James Brooks

Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

Background Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. Methods and Findings We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Conclusions Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Cross-species retroviral transmission from macaques to human beings

Cross-species transmission of simian foamy virus (SFV) to human beings from chimpanzees, baboons, and African green monkeys has been described. Although macaques are the non-human primate most often handled in research, human infection with SFV from macaques has not been reported. Two of 46 primate-facility workers tested positive for antibodies that reacted with an immunoblot that contained macaque foamy virus antigens. Phylogenetic assessment of a 96-bp fragment of amplified proviral DNA isolated from peripheral-blood mononuclear cells from one infected individual was consistent with SFV infection of macaque origin. Frequent use of macaques in biomedical research, and identification of persistent retroviral infection from macaques to human beings, could have implications for public-health policy and occupational health and safety.

Ebola viral load at diagnosis associates with patient outcome and outbreak evolution

BACKGROUND Ebola virus (EBOV) causes periodic outbreaks of life-threatening EBOV disease in Africa. Historically, these outbreaks have been relatively small and geographically contained; however, the magnitude of the EBOV outbreak that began in 2014 in West Africa has been unprecedented. The aim of this study was to describe the viral kinetics of EBOV during this outbreak and identify factors that contribute to outbreak progression. METHODS From July to December 2014, one laboratory in Sierra Leone processed over 2,700 patient samples for EBOV detection by quantitative PCR (qPCR). Viremia was measured following patient admission. Age, sex, and approximate time of symptom onset were also recorded for each patient. The data was analyzed using various mathematical models to find trends of potential interest. RESULTS The analysis revealed a significant difference (P = 2.7 × 10(-77)) between the initial viremia of survivors (4.02 log10 genome equivalents [GEQ]/ml) and nonsurvivors (6.18 log10 GEQ/ml). At the population level, patient viral loads were higher on average in July than in November, even when accounting for outcome and time since onset of symptoms. This decrease in viral loads temporally correlated with an increase in circulating EBOV-specific IgG antibodies among individuals who were suspected of being infected but shown to be negative for the virus by PCR. CONCLUSIONS Our results indicate that initial viremia is associated with outcome of the individual and outbreak duration; therefore, care must be taken in planning clinical trials and interventions. Additional research in virus adaptation and the impacts of host factors on EBOV transmission and pathogenesis is needed.

Longitudinal phylogenetic surveillance identifies distinct patterns of cluster dynamics.

Objective:Through the application of simple, accessible, molecular epidemiology tools, we aimed to resolve the phylogenetic relationships that best predicted patterns of cluster growth using longitudinal population level drug resistance genotype data. Methods:Analysis was performed on 971 specimens from drug naïve, first time HIV positive subjects collected in British Columbia between 2002 and 2005. A 1240bp fragment of the pol gene was amplified and sequenced with relationships among subtype B sequences inferred using Neighbour-Joining analysis. Apparent clusters of infections having both a mean within group distance <0.031 and bootstrap value >80% were systematically identified. The entire 2002-2005 dataset was then re-analyze to evaluate the relationship of subsequent infections to those identified in 2002. BED testing was used to identify recent infections (<156 days). Results:Among the 2002 infections, 136 of 300 sequences sorted into 52 clusters ranging in size from 2 to 9 members. Aboriginal ethnicity and intravenous drug use were correlated, and both were linked to cluster membership in 2002. Although cluster growth between 2002 and 2005 was correlated with the size of the original cluster, more related infections were found in clusters seeded from nonclustered infections. Finally, all large growth clusters were seeded from infections that were much more likely to be recent. Conclusions:This population level phylogenetic analysis suggests that a greater increase in cluster size is associated with recently infected individuals, which may represent the leading edge of the epidemic. The most impressive increase in cluster size is seen originating from initially nonclustered infections. In contrast, smaller existing clusters likely describe historical patterns of transmission and do not substantially contribute to the ongoing epidemic. Application of this method for cross-sectional analysis of existing sequences from defined geographic regions may be useful in predicting trends in HIV transmission.

Characterization of blood-borne transmission of simian foamy virus.

BACKGROUND: Simian foamy virus (SFV) is an endemic, nonhuman primate (NHP) retrovirus that is transmitted to individuals who work with or hunt NHPs. The cross‐species transmission of simian retroviruses is believed to be the etiology of human immunodeficiency virus and human T‐lymphotropic virus infections in humans. Although SFV is not pathogenic in the native host, the shared ancestry with other simian retroviruses has brought into question the potential for acquired pathogenicity after cross‐species transmission. This study examines whether SFV also shares the traits of transmissibility through the blood supply.

Genetic Diversity as a Marker for Timing Infection in HIV-Infected Patients: Evaluation of a 6-Month Window and Comparison With BED

BACKGROUND It has been reported that the increase in human immunodeficiency virus (HIV) sequence diversity in drug resistance surveillance specimens may be used to classify the duration of HIV infection as <1 or >1 year. We describe a mixed base classifier (MBC) optimized to categorize the duration of subtype B infections as <6 or >6 months on the basis of sequences for drug resistance surveillance specimens and compared MBC findings with those of serologic methods. METHODS The behavior of the MBC was examined across a range of thresholds for calling mixed bases. MBC performance was then evaluated using either complete pol sequences or sites reflecting evolutionary pressures (HLA selection sites, sites that increased in entropy over the course of infection, and codon positions). RESULTS The MBC performance was optimal when secondary peaks on the sequencing chromatogram accounted for at least 15% of the area of primary peaks. A cutoff of <0.45% mixed bases in the pol region best identified recent infections (sensitivity = 82.7%, specificity = 78.8%), with improvement achieved by analyzing only sites that increased in entropy. CONCLUSIONS In an extended data set of 1354 specimens classified by BED, the optimized MBC performed significantly better than a simple MBC (agreement, 68.98% vs 67.13%). If further validated, the MBC may prove beneficial for detecting recent infection and estimating the incidence of HIV infection.

HIV Drug Resistance Surveillance Using Pooled Pyrosequencing

Background Surveillance for HIV transmitted drug resistance (TDR) is performed using HIV genotype results from individual specimens. Pyrosequencing, through its massive parallel sequencing ability, can analyze large numbers of specimens simultaneously. Instead of using pyrosequencing conventionally, to sequence a population of viruses within an individual, we interrogated a single combined pool of surveillance specimens to demonstrate that it is possible to determine TDR rates in HIV protease from a population of individuals. Methodology/Principal Findings The protease region from 96 treatment naïve, HIV+ serum specimens was genotyped using standard Sanger sequencing method. The 462 bp protease amplicons from these specimens were pooled in equimolar concentrations and re-sequenced using the GS FLX Titanium system. The nucleotide (NT) and amino acid (AA) differences from the reference sequence, along with TDR mutations, detected by each method were compared. In the protease sequence, there were 212 nucleotide and 81 AA differences found using conventional sequencing and 345 nucleotide and 168 AA differences using pyrosequencing. All nucleotide and amino acid polymorphisms found at frequencies ≥5% in pyrosequencing were detected using both methods with the rates of variation highly correlated. Using Sanger sequencing, two TDR mutations, M46L and I84V, were each detected as mixtures at a frequency of 1.04% (1/96). These same TDR mutations were detected by pyrosequencing with a prevalence of 0.29% and 0.34% respectively. Phylogenetic analysis established that the detected low frequency mutations arose from the same single specimens that were found to contain TDR mutations by Sanger sequencing. Multiple clinical protease DR mutations present at higher frequencies were concordantly identified using both methods. Conclusions/Significance We show that pyrosequencing pooled surveillance specimens can cost-competitively detect protease TDR mutations when compared with conventional methods. With few modifications, the method described here can be used to determine population rates of TDR in both protease and reverse transcriptase. Furthermore, this pooled pyrosequencing technique may be generalizable to other infectious agents where a survey of DR rates is required.

Next-generation sequencing of dried blood spot specimens: a novel approach to HIV drug-resistance surveillance.

BACKGROUND HIV drug-resistance (DR) surveillance in resource-limited settings can be performed using dried blood spots (DBS) because of ease of collection, transportation and storage. Analysis of pooled specimens on next-generation sequencing (NGS)-based platforms, such as the 454 pyrosequencing, is an efficient sequencing method for determining HIV DR rates. In this study, we conducted HIV DR surveillance on DBS using NGS and identified minority variants in individual patients. METHODS A total of 48 extracts of DBS from an HIV DR surveillance study in Mexico City were re-amplified using primers tagged with multiplex identifiers, pooled and pyrosequenced. Consensus sequences were generated for each specimen with mixtures identified at positions where >20% of the reads contained a variant. Individual consensus sequences were then analysed for DR mutations and compared with those derived from Sanger sequencing. RESULTS DBS analysed with tagged pooled pyrosequencing (TPP) were highly concordant with Sanger sequencing genotypes from matching plasma and DBS (99.21% and 99.51%, respectively). An exception was an M184I mutation only detected with TPP of DBS at a frequency of 20.4%. Multiple specimens had minority variant reads below the 20% mixture threshold. CONCLUSIONS TPP using DBS is an effective method for HIV DR surveillance. TPP for genotyping results in cost savings of 40% over conventional in-house methods. The effect of low-abundance DR mutations, undetectable by conventional methods, remains to be determined. This technology might be applied to any HIV specimen (plasma/serum) and can also be used for other diagnostic assays where DNA sequencing is required.

Surveillance of HIV drug resistance transmission in Iran: experience gained from a pilot study

We performed a pilot surveillance study on transmitted HIV drug resistance (TDR) in Iran, with specimens collected and stored as dried blood spots (DBS). The protease region and relevant positions in the reverse transcriptase region of the pol gene were sequenced to detect mutations known to be associated with resistance to drugs in standard first-line regimens. Seventy-three specimens were collected, with 39 (53%) specimens yielding sequence from both protease and at least part of RT. Specimens were almost exclusively HIV-1 subtype CRF 35_A1D based on pol sequencing. Mutations were restricted to RT, with D67DG and V75AV each seen in a single specimen. An atypical protease inhibitor mutation, I47M, appeared at a resistance-associated position in protease from a single specimen. These preliminary data showed that the rate of transmitted drug resistance in Iran, within the areas sampled, was 5.1% (2/39). However, the small sample size makes this figure only an approximation. Due to the sampling strategy and resulting small sample size, we were unable to accurately calculate TDR rates for individual areas using the WHO HIV drug resistance threshold survey method. Increasing the sample size and improving the yield from DBS would improve the accuracy of drug resistance surveillance and facilitate wider application of this methodology in Iran.

No evidence of PERV infection in healthcare workers exposed to transgenic porcine liver extracorporeal support.

Background:  Clinical xenotransplantation holds great promise by providing one solution to the shortage of human organs for transplantation, while also posing a potential public health threat by facilitating transmission of infectious disease from source animals to humans. One potential vector for infectious disease transmission is healthcare workers (HCW) who are involved in administering xenotransplantation procedures. Methods: In this study, we studied 49 healthcare workers involved in the care of two subjects who participated in a study of porcine liver perfusion as treatment of fulminant hepatic failure. We looked for serologic and virologic evidence of transmission of porcine endogenous retrovirus, and found that HCW had no evidence of infection. Conclusions: Results of our survey demonstrate that application of standard precautions may be sufficient to prevent transmission of porcine endogenous retrovirus, an agent of concern in ex vivo xenotransplantation products.