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1.  Sequence Conservation of the Region Targeted by the Abbott RealTime HCV Viral Load Assay 
Journal of Clinical Microbiology  2014;52(4):1220-1221.
The Abbott RealTime (RT) HCV assay targets the 5′ untranslated region (UTR) of the HCV genome. Here, we analyzed the sequence variability of the assay target regions from 1,092 specimens. Thermodynamic modeling of the percentage of primers/probes bound at the assay annealing temperature was performed to assess the potential effect of sequence variability. An analysis of this large data set revealed that the primer and probe binding sites of the RealTime HCV viral load assay are highly conserved and that naturally occurring sequence polymorphisms are not expected to discernibly impact assay performance.
PMCID: PMC3993474  PMID: 24430453
2.  Cytochrome P450 Compound I in the Plane–Wave Pseudopotential Framework: GGA Electronic and Geometric Structure of Thiolate–Ligated Iron(IV)–Oxo Porphyrin 
Journal of computational chemistry  2013;34(19):1647-1660.
The cytochromes P450 constitute a ubiquitous family of metalloenzymes, catalyzing manifold reactions of biological and synthetic importance via a thiolate–ligated iron–oxo (IV) porphyrin radical species denoted Compound I (Cpd I). Experimental investigations have implicated this intermediate in a broad spectrum of biophysically interesting phenomena, further augmenting the importance of a Cpd I model system. Ab initio molecular dynamics (AIMD), including Car—Parrinello (CP) and path integral (PI) methods, conjoin electronic structure theory with finite temperature simulation, affording tools most valuable to approach such enzymes. These methods are typically driven by density functional theory (DFT) in a plane–wave pseudopotential framework, however, existing studies of Cpd I have been restricted to localized Gaussian basis sets. The appropriate choice of density functional and pseudopotential for such simulations is accordingly not obvious. To remedy this situation, a systematic benchmarking of thiolate–ligated Cpd I is performed using several generalized gradient approximation (GGA) functionals in the Martins—Troullier and Vanderbilt ultrasoft pseudopotential schemes. The resultant electronic and structural parameters are compared to localized–basis DFT calculations using GGA and hybrid density functionals. The merits and demerits of each scheme are presented in the context of reproducing existing experimental and theoretical results for Cpd I.
PMCID: PMC3711018  PMID: 23670855
3.  Large-Scale Analysis of the Prevalence and Geographic Distribution of HIV-1 Non-B Variants in the United States 
Journal of Clinical Microbiology  2013;51(8):2662-2669.
The genetic diversity of human immunodeficiency virus type 1 (HIV-1) has significant implications for diagnosis, vaccine development, and clinical management of patients. Although HIV-1 subtype B is predominant in the United States, factors such as global travel, immigration, and military deployment have the potential to increase the proportion of non-subtype B infections. Limited data are available on the prevalence and distribution of non-B HIV-1 strains in the United States. We sought to retrospectively examine the prevalence, geographic distribution, diversity, and temporal trends of HIV-1 non-B infections in samples obtained by ARUP Laboratories, a national reference laboratory, from all regions of the United States. HIV-1 pol sequences from 24,386 specimens collected from 46 states between 2004 and September 2011 for drug resistance genotyping were analyzed using the REGA HIV-1 Subtyping Tool, version 2.0. Sequences refractory to subtype determination or reported as non-subtype B by this tool were analyzed by PHYLIP version 3.5 and Simplot version 3.5.1. Non-subtype B strains accounted for 3.27% (798/24,386) of specimens. The 798 non-B specimens were received from 37 states and included 5 subtypes, 23 different circulating recombinant forms (CRFs), and 39 unique recombinant forms (URFs). The non-subtype B prevalence varied from 0% in 2004 (0/54) to 4.12% in 2011 (201/4,884). This large-scale analysis reveals that the diversity of HIV-1 in the United States is high, with multiple subtypes, CRFs, and URFs circulating. Moreover, the geographic distribution of non-B variants is widespread. Data from HIV-1 drug resistance testing have the potential to significantly enhance the surveillance of HIV-1 variants in the United States.
PMCID: PMC3719628  PMID: 23761148
4.  Use of a Multifaceted Approach to Analyze HIV Incidence in a Cohort Study of Women in the United States: HIV Prevention Trials Network 064 Study 
The Journal of Infectious Diseases  2012;207(2):223-231.
Background. Reliable methods for estimating the incidence of human immunodeficiency virus (HIV) infection are needed to monitor the epidemic, identify at-risk populations, and evaluate HIV prevention strategies. We used a multifaceted approach to estimate HIV incidence in the HIV Prevention Trials Network (HPTN) 064 study.
Methods. The HPTN 064 study enrolled 2067 HIV-seronegative women and 32 HIV-seropositive women with no prior HIV infection diagnosis. Women were followed for up to 12 months. HIV incidence estimates were based on (1) detection of acute HIV infection, (2) documentation of HIV seroconversion, and (3) detection of recent HIV infection, using a multiassay algorithm (MAA).
Results. Two women had acute HIV infection at enrollment, 4 seroconverted, and 2 were identified as recently infected at enrollment using the MAA. The annual HIV incidence estimate based on acute infection at enrollment (2.52% [95% confidence interval {CI}, .17%–9.33%], using a 14-day window period) was higher than the estimate based on seroconversion (0.24% [95% CI, .07%–.62%]; P = .027). Incidence estimates obtained using the MAA at enrollment and at the end of study were 0.25% (95% CI, .03%–.93%) and 0.13% (95% CI, .006%–.76%), respectively.
Conclusions. We detected a high frequency of acute infection at enrollment. Cross-sectional HIV incidence estimates obtained using the MAA were similar to the longitudinal estimate based on HIV seroconversion.
Clinical Trials Registration. NCT00995176.
PMCID: PMC3532822  PMID: 23129758
HIV-1; women; United States; incidence
5.  Performance of Rapid Point-of-Care and Laboratory Tests for Acute and Established HIV Infection in San Francisco 
PLoS ONE  2013;8(12):e80629.
Current laboratory and point-of-care tests for HIV detect different analytes and use different sample types. Some have fast turnaround times (<1 hour). We investigated how HIV test choice could impact case finding by testing programs.
We analyzed 21,234 consecutive HIV tests with venous blood obtained by San Francisco HIV testing programs from 2003 to 2008. For a subset, oral fluid (n = 6446) or fingerstick blood (n = 8127) samples were also obtained for rapid testing. In all cases, HIV status was determined using an HIV antibody-plus-RNA test algorithm. We assessed how the screening antibody tests performed individually versus the gold standard of the full algorithm. We then evaluated the potential ability of other tests (including new tests) to detect more cases, by re-testing all specimens that had negative/discrepant antibody results on initial screening.
The antibody-RNA algorithm identified 58 acute and 703 established HIV infection cases. 1st-generation (Vironostika) and 3rd-generation (Genetic Systems) immunoassays had 92 and 96 percent sensitivity, respectively. The Oraquick rapid test had clinical sensitivity of only 86 percent on oral fluid samples, but 92 percent on finger-stick blood. Newer 4th-generation, antigen-antibody combo rapid immunoassay (ARCHITECT) detected HIV in 87 percent of all the acute cases that had been missed by one of the previous screening assays. A point-of-care 4th generation antigen-antibody combo rapid test (Determine) detected about 54 percent of such acute cases.
Our study suggests that some rapid antibody blood tests will give similar case detection to laboratory antibody tests, but that oral fluid testing greatly reduces ability to detect HIV. New 4th-generation combo tests can detect the majority of acute infections detectable by HIV RNA but with rapid results. Using these tests as a primary screening assay in high-risk HIV testing programs could reduce or eliminate the need for HIV RNA testing.
PMCID: PMC3861178  PMID: 24349007
6.  The Perils of Pathogen Discovery: Origin of a Novel Parvovirus-Like Hybrid Genome Traced to Nucleic Acid Extraction Spin Columns 
Journal of Virology  2013;87(22):11966-11977.
Next-generation sequencing was used for discovery and de novo assembly of a novel, highly divergent DNA virus at the interface between the Parvoviridae and Circoviridae. The virus, provisionally named parvovirus-like hybrid virus (PHV), is nearly identical by sequence to another DNA virus, NIH-CQV, previously detected in Chinese patients with seronegative (non-A-E) hepatitis. Although we initially detected PHV in a wide range of clinical samples, with all strains sharing ∼99% nucleotide and amino acid identity with each other and with NIH-CQV, the exact origin of the virus was eventually traced to contaminated silica-binding spin columns used for nucleic acid extraction. Definitive confirmation of the origin of PHV, and presumably NIH-CQV, was obtained by in-depth analyses of water eluted through contaminated spin columns. Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture. The confirmation of PHV/NIH-CQV as laboratory reagent contaminants and not bona fide infectious agents of humans underscores the rigorous approach needed to establish the validity of new viral genomes discovered by next-generation sequencing.
PMCID: PMC3807889  PMID: 24027301
7.  Estimation of HIV Incidence in a Large, Community-Based, Randomized Clinical Trial: NIMH Project Accept (HIV Prevention Trials Network 043) 
PLoS ONE  2013;8(7):e68349.
National Institute of Mental Health Project Accept (HIV Prevention Trials Network [HPTN] 043) is a large, Phase III, community-randomized, HIV prevention trial conducted in 48 matched communities in Africa and Thailand. The study intervention included enhanced community-based voluntary counseling and testing. The primary endpoint was HIV incidence, assessed in a single, cross-sectional, post-intervention survey of >50,000 participants.
HIV rapid tests were performed in-country. HIV status was confirmed at a central laboratory in the United States. HIV incidence was estimated using a multi-assay algorithm (MAA) that included the BED capture immunoassay, an avidity assay, CD4 cell count, and HIV viral load.
Data from Thailand was not used in the endpoint analysis because HIV prevalence was low. Overall, 7,361 HIV infections were identified (4 acute, 3 early, and 7,354 established infections). Samples from established infections were analyzed using the MAA; 467 MAA positive samples were identified; 29 of those samples were excluded because they contained antiretroviral drugs. HIV prevalence was 16.5% (range at study sites: 5.93% to 30.8%). HIV incidence was 1.60% (range at study sites: 0.78% to 3.90%).
In this community-randomized trial, a MAA was used to estimate HIV incidence in a single, cross-sectional post-intervention survey. Results from this analysis were subsequently used to compare HIV incidence in the control and intervention communities.
Trial Registration NCT00203749
PMCID: PMC3708944  PMID: 23874597
8.  Coupled electron transfer and proton hopping in the final step of CYP19-catalyzed androgen aromatization† 
Biochemistry  2012;51(14):3039-3049.
Aromatase (CYP19) catalyzes the terminal step in estrogen biosynthesis, which requires three separate oxidation reactions, culminating in an enigmatic aromatization that converts an androgen to an estrogen. A stable ferric peroxo (Fe3+O22−) intermediate is seen by electron paramagnetic resonance but its role in this complex reaction remains controversial. Combining molecular dynamics simulation and hybrid quantum mechanics/molecular mechanics, we show that ferric peroxo addition to the 19-aldehyde initiates the reaction. Stepwise cleavage of the C10-C19 and O-O bonds of the peroxohemiacetal extrudes formate and yields Compound II, which in turn desaturates the steroid through successive abstraction of the 1β-hydrogen atom and deprotonation of the 2β-position. Throughout the transformation, a proton is cyclically relayed between D309 and the substrate to stabilize reaction intermediates. This mechanism invokes novel oxygen intermediates and provides a unifying interpretation of past experimental mechanistic studies.
PMCID: PMC3326664  PMID: 22439696
Aromatase; Cytochrome P450; Quantum Mechanics/Molecular Mechanics; Electron Transfer; Ferric Peroxo; Aromatase; Molecular Dynamics Simulation
9.  Development of sensitive single-round pol or env RT-PCR assays to screen for XMRV in multiple sample types 
Journal of virological methods  2011;179(1):127-134.
The potential association between xenotropic murine leukemia virus-related virus (XMRV) and prostate cancer and chronic fatigue syndrome (CFS) has been much debated. To help resolve the potential role of XMRV in human disease, it is critical to develop sensitive and accurate reverse transcriptase (RT)-PCR assays to screen for the virus.
Single-round RT-PCR assays were developed on the automated m2000™ system for detection of the pol or env regions of XMRV in whole blood, plasma, urine cell pellets and urogenital swab samples. Assay performance was assessed by testing two blinded panels, one comprised of whole blood and the other of plasma spiked with serial dilutions of XMRV-infected tissue culture cells and supernatant, respectively, prepared by the Blood XMRV Scientific Research Working Group (SRWG). For both whole blood and plasma panel testing, the assays showed excellent specificity and sensitivity as compared to the other tests included in the SRWG phase I study. Analytical specificity of the assays was also evaluated. Neither pol nor env PCR assays detected a panel of potential cross-reactive microorganisms, although some cross-reaction was observed with mouse genomic DNA. Screening of 196 normal human blood donor plasma, 214 HIV-1 seropositive plasma, 20 formalin-fixed paraffin-embedded (FFPE) prostate cancer specimens, 4 FFPE benign prostate specimens, 400 urine pellets from prostate cancer patients, 166 urine pellets from non-prostate cancer patients, and 135 cervical swab specimens, detected no samples as unequivocally XMRV positive.
PMCID: PMC3573700  PMID: 22057262
real-time RT-PCR assay; XMRV pol; XMRV env; Blood XMRV Scientific Research Working Group blinded panel
10.  Specialized brain regions and sensory inputs that control locomotion in leeches 
Locomotor systems are often controlled by specialized cephalic neurons and undergo modulation by sensory inputs. In many species, dedicated brain regions initiate and maintain behavior and set the duration and frequency of the locomotor episode. In the leech, removing the entire head brain enhances swimming, but the individual roles of its components, the supra- and subesophageal ganglia, in the control of locomotion are unknown. Here we describe the influence of these two structures and that of the tail brain on rhythmic swimming in isolated nerve cord preparations and in nearly-intact leeches suspended in an aqueous, “swim-enhancing” environment. We found that, in isolated preparations, swim episode duration and swim burst frequency are greatly increased when the supraesophageal ganglion is removed, but the subesophageal ganglion is intact. The prolonged swim durations observed with the anterior-most ganglion removed were abolished by removal of the tail ganglion. Experiments on the nearly intact leeches show that, in these preparations, the subesophageal ganglion acts to decrease cycle period but, unexpectedly, also decreases swim duration. These results suggest that the supraesophageal ganglion is the primary structure that constrains leech swimming; however, the control of swim duration in the leech is complex, especially in the intact animal.
PMCID: PMC3265633  PMID: 22037913
Hirudo; brain; swimming; motor control; sensory modulation
11.  In Vivo Hypermutation of Xenotropic Murine Leukemia Virus-Related Virus DNA in Peripheral Blood Mononuclear Cells of Rhesus Macaque by APOBEC3 Proteins 
Virology  2011;421(1):28-33.
The gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), replicates to high titers in some human cell lines and is able to infect non-human primates. To determine whether APOBEC3 (A3) proteins restrict XMRV infections in a non-human primate model, we sequenced proviral DNA from peripheral blood mononuclear cells of XMRV-infected rhesus macaques. Hypermutation characteristic of A3DE, A3F and A3G activities was observed in the XMRV proviral sequences in vivo. Furthermore, expression of rhesus A3DE, A3F, or A3G in human cells inhibited XMRV infection and caused hypermutation of XMRV DNA. These studies show that some rhesus A3 isoforms are highly effective against XMRV in the blood of a non-human primate model of infection and in cultured human cells.
PMCID: PMC3208790  PMID: 21982221
XMRV; APOBEC3; hypermutation; retrovirus; host restriction; innate immunity
12.  Failure to Confirm XMRV/MLVs in the Blood of Patients with Chronic Fatigue Syndrome: A Multi-laboratory Study 
Science (New York, N.y.)  2011;334(6057):814-817.
Murine leukemia viruses (MLV), including xenotropic-MLV-related virus (XMRV), have been controversially linked to chronic fatigue syndrome (CFS). To explore this issue in greater depth, we compiled coded replicate samples of blood from 15 subjects previously reported to be XMRV/MLV-positive (14 with CFS) and from 15 healthy donors previously determined to be negative for the viruses. These samples were distributed in a blinded fashion to nine laboratories which performed assays designed to detect XMRV/MLV nucleic acid, virus replication, and antibody. Only two laboratories reported evidence of XMRV/MLVs; however, replicate sample results showed disagreement and reactivity was similar among CFS subjects and negative controls. These results indicate that current assays do not reproducibly detect XMRV/MLV in blood samples and that blood donor screening is not warranted.
PMCID: PMC3299483  PMID: 21940862
13.  In-Depth Investigation of Archival and Prospectively Collected Samples Reveals No Evidence for XMRV Infection in Prostate Cancer 
PLoS ONE  2012;7(9):e44954.
XMRV, or xenotropic murine leukemia virus (MLV)-related virus, is a novel gammaretrovirus originally identified in studies that analyzed tissue from prostate cancer patients in 2006 and blood from patients with chronic fatigue syndrome (CFS) in 2009. However, a large number of subsequent studies failed to confirm a link between XMRV infection and CFS or prostate cancer. On the contrary, recent evidence indicates that XMRV is a contaminant originating from the recombination of two mouse endogenous retroviruses during passaging of a prostate tumor xenograft (CWR22) in mice, generating laboratory-derived cell lines that are XMRV-infected. To confirm or refute an association between XMRV and prostate cancer, we analyzed prostate cancer tissues and plasma from a prospectively collected cohort of 39 patients as well as archival RNA and prostate tissue from the original 2006 study. Despite comprehensive microarray, PCR, FISH, and serological testing, XMRV was not detected in any of the newly collected samples or in archival tissue, although archival RNA remained XMRV-positive. Notably, archival VP62 prostate tissue, from which the prototype XMRV strain was derived, tested negative for XMRV on re-analysis. Analysis of viral genomic and human mitochondrial sequences revealed that all previously characterized XMRV strains are identical and that the archival RNA had been contaminated by an XMRV-infected laboratory cell line. These findings reveal no association between XMRV and prostate cancer, and underscore the conclusion that XMRV is not a naturally acquired human infection.
PMCID: PMC3445615  PMID: 23028701
14.  Positive feedback loops sustain repeating bursts in neuronal circuits 
Journal of Biological Physics  2010;37(3):317-345.
Voluntary movements in animals are often episodic, with abrupt onset and termination. Elevated neuronal excitation is required to drive the neuronal circuits underlying such movements; however, the mechanisms that sustain this increased excitation are largely unknown. In the medicinal leech, an identified cascade of excitation has been traced from mechanosensory neurons to the swim oscillator circuit. Although this cascade explains the initiation of excitatory drive (and hence swim initiation), it cannot account for the prolonged excitation (10–100 s) that underlies swim episodes. We present results of physiological and theoretical investigations into the mechanisms that maintain swimming activity in the leech. Although intrasegmental mechanisms can prolong stimulus-evoked excitation for more than one second, maintained excitation and sustained swimming activity requires chains of several ganglia. Experimental and modeling studies suggest that mutually excitatory intersegmental interactions can drive bouts of swimming activity in leeches. Our model neuronal circuits, which incorporated mutually excitatory neurons whose activity was limited by impulse adaptation, also replicated the following major experimental findings: (1) swimming can be initiated and terminated by a single neuron, (2) swim duration decreases with experimental reduction in nerve cord length, and (3) swim duration decreases as the interval between swim episodes is reduced.
PMCID: PMC3101330  PMID: 22654180
Neuronal circuits; Leech; Reciprocal excitation; Mutual excitation; Episodic behavior
15.  Absence of XMRV and Closely Related Viruses in Primary Prostate Cancer Tissues Used to Derive the XMRV-Infected Cell Line 22Rv1 
PLoS ONE  2012;7(5):e36072.
The 22Rv1 cell line is widely used for prostate cancer research and other studies throughout the world. These cells were established from a human prostate tumor, CWR22, that was serially passaged in nude mice and selected for androgen independence. The 22Rv1 cells are known to produce high titers of xenotropic murine leukemia virus-related virus (XMRV). Recent studies suggested that XMRV was inadvertently created in the 1990's when two murine leukemia virus (MLV) genomes (pre-XMRV1 and pre-XMRV-2) recombined during passaging of the CWR22 tumor in mice. The conclusion that XMRV originated from mice and not the patient was based partly on the failure to detect XMRV in early CWR22 xenografts. While that deduction is certainly justified, we examined the possibility that a closely related virus could have been present in primary tumor tissue. Here we report that we have located the original prostate tumor tissue excised from patient CWR22 and have assayed the corresponding DNA by PCR and the tissue sections by fluorescence in situ hybridization for the presence of XMRV or a similar virus. The primary tumor tissues lacked mouse DNA as determined by PCR for intracisternal A type particle DNA, thus avoiding one of the limitations of studying xenografts. We show that neither XMRV nor a closely related virus was present in primary prostate tissue of patient CWR22. Our findings confirm and reinforce the conclusion that XMRV is a recombinant laboratory-generated mouse virus that is highly adapted for human prostate cancer cells.
PMCID: PMC3353988  PMID: 22615748
16.  Neuronal Control of Swimming Behavior: Comparison of Vertebrate and Invertebrate Model Systems 
Progress in neurobiology  2010;93(2):244-269.
Swimming movements in the leech and lamprey are highly analogous, and lack homology. Thus, similarities in mechanisms must arise from convergent evolution rather than from common ancestry. Despite over forty years of parallel investigations into this annelid and primitive vertebrate, a close comparison of the approaches and results of this research is lacking. The present review evaluates the neural mechanisms underlying swimming in these two animals and describes the many similarities that provide intriguing examples of convergent evolution. Specifically, we discuss swim initiation, maintenance and termination, isolated nervous system preparations, neural-circuitry, central oscillators, intersegmental coupling, phase lags, cycle periods and sensory feedback. Comparative studies between species highlight mechanisms that optimize behavior and allow us a broader understanding of nervous system function.
PMCID: PMC3034781  PMID: 21093529
17.  Infection, Viral Dissemination, and Antibody Responses of Rhesus Macaques Exposed to the Human Gammaretrovirus XMRV▿ 
Journal of Virology  2011;85(9):4547-4557.
Xenotropic murine leukemia-related virus (XMRV) was identified in association with human prostate cancer and chronic fatigue syndrome. To examine the infection potential, kinetics, and tissue distribution of XMRV in an animal model, we inoculated five macaques with XMRV intravenously. XMRV established a persistent, chronic disseminated infection, with low transient viremia and provirus in blood lymphocytes during acute infection. Although undetectable in blood after about a month, XMRV viremia was reactivated at 9 months, confirming the chronicity of the infection. Furthermore, XMRV Gag was detected in tissues throughout, with wide dissemination throughout the period of monitoring. Surprisingly, XMRV infection showed organ-specific cell tropism, infecting CD4 T cells in lymphoid organs including the gastrointestinal lamina propria, alveolar macrophages in lung, and epithelial/interstitial cells in other organs, including the reproductive tract. Of note, in spite of the intravenous inoculation, extensive XMRV replication was noted in prostate during acute but not chronic infection even though infected cells were still detectable by fluorescence in situ hybridization (FISH) in prostate at 5 and 9 months postinfection. Marked lymphocyte activation occurred immediately postinfection, but antigen-specific cellular responses were undetectable. Antibody responses were elicited and boosted upon reexposure, but titers decreased rapidly, suggesting low antigen stimulation over time. Our findings establish a nonhuman primate model to study XMRV replication/dissemination, transmission, pathogenesis, immune responses, and potential future therapies.
PMCID: PMC3126245  PMID: 21325416
18.  Performance of the Abbott RealTime HIV-1 Viral Load Assay Is Not Impacted by Integrase Inhibitor Resistance-Associated Mutations▿ 
Journal of Clinical Microbiology  2011;49(4):1631-1634.
The Abbott RealTime HIV-1 viral load assay uses primers and probes targeted to integrase, which is also the target of integrase inhibitors such as raltegravir. Viral loads of 42 raltegravir-susceptible and 40 raltegravir-resistant specimens were determined using RealTime HIV-1 and Roche Monitor (v1.5). The differences in viral load measurements between assays were comparable in the two groups, demonstrating that the RealTime HIV-1 assay can tolerate raltegravir-selected mutations.
PMCID: PMC3122809  PMID: 21289145
19.  HIV Surveillance in a Large, Community-Based Study: Results from the Pilot Study of Project Accept (HIV Prevention Trials Network 043) 
BMC Infectious Diseases  2011;11:251.
Project Accept is a community randomized, controlled trial to evaluate the efficacy of community mobilization, mobile testing, same-day results, and post-test support for the prevention of HIV infection in Thailand, Tanzania, Zimbabwe, and South Africa. We evaluated the accuracy of in-country HIV rapid testing and determined HIV prevalence in the Project Accept pilot study.
Two HIV rapid tests were performed in parallel in local laboratories. If the first two rapid tests were discordant (one reactive, one non-reactive), a third HIV rapid test or enzyme immunoassay was performed. Samples were designated HIV NEG if the first two tests were non-reactive, HIV DISC if the first two tests were discordant, and HIV POS if the first two tests were reactive. Samples were re-analyzed in the United States using a panel of laboratory tests.
HIV infection status was correctly determined based on-in country testing for 2,236 (99.5%) of 2,247 participants [7 (0.37%) of 1,907 HIV NEG samples were HIV-positive; 2 (0.63%) of 317 HIV POS samples were HIV-negative; 2 (8.3%) of 24 HIV DISC samples were incorrectly identified as HIV-positive based on the in-country tie-breaker test]. HIV prevalence was: Thailand: 0.6%, Tanzania: 5.0%, Zimbabwe 14.7%, Soweto South Africa: 19.4%, Vulindlela, South Africa: 24.4%, (overall prevalence: 14.4%).
In-country testing based on two HIV rapid tests correctly identified the HIV infection status for 99.5% of study participants; most participants with discordant HIV rapid tests were not infected. HIV prevalence varied considerably across the study sites (range: 0.6% to 24.4%).
Trial Registration registry number NCT00203749.
PMCID: PMC3198953  PMID: 21943026
20.  Failure to Detect XMRV-Specific Antibodies in the Plasma of CFS Patients Using Highly Sensitive Chemiluminescence Immunoassays 
Advances in Virology  2011;2011:854540.
In 2009, Lombardi et al. reported their startling finding that the gammaretrovirus xenotropic murine leukemia virus-related retrovirus (XMRV) is present in 67% of blood samples of patients suffering from chronic fatigue syndrome (CFS), as opposed to only 3.7% of samples from healthy individuals. However, we and others could not confirm these results, using a nested PCR assay. An alternative to this highly sensitive, but contamination-prone, technique is to measure the serological response to XMRV. Thus, we tested the plasma samples from our cohorts of CFS patients and healthy controls for the presence of XMRV-specific antibodies. Using two novel chemiluminescence immunoassays (CMIAs), we show that none of our samples have any XMRV-reactive antibodies. Taken together with our previous findings, we conclude that XMRV is not present in any human individual tested by us, regardless of CFS or healthy control.
PMCID: PMC3265317  PMID: 22312356
21.  Sexual Transmission of XMRV: A Potential Infection Route 
Advances in Virology  2011;2011:965689.
Although XMRV dissemination in humans is a matter of debate, the prostate of select patients seem to harbor XMRV, which raises questions about its potential route of transmission. We established a model of infection in rhesus macaques inoculated with XMRV. In spite of the intravenous inoculation, all infected macaques exhibited readily detectable XMRV signal in the reproductive tract of all 4 males and 1 female during both acute and chronic infection stages. XMRV showed explosive growth in the acini of prostate during acute but not chronic infection. In seminal vesicles, epididymis, and testes, XMRV protein production was detected throughout infection in interstitial or epithelial cells. In the female monkey, epithelial cells in the cervix and vagina were also positive for XMRV gag. The ready detection of XMRV in the reproductive tract of male and female macaques infected intravenously suggests the potential for sexual transmission for XMRV.
PMCID: PMC3265321  PMID: 22312360
25.  A Metagenomic Analysis of Pandemic Influenza A (2009 H1N1) Infection in Patients from North America 
PLoS ONE  2010;5(10):e13381.
Although metagenomics has been previously employed for pathogen discovery, its cost and complexity have prevented its use as a practical front-line diagnostic for unknown infectious diseases. Here we demonstrate the utility of two metagenomics-based strategies, a pan-viral microarray (Virochip) and deep sequencing, for the identification and characterization of 2009 pandemic H1N1 influenza A virus. Using nasopharyngeal swabs collected during the earliest stages of the pandemic in Mexico, Canada, and the United States (n = 17), the Virochip was able to detect a novel virus most closely related to swine influenza viruses without a priori information. Deep sequencing yielded reads corresponding to 2009 H1N1 influenza in each sample (percentage of aligned sequences corresponding to 2009 H1N1 ranging from 0.0011% to 10.9%), with up to 97% coverage of the influenza genome in one sample. Detection of 2009 H1N1 by deep sequencing was possible even at titers near the limits of detection for specific RT-PCR, and the percentage of sequence reads was linearly correlated with virus titer. Deep sequencing also provided insights into the upper respiratory microbiota and host gene expression in response to 2009 H1N1 infection. An unbiased analysis combining sequence data from all 17 outbreak samples revealed that 90% of the 2009 H1N1 genome could be assembled de novo without the use of any reference sequence, including assembly of several near full-length genomic segments. These results indicate that a streamlined metagenomics detection strategy can potentially replace the multiple conventional diagnostic tests required to investigate an outbreak of a novel pathogen, and provide a blueprint for comprehensive diagnosis of unexplained acute illnesses or outbreaks in clinical and public health settings.
PMCID: PMC2956640  PMID: 20976137

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