The Na-H exchanger [NHE] performs an electroneutral uptake of NaCl and water from the lumen of the gastrointestinal tract. There are several distinct NHE isoforms, some of which show an altered expression in the inflammatory bowel diseases (IBD). In this study, we examined a role of NHE-2 in experimental colitis.Colitis was induced in male Sprague-Dawley rats by intra-rectal administration of trinitrobenzenesulphonic acid (TNBS). On day 6 post-TNBS, the animals were sacrificed, colonic and ileal segments were taken out, cleaned with phosphate buffered saline and used in this study.in vitro TNF-α treatment of uninflamed rat colonic segment. These changes were selective and localized to the colon as actin, an internal control, remained unchanged. Confocal immunofluorescence microscopy revealed co-localization of NHE-2 and NHE-3 in the brush borders of colonic epithelial cells. Inflamed colon showed a significant increase in myeloperoxidase activity and colon hypertrophy. In addition, there was a significant decrease in body weight and goblet cells’ mucin staining in the TNBS treated colon. These changes were not conspicuous in the non-inflamed ileum.There was a significant decrease in the level of NHE-2 protein as measured by ECL western blot analysis and confocal immunofluorescence microscopy. The levels of NHE-2 mRNA and heteronuclear RNA measured by an end-point RT-PCR and a real time PCR were also decreased significantly in the inflamed colon. However, there was no change in the level of NHE-2 protein in response to These findings demonstrate suppression of NHE-2 expression on the brush borders in the colonic epithelial cells which is regulated transcriptionally. However a role of TNF-α in the regulation of NHE-2 is discounted in the present model of colitis. This decrease in the NHE-2 expression will lead to a loss of electrolyte and water uptake thus contributing to the symptoms associated with IBD. The NHEkappa-B [NF-kB] binding site makes it respond to proinflammatory cytokines, infectious agents, and oxidative stress. Therefore, high levels of pro-inflammatory cytokines, such as interferon-γ [IFN-γ], tumor necrosis factor-α [TNF-α] in IBD might regulate NHE-2 expression leading to alteration in the secretion and absorption of electrolytes in the inflamed colon  solution containing 30 mg TNBS dissolved in 250 μl of 50% ethanol intra-rectally about 8 cm from the anal margin, and the control group in which each animal received phosphate buffered saline [PBS] in a similar fashion –25. The 2O2 converted to water per min per mg tissue.Colitis was characterized by measuring myeloperoxidase activity [MPO] following standard methods –25. TissBody weight [BW] of each animal was recorded before induction of colitis on day 0 and on day 6 post-TNBS just before sacrifice. Changes in the BW on day 6 were calculated with respect to their initial BW at day 0. Colon length and weights without feces were also measured on day 0 and day 6 post-TNBS, and colon hypertrophy was expressed as weight [mg] per cm length.Standard methods were used to fix tissues in paraformaldehyde and for embedding in paraffin blocks . Colon sTissue segments were chopped finely with scissors using 10 ml [per gm tissue] of ice cold MOPS-sucrose (3 [N-Morpholino]propanesulfonic acid) buffer, pH 7.4 –25. The The samples containing 2–3 mg/ml protein were prepared using a 4 x sample buffer, heated in a boiling water bath for 5 min and then transferred directly onto an ice bath before loading onto an 8% polyacrylamide gel. The buffer and gel solutions were prepared following methods as used earlier in this laboratory –25. LysaLevels of NHE-2 protein were estimated by measuring the fluorescence units obtained by confocal immunofluorescence microscopy. Paraformaldehyde fixed colonic tissues were embedded in paraffin to prepare paraffin blocks. Tissue sections [7 μm thickness] were placed onto glass slides, deparaffinized, dehydrated and rehydrated using graded ethanol solutions . SectionTo understand the underlying mechanism of NHE-2 protein expression we quantitated the levels of NHE-2 transcript. Two approaches, an end-point cycle RT-PCR and a SYBR-green real time RT-PCR were used to estimate the level of NHE-2 mRNA. For this purpose upstream [PR1] and downstream [PR2] primers were designed using a published NHE-2 cDNA sequence . The mRNAn aliquot of total RNA (1μg) was reverse transcribed using 1 pmol of PR3 primer using a single step RT-PCR kit [Amersham], and subsequently amplified for 28 cycles using the primers PR1 and PR2 [100 pmoles each]. The following cycle settings were used: denaturation [94°C x 30 sec]; annealing [55°C x 30 sec]; extension [74°C x 30 sec]. The PCR product  was separated by the agarose gel electrophoresis, and purified. An optimal concentration of the purified CC measured separately was used to spike each sample for its co-amplification with the NHE-2 transcript. This process was called as competitive RT-PCR method because the same set of primers were used to amplify both the target and the control. In this method variations dependent on the structure and composition of the primers, the target and the control are minimized, and hence this quantitation is more accurate than the normal RT-PCR in which a house-keeping gene is amplified using two different sets of primers.Total RNA from rat colon was extracted using a Trizol RNA extraction kit following instructions from the supplier [GIBCO]. Concentration and purity of total RNA were estimated from optical densities obtained at 260 and 280 nm using a spectrophotometer [Pharmacia].The quality of total RNA was assessed using a formaldehyde agarose gel electrophoresis and a nano-drop method . An aliquot of total RNA [5–10 μg] was mixed with an equal volume of 2 x RNA buffer [Ambion] and heated at 80°C for 5 min. The samples were then separated electrophoretically on a 1.4% agarose gel containing 1% formaldehyde, or on 8% polyacrylamide gel as described earlier , 24–25. Different concentrations of CC were used to obtain an optimal concentration for quantitation. A known optimized concentration of CC was then used to spike both the control and colitis samples, and co-amplified by 28 cycles. Individual densities of both the target and the CC bands was obtained from each lane, and a ratio of NHE-2:CC was calculated. This ratio was therefore used to express the level of NHE-2 mRNA in this study. This data was further confirmed using a SYBR green real time PCR method.An aliquot of total RNA [1μg] was mixed with the primers PR1 and PR2 [100 pmoles of each], buffer and 1 unit of Platinum Taq DNA polymerase supplied with the kit [Promega]. Reactions were performed as follows: Reverse transcription: 42°C x 45 min → 30 . A standard curve between the cycle threshold [CT] and concentrations of purified CC was prepared separately. The Corresponding CT values were obtained for both the non colitis control and the colitis RNA samples. The level of NHE-2 mRNA was calculated using the CT curve. A change in the mRNA was calculated with respect to the control CT values.www.ncbi.nlm.nih.gov) with the published sequence.The gel-purified PCR fragment was sequenced using a non-radioactive DNA sequencing method [Perkin-Elmer], with the downstream primer using a standard capillary gel electrophoresis method  in the RCF, Health Sciences Center, Kuwait University, Kuwait. Nucleotide sequence was identified using the Blast program  in the steady state level of NHE-2 protein in the crude lysates from inflamed colon as compared to non-colitis controls Figs . On the Since the western blot analysis has multiple variables, it was necessary to support this data with confocal microscopy. The confocal data as shown in The quality and yield [μg/mg tissue] of total RNA were consistent in both the control (C) and colitis (T) samples . The NHEThe level of NHE-2 mRNA measured using the end-point RT-PCR  in inflaThe level of HnRNA was also significantly decreased in inflamed colon . This dein vitro did not show any difference in the expression of NHE-2 protein levels as compared to untreated controls [data not shown].TNF-α has been shown to suppress NHE-2 expression in the epithelial cell line through interaction with NFkB region present in the NHE-2 promoter region . In our + and Ca+2 play an important role in maintaining these functions. Sodium is transported through multiple mechanisms including Na-H exchanger which is located in the plasma membrane and in the inner compartment of eukaryotic cells  and known concentrations of the competitive control was constructed to calculate the level of NHE-2 mRNA expression. Interestingly, the real time PCR findings also showed a similar reduction in the level of NHE-2 mRNA, suggesting that the decrease in NHE-2 protein levels is due to a decrease in the NHE-2 mRNA abundance in inflamed tissue. Furthermore, the yield and the integrity of total RNA used in this study were not different in the inflamed colon than the controls . TherefoTo investigate if transcription has a role in regulating the level of mRNA in inflamed tissue, we measured the level of heteronuclear RNA using the real time RT-PCR with SYBR green. For this purpose we used an upstream primer (PR4) selected from an intron, and a downstream primer (PR5) selected from an adjoining exon of NHE-2 gene sequence . We usedin vitro. Since the level of these inflammatory mediators are induced in IBD [ex vivo findings discount a role of TNF-α in the suppression of NHE-2 in the experimental colitis (data not shown). Our findings are different than those reported earlier [in vivo for the action of TNF-α as compared to the present ex vivo study. In addition, recombinant cells are different in many respects including that they carried a recombinant NHE-2 gene promoter [ex vivo condition used in the present study. It is worth noting that TNF-α is commonly used to treat Crohn’s disease, and in this present model TNF-α and other cytokines have been shown to be upregulated [Studies using recombinant cells have shown that NHE-2 gene is immune responsive as it responds to TNF-α and INFγ d in IBD , 20, we d in IBD , 28–30.  earlier  which copromoter  which waegulated –19.In conclusion, taken together these findings suggest that NHE-2 is colocalized with NHE-3 on the apical domain of the colonic epithelial cells, and that NHE-2 is more abundant than the NHE-3 in the colonic epithelial cells. The decrease in NHE-2 expression together with NHE-3 and NHE-8 , 31–35 m


N=196). The results indicated that there were no statistically significant differences between the intervention and control groups on change in HRQOL, but the final regression models were statistically significant. SF-36 mental component summary (MCS) score at baseline, stress at baseline, and change in stress were significant predictors for predicting change in the SF-36 MCS. SF-36 physical component summary (PCS) at baseline and change in stress were significant predictors for predicting change in the SF-36 PCS. The findings suggest that the development of an effective intervention in improving HRQOL should be considered within individual, interpersonal, societal, and cultural factors for future research and clinical practice.The purpose of this study was to examine the effect of a six-month lifestyle-based intervention on health-related quality of life (HRQOL) in older adults with hypertension. A secondary analysis of a randomized controlled trial was conducted to test the differences between the intervention and control groups on HRQOL ( The older population is growing significantly in the United States and global society. In 2014, the number of Americans aged 65 and above was 46 million, representing 15% of the total U.S. population; by 2030, the older population is estimated to be about 21% of the total population and one in 13 will be older than 85 [The World Health Organization (WHO) has emphasized the importance of assessing and promoting people's quality of life . One of Studies suggested that stress and social support may impact HRQOL in older adults –16. FriaThe conceptual framework guiding the current study was derived from the Social Cognitive Theory  and liteThe purpose of this study was to test the effects of a 6-month lifestyle-based intervention on HRQOL in older American adults with hypertension, accounting for stress and social support as mediating variables. After receiving a 6-month lifestyle-based intervention, the intervention group was hypothesized to significantly improve in person (stress) and environment  (H1) and outcome (HRQOL) (H2) in older adults with hypertension from pretest (baseline) to posttest (6 months) compared to the control group.This study was approved by the Institutional Review Board (IRB) of the University of North Carolina at Greensboro (study#: 14-0428). The sample in the current study was drawn from the Well Elderly 2 Study. The data from the Well Elderly 2 Study were provided by the Inter-university Consortium for Political and Social Research , 31. BriIn the present secondary analysis, we used only data collected during the first 6 months of the study (baseline and the first 6-month time point). Participants who self-reported taking blood pressure medication at baseline in the Well Elderly 2 Study were selected as subjects.Demographic characteristics and medical history obtained by participants' self-report included gender, age, race, educational level, prescription medications, over-the-counter medicine, and diagnosis.Stress was measured using the adapted Perceived Stress Scale . The PerSocial support was assessed using the Lubben Social Network Scale (LSNS) . The LSNHRQOL was measured using the 36-Item Short-Form Health Survey  , 39. TheH1 and H2, multiple linear regression using a hierarchical regression model building approach was performed to test the effect of the intervention and make predictions on criterion variables [p value < 0.05 was considered statistically significant. All analyses were performed using SPSS version 23 .Descriptive statistics were initially calculated using means and standard deviations or frequencies and percentages. Continuous variables were checked for outliers and normality in univariate analysis. To test hypotheses ariables , 44. Demp ≥ 0.05).There were a total of 196 participants in this study. Of the 196 participants, 103 were randomly assigned to the intervention group and 93 to the control group. p ≥ 0.05).At baseline, the average stress score was 43.7 ± 10.7, and the average of social support score was 27.2 ± 9.1. The mean scores of the SF-36 mental component summary (MCS) and physical component summary (PCS) were 46.7 ± 11.4 and 39.6 ± 10.1, respectively. As shown in p < 0.001). In the final hierarchical regression model, demographic variables , stress at baseline, and intervention versus control significantly accounted for 25% of the variance in change in stress (R2=0.25). In addition, stress at baseline accounted for a significant amount of variance in change in stress after controlling for the effect of demographic variables based on the second step of the hierarchical model building . Four years of college or more, Hispanic/Latino versus White Americans, and stress at baseline were significant predictors. Adjusting for all other factors, participants who received four years of college or more education were associated with a 0.22 increase in standard deviation (SD) units of predicted change in stress compared to participants who received less than high school; Hispanic/Latino participants were associated with a 0.20 increase in SD units of predicted change in stress compared to White participants. For every 1 SD increase in stress at baseline, the predicted mean decrease in change in stress was 0.49 SD units, adjusting for all other factors.p < 0.01). In the final hierarchical regression model, demographic variables, social support at baseline, and intervention versus control significantly accounted for 18% of the variance in change in social support (R2=0.18). Additionally, social support at baseline accounted for a significant amount of variance in change in social support, after controlling for the effect of demographic variables . The only significant predictor was social support at baseline, where for every 1 SD increase in social support at baseline, the predicted mean decrease in change in social support was 0.37 SD units, adjusting for all other factors.p < 0.001). In the final hierarchical regression model, demographic variables, SF-36 MCS score at baseline, intervention versus control, stress at baseline, change in stress, social support at baseline, and change in social support significantly accounted for 39% of variance in change in the SF-36 MCS (R2=0.39). The SF-36 MCS at baseline accounted for a significant amount of variance in change in the SF-36 MCS, after controlling for the effect of demographic variables in the second step of modeling . In the last step, stress at baseline, change in stress, social support at baseline, and change in social support accounted for a significant amount of variance in change in the SF-36 MCS after controlling for the effect of demographic variables, SF-36 MCS score at baseline, and the effect of intervention . The SF-36 MCS score at baseline, stress at baseline, and change in stress were significant predictors in the final model. For every 1 SD increase in the SF-36 MCS at baseline, the predicted mean decrease in the change in the SF-36 MCS was 0.66 SD units; for every 1 SD increase in stress at baseline, the predicted mean decrease in change in the SF-36 MCS was 0.27 SD units; for every 1 SD increase in change in stress, the predicted mean decrease in change in the SF-36 MCS was 0.28 SD units, adjusting for all other factors.p < 0.05). In the final hierarchical regression model, demographic variables, SF-36 PCS score at baseline, intervention versus control, stress at baseline, change in stress, social support at baseline, and change in social support significantly accounted for 18% of the variance in change in the SF-36 PCS (R2=0.18). The SF-36 PCS score at baseline accounted for a significant amount of variance in the change in the SF-36 PCS after controlling for the effect of demographic variables . The SF-36 PCS at baseline and change in stress were significant predictors of change in PCS scores in the final model. For every 1 SD increase in the SF-36 PCS at baseline, the predicted mean decrease in the change in the SF-36 PCS was 0.38 SD units; for every 1 SD increase in change in stress, the predicted mean decrease in change in the SF-36 PCS was 0.18 SD units, adjusting for all other factors.This secondary analysis examined the effectiveness of a lifestyle-based intervention on HRQOL in older adults with hypertension and investigated stress and social support as mediating variables. As many older adults suffer from hypertension, developing effective interventions to enhance older adults' HRQOL is necessary for healthy aging. The results of this analysis provide empirical evidence, advance the scientific knowledge, and propose intervention recommendations for future research and clinical practice in older adults with hypertension.The findings of the study indicated that there were no statistically significant intervention effects on stress, social support, and HRQOL, but the final regression models were statistically significant in the last step of the hierarchical multiple regression analysis. According to Baron and Kenny (1986) criteria for a mediation analysis, social support and stress failed to function as mediators in the current study . This reIn this study, stress at baseline and change in stress were significant predictors in predicting the mental component of HRQOL; change in stress was a significant predictor in predicting the physical component of HRQOL. Gerber also found that higher perceived stress was significantly associated with poorer mental HRQOL in older adults . In addiAging is a multifaceted process and is related to reduced functional capacity and chronic diseases –49. ManyThe current study has several limitations. First, the effect of the lifestyle-based intervention was tested from pretest (baseline) to posttest (the 6-month time point). Hence, the lifestyle intervention may not have significant short-term effects on change in social support, stress, and HRQOL , 50, 51.There is limited research to test the effects of lifestyle interventions on HRQOL in older adults with hypertension. In this study, the results revealed that the regression model is statistically significant in predicting changes in HRQOL according to lifestyle-based intervention, stress, and social support. Educational levels, race, stress at baseline are significant predictors for predicting change in stress; social support at baseline is the significant predictor for predicting change in social support. In addition, SF-36 MCS score at baseline, stress at baseline, and change in stress are significant predictors of change in MCS scores in the final model. SF-36 PCS score at baseline and change in stress are significant predictors for predicting change in the SF-36 PCS. As many older adults have high blood pressure and reduced HRQOL, developing effective interventions in promoting hypertension self-management and improving HRQOL for older adults with hypertension is essential. This secondary analysis suggests that stress management and social support resources should be included in the lifestyle intervention for future research and clinical practice. The results indicate that the development of an effective intervention in improving HRQOL should be considered within individual, interpersonal, societal, and cultural factors when implementing the lifestyle-based interventions.


The United States Food and Drug Administration (FDA) reviews class III orthopedic devices submitted for premarket approval with pivotal clinical trials. The purpose of this study was to determine the types of orthopedic devices reviewed, the design of their pivotal clinical trials, and the subjective factors affecting the interpretation of clinical trial data.Meetings of the FDA Orthopaedic and Rehabilitation Devices Panel were identified from 2000–2016. Meeting materials were collected from FDA electronic archives and notes were made regarding the device-type and subsequent approval and recall, the design of pivotal clinical trials, and issues of trial interpretation debated during panel deliberations.The panel was convened on 29 separate occasions over the course of 35 days to deliberate 38 distinct topics. Of these, 23 topics included clinical data submitted for approval of a device, and two topics were excluded. Of the 23 devices, five were biologic, three were hip arthroplasty, three were disc arthroplasty, two were viscosupplementation, three were interspinous process devices, and seven were other devices. Of the 23 pivotal trials, 20 (87.0%) were randomized controlled trials (RCTs), consisting of 13 (65.0%) non-inferiority trials and 7 (35.0%) superiority trials, and all RCTs were two-arm trials. At panel, the most commonly debated issues were related to the design and interpretation of non-inferiority trials.A broad array of device types is reviewed by the FDA. The predominance of two-arm non-inferiority trials as pivotal studies indicates that the nuances of their design and interpretation are commercially important. Orthopedic surgery is one of the largest segments of the medical device industry. In the United States jurisdiction, orthopedic devices are regulated by the Division of Orthopedic Devices, Office of Device Evaluation, Center for Devices and Radiologic Health of the Food and Drug Administration (FDA). With few exceptions, many of the most complex, novel, or high-risk orthopedic devices (class III devices) are subject to premarket approval (PMA), the FDA’s most intensive review process for devices . TypicalThe FDA utilizes approximately 50 committees and panels that provide expert advice on matters of science and policy. Of these, the Orthopaedic and Rehabilitation Devices Panel provides advice on orthopedic devices, and PMA applications may be subject to review in a public forum. Though not all PMAs come before the panel , the PMA process including panel review represents the most intensive scrutiny and highest level of scientific deliberation that an orthopedic device may receive in consideration of regulatory approval.The purpose of the present study was to assess the design of pivotal trials of orthopedic devices and their interpretation at FDA panel meetings. This is important because IDE trials are frequently the best scientific evidence available for both regulatory affairs and clinical decision making. The relationship of device characteristics to the rate of device approval and recall was studied, since these are mechanismfigurs by which devices enter and exit the market for routine clinical use. Similarly, the characteristics of the IDE trials were reviewed including the trial designs and their relationship to approval and recall. Finally, those clinically relevant aspects of trial structure that affect panel deliberations were identified.Meetings of the Orthopaedic and Rehabilitation Devices Panel were identified from the beginning of the 2000 calendar year to 2016 from official announcements in the Federal Register. Meeting materials were obtained from the FDA electronic archives, including panel-packs, sponsor executive summaries, FDA executive summaries, rosters, postmeeting summaries, and official meeting transcripts. Notes were made regarding the purpose of the meeting as either reclassification, special advisory, PMA, or premarket notification ).For PMA and 510(k) topics, IDE trials were classified as a randomized clinical trial (RCT) versus observational/nonrandomized study, and superiority versus non-inferiority design. The number of arms and the type of control were recorded. Notes were made regarding comments and questions of panelists regarding complexities in the interpretation of clinical trial data, as reflected in the official transcripts when available. Comments were qualitatively categorized into three major categories: outcome measures, randomized trial conduct, and issues specific to non-inferiority trials. Among outcome measures, subtopics included time points, primary composite endpoint, secondary endpoints, surrogate radiographic endpoints, choice of clinical instruments, and time of final follow-up. For randomized trials, subtopics included power, randomization ratio, blinding, number of arms, choice of control procedure, and rate of crossover, dropout, and missing data. For issues specific to non-inferiority trials, subtopics included the choice of active control, the choice of non-inferiority margin, assay sensitivity, and analysis of datasubsets , modified ITT, as-treated (AT), and per-protocol (PP) analyses).For each device, a note was made of the device type—biologic, hip arthroplasty, disc arthroplasty, viscosupplementation, interspinous process device, or other device . The subsequent approval and recall of devices was noted up to the time of writing. The date of each conducted panel meeting was noted. For the purposes of hypothesis testing, the number of meetings per year was analyzed by dividing the investigation period (2000–2016) into two roughly comparable portions—years 2000–2008 and 2008–2016 inclusive. The rate of device approval was tested with the rate-ratio test for the intensity of Poisson processes. The subsequent approval and recall of devices was investigated up to the time of writing. For the purposes of hypothesis testing, devices were grouped in four dichotomous ways: spinal versus nonspinal, arthroplasty versus nonarthroplasty, biologic versus nonbiologic, and other devices versus non-other. Hypotheses were tested with the Fisher exact test.Between 20 July 2000 and 20 April 2016, the panel was convened on 29 separate occasions over the course of 35 days to deliberate 38 distinct topics. Table Of the 23 unique devices with pivotal IDE trial data, 20 (87.0%) were RCTs, consisting of 13 (65.0%) non-inferiority trials and seven (35.0%) superiority trials. The remaining three (13.0%) studies were observational/non-randomized. All non-inferiority studies (13) were two-arm trials versus an active control, and none was a three-arm study including a sham control. Table p > 0.05). The results do not change by excluding the middle year 2008 or by assigning it to the earlier or later period.In total, five devices were biologic, three were hip arthroplasty, three were disc arthroplasty, two were viscosupplementation, three were interspinous process devices, and seven were other devices. Figure p > 0.05). Regarding device type, there was no significant difference in the rate of approval between spinal versus nonspinal devices (p = 0.66), biologic versus nonbiologic devices (p = 0.28), and other devices versus non-other devices (p = 0.35). The only factor affecting approval was whether the device was an arthroplasty device of any type (p = 0.047), though this is no longer significant after multitest correction.Of 23 devices, the approval status was known for 21 as of the time of writing, as approval decisions and their disclosure takes time from the date of the panel meeting. Of these 21 devices, fourteen devices were approved subsequent to panel review (66.7%), and no device directly reviewed by the panel was subsequently recalled. The clinical trial type of superiority versus non-inferiority was not significantly associated with approval  premarket notification . SubsequRegarding device approval, approximately two-thirds of devices were approved at the time of writing. Arthroplasty devices had the highest rate of approval, and biologic devices had the lowest rate of approval. The only device-related factor associated with approval was whether the device was for arthroplasty; however, this classification represented a diverse group of devices from hip to cervical spine arthroplasty. Of note, it also included a number of hard-on-hard bearing surfaces for hip arthroplasty. Metal-on-metal bearing surfaces have become controversial, and were the subject of a special FDA advisory panel during the study period . DespiteA broad range of devices is reviewed by the FDA’s Orthopaedic Devices Panel. Although few device or trial-related factors affect approval or recall, panel review is associated with no recent recalls. The majority of devices are studied in pivotal two-arm non-inferiority trials which are subject to complex technical issues and nuances in their interpretation.


All six World Health Organization (WHO) regions have established measles elimination goals, and three regions have a rubella elimination goal. Each region has established a regional verification commission to monitor progress toward measles elimination, rubella elimination, or both, and to provide verification of eliminationCountries report data from measles and rubella cases identified through laboratory-supported case-based surveillance systems to WHO. Laboratory testing includes both serologic and molecular confirmation of suspected cases and genetic characterization of viruses from confirmed cases. Participating GMRLN laboratories report MeV and RuV sequence dataAccording to the monthly reports of 184 countries that reported measles and rubella case-based surveillance data in 2018, a total of 317,445 serum specimens were received by the participating GMRLN laboratories from patients with suspected cases, an increase of 101% compared with the number of specimens received in 2016. Among 275,020 (87%) specimens tested for measles immunoglobulin M, 78,950 (29%) were positive; 203,898 (64%) also were tested for rubella immunoglobulin M, and 11,874 (6%) were positive. By the end of 2018, MeaNS contained 47,521 MeV sequences, a 93% increase from the 24,571 sequences reported as of July 1, 2015 . Among the 10 most commonly reported named strains, two appeared in all six regions .During 2016–2018, five of the 13 recognized RuV genotypes were detected, and the number of detected RuV genotypes decreased from five in 2016 (58% of the sequences belonged to genotype 1E and 40% to genotype 2B) to two (1E and 2B) in 2018 . HoweverGMRLN continues to provide high-quality laboratory support to surveillance for measles and rubella virus transmission and critical evidence needed for the verification of elimination. The increase in serologic testing and the number of sequences reported to the databases reflect an expansion of the capacity of GMRLN as well as the resurgence of measles in many countries during 2018. With support of the molecular surveillance data provided by GMRLN, measles elimination has been verified by 81 (42%) of the 194 WHO member countries and rubella by 76 (39%) of the 194 countries.The MeaNS database recognizes distinct N450 sequences and assigns DSIds to enable the identification of related MeVs in different countries and regions. In addition, a convention of naming the MeV strains with the same DSId is used. However, when defining endemic circulation of a specific MeV strain, caution should be exercised in interpreting the significance of MeV N450 sequences with different DSIds, named strains, or both. Given the conserved nature of the MeV genome, even within the highly variable N450 coding region, identical N450 sequences can be detected over multiple years and thus might not be linked or in the same direct line of transmission within a country or region. Conversely, sequences with a single nucleotide difference within an identified short chain of MeV transmission will be given different DSIds, with different names, even though they might be epidemiologically linked.The current naming convention does not describe MeV lineages derived from sequence analysis of regions of the MeV genome other than N450. To further differentiate viral transmission chains, additional sequence information from other regions of the genome is needed. Using an expanded sequence window in addition to the N450 sequence has been proposed for countries and regions where measles has been eliminated or is nearing elimination  accounted for 95% of reported sequences. Of 13 rubella virus genotypes, reported genotypes declined from five to two.Diversity of measles and rubella viruses has decreased globally, consistent with progress toward elimination. Continued collection of specimens from all confirmed cases for genotyping and submission of wild-type virus sequences to global databases will strengthen case-based surveillance.


A new family of fluorescent thiophene and thienothiophene-containing squaraine dyes is described with tunable wavelengths that cover the absorption/emission range of 600–800 nm. The deep-red and near-infrared fluorescent compounds were easily prepared by simple synthesis and purification methods. Spectral studies showed that each squaraine was rapidly encapsulated by a tetralactam macrocycle, with nanomolar affinity in water, to produce a threaded supramolecular complex with high chemical stability, increased fluorescence quantum yield, and decreased fluorescence quenching upon dye self-aggregation. Energy transfer within the supramolecular complex permitted multiplex emission. That is, two separate dyes with fluorescence emission bands that match the popular Cy5 and Cy7 channels, could be simultaneously excited with a beam of 375 nm light. A broad range of practical applications is envisioned in healthcare diagnostics, microscopy, molecular imaging, and fluorescence-guided surgery. Organic dyes that emit fluorescence in the near infrared (NIR) window 650–900 nm) are needed for biological imaging as this wavelength region has relatively deep tissue penetration, minimal tissue auto fluorescence, and lower Rayleigh scattering of the light ,2,3. At 0–900 nm Squaraine dyes are a well-known family of NIR fluorescent dyes with intense and narrow absorption bands, high fluorescence quantum yields, and excellent photostabilities . BecauseS1–S3) that were easily prepared from commercially available materials in a few steps, and water-soluble analogues (S1PEG–S3PEG) that were obtained by appending long polyethylene glycol (PEG) chains. We find that the dyes form supramolecular complexes with the tetralactam macrocycles M1 or M2 to produce a suite of 12 fluorescent compounds that span the absorption window of 600–800 nm, making them a very attractive set of dyes for a wide range of future biological imaging applications.We decided to develop a new set of squaraine chromophores with incrementally extended absorption/emission wavelengths. We knew that squaraine structures containing aminophenyl or aminothiophene units would be useful for operation in the region of 600–700 nm . The reaS1 and S2, were synthesized by similar procedures using N,N-dibenzylaminophenyl semisquaraine 2 as a common building block  at one or both ends of the central chromophore, which ensures that macrocycle threading is facile   1 thiophene (4): Thienothiophene  was dissolved in acetic acid (10 mL). N-bromosuccinimide  was added and the reaction mixture was stirred at room temperature for 2 h. Solvent was removed and the residue was dissolved in diethyl ether (80 mL) and washed with NaOH , H2O (100 mL × 3) and brine (100 mL). The resulting solution was dried over Na2SO4, and the solvent was removed by rotary evaporation to obtain pure 4 as a light yellow liquid . 1H-NMR  δ 7.40 , 7.28 , 7.20–7.14 . 13C-NMR  δ 127.57, 126.78, 122.32, 121.98, 119.60, 119.24. HRMS-ESI m/z 217.8837 .2-amino)ethan-1-ol (5): Compound 4 , Cu0 , CuI  and K3PO4·H2O  were suspended in 2-(methylamino)ethanol (15 mL). The reaction mixture was stirred at 90 °C over 5 h. After cooling to room temperature, H2O (80 mL) was added, and the reaction mixture was extracted with diethyl ether (100 mL × 3). The organic phase was combined and dried over Na2SO4, the solvent was removed and the residue was purified by column chromatography  using 10–40% EtOAc/hexane to obtain pure 5 as a light yellow solid . 1H-NMR  δ 7.10 , 7.07 , 6.12 , 3.99 , 3.80 , 3.40 , 3.00 . 13C-NMR  δ 206.11, 160.53, 139.67, 120.16, 119.69, 93.96, 59.29, 57.85, 40.43, 29.67, 29.51, 29.36, 29.21, 29.05. HRMS-ESI m/z 214.0379 .N-methyl-N-(2-(prop-2-yn-1-yloxy)ethyl)thienothiophen-2-amine (6): Propargyl bromide  was added to a mixture of compound 5 , aqueous NaOH  and toluene (15 mL). TBA HSO4 (200 mg) was added to the mixture as a phase transfer reagent and the resulting solution was stirred at room temperature for 5 h. Toluene was removed by rotary evaporation and H2O (100 mL) was added to the residue. The resulting solution was extracted with diethyl ether (100 mL × 3). The organic phase was combined and dried over Na2SO4, the solvent was removed and the residue was purified by column chromatography  using 5–20% EtOAc/hexane to obtain pure 6 as a yellow liquid . 1H-NMR  δ 7.11 , 7.09 , 6.16 , 4.18 , 3.75 , 3.49 , 3.00 , 2.93 . 13C-NMR  δ 205.53, 160.12, 139.58, 125.88, 120.28, 119.63, 94.26, 80.10, 75.36, 67.20, 58.03, 54.88, 40.36. HRMS-ESI m/z 252.0540 .S2: Compound 6  and 3-(4-(dibenzylamino)phenyl)-4-hydroxycyclobut-3-ene-1,2-dione  were dissolved in a mixture of 1-butanol (15 mL) and benzene (45 mL). The reaction was heated to reflux with Dean-Stark distillation for 2 h. After cooling to room temperature, the solvent was removed and the residue was purified by column chromatography using 0–7% MeOH/CHCl3 to obtain pure S2 as green solid . 1H-NMR  δ 8.28–8.14 , 7.39–7.27 , 7.25–7.13 , 6.85 , 6.23 , 4.77 , 4.17 , 3.81 , 3.67 , 3.25 , 2.44 . 13C-NMR: Limited compound solubility prevented 13C analysis. HRMS-ESI m/z 602.1704 .S2PEG: Compound S2 , azido-mPEG453 , triethylamine (2 drops) and TBTACu(I)Br (2 mg) was dissolved in CHCl3 (5 mL) and the reaction mixture was stirred at room temperature for 12 h. After removing the solvent, the residue was purified by column chromatography (silica gel as stationary phase) using 0–10% MeOH/CHCl3 to obtain the crude product. The crude product was further purified by a second column  using 0–5% MeOH/CHCl3 to obtain pure S2PEG as green solid . 1H-NMR  δ 8.23–8.20 , 7.69 , 7.35 , 7.29 , 7.23–7.18 , 6.84 , 6.23 , 4.76 , 4.64 , 4.54–4.46 , 3.84–3.71 , 3.70–3.48 , 3.37 , 3.20 . MS-MALDI (DHBA as matrix) showed a set of peaks around 2421, which reflected the polydispersity of the PEG45 chains.2-amino)ethan-1-ol (7): Compound 4 , Cu0 , CuI  and K3PO4·H2O  was suspended in 2-(ethylamino)ethanol (15 mL). The reaction mixture was stirred at 90 °C over 5 h. After cooling to room temperature, H2O (80 mL) was added, and the reaction mixture was extracted with diethyl ether (100 mL × 3). The organic phase was combined and dried over Na2SO4, the solvent was removed and the residue was purified by column chromatography  using 10–40% EtOAc/hexane to obtain pure 7 as light yellow solid . 1H-NMR  δ 7.10 , 7.07 , 6.17 , 3.97–3.81 , 3.76 , 3.45–3.35 , 1.19 . 13C-NMR  δ 205.64, 159.34, 139.55, 120.06, 119.63, 94.31, 59.32, 55.29, 48.45, 29.53, 29.38, 29.22, 29.07, 28.92, 11.67. HRMS-ESI m/z 228.0534 .N-ethyl-N-(2-(prop-2-yn-1-yloxy)ethyl)thienothiophen-2-amine (8): Propargyl bromide  was added to a mixture of compound 7  in toluene (15 mL) and NaOH solution . TBA HSO4 (200 mg) as phase transfer reagent was added to the mixture and the resulting solution was stirred at room temperature for 5 h. Toluene was removed by rotary evaporation and H2O (100 mL) was added to the residue. The resulting solution was extracted with diethyl ether (100 mL × 3). Organic phase was combined and dried over Na2SO4, the solvent was removed and the residue was purified by column chromatography  using 5–20% EtOAc/hexane to obtain pure 8 as a yellow liquid . 1H-NMR  δ 7.11 , 7.08 , 6.18 , 4.18 , 3.72 , 3.48 , 3.39 , 2.93 , 1.19 . 13C-NMR  δ 205.55, 159.01, 139.54, 125.67, 120.24, 119.65, 94.55, 80.14, 75.39, 67.45, 58.09, 52.51, 48.43, 29.42, 29.27, 29.12, 11.82. HRMS-ESI m/z 266.0685 .S3: Compound 8 , 3,4-dihydroxycyclobut-3-ene-1,2-dione  was dissolved in a mixture of 1-butanol (15 mL) and benzene (45 mL). The reaction was heated to reflux with Dean-Stark distillation for 2 h. After cooling to room temperature, the solvent was removed and the residue was purified by recrystallization in MeOH to obtain pure S3 as a brown solid . 1H-NMR  δ 8.11 , 6.19 , 4.18 , 3.81 , 3.67 , 3.22 , 2.45 . 13C-NMR: Limited compound solubility prevented 13C analysis. HRMS-ESI m/z 608.0921 .S3PEG: Compound S3 , azido-mPEG453 , triethylamine (2 drops) and TBTACu(I)Br (2 mg) were mixed in CHCl3 (5 mL) and the reaction mixture was sonicated at 40 °C for 4 h. After removing the solvent, the residue was purified by column chromatography (silica gel as stationary phase) using 0–10% MeOH/CHCl3 to obtain the crude product. The crude product was further purified by a send column chromatography  using 0–5% MeOH/CHCl3 to obtain pure S3PEG as a brown solid . 1H-NMR  δ 8.00 , 7.67 , 6.13 , 4.60 , 4.47 , 3.79 , 3.74 , 3.66–3.39 , 3.33 , 1.22 . MS-MALDI (DHBA as matrix) showed a set of peaks around 4391 which reflected the polydispersity of the PEG45 chains.Association Measurements: Stock solutions of squaraine guest, generally 0.03–3 µΜ were prepared and stock solutions of the host (0.6–60 µM) were made by using the guest solution as the solvent (to keep the concentration of guest constant during the titration). A solution of guest (1 mL) was placed in the cuvette and host solution was titrated into guest solution. Spectra changes were recorded by fluorometer after each injection. Association constants were determined using Origin LabTM 8.6 software  that enabled non-linear least squares fitting of the titration data with an equation for 1:1 binding.Kinetic Measurements: Kinetic studies were performed by using a SFA-20M stopped flow device  . Equal volumes of host solution and guest solution were mixed by the stopped flow device. The spectral changes were monitored by fluorescence. Second order rates constants were determined using Origin LabTM 8.6 software that enabled non-linear least squares fitting of the titration data with an equation for second order kinetics.A new family of fluorescent thiophene and thienothiophene-containing squaraine dyes was prepared using simple synthesis and purification methods. Each squaraine can be rapidly encapsulated by a tetralactam macrocycle in organic or aqueous solution. The threaded supramolecular complexes have nanomolar affinity in water, increased stability towards chemical attack by nucleophiles, and favorable optical properties such as increased fluorescence quantum yield and decreased fluorescence quenching upon dye self-aggregation. Together, the set of squaraine dyes and their macrocycle complexes cover the absorption/emission range of 600–800 nm. A notable favorable attribute with the threaded complexes is the capability to excite each of them with 375 nm light and produce an emission band that is characteristic of the encapsulated squaraine. A wide set of single wavelength and multiplex detection techniques are envisioned using these new dyes for various practical applications in diagnostics, microscopy, molecular imaging, and fluorescence-guided surgery.


Coxiella burnetii is a Gram negative bacterium that survives and grows in a large Coxiella replicative vacuole (CRV), which displays lysosomal and autophagic features. In this report, we present evidence that both, EPAC and its downstream effector Rap2b, were recruited to the CRV. The transient over-expression of the Rap2b wt protein, but not its inactive mutant Rap2b ΔAAX, markedly inhibited the development of the large CRV. Additionally, Rap2b wtinhibited the fusion of early Coxiella phagosomes with the fully developed CRV, indicating that homotypic fusion events are altered in the presence of high levels of Rap2b wt. Likewise, the fusion of endosome/lysosomal compartments (heterotypic fusions) with the large CRV was also affected by the over-expression of this GTPase. Interestingly, cell overexpression of Rap2b wt markedly decreased the levels of the v-SNARE, Vamp7, suggesting that this down-regulation impairs the homotypic and heterotypic fusions events of the Coxiella vacuole.Cyclic Adenosine 3′,5′-monophosphate (cAMP) is a key second messenger known to directly regulate not only the protein kinase A (PKA) activity but also other important molecules such as the exchange protein activated by cAMP (EPAC), which is as a guanine nucleotide exchange factor (GEF) of the low molecular weight GTPase, Rap2.  Coxiella burnetii, the etiological agent of the Q-fever, is an obligate intracellular bacterium. This microorganism has a biphasic life cycle in which the small cell variant (SCV) and the large cell variant (LCV) alternate. The LCV form is generated in the lysosomal-like large replicative compartment  of infected cells, where the acidic microenvironment favors its replication . In . In Coxim (T4SS)  6]. The. TheCoxiynthesis . We haveynthesis . Furtherynthesis  9]..CoxiellaThe autophagy pathway is a highly conserved, physiological degradation process in eukaryotic cells. During autophagy, small portions of cytoplasm or damaged organelles are sequestered into double-membrane vesicles named autophagosomes. These vesicles then fuse with degradatives organelles which supply the hydrolytic enzymes for breaking down and eventual recycling of the sequestered material. The Microtubule-associated protein light chain 3 (LC3) has been shown to be an autophagosomal marker in mammals. There are two forms of LC3 called LC3-I and LC3-II, which are produced post-translationally in various cell types. LC3-I is a cytosolic protein, whereas LC3-II is membrane-bound and specifically associates with autophagosome membranes.Staphylococcus aureus and Legionella pneumophila, exploit the autophagy pathway for their own benefit in order to survive and replicate in the host cells.  [12 [1211] . Th. Th18]. S. aureus . Th. Th20]. . aureus . That st. aureus .C. burnetii co-opt to generate its replicative vacuole. The results obtained in this work offer a deeper insight into the molecular components of the host cell that are involved in the regulatory mechanism of the development of C. burnetii replicative vacuole.In this work, we aimed at studying the effectors EPAC and Rap2b as key regulators of CRV development. We have demonstrated that the cAMP modulated protein EPAC was recruited to the CRV. In addition by analyzing the EPAC downstream effector Rap2b, we determined that the latter factor, but not its inactive mutant Rap2b ΔAAX, is also recruited to the CRV from early times p.i. More importantly, we demonstrated that over-expression of Rap2b wt protein, but not Rap2b ΔAAX, significantly impaired the development of the large CRV. Interestingly, we have shown that the over-expression of Rap2b wt reduced both, the homotypic and the heterotypic fusion capacity of the CRV, and also, decreased the intracellular levels of the v-SNARE Vamp7. These results suggest that the over-expression of the active form of Rap2b affects molecular components of the fusion machinery that Coxiella antiserum and mCherry-Coxiella burnetii were generously provided by Dr. Robert Heinzen . Coxiella-GFP (Tn1832) and the DotA mutant GFP (Tn292) were kindly provided by Dr. Matteo Bonazzi . Texas Red-tagged dextran (3 kDa) was purchased from Molecular Probes. PEGFP-Rap2b wt and pEGFP-Rap2b ΔAAX plasmids were kindly provided by Dr. MauroTorti . Plasmids for EPAC-GFP, Δ(1–148)-EPAC-GFP, Δ(72–148)-EPAC-GFP were kindly provided by Dr. Xiaodong Cheng .D-MEM and alpha-MEM were obtained from Gibco Laboratories ; fetal bovine serum (FBS) was obtained from GIBCO BRL/Life Technologies . The anti-Rap2b antibody and Rap2b siRNA were purchased from Santa Cruz Biotechnology . Rabbit anti-2 atmosphere until 80% confluence was reached.Vero cells , HeLa cells  and Chinese hamster ovary cells (CHO)  were grown in 24-well plates in either D-MEM or α-MEM, supplemented with 15% FBS, and the antibiotics streptomycin (50μg/ml) and penicillin (50 μg/ml), at 37ºC in a 5% COCHO cells were transfected with the plasmids (1 μg/μl) using the Lipofectamine 2000 reagent  as previously described in . TransfeC. burnetii clone 4, phase II, Nine Mile strain was performed as previously described , . Th. ThS. auC. burnetii (homotypic fusion) or vesicles from the endocytic, autophagic and secretory pathways (heterotypic fusion). As demonstrated before, an inhibition of fusion causes a decrease in the size of the CRVs and therefore increases the number of smaller vesicles containing C. burnetii 35].[35].34][.[35].34] pathway . The mulC. burnetii was not able to fuse with lysosomes or with other CRV in the absence of Vamp7 . We . We 38].of Vamp7 . Since CCoxiella vacuole and that this effect might be at least in part, by modulating the levels of Vamp7. This is a very intriguing observation. However, further studies would be necessary to determine the critical molecular mechanisms involved in this modulation.In conclusion, in this report we have demonstrated that the cAMP effectors EPAC and Rap2b are key regulators of CRV development. We have proved that the small GTPase Rap2b has a critical role in the development of CRV by altering the fusion events of the S1 Table(PDF)Click here for additional data file.


Pre-exposure prophylaxis (PrEP) targeting high-risk men who have sex with men (MSM) has been shown to be a cost-effective HIV control measure. However, the approach could be a challenge in low HIV incidence places with a low proportion of high-risk MSM. To examine the impact of PrEP in such setting in Asia, we developed an epidemic model and conducted cost-effectiveness analysis using empirical multicentre clinical and HIV sequence data from HIV-infected MSM in Hong Kong, in conjunction with behavioural data of local MSM. Without PrEP, the HIV incidence (per 100 person-years) would increase from 1.1 to 1.6 between 2011 and 2021. PrEP could avert 3–63% of total new infections in a five-year period (2017–2021), the variability of which depends on the implementation strategies and combination with test-and-treat. However, under current market drug price in 2016, the incremental cost per quality-adjusted life-year gained (QALYG) of PrEP (USD1583136/QALYG) is almost 3 times higher than test-and-treat intervention alone (USD396874/QALYG). Assuming 93% fall of PrEP drug price and in combination with test-and-treat, putting 30% of MSM on non-targeting PrEP would be more feasible, cost-effective (USD268915/QALYG), and could avert more new infections (40%). PrEP could contribute to HIV epidemic control in a low incidence place. In Australia, the proportion of HIV-infected MSM receiving treatment increased from 84% in 2014 to 88% in 2016, and the number of new diagnoses decreased by 8% per year1. In UK, the number of new diagnoses among MSM declined by 1% from 2014 to 2015, and the treatment coverage was 95% in 20152. Besides treatment-as-prevention (TasP), the epidemic could further be controlled through addressing prevention needs of HIV negative individuals. The effectiveness of pre-exposure prophylaxis (PrEP), a biomedical preventive measure, was demonstrated in a number of studies, the results of which had been comprehensively reviewed3. In 2012, PrEP was approved by Food and Drug Administration (FDA). PrEP offers new opportunities to protecting HIV uninfected individuals including MSM, the HIV prevalence of which is often higher compared to other community groups in places like Europe and Asia Pacific5. PrEP as a preventive measure has been shown in modelling studies to avert a proportion of new infections and determined to be cost-effective in the estimations9. In a sub-study of iPrEX, a 99% transmission risk reduction was estimated under full adherence to PrEP usage10. The major challenge is to identify the most effective means for implementation.Despite a relatively high antiretroviral treatment (ART) coverage of over 80% in men who have sex with men (MSM) diagnosed with HIV infection since 2015, the annual number of new diagnoses has not fallen by more than 20%, as shown in some places like Australia and the United Kingdom (UK)11. In low HIV incidence setting, introduction of PrEP has been shown in modelling studies (without consideration of cost) to contribute to epidemic control, as reported in Australia6, rural Zambia7, UK12, and Canada13. Some of these modelling studies and a cost-effectiveness analysis study in the Netherlands concluded with the recommendation of prioritizing high-risk groups in low incidence locality for PrEP14. Naturally, with the small proportion of high-risk MSM in these places, the cost of identifying and managing hidden population at high-risk of infection could be very high, while the actual proportion of MSM subsequently put on PrEP would be even smaller. In addition, stigma might be introduced by the targeting approach adopted for PrEP. High-risk MSM might be discouraged to take PrEP, while the programme cannot stop low-risk MSM from taking PrEP if they self-claim to be at high risk of HIV infection. Such phenomena could constitute new challenges to PrEP implementations.In 2014, World Health Organization (WHO) recommended prioritizing populations at substantial risk of HIV infection when offering PrEP. WHO defined substantial risk based on local sub-population epidemiology (HIV incidence ≥3 per 100 person-years) in conjunction with personal assessment17. Hong Kong, an Asian city, has a relatively stable HIV epidemic after an initial expansion phase predominantly affecting MSM, while the rate of infection in heterosexuals has remained low. The situation is different from India and South Korea, where heterosexual transmission continued to predominate. As of 2015, the cumulative number of reported HIV infected MSM cases was 3151  in Hong Kong18. Among MSM, the HIV prevalence is around 5%, and the annual number of new diagnoses had increased from 170 in 2010 to 464 in 201518, despite the low incidence density of 1.1 per 100 person-year (http://www.chp.gov.hk/files/pdf/interim_statement_on_hiv_pre_exposure_prophylaxis.pdf). The rates of linkage to and retention in care, treatment initiation and viral load suppression are high . In such setting, it is a challenge to effectively achieve further control of the HIV epidemic in MSM, and PrEP offers a new opportunity.We hypothesize that the additional benefit of targeting high-risk MSM in low incidence places for PrEP could be very small. To prove the hypothesis, epidemic modelling parameterized by clinical, HIV-1 sequences and behavioural data is a useful approach that has been applied in previous studies. To date, numerous PrEP modelling studies for MSM have been conducted in the United States, Canada, Europe, South America, Africa, and Australia, but only a few were reported in Asian countries (India and South Korea)2, https://www.bycensus2016.gov.hk/en/bc-mt.html). A previous study has estimated that 4.1% of adult male in Hong Kong were MSM, and 47.1% of them were sexually active19. The estimated number of sexually active MSM was around 50,000 in 2017. Apart from a pilot study, PrEP is currently not available as a public health preventive service in Hong Kong, and the cost of self-financed PrEP is very high (~USD23 per dose in 2016) in the absence of programmatic support. While some degree of PrEP awareness has been shown (26.6%), the current number of MSM on PrEP is speculatively small (1%)20.Hong Kong is a metropolitan city, with a highly mobile population  and high population density  Ordinance. Individual consent was waived. The datasets used in the current study are not publicly available because the data are owned separately by third parties. Access to these data and permission could be inquired through Department of Health, Hong Kong Special Administrative Region Government, Queen Elizabeth Hospital and Princess Margaret Hospital.There are three HIV specialist clinics providing treatment to HIV-infected patients in the public service in Hong Kong. Clinical data (baseline and follow-up records) in 1985–2014, and HIV-1 genotype resistance testing (GRT) sequences in 1994–2012 of HIV-infected adult MSM were collected from all three clinics for model parameterization. The collected data accounted for 96% (1886/1958) of the cumulative reported HIV MSM cases by 2012. Results of our behavioural study on MSM were used to set behavioural parameter values22. Collected sequences  were aligned by MUSCLE in MEGA6. From the constructed neighbour-joining tree, clusters were identified . They were further classified into subgroups by size of the clusters (number of cases) and by HIV subtype. Each subgroup was represented by one sub-model, and each sub-model included high and low-risk MSM for both HIV negative and positive MSM. A MSM is classified as belonging to the low-risk category if he has lower partner exchange rate , or high-risk if there has been higher partner exchange rate  high-risk and low-risk susceptible MSM, (b) undiagnosed non-locally acquired infections, (c) undiagnosed individuals in acute infection, chronic infection categorized by CD4 levels of ≤200/μL, 201–350/μL, 351–500/μL, >500/μL, and AIDS, (d) diagnosed individuals in pre-treatment care with chronic infections and AIDS and (e) loss to follow-up before antiretroviral treatment (ART), (f) patients on treatment with non-suppressed (>500copies/mL) and suppressed viral load, and (g) loss to follow-up after treatment initiation. To incorporate the principles of TasP, we assumed minimal transmission risk for HIV-infected MSM with suppressed viral load Appendix .23). The average difference in the number of new diagnoses in 2011 between basecase and 100 simulations was 4.9 persons (SD 3.12 persons)  and Neisseria gonorrhoeae (NG). HIV-infected MSM were assumed to have 4 specialist clinic visits, CD4 and viral load measurements in a year. For those on treatment, the ART cost reported locally was included. We adopted the CD4 level specific utility list applied in a previous cost-effectiveness analysis among MSM in Australia26. We assumed 3.5% annual discounted rate in the analysis26. List of cost and utility parameters is shown in Table For each HIV negative MSM on PrEP, high adherence was defined as 87.5% (mid-point of 75% and 100%) usage and low adherence as 38% (mid-point of 1% and 75%) usage of daily oral PrEP, attending 4 visits at HIV specialist clinics with HIV testing, a yearly test for creatinine, syphilis, In the study, PrEP implementation was assumed to have started from 2017. We assumed that a proportion of susceptible MSM was on PrEP at the beginning of the year, with some dropping out in the remainder of the year. At the beginning of the following year, the total number of dropped-out users was replaced to maintain a constant coverage.basecase without PrEP (A1); with 10%, 30% and 90% coverage of PrEP (A2) involving both low-risk and high-risk MSM (i.e. non-targeting approach) with low or high adherence usage; and involving high-risk MSM only (i.e. targeting approach) (A3) with low or high adherence usage;test-and-treat without PrEP (B1); with 10%, 30% and 90% coverage of PrEP through a non-targeting approach (B2) with low or high adherence usage; and through a targeting approach (B3) with low or high adherence usage;To compare the relative impacts of PrEP implementation with improving engagement in the cascade of HIV care, we developed scenarios in the model and conducted cost-effectiveness analysis from 2017 onwards, with different coverage of PrEP in (A) basecase, and with (B) a high rate of diagnosis and treatment initiation :As it was obvious that low adherence PrEP would not be cost-effective, only scenarios with high adherence PrEP had been developed in the cost-effectiveness analysis.We performed two-way sensitivity analyses with different levels of PrEP coverage  in high- and low-risk MSM, stratified by high and low adherence in the epidemic model. We also developed hypothetical scenarios in high HIV incidence environment (≥3 per 100 person-years) to analyse the impact of PrEP implementation strategies. For cost-effectiveness analysis, we performed sensitivity analyses for different pricing scale of PrEP drug cost .The opinions and assertions contained herein are private views of the authors and do not necessarily reflect those of the affiliating institutions. Part of the preliminary model projection results have been presented at Health Research Symposium 2017 in Hong Kong, and at Asia Pacific AIDS & Co-infections Conference (APACC) 2018 in Hong Kong [oral presentations].27).Among 1886 MSM cases with clinical data, the median age was 34 (IQR = 28–42), and 1541 (82%) were Chinese. For those with subtyping performed (1157 MSM), 254 (22%) belonged to subtype CRF01_AE, 835 (72%) to subtype B, and 68 (6%) to other subtypes. A total of 841 (45%) had a negative HIV test result before HIV diagnosis. Among them, 579 (69%) underwent the test ≤3 years before HIV diagnosis. By 2012, 1443 (77%) of MSM cases had initiated ART, after a median interval between HIV diagnoses and treatment initiation of 8 months (IQR = 2–29 months). Among those on treatment, 1286 (89%) cases had ever achieved viral load suppression (≤500/mL) by 2012 , 44 small clusters (four to ten connected nodes), 88 dyads or very small clusters (two to three connected nodes) and 447 isolates. The distribution of these clusters by subtype is shown in Fig. 18.In the basecase model, the annual number of new diagnoses was projected to increase continuously from the year 2012 to 2021 Fig. . Model eIf PrEP is implemented from 2017, it could avert 8% (10% high adherence PrEP) to 55% (90% high adherence PrEP) of total new infections above basecase scenario in 2017–2021 Table . In the In the scenario of test-and-treat without PrEP, the proportion of new infections averted would increase from 6% in 2017 to 41% in 2021  and with high adherence, a non-targeting approach could avert an additional 5–24% of the total new infections in five years  to assess the situation in perspective. The impact is obviously high when targeting high-risk MSM for PrEP, as shown in the sensitivity analyses in Appendix 13, our results illustrated positive impact of PrEP intervention on HIV epidemic control among MSM, despite the low incidence in the locality.In the deterministic compartmental model, we estimated that PrEP implementation since 2017 could avert 3–63% of total new MSM infections above basecase in Hong Kong in 2017–2021, the extent of which would vary with PrEP coverage and the effectiveness of implementing mixed interventions with the incorporation of test-and-treat intervention. Similar to other studies28. Although both studies suggested targeting MSM with higher sexual activity for PrEP, its definition was set at having more than one new sexual partner a year, with the adoption of a low threshold approach28. Our simulation results were however completely different from other PrEP modelling studies in low HIV incidence places that had adopted a targeted approach. Elsewhere, the results supported the prioritization of high-risk MSM for PrEP usage29. The discrepancy might have arisen from the different assumptions adopted for population mixing. Assortative mixing was assumed in our models, the results of which fitted well with the reported annual number of new diagnoses. We have developed a separate model with the incorporation of proportionate mixing pattern  approach could avert more new infections than offering PrEP exclusively to MSM belonging to one distinct risk category. The finding was in line with those from UK’s modelling studiesAppendix , which s31. A modelling study in Los Angeles projected that 59% new infections could be averted by a mixed PrEP and test-and-treat strategy30, and a study in San Diego projected 50% reduction of new infections by the mixed approach31.In the implementation of PrEP, it is important to focus on adherence, as high adherence PrEP would avert additionally 10% or more new infections than low adherence Appendix ppendix A33. While targeting 30% high risk MSM for PrEP was 11% more cost-effective than non-targeting approach, the proportion of new infections averted would be 12% lower than that of non-targeting approach. Combined with test-and-treat intervention, a non-targeting approach with 30% coverage could avert 8% more new infections, and was 3% in Plan B or 15% in Plan C more cost-effective than targeting approach in the five-year period.Taking the current high drug cost of PrEP  into consideration, PrEP intervention alone in the low HIV incidence setting does not appear to be cost-effective (at least USD1583136/QALYG). In the case of Hong Kong, a 93% reduction of the drug cost  is desirable in order to demonstrate PrEP’s cost-effectiveness. Drug price as an important determinant of cost-effectiveness for PrEP is consistent with previous cost-effectiveness analyses in the published literature34, suggesting that a non-targeting approach might already be able to attract more high-risk than low-risk MSM for PrEP. Similar relationship between intention to use PrEP and risk behaviour was observed in people who inject drugs35. Naturally, informing MSM and connecting them with an accessible PrEP service should be prioritized, which can normally be achieved with the collaborative efforts of community organisation and strategic use of Internet36.It is important to note that the cost implicated in PrEP includes not just drug cost but the accompanying programme cost of establishing/running an accessible PrEP service (cost not included in this study), coupled with the means to enrol eligible persons, the latter depending naturally also on the approach to be adopted. Resources should also be spent to enhance adherence. For a targeting approach, the identification of people at high risk of infection, who may otherwise be hidden in the low HIV incidence population, is a challenge. It can be argued that if the impact of PrEP implementation via a targeting or non-targeting approach is similar, it might not be necessary to pay for this additional cost aiming to identify MSM with very high risk behaviours. From a previous study, high-risk MSM had higher intention for future PrEP use37. Third, only two types of sexual partnership, serial monogamy and random mixing were assumed, which might have underestimated the impact of concurrent partnership and group sex. We also assumed that the HIV transmission hazard of MSM was 1.2 times higher than heterosexuals. The value was determined under model calibration in our previous study22. As the basecase model estimation was close to the observed data , model fitness was implied. Fourth, the effectiveness of PrEP was categorized in two levels, corresponding to low and high adherence of PrEP. However, with different mode of PrEP usage, including daily oral, event-driven and time-driven, the adherence pattern and overall effectiveness of PrEP would be very different in real life situation38. The effectiveness of PrEP would likely increase with the optimization of regimens in the future. From our sensitive analysis, more new infections would be averted with the increasing effectiveness of PrEP. Fifth, as the occurrence of adverse events of PrEP is unlikely to be related to the implementation approach, it had not been included in the model. However, we acknowledged the impact of adverse events in risk benefit calculation, when the HIV infection risk is minimal with PrEP usage. Finally, cost-effective analyses have been conducted to provide a broader perspective in evaluating possible strategic approach to PrEP. We have included drug cost and monitoring cost in the analyses, on the assumption that programmatic cost would be similar irrespective of the approach adopted. This can be an over-simplification of the actual situation should PrEP become implemented. Furthermore, the cost-effectiveness analyses only estimate a five-year period instead of longer duration. The incremental cost per QALYG might have been overestimated.This study carries some limitations. First, bisexual individuals were subsumed as MSM for analytic simplicity. Theoretically they could function as the epidemiological link, allowing HIV to spread from MSM to heterosexual population. In models projecting both heterosexual and MSM population, especially in the presence of a high proportion of bisexuals, further adjustment would be needed when applying our model structure. Second, the impact of condom usage on transmission risk was considered during high and low risk group classification, and as such, condom intervention and risk compensation (i.e. less condom use after PrEP usage) had not been modelled in the analyses. The potential impacts of sexually transmitted infections (STI) had not been factored in, though we acknowledged the potentially higher transmission risk of STI among PrEP usersIn conclusion, for low incidence places such as Hong Kong, PrEP could contribute to the control of the HIV epidemic in MSM in addition to the benefits from adopting a test-and-treat strategy. Offering PrEP without prioritization by behavioural risk in moderate coverage (say 30%) would be a feasible and cost-effective means for averting more new infections if PrEP drug cost is low enough. This strategy would likely be applicable for places with similarly low HIV incidence in particular in Asia, on the assumption that the networking patterns of MSM were similar. Our study shows that it is important not just to establish the baseline HIV incidence but consider the strategic approach for PrEP implementation in its development as part of a city/country’s HIV prevention programme.Appendix A


Pre-exposure prophylaxis (PrEP) with daily oral tenofovir/emtricitabine dramatically reduces HIV risk in men who have sex with men (MSM). However, uptake is slow worldwide.We administered anonymous cross-sectional questionnaires to MSM presenting for anonymous HIV testing at a Toronto sexual health clinic at four successive time points during the period 2013–2016. We assessed trends in PrEP awareness, acceptability, and use over time using the Cochran-Armitage Trend Test, and identified barriers to using PrEP by constructing “PrEP cascades” using 2016 data. We assumed that to use PrEP, MSM must (a) be at risk for HIV, (b) be at objectively high risk (HIRI-MSM score ≥ 10), (c) perceive themselves to be at medium-to-high risk, (d) be aware of PrEP, (e) be willing to use PrEP, (f) have a family doctor, (g) be comfortable discussing sexual health with that doctor, and (h) have drug coverage/be willing to pay out of pocket.p < 0.0001), reaching 91.3% and 5.0%, respectively, in the most recent wave. Willingness to use PrEP rose to 56.5%, but this increase did not reach statistical significance (p = 0.06). The full cascade, ABCDEFGH, suggested few could readily use PrEP under current conditions (11/400 = 2.8%). The largest barriers, in descending order, were low self-perceived HIV risk, unwillingness to use PrEP, and access to PrEP providers.MSM participants were mostly white (54–59.5%), with median age 31 years (IQR = 26–38). PrEP awareness and use increased significantly over time (both To maximize its potential public health benefits, PrEP scale-up strategies must address self-perceived HIV risk and increase access to PrEP providers. Men who have sex with men (MSM) continue to see stable or increasing rates of HIV incidence worldwide  April–June 2013, (2) May–August 2014, (3) November 2014–April 2015, and (4) May–August 2016. Adult MSM who understood English were eligible to complete the survey immediately after their point-of-care HIV test or within 2 weeks if they returned to the study site. All participants were offered a CAD$10 gift card.Hassle Free Clinic is a busy sexual health clinic in downtown Toronto that conducts approximately 5400 point-of-care HIV tests among MSM every year. Of these, over half are performed at four community-based satellite clinics across the city. Demographic characteristics and HIV positivity rates are generally similar across sites. Collectively, Hassle Free Clinic has the highest positivity rate (1.7%) of all 38 anonymous HIV testing sites in Ontario .Questionnaire content varied slightly between waves 1 and 4, but included demographic characteristics, sexual practices, recreational drug use, and perceived HIV risk, as well as awareness of, willingness to use, and current use of HIV prevention technologies such as post-exposure prophylaxis (PEP) and PrEP.We quantified HIV risk using the HIV Incidence Risk Index for MSM (HIRI-MSM), a seven-item scale that incorporates (1) age, (2) number of male partners, (3) number of HIV-positive partners, (4) frequency of condomless receptive anal sex, (5) frequency of condomless insertive anal sex with HIV-positive partners, (6) amyl nitrate use, and (7) amphetamine use in the past 6 months  to reduce their risk of HIV infection. This pill contains two anti-HIV drugs (tenofovir/emtricitabine or Truvada®) and research suggests that it is relatively safe and is over 90% effective if taken consistently. It is much less effective if not taken every day and it does not protect against other STIs. Taking PrEP would require a visit to the doctor every 3 months in order to be tested for HIV, STIs and side effects. Truvada® was recently approved to be used for PrEP in Canada.” This description was modified for each wave of survey administration, in keeping with the approval status and available data on PrEP efficacy at the time. Each version was pilot-tested with five participants for clarity and face validity.Awareness of PrEP was defined as responding “yes” to the question, “Before today, had you ever heard of PrEP?” Willingness to use PrEP was defined as responding with either “agree” or “strongly agree” on a five-point Likert-type scale to the statement, “I would be interested in taking PrEP to reduce my current risk of HIV infection”. Participants were also asked to indicate their reasons for being interested or not interested in PrEP, using a list of options derived from literature review and discussions with community members, including a free-text “other” field.We summarized participant characteristics using descriptive statistics, with continuous variables summarized by measures of central tendency and dispersion and categorical variables by proportions. We used Kruskal-Wallis and chi-square tests to assess for differences in participant characteristics between survey waves.To achieve our primary objective, we calculated the proportion of respondents in each survey wave who had each outcome of interest , and conducted Cochran-Armitage Trend tests to assess for linear increasing trends in these proportions.p < 0.01), only one was considered for inclusion in the multivariable model, based on model fit (AIC). We constructed models using forward selection, where variables were added one at a time and retained if statistically significant at a threshold of alpha = 0.10  and analyzed them visually for gaps. We repeated this process using a higher HIRI-MSM score of ≥ 25 for defining high risk, as suggested by our prior work and by cost-effectiveness analyses  with reasonable precision, as previously described  to 58.3% (95% CI = 53.3–63.3%) to 71.8% (95% CI = 67.2–76.1%) to 91.3% (95% CI = 88.0–93.8%)  suggested that only 2.8% (11/400) participants had no individual or system barriers to using PrEP, similar to our previous findings Fig. . The greSince MSM at high objective HIV risk (HIRI-MSM ≥ 10) who indicate willingness to use PrEP represent an important group that should be prioritized for PrEP initiation, we tabulated perceived barriers to PrEP initiation in this group. While the most common barriers were related to financial access, including not having private drug insurance  or believing that PrEP was not covered by one’s private insurance , other common concerns included simply not having looked into getting PrEP yet , not having a doctor , and not feeling comfortable asking doctors for PrEP , suggesting that efforts to increase the availability of trained PrEP providers and more actively facilitate linkages with PrEP services could increase uptake.Those with high objective HIV risk who were unwilling to use PrEP are another group whose perspectives are important to understand Table . Common p = 0.09) and less likely to cite interest in condomless sex as reasons .Finally, we examined reasons for PrEP interest among those not at high objective HIV risk who nevertheless were willing to use PrEP Table . OverallIn this series of four surveys among Toronto MSM undergoing anonymous HIV testing during the period 2013–2016, we observed a significant increase in PrEP awareness over time, reaching 91.3% in 2016. Although PEP awareness, PEP use and higher objective HIV risk were predictors of PrEP awareness in univariable analyses, time remained the strongest independent predictor when building the multivariable model. This association with time did not, however, correspond to a significant increase in willingness to use PrEP over time, which was 56.5% in 2016. In addition, PrEP uptake was low, with only 5.0% in the most recent survey reporting ever using PrEP.The high degree of PrEP awareness in our 2016 sample is striking, and given the lack of concerted public health messaging about PrEP in Canada, is testament to the capacity of communities and healthcare providers to raise awareness about important health interventions at a population level. Conduct of Canada’s first PrEP demonstration project in the city from 2014 to 2016 , these changes reflect the changing contexts in which respondents formulated their opinions about PrEP, and were necessary to ensure that information provided to participants about PrEP was up-to-date. Wording changes also necessitated imputation of some values when calculating HIRI-MSM scores for survey waves 1 and 2; such changes may have contributed to the small observed differences in participant characteristics between waves but would not have affected our primary or secondary outcomes. Third, HIRI-MSM has limited sensitivity for identifying HIV seroconverters in some contexts, and thus may have underestimated the proportion of those who should use PrEP (Lancki et al. Our study identified increasing awareness and consistent, moderate willingness to use PrEP among at-risk Toronto MSM. To maximize the potential public health benefit or PrEP, next steps should include developing strategies for helping men better understand their HIV risk, publicizing the excellent safety and efficacy of PrEP, training providers for PrEP delivery, and identifying funding strategies to facilitate PrEP use.