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Steven D Reid, Sarah J Fidler, Graham S Cooke Department of Infectious Diseases, St Mary's Hospital, Imperial College London, London, UK Abstract: It is now around 30 years since the discovery of HIV, the virus that causes AIDS. More than 70 million people have been infected in that time and around 35 million have died. The majority of those currently living with HIV/AIDS are in low- and middle-income countries, with sub-Saharan Africa bearing a disproportionate burden of the global disease. In high-income countries, the introduction of antiretroviral therapy (ART) has drastically reduced the morbidity and mortality associated with HIV. Patients on ART are now predicted to have near-normal life expectancy and the role of treatment is increasingly recognized in preventing new infections. In low- and middle-income countries, treatment is now more widely available and around half of those who need ART are currently receiving it. Early diagnosis of HIV is essential if ART is to be optimally implemented. Lab-based diagnostics for screening, diagnosis, treatment initiation, and the monitoring of treatment efficacy are critical in managing the disease and reducing the number of new infections each year. The introduction of point-of-care HIV rapid tests has transformed the epidemic, particularly in low- and middle-income countries. For the first time, these point-of-care tests allow for the rapid identification of infected individuals outside the laboratory who can undergo counseling and treatment and, in the case of pregnant women, allow the timely initiation of ART to reduce the risk of vertical transmission. Although survival is markedly improved with ART even in the absence of laboratory monitoring, long-term management of people living with HIV on ART, and their partners, is essential to ensure successful viral suppression. The burden of disease in many resource-poor settings with high HIV prevalence has challenged the ability of local laboratories to effectively monitor those on ART. Diagnostics used to initiate and monitor treatment are now moving out of the laboratory and into the field. These new point-of-care tests for viral load and CD4 are poised to further transform the disease and shift the treatment paradigm in low- and middle-income countries. Keywords: point-of-care, diagnostics, HIV, CD4, viral load, resource-poor countries

Female crack cocaine users are at high risk for HIV infection. Data from 121 female substance abusers admitted to an all-female rehabilitation center in Trinidad and Tobago between 1996 and 2002 were reviewed retrospectively to determine human immunodeficiency virus (HIV) seroprevalence and associated risk factors. HIV seroprevalence was 19.8%, which is six times higher than in the general population. The univariate analysis identified the following factors associated with HIV infection: poor educational attainment, history of a sexually transmitted infection (STI), and use of crack cocaine. In the multivariate analysis, only poor educational attainment and history of an STI were independently associated with HIV seroprevalence. Female substance abusers, especially female crack cocaine users, are at high risk of acquiring and transmitting the HIV virus. To reduce risk of HIV infection, rehabilitation programs should address risky sexual behaviors and screen for STIs, and they also should improve educational attainment, develop skills, and provide vocational training. [ABSTRACT FROM AUTHOR]

In the Caribbean AIDS has become the leading cause of death among those aged 15-45. The homeless are at high risk due to the nexus of mobility, psychiatric disorders and substance use, particularly crack. Seventy-four homeless, out-of-treatment drug users were recruited in Saint Lucia and Trinidad to assess risk behaviour and healthcare needs. Information was collected in several domains: medical, mental health, substance use, sexual risk, and barriers to care. Ninety-five percent of the sample reported crack cocaine use within the previous 30 days. Approximately 35% of the sample had ever been diagnosed with a sexually transmitted disease and 25% of the sample reported being HIV+. Approximately 40% had a history of trading sex for crack or money. Due to the range of needs in this population, interventions should be multimodal. A focus on the individual, community, and structural levels is warranted to improve chances of program effectiveness. [ABSTRACT FROM AUTHOR]

Background: Since the onset of the coronavirus (COVID-19) pandemic its negative effects have been highlighted globally. There is little literature that examines the positive impact on daily living., Aims: To examine the positive influences of government-imposed COVID-19 public health restrictions on adults throughout Trinidad and Tobago (TT) and their associated factors., Method: A cross-sectional study, using an adapted pretested online survey instrument, was conducted amongst non-institutionalised, community-dwelling adults using convenience sampling., Results: Of 1287 completed and weighted responses, the mean age (S.D.) was 39.2 (14.6) years, 49% were males, and 61% completed secondary level education. The top 5 positive experiences were more time spent with family (80%), more down-time (78%), learning new skills (68%), more time for hobbies (66%), self-care (57%), more time spent with friends (55%) and eating healthier (50%). The importance of religion, working from home, and an increase in marijuana use emerged as positive correlates for certain positive experiences, while participants with chronic illness, and a positive anxiety or depression screen, were negatively associated with most positive experiences., Conclusions: Despite the negative consequences of the COVID19 pandemic, several positives were highlighted in this study. These findings have implications for social policies toward building resilience within the community.

Persons with chronic non-communicable diseases (NCDs) were identified as particularly at risk of severe morbidity and mortality during the COVID-19 pandemic. Little is written about the impact of COVID-19 on this sub-population in the Caribbean, where the prevalence of NCDs is disproportionately high. This study aimed to ascertain COVID-related concerns, and the mental health impact of the pandemic among persons with and without NCDs in Trinidad and Tobago, during the acute period of COVID-19 lockdowns early in the pandemic. An anonymous online survey collected cross-sectional data from a convenience sample nationwide. Of 1287 respondents, 219 self-identified as having an NCD. Findings suggest that the pandemic was experienced unequally by persons with NCDs, who were more likely to be concerned about health and wellbeing and to report health inequalities-unemployment, social isolation and negative effects of government restrictions. Compared to those without NCDs, they were more likely to increase use of marijuana during the lockdown period, and to report severe anxiety/depression that can result in exacerbation of NCDs. Interventions for persons with NCDs must address the mental health consequences of any pandemic, including increased drug use, and also address social inequalities to reduce sustained post-pandemic mental health impact and negative health outcomes.

This article contributes to the still limited analysis of gender and sexuality in the exploration of HIV infection in the Caribbean. In Trinidad and Tobago, child sexual abuse was identified as a prevalent behavior. This motivated a gendered exploration of the sociocultural factors underlying child sexual abuse and the link with HIV. Using a qualitative action research methodology, researchers sought to understand the patterns of behavior, sociocultural and gendered meanings, and significance of child sexual abuse; to sensitize stakeholders to these issues; and to develop multidisciplinary community-based interventions and policies. This article reports on the analysis of data collected from the interventions carried out as part of the action research methodology, as well as a range of national and community-specific action research activities including ethnographic case studies, national stakeholders meetings, and a study of service providers. It provides a nuanced understanding of the meanings, underlying assumptions, perceptions, and taboos associated with child sexual abuse in Trinidad and Tobago, as well as new ways of understanding child sexual abuse in the Caribbean. Findings showed that the social and gender context for child sexual abuse involves several interconnecting factors located within a framework of gender ideologies and expressions, sexual expectations and behaviors, and social norms based on patriarchal values. These findings would be of significance for the prevention of child sexual abuse and the associated HIV risk in Trinidad and Tobago, and the management of victims and perpetrators of child sexual abuse.

Background: Guinea reported its first case of COVID-19 on March 12, 2020. Soon thereafter, a national state of emergency was declared, all land borders were closed, schools were shut down, and public gatherings were limited. Many health activities, including field-based activities targeting neglected tropical diseases (NTDs), were paused. The World Health Organization (WHO) issued updated guidance on the resumption of NTD field-based activities on July 27, 2020. In response, the Guinea Ministry of Health (MoH) and its partners planned and resumed mass drug administration (MDA) in mid-August to September 2020 in 19 health districts., Methodology/principal Findings: A risk-benefit assessment was conducted to identify potential risks associated with the MDA in the COVID-19 context. Following this assessment, a risk mitigation plan with barrier measures was developed to guide MDA implementation. These measures included COVID-19 testing for all national staff leaving Conakry, mask wearing, social distancing of two meters, and hand washing/sanitizing. A checklist was developed and used to monitor compliance to risk mitigation measures. Data on adherence to risk mitigation measures were collected electronically during the MDA. A total of 120 checklists, representing 120 community drug distributor (CDD) teams (two CDDs per team) and 120 households, were completed. Results indicated that washing or disinfecting hands was practiced by 68.3% of CDD teams, compared to 45.0% among households. Face masks to cover the mouth and nose were worn by 79.2% of CDD teams, while this was low among households (23.3%). In 87.5% of households, participants did not touch the dose pole and in 88.3% of CDD teams, CDDs did not touch the hands of the participants while giving the drugs. A large majority of CDD teams (94.2%) and household members (94.2%) were willing to participate in the MDA despite the pandemic. The epidemiological coverage was ≥65% for lymphatic filariasis, onchocerciasis and soil-transmitted helminths in 10 out of 19 HDs and ≥75% for schistosomiasis for school-aged children in 7 out of 11 HDs., Conclusions/significance: Guinea was one of the first countries in Africa to resume MDA activities during the COVID-19 pandemic without causing an observed increase of transmission. The development of a risk mitigation plan and a method to monitor adherence to barrier measures was critical to this unprecedented effort. The rapid incorporation of COVID-19 barrier measures and their acceptance by CDDs and household members demonstrated both the adaptability of the National NTD Program to respond to emerging issues and the commitment of the MoH to implement NTD programs., Competing Interests: The authors declare that they have no competing interests.

Background: The control of lymphatic filariasis (LF) caused by Wuchereria bancrofti in the Central African Region has been hampered by the presence of Loa loa due to severe adverse events that arise in the treatment with ivermectin. The immunochromatographic test (ICT) cards used for mapping LF demonstrated cross-reactivity with L. loa and posed the problem of delineating the LF map. To verify LF endemicity in forest areas of Cameroon where mass drug administration (MDA) has not been ongoing, we used the recently developed strategy that combined serology, microscopy and molecular techniques., Methods: This study was carried out in 124 communities in 31 health districts (HDs) where L. loa is present. At least 125 persons per site were screened. Diurnal blood samples were investigated for circulating filarial antigen (CFA) by FTS and for L. loa microfilariae (mf) using TBF. FTS positive individuals were further subjected to night blood collection for detecting W. bancrofti. qPCR was used to detect DNA of the parasites., Results: Overall, 14,446 individuals took part in this study, 233 participants tested positive with FTS in 29 HDs, with positivity rates ranging from 0.0 to 8.2%. No W. bancrofti mf was found in the night blood of any individuals but L. loa mf were found in both day and night blood of participants who were FTS positive. Also, qPCR revealed that no W. bancrofti but L.loa DNA was found with dry bloodspot. Positive FTS results were strongly associated with high L. loa mf load. Similarly, a strong positive association was observed between FTS positivity and L loa prevalence., Conclusions: Using a combination of parasitological and molecular tools, we were unable to find evidence of W. bancrofti presence in the 31 HDs, but L. loa instead. Therefore, LF is not endemic and LF MDA is not required in these districts.

Background: Child sexual abuse (CSA) is a multi-dimensional problem. The search for best practice must consider the complexities surrounding CSA and its management in any particular society., Objective: Data previously gathered from service providers on CSA service provision in Trinidad and Tobago identified key deficient issues in policy and practice. In this paper, researchers aimed to bridge the gaps identified, and effect changes to improve services for CSA using an action research methodology., Participants and Setting: Service providers from all sectors in governmental and non-governmental organizations in Trinidad and Tobago, who work with children at risk of CSA were involved in the process., Methods: Researchers led the service providers into an awareness of their own practice through critical discussion of, and reflection on, the key deficient issues. The new knowledge generated, with guided input from evidenced-based best practice, led to the development of guidelines for management. Discussion of the practicability of the guidelines by service providers in multiple sectors generated more new knowledge that refined the management approach., Results: The contextual knowledge obtained from service providers resulted in best practice guidelines for service providers that were culturally relevant and context-sensitive, adaptive and implementable, and allowed a seamless multidisciplinary response to CSA in Trinidad and Tobago within prevailing constraints., Conclusions: Action research offers an effective approach to improve services for CSA through mobilization of service providers and changes in policy and practice. It is applicable in any setting and likely to be effective in any socio-cultural context., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The relationship between energy drink (ED) use and risky behaviours has not been explored in the Caribbean, where youth risky behaviours are prevalent. This paper examines the relationship between ED use and risky behaviours and the moderating effect of gender among 1994 undergraduate students in Trinidad and Tobago. Analyses examined the association between ED use and risky behaviours, and the significant predictors of risky behaviours. Analysis of variance determined whether there were differences in risky behaviours between those who used only alcohol or EDs, both alcohol and EDs, alcohol mixed with EDs and neither alcohol nor EDs; and the difference between those with high and low intensity of ED use. In both males and females current use of energy drinks was positively associated with harmful substance use, risky sexual behaviours, and risky non-sexual behaviours, more strongly for males. The strongest predictor of risky behaviours was gender. Males consuming both alcohol and EDs, independently or mixed, were most likely to have risky behaviours. Consistent with previous reports, harmful alcohol use and other risk taking health behaviours appear to be predicted by a combination of high intensity use of EDs and alcohol. In countries like Trinidad and Tobago where violence, alcohol use disorders, STI/HIV infection and teenaged pregnancy are prevalent, the assessment of consumption of EDs and alcohol may be an important potential predictor of health compromising behaviours.

Background: Over 60% of households (HHs) in Trinidad and Tobago (T&T) consume alcohol. These HHs were more likely to report illnesses, relationship problems, and behavioral problems with children. This study set out to determine what proportion of HHs were willing to support changes in specific policies, laws and regulations in a national alcohol campaign., Methods: A cross-sectional convenience sample of HHs were surveyed from a random sample of enumeration districts (ED) in T&T. An interviewer-applied, field pre-tested de novo questionnaire had 5 domains and was developed over 1 1/2 years after an extensive literature review and consultation. Many of the WHO 'best buys' recommendations were included., Results: One thousand six hundred ninety-five HHs (from 53 ED) responded from a total of 1837 HHs approached (response rate 92%). In a national campaign the following proportions of HHs would support: setting the legal age for drinking at 21 years (82.4%); restricting or banning alcohol advertising on TV and other media (73.1% and 54.4% respectively); banning all alcohol advertising at sports and cultural events (64.8%); banning radio stations playing songs with reference to alcohol use (71.3%); holding sellers of alcohol responsible for the amount of alcohol sold (79.5%); advocating that proof of age to be shown by persons buying alcohol (87.4%); placing more prominent warning labels on products displaying alcohol content (87.2%); placing more prominent warning labels on products showing harmful effects (88.5%); increasing taxes on alcohol sales (87.7%). Less than 50% of HH supported restrictions in density of outlets and reduction in opening times for alcohol outlets., Conclusions: Many HHs in T&T are willing to support changes in policies around alcohol, including many of the policies shown by the WHO to be effective in reducing the harmful consumption of alcohol.

Background: There is limited research into normative hair density values in individuals of Hispanic descent., Methods: In order to help establish baseline density values in these individuals, we evaluated the hair density in 99 Americans of Hispanic descent and compared them with the values in 44 individuals of African descent and 23 Caucasians. Participants were recruited from a New York City medical center and self-identified their ethnicity. Biracial individuals were excluded from the study., Results: The hair density means ± SD ranged from 169 ± 31 to 178 ± 33/cm 2 in Americans of Hispanic descent, from 148 ± 25 to 160 ± 27/cm 2 in individuals of African descent, and from 214 ± 28 to 230 ± 33/cm 2 in Caucasian individuals. All differences in hair densities between the ethnicities were statistically significant ( p values ranging from 3.03 × 10 -14 to 0.0249)., Conclusion: While trichoscopy is not as accurate as invasive histological assessment of hair density or phototrichogram, it is still a reliable and fast method for assessing hair density. Increased awareness of ethnic variances in hair density can help clinicians and researchers diagnose hair disorders, monitor response to therapy, and conduct hair-related research in these patients.

Background: Based on previous studies, historical records and risk factors, trachoma was suspected to be endemic in 31 health districts (HDs) in Guinea. To facilitate planning for the elimination of trachoma as a public health problem, national trachoma surveys were conducted between 2011 and 2016 to determine the prevalence of trachomatous inflammation-follicular (TF) and trachomatous trichiasis (TT) in all 31 endemic HDs., Methodology/principal Findings: A total of 27 cross-sectional surveys were conducted, each using two-stage cluster sampling (one survey in 2011 covered five HDs). Children aged 1-9 years and adults aged ≥15 years were examined for TF and TT, respectively, using the World Health Organization (WHO) simplified grading system. Indicators of household access to water, sanitation and hygiene (WASH) were also collected. A total of 100,051 people from 13,725 households of 556 clusters were examined, of whom 44,899 were male and 55,152 were female. 44,209 children aged 1-9-years and 48,745 adults aged ≥15 years were examined. The adjusted prevalence of TF varied between 1.0% (95%CI: 0.6-1.5%) to 41.8% (95%CI: 39.4-44.2%), while the adjusted prevalence of TT ranged from 0.0% (95%CI: 0.0-0.2%) to 2.8% (95%CI: 2.3-3.5%) in the 27 surveys. In all, 18 HDs had a TF prevalence ≥5% in children aged 1-9 years and 21 HDs had a TT prevalence ≥0.2% in adults aged ≥15 years. There were an estimated 32,737 (95% CI: 19,986-57,811) individuals with TT living in surveyed HDs at the time of surveys., Conclusions/significance: Trachoma is a public health problem in Guinea. 18 HDs required intervention with at least one round of mass drug administration and an estimated 32,737 persons required TT surgery in the country. The results provided clear evidence for Guinea to plan for national trachoma elimination., Competing Interests: The authors have declared that no competing interests exist.

Background: In 2009, three years after stopping mass treatment with azithromycin, a trachoma impact survey in four health districts in the Kayes region of Mali found a prevalence of trachomatous inflammation-follicular (TF) among children aged 1 to 9 years of >5% and a trachomatous trichiasis (TT) prevalence within the general population (≥1-year-old) of <1%. As a result, the government's national trachoma program expanded trichiasis surgery and related activities required to achieve trachoma elimination., Methodology/principal Findings: In 2015, to assess progress towards elimination, a follow-up impact survey was conducted in the Kayes, Kéniéba, Nioro and Yélimané health districts. The survey used district level two-stage cluster random sampling methodology with 20 clusters of 30 households in each evaluation unit. Subjects were eligible for examination if they were ≥1 year. TF and TT cases were identified and confirmed by experienced ophthalmologists. In total 14,159 people were enumerated and 11,620 (82%) were examined. TF prevalence (95% confidence interval (CI)) was 0.5% (0.3-1%) in Kayes, 0.8% (0.4-1.7%) in Kéniéba, 0.2% (0-0.9%) in Nioro and 0.3% (0.1-1%) in Yélimané. TT prevalence (95% CI) was 0.04% (0-0.25%) in Kayes, 0.29% (0.11-0.6%) in Kéniéba, 0.04% (0-0.25%) in Nioro and 0.07% (0-0.27%) in Yélimané., Conclusions/significance: Eight years after stopping MDA and intensifying trichiasis surgery outreach campaigns, all four districts reached the TF elimination threshold of <5% and three of four districts reached the TT elimination threshold of <0.1%.

Background: This study reports dose-response estimates for the odds ratio (OR) and population attributable risk of acute alcohol use and road traffic injury (RTI)., Methods: Data were analyzed on 1,119 RTI patients arriving at 16 emergency departments (EDs) in Argentina, Brazil, Costa Rica, Dominican Republic, Guatemala, Guyana, Mexico, Nicaragua, Panama, and Trinidad and Tobago. Case-crossover analysis, pair-matching the number of standard drinks consumed within the 6 hours prior to the RTI with 2 control periods (prior d/wk), was performed using fractional polynomial analysis for dose-response., Results: About 1 in 6 RTI patients in EDs were positive for self-reported alcohol 6 hours prior to the injury (country range 8.6 to 24.1%). The likelihood of an RTI with any drinking prior (compared to not drinking) was 5 times higher (country range OR 2.50 to 15.00) and the more a person drinks the higher the risk. Every drink (12.8 g alcohol) increased the risk of an RTI by 13%, even 1 to 2 drinks were associated with a sizable increase in risk of an RTI and a dose-response was found. Differences in ORs for drivers (OR = 3.51; 95% CI = 2.25 to 5.45), passengers (OR = 8.12; 95% CI = 4.22 to 15.61), and pedestrians (OR = 6.30; 95% CI = 3.14 to 12.64) and attributable fractions were noted. Acute use of alcohol was attributable to 14% of all RTIs, varying from 7% for females to 19% for being injured as a passenger., Conclusions: The finding that the presence of alcohol increases risk among drivers and nondrivers alike may further help to urge interventions targeting passengers and pedestrians. Routine screening and brief interventions in all health services could also have a beneficial impact in decreasing rates of RTIs. Higher priority should be given to alcohol as a risk factor for RTIs, particularly in Latin America and the Caribbean., (Copyright © 2017 by the Research Society on Alcoholism.)

There is an association between alcohol consumption and injuries in Latin America and the Caribbean. This cross-sectional study explores the socio-contextual factors of alcohol-related injuries in Trinidad and Tobago. Data on drinking patterns, injury type, drinking context prior to injury, and demographics were collected from patients presenting with injuries to the Emergency Departments (ED) of four hospitals. Findings show that 20.6% of patients had consumed alcohol, mainly beer, in the 6 h before injury. More than half were drinking at home (27%), or someone else's home (27%). Injury most commonly occurred outdoors (36%) while in transit. Alcohol-related injuries occurred mainly because of falling or tripping (31.7%); these patients recorded the highest mean alcohol consumption prior to injury. Most persons who fell (50%) did so at home. Findings highlight the previously unreported significant risk of non-drivers sustaining injures through falling and tripping because of heavy alcohol use. Current interventions to reduce alcohol-related injury have focused on drink driving but there is a need for interventions targeting pedestrians and those who drink at home. A comprehensive multi-component approach including secondary prevention interventions in the medical setting, community educational interventions, enforcement of current legislative policies concerning the sale of alcohol, and policy initiatives surrounding road safety and alcohol outlet density should be implemented., Competing Interests: The authors declare no conflict of interest. The study was conceived and designed by the World Health Organization Collaborative Study on Alcohol and Injuries. The funding sponsors had no role in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Background: To determine the patterns of alcohol use among households in Trinidad and Tobago (T&T) and to estimate the association between alcohol use and negative psychological, social, or physical events experienced by the household., Methods: A convenience sample of 1837 households across T&T. We identified bivariate correlates of alcohol use, and heavy episodic drinking using chi-square and t-test analyses and used multivariable logistic regression to estimate adjusted associations between household alcohol use and experiences within the past 12 months adjusted for sociodemographic covariates., Results: One thousand five hundred two households had complete data for all variables (82% response rate). Nearly two thirds (64%) of households included alcohol users; 57% of household that consumed alcohol also reported heavy episodic drinking. Households that reported alcohol consumption were significantly more likely to report illnesses within the households, relationship problems, and behavioral and antisocial problems with children. Among households where a member was employed, those who consumed alcohol were nearly twice as likely (OR = 1.98; 95% confidence interval (CI) 1.03, 3.82) to have a household member call in sick to work and 2.9 times as likely (OR = 2.9; CI 1.19, 7.04) to have a household member suffer work related problems compared with households who reported not consuming alcohol., Conclusions: Approximately two thirds of households in T&T reported using alcohol. These households were more likely to report psychological, physical, and social problems. These findings would support efforts to enforce current policies, laws, and regulations as well as new strategies to reduce the impact of harmful alcohol consumption on households in T&T.

Background: Substance abuse is a significant public health challenge in the Caribbean. It is important that health and allied professionals be adequately trained in this field. The Caribbean Institute on Alcoholism and other Drug Problems (CARIAD) was established to provide new knowledge and share successful best practices in substance abuse in the Caribbean. CARIAD brings together diverse participants from throughout the region in a structured community of practice and emphasizes local knowledge formation. This paper evaluates the acceptability and perceived impact of CARIAD to/on participants., Methods: The authors analyzed anonymous written evaluations of 458 CARIAD participants completed immediately after the program in 2003-2012. Questionnaires collected quantitative data on the acceptability of the program in meeting the learning needs of participants. Perception of program acceptability and impact on the participant were also obtained from descriptive content analysis of qualitative data obtained through open-ended questions. A 2013 online follow-up survey collected data about the utilization and dissemination of knowledge acquired at CARIAD from 141 graduates of the same period. Data analysis employed basic descriptive statistics and qualitative content analysis., Results: Participant evaluation immediately following CARIAD showed a high level of satisfaction with the organization, structure, content, and usefulness of the program. Participants valued the program's cultural relevance and the opportunity for networking and collaboration. They expressed a collective solidarity, and empowerment to address substance abuse problems. Participants reported that CARIAD had impacted positively on their professional practice and facilitated knowledge transfer to colleagues and communities., Conclusion: CARIAD is perceived as an effective community of practice, producing a network of graduates who use the acquired knowledge, skills, and motivation to lead in the field of substance abuse in the Caribbean. This process of learning can be recommended to countries where research is not prioritized and evidence-driven interventions are not always available.

Heading date in wheat (Triticum aestivum L.) and other small grain cereals is affected by the vernalization and photoperiod pathways. The reduced-height loci also have an effect on growth and development. Heading date, which occurs just prior to anthesis, was evaluated in a population of 299 hard winter wheat entries representative of the U.S. Great Plains region, grown in nine environments during 2011-2012 and 2012-2013. The germplasm was evaluated for candidate genes at vernalization (Vrn-A1, Vrn-B1, and Vrn-D1), photoperiod (Ppd-A1, Ppd-B1 and Ppd-D1), and reduced-height (Rht-B1 and Rht-D1) loci using polymerase chain reaction (PCR) and Kompetitive Allele Specific PCR (KASP) assays. Our objectives were to determine allelic variants known to affect flowering time, assess the effect of allelic variants on heading date, and investigate changes in the geographic and temporal distribution of alleles and haplotypes. Our analyses enhanced understanding of the roles developmental genes have on the timing of heading date in wheat under varying environmental conditions, which could be used by breeding programs to improve breeding strategies under current and future climate scenarios. The significant main effects and two-way interactions between the candidate genes explained an average of 44% of variability in heading date at each environment. Among the loci we evaluated, most of the variation in heading date was explained by Ppd-D1, Ppd-B1, and their interaction. The prevalence of the photoperiod sensitive alleles Ppd-A1b, Ppd-B1b, and Ppd-D1b has gradually decreased in U.S. Great Plains germplasm over the past century. There is also geographic variation for photoperiod sensitive and reduced-height alleles, with germplasm from breeding programs in the northern Great Plains having greater incidences of the photoperiod sensitive alleles and lower incidence of the semi-dwarf alleles than germplasm from breeding programs in the central or southern plains.

Importance: Frontal fibrosing alopecia has previously been reported as rare among patients of African descent. The authors present 18 cases of frontal fibrosing alopecia affecting African American patients and review all published cases of frontal fibrosing alopecia involving patients of African descent. Observations: Since 2010, there have been 66 published cases of frontal fibrosing alopecia among patients of African descent; 59 women, five men, and two cases of unknown gender. Frontal fibrosing alopecia is not uncommon among patients of African descent. In this study, the authors find that female African American patients may have fewer symptoms and unique clinical presentations. Conclusion and relevance: Frontal fibrosing alopecia is an entity that can be seen in patients with many different ethnic backgrounds, often with varying presentations. The diagnosis of frontal fibrosing alopecia must be considered in any patient of African descent who presents with frontotemporal alopecia. In the authors' patient population, there was a younger age of presentation. The presence of perifollicular hyperpigmentation along the hairline and concomitant facial hyperpigmentation may aid in making the diagnosis and distinguishing this entity from traction alopecia.

Alcohol use is deeply engrained in the socio-cultural fabric of the Caribbean where heavy episodic drinking is an accepted and common drinking pattern. This article aims to create awareness of the tremendous negative impact on population health in the English-speaking Caribbean from culturally customary levels of alcohol consumption. It advocates for the urgent development and implementation of a regional alcohol policy in the English-speaking Caribbean, and emphasizes the importance of crafting and implementing the policy without cultural offence.

Unlabelled: Infection with Streptococcus pyogenes is associated with a breadth of clinical manifestations ranging from mild pharyngitis to severe necrotizing fasciitis. Elevated levels of intracellular copper are highly toxic to this bacterium, and thus, the microbe must tightly regulate the level of this metal ion by one or more mechanisms, which have, to date, not been clearly defined. In this study, we have identified two virulence mechanisms by which S. pyogenes protects itself against copper toxicity. We defined a set of putative genes, copY (for a regulator), copA (for a P1-type ATPase), and copZ (for a copper chaperone), whose expression is regulated by copper. Our results indicate that these genes are highly conserved among a range of clinical S. pyogenes isolates. The copY, copA, and copZ genes are induced by copper and are transcribed as a single unit. Heterologous expression assays revealed that S. pyogenes CopA can confer copper tolerance in a copper-sensitive Escherichia coli mutant by preventing the accumulation of toxic levels of copper, a finding that is consistent with a role for CopA in copper export. Evaluation of the effect of copper stress on S. pyogenes in a planktonic or biofilm state revealed that biofilms may aid in protection during initial exposure to copper. However, copper stress appears to prevent the shift from the planktonic to the biofilm state. Therefore, our results indicate that S. pyogenes may use several virulence mechanisms, including altered gene expression and a transition to and from planktonic and biofilm states, to promote survival during copper stress., Importance: Bacterial pathogens encounter multiple stressors at the host-pathogen interface. This study evaluates a virulence mechanism(s) utilized by S. pyogenes to combat copper at sites of infection. A better understanding of pathogen tolerance to stressors such as copper is necessary to determine how host-pathogen interactions impact bacterial survival during infections. These insights may lead to the identification of novel therapeutic targets that can be used to address antibiotic resistance., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Otitis media is a prominent disease among children. Previous literature indicates that otitis media is a polymicrobial disease, with Haemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis and Moraxella catarrhalis being the most commonly associated bacterial pathogens. Recent literature suggests that introduction of pneumococcal conjugate vaccines has had an effect on the etiology of otitis media. Using a multiplex PCR procedure, we sought to investigate the presence of the aforementioned bacterial pathogens in middle ear fluid collected from children undergoing routine tympanostomy tube placement at Wake Forest Baptist Medical Center during the period between January 2011 and March 2014. In purulent effusions, one or more bacterial organisms were detected in ~90% of samples. Most often the presence of H. influenzae alone was detected in purulent effusions (32%; 10 of 31). In non-purulent effusions, the most prevalent organism detected was A. otitidis (26%; 63 of 245). Half of the non-purulent effusions had none of these otopathogens detected. In purulent and non-purulent effusions, the overall presence of S. pneumoniae was lower (19%; 6 of 31, and 4%; 9 of 245, respectively) than that of the other pathogens being identified. The ratio of the percentage of each otopathogen identified in purulent vs. non-purulent effusions was >1 for the classic otopathogens but not for A. otitidis.

Objective: There has been little inquiry addressing whether or not concerns about adverse effects of energy drink usage are relevant in the Caribbean. This survey investigated energy drink usage and adverse consequences among tertiary level students in Trinidad and Tobago., Methods: A cross-sectional survey of 1994 students from eight institutions was conducted using a de novo questionnaire based on findings from a focus group of students. Chi-squared analyses and logistic regression were used to assess relationships between energy drink usage, adverse effects and other factors affecting energy drink use, and to verify predictors of energy drink use., Results: Prevalence of use was 86%; 38% were current users. Males were more likely to use, used more frequently and at an earlier age. Energy drinks were used most commonly to increase energy (50%), combat sleepiness (45%) and enhance academic performance (40%), and occurred during sports (23%) and mixed with alcohol (22.2%). The majority (79.6%) consumed one energy drink per sitting; 62.2% experienced adverse effects, most commonly restlessness (22%), jolt and crash (17.1%) and tachycardia (16.6%). Awareness of adverse effects was associated with no use (p=0.004), but adverse effects were not a deterrent to continued use., Conclusion: Energy drink usage is prevalent among students. The use is not excessive, but associated with high rates of adverse effects and occurs in potentially dangerous situations like during exercise and with alcohol. There is a need to educate students about the potential adverse effects of energy drinks., (Copyright © 2014 Ministry of Health, Saudi Arabia. Published by Elsevier Ltd. All rights reserved.)

To determine if molybdenum (Mo) is a chemical stressor, fingerling and juvenile rainbow trout (Oncorhynchus mykiss) were exposed to waterborne sodium molybdate (0, 2, 20, or 1,000 mg l-1 of Mo) and components of the physiological (plasma cortisol, blood glucose, and hematocrit) and cellular (heat shock protein [hsp] 72, hsp73, and hsp90 in the liver, gills, heart, and erythrocytes and metallothionein [MT] in the liver and gills) stress responses were measured prior to initiation of exposure and at 8, 24, and 96 h. During the acute exposure, plasma cortisol, blood glucose, and hematocrit levels remained unchanged in all treatments. Heat shock protein 72 was not induced as a result of exposure and there were no detectable changes in total hsp70 (72 and 73), hsp90, and MT levels in any of the tissues relative to controls. Both fingerling and juvenile fish responded with similar lack of apparent sensitivity to Mo exposure. These experiments demonstrate that exposure to waterborne Mo of up to 1,000 mg l(-1) did not activate a physiological or cellular stress response in fish. Information from this study suggests that Mo water quality guidelines for the protection of aquatic life are highly protective of freshwater fish, namely rainbow trout.

Introduction: Point-of-care testing for CD4 cell count is considered a promising way of reducing the time to eligibility assessment for antiretroviral therapy (ART) and of increasing retention in care prior to treatment initiation. In this review, we assess the available evidence on the patient and programme impact of point-of-care CD4 testing., Methods: We searched nine databases and two conference sites (up until 26 October 2013) for studies reporting patient and programme outcomes following the introduction of point-of-care CD4 testing. Where appropriate, results were pooled using random-effects methods., Results: Fifteen studies, mainly from sub-Saharan Africa, were included for review, providing evidence for adults, adolescents, children and pregnant women. Compared to conventional laboratory-based testing, point-of-care CD4 testing increased the likelihood of having CD4 measured [odds ratio (OR) 4.1, 95% CI 3.5-4.9, n=2] and receiving a CD4 result (OR 2.8, 95% CI 1.5-5.6, n=6). Time to being tested was significantly reduced, by a median of nine days; time from CD4 testing to receiving the result was reduced by as much as 17 days. Evidence for increased treatment initiation was mixed., Discussion: The results of this review suggest that point-of-care CD4 testing can increase retention in care prior to starting treatment and can also reduce time to eligibility assessment, which may result in more eligible patients being initiated on ART.

In Trinidad and Tobago, little data exists on child sexual abuse, although there are many anecdotal reports of high prevalence. The Breaking the Silence Gender and Community Empowerment Model is a multidisciplinary intervention to prevent and respond to child sexual abuse in Trinidad and Tobago. It is an innovative, gender-sensitive intervention that uses a community based action research methodology anchored in a national framework. Preliminary evaluation of the Breaking the Silence model shows increased knowledge of child sexual abuse, increased willingness to discuss child sexual abuse, and an impact that goes beyond the target communities. This model can be replicated in communities to prevent and respond to child sexual abuse and adapted to address other sensitive social issues in the Caribbean.

Necrobiosis lipoidica (NL) is a rare chronic granulomatous disease that has historically been associated with diabetes mellitus. Debate exists regarding the etiology and pathogenesis of NL with a widely accepted theory that microangiopathy plays a significant role. NL typically presents clinically as erythematous papules on the front of the lower extremities that can coalesce to form atrophic telangiectatic plaques. NL is usually a clinical diagnosis, but if the clinical suspicion is uncertain, skin biopsy specimen can help differentiate it from sarcoidosis, necrobiotic xanthogranuloma, and granuloma annulare. NL is a difficult disease to manage despite a large armamentarium of treatment options that include topical and intralesional corticosteroids, immunomodulators, biologics, platelet inhibitors, phototherapy, and surgery. Randomized control trials are lacking to evaluate the many treatment methods and establish a standard regimen of care. Disease complications such as ulceration are common, and lesions should also be monitored for transition to squamous cell carcinoma, a less common sequelae., (Copyright © 2013 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.)

Background: Because previous studies have indicated that otitis media may be a polymicrobial disease, we prospectively analyzed middle ear effusions of children undergoing tympanostomy tube placement with multiplex polymerase chain reaction for four otopathogens., Methods: Middle ear effusions from 207 children undergoing routine tympanostomy tube placement were collected and were classified by the surgeon as acute otitis media (AOM) for purulent effusions and as otitis media with effusion (OME) for non-purulent effusions. DNA was isolated from these samples and analyzed with multiplex polymerase chain reaction for Haemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis, and Moraxella catarrhalis., Results: 119 (57%) of 207 patients were PCR positive for at least one of these four organisms. 36 (30%) of the positive samples indicated the presence of more than one bacterial species. Patient samples were further separated into 2 groups based on clinical presentation at the time of surgery. Samples were categorized as acute otitis media (AOM) if pus was observed behind the tympanic membrane. If no pus was present, samples were categorized as otitis media with effusion (OME). Bacteria were identified in most of the children with AOM (87%) and half the children with OME (51%, p < 0.001). A single bacterial organism was detected in middle ear effusions from children with AOM more often than those with OME (74% versus 33%, p < 0.001). Haemophilus influenzae was the predominant single organism and caused 58% of all AOM in this study. Alloiococcus otitidis and Moraxella catarrhalis were more frequently identified in middle ear effusions than Streptococcus pneumoniae., Conclusions: Haemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis, and Moraxella catarrhalis were identified in the middle ear effusions of some patients with otitis media. Overall, we found AOM is predominantly a single organism infection and most commonly from Haemophilus influenzae. In contrast, OME infections had a more equal distribution of single organisms, polymicrobial entities, and non-bacterial agents.

Central centrifugal cicatricial alopecia is a scarring alopecia that is predominantly seen in African American women, but occurs less frequently in men. The authors present three cases of African American men with biopsy-proven central centrifugal cicatricial alopecia and detail the clinical presentation, histological findings, and treatment regimens. Central centrifugal cicatricial alopecia should be considered in the differential diagnosis when evaluating male patients with vertex hair loss accompanied by scalp symptoms. Physicians should maintain a high index of suspicion in African American men with the appropriate clinical picture and confirm the diagnosis by scalp biopsy. Prompt and appropriate treatment can help halt or slow disease progression.

Background: Waterless antiseptic surgical hand scrub (1% chlorhexidine gluconate and 61% ethyl alcohol, Avagard™; 3M Health Care, St. Paul, MN), alcohol-only cleanser (62% ethyl alcohol), and traditional surgical scrub (5-minute scrub with 4% chlorhexidine soap using a sterile scrub brush with water) are techniques used for hand cleansing and disinfection. We hypothesized that alcohol-only cleanser and waterless antiseptic scrub (Avagard) would be as effective as a traditional surgical scrub for hand cleansing before placement of central venous catheters., Methods: Fingers of subjects were plate-cultured for 24 hours after 5 methods of hand cleansing: method 1: traditional surgical scrub (n = 49 plates produced by 14 subjects); method 2: traditional surgical scrub (5-minute scrub with water, brush, and 4% chlorhexidine soap) followed by a 15-minute break, then alcohol-only cleanser (62% alcohol) (n = 49 plates produced by 14 subjects); method 3: alcohol-only cleanser alone (n = 49 plates produced by 14 subjects); method 4: alcohol-only cleanser (62% alcohol), followed by a 15-minute break, then traditional surgical scrub (5-minute scrub with brush, and 4% chlorhexidine soap with water) (n = 49 plates produced by 14 subjects); and method 5: waterless surgical scrub (Avagard) alone (n = 116 plates produced by 38 subjects). The 15-minute break was introduced to allow a short period of recontamination, and to test for residual effects from prior cleansing., Results: Alcohol-only cleanser alone (method 3) was significantly less effective than the traditional surgical scrub (method 1) (P < 0.001; 82% plate growth). Waterless surgical scrub (Avagard) (method 5) had a 0% observed difference (95% confidence interval [CI]: -14% to 11%) compared with the traditional 5-minute scrub (method 1) (P = 0.99; 16% plate growth). When a traditional surgical scrub was used first followed by a 15-minute period of recontamination, there was a 6% observed difference in method 2 from reference (method 1) (95% CI: -10% to 22%), and 0% observed difference in method 4 from reference (95% CI: -15% to 15%)., Conclusion: As the initial cleansing method, the alcohol-only cleanser (method 3) was significantly less effective than the traditional surgical scrub (method 1) (P < 0.001).

Background: Group A Streptococcus (GAS) causes acute tonsillopharyngitis in children, and approximately 20% of this population are chronic carriers of GAS. Antibacterial therapy has previously been shown to be insufficient at clearing GAS carriage. Bacterial biofilms are a surface-attached bacterial community that is encased in a matrix of extracellular polymeric substances. Biofilms have been shown to provide a protective niche against the immune response and antibiotic treatments, and are often associated with recurrent or chronic bacterial infections. The objective of this study was to test the hypothesis that GAS is present within tonsil tissue at the time of tonsillectomy., Methods: Blinded immunofluorescent and histological methods were employed to evaluate palatine tonsils from children undergoing routine tonsillectomy for adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis., Results: Immunofluorescence analysis using anti-GAS antibody was positive in 11/30 (37%) children who had tonsillectomy for adenotonsillar hypertrophy and in 10/30 (33%) children who had tonsillectomy for recurrent GAS pharyngitis. Fluorescent microscopy with anti-GAS and anti-cytokeratin 8 & 18 antibodies revealed GAS was localized to the tonsillar reticulated crypts. Scanning electron microscopy identified 3-dimensional communities of cocci similar in size and morphology to GAS. The characteristics of these communities are similar to GAS biofilms from in vivo animal models., Conclusion: Our study revealed the presence of GAS within the tonsillar reticulated crypts of approximately one-third of children who underwent tonsillectomy for either adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis at the Wake Forest School of Medicine., Trial Registration: The tissue collected was normally discarded tissue and no patient identifiers were collected. Thus, no subjects were formally enrolled.

The HIV epidemic in Trinidad and Tobago is primarily heterosexual, fueled by a high level of risky sex, gender inequality, and alcohol and drug use; however, the influence of alcohol and drugs has been neglected in the literature. Research shows that current HIV prevention approaches have failed to substantially impact sexual behavior change. This may be so because they do not incorporate a comprehensive understanding of the sociocultural factors underlying sexual behavior. There is an urgent need to understand how socially accepted patterns of alcohol and drug use contribute to sexual behaviors and HIV risk in Trinidad and Tobago. Moreover, specialized, evidence-based interventions are needed for HIV-infected substance abusers. Using an adaptation of the cognitive behavioral stress management (CBSM) protocol, this intervention project aimed to assess effectiveness in reducing relapse and risky behaviors among recovering HIV-infected substance abusers in Trinidad and Tobago.

Background: Group A Streptococcus (GAS) is a human-specific pathogen responsible for a number of diseases characterized by a wide range of clinical manifestations. During host colonization GAS-cell aggregates or microcolonies are observed in tissues. GAS biofilm, which is an in vitro equivalent of tissue microcolony, has only recently been studied and little is known about the specific surface determinants that aid biofilm formation. In this study, we demonstrate that surface-associated streptococcal collagen-like protein-1 (Scl1) plays an important role in GAS biofilm formation., Results: Biofilm formation by M1-, M3-, M28-, and M41-type GAS strains, representing an intraspecies breadth, were analyzed spectrophotometrically following crystal violet staining, and characterized using confocal and field emission scanning electron microscopy. The M41-type strain formed the most robust biofilm under static conditions, followed by M28- and M1-type strains, while the M3-type strains analyzed here did not form biofilm under the same experimental conditions. Differences in architecture and cell-surface morphology were observed in biofilms formed by the M1- and M41-wild-type strains, accompanied by varying amounts of deposited extracellular matrix and differences in cell-to-cell junctions within each biofilm. Importantly, all Scl1-negative mutants examined showed significantly decreased ability to form biofilm in vitro. Furthermore, the Scl1 protein expressed on the surface of a heterologous host, Lactococcus lactis, was sufficient to induce biofilm formation by this organism., Conclusions: Overall, this work (i) identifies variations in biofilm formation capacity among pathogenically different GAS strains, (ii) identifies GAS surface properties that may aid in biofilm stability and, (iii) establishes that the Scl1 surface protein is an important determinant of GAS biofilm, which is sufficient to enable biofilm formation in the heterologous host Lactococcus. In summary, the GAS surface adhesin Scl1 may have an important role in biofilm-associated pathogenicity.

Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980's there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which GAS disease may transition from mild to severe through the Srv mediated dispersal of GAS biofilms.

Macrophages regulate immune responses during many viral infections, and can be a major determinant of pathogenesis, virus replication and immune response to infection. Here, we have addressed the question of the outcome of infection of primary human macrophages with parainfluenza virus 5 (PIV5) and a PIV5 mutant (P/V-CPI-) that is unable to counteract interferon (IFN) responses. In cultures of naïve monocyte-derived macrophages (MDMs), WT PIV5 established a highly productive infection, whereas the P/V-CPI- mutant was restricted for replication in MDMs by IFN-beta. Restricted replication in vitro was relieved in MDM that had been activated by prior exposure to heat killed Gram positive bacteria, including Listeria monocytogenes, Streptococcus pyogenes, and Bacillus anthracis. Enhanced replication of the P/V mutant in MDM previously activated by bacterial components correlated with a reduced ability to produce IFN-beta in response to virus infection, whereas IFN signaling was intact. Activated MDM were found to upregulate the synthesis of IRAK-M, which has been previously shown to negatively regulate factors involved in TLR signaling and IFN-beta production. We discuss these results in terms of the implications for mixed bacteria-virus infections and for the use of live RNA virus vectors that have been engineered to be attenuated for IFN sensitivity., (Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

In this paper, we discuss the reasons why we urgently need a point-of-care (POC) CD4 test, elaborate the problems we have experienced with the current technology which hampers CD4-count coverage and highlight the ideal characteristics of a universal CD4 POC test. It is high-time that CD4 technology is simplified and adapted for wider use in low-income countries to change the current paradigm of restricted access once and for all., (© 2010 Blackwell Publishing Ltd.)

Group A Streptococcus (GAS) is a human specific pathogen capable of causing both mild infections and severe invasive disease. We and others have shown that GAS is able to form biofilms during infection. That is to say, they form a three-dimensional, surface attached structure consisting of bacteria and a multi-component extracellular matrix. The mechanisms involved in regulation and dispersal of these GAS structures are still unclear. Recently we have reported that in the absence of the transcriptional regulator Srv in the MGAS5005 background, the cysteine protease SpeB is constitutively produced, leading to increased tissue damage and decreased biofilm formation during a subcutaneous infection in a mouse model. This was interesting because MGAS5005 has a naturally occurring mutation that inactivates the sensor kinase domain of the two component regulatory system CovRS. Others have previously shown that strains lacking covS are associated with decreased SpeB production due to CovR repression of speB expression. Thus, our results suggest the inactivation of srv can bypass CovR repression and lead to constitutive SpeB production. We hypothesized that Srv control of SpeB production may be a mechanism to regulate biofilm dispersal and provide a mechanism by which mild infection can transition to severe disease through biofilm dispersal. The question remained however, is this mechanism conserved among GAS strains or restricted to the unique genetic makeup of MGAS5005. Here we show that Srv mediated control of SpeB and biofilm dispersal is conserved in the invasive clinical isolates RGAS053 (serotype M1) and MGAS315 (serotype M3), both of which have covS intact. This work provides additional evidence that Srv regulated control of SpeB may mediate biofilm formation and dispersal in diverse strain backgrounds.

Background: Group A Streptococcus (GAS) is a Gram-positive human pathogen that is capable of causing a wide spectrum of human disease. Thus, the organism has evolved to colonize a number of physiologically distinct host sites. One such mechanism to aid colonization is the formation of a biofilm. We have recently shown that inactivation of the streptococcal regulator of virulence (Srv), results in a mutant strain exhibiting a significant reduction in biofilm formation. Unlike the parental strain (MGAS5005), the streptococcal cysteine protease (SpeB) is constitutively produced by the srv mutant (MGAS5005Δsrv) suggesting Srv contributes to the control of SpeB production. Given that SpeB is a potent protease, we hypothesized that the biofilm deficient phenotype of the srv mutant was due to the constitutive production of SpeB. In support of this hypothesis, we have previously demonstrated that treating cultures with E64, a commercially available chemical inhibitor of cysteine proteases, restored the ability of MGAS5005Δsrv to form biofilms. Still, it was unclear if the loss of biofilm formation by MGAS5005Δsrv was due only to the constitutive production of SpeB or to other changes inherent in the srv mutant strain. To address this question, we constructed a ΔsrvΔspeB double mutant through allelic replacement (MGAS5005ΔsrvΔspeB) and tested its ability to form biofilms in vitro., Findings: Allelic replacement of speB in the srv mutant background restored the ability of this strain to form biofilms under static and continuous flow conditions. Furthermore, addition of purified SpeB to actively growing wild-type cultures significantly inhibited biofilm formation., Conclusions: The constitutive production of SpeB by the srv mutant strain is responsible for the significant reduction of biofilm formation previously observed. The double mutant supports a model by which Srv contributes to biofilm formation and/or dispersal through regulation of speB/SpeB.

Group A Streptococcus (GAS) is a common causative agent of pharyngitis, but the role of GAS in otitis media is underappreciated. In this study, we sought to test the hypothesis that GAS colonizes the middle ear and establishes itself in localized, three-dimensional communities representative of biofilms. To test this hypothesis, the middle ears of chinchillas were infected with either a strain of GAS capable of forming biofilms in vitro (MGAS5005) or a strain deficient in biofilm formation due to the lack of the transcriptional regulator Srv (MGAS5005 Δsrv). Infection resulted in the formation of large, macroscopic structures within the middle ears of MGAS5005- and MGAS5005 Δsrv-infected animals. Plate counts, scanning electron microscopy, LIVE/DEAD staining, and Gram staining revealed a difference in the distributions of MGAS5005 versus MGAS5005 Δsrv in the infected samples. High numbers of CFU of MGAS5005 Δsrv were isolated from the middle ear effusion, and MGAS5005 Δsrv was found randomly distributed throughout the excised macroscopic structure. In contrast, MGAS5005 was found in densely packed microcolonies indicative of biofilms within the excised material from the middle ear. CFU levels of MGAS5005 from the effusion were significantly lower than that of MGAS5005 Δsrv early during the course of infection. Allelic replacement of the chromosomally encoded streptococcal cysteine protease (speB) in the MGAS5005 Δsrv background restored biofilm formation in vivo. Interestingly, our results suggest that GAS naturally forms a biofilm during otitis media but that biofilm formation is not required to establish infection following transbullar inoculation of chinchillas.

Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce α-d-glucosaminyl l-malate (GlcN-malate) from UDP-GlcNAc and l-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase (→GlcNAc-malate) and the BaBshB deacetylase (→GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 Å resolution, identifies several active-site interactions important for the specific recognition of l-malate, but not other α-hydroxy acids, as the acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-d-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, or germination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M. (2007) J. Bacteriol. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work.

CD8(+) T cells play a critical role in the clearance of respiratory pathogens. Thus, it is surprising that functional inactivation of lung effectors has been observed in many models of viral infection. Currently, the molecular defect responsible for the shut-off of function in these cells is unknown. In the present study, we addressed this question using a model of respiratory infection with the paramyxovirus SV5. Nonfunctional cells were found to exhibit decreases in SOCE, resulting in reduced NFAT1 activation. Notably, function could be restored by the provision of increased levels of extracellular calcium. The reduced ability to mobilize calcium was associated with reduced expression of ORAI1, the CRAC channel subunit. These findings reveal a previously unknown mechanism for the negative regulation of function in effector T cells.

The synthesis and structures of Fe, Co, and Zn halide complexes [MX(2)(H(2)L)] (M = Fe, X = Br; M = Co, Zn, X = Cl) of the N-donor extended dipyrromethane ligand H(2)L are described, from which it is clear that bond rearrangements from imine-pyrrole to amine-azafulvene tautomers occur on metal co-ordination, both in the solid state and in solution. In the structure of [FeBr(2)(H(2)L)], this H-migration results in a pendant amine that is involved in both inter- and intramolecular hydrogen bonds to the bromide ligands, so forming a dimer. As the tautomerisation renders the N-H protons less acidic, metal-based ligand substitution reactions can occur in favour of deprotonation. As such, the reaction between [MCl(2)(H(2)L)] (M = Co, Zn) and NaN(3) results in the formation of the bis(azide) complexes [M(N(3))(2)(H(2)L)] which for Co displays both inter- and intramolecular N-H...N(3)-Co hydrogen bonds in the solid state. In contrast, reactions of the dihalides with the lithium bases LiNMe(2) or LiMe (M = Fe), or reduction reactions with C(8)K (M = Fe, Co) result in the formation of the known dinuclear helicates [M(2)(L)(2)].

The angiotensin (Ang) type 1 receptor (AT(1)R) is highly expressed on renal nuclei and stimulates reactive oxygen species (ROS). It is not known whether other functional components of the Ang system regulate the nuclear Ang II-AT(1)R ROS pathway. Therefore, we examined the expression of Ang receptors in nuclei isolated from the kidneys of young adult (1.5 years) and older adult (3.0 to 5.0 years) sheep. Binding studies in renal nuclei revealed the AT(2)R as the predominant receptor subtype ( approximately 80%) in young sheep, with the Ang-(1-7) (AT(7)R; Mas protein) and AT(1)R antagonists competing for the remaining sites. Conversely, in older sheep, the AT(1)R accounted for approximately 85% of nuclear sites, whereas the Ang type 2 receptor and AT(7)R subtypes comprise approximately 20% of remaining sites. Ang II increased nuclear ROS to a greater extent in older (97+/-22%; n=6) versus young animals (7+/-2%; P=0.01; n=4), and this was abolished by an AT(1)R antagonist. The AT(7)R antagonist D-Ala(7)-Ang-(1-7) increased ROS formation to Ang II by approximately 2-fold (174+/-5% versus 97+/-22%; P<0.05) in older adults. Immunoblots of renal nuclei revealed protein bands for the AT(7)R and Ang-converting enzyme 2 (ACE2), which metabolizes Ang II to Ang-(1-7). The ACE2 inhibitor MLN4760 also exacerbated the Ang II-dependent formation of ROS (156+/-15%) and abolished the generation of Ang-(1-7) from Ang II. We conclude that an ACE2-Ang-(1-7)-AT(7)R pathway modulates Ang II-dependent ROS formation within the nucleus, providing a unique protective mechanism against oxidative stress and cell damage.