Repeat testing of low-level HIV-1 RNA: assay performance and implementation in clinical trials.

OBJECTIVE: Assess the performance of HIV-1 RNA repeat testing of stored samples in cases of low-level viremia during clinical trials. DESIGN: Prospective and retrospective analysis of randomized clinical trial samples and reference standards. METHODS: To evaluate assay variability of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test, v2.0, three separate sources of samples were utilized: the World Health Organization (WHO) HIV reference standard (assayed using 50 independent measurements at six viral loads <200 copies/ml), retrospective analysis of four to six aliquots of plasma samples from four clinical trial participants, and prospective repeat testing of 120 samples from participants in randomized trials with low-level viremia. RESULTS: The TaqMan assay on the WHO HIV-1 RNA standards at viral loads <200 copies/ml performed within the expected variability according to assay specifications. However, standards with low viral loads of 36 and 18 copies/ml reported values of ≥ 50 copies/ml in 66 and 18% of tests, respectively. In participants treated with antiretrovirals who had unexpected viremia of 50-200 copies/ml after achieving <50 copies/ml, retesting of multiple aliquots of stored plasma found <50 copies/ml in nearly all cases upon retesting (14/15; 93%). Repeat testing was prospectively implemented in four clinical trials for all samples with virologic rebound of 50-200 copies/ml (n = 120 samples from 92 participants) from which 42% (50/120) had a retest result of less than 50 copies/ml and 58% (70/120) retested ≥ 50 copies/ml. CONCLUSION: The TaqMan HIV-1 RNA assay shows variability around 50 copies/ml that affects clinical trial results and may impact clinical practice. In participants with a history of viral load suppression, unexpected low-level viremia may be because of assay variability rather than low drug adherence or true virologic failure. Retesting a stored aliquot of the same sample may differentiate between assay variability and virologic failure as the source of viremia. This retesting strategy could save time, money, and anxiety for patients and their providers, as well as decrease follow-up clinic visits without increasing the risk of virologic failure and resistance development.

Identification of annexin A13 as a regulator of chemotherapy resistance using random homozygous gene perturbation.

OBJECTIVE: To utilize a powerful new technology for target discovery, Random Homozygous Gene Perturbation (RHGP), and to identify novel targets that cause tumor cells to become chemoresistant. STUDY DESIGN: RHGP was used to identify and validate genetic changes that cause chemoresistance of tumor cells to Rapamycin. RESULTS: A series of targets was identified that allowed tumor cells to survive treatment with Rapamycin. We validated these targets and focused on Annexin A13, a target where decreased expression caused tumor cell insensitivity to Rapamycin. Ectopic overexpression of Annexin A13 was likewise sufficient to sensitize malignant breast cancer cells to treatment with Rapamycin. CONCLUSION: These findings expand our knowledge of mechanisms that allow tumor cell drug resistance and demonstrate the power of RHGP to identify novel targets and mechanisms.

Ribosomal RNA sequences of Clostridium piliforme isolated from rodent and rabbit: re-examining the phylogeny of the Tyzzer's disease agent and development of a diagnostic polymerase chain reaction assay.

We used polymerase chain reaction (PCR) technology to amplify the 16S rRNA gene, the intergenic spacer, and most of the 23S rRNA gene from 6 isolates (2 mice, 1 hamster, 1 rat, and 2 rabbit isolates) of the Tyzzer's disease agent (Clostridium piliforme) and C. colinum. Sequence similarity searches of GenBank identified 45 closely related bacteria, which we used for phylogenetic analysis by parsimony and maximum-likelihood methods using Escherichia coli to root the resulting phylogram. Microorganisms identified as C. piliforme form 3 clusters within a single clade; the nearest related distinguishable species is C. colinum. Other bacterial clades closely related to C. piliforme are clostridia previously identified by molecular methods in the bovine, porcine, and human gastrointestinal tracts. DNA sequence alignment highlighting sequence differences were used to design a rodent and rabbit C. piliforme-specific PCR assay, which targets a 639-basepair region at the 3' end of the 16S rRNA gene and the 5' end of the intergenic spacer. We used this PCR assay to examine 4 rat fecal samples from C. piliformeseropositive rats and reexamine 2 rabbit fecal samples previously identified as containing DNA sequences consistent with C. piliforme infection by 16S PCR assay. Our new assay did not detect the presence of C. piliforme DNA sequences in either the rat or rabbit fecal DNA samples, consistent with the absence of clinical disease in the colonies evaluated.