A fresh look at the concept of Genus Epidemicus: findings from the Clificol Project

The Clificol® COVID-19 Support Project is an innovative international data collection project aimed at tackling some of the core questions in homeopathy, including the notion of Genus Epidemicus. Aims:To shed some light on the notion of Genus Epidemicus in the context of this infection. Going beyond that, the project aims to use these data to tackle more fundamental questions, such as the role of symptoms and rubrics in treatment individualisation. Methodology:This online multi-national data-collection project is supported by the ECH, ECCH, ICH, HRI, LMHI, and other professional associations. The collected data includes demographic information, severity, conventional diagnosis and treatment, presenting symptoms as well as the remedies prescribed. The outcome of treatment was tracked using the ORIDL scale. The concept of Genus Epidemicus, including the role of treatment individualisation, was investigated by analysing whether presenting symptoms cluster into distinct groups (K-Means clustering approach). The symptom data originating from China was obtained using a questionnaire. Results and discussion: 20 Chinese practioners collected 359 cases, primarily in the first half of 2020 (766 consultations, 363 prescriptions). The cluster analysis found two to be the optimum number of clusters. These two symptomatic clusters had a high overlap with the two most commonly prescribed remedies in that population: In cluster 1 there were 297 prescriptions, 95.6% of which were Gelsemium sempervirens, incluster 2, there were 61 prescriptions, 95.1% of which were Bryonia alba. Under the assumption of a single genus epidemicuswe would expect to see a single cluster of symptoms. The data from the Chinese population were not compatible with this assumption. Conclusion:This was the first study that investigated the notion of Genus Epidemicus by using modern statistical techniques. These analyses identified at least two distinct symptom pictures. The notion of a single COVID-19 Genus Epidemicus did not apply to this population.

Update from the Clificol Project: Omicron in Hong-Kongand worldwide overview of the pandemic

The Clificol® COVID-19 Support Project is an innovative international data collection project aimed at documenting the presenting symptoms, use of homeopathy through the pandemic and tackling some of the core questions in homeopathy. The Covid-19 pandemic raised many questions and mobilised many resources. In order to make good use of resources, sound knowledge of the presenting complaints and demographics are required. In particular, we aimed to characterise the recent Omicron wave in Hong-Kong and to get an overall picture of the global pandemic as experienced by the homeopathic community. This online multi-national data-collection project is supported by the ECH, ECCH, ICH, HRI, LMHI, and other professional associations. The collected data includes demographic information, severity, conventional diagnosis and treatment, presenting symptoms as well as the remedies prescribed. The outcome of treatment was tracked using the ORIDL scale. More recently a 23-items prospective questionnaire was added to the input in order to gather targeted data about the presenting complaints. The recent wave of the Omicron variant in Hong-Kong, was analysed (N=372 cases) in terms of the symptomatology of this variant. The data from the 23-items questionnaire is compared to the longer questionnaire (150 items) used by the Hong-Kong team (21 practitioners). The most frequently reported common Clinical symptoms were extreme tiredness (60%), sore throat (46%), headache during fever (45%), dryness of mouth (37%), poor appetite (37%), runny nose (34%) and unusual muscle pains (31%).Also, the cases collected from around the world over the course of the pandemic (N=1300) were analysed, providing an overall picture of the pandemic and its specificities per country and over time. Clificol has shown and continues to show the value of data collection for the homeopathy community, providing important information for the management of future pandemics and opening new avenues for research in homeopathy.

Decreased diversion by doctor-shopping for a reformulated extended release oxycodone product (OxyContin).

BACKGROUND: Doctor-shopping (obtaining prescriptions from multiple prescribers/pharmacies) for opioid analgesics produces a supply for diversion and abuse, and represents a major public health issue. METHODS: An open cohort study assessed changes in doctor-shopping in the U.S. for a brand extended release (ER) oxycodone product (OxyContin) and comparator opioids before (July, 2009 to June, 2010) versus after (January, 2011 to June, 2013) introduction of reformulated brand ER oxycodone with abuse-deterrent properties, using IMS LRx longitudinal data covering >150 million patients and 65% of retail U.S. prescriptions. RESULTS: After its reformulation, the rate of doctor-shopping decreased 50% (for 2+ prescribers/3+ pharmacies) for brand ER oxycodone, but not for comparators. The largest decreases in rates occurred among young adults (73%), those paying with cash (61%) and those receiving the highest available dose (62%), with a 90% decrease when stratifying by all three characteristics. The magnitude of doctor-shopping reductions increased with increasing number of prescribers/pharmacies (e.g., 75% reduction for ≥2 prescribers/≥4 pharmacies). CONCLUSIONS: The rate of doctor-shopping for brand ER oxycodone decreased substantially after its reformulation, which did not occur for other prescription opioids. The largest reductions in doctor-shopping occurred with characteristics associated with higher abuse risk such as youth, cash payment and high dose, and with more specific thresholds of doctor-shopping. A higher prescriber and/or pharmacy threshold also increased the magnitude of the decrease, suggesting that it better captured the effect of the reformulation on actual doctor-shoppers.

A retrospective cohort study of long-term immediate-release hydrocodone/acetaminophen use and acetaminophen dosing above the Food and Drug Administration recommended maximum daily limit among commercially insured individuals in the United States (2008-2013).

UNLABELLED: Immediate-release (IR) hydrocodone/acetaminophen is the most prescribed opioid in the United States; however, patterns of use, including long-term treatment and dose, are not well described. Duration of use, including the percentage of patients on long-term treatment (>90 days of continuous use), was assessed for patients newly prescribed IR hydrocodone/acetaminophen compared to other opioid analgesics in a national commercial insurance database (January 2008-September 2013). Though only a small percentage of IR hydrocodone/acetaminophen patients continued treatment long-term (1.7%), the number was large (104,839) and was nearly 5 times the number receiving extended-release (ER) morphine (n = 22,338) and nearly 4 times the number receiving ER oxycodone (n = 26,946) long-term. Using a less conservative allowable gap in treatment increased the number of patients meeting the criteria for long-term use (approximately 160,000 for IR hydrocodone/acetaminophen vs <30,000 for ER morphine and ER oxycodone). Most patients meeting these criteria received IR hydrocodone doses between >20 and ≤60 mg/d (n = 56,220, 53.6%) in month 4; 5.5% (n = 5,743) received doses >60 mg/d. Moreover, approximately 15% of IR hydrocodone/acetaminophen patients (n > 900,000) were prescribed total daily acetaminophen doses exceeding 4 g (the limit recommended by the U.S. Food and Drug Administration) at their initial IR hydrocodone/acetaminophen prescription or any time during therapy. PERSPECTIVE: Although most patients were prescribed IR hydrocodone/acetaminophen for acute pain, the number of patients prescribed long-term therapy exceeds the number of patients prescribed ER opioids. It is important to consider the benefits and risks inherent with long-term opioid therapy, whether with IR or ER opioids, to ensure safe use of these products.

Development and impact of prescription opioid abuse deterrent formulation technologies.

BACKGROUND: Millions of patients are treated with opioid analgesics (OpAs) to relieve pain. Unfortunately, these medications are subject to abuse and/or unintended misuse. Abuse deterrent formulations (ADFs) represent an intervention strategy to decrease abuse/misuse without affecting patient access. The Food and Drug Administration (FDA) has issued Draft Guidance "Abuse deterrent opioids, Evaluation and Labeling" and is currently actively pursuing scientific input on this issue. METHODS: The development of ADF technologies was reviewed using peer reviewed journals describing OpA post marketing studies, web sites containing FDA announcements on product approvals and manufacturer product use profiles. RESULTS: Reviewed is the FDA recent approval of a product label describing the abuse deterrent characteristics of OxyContin(®) (physical barrier formulation), and the FDA determination that studies were insufficient for an Opana(®) (physical barrier) ADF label. Additional reviewed marketed OpAs with ADF technologies include: Suboxone(®) and Embeda(®) (opioid agonist/antagonist combinations), Oxecta(®) (aversion technology), and Nucynta(®) (physical barrier). Reviewed ADF technologies currently in development include: new physical barrier and aversion technologies, an innovative extended release formulation as well as novel polymer-opioid conjugates. As ADF technologies are part of a comprehensive intervention strategy to promote safe OpA use, additional components including governmental, community, and educational initiatives are reviewed. CONCLUSIONS: The outcomes of the recent ADF labeling applications for OxyContin(®) (Tier 3 approval) and Opana(®) (non-approval) suggest that the threshold for ADF labeling will be appropriately high. The presented findings indicate that ADF technologies can be a critical component of a comprehensive strategy to promote the safe and effective use of OpAs.

In vivo patterns of resistance to the HIV attachment inhibitor BMS-488043.

Attachment inhibitors (AI) are a novel class of HIV-1 antivirals, with little information available on clinical resistance. BMS-488043 is an orally bioavailable AI that binds to gp120 of HIV-1 and abrogates its binding to CD4(+) lymphocytes. A clinical proof-of-concept study of the AI BMS-488043, administered as monotherapy for 8 days, demonstrated significant viral load reductions. In order to examine the effects of AI monotherapy on HIV-1 sensitivity, phenotypic sensitivity assessment of baseline and postdosing (day 8) samples was performed. These analyses revealed that four subjects had emergent phenotypic resistance (a 50% effective concentration [EC(50)] >10-fold greater than the baseline value) and four had high baseline EC(50)s (>200 nM). Population sequencing and sequence determination of cloned envelope genes uncovered five gp120 mutations at four loci (V68A, L116I, S375I/N, and M426L) associated with BMS-488043 resistance. Substitution at the 375 locus, located near the CD4 binding pocket, was the most common (maintained in 5/8 subjects at day 8). The five substitutions were evaluated for their effects on AI sensitivity through reverse genetics in functional envelopes, confirming their role in decreasing sensitivity to the drug. Additional analyses revealed that these substitutions did not alter sensitivity to other HIV-1 entry inhibitors. Thus, our studies demonstrate that although the majority of the subjects' viruses maintained sensitivity to BMS-488043, substitutions can be selected that decrease HIV-1 susceptibility to the AI. Most importantly, the substitutions described here are not associated with resistance to other approved antiretrovirals, and therefore, attachment inhibitors could complement the current arsenal of anti-HIV agents.

Increased sensitivity of HIV variants selected by attachment inhibitors to broadly neutralizing antibodies.

Treatment with HIV attachment inhibitors (AIs) can select for escape mutants throughout the viral envelope. We report on three such mutations: F423Y (gp120 CD4 binding pocket) and I595F and K655E (gp41 ectodomain). Each displayed decreased sensitivity to the AI BMS-488043 and earlier generation AIs, along with increased sensitivity to the broadly neutralizing antibodies 2F5 and 4E10, without affecting the rate of viral entry or sensitivity to the entry inhibitors AMD-3100 and Enfuvirtide. We also observed that I595F did not substantially increase envelope sensitivity to HIV-infected patient sera. Based on these observations, we propose that although F423Y, I595F and K655E may all affect the presentation of the 2F5 and 4E10 epitopes, natural immune mimicry is rare only for the I595F effect. Thus, it seems that in addition to restricting AI resistance development, incorporation of I595F into an appropriate vehicle could elicit a novel antiviral response to improve vaccine efficacy.

Protection against HIV-envelope-induced neuronal cell destruction by HIV attachment inhibitors.

We demonstrate that HIV attachment inhibitors (AIs) prevent HIV envelope-induced destruction of two neuronal cell lines (SH-SY5Y and BE(2)-M17) at low nanomolar concentrations. The fusion inhibitor enfuvirtide and the CCR5 inhibitors UK427,857 and TAK779 do not display protection activity, suggesting the involvement of Env/cell interaction site(s) distinct from the sites involved in the viral entry process. We surmise that by inducing conformation changes in the envelope, AIs likely obstruct novel interactions with a neuronal cell factor(s) required for induction of apoptosis. This antiretroviral class may therefore have the potential to inhibit HIV-induced neuron damage, thereby curtailing the increasing incidence of HIV-associated cognitive impairment.

Inhibition of envelope-mediated CD4+-T-cell depletion by human immunodeficiency virus attachment inhibitors.

Human immunodeficiency virus type 1 (HIV-1) envelope (Env) binding induces proapoptotic signals in CD4(+) T cells without a requirement of infection. Defective virus particles, which represent the majority of HIV-1, usually contain a functional Env and therefore represent a potentially significant cause of such CD4(+)-T-cell loss. We reasoned that an HIV-1 inhibitor that prohibits Env-host cell interactions could block the destructive effects of defective particles. HIV-1 attachment inhibitors (AIs), which potently inhibit Env-CD4 binding and subsequent downstream effects of Env, display low-nanomolar antiapoptotic potency and prevent CD4(+)-T-cell depletion from mixed lymphocyte cultures, also with low-nanomolar potency. Specific Env amino acid changes that confer resistance to AI antientry activity eliminate AI antiapoptotic effects. We observed that CD4(+)-T-cell destruction is specific for CXCR4-utilizing HIV-1 strains and that the fusion blocker enfuvirtide inhibits Env-mediated CD4(+)-T-cell killing but is substantially less potent than AIs. These observations, in conjunction with observed antiapoptotic activities of soluble CD4 and the CXCR4 blocker AMD3100, suggest that this AI activity functions through a mechanism common to AI antientry activity, e.g., prevention of Env conformation changes necessary for specific interactions with cellular factors that facilitate viral entry. Our study suggests that AIs, in addition to having potent antientry activity, could contribute to immune system homeostasis in individuals infected with HIV-1 that can engage CXCR4, thereby mitigating the increased risk of adverse clinical events observed in such individuals on current antiretroviral regimens.

Hepatitis C virus infection among drug injectors in St Petersburg, Russia: social and molecular epidemiology of an endemic infection.

AIMS: To understand the epidemiology and transmission patterns of hepatitis C virus (HCV), the predominant blood borne-pathogen infecting injection drug users (IDUs), in a part of the former Soviet Union. DESIGN: Cross-sectional respondent-driven sample of IDUs. SETTING: St Petersburg, Russia. PARTICIPANTS: A total of 387 IDUs were recruited in late 2005 and throughout 2006. MEASUREMENTS: Participants were surveyed to collect demographic, medical and both general and dyad-specific drug injection and sexual behaviors. A blood sample was collected to detect antibodies to hepatitis C and to amplify viral RNA for molecular analysis. The molecular data, including genotypes, were analyzed spatially and linkage patterns were compared to the social linkages obtained by respondent-driven sampling (RDS) for chains of respondents and among the injection dyads. FINDINGS: HCV infection was all but ubiquitous: 94.6% of IDUs were HCV-seropositive. Among the 209 viral sequences amplified, genotype 3a predominated (n = 119, 56.9%), followed by 1b (n = 61, 29.2%) and 1a (n = 25, 11.9%). There was no significant clustering of genotypes spatially. Neither genotypes nor closely related sequences were clustered within RDS chains. Analysis of HCV sequences from dyads failed to find associations of genotype or sequence homology within pairs. CONCLUSIONS: Genotyping reveals that there have been at least five unique introductions of HCV genotypes into the IDU community in St Petersburg. Analysis of prevalent infections does not appear to correlate with the social networks of IDUs, suggesting that simple approaches to link these networks to prevalent infections, rather than incident transmission, will not prove meaningful. On a more positive note, the majority of IDUs are infected with 3a genotype that is associated with sustained virological response to antiviral therapy.

Virologic and host characteristics of human immunodeficiency virus type 1-infected pediatric long term survivors.

BACKGROUND: There are limited data concerning determinants of varying clinical progression rates in human immunodeficiency virus type 1 (HIV-1)-infected children. Therefore, we sought to determine whether viral or host factors associated with nonprogressive HIV-1 infection in adults play a role in limiting progression of infection in 5 vertically infected youths, ages 12-18 years, who have displayed no signs of advanced HIV-1 disease or acquired immunodeficiency syndrome despite having received minimal treatment with antiretroviral drugs. RESULTS: The 5 individuals, whom we characterize as long term survivors, have maintained low loads of HIV-1 RNA in plasma when compared to many of their peers, and have also maintained normal and stable CD4 T-lymphocyte numbers and percentages throughout their lives. Determination of their predominant HIV-1 sequences revealed that 4 of 5 patients harbor virus with markers of resistance to their therapy (one was never treated). Furthermore 2 harbored viral isolates that contained insertions in Gag or Vif that inhibit HIV-1 replication. Moreover, 2 were found to be heterozygous for the CCR2 polymorphism 64I, a genotype associated with slower progression to acquired immunodeficiency syndrome in adults. All 5 expressed the histocompatibility leukocyte antigen DQ1 and 2 had unusual DR/DQ1 phenotypes. CONCLUSIONS: We believe that the limited antiretroviral therapy received by the long term survivors cannot solely account for their benign clinical status. Therefore, we conclude that other factors, including gene polymorphisms that affect viral replicative capacity, account for the long term survival in some, and deduce that, as in adults, no single factor (virologic or host) can account for this clinical phenotype in all cases.

The contribution of HIV fitness to the evolution pattern of reverse transcriptase inhibitor resistance.

All currently recommended anti-retroviral therapy protocols employ reverse transcriptase inhibitors (RTIs). However, mutations within the reverse transcriptase (RT) domain can lead to resistance to these agents and treatment failure. The contribution of the fitness of drug-resistant species to the evolution of RTI resistance has not been elucidated despite its potential implications for therapeutic strategies. In this study we utilized a competitive fitness assay to assess the relative fitness of 13 drug-resistant HIV-1 mutants in the presence and absence of inhibitor. Among these mutants were thymidine analog mutations (TAMs) such as 41L/210W/215Y and 67N/70R/219Q, as well as single mutants such as 103N and 181C that confer high-level resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as nevirapine. These studies revealed that 67N/70R and 67N/70R/219Q were fitter than the 70R progenitor species, and the acquisition of 41L by 215Y substantially increased its fitness in the absence of drug. We also observed that 215Y was more fit than 70R and 67N/70R, and that 41L/215Y and 41L/210W/215Y were the most-fit species in the presence of zidovudine. Moreover, 103N was fitter than 181C without nevirapine but less fit with nevirapine. From these studies we conclude that viral fitness contributes substantially to the evolutionary pattern of TAMs suggesting that, as for protease inhibitor resistance, mutations can act in primary (increasing resistance) and secondary (increasing fitness) capacities. We also surmise that drug resistance and fitness are competing forces underlying the emergence of nevirapine resistant mutants 103N and 181C.

Recombinant vesicular stomatitis viruses encoding simian immunodeficiency virus receptors target infected cells and control infection.

We have constructed VSV recombinants lacking the viral glycoprotein gene and instead expressing rhesus macaque SIV receptors CD4 and CCR5 with or without the receptor DC-SIGN. The recombinant expressing CD4 and CCR5 specifically infected SIV envelope protein-expressing cells. Incorporation of DC-SIGN into the particles required deletion of the cytoplasmic domain. Inclusion of DC-SIGN in the particles definitely enhanced infection, indicating that the enhancement by coexpression of DC-SIGN with CD4 and CCR5 does not require internalization of the virus into cells. The recombinants also specifically infected, killed, and propagated in CEMx174 cells that were first infected with an SIV expressing EGFP. If cells were superinfected with either of the recombinants after the primary SIV infection, the numbers of SIV-infected cells and titers of infectious SIV in the cultures were significantly reduced. Such antivirals can now be tested in the SIV/non-human primate model for AIDS to determine their therapeutic value in vivo.

Endothelial cells promote human immunodeficiency virus replication in nondividing memory T cells via Nef-, Vpr-, and T-cell receptor-dependent activation of NFAT.

Human endothelial cells (ECs) enhance human immunodeficiency virus (HIV) replication within CD4(+) memory T cells by 50,000-fold in a Nef-dependent manner. Here, we report that EC-mediated HIV type 1 replication is also dependent on an intact vpr gene. Moreover, we demonstrate that despite a requirement for engaging major histocompatibility complex (MHC) class II molecules and costimulators, EC-stimulated virus-producing cells (p24(high) T cells) do not proliferate, nor are they arrested in the cell cycle. Rather, they are minimally activated, sometimes expressing CD69 but not CD25, HLA-DR, VLA-1, or effector cytokines. Blocking antibodies to interleukin 2 (IL-2), IL-6, IL-7, or tumor necrosis factor do not inhibit viral replication. Cyclosporine effectively inhibits viral replication, as does disruption of the NFAT binding site in the viral long terminal repeat. Furthermore, in the presence of ECs, suboptimal T-cell receptor (TCR) stimulation with phytohemagglutinin L supports efficient viral replication, and suboptimal stimulation with toxic shock syndrome toxin 1 leads to viral replication selectively in the TCR-stimulated, Vbeta2-expressing T cells. Collectively, these data indicate that ECs provide signals that promote Nef- and Vpr-dependent HIV replication in memory T cells that have been minimally activated through their TCRs. Our studies suggest a mechanism for HIV replication in vivo within the reservoir of circulating memory CD4(+) T cells that persist despite antiretroviral therapy and further suggest that maintenance of immunological memory by MHC class II-expressing ECs via TCR signaling may contribute to HIV rebound following cessation of antiretroviral therapy.

Human endothelial cells enhance human immunodeficiency virus type 1 replication in CD4+ T cells in a Nef-dependent manner in vitro and in vivo.

Infected CD4+ T cells are the primary sites of human immunodeficiency virus type 1 (HIV-1) replication in vivo. However, signals from professional antigen-presenting cells (APCs), such as dendritic cells and macrophages, greatly enhance HIV-1 replication in T cells. Here, we report that in cocultures, vascular endothelial cells (ECs), which in humans can also serve as APCs, can enhance HIV-1 production of both CCR5- and CXCR4-utilizing strains approximately 50,000-fold. The observed HIV-1 replication enhancement conferred by ECs occurred only in memory CD4+ T cells, required expression of major histocompatibility complex class II (MHC-II) molecules by the ECs, and could not be conferred by fixed ECs, all of which are consistent with a requirement for EC-mediated T-cell activation via T-cell receptor (TCR) signaling. Deletion of nef (Nef-) decreased HIV-1 production by approximately 100-fold in T cells cocultured with ECs but had no effect on virus production in T cells cocultured with professional APCs or fibroblasts induced to express MHC-II. Human ECs do not express B7 costimulators, but Nef- replication in CD4(+)-T-cell and EC cocultures could not be rescued by anti-CD28 antibody. ECs act in trans to enhance wild-type but not Nef- replication and facilitate enhanced wild-type replication in naive T cells when added to T-cell or B-lymphoblastoid cell cocultures, suggesting that ECs also provide a TCR-independent signal to infected T cells. Consistent with these in vitro observations, wild-type HIV-1 replicated 30- to 50-fold more than Nef- in human T cells infiltrating allogeneic human skin grafts on human huPBL-SCID/bg mice, an in vivo model of T-cell activation by ECs. Our studies suggest that ECs, which line the entire cardiovascular system and are, per force, in frequent contact with memory CD4+ T cells, provide signals to HIV-1-infected CD4+ T cells to greatly enhance HIV-1 production in a Nef-dependent manner, a mechanism that could contribute to the development of AIDS.

Impact of Nef-mediated downregulation of major histocompatibility complex class I on immune response to simian immunodeficiency virus.

Functional activities that have been ascribed to the nef gene product of simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) include CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, downregulation of other plasma membrane proteins, and lymphocyte activation. Monkeys were infected experimentally with SIV containing difficult-to-revert mutations in nef that selectively eliminated MHC downregulation but not these other activities. Monkeys infected with these mutant forms of SIV exhibited higher levels of CD8(+) T-cell responses 4 to 16 weeks postinfection than seen in monkeys infected with the parental wild-type virus. Furthermore, unusual compensatory mutations appeared by 16 to 32 weeks postinfection which restored some or all of the MHC-downregulating activity. These results indicate that nef does serve to limit the virus-specific CD8 cellular response of the host and that the ability to downregulate MHC class I contributes importantly to the totality of nef function.

Competitive fitness of nevirapine-resistant human immunodeficiency virus type 1 mutants.

Determining the fitness of drug-resistant human immunodeficiency virus type 1 (HIV-1) strains is necessary for the development of population-based studies of resistance patterns. For this purpose, we have developed a reproducible, systematic assay to determine the competitive fitness of HIV-1 drug-resistant mutants. To demonstrate the applicability of this assay, we tested the fitness of the five most common nevirapine-resistant mutants (103N, 106A, 181C, 188C, and 190A), with mutations in HIV-1 reverse transcriptase (RT), singly and in combination (for a total of 31 variants) in a defined HIV-1 background. For these experiments, the 27 RT variants that produced viable virus were cocultured with wild-type virus without nevirapine. The ratios of the viral species were determined over time by utilization of a quantitative real-time RT-PCR-based assay. These experiments revealed that all of the viable variants were less fit than the wild type and demonstrated that the order of relative fitness of the single mutants tested was as follows: 103N > 181C > 190A > 188C > 106A. This order correlated with the commonality of these mutants as a result of nevirapine monotherapy. These investigations also revealed that, on average, the double mutants were less fit than the single mutants and the triple mutants were less fit than the double mutants. However, the fitness of the single and double mutants was often not predictive of the fitness of the derivative triple mutants, suggesting the presence of complex interactions between the closely aligned residues that confer nevirapine resistance. This complexity was also evident from the observation that all three of the replication-competent quadruple mutants were fitter than most of the triple mutants, and in some cases, even the double mutants. Our data suggest that, in many cases, viral fitness is the determining factor in the evolution of nevirapine-resistant mutants in vivo, that interactions between the residues that confer nevirapine resistance are complex, and that these interactions substantially affect reverse transcriptase structure and/or function.