Clinical evaluation of the GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV PLUS combination test.

The GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV PLUS combination test (PLUS assay) received Health Canada approval in January 2022. The PLUS assay is similar to the SARS-CoV-2/Flu/RSV combination test, with modifications to improve assay robustness against circulating and emerging variants. The performance characteristics of the PLUS assay were assessed at the Lakeridge Health Oshawa Hospital Centre and the National Microbiology Laboratory of Canada. The PLUS assay was directly compared to the SARS-CoV-2/Flu/RSV combination test using SARS-CoV-2 culture from five variants and remnant clinical specimens collected across the coronavirus disease 2019 pandemic. This included 50 clinical specimens negative for all pathogens, 110 clinical specimens positive for SARS-CoV-2, influenza A, influenza B, RSVA, and(or) RSVB and an additional 11 mixed samples to screen for target interactions. The PLUS assay showed a high % agreement with the widely used SARS-CoV-2/Flu/RSV combination test. Based on these findings, the PLUS assay and the Xpert SARS-CoV-2/Flu/RSV combination test results are largely consistent with no observed difference in sensitivity, specificity, or time to result when challenged with various SARS-CoV-2 variants. The reported cycle threshold (Ct) values provided by the new PLUS assay were also unchanged, with the exception of a possible 1-2 decrease reported in Ct for RSVA across a limited sample size.

A Sensitive and Rapid Wastewater Test for SARS-COV-2 and Its Use for the Early Detection of a Cluster of Cases in a Remote Community.

Throughout the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has been used to monitor trends in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevalence in the community. A major challenge in establishing wastewater surveillance programs, especially in remote areas, is the need for a well-equipped laboratory for sample analysis. Currently, no options exist for rapid, sensitive, mobile, and easy-to-use wastewater tests for SARS-CoV-2. The performance of the GeneXpert system, which offers cartridge-based, rapid molecular clinical testing for SARS-CoV-2 in a portable platform, was evaluated using wastewater as the input. The GeneXpert demonstrated a SARS-CoV-2 limit of detection in wastewater below 32 copies/mL with a sample processing time of less than an hour. Using wastewater samples collected from multiple sites across Canada during February and March 2021, a high overall agreement (97.8%) was observed between the GeneXpert assay and laboratory-developed tests regarding the presence or absence of SARS-CoV-2. Additionally, with the use of centrifugal filters, the detection threshold of the GeneXpert system was improved to <10 copies/mL in wastewater. Finally, to support on-site wastewater surveillance, GeneXpert testing was implemented in Yellowknife, a remote community in Northern Canada, where its use successfully alerted public health authorities to undetected transmission of COVID-19. The identification of SARS-CoV-2 in wastewater triggered clinical testing of recent travelers and identification of new COVID-19 cases/clusters. Taken together, these results suggest that GeneXpert is a viable option for surveillance of SARS-CoV-2 in wastewater in locations that do not have access to established testing laboratories. IMPORTANCE Wastewater-based surveillance is a powerful tool that provides an unbiased measure of COVID-19 prevalence in a community. This work describes a sensitive wastewater rapid test for SARS-CoV-2 based on a widely distributed technology, the GeneXpert. The advantages of an easy-to-use wastewater test for SARS-CoV-2 are clear: it supports surveillance in remote communities, improves access to testing, and provides faster results allowing for an immediate public health response. The application of wastewater rapid testing in a remote community facilitated the detection of a COVID-19 cluster and triggered public health action, clearly demonstrating the utility of this technology. Wastewater surveillance will become increasingly important in the postvaccination pandemic landscape as individuals with asymptomatic/mild infections continue transmitting SARS-CoV-2 but are unlikely to be tested.

Characterization of the Drug Resistance Profiles of Integrase Strand Transfer Inhibitors in Simian Immunodeficiency Virus SIVmac239.

UNLABELLED: We previously showed that the simian immunodeficiency virus SIVmac239 is susceptible to human immunodeficiency virus (HIV) integrase (IN) strand transfer inhibitors (INSTIs) and that the same IN drug resistance mutations result in similar phenotypes in both viruses. Now we wished to determine whether tissue culture drug selection studies with SIV would yield the same resistance mutations as in HIV. Tissue culture selection experiments were performed using rhesus macaque peripheral blood mononuclear cells (PBMCs) infected with SIVmac239 viruses in the presence of increasing concentrations of dolutegravir (DTG), elvitegravir (EVG), and raltegravir (RAL). We now show that 22 weeks of selection pressure with DTG yielded a mutation at position R263K in SIV, similar to what has been observed in HIV, and that selections with EVG led to emergence of the E92Q substitution, which is a primary INSTI resistance mutation in HIV associated with EVG treatment failure. To study this at a biochemical level, purified recombinant SIVmac239 wild-type (WT) and E92Q, T97A, G118R, Y143R, Q148R, N155H, R263K, E92Q T97A, E92Q Y143R, R263K H51Y, and G140S Q148R recombinant substitution-containing IN enzymes were produced, and each of the characteristics strand transfer, 3'-processing activity, and INSTI inhibitory constants was assessed in cell-free assays. The results show that the G118R and G140S Q148R substitutions decreased Km' and Vmax'/Km' for strand transfer compared to those of the WT. RAL and EVG showed reduced activity against both viruses and against enzymes containing Q148R, E92Q Y143R, and G140S Q148R. Both viruses and enzymes containing Q148R and G140S Q148R showed moderate levels of resistance against DTG. This study further confirms that the same mutations associated with drug resistance in HIV display similar profiles in SIV. IMPORTANCE: Our goal was to definitively establish whether HIV and simian immunodeficiency virus (SIV) share similar resistance pathways under tissue culture drug selection pressure with integrase strand transfer inhibitors and to test the effect of HIV-1 integrase resistance-associated mutations on SIV integrase catalytic activity and resistance to integrase strand transfer inhibitors. Clinically relevant HIV integrase resistance-associated mutations were selected in SIV in our tissue culture experiments. Not only do we report on the characterization of SIV recombinant integrase enzyme catalytic activities, we also provide the first research anywhere on the effect of mutations within recombinant integrase SIV enzymes on drug resistance.

Simian-tropic HIV as a model to study drug resistance against integrase inhibitors.

Drug resistance represents a key aspect of human immunodeficiency virus (HIV) treatment failure. It is important to develop nonhuman primate models for studying issues of drug resistance and the persistence and transmission of drug-resistant viruses. However, relatively little work has been conducted using either simian immunodeficiency virus (SIV) or SIV/HIV recombinant viruses for studying resistance against integrase strand transfer inhibitors (INSTIs). Here, we used a T-cell-tropic SIV/HIV recombinant virus in which the capsid and vif regions of HIV-1 were replaced with their SIV counterparts (simian-tropic HIV-1 [stHIV-1](SCA,SVIF)) to study the impact of a number of drug resistance substitutions in the integrase coding region at positions E92Q, G118R, E138K, Y143R, S153Y, N155H, and R263K on drug resistance, viral infectivity, and viral replication capacity. Our results show that each of these substitutions exerted effects that were similar to their effects in HIV-1. Substitutions associated with primary resistance against dolutegravir were more detrimental to stHIV-1(SCA,SVIF) infectiousness than were resistance substitutions associated with raltegravir and elvitegravir, consistent with data that have been reported for HIV-1. These findings support the role of stHIV-1(SCA,SVIF) as a useful model with which to evaluate the role of INSTI resistance substitutions on viral persistence, transmissibility, and pathogenesis in a nonhuman primate model.

Effect of HIV-1 integrase resistance mutations when introduced into SIVmac239 on susceptibility to integrase strand transfer inhibitors.

UNLABELLED: Studies on the in vitro susceptibility of SIV to integrase strand transfer inhibitors (INSTIs) have been rare. In order to determine the susceptibility of SIVmac239 to INSTIs and characterize the genetic pathways that might lead to drug resistance, we inserted various integrase (IN) mutations that had been selected with HIV under drug pressure with raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) into the IN gene of SIV. We evaluated the effects of these mutations on SIV susceptibility to INSTIs and on viral infectivity. Sequence alignments of SIVmac239 IN with various HIV-1 isolates showed a high degree of homology and conservation of each of the catalytic triad and the key residues involved in drug resistance. Each of the G118R, Y143R, Q148R, R263K, and G140S/Q148R mutations, when introduced into SIV, impaired infectiousness and replication fitness compared to wild-type virus. Using TZM-bl cells, we demonstrated that the Q148R and N155H mutational pathways conferred resistance to EVG (36- and 62-fold, respectively), whereas R263K also displayed moderate resistance to EVG (12-fold). In contrast, Y143R, Q148R, and N155H all yielded low levels of resistance to RAL. The combination of G140S/Q148R conferred high-level resistance to both RAL and EVG (>300- and 286-fold, respectively). DTG remained fully effective against all site-directed mutants except G118R and R263K. Thus, HIV INSTI mutations, when inserted into SIV, resulted in a similar phenotype. These findings suggest that SIV and HIV may share similar resistance pathways profiles and that SIVmac239 could be a useful nonhuman primate model for studies of HIV resistance to INSTIs. IMPORTANCE: The goal of our project was to establish whether drug resistance against integrase inhibitors in SIV are likely to be the same as those responsible for drug resistance in HIV. Our data answer this question in the affirmative and show that SIV can probably serve as a good animal model for studies of INSTIs and as an early indicator for possible emergent mutations that may cause treatment failure. An SIV-primate model remains an invaluable tool for investigating questions related to the potential role of INSTIs in HIV therapy, transmission, and pathogenesis, and the present study will facilitate each of the above.

Translating knowledge from Pakistan's second generation surveillance system to other global contexts.

BACKGROUND: From 2004 to 2011, a collaborative project was undertaken to enhance the capacity of the Government of Pakistan to implement an effective second-generation surveillance system for HIV/AIDS, known as the HIV/AIDS Surveillance Project (HASP). In four separate rounds, behavioural questionnaires were administered among injection drug users, and female, male and hijra (transgender) sex workers. Dried blood spots were collected for HIV testing. METHODS: Through interviews with project staff in Pakistan and Canada, we have undertaken a critical review of the role of HASP in generating, using and translating knowledge, with an emphasis on capacity building within both the donor and recipient countries. We also documented ongoing and future opportunities for the translation of knowledge produced through HASP. RESULTS: Knowledge translation activities have included educational workshops and consultations held in places as diverse as Colombia and Cairo, and the implementation of HASP methodologies in Asia, the Middle East and sub-Saharan Africa. HASP methodologies have been incorporated in multiple WHO reports. Importantly, the donor country, Canada, has benefited in significant ways from this partnership. Operational and logistical lessons from HASP have, in turn, improved how surveillance is performed in Canada. Through this project, significant capacity was built among the staff of HASP, non-governmental organisations which were engaged as implementation partners, data coordination units which were established in each province, and in the laboratory. As is to be expected, different organisations have different agendas and priorities, requiring negotiation, at times, to ensure the success of collaborative activities. Overall, there has been considerable interest in and opportunities made for learning about the methodologies and approaches employed by HASP. CONCLUSIONS: Generally, the recognition of the strengths of the approaches and methodologies used by HASP has ensured an appetite for opportunities of mutual learning.

Gardnerella subgroup dominant microbiomes are associated with divergent cervicovaginal immune responses in a longitudinal cohort of Kenyan women.

Most cervicovaginal microbiome-immunology studies to date have relied on 16S rDNA microbial profiling which does not resolve the molecular subgroups of Gardnerella, believed to be central to the pathogenesis of bacterial vaginosis (BV) and subsequent risk of HIV acquisition. Here we used the cpn60 universal target which in addition to other microbial taxa, resolves four Gardnerella subgroups, for cervicovaginal microbial profiling in a longitudinal cohort of Kenyan women to examine associations with cellular and soluble markers of inflammation and HIV susceptibility. Participants (N = 41) were sampled, contributing 362 samples for microbiome analysis. All non-Lactobacillus dominant microbial communities were associated with high pro-inflammatory cytokine levels. Divergent associations were observed among different Gardnerella subgroup dominated communities with respect to the chemokine IP-10. Specifically, Gardnerella subgroup A dominant and polymicrobial communities were associated with reduced concentrations of IP-10 in adjusted linear mixed models (p<0.0001), compared to microbial communities dominated by Lactobacillus (non-iners) species. However, these associations did not translate to significant differences in the proportion or absolute number of CCR5, HLA-DR and CD38 expressed on cervical CD4+ T- cells. These findings suggest that some associations between Gardnerella subgroup dominant microbiomes and mucosal immunity differ and are relevant for the study of BV-pathogenesis and understanding the mechanisms of BV-associated HIV risk.

Clinical evaluation of the GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV combination test.

The Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV combination test received emergency use authorization approval by the United States Food and Drug Administration in December 2020, and Health Canada approval in January 2021. The performance characteristics of the GeneXpert Xpert Xpress SARS-CoV-2/Flu/RSV combination test were assessed at Lakeridge Health Oshawa and the National Microbiology Laboratory of Canada. The combination test was compared to the Xpert SARS-CoV-2 and Xpert Flu/RSV assays, and the BioFire FilmArray Respiratory Panel 2.1 (RP2.1) test kit. Materials evaluated were serial dilutions of chemically-inactivated SARS-CoV-2 and remnant clinical specimens (nasal or nasopharyngeal swabs) collected from patients. The limit of detection (LOD) for the SARS-CoV-2 component of the Xpert SARS-CoV-2/Flu/RSV combination test was determined to be <100 viral copies/mL when using chemically-inactivated SARS-CoV-2. In total, 86 clinical positive and 51 clinical negative samples were used for this study, with mixtures of clinical positives being used to mimic coinfection and screen for competitive inhibition. The combination test showed a high percent agreement with the Xpert SARS-CoV-2 and Xpert Flu/RSV tests, as well as the BioFire FilmArray RP2.1. Based on the findings from this study and a growing body of research, the Xpert SARS-CoV-2/Flu/RSV combination test will serve as an effective replacement for the Xpert SARS-CoV-2 and Xpert Flu/RSV assays.

Recommendations for sample pooling on the Cepheid GeneXpert® system using the Cepheid Xpert® Xpress SARS-CoV-2 assay.

The coronavirus disease 2019 (Covid-19) pandemic, caused by SARS-CoV-2, has resulted in a global testing supply shortage. In response, pooled testing has emerged as a promising strategy that can immediately increase testing capacity. In pooled sample testing, multiple samples are combined (or pooled) together and tested as a single unit. If the pool is positive, the individual samples can then be individually tested to identify the positive case(s). Here, we provide support for the adoption of sample pooling with the point-of-care Cepheid Xpert® Xpress SARS-CoV-2 molecular assay. Corroborating previous findings, the limit of detection of this assay was comparable to laboratory-developed reverse-transcription quantitative PCR SARS-CoV-2 tests, with observed detection below 100 copies/mL. The Xpert® Xpress assay detected SARS-CoV-2 after samples with minimum viral loads of 461 copies/mL were pooled in groups of six. Based on these data, we recommend the adoption of pooled testing with the Xpert® Xpress SARS-CoV-2 assay where warranted based on public health needs. The suggested number of samples per pool, or the pooling depth, is unique for each point-of-care testing site and can be determined by the positive test rates. To statistically determine appropriate pooling depth, we have calculated the pooling efficiency for numerous combinations of pool sizes and test rates. This information is included as a supplemental dataset that we encourage public health authorities to use as a guide to make recommendations that will maximize testing capacity and resource conservation.

Bayesian inference reveals ancient origin of simian foamy virus in orangutans.

Simian foamy viruses (SFVs) infect most nonhuman primate species and appears to co-evolve with its hosts. This co-evolutionary signal is particularly strong among great apes, including orangutans (genus Pongo). Previous studies have identified three distinct orangutan SFV clades. The first of these three clades is composed of SFV from P. abelii from Sumatra, the second consists of SFV from P. pygmaeus from Borneo, while the third clade is mixed, comprising an SFV strain found in both species of orangutan. The existence of the mixed clade has been attributed to an expansion of P. pygmaeus into Sumatra following the Mount Toba super-volcanic eruption about 73,000years ago. Divergence dating, however, has yet to be performed to establish a temporal association with the Toba eruption. Here, we use a Bayesian framework and a relaxed molecular clock model with fossil calibrations to test the Toba hypothesis and to gain a more complete understanding of the evolutionary history of orangutan SFV. As with previous studies, our results show a similar three-clade orangutan SFV phylogeny, along with strong statistical support for SFV-host co-evolution in orangutans. Using Bayesian inference, we date the origin of orangutan SFV to >4.7 million years ago (mya), while the mixed species clade dates to approximately 1.7mya, >1.6 million years older than the Toba super-eruption. These results, combined with fossil and paleogeographic evidence, suggest that the origin of SFV in Sumatran and Bornean orangutans, including the mixed species clade, likely occurred on the mainland of Indo-China during the Late Pliocene and Calabrian stage of the Pleistocene, respectively.

Detailed phylogenetic analysis of primate T-lymphotropic virus type 1 (PTLV-1) sequences from orangutans (Pongo pygmaeus) reveals new insights into the evolutionary history of PTLV-1 in Asia.

While human T-lymphotropic virus type 1 (HTLV-1) originates from ancient cross-species transmission of simian T-lymphotropic virus type 1 (STLV-1) from infected nonhuman primates, much debate exists on whether the first HTLV-1 occurred in Africa, or in Asia during early human evolution and migration. This topic is complicated by a lack of representative Asian STLV-1 to infer PTLV-1 evolutionary histories. In this study we obtained new STLV-1 LTR and tax sequences from a wild-born Bornean orangutan (Pongo pygmaeus) and performed detailed phylogenetic analyses using both maximum likelihood and Bayesian inference of available Asian PTLV-1 and African STLV-1 sequences. Phylogenies, divergence dates and nucleotide substitution rates were co-inferred and compared using six different molecular clock calibrations in a Bayesian framework, including both archaeological and/or nucleotide substitution rate calibrations. We then combined our molecular results with paleobiogeographical and ecological data to infer the most likely evolutionary history of PTLV-1. Based on the preferred models our analyses robustly inferred an Asian source for PTLV-1 with cross-species transmission of STLV-1 likely from a macaque (Macaca sp.) to an orangutan about 37.9-48.9kya, and to humans between 20.3-25.5kya. An orangutan diversification of STLV-1 commenced approximately 6.4-7.3kya. Our analyses also inferred that HTLV-1 was first introduced into Australia ~3.1-3.7kya, corresponding to both genetic and archaeological changes occurring in Australia at that time. Finally, HTLV-1 appears in Melanesia at ~2.3-2.7kya corresponding to the migration of the Lapita peoples into the region. Our results also provide an important future reference for calibrating information essential for PTLV evolutionary timescale inference. Longer sequence data, or full genomes from a greater representation of Asian primates, including gibbons, leaf monkeys, and Sumatran orangutans are needed to fully elucidate these evolutionary dates and relationships using the model criteria suggested herein.

A prospective study of 32 patients with neurosarcoidosis.

BACKGROUND AND AIM: There are few published prospective studies of neurosarcoidosis. To establish the incidence of neurological involvement and the response to treatment in patients presenting with sarcoidosis. METHODS: From 1991 to 1994, 123 patients were studied prospectively at the Prince Charles Hospital using a purpose designed computerised database (1). Consecutive patients were referred for neurological and psychiatric assessment when clinically indicated. Nerve conduction studies were done to confirm the presence of peripheral neuropathy. RESULTS: Neurological involvement was identified in 32/123 patients (15 male, 17 female, all white), mean age 48 years, age range 21-80 years. Of the 32 patients, the following frequencies of abnormalities were observed: papilloedema (6%), cranial neuropathy (59%), peripheral neuropathy (47%), mononeuropathy (25%), myopathy (25%), psychiatric disorders (19%), cerebellar ataxia (13%), and hydrocephalus (6%). A neurological improvement was seen in 16/19 (84%) of our patients as a result of therapy, and in 5/13 (38%) who were untreated. Corticosteroid treatment was used in 19/32 patients, with 6/32 requiring pulse intravenous methylprednisolone for initial poor response. The most predictable response incurred in patients with peripheral neuropathy; 12/14 treated patients responding. Only 1/8 patients who remained untreated for this, improved spontaneously. CONCLUSION: Neurological involvement was found more commonly than previously reported (26%). Corticosteroid treatment was found to be effective, although the response was often slow. High dose intravenous methylprednisolone was useful in poor responders (2). Peripheral neuropathy responded predictably to treatment. A formal neurological examination is recommended in all patients with sarcoidosis as neurological involvement may be overlooked.

Cognitive and language outcomes in HIV-uninfected infants exposed to combined antiretroviral therapy in utero and through extended breast-feeding.

OBJECTIVE: To determine whether there is a higher risk for cognitive or language delay among HIV-exposed uninfected (HEU) children exposed to cART (zidovudine/lamivudine/lopinavir/ritonavir) in utero and through 1 year of breast-feeding (World health Organization Option B+), compared with the control children born to HIV-uninfected mothers. DESIGN: This is a double cohort study from Lusaka, Zambia. METHODS: HEU (n = 97) and control (n = 103) children aged 15-36 months were assessed on their early nonverbal problem-solving and language skills using the standardized Capute Scales. A score of less than 85 on the Capute Full-Scale Developmental Quotient (FSDQ) was considered indicative of developmental delay and was the primary outcome of interest. RESULTS: An FSDQ of less than 85 was found in eight (8.3%) of HEU participants and 15 (14.6%) of controls. In univariate logistic regressions, lower income [odds ratio (OR) = 0.93, P = 0.02], older infant age (OR = 1.08, P = 0.03), lower birth weight (OR = 0.16, P < 0.001), and less maternal education (OR = 0.41, P = 0.047) were associated with the probability of FSDQ less than 85, whereas Group (control/HEU) was not (OR = 1.88, P = 0.16). In the multivariable analysis, only lower birth weight (OR = 0.15, P < 0.001) remained associated with FSDQ less than 85. CONCLUSIONS: Our study did not support the presence of an adverse effect on cognitive and language development with prolonged antepartum and postpartum cART e/xposure. Larger studies and studies of older HEU children will be required to confirm these reassuring findings.

The power and pitfalls of HIV phylogenetics in public health.

Phylogenetics is the application of comparative studies of genetic sequences in order to infer evolutionary relationships among organisms. This tool can be used as a form of molecular epidemiology to enhance traditional population-level communicable disease surveillance. Phylogenetic study has resulted in new paradigms being created in the field of communicable diseases and this commentary aims to provide the reader with an explanation of how phylogenetics can be used in tracking infectious diseases. Special emphasis will be placed upon the application of phylogenetics as a tool to help elucidate HIV transmission patterns and the limitations to these methods when applied to forensic analysis. Understanding infectious disease epidemiology in order to prevent new transmissions is the sine qua non of public health. However, with increasing epidemiological resolution, there may be an associated potential loss of privacy to the individual. It is within this context that we aim to promote the discussion on how to use phylogenetics to achieve important public health goals, while at the same time protecting the rights of the individual.

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.

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.

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.