Web-Based CBT for Childhood Anxiety: A Family-Led Approach

DESCRIPTION (provided by applicant): Successful management of HIV1 infection with anti-retroviral therapy (ART) is critically dependent on choosing effective drugs. The spread of drug resistance HIV mutations poses a challenge (1-3) and drug resistance testing (DRT) has become an integral part of HIV clinical care. It is recommended at initial presentation, before initiation of ART and when drug therapy fails. (1, 2, 4, 5). Current clinical genotyping methods for HIV drug resistance employ PCR followed by Sanger sequencing. The most commonly used Sanger sequencing tests, such as ViroSeq (Abbott/Celera), Trugene (Siemens), and GenoSure MG (LabCorp/Monogram) detect HIV genotypes in plasma when the resistant viral species are present at gt20% of the totalvirus population, have a turnaround time of about 1-2 weeks and a cost of ~ 350- 600 per test (4). One critical limitation of existing Sanger sequencing methods is their lack of sensitivity. Because capillary electrophoresis sequencing calls depend on theability to detect mixed bases a mutation must represent at least 20% of the total viral load (5). Therefore, less abundant resistant genotypes will remain undetected, and can replicate unchallenged leading to ART failure. The cost and technical expertise required for multistep Sanger sequencing protocol limits its use in resource-limited countries (6). Thus, there is a strong need to develop simple, more sensitive and less expensive HIV drug resistance tests. We propose to develop homogenous, single-well(for each drug), closed tube multiplexed qPCR assays that detect resistant Group M HIV at 1% of the total viral load to be used on any qPCR instrument for rapid close to point of care testing. The test will detect 25 mutations that cause resistance to ZDV, 3TC, TDF (NRTI), NVP and EFV (NNRTI) (7-9). Our qPCR drug resistance tests are based on combining two new technologies. First, a novel multiplex qPCR approach known as UniTaq (10, 11) permits simultaneous detection of several mutations. Mutations are encoded using primers with 5' universal tags, allowing detection in a single qPCR reaction using the same dye-labeled primer. Secondly, we will use rhPCR technology from IDT (12) that provides extraordinarily high mutation detection specificity, enabling calling a single mutant in a background of 10,000 normal alleles. rhPCR eliminates primer dimer formation allowing high PCR multiplexing. Unlike allele-specific PCR this high specificity does not require low primer Tm, thus enabling use of long primers that stably anneal to diverse HIV strains (gt90% identity in the HIV pol gene). Our rhPCR/qPCR multiplex genotyping test is a single closed tube test, simplifying workflow and reducing risk of contamination. We have demonstrated the feasibility of this approach detecting nine rifampicin drug resistance mutations in Mycobacteria tuberculosis (MTB) using artificial templates for mutations and wild type MTB. Our approach will enable rapid and cost effective (lt 1 per reagent cost) detection of multiple mutations at asensitivity of ~1% mutant in 99% wild-type background. We plan to develop a set of multiplexed rhPCR/qPCR assays for research use and partner with a molecular diagnostics vendor to develop both diagnostic and CLIA tests for use in developed and resource limited countries. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: HIV-1 drug resistance is increasingly becoming a significant impediment for successful ART. Drug resistance is either due to transmission of drug resistant strains (TDR) which occurs in ~5-15% of newly infected patients (1-3) or emerges over the course of therapy, especially when patients interrupt their regular treatment regimen. Therefore, effective therapeutic regimens critically depend on drug resistance testing. Current testsfor drug resistance use RT-PCR followed by Sanger sequencing. These tests have ~1-2 weeks turnaround time, are expensive and exhibit low sensitivity, unable to detect emerging drug resistance strains below 20% of viral population. The inability to detectat higher sensitivity allows resistant strains to 'escape' detection, leading to failure of ART. The proposed single closed tube qPCR assays will have ~5 hour turn-around time, use widely available qPCR instruments, have gt10x higher sensitivity and willbe less expensive than sequencing. Rather than proving a complete drug-resistance spectrum, these tests will provide a sensitive tool to detect known mutations and inform better therapeutic efficacy by choosing the appropriate drug regimen. The rhPCR/qPCRtests can enable, same day sensitive testing in developed countries and cost effective testing in resource limited countries. Using optimal drugs for each patient decreases the viral load, increases quality of life and decrease the chance of HIV transmission.