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At least 19 recordsLinked to original sources
PubMed1994

Recombinant virus assay: a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates.

Antiviral drug susceptibility assays for clinical human immunodeficiency virus type 1 (HIV-1) isolates are required to monitor the development of drug resistance during clinical trials and antiretroviral drug therapy. First-generation phenotypic assays possess a number of drawbacks, not least the selection of unrepresentative virus populations during cocultivation. Here we describe a rapid phenotypic assay for the assessment of the susceptibility of clinical isolates to reverse transcriptase (RT) inhibitors. This procedure, called the recombinant virus assay, allows the generation of viable virus by homologous recombination of a PCR-derived pool of RT coding sequences into an RT-deleted, noninfectious proviral clone, pHIV delta BstEII. A nested PCR procedure has been optimized to allow the amplification of an RT pool from both uncultured and cocultured infected patient peripheral blood lymphocyte (PBL) DNA for subsequent use in the creation of recombinant viruses. Analysis of two patients during the course of zidovudine therapy showed that this approach produced viruses which accurately exhibited the same genotype and phenotype as that of the original infected PBL DNA. The recombinant virus assay can be performed in approximately 3 weeks without the use of donor PBLs and therefore represents a rapid, nonselective procedure for the assay of clinical isolates.

Antiviral Agents↗
PubMed1996

Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase.

Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.

Base Sequence↗
PubMedDate not supplied

Role of HIV phenotypic assays in the management of HIV infection.

Contemporary phenotypic assays such as the PhenoSense HIV assay are more straightforward to interpret than genotyping results because they do not require the expert interpretation of complex mutation patterns. The drug susceptibility data provides information for the clinician to select a treatment regimen effective against the predominant viral population circulating in the patient's blood. Compared to traditional phenotypic assays, the PhenoSense HIV assay uses a virus vector and a luciferase reporter gene to provide a quick and sensitive measure of viral replication. The main use of phenotypic assays at present is to identify those antiretroviral drugs that still retain activity against the patient's virus. Phenotypic assays are useful to provide guidance after treatment failure and to select a proper combination of drugs prior to initiation of therapy. They are also useful to detect transmission of resistance virus and to monitor HIV patients during early viral rebound. In essence, phenotypic testing provides information to target antiretroviral therapy against the predominant HIV variant in the patient for a prolonged suppression of viral replication, decreased mortality, and lower health-care costs.

Anti-HIV Agents↗
PubMed2006

Determining human immunodeficiency virus coreceptor use in a clinical setting: degree of correlation between two phenotypic assays and a bioinformatic model.

Two recombinant phenotypic assays for human immunodeficiency virus (HIV) coreceptor usage and an HIV envelope genotypic predictor were employed on a set of clinically derived HIV type 1 (HIV-1) samples in order to evaluate the concordance between measures. Previously genotyped HIV-1 samples derived from antiretroviral-naïve individuals were tested for coreceptor usage using two independent phenotyping methods. Phenotypes were determined by validated recombinant assays that incorporate either an approximately 2,500-bp ("Trofile" assay) or an approximately 900-bp (TRT assay) fragment of the HIV envelope gp120. Population-based HIV envelope V3 loop sequences ( approximately 105 bp) were derived by automated sequence analysis. Genotypic coreceptor predictions were performed using a support vector machine model trained on a separate genotype-Trofile phenotype data set. HIV coreceptor usage was obtained from both phenotypic assays for 74 samples, with an overall 85.1% concordance. There was no evidence of a difference in sensitivity between the two phenotypic assays. A bioinformatic algorithm based on a support vector machine using HIV V3 genotype data was able to achieve 86.5% and 79.7% concordance with the Trofile and TRT assays, respectively, approaching the degree of agreement between the two phenotype assays. In most cases, the phenotype assays and the bioinformatic approach gave similar results. However, in cases where there were differences in the tropism results, it was not clear which of the assays was "correct." X4 (CXCR4-using) minority species in clinically derived samples likely complicate the interpretation of both phenotypic and genotypic assessments of HIV tropism.

Computational Biology↗
PubMed2004

Novel recombinant phenotypic assay for clonal analysis of reverse transcriptase mutations conferring drug resistance to HIV-1 variants.

OBJECTIVE: A novel rapid reverse transcriptase (RT) recombinant HIV-1 drug-susceptibility assay was developed to evaluate resistance to RT inhibitors. MATERIAL AND METHODS: HIV-1 RTs from five treatment-naive and 10 highly active antiretroviral therapy-experienced patients were evaluated. HIV-1 isolates recovered by culturing peripheral blood mononuclear cells from patients were used in the conventional isolate phenotype analysis. Recombinant HIV-1 strains were obtained by cloning the RT gene amplified from the supernatant of HIV-1 cultures in a plasmid carrying the HIV-1 strain HXB2 backbone, and the most represented clone for each virus isolate was then tested for antiviral drug susceptibility in parallel with HIV-1 isolates. RESULTS: Comparison of conventional virus isolate and the novel recombinant virus phenotypic assays showed a large concordance of results. However, some discrepant results were observed, in that higher drug-resistance levels were detected by the conventional isolate phenotypic assay in HIV-1 isolates showing the presence of a mixture of HIV-1 variants, whereas the novel recombinant phenotypic assay could more precisely detect the level of drug resistance of the single viral clones selected for the analysis. DISCUSSION: The novel recombinant phenotype assay, compared with the conventional virus isolate phenotype assay, showed widely overlapping results. The comparison of the two assays show that the conventional phenotypic assay is able to identify more efficiently the combined effect of drug-resistant viral variants, whereas the novel recombinant phenotypic assay is better able to define the level of drug resistance of the single viral variants. In addition, rapidity (2 weeks versus 4 weeks required by the reference recombinant assay and 6 weeks required by the conventional virus isolate phenotypic assay) is a major advantage of the novel assay.

Antiretroviral Therapy, Highly Active↗
PubMed2003

Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients.

A novel phenotypic assay, based on recombinant expression of the HIV-1-protease was developed and evaluated; it monitors the formation of resistance to protease inhibitors. The HIV-1 protease-encoding region from the blood sample of patients was amplified, ligated into the expression vector pBD2, and recombinantly expressed in Escherichia coli TG1 cells. The resulting recombinant enzyme was purified by a newly developed one-step acid extraction protocol. The protease activity was determined in presence of five selected HIV protease inhibitors and the 50% inhibitory concentration (IC(50)) to the respective protease inhibitors determined. The degree of resistance was expressed in terms of x-fold increase in IC(50) compared to the IC(50) value of an HIV-1 wild type protease preparation. The established test system showed a reproducible recombinant expression of each individual patients' HIV-1 protease population. Samples of nine clinically well characterised HIV-1-infected patients with varying degrees of resistance were analysed. There was a good correlation between clinical parameters and the results obtained by this phenotypic assay. For the majority of patients a blind genotypic analysis of the patients' protease domain revealed a fair correlation to the results of the phenotypic assay. In a minority of patients our phenotypic results diverged from the genotypic ones. This novel phenotypic assay can be carried out within 8-10 days, and offers a significant advantage in time to the current employed phenotypic tests.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Detection of fimbrial and toxin genes in Escherichia coli and their prevalence in piglets with diarrhoea. The application of colony hybridization assay, polymerase chain reaction and phenotypic assays.

Two different genotypic methods, colony hybridization and polymerase chain reaction (PCR), were applied to detect enterotoxin, verotoxin and fimbrial genes in 708 Escherichia coli (E. coli) strains from piglets with diarrhoea. The results were compared with those obtained by phenotypic methods. DNA fragments specific for each of the enterotoxins LT, STa and STb, the verotoxins VT1, VT2 and VT2v, and for each of the fimbrial genes K88 (F4), K99 (F5), 987P (F6), F41 and F107, respectively, were used as probes in a colony hybridization assay of the E. coli strains. A PCR assay was used as genotypic test for the verotoxin gene. An Enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was performed to test for the presence of K88 and K99 fimbriae, and a Vero cell test was performed to test for verotoxin production. Toxin detection kits were applied to detect the E. coli heat-labile enterotoxin (LT) and the heat-stable (STa) enterotoxin. The correlation between the results obtained by genotypic and phenotypic methods was 97.7-100%. The prevalence of the different fimbrial and toxin genes in E. coli strains from piglets with neonatal and postweaning diarrhoea were recorded. The verotoxin and the fimbrial F107 genes were found to be more frequent in postweaning E. coli strains than in neonatal E. coli strains.

Animals↗
PubMed2004

Complex phenotypic assays in high-throughput screening.

High-throughput screening (HTS), systematically testing thousands of small molecules to find candidates for lead optimization, primarily involves exposure of purified proteins to arrayed collections of small molecules. More complex phenotypic assays, such as cell-based or whole-organism assays, traditionally have flanked HTS, preceding it to validate new therapeutic targets, and following it to characterize new lead compounds in cellular contexts. Recently, however, cell- and organism-based phenotypic assays have increasingly been adopted as a primary screening platform for annotating small molecules.

Combinatorial Chemistry Techniques↗
PubMed2002

A rapid phenotypic assay for detecting multiple nucleoside analogue reverse transcriptase inhibitor-resistant HIV-1 in plasma.

Zidovudine and other nucleoside analogue reverse transcriptase inhibitors (NRTIs), like zalcitabine and didanosine used for treatment of individuals infected with HIV-1, can select for viruses with Q151M and other associated mutations (for example, A62V, S68G, V751, F77L, F116Y) in the reverse transcriptase (RT) enzyme. These mutations confer resistance to multiple nucleoside analogues, and thereby compromise the efficacy of this class of drugs. Presently available phenotypic assays for detection of multiple nucleoside analogue resistant (MNR) HIV-1 require testing for each NRTI individually. Here we report an enzymatic RT assay that uses resistance to zidovudine triphosphate (zidovudine-TP) as a diagnostic biochemical marker of MNR HIV-1. This assay exploits the different biochemical mechanisms for zidovudine-resistance conferred by either Q151 M or T215Y/F mutations and the inability of conventional RT assays to detect T215Y/F-associated zidovudine resistance. The assay detects RT activity directly in plasma by using Amp-RT, an ultra-sensitive PCR-based RT assay. We show that enzymatic resistance to zidovudine-TP is specific to MNR RT and is distinguishable from both wild-type (WT) and RT containing classical zidovudine-resistant mutations (D67N, K70R, T215Y/F, K219Q). Compared to WT, MNR HIV-1 RT had 5- to 36-fold increases in the concentration of drug required to inhibit 50% (IC50) of RT activity, depending on the presence of Q151 M alone or with additional MNR mutations. A screening assay utilizing 1 microM zidovudine-TP was developed and validated on 14 reference isolates, 37 plasma specimens, and seven patient-derived viruses. Twenty-three specimens were found to have reduced susceptibility to zidovudine-TP, and all had Q151 M. In contrast, 21 specimens were sensitive to zidovudine-TP, of which 12 had WT genotypes, four had T215Y/F, and five had T69S-insertions along with T215Y/F mutations. This RT-based phenotypic assay provides a specific and rapid tool for the direct identification and monitoring of Q151M-associated MNR HIV-1 in plasma.

Adenosine Monophosphate↗
PubMed2001

Rapid and simple phenotypic assay for drug susceptibility of human immunodeficiency virus type 1 using CCR5-expressing HeLa/CD4(+) cell clone 1-10 (MAGIC-5).

We describe a rapid and simple novel phenotypic assay for drug susceptibility of human immunodeficiency virus type-1 (HIV-1) using a CCR5-expressing HeLa/CD4(+) cell clone 1-10 (MAGIC-5). MAGIC-5 cells produced large amounts of HIV-1 in culture supernatants, which enabled us to perform the phenotypic resistance assay. Determination of HIV-1 susceptibility to various protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors was completed within 15 days in T-cell-tropic (X4) and macrophage-tropic (R5) viruses using fresh plasma samples containing at least 10(4) copies/ml. The nucleotide sequence of the envelope V3 region of HIV-1 in plasma was almost identical to that of the virus isolated by MAGIC-5 cells, suggesting a lack of selection bias in our assay. The assay variability was confined to within five-fold in all drugs examined. Accordingly, we used a 10-fold increase in the 50% inhibitory concentration as the cutoff value for viral resistance in the present assay. HIV-1 resistant to lamivudine, which was not detected by conventional genotypic assays, was isolated. In HIV-1 with PI-associated primary amino acid substitutions, our assay showed that drug resistance profiles correlated well with previously reported genotypic-assay data. Furthermore, our assay provided comprehensive results regarding PI resistance in the presence of multiple mutations. The novel assay successfully quantified the level of resistance of clinical HIV-1 isolates to a battery of anti-HIV drugs, indicating its clinical usefulness, particularly in patients who failed to respond to antiretroviral chemotherapy.

Amino Acid Sequence↗
PubMed1987

Phenotypic assay for excision of the maize controlling element Ac in tobacco.

We describe a phenotypic assay designed to detect excision of the maize controlling element Ac from a selectable marker gene, neomycin phosphotransferase II (NPT II). An NPT II gene which expresses kanamycin resistance in tobacco cells, and contains a unique restriction enzyme site in the untranslated leader region, was constructed. Ac, or a defective Ac element (Ac big up tri, open), was inserted into the leader region of this gene. The transposon insertions inactivated the NPT II gene as determined by transient NPT II expression assays. The three plasmids were inserted into the T DNA of Agrobacterium tumefaciens Ti plasmid vectors, and transferred to tobacco protoplasts. The transformed protoplasts were selected with 100 or 200 microg/ml kanamycin. Protoplasts transformed by the NPT II gene interrupted by Ac formed 25% as many calli resistant to 100 or 200 microg/ml kanamycin as protoplasts transformed by the uninterrupted NPT II gene. Protoplasts transformed by the NPT II gene interrupted by Ac big up tri, open did not form any calli resistant to 200 microg/ml of kanamycin when transformed under similar conditions. Southern blot hybridization analyses of seven kanamycin-resistant calli or plants obtained after transformation by the NPT II gene interrupted by Ac revealed that in all cases Ac had excised, restoring the structure of the NPT II gene. This assay is therefore useful to monitor the activity of a transposable element such as Ac and to define the regions of this element involved in transposition activity.

Journal Article↗
PubMed2006

Rapid identification of ascomycetous yeasts from clinical specimens by a molecular method based on flow cytometry and comparison with identifications from phenotypic assays.

This study was designed to compare the identification of ascomycetous yeasts recovered from clinical specimens by using phenotypic assays (PA) and a molecular flow cytometric (FC) method. Large-subunit rRNA domains 1 and 2 (D1/D2) gene sequence analysis was also performed and served as the reference for correct strain identification. A panel of 88 clinical isolates was tested that included representatives of nine commonly encountered species and six infrequently encountered species. The PA included germ tube production, fermentation of seven carbohydrates, morphology on corn meal agar, urease and phenoloxidase activities, and carbohydrate assimilation tests when needed. The FC method (Luminex) employed species-specific oligonucleotides attached to polystyrene beads, which were hybridized with D1/D2 amplicons from the unidentified isolates. The PA identified 81 of 88 strains correctly but misidentified 4 of Candida dubliniensis, 1 of C. bovina, 1 of C. palmioleophila, and 1 of C. bracarensis. The FC method correctly identified 79 of 88 strains and did not misidentify any isolate but did not identify nine isolates because oligonucleotide probes were not available in the current library. The FC assay takes approximately 5 h, whereas the PA takes from 2 h to 5 days for identification. In conclusion, PA did well with the commonly encountered species, was not accurate for uncommon species, and takes significantly longer than the FC method. These data strongly support the potential of FC technology for rapid and accurate identification of medically important yeasts. With the introduction of new antifungals, rapid, accurate identification of pathogenic yeasts is more important than ever for guiding antifungal chemotherapy.

Ascomycota↗
PubMed2002

Differential activation of protein kinase C isoforms by euxanthone, revealed by an in vivo yeast phenotypic assay.

The protein kinase C (PKC) modulatory effects of euxanthone, isolated from the wood of Cratoxylum maingayi, on isoforms alpha, betaI, delta, eta and zeta were characterised using an alternative in vivo yeast phenotypic assay. The present study shows that euxanthone can activate isoforms alpha, betaI, delta, eta and zeta, being more effective on PKC-betaI, -delta, -eta and -zeta than the established PKC activators used (the phorbol ester PMA and arachidonic acid for PKC-zeta). Furthermore, euxanthone presents differences on its potency towards individual PKC isoforms, showing a remarkable selectivity for PKC-zeta. These results can help to clarify the molecular basis of the euxanthone-mediated effects.

Clusiaceae↗
PubMed2026

Hybrid genome assembly and phenotypic assays reveal carbohydrate metabolism diversity in Lacticaseibacillus strains.

Investigation of carbohydrate metabolism in lactic acid bacteria is essential for the rational selection of strains for fermentation processes, particularly in emerging applications involving non-conventional substrates or building of synthetic microbial consortia. However, establishing robust genotype-phenotype relationships remains challenging, as gene presence alone often fails to explain observed metabolic traits without considering the genomic context and regulatory architecture. In the present study, we combined hybrid genome assembly (Illumina and Oxford Nanopore) with high-throughput phenotype profiling (Biolog GENIII and PM2A) to investigate carbohydrate utilization in five Lacticaseibacillus strains. Phenotypic assays revealed clear intra- and inter-specific variability in substrate utilization. We therefore investigated whether such differences could be attributed to the organization and regulatory context of carbohydrate-associated loci, rather than to gene presence alone. Functional annotation based on COG and CAZyme databases revealed candidate genomic regions potentially involved in carbohydrate metabolism. Comparative analysis between predicted and experimentally observed substrate usage highlighted specific loci associated with carbohydrate utilization profile. The trehalose (tre) operon was conserved across all strains, while at least two distinct cellobiose-associated loci were detected in each genome. Despite the presence of these loci, L. paracasei strains were unable to metabolize cellobiose, a phenotype likely linked to the presence of a downstream TetR-type transcriptional repressor within the cellobiose (cel) operon. Additionally, a genomic region uniquely found in L. rhamnosus strains was associated with gentiobiose utilization, consistent with phenotypic observations. Overall, these findings highlight the importance of integrating phenotypic validation with complete genome context to support the identification of candidate structural and regulatory determinants of carbohydrate utilization in lactic acid bacteria. KEY POINTS: • Phenotype microarrays reveal metabolic traits of interest in isolated strains. • Regulatory context is key to understanding carbohydrate metabolism differences. • Basis of subspecies-dependent cellobiose metabolism in L. paracasei is provided.

Carbohydrate Metabolism↗
PubMed2005

Comparison of standard phenotypic assays with a PCR method to discriminate Candida albicans and C. dubliniensis.

In 1995, Candida dubliniensis was described as a new species in the genus Candida. Its close relationship to C. albicans has proved problematic in the identification of C. dubliniensis in clinical specimens. The objective of this study was to determine if reproducible differentiation between both species can be obtained by phenotypic assays. Therefore, 100 strains from 86 patients with the ability to produce chlamydospores were examined with different methods including API ID 32 C, colour development on CHROMagar, chlamydospore formation on Staib agar, growth at different temperatures and germ tube formation at 39 degrees C. Additionally, polymerase chain reaction (PCR) was used as gold standard. Six of the investigated strains were C. dubliniensis. The results suggest that there is still no single phenotypic method satisfactory to distinguish between C. albicans and C. dubliniensis.

Agar↗
PubMed2004

Novel single-cell-level phenotypic assay for residual drug susceptibility and reduced replication capacity of drug-resistant human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1)-infected individuals who develop drug-resistant virus during antiretroviral therapy may derive benefit from continued treatment for two reasons. First, drug-resistant viruses can retain partial susceptibility to the drug combination. Second, therapy selects for drug-resistant viruses that may have reduced replication capacities relative to archived, drug-sensitive viruses. We developed a novel single-cell-level phenotypic assay that allows these two effects to be distinguished and compared quantitatively. Patient-derived gag-pol sequences were cloned into an HIV-1 reporter virus that expresses an endoplasmic reticulum-retained Env-green fluorescent protein fusion. Flow cytometric analysis of single-round infections allowed a quantitative analysis of viral replication over a 4-log dynamic range. The assay faithfully reproduced known in vivo drug interactions occurring at the level of target cells. Simultaneous analysis of single-round infections by wild-type and resistant viruses in the presence and absence of the relevant drug combination divided the benefit of continued nonsuppressive treatment into two additive components, residual virus susceptibility to the drug combination and selection for drug-resistant variants with diminished replication capacities. In some patients with drug resistance, the dominant circulating viruses retained significant susceptibility to the combination. However, in other cases, the dominant drug-resistant viruses showed no residual susceptibility to the combination but had a reduced replication capacity relative to the wild-type virus. In this case, simplification of the regimen might still allow adequate suppression of the wild-type virus. In a third pattern, the resistant viruses had no residual susceptibility to the relevant drug regimen but nevertheless had a replication capacity equivalent to that of wild-type virus. In such cases, there is no benefit to continued treatment. Thus, the ability to simultaneously analyze residual susceptibility and reduced replication capacity of drug-resistant viruses may provide a basis for rational therapeutic decisions in the setting of treatment failure.

Adult↗
PubMed2004

Identification and characterization of Sir2 inhibitors through phenotypic assays in yeast.

Yeast Sir2 is a defining member of a large family of protein deacetylases found in organisms ranging from bacteria to humans. SIR2 was discovered as a gene required for mating in S. cerevisiae 25 years ago, but it was only recently that Sir2's activity as an NAD-dependent protein deacetylase was established. However, years of extensive research did generate a large body of knowledge about the cellular roles of Sir2 in yeast long before its biochemical function was discovered. In addition to Sir2, yeast have four additional NAD-dependent histone deacetylases Hst1-4 (for homologue of Sir2), with distinct cellular roles. Detailed knowledge of the phenotypes of SIR2 and HST loss of function mutants has allowed design of a series of cell based screens that yielded the first inhibitors of NAD-dependent protein deacetylases. These phenotypic assays, amenable to high throughput screening, and coupled with transcript array analysis for evaluation of compound specificity, allowed the identification and detailed characterization of a series of Sir2 inhibitors, entirely bypassing traditional biochemical approaches.

Amino Acid Sequence↗
PubMed2004

Virological and immunological response to antiretroviral therapy in HIV-1 infected children: genotypic and phenotypic assays in monitoring virological failure.

Children differ from adults in both natural history of HIV-1 infection and their response to anti-retroviral therapy (ART). ART appears to be less successful in children than in adults at reducing HIV-1 RNA to below the level of detection of current assays. Nonetheless, children receiving ART frequently experience rises in CD4 cell counts, even in the absence of full virological suppression in plasma (discordant response). This immune repopulation in the presence of viral replication may increase the risk of emergence of drug-resistant viral variants. While the rationale for resistance testing is to optimise therapy, particularly when drugs are being changed following virological failure, it should be pointed out that currently available genotypic and phenotypic assays fail to detect drug resistance in about one third of viremic ART-treated children. This cannot be fully explained by the limitation of assays in detecting minor variants; factors other than resistance might be involved in the failure of therapy. Reduced fitness of drug-resistant viral variants is also unlikely to fully explain the discordant response to therapy, since immunological recovery is often observed even in the absence of detectable drug resistance. Rather, restoration of thymic function and higher thymic output may play a critical role in sustaining peripheral CD4 cell increases despite the persistence of viral replication. Such immune recovery might also drive the evolution of the replicating viruses.

Anti-HIV Agents↗