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BackgroundAs concerns about the prevalence of infections that are resistant to available antibiotics increase, attention has turned toward the use of these medicines both within and outside of formal healthcare settings. Much of what is known about use beyond formal settings is informed by survey-based research. Few studies to date have used comparative, mixed-methods approaches to render visible patterns of use within and between settings as well as wider points of context shaping these patterns.DesignThis article analyses findings from mixed-methods anthropological studies of antibiotic use in a range of rural and urban settings in Zimbabwe, Malawi and Uganda between 2018 and 2020. All used a ‘drug bag’ survey tool to capture the frequency and types of antibiotics used among 1811 households. We then undertook observations and interviews in residential settings, with health providers and key stakeholders to better understand the stories behind the most-used antibiotics.ResultsThe most self-reported ‘frequently used’ antibiotics across settings were amoxicillin, cotrimoxazole and metronidazole. The stories behind their use varied between settings, reflecting differences in the configuration of health systems and antibiotic supplies. At the same time, these stories reveal cross-cutting features and omissions of contemporary global health programming that shape the contours of antibiotic (over)use at national and local levels.ConclusionsOur findings challenge the predominant focus of stewardship frameworks on the practices of antibiotic end users. We suggest future interventions could consider systems—rather than individuals—as stewards of antibiotics, reducing the need to rely on these medicines to fix other issues of inequity, productivity and security.

© 2017 The Author(s). The frequency and duration of Chlamydia trachomatis (Ct) ocular infections decrease with age, suggesting development of partial immunity. However, there is a lack of clear correlates of immunity to Ct infection in humans. We screened sera from a cohort of Gambian children followed for six-months against a Ct-proteome microarray. At genome sequence level, we detected signatures of selection from a population of ocular Ct isolates from Guinea-Bissau. Together these approaches allowed us to highlight the focus of humoral responses and hypothesise new modes of pathogen immune evasion. Children who were susceptible to frequent and/or prolonged Ct infection had a less focussed antibody response, including preferential recognition of forty-two antigens. There was evidence of positive and purifying selection across the genome, but little balancing selection. In contrast, most antigens that were associated with susceptibility were under neutral selection. These data suggest an evasion strategy in which Ct presents a large panel of irrelevant antigens to the immune system to block or misdirect protective responses. Development of a focused immune response, possibly induced through vaccination, may be an effective strategy to promote protection to Ct infection.

Background There remains limited data on what variables affect risk of transmission of SARS-CoV-2 and developing symptomatic Covid-19 and in particular the relationship to viral load (VL). We analysed data from linked index cases and their contacts to explore factors associated with transmission of SARS-CoV-2. Methods Patients were recruited as part of a randomized control trial, conducted between March to April 2020, that aimed to assess if hydroxychloroquine reduced transmission of SARS-CoV-2. Non-hospitalised Covid-19 cases and their contacts were identified through the local surveillance system. VL, measured by quantitative PCR from a nasopharyngeal swab, was assessed at enrollment, at day 14, and whenever the participant reported Covid-19-like symptoms. Risk of transmission, developing symptomatic disease and incubation dynamics were evaluated using regression analysis. Findings We identified 314 cases, 282 of which had at least one contact (753 contacts in total). Ninety (33%) of 282 clusters had at least one transmission event. The secondary attack rate was 16% (125/753), with a variation from 12% to 24% for VL of the index case of 109 copies/mL, respectively (OR per log10 increase in VL 1.3 95%CI 1.1–1.6). Increased risk of transmission was also associated with household contact (OR 2.7; 1.4–5.06) and age of the contact (OR 1.02 per year; 1.01–1.04). The proportion of PCR positive contacts who developed symptomatic Covid-19 was 40.3% (181/449), with a variation from 25% to 60% for VL of the contact 109 copies/mL (HR log10 increase in VL 1.12; 95% CI 1.05 – 1.2). Time to onset of symptomatic disease decreased from a median of 7 days (IQR 5–10) for individuals with an initial viral load 109, respectively. Interpretation Viral load of index cases is a leading driver of SARS-CoV-2 transmission. The risk of symptomatic Covid-19 is strongly associated with viral load of contacts at baseline and shortens the incubation time in a dose-dependent manner., Crowdfunding campaign YoMeCorono (http://www.yomecorono.com/), and Generalitat de Catalunya. Support for laboratory equipment from Foundation Dormeur.

Background: Trachoma, a neglected tropical disease, is the leading infectious cause of blindness and visual impairment worldwide. Host responses to ocular chlamydial infection resulting in chronic inflammation and expansion of non-chlamydial bacteria are hypothesized risk factors for development of active trachoma and conjunctival scarring.Methods: Ocular swabs from trachoma endemic populations in The Gambia were selected from archived samples for 16S sequencing and host conjunctival gene expression. We recruited children with active trachoma and adults with conjunctival scarring, alongside corresponding matched controls.Findings: In children, active trachoma was not associated with significant changes in the ocular microbiome. Haemophilus enrichment was associated with antimicrobial responses but not linked to active trachoma. Adults with scarring trachoma had a reduced ocular bacterial diversity compared to controls, with increased relative abundance of Corynebacterium. Increased abundance of Corynebacterium in scarring disease was associated with innate immune responses to the microbiota, dominated by altered mucin expression and increased matrix adhesion.Interpretation: In the absence of current Chlamydia trachomatis infection, changes in the ocular microbiome associate with differential expression of antimicrobial and inflammatory genes that impair epithelial cell health. In scarring trachoma, expansion of non-pathogenic bacteria such as Corynebacterium and innate responses are coincident, warranting further investigation of this relationship. Comparisons between active and scarring trachoma supported the relative absence of type-2 interferon responses in scarring, whilst highlighting a common suppression of re-epithelialization with altered epithelial and bacterial adhesion, likely contributing to development of scarring pathology.

BackgroundTrachoma, a neglected tropical disease, is the leading infectious cause of blindness and visual impairment worldwide. Host responses to ocular chlamydial infection resulting in chronic inflammation and expansion of non-chlamydial bacteria are hypothesised risk factors for development of active trachoma and conjunctival scarringMethodsOcular swabs from trachoma endemic populations in The Gambia were selected from archived samples for 16S sequencing and host conjunctival gene expression. We recruited children with active trachoma and adults with conjunctival scarring, alongside corresponding matched controls.FindingsIn children, active trachoma was not associated with significant changes in the ocular microbiome.Haemophilusenrichment was associated with antimicrobial responses but not linked to active trachoma. Adults with scarring trachoma had a reduced ocular bacterial diversity compared to controls, with increased relative abundance ofCorynebacterium. Increased abundance ofCorynebacteriumin scarring disease was associated with innate immune responses to the microbiota, dominated by altered mucin expression and increased matrix adhesion.InterpretationIn the absence of currentC. trachomatisinfection, changes in the ocular microbiome associate with antimicrobial and inflammatory responses that impair epithelial cell health. In scarring trachoma, expansion of ‘non-pathogenic’ bacteria such asCorynebacteriumand innate responses are coincident, warranting further investigation of this relationship. Comparisons between active and scarring trachoma supported the relative absence of type-1 interferon responses in scarring, whilst highlighting a common suppression of re-epithelialisation with altered epithelial and bacterial adhesion, likely contributing to development of scarring pathology.

Yaws is a neglected tropical disease targeted for eradication by 2020. Kiribati, a Pacific Island nation, was previously endemic for yaws but lacks recent data from which its current endemicity status could be determined. This study tested antibody responses to Treponema pallidum to determine if transmission of yaws is taking place among children in Kiribati. Using a commercially available T. pallidum particle agglutination kit (Serodia®, Fujirebio Inc., Tokyo, Japan), we tested dried blood spots, collected during population-based trachoma prevalence surveys on Tarawa Atoll and Kiritimati Island, for long-lived treponemal antibodies. Dried blood spots from 1,420 children aged 1–9 years were tested. Only two were positive, suggesting T. pallidum is not being widely transmitted among children in the settings sampled. These data require support from additional surveys to demonstrate the absence of clinical signs of disease and molecular evidence of infection, to confirm that yaws is no longer endemic in Kiribati.

Background Ethnic and religious minorities have been disproportionately affected by SARS-CoV-2 worldwide. The UK strictly-Orthodox Jewish community has been severely affected by the pandemic. This group shares characteristics with other ethnic minorities including larger family sizes, higher rates of household crowding and relative socioeconomic deprivation. We studied a UK strictly-Orthodox Jewish population to understand transmission of COVID-19 within this community. Methods We performed a household-focused cross-sectional SARS-CoV-2 serosurvey between late-October and early December 2020 prior to the third national lockdown. Randomly-selected households completed a standardised questionnaire and underwent serological testing with a multiplex assay for SARS-CoV-2 IgG antibodies. We report clinical illness and testing before the serosurvey, seroprevalence stratified by age and sex. We used random-effects models to identify factors associated with infection and antibody titres. Findings A total of 343 households, consisting of 1,759 individuals, were recruited. Serum was available for 1,242 participants. The overall seroprevalence for SARS-CoV-2 was 64.3% (95% CI 61.6-67.0%). The lowest seroprevalence was 27.6% in children under 5 years and rose to 73.8% in secondary school children and 74% in adults. Antibody titres were higher in symptomatic individuals and declined over time since reported COVID-19 symptoms, with the decline more marked for nucleocapsid titres. Interpretation In this tight-knit religious minority population in the UK, we report one of the highest SARS-CoV-2 seroprevalence levels in the world to date, which was markedly higher than the reported 10% seroprevalence in London at the time of the study. In the context of this high force of infection, all age groups experienced a high burden of infection. Actions to reduce the burden of disease in this and other minority populations are urgently required. Funding This work was jointly funded by UKRI and NIHR [COV0335; MR/V027956/1], a donation from the LSHTM Alumni COVID-19 response fund, HDR UK, the MRC and the Wellcome Trust.

Made available in DSpace on 2019-10-06T16:55:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-01 Wisconsin Turfgrass Association National Institutes of Health Wellcome Trust Institute for National Strategic Studies Leukemia and Lymphoma Research Seventh Framework Programme Horizon 2020 Medical Research Council Canada National Institute on Drug Abuse National Institute of Child Health and Human Development Killer cell immunoglobulin-like receptors (KIRs) are expressed predominantly on natural killer cells, where they play a key role in the regulation of innate immune responses. Recent studies show that inhibitory KIRs can also affect adaptive T cell–mediated immunity. In mice and in human T cells in vitro, inhibitory KIR ligation enhanced CD8+ T cell survival. To investigate the clinical relevance of these observations, we conducted an extensive immunogenetic analysis of multiple independent cohorts of HIV-1–, hepatitis C virus (HCV)–, and human T cell leukemia virus type 1 (HTLV-1)–infected individuals in conjunction with in vitro assays of T cell survival, analysis of ex vivo KIR expression, and mathematical modeling of host-virus dynamics. Our data suggest that functional engagement of inhibitory KIRs enhances the CD8+ T cell response against HIV-1, HCV, and HTLV-1 and is a significant determinant of clinical outcome in all three viral infections. Department of Medicine Imperial College London Faculty of Engineering São Paulo State University—UNESP Institute for Infection and Immunity St. George’s University of London International AIDS Vaccine Initiative Human Immunology Laboratory Clinical Research Department London School of Hygiene and Tropical Medicine Divi-sion of Infection and Immunity Cardiff University School of Medicine Nuffield Department of Medicine University of Oxford Oxford NIHR Biomedical Research Centre Immunology Division Department of Pathology University of Cambridge Cancer and Inflammation Program Leidos Biomedical Research Inc. Frederick National Laboratory for Cancer Research Johns Hopkins University Rho San Francisco Department of Public Health Faculty of Medicine University of Southampton Division of Cancer Epidemiology and Genetics National Cancer Institute Ragon Institute of MGH, MIT and Harvard Faculty of Engineering São Paulo State University—UNESP Wisconsin Turfgrass Association: 090323/Z/09/Z National Institutes of Health: 100326Z/12/Z Wellcome Trust: 103865Z/14/Z Institute for National Strategic Studies: 105609/Z/14/Z Leukemia and Lymphoma Research: 15012 Seventh Framework Programme: 317040 Horizon 2020: 695551 Medical Research Council Canada: G1001052 Medical Research Council Canada: J007439 National Institute on Drug Abuse: K24-AI118591 Medical Research Council Canada: MR/L018373/L Medical Research Council Canada: MR/M019829/1 Medical Research Council Canada: MR/P001602/1 National Institutes of Health: R01 DA13324 National Institute on Drug Abuse: R01-DA-12568 National Institute of Child Health and Human Development: R01-HD-41224 National Institute on Drug Abuse: U01-DA-036297

Introduction Trachoma is endemic in several Pacific Island countries. The aims of this study were to (a) identify future trachoma mapping needs in the Pacific and (b) to examine whether any temporal trends in trachoma prevalence could be ascertained from the historical literature on trachoma in the Pacific Islands. Methods Human studies of trachoma and eye care in the Pacific Islands were identified from a systematic search of PubMed, EMbase, Scopus and Web of Science databases. A published quality assessment system for disease prevalence studies was modified to assess studies for quality and transparency. Results Few general ophthalmic studies in the Pacific mention trachoma. In targeted studies of trachoma, cases have consistently been identified throughout the Pacific since the early twentieth century. The largest number of studies come from Papua New Guinea and Fiji, whereas some countries have no published data on trachoma. The majority of studies identified were published before the Alliance for the Global Elimination of Trachoma 2020 was convened, so lack the standardisation of population-based mapping which has been implemented in the past decade. Conclusions Population-based trachoma prevalence estimates have been recently generated in Papua New Guinea, Solomon Islands, Vanuatu, Kiribati and Fiji. There is insufficient evidence to assess whether there has been temporal change in trachoma prevalence in these countries over the past century. Cases of trachoma have been identified in some countries (for example, Nauru and Samoa) which have no recent population-based mapping data, but may be at risk of trachoma endemcitiy. Deployment of appropriate mapping strategies is warranted to identify whether interventions are required.

Background: The low population prevalence of trachomatous trichiasis and high prevalence of trachomatous inflammation–follicular (TF) provide contradictory estimates of the magnitude of the public health threat from trachoma in the Solomon Islands. Improved characterisation of the biology of trachoma in the region may support policy makers as they decide what interventions are required. Here, age-specific profiles of anti-Pgp3 antibodies and conjunctival scarring were examined to determine whether there is evidence of ongoing transmission and pathology from ocular Chlamydia trachomatis (Ct) infection. Methods: A total of 1511 individuals aged ≥1 year were enrolled from randomly selected households in 13 villages in which >10% of children aged 1–9 years had TF prior to a single round of azithromycin mass drug administration undertaken six months previously. Blood was collected to be screened for antibodies to the Ct antigen Pgp3. Tarsal conjunctival photographs were collected for analysis of scarring severity. Results: Anti-Pgp3 seropositivity was 18% in 1–9 year olds, sharply increasing around the age of sexual debut to reach 69% in those over 25 years. Anti-Pgp3 seropositivity did not increase significantly between the ages of 1–9 years and was not associated with TF (p=0.581) or scarring in children (p=0.472). Conjunctival scars were visible in 13.1% of photographs. Mild (p

Background: The low population-prevalence of trachomatous trichiasis and high prevalence of trachomatous inflammation–follicular (TF) provide contradictory estimates of the magnitude of the public health threat from trachoma in the Solomon Islands. Improved characterisation of the biology of trachoma in the region may support policy makers as they decide what interventions are required. Here, age-specific profiles of anti-Pgp3 antibodies and conjunctival scarring were examined to determine whether there is evidence of ongoing transmission and pathology from ocular Chlamydia trachomatis (Ct) infection. Methods: A total of 1511 individuals aged ≥1 year were enrolled from randomly selected households in 13 villages in which >10% of children aged 1–9 years had TF prior to a single round of azithromycin mass drug administration undertaken six months previously. Blood was collected to be screened for antibodies to the Ct antigen Pgp3. Tarsal conjunctival photographs were collected for analysis of scarring severity. Results: Anti-Pgp3 seropositivity was 18% in 1–9 year olds, sharply increasing around the age of sexual debut to reach 69% in those over 25 years. Anti-Pgp3 seropositivity did not increase significantly between the ages of 1–9 years and was not associated with TF (p=0.581) or scarring in children (p=0.472). Conjunctival scars were visible in 13.1% of photographs. Mild (p

BackgroundSeveral non-chlamydial microbial pathogens are associated with clinical signs of active trachoma in trachoma-endemic communities with a low prevalence of ocular Chlamydia trachomatis (Ct) infection. In the Solomon Islands, the prevalence of Ct among children is low despite the prevalence of active trachoma being moderate. We therefore set out to investigate whether active trachoma was associated with a common non-chlamydial infection or with a dominant polymicrobial community dysbiosis in the Solomon Islands.MethodsWe studied DNA from conjunctival swabs collected from 257 Solomon Islanders with active trachoma and matched controls. Droplet digital PCR was used to test for pathogens suspected to be able to induce follicular conjunctivitis. Polymicrobial community diversity and composition were studied by sequencing of hypervariable regions of the 16S ribosomal ribonucleic acid gene in a subset of 54 cases and 53 controls.ResultsAlthough Ct was associated with active trachoma, the number of infections was low (cases: 3.9%, controls: 0.4%). Estimated prevalence (cases, controls) of each non-chlamydial infection was as follows: S. aureus (1.9%, 1.9%), Adenoviridae (1.2%, 1.2%), coagulase-negative Staphylococcus (5.8%, 4.3%), H. influenzae (7.4%, 11.7%), M. catarrhalis (2.3%, 4.7%) and S. pneumoniae (7.0%, 6.2%). There was no statistically significant association between clinical signs of trachoma and presence or load of any of the non-Ct infections that were assayed. Inter-individual variations in the conjunctival microbiome were characterised by differences in the levels of Corynebacterium, Proprionibacterium, Helicobacter and Paracoccus, but diversity and relative abundance of these specific genera did not differ significantly between cases and controls.DiscussionIt is unlikely that the prevalent trachoma-like follicular conjunctivitis in the Solomon Islands has a dominant bacterial aetiology. Before implementing community-wide azithromycin distribution for trachoma, policy makers should consider that clinical signs of trachoma can be observed in the absence of any detectable azithromycin-susceptible organism.

Chlamydia trachomatis is the most commonly diagnosed bacterial sexually transmitted infection and can lead to tubal factor infertility, a disease characterised by fibrosis of the fallopian tubes. Genetic polymorphisms in molecular pathways involving G protein-coupled receptor signalling, the Akt/PI3K cascade, the mitotic cell cycle, and immune response have been identified in association with the development of trachomatous scarring, an ocular form of chlamydia-related fibrotic pathology. In this case-control study, we performed genome-wide association and pathways-based analysis in a sample of 71 Dutch women who attended an STI clinic who were seropositive for Chlamydia trachomatis antibodies and 169 high-risk Dutch women who sought similar health services but who were seronegative. We identified two regions of within-gene SNP association with Chlamydia trachomatis serological response and found that GPCR signalling and cell cycle pathways were also associated with the trait. These pathway-level associations appear to be common to immunological sequelae of chlamydial infections in both ocular and urogenital tropisms. These pathways may be central mediators of human refractoriness to chlamydial diseases.

Trachoma is a blinding disease usually caused by infection withChlamydia trachomatis (Ct)serovars A, B, and C in the upper tarsal conjunctiva. Individuals in endemic regions are repeatedly infected withCtthroughout childhood. A proportion of individuals experience prolonged or severe inflammatory episodes that are known to be significant risk factors for ocular scarring in later life. Continued scarring often leads to trichiasis and in-turning of the eyelashes, which causes pain and can eventually cause blindness. The mechanisms driving the chronic immunopathology in the conjunctiva, which largely progresses in the absence of detectableCtinfection in adults, are likely to be multifactorial. Socioeconomic status, education, and behavior have been identified as contributing to the risk of scarring and inflammation. We focus on the contribution of host and pathogen genetic variation, bacterial ecology of the conjunctiva, and host epigenetic imprinting including small RNA regulation by both host and pathogen in the development of ocular pathology. Each of these factors or processes contributes to pathogenic outcomes in other inflammatory diseases and we outline their potential role in trachoma.

Chlamydia trachomatis is the most commonly diagnosed bacterial sexually transmitted infection and can lead to tubal factor infertility, a disease characterised by fibrosis of the fallopian tubes. Genetic polymorphisms in molecular pathways involving G protein coupled receptor signalling, the Akt/PI3K cascade, the mitotic cell cycle and immune response have been identified in association with the development of trachomatous scarring, an ocular form of chlamydia-related fibrotic pathology. In this case control study, we performed genome-wide association and pathways based analysis in a sample of 71 Dutch women who attended an STI clinic who were seropositive for Chlamydia trachomatis antibodies and 169 high risk Dutch women who sought similar health services but who were seronegative. We identified two regions of within-gene SNP association with Chlamydia trachomatis serological response and found that GPCR signalling and cell cycle pathways were also associated with the trait. These pathway level associations appear to be common to immunological sequelae of Chlamydial infections in both ocular and urogenital tropisms. These pathways may be central mediators of human refractoriness to chlamydial diseases.

The health impact of many copy number variants in our genome remains still largely to be discovered. Detecting and genotyping this often complex variation presents a technical challenge. Here we describe a 96-well format droplet digital PCR (ddPCR) protocol for genotyping a common copy variant in the human haptoglobin gene. ddPCR allows for high-throughput and accurate quantitation of gene copy numbers.

Objective In some Pacific Island countries, such as Solomon Islands and Fiji, active trachoma is common, but ocular Chlamydia trachomatis (Ct) infection and trachomatous trichiasis (TT) are rare. On Tarawa, the most populous Kiribati island, both the active trachoma sign “trachomatous inflammation—follicular” (TF) and TT are present at prevalences warranting intervention. We sought to estimate prevalences of TF, TT, ocular Ct infection, and anti-Ct antibodies on Kiritimati Island, Kiribati, to assess local relationships between these parameters, and to help determine the need for interventions against trachoma on Kiribati islands other than Tarawa. Methods As part of the Global Trachoma Mapping Project (GTMP), on Kiritimati, we examined 406 children aged 1–9 years for active trachoma. We collected conjunctival swabs (for droplet digital PCR against Ct plasmid targets) from 1–9-year-olds with active trachoma, and a systematic selection of 1–9-year-olds without active trachoma. We collected dried blood spots (for anti-Pgp3 ELISA) from all 1–9-year-old children. We also examined 416 adults aged ≥15 years for TT. Prevalence of TF and TT was adjusted for age (TF) or age and gender (TT) in five-year age bands. Results The age-adjusted prevalence of TF in 1–9-year-olds was 28% (95% confidence interval [CI]: 24–35). The age- and gender-adjusted prevalence of TT in those aged ≥15 years was 0.2% (95% CI: 0.1–0.3%). Twenty-six (13.5%) of 193 swabs from children without active trachoma, and 58 (49.2%) of 118 swabs from children with active trachoma were positive for Ct DNA. Two hundred and ten (53%) of 397 children had anti-Pgp3 antibodies. Both infection (p, Author summary Ocular infection with Chlamydia trachomatis causes trachoma. It is the leading infectious cause of blindness, and the target of an international campaign for elimination as a public health problem. Trachoma is endemic to Tarawa, the most populated island of Kiribati, housing approximately half the national population. However, the country has 20 inhabited islands and there were no previous trachoma prevalence data from Kiribati outside Tarawa. We set out to determine the prevalence of trachoma in the second most populated island, Kiritimati, located over 3000 km from Tarawa. In some other Pacific Island countries, ocular C. trachomatis infection is much less prevalent than the clinical signs that are used to guide interventions; we therefore looked for PCR-based evidence of current infection and antibodies to chlamydial proteins, in addition to recording clinical signs of trachoma. Our results indicate that trachoma and ocular C. trachomatis infection are prevalent on Kiritimati, and suggest that interventions are required here. The combined application of antibody, nucleic acid and clinical tools in an intervention-naïve population provides insight into their inter-relationships and the data are, therefore, of considerable interest to elimination programmes within and beyond the Pacific.

Trachoma, caused by Chlamydia trachomatis, is the world's leading infectious cause of blindness and remains a significant public health problem. Much of trachomatous disease pathology is thought to be caused indirectly by host cellular and immune responses, however the immune response during active trachoma and how this initiates progressive scarring is not clearly understood. Defining protective vs. pathogenic immune response to C. trachomatis is important for vaccine design and evaluation. This study reports the baseline results of a longitudinal cohort of Tanzanian children, who were monitored for 4 years in order to determine the immunofibrogenic and infectious correlates of progressive scarring trachoma. In this cohort baseline, 506 children aged 6–10 years were assessed for clinical signs, infection status and the expression of 91 genes of interest prior to mass azithromycin administration for trachoma control. C. trachomatis was detected using droplet digital PCR and gene expression was measured using quantitative real-time PCR. The prevalence of follicles, papillary inflammation and scarring were 33.6, 31.6, and 28.5%, respectively. C. trachomatis was detected in 78/506 (15.4%) individuals, 62/78 of whom also had follicles. C. trachomatis infection was associated with a strong upregulation of IFNG and IL22, the enrichment of Th1 and NK cell pathways and Th17 cell-associated cytokines. In individuals with inflammation in the absence of infection the IFNG/IL22 and NK cell response was reduced, however, pro-inflammatory, growth and matrix factors remained upregulated and mucins were downregulated. Our data suggest that, strong IFNG/IL22 responses, probably related to Th1 and NK cell involvement, is important for clearance of C. trachomatis and that the residual pro-inflammatory and pro-fibrotic phenotype that persists after infection might contribute to pathological scarring. Interestingly, females appear more susceptible to developing papillary inflammation and scarring than males, even at this young age, despite comparable levels of C. trachomatis infection. Females also had increased expression of a number of IFNγ pathway related genes relative to males, suggesting that overexpression of this pathway in response to infection might contribute to more severe scarring. Longitudinal investigation of these factors will reveal their relative contributions to protection from C. trachomatis infection and development of scarring complications.

Killer-cell immunoglobulin-like receptors (KIRs) are expressed predominantly on natural killer cells, where they play a key role in the regulation of innate immune responses. Recent studies show that inhibitory KIRs can also impact adaptive T cell-mediated immunity. In mice and in human T cells in vitro, inhibitory KIR ligation enhanced CD8(+) T cell survival. To investigate the clinical relevance of these observations, we conducted an extensive immunogenetic analysis of multiple, independent cohorts of HIV-1, hepatitis C virus (HCV) and human T cell leukemia virus (HTLV-1)-infected individuals in conjunction with in vitro assays of T cell survival, analysis of ex vivo KIR expression and mathematical modeling of host-virus dynamics. Our data suggest that functional engagement of inhibitory KIRs enhances the CD8(+) T cell response against HIV-1, HCV and HTLV-1 and is a significant determinant of clinical outcome in all three viral infections.

Introduction The presence of antibodies against Chlamydia trachomatis (Ct) is indicative of previous genital or ocular infection. Serology was introduced in the 1970s to support the diagnosis of pelvic inflammatory disease (PID), but fell out of favour due to its cross-reactivity with Chlamydophila (Chlamydia) pneumoniae bacteria. With the recent development of sensitive and specific assays, as well as the identification of immunogenic Ct antigens developed as recombinant proteins, serology holds the potential to be a useful tool in public health. To date, there has been no summary of the techniques used, their development and their potential usefulness in public and tropical medicine. Methods We searched PubMed, Cochrane, Lilacs, Scielo, Scopus and Web of Science for articles published on serological techniques and their use in a public health context. Studies were categorised by technique employed, antigen used and antibody detected. Results A total of 16 studies were included- 5 related to ocular Ct infection and 11 related to genital Ct infection. The trachoma studies were predominantly based on Tanzanian samples, while the genital studies were based on samples from an array of countries. The studies were heterogeneous in design, assay and antigen used, and immunoglobulin detected. The estimated prevalence of antibodies against Ct in trachoma studies ranged from 0%–62%; from 0%–88.9% in genital studies. For genital Ct infections, serology is commonly used to explore disease sequelae. For ocular Ct infections, serology is explored as a means to monitor elimination efforts. Conclusion Techniques used to measure the prevalence of antibodies against Ct have reported increased sensitivity and specificity. There is wide diversity in antigens and assays used and antibodies detected. The practicality of an assay depends on resources available, purpose of the study, and population being studied. There is wide scope for the development and refinement of techniques to increase the value of serology as it relates to development of new techniques, research and public health.

Trachoma is caused byChlamydia trachomatis(Ct). It is targeted for global elimination as a public health problem. In 2014, a population-based cross-sectional study was performed in two previously trachoma-endemic areas of The Gambia. Participants of all ages from Lower River Region (LRR) (N = 1028) and Upper River Region (URR) (N = 840) underwent examination for trachoma and had blood collected for detection of antibodies against the Ct antigen Pgp3, by ELISA. Overall, 30 (1.6%) individuals had active trachoma; the prevalence in children aged 1–9 years was 3.4% (25/742) with no statistically significant difference in prevalence between the regions. There was a significant difference in overall seroprevalence by region: 26.2% in LRR and 17.1% in URR (p

Droplet digital PCR (ddPCR) is an emulsion PCR process that performs absolute quantitation of nucleic acids. We developed a ddPCR assay for Chlamydia trachomatis infections and found it to be accurate and precise. Using PCR mixtures containing plasmids engineered to include the PCR target sequences, we were able to quantify with a dynamic range between 0.07 and 3,160 targets/μl ( r 2 = 0.9927) with >95% confidence. Using 1,509 clinical conjunctival swab samples from a population in which trachoma is endemic in Guinea Bissau, we evaluated the specificity and sensitivity of the quantitative ddPCR assay in diagnosing ocular C. trachomatis infections by comparing the performances of ddPCR and the Roche Amplicor CT/NG test. We defined ddPCR tests as positive when we had ≥95% confidence in a nonzero estimate of target load. The sensitivity of ddPCR against Amplicor was 73.3% (95% confidence interval [CI], 67.9 to 78.7%), and specificity was 99.1% (95% CI, 98.6 to 99.6%). Negative and positive predictive values were 94.6% (95% CI, 93.4 to 95.8%) and 94.5% (95% CI, 91.3 to 97.7%), respectively. Based on Amplicor CT/NG testing, the estimated population prevalence of C. trachomatis ocular infection was ∼17.5%. Receiver-operator curve analysis was used to select critical cutoff values for use in clinical settings in which a balance between higher sensitivity and specificity is required. We concluded that ddPCR is an effective diagnostic technology suitable for both research and clinical use in diagnosing ocular C. trachomatis infections.

Introduction Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), is the leading infectious cause of preventable blindness. Many commercial platforms are available that provide highly sensitive and specific detection of Ct DNA. However, the majority of these commercial platforms are inaccessible for population-level surveys in resource-limited settings typical to trachoma control programmes. We developed two low-cost quantitative PCR (qPCR) tests for Ct using readily available reagents on standard real-time thermocyclers. Methods Each multiplex qPCR test targets one genomic and one plasmid Ct target in addition to an endogenous positive control for Homo sapiens DNA. The quantitative performance of the qPCR assays in clinical samples was determined by comparison to a previously evaluated droplet digital PCR (ddPCR) test. The diagnostic performance of the qPCR assays were evaluated against a commercial assay (artus C. trachomatis Plus RG PCR, Qiagen) using molecular diagnostics quality control standards and clinical samples. We examined the yield of Ct DNA prepared from five different DNA extraction kits and a cold chain-free dry-sample preservation method using swabs spiked with fixed concentrations of human and Ct DNA. Results The qPCR assay was highly reproducible (Ct plasmid and genomic targets mean total coefficients of variance 41.5% and 48.3%, respectively). The assay detected 8/8 core specimens upon testing of a quality control panel and performed well in comparison to commercially marketed comparator test (sensitivity and specificity > 90%). Optimal extraction and sample preservation methods for research applications were identified. Conclusion We describe a pipeline from collection to diagnosis providing the most efficient sample preservation and extraction with significant per test cost savings over a commercial qPCR diagnostic assay. The assay and its evaluation should allow control programs wishing to conduct independent research within the context of trachoma control, access to an affordable test with defined performance characteristics., Graphical abstract Image 1, Highlights • Current WHO guidelines for trachoma control do not recommend tests for infection. • A Chlamydia trachomatis diagnostic more suited to low-income economies was developed. • A complete sample-to-result pipeline is evaluated. • The assay offers high throughput and comparable performance to commercial diagnostics. • The assay was significantly cheaper than commercial alternatives.

The health impact of many copy number variants in our genome remains still largely to be discovered. Detecting and genotyping this often complex variation presents a technical challenge. Here we describe a 96-well format droplet digital PCR (ddPCR) protocol for genotyping a common copy variant in the human haptoglobin gene. ddPCR allows for high-throughput and accurate quantitation of gene copy numbers.

Background Healthcare and other front-line workers are at particular risk of infection with Ebola virus (EBOV). Despite the large-scale deployment of international responders, few cases of Ebola virus disease have been diagnosed in this group. Since asymptomatic or pauci-symptomatic infection has been described, it is plausible that infections have occurred in healthcare workers but have escaped being diagnosed. We aimed to assess the prevalence of asymptomatic or pauci-symptomatic infection, and of exposure events, among returned responders to the West African Ebola epidemic 2014–2016. Methods and findings We used snowball sampling to identify responders who had returned to the UK or Ireland, and used an online consent and questionnaire to determine their exposure to EBOV and their experience of illness. Oral fluid collection devices were sent and returned by post, and samples were tested using an EBOV IgG capture assay that detects IgG to Ebola glycoprotein. Blood was collected from returnees with reactive samples for further testing. Unexposed UK controls were also recruited. In all, 300 individuals consented, of whom 268 (89.3%) returned an oral fluid sample (OFS). The majority had worked in Sierra Leone in clinical, laboratory, research, and other roles. Fifty-three UK controls consented and provided samples using the same method. Of the returnees, 47 (17.5%) reported that they had had a possible EBOV exposure. Based on their free-text descriptions, using a published risk assessment method, we classified 43 (16%) as having had incidents with risk of Ebola transmission, including five intermediate-risk and one high-risk exposure. Of the returnees, 57 (21%) reported a febrile or diarrhoeal illness in West Africa or within 1 mo of return, of whom 40 (70%) were not tested at the time for EBOV infection. Of the 268 OFSs, 266 were unreactive. Two returnees, who did not experience an illness in West Africa or on return, had OFSs that were reactive on the EBOV IgG capture assay, with similar results on plasma. One individual had no further positive test results; the other had a positive result on a double-antigen bridging assay but not on a competitive assay or on an indirect EBOV IgG ELISA. All 53 controls had non-reactive OFSs. While the participants were not a random sample of returnees, the number participating was high. Conclusions This is the first study, to our knowledge, of the prevalence of EBOV infection in international responders. More than 99% had clear negative results. Sera from two individuals had discordant results on the different assays; both were negative on the competitive assay, suggesting that prior infection was unlikely. The finding that a significant proportion experienced “near miss” exposure events, and that most of those who experienced symptoms did not get tested for EBOV at the time, suggests a need to review and standardise protocols for the management of possible exposure to EBOV, and for the management of illness, across organisations that deploy staff to outbreaks., In a cross-sectional study, Catherine Frances Houlihan and colleagues surveyed international responders to the West African Ebola crisis, after they returned to the UK or Ireland, to analyze their experience of risk., Author summary Why was this study done? A large number of international volunteers worked in West Africa during the largest Ebola outbreak ever seen, but only a small number of international staff were diagnosed with Ebola virus disease. Since asymptomatic or unrecognised infections can occur, it is possible that some infections in international responders to the epidemic were missed. What did the researchers do and find? We carried out an online survey and antibody testing of oral fluid samples from 268 individuals who returned to the UK and Ireland after working in West Africa during the Ebola outbreak. A high number of near miss events were reported, and less than a third of individuals who experienced illness whilst in West Africa or soon after return were tested for Ebola virus while they were unwell. Of the 268 who were tested for antibodies, two had reactive test results, but these were not reactive on further testing. What do these findings mean? Although our study could not reach every individual who returned to the UK after working in West Africa during the Ebola outbreak, we demonstrated that asymptomatic or pauci-symptomatic Ebola virus infection was rare in international healthcare and other workers in our study who responded to the epidemic in West Africa. The descriptions of near miss events and the finding that many of those who experienced illness were not tested at the time suggest that protocols for the management of possible exposure to Ebola virus and for the management of illness should be reviewed and standardised across organisations that deploy staff to outbreaks.

Background Trachoma, a neglected tropical disease, is caused by ocular infection with Chlamydia trachomatis (Ct). The World Health Organization (WHO) recommends three annual rounds of community mass drug treatment with azithromycin (MDA) if the prevalence of follicular trachoma in 1–9 year olds (TF1–9) exceeds 10% at district level to achieve an elimination target of district-level TF1–9 below 5% after. To evaluate this strategy in treatment-naïve trachoma-endemic island communities in Guinea Bissau, we conducted a cross-sectional population-based trachoma survey on four islands. The upper tarsal conjunctivae of each participant were clinically assessed for trachoma and conjunctival swabs were obtained (n = 1507). We used a droplet digital PCR assay to detect Ct infection and estimate bacterial load. We visited the same households during a second cross-sectional survey and repeated the ocular examination and obtained conjunctival swabs from these households one year after MDA (n = 1029). Results Pre-MDA TF1–9 was 22.0% (136/618). Overall Ct infection prevalence (CtI) was 18.6% (25.4% in 1–9 year olds). Post-MDA (estimated coverage 70%), TF1–9 and CtI were significantly reduced (7.4% (29/394, P < 0.001) and 3.3% (34/1029, P < 0.001) (6.6% in 1–9 year olds, P < 0.001), respectively. Median ocular Ct load was reduced from 2038 to 384 copies/swab (P < 0.001). Following MDA cases of Ct infection were highly clustered (Moran’s I 0.27, P < 0.001), with fewer clusters of Ct infection overall, fewer clusters of cases with high load infections and less severe disease. Conclusions Despite a significant reduction in the number of clusters of Ct infection, mean Ct load, disease severity and presence of clusters of cases of high load Ct infection suggesting the beginning of trachoma control in isolated island communities, following a single round of MDA we demonstrate that transmission is still ongoing. These detailed data are useful in understanding the epidemiology of ocular Ct infection in the context of MDA and the tools employed may have utility in determining trachoma elimination and surveillance activities in similar settings.

Robinsoniella peoriensis is a Gram-positive, strictly anaerobic, spore-forming, rod-shaped organism. Here, we report the draft genome of R. peoriensis 6600698, initially classified as Clostridium difficile due to growth on selective agar, a fecal gdh PCR-positive result, and clinical symptoms. R. peoriensis is a potential confounder of C. difficile diagnosis.

Background Efforts are underway to eliminate trachoma as a public health problem by 2020. Programmatic guidelines are based on clinical signs that correlate poorly with Chlamydia trachomatis (Ct) infection in post-treatment and low-endemicity settings. Age-specific seroprevalence of anti Ct Pgp3 antibodies has been proposed as an alternative indicator of the need for intervention. To standardise the use of these tools, it is necessary to develop an analytical approach that performs reproducibly both within and between studies. Methodology Dried blood spots were collected in 2014 from children aged 1–9 years in Laos (n = 952) and Uganda (n = 2700) and from people aged 1–90 years in The Gambia (n = 1868). Anti-Pgp3 antibodies were detected by ELISA. A number of visual and statistical analytical approaches for defining serological status were compared. Principal Findings Seroprevalence was estimated at 11.3% (Laos), 13.4% (Uganda) and 29.3% (The Gambia) by visual inspection of the inflection point. The expectation-maximisation algorithm estimated seroprevalence at 10.4% (Laos), 24.3% (Uganda) and 29.3% (The Gambia). Finite mixture model estimates were 15.6% (Laos), 17.1% (Uganda) and 26.2% (The Gambia). Receiver operating characteristic (ROC) curve analysis using a threshold calibrated against external reference specimens estimated the seroprevalence at 6.7% (Laos), 6.8% (Uganda) and 20.9% (The Gambia) when the threshold was set to optimise Youden’s J index. The ROC curve analysis was found to estimate seroprevalence at lower levels than estimates based on thresholds established using internal reference data. Thresholds defined using internal reference threshold methods did not vary substantially between population samples. Conclusions Internally calibrated approaches to threshold specification are reproducible and consistent and thus have advantages over methods that require external calibrators. We propose that future serological analyses in trachoma use a finite mixture model or expectation-maximisation algorithm as a means of setting the threshold for ELISA data. This will facilitate standardisation and harmonisation between studies and eliminate the need to establish and maintain a global calibration standard., Author Summary Trachoma is caused by the bacterium Chlamydia trachomatis (Ct). Individuals who have previously been infected with Ct carry specific antibodies in their blood. Recent studies have suggested that these antibodies may be a good way to estimate the intensity of transmission of this bacterium in a population. Among people who do have antibodies (seropositives) there is variation in the amount that is detectable in their blood. Some people have such low levels that differentiating them from those who don’t have antibodies (seronegatives) is challenging. We used a new test for Ct antibodies on blood specimens from three countries. Our test worked extremely well, giving reproducible results when we tested the same samples multiple times. We compared four different methods for setting the position of the threshold line between seronegatives and seropositives. The estimated transmission intensity in each country varied depending on the threshold method used, but two methods that used statistical modelling algorithms to define the two groups performed consistently across all three countries’ samples. We recommend that future studies should consider adopting the statistical modelling approaches, as they are objective tests that require no reference material and allow for standardisation between studies.

BACKGROUND: Leprosy is complicated by immunological reactions which can occur before, during and after successful completion of multidrug therapy. Genetic studies have suggested that polymorphisms in toll-like receptors (TLRs) may affect the susceptibility of an individual with leprosy to developing Type 1 reactions. OBJECTIVES: To examine the gene and protein expression of TLRs in the cutaneous lesions of leprosy Type 1 reactions at the onset of reaction and during systemic corticosteroid therapy. METHODS: Patients who were being treated for leprosy type 1 reactions with corticosteroids as part of a randomized controlled trial of corticosteroid treatment had skin biopsies performed before, during and at the end of treatment. The gene and protein expression of TLR2 and TLR4 were measured. RESULTS: We have demonstrated that the gene hARP-P0 is a suitable control gene for TLR gene expression studies in this population. The gene and protein expression of TLR2 and TLR4 were both reduced significantly during corticosteroid treatment. CONCLUSIONS: This is the first study to examine the expression of TLR2 and TLR4 in vivo in individuals experiencing leprosy Type 1 reactions. The data support the possibility of an important role for TLR2 and TLR4 in the pathogenesis of this important complication of leprosy.

Sequence-based typing (SBT) is a powerful method of genotyping in highly polymorphic gene systems. In standard SBT methods, both strands of a double-stranded template amplicon are sequenced in separate reactions in order to achieve high quality data across the region of interest. The amount of informative data that is obtained from the second strand sequence is often low, whilst the impact of performing second strand sequencing on costs and throughput are significant. Here we present short template amplicon and multiplex megaprimer-enabled relay (STAMMER) sequencing, a novel simultaneous sequence-based typing methodology that allows the detection of any practical amount of useful sequence from a plurality of distinct polymerase chain reaction products in a single sequencing reaction. In addition to simultaneous bidirectional sequencing, we show how the STAMMER approach can be used to simultaneously sequence a number of regions of interest that are not physically linked within the range of a single sequencing reaction. The efficiencies of this method could impact significantly on the output of SBT laboratories.

The European Searchable Tumour line Database (ESTDAB) ( http://www.ebi.ac.uk/ipd/estdab ) is a freely available and fully searchable database of melanoma-derived cell lines, which have been characterised for over 250 immunologically relevant markers by a consortium of European scientists. The database is linked to a cell bank, which can provide melanoma cell lines to non-profit investigators for a nominal handling charge. All cells are fully HLA typed at the genomic and surface expression levels. The expression of a number of surface antigens, apoptotic markers, tumour-associated antigens and extracellular matrix proteins has also been determined. Cytokine secretion has been tested and polymorphisms in cytokine genes have been identified. Glycans at the cell surface were identified and glycosyltransferase activity quantified. Cell lines with a particular constellation of these parameters can be sought online via the ESTDAB interface, which is included as part of the Immuno-Polymorphism Database (IPD) section of the European Bioinformatics Institute's (EBI) website.

Killer-cell immunoglobulin-like receptor (KIR) genes are highly polymorphic and polymorphisms have been found in all KIR exons. Although less is known of the introns, these also appear to be polymorphic. To generate a comprehensive database of KIR genomic sequences, which will aid in the design of KIR typing reagents, we have established a method for cloning and sequencing of KIR genes from genomic DNA. We cloned and sequenced the entire KIR2DL4 gene from genomic DNA using long template touchdown PCR and high capacity cloning vectors. Overlapping secondary amplicons were modified to include a nucleotide analogue that reduced sequencing problems associated with secondary structure formation in the KIR sequence. Using a modified sequencing chemistry we were able to sequence approximately 11,000 bases confirming the previously published KIR2DL4*005 allele sequence.

Chlamydia trachomatis causes both trachoma and sexually transmitted infections. These diseases have similar pathology and potentially similar genetic predisposing factors. We aimed to identify polymorphisms and pathways associated with pathological sequelae of ocular Chlamydia trachomatis infections in The Gambia. We report a discovery phase genome-wide association study (GWAS) of scarring trachoma (1090 cases, 1531 controls) that identified 27 SNPs with strong, but not genome-wide significant, association with disease (5 × 10−6 > P > 5 × 10−8). The most strongly associated SNP (rs111513399, P = 5.38 × 10−7) fell within a gene (PREX2) with homology to factors known to facilitate chlamydial entry to the host cell. Pathway analysis of GWAS data was significantly enriched for mitotic cell cycle processes (P = 0.001), the immune response (P = 0.00001) and for multiple cell surface receptor signalling pathways. New analyses of published transcriptome data sets from Gambia, Tanzania and Ethiopia also revealed that the same cell cycle and immune response pathways were enriched at the transcriptional level in various disease states. Although unconfirmed, the data suggest that genetic associations with chlamydial scarring disease may be focussed on processes relating to the immune response, the host cell cycle and cell surface receptor signalling.

Background Trachoma, a preventable blinding eye disease, is initiated by ocular infection with Chlamydia trachomatis (Ct). We previously showed that microRNAs (miR) -147b and miR-1285 were up-regulated in inflammatory trachomatous scarring. During the initial stage of disease, follicular trachoma with current Ct infection, the differential expression of miR has not yet been investigated. Methods Conjunctival samples were collected from 163 children aged 1–9 years old living in a trachoma-endemic region of Guinea Bissau, West Africa. Small RNA sequencing (RNAseq) was carried out on samples from five children with follicular trachoma and current Ct infection and five children with healthy conjunctivae and no Ct infection. Small RNAseq was also carried out on human epithelial cell lines infected with ocular Ct strains A2497 and isogenic plasmid-free A2497 in vitro. Results were validated by quantitative PCR (qPCR) in 163 clinical samples. Results Differential expression of RNAseq data identified 12 miR with changes in relative expression during follicular trachoma, of which 9 were confirmed as differentially expressed by qPCR (miR-155, miR-150, miR-142, miR-181b, miR-181a, miR-342, miR-132, miR-4728 and miR-184). MiR-155 and miR-184 expression had a direct relationship with the degree of clinical inflammation. MiR-155 was up-regulated (OR = 2.533 ((95 % CI = 1.291–4.971); P = 0.0069) and miR-184 was down-regulated (OR = 0.416 ((95 % CI = 0.300–0.578); P = 1.61*10−7) as the severity of clinical inflammation increased. Differential miR expression was not detected in HEp-2 or HCjE epithelial cells 48 h post infection with Ct in vitro. HCjE cells, a conjunctival epithelial cell line, had a markedly different miR background expression compared to HEp-2 cells. Conclusions In follicular trachoma, expression of miR-155 and miR-184 is correlated with the severity of inflammation. This likely reflects host regulation of the immune response and a prolonged period of wound healing following the clearance of Ct. Prolonged healing may be associated with subsequent development of scarring trachoma. Electronic supplementary material The online version of this article (doi:10.1186/s12879-016-1367-8) contains supplementary material, which is available to authorized users.

Malignant transformation of cells is frequently associated with abnormalities in human leukocyte antigen (HLA) expression. MHC class I loss or down-regulation in cancer cells is a major immune escape route used by a large variety of human tumours to evade antitumour immune responses mediated by cytotoxic T lymphocytes. The goal of our study was to explore HLA genotyping and phenotyping in a variety of melanoma tumour cell lines. A total of 91 melanoma cell lines were characterised for HLA class I and II genotype. In addition, 61 out of the 91 cell lines were also analysed for HLA class I and II cell surface molecule expression by flow cytometry. Unexpectedly, we found that 19.7% of the melanoma cell lines were homozygous for HLA class I genotypes, sometimes associated with HLA class II homozygosity (8.79%) and sometimes not (10.98%). The frequency of homozygosity was significantly higher compared with the control groups (1.6%). To identify the reasons underlying the high frequency of HLA homozygosity we searched for genomic deletions using eight pairs of highly polymorphic microsatellite markers covering the entire extended HLA complex on the short arm of chromosome 6. Our results were compatible with hemizygous deletions and suggest that loss of heterozygosity on chromosome arm 6p is a common feature in melanoma cell lines. In fact, although autologous normal DNA from the patients was not available and could not be tested, the retention in some cases of heterozygosity for a number of microsatellite markers would indicate a hemizygous deletion. In the rest of the cases, markers at 6p and 6q showed a single allele pattern indicating the probable loss of part or the whole of chromosome 6. These results led us to conclude that loss of heterozygosity in chromosome 6 is nonrandom and is possibly an immunologically relevant event in human malignant melanoma. Other well-established altered HLA class I phenotypes were also detected by flow cytometry that correspond to HLA class I total loss and HLA-ABC and/or specific HLA-B locus down-regulation.

Vitamin D deficiency may increase susceptibility to opportunistic infections in HIV-infected individuals. We found no evidence that vitamin D deficiency increases risk of cryptococcal meningitis or leads to impaired immune responses or microbiological clearance in HIV-infected patients with cryptococcal meningitis., Background. Vitamin D deficiency is associated with impaired immune responses and increased susceptibility to a number of intracellular pathogens in individuals infected with human immunodeficiency virus (HIV). It is not known whether such an association exists with Cryptococcus neoformans. Methods. Levels of 25-hydroxyvitamin D (25[OH]D) were measured in 150 patients with cryptococcal meningitis (CM) and 150 HIV-infected controls in Cape Town, South Africa, and associations between vitamin D deficiency and CM were examined. The 25-hydroxyvitamin D levels and cryptococcal notifications were analyzed for evidence of reciprocal seasonality. Associations between 25(OH)D levels and disease severity, immune responses, and microbiological clearance were investigated in the patients with CM. Results. Vitamin D deficiency (plasma 25[OH]D ≤50 nmol/L) was present in 74% of patients. Vitamin D deficiency was not associated with CM (adjusted odds ratio, 0.93 [95% confidence interval, .6–1.6]; P = .796). Levels of 25(OH)D showed marked seasonality, but no reciprocal seasonality was seen in CM notifications. No significant associations were found between 25(OH)D levels and fungal burden or levels of tumor necrosis factor α, interferon γ, interleukin 6, soluble CD14, or neopterin in cerebrospinal fluid. Rates of fungal clearance did not vary according to vitamin D status. Conclusions. Vitamin D deficiency does not predispose to the development of CM, or lead to impaired immune responses or microbiological clearance in HIV-infected patients with CM.

Background Chlamydia trachomatis is globally the predominant infectious cause of blindness and one of the most common bacterial causes of sexually transmitted infection. Infections of the conjunctiva cause the blinding disease trachoma, an immuno-pathological disease that is characterised by chronic conjunctival inflammation and fibrosis. The polymorphic Killer-cell Immunoglobulin-like Receptors (KIR) are found on Natural Killer cells and have co-evolved with the Human Leucocyte Antigen (HLA) class I system. Certain genetic constellations of KIR and HLA class I polymorphisms are associated with a number of diseases in which modulation of the innate responses to viral and intracellular bacterial pathogens is central. Methodology A sample of 134 Gambian pedigrees selected to contain at least one individual with conjunctival scarring in the F1 generation was used. Individuals (n = 830) were genotyped for HLA class I and KIR gene families. Family Based Association Tests and Case Pseudo-control tests were used to extend tests for transmission disequilibrium to take full advantage of the family design, genetic model and phenotype. Principle findings We found that the odds of trachomatous scarring increased with the number of genome copies of HLA-C2 (C1/C2 OR = 2.29 BHP-value = 0.006; C2/C2 OR = 3.97 BHP-value = 0.0004) and further increased when both KIR2DL2 and KIR2DL3 (C2/C2 OR = 5.95 BHP-value = 0.006) were present. Conclusions To explain the observations in the context of chlamydial infection and trachoma we propose a two-stage model of response and disease that balances the cytolytic response of KIR expressing NK cells with the ability to secrete interferon gamma, a combination that may cause pathology. The data presented indicate that HLA-C genotypes are important determinants of conjunctival scarring in trachoma and that KIR2DL2/KIR2DL3 heterozygosity further increases risk of conjunctival scarring in individuals carrying HLA-C2., Author Summary Chlamydia trachomatis is a pathogen that causes sexually transmitted infections (STIs) and the blinding disease trachoma. Natural Killer (NK) cells are part of the host immune system's first line of defence against infection. NK cell functions are genetically encoded and differences between individuals mean that some people are better able to respond to infections than others. We found that in certain combinations, specific variants of the gene HLA-C (Human Leucocyte Antigen, C) and of a complex set of genes called the Killer-cell Immunoglobulin-like Receptors (KIR) were associated with a six-fold increase in the relative risk of scarring tissue damage resulting from ocular C. trachomatis infection (trachoma). This combination of genetic variants may reduce the host's ability to effectively resolve infections and result in a harmful immune response that ultimately leads to tissue damage and scarring. KIR+ NK cells are potential cellular mediators of the damaging immune response. Previous studies have identified that the same HLA-KIR genetic constellation that associates with trachoma is actually protective against infectious diseases such as malaria and tuberculosis. The high frequency of the trachoma-associated constellation in African populations may therefore be explained by the evolutionary benefits of protection from the complications of severe disease.

In ophthalmological studies, the need to take photographs as evidence introduces additional complications to fieldwork. A recent report1 highlighted the usefulness of smartphones in the collection and grading of photographic evidence of trachoma. We have recently considered the wider potential of portable computerised equipment to integrate the recording and management of trachoma photographs with field data and biological specimens. While the overall resolution of smartphone cameras increases rapidly, an outstanding issue with smartphone photography is the phenomenon of shutter lag, a significant time interval between actuation and the recording of an image. Shutter lag is associated with image blurring as the subject or camera may have moved out of focus by the time of image recording. Recently released smartphones such as the HTC One X and Samsung Galaxy S3 are robust against shutter lag, but are currently the exceptions in a telephony market that has responded slowly to the problem. Light Field Photography (LFP) technology (http://www.lytro.com) has the potential to eliminate blurring …

In the last fifteen years published reports have described KIR gene-content frequency distributions in more than 120 populations worldwide. However, there have been limited studies examining these data in aggregate in order to detect overall patterns of variation at regional and global levels. Here, we present a summary of the collection of KIR gene-content data for 105 worldwide populations collected as part of the 15th and 16th International Histocompatibility and Immunogenetics Workshops, and preliminary results for data analysis.

Background The clinical signs of active trachoma are often present in the absence of ocular Chlamydia trachomatis infection in low prevalence and mass treated settings. Treatment decisions are currently based on the prevalence of clinical signs, and this may result in the unnecessary distribution of mass antibiotic treatment. We aimed to evaluate the diagnostic accuracy of a prototype point-of-care (POC) test, developed for field diagnosis of ocular C. trachomatis, in low prevalence settings of The Gambia and Senegal. Methodology/Principal Findings Three studies were conducted, two in The Gambia and one in Senegal. Children under the age of 10 years were screened for the clinical signs of trachoma. Two ocular swabs were taken from the right eye. The first swab was tested by the POC test in the field and the result independently graded by two readers. The second swab was tested for the presence of C. trachomatis by Amplicor Polymerase Chain Reaction. In Senegal, measurements of humidity and temperature in the field were taken. A total of 3734 children were screened, 950 in the first and 1171 in the second Gambian study, and 1613 in Senegal. The sensitivity of the prototype POC test ranged between 33.3–67.9%, the specificity between 92.4–99.0%, the positive predictive value between 4.3–21.0%, and the negative predictive value between 98.0–99.8%. The rate of false-positives increased markedly at temperatures above 31.4°C and relative humidities below 11.4%. Conclusions/Significance In its present format, this prototype POC test is not suitable for field diagnosis of ocular C. trachomatis as its specificity decreases in hot and dry conditions: the environment in which trachoma is predominantly found. In the absence of a suitable test for infection, trachoma diagnosis remains dependent on clinical signs. Under current WHO recommendations, this is likely resulting in the continued mass treatment of non-infected communities., Author Summary Trachoma, caused by infection of the eye with the bacterium Chlamydia trachomatis, is the leading infectious cause of blindness and is associated with poverty. Antibiotic treatment of all community members is one of the recommended control strategies for trachoma. However, in places where the prevalence of clinical signs is low, C. trachomatis eye infection is often absent. Laboratory testing for C. trachomatis infection by polymerase chain reaction (PCR) is highly sensitive but expensive and requires well-trained staff. A simple point-of-care (POC) test that can be used in trachoma-affected communities could help trachoma control efforts. We evaluated a POC test for C. trachomatis eye infection. Children under 10 years of age were screened for clinical signs of trachoma and C. trachomatis eye infection. The POC test result was compared with laboratory PCR test results. The POC test detected just over half of PCR test positives correctly. However, the POC test tended to give false-positive results in hot and dry conditions, which is the typical environment of trachoma. The POC test requires high specificity since it would be used to make treatment decisions at the community level. Therefore, its present format requires improvement before it can be utilized in trachoma control.

Background Trachoma has been endemic in The Gambia for decades. National trachoma control activities have been in place since the mid-1980's, but with no mass antibiotic treatment campaign. We aimed to assess the prevalence of active trachoma and of actual ocular Chlamydia trachomatis infection as measured by polymerase chain reaction (PCR) in the two Gambian regions that had had the highest prevalence of trachoma in the last national survey in 1996 prior to planned national mass antibiotic treatment distribution in 2006. Methodology/Principal Findings Two stage random sampling survey in 61 randomly selected Enumeration Areas (EAs) in North Bank Region (NBR) and Lower River Region (LRR). Fifty randomly selected children aged under 10 years were examined per EA for clinical signs of trachoma. In LRR, swabs were taken to test for ocular C. trachomatis infection. Unadjusted prevalences of active trachoma were calculated, as would be done in a trachoma control programme. The prevalence of trachomatous inflammation, follicular (TF) in the 2777 children aged 1–9 years was 12.3% (95% CI 8.8%–17.0%) in LRR and 10.0% (95% CI 7.7%–13.0%) in NBR, with significant variation within divisions (p, Author Summary Trachoma is the leading infectious cause of blindness worldwide, and is mainly found in tropical and poor countries. It is caused by infection of the eyes with the bacterium Chlamydia trachomatis. However, sometimes the clinical signs of disease can be present without infection being detected. Control efforts involve surgery, antibiotic treatment, face washing, and environmental improvement for better hygiene. Surveys of trachoma help countries to know whether and where they should implement control interventions. The Gambia is found in West Africa and has suffered from trachoma for decades. We conducted a survey of two Gambian regions to look at how much trachoma disease and C. trachomatis infection there is in the eyes. We found that although there was enough disease (≥10%) to warrant antibiotic treatment for everyone in the regions, there was nearly no infection (0.3%). This means that using clinical signs alone to make treatment decisions in low prevalence settings like The Gambia can lead to the waste of scarce resources. Our results also suggest that since less than 1% of children are infected with C. trachomatis, The Gambia is on course to achieve the World Health Organization's aim of eliminating blinding trachoma by the year 2020.

DNA enrichment by allele-specific hybridization (DEASH) was used as a means to isolate individual alleles of the killer cell immunoglobulin-like receptor (KIR2DL4) gene from heterozygous genomic DNA. Using long-template polymerase chain reaction (LT-PCR), the complete KIR2DL4 gene was amplified from a cell line that had previously been characterized for its KIR gene content by PCR using sequence-specific primers (PCR-SSP). The whole gene amplicons were sequenced and we identified two heterozygous positions in accordance with the predictions of the PCR-SSP. The amplicons were then hybridized to allele-specific, biotinylated oligonucleotide probes and through binding to streptavidin-coated beads, the targeted alleles were enriched. A second PCR amplified only the exonic regions of the enriched allele, and these were then sequenced in full. We show DEASH to be capable of enriching single alleles from a heterozygous PCR product, and through sequencing the enriched DNA, we are able to produce complete coding sequences of the KIR2DL4 alleles in accordance with the typing predicted by PCR-SSP.

complaints of diplopia, pain, edema and conjuctivitis on the right eye. Steroid treatment was started and she was referred to our setting because of total ophtalmoplegia on right eye. In addition to right eye chemosis and edema, eye movements were restricted in all directions with fix-dilated pupilla. Orbital computer tomography was interpreted as preorbital cellulitis. The patient was internalized in the ophthalmology department with piperacilintazobactam plus linezolid. After 5 days of treatment control MRI revealed a fungal infection which infiltrated all of the compartments of right eye, right cavernous sinus plus occludes right internal carotid artery. After this, the patient was transferred to infectious diseases department with the diagnosis of mucormycosis. Piperacillin-tazobactamwas switched to meropenem (1gr q8h i.v.). Also, amphotericin B deoxycholate 1mg/kg was started but due to allergic reaction, it was changed with LAMB (5mg/kg/day). Ethmoidal and sphenoidal sinus biopsies revealed mucormycosis in pathology. Results: The surgical debridement was not found applicable by surgical departments. The dose of LAMB was increased to 7mg/kg/day on the 10th day of LAMB. After six days posaconazole (400mg/day q12h p.o.) was added due to no clinical improvement.and worsening of the patient’s headache. Control Cranial MRI showed progression. Therefore LAMB dose was increased to 10mg/kg/day and deferasirox was added (20mg/kg/day p.o.) at the 15th day of treatment and contiunued for 14 days. After that, clinical findings and control cranial MRI stable when compared to previous MRI. On the 30th day of treatment LAMB dose was decreased to 7mg/kg/day andmeropenemplus linezolid treatment was stopped. The patient was discharged with posaconazole after 47 days of LAMB. Therewas no relapse on 3month follow-up of the patient with posaconazole treatment which was given 2months at all. Conclusion: Combination of antifungal antibiotics with deferasirox may be successful in the salvae tehrapy of mucormycosis.