Genotypic patterns associated with nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance in the absence of well-characterized resistance mutations were identified using a database (n > 47,000) of phenotypegenotype data. Among samples with no known NNRTI mutations, the most resistant samples contained K101P (n ؍ 35) or a combination of K103R and V179D (n ؍ 41). Site-directed mutagenesis confirmed the importance of these mutations.Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) of human immunodeficiency virus type 1 (HIV-1) are an important component of highly active antiretroviral therapy regimens (14). Resistance to NNRTIs is common in virus from treated patients and is typically accompanied by the development of one or more recognized mutations in the target enzyme. These mutations, derived from a combination of in vitro and in vivo data, form the basis for predicting NNRTI resistance in genotype-based resistance assays (13).The availability of large databases of matched protease-RT genotype and drug susceptibility data, as well as the increasing clinical use of combined phenotype-genotype resistance assays, enables more thorough and comprehensive analyses of concordance between the interpretation of both types of assays (6,8,12). While concordance rates are high for NNRTIs, infrequent cases of reduction in phenotypic susceptibility not explained by known mutations can be dramatic (10-fold to more than 100-fold change in 50% inhibitory concentration [IC 50 ]), in a range that is clinically significant. We investigated the underlying causes for such discordance in a large database of genotype and phenotype resistance assay results from clinical samples submitted to the Monogram Biosciences (formerly ViroLogic) Clinical Reference Laboratory for routine testing.As of April 2005, the Monogram Biosciences phenotypegenotype database contained data for more than 47,000 individual clinical samples, 24,151 of which lacked any commonly recognized NNRTI resistance mutations (A98G, K101E, K103N/S, V106A/M, P225H, M230L, P236L, or any substitution at position L100, Y181, Y188, G190, or F227). There were 577 samples that displayed a fold change in IC 50 (FC) for at least one NNRTI of fivefold or greater, and 196 had an FC of 10-fold or greater. Several samples had IC 50 s above the highest concentration tested in the assay: 28 samples for nevirapine (NVP), 10 samples for delavirdine (DLV), and 1 sample for efavirenz (EFV). For statistical purposes, these samples were assigned FC values of 400-, 250-, and 700-fold for NVP, DLV, and EFV, respectively. These maximum FC values are derived from the ratio of the highest drug concentration tested to the distribution of IC 50 s for the drug-sensitive reference virus over a 1-year time period.To explore the genotypic correlates of this unexplained NNRTI resistance, we performed univariate analysis, testing for the association of all mutations in RT which occur in more than 1% of the samples with an elevated NNRTI FC. We used an arbitrary threshold of five...