Little is known about the natural history of infection with this organism and its ability to be transmitted in the community.Methods. The bacterial load was estimated in sequential sputum samples from 9 patients by quantitative polymerase chain reaction. The genomes of all available isolates, originating from patients in the United Kingdom, France, and Tunisia, were sequenced along with the type strain. Genomic data were assembled and annotated, and a high-resolution phylogenetic tree was constructed.Results. By using high-resolution whole-genome sequencing (WGS) data, we show that patients can be chronically infected with M. amphoriforme manifesting as a relapsing-remitting bacterial load, interspersed by periods when the organism is undetectable. Importantly, we demonstrate transmission of strains within a clinical environment. Antibiotic resistance mutations accumulate in isolates taken from patients who received multiple courses of antibiotics.Conclusions. Mycoplasma amphoriforme isolates form a closely related species responsible for a chronic relapsing and remitting infection in PAD patients in the United Kingdom and from immunocompetent patients in other countries. We provide strong evidence of transmission between patients attending the same clinic, suggesting that screening and isolation may be necessary for susceptible patients. This work demonstrates the critical role that WGS can play in rapidly unraveling the biology of a novel pathogen. Mycoplasma amphoriforme was first isolated from a patient with X-linked agammaglobulinemia with chronic bronchitis in 1999. The patient was expectorating a large volume of sputum that tested negative for all other recognized lower respiratory tract pathogens ) [Supplementary Table 1) [The Ethics Committee of the Royal Free London National Health Service Foundation Trust (RFL) approved these studies. Sputum samples were collected from a total of 88 adult patients with PAD attending the PAD Clinic at the RFL and tested for MAM using  [qPCR]) . A totalTable 1) . The cliExtraction of DNA from sputum samples was performed using a Chelex-based method as previously described . DNA forudg was performed retrospectively on DNA extracts from PAD patient samples as described previously [The MAM-specific conventional 16S PCR was performed prospectively, and the real-time qPCR targeting eviously .The genome of the reference MAM strain A39 was Sanger sequenced to a depth of 8 times coverage using cloning vector p0TWI2 with a selection of insert sizes  using dye terminator chemistry on ABI3700 automated sequencers . Repetitive regions of the genome were spanned and the assembly manually was finished using long-range PCR and pair read information. The genome was annotated as described previously, using Artemis  and the http://www.sanger.ac.uk/resources/software/smalt/). Single-nucleotide polymorphisms (SNPs) were identified using SamTools Mpileup [WGS was performed on single isolates on a MiSeq Instrument . A 300-cycle MiSeq Reagent Kit version 2 was used to generate 75-bp paired-end reads. The reads obtained for each isolate were mapped against the MAM reference strain A39 with SMALT (Available at:  Mpileup  and bcft Mpileup .The raw sequence data are available under the accession number ERP000340. The sequence and annotation for MAM A39 have been deposited with EMBL (accession number HG937516).Regions with high-SNP density or SNPs within repetitive regions were excluded from the phylogenetic analysis as described previously . MaximumP value of at least .05, depth of coverage within a normal range (± 50% of the average), and a distance of at least 200 bp from another variant. As depth of coverage varies across the genome, we corrected each count of a minority variant by dividing it by the depth. For example, for a particular position with a depth of coverage of 100 reads, we recorded 0.01. We summed these values to produce a minority variant score for each sample, with higher scores indicating higher diversity in the sample.Minority variants were extracted from the mapping data using stringent filters to distinguish true variants from sequencing or mapping errors. A variant was only counted if confirmed by at least 4 reads with at least 2 reads on each strand, and a base and mapping quality of 50 and 30, respectively. The variants required strand bias Haemophilus influenzae, 6 Streptococcus pneumoniae, and 3 Moraxella catarrhalis  were found to be positive for MAM by culture, 85 of 92 (92.4%) were positive by 16S PCR, and 81 of 89 (91%) were positive by qPCR. No other recognized LRTI bacterial pathogen was identified in 51 of the 64 samples for which routine sputum culture results were available. Of those samples positive for MAM where another LRTI pathogen was cultured, 3 samples contained Supplementary Figure 1). We use patient 1, from whom the original isolate and now the type strain, A39, was isolated [M. catarrhalis and H. influenzae . The overall level of sequence-based variation is low, with distances between clades ranging from 159 to 254 SNPs, and a maximum of 76 SNPs (minimum = 4) separating any pair of isolates within 1 clade. It is clear that the UK isolates from the RFL are more similar to each other than the French out-group strains. Isolates from an individual are more closely related to each other than to those from other patients . Where we have multiple longitudinal samples taken from a single patient , the phylogeny does not appear to be entirely consistent with the dates of sample collection. Some isolates that appear more basal in the tree were collected more recently , Across the strain set, we detected 636 minority variants, and in patient 1 there was a trend toward increasing numbers of numbers of minority variants over time .The phylogenetic tree Figure  shows thA). The collection dates of the specimens indicate that patient 1 and patient 8 attended the outpatient clinic on the same day on 2 occasions. The 2003 isolates from patient 1 share ≥37 SNPs, with the latter isolate from patient 8; the highest number of shared SNPs between the isolates of these 2 patients is found in early 2004, suggesting transmission around this date .There were several different patterns of infection Figure : patient3tetM). A total of 64 SNPs were identified in all isolates in gyrA, 11 of which were nonsynonymous, but none were found in the quinolone-resistance determining region (QRDR). Among 43 SNPs detected in gyrB, there were 3 nonsynonymous amino acid changes at positions V17I, P389Q, and V620A. A total of 23 nonsynonymous SNPs were identified in the parC gene, 1 at position 89 (position 80 in Escherichia coli numbering) found within the QRDR. The 3 isolates from France and Tunisia had a serine residue at the position 89 (associated with a susceptible phenotype), whereas all UK isolates, including the reference isolate A39, had either F or Y substitutions, implying that the UK isolates were quinolone resistant. A total of 22 mutations were detected in the 23S rRNA gene, 1 of which was located within a region associated previously with macrolide resistance. Two strains from patient 2 (both isolated in 2001) both carry an A2059G substitution in the 2 latest isolates of this series. In vitro susceptibility testing of this isolate showed that this strain had a minimum inhibitory concentration of 0.2 µg/L and 1.5 µg/L for doxycycline and ciprofloxacin, respectively.From sequence analysis, there were no whole-gene acquisitions within the course of any of the UK PAD patient infections, providing no evidence of resistance acquisition by horizontal gene transfer , indicating a relapsing-remitting course in patients with PAD, lasting at least 1626 days . WGS data demonstrate that relapse was with the same strain, suggesting that intervening qPCR negatives were false negatives or below the limit of qPCR detection.The genome of MAM has a similar G + C content to other Relapsing respiratory symptoms are common in PAD patients and, in the absence of other recognized respiratory pathogens and the continued presence of symptoms, it suggests that MAM is causing bronchial inflammation. A similar relapsing course is seen in immunocompetent patients with chronic obstructive pulmonary disease where bacterial load is higher during exacerbation than during stable state . ControlS. aureus in a neonatal care unit, the strain carried by a staff member differed by up to 27 SNPs when different colonies from a sample were sequenced independently [A trend for an increase in minority variants in a chronically infected individual who failed to achieve statistical significance may suggest that organisms exist as an increasingly heterogeneous mixture in chronic infection. This result should be treated with caution as it is unknown what affect whole-genome amplification may have on the error rate of the sequencing reads. In addition, we do not know how many variants may have been generated or selected for during in vitro growth of these samples. The limited number of samples and sampling depth is insufficient to capture the full diversity of isolates infecting a single person at a particular time-point. However, this observation is consistent with studies from chronic infections  and explendently , and is Mycobacterium tuberculosis in human immunodeficiency virus–positive patients and Burkholderia cepacia in patients with cystic fibrosis [M. amphoriforme strains were isolated at a single hospital, it was possible that this represented an unrecognized nosocomial outbreak. WGS has enabled us to answer this question unequivocally. Almost all of the patients investigated in this collection had a genetically distinct strain in their lungs, which excludes the possibility of this being a point source outbreak. The interaction between patients 1 and 8 is significant, as these patients were in the clinic on the same day on at least 2 occasions in 2002 and 2003. The strain isolated from patient 8 in 2002 is distinct from the 2 strains isolated in 2004 and 2005, and the latter strains are sufficiently similar to the 2004 strain from patient 1 to indicate that transmission had occurred. The map of SNP accumulation for patient 1  suggests that transmission was associated with clinic visits that occurred in early 2004.Patients with anatomical or genetic deficits or immunocompromise are susceptible to cross-infection in the hospital environment and respiratory pathogens in a clinic setting such as fibrosis –31. As mIt is notable that all but 2 of the isolates from UK PAD patients and 2 from France and Tunisia, although distinct, are closely related. The other isolates are more diverse but part of the same species. More highly divergent strains may form distinct lineages or may ultimately be described as distinct taxa, but deeper understanding of the population genetics of this organism will only be achieved when more strains are sequenced.Supplementary Figure 1) and received immunoglobulin regularly. Changing bacterial load may be related to treatment, but the incomplete record makes the relationship between treatment and bacterial load uncertain. We detected resistance mutations in the QRDR of parC of all the UK isolates, indicating that these strains are likely to be resistant. This provides genetic evidence for the previously reported clinical finding in patient 1, who was treated with courses of antibiotics, including quinolones, without improvement and who only responded to the pleuromutilin agent valnemulin (Econor) [E. coli numbering) [M. hominis [PAD patients received multiple antibiotic treatment courses  . The submbering)  is known hominis . Mutatio hominis .M. amphoriforme infects immunocompetent and immunocompromised patients [In summary, we have used genomics to differentiate between persistence and reinfection, providing evidence that patients , 18, is Supplementary materials are available at Clinical Infectious Diseases online . Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.


Few studies have compared the prognosis and liver-related mortality in patients with NAFLD  and AFLD . We aimed to investigate the etiology and liver-related mortality of patients with liver biopsy verified fatty liver disease in a population based setting.In this retrospective study, all patients who underwent a liver biopsy 1984–2009 at the National University Hospital of Iceland were identified through a computerized pathological database with the code for fatty liver. Only patients with NAFLD and AFLD were included and medical records reviewed. The patients were linked to the Hospital Discharge Register, the Causes of Death Registry and Centre for Addiction Medicine.A total of 151 had NAFLD and 94 AFLD with median survival of 24 years and 20 years, respectively (p = NS). A total of 10/151 (7%) patients developed cirrhosis in the NAFLD group and 19/94 (20%) in AFLD group (p = 0.03). The most common cause of death in the NAFLD group was cardiovascular disease (48%). Liver disease was the most common cause of death in the AFLD group (36%), whereas liver-related death occurred in 7% of the NAFLD group. The mean liver-related death rate among the general population during the study period was 0.1% of all deaths. There was a significantly worse survival for patients in the AFLD group compared to the NAFLD group after adjusting for gender, calendar year of diagnosis and age at diagnosis . The survival for patients with moderate to severe fibrosis was significantly worse than for patients with mild fibrosis after adjusting for gender, calendar year of diagnosis and age at diagnosis .Patients with fatty liver disease showed a markedly higher risk of developing liver-related death compared to the general population. The AFLD group had higher liver-related mortality and had a worse survival than the NAFLD group. Patients with more severe fibrosis at baseline showed a worse survival than patients with none or mild fibrosis at baseline. Fatty liver disease is clinically categorized into two main groups, alcoholic fatty liver disease (AFLD) and non-alcoholic fatty liver disease (NAFLD). NAFLD is becoming one of the most common liver diseases worldwide  with a p. 2) Compare the prognosis and liver-related mortality in patients with NAFLD and AFLD, and compare these patients to the liver-related mortality in the general population. Our hypothesis was that these patients suffer from increased liver-related morbidity compared to the general population.The aims of this study were 1) to examine the natural history and outcome of patients with fatty liver disease with non-alcoholic and alcoholic etiologiesIn this retrospective study, a search was undertaken in a computerized diagnoses database from 1984 (when the pathology registry commenced their electronic registration) to 2009, in the Department of Pathology at the National University Hospital (NUH) of Iceland and identified all liver biopsies analysed and registrated in the SNOMED coding-system, T-56000 and the M-50080 as having fatty change. The SNOMED (Systemized Nomenclature of Medicine) is a coding system used in the pathology laboratories in Iceland to specify: Procedure, Topography, Morphology, Disease and Etiology. This is a very valuable coding system for retrieving data and pathology reports from past years, like biopsies of the liver (T-56…) showing fatty change (M-50080).The catchment area of the NUH covers >95% of the population in Iceland.All medical records from these patients were examined with respect to the following exclusion criteria: 1) presence of acute or chronic liver disease: PBC, autoimmune hepatitis, alfa-1-antitrypsin deficiency, hemochromatosis and viral hepatitis. 2) jejunoileal bypass operation. 3) use of drugs known to be associated with fatty liver disease such as methotrexate, amiodarone, tamoxifen and high doses of corticosteroids. 4) malignancy at the time of index liver biopsy. 5) age under eighteen years at the time of index liver biopsy. 6) gallstone surgery at the time of index liver biopsy. The patients with gallstone surgery at the time of index liver biopsy were excluded to better represent the patients who would undergo a liver biopsy in clinical practice and not just the incidental finding of fatty liver during an operation.Indications for the index biopsy in the cohort were elevated liver tests, mainly serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) and/or hepatomegaly or suspected alcoholic liver disease. A total of 420 patients met the inclusion criteria and were divided into two groups, non-alcoholic and alcoholic group respectively Figure .Figure 1The following information was obtained from the medical records and recorded at baseline: gender, age at diagnosis, height, weight and body mass index (BMI), history of diabetes mellitus, hyperlipidemia, hypertension, cardio- and cerebrovascular disease, liver disease and malignancy. Data on drug and alcohol intake was noted in the medical records. Patient with a reported alcohol problem or abuse or an alcohol-related diagnosis before or at the time of liver index biopsy were considered to have alcoholic fatty liver disease. The diagnosis of cirrhosis was accepted in patients who had a discharge diagnosis, a death certificate diagnosis and/or histological confirmation in the follow-up period indicating cirrhosis. Laboratory data included at baseline were: AST, ALT, bilirubin, albumin, alkaline phosphatase (ALP), prothrombin time (PT), glucose, platelets count (PLT), serum cholesterol, serum triglycerides (TG) and mean corpuscular volume (MCV). The last follow-up date was the 30th of November 2011.All members of the cohort were linked through their unique personal identification number to the centralized Hospital Discharge Register at the Directorate of Health and the Registry of Causes of Death (RCD) at Statistics Iceland. Information was obtained from National Centre of Addiction Medicine in Iceland on whether or not the patients had undergone addiction therapy before or after the time of the index liver biopsy. This information was obtained from the addiction centre without knowledge of the liver biopsy results.Patients were excluded if they were lost to follow-up in the registries. 8 patients were lost to follow-up.The study was approved by the National Bioethics Committee (10-029-V1-S1) and the Icelandic Data Protection Authority (S4766/2010).The index liver biopsies were formalin fixed and treated routinely in the pathology laboratory. They were paraffin embedded and cut in 4–5 micrometer thick sections. The sections were stained with hematoxylin and eosin, periodic acid Schiff reagent (PAS) with diastase and for reticulin. In addition a connective tissue stain, Weigert van Gieson or most commonly Masson Trichrome stain was performed. All histological slides were reviewed by experienced pathologists without knowledge of the clinical or biochemical data of the patients. Three pathologists  divided the slides between them and reviewed all of the samples over a period of 2 years. The assessment was blinded to the clinical details. Second review to examine inter-observer variability was not undertaken. Morphological findings were recorded in a semi-quantitative manner regarding steatosis and fibrosis, which was initially proposed by Brunt et al. in 1999 and developed further by Kleiner et al. , 12. GraU-test was used to test for differences between groups. Dichotomous variables were compared by using the Chi-square (X2) or Fischer’s exact test. A p-value of less than 0.05 was considered statistically significant. The survival curves were estimated by the Kaplan-Meier estimator, using years as the time-scale and taking delayed entry into account. Differences between groups were investigated by Wald test in Cox proportional hazards model, with age as the time-scale, adjusting for gender, calendar year of diagnosis and age of diagnosis. The primary end-point was death from all causes.Statistical testing was performed using R-software. The results were presented as medians, interquartile range and range and as number (%). The Mann–Whitney Overall 2262 liver biopsies were performed at the University Hospital of Iceland during the study period. A total of 420 patients with at least one index biopsy were identified by the computerized search in the pathology database as having fatty liver on biopsy. Those who were misclassified and did not have fatty liver on review were excluded Figure . A totalMedical records from these patients were traced and the biopsies reviewed by experienced pathologists. A total of 245 patients constituted the study group, 136 (56%) women and 109 men (44%). Women were in the majority in the NAFLD group, 105/151 (70%) compared to 46/151 (30%) men (p < 0.001). The proportion of men was higher in the AFLD group, that is 67% (63/94) whereas the proportion of women was 31/94 (33%).Clinical and biochemical data at the time of index liver biopsy in the 245 patients of the two study groups Table . Informa). At the end of follow-up, ALT, AST and MCV were the only biochemical markers still significantly higher in the AFLD group compared to the NAFLD group  patients in the NAFLD group and 19 (20%) patients in the AFLD group (p = 0.003).Among patients developing cirrhosis in the follow-up period the histological diagnosis at baseline was as follow: one patient had no fibrosis (in the NAFLD group), three patients had stage 1A fibrosis , one patient had portal fibrosis (in the NAFLD group) and six patients bridging fibrosis (four in the AFLD group and two in the NAFLD group).Patients developing liver cirrhosis and liver related complications are demonstrated in Table The median survival was 24.2 (range 0.2-26.1) years in the NAFLD group and 19.5 (range 0.2-24.2) years in the AFLD group (p = 0.0007). Median follow-up time for the non-alcoholic group was 9.9 years (range 0.2-26 years) and 9.2 years (0.2-25 years) for the alcoholic group. There was no significant difference in overall survival between the two study groups and no significant difference between genders (data not shown). Patients in the AFLD group diagnosed with cirrhosis had a higher death rate compared to the NAFLD group; 10 patients (40%) in the AFLD group compared with 7 patients (17%) in the NAFLD group (NS). Using Cox analysis the survival was significantly worse for patients in the AFLD group compared to the NAFLD group after adjusting for gender, calendar year of diagnosis and age at diagnosis   Figure .Figure 2A total of 67 patients died during the follow-up period; 41 women (61%) and 26 men (39%). Of these 12 (18%) were liver related in both the NAFLD and AFLD group Table . The mosThe most common cause of death in the NAFLD group was of cardiovascular disease 20/42 (48%), followed by malignancy 6/42 (14%) and other chronic medical conditions 4/42 (9.5%). A total of 12 patients (18%) died of malignancy. In the AFLD group, 4 died of breast cancer and one each of renal cancer, malignant brain tumor, colon cancer, nasopharyngeal cancer and pancreas cancer. In the NAFLD group, two died of breast cancer and one each of prostate cancer, multiple myeloma, endometrial cancer and one of small cell lung cancer.Few studies have determined the natural history of biopsy-proven fatty liver disease and compared the long-term prognosis of these two major groups of fatty liver disease due to alcoholic and non-alcoholic fatty liver disease.. The fact that all the patients underwent a liver biopsy is also a weakness, especially when evaluating disease outcome. Previous studies have shown that NAFLD patients diagnosed with liver biopsy have a worse prognosis compared with patients diagnosed with ultrasonography. Therefore, studying patients recruited from the pathology registry involves a selection bias. It must be stressed that the indications for the liver biopsy was not always clear, although most cases were because of elevated liver function tests and/or hepatomegaly or suspected alcoholic liver disease, and this in turn can give a selection bias. Also the indication for biopsy in NAFLD and AFLD may differ between conditions and between practitioners and might explain some of the differences in disease outcome.Our study has several methodological strengths. First, all patients had biopsy proven fatty liver disease and histology was re-evaluated based on validated scores. We believe we have included the vast majority of patients with biopsy-proven fatty liver disease in the whole population of Iceland during this period as at least 95% of the population lives in the catchment area of the National University HospitalThe main limitation of the study was its retrospective design and data was not systematically registrered and was therefore sometimes missing or unavailable. In addition the search for the code M-50080 (fatty liver) is limiting in itself as the more serious steatosis with fibrosis and even cirrhosis might be coded as something else than just fatty liver. Since this was a retrospective study on liver biopsies it is not possible to standardize the size of the needle biopsies. Therefore the size is very variable and the range can be considerable, but should be similar to the standards observed in pathology departments in general. Samples less than 2 mm in diameter would have been excluded from the study, but no such samples came into the study.The slides used for pathological estimation were the original slides and recuts or restaining of slides was not done except for a few exceptional cases where the original slides were unavailable or when a Masson-Trichrome stain had not been performed originally. Occationally the colours of the slides had faided somewhat. This however we do not anticipate having significant effect on the results, especially since ballooning degeneration increases cell size and this is not greatly affected by fading colours.Another limitation is the small number of hard endpoints with only four patients in the NAFLD group who developed cirrhosis over the follow-up period. There is also a potential uncertainty in the non-histological diagnosis of cirrhosis which must be taken into consideration when reviewing the results.The information on ASH should be interpreted with caution as NAFLD activity score has to our knowledge not yet been validated in AFLD, but we chose to use it for comparison as the histolopathological development is similar in the two conditions and there is no difference morphologically between NAFLD and AFLD.One of the main findings in this study was that patients with fatty liver disease showed a markedly higher risk of developing liver-related death compared to the general population. Although significantly higher in the AFLD group liver-related death in the NAFLD group was 7% of all deaths. These findings are in contrast with liver-related death rate in Iceland  which waOur results show that patients with NAFLD had a rather benign course in terms of liver-related morbidity and mortality. Only 7% developed cirrhosis after a mean of 13 years of follow-up which is similiar to what previous studies on the prognosis of NAFLD have shown –17. A hiIn the AFLD group the most common cause of death was liver-related (35%).Other studies have shown that obesity in both NAFLD and AFLD predispose to the development of fatty liver and chronic liver disease , 20. In In the current study women were in the majority in the NAFLD group but the high proportion of women with fatty liver compared to men may reflect a higher disease burden in women. A recent study from the US also found a higher proportion of women in the NAFLD group  which isProgression of NAFLD has been found to be slow and seems to depend a great deal on the initial fibrosis stage , 14. PatIn the total study cohort patients with more severe fibrosis at baseline showed a worse overall survival than patients with none or mild fibrosis at baseline. Based on this we were able to show an association in the total study cohort between the stage of fibrosis and the prognosis. However more NAFLD than AFLD patients had mild or no fibrosis at baseline. This is in agreement with results from a recent study showing that advanced fibrosis in the index liver biopsy was the most important predictor of the prognosis in these patients . A recenA Swedish cohort study of patients with biopsy-proven NAFLD and elevated liver tests showed that they had a similar survival compared to the Swedish population . InteresA study from Minnesota, identified 420 patients with NAFLD by imaging or liver biopsy found liver-related complications to be the third most common cause of death among NAFLD patients . This isIn the current study, no patient in the NAFLD group died of hepatocellular carcinoma and only one patient in the AFLD group. The absence of HCC among the NAFLD group differs from previous cohort studies showing that 3/129 (2%)  and 2/42In agreement with many previous studies a markedly higher proportion of our women in the NAFLD group died of cardiovascular disease compared to women in the AFLD group, 17 patients vs. one patient respectively. A previous study  showed tThe most common cause of death in the NAFLD group was from cardiovascular disease, followed by malignancy which is in agreement with findings of other cohort studies . One study found 34% increased risk of cardiovascular mortality among NAFLD patients compared to the general Minnesota population over a 7.6 year follow-up [. Patients in the AFLD group showed a significantly worse survival compared to patients in the NAFLD group. Patients with more severe fibrosis at baseline showed a worse survival than patients with none or mild fibrosis at baseline. Patients with fatty liver disease showed a markedly higher risk of developing liver-related death compared to the general population.In conclusion a higher proportion of patients with AFLD developed liver cirrhosis and had liver-related death compared to patients with NAFLD in this population based setting and had also more severe histological changes in the liver biopsy at baseline


Leptospirosis is a zoonosis caused by highly motile, helically shaped bacteria that penetrate the skin and mucous membranes through lesions or abrasions, and rapidly disseminate throughout the body. Although the intraperitoneal route of infection is widely used to experimentally inoculate hamsters, this challenge route does not represent a natural route of infection.Leptospira interrogans serovar Copenhageni, strain Fiocruz L1-130. Histopathologic changes in and around the kidney, including glomerular and tubular damage and interstitial inflammatory changes, began on day 5, and preceded deterioration in renal function as measured by serum creatinine. Weight loss, hemoconcentration, increased absolute neutrophil counts (ANC) in the blood and hepatic dysfunction were first noted on day 6. Vascular endothelial growth factor, a serum marker of sepsis severity, became elevated during the later stages of infection. The burden of infection, as measured by quantitative PCR, was highest in the kidney and peaked on day 5 after intradermal challenge and on day 6 after subcutaneous challenge. Compared to subcutaneous challenge, intradermal challenge resulted in a lower burden of infection in both the kidney and liver on day 6, lower ANC and less weight loss on day 7.Here we describe the kinetics of disease and infection in hamster model of leptospirosis after subcutaneous and intradermal inoculation of L. interrogans serovar Copenhageni strain Fiocruz L1-130 at an experimental dose of 2×106 leptospires. These results provide new information regarding infection kinetics in the hamster model of leptospirosis.The intradermal and subcutaneous challenge routes result in significant differences in the kinetics of dissemination and disease after challenge with  Leptospira interrogans, frequently result in a life-threatening illness characterized by liver and kidney failure. In the hamster model of leptospirosis, signs of hepatic and renal dysfunction developed on days 6 and 7, respectively, after intradermal and subcutaneous inoculation of L. interrogans. Renal dysfunction was preceded by the development of inflammatory changes and the appearance of large numbers of leptospires in the kidney on day 5. On day 6, animals began to lose weight, became dehydrated, and had elevated numbers of neutrophils circulating in their bloodstream. Importantly, animals inoculated intradermally had lower numbers of bacteria in their liver and kidneys on day 6 than animals inoculated subcutaneously and lower weight loss and circulating neutrophil levels on day 7. These studies show that the hamster model of leptospirosis is similar to human infection and indicate that the route of infection has significant effects on the course of the illness.Leptospirosis is the most widespread bacterial infection transmitted from animals to man. Humans are exposed to infection when host animals that harbor the bacteria in their kidneys shed them in their urine. Human infections, caused by the bacterium  Leptospirosis is a zoonosis with worldwide distribution caused by spirochetes that are spread by reservoir animals to humans and other accidental hosts. Up to 500,000 severe human infections are documented annually in tropical areas with an incidence of >10 cases per 100,000 population Acquisition of infection by a new host can occur through several possible routes, with cutaneous lesions as a major portal of entry IP inoculation is the most common method employed in animal models of leptospirosis including hamsters and guinea pigs. Although this challenge route has certain technical advantages, it is not a natural route of infection as it bypasses relevant mucosal and cutaneous defense mechanisms. Other routes of inoculation such as epicutaneous, conjunctival, subcutaneous, intradermal, oral, intracardiac and intracranial have been reported Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 was cultivated in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium 6 leptospires or EMJH alone on day 0. For intradermal inoculation, fur was removed with a pair of electric clippers to better visualize the injection site , the National Institutes of Health's Plan for Use of Animals in Research , U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training, Public Health Service Policy on Humane Care and Use of Laboratory Animals, the United States Department of Agriculture's Animal Welfare Act & Regulations, and Veterans Health Administration Handbook 1200.7.Female Syrian hamsters, 5 to 6 weeks of age , were inoculated either subcutaneously (0.5 mL) or intradermally (0.05 mL) using a 25 gauge needle with 2×10ion site . From 3 5′-CGCTTGTGGTGCTTTCGGTG-3′, and LipL32-r, 5′- GCGCTTGTCCTGGCTTTACG-3′. The resulting amplicon was 152 bp. The PCR protocol consisted of an initial incubation step at 95°C for 12.5 min followed by 40 cycles of amplification . Standard curves were generated ranging from 10 up to 1.6×106 copies of Leptospira (20-fold dilutions). DNA (100 ng) from 4 uninfected hamsters were used as negative controls.Tissue DNA was extracted using either the FastDNA SPIN Kit , accordingly to the manufacturer instructions or with the DNeasy Blood and Tissue kit , with modifications as previously described 9 heat-inactivated leptospires/mL diluted in PBS, pH 7.2 , by overnight incubation at 4°C as described previously 2SO4 and plates were immediately read at 450 nm in a Bio-Rad 550 Microplate Reader.96-well ELISA microtiter plates  were coated with 1×10One-way analysis of variance (ANOVA) with post-hoc Tukey and Šídák tests was used to test for differences between multiple (≥3) groups.6 leptospires via either the subcutaneous (SQ) or intradermal (ID) routes. Hamster body weight was included in the daily clinical examination. Uninfected hamsters gained an average of 3.6% of body weight per day during the course of the study. As shown in We compared uninfected hamsters with animals challenged with 2×10At the same time that hamsters began losing weight, total serum protein became elevated . The obs3 copies/µg of tissue DNA, after which the burden decreased. The leptospiral burden in the kidney after SQ challenge was similar to that after ID challenge through day 5. However, the leptospiral burden in the kidney continued to increase on day 6 after SQ challenge, peaking at a level six-fold higher (5.0×104 copies/µg of tissue DNA), whereas it decreased on day 6 after ID challenge and was significantly lower than after SQ challenge (P<0.01). The leptospiral burden in the liver had a similar pattern to that in the kidney, reaching a peak on days 5 and 6 after ID and SQ challenge, respectively. However, in both challenge models, the peak leptospiral burden in the liver was two logs lower than it was in the kidney.The leptospiral burden in kidneys and liver after SQ and ID challenge as measured by qPCR, in terms of DNA copies per microgram of tissue DNA, is presented in The histopathology findings were similar in the animals inoculated subcutaneously and intradermally. Kidney histology appeared normal until day 4 after challenge when mild focal tubular damage was noted in some animals. This early renal inflammation was mirrored by inflammatory changes in the perinephric  and periAnimals challenged by the SQ route did not show an increase in antibody level during the 8 days of the experiment. As shown in Leptospira strains The hamster is one of most widely used animal models of acute leptospirosis infection because of its reproducibility and the susceptibility of hamsters to a wide variety of pathogenic The peak in the burden of infection on days 5 and 6, followed closely by or concurrent with the onset of weight loss, elevation of inflammatory markers, and changes in blood analytical chemistry results indicative of renal and hepatic dysfunction, implies a causal relationship. Elevation in the absolute neutrophil count (ANC) is a typical response to systemic infection that is also observed in human leptospirosis Levels of vascular endothelial growth factor (VEGF) were examined as an additional marker of inflammation. Elevated VEGF levels, as documented in our study, have been found to be an important determinant of morbidity and mortality in the mouse model of sepsis et al. The qPCR approach allowed us to document striking 8- and 13-fold increases in leptospiral burden in the kidney between days 3 and 4 after ID challenge and between days 4 and 5 after SQ challenge, respectively. As shown in A limitation of this study is that leptospiremia levels were not measured in the blood. However, bacterial burdens were measured in the spleen, and given its role in blood filtration, the spleen can be considered as a surrogate for the blood. Another limitation is that some of the bacterial burden found in organs was due to residual blood, given that animals were not perfused with saline prior to organ harvesting. Nevertheless, bacterial burdens were higher in the kidney than in either the spleen or liver by more than an order of magnitude. These data indicate that leptospires selectively target the kidneys during infection and substantiate the contribution of the higher bacterial burdens to the pathogenesis of kidney dysfunction. The predilection for kidney infection was observed at early time points. Organisms were detected in the kidney by qPCR at levels significantly above background by day 1 and 3 after infection by the SQ and ID routes, respectively. Homing to the kidney by leptospires may be mediated either by leptospiral factors such as kidney-specific adhesins or by production of anti-leptospiral antibodies. Colonization of the lumen of proximal renal tubules has been documented by immunohistochemistry as early as 4 days after intraperitoneal challenge A novel aspect of our study was the comparison of the SQ and ID challenge routes with the finding of significant differences in the kinetics of infection. The differences in leptospiral tissue burden were most pronounced on day 6, when we observed 31- and 36-fold higher levels of leptospiral DNA in kidney and liver tissues respectively, after SQ compared to ID inoculation. The lower leptospiral burden in kidney and liver was reflected in less weight loss on day 7 and better survival in ID challenged animals. The ID route is of interest because it is a biologically relevant route of infection in individuals with abrasions and cutaneous exposure. An ID challenge model incorporates interactions between leptospires and defense mechanisms of the skin, such as the dendritic cells, that may function as an early warning system for the immune response. Prompt immune activation during the earliest stages of the transmission process is to the advantage of the host if it results in early clearance of organisms and a lower leptospiral burden. The intradermal route may also be of interest as a more reliable way to evaluate transmission-blocking vaccines using surface antigens that are either expressed in the environmental phase or early in infection.Figure S1Intradermal inoculation of hamsters. Hamsters were anesthetized by isoflurane inhalation, shaved on the left flank of their abdomen then infected intradermally with 100 µl of leptospiral culture. ID injection induces a bleb at the site of inoculation.(TIF)Click here for additional data file.Table S1Data compilation of serum chemistry values, blood counts, and vascular endothelial growth factor levels for control animals and animals inoculated either subcutaneously (SQ) or intradermally (ID) on each day of the study.(PDF)Click here for additional data file.


VEGF-A, for the treatment of cardiovascular disease.This review discusses recent developments in the use of non-coding RNAs (ncRNAs) for the regulation of therapeutically relevant genes, with special focus on applications for the treatment of cardiovascular diseases. The interest in using ncRNAs as therapeutics has steadily increased since the discovery of RNA interference. During the last decade it has become evident that these RNAs, delivered either as oligos or expressed as small hairpin RNAs (shRNAs) from vectors, can either upregulate  or downregulate  gene expression, typically inducing epigenetic changes in their target sites in the chromatin. Also, the important role of naturally occurring long non-coding RNAs (lncRNAs) has been recently discovered and will likely provide new insights into cardiovascular pathology and provide new treatment strategies based on the manipulation of their expression. In this review, we discuss the possibility of using ncRNAs for activating or silencing therapeutically relevant genes, such as  Traditional pharmacological treatments for CVD have limited efficiency and an unmet clinical need exists for novel treatment strategies, such as gene therapy. The recently published ENCODE project1 has revealed the existence of vast amount of non-coding RNAs (ncRNAs) in human cells, and the role of ncRNAs in both basic cellular processes and diseases has in recent years gained much interest. Understanding the complexity of various RNA species, their interactions and intra- and extracellular trafficking will provide a better understanding of the biological basis of various diseases. Furthermore, manipulation of these ncRNAs represents a promising new gene therapy strategy for the treatment of various diseases, including CVD.2According to the reports by WHO, cardiovascular diseases (CVDs) are responsible for the majority of the deaths worldwide and their prevalence is increasing .4 Various reports have related increased levels of active chromatin markers, such as H3K4me2/me3, and increased RNA polymerase II occupancy to TGA and similarly increased levels of repressive chromatin markers, such as HK9me2 and lysine 27 trimethylation (H3K27me3), and decreased polymerase occupancy to TGS.9 Involvement of other proteins has also been shown in these processes, for example, the Argonaute proteins (both AGO1 and AGO2) have been shown in many reports of TGS10 and TGA,14 and in TGA the recruitment of HP1γ  to the promoter is decreased.15Many of the small ncRNAs have been considered to function at the post-transcriptional level in the cytoplasm, mediating, for example, mRNA degradation and inhibition of mRNA translation. The RISC protein complex, guided by the small RNA (eg miRNA or siRNA) to the target mRNA sequence, includes the effector proteins such as Argonaute protein family member AGO2 that possesses endonuclease activity. Recently many of these components have been observed also in the nucleus16 or they might bind to non-coding sense or antisense transcripts located at the target site forming RNA-RNA hybrids.17 It is also possible that the antisense transcripts could serve as a scaffold where the small RNA guided complex recruit chromatin-modifying proteins.18 It has been shown that the same gene can be either activated or suppressed by a small RNA in a sequence-dependent manner.5 These effects are also cell type specific and may depend on the basal expression level of the targeted gene and the presence of the antisense transcripts on the promoter.19 For example, MS1 and C166 are both mouse endothelial cells, but typically only C166 cell line is responsive to TGS/TGA by promoter-targeted small hairpin RNAs (shRNAs). However, if these cells are pretreated with 5-azacytidine, a DNA methyltransferase inhibitor, MS1 cells become responsive to TGS, whereas C166 cells turn unresponsive.20 Therefore, the preexisting epigenetic state of the cell is important for the function of nuclear small RNA-mediated gene regulation.20The exact mechanisms for TGS and TGA are still not clear but a few different mechanisms have been suggested; small RNAs targeted to the promoter sequences may be in direct contact with the chromatin forming DNA–RNA hybrids21 This involved an increase in histone 3 lysine 9 dimethylation (H3K9me2) and H3K27me3 levels at the target site within the gene, AGO1 and HP1α recruitment and a change in the RNA polymerase II elongation rate.21 This finding was supported by another study where it was shown that the nuclear AGO1 and AGO2 directly associate with both chromatin-modifying proteins and regulators of alternative splicing, and these chromatin-associated complexes increase H3K9me3 amount and HP1γ binding, thus slowing down the RNA polymerase II elongation rate and facilitating inclusion of variant exons.22 It is interesting to note that in the study by Alló et al., the presence of the antisense transcripts on the siRNA target site also affected the activity of siRNA and contributed to the differences between cell lines.21Small RNAs have also been shown to affect alternative splicing events, in a mechanism similar to TGS. siRNAs targeted to sequences near an alternative exon regulated the splicing of that exon.24 There are also some experimental data supporting these computational analyses. For example, Place et al. identified a target site for miR-373 on E-cadherin promoter and both miR-373 mimic and synthetic oligo fully complementary to the same sequence were able to induce E-cadherin expression.6 Younger and Corey screened the progesterone receptor (PR) promoter antisense transcript for potential miRNA binding sites and found four different miRNAs that were able to inhibit PR expression when transfected to cells.25 These experiments demonstrated that while synthetic siRNAs inducing either TGS or TGA need to be fully complementary to their target promoter, the endogenous miRNAs can include even extensive mismatches and still achieve to regulate the gene transcription, making the prediction of functional target sites of miRNAs especially difficult. A study by Dharap et al.26 found that miR-324-3p is induced 6 hours after focal cerebral ischemia, and the in silico study predicted a strong binding site for this miRNA on the RelA promoter. RelA is a subunit of NF-κB and is activated after cerebral ischemia.27 The in vitro studies showed that the expression of RelA was indeed induced with transfection of pre-miR-324-3p to cells of neural origin, indicating that miR-324-3p may be regulating RelA expression in ischemia in TGA-like manner.28 It has also recently been shown that miRNAs regulate the function and expression of both other miRNAs and their own and for example, in Caenorhabditis elegans, it was also shown that miRNA lin-4 is able to autoregulate its own expression by increasing the transcription through a complementary target site on its promoter.29Most of the reports have utilized synthetic small double-stranded RNAs, but these studies imply that there may also be a similar endogenous gene regulation mechanism. Some bioinformatic studies have already predicted that miRNAs have target sites on gene promoters in similar quantity as in the 3'UTRs and with very high complementary.et al.30 showed that lncRNAs are bound by PCR2, a protein complex responsible for trimethylation of H3K27, and other chromatin-modifying complexes such as CoREST, suggesting that lncRNAs regulate gene transcription by directing these proteins to specific sites in the genome. In mouse embryonic stem cells, lncRNAs Evx1as and Hoxb5/6as were shown to be associated with MLL1, a histone methyltransferase that trimethylates H3K4, suggesting that they are involved in epigenetically directing differentiation.31 ANRIL, a lncRNA produced from a genomic locus considered a hotspot for disease-associated polymorphisms that have been often associated with, e.g., CVD and diabetes, has been shown to associate with both CBX7 (a member of PRC1 complex) and SUZ12 (a PRC2 member).33 The overexpression of ANRIL also downregulates the expression of a set of chromatin-modifying proteins such as histone acetyltransferase p300.34 In mouse embryonic stem cells, an ANRIL variant p15AS (p15 antisense transcript) induced p15 silencing through heterochromatin formation and DNA methylation after differentiation.35 Experiments in human aortic vascular smooth muscle cells showed that ANRIL may have different splice variants that guide distinct gene regulation patterns, modulating the expression of several genes related to the pathogenesis of atherosclerosis.36 LncRNA Fendrr regulates heart development in embryos by epigenetic mechanisms.37 This involves the action of histone-modifying complexes PRC2 and TrxG/MLL that regulate the activation of genes that are pivotal to developmental functions. Specifically, Fendrr anchors PRC2 at its target promoters and hence increases PRC2 occupancy and H3K27 trimethylation leading to the silencing of target gene expression.37 In a recent study of mouse cardiac long non-coding transcriptome after myocardial infarction, many novel lncRNAs were identified and the differential expression was correlated with physiology.38 These heart-specific novel lncRNAs were often associated with active enhancers, which act as cell type-specific regulatory elements, and also with miRNA networks previously shown to influence cardiac stress response, indicating a complex regulatory system of different ncRNAs.Additionally, lncRNAs have been reported to regulate gene transcription and promoter epigenetics. A study by Khalil 39 In addition to these classical RNAi treatment strategies, new therapy concepts based on nuclear action of ncRNAs have evolved during the past years. We have recently shown that the upregulation of VEGF-A gene using a promoter-targeted shRNA significantly decreases the infarct size in mouse myocardial infarction model20 and improves blood flow in ischemic hindlimb through increased vasculature.5 The observed therapeutic efficiencies in both studies were surprisingly good, considering the limited in vivo transduction efficiency of lentiviral vectors. There are several possible reasons for these observed effects. First, VEGF-A is a secreted protein and can exert its effects from transduced cells to the surrounding tissue.40 Second, all isoforms of VEGF-A are upregulated in response to the epigenetic regulation of endogenous gene20 and this will likely provide more natural responses in the target tissue as compared with traditional gene therapy approach where usually only one isoform of the therapeutic gene is delivered, typically driven by a strong promoter. The endogenous gene encoding human VEGF-A consists of eight exons and alternative splicing produces at least four different isoforms . These different isoforms differ in their biological functions, such as in heparin binding that in turn determines how strongly the isoform binds to extracellular matrix and can therefore be secreted to neighboring cells.41 The third and most intriguing possibility  is that the expressed shRNAs are secreted out of the cells and taken up by neighboring recipient cells, where they pass on the regulatory effects. These shRNAs structurally resemble miRNAs that have been shown to facilitate communication between endothelial cells and smooth muscle cells via an extracellular-vesicle-mediated mechanism in response to atheroprotective stimuli.41 Furthermore, Vickers et al.42 showed that miRNAs are transported in plasma by high-density lipoprotein and taken up by the recipient cells. They found several interesting miRNAs of which miR-223 was one of the most abundant and it's expression was increased in atherosclerosis. This miRNA is highly conserved43 and is involved in the regulation of many biological processes.45 miRNAs can also be secreted in apoptotic bodies by endothelial cells during atherosclerosis, which in turn induces the recruitment of progenitor cells in atherosclerotic mice.46 The secretion of miRNAs in exosomes has been shown and for example endothelial cells can transfer miRNAs to cardiomyocytes in some pathological conditions.47 It is tempting to think that therapeutic ncRNAs could be delivered in vivo using similar, even synthetic, vehicles as carriers. It has already been shown that exosomes produced in vitro can be delivered in vivo in an acute mouse myocardial ischemia/reperfusion model.48A common pathological feature of CVD is the harmful proliferation of smooth muscle cells. It has been shown that the proliferation of smooth muscle cells is at least partly an epigenetic process and that this proliferation can be inhibited by preventing histone deacetylase activity using siRNAs.2 However, the emergence of CVDs on global scale is constantly growing and these pathologies can be partially related to epigenetic regulation of genes and regulatory RNAs, such as miRNAs or other ncRNAs. Therefore, it is an intriguing approach to target these mechanisms by gene therapy strategies. One such method for regulating the expression of a specific gene is to use small RNAs that are complementary to gene promoters, delivered either as RNA oligonucleotides or as hairpin RNAs using vectors. Even though the gene therapy field still has many obstacles to be solved, such as limited efficiency of transfection/transduction and immune responses against vectors and/or transgenes,49 the manipulation of naturally occurring, ncRNA-mediated, regulatory mechanisms may provide more efficient biological responses and allow using lower vector amounts in vivo. When using the traditional gene delivery as a treatment strategy, there often exists a need to regulate the expression of the transgene to avoid unwanted responses, such as cancer when using potent growth factors. Short-term delivery of small RNAs that mediate their effects by epigenetic mechanisms may also help to avoid these problems, since the therapy will merely give a push in the cell towards the desired response and the epigenetic status of the cell will later better accommodate to the long-term status and needs of the tissue. Thus, characterizing the nature and functions of ncRNAs will increase the overall understanding of the mechanisms involved in CVDs and help to develop novel treatment strategies based on gene therapy.Epigenetic gene regulation allows organisms, including humans, to adapt to the environmental stress and changing living conditions.


Resistance training of peripheral muscles has been recommended in order to increase muscle strength in patients with Chronic Obstructive Pulmonary Disease (COPD). However, whether peripheral muscle strength is associated with exercise performance (EP) and physical activity in daily life (PADL) in these patients needs to be investigated.The aim of this study is to evaluate whether strength of the quadriceps muscle (QS) is associated with EP and daily PADL in patients with COPD.We studied patients with COPD (GOLD A-D) and measured maximal isometric strength of the left QS. PADL was measured for 7 days with a SenseWear-Pro® accelerometer. EP was quantified by the 6-minute walk distance (6MWD), the number of stands in the Sit-to-Stand Test (STST), and the handgrip-strength. Univariate and multivariate analyses were used to examine possible associations between QS, PADL and EP.1 of 37.6 (17.6)% predicted, QS was associated with 6MWD, STST, and handgrip-strength but not with PADL. Multiple linear regression analyses showed that QS was independently associated with the 6MWD , STST  and with handgrip-strength .In 27 patients with COPD with a mean (SD) FEVPeripheral muscle strength may be associated with exercise performance but not with physical activity in daily life. This may be due to the fact that EP tests evaluate patients’ true abilities while PADL accelerometers may not. Physical inactivity in daily life is a prominent feature in patients with Chronic Obstructive Pulmonary Disease (COPD) -4 and thOne of the extrapulmonary manifestations of COPD is skeletal muscle dysfunction -15 and tInvestigating the role of peripheral muscle strength and its contribution to exercise tolerance may be important for understanding the pathophysiology of exercise limitation and reduced PADL in patients with COPD and may help to define the best therapeutic approach in these patients. Therefore, the purpose of the present study was to examine the relationship between QS and EP , the number of stands in the Sit-to-Stand Test (STST), handgrip-strength) and PADL as assessed by a multi-dimensional accelerometer (the SenseWear Pro® armband) in patients with COPD.A cross-sectional study in patients with COPD (GOLD-classification A-D) was performed.Patients with COPD referred to the Pulmonary Division, University Hospital of Zurich, Switzerland between March and September 2012 were considered for participation in the study. The inclusion criteria for patients were: male and female subjects aged 40–75 years and confirmed COPD according to GOLD-guidelines . The parThe sample size calculation was based on the results reported by Mador and colleagues .We calculated that the minimum sample size needed to detect a significant association between QS and PADL with 80% power, a significance level of .05 and a hypothetical drop-out-rate due to technical problems of 25%, would be 23 subjects.Spirometry, whole-body plethysmography and diffusion capacity measurements were performed according to the American Thoracic Society (ATS) and the European Respiratory Society (ERS) guidelines with a commercially available system ,33.Patients performed the 6MWT following pulmonary function testing. The 6MWT was conducted in accordance with the ATS guidelines . None ofThe STST was performed in a distraction-protected environment. A standard chair (46 cm height) without arm supports was used. Subjects were asked to stand up from and sitting down on the chair with arms stationary on the hips, repeating the procedure as many times as possible within 1 minute at a patient-defined pace. The patients had to stand in full extension (knee and hip extension) and sitting in a position with their knees at 90° flexion. The number of completed repetitions was counted .Handgrip-strength of the dominant hand was measured with a dynamometer  as described elsewhere .Isometric strength of the left quadriceps muscle group was measured during maximum voluntary contraction (QMVC) with the hips and knees in 90° flexion. QMVC was defined as the highest mean strength that could be sustained over 3 s and expressed in Newton-meters (Nm).Strength was measured with a strap looped around the left leg just proximal to the ankle and connected to a strain gauge. The strain gauge was connected to the Interface series SM S-Type Load Cell (U.S. & Metric) and the Nexus-10™ device . All subjects were studied while seated in an adjustable straight-backed chair. Three maximum voluntary contractions were recorded for each patient. The average of the three scores was used for further analysis. After each test the patients were able to rest for a period of two minutes.Daily Physical Activity was measured by a multisensor accelerometer , which was worn on the upper right arm. The device estimates energy expenditure (EE) using measurements from a biaxial accelerometer and sensors that quantify galvanic skin response, heat flux and skin temperature. The biaxial accelerometer records the number of steps per day and the duration of PADL . The num1) and age. A p < 0.05 was considered to indicate statistical significance.A statistical software package was used for all calculations . Descriptive data for continuous variables are expressed as mean, standard deviation and percentages for frequencies. Variables were tested for parametric distribution by applying the Shapiro-Wilk Test (null hypothesis rejection set at p < 0.25). Univariate regression was performed to evaluate a possible association between QS and measures reflecting PADL derived from accelerometry, 6MWD, the number of stands during the STST, and handgrip-strength. The multivariate analysis was performed to analyse if QS is independently associated with these measures reflecting PADL and EP. The multivariate analysis included measures of PADL that showed significant associations in the univariate regression analysis, Forced Expiratory Volume in 1 second   and handgrip-strength . Both FEV1 and QS were independently associated with the 6MWD . 52.4% of the variance in 6MWD could be explained by the model  QS was independently associated with the 6MWD, the number of stands in the STST, handgrip-strength but not with PADL as assessed by the SenseWear Pro® armband.It has been recognized that skeletal muscle dysfunction is a common feature in patients with COPD, and may play a significant role in morbidity and mortality . SeveralInternational guidelines recommend exercise training to improve skeletal muscle function as an essential component of PR in patients with COPD ,16. HoweAfter reviewing the literature, a total of nine studies, which investigated the association between QS and PADL in patients with COPD were found. Out of nine studies only two ,27 used In this study QS was independently associated with EP but not with PADL as assessed by the SenseWear Pro® armband. Although the design of this study does not allow to establish a causal relationship between QS and PADL, these results need to be elucidated. In a previous research we demonstrated that EP tests do not necessarily reflect daily activities in patients with COPD accurately . This maIt should be stressed that correlations do not prove causal relationships. Interventional studies investigating the effect of PR- induced improvements in peripheral muscle strength on daily PADL allow for a more precise and meaningful examination of the association between QS and PADL. Although the main purpose of PR should be enhancing PADL , only fe2-agonists, which may have a small effect on exercise performance and PADL. Furthermore, because cardiopulmonary exercise testing (CPET) is regarded as the gold standard assessment for evaluating exercise capacity, the tests used in this study may not reflect the true exercise capacity. It should be stressed that the results of the STST mainly reflect the performance of lower extremity muscles.The present study has certain limitations that need to be taken into account. The number of subjects is small given the variance in data. In addition, the majority of the study population represents GOLD D, which may affect outcomes. We did not investigate potential exercise performance limiting factors such as hemodynamic factors, cardiac autonomic nervous system factors, and psychological factors . Neither did we control for the use of long-acting βWe conclude that quadriceps strength may be associated with exercise capacity as assessed by several laboratory-based tests such as the 6-minutes walking test, the sit-to-stand test and handgrip-strength. Future well-designed interventional trials investigating the predominant role of peripheral muscle strength on daily physical activity are needed to establish the preliminary finding of the current study. However, quadriceps strength was not associated with physical activity levels in patients with COPD. This may be due to the fact that patients with COPD do not exhaust their functional abilities during daily living, as they do during laboratory-based testing.None of the authors has a conflict of interest related to the content of the manuscript.


In 2015 South Africa established a national cryptococcal antigenemia (CrAg) screening policy targeted at HIV-infected patients with CD4+ T-lymphocyte (CD4) counts <100 cells/ μl who are not yet on antiretroviral treatment (ART). Two screening strategies are included in national guidelines: reflex screening, where a CrAg test is performed on remnant blood samples from CD4 testing; and provider-initiated screening, where providers order a CrAg test after a patient returns for CD4 test results. The objective of this study was to compare costs and effectiveness of these two screening strategies.We developed a decision analytic model to compare reflex and provider-initiated screening in terms of programmatic and health outcomes  and screening and treatment costs (2015 USD). We estimated a base case with prevalence and other parameters based on data collected during CrAg screening pilot projects integrated into routine HIV care in Gauteng, Free State, and Western Cape Provinces. We conducted sensitivity analyses to explore how results change with underlying parameter assumptions.In the base case, for each 100,000 CD4 tests, the reflex strategy compared to the provider-initiated strategy has higher screening costs  but lower treatment costs , so overall costs of screening and treatment are $17,629 less with the reflex strategy. The reflex strategy saves more lives . Sensitivity analyses suggest that reflex screening dominates provider-initiated screening  or saves additional lives for small additional costs (< $125 per life year) across a wide range of conditions .In countries with substantial numbers of people with untreated, advanced HIV disease such as South Africa, CrAg screening before initiation of ART has the potential to reduce cryptococcal meningitis and save lives. Reflex screening compared to provider-initiated screening saves more lives and is likely to be cost saving or have low additional costs per additional year of life saved. Cryptococcal meningitis (CM) is a leading cause of death among HIV-infected patients with low CD4+ T-lymphocyte (CD4) counts. Further, patients with undiagnosed cryptococcal disease at time of antiretroviral treatment (ART) initiation are at high risk for death from immune reconstitution inflammatory syndrome (IRIS) –3. CryptAs of 2015, the South African government recommended that “HIV-positive adults with a CD4 count <100 cells/μl should be screened for cryptococcal disease before ART is started” . The relIn this paper, we present a decision-analytic model to compare reflex and provider-initiated CrAg screening strategies based on costs (2015 USD), disaggregated into screening, preemptive treatment to avoid hospitalization, hospital, and post-hospital costs, and health outcomes (lives saved and years of life saved using a 3% discount rate). Although prior research has shown that CrAg screening prior to ART initiation is cost effective, alternative strategies for CrAg screening have not been compared systematically. Thus, the focus here is on the difference in costs between the two strategies, the difference in health outcomes achieved, and the incremental cost effectiveness of reflex compared to provider-initiated screening.Of particular interest is the impact of provider and patient adherence to the different strategy protocols. The provider-initiated strategy places responsibility on providers to assess correctly patient eligibility for CrAg screening, while the reflex strategy does not. Both strategies require patients to return for a second visit. High rates of loss-to-follow-up after HIV diagnosis are well documented in South Africa and elsewhere –18, whicWe parameterize the model for a base case using program evaluation data observed during large- scale pilot CrAg screening programs in Gauteng, Free State, and Western Cape Provinces . AdditioReflex and provider-initiated strategies were modeled in two stages: the screening stage and the treatment stage. The screening stage identifies two main categories of patients for the treatment stage: (i) incident CrAg positive patients identified for preemptive treatment; and (ii) incident CrAg positive patients missed by the screening program who also develop CM (discussed in more detail below).In the treatment stage, costs include outpatient fluconazole treatment for patients identified for preemptive treatment in the screening stage. Treatment costs also include hospitalization and post-hospital treatment for patients who develop CM, which includes some patients missed by the screening program and patients identified for preemptive treatment who may still develop CM. A patient’s outcome in the screening stage determines where they enter the treatment stage. The structure of the treatment cost module is the same for both strategies; only the starting numbers of patients in each arm of the model differ (based on the results of the screening program).A key starting point for this analysis is CrAg prevalence in the target population at the time of CD4 count testing. While CrAg prevalence in some populations has been reported in many prior studies , 20, 21,The remainder of this section works through the screening and treatment cost modules. Tables The screening stage for the reflex strategy is described in Figs Under the reflex strategy, 10% of blood samples have a CD4 count < 100 cells/uL; 95% of these are tested for CrAg, with the remaining 5% not tested due to non-adherence to guidelines by lab personnel . In 2014Among the 5% of samples that are not tested, the base case assumption is that 80% are from patients who are pre-ART . This is based on CD4 results from eight Right to Care-supported public clinics in Gauteng Province from 2013. Although the South African CrAg screening guidelines specifically target patients who are not yet on ART, reflex lab screening procedures do not identify patients with low CD4 counts who are on ART. Since patients on ART are not the intended targets for CrAg screening , arm 1, Among those not screened, we use existing program data to estimate that 95.4% are CrAg negative , arm 3, Among these CrAg positive patients who are missed by the screening program, because they were not screened, the base case assumption in Actual data on the proportion of CrAg positive individuals, with low CD4 counts and CSF negative at the time of CD4 testing, who then develop CM does not exist for a large population of HIV-infected patients (whether there is rapid initiation of ART or not). The implications of these assumptions are explored further through sensitivity analysis following the presentation of main results.Among the 95% of reflex samples tested for CrAg , 80% areFor the base case , among tAmong the 60% of pre-ART patients who return ‘quickly enough’ to the clinic for a second visit, 95.4% are CrAg negative, 1.6% are positive and have had prior CM, and 3% are incident CrAg positive patients . The incBased on the model outlined in Figs The logic of the provider-initiated screening module, presented in Figs The first key difference between the two strategies is that, with provider-initiated screening, patients need to return ‘quickly enough’ for their CD4 results, and also then return ‘quickly enough’ to collect CrAg results (two return visits to complete CrAg screening are needed with the provider-initiated policy rather than the one needed for the reflex policy). In A second key difference between the two screening strategies is that provider-initiated screening allows clinicians to target CrAg screening to the intended group of patients: those who are pre-ART and with CD4 count < 100 cells/μl. In A third key difference between the two strategies is provider adherence to screening guidelines. With the reflex policy, 95% of all patients who should be screened for CrAg are screened (in addition to patients who are not the target of the screening program), and CD4 count and CrAg results are available to the clinician simultaneously. With the provider-initiated policy, which depends on provider decision-making, such high levels of adherence to guidelines cannot be assumed. Experience from the Western Cape Province, which implemented the provider-initiated policy, showed low adherence to guidelines, with some modest improvement over time  . AssuminReturning to Of the 48% of patients with CD4 count < 100 cells/μl who return for CD4 results, providers request CrAg tests on 40% of these patients. For the remaining 60% not tested for CrAg, again 3% are incident CrAg positive . As withThe patients actually screened for CrAg need to return quickly enough (their third visit to the clinic within 28 days) to obtain their CrAg results. Data are currently not available to be able to provide a reasonable estimate for this parameter. As a base case, we assume that 80% of these patients return within 28 days, and 20% do not. A high proportion, such as 80%, seems plausible because these are patients who already returned quickly for CD4 results, know their low CD4 results, and are likely to be motivated to return again quickly.Among patients tested for CrAg who do not return quickly enough (20% of those tested), 3.0% are incident CrAg positive with the same structure of outcomes (missed and developed CM) as in Based on the model outlined in Figs Far fewer patients will be screened for CrAg with the provider-initiated policy in part because screening is avoided for patients already on ART. In addition, patients who should be screened are not because they do not return for CD4 results and providers do not order the CrAg test.missed in the screening stage who develop CM . A small number of patients have this endpoint, so the additional treatment costs and health outcomes are essentially irrelevant for a policy comparison.Most patients identified for preemptive treatment initiate treatment (90%). Current guidelines in South Africa for preemptive care of identified CrAg positive patients are 800mg of fluconazole for 2 weeks prior to the initiation of ART, followed by initiation of ART and continuation of daily fluconazole, 400mg for 8 weeks and then 200mg daily for at least 1 year until CD4 counts are > 200 cells/μl., 27 The Some incident CrAg positive patients will also receive a lumbar puncture to assess CSF status, whether symptomatic or asymptomatic, after receiving their CrAg test results, at the same visit or a future visit perhaps to another facility. The costAmong the patients initiated on preemptive fluconazole, some will be treated successfully and some will fail on preemptive treatment and develop CM (or be diagnosed as CSF positive after a lumbar puncture). Among those initiated on preemptive treatment, an estimated 39.7% will fail preemptive treatment, which combines the 33% of patients already CSF positive at time of preemptive treatment initiation as well as a small share (10%) of those CSF negative. The remaining 60.3% initiated on preemptive treatment will not develop CM. Of the patients who develop CM after initiating preemptive treatment, 80% are hospitalized, of whom 68% survive. Of the patients who survive hospitalization, 75% are successful on maintenance fluconazole. The 20% of patients who fail preemptive treatment, but are never hospitalized and die at home, may have failed on preemptive fluconazole due to being CSF positive at time of fluconazole initiation of lack of patient adherence to fluconazole.Parameter assumptions related to the costs of screening and treatment as well as health outcomes are presented in Under the provider-initiated strategy, the CrAg test is completed as a separate test, which requires additional provider time, an additional blood draw, additional transport to lab, additional data entry into electronic data systems, etc. Thus, the unit cost for one CrAg test will be higher with the provider-initiated policy. As a base case, we assume a 25% cost differential between the two strategies for a cost of $6.19 for provider initiated CrAg testing.Patients started on preemptive fluconazole treatment, and who fully adhere to a one-year treatment course, require 533 tablets (200mg each). The cost per 200mg tablet is $0.056, for a total cost of $30.09. The price per tablet is based on the most recent negotiated fluconazole tender in South Africa at the time of this analysis (2015). The cost in USD looks especially low because of the very weak exchange rate in 2015 (ZAR/$15). Not all patients will complete the full year of preemptive treatment. For the base case, partial adherence is defined as taking 66% of the total prescribed annual amount .For patients with CM successfully treated in the hospital (with 14 days of amphotericin B and related services and hotel costs), we use recently published data on hospitalization costs per day in South Africa to estimate the costs of a full 15-day hospital stay at $1950 ($130 per day). Compared to basic hotel costs, the costs of services provided in the hospital  are relatively minor. As an alternative, hospital costs would be slightly lower if we used the detailed information on hospital services and costs reported in Jarvis et al. (see ), and thThose who do not survive the hospital are assumed to require six hospitalized days on average , at a toPatients can have three primary outcomes at the end of the treatment module: survived, died, unknown. In the treatment cost module, patients in For comparing the two screening strategies, health outcomes are based on known lives saved from screening and treatment as well as patients missed by the screening program who do not develop CM. When comparing the two strategies, the difference in this outcome is the additional lives saved from reflex compared to provider-initiated screening. For patients who die from CM, the base case assumption is that the average age of death is between 35–39 years. For this age range, average life expectancy is 32.6 additional years . With a 3% discount rate and 32.6 years of life lost from a death, 21.6 discounted years of life are lost per death, or conversely 21.6 years of life are saved per avoided death.Patients with CM experience serious symptoms and disability whether receiving treatment or not. Thus, in general, the period of time lived disabled due to CM disease prior to and during treatment could be added into the analysis (with disability adjusted life years (DALYs) lost as the combined health outcome). However, the time period for disability for these patients is fairly short (they either respond to treatment or die within one year), so the “years lived disabled” component of DALYs before completion of treatment will be minor for CM compared to the “years of life lost” component .In addition, patients who survive their hospital stay and complete the post-hospital fluconazole treatment could have long-term impairment from CM despite surviving. For example, disability weights for meningitis-related conditions used previously in the Global Burden of Disease estimates are, for example, deafness = 0.229; intellectual impairment = 0.456; and motor deficit = 0.380.[In the base case population of 100,000 patients who receive a CD4 test, 8,000 have a CD4 count <100 cells/μl and are not yet on ART. The goal of screening is to identify the 240 patients out of 8,000 who are not yet on ART, with incident CrAg (no prior CM). In the absence of a screening program, all of these patients are missed. In the presence of a screening program, with the base case assumptions and analysis presented in Figs From From In the base case summarized in In terms of health outcomes, the reflex policy is also estimated to save an additional 30 lives per 100,000 CD4 tests . Note that these are estimates of additional patients “known” to be living at the end of the treatment stage; any patients in the treatment arms with indeterminate final health outcomes (CM relapse) are not included in this estimate, but their treatment costs are included. For reference, there are 21 patients in the reflex policy treatment module and 28 patients in the provider-initiated treatment module with this indeterminate health outcome . We first consider 5 initial scenarios (denoted as SA1-SA5) beyond the base case, and key results are summarized in As noted in Compared to no screening, previous studies have shown that CrAg screening prior to the initiation of ART for patients with baseline CD4 counts < 100 cells/μl is a relatively low cost or cost-saving policy that also saves lives . This will remain true as long as a baseline CD4 count continues to be required prior to the initiation of ART, even if in the future, CD4 thresholds disappear as an eligibility criterion for ART initiation.The question addressed in this analysis is: how best to organize a screening program in a given setting? The model developed for this analysis and the results reported in Tables As with any policy, efficient operational implementation is critical to achieving program targets. In particular, the important first step is to support patients to know their HIV status and present for care when their CD4 count is still well above 100 cells/μL. The second key step is to get patients to return for CD4 results relatively quickly. In South Africa, patients are typically counseled to return in one week, which if adhered to, provides a good opportunity for CrAg-positive patients to benefit from early treatment. Given that NHLS typically completes CD4 tests within 48 hours, opportunities to shorten the return-for-CD4 results period could be evaluated further.Third, screening and provision of preemptive fluconazole treatment must be simultaneously implemented. While a relatively small number of NHLS laboratories provide the bulk of CD4 tests completed in the country, thousands of Primary Health Centers (PHCs) initiate patients on ART. Based on estimates of 200 mg fluconazole tablets needed per 100,000 CD4 tests in As with most modeling analyses, limitations follow from the need to develop model parameters based on data from several sources . As much as possible, parameters for this analysis were generated from a recent evaluation of CrAg screening programs in South Africa . As showBesides the reflex and provider-initiated CrAg screening policies currently included in South Africa’s HIV guidelines, additional strategies have been discussed or may be relevant in the future. First, for example, point-of-care (POC) CrAg screening as part of a provider-initiated approach, using a lateral flow assay (LFA) method when a patient returns for CD4 results, is possible. However, unless paired with a POC CD4 test, this approach does not provide CrAg results to patients any sooner than the reflex policy, but would eliminate the need for a third return visit under a provider-initiated policy. While the CrAg LFA can be performed relatively easily as a POC test, a large scale program of training, quality control, supply chain management, and data systems would need to be developed for POC CrAg screening to be implemented across thousands of PHCs with equivalent quality to current lab-based CrAg screening . The addSecond, in the future quantitative CrAg tests, reporting a CrAg titer to better differentiate disease status, may help target those who should receive preemptive treatment with fluconazole and who should be admitted and treated with amphotericin. While beyond the scope of the current paper, the existing model could be adapted to such strategies for assessing costs and effectiveness.prior to the initiation of ART as is the case for countries considering “test and treat” policies.And third, all patients testing positive for HIV could be prescribed an initial short-course of fluconazole, or perhaps only patients clinically suspected to have a low CD4 cell count based on WHO staging or some other criteria, while awaiting CD4 results. When these patients return for their CD4 result and CrAg result (if CD4 < 100 cells/μl and with reflex screening), those testing CrAg negative would stop fluconazole treatment and immediately initiate ART, while those CrAg positive would continue with preemptive treatment prior to initiating ART. Future research is needed to evaluate the health risks and benefits of such an approach. However, the logic of this third approach becomes more relevant if CD4 test results are not required as long as a baseline CD4 count continues to be included in national guidelines prior to the initiation of ART. Even if all HIV patients are eligible for ART, regardless of CD4 counts, the question remains if a baseline CD4 count will be required prior to initiation of ART or not.CrAg screening prior to the initiation of ART is a relatively low cost or potentially cost saving policy prior to initiation of ART, then a reflex CrAg screening strategy imposes no additional delays on ART initiation for the vast majority of patients. For the relatively few total HIV-infected patients with low CD4 counts who are CrAg positive, appropriate treatment  can still begin prior to initiation of ART.If guidelines under new test-and-treat policies still include baseline CD4 counts and other laboratory tests prior to the initiation of ART becomes more complicated if policies change to same-day ART initiation after an HIV diagnosis . If an immediate test-and-treat approach is implemented as a national policy, CrAg screening before initiation of ART would require both provider-initiated POC CD4 and CrAg testing. Alternatively, CrAg screening after initiation of ART (perhaps implemented discussed above) would need to be evaluated further.However, CrAg screening To date, all analyses of CrAg screening have assumed the presence of CD4 results to target screening. In the absence of such information, future research is needed to compare non-CD4 based strategies for screening, as well as the benefits of CrAg screening for the small proportion of newly-diagnosed HIV-infected patients with low CD4 counts who are incident CrAg positive to the possible costs of delaying ART initiation for the vast majority of all patients (those with CD4 counts > 100 cells/μl and those with low CD4 counts who are CrAg negative).This costing analysis was based on routinely-collected aggregated program data, publically available information, and aggregated results from evaluation studies. No human subjects data were used.S1 Appendix(DOCX)Click here for additional data file.