Sepsis caused by gram positive and gram negative bacteria is the leading cause of death in noncoronary ICUs and the tenth leading cause of death in the United States. We have developed a microfluidic sample preparation platform for rapid on-chip detection of infectious organisms for point-of-care diagnostics. The microfluidic chips are made of a robust thermoplastic and can be easily multiplexed for high throughput applications. Bacteria are lysed on-chip via hybrid chemical/mechanical method. Once lysed, the bacterial DNA is isolated using a microscale silica bead/polymer composite solid-phase-extraction (SPE) column. Lysis was confirmed using off-chip real time PCR. We isolated and detected both gram-negative (Escherichia coli) and gram-positive (Bacillussubtilis and Enterococcus faecalis) bacterial genomic DNA from microliter scale spiked whole human blood samples. The system performs better for gram-negative bacteria than it does for gram-positive bacteria, with limits of detection at 10(2) CFU/ml and 10(3)-10(4) CFU/ml, respectively. Total extraction times are less than one hour and can be further decreased by altering the channel geometry and pumping configuration.

We assessed differences in the character and specificity of autologous neutralizing antibodies (ANAbs) against individual viral variants of the quasispecies in a cohort of drug-naïve subjects with long-term controlled human immunodeficiency virus type 1 (HIV-1) infection and moderate levels of broad heterologous neutralizing antibodies (HNAb). Functional plasma virus showed continuous env evolution despite a short time frame and low levels of viral replication. Neutralization-sensitive variants dominated in subjects with intermittent viral blips, while neutralization-resistant variants predominated in elite controllers. By sequence analysis of this panel of autologous variants with various sensitivities to neutralization, we identified more than 30 residues in envelope proteins (Env) associated with resistance or sensitivity to ANAbs. The appearance of new sensitive variants is consistent with a model of continuous selection and turnover. Strong ANAb responses directed against autologous Env variants are present in long-term chronically infected individuals, suggesting a role for these responses in contributing to the durable control of HIV replication.

Human immunodeficiency virus type 1 (HIV-1) is a difficult target for vaccine development, in part because of its ever-expanding genetic diversity and attendant capacity to escape immunologic recognition. Vaccine efficacy might be improved by maximizing immunogen antigenic similarity to viruses likely to be encountered by vaccinees. To this end, we designed a prototype HIV-1 envelope vaccine using a deduced ancestral state for the env gene. The ancestral state reconstruction method was shown to be >95% accurate by computer simulation and 99.8% accurate when estimating the known inoculum used in an experimental infection study in rhesus macaques. Furthermore, the deduced ancestor gene differed from the set of sequences used to derive the ancestor by an average of 12.3%, while these latter sequences were an average of 17.3% different from each other. A full-length ancestral subtype B HIV-1 env gene was constructed and shown to produce a glycoprotein of 160 kDa that bound and fused with cells expressing the HIV-1 coreceptor CCR5. This Env was also functional in a virus pseudotype assay. When either gp160-or gp140-expressing plasmids and recombinant gp120 were used to immunize rabbits in a DNA prime-protein boost regimen, the artificial gene induced immunoglobulin G antibodies capable of weakly neutralizing heterologous primary HIV-1 strains. The results were similar for rabbits immunized in parallel with a natural isolate, HIV-1 SF162. Further design efforts to better present conserved neutralization determinants are warranted.

To evaluate human immunodeficiency virus type 1 (HIV-1) replication and selection of drug-resistant viruses during seemingly effective highly active antiretroviral therapy (HAART), multiple HIV-1 env and pol sequences were analyzed and viral DNA levels were quantified from nucleoside analog-experienced children prior to and during a median of 5.1 (range, 1.8 to 6.4) years of HAART. Viral replication was detected at different rates, with apparently increasing sensitivity: 1 of 10 by phylogenetic analysis; 2 of 10 by viral evolution with increasing genetic distances from the most recent common ancestor (MRCA) of infection; 3 of 10 by selection of drug-resistant mutants; and 6 of 10 by maintenance of genetic distances from the MRCA. When four-or five-drug antiretroviral regimens were given to these children, persistent plasma viral rebound did not occur despite the accumulation of highly drug-resistant genotypes. Among the four children without genetic evidence of viral replication, a statistically significant decrease in the genetic distance to the MRCA was detected in three, indicating the persistence of a greater number of early compared to recent viruses, and their HIV-1 DNA decreased by >0.9 log 10 , resulting in lower absolute DNA levels (P ‫؍‬ 0.007). This study demonstrates the variable rates of viral replication when HAART has suppressed plasma HIV-1 RNA for years to a median of <50 copies/ml and that combinations of four or five antiretroviral drugs suppress viral replication even after short-term virologic failure of three-drug HAART and despite ongoing accumulation of drug-resistant mutants. Furthermore, the decrease of cellular HIV-1 DNA to low absolute levels in those without genetic evidence of viral replication suggests that monitoring viral DNA during HAART may gauge low-level replication.

A sensitive, specific, and high-throughput oligonucleotide ligation assay (OLA) for the detection of genotypic human immunodeficiency virus type 1 (HIV-1) resistance to Food and Drug Administration-approved protease inhibitors was developed and evaluated. This ligation-based assay uses differentially modified oligonucleotides specific for wild-type or mutant sequences, allowing sensitive and simple detection of both genotypes in a single well of a microtiter plate. Oligonucleotides were designed to detect primary mutations associated with highlevel resistance to amprenavir, nelfinavir, indinavir, ritonavir, saquinavir, and lopinavir, including amino acid substitutions D30N, I50V, V82A/S/T, I84V, N88D, and L90M. Plasma HIV-1 RNA from 54 infected patients was amplified by reverse transcription-PCR and sequenced by using dideoxynucleotide chain terminators for evaluation of mutations associated with drug resistance. These same amplicons were genotyped by the OLA at positions 30, 50, 82, 88, 84, and 90 for a total of 312 codons. The sensitivity of detection of drug-resistant genotypes was 96.7% (87 of 90 mutant codons) in the OLA compared to 92.2% (83 of 90) in consensus sequencing, presumably due to the increased sensitivity of the OLA. The OLA detected genetic subpopulations more often than sequencing, detecting 30 mixtures of mutant and wild-type sequences and two mixtures of drug-resistant sequences compared to 15 detected by DNA sequencing. Reproducible and semiquantitative detection of the mutant and the wild-type genomes by the OLA was observed by analysis of wild-type and mutant plasmid mixtures containing as little as 5% of either genotype in a background of the opposite genome. This rapid, simple, economical, and highly sensitive assay provides a practical alternative to dideoxy sequencing for genotypic evaluation of HIV-1 resistance to antiretrovirals.