Nevirapine-induced DRESS syndrome is uncommon but a potentially life-threatening condition, with significant morbidity and mortality rates due to multiple-organ involvement. The authors report a case of a 47-year-old HIV-infected female patient who presented with fever, right hypochondrium pain, jaundice, and skin rash. The Nevirapine-induced DRESS syndrome was suspected based on clinical presentation and RegiSCAR scoring system. This case highlights the need for a high index of clinical suspicion among HIV-infected patients with severe skin eruption and systemic symptoms. The Drug Reaction with Eosinophilia and Systemic Symptom (DRESS) is a severe idiosyncratic adverse drug reaction with multiple-organ involvement, which was first described by Bocquet et al. in 1996 and has been reported mostly in association with aromatic anticonvulsants, sulfonamides, allopurinol, and Nevirapine . The acronym designated by Bocquet et al.  describeWe describe a 47-year-old woman infected with HIV whose chronology of signs and symptoms and RegiSCAR score suggest a case of adverse drug reaction associated with Nevirapine compatible with diagnosis of DRESS syndrome. She was successfully treated with intravenous methylprednisolone.A 47-year-old woman with human immunodeficiency virus 1 (HIV-1) infection was admitted to the São Paulo Hospital because of fever, right hypochondrium pain, jaundice, and skin rash. In the emergency department, the patient reported fever , myalgias and arthralgias, choluria, pruritus, and gradual onset of right hypochondrium pain. The patient had been well until twenty days before admission, when a diffuse maculopapular rash accompanied by discrete yellow discoloration of the conjunctive of the eyes developed, followed by a myalgias and arthralgias, choluria, pruritus, and vague pain in the abdomen and lower back. Three days before admission, nausea and vomiting developed, and oral intake decreased.Mycobacterium tuberculosis, or history of opportunistic infections. Positive IgG but negative IgM antibodies to cytomegalovirus (CMV) and Toxoplasma gondii were detected in the patient's serum. After the diagnosis of HIV-1 infection, it was started with Zidovudine, Lamivudine, and Lopinavir boosted with Ritonavir; subsequently, CD4 T-cell counts remained consistently above 500 cells per cubic millimeter, and there was a fall in the plasma HIV load to less than 50 RNA copies per milliliter. One year before admission, daily gastrointestinal intolerance developed, which gradually worsened. Forty-five days before admission the patient was admitted to her reference outpatient care clinic, when the medical assistant changed the antiretroviral therapy to Nevirapine, Zidovudine, and Lamivudine. She was admitted to our hospital twenty days after initiating the new regimen because of a diffuse maculopapular rash accompanied by discrete yellow discoloration of the whites of the eyes that has evolved in the past ten days due to intensification of the yellow discoloration of the skin and mucous membranes, fever , myalgias and arthralgias, choluria, pruritus, and gradual onset of right hypochondrium pain. She did not take other forms of medication.The HIV-1 infection was detected on a routine testing three years earlier. The CD4 T-lymphocyte count was 436 cells per cubic millimeter, and the HIV viral load was not available. There was no evidence of active coinfection with hepatitis B or C, syphilis, On examination, the patient was awake, alert, and oriented. The blood pressure was 95/66 mm Hg; other vital signs were normal. There was marked conjunctival and skin icterus, and the skin was yellow, with diffuse maculopapular rash but without spider angiomas, palmar erythema, or telangiectasias. There was no anathema in her oral cavity nor lymph nodes enlargement. The abdomen was soft, with tenderness on right hypochondrium, without distention or organomegaly. Neurologic examination revealed normal speech and attention and no asterixis. The remaining of the physical examination was unremarkable.Results of a complete blood count and differential count were normal as were serum levels of glucose, calcium, phosphorus, magnesium, total protein, albumin, globulin, amylase, and lipase. There was important elevation of the liver enzymes combined with elevation of bilirubins, alkaline phosphatase, and gamma-glutamyl transpeptidase. Also, according to the RIFLE criteria, there was renal failure determined by a threefold increase in the serum creatinine combined with decrease of glomerular filtration rate by 75 percent. Testing was negative for cytomegalovirus (CMV) antigenemia, antibodies to HBV and HCV, and IgM antibodies to hepatitis A virus (HAV); other test results are shown in Based on a RegiSCAR's scoring system of 5 points, a probable diagnosis of Nevirapine-induced DRESS syndrome was made. The rapid development of hepatocellular and renal dysfunction determined severity to this case, and the patient was admitted to a semi-intensive care unit. All antiretroviral treatment use was discontinued, and methylprednisolone 1 mg/kg/day was started. Laboratory followup was also shown in Nevirapine is an oral medication that is used in combination with other antiretroviral agents for the treatment of human immunodeficiency virus disease. It belongs to a class of drugs known as nonnucleoside reverse transcriptase inhibitors (a “nonnuke” or NNRTI). The drug exerts a virustatic effect by acting as a specific, noncompetitive HIV-1 reverse transcriptase (RT) inhibitor. The drug binds directly to heterodimeric HIV-1 RT and inhibits the RT activity by disrupting the catalytic site of the enzyme . To establish its safety profile, Pollard et al. conducted a review of data from prospective US and international clinical trials involving a total of 906 adult patients and 468 pediatric patients treated with NVP. Drug-related adverse events were similar in adults and children, with rash and nausea most frequently reported in adults. A separate analysis of rash based on data from adult patients in controlled trials demonstrated a 16% rate of NVP-attributable rash in these patients. Of patients with NVP-associated rash, 65% developed rash within the first 6 weeks of therapy. No cases of DRESS syndrome were reported . Yet this drug is associated with a drug hypersensitivity syndrome in approximately 5% of those starting the drug and Stevens-Johnson syndrome/toxic epidermal necrolysis in 0.3% or less of those initiating the drug . DRESS sIn an effort to define more accurately the DRESS syndrome, a scoring system has been recently developed: the RegiSCAR scoring system , 6. RegiRecently, Wongkitisophon et al.  reviewedP = 0.029). Nevertheless, Umapathy et al. [P value 0.0032) with Nevirapine-induced skin rash. Further, significant HLA B7 , HLA B8 , and HLA B15  were also found to be decreased significantly among the Nevirapine-hypersensitive patients. Further investigations are needed to understand whether these HLA alleles are truly causative or acting as surrogate markers of predisposition to Nevirapine hypersensitivity reaction.When available, molecular typing methods and the ability to analyze the whole genome in an unbiased fashion can lead to some remarkable findings of the role of the human leukocyte antigen (HLA) genes as genomic biomarkers of predisposition. Recently, HIV-positive Chinese patients treated with a Nevirapine-based regimen were investigated for the association between Nevirapine hypersensitivity reaction and HLA allele. More HLA-Cw*04 alleles were observed in Nevirapine hypersensitivity reaction cases than in Nevirapine-tolerant cases  associated with Nevirapine therapy who was successfully treated with intravenous methylprednisolone . Lanzafa3 or in adult males with CD4 counts greater than 400 cells/mm3 unless the benefit outweighs the risk [Nevertheless, based on hepatotoxicity observed in controlled and uncontrolled studies, now it is established in the literature that Nevirapine, including extended-release Nevirapine, should not be initiated in adult females with CD4 counts greater than 250 cells/mmthe risk , 15. Thithe risk .In conclusion, DRESS is potentially life-threatening, with significant morbidity and estimated mortality of 10% . Our cas


In addition to the negative effects described for HIV itself and/or HAART on mitochondria, HIV-infected patients are more prone to develop a premature aging and, therefore, to present an increased oxidative state that could lead to the development of these metabolic disturbances observed in HIV-infected patients.Highly active antiretroviral therapy (HAART) has considerably improved the prognosis of HIV-infected patients. However, prolonged use of HAART has been related to long-term adverse events that can compromise patient health such as HIV-associated lipodystrophy syndrome (HALS) and nonalcoholic fatty liver disease (NAFLD). There is consistent evidence for a central role of mitochondrial dysfunction in these pathologies. Nucleotide reverse transcriptase inhibitors (NRTIs) have been described to be mainly responsible for mitochondrial dysfunction in adipose tissue and liver although nonnucleoside transcriptase inhibitors (NNRTIs) or protease inhibitors (PIs) have also showed mitochondrial toxicity, which is a major concern for the selection and the long-term adherence to a particular therapy. Several mechanisms explain these deleterious effects of HAART on mitochondria, and evidence points to other mechanisms beyond the “Pol- Human immunodeficiency virus (HIV) infection is a serious public health disorder that affects up to 34 million people in the world . Since tMitochondria are intracellular organelles present in all mammalian cells (except red blood cells). They contain an outer membrane, an intramembrane space, an inner membrane, and the matrix where the mitochondrial genome is found. Each mammalian cell contains several hundreds to more than a thousand mitochondria. The size, shape, and abundance of mitochondria vary dramatically in different cell types and may change under different energy demands and different physiological or environmental conditions. The main function of mitochondria is to synthesize ATP through electron transport and oxidative phosphorylation (OXPHOS) in conjunction with the oxidation of metabolites by tricarboxylic acid cycle and catabolism of fatty acids by beta-oxidation. In this sense, the mitochondrion had been, for years, considered to be simply the fuel provider for the most basic energy demands of the cells. However, mitochondria are now recognised as being critical components in the control of multiple key cellular processes, being the main arbitrator in the initiation and execution of apoptosis and, therefore, playing a pivotal role in the determination of life and death of the mammalian cells. In addition, mitochondria are the main intracellular source and immediate target of reactive oxygen species (ROS) . During Several factors can affect the functionality of mitochondria, such as aging, infections, or certain antiretroviral drugs. These factors can damage the mitochondria, affecting the normal functioning of the cell. Mitochondrial toxicity is a general term that refers to these changes, it can cause different symptoms in the heart, nerves, muscles, pancreas, kidneys, and liver, and it is involved in several disorders. α) production, which can directly affect the mitochondrial function, has been observed in patients with HIV infection and under antiretroviral treatment [It is plausible that chronic infection and inflammation and/or drugs with adverse effects on mitochondrial function would contribute to long-term complications in HIV-infected persons. A large list of clinical manifestations of mitochondrial toxicity has been described within HAART-associated adverse events, which is a major concern for the selection and the long-term adherence to a particular therapy. For example, the increase in tumor necrosis factor  [In vivo studies with AZT demonstrated a disrupted cardiac mitochondrial ultrastructure, diminished expression of mitochondrial cytochrome b mRNA, and the presence of OS in mtDNA. Mitochondrial ROS generation has also been suggested to accompany or even constitute a separate mechanism of NRTI-induced mitochondrial toxicity. It is also important to state that tissue and compound-specific patterns of drug import, compartmentalization, and activation may account, at least in part, for variation in mitochondrial toxicity according to specific NRTIs and/or tissue. Furthermore, not all the drugs of this group exert their deleterious effects on mitochondria with the same intensity. In fact, what became apparent from several in vitro studies is that the order of ranking of toxicity differs depending on the cell line and the method used for measuring mitochondrial dysfunction  [in vivo state that ddC component of antiretrovirals (and possibly ddI) rather than d4T or AZT mediates mtDNA depletion in PBMCs in HIV-infected patients [γ or reduce mtDNA content. In vitro studies revealed an increase in mtDNA in murine adipocytes treated with indinavir (IDV) [NRTIs have long been considered the main source of HAART-related mitochondrial toxicity. These toxicities can be difficult to reverse and can be life-threatening. The exact pathogenesis underlying NRTI-induced mitochondrial toxicity remains unclear and likely differs for specific NRTI drugs . NRTIs a., 2011) . Thus, i., 2011) . AZT als., 2011) , 22. NRT., 2011) , 23, 24.n, 2007) . Thus, Apatients . Other apatients . Interesir (IDV)  or treatir (IDV) . Recent ir (IDV) . Althougir (IDV) .It is also important to take into account that the progressive aging of the HAART-treated population and the subsequent appearance of age-related diseases are relevant. In fact, it has been demonstrated that antiretroviral therapy through the clonal expansion of mtDNA mutations accelerates mitochondrial aging and, therefore, induces mitochondrial dysfunction . per se seems to undermine mtDNA and provoke other mtDNA-independent mitochondrial alterations [Not only antiretrovirals but also HIV itself contributes to mitochondrial dysfunction. HIV erations . Thus, Herations –34. The erations . A studyerations . Howevererations –41. In terations . Thus, dIn summary, it seems quite clear that HAART and HIV itself contribute to mitochondrial dysfunction although the underlying mechanisms remain partially understood. In this context, we cannot forget the importance of patient genetics, the presence of mitochondrial pathologies, and/or pathological settings with compromised mitochondrial function as they can all magnify the adverse effects related to mitochondria.Liver diseases constitute a major health problem nowadays. Among distinct diseases, nonalcoholic fatty liver disease (NAFLD) is a multifactorial condition, which affects both young and adult patients and which is associated with several risk factors  . NAFLD iNowadays, NAFLD is one of the most prevalent chronic liver diseases in industrialized countries. This is mainly due to its association with obesity, so it is prevalent in these countries . In factPrevalence of NAFLD is higher in individuals with HIV infection (30–40%) than in the general population (14–31%) , 49. In The pathogenesis of NAFLD was previously based on the “two-hit hypothesis” considering steatosis as the result of the first step in the development of NASH . The newper se an increased demand of mitochondrial oxidation. On the other hand, an increase in fatty acid beta-oxidation, as a response to increase hepatocyte influx, can damage normally functional mitochondria through several mechanisms, including uncoupling, induction of the mitochondrial permeability transition (MPT) and OS . The loss of mitochondrial function will affect fatty acid beta-oxidation, which will lead to the accumulation of nonmetabolized fatty acids in the cytosol. This is further complicated in the presence of obesity and higher circulating levels of fatty acids, which may determine ) and OS   have been the main insulin-sensitizing molecules tested for the treatment of NASH, but the poor benefit of metformin has been confirmed in a recent meta-analysis . HoweverHIV-associated lipodystrophy syndrome is characterised by alterations in adipose tissue distribution in association with systemic metabolic complications. The alterations of adipose tissue consist of loss of subcutaneous fat in face, limbs and buttocks , accumulThe prevalence of HALS is about 50%, although reported percentages vary in different studies (20–80%) , 81, 82.HAART might play an important role in the development of HALS. In this sense, NRTIs favour lipoatrophy whereas viral PIs tend to promote visceral lipohypertrophy and systemic metabolic disturbances. However, recent studies suggest that drugs of the three main classes  act in synergy. Duration of HIV infection, age, and gender may also contribute to the risk of development of HALS. A role for genetics is also probable but has been only partly evaluated. The toxicity of antiretroviral also depends on patients metabolism, which is partly genetically determined . γ [The importance of mitochondria in most cells is known. In adipocytes, mitochondria play an important role in their differentiation and function. Preadipocytes mature in two steps: differentiation and then hypertrophy. During the early maturation stage, an increased number of mitochondria are required , 85, resγ  and induγ , 87, whiγ .in vitro studies have shown that NRTIs such as zidovudine, stavudine, and didanosine result in mtDNA depletion [Longitudinal studies comparing subcutaneous fat in HIV-infected patients before and after HAART initiation have provided important clues. Thus, six to eight months of treatment with NRTIs alter the expression of several key mitochondrial electron transport genes in subcutaneous fat, which leads to increased OS . A reducepletion . Thus, Nepletion   , which aAs stated before, mitochondria are the main intracellular source and immediate target of ROS. These reactive species, if not counterbalanced by antioxidant defenses, could damage DNA, proteins, carbohydrates, and lipid constituents and compromise cell function leading to the development of a number of diseases such as AIDS . OxidatiAging is also associated with a chronic low level inflammation  and makeNigella sativa with potent antioxidant properties, is able to inhibit the effect of nelfinavir on augmented ROS production and suppressed SOD levels in pancreatic β-cells [β-gal cells (MAGI) via NAD(+)-dependent SIRT1 activity suggesting that this antioxidant could be a novel therapeutic approach in anti-HIV-1 therapy [If an increased oxidative stress is observed in HIV-infected patients, actions that could decrease it should be therapeutically beneficial , 104. In β-cells . A recen therapy . Similar therapy . Plasma  therapy . Other s therapy . Because therapy . However therapy . Further therapy . Several therapy , which c therapy . Thus, w therapy . Other potential strategy to ameliorate OS caused by HAART is switching to a new drug with a better profile. Thus, atazanavir increases bilirubin plasma levels. This increase has been associated with improved endothelial function in patients with type 2 diabetes mellitus . Howeverγ hypothesis. NRTIs have long been considered the main source of mitochondrial toxicity. In this context, in many parts of the world, persons have been or are currently treated with older NRTIs and remain at risk for overt mitochondrial toxicities. Therefore, mitochondrial toxicity management during HIV therapy has become an important challenge with growing interest in developing new compounds and identifying combinations of available antiretroviral drugs that are clinically effective while eliciting minimal mitochondrial interference in these patients [In the last years, several studies highlighted the central role for mitochondrial impairment in HIV infection and associated disturbances such as NAFLD and HALS. Mitochondrial dysfunction seems to be associated with premature aging in these patients which could contribute to the development of several metabolic pathologies such as cardiovascular events, diabetes, and so forth. The underlying mechanisms that could explain the relationship between mitochondrial dysfunction and HIV, NASH, or HALS are not fully understood despite accumulating evidence points to other mechanisms beyond the Pol-patients . Therefo


Background. Current treatment of chronic hepatitis C with pegylated interferon and ribavirin has the ability to eliminate viral infection in about half of the patients treated. Therapeutic options, for those with remaining chronic hepatitis, will remain limited untilnovel antivirals become available in the future. Consensus interferon is currently available and has demonstrated clinical efficacy with superior invitro antiviral activity, but the maximum tolerated dose is not defined. Methods. We assessed the efficacy of daily high-dose (24 ug) consensus interferon with weight-based (1000–1200 mg daily) ribavirin in HCV genotype 1-infected non-responder patients. Results. Six adverse events were documented in five patients, and the trial was terminated with no subject achieving viral clearance. Conclusions. The occurrence of serious adverse events effectively defined the upper limit of acceptable dose, while also revealing that this dose did not offer enhanced sustained viral clearance.  The most commonly employed treatment of patients with hepatitis C virus (HCV) infection who are treatment-naïve consists of pegylated interferon-alfa and ribavirin with sustained viral response (SVR) rates in genotype 1 patients of up to 52% . Consensinvitro activity compared to other interferons [μg followed by 9 μg for 40 weeks did not result in a higher SVR compared to 9 μg for 48 weeks, presumably due to dose modifications in subjects receiving the induction dose of CIFN. When the subgroup of patients receiving the higher dose of CIFN, without early dose modification, was evaluated, 40% achieved SVR [μg or 15 μg with weight-based ribavirin administration in the treatment of patients unresponsive to prior pegylated interferon and ribavirin treatment. Viral clearance was achieved in a dose-dependent manner with greatest loss of HCV in the 15 μg group [CIFN has more potent antiviral erferons , 5. Currerferons –8. Cornbμg) with weight-based ribavirin dosage given with maximal support of patient adherence including blood cell growth factors. Previous clinical trials did not evaluate this high dose of daily CIFN in the treatment of non-responders to pegylated interferon and ribavirin beyond an 8-week induction period [The aim of our clinical trial was to evaluate the efficacy, safety and tolerability of high-dose daily CIFN  from baseline at 12 weeks of therapy or the presence of detectable HCV RNA at 24 weeks of therapy. All subjects must have previously received a weight-based dosage of ribavirin. At least one liver biopsy, consistent with chronic hepatitis C without significant alternative liver disease, must have been performed prior to screening for enrollment into the trial. All subjects, prior to participation, signed a consent form approved by each institutional review board.μg dose of CIFN  given subcutaneously and a daily weight-based dose of ribavirin (1000–1200 mg per day). All patients were evaluated at weeks one, two, and four of therapy and at subsequent four week intervals unless additional urgent clinical evaluation was needed. At each visit, subjects underwent physical examination and clinical history detailing adverse events and completion of the Beck Depression Inventory second edition (BDI-II). Treatment was discontinued and the subject considered a treatment failure if the patient failed to achieve early virologic response (EVR) defined as less than a 2-log drop of HCV RNA from baseline at Week 12 of therapy, or had detectable HCV RNA at or after Week 24 of therapy. Sustained viral response (SVR) was defined as an undetectable HCV RNA level using Quest Diagnostics  Heptimax and TMA serum assays 24 weeks after completion of 48 weeks of treatment.This study was an open-label pilot trial. All study subjects were treated with a daily 24 Potential subjects for this clinical trial were recruited from three hepatology practices in New York. Compliance with the experimental protocol was assessed by the subjects' self-reported administration of ribavirin and the return of used CIFN vials. In addition, the trial had a data safety monitor (DSM), who assessed the safety of this trial at specified time points during the study. The DSM was an independent physician with extensive clinical experience with treatment of patients with hepatitis C infection. t-test, chi-square and Fisher's exact tests. Response rates between partial and null responders were compared using chi-square and Fisher's exact tests.The subject's baseline demographics were compared using Student's Thirteen subjects (8 men) with a mean age of 55-years were enrolled in this study. The median baseline HCV RNA level was 3,300,000 IU/mL with only two subjects with levels less than 1,000,000 IU/mL. Ten patients had advanced fibrosis . During n = 6) in five patients. These serious adverse events included abdominal wall abscess requiring hospitalization, severe dehydration, and hypotension resulting in metabolic derangements, loss of consciousness, anemia, and ventricular tachycardia. In addition, one subject discontinued therapy due to disabling fatigue. The trial had initially been planned to enroll thirty subjects at this interferon dose. The study termination decision was made by the Principal Investigator on the advice of the DSM after assessing the frequency of serious toxicity while no patient had achieved a sustained loss of HCV RNA. The severity and frequency, of serious adverse events, are evidence that the high-dose aggressive treatment regimen of this study defined an upper dose limit of tolerability for CIFN-ribavirin therapy. The trial was terminated early due to the occurrence of serious adverse events  had greater than a 2-log drop from baseline HCV RNA while on treatment. One of these had transient loss of HCV but subsequently experienced a viral breakthrough. The four subjects who received 24 weeks of therapy all had partial viral response after 12 weeks of therapy, but had detectable hepatitis C virus at week 24 and therapy was discontinued as they were deemed non-responders .Three subjects with partial viral response prematurely discontinued therapy due to serious adverse events. There were three subjects still on therapy when the trial was halted. One subject had greater than a 2-log drop in HCV RNA level from baseline by week 8 of therapy, but the HCV RNA level at the time of termination had markedly increased, and there was no longer a 2-log drop in the HCV RNA level from baseline at that point. The second subject did have a greater than 2-log decrease from baseline HCV RNA level after 16 weeks of therapy, but by 20 weeks the hepatitis C viral level had increased over 1-log from the nadir level. The third subject did not have a 2-log decline from baseline viral level after 12 weeks of therapy and was deemed a non-responder. The final three subjects were non-responders to the study medications at the time of discontinuation of therapy due to adverse events.P < .04).There were three subjects with greater than a 2-log drop in viral level by four weeks of therapy. One of these subjects could not tolerate therapy and did not complete 12 weeks of therapy. Another was a nonresponder at 24 weeks of therapy. The third subject was still on study medication when the clinical trial was halted, however, while on therapy there was marked increase in HCV RNA level. All three subjects who had greater than a 2-log drop from the baseline viral level after four weeks of therapy were partial responders to previous pegylated interferon and ribavirin therapy compared to none of the prior null responders (n = 12) and flu-like symptoms (n = 12), anorexia (n = 10), anxiety/depression (n = 7), and insomnia (n = 7). The mean decrease in body weight during the experimental protocol was 8.1 ± 5.8 kilograms. The median increase in BDI-II score was 9 ± 10.1. Serious adverse events (n = 6) occurred in five patients and included abdominal wall abscess, severe dehydration, hypotension, and ventricular tachycardia all requiring hospitalization and loss of consciousness and anemia. These events occurred within the first six to 16 weeks of therapy. Two subjects (15%) had their dose of CIFN reduced during the trial from 24 μg to 15 μg daily. Five subjects (38.5%) had a dose reduction of ribavirin and one of the five subjects had ribavirin discontinued. The mean decrease in hemoglobin level from baseline to nadir was 3.6 ± 1.6 g/dL with eight subjects receiving epoetin alpha injections. The most common adverse events were fatigue  to lower-dose treatment  in those patients who had previously failed treatment with interferon and ribavirin therapy. The week 12 and 24 responses were superior in the high- versus low-dose group (P < .05), but the SVR rates were identical at 26%. This suggests that the initial positive effect of the 24 weeks of high-dose daily induction was lost after dose reduction to three times per week treatment [Previous studies demonstrate promising SVR rates of 23% to 37% using daily CIFN and ribavirin in the retreatment of non-responders to the combination of standard or pegylated interferon and ribavirin –8. HCV cμg for four weeks followed by 18 μg for eight weeks versus 18 μg for 12 weeks in 120 patients infected with HCV who were previous non-responders to pegylated interferon and weight-based ribavirin [Kaiser etal. from Germany evaluated an induction dose of daily CIFN of 27 ibavirin . No ribaμg for the first 8-weeks of treatment resulted in an enhanced first-phase HCV-RNA decay suggesting higher antiviral efficacy of a higher dose of CIFN, but this did not translate to a better SVR, presumably due to dose modifications [Another study by Cornberg and colleagues investigated the efficacy of CIFN plus ribavirin in HCV patients who were non-responders to standard interferon and ribavirin . ConsensPrevious partial responders to pegylated interferon and weight-based ribavirin as compared to null responders had better viral kinetic responses with all three partial responders having greater than a 2-log drop in HCV RNA from baseline viral level by week four. This response, however, did not translate into a higher overall efficacy since these subjects could not tolerate continued therapy and viral response did not decline to undetectable levels.μg daily dose when used in combination with weight-based ribavirin. This observation is important since the efficacy results of the prior studies left open the possibility of greater efficacy of high-dose CIFN.This trial used a sustained high daily dose (24 mg) of CIFN, a treatment regimen which showed promising early results in achieving a decline in HCV RNA levels in a population of subjects resistant to prior therapy. The major factor leading to termination of this study was the occurrence of serious adverse events. Although each event was not unique in patients receiving interferon ribavirin treatment, the occurrence of frequent serious adverse events in a small patient population raised significant concern. The abdominal wall abscess was related to injection site infection. The development of dehydration and hypotension was related to anorexia and severe lassitude. The episode of loss of consciousness may have been related to anorexia and anemia. The observed adverse events, even including cardiac arrhythmias, have been reported previously in patients receiving interferon alfa and ribavirin. The frequency and severity of adverse events, however, effectively determined the upper limit of tolerability of daily consensus interferon to be less than the 24 Despite use of blood cell growth factors and maximal support of experienced investigators, this high-dose of daily CIFN therapy in combination with a weight-based ribavirin dose was not tolerated due to serious adverse events. Most of the subjects required epoetin alfa injections for therapy-associated anemia. Even in the subjects who could tolerate high-dose CIFN therapy with weight-based ribavirin, sustained loss of HCV was not achieved by 24 weeks. μg dose of CIFN in combination with weight-based ribavirin. Our study effectively defines the upper limit of tolerability of daily CIFN dosage when used in combination with ribavirin. Furthermore, this high dosage of CIFN and weight-based ribavirin combination treatment was ineffective in eradicating hepatitis C virus in patients who failed previous pegylated interferon and ribavirin therapy.We cannot recommend this higher daily 24


Severe acute exacerbation in patients with chronic hepatitis B is a unique clinical presentation that results in high mortality. To compare the efficacy of 4 weeks of entecavir or lamivudine therapy in patients with severe acute exacerbation caused by chronic hepatitis B, 65 patients were retrospectively analyzed. The groups received 0.5 mg entecavir (group A) or 100 mg lamivudine (group B) daily. After 4 weeks of treatment, virological and biochemical responses and the deterioration rates were determined. The average age and alanine aminotransferase (ALT), bilirubin, albumin, creatinine (Cr), and HBV DNA levels were similar in groups A and B prior to treatment, but the prothrombin time (PT) was shorter in group A. Following treatment, ALT and bilirubin levels were improved while there were no significant changes in PT, albumin or Cr levels at week 4 in group B. Only ALT was markedly upregulated following treatment in group A. There were no significant differences between the virological and biochemical responses or the deterioration rates of the two groups. These results suggested that short-term treatment with lamivudine markedly alleviated the increased bilirubin (TB) levels in patients with severe acute exacerbation of chronic hepatitis B and that there was no significant difference in the deterioration rate between patients treated by the two types of medication. Chronic hepatitis B virus (HBV) infection is a public health concern worldwide and is responsible for 1 million deaths annually. The virus significantly increases the risk of hepatitis, cirrhosis and hepatocellular carcinoma . DeterioLamivudine was the first registered oral antiviral drug and has been widely used due to its efficacy during short-term administration . OutcomeThe present study retrospectively compared the effect of entecavir and lamivudine in the short-term treatment (30 days) of patients with severe acute exacerbation of chronic hepatitis.μmol/l). All 65 patients had been recorded as positive for the hepatitis B surface antigen (HBsAg) for >6 months. Patients coinfected with hepatitis A, C and E viruses were excluded by serological assays. An abdominal ultrasound was performed at admission to exclude cases of hepatocellular carcinoma and biliary obstruction. There were no patients with hepatic encephalopathy, ascites or abnormal renal function tests at baseline. Patients who received immunosuppressants or systemic corticosteroids were also excluded. Cirrhosis in patients was defined by an ultrasound revealing a coarse liver echotexture with nodularity and small liver size and the presence of portal hypertension . Prior written and informed consent was obtained from each patient and the study was approved by the ethics review board of Shandong University.A total of 65 patients who were consecutively admitted to the First Affiliated Hospital of the College of Medicine, Zhejiang University, China, between May, 2008 and May, 2011 with spontaneous severe acute exacerbation of chronic hepatitis B, were retrospectively studied. Severe acute exacerbation of chronic hepatitis B was defined as an elevation of ALT to a level ≥10-fold higher than the upper limit of normal  and an elevation of bilirubin to a level ≥3-fold higher than the ULN  and HBV DNA titer were analyzed at week 4 following the treatment. The deterioration rate, defined as the rate of disease progression in a patient taken from the date when the first dose of lamivudine or entecavir was administered to the date of acute-on-chronic liver failure or mortality, was also calculated.HBV DNA was measured by real-time polymerase chain reaction on a LightCycler 2.0 . The fluorescence detection kit and probes were purchased from Piji Biological Engineering Co., Ltd. . The detection limit of the HBV DNA was 1,000 copies/ml and the level of negative HBV DNA was set as 500 copies/ml.Statistical analysis was performed by SPSS software (version 13). In the present study, continuous variables were expressed as the mean ± standard deviation (SD) or median (range) values. The level of HBV DNA was logarithmically transformed to a normal distribution for analysis. The significance of the differences between the results of Levene’s homogeneity examination of the normalized continuous variables was determined by the Student’s t-test. The significance of the differences between the categorical variables was analyzed by the Chi-square test. Non-conformity in the baseline between the two groups was eliminated by covariance analysis of the prothrombin time (PT). P<0.05 was considered to indicate a statistically significant difference.To determine whether the baseline conditions of subjects in groups A and B  were the same prior to the treatment, biochemical markers were analyzed. No significant differences were detected in gender or age between the two groups  . The basTo study the changes in patient responses prior to and following treatment, levels of ALT, bilirubin, albumin, Cr, HBV DNA and PT were analyzed. Following the 4-week treatment of enticavir, patients in group A showed a marked downregulation of ALT P=0.000, , with a The present study investigated the difference in the efficiency of enticavir and lamivudine treatment. There were no significant differences in PT or in the levels of ALT, TB, ALB and Cr between the two groups following the 4-week treatment . MoreoveAcute chronic liver failure (ACLF) developed in 2 patients (6.45%) in group A, while 2 patients (5.88%) in group B deteriorated into hepatic failure, with one patient undergoing liver transplantation. Therefore the deterioration rates of the two groups were similar (P=0.385).et al showed that while entecavir was the better inhibitor of hepatitis B, the mortality rate within 30 days was significantly higher than when using lamivudine (p=0.028)  . In the et al(The study of Wong et al also demet al. Therefoet al(The present study identified that besides being a virological indicator, lamivudine was able to significantly reduce the levels of ALT and bilirubin, while entecavir was only able to decrease the ALT level. These results showed that lamivudine was superior in decreasing bilirubin levels as compared to entecavir, as observed by Wong et al, but thaet al(The main purpose of the present study was not to observe the short-term effects of entecavir treatment, but to prove whether entecavir would significantly increase the short-term deterioration rate and thus cause its long-term application to be restricted. Current studies have yet to demonstrate the manner in which the anti-viral therapy may improve the short-term prognosis of patients with severe acute exacerbation of chronic hepatitis B ,16. Certet al which de


Data on which to base definitive recommendations on the doses and duration of therapy for genotype 3 HCV/HIV-coinfected patients are scarce. We evaluated the efficacy of a lower peginterferon-α 2a dose and a shorter duration of therapy than the current standard of care in genotype 3 HCV/HIV-coinfected patients.Pilot, open-label, single arm clinical trial which involved 58 Caucasian HCV/HIV-coinfected patients who received weekly 135 µg peginterferon-α 2a plus ribavirin 400 mg twice daily during 20 weeks after attaining undetectable viremia. The relationships between baseline patient-related variables, including IL28B genotype, plasma HCV-RNA, ribavirin dose/kg, peginterferon-α 2a and ribavirin levels with virological responses were analyzed.Only 4 patients showed lack of response and 5 patients dropped out due to adverse events related to the study medication. Overall, sustained virologic response (SVR) rates were 58.3% by intention-to-treat and 71.4% by per protocol analysis, respectively. Among patients with rapid virologic response (RVR), SVR and relapses rates were 92.6% and 7.4%, respectively. No relationships were observed between viral responses and ribavirin dose/kg, peginterferon-α 2a concentrations, ribavirin levels or rs129679860 genotype.Weekly 135 µg pegIFN-α 2a could be as effective as the standard 180 µg dose, with a very low incidence of severe adverse events. A 24-week treatment duration appears to be appropriate in patients achieving RVR, but extending treatment up to just 20 weeks beyond negativization of viremia is associated with a high relapse rate in those patients not achieving RVR. There was no influence of IL28B genotype on the virological responses.NCT00553930ClinicalTrials.gov  Although data on which to base definitive recommendations on the dosing and duration of therapy for co-infected patients are limited, the current standard therapy for chronic hepatitis C (CHC) genotype 3 (G3) is the combination of weekly 180 µg pegylated interferon-α (pegIFN-α) 2a or 1.5 µg/kg pegIFN-α 2b and 800 mg daily ribavirin (Rbv) for 48 weeks Besides, both pegIFN-α formulations are associated with large interpatient variability in plasma concentrations after specific doses On the other hand, the current standard treatment is associated with frequent and, sometimes, severe adverse events (AEs) and requires an extended duration of therapy. Therefore, a goal must be to find the minimal dose and duration that is effective for achieving SVR, decreasing the incidence or severity of AEs and enhancing adherence, particularly in HIV-coinfected patients in whom the treatment of HCV infection has been associated with a high rate of intolerance. In this study we evaluated the efficacy and safety of a lower pegIFN-α 2a dose (135 µg) plus flat dose ribavirin with shorter therapy duration (20 weeks after attaining undetectable serum HCV-RNA) in a cohort of HIV-coinfected patients with CHC G3. In this study we evaluated the efficacy and safety of a lower pegIFN-α 2a dose (135 µg) plus flat dose ribavirin with shorter therapy duration (20 weeks after attaining undetectable serum HCV-RNA) in a cohort of HIV-coinfected patients with CHC G3. After the study had begun, it was known that single nucleotide polymorphisms (SNPs) nearby the IL28B gene are strong predictors of response to pegIFN-α-2a plus Rbv treatment in CHC The protocol for this trial and supporting CONSORT checklist are available as supporting information; see This was a pilot, open-label, single arm, investigator-initiated clinical trial in which Caucasian HCV treatment naive patients were prospectively enrolled in four infectious disease units in Spain from January 2008 to August 2009. The trial ended after the completed follow-up of the last enrolled patient. Patients were eligible if they were older than 18 years, HIV-coinfected, and had CHC or compensated cirrhosis by HCV G3. Although the initial protocol contemplated the inclusion of patients carrying HCV G2, these were excluded from analysis as only two of these patients were enrolled. Patients were treated under routine clinical care conditions, and liver biopsy was not mandatory for inclusion in the study. Patients were excluded if they had active HIV-related opportunistic infection, cancer, hepatitis B coinfection, other causes of liver disease, hemoglobinopathies, previous history of severe psychiatric illness, autoimmune diseases, abnormal renal function, or pregnancy. There were no exclusion criteria regarding methadone use, CD4 cell counts, plasma HIV-RNA, or concurrent antiretroviral therapy.HCV genotypes were determined using a reverse hybridization assay . Plasma HCV-RNA loads were measured by a quantitative PCR assay  at baseline, after weeks 1, 2, 4, and monthly thereafter until the end of treatment, and again at 6 months after completing treatment. Hematological and biochemical profiles were also assessed at the same time points. CD4 counts (standard flow cytometry) and plasma HIV-RNA  were measured at baseline and every three months thereafter. Hepatic fibrosis was evaluated by pretreatment liver biopsy according to the Scheuer's scoring system Genomic DNA was extracted from whole blood provided in EDTA tubes using the QIAmp DNA Blood Kit (Qiagen). The rs129679860 SNP was genotyped with a custom TaqMan genotyping assay (Applied Biosystems) on DNA isolated from whole blood samples. The DNA was genotyped according to the manufacturer's instructions on a MX3005 thermocycler using MXpro software (Stratagene).min determinations were drawn on the morning of the day scheduled for weekly administration of pegIFN-α-2a after 11.45 to 12.15 hours to the previous Rbv dose  at weeks 1, 2, 4, and monthly afterwards until the end of therapy. Samples were stored at −80°C until tested. Plasma pegIFN-α-2a and Rbv concentrations were assayed by enzyme-linked immunosorbent assay Hu-INF-α , and reverse-phase high-performance liquid chromatography (HPLC-UV) as previously described Plasma samples for PegIFN-α-2a and Rbv CThe primary efficacy end-point was SVR, defined as undetectable serum HCV-RNA at 24 weeks after completion of treatment. Efficacy data were assessed by both intention-to-treat (ITT) and by per-protocol analysis. Secondary end-points were to assess the influence of IL28B polymorphisms on SVR and the relationship between the plasma pegIFN-α-2a and Rbv concentrations and virological responses in patients receiving treatment.10 IU/ml at week 12 of treatment or earlier, respectively. End of treatment response (ETR) was defined as undetectable serum HCV-RNA at the end of therapy. Relapses were defined as a detectable serum HCV-RNA at any time point after attaining ETR.Rapid virologic response (RVR) was defined as a plasma HCV-RNA<15 IU/mL at week 4 of treatment. Early virologic response (EVR) was defined as a plasma HCV-RNA<15 IU/mL or a decrease of ≥2 logSafety was assessed by means of AEs reported by patients or detected by investigators, and laboratory results at scheduled visits on weeks 1, 2, 4, 8, 12, and monthly thereafter until the end of therapy, and categorized via a standardized toxicity grade scale . For toxicity and response investigations, analysis dropouts were considered until the last available visit.Patients started treatment with the combination of weekly 135 µg pegIFN-α-2a plus oral Rbv (400 mg twice daily), and a planned duration of 20 weeks after attaining undetectable serum RNA-HCV. Treatment was discontinued at week 12 in patients not achieving EVR, and these cases were considered as virological failures. Antiretroviral treatment and use of erythropoietin remained at the responsible physicians' judgment.This pilot study was designed as a single-arm, uncontrolled trial, with a historical comparison. For ‘a priori’ sample size calculations, the Apricot trial, the largest clinical trial with pegIFN-α-2a plus Rbv conducted in HCV/HIV-coinfected patients  so far, was considered as historical reference. The SVR rate observed in patients harbouring HCV genotype 2 or 3 in the Apricot trial was 62%. Assuming a 57% SVR rate in our patients, a sample size of 60 patients would be required to reach a 80% statistical power with an asymptotic unilateral confidence interval of 95% and a 10% noninferiority margin .Continuous variables are expressed as median (range) and categorical variables are expressed as the number of cases (percentage). For this analysis, the level of 800000 IU/mL was chosen as the cut-off point for plasma HCV-RNA concentration.95) around single proportions were calculated as p±1.96 q = 1 − p. The relationships between virological responses and potentially predictive variables were examined using χ2 tests. To assess the independence of these factors, a logistic regression analysis was used in which all variables with P values below 0.2 in the initial analyses were included. All tests were two-tailed, and the differences were considered significant when P was<0.05. Statistical calculations were performed with the Statistical Product and Service Solutions for Windows .Intraindividual variability in plasma concentrations was assessed by measuring the coefficient of variation (CV = standard deviation/mean×100) of all the available values from each patient throughout the follow-up. Interindividual variability was calculated using the CV for the geometric mean of each subject. Ninety-five percent confidence intervals  and in those carrying the T allele .A total of 60 Caucasian HCV/HIV-1-coinfected patients  were included in the study in four infectious disease units in Spain from January 2008 to August 2009. The trial ended after the completed follow-up of the last enrolled patient. The two patients with HCV G2 were excluded from analysis for homogenizing results. Baseline characteristics are summarized in The study flow diagram and virological responses are shown in 95: 43.8 – 70.0%) by ITT, and 70.2% (CI95: 56.6 – 83.8%) by per protocol analysis, with a relapse rate of 23.2% (CI95: 10.1 – 36.4%). Among those 28 patients who had RVR, 24 (85.7%) attained SVR by ITT analysis, with rates of SVR and relapses of 92.3% and 7.7%, respectively, by per-protocol analysis. On the other hand, among the 32 patients who did not achieve RVR, 9 of them (28.1%) attained SVR and 8 relapsed.Overall, SVR rates were 56.9% . The median Rbv dose was 11.6 mg/kg . The results of drug plasma levels are shown in No relationship was observed between Rbv levels and body weight , the administered dose/kg  or the estimated glomerular filtration rate .95: 1.04 – 27.81; P = 0.04), SVR , and relapses . Specifically, no relationships were observed between any virological responses or relapses and rs129679860 genotype , 0.811 , 0.697 , and 0.587 , respectively. For Rbv these values were 0.673 , 0.380 , 0.375 , and 0.632 , respectively. Nevertheless, the regression model based on the selected variables explained only moderately the observed variability in the SVR as Nagelkerke's R2 was 0.439.Receiver-operating characteristic curve analysis showed that the ability of PegIFN-α 2a and Rbv plasma concentrations to identify viral responders was null. Particularly, the areas under the curve for RVR, EVR, ETR and SVR for PegIFN-α 2a levels were 0.505  achieved similar SVR rates in HCV monoinfected patients with G 3, both as a single agent Our results suggest that pegIFN-α-2a given at 135 µg once weekly might be as effective as the standard 180 µg dose, when administered together with 800 mg daily of Rbv in G3 HCV/HIV-coinfected patients, since both on treatment RVR and EVR rates in our study  were similar to those observed in other studies as the Apricot trial  Our hypothesis is also supported by the lack of relationship between pegIFN-α 2a plasma levels and the rate of viral response, despite those being lower than that observed previously with pegIFN-α 2a at 180 µg once weekly  No relationship was observed between Rbv plasma levels and viral responses during therapy or SVR rate, similar to results in our previous study Likewise, we observed no relationship between rs129679860 IL28B genetic polymorphisms and virological responses to pegIFN-α 2a plus Rbv in our patients with CHC G3, as has been previously reported in both G3 monoinfected and HCV/HIV-coinfected patients Regarding the incidence of toxicity, it is remarkable that both clinical and laboratory AEs were just mild and did not motivate dose reductions in patients receiving the 135 µg dose. Four out of the 8 cases of AEs which motivated treatment interruption were considered unrelated to study treatment. Thus, the rate of drop-outs due to treatment-related toxicity was only 6.9%, which compare favorably with previous studies in which the rate of serious AEs varied between 10 and 18% The main limitation of our study is the fact that it is a single-arm trial in which the results were compared with those observed in earlier clinical trials. However, we believe such a study had to be carried out prior to the design of a randomized controlled trial allowing for a one-to-one comparison between efficacy and safety of weekly 135 and 180 µg doses of pegIFN-α-2a in G3 HCV/HIV-coinfected patients. Furthermore, a high drop-out rate (18.9%), mainly due to low grade AEs and those unrelated to the study medication, penalized the intention-to-treat analysis results.In conclusion, our results suggest that weekly 135 µg pegIFN-α 2a could be as effective as the standard 180 µg dose, with lower incidence of severe AEs, supporting the design of a randomized controlled trial allowing for a one-to-one comparison between efficacy and safety of weekly 135 and 180 µg doses of pegIFN-α-2a in G3 HCV/HIV-coinfected patients. Our data evidence that neither the rs129679860 IL-28B genetic polymorphisms nor plasma pegIFN-α-2a or Rbv levels influence the virological responses in G3 HCV/HIV-coinfected patients. On the other hand, while a 24-week treatment duration appears to be appropriate in patients achieving negative viremia on week 4, extending treatment duration up to just 20 weeks beyond negativization of viremia is associated with a high relapse rate in those patients not achieving RVR, and therefore a more prolonged treatment is warranted in these patients.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial protocol.(PDF)Click here for additional data file.


The use of combination antiretroviral therapy (cART) has become a standard of care for the treatment of HIV infection. However, cost and resistance to cART are major obstacles for access to treatment especially in resource-limited settings. In this study, we aimed to determine the incidence and risk factors of treatment failure in a cohort of treatment-naïve Thai HIV-infected patients.A retrospective cohort study was conducted among HIV-infected patients initiating their first cART at Chiang Mai University Hospital, Thailand.3 . GPO-VIR®  was the most common prescribed cART . Seventy-six patients developed virological failure given the cumulative incidence of 9.6%. The incidence of virological failure was 2.79  cases per 100 person years. Poor adherence was the strongest predictor for virological failure. Of 535 immunologically evaluable patients, 179 (33.5%) patients developed immunological failure. A low CD4 cell count at baseline (< 100 cells/mm3) and the increment of CD4 cell count of < 50 cell/mm3 after 6 months of cART were the predictors for immunological failure (p < 0.001).From January 2002 to December 2008, 788 patients were enrolled; 365 were male (46.3%), and the mean age was 37.9 ± 8.6 years. The median baseline CD4 count was 57.7 cells/mmThis study demonstrated that even in resource-limited settings, the high rate of success could be expected in the cohort with good and sustainable drug adherence. Poor adherence, older age, and low baseline CD4 cell count are the predictors for unfavorable outcome of cART. During the study period of 6 years and a median follow-up duration of 3.63 years , the cumulative incidence of treatment failure was 12.3% and 9.6% by intention-to-treat and on treatment analyses, respectively. This result is similar to failure rates found in a previous study by Anekthanonon et al from Bangkok, Thailand  and in tWe found that adherence of < 95% and age < 40 years were the independent predicting factors for virological failure. Although adherence has been demonstrated to be important in many clinical cohorts, -21there 3 [3 after 2 years of cART between those with baseline CD4 cell count ≤ 100 cell/mm3 and those with higher baseline CD4 count (59.0% vs. 79.8%). It has been hypothesized that patients who initiated cART late in the course of disease may have thymic damage and limitation of T cell production and proliferation [3 after the first 6 month of cART may predict immunological failure. Failure to achieve early CD4 recovery could be another signal of thymic damage. Our study did not find the difference in CD4 recovery between sexes, which was similar to other reports [The SWISS HIV Cohort Study showed that only 39% of individuals treated with cART reached CD4 T-lymphocyte count of 500 cells/mm3 . Many HI3 -10,13,24feration ,26. We c reports ,16,25,27 reports ,28. Whet reports ,30. In tOur study has several limitations. First, HIV-1 RNA was not monitored at baseline in all patients so we couldn't evaluate the higher baseline HIV-1 RNA viral load as a predictor for virological failure. Second, the HIV-1 RNA monitoring was not regularly performed in all patients, therefore, we could not calculate the accurate time of treatment failure. It is also possible that some patients may already have virological failure at the time we evaluated the immunological criteria and thus misinterpret the true CD4 cell count response. Third, assessment and documentation of patient's adherence to cART was not standardized. However, this reflected the usual clinical practice and adherence was still the strongest predictor for virological failure. Fourth, this is a retrospective cohort study that the accuracy of analysis depends on the completeness of data entry into the database. Finally, this study is limited to a single cohort, the findings should be confirmed in other clinical settings.In summary, this analysis of a cohort of HIV-infected patients under usual clinical care showed that predictors of immunological and virological failure can be identified using data routinely recorded. The risk factors of virological failure were poor adherence of cART and younger age, whereas those of immunological failure were low baseline CD4 count and slow CD4 cell recovery. This study demonstrated that even in resource-limited settings, the high rate of success could be expected in the cohort with good and sustainable drug adherence.The authors declare that they have no competing interests.NK participated in data collection, performed the statistical analysis, and drafted the manuscript. RC participated in the design of the study, performed the statistical analysis, and drafted the manuscript. KS and TS revised manuscript critically for important intellectual content. All authors read and approved the final manuscript.


Treatment outcomes of HIV patients receiving antiretroviral therapy (ART) in Rwanda are scarcely documented. HIV viral load (VL) and HIV drug-resistance (HIVDR) outcomes at month 12 were determined in a prospective cohort study of antiretroviral–naïve HIV patients initiating first-line therapy in Kigali. Treatment response was monitored clinically and by regular CD4 counts and targeted HIV viral load (VL) to confirm drug failure. VL measurements and HIVDR genotyping were performed retrospectively on baseline and month 12 samples. One hundred and fifty-eight participants who completed their month 12 follow-up visit had VL data available at month 12. Most of them (88%) were virologically suppressed (VL≤1000 copies/mL) but 18 had virological failure (11%), which is in the range of WHO-suggested targets for HIVDR prevention. If only CD4 criteria had been used to classify treatment response, 26% of the participants would have been misclassified as treatment failure. Pre-therapy HIVDR was documented in 4 of 109 participants (3.6%) with an HIVDR genotyping results at baseline. Eight of 12 participants (66.7%) with virological failure and HIVDR genotyping results at month 12 were found to harbor mutation(s), mostly NNRTI resistance mutations, whereas 4 patients had no HIVDR mutations. Almost half (44%) of the participants initiated ART at CD4 count ≤200cell/µl and severe CD4 depletion at baseline (<50 cells/µl) was associated with virological treatment failure (p = 0.008).Although the findings may not be generalizable to all HIV patients in Rwanda, our data suggest that first-line ART regimen changes are currently not warranted. However, the accumulation of acquired HIVDR mutations in some participants underscores the need to reinforce HIVDR prevention strategies, such as increasing the availability and appropriate use of VL testing to monitor ART response, ensuring high quality adherence counseling, and promoting earlier identification of HIV patients and enrollment into HIV care and treatment programs. Improved access to combined antiretroviral therapy (ART) has significantly reduced HIV-related morbidity and mortality worldwide However, ART scale up in resource-poor settings could accelerate HIVDR emergence HIVDR can develop because of the error prone nature of HIV replication resulting in a high mutation rate in combination with the ongoing presence of drug-selective pressures. HIVDR strains that emerge after treatment initiation (referred to as acquired or secondary HIVDR) can subsequently be transmitted to previously uninfected patients (referred to as transmitted or primary HIVDR) HIVDR increases direct medical and laboratory costs associated with treatment failure as well as indirect health care costs associated with having to switch patients to more expensive second-line therapy and the ongoing need to develop new drugs. Therefore, HIVDR prevention should be an important public health goal in countries with limited resources.Recent reports indicate that the lack of VL monitoring during ART leads to late detection of virological failure. Long-term exposure to failing regimens facilitates the emergence and accumulation of acquired HIVDR mutations Since Rwanda initiated ART roll-out in 2004, few data have been generated on treatment outcomes in general, and prevalence and incidence of transmitted and acquired HIVDR in particular. Here, we describe transmitted HIVDR at baseline, and treatment outcomes and acquired HIVDR one year after ART initiation in a cohort of Rwandan HIV patients. Treatment outcomes were specifically examined as a function of immunological status at ART initiation, and other potential determinants of virological failure after 12 months of treatment were also examined.Ethical clearance was obtained from the Rwandan National Ethics Committee. All study participants provided written informed consent prior to study enrolment. Participants had the right to withdraw from the study at any time.th of September 2010 were analyzed.The present study was part of a larger prospective investigation addressing the secondary effects of HIV treatment and its impact on reproductive health in a cohort of patients initiating ART . The study was conducted at the Treatment and Research AIDS Center (TRAC-Plus) outpatient clinic. Patients were enrolled between November 2007 and January 2010, and were followed up for a maximum of 24 months per person. In this report, only data up to month 12 of follow-up and collected before the 15Medical visits were scheduled at baseline (ART initiation), week 2, month 1, month 3 and every three months thereafter until month 12. Information on demographics, sexual behavior, clinical history and physical examination was collected at these visits. Pharmacy visits were scheduled at baseline, every week during the first four weeks of ART (week 1 to 4), and every month thereafter (month 2 to 12). During these visits, medications were delivered and treatment adherence was monitored. Laboratory tests  were scheduled at baseline, month 6 (CD4 count only) and month 12. Study staff minimized loss-to-follow-up by actively contacting participants who had missed a scheduled clinic visit and by providing travel reimbursements to participants.HIV-positive patients attending the TRAC-Plus clinic, who were ARV-naive and immediately eligible for ART according to the Rwandan national guidelines ART was provided through the national HIV treatment program. Participants received a first-line regimen in accordance with the 2007 Rwanda national ART treatment guidelines, which were in line with WHO recommendations at that time ART adherence was captured in three different ways. First, a standardized questionnaire including questions on frequency of dosing, missed doses, drug sharing and reasons for poor adherence was administered by the study nurse at month 1 and at every three-month visit thereafter. Second, pill counts were recorded at each monthly pharmacy visit. Third, the attendance rates of medical and pharmacy appointments (with a 7 day window) were recorded. Participants were classified as fully adherent if they took more than 95% of their prescribed ART regimen doses and as poorly adherent otherwise.Blood samples for laboratory testing were collected at baseline, month 6 (CD4 count only) and month 12. Five milliliters (mL) of whole blood was collected in EDTA vacutainer tubes . Fifty microliters (µl) of fresh blood was used for CD4 cell enumeration. Plasma was separated from the cellular fraction by centrifugation and collected into three aliquots of one mL each, within four hours after blood collection. Plasma samples were stored at −80°C until further analysis.CD4 count was determined at baseline, month 6 and month 12 in whole blood on a single flow-cytometry platform using TruCOUNT® tubes on a FACScalibur instrument  and according to the manufacturer's instructions.Immunological failure was defined as failure to achieve a CD4 gain of at least 50 cells above pre-therapy levels or having an absolute CD4 count of less than 100 cells/µl after one year of therapy HIV RNA VL was measured at baseline and month 12. Viral RNA quantification was performed on thawed plasma using the Roche CobasAmpliPrep/CobasTaqMan HIV-1 version 2  and according to the manufacturer's instructions. The lower limit of detection was 40 copies of HIV RNA/mL. Virological treatment failure was defined as a confirmed VL>1000 copies/mL at month 12.HIVDR genotyping was done retrospectively on samples from participants that had completed their month 12 visits and had available VL data at baseline and month 12. Baseline and month 12 samples with a VL>1000 RNA copies/mL were analyzed at the Department of Medical Microbiology  using the Viroseq HIV genotyping kit version 2  on an automated sequencer ABI 3130 XL . Sequences of amplified viral genes coding for the HIV-1 protease and reverse transcriptase enzymes were assembled, edited using the software provided by Viroseq, and submitted to the Stanford University database. Baseline sequences were categorized according to the WHO list of mutations for surveillance of transmitted drug resistant HIV strains (2009 update) Analyses were conducted with STATA version 11 .Baseline characteristics were reported as percentages for categorical data and means with standard deviations (SD) for continuous data. Differences between the groups were tested using the Pearson Chi-square test, the student's t-test and the Fisher's exact test, as appropriate. Stem and leaf plots and Shapiro-Wilks test were used to investigate the normality of data distribution. If variables deviated from the normal distribution, medians and interquartile ranges (IQRs) and non-parametric tests were used.Potential risk factors for virological treatment failure at month 12 were examined by bivariable and multivariable logistic regression analysis. Factors were included in the multivariable analysis when p-values<0.2 in bivariable analysis Two hundred and eighteen HIV-1 positive participants (52.3% women) visited the TRAC-Plus clinic, were eligible for ART and consented to participate in the study . Of thesAt ART initiation, the 158 participants with 12 months of follow-up who were included in this analysis did not significantly differ from the 213 participants who enrolled in the SEARCH study for any of the baseline parameters collected (data not shown). The mean age was 37.9 years (SD = 7.6) and 55.1% were females . Of the All participants were prescribed an appropriate ART regimen as per the 2007 Rwanda ART National guidelines. A combination of zidovudine (AZT), lamivudine (3TC), and nevirapine or efavirenz (NVP/EFV) was the most commonly (89%) prescribed first-line regimen. The other first-line regimens contained tenofovir  or stavudine  instead of zidovudine. Women with a previous history of PMTCT all received NNRTI-based regimen. Participants with virological failure at month 12 were more likely to have initiated ART at a more advanced WHO stage 3 or 4 (38.9%) than participants with virological suppression at month 12 . Participants with virological treatment failure also had lower baseline CD4 count  and a higher proportion of baseline HIVDR mutations  than participants with virological suppression.HIVDR genotyping was performed in pre-ART plasma specimens from 109 participants and HIVDR mutations were identified in 4 of them . NNRTI HIVDR mutations were detected in all 4 participants. The K103N mutation was the single NNRTI mutation detected in 3 of 4 patients, with one of them also harboring the M184V NRTI mutation . One patThe HIV-1 subtype distribution in the SEARCH cohort has been described elsewhere Among the 4 participants with baseline HIVDR, the observed proportions of subtype C (2/4), A1 (1/4) and A1/C (1/4) were significantly different than those observed in the 105 participants with no HIVDR mutation at baseline (Fisher's exact test p = 0.036).Forty-one substitutions within the first-line, and two switches from first- to second-line regimens, occurred during the study period . The mosThe proportion of patients with virological failure was higher, although not significantly so, in the group of participants that initiated ART at CD4 count ≤200 cell/µl as compared to the group that initiated therapy at CD4 count >200 cells/µl . None of the women that had received NVP or AZT for PMTCT experienced virological treatment failure at month 12. Only nine of the 18 participants with virological failure (50%) also experienced immunological failure at month 12 and would have been correctly identified as failing their treatment (sensitivity = 50%). Conversely, 32 of 41 participants with virological suppression (23.8%) would have been misclassified as experiencing treatment failure based on immunological criteria at month 12 (specificity = 77%). Although the difference was not significant, acquired HIVDR mutations at month 12 were more frequent in the group of participants with baseline CD4 count ≤200 cells/µl, as compared to participants with baseline CD4 count >200 cells/µl .Samples from 12 of the 18 participants with HIV VL>1000 copies/mL at month 12 were successfully genotyped. Eight participants harbored major mutations while no evidence of acquired HIVDR was found in 4 participants . HIVDR gAll cases of acquired HIVDR involved at last one NNRTI mutation, with the K103N mutation being the most frequently observed (6/8), followed by the G190A mutation (3/8) and the Y181I mutation (2/8). The K101Q, V108I and V106M mutations were each observed once as a single mutation . CombinePatients with virological failure at month 12 were not less adherent than the rest of the group regardless of the method used to measure adherence. Levels of antiretroviral treatment adherence in this cohort have been analyzed elsewhere Bivariable analyses indicated that treatment adherence by pill count  and baseline viral load  were not significant risk factors for virological failure at 12 months. HIV subtype was not a significant risk factor for virological failure at month 12 either when comparing subtype C to subtype A1(OR 3.65 (95% CI 0.34–39.09) and when comparing recombinant subtype A1/C to subtype A1 (OR 3.65 (95% CI 0.34–39.09).In the multivariable model, participants with advanced HIV disease defined by WHO HIV clinical stage 3 and 4 were more than 5 times more likely to have virological failure compared to those with WHO HIV clinical stage 1 . In addition, severe immunosuppression at ART initiation (CD4 count <50 cells/µl) was significantly associated with virological failure at month 12 . In addition, higher frequency of virological failure and acquired HIVDR mutations was associated with lower CD4 count at baseline. Initiation of ART at a more advanced stage of HIV disease is common in sub-Saharan Africa and negatively impacts HIV care and treatment program outcomes As previously documented, immunological criteria were poorly correlated with virological treatment failure The 11% of participants that developed virological failure after 12 month of treatment is comparable to findings from 12 low- and middle-income countries of Asia and Africa, reporting an average of 9.4% patients experiencing treatment failure after one year Among the 18 participants failing therapy, at least four (22%) did not show any evidence of HIVDR mutations, indicating that they are failing therapy for reasons other than drug resistance. One reason might have been sub-optimal adherence although none of the treatment adherence measures collected in this study was identified as a predictor for treatment failure. Eight of the 12 study participants (66.6%) genotyped at treatment failure had evidence of HIVDR, which is comparable to the 63.7% reported in more than 2000 HIV patients initiating first-line ART using the WHO approach in Eastern Africa between 2006 and 2010 Overall, the NRTI and NNRTI transmitted and acquired mutation patterns that we identified were consistent with previous reports in similar settings In 2 of 4 participants with HIVDR mutations at baseline, exposure to a failing regimen during 12 months was associated with the accumulation of additional HIVDR mutations, including the emergence of TAMs, also in association with M184V. M184V and TAMs confer cross-resistance to NRTIs and their relatively high overall prevalence in this cohort may have consequences for second line treatment responses in Rwanda. NRTI cross-resistance has the potential to significantly reduce the activity of the NRTI backbone of standard second line regimens. More specifically, TAMs have the capacity to reduce the efficacy of TDF containing-NRTI backbones. Functional PI monotherapy will lower the barrier for PI resistance Although our findings may not be generalized to all HIV clinics in Rwanda, they indicate that efforts to minimize HIVDR are needed. These should include improved availability and utilization of VL-based monitoring of ART response, and evaluation of the potential added value of HIV genotyping at treatment failure. In addition, high quality patient support for treatment adherence as well as earlier initiation of therapy will contribute to protecting the efficacy of second line and subsequent therapy and improving overall treatment outcome.


World Health Organization guidelines now recommend routine use of viral load testing, where available, for patients receiving antiretroviral treatment (ART). However, its use has not been routinely implemented in many resource-limited settings due to cost, availability and accessibility. Viral load testing is complex, making its application in resource-limited settings challenging. We describe the issues encountered by Médecins Sans Frontières (MSF) when using routine viral load testing in a large HIV programme in sub-Saharan Africa.Between October 2005 and August 2006, more than 1200 patients on ART had viral load tests at baseline and at three-month intervals performed by a local reference laboratory that was quality assured by an experienced international institution. Concerns with reliability of results halted testing. The quality control measures instituted with a second laboratory and outcomes of these were documented.In 2005 and 2006, only 178 of 334 (53%) previously ART-naïve patients tested after six to 12 months of treatment had viral loads of less than 1000 copies/mL. Similar MSF programmes elsewhere demonstrated virological suppression rates of more than 85%, and duplicate testing showed unacceptable discordance. Laboratory problems encountered included: disregarded quality control; time delays; requirement for retesting; and duplicate sample variations. Potentially harmful clinical outcomes of inaccurate viral load results include: unnecessary ART regimen changes; unnecessary enhanced adherence counselling after "false failures"; and undetected virological failure.Viral load testing performed without rigorous quality control carries the risk of erroneous and potentially damaging results. Viral load testing should be utilized only if robust quality assurance has been implemented. Our experience in this and other settings led to the development of a guide for assessing the suitability of a laboratory for viral load testing that can be used to help achieve reliable results. Viral load (VL) testing is the only definitive method for early detection of antiretroviral treatment (ART) failure ,2. HIV tAn HIV programme run by Médecins Sans Frontières (MSF) in an urban resource-limited setting in sub-Saharan Africa offered free medical care and psychosocial support to people living with HIV/AIDS. Patients could self-refer for on-site counselling and testing, or were referred by other healthcare providers. Over a period of more than five years, MSF registered more than 2700 adults living with HIV/AIDS and provided ART to almost 2000. Most were women (63%), and many survived on less than US$2 per day. We describe the experience of routine VL testing for patients receiving ART in this HIV programme in two separate series of events between 2005 and 2008 Figure .Between October 2005 and August 2006, more than 1200 patients on ART had VL tests at baseline, and every three months thereafter. Samples were collected in EDTA tubes, centrifuged for 10 minutes at 800 × g and the plasma stored in sterile tubes at -20°C for less than eight weeks. Samples were measured at a local reference laboratory (Lab 1) using Roche Cobas Amplicor HIV-1 Monitor Test v1.5 and Cobas Amplicor Analyser , with preparation of frozen plasma in accordance with the Amplicor v1.5 manual guideline.Prior to use, Lab 1 was assessed by an MSF laboratory scientist for: training of personnel; appropriate laboratory facilities; workflow; separation of areas for sample preparation, reagent preparation and sample analysis; backup power for laboratory freezers and refrigerators; temperature monitoring guidelines; cleaning guidelines for different laboratory work areas; and access to water of appropriate quality. The laboratory was found to be of a high standard, and was supported and quality assured by an international institution with experience in VL testing, with tests performed by trained staff already experienced with the procedures. No external quality control was organized by MSF.Due to concerns regarding the validity of results, sample collection and preparation procedures were reviewed and reinforced, and additional samples were tested as blinded duplicates: 21 duplicates were sent to Lab 1 and another laboratory in-country . A further 29 duplicates were sent to Lab 1 and a reference laboratory in another sub-Saharan African country (Lab 3) that used Roche Amplicor. The differences between paired log VL results and the mean absolute differences for all samples were calculated, and the limits of agreement between laboratories compared using the method of Bland and Altman . The resIn mid 2007, the internationally supported Lab 2 was re-assessed by an MSF laboratory scientist, and external quality control of VL performance was undertaken with the lab's consent. Ten triplicate specimens, obtained with patient consent and prepared as previously, were sent to laboratories in Europe (Lab 4 and Lab 5) that used COBAS AmpliPrep/COBAS TaqMan HIV-1 Tests . This process involved approximately one week of staff time, cost around US$1500 for testing and sending frozen samples with dry ice and triple-packaging by courier, and incurred considerable difficulties in ensuring unbroken cold chain conditions.Results were compared as for the earlier duplicate testing. After an acceptable outcome of this quality review, VL tests were performed on all patients after at least six months on ART. Specimens were labelled with patient name, ID code and date of collection. Samples were sent in large batches with both electronic and printed lists of these details, all of which were cross checked at the time of specimen storage and again when samples were shipped frozen on ice. Results were provided on these lists, both electronically and printed.After consideration of the MSF Ethics Review Board framework criteria, this paper was assessed as not needing ethics approval. The work described was undertaken as part of routine programme work and analyzed retrospectively. Neither the programme nor individuals have been identified.In 2005 and 2006, 178 of 334 (53%) previously ART-naïve patients tested by Lab 1 after six to 12 months of treatment had VLs of less than 1000 copies/mL. However, only 4% showed evidence of immunological failure according to 2006 WHO guidelines . VirologTolerating variation of up to 1.0 log difference in results , there was substantial discordance in results of duplicate samples tested by Lab 1 and comparison laboratories  performed by Lab 2 in 2007 were released to the programme as valid, but quality control data to verify this were not released concurrently. MSF received VL results from the test runs on average 27 days (range 5-69) after sample receipt by Lab 2, but the internal quality control data were received a further 34 days (range 25-42) later, despite repeated requests. Reasons for VL result delays included laboratory stock shortages caused by delays in importing test kits and a lack of in-country technical support for instrument maintenance and repairs. As a result, more than 300 of the 614 patients tested in the first batch did not receive their VL results at their scheduled follow-up appointment, and clinicians could not use the information for optimum care.When eventually released, quality control data showed 363 (59%) of the initial 614 samples were invalid because calibration curves or positive controls were outside acceptable limits. These faults were only recognized when data were examined by MSF staff and external international advisers; the expert interpretation was communicated to Lab 2 and the service agreement conditions were further clarified. The official release of results by Lab 2 combined with the delay in receiving quality control data meant that some patients received inaccurate results. Seventy-eight of the 363 patients from these invalid test runs were told that their VLs were greater than 1000 copies/mL, which was the project criteria for enhanced adherence counselling, whereas other results might have been inaccurately low. Lab 2 blamed calibration errors on power failures and an inverter too weak to prevent interruptions when switching from national power to generator. The initial sample volume requested by Lab 2 was inadequate for retesting, and the 363 patients had to be recalled for repeat venepuncture. Of the first 124 of these samples retested, 52 (42%) were in a second invalid run and required further repeat testing.Sending samples internationally for ongoing quality control was not feasible because of the time, logistics and expense involved. Instead, around 10% of samples after the initial eight test runs were sent in blinded duplicate to Lab 2. Of 46 initial sample pairs in test runs with acceptable quality control, the result for seven (15%) duplicate pairs was greater than 1.0 absolute log difference (maximum 2.7 log).Although we support the WHO recommendations on routine VL testing in principle, our results question the feasibility of safe implementation in resource-limited settings with current technology in the absence of real-time review of quality control data by an experienced third party. Inaccurate results and quality control delays created the potential for HIV patients to be attributed with "false virological failures". This caused confusion and distress for patients, since ART appeared to be failing despite potentially optimal adherence.Extra appointments were also needed, which increased time and transport costs. ART might also have been switched to second-line unnecessarily. No regimens were changed on the basis of invalid 2007 results; however, on file review, three changes in 2006 were possibly influenced by VL results later deemed invalid. Second-line treatment carries an increased pill burden and cost, and if true virological failure occurred later, patients might be assumed to have no effective regimens in the absence of third-line ART. Importantly, poor VL performance may have resulted in false negative results, where patients with virological failure were not detected, putting them at risk of severe clinical illness and antiviral resistance.Retesting patients because of quality control problems was a burden for them and for programme staff, especially as some patients had to be recalled to discuss released, then invalidated, results. This led to apparent frustration, loss of confidence, and inconvenience for patients. Perceived poor VL outcomes led to inaccurate assessments of programme quality, with consequent unnecessary programmatic changes.There are potential limitations with the quality control analyses with regard to the number of externally tested samples, time of storage, and comparison of VL on duplicate samples using different tests in different laboratories and where subtype was not tested. However, these differing test methods would be expected to give comparable results -17. In vWe cannot assume that a laboratory will provide accurate results simply because it is supported by a reputable international laboratory and participates in an external quality assurance programme. Sophisticated equipment does not guarantee accurate results . ContracBarriers to obtaining reliable laboratory results for complex tests in resource-limited settings include: unpredictable power supplies; shortages of service engineers; expense of reagents in performing repeat test runs; lack of motivation and accountability; and shortages of highly qualified scientific staff ,18,20. IThe resources and expertise needed to ensure accuracy make it unlikely that all programmes will be able to achieve sufficient confidence in VL results. Worryingly, many programmes are already using VL technology routinely in settings where it is difficult to ensure the quality of laboratory services . Our resOur experience of the difficulties encountered in this programme and other programmes in eastern Europe, Asia and other countries in sub-Saharan Africa led to development of a guide for assessing a laboratory's suitability for VL (or other) testing (See additional file Although the quality of VL testing may be reliable in many resource-limited settings, the need to ensure that this is true applies to all laboratories in these settings. Despite cost and logistical issues, blinded duplicate samples should be sent to the testing laboratory, with investigation triggered by a simple rule violation of any identical samples being outside the expected range of difference.PRH declares grant funding and/or consulting relationships with Virco, Viiv, Quest, Pfizer, Abbott and Merck. The other authors declare that they have no competing interests.JG, PDC, CM, DK and DOB contributed to the study conception and design. JG, DK and DOB collected and analyzed data. JG and DOB wrote the first draft of the paper. DK and PDC contributed to the writing of the paper. CM, SJ and PRH reviewed the paper. All authors read and approved the final manuscript.ChecklistClick here for file