Caenorhabditis elegans as a novel model system for studying these effects.Highly active antiretroviral therapy (HAART) has significantly increased life expectancy of the human immunodeficiency virus (HIV)-positive population. Nevertheless, the average lifespan of HIV-patients remains shorter compared to uninfected individuals. Immunosenescence, a current explanation for this difference invokes heavily on viral stimulus despite HAART efficiency in viral suppression. We propose here that the premature and accelerated aging of HIV-patients can also be caused by adverse effects of antiretroviral drugs, specifically those that affect the mitochondria. The nucleoside reverse transcriptase inhibitor (NRTI) antiretroviral drug class for instance, is known to cause depletion of mitochondrial DNA via inhibition of the mitochondrial specific DNA polymerase-γ. Besides NRTIs, other antiretroviral drug classes such as protease inhibitors also cause severe mitochondrial damage by increasing oxidative stress and diminishing mitochondrial function. We also discuss important areas for future research and argue in favor of the use of  Retrovirus of the Lentivirus genus that primarily infects cells of the host immune system. Once an individual is infected, HIV-1 replication takes place in several steps. In the first step, the virion attaches itself to the host cell with the help of co-receptors, whereupon it fuses with the host cell membrane and the two single-stranded RNA molecules and three different viral enzymes are released into the host cell cytoplasm. The viral reverse transcriptase transcribes the viral RNA into DNA, at which point the viral DNA is transported into the nucleus. With the aid of the viral integrase the viral DNA is processed and incorporated into the host genome. The integrated viral DNA, now known as a provirus, is transcribed and translated by the host machinery to synthesize viral proteins and single-stranded RNA for new virions. After assembly of these components at the plasma membrane, the new virions bud off and mature using the viral protease, completing the HIV-1 life cycle  is a e Figure . HIV infFigure 1), and are used in unison to increase therapy efficacy, overcome problems of tolerance, and decrease emergence of viral resistance. The major classes include the entry inhibitors (EIs), the nucleoside reverse transcriptase inhibitors (NRTIs), the non-nucleoside reverse transcriptase inhibitors (NNRTIs), and the protease inhibitors (PIs). The additional two anti-HIV drug classes are the maturation inhibitors (MIs) and integrase inhibitors (IIs), of which most compounds are still in clinical development.For the treatment of HIV-1 infection there are currently six different classes of anti-HIV drugs. Each class of drug acts on a particular aspect of the viral life cycle (Table 1) and often supplemented with one drug from another class . Due to the large variety in drug combinations, standard HAART has been defined as one or more NRTIs combined with a PI  homeostasis. Therefore, a perturbation of any of these functions impairs cellular life-expectancy and has been shown to have tissue and systemic repercussions including accelerated aging . In consBecause mitochondria contain their own DNA, mitochondrial genome integrity is essential for organelle function. The mtDNA encodes vital components of the mitochondrial respiratory chain and therefore damage to mtDNA is directly detrimental to energy metabolism and organelle fitness. Not surprisingly, cell senescence and aging are associated with an increase in the amount of damaged mtDNA. Additionally, accumulation of mutations in mtDNA is known to increase with age, and aberrant mtDNA replication contributes to premature-and-accelerated-aging phenotypes .Figure 2; alovudine or FLT), known for its high toxicity, can cause DNA fragmentation and induce apoptosis (zidovudine or AZT) and 2′,3′-didehydro-2′,3′-deoxythymidine (stavudine or d4T) also disrupt telomerase maintenance and have telomere shortening effects, properties often related to cell senescence and aging  is used in the HAART cocktail. RTV impedes the enzyme cytochrome P450-CYP3A4, which is responsible for the metabolism of xenobiotics, and therefore RTV induces an increase in intracellular drug concentrations in the patient  induced ROS production, independent of mtDNA depletion or damage, a finding that emphasizes the impact of antiretroviral-induced ROS toxicity , two frequently prescribed PIs, can increase ROS production in human arterial endothelial cells (Indinavir (IDV) and nelfinavir (NFV) have been shown to elicit ROS production in skin fibroblast cultures in vitro and in patients’ adipose tissue in vivo (Saquinavir (SQV) however, was shown to cause apoptosis in human umbilical vein endothelial cells via higher levels of ROS production -treated human hepatocytes show widespread mitochondrial swelling with poorly organized cristae . C. elegans demonstrated that NRTIs decrease mtDNA copy number, disrupt both structure and function of mitochondria and shorten the average lifespan. Using oxygen consumption as a measure of mitochondrial function, NRTIs were shown to induce a rapid decrease in mitochondrial fitness. These findings compare well to earlier studies wherein abnormal mitochondrial respiratory activity was correlated with altered expression or deficiency in various respiratory chain complexes .omplexes . AdditioFigure 3). However, there are a number of questions that remain unanswered, simply because we do not fully understand the effects of antiretroviral drugs individually, let alone in combination.With the increase in life expectancy it has only recently become clear that HIV-1 patients are suffering from symptoms of aging ahead of time. In this review we postulate that strong correlations exist between antiretroviral drug-induced mitochondrial toxicity and premature and accelerated aging (For example, questions remain about mitochondrial toxicity of NRTIs beyond the polymerase-γ theory. Various cellular transport systems interact with NRTIs and once inside the cell NRTIs are actively phosphorylated from their pro-drug form . ChangesC. elegans as a model system to study the effects of antiretroviral therapy on premature and accelerated aging. Using C. elegans we can begin to study the effects of specific genetic backgrounds on HAART toxicities. Effects of HAART seen in the nematode can direct more specific research into human conditions. In addition, C. elegans could provide an easy platform, not just for toxicity studies of various antiretroviral drugs, but also to screen for suitable compounds that neutralize toxic effects of HAART, which remains crucial as long as total HIV eradication is not possible. Combining these genetic and toxicology approaches we can initiate research leading to efficient, personalized, anti-HIV treatment in humans.We propose the use of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aim of our study is to investigate the clinical and immunological outcomes according to first-line HAART adherence in a large cohort of HIV-infected patients in Burkina Faso.A retrospective study was conducted between 2001 and 2009 among patients from two urban medical centers [St. Camille Medical Center (CMSC) and “Pietro Annigoni” Biomolecular Research Center (CERBA)] and 1 in the rural District of Nanoro . Socio-demographical and clinical data were analyzed. Adherence was evaluated through a questionnaire investigating 5 key points related to drugs, consultations and blood exams, by assigning 0 to 2 points each up to 10 points overall. Data were collected at baseline and regularly thereafter. Adherence score was considered as a continuous variable and classified in optimal (8–10 points) and sub-optimal (0–7 points). Immunological outcome was evaluated as modification in CD4+ T-cell count over time, while predictors of death were explored by a univariate and multivariate Cox model considering adherence score as a time-varying covariate.vs 0–7, Δ=53.8, p=0.004). Survival multivariate analysis revealed that covariates significantly related to death included being followed at CERBA (urban area) or Nanoro , and receiving a regimen not including fixed dose combinations, ; conversely, an increasing adherence score as well as an optimal adherence score were significantly related to survival (p<0.001).A total of 625 patients were included: 455 (72.8%) were females, the median age was 33.3 (IQR 10.2) years, 204 (32.6.%) were illiterates, the median CD4+ T-cell count was 149 (IQR 114) cells/μl at baseline. At the end of the observation period we recorded 60/625 deaths and 40 lost to follow-up. The analysis of immunological outcomes showed a significant variation in CD4+ T-cell count between M12 and M24 only for patients with optimal adherence , with a significant Δ between the two adherence groups at M24 (8–10 Adherence to HAART remains pivotal to build up a good therapeutic outcome. Our results confirm that, according to our adherence system evaluation, less adherent patients have a higher risk of death and of inadequate CD4+ count recovery. It is estimated that about 32.2 million people were living with HIV at the end of 2012. Two thirds of HIV infections (approximately 23.5 million people) were in Sub-Saharan Africa, where a high prevalence of HIV/AIDS is reported (4.7%)  and 1 in the rural District of Nanoro . We enrolled all HIV-infected adults who started HAART according to WHO international guidelines available in the study period ,19, betwAll data were collected in Burkina Faso (a West-African country with a population of about 13.2 million people) which is among the poorest countries in the world (181st position out of 187) . Notably0), on day 15 (D15), each month during the first semester (M1-M6) and thereafter every three months since HAART initiation. Each time, WHO clinical stage and adherence score (from D15 on) were registered. Furthermore, CD4+ T cell count was scheduled on D0 and then at M6, M12 and M24, together with some routine laboratory tests . During the study, patients were categorized into (i) followed, (ii) lost to follow-up, (iii) dead and (iv) referred to other Medical Centers. A patient was considered lost to follow up if he/she had missed 2 consecutive visits without a further contact. Demographical and clinical variables, social behaviors, family conditions and literacy were also analyzed.Consultations and data collection were performed by a medical doctor, available for both scheduled and emergency visits. Routine clinical visits where conducted according to WHO international guidelines ,19. PatiAdherence was assessed through the administration of a questionnaire regarding 5 different aspects: (1) pills count, (2) delays and omissions in taking pills referred from patient, (3) knowledge of posology, (4) punctuality on consultations, (5) proper execution of blood tests as planned at the previous consultation. A score was estimated by the medical doctor who performed the visit through the assignment of 0 , 1  or 2  points to each feature, up to 10 points overall. This score has not been validated. Treatment interruption was determined as a referred and/or planned history of stopping and resuming treatment, and corresponds to a zero score. On the other hand, a full adherence score was attributed to patients (1) who took at least 95% of pills (as stated through direct count and self report of missed doses), (2) at the right time of the day (with 30 minutes tolerance), (3) who properly knew the regimen posology, (4) presented on scheduled consultations  and (5) underwent blood tests as planned. If a patient was lost to follow up (see above), no value was assigned to the adherence score, and the patient was considered in the statistical analysis only according to the period of follow-up.Associations between categorical variables were evaluated using Chi-square tests or Fisher exact tests where needed. Overall survival was modeled using Cox models considering adherence score as time-varying covariate. The following variables were considered in univariate analyses: center, age (continuous), gender, religion, living area, HIV type infection, literacy, CD4+ T-cell count, WHO clinical stage, HAART regimen including fixed dose combination (FDC), calendar year and adherence score  and those significant were included in a multivariate Cox model. Model selection for the multivariate Cox model was based on likelihood ratio tests. The final model included center, HAART regimen including FDC, calendar year, WHO clinical stage and adherence score . Both calendar year and WHO clinical stage were included as a stratification variable. When performing the survival analysis, all patients lost to follow-up were censored.CD4+ T-cell count variation over time was modeled using a random-intercept linear mixed model  with CD4Considering the possibility of a missing not at random pattern and its potential effects on model estimates we conducted a sensitivity analysis fitting a linear mixed model as specified above only on subjects that had a visit at 24 months. Results did not differ from the model with all data (data not shown).survival[nlme[All analysis were performed using the R statistical software  and specsurvival and nlmeival[nlme. All staThe Study was conducted in compliance with the Helsinki declaration. The Joint Centre Médicale Saint Camille and Centre de Recherche Biomoléculaire Pietro Annigoni Ethics Committee were duly informed about the ongoing research and gave their permission. In fact, the CNERS  is questioned only in case of interventional clinical trials as well as in international or national pharmacological studies: therefore in this case it was not informed. Moreover, being a retrospective non-interventional study founded on post hoc analysis of data already present in patients’ files, and collected only for clinical indications, no written informed consent was asked to patients.A total of 625 HIV-infected patients who started HAART between January 2001 and February 2009, were included. Socio-demographic and clinical features of this cohort are showed in Tables At the beginning of antiretroviral treatment, a significant proportion of patients belonged to an advanced WHO clinical stage  and the median CD4+ absolute count was 149 cells/μl (IQR 114). Overall, 63/625 patients (10.1%) had a concurrent HBV or HCV chronic infection. The 87.8% (549/625) of prescribed HAART regimens consisted of two nucleoside reverse transcriptase inhibitors (NRTIs) and a non-nucleoside reverse transcriptase inhibitor (NNRTI), while the remaining 12.2% (76/625) were made up of two NRTIs and a protease inhibitor (PI). Moreover, 75.8% (474/625) of patients received FDC .By the end of follow-up, 9.6% of patients (60/625) had died, 5.9% (37/625) had been referred, 6.4% (40/625) were lost to follow-up and 78.1% (487/625) were still under active monitoring. Median follow-up for subject censored before the end of the study (24 months) was 8.97 months, in particular median follow-up for dead patients was 6.54 months.vs 0–7, Δ = 53.8, p = 0.004). Figure To evaluate the immunological outcome, we considered two different groups of patients based on adherence score considered at each time-point. We divided patients with optimal adherence 8–10 points) from those with sub-optimal adherence (0–7 points). We observed a significant increase in CD4+ T-cell count from baseline to M6, both in the 0–7  and 8–10 groups , while the difference between the two groups at M6 was not significant (p = 0.56). Moreover, from M6 to M12 we found a CD4+ T-cell count significant variation in both adherence groups (Δ = 62.6 p < 0.001 and Δ = 38.1 p < 0.001 in the 0–7 and 8–10 group respectively), and a non-significant difference was observed between the two groups at M12 (p = 0.39). A significant variation was observed between M12 and M24 only in the 8–10 group  with a significant Δ between the two groups at M24  and adherence evaluated through our score: our data substantially confirm previous findings ,26 suggeA significant proportion of our study population was represented by women (72.8%), since CMSC is a medical center devoted to “mother and child health-care” where patients are predominantly pregnant women and their HIV-infected partners . Hence, Moreover, people coming from Nanoro rural District often emigrate particularly to Ivory Coast, delaying access to HIV screening and care . The higThe lack of a gold standard method to evaluate adherence brought us to use a multi-parametric strategy based on a combination of questionnaires, pills count, punctuality on clinical consultations and blood tests. This method allowed us to bypass single measurements–related biases, by taking into account several aspects of treatment adherence according to WHO recommendations . PreviouThe increase in CD4+ T-cell count was similar in patients with optimal and suboptimal adherence (0–7 and 8–10 group) during the first year of follow-up, while a better adherence (8–10 group) during the second year was significantly related to a progressive increase in CD4+ T-cell count. Our findings are in line with those described in the DART trial where investigators observed a worse immunological recovery (assessed through the CD4+ T-cell count evolution) among less-adherent patients.vs 0–7 points).We also found that good adherence is strongly related to a lower risk of death in univariate and multivariate models, given that the adherence score was considered as continuous variable or as a binomial variable (8–10 vs >50 cells/μl) did not.The univariate analysis of death predictors revealed that living in urban areas was associated to a better survival, while the other socio-demographic features  were not. In fact, living in a urban area is likely to be associated with fewer disruptions in access to medicines, which seem to facilitate adherence -37. MoreThe multivariate analysis showed an increased risk of death among patients who were followed at CERBA or Nanoro as compared to those followed at CMSC, as a consequence of the above-mentioned differences between patients enrolled in the three healthcare facilities.In addition, receiving a regimen including FDCs was confirmed to be significantly related to survival, suggesting the importance of a wider access to HAART in low-income Countries with a particular attention to compact regimens .At any rate, some limitations should b e taken into consideration while interpreting our study. First of all, it was not possible to collect full data for the whole cohort since medical records were sometimes incomplete or because patients had died, had been lost to follow-up or transferred elsewhere. Second, our adherence score has not been validated and we are not able to establish the relative weight of each component of the score. Moreover, we did not compare this score with other methods to evaluate adherence since validation of this score was not the aim of this study. Lastly, the assignation of our score is purely at the discretion of the physician who performed the visit, and it should not be interpreted as a objective measure. Although the scores assigned at each visit were equally weighted, despite the variability in the length of assessment, the patients were followed in accordance with current WHO guidelines and all data were analyzed only retrospectively.In summary, our findings confirm that optimal adherence is strongly related to better immunological recovery and survival, though assessed through a non-validated score. Our results reinforce the need of additional support such as reminders and enhanced community education . In factEF have received travel grants or speakers honoraria from several Pharmaceuticals Companies producing antiretroviral drugs and consultancy fees from Gilead and Abbvie but this did not influence the content of this paper. FC is acting as Principal Investigator in investigational Clinical Trials sponsored by Pharmaceuticals Companies producing antiretroviral drugs. The remaining authors declare that they have no competing interests.Study concept and design: EF, SO, VP. Acquisition of data: EF, SO, AN. Analysis and interpretation of data: EF, SO, GS, SC, VP. Drafting of the manuscript: EF, SO, GS, PFG. Statistical Analysis: SC. Critical revision of the manuscript for important intellectual content: EF, SO, GS, SC, VP, PR, PFG, AN, PO, JS, SP, FC. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/153/prepub


We collected data on feasibility and acceptability of a real-time web-linked adherence monitoring container among HIV-positive injection drug users (IDU) in China. “Wisepill” uses wireless technology to track on-time medication dosing. Ten patients on antiretroviral therapy (ART) at the Guangxi CDC HIV clinic in Nanning, China, used Wisepill for one ART medication for one month. We monitored device use and adherence and explored acceptability of the device among patients. Mean adherence was 89.2% (SD 10.6%). Half of the subjects reported a positive overall experience with Wisepill. Seven said that it was inconvenient, supported by comments that it was large and conspicuous. Five worried about disclosure of HIV status due to the device; no disclosures were reported. Twelve signal lapses occurred (5.4% of prescribed doses), of which one was due to technical reasons, nine to behavioral reasons , and two to unclear reasons. Although the technical components must be monitored carefully, and acceptability to patients presents challenges which warrant further exploration, the Wisepill device has potential for adherence interventions that deliver rapid adherence-support behavioral feedback directly to patients, including IDU. The use of wireless technology appears uniquely promising for providing time-sensitive communication on patient behavior that can be harnessed to maximize the benefits of HIV treatment. Interventions to improve adherence to antiretroviral therapy (ART) are urgently needed among HIV-positive patients, particularly in developing countries, where most of the world's HIV-infected population lives. China has experienced a major HIV/AIDS epidemic since the 1990s, with an estimated 780,000 persons living with HIV in 2011 .Recently, wireless technology has emerged as a potential tool for monitoring medication adherence in real time –8. This In a previous intervention study (Adherence for Life (AFL)), which tested the use of electronic drug monitoring (EDM) data as an information and counseling tool among primarily injection drug using (IDU) ART patients in China's Yunnan province, we found that monthly EDM-informed counseling significantly improved mean ART adherence and CD4 counts . These fTo lay the groundwork for a larger intervention study in China using wireless technology to monitor adherence and intervene in real time among injection drug users (IDU), we conducted an in-depth feasibility and acceptability study on the use of the device in a small cohort of IDU patients. This approach allowed us to glean important information about the potential for use of the device given the sociocultural circumstances of provision of ART in China, including the following: HIV is highly stigmatized; ART is relatively new, and policies on provision have changed in recent years; use of cellphone technology, while ubiquitous and familiar, is in rapid transition; and the clinician-patient relationship, particularly when the patient is an IDU, is extremely hierarchical. The study was led by Boston University's Center for Global Health and Development (CGHD), with enrollment at the Guangxi Centers for Disease Control (CDC) antiretroviral therapy (ART) clinic in Nanning, China, a large clinic that currently treats over 1,400 patients, including 1,040 adults, 370 children, and a high proportion of IDU among the adult patients. We provided ten current or former IDU patients who were being treated with ART at the clinic with a Wisepill device for one or more of their ART medications. We then monitored their use of the device for one month without the subjects receiving any information about their adherence data. This allowed us to collect pilot data on (1) acceptability of use of the Wisepill device among a Chinese patient population and (2) feasibility of monitoring patient adherence using the device in China. We limited the sample size to ten subjects due to budget constraints and to permit a rapid assessment prior to initiating the larger study, but often formative feasibility studies of this type have used similar sample size , 15.The study involved the following sources of patient data: (1) a baseline sociodemographic and health history questionnaire; (2) a brief self-report of adherence and use of Wisepill from subjects after 1 month of use; (3) continuous adherence data from the Wisepill device; (4) continuous data on reasons for signal lapses; and (5) CD4 and VL test results from patients' medical charts.The baseline and monthly interviews were administered in Mandarin Chinese by trained clinic staff. In addition to sociodemographic information and health history, the baseline instrument also covered route and duration of HIV infection, history of depression, and alcohol/drug use. Besides self-reported adherence, the monthly form included quantitative and open-ended questions covering acceptability and usability of the Wisepill device , as well as perceptions of a possible intervention that makes use of Wisepill.The Wisepill device measures 30 × 60 × 130 mm and holds up to 60 small pills in two inner compartments see . It is pData on Wisepill openings were transmitted automatically and continuously over the month, as described previously. Investigators downloaded the data from a password-protected account on the Wisepill website. From these data, we calculated mean adherence over the one-month period using the following formula: (number of doses taken ± one hour of dose time)/(number of prescribed doses); the approach used in the AFL study based on the adherence measure that was most significantly associated with viral load . We alsoWe investigated all Wisepill signal lapses by a phone call to the subject within 2-3 days to determine whether a lapse was due to technical failure  or had a behavioral cause . This information was recorded in a “signal lapse” report that we created for each subject over the month of data collection. We then calculated separately the proportion of technical issues and behavioral reasons among all expected Wisepill signal lapses.CD4 and viral load test results were collected from subjects' medical charts as background information on subjects; no additional blood draws were required for the study. For all quantitative data, we calculated descriptive statistics . Qualitative data from the open-ended questions were analyzed using a thematic approach. All quantitative analyses were conducted using SAS 9.1 . http://ClinicalTrials.gov/ was not required.The study was approved by the Institutional Review Boards at Boston University Medical Center and the Guangxi Provincial Center for Disease Control, Nanning, China. All subjects provided written informed consent prior to enrollment. Because it was not a clinical trial, registration with μL (SD = 170 cells/μL). Seven had a history of depression, and two were alcoholic. Four reported having hepatitis C, and two reported having had syphilis. Nine of the ten had an educational level of middle school or lower. Most subjects had a low monthly income, typical of IDU in China. While one did not know and one reported a monthly income of 1001–5000 Yuan (approximately $ 159–794), eight had a monthly income of 1000 Yuan (approximately $ 156) or less. The ten subjects were current and former injection drug users, and most (7) reported that they had been infected via shared needles. The mean age of the sample was 32.7 years (SD = 5.3); seven of the ten were men, and six were married. On average, the subjects had been on ART 40.9 months (SD = 29.4) and had a mean baseline CD4 count of 383 cells/The total prescribed (expected) number of device openings was 614 for the month of study. Twelve lapses occurred over the month ; thus 33/614 or 5.4% of prescribed doses were not recorded in real time. The mean duration of real-time lapses was 2.75 doses (range 1 dose–21 doses). Of the lapses, one was due to technical reasons, nine to behavioral reasons, and two to unclear reason. The technical problem was a lack of airtime on the SIM card, but this resulted in 21 consecutive openings missed because the subject could not be contacted. After the SIM card was reactivated, these initially missed doses were eventually received by the server, so they could be included in the subject's adherence calculation. If these 21 openings not recorded in real time are included, the proportion of openings not measured by the device was 2.0% (12/614). Of the nine behavioral lapses, seven were due to a subject forgetting to take a dose, and one was due to a subject forgetting to close the device after use. The final behavioral lapse was due to a patient purposely not using the box . Using Wisepill data, adherence was 97.2% (SD = 3.5%) of prescribed doses taken and 89.2% (SD = 10.6%) using a measure that incorporates dose timing (detailed previously). Using a visual analogue scale at the monthly visit, self-reported adherence was 98.5% (SD = 3.2%). In quantitative questions, half of the subjects reported a positive or very positive overall experience with Wisepill; the other five reported a “somewhat negative” overall experience. Seven were willing or very willing to participate in a larger intervention study. Eight found the device very easy to use. However, seven said that it was inconvenient or very inconvenient. Five were somewhat or very worried about disclosure of their HIV status due to the device; no disclosures were reported.  Knowing that someone is monitoring my medication spurs me to take my medication better. The pill box is just a normal drug container; there is nothing good or bad about it. I like the pill box, because first of all, there is no special label on the pillbox , so no one will know what medicine I am taking; second, it records the time of medicine taking, which helps me take doses better.Three reported a negative experience. One said the device was inconvenient to carry and therefore a burden, and one did not like the feeling of being watched .In the open-ended questions exploring acceptability and usability of the Wisepill device , six patients reported a positive feeling about the device, of whom four said knowing that someone was monitoring their adherence helped them take their medications more regularly. As two subjects explained: It is too big to carry. I have a feeling of unease using the Wisepill in front of other people. I never take the pillbox outside. The pillbox is too big. I have a big concern that using this pill box could disclose my HIV status. When asked about difficulties encountered using the Wisepill device, one subject reported having no problem with the device at all, while another had no major problem because he/she “always carried it in a bag.” However, eight indicated that the device was inconvenient or felt uncomfortable using it. Many found it too big; one patient thought it should be wider. Additional feedback included the following: two subjects did not like that it could only hold one drug; two worried that drugs would be damaged while carrying the device; two were concerned about others seeing it. These were typical statements: I have no job in the past one month. I always take my medicine at home. Even when I go to parties or meeting friends, I did not bring the pill box with me. That's why, sometimes when I returned home late, I also took my medications late. Subjects reported a variety of ways to manage their use of the device. One compensated for the inconvenience by always taking doses at home, another changed his/her dose time to avoid carrying the device to work, and one told friends that the drugs were a hepatitis medicine instead of HIV-related medications. Seven of the patients reported keeping their device at home exclusively; six seemed to keep it hidden, whether at home or outside. Patients were also asked what they did with the device when they traveled. Half reported never taking the device away from home; the other half reported having found ways to travel with the device. One of the former elaborated thatWhen asked specifically about text messages, only four patients thought reminder messages would be helpful. Of the six who did not, one was a truck driver and could not read messages while driving, one did not read text messages at work, and one thought that text message reminders are not much more useful than an alarm. Yes, the reminder message could help. I usually read short messages. I hope the message could be as simple as possible, like a symbol would be good.  Yes, the reminder message could help. The SMS could just be “It's time to take your medication, do not forget”. I do not worry about other people (knowing) my health status through this message.Three subjects were not willing to participate in a subsequent larger study. All three did not think text messages would work; two said the box was too inconvenient. One was concerned about possible disclosure of status via messages, and one did not use text messages.When asked about possibly participating in a larger study using Wisepill with text message reminders, six said they would be willing to participate, of whom four said it would help them take their ART medications on time. As two explained: This study has demonstrated that use of a real-time, SMS-enabled web-linked ART adherence monitoring system is technically feasible in an urban Chinese clinical setting among predominantly IDU HIV patients. Only a few minor technical difficulties were encountered and easily addressed; the issue of acceptability is more complex.The technical lapses due to inadequate time on the SIM card bear some discussion. In terms of adherence measurement, these types of lapses do not represent a major concern; as long as there is adequate battery power in the device, the openings are recorded but are just not transmitted to the server until the airtime on the SIM card is replenished. In other words, the transfer of information is delayed, but the data needed for characterizing adherence rates are not affected. Because the system did not work perfectly for capture of data in real time, however, this presumably would have an impact on its effectiveness as a tool for promoting adherence, including triggered SMS reminders. Moreover, if an intervention was designed to send an SMS message when the server does not receive a signal within a set time window, then the server would send an SMS reminder to the participant regardless of the reason for the lapse. In such an intervention context, the subject with the lapse of 21 doses in the present study would have received 21 reminder messages even though she/he actually used the device correctly and took all of the doses on time. Readers in the USA should also note that in most other countries including China, a mobile phone that has run out of SIM card airtime can still receive text messages. The main risk here is that patients might become annoyed. In short, the logistical requirements of the device are real  and can cause some problems. A technical lapse due to the airtime issue does not preclude us from measuring and understanding adherence behavior, but to take full advantage of the technical capacity of the device, researchers and clinicians must pay close attention to these logistical requirements.The study raises greater concerns about the acceptability of the device to patients. Several subjects complained about the size and inconvenience of the device. To overcome these concerns, consideration should be given to the design and appearance of the device. Some ideas might include devising a carrying case or bag to make the device less conspicuous or creating a cell phone case that would hold both the phone and the monitoring device. A smaller less conspicuous box, perhaps retaining the removable pill containers for easy refilling that Wisepill currently offers, might also be considered. In addition, although no disclosures were reported during the pilot, subjects did raise concerns about disclosure and stigma due to the device in both closed and open-ended questions. This is a potential problem with the device which deserves further attention . That saA limitation of the study is the small sample of ten individuals who used the device, with no accompanying intervention that might foster more positive feelings about the device. The full wireless capabilities of the device were not tested and could not be appreciated. It is possible that subjects' attitudes might change after having the experience of receiving SMS reminders, particularly if they could see their adherence increase or their health improve as a result. The technical findings from the study are more persuasive, but the qualitative aspects are by definition limited and subjective. Therefore, we revised aspects of the subsequent larger randomized controlled trial currently underway in the same clinic, including our data collection instruments, to allow more extensive collection of qualitative data. This will allow us to obtain more conclusive data from the larger study, in which intervention subjects receive a tailored SMS reminder message if they are more than 30 minutes late taking a dose. Those who are suboptimal adherers (<95%) also review a printout of their adherence behavior over the previous month in counseling sessions. The design of this larger study will permit us to collect quantitative and qualitative data on acceptability over time for both intervention and control groups and thereby gain a deeper understanding of the evolution of acceptability of the device over time in this Chinese patient population.Although the technical logistical requirements must be monitored carefully, and acceptability to patients is not perfect, the Wisepill device shows potential for adherence interventions that deliver rapid adherence-support behavioral feedback directly to patients, including IDU, as well as in clinical settings. The fact is that each current adherence monitoring device or technology has its advantages and disadvantages, as well as a certain measure of intrusion for patients. Wisepill involves a high degree of intrusion but also a high degree of accuracy as well as the unique benefit of real-time monitoring which allows for real-time interventions to improve adherence before the substantial negative impact of poor adherence can accumulate to cause substantial harm. Other adherence measures  may involve a lesser burden for patients, but they are less accurate and do not permit real-time interventions. Electronic drug monitoring reviewed at the time of clinic visit (such as the Medication Event Monitoring System (MEMS)) imposes a similar degree of intrusion on patients compared to Wisepill, but again, there is no opportunity to intervene in real time. Thus, while the Wisepill delivery system is not perfect, we believe that it holds substantial advantages over other currently available adherence monitoring options. Just as so many recent technological advances offer the possibility of client-centered approaches, the use of wireless technology appears uniquely promising for providing time-sensitive communication on patient behavior that can be harnessed to maximize the benefits of HIV treatment.


There is limited data on antiretroviral therapy (ART) adherence among patients in Eastern Europe, despite the high incidence of HIV infection and the growing number of HIV-infected individuals who are being prescribed ART. We conducted a study to measure rates of adherence to ART and factors associated with non-adherence among patients receiving care at an outpatient HIV clinic in Estonia.The study was based on cross-sectional data from a convenience sample of 161 patients receiving outpatient HIV care. Data were obtained via interviewer administered surveys and data abstraction from clinical records. Adherence was measured from 3-day patient self-report.Among the 161 participants , two thirds (64%) had been infected with HIV through intravenous drug use. Most (74%) were co-infected with hepatitis C (HCV). Perfect adherence over the last 3-days was commonly reported [87% (95% CI 80 − 92%)], with non-perfect adherence associated with greater concerns about the potential negative consequences of taking ART [AOR 5.8 (95% CI 1.3 − 45.7)] and fewer antiretroviral medications (ARVs) in one´s current regimen (2 or fewer different ARVs vs 3 or more different ARVs: AOR 17.0 (95% CI 3.7 – 97.6).Self-reported ART adherence in this sample of Estonian HIV-infected patients in clinical care was similar to rates observed in Western Europe and other developed countries. The results suggest that adherence education and support could be most beneficial, if specifically targeting the development of positive beliefs, reduction of negative expectations towards ART and when helping patients manage negative treatment experience, particularly with regimens including multiple ARVs.


There is a paucity of prospective evaluations of bone markers after the initiation of drugs currently recommended to treat HIV infection and results on the evolution of these markers are conflicting. Lastly, the effect of tenofovir on 1,25-(OH)2 vitamin D before and after standard antiretroviral regimens. This was a sub-study of a trial conducted in antiretroviral-naïve patients randomized to tenofovir + emtricitabine in combination with either atazanavir/ritonavir (ATV/r) or efavirenz (EFV). Follow-up lasted 48 weeks. The following bone markers were analyzed: C-terminal cross-laps (CTx), osteocalcin (OC), osteoprotegerin (OPG), and receptor activator of nuclear factor κB ligand (RANKL). Mixed-factorial analysis of variance with random-coefficient general linear model was used to compare their trends over time and linear multivariable regression was performed with a backward selection method to assess predictors of their variations from baseline to week 48. Trends of parathormone and 1,25-(OH)2 vitamin D were also evaluated.We performed a prospective study on the evolution of bone markers, parathormone and 1,25-(OH)2 vitamin D remained stable, though a seasonality variation was demonstrated.Seventy-five patients were studied: 33 received EFV and 42 ATV/r. Significant increases were found for all markers except for RANKL. There was a significant direct association between CTx and OC increases. Multivariable analysis showed that higher glomerular filtration rate (estimated through cystatin C clearance) predicted greater OPG increase, while older age, higher HIV RNA at baseline and use of ATV/r predicted greater CTx increase. A significant increase of parathormone accompanied the evolution of the study markers. 1,25-(OH)These data demonstrate CTx increase (bone resorption marker) corresponding to OC increase (bone formation marker) early upon HAART initiation. Moreover, predictors of bone marker increases have been suggested, possibly indicating that a stricter monitoring of bone health and pro-active interventions are needed in older patients, those with higher HIV RNA, prescribed ATV/r rather than EFV, and with decreased renal function at baseline. Further studies are needed to clarify the mechanisms responsible for up-regulation of bone turnover markers, as well as to understand if and what markers are best correlated or predictive of pathological fractures. Highly active antiretroviral therapy (HAART) consisting of 2 nucleoside reverse transcriptase inhibitors (NRTI)  combined with either a protease inhibitor (PI) or a non nucleoside reverse transcriptase inhibitor (NNRTI)  have dramatically reduced opportunistic infections and death in HIV infected patients. However, despite these benefits, several co-morbidities, including osteoporosis , have emSeveral biochemical markers of bone turnover have been proposed to detect early-onset modifications in bone formation  or bone resorption  . These mThe mechanisms responsible for osteopenia and osteoporosis are uncertain but exposure to certain antiretroviral drugs (in particular a NRTI: tenofovir--TDF--and the PI class), aging, HIV by itself, parathormone (PTH) increase, and vitamin D deficiency may be implicated -10.Discordant results regarding levels and evolution of bone turnover markers in HIV patients on HAART have been demonstrated -16. MoreTherefore, the main objective of this study was to describe the evolution of bone markers in HIV infected patients starting the HAART regimens currently recommended ,18. We aThis was a sub-study of a pilot open-label, multicenter, randomized controlled trial conducted in 91 patients without chronic kidney diseases to evaluate renal function after TDF + emtricitabine (FTC) plus either atazanavir boosted by ritonavir (ATV/r) or efavirenz (EFV) in HIV-infected patients naive to antiretroviral therapy (EudraCT number: 2007-007934-21) .Seventy-five patients who remained in the randomized treatment arms were included. Baseline data included clinical history, CD4+ T-cell count, HIV RNA  and HBV/HCV serostatus. Clinical examination, HIV RNA, CD4+ T-cell count and routine laboratory tests (including creatinine and electrolytes) were assessed during follow-up. Plasma samples were taken and stored at -80°C until they were de-frozen for the present analyses.2 vitamin D  were determined with EIA kits by IDS ; OPG  and RANK-L  were determined with EIA kits by Peprotech ; lastly, PTH was determined with a DIAsource ImmnoAssays S.A. EIA kit . Manufacturers' procedures were followed as indicated in the instruction manuals. All analyses were performed at the Microbiology Section, University of Bologna, Bologna, Italy. The following ranges of normality were considered: OC in women 8.4-33.9 ng/mL, in men 9.6-40.8 ng/mL; CTx in women 0.112-0.738 ng/mL, in men 0.115-0.748 ng/mL. Hyperparathyroidism cut-off was 65 pg/ml, and vitamin D deficiency was defined as < 11 pg/mL. Clinically validated ranges of normality for OPG and RANKL currently do not exist.Enzyme immunoassay techniques were used for all markers: OC , CTx  and 1,25-(OH)Glomerular filtration rate (GFR) was estimated by the CKD-EPI formulae since they are more suitable for patients without chronic kidney disease using both creatinine  and cystThe study was conducted in accordance with good clinical practice (ICH-E6) . The pro2 test (Fisher's exact test) for qualitative variables.Descriptive statistics was calculated for quantitative variables  and qualitative variables (absolute and percentage frequencies). Ninety-five percent confidence intervals (95%CI) were calculated as appropriate. The unpaired Student's t test was used to compare quantitative variables (Wilcoxon's Rank Sum test for variables that were not Gaussianly distributed) and the χThe overall time-variation of the markers was evaluated by a mixed-factorial analysis of variance (ANOVA) with random-coefficient general linear model.[wk 48-bl]) for each marker. Pearson's correlation coefficient was used to assess the relationship of the markers' Δ[wk 48-bl] between each other.Differences between week-48 and baseline values were calculated as delta . Factors that resulted associated with the outcomes at univariate analysis with a p-value < 0.10, as well as factors of clinical importance, such as antiretroviral treatment received, were included in a multivariable regression model with backward selection in order to obtain the most parsimonious set of the variables that predicted Δ[wk 48-bl] for each of the bone turnover markers as outcome measures.Demographical, clinical and laboratory characteristics at baseline . All patients resided in a region between 41° and 45° latitude North; these periods were selected because 1,25-(OH)2 vitamin D production is impossible during the not-sunny months owing to the solar zenith angle, which influences the incident UVB radiation  were as follows: 18.78 (SD: 15.5) ng/mL for OC, 0.33 (SD: 0.31) ng/mL for CTx, and 0.09 (SD: 0.36) ng/mL for OPG. Up to week 48, 32/75 (42.7%) patients for OC and 24/75 (32%) for CTx exceeded the respective cut-offs of normality. Therefore, with respect to baseline, there were significant increases in proportion of patients above the cut-offs (p < 0.01).There was a significant increase in bone markers during the follow-up Figure . Mean Δ[[wk 48-bl] of the study markers showed that OC increased with increasing CTx , while no other significant correlations between each other were found.RANKL remained or became undetectable in 87% patients throughout the follow-up. Linear correlation analysis to explore relationship between Δ[wk 48-bl]) of 15.05 (SD: 35.76) pg/mL  (p = 0.2364); however, as shown in Figure not-sunny period, while a decrease in those who started in the sunny period was found (p < 0.0001). At week 48 patients with a deficit of vitamin D were 19 (25.3%). None of the patients received vitamin D supplementation during the study.There were no significant variations in 1,25-(OH)[wk 48-bl] with higher CD4+/CD8+ T-cell ratio , and with greater eGFR estimated by CKD-EPI based on cystatin C values at baseline , but only the latter maintained a borderline significance at multivariable model , lower CD4+/CD8+ T-cell ratio at baseline , smaller eGFR Creatinine Δ[wk 48-bl] , older age , and higher HIV RNA at baseline . At multivariable analysis including treatment group as clinically significant variable, older age, higher HIV RNA at baseline, and the use of ATV/r were significantly associated with CTx increase. Particularly, as for treatment received, patients prescribed ATV/r had a mean of 0.22 ng/mL CTx increase, independently from the other two factors.CTx Δ[wk 48-bl], no significant associations besides that with CTx Δ[wk 48-bl] were found.For OC ΔThere is a paucity of data on bone turnover markers in HIV-infected patients after HAART. Some studies demonstrated an increase in OC and CTx ,15 and tWe found a significant increase of OPG after HAART, differently from the study by Brown et al.  who founHigher eGFR at baseline (measured by cystatin C using the CKD-EPI formula) predicted OPG increase from baseline to week 48. Glomerular filtration rate estimated through cystatin C may be a reliable method to assess renal function in HIV infected patients because it is less influenced by muscle mass and liver function than the methods based on creatinine ,27. Ther[wk 48-bl] of the CD4+ T-cell count and CTx increase was found in our study (data not shown), and this seems to contradict the previous hypothesis [Older age and increasing HIV RNA at baseline were independently predictive of CTx increase during the follow-up. Since the increase in CD4+ T-cells is greater in patients with higher HIV RNA at baseline ,32 the cpothesis . Therefopothesis ,35. HoweNotably, the use of ATV/r was associated with greater CTx increase in our study. Brown et al.  found th2 vitamin D in order to assess the availability of the final product exerting biological activity. We found that 1,25-(OH)2 vitamin D remained stable, while in other studies TDF seemed to increase it [2 vitamin D needs further considerations.We found a significant PTH increase after HAART, confirming previous studies ,37,38. Trease it ,39. Thisrease it . Therefo2 vitamin D levels are unstable and its values are of difficult interpretation [This study suffers from several limitations. First of all, BMD was not assessed; therefore the early activation of bone markers could not be coupled with BMD reduction. Second, 1,25-(OH)retation . Third, The rapid and significant increase in bone markers observed in the present study reinforce that a strict monitoring is necessary to detect early signs of bone damage as already stated by recent guidelines . MoreoveSome authors  received grants from several Pharmaceutical Companies for participating to advisory board and scientific conferences but the received supports did not influence the content of this paper. The remaining authors declare that they have no competing interests.Study concept and design: CT, EF, DM. Acquisition of data: ACa, II, RB, PN, LS, EQR, NB. Laboratory analyses: ACl, MCR, DG. Analysis and interpretation of data: CT, EF, DM, BMC. Drafting of the manuscript: CT, EF, DM. Critical revision of the manuscript for important intellectual content: MB, II, RB, PN, LS, EQR, DG, NB, LA, BMC. Statistical analysis: BMC. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/38/prepub


Chronic invasive fungal sinusitis is a rare condition, and the conventional treatment surgical debridement and systemic antifungal therapy such as amphotericin B. Recently, voriconazole has demonstrated superior efficacy in the treatment of invasive aspergillosis, compared to amphotericin B. However, its use in invasive fungal sinusitis in an HIV patient has not been reported.We describe a case of chronic invasive fungal sinusitis in a patient with HIV infection successfully treated with surgical clearance and a prolonged course of oral voriconazole.Streptococcal pyogenes and Aspergillus flavus. MRI sinuses in May 2009 demonstrated extensive soft tissue mass involving right maxillary, bilateral ethmoid and frontal sinuses, causing right axial proptosis. Dural enhancement suggested intracranial extension, and there was erosion into pituitary fossa posteriorly. Her only complaint was epiphora, with no visual disturbance. An HIV test was positive. Baseline CD4 count was 395 cells/cm3, and viral load 103,469 copies/ml. She underwent endoscopic sinus surgery to debulk tissue from her sinuses. Grocott staining of biopsy specimen demonstrated presence of fungal hyphae, and tissue culture grew Aspergillus flavus. She was commenced on oral voriconazole 300mg b.d. Due to potential drug interactions with non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI), she was treated with tenofovir, emtricitabine and raltegravir. Follow-up MRI after 4 months of therapy showed marked improvement with resolution of proptosis. She has been on voriconazole for 12 months with no progression.A 39 year old Malawian lady presented to the Ear, Nose and Throat (ENT) surgeons with recurrent nasal polyps and was noted to have proptosis in April 2009. She had had a previous polypectomy in 2007, which grew Invasive fungal sinusitis has been rarely reported in HIV patients, but recent case reports in non-HIV immunocompromised patients have shown a favourable response to new triazoles. It has previously been associated with high mortality even with amphotericin therapy. There are important potential interactions with antiretroviral (ARV) drugs. The optimal duration of treatment for immunocompromised patients is unclear.


Human immunodeficiency virus type 1 (HIV-1) genomes often carry one or more mutations associated with drug resistance upon transmission into a therapy-naïve individual. We assessed the prevalence and clinical significance of transmitted drug resistance (TDR) in chronically-infected therapy-naïve patients enrolled in a multi-center cohort in North America. Pre-therapy clinical significance was quantified by plasma viral load (pVL) and CD4+ cell count (CD4) at baseline. Naïve bulk sequences of HIV-1 protease and reverse transcriptase (RT) were screened for resistance mutations as defined by the World Health Organization surveillance list. The overall prevalence of TDR was 14.2%. We used a Bayesian network to identify co-transmission of TDR mutations in clusters associated with specific drugs or drug classes. Aggregate effects of mutations by drug class were estimated by fitting linear models of pVL and CD4 on weighted sums over TDR mutations according to the Stanford HIV Database algorithm. Transmitted resistance to both classes of reverse transcriptase inhibitors was significantly associated with lower CD4, but had opposing effects on pVL. In contrast, position-specific analyses of TDR mutations revealed substantial effects on CD4 and pVL at several residue positions that were being masked in the aggregate analyses, and significant interaction effects as well. Residue positions in RT with predominant effects on CD4 or pVL (D67 and M184) were re-evaluated in causal models using an inverse probability-weighting scheme to address the problem of confounding by other mutations and demographic or risk factors. We found that causal effect estimates of mutations M184V/I ( An aggregate approach sums across mutations occupying different positions in HIV-1 protease and reverse transcriptase, implicitly assuming that every mutation has a similar effect (in both direction and magnitude) on baseline CD4 or pVL. In contrast, a position-specific analysis recognizes the possibility that some TDR mutations will have a disproportionately large effects while others have negligible effects. Furthermore, it enables us to identify compensatory interactions, such that the effect of a specific mutation on CD4 or pVL depends on the presence or absence of other mutations; such interactions are an important aspect of the evolution of drug resistance in HIV-1 All patients in the CNICS database have provided informed written consent, as reviewed and approved by the institutional review boards of all participating sites – University of Alabama, Birmingham (UAB); University of California, San Diego (UCSD); University of California, San Francisco (UCSF); University of Washington (UW); Case Western Reserve University (CWRU); and Harvard University (FENWAY) – to have their clinical information used for the purposes of research. Patients were informed on the minimum information being collected, alternatives to participation, and possible uses of the data. In addition, CNICS has received a Certificate of Confidentiality from the National Institutes of Health (NIH).pol sequences in the region encoding protease and/or part of reverse transcriptase  were obtained at each site by conventional Sanger sequencing directly from RT-PCR amplification products from sample extractions of viral RNA. The exact bulk sequencing protocol varied among sites, as some sites used commercial laboratory services  whereas others used in-house assays based on commercial kits. At the time of this study, 4914 codon or amino acid sequences were available in the CNICS database but many were obtained when patients were on-therapy. Amino acid sequences were reconstituted from reports of residue polymorphisms relative to the HXB2 reference sequence. Sequences were linked to: unique anonymized patient identifiers; CNICS site; sampling date; age; ARV regimens; demographic factors ; HIV risk factors ; baseline plasma HIV RNA titres ; and baseline CD4 cell counts (cells/mL). A patient record was included in this study if: (1) a sequence was sampled before therapy, and (2) CD4 and/or pVL were sampled within 120 days of the sequence sample date. To normalize the distributions of pVL and CD4, we used The study population comprised individuals from 6 CNICS sites: University of Alabama, Birmingham; University of California, San Diego; University of California, San Francisco; University of Washington; Case Western Reserve University; and Harvard University pol reference sequence using an implementation of the Gotoh algorithm in HyPhy e.g., bulk sequence mixtures) were resolved to the resistant residue when applicable.Sequences were aligned pairwise against the NL4-3 stepAICResistance scores by drug class  were calculated according to the Stanford HIV Drug Resistance database algorithm  stepAIC to selecmultinom function in the R package nnet. Because weighting observations by the inverse of svyglm function in the R package surveyTo estimate causal effects of individual SDRMs, we used inverse probability-of-treatment weight (IPTW) estimators i.e., the presence/absence of TDR mutations), where ‘joint’ implies that the probability distribution of a variable may be conditional on other variables in the network A Bayesian network analysis to detect co-transmission of TDR mutations was carried out using a Markov chain Monte Carlo (MCMC) procedure in HyPhy e.g., the sample included a disproportionately greater number of naïve sequences from white males  comprised mutations associated with PI resistance, the latter group being associated with resistance to nelfinavir in particular. The remaining group (K101/G190) comprised mutations associated with resistance to NNRTIs. In sum, co-transmitted SDRMs tended to confer resistance to the same drug or drug class.Overall, 225 out of 1585 naïve sequences (14.2%) contained one or more SDRMs. Of these 225 sequences, 66 (4.2%) contained at least one PI-associated SDRM, 130 (8.2%) contained at least one NRTI-associated SDRM, and 131 (8.3%) contained at least one NNRTI-associated SDRM. In addition, 17 (1.1%) sequences contained SDRMs in all three drug classes, which is substantially greater than the number expected by chance .Although aggregate SDRM-based statistics such as Stanford scores are convenient and easy to interpret as proximate measures of drug class-specific resistance, they may mask the effects of individual mutations on clinical outcome. Consequently, we assessed the effects of position-specific SDRMs in linear models of baseline CD4 and pVL. To minimize the number of predictor variables and avoid over-fitting of the data, we limited our analysis to positions with a minimum SDRM frequency of 1% in naïve sequences see . These pD67X and K219X were each associated with significantly lower baseline CD4 ; ; both seunits  ; . This moM184X was significantly associated with an approximately ten-fold lower pVL at baseline ; . M46X ani.e., wildtype, uncompensated M184X, and M184X compensated by K103X and/or K219X). In an unweighted univariate linear model, M184X was significantly associated with a We used an inverse probability weighting scheme (using stabilized weights) to disentangle the causal effects of SDRMs from confounding variables that influence both the probability of the ‘treatment’ (an SDRM at the position in question) and CD4 or pVL. Based on the results of the preceding section, we focussed on the compensated and uncompensated effects of M184X on baseline pVL, and of D67X on both pVL and or K219X . Stabilie.g., M41L, K103N, and T215X in RT and L90M in protease), implying a negligible cost to fitness The overall prevalence of transmitted drug resistance (TDR) mutations in therapy-naïve patients has been reported in a large number of study populations around the world and ranges from 0 to 25% e.g., D67N/G and K219Q/E).The foremost and best -documented clinical implication of TDR is that it impedes the virological response to subsequent drug therapy Because this study population was comprised of seroprevalent rather than seroincident individuals , each baseline measurement of CD4 and pVL was sampled at a point in time following an unknown date of transmission. Consequently, there is no guarantee that these baseline measurements were taken at a consistent stage of infection. In other words, the CD4 count or pVL at baseline is the result of dynamics over an unknown period of time, starting from an unknown value upon infection. These missing data would be required to calculate rates of change, which would be easier to generalize to specific cases in clinical practice where these quantities (time since infection and prognostic markers at infection) may be known. This is a common problem of observational cohort studies and may explain why a substantial fraction (Some of these issues might be resolved by the analysis of longitudinal data that are available for this study population in vitro are well-characterized in vivo fitness is reduced in viruses carrying the M184V mutation in the absence of lamivudine in vivo is confounded by the effects of other SDRMs and demographic/risk factors that should be handled using a causal modeling approach in vitroin vivo from in vitro measures of its effect on replication efficiency as there are other components of RT functionality that can be affected Of all position-specific effects of SDRMs on baseline prognostic markers inferred from these data, one of the largest and most statistically significant was M184V/I. The effects of these substitutions on the kinetics and fidelity of HIV-1 reverse transcriptase and replication capacity i.e., substitutions at D67 were causally associated with a 2.5-fold increase in pVL that was only partially compensated by substitutions at K219. These residues potentially form a salt bridge and likely affect the formation of the RT 3′ pocket during polymerization in vitroIn contrast, the clinical consequences of D67N/G and K219Q/E are not as well known. The effects of D67N/G and K219Q/E on CD4 in our study were mirrored by significant effects on pVL;


The efficacy of raltegravir (RAL) in salvage regimens without protease inhibitors (PI) has not been evaluated in randomized trials, and information about its efficacy and reasons for its initiation in cohort studies has received scant attention. In some particular scenarios physicians may be forced to use RAL without PI due to advanced protease resistance, toxicity or patient refusal.Systematic multicenter search of databases in University-affiliated hospitals in Spain to identify all pre-treated patients with limited options due to resistance or intolerance to multiple antiretrovirals, starting a regimen including raltegravir and not a PI for any given reason, with a baseline plasma HIV-1 viral load (VL) >500 copies/mL. Primary endpoint: proportion achieving a VL<50 c/mL at 48 weeks.We identified 55 patients, 69% male, with a median age of 45,8 y., 40% IVDU, 45% with chronic hepatitis C, 40% in stage CDC C. Fourteen (25%) were diagnosed of dyslipidemia. 19/20 (95%) patients with results available had a CCR5 tropism. The main reasons for initiating a regimen with RAL and without PI were advanced protease resistance (44%), toxicity (15%), pharmacokinetic interactions (11%), and patient refusal (7%). The most frequently used drugs on board were tenofovir in 43 (78%) patients, maraviroc in 16 (29%) patients, and etravirine in 11 (20%). Eight (14%) patients did not use any nucleoside analogue, all of them due to complete resistance or toxicity. At 48 weeks, 66% of them had an HIV-1 VL < 50 c/mL. There was a significant increase in the median CD4 cell count from 257 cells/<micro>L at baseline to 415 cells/µL at 48 weeks (p=0.01), with only 19% of patients remaining with <200 CD4 cells/µL at 48 weeks. Virological failure was documented in 4 (7%) patients. There were no unexpected adverse events related to RAL when used without PI.Salvage regimens including RAL but not PI may be used in selected patients mainly due to advanced protease resistance, toxicity, pharmacokinetic interactions or patient refusal to PI. The efficacy of raltegravir when used with a background regimen without a protease inhibitor is high, but the inclusion of 2 further active drugs must be strongly pursued. This cohort analysis supports further study of RAL with novel combinations in this difficult-to-treat population.


Studies have shown that transmitted drug resistance mutations involving major antiretroviral classes appear to persist for months, even years, after infection. Efficacy of antiretroviral treatment in recently infected individuals imply resistance testing prior to initialize treatment. The prevalence of drug resistance mutations in patients newly infected with HIV-1 is high throughout the world and little is known about transmission of resistant virus in non B subtypes. In Romania from 1992 to 2009, analysis of the relative incidence of different HIV-1 circulating strains revealed a stable profile with high prevalence of subtype - F, in both long term survivors and recently infected adults.To study the prevalence of transmitted drug resistance and asses the type of mutations fond in F subtype recently diagnosed and naïve patients.Sequencing of the pol gene was carried out using Trugene genotyping kits, (Bayer diagnostics) in plasma samples from 10 recently diagnosed adults, average age 28.9±5.2 years, parenterally HIV infected. The nucleotide sequences were submitted to the Stanford database, and all strains were found to belong to the F subtype.All patients had clinical progression, median CD4 count was 143.6cells/mmc (range 8-490); median HIV ARN was 380278.5copies/ml plasma (range38000-1020000) and they need to initialize antiretroviral treatment. We found PI major resistance mutations in 2 patients: the mutations were V82S, V82 F, I84L; NRTI resistance mutations in 7 patients, the mutations were: M41L, A62P, D67G, F77L, Q151R, K219P, M41V, T215N, K219Q, D67A, T69N, K70R, T215I, K219N and NNRTI resistance mutations in 3 patients, the mutations were: L100S, K103N, F227L, V179D, Y181S, . All patients presented other mutations in average number of 11.5±2 for protease and 26.7±19.8 for reverse transcriptase. From the type 2 TAMs recognized to confer NRTI drug resistance only K70R was found in 2 patients also K103N associated with primary NNRTI resistance was found in one patient. The L89M polymorphism, the most prevalent signature among treatment-naïve non-subtype B isolates, was found in 7 patients.Due to transmitted drug resistance only 3 of the tested patients could have been treated with any kind of antiretroviral therapy, the rest, 70%, have limited therapy option. If genotyping is not performed prior choosing an antiretroviral combination the chances of therapy efficacy are low.


Cryptococcal meningitis is the most common neurological infection in HIV infected patients in Sub Saharan Africa, where gold standard treatment with intravenous amphotericin B and 5 flucytosine is often unavailable or difficult to administer. Fluconazole monotherapy is frequently recommended in national guidelines but is a fungistatic drug compromised by uncertainty over optimal dosing and a paucity of clinical end-point outcome data.From July 2010 until March 2011, HIV infected adults with a first episode of cryptococcal meningitis were recruited at Queen Elizabeth Central Hospital, Blantyre, Malawi. Patients were treated with oral fluconazole monotherapy 800 mg daily, as per national guidelines. ART was started at 4 weeks. Outcomes and factors associated with treatment failure were assessed 4, 10 and 52 weeks after fluconazole initiation.Sixty patients were recruited. 26/60 (43%) died by 4 weeks. 35/60 (58.0%) and 43/56 (77%) died or failed treatment by 10 or 52 weeks respectively. Reduced consciousness (Glasgow Coma Score <14 of 15), moderate/severe neurological disability (modified Rankin Score >3 of 5) and confusion  were all common at baseline and associated with death or treatment failure. ART prior to recruitment was not associated with better outcomes.Mortality and treatment failure from cryptococcal meningitis following initiation of treatment with 800 mg oral fluconazole is unacceptably high. To improve outcomes, there is an urgent need for better therapeutic strategies and point-of-care diagnostics, allowing earlier diagnosis before development of neurological deficit. Cryptococcus neoformans, occurs most commonly in HIV infected patients with CD4 counts of 100 cells/µl or less Cryptococcal meningitis, caused by the encapsulated saprophytic yeast Unacceptable differences in mortality exist between developed and developing countries. Ten week case fatality rates have been estimated at 9% in Western Europe and North America, but as high as 70% in SSA Fluconazole is a fungistatic drug and past studies have shown poor clinical outcomes from monotherapy; in Uganda and Zambia administration of 200 mg/day resulted in 1–2 month survival of only 10–12% Increasingly, early ART introduction has been advocated in patients suffering from opportunistic infections. Therefore, several African countries including Malawi now recommend commencing ART within a month of diagnosis of cryptococcal meningitis Within the last 5 years evidence has emerged that regimens incorporating short courses of amphotericin B may be safely and effectively used in South Africa and Uganda From July 2010 until March 2011, 60 consecutive patients with confirmed cryptococcal meningitis in Blantyre, Malawi, were recruited and treated according to national guidelines The study was prospectively approved by the University of Malawi, College of Medicine Research Ethics Committee (COMREC protocol P.04/10/926). Cryptococcal meningitis may impair a patient’s cognitive function to the extent that assistance of a guardian was required for discussions with the study team and therefore verbal consent was requested to approach all patients on diagnosis of the condition. Informed written consent to enter the study was obtained from patients or their guardians if unconscious. The study itself was completely observational: all procedures performed and medications commenced by the authors were consistent with their roles as attending physicians within the hospital. National and local guidelines and protocols for the management of cryptococcal meningitis and HIV were strictly adhered to by the study team in the routine care of the patients.Queen Elizabeth Central Hospital (QECH) is a 1,500 bed hospital in Blantyre, Malawi. It serves a population of approximately 1 million in the Blantyre District and takes tertiary referrals from further afield. Inpatient prevalence of HIV has been estimated at 70% amongst adult patients Patients were eligible for recruitment if they received a first microbiological diagnosis of cryptococcal meningitis following either positive India-Ink microscopy or positive culture of CSF. Malawian national guidelines for the treatment of Cryptococcal meningitis were followed; treatment was initiated with oral fluconazole 800 mg once daily (od) for 2 weeks, followed by 400 mg od for 6 weeks, then secondary prophylaxis with 200 mg od indefinitely Baseline demographic and clinical data were recorded including presence of fever (body temperature >37.5°C) and cranial nerve palsies or focal neurological signs. Prior use of ART and CD4 count at baseline were noted. Three aspects of overall neurological function were assessed; consciousness by Glasgow Coma Score (GCS), physical disability by modified Rankin Score (mRS) and cognition by Abbreviated Mental Test Score (AMTS). mRS is a 6 point scale , which has previously been used in Malawi for stroke patients It was not possible to measure CSF pressure and LPs were only repeated if patients developed clinical features of raised intra-cranial pressure.Patients were reviewed 4, 10 and 52 weeks after initiation of fluconazole, and encouraged to contact the study team at any time if clinical deterioration occurred. ART was commenced 4 weeks into fluconazole therapy. The study team ensured continuous access to fluconazole and ART.Treatment outcomes were defined as survival or treatment failure . This pragmatic approach to refractory cases was necessitated by a limited supply of amphotericin B, a shortage of trained staff to administer the drug and a lack of routine diagnostic biochemistry to monitor side effects.All CSF diagnostic testing was performed at MLW. Cell counts were performed using a FastRead (Immune Systems) disposable counting chamber. India-Ink microscopy was performed on all unclotted samples of adequate volume. All samples were cultured on sheep blood and chocolate agar for 48 hours under aerobic and microaerophilic conditions and sub-cultured onto sabouraud agar and urea slopes if they were India-Ink positive or culture yielded yeasts. Bacteria and yeasts were identified using standard methods Clinical and microbiological data was stored in a Microsoft Excel file and analysed using “R” version 2.12.1. Baseline characteristics and treatment outcomes were reported by simple descriptive statistics. A two-sample Wilcoxon test was used to assess the relationship between baseline CD4 count and prior ART. Kaplan-Meier plots were used to demonstrate survival on fluconazole by 52 weeks. Data from previous studies suggest that deaths within the first month of treatment are directly attributable to cryptococcal meningitis whilst later deaths are multi-factorial Sixty patients with a first episode of cryptococcal meningitis were recruited. Thirty-three (55%) were male and the median age was 32 years (range: 15–62). All patients complained of headache at presentation, with a median duration of 14 days (range: 1–150). 21 (37%) were febrile.Baseline GCS was <14 of 15 in 14/58 (24%) patients. mRS was >3 (moderate disability) in 24/60 (40%) patients. 13/44 (30%) patients who completed an AMTS on admission obtained a score of <8/10. Five (10%) patients had localising neurological signs .All patients were HIV-infected. Thirty-two had a CD4 count result available, the median result was 37cells/µl (range: 2–234 cells/µl). Thirteen (22%) patients were on ART at presentation. Their median CD4 count was 55 cells/µl (range: 2–234cells/µl). There was no association between CD4 count and prior ART .All 60 patients had evidence of cryptococcal infection in their CSF; 57 (95%) were culture positive and the remaining 3 (5%) had positive microscopy but no culture result available. White cells were seen in the CSF of 45 (75%) patients. The median white cell count was 20 cells/µl (range: 0–520).Forty-six patients had a documented CSF protein result and 46 a documented CSF glucose. Median CSF protein was 0.78 g/L  and median CSF glucose was 1.97 mmol/L . 39/46 (85%) patients had high CSF protein and 27/44 (61%) had low CSF glucose values.26/60 (43%) patients died in the first 4 weeks. A total of 33 (55%) died by 10 weeks, and a further 2 failed fluconazole monotherapy so were switched to intravenous amphotericin B between weeks 2 and 10. In total, 35/60 (58%) patients had fluconazole treatment failure by 10 weeks.Between weeks 10 and 52, a further 8 patients died and 4 were lost to follow-up. There were only 13 known survivors one year after presentation. Of these, six were reviewed in person and seven were interviewed by telephone. The six presenting for review were taking both ART and secondary fluconazole prophylaxis. All had good neurological recovery . In total 43/56 (77%) of patients had failure of treatment of cryptococcal meningitis at one year following treatment induction with 800 mg oral fluconazole. When the univariate analysis was repeated with end-points of treatment failure by 10 or 52 weeks , the impPrior ART was not associated with better outcomes. Of the 13 patients on ART at baseline, 9 (69%) had died at 10 weeks and 11 (84%) at one year. As HIV viral loads, quantitative CSF fungal culture and CSF mycobacterial culture were unavailable, it was impossible to distinguish cryptococcal IRIS, HIV treatment failure and other causes of cerebral infection or IRIS.CD4 counts were unavailable for 28 patients, 11 of who died within the first 4 weeks so were not used in the analysis.Clinical outcomes from cryptococcal meningitis in HIV infected Malawian adults following treatment induction with 800 mg oral fluconazole were poor, with 43% mortality at 4 weeks, 58% treatment failure at 10 weeks and 77% treatment failure at 1 year. These data are consistent with prior studies of fluconazole monotherapy at both lower The contribution of baseline clinical and laboratory variables to poor outcomes by 4, 10 and 52 weeks was assessed. Although AMTS provides an imperfect assessment of cognitive function, a score of <8/10 was associated with earlier death or treatment failure at all time-points. Prior studies have reported similar results Fever (temperature >37.5°C) and low CSF glucose measurements (<2.2 mmol/l) were associated with poor outcomes by 4 weeks but were insignificant by later time-points. This finding is consistent with data suggesting the longer an individual survives, the less likely that subsequent death is due to cryptococcal meningitis The high incidence of altered mental status at baseline suggests that many patients presented with advanced disease. As the study contained no comparator arm, it is impossible to say whether better anti-fungal therapy would have improved outcomes in late presenters with worse neurological deficits. However, it is likely that public health interventions to encourage earlier presentation of patients with symptoms of meningitis will augment optimal anti-fungal treatments.CD4 count data were incomplete, however several patients with extremely low CD4 counts survived to one year (median CD4 count amongst 13 long-term survivors: 13 (IQR 4–41) cells/µl). This illustrates the potential for full recovery from cryptococcal meningitis provided an adequate framework is in place to ensure uninterrupted supply of antifungals and timely commencement of ART. Without the intensive follow-up afforded by study participation, mortality in this group may have been higher.Prior ART was not associated with improved survival and CD4 counts amongst ART-experienced patients were no higher than the rest of the cohort. This may represent initiation of ART during severe immune-suppression (i.e. CD4 count has risen from an unknown nadir) or ART failure. Irrespective, these data reinforce the well documented association between late ART initiation and higher mortality There were some limitations to this work. The sample size was small. As the study was conducted amidst routine clinical practice some data were incomplete, precluding multivariate analysis. Given the constraints of our setting, standardised management of high CSF pressure was not possible and it was difficult to characterise all co-morbidities, episodes of IRIS or causes of death after 4 weeks.We have described clinical outcomes from cryptococcal meningitis in patients initiated on 800 mg oral fluconazole monotherapy. Despite timely ART initiation and uninterrupted drug supply through near complete follow up, there was unacceptably high mortality. There is an urgent need to assess the efficacy of faster acting, combination fungicidal regimens, particularly in light of recent work on those containing flucytosine


For settings with limited laboratory capacity, 2013 World Health Organization (WHO) guidelines recommend targeted HIV-1 viral load (VL) testing to identify virological failure. We previously developed and validated a clinical prediction score (CPS) for targeted VL testing, relying on clinical, adherence and laboratory data. While outperforming the WHO failure criteria, it required substantial calculation and review of all previous laboratory tests. In response, we developed four simplified, less error-prone and broadly applicable CPS versions that can be done ‘on the spot’.Findings From May 2010 to June 2011, we validated the original CPS in a non-governmental hospital in Phnom Penh, Cambodia applying the CPS to adults on first-line treatment >1 year. Virological failure was defined as a single VL >1000 copies/ml. The four CPSs included CPS1 with ‘current CD4 count’ instead of %-decline-from-peak CD4; CPS2 with hemoglobin measurements removed; CPS3 having ‘decrease in CD4 count below baseline value’ removed; CPS4 was purely clinical. Score development relied on the Spiegelhalter/Knill-Jones method. Variables independently associated with virological failure with a likelihood ratio ≥1.5 or ≤0.67 were retained. CPS performance was evaluated based on the area-under-the-ROC-curve (AUROC) and 95% confidence intervals (CI). The CPSs were validated in an independent dataset. A total of 1490 individuals ); median baseline CD4 count: 94 cells/µL (IQR 28–205), median time on antiretroviral therapy 3.6 years (IQR 2.1–5.1)), were included. Forty-five 45 (3.0%) individuals had virological failure. CPS1 yielded an AUROC of 0.69 (95% CI: 0.62–0.75) in validation, CPS2 an AUROC of 0.68 (95% CI: 0.62–0.74), and CPS3, an AUROC of 0.67 (95% CI: 0.61–0.73). The purely clinical CPS4 performed poorly .Simplified CPSs retained acceptable accuracy as long as current CD4 count testing was included. Ease of field application and field accuracy remains to be defined. Scaling-up of antiretroviral treatment (ART) is currently ongoing in low and middle income countries (LMIC), aiming to initiate 15 million individuals on ART by 2015 Routine viral load (VL) testing is now recommended by the World Health Organization (WHO) Targeted VL testing, whereby VL testing is done only in individuals meeting failure criteria, aims to avoid unnecessary and costly switches to second line treatment for patients with false-positive ‘screening’ tests We previously developed a clinical prediction score (CPS) for virological failure integrating clinical, adherence and laboratory data Two limitations of the original CPS were identified during the validation study in Cambodia Sihanouk Hospital Center of HOPE (SHCH) is a non-governmental hospital in Phnom Penh Cambodia. Since 2003, the hospital has provided ART at no cost as part of the national program. Patients were initiated and treated according to WHO recommendations Details of the original validation study have been previously reported The following simplified CPSs were evaluated with a progressive increase in simplification by removing: 1) the ‘decline from peak CD4 count values’ and only using ‘current’ CD4 count (CPS1) - ie the value of the CD4 count at the time of assessment for virological failure; 2) hemoglobin as item in the CPS (CPS2); 3) ‘decrease in CD4 count below baseline value’ as item in the CPS (CPS3); and 4) all laboratory items from the CPS (CPS4).The same methodology for score development was used as for the original score derivation The VL validation study was approved by the National Ethics Committee for Health Research, Phnom Penh, Cambodia; the institutional review board (IRB) of the Institute of Tropical Medicine, Antwerp, Belgium; and the Ethics Committee of the University Hospital of Antwerp, Belgium. All study participants provided written consent.A total of 1490 adults on first-line ART for at least one year were included. As previously described, median time on ART at the time of evaluation of virological treatment response was 3.6 (IQR 2.5–5.1) years. The median age at study inclusion was 38 (IQR 33–44) years and the median CD4 count was 379 (IQR 265–507) cells/µL. Approximately half (56.7%) were female and >90% were on a zidovudine or stavudine-based regimen at the time of evaluation. A total of 45 patients (3.0%) were detected with virological failure.In a first step (CPS1), we removed CD4 decline from peak value, since it requires substantial calculation and complete on treatment CD4 count data, but retained ‘current CD4 count’. The AUROC of CPS1 was 0.78 (95% CI 0.70–0.85) in derivation and 0.69 (95% CI 0.62–0.75) in validation . With thWe developed four simplified CPSs, of which three performed relatively well and have a number of advantages. Compared to the original CPS, no regular laboratory monitoring while on ART is required. Moreover, with a choice of CPS, programs can decide which is most appropriate and feasible to apply in their program settings. Performing hemoglobin measurement as part of an ART monitoring strategy can be cumbersome in many settings, comes with an additional cost and is not required in the latest WHO guidelines. Moreover, the added value in terms of diagnostic performance was limited, especially at lower cut-off values. CPS2 and CPS3 seem most useful and practical. CPS2 could be used when a baseline CD4 cell count is available, while CPS3 would apply when patients have initiated ART without CD4 guidance. With only a limited amount of key clinical information and minimal calculation required, these CPSs are also substantially simpler and possibly more adapted for use by non-physicians.Some resource-constrained countries predominantly use clinical monitoring to detect treatment failure and CD4 cell counts are not always regularly done while on ART, especially in remote areas. However, we found that, in line with previous studies, CPS4 - relying on clinical information only - performed poorly Further study is required to determine at what time points during ART these scores should be applied, and in which different health care settings and patient populations. Moreover, these algorithms were designed for patients on ART for at least one year. One approach could consist of a routine VL early after ART initiation (eg six months after ART initiation) followed by application of the score at predefined intervals, combined with targeted VL testing. Whereas applying the score every six months would equate to routine CD4 count monitoring, the current CPS would still have the advantage of not requiring interpretation of a trend of laboratory tests. While these simplifications would likely render them easier to apply and less error-prone compared to the original CPS, this is subject to further study.A number of issues remain to be assessed before implementing these CPSs in routine care. Validation in other study populations and different settings should be conducted. Additional studies on the clinical utility and impact are warranted Additional limitations include the fact that treatment failure was based on a single VL measurement, in line with the previous derivation and validation study. Since a substantial proportion might have an undetectable VL on repeat measurement, only a proportion of those with virological failure in our study would be true treatment failures. On the other hand, the main purpose of targeted VL testing is early detection of active viral replication, since these patients need enhanced adherence counseling followed by a repeat VL. We did not evaluate whether individual opportunistic infections should be included as predictors in the CPS, but used ‘pooled’ WHO clinical staging groups. While this might be a more ‘crude’ approach, it has the advantage of being simpler. Finally, the failure rate in our study was clearly lower than what has been observed in most other programs. Consequently, positive predictive values will be higher and negative predictive values lower in settings with higher failure rates.eg at the country or regional level) might be a reasonable alternative, and might also be valuable for a wider range of diagnostic tests or health care interventions. Targeted evaluation using evidence-based tools might be a rational way to optimize the use of often scarce resources available for health delivery in LMIC We acknowledge that the present algorithms are unlikely to be universally applicable and generalizable across a wide range of geographical regions, populations and health care settings. Derivation and validation of algorithms for local use (In conclusion, we have developed three simplified CPSs for targeted VL testing that retained an acceptable diagnostic performance as long as ‘spot’ CD4 testing was included. They have the advantages of not requiring regular CD4 cell count or hemoglobin testing, or complete and reliable data collection while on ART, or the need to interpret trends in laboratory tests. They should, therefore, be less prone to error and more readily adopted in field conditions. Their use would enable more accurate targeted VL testing in contexts where it is too expensive to be done routinely. We suggest that they be tested and tailored to the other health care settings.