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Background: Viral rebound during antiretroviral treatment (ART) is most often driven by suboptimal adherence in the absence of drug resistance. We assessed the diagnostic performance of point-of-care (POC) tenofovir (TFV) detection in urine for the prediction of viral rebound and drug resistance during ART., Methods: We performed a nested case-control study within the ADVANCE randomized clinical trial (NCT03122262) in Johannesburg, South Africa. Adults with human immunodeficiency virus (HIV) and newly initiating ART were randomized to receive either dolutegravir or efavirenz, tenofovir disoproxil fumarate or alafenamide, and emtricitabine. All participants with rebound ≥200 copies/mL between 24 and 96 weeks of follow-up were selected as cases and matched to controls with virological suppression <50 copies/mL. Rapid POC urine-TFV detection was performed retrospectively., Results: We included 281 samples from 198 participants. Urine-TFV was detectable in 30.7% (70/228) of cases and in 100% (53/53) of controls. Undetectable urine-TFV predicted rebound with a sensitivity of 69% [95% confidence interval {CI}: 63-75] and specificity of 100% [93-100]. In cases with virological failure and sequencing data (n = 42), NRTI drug resistance was detected in 50% (10/20) of cases with detectable urine-TFV versus in 8.3% (2/24) of cases with undetectable urine-TFV. Detectable urine-TFV predicted NRTI resistance (odds ratio [OR] 10.4 [1.8-114.4] P = .005) with a sensitivity of 83% [52-98] and specificity of 69% [50-84]., Conclusions: POC objective adherence testing using a urine-TFV test predicted viral rebound with high specificity. In participants with rebound, urine-TFV testing predicted the selection of drug resistance. Objective adherence testing may be used to rapidly provide insight into adherence, suppression, and drug resistance during ART., Competing Interests: Potential conflicts of interest. S. K. H. was employed by OraSure Technologies Inc and provided technical assistance during the study. OraSure Technologies Inc. was not involved in the design, execution, or analysis of this study. S. K. H. also reports grants from NIMH (Assay development was funded in part through SBIR grants from NIMH); Stock or stock options for OraSure Technologies (RSUs and options as part of general compensation from OraSure). A. M. J. W. received funding for this study through her institution from the Dutch AIDS Fund. A. M. J. W. reports grants or contracts from an Investigator-initiated grant from Gilead global for the study of HIV Integrase resistance (Rosetta) (pending, grant to institution), Health Holland ICD-ICK4HIVCure (paid to institution), and ZonMW (dutch government, paid to institution); consulting fees from Gilead, ViiV/GSK, and Janssen (paid to institution); payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Virology Education and the Southern African HIV Clinicians Society (paid to institution). A. M. J. W. Serves as a CEO for the European Society for Antiviral Research (ESAR) (unpaid), as governing Board member European AIDS Clinical Society, Chair of the European AIDS conference 2021 (unpaid, travel expenses paid to Institution), Member of the WHO Resnet (unpaid, travel expenses paid to Institution), Chair of the IAS-USA HIV drug resistance panel (unpaid), and as an Organizing Committee member of the European HIV and Hepatitis Meeting (unpaid, travel expenses paid to Institution). W. D. F. V. received funding for the ADVANCE RCT through his institution from Unitaid, USAID, and SAMRC, and received study drug from ViiV Healthcare and Gilead Sciences. W. D. F. V. also reports funding for his unit from the Bill and Melinda Gates, Foundation, National Institutes for Health, Unitaid, Foundation for Innovative New Diagnostics (FIND) and the Children's Investment Fund Foundation (CIFF), and received drug donations from Merck and J&J Sciences for investigator-led clinical studies. The unit leads investigator-led studies that receive financial support from Merck and ViiV and is involved in commercial drug studies for Merck. The unit performs evaluations of diagnostic devices for multiple biotech companies. W. D. F. V. also receives honoraria for educational talks and advisory board membership for Gilead, ViiV, Mylan, Merck, Adcock-Ingram, Aspen, Abbott, Roche, J&J, Sanofi and Virology Education; participates on DSMB for NIH International; is currently an unpaid board member for Dira Sengwe and was an unpaid board member for SAHCS. None of the ADVANCE RCT funders were involved in the design, execution, or analysis of this study. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The most studied HIV eradication approach is the "shock and kill" strategy, which aims to reactivate the latent reservoir by latency reversing agents (LRAs) and allowing elimination of these cells by immune-mediated clearance or viral cytopathic effects. The CNS is an anatomic compartment in which (persistent) HIV plays an important role in HIV-associated neurocognitive disorder. Restriction of the CNS by the blood-brain barrier is important for maintenance of homeostasis of the CNS microenvironment, which includes CNS-specific cell types, expression of transcription factors, and altered immune surveillance. Within the CNS predominantly myeloid cells such as microglia and perivascular macrophages are thought to be a reservoir of persistent HIV infection. Nevertheless, infection of T cells and astrocytes might also impact HIV infection in the CNS. Genetic adaptation to this microenvironment results in genetically distinct, compartmentalized viral populations with differences in transcription profiles. Because of these differences in transcription profiles, LRAs might have different effects within the CNS as compared with the periphery. Moreover, reactivation of HIV in the brain and elimination of cells within the CNS might be complex and could have detrimental consequences. Finally, independent of activity on latent HIV, LRAs themselves can have adverse neurologic effects. We provide an extensive overview of the current knowledge on compartmentalized (persistent) HIV infection in the CNS and on the "shock and kill" strategy. Subsequently, we reflect on the impact and promise of the "shock and kill" strategy on the elimination of persistent HIV in the CNS., (© 2022 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals LLC on behalf of Society for Leukocyte Biology.)

Allogeneic stem cell transplantation (alloSCT) of homozygous CCR5 Δ32 stem cells once resulted in the cure of human immunodeficiency virus (HIV) infection. We have recently reported a viral breakthrough in a similar setting. Here, we demonstrate that the rapid rebound after alloSCT was related to a highly replicative CXCR4-tropic HIV variant, which could already be detected before alloSCT.

Background: In Western countries emergence of human immunodeficiency virus (HIV) drug resistance has tremendously decreased, and transmission of drug resistance has merely stabilized in recent years. However, in many endemic settings with limited resources rates of emerging and transmitted drug resistance are not regularly assessed., Methods: We performed a survey including all HIV-infected individuals who received resistance testing in 2010-2015 in Aruba, a highly endemic HIV area in the Caribbean. Transmitted HIV drug resistance was determined using World Health Organization (WHO) criteria. Transmission dynamics were investigated using phylogenetic analyses. In a subset, baseline samples were re-analyzed using next generation sequencing (NGS)., Results: Baseline resistance testing was performed in 104 newly diagnosed untreated individuals (54% of all newly diagnosed individuals in 2010-2015): 86% were men, 39% were foreign-born, and 22% had AIDS at diagnosis. And 33% (95% CI: 24-42%) was infected with a drug-resistant HIV variant. The prevalence of resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) reached 45% (95% CI: 27-64%) in 2015, all based on the prevalence of mutation K103N. NGS did not demonstrate additional minority K103N-variants compared to routine resistance testing. K103N-harboring strains were introduced into the therapy-unexposed population via at least 6 independent transmissions epidemiologically linked to the surrounding countries. Virological failure of the WHO-recommended first-line NNRTI-based regimen was higher in the presence of K103N., Conclusions: The prevalence of resistant HIV in Aruba has increased to alarming levels, compromising the WHO-recommended first-line regimen. As adequate surveillance as advocated by the WHO is limited, the Caribbean region could face an unidentified rise of NNRTI-resistant HIV., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

Objectives: In ∼10% of newly diagnosed HIV-1 patients, drug-resistant viral variants are detected. In such transmitted HIV-1 variants, the thymidine analogue mutation (TAM) M41L is frequently observed as a single resistance mutation and these viral variants often belong to phylogenetic transmission clusters. The presence of at least three TAMs, in particular patterns with M41L/L210W, impairs the efficacy of the extensively used drug tenofovir. We investigated whether the presence of a single M41L mutation at baseline influences the selection of resistance to tenofovir and emtricitabine in vitro and in vivo., Methods: The impact of M41L on the development of drug resistance to tenofovir and emtricitabine was determined by extensive in vitro selection experiments and investigation of the virological outcome of patients on a first-line regimen., Results: The presence of a single M41L mutation did not influence the selected mutational profile or the genetic barrier to resistance to tenofovir and/or emtricitabine during long-term in vitro selection experiments. In vivo, virological outcome of first-line regimens containing tenofovir and emtricitabine was comparable between patients diagnosed with HIV-1 harbouring M41L (n=17, 16 were part of one transmission cluster) and WT virus (n=248)., Conclusions: Detection of a single M41L reverse transcriptase mutation at baseline did not influence the development of resistance in vitro or virological outcome on tenofovir-containing regimens in patients belonging to a large transmission cluster. Our results indicate that a high genetic barrier regimen may not be required when patients are diagnosed with HIV variants containing a single M41L mutation in reverse transcriptase., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The "Berlin patient" is the first patient cured of HIV-1 infection after allogeneic transplantation with nonfunctional CCR5 coreceptor stem cells. We demonstrate that CXCR4-predicted minority viruses present prior to transplantation were unable to rebound after transplantation due to their dependence on CCR5 for replication and high genetic barrier toward CXCR4 usage., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Using deep sequencing methods, we intensively investigated the selective pressure of maraviroc on the viral population in four patients with dual/mixed HIV-1 experiencing treatment failure., Methods: Patients received maraviroc add-on therapy (n = 4). Tropism was determined by Monogram's Trofile assay and/or 'deep' sequencing. Longitudinal 'deep' sequence analysis used triplicate HIV V3 RT-PCR on plasma samples. Sequences were interpreted using the geno2phenocoreceptor algorithm with a 3.5% false-positive rate (FPR) cut-off., Results: Patients had a median viral load of 4.7 log10 HIV RNA copies/mL with a median of 24% chemokine (C-X-C motif) receptor 4 (CXCR4)-using virus at baseline. Following maraviroc exposure, the chemokine (C-C motif) receptor 5 (CCR5)-using virus (R5) plasma viral load decreased by at least 1 log10, and only non-R5 variants with extremely low FPR values predominated after 21 days. Virus with an FPR ≤1.8% accounted for more than 90% of the circulating virus, having expanded to occupy the 'space' left by the suppression of R5 variants. Population genetic estimates of viral fitness in the presence of maraviroc showed a steep rise around an FPR value of 2%., Conclusions: Longitudinal analysis of independent R5 and non-R5 HIV populations shows that maraviroc selects viruses with an extremely low FPR, implying that the antiviral activity of maraviroc may extend to a broader range of HIV variants than previously suspected.

Background: Transmitted resistant HIV may revert to wild-type in the absence of drug pressure due to reduced replication capacity (RC). We observed eight therapy-naive patients infected with HIV harbouring four mutations at nucleoside reverse transcriptase inhibitor (NRTI) resistance-related positions: M41L, T69S, L210E and T215S. If partial reverted resistance patterns like these are detected at baseline, concerns for more extensive resistance in the quasispecies often directs selection of first-line combination antiretroviral therapy (cART) towards more complex regimens., Methods: Genotypic resistance testing and phylogenetic analysis was performed using pol sequences of 400 therapy-naive patients and 1322 patients with at least one NRTI-related mutation. Reverse transcriptase (RT) genes were cloned into a reference strain and RC was investigated., Results: Phylogenetic analysis showed that all eight patients are part of a transmission cluster (bootstrap value 92%). The patients resided in three distinct geographical regions and were either homosexually or heterosexually infected. Prior negative serology and analysis of base ambiguity demonstrated circulation for at least 7 years. In vivo evolution showed a mixture with wild-type (T215S/T) in only one untreated patient more than 6 years after diagnosis. The reverted resistance pattern did not confer a substantial reduction in RC compared with wild-type, explaining its persistence in vivo and long-term circulation in the population. Four patients started cART; three of them received quadruple cART. All patients showed good virological and immunological response., Conclusions: Long-term circulation transcending distinct regions and transmission groups suggests reversion occurred in previous hosts in the transmission chain. Identification of clusters using epidemiological data and active-partner tracing may broaden therapeutic options in cases of transmitted resistance.

Several large studies in Europe and the USA revealed that approximately 10% of all newly diagnosed patients harbour HIV-1 variants with at least one major resistance-associated mutation. In this review we discuss the underlying mechanisms that drive the evolution of drug-resistant viruses after transmission to the new host. In a comprehensive literature search 12 papers describing the evolution of 58 cases of transmitted resistant HIV-1 variants were found. Based on observations in the literature we propose three pathways describing the evolution of resistant HIV-1 after transmission to a new host. Firstly, reversion of the resistance mutation towards wild-type may rapidly occur when drug resistance mutations severely impact replicative capacity. Alternatively, a second pathway involves replacement of transmitted drug resistance mutations by atypical amino acids that also improve viral replication capacity. In the third evolutionary pathway the resistance mutations persist either because they do not significantly affect viral replication capacity or evolution is constrained by fixation through compensatory mutations. In the near future ultra-sensitive resistance tests may provide more insight into the presence of archived and minority variants and their clinical relevance. Meanwhile, clinical guidelines advise population sequence analysis of the baseline plasma sample to identify transmission of resistance. Given the limited sensitivity of this technique for minority populations and the delay between the moment of infection and time of analysis, knowledge of the described evolutionary mechanisms of transmitted drug resistance patterns is essential for clinical management and public health strategies.

Objectives: Maraviroc is the first licensed chemokine co-receptor 5 (CCR5) co-receptor antagonist in clinical practice. It is currently being used in patients harbouring exclusively CCR5-tropic virus. The objective of the study was to investigate the impact of maraviroc on viruses with different co-receptor preferences in a patient with a dual/mixed (D/M) infection., Methods: We present a case report of an HIV-1 patient infected with a D/M virus population. Co-receptor tropism was determined by phenotypic and genotypic tests. Biological clones from pre- and post-maraviroc therapy were generated. Tropism of these infectious clones was investigated in U373-MAGI cells expressing CD4+ CCR5+ or CD4+ CXCR4+. Maraviroc susceptibility and viral replication were determined using donor peripheral blood mononuclear cells (PBMCs)., Results: In-depth clonal genotypic analysis revealed the presence of both R5-tropic variants and X4-tropic viruses before the start of maraviroc. During maraviroc therapy all R5-predicted viruses were suppressed. Phenotypic analyses revealed that all biological clones before maraviroc therapy could infect both CCR5- and CXCR4-bearing U373-MAGI cells, demonstrating dual tropism. The baseline biological clones preferentially infected the CCR5 cell line and were fully susceptible to maraviroc in PBMCs (dual-R5). In contrast, during maraviroc therapy the dual-R5-tropic viruses were replaced by more X4-tropic viruses (dual-X4), which could not be inhibited by maraviroc., Conclusions: This case report demonstrates that dual-tropic viruses, capable of using both co-receptors in phenotypic assays, can be inhibited by maraviroc if they have a CCR5 co-receptor preference in vivo.

Objectives: The genetic barrier to development of raltegravir resistance is considered to be low, requiring at least one primary integrase mutation: Y143C, Q148H/K/R or N155H to confer raltegravir therapy failure. However, during continued raltegravir treatment failure, additional mutations may be selected. In a patient failing raltegravir therapy, we investigated the impact of multiple integrase mutations on resistance and viral replication. Furthermore, in vivo fitness was investigated during failure of raltegravir-containing highly active antiretroviral therapy and after raltegravir was discontinued from the regimen., Methods: Patient-derived viral integrase genes were cloned into a reference strain. These recombinant viruses were used to determine the contribution of individual integrase mutations to raltegravir resistance and replication capacity in vitro. To determine in vivo fitness, the relative proportion of specific integrase mutations was monitored over time by in-depth clonal analysis of the viral integrase at baseline, during and after raltegravir treatment., Results: Raltegravir therapy failure was associated with the initial selection of primary resistance mutation N155H. This mutation conferred a 3.8-fold reduction in raltegravir susceptibility and a severe reduction in viral replication. Acquisition of integrase mutation Q95K increased resistance (6.2-fold) and partly restored viral replication. Selection of a third mutation, V151I, further increased raltegravir resistance (20-fold), but decreased viral replication. After prolonged raltegravir interruption, raltegravir resistance mutations were lost, demonstrating the reduced replication capacity of the resistant virus., Conclusions: We describe selection of Q95K as a secondary resistance mutation during raltegravir therapy failure. In the background of N155H, Q95K enhances raltegravir and elvitegravir resistance and improves the impaired replication of the virus.

Background: Factors promoting the emergence of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) connection domain mutations and their effect on antiretroviral therapy (ART) are still largely undetermined. We investigated this matter by analyzing genotypic resistance tests covering 400 amino acid positions in the RT of HIV-1 subtype B viruses and corresponding treatment histories and laboratory measurements., Methods: The emergence of connection domain mutations was studied in 334 patients receiving monotherapy or dual therapy with thymidine analogues at the time of the genotypic resistance test. Response to subsequent combination ART (cART) was analyzed using Cox regression for 291 patients receiving unboosted protease inhibitors. Response was defined by ever reaching an HIV RNA level <50 copies/mL during the first cART., Results: The connection domain mutations N348I, R356K, R358K, A360V, and A371V were more frequently observed in ART-exposed than ART-naive patients, of which only N348I and A360V were nonpolymorphic (with a prevalence of <1.5% in untreated patients). N348I correlated with M184V and predominantly occurred in patients receiving lamivudine and zidovudine concomitantly. A360V was not associated with specific drug combinations and was found to emerge later than M184V or thymidine analogue mutations. Nonpolymorphic connection domain mutations were rarely detected in the absence of established drug resistance mutations in ART-exposed individuals (prevalence, <1%). None of the 5 connection domain mutations associated with treatment showed a statistically significant effect on response to cART., Conclusions: Despite their frequent emergence, connection domain mutations did not show large detrimental effects on response to cART. Currently, routine implementation of connection domain sequencing seems unnecessary for developed health care settings.

Background: The connection domain mutation N348I confers resistance to zidovudine (AZT) and is associated with the lamivudine (3TC) mutation M184V. We explored the biochemical and virological influence of N348I in the context of M184V., Methods: Genotypic resistance data for patients receiving monotherapy or dual therapy with AZT, lamivudine (3TC), or AZT/3TC were analyzed. Rates of N348I emergence were compared between treatment groups. Mutant reverse transcriptases (RTs) containing M184V and/or N348I were generated to study enzymatic and virological properties., Results: We included 50 AZT-treated, 11 3TC-treated, and 10 AZT/3TC-treated patients. N348I was observed in 3 (6%), 0, and 4 (40%) of these patients, respectively. The rate of N348I emergence was increased by 5-fold in the AZT/3TC group (11.7 instances [95% confidence interval {CI}, 3.2-30.1 instances] per 100 person-years of receipt of AZT), compared with the rate noted for the AZT group (2.3 instances [95% CI, 0.4-6.8 instances] per 100 person-years of receipt of AZT; P = .04). Biochemical data show that N348I can partially compensate for the diminution in processive DNA synthesis and the reduction in AZT excision associated with M184V. Furthermore, virological analyses demonstrate that N348I confers low-level resistance to AZT and partly restores the reduced RT activity of the M184V variant., Conclusion: In vivo selection of N348I is driven by AZT and is further facilitated when 3TC is coadministered. Compensatory interactions between N348I and M184V help to explain these findings.

We report the selection of enfuvirtide-resistant human immunodeficiency virus type 1 in cerebrospinal fluid, resulting in subsequent loss of viral suppression in the plasma. This case report emphasizes the potential danger of low-level penetration of entry inhibitors into the central nervous system.

Background: Virological failure of first-line antiretroviral therapy based on lopinavir boosted with ritonavir (lopinavir/r) has rarely been associated with resistance in protease. We identified a new genotypic resistance pathway in 3 patients who experienced failure of first-line lopinavir/r treatment., Methods: Viral protease and the C-term part of Gag were sequenced. The observed mutations were introduced in a reference strain to investigate impact on protease inhibitor susceptibility and replication capacity., Results: A detailed longitudinal analysis demonstrated the selection of the M46I+L76V protease mutations in all 3 patients. The L76V conferred a solitary 3.5-fold increase in one-half the maximal inhibitory concentration to lopinavir but severely hampered viral replication. Addition of M46I, which did not confer any lopinavir resistance on its own, had a dual effect. It partly compensated for the loss in replication capacity and increased the one-half maximal inhibitory concentration to above the lower clinical cutoff (11-fold). Analysis of a large clinical database (>180,000 human immunodeficiency virus [HIV] sequences) demonstrated a significant association (Spearman rho, 0.93) between the increased presence of L76V in clinical samples (0.5% in 2000 to 3.4% in 2006) and lopinavir prescription over time., Conclusions: The HIV protease substitution L76V, in combination with M46I, confers clinically relevant levels of lopinavir resistance and represents a novel resistance pathway to first-line lopinavir/r therapy.

Objective: To investigate the mechanism explaining the persistence of human immunodeficiency virus (HIV) type 1 variants with multiple protease inhibitor (PI)-resistance mutations in the absence of PI therapy., Methods: Longitudinal genotypic analyses were performed on sequential samples obtained from 2 HIV-1-infected patients who had stopped PI therapy for 4 years. Replication capacity (RC) was determined using recombinant viruses. Subsequently, the effect that changing individual protease mutations back to wild type has on RC was analyzed., Results: We observed prolonged persistence (up to 4 years) of viruses with multiple protease mutations after PI therapy was stopped, despite the fact that the RC of the viruses was severely reduced. Forcing the virus to evolve toward wild type by changing individual protease mutations to wild type was unsuccessful, because all variants displayed a decreased RC in comparison with that of their predecessors., Conclusions: We propose compensatory fixation as a mechanism for the in vivo persistence of variants with multiple PI-resistance mutations in the absence of PI therapy. Viruses with multiple PI mutations have (partially) compensated for the initial loss in RC. Therefore, reversion of a single mutation causes a (further) reduction in RC and, as a consequence, the route to wild type is blocked.

Background: Airway infection with influenza virus induces local expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO), which has been shown to enhance inflammatory mediator responses in vitro. Because secondary pneumococcal infections occurring shortly after recovery from influenza are associated with enhanced inflammatory responses, we hypothesized that IDO activity contributes to the enhanced response to bacterial challenges in mice previously infected with influenza virus., Methods: On day 14 after influenza virus infection (with strain A/PR/8/34), C57Bl/6 mice were intranasally inoculated with 1 x 10(4) colony-forming units of S. pneumoniae (serotype 3). Matrix-driven delivery pellets that contained 70 mg of the IDO inhibitor 1-methyl-DL-tryptophan (MeTrp) released over a period of 7 days were subcutaneously implanted 48 h before pneumococcal infection., Results: MeTrp treatment resulted in a 20-fold reduction in pneumococcal outgrowth 48 h after bacterial inoculation. Remarkably, pulmonary levels of interleukin-10 and tumor necrosis factor-alpha were significantly reduced in mice treated with MeTrp., Conclusions: Our data suggest that IDO expression during influenza virus infection alters the inflammatory response and facilitates the outgrowth of pneumococci during secondary bacterial pneumonia.

Background: Respiratory virus infections have been recognized as important causes of severe pneumonia in patients who have undergone stem cell transplantation (SCT). Reported incidences of respiratory virus infection in adult SCT recipients vary in the literature from 3.5% to 36% when determined by viral culture. However, a more sensitive method to assess the presence of respiratory viruses in the lower airways may be important for delineation of the true incidence of respiratory virus-associated pneumonia and may be essential for guidance on implementation of antiviral therapy and prevention or limitation of nosocomial spread of infection with respiratory viruses., Methods: To determine the incidence and severity of respiratory tract illness (RTI) and to assess the diagnostic value of real-time reverse-transcriptase polymerase chain reaction (RT-PCR) versus viral culture, 72 SCT recipients were monitored during a 6-month period., Results: A respiratory virus was detected in 21% of episodes of RTI by viral culture and in 63% of RTI episodes by real-time RT-PCR (P<.0001). In lower respiratory tract illness, real-time RT-PCR was much more sensitive than viral culture for detection of respiratory virus (73% vs. 9%; P=.008). The mortality rate for patients with respiratory virus-associated lower respiratory tract illness (25%) was similar to rates reported elsewhere. Respiratory viruses (predominantly rhinovirus) were detected by real-time RT-PCR in 9% of samples obtained from symptom-free SCT recipients at predetermined times by real-time RT-PCR and by viral culture in 1% (P<.0001), indicating that asymptomatic shedding of respiratory viruses also occurs., Conclusion: We conclude that, although asymptomatic shedding of respiratory virus occurs, respiratory viruses are frequent causes of RTI in SCT recipients.

During the past years, human coronaviruses (HCoVs) have been increasingly identified as pathogens associated with more-severe respiratory tract infection (RTI). Diagnostic tests for HCoVs are not frequently used in the routine setting. It is likely that, as a result, the precise role that HCoVs play in RTIs is greatly underestimated. We describe a rapid, sensitive, and highly specific quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) for the detection of HCoV that can easily be implemented in the routine diagnostic setting. HCoV was detected in 28 (11%) of the 261 clinical specimens obtained from patients presenting with symptoms of RTI ranging from common cold to severe pneumonia. Only 1 (0.4%) of the 243 control specimens obtained from patients without symptoms of RTI showed the presence of HCoV. We conclude that HCoVs can be frequently detected in patients presenting with RTI. Real-time RT-PCR provides a tool for large-scale epidemiological studies to further clarify the role that coronavirus infection plays in RTI in humans.

During summer and fall, enterovirus infections are responsible for a considerable proportion of hospitalizations of young infants. We prospectively studied the incidence of enterovirus infections via real-time polymerase chain reaction (PCR) in blood, feces, and cerebrospinal fluid samples from infants

The relationship between clinical changes in stavudine activity and stavudine resistance was investigated in 16 human immunodeficiency virus (HIV)-infected children who received stavudine monotherapy for 18 months. Seven patients responded well to stavudine therapy, 3 experienced transient reductions in virus load, and all others had no detectable virologic response. In both the responders and nonresponders, no changes in stavudine susceptibility or specific baseline/emergent mutations in reverse transcriptase were observed. Only posttherapy HIV isolates from transient responders had elevated IC(50) values for stavudine. In 2 of the 3 transient responders, substitutions at codons 41, 210, and 215 were selected. The significance of these mutations was confirmed in viral competition experiments, site-directed mutagenesis, and in vitro selection. Selection of mutations previously associated with zidovudine resistance can be an important mechanism through which HIV may escape stavudine. The effect of these mutations on phenotypic stavudine susceptibility is relatively small but apparently large enough to be clinically significant.

We retrospectively analyzed the value of polymerase chain reaction (PCR) for the detection of respiratory viral infections in 43 patients with hematological cancer whose bronchoalveolar lavage (BAL) samples had been stored. In addition, 17 nose-throat (NT) swabs and 29 blood samples had been obtained. PCR was performed to detect parainfluenza viruses 1-3, respiratory syncytial virus, rhinovirus, influenza viruses A and B, enteroviruses, and coronaviruses. Viral cultures or antigen testing of BAL samples revealed 9 respiratory viruses in 8 patients. By use of PCR, 8 more respiratory viruses were detected in another 7 patients, increasing the rate of identification from 19% to 35% (P<.0005). Available NT swabs yielded the same results with PCR as did BAL samples. We conclude that PCR is more sensitive than viral culture or antigen or serologic testing for detection of respiratory viruses in patients with hematological malignancies, and that it offers the possibility for early, more rapid diagnosis.

Use of human immunodeficiency virus (HIV) drug-resistance testing in therapeutic decision making may be aided by understanding the relationship between results of genotypic and drug-susceptibility phenotypic assays. We investigated this relationship by applying 3 different statistical methods-cluster analysis, recursive partitioning, and linear discriminant analysis-to results for 72 patients followed in the Adult AIDS Clinical Trials Group (ACTG) protocol 333. ACTG 333 was a multicenter, randomized trial comparing 2 formulations of saquinavir (SQV) to indinavir (IDV) in patients with extensive hard-gel SQV experience. Data include protease amino acid sequences and 50% inhibitory concentrations for SQV and IDV at baseline. The 3 methods give similar results showing the association of mutations at codons 10, 63, 71, and 90 with in vitro resistance to IDV and SQV. Recursive partitioning is especially useful because it can identify interactions among mutations at different codons and accommodates many types of data as well as missing observations.

Exposure of human immunodeficiency virus to the nucleoside analogue lamivudine (3TC) rapidly selects for resistant variants with a valine at codon 184 (M184V) in the catalytic site of reverse transcriptase. In vitro, 184V demonstrated increased enzyme fidelity and suppressed zidovudine resistance. Clinical trials demonstrated that 3TC-zidovudine combination therapy results in a strong and sustained antiviral response. To investigate the role of 184V on in vivo virus evolution, the effect of zidovudine addition in 3TC-pretreated patients harboring 184V was studied. In vivo, no significant change in fidelity was observed with 184V, shown by generation of the classical pattern of zidovudine mutations. Of interest, in contrast to zidovudine monotherapy, in which just one substitution is sufficient for in vivo development of significant zidovudine resistance, multiple substitutions are required for the same level of zidovudine resistance in strains harboring 184V. This need for multiple substitutions may be one of the mechanisms explaining the sustained antiretroviral response of the 3TC-zidovudine combination.

The effect of the appearance of drug-resistant human immunodeficiency virus type 1 (HIV-1) on viral RNA load was studied in patients treated with the reverse transcriptase inhibitor lamivudine. During the first 12 weeks of treatment, HIV-1 RNA concentrations and amino acid changes in codon 184, causing high-level resistance to lamivudine, were determined in longitudinal serum samples from HIV-1 p24 antigen-positive and -negative patients. A marked decline in the amount of HIV-1 RNA (approximately 95% below baseline) and HIV-1 p24 antigen was observed within 2 weeks, followed by a rise that coincided with the appearance of lamivudine-resistant viruses in serum (isoleucine mutants initially, which were subsequently replaced by valine variants). After 12 weeks, a partial antiviral effect was observed despite the presence of a complete codon 184 mutant virus population in serum. This study shows that the rapid appearance of drug-resistant virus in serum is followed by an increase in viral RNA load.

The substrate-binding region of the cell-envelope proteinase of Lactococcus lactis strain SK11 was modelled, based on sequence homology of the catalytic domain with the serine proteinases subtilisin and thermitase. Substitutions, deletions and insertions were introduced, by site-directed and cassette mutagenesis of the prtP gene encoding this enzyme, based on sequence comparison both with subtilisin and with the homologous L.lactis strain Wg2 proteinase, which has different proteolytic properties. The engineered enzymes were investigated for thermal stability, proteolytic activity and cleavage specificity towards small chromogenic peptide substrates and the peptide alpha s1-casein(1-23). Mutations in the subtilisin-like substrate-binding region showed that Ser433 is the active site residue, and that residues 138 and 166 at either side of the binding cleft play an important role in substrate specificity, particularly when these residues and the substrate are oppositely charged. The K748T mutation in a different domain also affected specificity and stability, suggesting that this residue is in close proximity to the subtilisin-like domain and may form part of the substrate-binding site. Several mutant SK11 proteinases have novel properties not previously encountered in natural variants. Replacements of residues 137-139AKT along one side of the binding cleft produced the 137-139GPP mutant proteinase with reduced activity and narrowed specificity, and the 137-139GLA mutant with increased activity and broader specificity. Furthermore, the 137-139GDT mutant had a specificity towards alpha s1-casein(1-23) closely resembling that of L.lactis Wg2 proteinase. Mutants with an additional negative charge in the binding region were more stable towards autoproteolysis.

Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains Wg2 and SK11. These plasmids were introduced into the plasmid-free strain L. lactis MG1363. The serine proteinases produced by these L. lactis strains were isolated, and their cleavage specificity and rate towards alpha s1- and beta-casein was investigated. The catalytic properties of both the SK11 and Wg2 proteinases, which differ in 44 out of 1902 amino acid residues, could be changed dramatically by the reciprocal exchange of specific fragments between the two enzymes. As a result, various L. lactis strains were constructed having new proteolytic properties that differ from those of the parental strains. Furthermore, two segments in the proteinase could be identified that contribute significantly to the cleavage specificity towards casein; within these two segments, several amino acid residues were identified that are important for substrate cleavage rate and specificity. The results also indicate that the lactococcal proteinase has an additional domain involved in substrate binding compared with the related subtilisins. This suggests that the 200 kd L. lactis proteinase may be the representative of a new subclass of subtilisin-like enzymes.