Dean L. Winslow

Accuracy of the TRUGENE HIV-1 Genotyping Kit

ABSTRACT Drug resistance and poor virological responses are associated with well-characterized mutations in the viral reading frames that encode the proteins that are targeted by currently available antiretroviral drugs. An integrated system was developed that includes target gene amplification, DNA sequencing chemistry (TRUGENE HIV-1 Genotyping Kit), and hardware and interpretative software (the OpenGene DNA Sequencing System) for detection of mutations in the human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase sequences. The integrated system incorporates reverse transcription-PCR from extracted HIV-1 RNA, a coupled amplification and sequencing step (CLIP), polyacrylamide gel electrophoresis, semiautomated analysis of data, and generation of an interpretative report. To assess the accuracy and robustness of the assay system, 270 coded plasma specimens derived from nine patients were sent to six laboratories for blinded analysis. All specimens contained HIV-1 subtype B viruses. Results of 270 independent assays were compared to “gold standard” consensus sequences of the virus populations determined by sequence analysis of 16 to 20 clones of viral DNA amplicons derived from two independent PCRs using primers not used in the kit. The accuracy of the integrated system for nucleotide base identification was 98.7%, and the accuracy for codon identification at 54 sites associated with drug resistance was 97.6%. In a separate analysis of plasma spiked with infectious molecular clones, the assay reproducibly detected all 72 different drug resistance mutations that were evaluated. There were no significant differences in accuracy between laboratories, between technologists, between kit lots, or between days. This integrated assay system for the detection of HIV-1 drug resistance mutations has a high degree of accuracy and reproducibility in several laboratories.

HIV-1 Protease and Reverse Transcriptase Mutation Patterns Responsible for Discordances Between Genotypic Drug Resistance Interpretation Algorithms

Several rules-based algorithms have been developed to interpret results of HIV-1 genotypic resistance tests. To assess the concordance of these algorithms and to identify sequences causing interalgorithm discordances, we applied four publicly available algorithms to the sequences of isolates from 2,045 individuals in northern California. Drug resistance interpretations were classified as S for susceptible, I for intermediate, and R for resistant. Of 30,675 interpretations (2,045 sequences x 15 drugs), 4.4% were completely discordant, with at least one algorithm assigning an S and another an R; 29.2% were partially discordant, with at least one algorithm assigning an S and another an I, or at least one algorithm assigning an I and another an R; and 66.4% displayed complete concordance, with all four algorithms assigning the same interpretation. Discordances between nucleoside reverse transcriptase inhibitor interpretations usually resulted from several simple, frequently occurring mutational patterns. Discordances between protease inhibitor interpretations resulted from a larger number of more complex mutation patterns. Discordances between nonnucleoside reverse transcriptase inhibitor interpretations were uncommon and resulted from a small number of individual drug resistance mutations. Determining the clinical significance of these mutation patterns responsible for interalgorithm discordances will improve interalgorithm concordance and the accuracy of genotypic resistance interpretation.

Performance Characteristics of the TRUGENE HIV-1 Genotyping Kit and the Opengene DNA Sequencing System

ABSTRACT The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System are designed to sequence the protease (PR)- and reverse transcriptase (RT)-coding regions of human immunodeficiency virus type 1 (HIV-1) pol. Studies were undertaken to determine the accuracy of this assay system in detecting resistance-associated mutations and to determine the effects of RNA extraction methods, anticoagulants, specimen handling, and potentially interfering substances. Samples were plasma obtained from HIV-infected subjects or seronegative plasma to which viruses derived from wild-type and mutant infectious molecular clones (IMC) of HIV-1 were added. Extraction methods tested included standard and UltraSensitive AMPLICOR HIV-1 MONITOR, QIAGEN viral RNA extraction mini kit, and QIAGEN Ultra HIV extraction kit, and NASBA manual HIV-1 quantitative NucliSens. Sequence data from test sites were compared to a “gold standard” reference sequence to determine the percent agreement. Comparisons between test and reference sequences at the nucleotide level showed 97.5 to 100% agreement. Similar results were obtained regardless of extraction method, regardless of use of EDTA or acid citrate dextrose as anticoagulant, and despite the presence of triglycerides, bilirubin, hemoglobin, antiretroviral drugs, HIV-2, hepatitis C virus (HCV), HBV, cytomegalovirus, human T-cell leukemia virus type 1 (HTLV-1), or HTLV-2. Samples with HIV-1 RNA titers of ≥1,000 copies/ml gave consistent results. The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System consistently generate highly accurate sequence data when tested with IMC-derived HIV and patient samples.

Aphthous ulceration of the gastrointestinal tract in patients with the acquired immunodeficiency syndrome (AIDS).

Excerpt Patients with steroid-responsive, severe aphthous ulceration involving the mouth, hypopharynx, and esophagus have been described in a previous report (1). In these patients, serious morbidi...

Clinical Resistance Patterns and Responses to Two Sequential Protease Inhibitor Regimens in Saquinavir and Reverse Transcriptase Inhibitor—Experienced Persons

The efficacy of sequential protease inhibitor therapy was studied in 16 human immunodeficiency virus (HIV) 1-infected persons in whom saquinavir with multiple nucleoside reverse transcriptase (RT) inhibitors (NRTI) had failed. Nelfinavir plus two NRTIs (new or continued) resulted in minimal (0.59 log RNA copies/mL) and transient (8 weeks) suppression of plasma HIV RNA levels. Rapid failure was surprisingly associated with baseline presence of protease gene mutation L90M (P=.04) in the absence of D30N and with RT mutations D67N (P<.01), K70R/S (P=.02), and K219Q/W/R/E (P<.01). Ten patients were subsequently switched to indinavir plus nevirapine and 2 NRTIs, resulting in a median 1.62 log reduction in plasma HIV RNA, with 3 patients maintaining 400 copies/mL for 24 weeks. These results suggest that nelfinavir may have limited utility after saquinavir failure, particularly without potent concomitant therapy. Combining an NRTI with a new protease inhibitor for rescue may improve response.

Selection conditions affect the evolution of specific mutations in the reverse transcriptase gene associated with resistance to DMP 266

OBJECTIVE To monitor the appearance of HIV-1 variants resistant to inhibition by DMP 266, a benzoxazinone non-nucleoside reverse transcriptase inhibitor using two different protocols for applying drug selective pressure in tissue culture. To compare the phenotype and genotype of viral isolates selected by each method. METHODS MT-2 cells and fresh donor peripheral blood mononuclear cells (PBMC) were infected with HIV-1 strain RF. The MT-2 cells were infected in the presence of a 50% inhibitory concentration (IC50) of DMP 266 and the concentration was slowly increased during the selection period. The PBMC were infected for 1 week in the absence of inhibitor and then a single concentration was maintained throughout the selection period. Both cultures were passaged for approximately 4 months. Virus and cell pellets were harvested over this in vitro selection period, the RT genes amplified by polymerase chain reaction from the cell pellets, and the proviral DNAs sequenced. Isolated virus was tested for DMP 266 susceptibility in either the AIDS Clinical Trials Group/Department of Defense consensus assay or MT-2 yield reduction assay. RESULTS Passage in MT-2 cells resulted in accumulation of three substitutions in RT (V179D, L1001, Y181C) after 24 passages associated with 1000-fold reduced susceptibility to DMP 266. In PBMC cultures treated with 0.96 microM DMP 266, virus replication was completely suppressed after 2 weeks; no regrowth occurred in the presence of compound after 10 weeks or in the absence of compound for 3 additional weeks. The 0.096 microM treated cultures had an initial 2.5-log reduction in infectious virus titre followed by rapid regrowth. Virus obtained at week 6 displayed a 28-fold reduction in susceptibility with an L1001 substitution in RT, and by week 11 displayed a 1000-fold reduction in susceptibility with an additional V1081 substitution. CONCLUSIONS High-level resistance to DMP 266 may develop by at least two pathways and experimental conditions influence the genotype selected. The continued absence of detectable virus in the PBMC cultures grown at 0.96 microM is supportive evidence that maintaining trough plasma levels of DMP 266 which result in sustained antiviral activity in vivo may delay emergence of highly resistant viral variants. Confirmation of this hypothesis will require clinical trials.

Efavirenz Plasma Concentrations and Cytochrome 2B6 Polymorphisms

Background: Interpatient variability in efavirenz concentrations may be due to CYP2B6 genetic polymorphisms. Efavirenz concentration and pharmacogenomic data are scarce in Latino patients. Objective: To evaluate the difference in trough and midpoint efavirenz plasma concentrations between HIV-positive Latino and white patients. In addition, this study evaluated the association between efavirenz concentrations and CYP2B6 polymorphisms in Latino and white HIV-positive subjects. Methods: This pilot study included 10 Latinos and 10 whites. Two efavirenz blood concentrations were determined: a trough and a midpoint. CYP2B6 genetic polymorphisms were analyzed at the 516 (G to T) and 785 (A to G) codons. The Mann-Whitney test was used to determine whether efavirenz concentrations varied with ethnicity. The Kruskal-Wallis test was used to determine whether efavirenz concentrations varied with CYP2B6 genetic polymorphisms. Efavirenz concentrations were expressed as medians (minimum, maximum). Results: Midpoint concentrations were 1.58 μg/mL (1.36, 6.02) and 3.14 μg/mL (1.74, 7.72) for whites and Latinos, respectively (p < 0.05). Trough concentrations did not vary as a function of ethnicity. Ten percent of Latinos and whites tested positive for homozygous variants of CYP2B6-516 and CYP2B6-785. One white subject tested positive for the homozygous variant of CYP2B6-1459. Trough concentrations for 516TT, 516GT, and 516GG (wild type) were 5.13 μg/mL (4.13, 6.12), 2.13 μg/mL (1.33, 3.37), and 1.44 μg/mL (0.59, 2.92), respectively (p < 0.05). Trough concentrations for 785GG, 785AG, and 785AA (wild type) were 5.12 μg/mL (4.13, 6.12), 1.98 μg/mL (1.33, 3.37), and 1.27 μg/mL (0.59, 2.92), respectively (p < 0.05). None of the patients took concomitant medications that impacted CYP2B6 metabolism. Conclusions: Trough efavirenz concentrations were significantly higher in patients with the 785 (A to G) and 516 (G to T) variants. Midpoint efavirenz concentrations in Latinos were significantly higher than those of whites.

Efavirenz-Induced Hypersensitivity Reaction Manifesting in Rash and Hepatitis in a Latino Male

Objective: To report a case of hypersensitivity manifesting in a rash, fever, and life-threatening hepatitis in a patient initiated on efavirenz therapy. Case Summary: A 30-year-old Latino male newly diagnosed with HIV was started on efavirenz-based highly active antiretroviral therapy (HAART) using tenofovir 300 mg, emtricitabine 200 mg, and efavirenz 600 mg once daily. Eleven days after beginning therapy, he developed a hypersensitivity reaction manifesting in rash and fever preceding severe drug-induced hepatitis. Liver enzyme peak values were aspartate transaminase 3410 U/L and alanine transaminase 2132 U/L. Hepatitis resolved with discontinuation of the HAART. The patient was rechallenged with tenofovir and emtricitabine one year later; no adverse reactions occurred. Discussion: The Naranjo probability scale demonstrated a probable relationship between this adverse reaction and efavirenz. A MEDLINE search (2004 to September 2007) revealed 2 cases of rash preceding hepatitis with the initiation of efavirenz. Both cases were in women; there were no prior reported cases of efavirenz hypersensitivity in men. Although the mechanism of this reaction is unknown, a few factors may have contributed to this reaction. The half-life and the auto-induction of efavirenz may explain the continued rise in liver enzymes and severe hepatitis that continued to occur once the drug was discontinued. Another cause that may have contributed is the metabolism of the medication. CYP2B6 is responsible for almost 90% of the clearance of efavirenz. Data from a recent pharmacokinetic study showed that efavirenz concentrations were higher in both black and Latino patients when compared with those of white patients. In addition, it is highly probable that this patients liver function was impaired when transaminase levels peaked, resulting in decreased clearance of efavirenz. Conclusions: Although such a hypersensitivity reaction is rare, efavirenz is the most probable cause of the erythematous maculopapular rash and acute hepatitis in this patient. Monitoring of liver function in patients who present with a rash following initiation of efavirenz-based HAART is recommended. In addition, clinicians should exercise caution in patients presenting with rash, fever, and increased liver enzymes (>3 times the upper limit of normal or patient baseline). It is strongly recommended that efavirenz therapy be withheld in such cases and reevaluated once liver enzyme levels stabilize.

In vitro susceptibility of clinical isolates of HIV-1 to XM323, a non-peptidyl HIV protease inhibitor.

ObjectiveTo determine the in vitro susceptibility of primary clinical isolates and laboratory strains of HIV-1 to XM323. MethodsThe AIDS Clinical Trials Group/US Department of Defense p24 antigen-based consensus assay was used to determine in vitro susceptibility of 57 primary clinical isolates and three laboratory strains of HIV-1 to XM323, zidovudine, zalcitabine (ddC), and didanosine (ddl). ResultsThe concentrations of compound required to inhibit viral p24 antigen production by 50% [median inhibitory concentration (IC50)] for nucleosides were as follows: zidovudine, 0.001->5μM; ddC, < 0.01–0.23μM; ddl, 0.2-25μM). Against both nucleoside susceptible and resistant isolates XM323 exhibited potent inhibition with IC50 values of < 0.02–0.27 μM and IC90 values of 0.03–1.17 μM. ConclusionsXM323 is a potent inhibitor of diverse clinical isolates of HIV-1 in vitro and represents a novel class of non-peptidyl inhibitors of HIV-1 protease.

Wind, rain, flooding, and fear : Coordinating military public health in the aftermath of hurricane katrina

On 29 August 2005, a category 4 hurricane struck the Gulf Coast of Mississippi and southeast Louisiana, resulting in widespread destruction caused by winds in excess of 190 km/h (120 miles/h), heavy rain, and flooding. Communication, electricity, and fresh water supplies were disrupted throughout the region, rendering much of the area uninhabitable. Despite tremendous obstacles, the US military spearheaded the eventually successful rescue, recovery, and relief operations. This article describes the challenges of protecting the health and safety of these personnel in the immediate aftermath of Hurricane Katrina.