To the Editor: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has been reported from almost every continent (–mec (SCCmec) IVa, a novel smaller variant of the methicillin-resistance locus , and production of clumping factor and protein A . Methicillin resistance was determined by susceptibility testing and confirmed by latex agglutination for penicillin binding protein-2a –based methods. Comparisons with CA-MRSA strains worldwide in terms of toxin profile and PFGE patterns were also performed in France; the latter was achieved by using Taxotron software  to digitize and analyze SmaI macrorestriction patterns.The medical records of patients infected by these MRSA were reviewed, and strains were labeled community-associated if they had been isolated within 48 hours of hospitalization from patients who had not been in any hospital for >1 year. Community-associated strains were tested for PVL genes  had been archived. The demographic and clinical data of the patients are shown in the mec IV. Isolates 1, 6, and 8 were mecA positive, but their SCCmec belonged to none of the 4 major structural types. Comparisons with published data on CA-MRSA strains showed that isolate 7 was identical to the European strain of CA-MRSA in terms of PFGE pattern, toxin profile, and sequence type (ST 80)  and SCCmec. It belonged to agr 1 and tested positive for γ-hemolysin gene as well as for the enterotoxin gene cluster.Isolate 6 had a PFGE pattern that may be distantly related to U.S. strains; the similar sequence type (ST 1) served to emphasize this, although the presence of nontypable SCC–Widely diversified CA-MRSA strains exist in Singapore. The demographic profile and clinical symptoms of local patients infected with these strains were consistent with published literature (–In contrast to previous reports (CA-MRSA has only been isolated sporadically in Singapore, and no dominant clone was seen among our isolates. Singapore may be in an early phase of CA-MRSA emergence, and healthcare workers should remain vigilant for future outbreaks.


The bovine equivalent of growth hormone, called recombinant bovine somatotropin (rbST) is (il)legally administered to enhance milk production in dairy cows. In this study, first a generic sample pre-treatment and 4-plex flow cytometric immunoassay (FCIA) were developed for simultaneous measurement of four candidate biomarkers selected from literature: insulin-like growth factor 1 (IGF-1), its binding protein 2 (IGFBP2), osteocalcin and endogenously produced antibodies against rbST. Next, bovine serum samples from two extensive controlled rbST animal treatment studies were used for in vivo validation and biomarker evaluation. Finally, advanced statistic tools were tested for the assessment of biomarker combination quality aiming to correctly identify rbST-treated animals. The statistical prediction tool k-nearest neighbours using a combination of the biomarkers osteocalcin and endogenously produced antibodies against rbST proved to be very reliable and correctly predicted 95% of the treated samples starting from the second rbST injection until the end of the treatment period and even thereafter. With the same biomarker combination, only 12% of untreated animals appeared false-positive. This reliability meets the requirements of Commission Decision 2002/657/EC for screening methods in veterinary control. From the results of this multidisciplinary study, it is concluded that the osteocalcin – anti-rbST-antibodies combination represent fit-for-purpose biomarkers for screening of rbST abuse in dairy cattle and can be reliably measured in both the developed 4-plex FCIA as well as in a cost-effective 2-plex microsphere-based binding assay. This screening method can be incorporated in routine veterinary monitoring programmes: in the European Union for detection of rbST abuse and in the control of rbST-free dairy farms in the United States of America and other countries.Biomarker profiling, as a rapid screening approach for detection of hormone abuse, requires well selected candidate biomarkers and a thorough  Many different techniques are available for detection of hormone abuse in sports doping and veterinary control, which all have to fulfil the requirements to be reliable, comparably fast and affordable. Biomarker profiling was suggested as a rapid screening approach for detection of doping practices because of its many advantages over the direct detection of the particular abused substances Extensive studies were done for the biomarker-based detection of recombinant somatotropin  in sports doping, where ST is abused by athletes for their performance enhancement Biomarkers indicative for ST abuse are described in detail in literature and several of them are listed and referenced in vice versa. For the anti-rbST-antibodies, a direct assay format with an rbST-coupled colour-encoded microsphere set was used, where the anti-rbST-antibodies bind and can be detected by fluorescently labelled anti-bovine detection antibodies. Here, a high biomarker level leads to a high fluorescence signal. With this 4-plex FCIA, biomarker profiles were measured in serum samples. Based on the biomarker profiles of 67 untreated animals from different origins, we assessed the inter-individual and physiological variability of these biomarkers within dairy cattle and determined decision limits, beyond which a sample could be classified rbST-treated. Then, we used a large set of serum samples obtained from two independent controlled rbST animal treatment studies to evaluate the discriminative power of each candidate biomarker and of all combinations of biomarkers for distinguishing rbST-treated from untreated cows. Following thorough statistical evaluations, the value of individual and multiple biomarkers was assessed for the prediction of rbST abuse in dairy cows.To be able to screen for these four candidate biomarkers in serum, we developed a 4-plex flow cytometric immunoassay (FCIA) enabling parallel biomarker analysis in a single sample. For IGF-1, IGFBP2 and osteocalcin, a competitive inhibition assay format was chosen, where the respective candidate biomarker is covalently coupled to one set of colour-encoded microspheres. The different microsphere sets can be discriminated by a red laser . BiomarkThe overall aim of the study was the development and validation of a chemical analytical method for rbST-dependent biomarker detection according to European legislation for screening methods For the prediction of rbST abuse in dairy cows, we selected candidate biomarkers based on information found in literature . These w−1 for IGF-1 and 32–320 ng mL−1 for osteocalcin . For IGFBP2, the standard protein could not completely inhibit the B0 signal; therefore, we decided to work with normalized responses (B/B0) for the data analysis. For the induced anti-rbST-antibodies, we worked with the responses normalized to a single standard serum (B/Bd).For the simultaneous detection of these four candidate biomarkers, we developed a generic sample pre-treatment and 4-plex flow cytometric immunoassay (FCIA). To this end, our previously reported 3-plex assay The generic sample pre-treatment was necessary for releasing IGF-1 from its binding protein-complex and preventing non-specific binding in the detection of anti-rbST-antibodies. The rather harsh pre-treatment protocol did not affect the detection quality of osteocalcin, thus it could be adopted for the combined 4-plex FCIA. Note that adding IGF-2 in excess, as done in commercially available human IGF-1 immunoassays, improved neither the normalized standard curves nor the detection of biomarker level differences in between treated and untreated animals. The developed assay showed high reproducibility for all measured candidate biomarkers  and a coAfter successful development of the 4-plex flow cytometric immunoassay, decision limits for each single candidate biomarker were calculated by analysis of sera from 67 untreated dairy cows . Compared to the number of tested athletes in human studies, the number of tested control animals may seem to be rather low, but the variation within the dairy population is expected to be much lower, because of several reasons: First, only female cows have to be taken into account. Second, milking only occurs after first calving , thus after puberty, in which levels of IGF-1, IGFBP2 and osteocalcin are mainly changed due to growth and are more stable thereafter −1 for IGF-1, 0.52 B/B0 for IGFBP2, 1.62 B/Bd for anti-rbST-antibodies and 160 ng mL−1 for osteocalcin and are shown as green horizontal lines in Decision limits were 216 ng mLThen, biomarker profiles of the dairy cows from both animal studies were measured . ResultsIGF-1 levels were found to be elevated directly after rbST treatment  and retuIGFBP2 levels  are expeet al. reported an increase of antibody formation within the first three months of rbST treatment and a decline thereafter, but the amount of rbST administered in their study was much higher than recommended by the manufacturer and applied here For the antibodies, endogenously produced by the cow as an immunological response upon rbST treatment For osteocalcin, a slow increase in concentration was observed after rbST treatment  comparedFor all of the candidate biomarkers large inter-individual physiological differences in biomarker levels were apparent as for example seen in the adaptation period of the treated animals. IGF-1, IGFBP2 and osteocalcin levels differed quite a lot between individual animals. Biomarker levels are known to be influenced by many factors such as age and state of lactation. Nevertheless, the expected variation is much smaller than in athletes tested for ST abuse as already discussed above. Note that we accounted for the variation in our untreated reference population used to assess the decision limits. Also the response upon rbST treatment differed in every individual cow. Some cows showed a big increase in IGF-1 levels short after injection while others did not show any response above decision limit (non-responders). Also for osteocalcin, some cows hardly showed any response after treatment.The predictive power of each candidate biomarker was assessed by calculating true-positive rates for all samples from rbST-treated cows in their treatment and withdrawal period . False-pSince no single candidate biomarker was capable of predicting 95% of the rbST-treated samples correctly, we tested different possibilities of combining biomarker results for improvement of the predictive power of our 4-plex FCIA. One approach to do this is the additive biomarker analysis. In Since the single biomarker analysis and additive biomarker analysis, which were both based on decision limits, did not deliver satisfying results for predicting rbST abuse, a different biomarker-combining approach was chosen for analysis of the data. K-nearest neighbours (kNN), a statistical prediction tool, was used to build a model from one group of data  and predict the results of Group B  on basis of the built model. Eleven different models (one for every possible combination of two to four biomarkers) were evaluated to find the optimal biomarker combination for rbST abuse prediction. True-positive rates of Group B data were calculated for every biomarker combination over the time of the whole animal study and are shown in Since we used all of the untreated animals of both animal studies for model building, false-positive rates for the eleven different models were calculated based on the results of the 67 independent untreated cows . For theWe concluded from the results of the here presented studies that the AO biomarker combination is the preferred model for predicting rbST abuse. It yielded seven out of eleven time points above the 95% target and if two biomarkers are equally well-suited for prediction as three biomarkers, the simpler version is favoured.in vivo evaluation and combined with a thorough statistical multiple biomarker analysis can detect more than 95% of the rbST-treated cows truly positive directly after the second rbST injection until the end of their treatment period and even thereafter. This meets the requirements of Commission Decision 2002/657/EC for a screening assay for the detection of banned veterinary drugs such as rbST The results obtained proof that the developed 4-plex FCIA reduced to an AO biomarker combination 2-plex FCIA, applied to an When comparing with previously reported results of a 3-plex FCIA combining IGF-1, IGFBP2 and anti-rbST-antibodies in vivo validation with two independent rbST animal treatment studies followed by statistical analysis revealed that a combination of just two candidate biomarkers is actually adequate for detection of rbST treatment. Therefore, even a 2-plex version  of our assay would already be fit-for-purpose based on the data presented here.For the first time, to the best of our knowledge, a 4-plex biomarker assay development and data evaluation model is presented for the detection of rbST abuse, which fulfils the requirements of Commission Decision 2002/657/EC for screening assays et al. and Zwickl et al. reported that repeated treatment periods did not cause an immunological memory effect with enhanced antibody production in the second treatment period Nevertheless some issues should be considered. First of all, for obvious ethical and cost reasons, the rbST treatment period was limited to 8 weeks in our animal studies, so we do not know yet how the prediction models would perform for long-term treated animals. As it can be seen in in vivo validated screening assay for rbST abuse in dairy cows. Four candidate biomarkers for rbST abuse were assessed using a newly developed 4-plex flow cytometric immunoassay, in vivo validation studies and advanced statistics. Biomarkers indicative for rbST administration were evaluated based on two extensive animal studies with rbST-treated and untreated animals and an additional untreated reference population. Different data evaluation approaches were tested. The prediction tool kNN using a biomarker combination endogenously produced antibodies against rbST and osteocalcin proved to be very reliable and correctly predicted 95% of the treated samples starting from the second rbST injection until the end of the treatment period and even thereafter. This reduced 2-plex FCIA method  combined with the statistical analysis approach was shown to be a fast, reliable and cost-effective approach to screen for rbST abuse in dairy cattle. These methods and models can be included in routine veterinary control programmes in the European Union for detection of rbST abuse and also in the control of rbST-free dairy farms in the United States of America and other countries.In this study a multidisciplinary approach was used for the development of an Permission for animal study I (EC2007/71) was obtained from the Ethical Commission of the Faculty of Veterinary Medicine of Ghent University (Belgium) on basis of the Dutch law on animal studies (Wet op de Dierproeven). For animal study II, permission (EC2010-21) was obtained from the Ethical Commission of the Animal Science Group of Wageningen University and Research Centre in Lelystad (The Netherlands).2PO4×H2O), potassium dihydrogen phosphate (KH2PO4), sodium chloride (NaCl), sodium azide (NaN3) and Tween 20 were obtained from VWR International . Microcentrifuge Model 16K was purchased from Bio-Rad . Protein LoBind Tubes, Safe Lock Tubes (amber) and Centrifuge 5810R were obtained from Eppendorf . 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 2-(N-morpholino)ethanesulfonic acid (MES) hydrate, ovalbumin and bovine serum albumin (BSA) were obtained from Sigma-Aldrich . MultiScreen HTS filter plates were purchased from Millipore . Purified bovine osteocalcin and mouse anti-bovine osteocalcin antibodies were obtained from Haematologic Technologies, Inc. . Insulin-like growth factor-I  was purchased from Fitzgerald Industries International . Insulin-like growth factor binding protein-2  was purchased from IBT . Mouse anti-IGF-1 was supplied by LifeSpan BioSciences, Inc.  and the rabbit anti-IGFBP-2 was from United States Biological . Monsanto rbST standard was obtained from the National Hormone & Peptide Program (NHPP) of Dr Parlow . R-Phycoerythrin (PE)-labelled goat anti-bovine immunoglobulins (GAB-PE) were from Santa Cruz Biotechnology  and R-Phycoerythrin (PE)-labelled goat anti-mouse immunoglobulins (GAM-PE) and goat anti-rabbit immunoglobulins (GAR-PE) were purchased at Prozyme . Donor adult bovine serum was from HyClone . Sodium hydroxide (NaOH), disodium hydrogen phosphate dihydrate (Na2HPO4×2 H2O) and hydrochloric acid (HCl) were purchased from Merck . SeroMAP microspheres  and sheath fluid were obtained from Luminex . The Luminex 100 IS 2.2 system consisting of a Luminex 100 analyser and a Luminex XY Platform was purchased from Applied Cytometry Systems . Snijder Test tube rotator was from Omnilabo International . 10 mL polypropylene tubes were obtained from Greiner Bio-One . Glycine was purchased from Duchefa  and sulfo-N-Hydroxysuccinimide (Sulfo-NHS) from Fluka . Sodium dodecyl sulphate (SDS) was obtained from Serva . The microtiter vari-shaker was purchased from Dynatech . Posilac® (rbST) 500 mg single dose syringes and syringes with only the slow release formula were obtained from Monsanto Company  for animal study I and from Elanco Animal Health  for animal study II.Ultrasonic bath, monosodium phosphate monohydrate , PBST , PBSTB (0.1% w/v BSA in PBST), glycine solution I , glycine solution II , MES buffer , blocking buffer .Buffers and solutions were prepared as follows: phosphate-buffered saline . Since we did not know for sure whether we would see any response, the cows were thereafter treated two times more but with a weekly interval, followed by a final 4-week withdrawal period. In animal study II, 10 Holstein dairy cows were divided in two groups. In contrast to animal study I, these cows were of different age (2–8 years). After a 2-week adaptation period, 8 cows were treated every second week with 500 mg rbST in a slow-release formula during 8 weeks and 2 control cows were treated with the slow-release formula only. The biweekly treatment period according to manufacturers' guidelines was directly followed by a 4-week withdrawal period. In both studies, blood sampling was scheduled similarly: During the two week adaptation period, blood samples were collected weekly; during the treatment period, blood samples were collected a day before, a day after and a week after injection and during withdrawal, blood samples were collected weekly for four more weeks, which yielded 21 serum samples per cow in animal study I and 18 serum samples per cow in animal study II. The treatment schedule and blood sampling time points are shown in Samples from different sources were used for analysis. Serum samples from two independent controlled animal treatment studies were used. In animal study I, eight Holstein cows were selected. These cows were all about 5 years old, divided in two groups of 4 animals each and treated with 500 mg rbST in slow-release formula or slow-release formula only. After a two-week adaptation period, they received an injection every second week, in total 4 times in accordance with the suggested treatment schedule by the manufacturer (−1 BSA in PBS), were used for standard curves ranging from 0.08 to 20 ng mL−1 for IGF-1 and osteocalcin and from 0.2 to 50 ng mL−1 for IGFBP2. Also blank standard samples  were measured. Note that no standards are commercially available for anti-rbST-antibodies.Protein standards of IGF-1, IGFBP2 and osteocalcin, prepared in serum-matched buffer  was described before −1 osteocalcin to microspheres (set 084) was done following the same procedure.Covalent coupling of 100 µg mL−1 until 50 microspheres per set were counted, up to a maximum of 50 µL per sample. A typical analysis of a full 96 well microtiter plate takes 3.5 hours starting from raw serum until the results are obtained.The assay procedure for detection of three biomarkers was described before Raw median fluorescence intensities (MFIs) were measured by the flow cytometer for every single candidate biomarker. Every sample was measured in duplicate and MFIs were averaged before further analysis. For IGF-1, IGFBP2 and osteocalcin, B/B0 values were calculated per sample by dividing the measured MFI by the MFI of a blank biomarker-free standard. Then, concentrations were recalculated from standard curves (non-linear four-parameter curve fit) using GraphPad Prism program  for IGF-1 and osteocalcin. For IGFBP2, no complete inhibition could be obtained with the available standard protein, therefore, no actual concentrations were determined and B/B0 values were simply used. For anti-rbST-antibodies, which are endogenously produced by the cow in response to rbST treatment, no standard was available. To be able to normalize, measured sample MFIs were divided by the MFI of one serum sample, which was measured every time (B/Bd). This serum was donor adult bovine serum which was a mixture of sera from many cows from one herd. Since this is produced in large amounts, it can be used for a long time with constant quality.50, inter-assay and intra-assay variation (describing precision and ruggedness). For IGF-1, IGFBP2 and osteocalcin, IC50 was read from standard curves at 50% inhibition of the signal of the blank. For all candidate biomarkers, inter-assay variation was determined by measuring 8 different serum samples on 8 days. Mean of results , standard deviation and percentaged standard deviation (%CV) were calculated for every serum. The average of the 8 percentaged standard deviations was the inter-assay deviation. Intra-assay variation was calculated the same way from 8 repetitions of 8 sera within one microtiter plate.To assess the 4-plex FCIA quality and compare it to other methods, assay performance characteristics were calculated, such as ICUsing a single biomarker for prediction of unknown samples as rbST-treated or untreated, the calculation of decision limits for each biomarker was necessary. These were based on the results obtained from a population of 67 untreated dairy cows being diverse in age, in lactation stage and in origin. For every biomarker, results were averaged and two-times the standard deviation was added to obtain the decision limit. Samples found to show concentrations , B/B0 (for IGFBP2) or B/Bd (for anti-rbST antibodies) beyond the respective calculated decision limit, were considered as rbST-treated (positive). True-positive and false-positive rates could be calculated for every single biomarker from the results of the controlled animal studies.After evaluating biomarker profiles and true-positive rates based on single biomarkers, an additive biomarker approach was tested. Here, a sample was considered as rbST-treated when at least one of the candidate biomarkers reacted above decision limit and also here, true-positive and false-positive rates were calculated.After evaluating single candidate biomarkers and testing the additive biomarker approach, we assessed how well a statistical combination of two to four markers was capable to predict rbST abuse. Therefore, a k-nearest neighbours prediction model (kNN) in the R environment First, the whole data set was divided into two groups: Group A data were used to build the time-point-independent prediction model. Therefore and to use sufficient sample numbers for the model building, this group contained all data from animal study II (diverse population with biweekly treatment only). Furthermore, since two control animals were not enough to represent untreated cows, Group A also contained the data from the untreated animals of animal study I. In total, 98 samples from treated and 119 samples from untreated cows were used for model building. Group B data were used for prediction based on the Group A model. Group B contained the data from the rbST-treated cows of animal study I  and the 67 untreated cows. Note that these are sample data independent from Group A data.For model building of the Group A data, a training and test set were chosen by using a stratified repeated random sub-sampling approach, which means that 70% of the rbST-treated and 70% of the untreated samples were selected for the training set and the remaining 30% of both groups for the test set for internal validation, which is necessary to build a strong model. Subsequently, concentrations, B/B0 and B/Bd values of the training set were auto-scaled and a kNN model was built on the training set data. The optimal number of k (1≤k≤10) was chosen based on the bootstrapping approach Figure S1Standard curves of the three rbST-dependent biomarkers IGF-1, IGFBP2 and osteocalcin. Each data point is the mean of 8 separate measurements in a serum-matched buffer (80 mg mL−1 BSA in PBS solution). All curves relate to 80-times diluted sera.(TIF)Click here for additional data file.Figure S2Treatment schedule and sampling time points for animal studies I and II. Arrows indicate the treatment of the cows with rbST in slow-release formula or the slow-release formula only; bold vertical lines indicate blood sampling time points.(TIF)Click here for additional data file.Materials S1Serum preparation, generic serum pre-treatment and 4-plex FCIA for serum candidate biomarkers.(DOCX)Click here for additional data file.Table S1True-positive rates of the statistical multiple biomarker analysis. True-positive rates, obtained with the prediction models based on the eleven different biomarker combinations, were calculated for rbST-treated cows from animal study I in their treatment (day 16–71) and withdrawal period (day 72–99).(DOCX)Click here for additional data file.Table S2False-positive rates of the statistical multiple biomarker analysis. Results were calculated for 67 independent untreated cows predicted with the eleven different biomarker combination models.(DOCX)Click here for additional data file.


Subtotal cystectomy  elicits a robust proliferative response resulting in complete structural and functional bladder regeneration within 8-weeks. The goal of these studies was to characterize the early cellular response that mediates this regenerative phenomenon, which is unique among mammalian organ systems. STC was performed on eighteen 12-week-old female Fischer F344 rats. At 1, 3, 5 and 7-days post-STC, the bladder was harvested 2-hours after intraperitoneal injection of bromodeoxyuridine (BrdU). Fluorescent BrdU labeling was quantified in cells within the urothelium, lamina propria (LP), muscularis propria (MP) and serosa. Cell location was confirmed with fluorescently co-labeled cytokeratin, vimentin or smooth muscle actin (SMA), to identify urothelial, interstitial and smooth muscle cells, respectively. Expression of sonic hedgehog (Shh), Gli-1 and bone morphogenic factor-4 (BMP-4) were evaluated with immunochemistry. Three non-operated rats injected with BrdU served as controls. Less than 1% of cells in the bladder wall were labeled with BrdU in control bladders, but this percentage significantly increased by 5-8-fold at all time points post-STC. The spatiotemporal characteristics of the proliferative response were defined by a significantly higher percentage of BrdU-labeled cells within the urothelium at 1-day than in the MP and LP. A time-dependent shift at 3 and 5-days post-STC revealed significantly fewer BrdU-labeled cells in the MP than LP or urothelium. By 7-days the percentage of BrdU-labeled cells was similar among urothelium, LP and MP. STC also caused an increase in immunostaining for Shh, Gli-1 and BMP-4. In summary, the early stages of functional bladder regeneration are characterized by time-dependent changes in the location of the proliferating cell population, and expression of several evolutionarily conserved developmental signaling proteins. This report extends previous observations and further establishes the rodent bladder as an excellent model for studying novel aspects of mammalian organ regeneration. In fact, the liver has an extensive regenerative potential in response to tissue loss.These well established observations regarding liver re-growth stand in contrast to rodent bladder regeneration, which occurs over a longer time frame (8 weeks rather than 2 weeks), and moreover, results in a regenerated bladder that structurally and functionally is essentially identical to the native bladder which it replaced.in vivo. The current findings extend previous work The rather remarkable regenerative capacity of the bladder has been recognized in rodents, canines and humans for decades.The animal protocol was approved by the Animal Care and Use Committee of Wake Forest University and carried out with strict adherence to the guidelines set fourth. All surgeries were performed under appropriate anesthesia with postoperative pain medication. A total of 18 female Fisher F344 rats weighing 154–189 g underwent subtotal cystectomy (STC), with three animals dying after surgery due to surgical complications. Three animals were removed from the study due to technical artifacts that prevented obtaining accurate histologic tissue sections . The remaining 12 animals were utilized in this study, with 3 age-matched control animals run in parallel .Animals were anesthetized with 2–5% isoflurane chamber induction and maintained with 2–3% via mask inhalation with spontaneous breathing. The lower abdomen was shaved and decontaminated with betadine. A low midline abdominal incision was made with sufficient exposure to identify and deliver the bladder. After bladder exposure, two stay sutures were placed just above the ureterovesical junction (UVJ) using 6–0 polyglycolic acid sutures for traction and alignment. The bladder dome (approximately 70–80% of the whole bladder) superior to the UVJs was then removed, sparing the trigone and ureters. The open bladder was then sutured closed in a continuous fashion using one of the original stay sutures and ensuring adequate coaptation of the anterior and posterior bladder walls as previously described.2 asphyxiation followed by cervical dislocation. An additional three age-matched control animals were also injected and euthanized.Two hours prior to sacrifice, rats were injected intraperioneally with 50 mg/kg of 5-Bromo-2′-deoxyuridine . Three animals were injected and sacrificed at 1, 3, 5, and 7-days after STC using COAfter sacrificing the animals, the abdomen was reopened and the bladder identified. The remaining bladder from the site of STC closure to the distal trigone was explanted, fixed in 10% buffered formalin overnight, processed and embedded in paraffin. Serial 6 µm sagittal tissue sections were sliced along the cranial-caudal axis of the bladder. Fluorescent immunohistochemistry for each time point was performed as follows: heat activated antigen retrieval was performed in 0.01 molar, pH 6.0 citrate buffer. After cooling for 30–60 minutes, the slides were washed in PBS, followed by 15-minute incubation with Serum-Free Protein Block . For quantitative analysis of DNA-replicating cells, double stains were performed for the following primary antibodies/incubation times: anti-BrdU  for 5 hours at room temperature and anti-SMA  overnight at 4°C. Following primary antibody incubation, slides were washed with PBS and a second protein block for 5 minutes was performed. Secondary antibodies Texas Red anti-mouse monoclonal  and Fluorescein anti-rabbit polyclonal  for 30 minutes at room temperature in a dark chamber recognized BrdU and SMA primary antibodies, respectively. For confirmation of BrdU-labeled cell location a subset of additional double stains were performed using the following antibodies/incubation times: anti-BrdU primary and secondary antibodies as described above with anti-cytokeratin  for one hour at room temperature or anti-vimentin  for one hour at room temperature. Fluorescein anti-mouse and anti-goat polyclonal antibodies  for 30 minutes at room temperature in the dark recognized cytokeratin and vimentin primary antibodies, respectively. For all slides, nuclei were nonspecifically counterstained with 4′–6′diamidino-2-phenylindole (DAPI) mounting medium  and coverslipped.Bright field immunohistochemistry probing Shh, Gli-1, and bone morphogenic protein-4 (BMP-4) was performed similarly. Following antigen retrieval, endogenous peroxidase activity was blocked using 10∶1 dilution of 70% methanol and 30% hydrogen peroxide. Primary antibodies against Shh , Gli-1 , and BMP-4  were incubated 1 hour at room temperature. Following primary antibody incubation and protein block, slides were treated for 30 minutes with secondary antibody biotinylated anti-rabbit  and ABC reagent . Shh and BMP-4 slides were then incubated with diaminobenzidine (DAB) chromagen  for 5–10 minutes, except for BMP-4 slides which were incubated with Nova Red  for 5–10 minutes. Lastly, slides were lightly counterstained with hematoxylin and mounted.Fluorescent microscopy was used to examine the number of BrdU-labeled cells and their location within the tissue layers of the bladder at each time point after STC. A Leica (model no. DM4000B) upright microscope with ImagePro software 6.3  was used to take eight 400x images of each tissue section per animal. Suture material at the STC closure site was used to orient the sagittal tissue sections, and systematic images were taken focusing on the upper half of the full tissue section . Four sets of paired images spanning the width of the bladder wall were taken for each animal  and captTo quantify BrdU-labeled cells, fluorescent staining for smooth muscle actin (SMA) helped define the MP layer. The LP was defined as the area between the basal lamina underlying the urothelium and the MP (denoted with SMA). The area beyond the MP was considered serosa. To further confirm the location BrdU-labled cells and better define their cell type, an Olympus FV1000 laser scanning confocal microscope with a 60x oil objective and Fluoview software (Olympus) allowed for high power, reconstructed z-stack examination of a subset of BrdU-cytokeratin, BrdU-vimentin and BrdU-SMA dual fluorescence images. Cytokeratin defined the urothelium, vimentin highlighted LP interstital cells, and SMA identified MP.p-value of less that 0.05 was considered significant. All results were expressed as the mean ± standard error of the mean.Statistical analysis was performed using GraphPad Prism software. For BrdU-labeling quantification a one-way ANOVA was used compare the total percentage of labeled cells for each time point, and a two-way ANOVA with Bonferroni post-hoc testing was used to compare the time-dependent location changes in BrdU-labeled cells. A BrdU labeling was used to evaluate the spatiotemporal characteristics of the cellular response to STC. p = 0.004), and, although not significant, there was a tendency for the percent of BrdU-labeled cells to decrease with time after STC (fter STC -Ι.p<0.001). Interestingly, a time-dependent shift in the location of BrdU-labeled cells was noted. More specifically, at 3 and 5-days post-STC the vast majority of BrdU-labeled cells (80–90%) are found in the urothelium  and LP . Significantly fewer labeled cells were detected in the MP and serosa at 3 and 5-days post-STC . At 7-days post-STC, the percentage of BrdU-labeled cells was similar across the urothelium, LP and MP . The proportion of labeled cells in the serosa remained relatively low at all time points (1.1±0.4% to 5.9±3.6%).In order to compare the impact of STC on BrdU labeling in the various layers of the bladder wall, the amount of BrdU-labeling was expressed as a percentage of the total number of BrdU-labeled cells throughout the bladder wall -ΙΙ. As iCo-labeling experiments were also performed to provide further detail about the location and nature of the BrdU-labeled cells in the various layers of the bladder wall following STC. Confocal microscopy of dual fluorescently-labeled tissue sections was performed. Immunohistochemical staining for Shh and its downstream effectors Gli-1 and BMP-4 A previous report documented a robust proliferative remodeling of the bladder wall following removal of ∼75% of the rat bladder, resulting in complete structural and functional bladder regeneration within 8 weeks.A literature review reveals several studies that have characterized increased urothelial proliferation in response to injury .same control animal demonstrate that the low levels of DNA synthesis/proliferation in the native bladder urothelium stand in contrast to much greater degree of BrdU labeling observed in the regenerating urothelium at 1-day post-STC, as well as the higher proliferation expected of normal gastrointestinal epithelium from the same animal.Consistent with these observations, we report here that under normal conditions the bladder urotheilum has a low proliferative rate  & 5, butAt 1-day post-STC significantly fewer BrdU-labeled cells are present in the MP and LP, as the urothelium accounts for ∼65% of the BrdU-labeling. After this initial burst of urothelial proliferation there is a gradual shift in the location of BrdU labeling to other layers in the bladder wall. Specifically, at 3 and 5-days post-STC, there is a significant increase in the extent of BrdU labeling in the LP such that approximately 80–90% of all labeled cells are contained within these two tissue layers. However, at 7-days post-STC the percentage of BrdU-labeled cells were equivalent across the urothelium, LP and MP .In terms of identifying the nature of the BrdU-labeled cells, we can confirm that differentiated cells in the urothelium, MP and LP contribute to the overall proliferative response. For example, urothelial cells are proliferating given the morphologic characteristics and co-labeling of these cells with cytokeratin . In addiIt is interesting to compare our findings with that of other well-established models of mammalian organ regeneration, specifically, liver regeneration.In summary, bladder regeneration appears to be a process that shares common features with both liver regeneration  and limb regeneration . Given the relatively complete structural and functional regeneration observed in the liver, it may more closely resemble blastema formation than liver regeneration. More specifically, liver regeneration is characterized by a strictly/largely hyperplastic response, while the salamander regenerates a limb via blastema formation. The blastema, in turn, may be the result of dedifferentiation, transdifferentiation, expansion of tissue-specific progenitor cells, or some combination of all of these.Choi and Diehl discussed the EMT/MET process in regards to liver regeneration and how the balance between the two processes can modulate the outcome of repair/regeneration. When EMT activity surpasses MET activity, repair is primarily fibrogenic, causing liver fibrosis. On the other hand, when the activity of the MET process out weights the activity of EMT the outcome is more of a normal liver regeneration.in vivo.Although the precise cellular mechanism for the proliferative response remains uncertain, these observations can be collectively interpreted several ways. (1) Epithelial to mesenchymal transitioning (EMT) occurs allowing the cells to migrate from the urothelium into the LP and incorporate into the MP. EMT is an essential component of bladder development such that embryonic bladder mensenchyme does not differentiate into smooth muscle without adjacent urothelium, and embryonic urothelium causes ingrowth of fibroblasts into an acellular matrix.We also conducted initial investigations into the potential role of hedgehog signaling in STC-induced bladder regeneration. The hedgehog pathway is a well-described and evolutionally conserved component of bladder development that is up regulated in response to bladder injury and therefore might be involved in STC-induced bladder regeneration as well.Shin et al. found that Shh expression and cell proliferation increased dramatically throughout the urothelium after bacterial injury to the mouse bladder. This study confirmed Shh activity and up-regulated Gli-1 staining within 24 hours of the injury, similar to our results.Cao et al. highlighted the importance of Shh signaling to mesenchymal-epithelial interactions during proper bladder development in mice with higher concentrations of Shh increasing both the number of cells and bladder size at 12-days gestation. However the presence of Shh without adjacent urothelium did not give rise to appropriate bladder development.Baskin and colleagues suggests that the Shh pathway may be responsible, at least in part, for aspects of functional bladder regeneration.In this regard, We also evaluated the expression of BMP-4 after STC. As part of the TGF-β superfamily, BMP-4 is a downstream signaling protein of the Shh pathway mediating SMAD transcriptional factors and promoting entry into the cell cycle.In conclusion, this initial study describes early quantitative and time-dependent changes in the location of DNA synthesizing/proliferating cells that occur with the corresponding increased expression of several components of the hedgehog signaling pathway during complete structural and functional bladder regeneration in the rat. Presumably, this proliferative response explains the rapid increase in bladder size and structural/functional recovery after STC, as demonstrated in our previous study.


Cervical squamous intra-epithelial lesions (SIL) are more frequent in HIV-positive women overall. However the appropriate age at which to begin and end cervical cancer screening for early detection of lesions in HIV-positive women is not clear. We assessed the age-specific prevalence of any SIL and SIL requiring colposcopy in HIV-positive women in Cameroon.We enrolled, interviewed and conducted conventional cervical cytology in 282 women, aged 19–68 years, initiating antiretroviral therapy in three clinics in Cameroon. In bivariable analyses, the crude relationship between age and the presence of lesions was assessed using locally weighted regression (LOWESS) methods. In multivariate analyses, generalized linear models with prevalence as the outcome, an identity link and a binomial distribution, were used to estimate prevalence differences. Bias analyses were conducted to assess the potential effect of inaccuracies in cytology.SIL were detected in 43.5% of the 276 women with satisfactory samples, 17.8% of whom had ASC-H/HSIL. On average, women aged 26 to 59 tended to have a slightly higher prevalence of any SIL than other women . This PD was a function of CD4 count : amongst patients with CD4 counts less than 200cells/uL, the prevalence was higher in patients aged 26–59, while there was essentially no difference amongst women with CD4 counts greater than 200 cells/uL. ASC-H/HSIL were present in women as young as 19 and as old as 62. Overall the prevalence of ASC-H/HSIL increased by 0.7%  per decade increase in age.Both severe and less severe lesions were prevalent at all ages suggesting little utility of age-targeted screening among HIV-positive women. Nevertheless, the long-term evolution of these lesions needs to be assessed in prospective studies. Cervical cancer is the second most common cancer in women worldwide . AlthougAge has been a common consideration in the targeted screening for precancerous lesions in the general population. Current guidelines for screening the general population of women in the United States (US) suggest screening commence no later than age 21 years, reducing the frequency of screening at age 30 among women with previously negative cytology results and stopping screening at age 65 (or 70 in some guidelines) . World HWe describe here the age-specific prevalence of lesions in HIV-positive women initiating antiretroviral therapy in Cameroon, with the aim of estimating the minimum age at which lesions occur, the age with maximum occurrence and the latest age at which lesions occur.In this cross-sectional study, HIV-positive women were recruited from three HIV-care clinics in Cameroon: the Bamenda Provincial Hospital AIDS Treatment Center (ATC), the Limbe Provincial Hospital ATC and the Nylon District Hospital ATC in Douala. These are all located in urban areas in Cameroon and provide regular care to patients from surrounding urban areas and peripheral rural areas. Consecutive HIV-positive women receiving care in these clinics, between August and September 2008, were invited to participate in the study. Women aged 18 years or more, who initiated HAART within a year of study enrollment and consenting to study procedures were eligible. Women who were either pregnant, bleeding due to menses or had a previous total hysterectomy were excluded. After obtaining written consent from each participant, socio-demographic and clinical data were collected using a structured interview, a clinical examination and a review of medical records. Cervical cell samples were then collected using Ayre’s spatula, and smeared into two pre-labeled slides.Conventional cytology slides collected were transported to the laboratory of the Center for the Study and Control of Communicable Diseases (CSCCD) in Yaounde, Cameroon where they were stained by the Papanicolau’s method and examined under the microscope by a trained cytologist. The stained slides were observed under the microscope (at 400X) and then scored according to the Bethesda 2001 system, as unsatisfactory; negative; atypical squamous cells of uncertain significance (ASCUS); low-grade squamous intraepithelial lesions (LSIL); atypical squamous cells, cannot exclude high grade lesions (ASC-H); high-grade SIL (HSIL); or invasive cervical cancer . For quaThe study was approved by the Cameroon National Ethics Committee and the University of North Carolina at Chapel Hill's Biomedical IRB (USA).Data collected were entered into MS Access interface on Epi-info 2000. Statistical analysis were conducted using SAS version 9.2  and Stata version 10 . Two outcomes were considered for this analysis: 1) Prevalent cervical lesions ; 2) Prevalent ASC-H/HSIL. Age (in years) was the independent predictor considered. Other covariates considered in this analysis included marital status, education level, parity, history of hormonal contraception, smoking history, CD4 count, and AIDS clinical stage. Univariable distributions of these characteristics were determined by computing means, median and ranges (for continuous variables) and proportions at different levels .In bivariable analyses, the crude relationship between each outcome and age was assessed using locally weighted regression (LOWESS) methods with a smoothing parameter of 0.5 . In subsIn multivariable analysis all covariates other than age and the outcomes were assessed as potential modifiers of the prevalence difference. Each covariate was coded as a binary variable and a product interaction term created between age and each covariate. Covariates were considered modifiers if a likelihood ratio test of the product interaction term had a p-value less than 0.1, (a higher cut-off point set a priori to account for the low power associated with tests of homogeneity) or if the stratum-specific prevalence differences varied by 20% or more.Although all covariates were also considered as potential confounders, a Directed Acyclic Graph (DAG) analysis revealed that none of the variables should be considered a confounder . We atteAltogether 282 women were enrolled in this study. Participants’ age ranged from 19 to 68 years (with a mean of 36 years). The median CD4 count was 179 cells/microliter (interquartile range: 100 to 271). SIL were detected in 43.5% of the 276 women with satisfactory samples: 0.7% as ASCUS, 25.0% as LSIL, 14.5% as ASC-H, and 3.3% as HSIL.The age of participants with no lesions ranged from 19 to 68 (with a mean of 36.3) years while that of participants with any lesion ranged from 19 to 62 (with a mean of 35.7) years. The prevalence of any lesion tended to increase from age 19 to a peak at about 25 years . However this PD was a function of CD4 count . Amongst patients with CD4 counts less than 200cells/uL, women aged 25–59 had a substantially higher prevalence . In contrast, there was only a little difference in prevalence by age among women with CD4 counts greater than 200 cells/uL .We conducted bias analyses assessing what the true population prevalence difference of lesions could be considering the inaccuracies in conventional cytology. The misclassification of the outcome resulting from these inaccuracies was assumed to be non-differential as the cytologist was masked from participants’ ages. Our analysis showed that a lower cytology sensitivity or specificity would mean that the study tended to underestimate the magnitude of the prevalence difference between age groups Figure . For exaAssuming a quadratic relationship between age and the prevalence of SIL, the age with maximum prevalence was estimated to be 34.9  years.The age of participants with ASC-H/HSIL ranged from 19 to 62 (with a mean of 36.5) years. In contrast to any lesion, the age-specific prevalence of ASC_H/HSIL increased slowly but more or less monotonically with age  women has been at the origin of age-targeted screening in these women. Studies conducted in the 1980s, documented the prevalence of lesions in young women -16. SubsIn this study limited to HIV-positive women, the prevalence of lesions was only slightly higher in all women aged 25–59 compared to other women. This suggests that, unlike in HIV-negative women, age only may not be a good criterion for targeted screening. Age differences in the prevalence of lesions, however, appeared to depend on CD4 counts. Amongst women with low CD4 counts, middle-aged women had a higher prevalence than younger or older women, suggesting that screening efforts are particularly needed in these women. To the best of our knowledge few studies have discussed the age-specific prevalence of lesions and severe lesions in HIV-positive women. Unlike our study in which the prevalence of ASC-H/HSIL lesions increased with age, Parham et al.  describeWhile we document prevalent ASC-H/HSIL at all ages in HIV-positive women, it is not clear what the long-term outcome of these lesions would be and this may depend on age. It is conceivable that if lesions were less likely to progress in younger versus older women then targeting older women would be justified or vice versa. Prospective studies in HIV-negative women have had inconsistent results: while lesions were more likely to progress in older women in some studies  the majoOur findings are susceptible to bias from misclassification of outcomes as conventional cytology typically has a low sensitivity . NonetheSecondly, because of the cross-sectional design of the study, the age-specific prevalence described here reflects the age of lesion detection and not necessarily the age of incidence or the age-specific prevalence in the population. Age differences in access to clinics may result in artificially increased prevalence in older women who are more likely to be in the health care system. The latter detection bias is however expected to be minimal in a study population of HIV-positive women in whom access to care is largely driven by worsening HIV disease rather than age. Four in five women in this study had advanced HIV diseases (WHO stage III or IV) and the small number of women aged 50 or more than limited the influence of these women on study estimates.In conclusion, cervical precancerous lesions were prevalent at all ages in this population of HIV-positive women, suggesting little utility of age-targeted screening. A better understanding of the value of age-targeted screening would require an assessment of the age-specific long-term evolution of untreated non-severe lesions and treated severe lesions using prospective studies. The potential costs and benefits associated with age-targeted screening will also need to be evaluated in formal cost-effectiveness analyses.ASC-H: Atypical Squamous cells, cannot exclude high grade lesions; ASCUS: Atypical Squamous Cells of Uncertain Significance; ATC: AIDS Treatment Center; CI: Confidence Interval; HAART: Highly Active Antiretroviral Therapy; HIV: Human Immunodeficiency Virus; HPV: Human Papilloma Virus; HSIL: High-grade Squamous Intra-epithelial Lesions; LOWESS: Locally weighted regression; LSIL: Low-grade Squamous Intra-epithelial Lesions; PD: Prevalence difference; SIL: Squamous Intra-epithelial Lesions; WHO: World Health Organisation.om Digene. JSS has received research grants, honoraria, or consulting fees during the last three years from GSK, Digene and GenProbe. EM has received research funding and done consulting for Merck & Co.JA has received honoraria from GSK and research supplies frStudy conception and design: JA, JSS, AAA, JE, WCM, EM. Study implementation: JA, JSS, PMN, GMI, ACR. Analysis and review: JA, GMI, ACR, JSS, WCM. Manuscript writing and revisions: JA, PMN, GMI, JSS, AAA, JE, WCM, EM. All authors read and approved the final manuscript.


The prevalence and the patterns of ocular inflammatory disease and ocular tuberculosis (TB) are largely undocumented among Multidrug Resistant TB (MDR-TB) patients co-infected with Human Immunodeficiency Virus (HIV) and on antituberculosis and antiretroviral therapy (ART).Lilavati Hospital and Research Center and Médecins Sans Frontières (MSF) organized a cross-sectional ophthalmological evaluation of HIV/MDR-TB co-infected patients followed in an MSF-run HIV-clinic in Mumbai, India, which included measuring visual acuity, and slit lamp and dilated fundus examinations.Between February and April 2012, 47 HIV/MDR-TB co-infected patients (including three patients with extensively drug-resistant TB) were evaluated. Sixty-four per cent were male, mean age was 39 years (standard deviation: 8.7) and their median (IQR) CD4 count at the time of evaluation was 264 cells/μL (158–361). Thirteen patients (27%) had detectable levels of HIV viremia (>20 copies/ml). Overall, examination of the anterior segments was normal in 45/47 patients (96%). A dilated fundus examination revealed active ocular inflammatory disease in seven eyes of seven patients ; 5.1-25.8%). ‘These included five eyes of five patients (10%) with choroidal tubercles, one eye of one patient (2%) with presumed tubercular chorioretinitis and one eye of one patient (2%) with evidence of presumed active CMV retinitis. Presumed ocular tuberculosis was thus seen in a total of six patients . Two patients who had completed anti-TB treatment had active ocular inflammatory disease, in the form of choroidal tubercles (two eyes of two patients). Inactive scars were seen in three eyes of three patients (6%). Patients with extrapulmonary TB and patients <39 years old were at significantly higher risk of having ocular TB [Risk Ratio: 13.65 (95% CI: 2.4-78.5) and 6.38 (95% CI: 1.05-38.8) respectively].Ocular inflammatory disease, mainly ocular tuberculosis, was common in a cohort of HIV/MDR-TB co-infected patients in Mumbai, India. Ophthalmological examination should be routinely considered in HIV patients diagnosed with or suspected to have MDR-TB, especially in those with extrapulmonary TB. Ocular tuberculosis (OTB) results from the haematogenous dissemination of mycobacteria and may affect virtually any intraocular tissue. Clinical features depend on the specific tissue involved and may be due to both, direct tissue infection or due to hypersensitivity reactions. The characteristic findings include tubercles, tuberculomas and serpiginous-like choroiditis. These represent direct choroidal infection via the hematogenous route. Less common lesions include lupus vulgaris of the eyelids, conjunctivitis, corneal ulcers and phlyctenulosis, and scleritis. Neuro-opthalmological lesions include orbital apex syndrome, disc edema and sixth nerve palsies.Non-invasive ocular examinations to detect ocular tuberculosis have several potential advantages. They may suggest a diagnosis in a subset of patients, thus allowing for a more focused investigational approach. This may be more important in immunocompromised patients who tend to have abnormal systemic findings (absence of fever) or investigational findings . These findings may delay a diagnosis of systemic tuberculosis with the attendant risks of poor outcomes.The association between systemic TB disease and ocular dissemination has been widely reported. The ocular morbidity pattern in 2010 eyes of 1005 patients with active pulmonary and extrapulmonary tuberculosis disease was studied prospectively in India; 1.4% of patients had ocular lesions .It is unclear whether the dual HIV-TB epidemic has lead to an increase in the prevalence and incidence of ocular tuberculosis in patients with active TB disease and co-infected with HIV. In various studies from Africa, differing proportions of ocular tuberculosis in association with HIV infection have been reported, varying between 0 to 2.8%, depending on the size and clinical characteristic of the studied cohort ,5. OpporMultidrug-resistant tuberculosis (MDR-TB) is defined as TB that is resistant to both isoniazid and rifampicin. Extensively drug-resistant tuberculosis (XDR-TB) is defined as MDR-TB with additional resistance to any fluoroquinolone and to at least one of three injectable second-line anti-tuberculosis drugs used in treatment .To-date, no data are available on the overall prevalence or common presentations of ocular inflammatory disease (OID), especially ocular tuberculosis, in patients with MDR-TB including those co-infected with HIV.The objectives of this study were 1) to assess the prevalence and presentations of ocular inflammatory disease and ocular tuberculosis, in particular, in a cohort of MDR-TB patients co-infected with HIV and 2) to identify possible risk factors for developing ocular tuberculosis, including those factors related to HIV infection and to drug resistance.The study was approved by the Institutional Review Board of Lilavati Hospital & Research Center, Mumbai, India and by the Médecins Sans Frontières independent Ethics Review Board, Geneva, Switzerland. Patients were counselled about the study and an informed consent was obtained.This was a cross-sectional study in a cohort of MDR-TB patients co-infected with HIV receiving care and treatment at an HIV clinic in Mumbai, India.Médecins Sans Frontières (MSF) has been operating an HIV clinic in Mumbai since 2006 and has been treating MDR-TB among HIV-infected individuals since May 2007. MDR-TB treatment became available in the public sector in Mumbai only in late 2010. Patients received empirical or when possible individualized therapy through an ambulatory, community-based program that we have described elsewhere . In summThe study was conducted from February 2012 to April 2012. All MDR-TB patients co-infected with HIV who were followed in the MSF clinic at the time of the study were eligible to be included in the study, regardless if they were on treatment during the study period or had completed the treatment earlier. There were no exclusion criteria for this study; only patients who died, were lost to follow-up or were transferred out were not included in the study.Patients underwent a detailed clinical evaluation: patients were asked for systemic and ocular complaints, including self-reported ones that may have been due to antiretroviral and/or antituberculosis therapy. Systemic evaluation was conducted as per the HIV clinic follow-up protocol: this consisted of clinical assessment and laboratory monitoring, including complete and differential blood counts, baseline hepatic and renal function tests, HIV markers , sputum collection on two consecutive days or extrapulmonary biopsy (commonly lymph nodes) as necessary. All collected samples underwent smear studies (with Ziehl Neelsen stain), culture and drug susceptibility testing for first and second line anti-TB drugs when culture was positive. Appropriate radiography was also carried out.Patients underwent a full ophthalmological evaluation by a consultant ophthalmologist familiar with ocular complications of HIV/AIDS, including visual acuity screening, slit lamp examination and dilated fundus examination of the entire retina in all patients using an indirect ophthalmoscope. The presence and severity of ocular inflammatory disease was assessed by documenting the presence, grade and type of anterior uveitis, presence and grade of vitreous cells , “snow-balls”, and the presence of cystoid macular oedema (CME), periphlebitis and intraocular pressure evaluation. We followed the currently accepted SUN (standardized uveitis nomenclature ) for thet-test, chi-square and Fisher’s exact test to assess differences between variables, as appropriate. Univariate and multivariate logistic regression and generalized linear models (GLM) were fitted to assess risk factors associated with the occurrence of ocular TB. Factors and covariates used as predictors included: age, sex, nadir CD4 count and CD4 count at the time of evaluation, HIV viral load, tuberculosis site (pulmonary/extrapulmonary), drug resistance pattern (MDR/XDR) and time on antiretroviral and anti-Tb treatment. We used the cohort’s mean age as a cut-off point in the modelling. For the parameter estimation in the generalized linear models we used the hybrid method and a robust estimator. The Wald statistic was used for the main analysis in the final model and the Wald type for the 95% confidence intervals. A p-value of less than 0.05 was considered to indicate statistical significance. The computer software SPSS  was used for analysis.Demographic and clinical information were systematically recorded in standardized clinical files. Information on HIV and antiretroviral therapy was entered into a specifically designed database. Data related to clinical evaluation of eyes were entered in a specific simple database. Data from all patients evaluated between February 2012 and April 2012 were included in the analyses. Patient characteristics were summarized using descriptive statistics. We used A total of 51 patients were examined during the course of this study. No patient refused consent to participate. Four patients were excluded from the analyses; two HIV-infected patients were found to have pan-susceptible TB and two patients with MDR-TB were not HIV-infected, but were followed in the HIV clinic because they were household contacts of co-infected patients. Thus, forty-seven patients were included in the analyses: 28 (59.6%) males, 17(36.2%) females and two (4.2%) transgenders. Ages ranged from 12 years to 54 years . The commonest subtype of HIV infection was type HIV-1  followed by HIV-2  and mixed HIV/1-2 infection . The median nadir CD4-count and the median CD4-count at the time of the evaluation were 153 cells/μL (IQR: 85–247) and 264 cells/μL (IQR: 158–361) respectively. Thirteen patients (27%) had detectable levels of HIV viremia (>20 copies/ml) at the time of the evaluation. Fourteen patients (24.8%) had been diagnosed with extrapulmonary TB (EPTB). The drug resistance patterns included 44 patients (93.6%) with multidrug resistance, and three patients (6.6%) with extensive drug resistance. Patients were on antiretroviral treatment for a median of 17.5 months (IQR: 4–32.5) and on second-line anti-TB treatment for a median of 13 months (IQR: 4–21) . The examination of one patient (2%) showed a corneal opacity and one patient (2%) had a well functioning bleb from prior glaucoma surgery. Both patients were on ART and anti-TB treatment at the time of the study. Among 36 patients on ART and anti-TB treatment at the time of clinical evaluation and among 11 patients who had completed anti-TB treatment prior to the study, the examination of the anterior chambers was normal in 34 patients (94.4%) and in 11 patients (100%) respectively.A dilated fundus examination revealed active or inactive ocular inflammatory disease in seven eyes of seven patients : 5.1-25.8%). These included five eyes of five patients (10.6%) with presumed choroidal tubercles : one scar in one eye of a patient on treatment and two scars in two eyes of two patients who had completed treatment . Other pathology seen included HIV retinopathy  and secondary open angle glaucoma  not related to TB or HIV-infection. No cases of multifocal choroidopathy, vasculitis, anterior uveitis, intermediate uveitis or panuveitis were seen in the overall cohort. Table Visual screening showed acuities ranging from 6/6 to hand movements. Causes of visual loss included CMV retinitis, presumed tubercular chorioretinitis, corneal opacities and uncorrected refractive errors. All the patients with choroidal tubercles were visually asymptomatic whereas the patient with chorioretinitis had profound visual loss due to the macular location of the disease. No cases of elevated intraocular pressure were found. One patient (2.1%) was on topical antiglaucoma medication but the intraocular pressure was normal. We found no patients with ocular adverse events due to antiretroviral or antituberculosis drugs in this Mumbai cohort as previously reported .In univariate analyses, co-infected patients with extrapulmonary tuberculosis or patients with both pulmonary and extrapulmonary disease were eleven times more at risk of ocular TB compared with those having only pulmonary TB . The only other factor independently associated with ocular TB was age below 39 years . The final multivariate model showed that immune status and duration of anti-TB treatment were not associated with the risk of ocular TB  were not seen in this study, probably due to effective antiretroviral therapy.In this Mumbai cohort, we observed a high yield of ocular tuberculosis that is possibly due to the greater propensity of HIV/AIDS patients to develop tuberculosis, along with the hyperendemic disease patterns of tuberculosis seen in India. Patients who develop secondary MDR-TB (due to incorrect or inadequate regimens), as well those who contract primary MDR-TB, are likely to have a delay in diagnosis, with a corresponding extended period of mycobacteraemia, that increases the risk of ocular infection.Our study reveals a higher prevalence of presumed ocular tuberculosis than that reported by Biswas et al. in a cohort of pulmonary tuberculosis patients in an era where drug resistance was presumably far less common, but less than that reported by Mehta et al. in a cohort of HIV/TB patients ,6. Both The commonest pattern of ocular inflammation encountered in this Mumbai cohort was choroidal tubercles representing presumed choroidal tubercular infection, followed by presumed tubercular chorioretinitis. Posterior segment ocular tuberculosis in the form of multifocal choroidopathy or vasculitis was not seen in this cohort. Similarly, the classical patterns of granulomatous or non-granulomatous anterior, intermediate or panuveitis were not seen in any of the patients in our cohort. Our study differs significantly from Babu et al. who noticed a lower prevalence and a distinctly different clinical pattern. This is probably because we studied all patients irrespective of visual symptomatology, allowing us to detect asymptomatic patients having ocular tuberculosis without visual morbidity. The large institutional cohort reported by Babu and co-workers consisted of visually symptomatic referrals to a large tertiary care center, often after a significant delay.Earlier reports from the developed world suggested mycobacterial ocular infections were much less common. In a study in 1987, tuberculosis was responsible for 0.2% of cases of posterior uveitis and no cases of anterior uveitis in Southern California . IncreasAmong the risk factors we studied with univariate and multivariate models, age and extrapulmonary tuberculosis were independently and significantly associated with the occurrence of ocular TB. Widespread dissemination of mycobacterial infection, which is often seen in patients with extrapulmonary tuberculosis, may explain the higher risk for ocular TB possible risk factor. Interestingly, in this cohort of co-infected patients, severe immunosuppression, as evidenced in the nadir CD4 T-cell counts, was not associated with heightened prevalence of ocular TB.Findings suggestive of ocular tuberculosis were recorded in two patients who had successfully completed their second line anti-TB regimen. These two patients underwent fluorescein angiography after an informed consent, and increased hyperfluorescence was seen suggestive of active intraocular inflammation. These patients, followed in our HIV clinic, are currently in good health, and are being closely monitored for the need for retreatment. Potentially, the use of ocular evaluation, in these groups of patients, may serve as a marker to monitor the therapeutic response. Larger cohorts that assess the ongoing ocular response of these patients may furnish data whether such patients may be better served by non-standard treatment regimens (therapeutic composition or duration) but such data are currently not available.There are some limitations to this study. First, the study design was cross-sectional and the data were collected from a cohort in a specific programmatic setting. Our findings may not be applicable to other populations with HIV/MDR-TB co-infection. Second, even though this Mumbai cohort is relatively large, we suggest caution in the generalization of these study findings and the study of risk factors. However, considering the small size of the global MDR-TB cohort on treatment ,13, our The detection of ocular inflammatory disease in these patients has several potential applications. First, a protocol that mandates retinal screening of all those presenting with low CD4 counts, and at subsequent regular intervals in certain cases, may help in the early diagnosis of ocular infections (tuberculosis and other opportunistic infections) as well as assess for drug toxicity (e.g. linezolid-induced optic neuropathy). Since specialty ophthalmologic consultation is rarely available in resource-limited settings , we haveSecond, assessment of tubercular healing may act as a clinical in vivo measure of systemic healing, even as an in vivo clinical tool for timing antitubercular therapy cessation, as compared to the current standard “one size fits all” duration of therapy. Investigating this possibility will require a prospective study correlating clinical outcomes with ocular outcomes documented with retinal photography.Ophthalmologists will increasingly encounter drug-resistant forms of TB disease and need to be familiar with the disease patterns and second and third lines of anti-TB therapy. Clinicians providing care to MDR-TB patients co-infected with HIV and public health officials involved in programs for such patients should be aware of the ocular manifestations and the implications for them.In conclusion, based on our findings, we suggest that ophthalmological examination should be routinely considered in HIV patients diagnosed with or suspected to have MDR-TB, especially in those with extrapulmonary TB.The authors declare that they have no conflict of interest.SM & PI conceived the study and wrote the first study protocol. PI & SM designed the study. SM collected the data with additional help from HM & SK. PI & SM analysed the data. SM & PI drafted the manuscript, and edited multiple versions with inputs from PS, HM & SK. All authors reviewed and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/225/prepubA fundus photo of 40 year old male with HIV 1 and extrapulmonary and pulmonary multidrug resistant tuberculosis showing a choroidal tubercle close to the optic nerve head.Click here for file


Catfish has a male-heterogametic (XY) sex determination system, but genes involved in gonadogenesis, spermatogenesis, testicular determination, and sex determination are poorly understood. As a first step of understanding the transcriptome of the testis, here, we conducted RNA-Seq analysis using high throughput Illumina sequencing.A total of 269.6 million high quality reads were assembled into 193,462 contigs with a N50 length of 806 bp. Of these contigs, 67,923 contigs had hits to a set of 25,307 unigenes, including 167 unique genes that had not been previously identified in catfish. A meta-analysis of expressed genes in the testis and in the gynogen  allowed the identification of 5,450 genes that are preferentially expressed in the testis, providing a pool of putative male-biased genes. Gene ontology and annotation analysis suggested that many of these male-biased genes were involved in gonadogenesis, spermatogenesis, testicular determination, gametogenesis, gonad differentiation, and possibly sex determination.We provide the first transcriptome-level analysis of the catfish testis. Our analysis would lay the basis for sequential follow-up studies of genes involved in sex determination and differentiation in catfish. Sex is one of the most fundamental features of life. In teleost, sex determination mechanism exhibits extraordinary plasticity and diversity during evolution. Fish represent over 50% of all vertebrates with over 32,400 species. The vast majority of fish species are gonochoristic although some fish are hermaphroditic. The latter is unique in fish among vertebrates Oryzias latipes) Oncorhynchus mykiss) Scophthalmus maximus) Cynoglossus semilaevis) O. latipes) Odontesthes hatcheri) O. luzonensis) O. latipes), DMY gene was found to be the sex determination gene, which is a duplicated gene of Dmrt1 on the Y-chromosome. In Fugu, there was no Y-specific gene that controls sex. Instead, a single nucleotide polymorphism within the anti-Mullerian hormone receptor type II (Amhr2) gene was found to control the sex amh) was suggested as a strong candidate of the master sex-determining gene in this species The most common sex determination system in fish is the XY system, i.e., male heterogamety. However, in many species, the sex determination system is the ZW system, i.e., female heterogamety. For instance, medaka  is the most important aquaculture species in the United States, and also an excellent model organism for teleost genetics and genomics studies Channel catfish (Two major approaches have been used for studies of sex-determining genes in teleost fish (using XY system as an example): 1) Identification of Y-specific sequences; 2) Identification of male-specific transcripts. In both cases, the sex-determining gene must be validated by functional analysis of the Y-specific sequences and male specific transcripts for their necessity and sufficiency for sex determination. Apparently, when the Y-chromosome is highly divergent from the X chromosome, the first approach is quite advantageous. However, no significant difference in DNA content was detected from male and female cells in channel catfish, which is consistent with the reports in many other fish species in which the karyotype of X chromosome and Y chromosome is very similar Nevertheless, analysis of genes expressed in male-specific organs has been utilized as one of the approaches to identify male-specific transcripts. Sex-biased genes expressed predominantly or exclusively in one sex were thought to drive the phenotypic differences between males and females In catfish, we have previously made major efforts for transcriptome analysis. Early efforts used EST analysis A total of 294.6 million reads of 100 bp long were generated. Ambiguous nucleotides, low-quality sequences  and short reads (length <30 bp) were removed, and the remaining high-quality reads (91.5%) were carried forward for transcriptome assembly and analysis .ABySS & Trans-ABySS was used for the assembly of the RNA-Seq short reads because they provided a superior assembly when compared with Velvet and CLC Genomics Workbench Gene identification of the assembled contigs was performed using BLASTX searches against the reference proteins available at NCBI zebrafish RefSeq and Uniprot protein databases to annotate the channel catfish testis transcriptome. After annotation, approximately 68,000 assembled contigs had hits in non-redundant database. A total of 55,521 assembled contigs had significant  hits to the zebrafish RefSeq database, corresponding to 17,180 unique proteins. In searches against the Uniprot database, 47,383 catfish contigs had significant hits, accounting for a total of 21,877 unique proteins. Cumulatively, a total of 67,923 catfish contigs had at least one significant hit against the two databases, allowing 25,307 unigenes to be identified in channel catfish testis transcriptome .Unique gene-coding contigs from the channel catfish testis reference assembly were then used as inputs to perform gene ontology (GO) annotation by Blast2GO To identify transcripts that were newly identified from the testis, all assembled contigs from testis transcriptome were used as queries to perform BLASTN searches against the existing catfish transcriptome. A total of 335 unique novel contigs from testis transcriptome had no blast hits against the existing catfish transcriptome assemblies. As more than one contigs had hits to a single unique gene, these 335 contigs corresponded to 167 putative unique novel genes when searched against the NR database using BLASTX , suggestIn silico mapping of RNA-Seq reads allowed 81% high quality sequencing reads of the testis RNA-Seq to be mapped onto the testis transcriptome, while 71% high quality sequencing reads from the gynogen catfish RNA-Seq In order to identify male-biased genes, i.e., genes that are preferentially expressed in the male, a meta-analysis of RNA-Seq data was conducted to compare expression ratios in the testis  and in the gynogen . The gynogen was a doubled haploid fish that are homozygous without the Y-chromosome BLASTX analysis indicated that many male-biased genes were involved in gonadogenesis, spermatogenesis, testicular determination, gametogenesis, gonad differentiation and sex determination . Some exGenomic locations of the male-biased genes were analyzed by sequence homology searches for their associated genomic draft sequence contigs. If the linkage group information is known for their associated genomic contigs, then by inference, their genomic locations are known. Analysis of genomic locations of the male-biased genes indicated that they are distributed all over the genome among all 29 linkage groups . Of the In this study, we conducted RNA-Seq analysis of the catfish testis that allowed expression profiling of genes expressed in the male-specific organ. In this process, over 25,307 unigenes were identified from the catfish testis, of which 167 unigenes were identified for the first time in catfish. This RNA-Seq analysis enhanced the transcriptome assembly in catfish, and will support the whole genome annotation of catfish.Our long term goal is to determine the sex determination mechanisms in catfish. In addition to transcriptome profiling, we were interested in exploring the possibility of identifying male-biased genes. We therefore compared the transcriptome expression profiles in the testis to those of the gynogen female catfish. The gynogen female, a doubled haploid female, has two identical copies of each chromosome but without the Y-chromosome A large number of genes were identified to express significantly higher in the male testis than in the gynogen fish. Overall, more than 5,000 had an expression ratio of greater than 5 fold in the testis than in the gynogen, however, only 637 genes were expressed much higher (>30 times) in the testis than in the gynogen fish. These putative male-biased genes can be interesting because they could be candidates contributing to gonadogenesis or testicular development and differentiation, or even sex determination. However, we realized the limitation of meta-analysis for the comparison of expression in different sexes. On the one hand, the genes important for sex differentiation are likely to be expressed in sex-specific organs such as the testis under this study. We understand that there is no direct control for such experiments because only male fish have testis. Therefore, we accepted the compromise in this initial study in order to more broadly capture the male-biased genes. In a way, the putative male-biased genes identified here can only serve as proxies of sex-biased genes. Nonetheless, a large number of genes with identities to known genes involved in gonadogenesis, spermatogenesis, testicular development and differentiation and sex determination were identified among the male-biased genes . InteresSilurus meridionals, North African catfish Clarias gariepinus, and Danio rerio, suggesting the importance of this gene family during gonad development, testis sex determination, and testis differentiation Of great interest in this study was the detection of the extremely high fold ratio in testis for three genes Dmrt1, Dmrta2 and Dmrt3a belonging to the Dmrt gene family. Characterized by a conserved DNA-binding motif known as the Doublesex- and Mab-3-related (DM) domain, Dmrt genes have been reported to be actively involved in sex determination and/or differentiation. These genes stimulate male-specific differentiation, but repress female-specific differentiation TDRD1 and TDRD7, both essential proteins for spermatogenesis, were found to be male-biased genes in this study. They were previously shown to be preferentially expressed in the murine testis In this study, we also identified PIWIL1 and PIWIL2 as male-biased genes. PIWIL1 and PIWIL2 are members of the mouse Piwi family proteins  that play important roles in spermatogenesis through transcriptional and post-transcriptional regulation. Acting as a functional partner of Piwi family proteins, TDRD group of Tudor proteins were reported to be physiological binding partners of Piwi family proteins and coordinately work together in the regulation of spermiogenesis in situ hybridization and detected very strong signal for DDX4 in rainbow trout testis, confirming its function in spermatogenesis regulation DDX4 and DDX11 exhibited high level of expression in the catfish testis, and were both identified as male-biased genes. DDX4 and DDX11, both members of the DEAD/DEAH box family of helicases, were believed to be involved in embryogenesis, spermatogenesis, and cellular growth and division Interestingly, Sox9, one of the most important genes expressed during testis determination in mammals All procedures involving the handling and treatment of fish used during this study were approved by the Auburn University Institutional Animal Care and Use Committee (AU-IACUC) prior to initiation of experiments. The fish used for this project were 25 male channel catfish including 13 one-year-old juveniles, 8 two-year-old adults and 4 four-year-old sexual maturation adults. All fish were sexed based on external genitalia, followed by anatomical confirmation. The fish were euthanized with tricaine methanesulfonate (MS 222) at 300 mg/L (buffered with sodium bicarbonate) before sample collection. Testis tissues from the 3 different ages were placed into 5 mL RNA later™  respectively. After one day of temporary storage at 4°C, samples immersed in the RNA later™ were transferred to a −80°C ultra-low freezer until preparation of RNA.Prior to RNA extraction, samples were removed from the −80°C freezer and ground with sterilized mortar and pestle in the presence of liquid nitrogen to a fine powder. Total RNA was extracted from testis powder using the RNeasy Plus Kit (Qiagen) treated with RNase free DNase I (Qiagen) to remove genomic DNA. RNA concentration and integrity was measured on an Agilent 2100 Bioanalyzer using a RNA Nano Bioanalysis chip. Equal amount of total RNA from catfish testis of each age was pooled together into only one sample for use in RNA-Seq.RNA-Seq library preparation and sequencing was carried out by HudsonAlpha Genomic Services Lab  as previously described by Li et al. and Sun et al. de novo assembly, raw sequencing reads were trimmed by removing adaptor sequences, ambiguous nucleotides (‘N’ at the end of reads), low quality sequences , and short read length sequences (length below 30 bp) with CLC Genomics Workbench  as previously described de Brujin graph approach with ABySS (version 1.3.4) Before The assembled contigs including both unique transcripts and singletons were used as queries for BLAST searches against the zebrafish RefSeq protein database, UniProtKB/SwissProt database and non-redundant database, respectively. Searches were conducted using the BLASTX program, with an E-value cut-off of 1e-10 and matching to the top hits. Gene ontology (GO) annotation analysis was performed using the zebrafish BLAST results in Blast2GO version 2.5.1, which is an automated tool for the assignment of gene ontology terms. The zebrafish BLAST result was imported to Blast2GO. The final annotation file was produced after GO-mapping, GO term assignment, annotation augmentation and generic GO-Slim process. The annotation result was categorized with respect to Biological Process, Molecular Function, and Cellular Component at level 2.in silico substraction were identified as putative newly identified transcripts.In order to identify putatively novel transcripts from the currently assembled testis transcriptome, the assembled contigs were used as queries to search against the existing catfish transcriptome databases To identify the sex-biased transcripts, high quality sequence reads from the male catfish testis RNA-Seq reads (current work) and the female doubled haploid catfish RNA-Seq reads Gene ontology (GO) annotation analysis was performed on the preferentially expressed genes in testis using Blast2GO version 2.5.1. The final annotation file was produced after GO-mapping, GO term assignment, annotation augmentation and generic GO-Slim process. The annotation result was categorized with respect to Biological Process, Molecular Function, and Cellular Component at Level 2.In order to determine the genome distribution, the male-biased genes were mapped to the catfish linkage groups by taking advantage of the existing genomic resources, especially the integrated genetic linkage and physical map Figure S1GO annotations of preferentially expressed genes in the testis(TIF)Click here for additional data file.Table S1Catfish testis transcriptome contigs and gene annotation.(XLSX)Click here for additional data file.Table S2Putative novel identified transcripts identified by channel catfish testis RNA-Seq.(XLSX)Click here for additional data file.Table S3Sex-biased genes detected by RNA-Seq analysis of channel catfish testis transcriptome(XLSX)Click here for additional data file.