The new massively parallel sequencing methods are so astonishing that one wonders whether space aliens are secretly behind them. One technician, running a single instrument, can obtain up to approximately 1 billion bases of DNA sequence in a few days. Here we describe the new sequencing methods, briefly present a few applications in HIV research, and then speculate on future directions. The new massively parallel sequencing methods Several methods for massively parallel pyrosequencing have recently been commercialized. As an example, consider the use of the 454 Life Sciences pyrosequencing method for metagenomic analysis of woolly mammoth DNA [1,2]. DNA from a mammoth carcass was purified and fragmented, and DNA linkers were ligated to the free ends. DNAs were then denatured and strands annealed to beads conjugated with oligonucleotides complementary to the linker sequences. This step is carried out with very low DNA concentrations so that on average only one strand binds to each bead. Bead-bound DNA is then PCR amplified in an oil–water emulsion, where each water droplet in the emulsion contains on average a single bead. The amplified DNA strands anneal to the beads, yielding beads with many copies of homogeneous PCR products. Pools of up to 400 000 beads are then distributed in a picotiter plate and further manipulations carried out in a custom fluidics station (Fig. 1). A polymerase is used to extend a DNA chain from a bound primer on each strand. The four nucleoside triphosphates are sequentially flowed over the picotiter plate. With each incorporation event, pyrophosphate is liberated into solution (hence 'pyrosequencing'). An enzyme system is present in the aqueous phase that directs incorporation of pyrophosphate into ATP, which in turn activates purified luciferase, also present in the aqueous phase, to produce a flash of light. Each flash from each well is quantified by a charge coupled device camera and the signals detected and stored in a computer. Sequential application of the four nucleotides (nts) allows DNA sequences of approximately 100 nt to be built up several hundred thousand fold at a time. Using this method a detailed comparison of the mammoth and elephant genomes can be carried out (yes, there is also an elephant genome project).Fig. 1: The 454 GS FLX sequencing station.With the improved 454 technology released recently, it is possible to generate reads of approximately 260 nt on approximately 400 000 beads per run, yielding a whopping 100 million bases of DNA sequence in a day or two. An illustrated description of the method can be found at http://www.454.com/enabling-technology/index.asp. A second pyrosequencing technology, commercialized by Solexa/Illumina (San Diego, California, USA), yields shorter sequence reads, only approximately 35 bp, but a single run yields up approximately 1 billion bases of DNA sequence [3,4] (see www.illumina.com/downloads/SS_DNAsequencing.pdf). Table 1 compares the Sanger, 454/Roche (Branford, Connecticut, USA), and Solexa/Illumina methods. A variety of additional technologies are also under development [5].Table 1: Comparison of sequencing methods.Pyrosequencing to analyze HIV diversity The pyrosequencing methods are well suited to addressing questions on the dynamics of HIV quasi-species in response to selective pressures. HIV reverse transcriptase is very error prone, making roughly one base pair substitution mutation per round of replication [6]. The viral populations in infected individuals are also very large, with some 1010 virions produced and destroyed per day [7–9]. After infection, this activity quickly results in the formation of a diverse pool, or quasispecies, in which most viral sequences differ from all others. Pyrosequencing offers an improved means of characterizing sequence variation present in such large populations. One application is quantification of rare drug-resistant mutations in treated individuals failing antiretroviral therapy. Current guidelines recommend that whenever an HIV-positive individual is initiating or changing therapy, possible drug-resistant alleles should be assayed and treatment choices adjusted accordingly. However, the most commonly used genotyping methods only provide information on the most abundant sequence variants. Evidence suggests that rare drug-resistant variants, when subjected to the selective pressures of drug treatment, can quickly grow out and become the predominant form, leading to treatment failure [10,11]. Two reports have applied pyrosequencing to the detection and characterization of rare drug-resistant variants [12,13]. Shafer and colleagues [13] purified RNA from patient virions, reverse transcribed to generate cDNA, sheared the cDNA product, and carried out pyrosequencing as described for mammoth DNA. Hoffmann et al.[12] took a different approach, PCR amplifying short regions of interest, then pyro-sequencing the amplicons directly. Both groups also analyzed control cloned viral stocks, allowing empirical determination of the rates of false-positive calls and thereby permitting convincing demonstrations of detection of drug-resistant mutations present as less than 1% of the population. Another study [14] applied pyrosequencing to analyzing viral tropism in quasispecies inferred from V3 loop sequences. As inhibitors that block coreceptor binding come into widespread use, there will be an increased need for deep profiling of coreceptor usage prior to initiating therapy, and pyrosequencing would be an ideal tool for this application. The Hoffmann approach [12] applied a twist that is likely to be useful in many applications. They indexed PCR products by adding short DNA bar codes to their PCR primers. Sequence reads extended across the bar codes, allowing different samples to be distinguished after sequencing in a mixed pool. Running a single 454 plate is fairly expensive, but bar coding allows many samples to be run on the same plate, thereby reducing the cost per sample [12,15]. Pyrosequencing to analyze HIV integration targeting Detailed studies of the placement of HIV integration sites in the human genome [16–19] have yielded a wealth of new insights into HIV biology. Related studies [20,21] on integration site distributions are also important in the gene therapy field, in which integration of therapeutic retroviral vectors has resulted in insertional activation of proto-oncogenes and clinical adverse events. The first genome-wide study of HIV integration targeting [22] used Sanger sequencing and reported approximately 500 integration sites [16]. A recent study using pyrosequencing yielded 40 000 sites. Each boost in the number of integration sites has led to new insights into mechanism. The pyrosequencing study of HIV integration, for example, yielded data indicating that integration in chromosomes in vivo usually takes place on nucleosome-bound DNA (Fig. 2)[23–25].Fig. 2: Use of pyrosequencing data to show that HIV integration takes place on nucleosomal DNA in vivo. (a) Schematic diagram of DNA wrapped on a nucleosome. The dyad axis (center of two-fold symmetry) is shown by the diamond. (b) Data from pyrosequencing analysis of 40 000 sites of HIV DNA integration, showing frequency as a function of position on the nucleosome. For each of the integration sites, the position of the underlying nucleosome was predicted from the primary sequence using the method of Segal et al. [23]. Nucleosomes were aligned at their centers of symmetry and the integration frequency quantified across the full data set moving outward from the dyad axis. A periodic pattern of high and low frequency integration was found with a period of about 10.5 bases. This pattern is exactly as expected for DNA bound on the nucleosome surface, where integration is favored in sequential out-ward facing major grooves [24,25].Pyrosequencing data sets are just beginning to be generated for samples from gene therapy trials, including trials to treat HIV [26], which will allow much deeper investigation of vector integration site distributions. One question for future studies will be to what degree patterns in deep integration site data help forecast impending clinical adverse events. Pyrosequencing to characterize unknown opportunistic infections Clinicians are occasionally confronted with apparent infections that cannot be easily attributed to a known pathogen. Given concerns about bioterrorism, there is an urgent need to identify the infectious agents quickly in such cases. For AIDS patients, opportunistic infections that would be cleared in immunocompetent individuals can cause morbidity in the immunocompromised, potentially resulting in challenges in identifying the agent. A wealth of new molecular methods are becoming available for identifying new pathogens, with pyrosequencing a major addition. The discovery of a virus potentially responsible for colony collapse disorder (CCD) of honey bees provides a striking example [27]. North American bee colonies have been failing at alarming rates, and an infectious agent implicated. Pyrosequencing of a large number of samples from affected and unaffected colonies revealed that the presence of Israeli acute paralysis virus was strongly associated with CCD, and follow-up quantitative PCR studies strengthened the connection. Experimental infection studies are now needed to test causality. Pyrosequencing also provides potent new methods for analyzing bacterial populations. Many bacterial diseases likely involve not only single pathogens but also the full microbial community in which that pathogen resides – for example, obesity and Crohn's disease are proposed to involve community-wide alterations in gastrointestinal microbiota [28–30]. Pyrosequencing of DNA samples from uncultured bacterial communities can identify the members of a community and their relative abundance in a rapid and cost-effective fashion [31,32]. For example, 454 pyrosequencing of segments of the 16S RNA genes from uncultured communities of marine bacteria has revealed numerous previously undiscovered bacterial taxa [31,33]. Of particular interest to HIV research, a pyrosequencing study [34] of the gastrointestinal microbiota present in simian immunodeficiency virus (SIV)-infected macaques has shown consistent changes associated with chronic colitis accompanying disease progression. These findings set the stage for an investigation of pathogenic mechanisms, particularly in connection with recent proposals for involvement of gastrointestinal flora in inflammation and lentiviral disease progression [35]. Pyrosequencing in hypothesis testing Pyrosequencing can be used not just to sequence genomes, but also as an end-point assay in a mechanistic experiment, as one might once have used a p24 assay or Southern blot. For example, in an application in the integration targeting field, HIV integration in cells containing or lacking the targeting factor PC4 and SFRS1 interacting protein 1/lens epithelium-derived growth factor/p75 (PSIP1/LEDGF/p75) was compared using pyrosequencing. The analysis revealed that cells lacking the cofactor showed reduced integration in transcription units ([18]; see also [17,19]). In another example, Shuman and colleagues [36] identified mitoxantrone as an antipoxviral agent and sought to identify the viral target of the inhibitor. They picked mutant vaccinia viruses insensitive to the drug and then they sequenced the entire 195 kb mutant genomes from mutants and compared them to the wild type. A mutation in the ligase gene was found to be selectively present in the genomes of the drug-resistant variants. Thus, pyrosequencing allowed efficient identification of the molecular target of a new antiviral agent in a large viral genome. Looking ahead In just approximately 2 years since the 454 sequencing system has been commercially available, the technology has improved considerably and other companies are introducing competing platforms. It is virtually certain that the already amazing new sequencing technologies will be further improved in the years to come and that these new methods will transform HIV research. Some present and possible future applications of pyrosequencing to HIV research are summarized as follows: Quantifying rare drug-resistant mutations in HIV quasispecies Quantifying immune escape mutations in quasispecies under immune pressure Quantifying coreceptor usage in quasispecies Genome-wide monitoring of integration site selection Identification of novel pathogens Analysis of effects of lentiviral infection on gastrointestinal microbiota Affordable genotyping for resource-limited settings. For any virus, when understanding the structure of a complex swarm is important, pyrosequencing is an attractive tool. Interactions of HIV with other viruses could be explored; for example, interactions with hepatitis C virus (HCV). HCV quasispecies are probably more complex than HIV quasispecies and understanding of HCV swarms is less advanced than for HIV. Pyrosequencing of HCV populations from well chosen patient populations should be highly informative and studies of the effects of coinfection with HIV are of considerable interest. Studies of human genetic determinants of HIV disease course will likely be accelerated by the new methods. A couple of examples of complete sequencing of individual human genomes have been reported and more are on the way. Such studies on AIDS patients with different disease responses should help identify new human determinants of disease transmission and progression. Lastly, pyrosequencing may make some molecular diagnostic methods affordable in resource-limited settings. Methods for characterizing HIV drug resistance are unavailable in many areas of the developing world. Single pyrosequencing runs are expensive (for the 454 method, approximately US$ 15 000 per plate), but using DNA bar coding, several hundred samples may readily be analyzed per plate and larger numbers should be accessible with further refinement. There would be many challenges to implementation, but ultimately pyrosequencing technology could make a variety of molecular diagnostics affordable for those who presently lack access.
BackgroundTo replicate, lentiviruses such as HIV must integrate DNA copies of their RNA genomes into host cell chromosomes. Lentiviral integration is favored in active transcription units, which allows efficient viral gene expression after integration, but the mechanisms directing integration targeting are incompletely understood. A cellular protein, PSIP1/LEDGF/p75, binds tightly to the lentiviral-encoded integrase protein (IN), and has been reported to be important for HIV infectivity and integration targeting.MethodologyHere we report studies of lentiviral integration targeting in 1) human cells with intensified RNAi knockdowns of PSIP1/LEDGF/p75, and 2) murine cells with homozygous gene trap mutations in the PSIP1/LEDGF/p75 locus. Infections with vectors derived from equine infections anemia virus (EIAV) and HIV were compared. Integration acceptor sites were analyzed by DNA bar coding and pyrosequencing.Conclusions/SignificanceIn both PSIP1/LEDGF/p75-depleted cell lines, reductions were seen in lentiviral infectivity compared to controls. For the human cells, integration was reduced in transcription units in the knockdowns, and this reduction was greater than in our previous studies of human cells less completely depleted for PSIP1/LEDGF/p75. For the homozygous mutant mouse cells, similar reductions in integration in transcription units were seen, paralleling a previous study of a different mutant mouse line. Integration did not become random, however–integration in transcription units in both cell types was still favored, though to a reduced degree. New trends also appeared, including favored integration near CpG islands. In addition, we carried out a bioinformatic study of 15 HIV integration site data sets in different cell types, which showed that the frequency of integration in transcription units was correlated with the cell-type specific levels of PSIP1/LEDGF/p75 expression.
A reservoir of HIV-infected cells that persists despite suppressive antiretroviral therapy (ART) is the source of viral rebound upon ART cessation and the major barrier to a cure. Understanding reservoir seeding dynamics will help identify the best timing for HIV cure strategies. Here we characterize reservoir seeding using longitudinal samples from before and after ART initiation in individuals who sequentially became infected with genetically distinct HIV variants (superinfected). We previously identified cases of superinfection in a cohort of Kenyan women, and the dates of both initial infection and superinfection were determined. Six women, superinfected 0.2–5.2 years after initial infection, were subsequently treated with ART 5.4–18.0 years after initial infection. We performed next-generation sequencing of HIV gag and env RNA from plasma collected during acute infection as well as every ~2 years thereafter until ART initiation, and of HIV DNA from PBMCs collected 0.9–4.8 years after viral suppression on ART. We assessed phylogenetic relationships between HIV DNA reservoir sequences and longitudinal plasma RNA sequences prior to ART, to determine proportions of initial and superinfecting variants in the reservoir. The proportions of initial and superinfection lineage variants present in the HIV DNA reservoir were most similar to the proportions present in HIV RNA immediately prior to ART initiation. Phylogenetic analysis confirmed that the majority of HIV DNA reservoir sequences had the smallest pairwise distance to RNA sequences from timepoints closest to ART initiation. Our data suggest that while reservoir cells are created throughout pre-ART infection, the majority of HIV-infected cells that persist during ART entered the reservoir near the time of ART initiation. We estimate the half-life of pre-ART DNA reservoir sequences to be ~25 months, which is shorter than estimated reservoir decay rates during suppressive ART, implying continual decay and reseeding of the reservoir up to the point of ART initiation.
BACKGROUND Adolescents and young adults aged <25 years (youth) are at a higher risk of perinatal depression than older adults, and they experience elevated barriers to in-person care. Digital platforms such as social media offer an accessible avenue to deliver group cognitive behavioral therapy (CBT) to perinatal youth. OBJECTIVE We aim to develop the Interactive Maternal Group for Information and Emotional Support (IMAGINE) intervention, a facilitated social media group CBT intervention to prevent perinatal depression in youth in the United States, by adapting the Mothers and Babies (MB) course, an evidence-based in-person group CBT intervention. In this study, we report perspectives of youth and health care providers on perinatal youths’ mental health needs and document how they informed IMAGINE design. METHODS We conducted 21 semistructured in-depth individual interviews with 10 pregnant or postpartum youths aged 14-24 years and 6 health care workers. All interviews were recorded, transcribed, and analyzed using deductive and inductive approaches to characterize perceptions of challenges and facilitators of youth perinatal mental health. Using a human-centered design approach, stakeholder perspectives were incorporated into the IMAGINE design. We classified MB adaptations to develop IMAGINE according to the Framework for Modification and Adaptation, reporting the nature, timing, reason, and goal of the adaptations. RESULTS Youth and health care workers described stigma associated with young pregnancy and parenting, social isolation, and lack of material resources as significant challenges to youth mental wellness. They identified nonjudgmental support, peer companionship, and access to step-by-step guidance as facilitators of youth mental wellness. They endorsed the use of a social media group to prevent perinatal depression and recommended that IMAGINE facilitate peer support, deliver content asynchronously to accommodate varied schedules, use a confidential platform, and facilitate the discussion of topics beyond the MB curriculum, such as navigating support resources or asking medical questions. IMAGINE was adapted from MB to accommodate stakeholder recommendations and facilitate the transition to web-based delivery. Content was tailored to be multimodal (text, images, and video), and the language was shortened and simplified. All content was designed for asynchronous engagement, and redundancy was added to accommodate intermittent access. The structure was loosened to allow the intervention facilitator to respond in real time to topics of interest for youth. A social media platform was selected that allows multiple conversation <i>channels</i> and conceals group member identity. All adaptations sought to preserve the fidelity of the MB core components. CONCLUSIONS Our findings highlight the effect of stigmatization of young pregnancy and social determinants of health on youth perinatal mental health. Stakeholders supported the use of a social media group to create a supportive community and improve access to evidence-based depression prevention. This study demonstrates how a validated intervention can be tailored to this unique group. CLINICALTRIAL
Perinatal depression is broadly defined as depressive symptoms during pregnancy or within the 12 months following delivery, affecting approximately 20-25% of pregnant and postpartum women in low- and middle-income countries. The wide accessibility of mobile phones allows mobile health (mHealth) interventions to be considered a solution to identify perinatal depression and provide appropriate referrals for treatment. This study, nested in a larger SMS communication project, examined the prevalence and correlates of perinatal depression, determined the association between antenatal depression and infant morbidity and mortality, and compared SMS communication patterns between women with and without perinatal depression.This was a prospective longitudinal cohort study of pregnant women seeking antenatal services at two public sector health clinics in Kenya. SMS messages were sent to participants with educational content related to their pregnancy and infant health and two-way SMS communication occurred with a nurse. Sociodemographic and obstetric characteristics, SMS messaging behaviors, infant health status, and depressive symptoms were assessed by a standardized questionnaire administered at enrollment (30-36 weeks gestation) and follow-up (14 weeks postpartum). Generalized estimating equation (GEE) with Poisson link was used to evaluate correlates of perinatal depressive symptoms, infant outcomes, and frequency of SMS messaging.Of the 572 women with complete follow-up information, 188 (32.9%) screened positive for elevated depressive symptoms (≥10 by EPDS scale) at some time point during pregnancy or postpartum. The strongest predictors of any depressive symptoms included interpersonal abuse during pregnancy, fewer years of schooling, and maternal unemployment. Antenatal depressive symptoms were associated with an increased risk of infant illness or hospitalization (RR = 1.12, 95% CI: 1.11, 1.13). Women with antenatal or persistent perinatal depressive symptoms sent fewer SMS messages during the study period than their counterparts without depression.Prevalence of elevated perinatal depressive symptoms was high in this cohort of Kenyan women. Our findings highlight the importance of screening perinatal women for experiences of symptoms of depression as well as abuse. Differences in messaging frequency between women with vs. without depressive symptoms presents an opportunity to provide more tailored support for those perinatal depression.
HIV superinfection (reinfection) has been reported in several settings, but no study has been designed and powered to rigorously compare its incidence to that of initial infection. Determining whether HIV infection reduces the risk of superinfection is critical to understanding whether an immune response to natural HIV infection is protective. This study compares the incidence of initial infection and superinfection in a prospective seroincident cohort of high-risk women in Mombasa, Kenya. A next-generation sequencing-based pipeline was developed to screen 129 women for superinfection. Longitudinal plasma samples at <6 months, >2 years and one intervening time after initial HIV infection were analyzed. Amplicons in three genome regions were sequenced and a median of 901 sequences obtained per gene per timepoint. Phylogenetic evidence of polyphyly, confirmed by pairwise distance analysis, defined superinfection. Superinfection timing was determined by sequencing virus from intervening timepoints. These data were combined with published data from 17 additional women in the same cohort, totaling 146 women screened. Twenty-one cases of superinfection were identified for an estimated incidence rate of 2.61 per 100 person-years (pys). The incidence rate of initial infection among 1910 women in the same cohort was 5.75 per 100 pys. Andersen-Gill proportional hazards models were used to compare incidences, adjusting for covariates known to influence HIV susceptibility in this cohort. Superinfection incidence was significantly lower than initial infection incidence, with a hazard ratio of 0.47 (CI 0.29-0.75, p = 0.0019). This lower incidence of superinfection was only observed >6 months after initial infection. This is the first adequately powered study to report that HIV infection reduces the risk of reinfection, raising the possibility that immune responses to natural infection are partially protective. The observation that superinfection risk changes with time implies a window of protection that coincides with the maturation of HIV-specific immunity.
BACKGROUND Prevention of mother-to-child HIV transmission (PMTCT) relies on long-term adherence to antiretroviral therapy (ART). Mobile health approaches, such as text messaging (short message service, SMS), may improve adherence in some clinical contexts, but it is unclear what SMS content is desired to improve PMTCT-ART adherence. OBJECTIVE We aimed to explore the SMS content preferences related to engagement in PMTCT care among women, male partners, and health care workers. The message content was used to inform an ongoing randomized trial to enhance the PMTCT-ART adherence. METHODS We conducted 10 focus group discussions with 87 HIV-infected pregnant or postpartum women and semistructured individual interviews with 15 male partners of HIV-infected women and 30 health care workers from HIV and maternal child health clinics in Kenya. All interviews were recorded, translated, and transcribed. We analyzed transcripts using deductive and inductive approaches to characterize women’s, partners’, and health care workers’ perceptions of text message content. RESULTS All women and male partners, and most health care workers viewed text messages as a useful strategy to improve engagement in PMTCT care. Women desired messages spanning 3 distinct content domains: (1) educational messages on PMTCT and maternal child health, (2) reminder messages regarding clinic visits and adherence, and (3) encouraging messages that provide emotional support. While all groups valued reminder and educational messages, women highlighted emotional support more than the other groups (partners or health care workers). In addition, women felt that encouraging messages would assist with acceptance of their HIV status, support disclosure, improve patient-provider relationship, and provide support for HIV-related challenges. All 3 groups valued not only messages to support PMTCT or HIV care but also messages that addressed general maternal child health topics, stressing that both HIV- and maternal child health–related messages should be part of an SMS system for PMTCT. CONCLUSIONS Women, male partners, and health care workers endorsed SMS text messaging as a strategy to improve PMTCT and maternal child health outcomes. Our results highlight the specific ways in which text messaging can encourage and support HIV-infected women in PMTCT to remain in care, adhere to treatment, and care for themselves and their children. CLINICALTRIAL ClinicalTrials.gov NCT02400671; https://clinicaltrials.gov/ct2/show/NCT02400671 (Archived by WebCite at http://www.webcitation.org/70W7SVIVJ)
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