treatment with 0.2% BPL, 0.05% sodium cholate, 10 mM BEI and 0.02% formaldehyde was designed to inactivate HIV-1 for use as an experimental vaccine. Complete inactivation of virus infectivity was confirmed by prolonged cell culture. The experimental vaccine preparation was analysed for the presence of HIV-1 proviral DNA utilizing the polymerase chain reaction. After treatment with both BPL and BEI proviral DNA was detected in one of four samples using primers encoding a 244 bp segment of the pol region of the viral genome. Proviral DNA could not be detected in any of the four samples using primers encoding segments of > 400 bp in the gag and reverse transcriptase region.

Cardiac rehabilitation based on group light exercise and discussion. An Australian hospital model.

Patients with cardiac disease potentially have many physical, psychological, and social problems. With adequate rehabilitation, most patients can return to enjoyable, productive lives. If rehabilitation programs can be provided at relatively low cost, they can be introduced more widely. It is obviously important that low-cost programs should include most of the effective components of more sophisticated programs. The program presented here, based on group light exercise and discussion, is effective and inexpensive. Therefore, it provides a potential model for group cardiac rehabilitation in situations where cost-containment is an important consideration.

An experimental chemically inactivated HIV-1 vaccine induces antibodies that neutralize homologous and heterologous viruses.

We have developed a unique multiple step procedure to inactivate human immunodeficiency virus chemically with a very high safety margin while retaining antigenically active structural virion proteins, including gp120, in the final immunogen. The whole virus preparation (1-10 micrograms per dose) was highly immunogenic in a variety of small mammals and induced antibodies that recognized homologous and heterologous strains of HIV-1. Sera from immunized animals bound to peptides representing the entire sequence of the external glycoprotein gp120. Neutralizing antibodies active against the homologous immunizing strain and against heterologous HIV-1 strains were also elicited. Sera with virus neutralizing activity did not bind to MHC class I proteins derived from the human cell line used to grow the virus.

Differential growth kinetics are exhibited by human immunodeficiency virus type 1 TAR mutants.

The human immunodeficiency virus type 1 (HIV-1) TAR element is critical for the activation of gene expression by the transactivator protein, Tat. Mutagenesis has demonstrated that a stable stem-loop RNA structure containing both loop and bulge structures transcribed from TAR is the major target for tat activation. Though transient assays have defined elements critical for TAR function, no studies have yet determined the role of TAR in viral replication because of the inability to generate viral stocks containing mutations in TAR. In the current study, we developed a strategy which enabled us to generate stable 293 cell lines which were capable of producing high titers of different viruses containing TAR mutations. Viruses generated from these cell lines were used to infect both T-lymphocyte cell lines and peripheral blood mononuclear cells. Viruses containing TAR mutations in either the upper stem, the bulge, or the loop exhibited dramatically decreased HIV-1 gene expression and replication in all cell lines tested. However, we were able to isolate lymphoid cell lines which stably expressed gene products from each of these TAR mutant viruses. Though the amounts of virus in these cell lines were roughly equivalent, cells containing TAR mutant viruses were extremely defective for gene expression compared with cell lines containing wild-type virus. The magnitude of this decrease in viral gene expression was much greater than previously seen in transient expression assays using HIV-1 long terminal repeat chloramphenicol acetyltransferase gene constructs. In contrast to the defects in viral growth found in T-lymphocyte cell lines, several of the viruses containing TAR mutations were much less defective for gene expression and replication in activated peripheral blood mononuclear cells. These results indicate that maintenance of the TAR element is critical for viral gene expression and replication in all cell lines tested, though the cell type which is infected is also a major determinant of the replication properties of TAR mutant viruses.

Challenges and strategies for AIDS vaccine development.

The major topics covered at this conference included the relevance of the SIV-macaque model for HIV vaccine development, an evaluation of the methods for producing inactivated vaccine, and the possibility of developing attenuated retrovirus vaccines. The possibility that there might be a Graft Versus Host Disease component in human AIDS was discussed, and also the recent discovery that the 'original antigenic sin' phenomenon encountered in influenza vaccines may also be seen in AIDS. It was concluded that results from animal models were encouraging in the quest for an HIV-1 vaccine.