Local phylogenetic analysis identifies distinct trends in transmitted HIV drug resistance: implications for public health interventions.

BACKGROUND: HIV transmitted drug resistance (TDR) surveillance is usually conducted by sampling from a large population. However, overall TDR prevalence results may be inaccurate for many individual clinical setting. We analyzed HIV genotypes at a tertiary care setting in Ottawa, Ontario in order to evaluate local TDR patterns among sub-populations. METHOD: Genotyping reports were digitized from ART naïve patients followed at the Immunodeficiency Clinic at the Ottawa Hospital, between 2008 and 2010. Quality controlled, digitized sequence data were assessed for TDR using the Stanford HIV Database. Patient characteristics were analyzed according to TDR patterns. Finally, a phylogenetic tree was constructed to elucidate the observed pattern of HIV TDR. RESULTS: Among the 155 clinic patients there was no statistically significantly difference in demographics as compared to the Ontario provincial HIV population. The clinic prevalence of TDR was 12.3%; however, in contrast to the data from Ontario, TDR patterns were inverted with a 21% prevalence among MSM and 5.5% among IDU. Furthermore, nearly 80% of the observed TDR was a D67N/K219Q pattern with 87% of these infections arising from a distinct phylogenetic cluster. CONCLUSIONS: Local patterns of TDR were distinct to what had been observed provincially. Phylogenetic analysis uncovered a cluster of related infections among MSM that appeared more likely to be recent infections. Results support a paradigm of routine local TDR surveillance to identify the sub-populations under care. Furthermore, the routine application of phylogenetic analysis in the TDR surveillance context provides insights into how best to target prevention strategies; and how to correctly measure outcomes.

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.

Foodborne protozoan parasites in fresh mussels and oysters purchased at retail in Canada.

Studies worldwide have reported the presence of protozoan parasites in a variety of commercial bivalve shellfish. The uptake of these parasites by shellfish occurs during filter feeding in faecally-contaminated waters. The objective of the present study was to determine the prevalence of Giardia, Cryptosporidium and Toxoplasma in fresh, live shellfish purchased in three Canadian provinces as part of the retail surveillance activities led by FoodNet Canada (Public Health Agency of Canada). Packages containing mussels (n = 253) or oysters (n = 130) were purchased at grocery stores in FoodNet Canada sentinel sites on a biweekly basis throughout 2018 and 2019, and shipped in coolers to Health Canada for testing. A small number of packages were not tested due to insufficient quantity or poor quality. Following DNA extraction from homogenized, pooled tissues, nested PCR and DNA sequencing were used to detect parasite-specific sequences. Epifluorescence microscopy was used to confirm the presence of intact cysts and oocysts in sequence-confirmed PCR-positive samples. Giardia duodenalis DNA was present in 2.4 % of 247 packages of mussels and 4.0 % of 125 packages of oysters, while Cryptosporidium parvum DNA was present in 5.3 % of 247 packages of mussels and 7.2 % of 125 packages of oysters. Toxoplasma gondii DNA was only found in mussels in 2018 (1.6 % of 249 packages). Parasite DNA was detected in shellfish purchased in all three Canadian provinces sampled, and there was no apparent seasonal variation in prevalence. While the present study did not test for viability, parasites are known to survive for long periods in the marine environment, and these findings suggest that there is a risk of infection, especially when shellfish are consumed raw.

No evidence of cross-species transmission of mouse retroviruses to animal workers exposed to mice.

BACKGROUND: Although recent data have brought into question the association between xenotropic murine leukemia virus-related virus (XMRV) and chronic fatigue syndrome, one group has reported evidence of human infection with distinct polytropic murine leukemia viruses (MLVs). Occult retroviral infection among humans poses a significant public health risk should it be introduced into the blood supply. To explore the possibility of cross-species transmission of MLVs to humans, we sought molecular and serologic evidence of XRMV/MLV infection among a cohort of animal workers highly exposed to mice. STUDY DESIGN AND METHODS: Before the commencement of the study, the laboratory and equipment were demonstrated to be free of XMRV/MLV DNA sequences. DNA extracted from 43 animal workers was tested using nested polymerase chain reaction (PCR) with published primer sets, targeting regions of XMRV and MLV gag. Negative controls were assayed in a 1:1 ratio with specimens. Serum specimens were tested using a validated immunoblot assay containing cross-reactive XMRV antigens. RESULTS: Initial molecular assays demonstrated that the physical space and laboratory equipment were free of MLV and XMRV DNA sequences. Nested PCR assays using multiple primer sets successfully amplified XMRV and MLV sequences from positive controls with high sensitivity. A single, nonreproducible, false-positive result from one specimen was shown to be the result of subsequent contamination. Immunoblotting of all subjects' sera failed to demonstrate any evidence of seroreactivity to XMRV proteins. CONCLUSIONS: There was no evidence of human infection with XMRV/MLV among a cohort of individuals highly exposed to mice. These data suggest that the likelihood of cross-species transmission events of MLV from mice to humans is low.

A cloth-based hybridization array system for rapid detection of the food- and waterborne protozoan parasites Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii.

Protozoan parasites in food or water samples are generally detected using microscopy or PCR followed by Sanger sequencing. However, microscopy is subjective, requires a high degree of expertise and has limited sensitivity, while DNA sequencing requires expensive and specialized equipment and facilities. This study describes a cloth-based hybridization array system (CHAS) that is an alternative to Sanger sequencing to confirm PCR-positive samples. CHAS is an inexpensive, rapid and reliable method for the simultaneous detection of multiple protozoan parasite species based on the colorimetric detection of PCR amplicons on a polyester cloth. PCR primers and CHAS hybridization probes were developed to detect the protozoan parasites Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii. In addition, CHAS probes were designed for the differentiation of G. duodenalis Assemblages A and B. In artificially contaminated fresh produce (lettuce, parsley) and water samples (river water, wastewater), this CHAS assay allowed for the successful detection of G. duodenalis, Cryptosporidium spp., and T. gondii. The present study demonstrates that the CHAS detection method is a simple and inexpensive alternative to DNA sequencing for the confirmation of PCR-positive results in laboratories testing for parasites in food or water samples. This assay may also be beneficial in developing countries, where DNA sequencing facilities may not be readily available.

Toxoplasma gondii, Sarcocystis sp. and Neospora caninum-like parasites in seals from northern and eastern Canada: potential risk to consumers.

Zoonotic parasites of seals that are harvested for food may pose a health risk when seal meat or organ tissues of infected animals are eaten raw or undercooked. In this study, 124 tissue samples from 81 seals, comprising four species, were collected from northern and eastern Canada. Tissues from 23 ringed seals (Pusa hispida), 8 hooded seals (Cystophora cristata), 21 harp seals (Pagophilus groenlandicus), and 29 grey seals (Halichoerus grypus) were tested for parasites of the Sarcocystidae family including Toxoplasma gondii, Sarcocystis spp., and Neospora spp. using nested PCR followed by Sanger sequencing. Toxoplasma gondii DNA was present in 26% of ringed seals, 63% of hooded seals, 57% of harp seals, and 31% of grey seals. Sarcocystis sp. DNA was found in 9% of ringed seals, 13% of hooded seals, 14% of harp seals, and 4% of grey seals, while N. caninum-like DNA was present in 26% of ringed seals. While it is unclear how pinnipeds may become infected with these protozoans, horizontal transmission is most likely. However, one harp seal pup (4 days old) was PCR-positive for T. gondii, suggesting vertical transmission may also occur. Phylogenetic analysis of the 18S gene region indicates that Sarcocystis sp. in these seals belongs to a unique genotype. Furthermore, this study represents a new host report for T. gondii in harp seals, a new host and geographic report for N. caninum-like parasites in ringed seals, and four new hosts and geographic reports for Sarcocystis sp. These results demonstrate that parasites of the Sarcocystidae family are prevalent in northern and eastern Canadian seals. While the zoonotic potential of Sarcocystis sp. and the N. caninum-like parasite are unclear, consumption of raw or undercooked seal meat or organ tissues pose a risk of T. gondii infection to consumers.

Low abundance drug resistance variants in transmitted HIV drug resistance surveillance specimens identified using tagged pooled pyrosequencing.

HIV drug resistance (DR) testing using Sanger sequencing (SS) is limited by the inability of the method to identify low abundance drug resistance variants. The application of tagged pooled pyrosequencing (TPP) for HIV DR surveillance is described and the results compared with SS. HIV(+) serum specimens were genotyped using both SS and TPP. Surveillance drug resistance mutations were identified using SS and TPP consensus reads at multiple mixed base identification thresholds (MBITs). Drug resistance patterns were highly concordant between SS and TPP when the MBIT was set at 20%. DR mutations were detected in 7.1% of the subjects, with 1.6% of individuals harboring resistance to NRTI, 3.3% NNRTI and 2.7% PI. Analyzing the TPP reads for each subject confirmed that drug resistance mutations with frequencies <20% were inconsistently detected by SS. Conversely, low abundance drug resistant variants were easily identified using TPP with mixed base identification threshold set at low value. In conclusion, at considerable savings when compared to commercial assays, TPP produces HIV DR profiles that are concordant with those from SS, furthermore, these same data can be used to identify low abundance drug resistant variants.

Baseline clinical HIV genotypes are a valid measure of transmitted drug resistance within the treatment-naive population.

OBJECTIVE: To examine whether baseline clinical genotypes are equivalent to diagnostic serum genotypes for surveillance of HIV transmitted drug resistance (TDR). DESIGN: Current HIV TDR surveillance in Canada is conducted through genotyping remnant diagnostic sera from new HIV diagnoses. As part of routine care, baseline genotyping is now conducted on all newly diagnosed HIV infections, with TDR data being generated a second time on the same patients. METHODS: Surveillance genotyping, on HIV diagnostic serum, was performed on newly diagnosed HIV cases from 2007 to 2010 in Alberta, Canada. All subjects with a baseline clinical genotype result on file, and no evidence of antiretroviral therapy, were studied further. The HIV sequences from diagnosis and from the first clinical genotype were compared according to elapsed time between testing and by evaluating timing of infection based on BED capture enzyme immunoassay (BED-CEIA, abbreviated as BED in this article). RESULTS: Eighty-seven genotype pairs were available for analysis, most of which were subtype B. The time between genotypes ranged from 0 to 755 days, with a median of 36 days and an interquartile range of 155.25 days. Genetic distance between genotypes varied between 0 and 0.03389 substitutions per site and did not correlate with sampling times. There was a tendency for the genotypes of infections classified as recent by BED to be more similar to their clinical genotypes but this effect was lost when adjusted for elapsed time between tests. There was no difference in the identified drug resistance. CONCLUSIONS: Baseline clinical genotypes from treatment-naive patients may be used for HIV TDR surveillance.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Adaptive evolution of HIV at HLA epitopes is associated with ethnicity in Canada.

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 pol gene from 1248 subtype B infected, treatment-naïve individuals in Canada. Phylogenetic analysis showed no separation between pol sequences from five self-identified ethnic groups, yet fixation index (F(ST)) values showed significant divergence between ethnicities. A total of 17 amino acid sites showed an ethnic-specific fixation pattern (0.015

Towards targeted screening for acute HIV infections in British Columbia.

BACKGROUND: 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. METHODS: 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 pol sequences from residual sera from newly infected individuals. RESULTS: 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. CONCLUSIONS: 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.

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.

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.

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. STUDY DESIGN AND METHODS: Within a controlled environment, blood from an SFV-infected monkey was transfused into an SFV-uninfected monkey. Evidence of infection, pathogenic effects, immune correlates, and viral shedding were followed for 6 months after transfusion. RESULTS: Molecular evidence of SFV infection manifested 8 weeks after transfusion followed by seroconversion 1 week later. Quantitative analysis demonstrated that the highest level of detectable virus was concomitant with seroconversion followed by establishment of a viral "set-point." Analysis of circulating lymphocytes revealed changes early in infection. Potential routes of transmission of SFV and roles of site-specific immune response are suggested by the late appearance of SFV shedding in the saliva of the transfused animal. CONCLUSION: The blood supply has historically provided a portal through which novel, occult viruses can become disseminated among humans. The demonstration of transmissibility of SFV through whole-blood transfusion, in an NHP model, contributes to the understanding of potential risks associated with blood donation by SFV-infected humans.

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.