Background: Current HIV-1 viral-load assays are too expensive for resource-limited settings. In some countries, monitoring of antiretroviral therapy is now more expensive than treatment itself. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes. Methods: We evaluated real-time reverse transcription-PCR with internal control targeting the conserved long terminal repeat (LTR) domain of HIV-1 on reference panels and patient samples from Brazil (n ؍ 1186), South Africa (n ؍ 130), India (n ؍ 44), and Germany (n ؍ 127). Results: The detection limit was 31.9 IU of HIV-1 RNA/mL of plasma (>95% probability of detection, Probit analysis). The internal control showed inhibition in 3.7% of samples (95% confidence interval, 2.32%-5.9%; n ؍ 454; 40 different runs). Comparative qualitative testing yielded the following: Roche Amplicor vs LTR assay (n ؍ 431 samples), 51.7% vs 65% positives;
An acute and often severe respiratory illness emerged in southern China in late 2002 and rapidly spread to different areas of the Far East as well as several countries around the globe. When the outbreak of this apparently novel infectious disease termed severe acute respiratory syndrome (SARS) came to an end in July 2003, it had caused over 8000 probable cases worldwide and more than 700 deaths. Starting in March 2003, the World Health Organization (WHO) organised an unprecedented international effort by leading laboratories working together to find the causative agent. Little more than one week later, three research groups from this WHO-coordinated network simultaneously found evidence of a hitherto unknown coronavirus in SARS patients, using different approaches. After Koch's postulates had been fulfilled, WHO officially declared on 16 April 2003 that this virus never before seen in humans is the cause of SARS. Ever since, progress around SARS-associated coronavirus (SARS-CoV) has been swift. Within weeks of the first isolate being obtained, its complete genome was sequenced. Diagnostic tests based on the detection of SARS-CoV RNA were developed and made available freely and widely; nevertheless the SARS case definition still remains based on clinical and epidemiological criteria. The agent's environmental stability, methods suitable for inactivation and disinfection, and potential antiviral compounds have been studied, and development of vaccines and immunotherapeutics is ongoing. Despite its grave consequences in humanitarian, political and economic terms, SARS may serve as an example of how much can be achieved through a well-coordinated international approach, combining the latest technological advances of molecular virology with more "traditional" techniques carried out to an excellent standard.
We describe a new human immunodeficiency virus type 1 (HIV-1) mutational pattern associated with phenotypic resistance to lamivudine (3TC) in the absence of the characteristic replacement of methionine by valine at position 184 (M184V) of reverse transcriptase. Combined genotypic and phenotypic analyses of clinical isolates revealed the presence of moderate levels of phenotypic resistance (between 4-and 50-fold) to 3TC in a subset of isolates that did not harbor the M184V mutation. Mutational cluster analysis and comparison with the phenotypic data revealed a significant correlation between moderate phenotypic 3TC resistance and an increased incidence of replacement of glutamic acid by aspartic acid or alanine and of valine by isoleucine at residues 44 and 118 of reverse transcriptase, respectively. This occurred predominantly in those isolates harboring zidovudine resistance-associated mutations (41L, 215Y). The requirement of the combination of mutations 41L and 215Y with mutations 44D and 44A and/or 118I for phenotypic 3TC resistance was confirmed by site-directed mutagenesis experiments. These data support the assumption that HIV-1 may have access to several different genetic pathways to escape drug pressure or that the increase in the frequency of particular mutations may affect susceptibility to drugs that have never been part of a particular regimen.The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) variants is almost always observed during the course of treatment of patients with antiretroviral drugs (3, 10, 14-16, 18, 21, 27 Strategies, abstr. 19, p. 15, 1998). The mutational profile of the resistant viruses generally is characteristic for the particular drug(s) taken. For example, mutations at codons 41, 67, 70, 210, 215, and 219 of reverse transcriptase (RT) typically confer resistance to zidovudine (ZDV) (6,12,13,27). Similarly, mutation M184V in RT has been shown to be specifically associated with high-level (Ն50-fold) phenotypic resistance to lamivudine (3TC) (1,22,28). No "specific" mutation(s) associated with moderate levels of phenotypic resistance (4-to Ͻ50-fold) to 3TC has been described before. Those mutations that confer moderate (4-to Ͻ50-fold) levels of phenotypic resistance to 3TC reported previously always appeared in the context of a constellation of mutations that confer resistance to multiple nucleoside analogues or as a cross-resistance phenomenon that appears with the emergence of resistance to another nucleoside analogue. This has been the case for the nucleoside multidrug resistance complex of mutations Q151M, F77L, F116Y, A62V, and V75I, although the increase in the level of phenotypic resistance to 3TC in viruses that harbor those mutations is slight (9,20,24,25). In the case of the insertion mutations near position 69 of RT, a notable increase in the frequency of 3TC resistance has been reported together with an increased frequency of phenotypic resistance to other nucleosides (2, 17, 29). The K65R mutation appears infrequently during the course of tr...
To investigate the occurrence of multinucleoside analog resistance during therapy failure, we surveyed the drug susceptibilities and genotypes of nearly 900 human immunodeficiency virus type 1 (HIV-1) samples. For 302 of these, the 50% inhibitory concentrations of at least four of the approved nucleoside analogs had fourfold-or-greater increases. Genotypic analysis of the reverse transcriptase (RT)-coding regions from these samples revealed complex mutational patterns, including the previously recognized codon 151 multidrug resistance cluster. Surprisingly, high-level multinucleoside resistance was associated with a diverse family of amino acid insertions in addition to “conventional” point mutations. These insertions were found between RT codons 67 and 70 and were commonly 69Ser-(Ser-Ser) or 69Ser-(Ser-Gly). Treatment history information showed that a common factor for the development of these variants was AZT (3′-azido-3′-deoxythymidine, zidovudine) therapy in combination with 2′,3′-dideoxyinosine or 2′,3′-dideoxycytidine, although treatment patterns varied considerably. Site-directed mutagenesis studies confirmed that 69Ser-(Ser-Ser) in an AZT resistance mutational background conferred simultaneous resistance to multiple nucleoside analogs. The insertions are located in the “fingers” domain of RT. Modelling the 69Ser-(Ser-Ser) insertion into the RT structure demonstrated the profound direct effect that this change is likely to have in the nucleoside triphosphate binding site of the enzyme. Our data highlight the increasing problem of HIV-1 multidrug resistance and underline the importance of continued resistance surveillance with appropriate, sufficiently versatile genotyping technology and phenotypic drug susceptibility analysis.
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