Prospective Study of Chikungunya Virus Acute Infection in the Island of La Réunion during the 2005–2006 Outbreak

Background

Chikungunya virus (CHIKV) is a recently re-emerged arthropod borne virus responsible for a massive outbreak in the Indian Ocean and India, and extended to Southeast Asia as well as Italy. CHIKV has adapted to Aedes albopictus, an anthropophilic mosquito species widely distributed in Asia, Europe, Africa and America. Our objective was to determine the clinical and biological features of patients at the acute phase of CHIKV infection.

Methods and Findings

A prospective study enrolled 274 consecutive patients with febrile arthralgia recorded at the Emergency Department of the Groupe Hospitalier Sud-Réunion between March and May 2006. Three groups were defined: one group of 180 viremic patients (positive CHIKV RT-PCR), one group of 34 patients with acute post-viremic infection (negative CHIKV RT-PCR, positive anti-CHIKV IgM and negative IgG), and one group of 46 uninfected patients (negative CHIKV RT-PCR, anti-CHIKV IgM and IgG). Bivariate analyses of clinical and biological features between groups were performed. Patients with CHIKV viremia presented typically with asymmetrical bilateral polyarthralgia (96.5%) affecting the lower (98%) and small joints (74.8%), as well as asthenia (88.6%), headache (70%), digestive trouble (63.3%), myalgia (59%), exanthems (47.8%), conjunctival hyperhemia (23%) and adenopathy (8.9%). Vertigo, cutaneous dysesthesia, pharyngitis and haemorrhages were seldom observed. So far unreported symptoms such as chondrocostal arthralgia (20%), entesopathies (1.6%), talalgia (14%) were also noted. Prurit was less frequent during the viremic than post-viremic phase (13.9% vs. 41.2%; p<0.001), whereas lymphopenia was more frequent (87.6% vs. 39.4%; p<0.001). Others biological abnormalities included leukopenia (38.3%), thrombocytopenia (37.3%), increased ASAT and ALAT blood levels (31.6 and 7.3%, respectively) and hypocalcemia (38.7%). Lymphopenia <1,000/mm³ was very closely associated with viremic patients (Yule coefficient 0.82, positive predictive value 92.3%). Age under 65 was associated with a benign course, as no patients younger than 65 had to be hospitalized (Yule coefficient 0.78).

Conclusions

The diagnosis of CHIKV infection in acute phase is based on commonly accepted clinical criteria (fever and arthralgia), however clinical and biological diffrences exist in acute phase depending on whether or not the patient is within the viremic phase of the infection.     

Host Immune Response to Mosquito-Transmitted Chikungunya Virus Differs from That Elicited by Needle Inoculated Virus

Background

Mosquito-borne diseases are a worldwide public health threat. Mosquitoes transmit viruses or parasites during feeding, along with salivary proteins that modulate host responses to facilitate both blood feeding and pathogen transmission. Understanding these earliest events in mosquito transmission of arboviruses by mosquitoes is essential for development and assessment of rational vaccine and treatment strategies. In this report, we compared host immune responses to chikungunya virus (CHIKV) transmission by (1) mosquito bite, or (2) by needle inoculation.

Methods and Findings

Differential cytokine expression was measured using quantitative real-time RT-PCR, at sites of uninfected mosquito bites, CHIKV-infected mosquito bites, and needle-inoculated CHIKV. Both uninfected and CHIKV infected mosquitoes polarized host cytokine response to a T_H2 profile. Compared to uninfected mosquito bites, expression of IL-4 induced by CHIKV-infected mosquitoes were 150 fold and 527.1 fold higher at 3 hours post feeding (hpf) and 6 hpf, respectively. A significant suppression of T_H1 cytokines and TLR-3 was also observed. These significant differences may result from variation in the composition of uninfected and CHIKV-infected mosquito saliva. Needle injected CHIKV induced a robust interferon-γ, no detectable IL-4, and a significant up-regulation of TLR-3.

Conclusions

This report describes the first analysis of cutaneous cytokines in mice bitten by CHIKV–infected mosquitoes. Our data demonstrate contrasting immune activation in the response to CHIKV infection by mosquito bite or needle inoculation. The significant role of mosquito saliva in these earliest events of CHIKV transmission and infection are highlighted.     

Nowcasting the Spread of Chikungunya Virus in the Americas

Background

In December 2013, the first locally-acquired chikungunya virus (CHIKV) infections in the Americas were reported in the Caribbean. As of May 16, 55,992 cases had been reported and the outbreak was still spreading. Identification of newly affected locations is paramount to intervention activities, but challenging due to limitations of current data on the outbreak and on CHIKV transmission. We developed models to make probabilistic predictions of spread based on current data considering these limitations.

Methods and Findings

Branching process models capturing travel patterns, local infection prevalence, climate dependent transmission factors, and associated uncertainty estimates were developed to predict probable locations for the arrival of CHIKV-infected travelers and for the initiation of local transmission. Many international cities and areas close to where transmission has already occurred were likely to have received infected travelers. Of the ten locations predicted to be the most likely locations for introduced CHIKV transmission in the first four months of the outbreak, eight had reported local cases by the end of April. Eight additional locations were likely to have had introduction leading to local transmission in April, but with substantial uncertainty.

Conclusions

Branching process models can characterize the risk of CHIKV introduction and spread during the ongoing outbreak. Local transmission of CHIKV is currently likely in several Caribbean locations and possible, though uncertain, for other locations in the continental United States, Central America, and South America. This modeling framework may also be useful for other outbreaks where the risk of pathogen spread over heterogeneous transportation networks must be rapidly assessed on the basis of limited information.     

Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication

Background

Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection.

Methods

Plasmid-based small hairpin RNA (shRNA) was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection.

Results

Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1.

Conclusion

Taken together, these data suggest the promising efficacy of anti-CHIKV shRNAs, in particular, plasmid-shRNA E1, as a novel antiviral strategy against CHIKV infection.     

Serological Evidence of Chikungunya Virus among Acute Febrile Patients in Southern Mozambique

Background

In the last two decades, chikungunya virus (CHIKV) has rapidly expanded to several geographical areas, causing frequent outbreaks in sub-Saharan Africa, South East Asia, South America, and Europe. Therefore, the disease remains heavily neglected in Mozambique, and no recent study has been conducted.

Methods

Between January and September 2013, acute febrile patients with no other evident cause of fever and attending a health center in a suburban area of Maputo city, Mozambique, were consecutively invited to participate. Paired acute and convalescent serum samples were requested from each participant. Convalescent samples were initially screened for anti-CHIKV IgG using a commercial indirect immunofluorescence test, and if positive, the corresponding acute sample was screened using the same test.

Results

Four hundred patients were enrolled. The median age of study participants was 26 years (IQR: 21–33 years) and 57.5% (224/391) were female. Paired blood samples were obtained from 209 patients, of which 26.4% (55/208) were presented anti-CHIKV IgG antibodies in the convalescent sample. Seroconversion or a four-fold titer rise was confirmed in 9 (4.3%) patients.

Conclusion

The results of this study strongly suggest that CHIKV is circulating in southern Mozambique. We recommend that CHIKV should be considered in the differential diagnosis of acute febrile illness in Mozambique and that systematic surveillance for CHIKV should be implemented.     

Neglected Tropical Diseases outside the Tropics

Background

The neglected tropical diseases (NTDs) cause significant morbidity and mortality worldwide. Due to the growth in international travel and immigration, NTDs may be diagnosed in countries of the western world, but there has been no specific focus in the literature on imported NTDs.

Methods

Retrospective study of a cohort of immigrants and travelers diagnosed with one of the 13 core NTDs at a Tropical Medicine Referral Unit in Spain during the period April 1989-December 2007. Area of origin or travel was recorded and analyzed.

Results

There were 6168 patients (2634 immigrants, 3277 travelers and 257 VFR travelers) in the cohort. NTDs occurred more frequently in immigrants, followed by VFR travelers and then by other travelers (p<0.001 for trend). The main NTDs diagnosed in immigrants were onchocerciasis (n = 240, 9.1%) acquired mainly in sub-Saharan Africa, Chagas disease (n = 95, 3.6%) in immigrants from South America, and ascariasis (n = 86, 3.3%) found mainly in immigrants from sub-Saharan Africa. Most frequent NTDs in travelers were: schistosomiasis (n = 43, 1.3%), onchocerciasis (n = 17, 0.5%) and ascariasis (n = 16, 0.5%), and all were mainly acquired in sub-Saharan Africa. The main NTDs diagnosed in VFR travelers were onchocerciasis (n = 14, 5.4%), and schistosomiasis (n = 2, 0.8%).

Conclusions

The concept of imported NTDs is emerging as these infections acquire a more public profile. Specific issues such as the possibility of non-vectorial transmission outside endemic areas and how some eradication programmes in endemic countries may have an impact even in non-tropical western countries are addressed. Recognising NTDs even outside tropical settings would allow specific prevention and control measures to be implemented and may create unique opportunities for research in future.     

Evaluation of Chikungunya Diagnostic Assays: Differences in Sensitivity of Serology Assays in Two Independent Outbreaks

Background

The sensitivity and specificity of two in-house MAC-ELISA assays were tested and compared with the performance of commercially-available CTK lateral flow rapid test and EUROIMMUN IFA assays for the detection of anti-Chikungunya virus (CHIKV) IgM. Each MAC-ELISA assay used a whole virus-based antigen derived from genetically distinct CHIKV strains involved in two chikungunya disease outbreaks in Singapore (2008); a January outbreak strain with alanine at amino acid residue 226 of the E1 glycoprotein (CHIKV-A226) and a May-to-September outbreak strain that possessed valine at the same residue (CHIKV-226V). We report differences in IgM detection efficacy of different assays between the two outbreaks. The sensitivities of two PCR protocols were also tested.

Methods and Findings

For sera from January outbreak, the average detection threshold of CTK lateral flow test, MAC-ELISAs and EUROIMMUN IFA assays was 3.75, 4.38 and 4.88 days post fever onset respectively. In contrast, IgM detection using CTK lateral flow test was delayed to more than 7 days after fever onset in the second outbreak sera. However, MAC-ELISA using CHIKV-226V detected IgM in the second outbreak sera 3.96 days after fever onset, which was approximately one day earlier compared to the same assay using CHIKV-A226 (4.86 days). Specificity was 100% for both commercial assays, and 95.6% for the in-house MAC-ELISAs. For sensitivity determination of the PCR protocols, the probe-based real time RT-PCR method was found to be 10 times more sensitive than one based on SYBR Green.

Conclusion

Our findings suggested that the two strains of CHIKV using variants A226 and 226V resulted in variation in sensitivities of the assays evaluated. We postulated that the observed difference in antigen efficacy could be due to the amino acid substitution differences in viral E1 and E2 envelope proteins, especially the E1-A226V substitution. This evaluation demonstrates the importance of appraisal of different diagnostic assays before their application in clinical and operational settings.     

Differential Proteome Analysis of Chikungunya Virus Infection on Host Cells

Background

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach.

Methodology and Principal Findings

The whole cell proteome profiles of CHIKV-infected and mock control WRL-68 cells were compared and analyzed using two-dimensional gel electrophoresis (2-DGE). Fifty-three spots were found to be differentially modulated and 50 were successfully identified by MALDI-TOF/TOF. Eight were significantly up-regulated and 42 were down-regulated. The mRNA expressions of 15 genes were also found to correlate with the corresponding protein expression. STRING network analysis identified several biological processes to be affected, including mRNA processing, translation, energy production and cellular metabolism, ubiquitin-proteasome pathway (UPP) and cell cycle regulation.

Conclusion/Significance

This study constitutes a first attempt to investigate alteration of the host cellular proteome during early CHIKV infection. Our proteomics data showed that during early infection, CHIKV affected the expression of proteins that are involved in mRNA processing, host metabolic machinery, UPP, and cyclin-dependent kinase 1 (CDK1) regulation (in favour of virus survival, replication and transmission). While results from this study complement the proteomics results obtained from previous late host response studies, functional characterization of these proteins is warranted to reinforce our understanding of their roles during early CHIKV infection in humans.     

Clinical Prediction Rule for Stratifying Risk of Pulmonary Multidrug-Resistant Tuberculosis

Background

Multidrug-resistant tuberculosis (MDR-TB), resistance to at least isoniazid and rifampin, is a worldwide problem.

Objective

To develop a clinical prediction rule to stratify risk for MDR-TB among patients with pulmonary tuberculosis.

Methods

Derivation and internal validation of the rule among adult patients prospectively recruited from 37 health centers (Perú), either a) presenting with a positive acid-fast bacillus smear, or b) had failed therapy or had a relapse within the first 12 months.

Results

Among 964 patients, 82 had MDR-TB (prevalence, 8.5%). Variables included were MDR-TB contact within the family, previous tuberculosis, cavitary radiologic pattern, and abnormal lung exam. The area under the receiver-operating curve (AUROC) was 0.76. Selecting a cut-off score of one or greater resulted in a sensitivity of 72.6%, specificity of 62.8%, likelihood ratio (LR) positive of 1.95, and LR negative of 0.44. Similarly, selecting a cut-off score of two or greater resulted in a sensitivity of 60.8%, specificity of 87.5%, LR positive of 4.85, and LR negative of 0.45. Finally, selecting a cut-off score of three or greater resulted in a sensitivity of 45.1%, specificity of 95.3%, LR positive of 9.56, and LR negative of 0.58.

Conclusion

A simple clinical prediction rule at presentation can stratify risk for MDR-TB. If further validated, the rule could be used for management decisions in resource-limited areas.     

Cytokines in Acute Chikungunya

Introduction

Acute chikungunya (CHIKV) is predominantly an acute onset of excruciatingly painful, self-limiting musculoskeletal (MSK) arbovirus illness and this was further reported by us during the 2006 Indian epidemic [Chopra et al. Epidemiol Infect 2012]. Selected serum cytokines profile in subjects within one month of onset of illness is being presented.

Methods

Out of 509 clinical CHIKV cases (43% population) identified during a rural population survey, 225 subjects consented blood investigations. 132 examined within 30 days of febrile onset are the study cohort. Anti-CHIKV IgM and IgG antibodies tested by immunochromatography and indirect immunofluorescence respectively. Interferons (IFN)-α, -β and -γ, Interferon Gamma-Induced Protein-10 (CXCL-10/IP-10), Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Interleukin-13 (IL-13), Monocyte Chemoattractant Protein-1 (MCP-1), Interleukin–4 (IL-4) and Interleukin–10 (IL-10) performed by ELISA. Samples collected from neighboring community a year prior to the epidemic used as healthy controls.

Results

Seropositivity for anti-CHIKV IgM and IgG was 65% and 52% respectively. IFN-α, IFN-β, IFN-γ, CXCL10/IP-10 and IL-1β showed intense response in early acute phase. Cytokines (particularly TNF-α, MCP-1, IL-4, IL-6 and IL-10) was maximum in extended symptomatic phase and remained elevated in recovered subjects. Higher (p<0.05) IFN and IL-4 seen in patients seropositive for anti-CHIKV IgG. Elderly cases (≥65 years) showed elevated cytokines (except IFN) and anti-CHIKV antibodies near similar to younger subjects. Significant correlations (p<0.05) found between cytokines and clinical features (fatigue, low back ache, myalgia) and anti-CHIKV antibodies.

Conclusion

An intense cytokine milieu was evident in the early and immediate persistent symptomatic phase and in recovered subjects. Early persistent IgM and lower IgG to anti-CHKV and intense Th2 cytokine phenotype seem to be associated with delay in resolution of MSK symptoms. Intriguingly, maximum TNF-α, IL-6 and IL-13 with low anti-CHIKV IgM response found in subjects recovered from CHIKV within one month of illness.     

Norwegian Physicians' Knowledge of and Opinions about Evidence-Based Medicine: Cross-Sectional Study

Objective

To answer five research questions: Do Norwegian physicians know about the three important aspects of EBM? Do they use EBM methods in their clinical practice? What are their attitudes towards EBM? Has EBM in their opinion changed medical practice during the last 10 years? Do they use EBM based information sources?

Design

Cross sectional survey in 2006.

Setting

Norway.

Participants

966 doctors who responded to a questionnaire (70% response rate).

Results

In total 87% of the physicians mentioned the use of randomised clinical trials as a key aspect of EBM, while 53% of them mentioned use of clinical expertise and only 19% patients'' values. 40% of the respondents reported that their practice had always been evidence-based. Many respondents experienced difficulties in using EBM principles in their clinical practice because of lack of time and difficulties in searching EBM based literature. 80% agreed that EBM helps physicians towards better practice and 52% that it improves patients'' health. As reasons for changes in medical practice 86% of respondents mentioned medical progress, but only 39% EBM.

Conclusions

The results of the study indicate that Norwegian physicians have a limited knowledge of the key aspects of EBM but a positive attitude towards the concept. They had limited experience in the practice of EBM and were rather indifferent to the impact of EBM on medical practice. For solving a patient problem, physicians would rather consult a colleague than searching evidence based resources such as the Cochrane Library.     

Chikungunya Virus Glycoproteins Pseudotype with Lentiviral Vectors and Reveal a Broad Spectrum of Cellular Tropism

Background

Outbreaks of the Chikungunya virus (CHIKV) infection has been documented in over 40 countries, resulting in clinical symptoms characterized by fever and joint pain. Diagnosing CHIKV in a clinical lab setting is often omitted because of the high lab safety requirement. An infection system that mimics CHIKV infection will permit clinical evaluation of the production of neutralizing antibody for both disease diagnostics and treatment.

Methodology/Principal Findings

We generated a CHIKV construct expressing CHIKV structural proteins. This construct permits the production of CHIKV pseudo-viral particles with a luciferase reporter. The pseudo-virus was able to infect a wide range of cell lines. The pseudovirus could be neutralized by the addition of neutralizing antibodies from patients.

Conclusions

Taken together, we have developed a powerful system that can be handled at biosafety level 2 laboratories for evaluation of existence of CHIKV neutralizing antibodies.     

Rhabdomyolysis in Community Acquired Bacterial Sepsis – A Retrospective Cohort Study

Background and Objectives

Rhabdomyolysis is often associated with sepsis and gram positive bacterial pathogens are reported to be the most frequent cause of sepsis induced rhabdomyolysis. We report the pattern of infecting bacterial pathogens and associated causal factors in a South-Indian cohort.

Design, Setting, Participants & Measurements

Retrospective cohort study of adult patients with community acquired bacterial sepsis complicated by rhabdomyolysis from March 2003 - August 2008. Rhabdomyolysis was defined as serum creatine kinase >2000 IU/L. The study population was divided into group-I (sepsis with gram positive pathogens), group–II (sepsis with gram negative pathogens) and group-III (culture negative sepsis).

Results

103 patients (group I -15, group II- 34 and group III- 54) formed the study cohort. Mean age was 55 years and two-third had diabetes. Mean creatine kinase was 7114 IU/L and mean serum creatinine on admission was 2.4 mg/dl. Causative pathogen of sepsis was identified in 47.5%. Gram negative pathogens were more frequently (33%) associated with rhabdomyolysis than gram positive pathogens (14.5%). Lung was the commonest foci of sepsis (38.8%). 78.6% of the study population had one or more additional causal factor for rhabdomyolysis like statin intake, chronic alcoholism, hypokalemia, hypernatremia and hypophosphatemia. Mortality was 59%.

Conclusions

Gram negative bacterial pathogens were more frequently associated with rhabdomyolysis than gram positive pathogens. Rhabdomyolysis in patients with sepsis is multifactorial and is associated with high mortality.     

Taenia solium Cysticercosis Hotspots Surrounding Tapeworm Carriers: Clustering on Human Seroprevalence but Not on Seizures

Background

Neurocysticercosis accounts for 30%–50% of all late-onset epilepsy in endemic countries. We assessed the clustering patterns of Taenia solium human cysticercosis seropositivity and seizures around tapeworm carriers in seven rural communities in Peru.

Methodology

The presence of T. solium–specific antibodies was defined as one or more positive bands in the enzyme-linked immunoelectrotransfer blot (EITB). Neurocysticercosis-related seizures cases were diagnosed clinically and had positive neuroimaging or EITB.

Principal Findings

Eleven tapeworm carriers were identified by stool microscopy. The seroprevalence of human cysticercosis was 24% (196/803). Seroprevalence was 21% >50 m from a carrier and increased to 32% at 1–50 m (p = 0.047), and from that distance seroprevalence had another significant increase to 64% at the homes of carriers (p = 0.004). Seizure prevalence was 3.0% (25/837) but there were no differences between any pair of distance ranges (p = 0.629, Wald test 2 degrees of freedom).

Conclusion/Significance

We observed a significant human cysticercosis seroprevalence gradient surrounding current tapeworm carriers, although cysticercosis-related seizures did not cluster around carriers. Due to differences in the timing of the two outcomes, seroprevalence may reflect recent T. solium exposure more accurately than seizure frequency.     

Chikungunya Virus Exploits miR-146a to Regulate NF-κB Pathway in Human Synovial Fibroblasts

Objectives

Chikungunya virus causes chronic infection with manifestations of joint pain. Human synovial fibroblasts get infected with CHIKV and could lead to pro-inflammatory responses. MicroRNAs have potentials to regulate the gene expression of various anti-viral and pro-inflammatory genes. The study aims to investigate the role of miR-146a in modulation of inflammatory responses of human synovial fibroblasts by Chikungunya virus.

Methods

To study the role of miR-146a in CHIKV pathogenesis in human synovial cells and underlying inflammatory manifestations, we performed CHIKV infection in primary human synovial fibroblasts. Western blotting, real-time PCR, luciferase reporter assay, overexpression and knockdown of cellular miR-146a strategies have been employed to validate the role of miR-146a in regulation of pro-inflammatory NF-κB pathway.

Results

CHIKV infection induced the expression of cellular miR-146a, which resulted into down-regulation of TRAF6, IRAK1, IRAK2 and increased replication of CHIKV in human synovial fibroblasts. Exogenous expression of miR-146a in human synovial fibroblasts led to decreased expression of TRAF6, IRAK1, IRAK2 and decreased replication of CHIKV. Inhibition of cellular miR-146a by anti-miR-146a restored the expression levels of TRAF6, IRAK1 and IRAK2. Downregulation of TRAF6, IRAK1 and IRAK2 led to downstream decreased NF-κB activation through negative feedback loop.

Conclusion

This study demonstrated the mechanism of exploitation of cellular miR-146a by CHIKV in modulating the host antiviral immune response in primary human synovial fibroblasts.     

Comparison of Prognostic Gene Profiles Using qRT-PCR in Paraffin Samples: A Retrospective Study in Patients with Early Breast Cancer

Introduction

Gene profiling may improve prognostic accuracy in patients with early breast cancer, but this technology is not widely available. We used commercial assays for qRT-PCR to assess the performance of the gene profiles included in the 70-Gene Signature, the Recurrence Score and the Two-Gene Ratio.

Methods

153 patients with early breast cancer and a minimum follow-up of 5 years were included. All tumours were positive for hormonal receptors and 38% had positive lymph nodes; 64% of patients received adjuvant chemotherapy. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) specimens using a specific kit. qRT-PCR amplifications were performed with TaqMan Gene Expression Assays products. We applied the three gene-expression-based models to our patient cohort to compare the predictions derived from these gene sets.

Results

After a median follow-up of 91 months, 22% of patients relapsed. The distant metastasis-free survival (DMFS) at 5 years was calculated for each profile. For the 70-Gene Signature, DMFS was 95% -good prognosis- versus 66% -poor prognosis. In the case of the Recurrence Score, DMFS was 98%, 81% and 69% for low, intermediate and high-risk groups, respectively. Finally, for the Two-Gene Ratio, DMFS was 86% versus 70%. The 70-Gene Signature and the Recurrence Score were highly informative in identifying patients with distant metastasis, even in multivariate analysis.

Conclusion

Commercially available assays for qRT-PCR can be used to assess the prognostic utility of previously published gene expression profiles in FFPE material from patients with early breast cancer. Our results, with the use of a different platform and with different material, confirm the robustness of the 70-Gene Signature and represent an independent test for the Recurrence Score, using different primer/probe sets.     

Fulfilling the Promise of Personalized Medicine? Systematic Review and Field Synopsis of Pharmacogenetic Studies

Background

Studies of the genetic basis of drug response could help clarify mechanisms of drug action/metabolism, and facilitate development of genotype-based predictive tests of efficacy or toxicity (pharmacogenetics).

Objectives

We conducted a systematic review and field synopsis of pharmacogenetic studies to quantify the scope and quality of available evidence in this field in order to inform future research.

Data Sources

Original research articles were identified in Medline, reference lists from 24 meta-analyses/systematic reviews/review articles and U.S. Food and Drug Administration website of approved pharmacogenetic tests.

Study Eligibility Criteria, Participants, and Intervention Criteria

We included any study in which either intended or adverse response to drug therapy was examined in relation to genetic variation in the germline or cancer cells in humans.

Study Appraisal and Synthesis Methods

Study characteristics and data reported in abstracts were recorded. We further analysed full text from a random 10% subset of articles spanning the different subclasses of study.

Results

From 102,264 Medline hits and 1,641 articles from other sources, we identified 1,668 primary research articles (1987 to 2007, inclusive). A high proportion of remaining articles were reviews/commentaries (ratio of reviews to primary research approximately 25∶1). The majority of studies (81.8%) were set in Europe and North America focussing on cancer, cardiovascular disease and neurology/psychiatry. There was predominantly a candidate gene approach using common alleles, which despite small sample sizes (median 93 [IQR 40–222]) with no trend to an increase over time, generated a high proportion (74.5%) of nominally significant (p<0.05) reported associations suggesting the possibility of significance-chasing bias. Despite 136 examples of gene/drug interventions being the subject of ≥4 studies, only 31 meta-analyses were identified. The majority (69.4%) of end-points were continuous and likely surrogate rather than hard (binary) clinical end-points.

Conclusions and Implications of Key Findings

The high expectation but limited translation of pharmacogenetic research thus far may be explained by the preponderance of reviews over primary research, small sample sizes, a mainly candidate gene approach, surrogate markers, an excess of nominally positive to truly positive associations and paucity of meta-analyses. Recommendations based on these findings should inform future study design to help realise the goal of personalised medicines.

Systematic Review Registration Number

Not Registered     

A Randomized,Controlled, Trial of Short Cycle Intermittent Compared to Continuous Antiretroviral Therapy for the Treatment of HIV Infection in Uganda

Background

Short cycle treatment interruption could reduce toxicity and drug costs and contribute to further expansion of antiretroviral therapy (ART) programs.

Methods

A 72 week, non-inferiority trial enrolled one hundred forty six HIV positive persons receiving ART (CD4+ cell count ≥125 cells/mm³ and HIV RNA plasma levels <50 copies/ml) in one of three arms: continuous, 7 days on/7 days off and 5 days on/2 days off treatment. Primary endpoint was ART treatment failure determined by plasma HIV RNA level, CD4+ cell count decrease, death attributed to study participation, or opportunistic infection.

Results

Following enrollment of 32 participants, the 7 days on/7 days off arm was closed because of a failure rate of 31%. Six of 52 (11.5%) participants in the 5 days on/2 days off arm failed. Five had virologic failure and one participant had immunologic failure. Eleven of 51 (21.6%) participants in the continuous treatment arm failed. Nine had virologic failure with 1 death (lactic acidosis) and 1 clinical failure (extra-pulmonary TB). The upper 97.5% confidence boundary for the difference between the percent of non-failures in the 5 days on/2 days off arm (88.5% non-failure) compared to continuous treatment (78.4% non failure) was 4.8% which is well within the preset non-inferiority margin of 15%. No significant difference was found in time to failure in the 2 study arms (p = 0.39).

Conclusions

Short cycle 5 days on/2 days off intermittent ART was at least as effective as continuous therapy.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00339456","term_id":"NCT00339456"}}NCT00339456     

Association of HLA Class-I and Inhibitory KIR Genotypes in Gabonese Patients Infected by Chikungunya or Dengue Type-2 Viruses

Background

Natural killer (NK) cells provide defense in the early stages of the immune response against viral infections. Killer cell immunoglobulin-like receptors (KIR) expressed on the surface of NK cells play an important role in regulating NK cell response through recognition of human leukocyte antigen (HLA) class I molecules on target cells. Previous studies have shown that specific KIR/ligand combinations are associated with the outcome of several viral infectious diseases.

Methods

We investigated the impact of inhibitory and activating KIR and their HLA-class I ligand genotype on the susceptibility to Chikungunya virus (CHIKV) and Dengue virus (DENV2) infections. From April to July 2010 in Gabon, a large outbreak of CHIKV and DENV2 concomitantly occurred in two provinces of Gabon (Ogooué-Lolo and Haut-Ogooué). We performed the genotypic analysis of KIR in the combination with their cognate HLA-class I ligands in 73 CHIKV and 55 DENV2 adult cases, compared with 54 healthy individuals.

Results

We found in CHIV-infected patients that KIR2DL1 and KIR2DS5 are significantly increased and decreased respectively, as compared to DENV2⁺ patients and healthy donors. The combination of KIR2DL1 and its cognate HLA-C2 ligand was significantly associated with the susceptibility to CHIKV infection. In contrast, no other inhibitory KIR-HLA pairs showed an association with the two mosquito-borne arboviruses.

Conclusion

These observations are strongly suggestive that the NK cell repertoire shaped by the KIR2DL1:HLA-C2 interaction facilitate specific infection by CHIKV.