New Delhi metallo-β-lactamase (NDM)-producing Gram-negative bacteria have spread globally and pose a significant public health threat. There is a need to better define risk factors and outcomes of NDM-1 clinical infection. We assessed risk factors for nosocomial infection with NDM-1-producers and associated in-hospital mortality.A matched case-control study was conducted during a nosocomial outbreak of NDM-1-producers in an adult intensive care unit (ICU) in South Africa. All patients from whom NDM-1-producers were identified were considered (n=105). Cases included patients admitted during the study period in whom NDM-1 producing Gram-negative bacteria were isolated from clinical specimens collected ≥48 hours after admission, and where surveillance definitions for healthcare-associated infections were met. Controls were matched for age, sex, date of hospital admission and intensive-care admission. Conditional logistic regression was used to identify risk factors for NDM-1 clinical infection and associated in-hospital mortality.Klebsiella pneumoniae was the most common NDM-1-producer . Cases had longer mean hospital stays  and ICU stays . Adjusting for co-morbid disease, the in-hospital mortality of cases was significantly higher than controls . Higher Charlson co-morbidity index score , mechanical ventilation days  and piperacillin/tazobactam exposure  were identified as risk factors on multivariate analysis. Cases had a significantly higher likelihood of in-hospital mortality when the NDM-1-producer was Klebsiella pneumoniae , or when they had a bloodstream infection .38 cases and 68 controls were included. NDM-1 infection is associated with significant in-hospital mortality. Risk factors for hospital-associated infection include the presence of co-morbid disease, mechanical ventilation and piperacillin/tazobactam exposure. Resistance to β-lactams is a long recognised problem in Gram-negative bacteria and withblaNDM-1 gene is plasmid mediated and associated with numerous other resistance determinants conferring resistance to β-lactams, fluoroquinolones and aminoglycosides resulting in significant treatment option limitations.[In 2008 a novel metallo-β-lactamase designated New Delhi metallo-β-lactamase (NDM-1) was identified in a Swedish patient returning from India. The firsitations.,8 Sensititations. Compareditations.,11 NDM-1itations.–14 with itations.,16blaNDM-1 through an understanding of risk factors for its acquisition is essential. However, there is a paucity of published epidemiological studies reporting on risk factors for NDM-1 clinical infection. In order to evaluate risk factors for NDM-1 clinical infection and associated in-hospital mortality, we conducted a matched case-control study during a prolonged outbreak of NDM-1 producing Gram-negative bacteria in a South African hospital. We hypothesised that exposure to antibiotics and medical devices would be risk factors for NDM-1 clinical infection and would be associated with greater in-hospital mortality.With limited treatment options available, slowing and preventing the spread of Study participant age ranged from 20 years to 90 years, with a mean age of 61.3 and a median age of 64. Verbal informed consent was obtained from all patients or their next of kin prior to conducting telephonic interviews which collected information on past hospitalization/chronic care admission and travel history. Verbal consent was obtained as this was a retrospective study and patients had subsequently relocated to various parts of the country. Consent was captured on a consent form by the researchers. Consent to review clinical records were obtained from the hospital and all patient data were anonymized and de-linked from unique identifiers prior to analysis. Ethics approval for this study, including the consent procedure, was obtained from the Human Research Ethics Committee  at the University of the Witwatersrand, Johannesburg. (M130248)The outbreak occurred across three private hospitals in South Africa with strong referral links. This study was confined to the hospital where the majority of cases  were detected, and all cases and controls were from the adult ICU. The hospital is located in the greater Johannesburg area, has a total of 322 beds including a 37-bed ICU, and offers tertiary-level specialist care.Klebsiella pneumoniae isolated from an 86-year-old male admitted following a hip fracture was found to harbour blaNDM-1. In response to this, the first case of NDM-1 both in the hospital and the country, a rectal screening programme was instituted to identify patients colonised with NDM-1-producers, with screening criteria revisions throughout the course of the outbreak. The method of screening employed by all diagnostic laboratories throughout the outbreak was direct real-time polymerase chain reaction (RT-PCR) testing for blaNDM-1 on dry rectal swabs. Clinical isolates demonstrating phenotypic resistance to carbapenems were further tested for blaNDM-1 using RT-PCR. The LightCycler 480 II (Roche Applied Science) instrument was used for the RT-PCR assay. The blaNDM-1 gene was amplified by a real time polymerase chain reaction (PCR) using the LightCycler 480 Probes Master kit  and the LightMix Modular NDM (ESBL) kit . The positive control was provided with the LightMix Modular kits and sterile water was used as a negative control. An internal control, the LightMix Modular PhHV Internal Control kit  was also included in the run.In early August 2011 All microbiological testing was conducted in routine private diagnostic laboratories servicing the private healthcare sector. Thirteen NDM-positive isolates  were subjected to DNA fingerprinting by macro-restriction analysis on pulsed-field gel electrophoresis (PFGE) at the Infection Control Services Laboratory, National Health Laboratory Services. PFGE was performed as described previously.The study design is summarized in blaNDM-1 was detected on an isolate from a specimen collected at least 48 hours after admission and the infection was categorised as a healthcare-associated infection as per the Centers for Disease Control and Prevention/National Healthcare Safety Network definitions.[blaNDM-1 was detected on rectal screening alone, where rectal screening was positive within the first 48 hours of admission, or where clinical records were incomplete. After exclusion of cases not fulfilling the inclusion criteria, 40 cases remained and three controls were matched to each case for sex , age (+/- 5 years), date of hospital admission (+/- 14 days) and ICU admission (yes/no). Where more than three eligible controls were identified on the hospital’s electronic database, three controls were randomly selected. Controls were excluded if they had blaNDM-1 detected on any sample during the hospitalisation period, if patient records were incomplete or missing, or if the patient was admitted for less than 48 hours.All patients admitted between 1 July 2011 and 31 October 2012 were eligible for inclusion. We included cases where initions. PotentiaNo controls could be found meeting the matching criteria for two cases and for three cases only two matching controls could be identified. Another 52 controls were excluded for missing/incomplete medical records (n = 26), record of screening NDM-1 positive on dry rectal swab (n = 13) or being admitted for less than 48 hours (n = 13). The final sample consisted of 38 cases and 68 controls.Clinical data were collected for cases and controls from clinical records, laboratory results and hospital billing data. Exposure to antibiotics  and corticosteroids were recorded as total number of doses received. Exposure to medical devices  as well as selected medical interventions  was recorded as total number of days exposed. Patients who underwent laparotomy or thoracotomy were grouped and compared to patients who received other (mainly orthopaedic) or no surgery. Co-morbid disease and severity of illness on admission was measured by calculating Charlson co-morbidity index and Mortality Probability Model III (MPM III) scores respectively.,20 ExposData on past hospital or long-term care facility admission and international travel history in the year leading up to the admission of interest were collected through telephonic interviews for both cases and controls. All data were collected between November 2012 and November 2013 by trained professional nurses and medical doctors.We evaluated risk factors associated with case status and compared in-hospital mortality between cases and controls. Except for MPM-III scores, where its calculation would have been invalid, there was no missing clinical data in the final sample used for analysis. Where past admission, travel history or MPM-III scores were missing, observations were excluded from the analysis.P < 0.20 at the univariate level were considered in the final multiple regression model. Significance was taken at a level of 0.05. Conditional logistic regression was further undertaken to calculate the odds of in-hospital mortality for cases and controls as well as for different sites of infection and clinical isolates. Adjusted odds ratios were calculated using multivariable conditional logistic regression.Data were entered into Epi-Info version 7 and exported to Excel 2007 where it was inspected for errors before being imported to Stata Version 12 for statistical analysis. Continuous variables such as length of hospital stay, MPM-III and Charlson scores, are described through the reporting of means and standard deviations. Two sided t-test for two groups (cases and controls) was used to compare means of continuous variables with normal distributions. Where data were not normally distributed Mann-Whitney U test was used. For differences in proportions such as previous hospitalisation or travel history, Mantel–Haenszel Chi square test was used. Bivariate conditional logistic regression analysis was undertaken to calculate crude odds ratio’s for exposure to medical devises and interventions, antibiotics and duration of stay. Stepwise conditional logistic regression was conducted to identify factors associated with case status. All exposure variables with a Klebsiella pneumoniae  followed by Enterobacter cloacae , Klebsiella oxytoca , Serratia marcescens  and Citrobacter amalonaticus . The most common clinical specimen types yielding NDM-1 were sputum , blood  and urine  followed by pus , broncho-alveolar lavage  and pleural fluid .The most common NDM-1-producing isolate among the 38 cases was PFGE showed two closely related clusters: cluster A comprised three case isolates and six environmental isolates, whilst cluster B comprised three case isolates. Given the protracted course of the outbreak, this suggests that these isolates are all related.P < 0.001) and longer mean durations of time at risk, particularly mean ICU time at risk  than controls  or being transferred from a referral hospital  compared to controls . No assoOn univariate analysis exposure to aminoglycosides, piperacillin/tazobactam and corticosteroids were significantly associated with case status . Each adblaNDM-1 was Klebsiella pneumoniae  had a significantly higher likelihood of in-hospital mortality.Of the 68 controls 10 died in hospital (14.7%), while 21 of the 38 cases died in hospital (55.3%);this translates to an attributable mortality of 47.5% . After aKlebsiella pneumoniae isolates from cases and the environment supported the hypothesis of horizontal transmission occurring in the ICU.To our knowledge this is the largest epidemiological study investigating risk factors and in-hospital mortality associated with clinical infection during an outbreak of NDM-1-producers, and adds evidence to support rational preventive and control measures. We found that higher Charlson co-morbidity scores, mechanical ventilation and piperacillin/tazobactam exposure were independently associated with infection with NDM-1-producers. Secondly, in-hospital mortality was found to be significantly higher in patients with clinical infection due to NDM-1-producers. Molecular strain typing of NDM-1-producing blaNDM-1 in Canada.[et al.[We identified three previously published papers reporting on risk factors for infection with NDM-1-producers. The first was a review of reported cases (n = 77) across the European Union which found travel to India, Pakistan or the Balkans to be associated with NDM-1 acquisition. The secon Canada. The thira.[et al. investiga.[et al. We foundOur findings that an increased duration of exposure to central venous lines, urinary catheters, mechanical ventilation and haemodialysis were associated with an increased risk of infection with NDM-1-producers are consistent with risk factors for the acquisition of carbapenemase-producers identified by previous investigators. Medical devices such as urinary catheters,26 and cblaNDM-1.[blaNDM-1 had been present in clinically-relevant bacteria for some time before the index case was identified.Of the early NDM-1 cases detected in the United States and United Kingdom, many had epidemiological links to India and Pakistan.,15 We folaNDM-1. SimilarlKlebsiella pneumoniae at between 44% and 48%.[Klebsiella pneumoniae is estimated at 53%.[In-hospital mortality for extended-spectrum beta-lactamase producers has been reported at around 37% and amon and 48%.,28 Cruded at 53%. Given thThe hospital undertook a range of interventions to control the outbreak. Patients found to be colonized with NDM-1-producers through the rectal screening program were cohorted and assigned dedicated nursing staff and medical equipment. Healthcare workers and cleaning staff received targeted education about the importance of hand hygiene, and stringent hand washing protocols were instituted throughout the hospital. Hand hygiene practice in the adult ICU was monitored for compliance through closed circuit television. Infrastructural alterations to the adult ICU increased the ICU’s capacity to effectively isolate patients. Weekly meetings were attended by multidisciplinary role-players, including the hospital infection prevention and control practitioners, hospital management staff, medical microbiologists, hospital clinicians, and members of the National Institute for Communicable Diseases’ Outbreak Response Unit. Controlling the outbreak was resource intensive and demanded a concerted effort from all role-players, with critical review of the outbreak situation and re-evaluation of interventional strategies throughout. Although sporadic cases of colonisation or infection with NDM-1-producers continue to be reported, no clusters or epidemiologically-linked cases have been identified since the end of the outbreak.This study has a number of limitations. Due to the inherent nature of outbreak investigations, there were a limited number of potential cases. All potential cases were reviewed and as many matching controls as were available were included. However, the small sample size limits the study’s power to detect other antimicrobial agents as risk factors for infection with NDM-1-producers. The outbreak was confined to the adult ICU, limiting generalisability to a paediatric population. Missing clinical records and missing data on international travel and previous admissions in the year leading up to the admission of interest reduced our sample size and ability to evaluate pre-hospitalization risk factors. The fluctuating point prevalence of NDM-1-producers and the clinicians’ enhanced diagnostic suspicion of infection with NDM-1-producers as the outbreak evolved may bias findings. We addressed this, however, by matching controls for date of hospital admission.Given the dearth of new antimicrobials in the drug development pipeline, the burgeoning threat of conquer by virtually untreatable multidrug-resistant organisms of clinical relevance is becoming realised thanks to the emergence and rapid spread of, amongst others, the carbapenemases.,35 Throu


Early identification and entry into care is critical to reducing morbidity and mortality in children with HIV. The objective of this report is to describe the impact of the Tingathe programme, which utilizes community health workers (CHWs) to improve identification and enrolment into care of HIV-exposed and -infected infants and children.Three programme phases are described. During the first phase, Mentorship Only (MO) (March 2007–February 2008) on-site clinical mentorship on paediatric HIV care was provided. In the second phase, Tingathe-Basic (March 2008–February 2009), CHWs provided HIV testing and counselling to improve case finding of HIV-exposed and -infected children. In the final phase, Tingathe-PMTCT (prevention of mother-to-child transmission) (March 2009–February 2011), CHWs were also assigned to HIV-positive pregnant women to improve mother-infant retention in care. We reviewed routinely collected programme data from HIV testing registers, patient mastercards and clinic attendance registers from March 2005 to March 2011.p<0.001).During MO, 42 children (38 HIV-infected and 4 HIV-exposed) were active in care. During Tingathe-Basic, 238 HIV-infected children (HIC) were newly enrolled, a six-fold increase in rate of enrolment from 3.2 to 19.8 per month. The number of HIV-exposed infants (HEI) increased from 4 to 118. During Tingathe-PMTCT, 526 HIC were newly enrolled over 24 months, at a rate of 21.9 patients per month. There was also a seven-fold increase in the average number of exposed infants enrolled per month (9.5–70 patients per month), resulting in 1667 enrolled with a younger median age at enrolment  in children aged 18 months to 15 years; 351 (4.6%) tested HIV-positive. Over this time, 1781 HEI were enrolled, with 102 (5.7%) found HIV-infected by positive PCR. Additional HIC entered care through various mechanisms (including positive linkage by CHWs and transfer-ins) such that by February 2011, a total of 866 HIC were receiving care, a 23-fold increase from 2008.A multipronged approach utilizing CHWs to conduct HIV testing, link HIC into care and provide support to PMTCT mothers can dramatically improve the identification and enrolment into care of HIV-exposed and -infected children. Within the past 10 years, great progress has been made in the prevention of mother-to-child transmission (PMTCT) of HIV infection, early infant diagnosis (EID) of HIV and care and treatment of HIV-exposed infants (HEI) and HIV-infected children (HIC). As currently available PMTCT strategies capable of reducing transmission rates to less than 5% are possible even in breastfeeding populations, UNAIDS has declared a goal of “virtual elimination” by 2015 . With thDespite these advances, considerable challenges remain, with the most significant being access to care. HIV-infected infants suffer from rapid immunologic deterioration, disease progression and high mortality without early ART initiation ,10, yet Improving identification and enrolment into care is also critical for older children and adolescents. Up to one third of infected infants have slower progressing disease and may survive into their teenage years without treatment . There aCommunity-based HIV testing has been proposed as one strategy to improve paediatric case finding . ResultsThe Baylor College of Medicine Children's Foundation Malawi (BCM-CFM) is the largest provider of paediatric HIV care in Malawi, with over 20,000 patients enrolled at the Clinical Center of Excellence and satellite clinics. In 2007, BCM-CFM partnered with the Malawi Ministry of Health (MOH) and Lilongwe District Health Office to initiate an outreach programme, called Tingathe , which utilizes CHWs to improve uptake and utilization of PMTCT, EID and paediatric HIV care services. The objective of this report is to summarize these efforts and examine their impact on identification and enrolment into care of HIV-exposed and -infected infants and children.This report describes Tingathe activities from 2007 to 2011 at three MOH health centres in Lilongwe, Malawi: Area 25, Area 18 and Kawale. These clinics serve a combined catchment area of close to 500,000 people, with 12,000 women attending antenatal clinic annually, and an antenatal HIV prevalence of 10.1% ,35. DuriThree programme phases are described within this report .In March 2007, BCM-CFM started a programme of clinical mentorship at these sites. Paediatricians trained in the United States provided on-site mentorship of government clinical care providers. Mentorship was provided during a dedicated, once weekly paediatric HIV clinic where both HEI and HIC were seen and regular pre-ART care was offered including clinical staging and CD4 measurements. All HIV care including rapid HIV testing, HIV DNA PCR testing via dried blood spot (DBS), WHO staging , CD4 tesIn March 2008, BCM-CFM initiated the Tingathe Community Outreach Programme. CHWs were recruited and trained in community sensitization and education, health-centre- and community-based HIV testing and counselling (HTC), as well as active case finding of patients. Details of the Tingathe intervention, including how CHWs were recruited, selected, trained, supervised and remunerated are summarized in All CHWs were trained in HTC, utilizing rapid antibody-based testing. CHWs conducted both health-centre-based and home-based testing. When the programme was initiated, testing was restricted to the health centre. As counsellors became more proficient in HTC, they began to solicit referrals for home-based testing of untested family members of current ART patients. After several months of referral-based testing, door-to-door testing was initiated. The majority of testing was conducted in either the home or health centre; with some additional testing conducted in other venues including community sensitization events, orphanages and youth services facilities .All of the children tested by Tingathe CHWs had an indication for testing such as a parent being infected, being an orphan or a specific request from a guardian for testing. Adults were usually tested first, and only if the parent tested positive were the children tested. If the mother was uninfected and there were no other indications for testing, the children were not tested. Children older than 18 months were tested with antibody-based rapid tests using finger-pricks. Exposed infants under the age of 18 months were tested using DBS for HIV DNA PCR testing through the national EID programme. Consent/assent for testing was obtained according to MOH guidelines .CHWs also attempted to identify children with known HIV-infected status who were not in clinical care, a process we referred to as positive linkage. Positive linkage was based on the premise that many guardians knew that their children were infected but were not aware that services were available for infected children. Strategies for positive linkage included asking adult ART patients if they had HIC and checking the status  of children at potentially high-prevalence settings such as tuberculosis and outpatient malnutrition clinics, as well as orphanages located within the community . If theyCHWs were not only responsible for identifying HIC but also enrolling them into clinical care and providing adherence support. When children and adolescents with HIV infection were identified, they were enrolled into the programme and assigned a CHW. The CHW would follow the child at home and in clinic to ensure that he or she enrolled into care, maintained good adherence and received all available services including clinical and immunologic staging, CPT and ART, if appropriate. CHWs utilized one-page patient mastercards to document and track clinic appointments, home visits, services utilized, new diagnoses and test results.Clinical mentorship by Baylor clinicians continued during this time period.During the T-Basic phase, it was noted that effective linkage of HEI into care was still suboptimal. Therefore, in March 2009, a PMTCT component was added that assigned CHWs to HIV-positive pregnant women at antenatal care. CHWs supported mother-infant pairs to access all available PMTCT services including CD4 testing, initiation of ART for the mother, provision of ARV prophylaxis for the child, testing and diagnosis of HEI, counselling on infant feeding and enrolment of infected infants into care. They followed the women at their homes and at the health centres, from initial diagnosis up until cessation of breastfeeding and final testing of infants, with either negative diagnosis and discharge or successful enrolment of infected infants into care. HIC were followed regularly to ensure that they were receiving appropriate services. Full details of this PMTCT intervention are described elsewhere ,37.All the activities of the MO and T-Basic phases continued during this time period.Identification of HIV-positive persons via HIV testing efforts. Data from HIV testing registers were analyzed.New enrolment of HEI and HIC into care. Clinic enrolment numbers and rates were calculated from clinic rosters. For clarity and to prevent double counting, all patients were categorized as either exposed (HEI) or infected (HIC) by their status at enrolment. Even if a HEI was later determined to be HIV-infected, he or she was not included in the HIC group in this analysis of new enrolment.Changes in patient characteristics. Patient level data  from mastercards and clinic registers were analyzed. Clinical and immunologic staging were determined using WHO classification guidelines .Total enrolment and mechanism of identification of HIC. For this analysis, in addition to all patients who were HIC at enrolment, those who were HEI at enrolment, but later found to be HIV-infected with a positive PCR, were also included. Patient mastercards and registers provided information on mechanism of identification.We reviewed routinely collected programme data from patient mastercards, clinic rosters, PMTCT registers and HIV testing registers from March 2007 to March 2011. All data were de-identified prior to analysis. The overall objective was to assess the impact of Tingathe activities on identification and enrolment. Four main outcomes were examined:Aggregate data were reported as mean with standard deviation or median with interquartile range (IQR) based on normality. Continuous non-parametric variables were evaluated with the Mann–Whitney U test. For categorical parameters, data were reported as raw value and percentage of the respective group. Analyses were performed using IBM SPSS Statistics .Both the Malawi National Health Sciences Research Committee and the Baylor College of Medicine institutional review board approved this study.p<0.001). However, four times more children were tested through the home-based strategy, resulting in roughly equivalent numbers of HIC being identified (163 vs. 159).During the three years of the T-Basic and T-PMTCT phases, CHWs conducted 44,388 rapid HIV tests, 7658 (17.3%) in children aged 18 months to 15 years . TestingDuring the MO phase, there were 42 children active in care at the three centres. Of these, 38 were HIV-infected and four were HIV-exposed.During the T-Basic phase, 238 HIC were enrolled at the three clinics, a six-fold increase in rate of enrolment for HIC from 3.2 to 19.8 per month from pre- to post-intervention periods. The number of HEI increased from 4 to 118.–70 patients per month) and in younger median age at enrolment . A total of 1667 HEI enrolled during the two-year period.During the T-PMTCT phase, 526 HIC were enrolled over 24 months, with a slight increase in the rate of enrolment from 19.8 to 21.9 patients per month. However, the addition of PMTCT activities resulted in a greater than seven-fold increase in the average number of HEI enrolled per month  and 97 (5.9%) children testing PCR positive during the T-Basic and T-PMTCT periods, respectively.Over the three time periods, the median age of enrolment of HEI decreased from 7.0 months (MO) to 5.2 months (T-Basic) to 2.5 months (T-PMTCT); Characteristics of the children who were HIV-infected (HIC) at enrolment are presented in p=0.016 for the global comparison.During the MO phase, the median age of enrolment was 2.1 years with 73% of staged patients WHO III or IV and 53.3% of those with a CD4 cell count having advanced or severe immunosuppression. During the T-Basic phase, the median age was 3.9 years with 37.8% of staged patients WHO III or IV and 57.0% of those with a CD4 cell count having advanced or severe immunosuppression. During the T-PMTCT phase, the median age was 5.0 years with 42.6% of staged patients WHO III or IV and 45.0% with a CD4 cell count having advanced or severe immunosuppression. Over the three time periods, the median age of enrolment increased from 2.1 years (MO) to 3.9 years (T-Basic) to 5.0 years (T-PMTCT); By February 2011, a total of 866 HIC were receiving care at the three combined clinics, a 23-fold increase from 2008. Of these, 764 (88.2%) were known to be HIV-infected at enrolment into care. Another 102 (11.8%) were HEI at enrolment, but were later found to be HIV-infected by DNA PCR. Although, as described above, the rate of new enrolment of HIC did not significantly increase from T-Basic to T-PMTCT, when including those enrolled as HEI who later tested PCR positive, the rate of total enrolment did increase from 20.3 per month during T-Basic to 26.0 during T-PMTCT.HIV testing with rapid tests identified 321 HIC (37%); 102 (12%) were enrolled as HEI but later found to be HIV-infected by PCR; 35 (4%) were referred with a positive PCR test directly from EID services; 146 (17%) were transferred from the Baylor Centre of Excellence or other healthcare facilities; 253 (29%) were identified through positive linkage; and 9 (1%) were identified by other means.Similar to other countries in the region, identification and enrolment into care of HEI and HIC has been a major challenge in Malawi. Approximately 83,000 HIV-positive pregnant women deliver each year  but onlyOur work demonstrates that strategies utilizing CHWs can improve identification and enrolment into care of HEI and HIC. We used a multipronged approach combining traditional health-centre-based testing, home-based testing, testing children of adults on ART, positive linkage for patients with known HIV-infected status but not in clinical care and follow-up of mother-infant pairs from PMTCT services. Although several studies have demonstrated the utility of home and community-based testing for adults ,31,32,34Following the addition of CHW support of HIV-positive pregnant women from diagnosis at antenatal clinic, there was a dramatic improvement in HEI enrolment; with a greater than seven-fold increase in average rate of monthly HEI enrolment, and earlier median age of enrolment for the infants. This finding supports results from other studies demonstrating improved follow-up of HEI with integration of PMTCT and EID services ,44. EarlOur results also demonstrate the impact of a comprehensive strategy for identification of HIC. Of the 764 HIC newly enrolled into care during the observation period, only 321 were found directly through HIV testing. The remainder either transferred in from other facilities or were children with known HIV status but not in clinical care, found through the process we refer to as positive linkage. These results suggest that although diagnosis and identification of unknown cases is important, it may be equally critical to identify children who have previously been diagnosed but failed to engage in care. The suboptimal linkage of children to clinical care may have been a reflection of a lack of awareness about availability of HIV services for children. Within Malawi, paediatric HIV care lagged behind adult services, with paediatric treatment guidelines first released in 2005 and paediatric ART formulations not available until 2009. Repeat testing may also be an important service, as 15.1% of those testing HIV-positive had previously been tested. For many of these children, guardians were only willing to start their children in care after previous test results had been independently verified. Interestingly, the median age of enrolment increased steadily over the course of the programme from 2.1 years (MO) to 5.0 years (T-PMTCT). This increase likely reflects improved enrolment of HEI, resulting in fewer young children with an HIV-infected diagnosis at enrolment, and more effective PMTCT service delivery resulting in fewer infections overall .The addition of PMTCT activities did not have a significant impact on new enrolment of HIC. This was not surprising, as the case finding strategies for HIC did not change dramatically from T-Basic to T-PMTCT. In fact, case finding likely became more challenging as the easy to find cases had already been enrolled. However, the dramatic increased enrolment of HEI following the addition of PMTCT support services not only increased the number of HEI in care but also the total number of HIC. During the T-PMTCT period, of 1667 HEI enrolled, 97 had a positive PCR. As a result, the rate of total enrolment of HIC increased from 20.2 per month during T-Basic to 26.0 during T-PMTCT. By February 2011, a total of 866 HIC were receiving care at the three combined clinics, a 23-fold increase from 2008.Our results suggest a significant unmet need for HIV testing of children in high-prevalence settings like Malawi. All of the children within our testing cohort had an indication for testing such as a parent being infected, being an orphan or a specific request from a guardian for testing. With a median age greater than four years at enrolment, the majority should have been screened through the PMTCT and EID programmes. However, the majority were being tested for the first time. These results are similar to those from a survey conducted in Blantyre, Malawi, that found greater than 80% of children of adult patients on ART had not been tested .Our study also demonstrates that while a higher HIV prevalence was seen in health-centre-based testing, the overall yield of the home versus health-centre-based testing strategies was comparable because the absolute number tested through home-based testing was four times higher than through health-centre-based testing. The children identified through the home-based strategy may have been found earlier in their disease course, but further studies are necessary to compare clinical characteristics and outcomes of children identified through these strategies. Both strategies will be important for a comprehensive approach to identification and enrolment of HIC that includes provider-initiated testing and counselling, screening at immunization clinics, and school-based campaigns ,21. Our There are several limitations to the data in this report. The Tingathe programme utilized multiple concurrent identification strategies and did not track exactly where and how each newly registered patient was identified. For example, in our data collection forms we grouped health centre testing and did not track if the testing was done at tuberculosis clinic, malnutrition clinic or through routine voluntary counselling and testing. Similarly, for positive linkage, we did not track exactly where each patient was found or how they were originally diagnosed. Moving forward, the programme will more closely record the mechanism of identification for all newly enrolled patients, thereby providing opportunities for more detailed analysis. Prospective studies evaluating the yield, cost and clinical outcomes of the individual strategies are also needed. At the time of our study, our programme was one of the few in Malawi that routinely provided longitudinal care for HEI and pre-ART patients. As such, there were no contemporary control groups with which to compare our intervention. The rise in patient enrolment could potentially reflect broader national improvements in EID and paediatric HIV testing. However, several observations support our assertion that the increase in patient enrolment was mainly due to our programmatic activities. First, the number of paediatric patients on ART rose more than 10-fold at the three participating clinics from 2007 to 2011. During this same period, nationally, the number only doubled ,47. FurtIn summary, this report is one of the first to describe a comprehensive approach to case finding, demonstrating that a multipronged approach utilizing CHWs can dramatically improve identification and enrolment into care of HEI and HIC. This is an area that urgently warrants attention. In the quest for “virtual elimination” of paediatric HIV, much international attention has been focused on the laudable goal of reducing mother-to-child transmission of HIV . However


Chronic obstructive pulmonary disease (COPD) is a known risk factor for lung cancer and a leading cause of mortality in the U.S., but its impact may not be fully appreciated, especially among low-income populations in the southeast where COPD prevalence and lung cancer incidence are elevated.We conducted a prospective study among 26,927 low-income adults age 40–79 in the Southern Community Cohort Study who had a Center for Medicare and Medicaid Services (CMS) encounter prior to enrollment and were followed for a median of over 6 years. Using a validated algorithm for assessing COPD from CMS claims data, we estimated COPD prevalence and potential misreporting. From Cox proportional hazard models, we computed overall and lung cancer-specific mortality according to COPD status.The overall prevalence of CMS-diagnosed COPD was 16%, but was twice as high among whites as blacks. Only 35% of these individuals, however, self-reported having COPD, with underreporting significantly greater for blacks than whites. Smoking-adjusted all-cause mortality was increased by 1.7-fold and lung cancer mortality by 2.3-fold among those with a CMS COPD diagnosis, with similar patterns in blacks and whites, but no excess was found among those self-reporting COPD and without CMS confirmation.The prevalence of COPD in this low-income population may be greater than previously recognized and misreporting is common. COPD is associated with elevated lung cancer mortality, even among those not self-reporting the condition. Chronic obstructive pulmonary disease (COPD) is a well-known risk factor for lung cancer ,5,6,7,8 Individuals often underreport COPD and the condition may be underdiagnosed in as many as 60–85% of patients, primarily those with mild to moderate disease ,17,18. FThe Southern Community Cohort Study (SCCS) is an ongoing prospective observational cohort study established to examine health disparities amongst a predominantly low-income population. From March 2002 to September 2009, 72,532 adults were enrolled into the SCCS at community health clinics, institutions providing basic health care and preventative services in medically underserved geographic areas, across a 12-state area of the Southeast . Details of the SCCS study are provided elsewhere ,20. In bBaseline epidemiologic data were collected during in-person computer-assisted personal interviews conducted by trained interviewers at the community health centers. Self-reported information was ascertained on demographic characteristics and exposure histories, including race/ethnicity, income, tobacco smoking history, medical history, and health insurance status. Participants were also given the 10-item Center for Epidemiologic Studies Depression Scale (CESD-10) . Medicalth birthday. End date for CMS follow-up was the date of SCCS enrollment. CMS coverage time is defined as time between the start of CMS enrollment and date of enrollment in the SCCS cohort. To ensure internal validity, only those SCCS participants with at least one CMS encounter prior to cohort entry were included in the analyses. Among these, 49% had only a Medicaid claim, 22% only a Medicare claim, and 29% both a Medicaid and a Medicare claim; 61% of Medicare claims occurred among persons below age 65.The roster of SCCS participants was linked with the CMS Research Identifiable Files from January 1, 1999 through December 31, 2008 to identify all persons who had a Medicaid or Medicare encounter prior to their entry into the SCCS. We defined the start of CMS enrollment as the minimum of either the date of their first CMS claim or the first day of the month of their 65et al., we required participants to have at least one inpatient hospitalization or emergency room encounter with a COPD diagnostic code  or at least two professional claims having different dates of service with a COPD diagnostic code [et al. [COPD diagnoses prior to entry into the SCCS were defined using two previously published algorithms for identification of patients with COPD ,23. Usin [et al. . We obtaThe cohort was followed for all-cause and lung cancer (ICD10 C33-34) mortality, with the follow up time defined as the time from SCCS enrollment until date of death, loss to follow-up, or censoring through December 31, 2011, by linkage with the National Death Index and the Social Security Administration.2) and summary CESD-10 score . We employed logistic regression to calculate adjusted prevalence odds ratios (ORs) and corresponding 95% confidence intervals (95% CI) of CMS-identified COPD associated with these factors. Kaplan-Meier curves were plotted to visualize differences in crude overall survival by COPD status and were compared using the log-rank test. We estimated hazard ratios (HRs) and accompanying 95% CIs for all-cause and lung cancer mortality using Cox proportional hazard models with age as the time scale, adjusted for race, sex, income, education, BMI, number of co-morbidities, CESD-10 score, CMS coverage time, and smoking status, to evaluate whether mortality differed by COPD diagnosis and between blacks and whites. We tested for differing patterns between black and white women and men by comparing models with and without cross-product terms for sex-race-COPD status. The proportional hazards assumption was assessed by including an interaction term between COPD and time and we found hazards remained constant over time. Because individuals reporting their race as other than black/African American or white were too few for stable statistical analysis, only blacks and whites are included herein. All analyses were conducted using SAS software, version 9.3  or R version 2.15.0. Statistical tests were two-sided and an alpha of 0.05 was used to assess statistical significance.SCCS participants with unknown self-reported COPD information (N = 488) were excluded from analyses leaving 26,927 participants. Individuals were classified according to their COPD status at entry into the SCCS in the following groups: no indication of COPD; self-report only COPD; CMS diagnosis only COPD; both self-report and CMS diagnosis of COPD. Contingency table analyses were used to compare percentages across the four groups with respect to self-reported race, gender, age, education, household income, employment status, health insurance status, self-reported prevalence (yes/no) of asthma, cardiovascular disease (history of heart attack or coronary artery bypass surgery), diabetes or depression, number of such morbidities reported (0–4), smoking status , body mass index  and higher among men (19%) than women (14%) (P < 0.0001) Underreporting of CMS-diagnosed COPD was significantly greater in blacks than whites  and males than females , but did not differ greatly by age. A total of 26,927 participants  had a CMS encounter prior to enrollment into the SCCS and had self-reported information on COPD status. Median coverage time on Medicaid or Medicare for SCCS participants prior to entry into the SCCS was 4.5 years (IQR: 3.1–6.3 years). We identified 4,213 patients (16%) with COPD diagnostic codes; only 1,463 (35%) of these individuals also self-reported COPD . Among tSCCS participants with self-reported or CMS-ascertained COPD had lower education (less than 12 years) and income  compared to individuals not having a COPD diagnosis . IndividIn multivariate logistic regression analyses, significant associations were found between COPD prevalence and race, sex, age, education, BMI, smoking, and comorbidities . PronounFollow up of the cohort identified 3,936 deaths overall and 318 lung cancer deaths. All-cause mortality was 1.7-fold  greater and lung cancer 2.3-fold greater  for those having a CMS code for COPD. P = 0.10), with no suggestion of an interaction (P = 0.94) for lung cancer mortality. In assessing potential risk factors for subsequent overall survival among those with CMS-diagnosed COPD, higher mortality was observed among men than women, but differences by race and socioeconomic status were not marked after adjusting for smoking status and number of comorbidities, and little differences by sex or race were seen for lung cancer . We note these data are for a low-resource population participating in CMS, but our finding of substantial underreporting is similar to that noted in other populations ,17,18,28The overall prevalence of self-reported COPD in the SCCS population was 12%, greater than the recently published BRFSS national average of 9.6% for adults 45 years and older, also obtained from self-reports , but a het al. noted that their algorithm developed for the purpose of identifying patients hospitalized for acute exacerbations of COPD had a low sensitivity, although high positive and negative predictive values suggest the algorithm has some clinical value for identifying primary acute exacerbation of COPD [This study is subject to several limitations. The population we studied is not necessarily representative of southern residents overall or those with CMS enrollment, having lower socioeconomic status and greater co-morbidities. We did not assess pulmonary function measurements or use CT scans to evaluate emphysema. It is important to consider that ICD-9 codes reported in CMS claims are used for physician reimbursement and nuances in ICD-9 coding can complicate findings . The use of COPD . Our covStrengths of this study are numerous. One important aspect of our study is that the SCCS includes a large low-income population with a high percentage of blacks which enabled a comprehensive assessment of COPD among blacks and whites of similar socioeconomic status. Our findings extend to other U.S. studies of COPD which have primarily focused on whites and higher income populations ,31,32 anOur findings suggest that COPD is a major public health burden, with elevated overall and lung cancer mortality. The NHLBI estimates 12 million people in the United States are living with COPD, resulting in significant health care expenditures estimated to exceed $72 billion annually ,34. Thes


In Poland, the HIV epidemic has shifted recently from being predominantly related to injection drug use (IDU) to being driven by transmissions among men-who-have-sex-with-men (MSM). The number of new HIV cases has increased in the recent years, while no current data on the transmitted drug resistance associated mutations (tDRM) frequency trend over time are available from 2010. In this study, we analyze the temporal trends in the spread of tDRM from 2008 to 2013.Partial pol sequences from 833 antiretroviral treatment-naive individuals of European descent (Polish origin) linked to care in 9 of 17 Polish HIV treatment centres were analyzed. Drug resistance interpretation was performed according to WHO surveillance recommendations, subtyping with REGA genotyping 2.0 tool. Time trends were examined for the frequency of t-DRM across subtypes and transmission groups using logistic regression .Frequency of tDRM proved stable over time, with mutation frequency change from 11.3% in 2008 to 8.3% in 2013  a. Also, The frequency of t-DRM in Poland is generally stable over time. Decrease in the overall tDRM frequency in heterosexual infected cases and NNRTI resistance in subtype B infected MSM may be related to the higher treatment efficacy of current cART.


Cutaneous squamous cell carcinoma of the ear represents a high-risk tumor location with an increased risk of metastasis and local tissue invasion. However, it is uncommon for these cancers to invade through nearby cartilage. Cartilage invasion is facilitated by matrix metalloproteases, specifically collagenase 3. We present the unusual case of a 76-year-old man with an auricular squamous cell carcinoma that exhibited full-thickness perforation of the scapha cartilage. Permanent sections through the eroded cartilage confirmed tumor invasion extending to the posterior ear skin. Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer of the skin in the United States. The ears are a frequent site of these cancers and represent a high-risk location with an increased risk of metastasis. Invasion of cartilage by cSCC is rare and considered to be a significant risk factor for the development of metastatic disease. Current treatment guidelines recommend surgical excision with histologic confirmation of negative margins and close follow-up to monitor for recurrence and metastasis. We present the case of a 76-year-old man with a cSCC on his anterior left ear with tumor perforation through his auricular cartilage that permitted tumor spreading to posterior ear skin.A 76-year-old Caucasian man with advanced dementia who was anticoagulated daily with 81 mg of aspirin and 75 mg of clopidogrel due to a history of cerebrovascular accident presented to the dermatology department with a biopsy proven moderately differentiated squamous cell carcinoma on the left ear. The tumor measured 2 by 2.7 cm, involved the left scapha, and clinically extended to the superior crus of the antihelix . It was After a surgical site preparation and infiltration of 1% buffered lidocaine with epinephrine 1 : 400,000, a curette was used to gently debride the clinical tumor. A 2 mm full-thickness defect in the cartilage of the scapha was noted . Due to Although the classification of high-risk cSCC is not uniform among dermatologists, most agree on a few key clinical and histopathologic features. High-risk clinical features include clinical size ≥2 cm, involvement of the lip, ear, or mid face, poorly defined clinical borders, neurologic symptoms, tumor destruction of cartilage, immunocompromised host, recurrent or rapidly growing tumor, and tumor arising in a scar, chronic ulcer, or site of prior radiotherapy. The histopathologic elements that are associated with increased tumor risk include depth >4 mm, moderate or poor cytologic differentiation, adenoid, acantholytic, adenosquamous or desmoplastic subtypes, and perineural or lymphovascular invasion . Poorly Matrix metalloproteases (MMP) play an integral role in tumor growth and metastasis. MMPs are a family of zinc-dependent endopeptidases that function to remodel the extracellular matrix (ECM). They allow tumors to grow by degrading matrix barriers and promoting angiogenesis as well as releasing active growth factors and modulating apoptosis. Direct invasion of local structures by tumors is facilitated by MMP-mediated proteolytic degradation of the ECM. Markers of malignant transformation of keratinocytes include MMP-13, MMP-7, MMP-12, and MMP-14.Specifically, MMP-13 is associated with greater metastatic capacity and MMP-11 is linked to increased local invasiveness of SCC of the head and neck .Cutaneous squamous cell carcinoma with auricular involvement has a metastatic rate of approximately 15.5% and destruction of cartilage is a significant risk factor for metastatic disease . The mosCurrent guidelines by the National Comprehensive Cancer Network (NCCN) for high-risk cSCC advocate surgical excision by either standard excision with postoperative margin assessment or MMS. Primary radiation therapy is also an option for nonsurgical candidates. MMS results in a 5-year disease-free survival rate of 97% for patients with cSCC . PostopeHigh-risk features for cSCC include clinical tumor size ≥2 cm, tumor depth >4 mm, moderate to poor histologic grade, high-risk anatomic location, perineural involvement, and invasion of cartilage. Treatment consists of surgical excision with adjuvant radiation therapy in the setting of large nerve perineural invasion by cSCC, other high-risk tumor features, or inability to obtain negative margins. The role of sentinel lymph node biopsy and elective neck dissection in treatment of cutaneous squamous cell carcinoma remains controversial and without current proven clinical efficacy. This case report represents a pathologically confirmed observation of an anteriorly positioned primary cutaneous squamous cell carcinoma with full-thickness auricular cartilage perforation leading into tumor invasion of the posterior ear skin.


Elevated depressive symptoms that do not reach criteria for a clinical diagnosis of depression are highly prevalent in persons with diabetes. This study was aimed at determining the efficacy of psychoeducation and physical exercise compared with enhanced treatment as usual on 1-year changes in depressive symptoms, diabetes distress and self-management, and quality of life and metabolic control in type 2 diabetes patients with subsyndromal depression.Adult type 2 diabetes patients who screened positively for depression and expressed a need for professional help with mood-related issues were eligible. Exclusion criteria were clinical depression, current psychiatric treatment and advanced diabetes complications. Out of 365 eligible patients 209 consented to either 6 weekly sessions of psychoeducation (A) and physical exercise (B), or to enhanced treatment as usual (C). Computer-generated sequences for block randomisation stratified by gender were used. Depressive symptoms (primary outcome) and diabetes distress, diabetes self-care, metabolic control and health-related quality of life (secondary outcomes) were analysed at 6-month and 12-month follow-up using repeated-measures ANOVAs.p < 0.001, η2 = 0.07 and F = 0.609, p = 0.656, η2 = 0.007 respectively), as did diabetes distress and quality of life , diabetes self-care (p < 0.001 to < 0.05), triglycerides, and total cholesterol and LDL-cholesterol (p < 0.001).Out of the 74 patients randomised into group A, 66 into B and 69 into group C, 203 completed the interventions, and 179 patients with all 3 assessments were analysed. Depressive symptoms in participants from the psychoeducational, physical exercise and the enhanced treatment as usual groups improved equally from baseline to 12-month follow-up  contains supplementary material, which is available to authorized users. Subsyndromal depression, defined as the presence of depressive symptoms that do not meet full diagnostic criteria for major depression or dysthymia, prevails in diabetic patients with depressive mood disorders. Among the approximately 30 % of diabetic patients who report elevated depressive symptoms, around two thirds do not reach criteria for a clinical diagnosis of depression, implying that 20 % of these patients are affected by subsyndromal depression . HoweverFindings from the general population indicate low spontaneous remission rates of subsyndromal depression . Recent Patients who report mild to moderate depressive symptoms frequently also report emotional problems related to diabetes –18, suggResearch on subsyndromal depression treatment in patients with diabetes is scarce, allowing no reliable conclusion on its effects on depression-related and diabetes-related outcomes. Since trials relying on pharmacological treatment have not justified the use of antidepressants in patients with minor depression , psycholThis study explored the significance of treating subsyndromal depression in type 2 diabetes patients while examining the effects of three behavioural interventions – psychoeducation, physical exercise and enhanced treatment as usual – on depressive symptoms, diabetes distress, diabetes self-management, health-related quality of life and metabolic control at 1 year. Our hypothesis was that a psychoeducational course and physical activity intervention, both consisting of 6 weekly 90-minute group sessions would be superior to enhanced treatment as usual, consisting of one 90-minute re-educational session, in improving psychological and diabetes-related outcomes.Diagnostic and Statistical Manual of Mental Disorders, version IV (DSM-IV) Axis I disorders (SCID-I) [The study was carried out at the Vuk Vrhovac University Clinic for Diabetes in Zagreb. Data on potential study participants were extracted from the patient registry  accordin(SCID-I) , currentThe participants were recruited from September 2010 to April 2012, and a 1-year follow-up of the latest groups lasted till June 2013. Assessments of psychological and biochemical variables were repeated at 8-week, and at 6 and 12-monthfollow-ups. A 1-year follow-up assessment included a repeated clinical interview to determine hypothetical conversion of depressive symptoms into clinical depression. A more detailed study procedure was described elsewhere , 32.A randomised, repeated measures experimental design was used. Eligible patients willing to participate in the interventions were randomised to psychoeducation and physical exercise or to the enhanced treatment as usual prior to baseline assessment. A computer-generated algorithm stratified by gender  providedThe outcome assessors were not blinded for the patients’ group assignment, since the included measures  were not considered likely to cause bias.1c) and lipid profile, were secondary outcomes. The sample size calculation was based on the absolute change in depressive symptoms as measured by the Center for Epidemiological Studies Depression Scale (CES-D) questionnaire. An improvement of 0.5 standard deviations was considered clinically relevant [n = 59 per group were shown to be needed to have 80 % power in demonstrating statistically significant differences in depressive symptoms.Depressive symptoms were primary study outcome, and diabetes-related distress, health-related quality of life, diabetes self-care and metabolic control, as assessed by glycosylated haemoglobin  in treating diabetes; 2. effects of exercise on glycaemic control and the cardiovascular system; 3. PA and energy expenditure; 4. effects of PA on mobility, muscles and peripheral nerves; 5. effects of exercise on mood; 6. acquiring strategies to maintain physical activities, and developing a personal plan for regular exercise. Educational topics were presented in the first 10–15 minutes of each session including a possibility to exchange personal experiences. The programme was developed and run by a physiotherapist experienced in working with a diabetic population. It was tested within regular educational work with patients in day hospital care.Exercise intensity was measured by a heart rate monitor and maintained in a light to medium intensity range. Blood glucose and blood pressure were measured before and after each session.Meetings were held in weekly intervals in a room at the Outpatient Clinic equipped for physical exercise.Written materials which were given to the patients as a reminder of the practised exercises and the PowerPoint presentation are available in Croatian language at the Vuk Vrhovac Clinic.Attending at least four sessions of psychoeducation and physical exercise was considered a criterion for receiving the planned treatment.1c and lipid values; b) patients’ goals in self-managing diabetes; c) patients’ concerns caused by diabetes in general, and the current laboratory findings. A method of delivery was small-group patient-centred counselling. In addition, patients were provided with written self-help instructions to cope with mood difficulties. This brief intervention was given in a group format and run by a diabetologist experienced in diabetes self-management education.No usual care arm was included in the trial because of concerns about leaving patients with depressive symptoms and a need for professional help in a non-interventional study arm. Instead, 1 re-educational intervention of 90 minutes duration was offered. It addressed: a) patients’ understanding of their current HbAA summary of programmes’ objectives and contents are delineated in Table Patient demographic variables included age, sex, education, family status, employment, and economic status. Diabetes-related variables included years of diabetes duration and diabetes treatment with or without insulin.Screening for mood difficulties was done using the adapted PHQ-2. The participants were asked to indicate if they experienced depressed mood or loss of interest and pleasure in everyday activities in the past month . An addiThe presence or absence of clinical depression was determined by phone-administered structured clinical interview for the DSM-IV Axis 1 disorders, as this method was demonstrated to be comparable to face-to-face interview . The SCIDepressive symptoms were measured by the Center for Epidemiological Studies Depression Scale (CES-D), a 20-item self-report instrument . The resDiabetes-specific emotional distress was measured by the Problem Areas in Diabetes scale (PAID) , a 20-itDiabetes self-care behaviours were measured by the Summary of Diabetes Self-Care Activities (SDSCA) . RespondHealth-related quality of life was measured by the Version 2 of the 12-Item Short Form Health Survey (SF-12v2) . The ins1c was measured by an automated immunoturbidimetric assay with dual reporting traceable to National Glycohaemoglobin Standardisation Programme (NGSP) (%) and International Federation of Clinical Chemistry (IFCC) (mmol/mol) reference systems  and a total imprecision, expressed as coefficient of variation (CV) < 2 %. Serum lipids were measured in the morning serum samples, obtained after an overnight fast, by routine automated enzymatic assays  with a total imprecision (CV) of 1.16 %, 1.48 % and 1.18 % for the total cholesterol, high-density lipoprotein (HDL)-cholesterol and triglycerides, respectively.HbAOne-way analysis of variance (ANOVA) and chi-squares were used to test for baseline differences across the three treatment arms and to examine differences between continuing participants and dropouts. Missing questionnaires’ scores were replaced by average individual scores , or by average group results, in particular subscales . Replacement was carried out if a number of missing values was not greater than two.Data were analysed per-protocol, after exclusion of participants who dropped out, died, did not attend all three follow-up meetings or initiated psychopharmacological treatment; these results are presented throughout the manuscript. Intention-to-treat (ITT) analysis was performed to validate these results; all participants who were randomised, with the exclusion of three persons who died during the trial, were analysed despite deviations from protocol. To perform ITT, missing measurements were imputed using the baseline-observation-carried-forward approach.2 coefficients ranging from 0.01–0.05 were regarded small, 0.06–0.12 moderate and ≥ 0.13 large. The outcomes were controlled for gender, age, education, diabetes duration, diabetes treatment, and baseline values of depressive symptoms, diabetes-related distress, HbA1c and low-density lipoprotein (LDL)-cholesterol. If significant interaction between main effects and covariates occurred, separate ANOVA models for divided subgroups were employed. Post-hoc ANOVA’s comparing psychological-related and diabetes-related outcomes in patients with high versus low baseline values of depressive symptoms, diabetes distress, HbA1c and cholesterol were used to test relative benefits of the interventions.Repeated-measures ANOVA was used to test for change in outcome variables across time and between the groups. Cohen’s ηp value of < 0.05 was considered significant in all analyses. Statistical analyses were performed by using the SPSS 17 A The study was approved by the Vuk Vrhovac University Clinic for Diabetes Ethics Committee on 30 June 2010.Among the 4858 patients identified from the registry of diabetic patients on the basis of the inclusion criteria, 365 were considered eligible and 265 agreed to participate in the interventions (72 %); 209 of them completed baseline laboratory and psychological assessments (79 %), with 74 randomised to psychoeducational intervention, 66 to physical exercise and 69 to diabetes re-education. Fifty-six patients withdrew their previous agreement to participate because of missing baseline assessments or reporting competitive life demands as an obstacle.Attrition rate was 3 % during the interventions (6 of 209 patients), 3 % from baseline to 6-months (7 of 203 patients), and 2 % from 6-month to 12-month follow-up period (4 of 196 patients). Of the seven dropouts who completed the intervention but missed both follow-up assessments, two missed the follow-up appointments due to health problems, four were unwilling to come and one patient died. Four patients were excluded from per-protocol analyses due to the initiation of pharmacological therapy or discovery of psychiatric co-morbidities that were not reported during the recruitment period. No differences between the participants and dropouts across the three study groups were observed. The patients’ flow is presented in Fig. p = 0.012).Table The effects of interventions on psychological, behavioural and metabolic outcomes based on repeated measures ANOVA are given in Table p = 0.656, p = 0.796, and p = 0.288, respectively), indicating comparable efficacy of psychoeducation, physical exercise and enhanced treatment as usual on these outcomes.No significant between-subject effects were obtained with respect to depressive symptoms, diabetes distress and health-related quality of life . The size of the time effect indicated a moderate effect of the three treatments on the primary study outcome.Significant reductions in depressive symptoms occurred across all 3 study groups from baseline to 12 months  occurred in all 3 groups from baseline to 12 months.Statistically significant time effects of interventions on diabetes-related distress  during the 1-year follow-up period, while physical quality of life aspect remained unchanged (p = 0.711).Mental component of health-related quality of life improved significantly in all treated individuals .No significant between-subject effects were obtained with respect to self-care behaviours as determined by self-reported healthy eating, maintaining a diabetic diet, exercising, blood glucose self-monitoring and foot self-care  occurred in all 3 groups from baseline to 12 months, as did self-reported exercising . Moderate main time effects were registered with respect to blood glucose self-monitoring and foot self-care .A small effect of interventions on healthy eating and diabetic diet .No significant between-subject effects occurred with respect to glycaemic control and lipid profile , while changes in total cholesterol, LDL-cholesterol and triglycerides reached significant and clinically meaningful improvement during the 1-year follow-up .In all treated individuals, changes in HbAThe results remain significant after controlling for changes in lipid-lowering therapy during the follow-up period.1c higher/lower than 7 % (53 mmol/mol) and LDL-cholesterol higher/lower than 2.6 mmol/l – are presented in Table Time changes of psychological and metabolic variables in the 2 subgroups of patients – with baseline CES-D and PAID scores above/below cut-offs of 16 and 40, and with baseline HbA2 = 0.16 and η2 = 0.27, respectively).Emotional symptoms in individuals with milder depressive symptoms (CES-D < 16) and no serious problems related to diabetes (PAID < 40) did not improve but remained within subclinical scores’ range. Patients with baseline CES-D and PAID scores above the cut-points, however, demonstrated continuous and clinically relevant improvement over a one-year period , and LDL-cholesterol lower than 2.6 mmol/l remained within reference values over a 1-year follow-up period, although variability of these indicators was shown to be statistically significant. Patients with unsatisfactory metabolic control at baseline reached significant and clinically relevant improvement in both glycaemic and lipid control .The same pattern of change was observed with respect to metabolic indicators. Patients with baseline HbAThe main finding of this 1-year study was that subclinically depressed patients treated by psychoeducation, physical exercise and enhanced treatment as usual comparably improved depressive symptoms, diabetes distress, self-management of diabetes, health-related quality of life and metabolic control. The absence of a significant treatment effect suggested that patterns of change might be similar in all treatment groups. Psychoeducation and physical exercise as more complex treatments were not superior to enhanced treatment as usual – a short diabetes re-education focused on patients’ diabetes-related concerns, but were equally effective.This might be associated with the nature of depressive symptoms in the treated individuals on the one hand, and with inter-relations of depressive symptoms and diabetes-related distress on the other hand. Mild depression, common in all treated individuals, could imply that they were only partially aware of having a problem, which is relevant and can be approached actively. Once clinical attention to their difficulties was established, patients’ self-awareness might be increased and their readiness to put efforts into a more active approach empowered, regardless whether a structured anti-depressive programme or an unspecific support was offered.p < 0.01) and 53 % also reported serious problems caused by diabetes. As psychoeducational and physical exercise treatments tended to focus on diabetes-related problems as did the enhanced treatment as usual, addressing diabetes distress can be supposed to be a pathway likely to improve outcomes in all three groups.In the study participants, elevated depressive symptoms were strongly associated with diabetes distress (r = 0.353, The obtained results can be put into a context of the study by Fisher et al.  on treatA comparable improvement in depressive symptoms in all treatment groups might be hypothesised to be attributable to spontaneous remission of depression. However, reports on the persistence of elevated depressive symptoms in diabetic population , 43, as Another relevant finding of this study is that even minimally intensified treatment as usual was associated with changes on multiple levels, including psychological, behavioural and biochemical ones, and continued over time. The effects remained significant after controlling for age, education, diabetes duration and insulin treatment, suggesting that the findings may be generalised to most type 2 diabetes patients suffering from subsyndromal depression.1c and LDL-cholesterol. In contrast, patients who were below standard cut-offs at baseline, remained so during the 1 year follow-up. The latter corresponds with well-known findings that individuals who entered a programme with better values on a variety of indicators improve less on these indicators [The improvement in emotional symptoms and metabolic control is even more apparent when viewing the subgroups of patients with higher depressive symptoms, more serious emotional problems related to diabetes and suboptimal metabolic control at baseline. Compared with patients with milder baseline symptoms and satisfactory metabolic control, these patients achieved greater reduction in depressive symptoms and diabetes distress, and reached significant and clinically meaningful improvement in HbAdicators . Howeverdicators .Reviews on treating subclinical depression in the general population  report pThis study has several strengths. It was focused on highly prevalent population of type 2 diabetes patients with depressive symptoms but no clinical depression, relied on a randomised comparative design, investigated samples large enough to demonstrate statistically significant changes, and used structured interventions with reasonable requirements in terms of staff, duration and costs. Its limitation is the lack of possibility to investigate the course and outcomes of subclinical depression without any treatment, as no usual care was included in the trial because of concerns about leaving patients without professional help. Also, the study was carried out in only one centre – a tertiary diabetes clinic – which does not allow concluding about its feasibility in other clinical settings. Allocating patients to the treatment groups was not concealed to researchers and the outcome assessors were not blinded. Both could be considered as the study weaknesses although a computer-generated randomisation, and highly standardised outcome measures substantially reduce hypothetical source of bias.Depressive symptoms that are not severe enough to warrant a diagnosis of clinical depression are highly prevalent in diabetic population and are associated with both poor well-being and poor diabetes self-management. Our study showed that psychoeducation, physical exercise and enhanced treatment as usual had comparable effects on long-term improvement of psychological, behavioural and metabolic outcome suggesting that even minimal intervention addressing patients’ diabetes-related problems and concerns had favourable clinical implications. Further investigation is warranted to clarify possible mechanisms of improvement.


Sarcoidosis is a systemic chronic granulomatous disease of unknown etiology. It predominantly involves the lungs but can affect many organs or tissues in the body, such as the lymphatic system, skin, eyes, and liver. Typical histopathological lesions are noncaseating granulomas in the affected organ or tissue. Indications, type of treatment, and duration of sarcoidosis treatment is currently debated. Despite studies showing that anti–tumor necrosis factor-α (TNF-α) treatment can successfully be used in refractory sarcoidosis, there are some case reports regarding the development of sarcoidosis with these agents. There have been reports of 47 anti-TNF-associated cases of sarcoidosis until 2012. The patient is a 54-year-old Caucasian male. During routine examinations of the patient who had been followed for psoriasis vulgaris for 20 years and who had been on several anti-TNF regimens thereafter, new pulmonary pathologies due to sarcoidosis were detected. We present here a case of sarcoidosis that developed after infliximab treatment and showed obvious radiologic regression with discontinuation of treatment. During anti-TNF treatment, it should be kept in mind that autoimmune and granulomatous diseases may develop and particular care should be given to patient follow-ups. Sarcoidosis is a multisystem granulomatous disease that generally involves lungs and intrathoracic lymph nodes, and it has an unknown etiology. While it is more common in adults, children can also be affected. It is most frequently observed in adults in their 30s and peaks in their 50s. Disease involvement and clinical course differ by ethnicity. Its etiology is unknown; however, genetic transition is thought to be influential. Although debates regarding the treatment still continue, there is some information about sarcoidosis development, thanks to anti–tumor necrosis factor (TNF) agents used in steroid-resistant sarcoidosis and relapses during systematic involvement. There have been reports of 47 anti-TNF-associated cases of sarcoidosis until 2012.1) 2860 cc 87%; forced vital capacity (FVC) 3370 cc 83%; FEV1/FVC 85%; diffusing capacity for carbon monoxide 77%; DLCO/alveolar volume (VA) 112; RV  130%; TLC  99%; and VC  85%. High-resolution CT revealed peribronchovascular nodules in the bilateral upper and middle zones that were centrally located  treatment due to exacerbation of his disease. The ETN treatment was discontinued after 59 cycles due to elevated carbohydrate antigen 199 levels in the follow-up examinations. The patient was scheduled for infliximab (IFX) therapy due to increased symptoms in March 2010. There was no sign of infiltration in the chest x-ray , and the located -5; hilar located  and 7.The patient underwent fiberoptic bronchoscopy. Bronchoalveolar lavage (BAL) was obtained from the anterior segment of the upper left lung. Mucosal biopsy specimens were obtained from the upper left lung and tracheal carinas of the upper left lung. Transbronchial biopsy (TBB) specimen were obtained from the anterior segment of the upper left lung. Fifty-five percent lymphocytes, 40% macrophages, and 5% neutrophils were detected in the BAL specimen, and non-necrotizing granulomatous lesions were detected in the TBB specimens. During histopathological examination of the biopsy specimens, Ehrlich-Ziehl-Neelsen and Kinyoun-specific microorganisms were not detected. The tuberculosis culture from the tissue biopsy was negative. The examination of histopathological and microbiological specimens did not yield any fungal elements or microorganisms including histoplasmosis. The BAL panfungal polymerase chain reaction was negative for the fungi. As no cases of histoplasmosis have been reported in our center, we did not specifically study histoplasmosis.Considering the radiological and pathological findings, the patient was diagnosed with stage 2 sarcoidosis and was suggested to discontinue anti-TNF treatment. After discontinuation, the patient had been followed for 4 months without any treatment and showed no symptoms. Thereafter, we observed regression in the control chest x-ray and high-resolution CT -11, and 2 Sarcoidosis, with unknown etiology, is a disease characterized by non-necrotizing granulomas and might affect all the organs and systems, particularly adenoids and the lungs. Regarding sarcoidosis treatment, the indications, type of treatment, and duration are currently debated. However, according to case reports anti-TNF agents, which are among the possible options for refractory sarcoidosis treatment, cause granulomatous lesions.4Anti-TNF-α agents are used to treat various chronic inflammatory diseases . In recent years, there have been many publications indicating that various autoimmune diseases and interstitial lung diseases develop or preexisting lung diseases deteriorate depending on these agents.5 The paradoxical aspect of this phenomenon is that TNF is one of the molecules involved in the induction of psoriatic skin lesions, and all TNF antagonists have repeatedly been shown to be efficacious in both cutaneous and articular disease. The appearance of psoriasis with all 3 anti-TNF agents is considered to be a “class-dependent” effect rather than an immuno-allergic reaction.6 Therefore, it is possible that both phenomena could be viewed in the context of a cytokine imbalance due to prolonged TNF suppression. Indeed, the occurrence of autoimmune diseases related to anti-TNF agents, such as “lupus-like” syndrome and vasculitis, is well known with such therapy.7A possible hypothesis to explain the pathogenesis of anti-TNF-induced granulomatous disease could rise from another paradoxical phenomenon linked to these drugs, that is, the psoriatic lesions that develop during the course of anti-TNF therapy.Here, we present a case of pulmonary sarcoidosis that developed during anti-TNF-α treatment for psoriasis and showed spontaneous regression after discontinuation of anti-TNF treatment.8The study by Ramos-Casals et al, which is regarded as one of the most comprehensive studies on the anti-TNF-α-associated sarcoidosis cases, evaluated 10 cases with sarcoidosis. Eight of the cases developed sarcoidosis during ETN treatment, while 2 cases developed sarcoidosis during IFX treatment. Treatment of ILD involved the withdrawal of anti-TNF therapy in 8 cases, and corticosteroids were used in 5 patients. Resolution was observed in 7 patients, improvement in 2 patients, and there was no resolution in 1 patients; no deaths were reported.9According to a literature search by Tong et al, 37 cases of anti-TNF treatment-associated sarcoidosis were determined between 2000 and 2009, and the authors reported 3 additional cases that were diagnosed by themselves.10 Nine of these cases received IFX, 8 cases received adalimumab, and 30 cases received ETN. In 5 of the cases that developed sarcoidosis during IFX treatment, clinical and radiological resolution (full resolution) was achieved, whereas only clinical resolution was achieved in 1 case. In 2 out of 9 cases, additional systemic steroid treatment (to discontinued IFX treatment) led to complete resolution, while clinical resolution was observed in 1 case. In our case, discontinuation of the IFX treatment led to almost complete clinical and radiological resolution during the 4-month follow-up period.There have been reports of 47 anti-TNF-associated cases of sarcoidosis until 2012.11 In our case, we excluded the possible tuberculosis diagnosis as there were no pulmonary symptoms and tuberculosis bacilli were not detected in the BAL and lung tissue biopsy cultures. Considering the current laboratory findings and clinical, radiological, and pathological features, the case was regarded as sarcoidosis and determined to have resolution after IFX treatment was discontinued. During anti-TNF treatment, it should be kept in mind that autoimmune and granulomatous diseases may develop and particular care should be given to patient follow-ups.During anti-TNF treatment, various infectious complications, particularly tuberculosis, may occur.


In breast cancer, recent clinical trials have shown that sentinel lymph node biopsy (SLNB) alone without axillary lymph node dissection results in excellent prognosis if there is sentinel lymph node (SLN) metastasis in two or fewer nodes. The aim of the present study was to investigate the association between non-SLN metastasis and clinicopathological factors in case of SLN metastasis in two or fewer nodes in breast cancer.Patients who underwent SLNB for invasive breast cancer and were found to have positive SLN in two or fewer nodes were evaluated. The associations between non-SLN metastasis and clinicopahological factors were examined. Statistical analyses were performed using the Mann-Whitney and Chi-square tests, with statistical significance set at P < 0.05.A total of 358 patients were enrolled during the study period and all of these patients were female and 54 patients had SLN metastasis (15%). Positive SLN in two or fewer nodes was identified in 44 patients (81.5%). Among these patients, 17 (38.6%) were found to have non-SLN metastasis. Non-SLN metastasis was associated with invasive tumor size (P = 0.015) and lymphatic involvement (P = 0.035). Multivariate analysis showed that tumor size (P = 0.011) and lymphatic involvement (P = 0.019) remained significant independent predictors of non-SLN metastasis, and that an invasive tumor size cut-off point of 28 mm was useful for dividing patients with positive SLN in two or fewer nodes into non-SLN-positive and non-SLN-negative groups.Non-SLN metastasis was found in more than 30% of patients with SLN metastasis present in two or fewer nodes. Large tumor size and the presence of lymphatic involvement were significantly associated with non-SLN metastasis. Non-sentinel lymph node (SLN) metastasis has been an important issue for planning further treatment of node positive breast cancer, because axillary lymph node metastasis has been a strong prognostic indicator for the patients with invasive breast cancer while new tumor markers have been widely studied . AxillarRecent clinical trials suggest that there is no difference in outcome between patients with positive SLN if they are treated with ALND or given no further axillary surgery , 7. ThesThe aim of the present study was to investigate the association between non-SLN metastasis and clinicopathological factors, particularly in the case of SLN metastasis in two or fewer nodes, in breast cancer.Patients with invasive breast cancer who underwent SLNB at Niigata University Hospital between January 2010 and December 2014 were eligible for inclusion in the study, which was a retrospective chart review. ALND was performed in patients with macro- and micrometastasis in SLN; however, ALND was avoided in patients with isolated tumor cells in SLN. Of the patients who were positive for SLN metastasis, those with SLN metastases in two or fewer nodes were evaluated further. Only those patients with complete data for clinicopathological factors  status, and Ki-67 labeling index) were enrolled in the study (n = 44). The data for these patients were analyzed following approval from the Institutional Review Board.in situ hybridization (FISH) was used in cases when it was difficult to decide the HER2 status by IHC. Ki-67 was also examined by IHC, and the results are expressed as the percentage of tumor cells stained by the antibody, as described previously [Immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PR) status was assessed and tumors were deemed positive for each receptor if at least 10% of the invasive tumor cells in a section exhibited nuclear staining for that particular receptor. HER2 expression was examined by IHC. A gene amplification assay using fluorescence eviously . Hematoxeviously . SLN metPatients were assigned into four subgroups, as proposed in the St Gallen International Expert Consensus  based onThe associations between non-SLN metastasis and clinicopahological factors  were examined. Statistical analyses were performed using Mann-Whitney’s U-test and the Chi-squared test, and multivariate analysis was performed using the logistic regression model. The diagnostic accuracy of invasive tumor size was assessed by receiver operating characteristic (ROC) analysis. The area under the ROC curve (AUC) was used to measure model discrimination. The AUC can range from 0.5 (which indicates a test with no information) to 1.0 (which indicates a perfect test). Statistical significance was set at P < 0.05.A total of 358 patients were enrolled during the study period. All patients were female and 54 had SLN metastasis (15%). Of the patients who were positive for SLN metastases, positive SLN in two or fewer nodes were obseved in 44 patients (81.5%). Among these 44 patients, non-SLN metastasis was observed in 17 patients (38.6%). Thirty-four patients (72.3%) had positive SLN in one node and 10 patients (22.7%) had positive SLN in two nodes.Mean patient age was 51.6 years; however, there was no association between age and non-SLN metastasis . UnivariConversely, median invasive tumor size was significantly larger in patients who were positive for non-SLN metastasis compared with metastasis negative patients : 7.255; 95% confidence interval (CI): 1.388 - 37.929) and invasive tumor size  were significant factors predicting non-SLN metastasis .SLNB for clinically node-negative breast cancer has become a standard procedure worldwide and it is important to determine pathologically whether the node is negative before surgery. Although ALND has been the standard procedure if patients are positive for SLN metastasis, recent clinical trials suggest that ALND is unnecessary if positive SLN metastasis is detected in one or two nodes. The ACOSOG Z0011 study  showed tPrevious studies have reported that younger age, higher pT stage, or lymphovascular involvement are independent predictors of SLN metastasis , with HRThe main limitations of the present study include the retrospective nature of the analysis and the small number of patients who have had positive SLN metastasis at two or fewer nodes. We believe, however, that these limitations did not greatly affect the results of the study as the differences between non-SLN positive and negative were too marked to have resulted from bias. Our results thus provide useful information on the risk factors for renmant non-SLN metastasis under the condition of SLN metastasis at two or fewer nodes.Finally, both lymphatic involvement and invasive tumor size (cut-off point 28 mm) were independent predictors of non-SLN metastasis in the case of SLN metastasis in two or fewer nodes. Systemic therapies, including hormone therapy, cytotoxic chemotherapy, and/or molecular targeting drugs and/or radiation therapy, remain extremely important, particularly in the treatment of node-positive breast cancer. Therefore, the results of the present study will contribute to decision-making with regard to the addition of ALND, systemic therapy, and/or radiation therapy in the case of SLN metastasis in two or fewer nodes.Non-SLN metastasis was found in more than 30% of patients in the present study, even if SLN metastasis was present in two or fewer nodes, and large tumor size and the presence of lymphatic involvement were significanly associated with non-SLN metastasis.


Herein, we used this dataset to demonstrate 1) why a manually curated, phylogeny-based dataset is more appropriate for benchmarking orthology than other popular practices and 2) how it guides database design and parameterization through careful error quantification. More specifically, we illustrate how function-based tests often fail to identify false assignments, misjudging the true performance of orthology inference methods. We also examined how our dataset can instruct the selection of a “core” species repertoire to improve detection accuracy. We conclude that including more genomes at the proper evolutionary distances can influence the overall quality of orthology detection. The curated gene families, called Reference Orthologous Groups, are publicly available at http://eggnog.embl.de/orthobench2.Accurate orthology prediction is crucial for many applications in the post-genomic era. The lack of broadly accepted benchmark tests precludes a comprehensive analysis of orthology inference. So far, functional annotation between orthologs serves as a performance proxy. However, this violates the fundamental principle of orthology as an evolutionary definition, while it is often not applicable due to limited experimental evidence for most species. Therefore, we  Currently, more than 6,868 complete bacterial genomes are available in public repositories (GOLD Database v4.0) et al.et alcommon proteomes). The latter is particularly important as orthology is always defined relative to the most recent common ancestor (speciation event) of the genomes under consideration, i.e. the same gene can be characterized as an ortholog or paralog to other genes depending on whether more strictly or more broadly defined taxonomic groups are considered However, the identification of genome-wide sets of orthologs for a large number of distantly related organisms is an enormous task due to the complex evolutionary history of gene families shaped by multiple events, such as horizontal gene transfer (HGT), gene duplication, gene loss or gene fusion. Often, an additional level of complexity rises due to protein domain reshuffling hindering the classification of full-length genes So far, the majority of benchmarks are either based on the functional conservation of detected orthologs - e.g. experimentally verified functional annotations, gene expression, enzymatic activity or other genomic features, such as gene order conservation Here, we present the first phylogeny-based benchmark for 238 bacterial species. We constructed a high quality “reference” orthologous groups (RefOGs) through intensive manual curation: inspecting multiple sequence alignments, evaluating the quality of phylogenetic trees and removing false assignment of orthologs. The main objective of our study is not the systematic analysis of orthology databases, as there is a poor overlap between the species repertoires of existing databases, as well as the dataset presented. Instead, we aim to demonstrate the benefits of phylogeny-based benchmark sets: 1) to disconnect orthology validation from gene function, which can have a great impact for functional genomics and 2) to explore the impact of various technical parameters (i.e. species selection) on the robustness of inference.As mentioned above, identification of genome-wide sets of orthologous groups for a large number of distantly related organisms is an arduous task due to the complexity of the genomic events that shape the evolution of gene families. We chose to delineate the evolutionary history of 49 bacterial gene families , resulting in paralogs (duplication-derived homologs) and xenologs (HGT-derived homologs), and can contract through gene loss or pseudogenization Although our benchmark set is restricted to only 49 families, it has the potential to identify xenologs, flag them as false assignments and occasionally cluster them separately. After manual inspection of the phylogenetic trees for the selected families, we identified 110 proteins . Only in 3 out of these 19 cases do the xenologs have different domain content compared to the rest of the members (Table S6 in C.elegans) tend to accumulate more errors. A careful inspection of the false assignments in this study revealed a similar pattern. Therefore, to investigate further this observation, especially since a substantial species collection  is available, we repeated a similar analysis using the bacterial-specific RefOGs and exemplifying how to select a species repertoire that most improves overall performance of an orthology inference method. Using the eggNOG pipeline, we generated two separate orthology datasets. The new datasets contain a smaller number of species; 104 and 152 species respectively, which overlap with the 238 proteomes of gamma-proteobacteria in the public eggNOG database. We refer to these two new datasets ToL-Species . Often, missing orthologs represent such bona-fide groups that fail to cluster together during the graph-mining step to define a gamma-Proteobacteria (deeper ancestor) orthologous groups, instead they cluster as a species/genus-specific cluster  accumulate a large number of false assignments  is a nice example. Although, this has been primarily supported by theory, available empirical studies are controversial. Systematic error quantification based on a “gold-standard” will allow the improvement of orthology inference and the study of functional evolution of proteins.C. elegans or A. baumannii) accumulate the larger number of errors. At the same time, introducing more species improves the overall performance of the database; i.e. benchmarking the two more recent releases of eggNOG database against the animal RefOGs http://eggnog.embl.de/orthobench2.We used the latest version of gamma-proteobacteria-specific orthologous groups from the eggNOG database to demonstrate the advantages of RefOGs against previously used tests to 1) identify errors concealed to functional benchmarks, 2) deal with xenologs  and 3) determine how species selection effects orthology reconstruction. This bacterial-specific benchmark set can be combined with our previously published animal-specific benchmark set. Currently, there are 119 publically available RefOGs (70 in eukaryotic and 49 in prokaryotic species with 19 spanning both domains of life). Similar to our previous findings –based on the animal-specific dataset Finally, we need to stress the limitations of manually curated, phylogeny-based benchmarking tests. Despite all our effort to provide accurate RefOGs, a reference tree still remains a tentative model of the evolutionary history of the studied species. This implies that 1) update and maintenance are essential when novel related sequences/genomes and improved tree-building approaches become available and 2) the dataset is limited to the orthology relationships of the reference species. Regarding the former conclusion, we are committed to supporting this effort; for instance, we recently updated the metazoan dataset and investigated errors that were pointed out from users. Still, we welcome any community effort for this project: sequences derived from incomplete or erroneous gene models or questionable robustness of tree topologies after adding or removing species are challenges for a long-lasting dataset that should be tackled on a collaborative manner. The Quest for Orthologs (QfO) consortium has shown that the orthology community cares for standardization and quality control In conclusion, we wholeheartedly support phylogeny-based benchmarking sets for orthology. As we demonstrated herein, they can provide guidance on inferring robust orthology predictions and disassociating function from the technical validation of orthology. Nevertheless, they have limitations that can be overcome through the collective efforts of this community.Starting with orthologous groups at the level of last universal ancestor (COGs) in the eggNOG database  Gene order conservation analysis: For each gene in the RefOGs, we inspected 10 adjacent genes . Although this number is significantly larger from the average operon size (3 genes) Enzymatic activity conservation analysis: For each protein, its closest BLAST hit in version 66.0 of the KEGG database Xylella fastidiosa is a ToL species and belongs both at the ToL-Species and ToL-Genus dataset, but Xylella oryzae exists only in the ToL-Genus dataset). Both the ToL-Species and ToL-Genus proteome are subsets of the set of 238 eggNOG gamma-proteobacterial species.To investigate the impact of species selection, we used the eggNOG pipeline to generate two new datasets (available upon request). The new datasets, referred to as ToL-Species and ToL-Genus, are gamma-proteobacterial orthologous groups generated using only a subset of species included in version 3 of the eggNOG database. The Ref.Species dataset provides predictions for 104 gamma-proteobacterial proteomes found in the Tree of Life (ToL) resource File S1Supporting figures.Figure S1, Gene order as discriminator of false-, missing- and true-assignments. Figure S2, Enzymatic activity as discriminator of false-, missing- and true-assignments. Figure S3, Protein domain architecture as discriminator of false-, missing- and true-assignments. Figure S4, Error distribution for eggNOG v3 and ToL species-specific orthologous groups. Figure S5, Species-specific distribution of errors for the three different datasets of orthologous groups .(DOC)Click here for additional data file.Data S1Supporting tables.Table S1, Information about the eukaryotic- and prokaryotic- Reference Orthologous Groups. Table S2, Benchmarking eggNOG v3 using OrthoBench. Table S3, Benchmarking eggNOG v3 using gene order. Table S4, Benchmarking eggNOG v3 using enzymatic activity. Table S5, Benchmarking eggNOG v3 using protein domain architecture. Table S6, Table of the Pfam domain composition for xenologs and orthologs (true assignments). Table S7, Benchmarking eggNOG v3, ToL Species, and ToL Genus orthologous groups using the reference orthologs for the 104 overlapping species. Table S8, Table of the error distribution of each species at the gene- and group-level.(XLS)Click here for additional data file.