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

Towards targeted screening for acute HIV infections in British Columbia

<p>Abstract</p> <p>Background</p> <p>Our objective was to describe the characteristics of acute and established HIV infections diagnosed in the Canadian province of British Columbia. Province-wide HIV testing and surveillance data were analyzed to inform recommendations for targeted use of screening algorithms to detect acute HIV infections.</p> <p>Methods</p> <p>Acute HIV infection was defined as a confirmed reactive HIV p24 antigen test (or HIV nucleic acid test), a non-reactive or reactive HIV EIA screening test and a non-reactive or indeterminate Western Blot. Characteristics of unique individuals were identified from the British Columbia HIV/AIDS Surveillance System. Primary drug resistance and HIV subtypes were identified by analyzing HIV <it>pol </it>sequences from residual sera from newly infected individuals.</p> <p>Results</p> <p>From February 2006 to October 2008, 61 individuals met the acute HIV infection case definition, representing 6.2% of the 987 newly diagnosed HIV infections during the analysis period. Acute HIV infection cases were more likely to be men who have sex with men (crude OR 1.71; 95% CI 1.01-2.89], to have had a documented previous negative HIV test result (crude OR 2.89; 95% CI 1.52-5.51), and to have reported a reason for testing due to suspected seroconversion symptoms (crude OR 5.16; 95% CI 2.88-9.23). HIV subtypes and rates of transmitted drug resistance across all classes of drugs were similar in persons with both acute and established HIV infections.</p> <p>Conclusions</p> <p>Targeted screening to detect acute HIV infection is a logical public health response to the HIV epidemic. Our findings suggest that acute HIV infection screening strategies, in our setting, are helpful for early diagnosis in men who have sex with men, in persons with seroconversion symptoms and in previously negative repeat testers.</p

Intestinal parasites of wolves (Canis lupus L. 1758) in northern and western Canada

Gray wolves (Canis lupus L., 1758) are mobile opportunistic predators that can be infected by a wide range of parasites, with many acquired via predator-prey relationships. Historically, many of these parasites were identified only to genus or family, but genetic tools now enable identification of parasite fauna to species and beyond. We examined 191 intestines from wolves harvested for other purposes from regions in the Northwest Territories, British Columbia, Saskatchewan, and Manitoba. Adult helminths were collected from intestinal contents for morphological and molecular identification, and for a subset of wolves, fecal samples were also analyzed to detect helminth eggs and protozoan (oo)cysts. Using both detection methods, we found that 83% of 191 intestines contained one or more parasite species, including cestodes (Taenia spp., Echinococcus spp., and Diphyllobothrium sp.), nematodes (Uncinaria stenocephala, Trichuris spp., Physaloptera spp., and Toxascaris leonina), a trematode (Alaria sp.), and protozoa (Sarcocystis spp., Giardia spp., and Cryptosporidium spp.). Molecular characterization identified one species of Diphyllobothrium (D. latum), three species of Taenia (T. krabbei, T. hydatigena, and T. multiceps), and two Giardia assemblages (B and C). These results demonstrate the diverse diet of wolves, and illustrate the possibility of parasite spillover among wildlife, domestic animals, and people.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Adaptive Evolution of HIV at HLA Epitopes Is Associated with Ethnicity in Canada

<div><p>Host immune selection pressure influences the development of mutations that allow for HIV escape. Mutation patterns induced in HIV by the human leukocyte antigen (HLA) are HLA-allele specific. As ethnic groups have distinct and characteristic HLA allele frequencies, we can expect divergent viral evolution within ethnicities. Here, we have sequenced and analyzed the HIV <em>pol</em> gene from 1248 subtype B infected, treatment-naïve individuals in Canada. Phylogenetic analysis showed no separation between <em>pol</em> sequences from five self-identified ethnic groups, yet fixation index (<em>F_ST</em>) values showed significant divergence between ethnicities. A total of 17 amino acid sites showed an ethnic-specific fixation pattern (0.015<<em>F_ST</em> <0.060, <em>p</em><0.01), and 27 codons were inferred to be under positive selection (<em>p</em><0.01), with each set of sites strongly associated with HLA sites (<em>p</em> = 1.78×10⁻⁶ and <em>p</em> = 1.91×10⁻⁷, respectively). Within the <em>pol</em> gene, eight sites under HLA selective pressure were correlated with ethnicity, indicating ‘adaptive divergence’ between the groups studied. Our findings highlight challenges in HIV vaccine design in ethnically diverse countries with subtype B epidemics.</p> </div

Evolution of primary HIV drug resistance in a subtype C dominated epidemic in Mozambique.

In Mozambique, highly active antiretroviral treatment (HAART) was introduced in 2004 followed by decentralization and expansion, resulting in a more than 20-fold increase in coverage by 2009. Implementation of HIV drug resistance threshold surveys (HIVDR-TS) is crucial in order to monitor the emergence of transmitted viral resistance, and to produce evidence-based recommendations to support antiretroviral (ARV) policy in Mozambique.World Health Organization (WHO) methodology was used to evaluate transmitted drug resistance (TDR) in newly diagnosed HIV-1 infected pregnant women attending ante-natal clinics in Maputo and Beira to non-nucleoside reverse transcriptase inhibitors (NNRTI), nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI). Subtypes were assigned using REGA HIV-1 subtyping tool and phylogenetic trees constructed using MEGA version 5.Although mutations associated with resistance to all three drug were detected in these surveys, transmitted resistance was analyzed and classified as <5% in Maputo in both surveys for all three drug classes. Transmitted resistance to NNRTI in Beira in 2009 was classified between 5-15%, an increase from 2007 when no NNRTI mutations were found. All sequences clustered with subtype C.Our results show that the epidemic is dominated by subtype C, where the first-line option based on two NRTI and one NNRTI is still effective for treatment of HIV infection, but intermediate levels of TDR found in Beira reinforce the need for constant evaluation with continuing treatment expansion in Mozambique

Differences in strength of selective pressure.

<p>The left hand side of the table (below the diagonal) indicates sites found to differ in strength of selective pressure across the whole tree. The right hand side of the table (above the diagonal) indicates sites found to differ in strength of selective pressure only at internal branches. Analyses compared populations two by two and a level of significance α≤0.005 was necessary for significance. Ethnicities are denoted by two letter codes: Aboriginal (Ab), African-Caribbean (AC), Asian (As), Caucasian (Ca), and Latin-American (LA).</p

Phylogenetic subtree 2.

<p>The phylogenetic relationships between taxa in the subtree were reconstructed in BEAST for subsequent analysis in BaTS. For graphic visualization, a maximum clade credibility tree was generated in TreeAnnotator and annotated in FigTree. Taxa are colored by ethnicity: Aboriginal (Ab, green), African-Caribbean (AC, orange), Asian (As, pink), Caucasian (Ca, blue), Latin-American (LA, red).</p

Epidemiological characteristics of the population.

<p><b>MSM</b> men who have sex with men, <b>IDU</b> intravenous drug users, <b>HET</b> heterosexual.</p

Amino acid frequency distributions at sites PR93 and RT277.

<p>For each ethnic group, the frequencies of alternative amino acids are shown for two sites in protease (PR) and reverse transcriptase (RT). Amino acids represented are: leucine (L), isoleucine (I), glutamine (Q), arginine (R) and lysine (K). <i>F_ST</i> values at PR93 and RT277 are 0.06 and 0.0521, respectively. Notation of ethnicities is as follows: Aboriginal (Ab), African-Caribbean (AC), Asian (As), Caucasian (Ca), and Latin American (LA).</p

Overlap between sites of interest.

<p>Of a total of 413 amino acid sites, considerable overlap is observed between HLA-associated sites, sites under positive selection and sites divergent between ethnicities. Associations were tested using Fisher’s exact test. Note that all three tests take into account the 7 sites in the center of the diagram.</p