A pathologist-AI collaboration framework for enhancing diagnostic accuracies and efficiencies.

In pathology, the deployment of artificial intelligence (AI) in clinical settings is constrained by limitations in data collection and in model transparency and interpretability. Here we describe a digital pathology framework, nuclei.io, that incorporates active learning and human-in-the-loop real-time feedback for the rapid creation of diverse datasets and models. We validate the effectiveness of the framework via two crossover user studies that leveraged collaboration between the AI and the pathologist, including the identification of plasma cells in endometrial biopsies and the detection of colorectal cancer metastasis in lymph nodes. In both studies, nuclei.io yielded considerable diagnostic performance improvements. Collaboration between clinicians and AI will aid digital pathology by enhancing accuracies and efficiencies.

Molecular classification of metastatic and recurrent endometrial endometrioid carcinoma: prognostic relevance among low- and high-stage tumours.

AIMS: Molecular classification according to The Cancer Genome Atlas (TCGA) improves endometrial endometrioid carcinoma (EEC) prognostication and has specific treatment implications; however, original data were skewed towards low-grade and low-stage tumours. Herein, we molecularly classify EECs metastatic at the time of diagnosis or with subsequently documented recurrent/metastatic disease to examine correlation with clinical outcomes. METHODS: TCGA categories include POLE-mutated, microsatellite instability (MSI), p53 abnormal (p53 abnl) and no specific molecular profile (NSMP). POLE targeted sequencing at exons 9, 11, 13 and 14 and immunohistochemistry (IHC) for PMS2, MSH6 and p53 were performed to establish molecular classification. RESULTS: The distribution in our cohort of 141 EECs was similar to that generally reported in EEC, with nine POLE-mutated (6%), 45 MSI (32%), 16 p53 abnl (11%) and 71 NSMP (50%), with similar distributions between low- and high-stage cohorts. We demonstrate that when stratified by molecular subtype, disease-specific survival from the time of high-stage (stages III-IV) presentation or time of recurrence in low-stage (stages I-II) disease among metastatic and/or recurrent EEC is strongly associated with TCGA classification (high-stage P = 0.02, low-stage P = 0.017). Discordant molecular classification between primary and metastatic/recurrent tumours occurred in four of 105 (3.8%) patients, two related to PMS2/MSH6 IHC and two related to p53 IHC. CONCLUSIONS: We demonstrate that molecular classification is prognostically relevant not only at the time of diagnosis, but also at the time of recurrence and in the metastatic setting. Rare subclonal alterations occur and suggest a role for confirming TCGA classification in recurrent/metastatic tumours.

Molecular Classification Outperforms Histologic Classification in Prognostication of High-grade Endometrial Carcinomas With Undifferentiated and Sarcomatous Components.

Since the establishment of 4 molecular subgroups of endometrial carcinoma (EC), there has been significant interest in understanding molecular classification in the context of histologic features and diagnoses. ECs with undifferentiated, spindle, and/or sarcomatous components represent a diagnostically challenging subset of tumors with overlapping clinical and histologic features. We examined the clinicopathologic, morphologic, immunohistochemical, and molecular features of these tumors identified in our institutions' pathology databases using immunohistochemistry and targeted sequencing. Disease-specific survival (DSS) and progression-free survival (PFS) were analyzed using Kaplan-Meier curves and log-rank tests. One hundred sixty-two ECs were included: carcinosarcomas (UCS; n=96), dedifferentiated/undifferentiated EC (DDEC/UDEC; n=49), and grade 3 endometrioid EC with spindled growth (GR3spEEC) (n=17). All molecular subgroups were represented in all histologic subtypes and included 12 (7%) POLE -mutated ( POLE mut), 43 (27%) mismatch repair-deficient (MMRd), 77 (48%) p53-abnormal (p53abn), and 30 (19%) no specific molecular profile (NSMP) tumors. However, the molecular classification (irrespective of histologic diagnosis) was a significant predictor for both DSS ( P =0.008) and P≤0.0001). POLE mut EC showed an excellent prognosis with no recurrences or deaths from the disease. MMRd tumors also showed better outcomes relative to NSMP and p53abn tumors. In conclusion, molecular classification provides better prognostic information than histologic diagnosis for high-grade EC with undifferentiated and sarcomatous components. Our study strongly supports routine molecular classification of these tumors, with emphasis on molecular group, rather than histologic subtyping, in providing prognostication.

The Evolving Spectrum of Endometrial Glandular Proliferations With Corded and Hyalinized Features.

We present the clinicopathologic and immunohistochemical features of 14 endometrial glandular proliferations with conspicuous corded and hyalinized (CH) features comprised entirely or predominantly of endometrial hyperplasia. Endometrial glandular lesions ranged in severity from endometrial hyperplasia with and without cytologic atypia (5/14 [36%]) to hyperplasia with architectural complexity bordering on well-differentiated endometrioid adenocarcinoma (3/14 [21%]) to frank corded and hyalinized endometrial carcinoma ("CHEC") (6/14 [43%]). In addition to sex cord-like growth and hyalinized stroma, other common histologic features included prominent spindle cells (11/14 [79%]), keratinizing and/or morular squamous differentiation (10/14 [71%]), and osseous metaplasia (6/14 [43%]). Immunohistochemical characterization revealed aberrant nuclear beta-catenin in all cases (14/14 [100%]); additionally, all cases demonstrated positive estrogen receptors, intact PTEN, PMS2 and MSH6, and wild-type p53 expression. Patients ranged in age from 24 to 58 (mean 38) years. Of 5 patients with hyperplasia with CH features, 2 experienced complete resolution after progestin therapy and none progressed to adenocarcinoma (mean follow-up 15.6 mo, range 2 to 64). By contrast, of 2 patients with hyperplasia bordering on CHEC and with available follow-up, both subsequently developed adenocarcinoma, suggesting that even focal increased architectural complexity may predict an elevated risk of malignancy. We conclude that CH morphology is not limited to endometrioid carcinoma and may occur across a spectrum of neoplastic proliferations, including those without sufficient architectural complexity or cytologic atypia to warrant classification as adenocarcinoma. We propose the term "corded and hyalinized endometrial hyperplasia" to describe this precursor lesion and report favorable outcomes with conservative treatment.

High-speed reflectance confocal microscopy of human skin at 1251-1342 nm.

OBJECTIVES: Reflectance confocal microscopy (RCM) is an imaging method that can noninvasively visualize microscopic features of the human skin. The utility of RCM can be further improved by increasing imaging speed. In this paper, we report high-speed RCM imaging of human skin with a frame rate that is over 10 times faster and an area imaging rate that is 6-9 times faster than those of commercially available RCM devices. METHODS: The higher imaging speed was achieved using a high-speed RCM technique, termed spectrally encoded confocal microscopy (SECM). SECM uses a diffraction grating and a high-speed, wavelength-swept source to conduct confocal imaging at a very high rate. We developed a handheld SECM probe using a scanned-grating approach. The SECM probe was used in conjunction with a wavelength-swept source with a spectral band of 1251-1342 nm. RESULTS: The SECM probe achieved high lateral resolution of 1.3-1.6 µm and an axial resolution of 3.5 µm. SECM images of the human skin (image size = 439 × 439 µm2 ) obtained at 100 frames/s clearly show previously reported RCM features of the human skin in vivo with adequate image quality. The fast imaging speed allowed for the rapid acquisiton of volumetric SECM image data (200 frames covering a depth range of 200 µm) within 2 s. The use of 1251-1342 nm provided sufficient signal level and contrast required to visualize key cellular morphologic features. CONCLUSIONS: These preliminary results demonstrate that high-speed SECM imaging of the human skin at 1251-1342 nm is feasible.

The menstrual cycle phase impacts the detection of plasma cells and the diagnosis of chronic endometritis in endometrial biopsy specimens.

OBJECTIVE: To assess the impact of menstrual cycle phase on the detection of plasma cells. DESIGN: A retrospective cohort study. SETTING: Fertility clinic. PATIENT(S): Biopsies from 157 patients met criteria for inclusion, 91 in the follicular phase and 60 in the luteal phase. Patient groups were similar in body mass index and number of previous live births; however, differed in terms of age, infertility history, and biopsy indication. INTERVENTIONS: Endometrial biopsies from patients at a fertility clinic from 2018-2020 were retrospectively reviewed. Biopsies were excluded if patients had a previous chronic endometritis diagnosis, abnormal uterine cavity or were on hormone therapy. Each case was reviewed by a gynecologic pathologist for plasma cells by hematoxylin and eosin and CD138 staining. Demographic and clinical data were collected. Continuous variables were compared using Welch t test and Wilcoxon's rank sum test, and categorical variables using Pearson's χ2 test. Logistic regression was used to calculate odds ratio and 95% confidence intervals for the association between the presence of plasma cells and cycle phase. Multinomial logistic regression was used to estimate the odds ratios for nominal outcomes. Pathology reports were reviewed. Plasma cell enumeration using hematoxylin and eosin-stained sections and CD138 immunohistochemical stains (performed at the time of biopsy by a gynecologic pathologist) was recorded. MAIN OUTCOME MEASURE(S): Presence and density of plasma cells. RESULT(S): We found a higher likelihood of finding plasma cells in the follicular than in luteal phase (59.3% vs. 19.7%). There was a higher likelihood of finding plasma cells in the early (cycle days 5-8, 29 cases or 76.3% of cases with plasma cells) than in the late follicular phase (cycle days 9-14, 25 cases or 47.2%). There was a higher density of plasma cells in the follicular phase group than in the luteal phase group (25.3% vs. 1.5% scattered and 13.2% vs. 0 clusters). CONCLUSION(S): Plasma cells are more likely to be present during the follicular phase compared with the luteal phase and in the early compared with the late follicular phase. Further studies are needed to identify the optimal timing of biopsy to standardize the diagnosis.

Passively scanned, single-fiber optical coherence tomography probes for gastrointestinal devices.

BACKGROUND/OBJECTIVES: Optical coherence tomography (OCT) uses low coherence interferometry to obtain depth-resolved tissue reflectivity profiles (M-mode) and transverse beam scanning to create images of two-dimensional tissue morphology (B-mode). Endoscopic OCT imaging probes typically employ proximal or distal mechanical beam scanning mechanisms that increase cost, complexity, and size. Here, we demonstrate in the gastrointestinal (GI) tracts of unsedated human patients, that a passive, single-fiber probe can be used to guide device placement, conduct device-tissue physical contact sensing, and obtain two-dimensional OCT images via M-to-B-mode conversion. MATERIALS AND METHODS: We designed and developed ultrasmall, manually scannable, side- and forward-viewing single fiber-optic probes that can capture M-mode OCT data. Side-viewing M-mode OCT probes were incorporated into brush biopsy devices designed to harvest the microbiome and forward-viewing M-mode OCT probes were integrated into devices that measure intestinal potential difference (IPD). The M-mode OCT probe-coupled devices were utilized in the GI tract in six unsedated patients in vivo. M-mode data were converted into B-mode images using an M-to-B-mode conversion algorithm. The effectiveness of physical contact sensing by the M-mode OCT probes was assessed by comparing the variances of the IPD values when the probe was in physical contact with the tissue versus when it was not. The capacity of forward- and side-viewing M-mode OCT probes to produce high-quality B-mode images was compared by computing the percentages of the M-to-B-mode images that showed close contact between the probe and the luminal surface. Passively scanned M-to-B-mode images were qualitatively compared to B-mode images obtained by mechanical scanning OCT tethered capsule endomicroscopy (TCE) imaging devices. RESULTS: The incorporation of M-mode OCT probes in these nonendoscopic GI devices safely and effectively enabled M-mode OCT imaging, facilitating real-time device placement guidance and contact sensing in vivo. Results showed that M-mode OCT contact sensing improved the variance of IPD measurements threefold and side-viewing probes increased M-to-B-mode image visibility by 10%. Images of the esophagus, stomach, and duodenum generated by the passively scanned probes and M-to-B-mode conversion were qualitatively superior to B-mode images obtained by mechanically scanning OCT TCE devices. CONCLUSION: These results show that passive, single optical fiber OCT probes can be effectively utilized for nonendoscopic device placement guidance, device contact sensing, and two-dimensional morphologic imaging in the human GI tract in vivo. Due to their small size, lower cost, and reduced complexity, these M-mode OCT probes may provide an easier avenue for the incorporation of OCT functionality into endoscopic/nonendoscopic devices.

Feasibility and Safety of Tethered Capsule Endomicroscopy in Patients With Barrett's Esophagus in a Multi-Center Study.

BACKGROUND & AIMS: Tethered capsule endomicroscopy (TCE) involves swallowing a small tethered pill that implements optical coherence tomography (OCT) imaging, procuring high resolution images of the whole esophagus. Here, we demonstrate and evaluate the feasibility and safety of TCE and a portable OCT imaging system in patients with Barrett's esophagus (BE) in a multi-center (5-site) clinical study. METHODS: Untreated patients with BE as per endoscopic biopsy diagnosis were eligible to participate in the study. TCE procedures were performed in unsedated patients by either doctors or nurses. After the capsule was swallowed, the device continuously obtained 10-µm-resolution cross-sectional images as it traversed the esophagus. Following imaging, the device was withdrawn through mouth, and disinfected for subsequent reuse. BE lengths were compared to endoscopy findings when available. OCT-TCE images were compared to volumetric laser endomicroscopy (VLE) images from a patient who had undergone VLE on the same day as TCE. RESULTS: 147 patients with BE were enrolled across all sites. 116 swallowed the capsule (79%), 95/114 (83.3%) men and 21/33 (63.6%) women (P = .01). High-quality OCT images were obtained in 104/111 swallowers (93.7%) who completed the procedure. The average imaging duration was 5.55 ± 1.92 minutes. The mean length of esophagus imaged per patient was 21.69 ± 5.90 cm. A blinded comparison of maximum extent of BE measured by OCT-TCE and EGD showed a strong correlation (r = 0.77-0.79). OCT-TCE images were of similar quality to those obtained by OCT-VLE. CONCLUSIONS: The capabilities of TCE to be used across multiple sites, be administered to unsedated patients by either physicians or nurses who are not expert in OCT-TCE, and to rapidly and safely evaluate the microscopic structure of the esophagus make it an emerging tool for screening and surveillance of BE patients. Clinical trial registry website and trial number: NCT02994693 and NCT03459339.

Antimicrobial and degradable triazolinedione (TAD) crosslinked polypeptide hydrogels.

Hydrogels are perfectly suited to support cell and tissue growth in advanced tissue engineering applications as well as classical wound treatment scenarios. Ideal hydrogel materials for these applications should be easy to produce, biocompatible, resorbable and antimicrobial. Here we report the fabrication of degradable covalent antimicrobial lysine and tryptophan containing copolypeptide hydrogels, whereby the hydrogel properties can be independently modulated by the copolypeptide monomer ratio and chiral composition. Well-defined statistical copolypeptides comprising different overall molecular weights as well as ratios of l- and d-lysine and tryptophan at ratios of 35 : 15, 70 : 30 and 80 : 20 were obtained by N-carboxyanhydride (NCA) polymerisation and subsequently crosslinked by the selective reaction of bifunctional triazolinedione (TAD) with tryptophan. Real-time rheology was used to monitor the crosslinking reaction recording the fastest increase and overall modulus for copolypeptides with the higher tryptophan ratio. Water uptake of cylindrical hydrogel samples was dependent on crosslinking ratio but found independent of chiral composition, while enzymatic degradation proceeded significantly faster for samples containing more l-amino acids. Antimicrobial activity on a range of hydrogels containing different polypeptide chain lengths, lysine/tryptophan composition and l/d enantiomers was tested against reference laboratory strains of Gram-negative Escherichia coli (E. coli; ATCC25922) and Gram-positive, Staphylococcus aureus (S. aureus; ATCC25923). log reductions of 2.8-3.4 were recorded for the most potent hydrogels. In vitro leachable cytotoxicity tests confirmed non-cytotoxicity as per ISO guidelines.

Virologic and Immunologic Features of Simian Immunodeficiency Virus Control Post-ART Interruption in Rhesus Macaques.

Antiretroviral therapy (ART) cannot eradicate human immunodeficiency virus (HIV) and a rapid rebound of virus replication follows analytical treatment interruption (ATI) in the vast majority of HIV-infected individuals. Sustained control of HIV replication without ART has been documented in a subset of individuals, defined as posttreatment controllers (PTCs). The key determinants of post-ART viral control remain largely unclear. Here, we identified 7 SIVmac239-infected rhesus macaques (RMs), defined as PTCs, who started ART 8 weeks postinfection, continued ART for >7 months, and controlled plasma viremia at <104 copies/ml for up to 8 months after ATI and <200 copies/ml at the latest time point. We characterized immunologic and virologic features associated with post-ART SIV control in blood, lymph node (LN), and colorectal (RB) biopsy samples compared to 15 noncontroller (NC) RMs. Before ART initiation, PTCs had higher CD4 T cell counts, lower plasma viremia, and SIV-DNA content in blood and LN compared to NCs, but had similar CD8 T cell function. While levels of intestinal CD4 T cells were similar, PTCs had higher frequencies of Th17 cells. On ART, PTCs had significantly lower levels of residual plasma viremia and SIV-DNA content in blood and tissues. After ATI, SIV-DNA content rapidly increased in NCs, while it remained stable or even decreased in PTCs. Finally, PTCs showed immunologic benefits of viral control after ATI, including higher CD4 T cell levels and reduced immune activation. Overall, lower plasma viremia, reduced cell-associated SIV-DNA, and preserved Th17 homeostasis, including at pre-ART, are the main features associated with sustained viral control after ATI in SIV-infected RMs.IMPORTANCE While effective, antiretroviral therapy is not a cure for HIV infection. Therefore, there is great interest in achieving viral remission in the absence of antiretroviral therapy. Posttreatment controllers represent a small subset of individuals who are able to control HIV after cessation of antiretroviral therapy, but characteristics associated with these individuals have been largely limited to peripheral blood analysis. Here, we identified 7 SIV-infected rhesus macaques that mirrored the human posttreatment controller phenotype and performed immunologic and virologic analysis of blood, lymph node, and colorectal biopsy samples to further understand the characteristics that distinguish them from noncontrollers. Lower viral burden and preservation of immune homeostasis, including intestinal Th17 cells, both before and after ART, were shown to be two major factors associated with the ability to achieve posttreatment control. Overall, these results move the field further toward understanding of important characteristics of viral control in the absence of antiretroviral therapy.

Collagen scaffolds functionalised with copper-eluting bioactive glass reduce infection and enhance osteogenesis and angiogenesis both in vitro and in vivo.

The bone infection osteomyelitis (typically by Staphylococcus aureus) usually requires a multistep procedure of surgical debridement, long-term systemic high-dose antibiotics, and - for larger defects - bone grafting. This, combined with the alarming rise in antibiotic resistance, necessitates development of alternative approaches. Herein, we describe a one-step treatment for osteomyelitis that combines local, controlled release of non-antibiotic antibacterials with a regenerative collagen-based scaffold. To maximise efficacy, we utilised bioactive glass, an established osteoconductive material with immense capacity for bone repair, as a delivery platform for copper ions (proven antibacterial, angiogenic, and osteogenic properties). Multifunctional collagen-copper-doped bioactive glass scaffolds (CuBG-CS) were fabricated with favourable microarchitectural and mechanical properties (up to 1.9-fold increase in compressive modulus over CS) within the ideal range for bone tissue engineering. Scaffolds demonstrated antibacterial activity against Staphylococcus aureus (up to 66% inhibition) whilst also enhancing osteogenesis (up to 3.6-fold increase in calcium deposition) and angiogenesis in vitro. Most significantly, when assessed in a chick embryo in vivo model, CuBG-CS not only demonstrated biocompatibility, but also a significant angiogenic and osteogenic response, consistent with in vitro studies. Collectively, these results indicate that the CuBG-CS developed here show potential as a one-step osteomyelitis treatment: reducing infection, whilst enhancing bone healing.

Impact of Genetic Variability on Physiological Responses to Caffeine in Humans: A Systematic Review.

Emerging research has demonstrated that genetic variation may impact physiological responses to caffeine consumption. The purpose of the present review was to systematically recognize how select single nucleotide polymorphisms (SNPs) impact habitual use of caffeine as well as the ergogenic and anxiogenic consequences of caffeine. Two databases (PubMed and EBSCO) were independently searched using the same algorithm. Selected studies involved human participants and met at least one of the following inclusion criteria: (a) genetic analysis of individuals who habitually consume caffeine; (b) genetic analysis of individuals who underwent measurements of physical performance with the consumption of caffeine; (c) genetic analysis of individuals who underwent measurements of mood with the consumption of caffeine. We included 26 studies (10 randomized controlled trials, five controlled trials, seven cross-sectional studies, three single-group interventional studies and one case-control study). Single nucleotide polymorphisms in or near the cytochrome P450 (CYP1A2) and aryl hydrocarbon receptor (AHR) genes were consistently associated with caffeine consumption. Several studies demonstrated that the anxiogenic consequences of caffeine differed across adenosine 2a receptor (ADORA2A) genotypes, and the studies that investigated the effects of genetic variation on the ergogenic benefit of caffeine reported equivocal findings (CYP1A2) or warrant replication (ADORA2A).

CD4 depletion in SIV-infected macaques results in macrophage and microglia infection with rapid turnover of infected cells.

In rhesus macaques (RMs), experimental depletion of CD4+ T-cells prior to SIV infection results in higher viremia and emergence of CD4-independent SIV-envelopes. In this study we used the rhesus recombinant anti-CD4 antibody CD4R1 to deplete RM CD4+ T-cells prior to SIVmac251 infection and investigate the sources of the increased viral burden and the lifespan of productively infected cells. CD4-depleted animals showed (i) set-point viral load two-logs higher than controls; (ii) macrophages constituting 80% of all SIV vRNA+ cells in lymph node and mucosal tissues; (iii) substantial expansion of pro-inflammatory monocytes; (iv) aberrant activation and infection of microglial cells; and (v) lifespan of productively infected cells significantly longer in comparison to controls, but markedly shorter than previously estimated for macrophages. The net effect of CD4+ T-cell depletion is an inability to control SIV replication and a shift in the tropism of infected cells to macrophages, microglia, and, potentially, other CD4-low cells which all appear to have a shortened in vivo lifespan. We believe these findings have important implications for HIV eradication studies.

Manganese-based superoxide dismutase mimics modify both acute and long-term outcome severity in a Drosophila melanogaster model of classic galactosemia.

AIMS: The goal of this study was to use two manganese (Mn)-based superoxide dismutase (SOD) mimics to test the hypothesis that reactive oxygen species contribute to both acute and long-term outcomes in a galactose-1P uridylyltransferase (GALT)-null Drosophila melanogaster model of classic galactosemia. RESULTS: We tested the impact of each of two Mn porphyrin SOD mimics, MnTnBuOE-2-PyP(5+), and MnTE-2-PyP(5+), (i) on survival of GALT-null Drosophila larvae reared in the presence versus absence of dietary galactose and (ii) on the severity of a long-term movement defect in GALT-null adult flies. Both SOD mimics conferred a significant survival benefit to GALT-null larvae exposed to galactose but not to controls or to GALT-null larvae reared in the absence of galactose. One mimic, MnTE-2-PyP(5+), also largely rescued a galactose-independent long-term movement defect otherwise seen in adult GALT-null flies. The survival benefit of both SOD mimics occurred despite continued accumulation of elevated galactose-1P in the treated animals, and studies of thiolated proteins demonstrated that in both the presence and absence of dietary galactose MnTE-2-PyP(5+) largely prevented the elevated protein oxidative damage otherwise seen in GALT-null animals relative to controls. INNOVATION AND CONCLUSIONS: Our results confirm oxidative stress as a mediator of acute galactose sensitivity in GALT-null Drosophila larvae and demonstrate for the first time that oxidative stress may also contribute to galactose-independent adult outcomes in GALT deficiency. Finally, our results demonstrate for the first time that both MnTnBuOE-2-PyP(5+) and MnTE-2-PyP(5+) are bioavailable and effective when administered through an oral route in a D. melanogaster model of classic galactosemia.

Biochemical, inhibition and inhibitor resistance studies of xenotropic murine leukemia virus-related virus reverse transcriptase.

We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (k(off)) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1.