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he use of combinations of antiretroviral drugs has proven remarkably effective in controlling the progression of human immunodeficiency virus (HIV) disease and prolonging survival, 1 but these benefits can be compromised by the development of drug resistance. 2,3 Resistance is the consequence of mutations that emerge in the viral proteins targeted by antiretroviral agents. In the United States, as many as 50 percent of patients receiving antiretroviral therapy are infected with viruses that express resistance to at least one of the available antiretroviral drugs. 4 Consequently, the transmission of drug-resistant strains is also a growing concern. 5-7 Because drug-resistant HIV often exhibits resistance to several classes of antiretroviral drugs 8 and because cross-resistance between drugs within a class is frequent, 9-12 the emergence of resistance always complicates further efforts to control viral replication. This review focuses on the mechanisms underlying the selection of drug-resistant HIV and on the consequences of viral resistance with respect to the evolution of HIV infection. The drugs currently used to treat HIV type 1 (HIV-1) infection (Table 1) belong to four distinct classes: nucleoside and nucleotide analogues, which act as DNA-chain terminators and inhibit reverse transcription of the viral RNA genome into DNA, a crucial event occurring at an early stage of the viral life cycle; nonnucleoside reverse-transcriptase inhibitors, which bind and inhibit reverse transcriptase, the viral enzyme that conducts reverse transcription; protease inhibitors, which target the viral protease, the enzyme required for the cleavage of precursor proteins (gag and gag-pol), permitting the final assembly of the inner core of viral particles; and entry inhibitors, which block the penetration of HIV virions into their target cells. Combinations of antiretroviral drugs are now used for the treatment of HIV infection-so-called highly active antiretroviral therapy (HAART). Current HAART regimens generally comprise three antiretroviral drugs, usually two nucleoside analogues and either a protease inhibitor or a nonnucleoside reverse-transcriptase inhibitor. 13 The use of agents from different classes is instrumental in controlling the development of resistance, but whereas some drug combinations have been shown to be antagonistic, there is no evidence that any combinations of currently available drugs are strongly synergistic in vitro. induced resistance Two concepts are important to an understanding of the development of drug resistance. First, HIV infection is characterized by high levels of virus production and turnover. In most untreated patients, the total number of productively infected cells in the lymphoid tissue has been estimated to be approximately 10 7 to 10 8 cells. 14 During the chronic phase of HIV infection, this number is relatively stable, reflecting the balance between t principles of drug therapy for hiv development of resistance

Endogenous retroviruses are multicopy retroelements accounting for nearly 10% of murine or human genomes. These retroelements spread into our ancestral genome millions of years ago and have acted as a driving force for genome evolution. Endogenous retroviruses may also be deleterious for their host, and have been implicated in cancers and autoimmune diseases. Most retroelements have lost replication competence because of the accumulation of inactivating mutations, but several, including some murine intracisternal A-particle (IAP) and MusD sequences, are still mobile. These elements encode a reverse transcriptase activity and move by retrotransposition, an intracellular copy-and-paste process involving an RNA intermediate. The host has developed mechanisms to silence their expression, mainly cosuppression and gene methylation. Here we identify another level of antiviral control, mediated by APOBEC3G, a member of the cytidine deaminase family that was previously shown to block HIV replication. We show that APOBEC3G markedly inhibits retrotransposition of IAP and MusD elements, and induces G-to-A hypermutations in their DNA copies. APOBEC3G, by editing viral genetic material, provides an ancestral wide cellular defence against endogenous and exogenous invaders.

Human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors (PI) results from mutations in the viral protease (PR) that reduce PI binding but also decrease viral replicative capacity (RC). Additional mutations compensating for the RC loss subsequently accumulate within PR and in Gag substrate cleavage sites. We examined the respective contribution of mutations in PR and Gag to PI resistance and RC and their interdependence using a panel of HIV-1 molecular clones carrying different sequences from six patients who had failed multiple lines of treatment. Mutations in Gag strongly and directly contributed to PI resistance besides compensating for fitness loss. This effect was essentially carried by the C-terminal region of Gag (containing NC-SP2-p6) with little or no contribution from MA, CA, and SP1. The effect of Gag on resistance depended on the presence of cleavage site mutations A431V or I437V in NC-SP2-p6 and correlated with processing of the NC/SP2 cleavage site. By contrast, reverting the A431V or I437V mutation in these highly evolved sequences had little effect on RC. Mutations in the NC-SP2-p6 region of Gag can be dually selected as compensatory and as direct PI resistance mutations, with cleavage at the NC-SP2 site behaving as a rate-limiting step in PI resistance. Further compensatory mutations render viral RC independent of the A431V or I437V mutations while their effect on resistance persists.

To determine the usefulness of samples obtained by bronchoscopy using a protected specimen brush and evaluated by quantitative culture techniques in establishing the diagnosis of nosocomial pneumonia in patients requiring mechanical ventilation, we prospectively studied 147 ventilated patients suspected of having nosocomial pneumonia because of the presence of a new pulmonary infiltrate and purulent tracheal secretions. Positive cultures of protected brush specimens (greater than 10(3) cfu/ml) were found in only 45 patients (31%). Subsequent follow-up confirmed the diagnosis of pneumonia in 34 of 45 patients, and in only 4 of 45 patients was a positive culture firmly established to be a false positive result. No patient with less than 10(3) cfu/ml was subsequently shown to have had pneumonia, and the diagnosis was definitely excluded in 72 of 102 patients by the absence of pneumonia at autopsy or recovery without antibiotic therapy. In contrast, when 16 clinical variables were evaluated by stepwise logistic regression analysis, no combination could be identified that was useful in distinguishing patients with bacterial pneumonia. Furthermore, when the actual costs of evaluation and therapy of our patients were compared with the projected costs entailed in treating all patients suspected of having pneumonia with antibiotics, evaluation using the protected specimen brush and quantitative cultures was less expensive after only 6 days of treatment. These results suggest that the appearance of pulmonary infiltrates and purulent tracheal secretions does not result from bacterial pneumonia in a majority of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

A 383bp segment of the gene coding for the 65kD mycobacterial antigens from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium fortuitum was amplified using Taq polymerase and synthetic oligonucleotide primers and the amplified DNAs from four of these species were compared by nucleotide sequencing. Although the gene segments from these species showed considerable similarity, oligonucleotide probes which could distinguish M. tuberculosis/M. bovis, M. avium/M. paratuberculosis and M. fortuitum could be identified. Samples containing 10(6) human cells and serial dilutions of a suspension of intact mycobacteria were prepared, DNA was extracted, the segment of the mycobacterial DNA sequence amplified, and the amplified DNA hybridized with oligonucleotide probes. In two independent experiments, this procedure permitted the detection and identification of less than 100 mycobacteria in the original sample. These results suggest that this approach may prove useful in the early diagnosis of mycobacterial infection.

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