Streptococcus pneumoniae, the pneumococcus, remains a major source of illness in older persons. Globally, it remains the most important pathogen in respiratory infection deaths.Disease due to Conjugated pneumococcal vaccines are used extensively in national pediatric programs, whereas a polysaccharide vaccine is used in all age groups, but mainly in the elderly and for high-risk groups.Recent data from the Netherlands led to the licensing in many countries of conjugated pneumococcal vaccines for older persons. There are substantial differences in recommendations from various national immunization technical advisory groups, which owe at least as much to differing assessments of available studies as to differences in local epidemiology.This review examines those differences and proposes a way forward. Streptococcus pneumoniae, the pneumococcus, may involve a normally sterile site; for example, blood or joint fluid ], or more commonly a local mucosal infection; for example, community-acquired non-bacteraemic pneumonia (CAP). Pneumococcal infection remains a major source of illness in the elderly. Globally, across all age groups, pneumococcus remains the most important pathogen in deaths due to respiratory infections [Infections caused by fections . Data onTwo types of vaccine are available and in widespread use in many countries. Conjugated vaccines (PCVs), covering 7 , 10 (Synflorix® GlaxoSmithKline), or 13 serotypes , are used extensively in national pediatric programs, whereas a polysaccharide vaccine containing 23 serotypes  is used mainly in the elderly and for high-risk groups.Neither the 7-valent nor the 10-valent conjugated vaccines are commonly used in current immunization programs; therefore, these vaccines are not considered further in this review. Both vaccines contain a subset of serotypes in 13vPCV.Recent data from the Netherlands Community-Acquired Pneumonia Immunisation Trial in Adults (CAPiTA) led to the licensing in many countries of conjugated vaccine for older persons and also assessment of the role of both vaccines in this age group . HoweverThere are substantial differences in the recommendations from various national immunization technical advisory groups (NITAGs), which the authors believe owes at least as much to differing assessments of available studies as to differences in local epidemiology.This review examines those differences and proposes a way forward.Assessing data for CAP of any cause and, more specifically, pneumococcal CAP is challenging—there is often no surveillance mechanism in place and published studies have used various combinations of diagnostic tests including blood culture, urinary antigen test, and sputum culture. Additionally, some historical studies used a pneumolysin test that no longer is regarded as diagnostic due to its poor specificity .A review of national databases from 2004 to 2012 and of published studies in Australia set the pneumococcal pneumonia hospitalization rate in those aged ≥65 years at 274/100,000 population, or 20% of all CAP hospitalisations. GP visits for pneumococcal CAP averaged 455 per 100,000 per year. Hospitalizations for IPD in 2012 were only found to be 19/100,000; thus pneumococcal CAP hospitalization rates were 15-fold higher than for IPD and the costs to the healthcare system were determined to be around 30 fold higher [Similarly, a three-year Canadian Serious Outcomes Surveillance (SOS) Network review found 23% of all-cause pneumonia admissions to be due to pneumococcus with a higher percentage in older patients and patients with a higher disease burden and mortality . The numAn Australian review of IPD trends in non-indigenous elderly people showed an ongoing substantial decrease in IPD due to serotypes found in the 7 V conjugated vaccine since its introduction in 2004. A similar trend was evident after only about 3 years of use of 13vPCV , and further decline continues. Conversely, likely as a result of serotype replacement, the IPD proportion attributable to 23v–non13v serotypes is increasing, with a rise from 19% to 27% of IPD in Australia [A significant proportion of the residual 13vPCV IPD currently seen is due to serotype 3; neither 13vPCV nor 23vPPV appear to have much effectiveness against this serotype. Studies in many countries have shown both poor vaccine effectiveness (VE) and no, or low, impact on disease rates with this serotype –9. In thA meta-analysis of the indirect effects of conjugated vaccines found the mean time taken to attain a 90% reduction in VT IPD due to 7vPCV serotypes was 8.9 years, and 9.5 years for the additional serotypes in 13vPCV, but not in 7vPCV .While data are more limited and somewhat inconsistent for CAP, a decline in VT 13vPCV CAP is also expected from the childhood program and has already been seen in unvaccinated young adults and older persons in some studies.Both invasive and non-invasive disease rates due to serotypes covered in the childhood 13vPCV program are declining in older persons.A cohort study of non-bacteremic pneumococcal pneumonia cases in adults in Nottingham described a 30% reduction of the proportion of 13vPCV serotypes within 3 years of the switch from 7vPCV to 13vPCV in the childhood program, having already seen an 88% decrease in CAP due to serotypes in 7vPCV . A DutchPublished estimates place the burden of disease due to CAP  as at least an order of magnitude greater than that due to IPD. Thus the effectiveness of both vaccine groups against CAP is important in the comparative assessment of the two vaccines, even if the burden of disease is decreasing due to serotypes in 13vPCV vaccine.VE data against CAP with 13vPCV is available from the CAPITA study, which was a randomised controlled trial (RCT). The study found a VE of 45% against VT pneumococcal CAP, 22% against all-type pneumococcal CAP and 5% against all-cause CAP [Reviews of available studies, including RCTs and observational studies, have come to differing conclusions about 23vPPV effectiveness against pneumococcal CAP. The variation occurs in both the estimated VE and the duration of effectiveness. The differences in outcomes of these meta-analyses are due to important variations in study inclusion criteria and variations in the quality and focus of the studies reviewed.Clinical studies and review documents have variously ascribed impacts of 23vPPV against pneumococcal CAP from no effect through to around 50% in many studies. While the studies are beset with methodological challenges and difficult to compare, the weight of evidence from the ‘better’ studies suggests that the attributable VE is non-zero and somewhere in this range. Protection against all cause CAP with both conjugate and polysaccharide vaccine types is similar and low, around 5%.Two of the studies , 16 inclThe observational studies in favour of 23vPPV being effective against VT CAP include a multi-centre Japanese prospective cohort study, which found 33% VE against VT CAP, 27% against all pneumococcal CAP and 2% against all CAP ; and a SSupporting a duration of protection with 23vPPV against CAP out to 5 years, Vila-Corcoles found a VE of 46% for pneumococcal CAP analysing subjects who were vaccinated up to 5 years previously . Meta-anThe 2013 Cochrane review of 23vPPV vaccine effectiveness against pneumococcal pneumonia derived a pooled VE of 54% (CI 16–75%) but this review included some older PPV formulations with higher antigen contents per serotype . The stuOther studies have found no effect of 23vPPV on pneumonia rates. A recent very large study in 152,000 healthy adults demonstrated no vaccine effect on pneumonia incidence . HoweverThe assessment of the cost effectiveness of pneumococcal vaccines in older persons is critically affected by: 1) the estimate used for herd protection produced by the conjugated childhood program against CAP; 2) the estimate used of VE for 23vPPVagainst pneumococcal pneumonia; 3) the VE for all-cause CAP (for each vaccine); and 4) the duration of effectiveness allocated to each vaccine.A British review, influenced heavily by the herd protection estimates, suggested that for 13vPCV to be cost effective the vaccine price needed to be negative . In GermWhen making the decision in the US to use both conjugate and polysaccharide vaccines sequentially for 3 years and then review the use of 13vPCV in 2018 , the AdvA review of the evidence assessed in making this decision highlighted the changing epidemiology of pneumococcal disease and the need to evaluate the utility and effectiveness of the new strategy .The burden of pneumococcal infections remains significant and more effective prevention could be achieved. Both conjugated and polysaccharide vaccines appear to be equally ineffective (around 4–5% VE) against all-cause CAP, in part because of a low contribution of pneumococcal CAP to all-cause CAP but also the increase in non-VT pneumococcal disease being seen.While some data exists to suggest that 23vPCV is not effective against pneumococcal CAP, enough recent studies suggest that VE with 23vPCV could even be as high as 50% and the duration of effectiveness may stretch to 5 years. There is no case to cease 23vPPV programs in older persons and, despite some hypo-responsiveness concerns, a case can be made for revaccination of older persons with chronic conditions every 5 years. Disease rates increase with increasing age and revaccination is likely to produce long-term antibody levels consistent with primary vaccination . The 5-yThe case for the additional use of 13vPCV is harder to make on a cost basis. Strong data supports the very significant reduction in VT disease due to 13vPCV in older persons after pediatric conjugate vaccine programs, thereby reducing the cost effectiveness of 13vPCV use for the elderly. An opportunity exists now to await the upcoming US review of using both vaccines sequentially to clarify some of the uncertainties.Current coverage rates of 23vPPV in older person programs need to be increased. It is likely that the increase in disease in the elderly due to the 11 serotypes contained in 23vPPV (but not in 13vPCV) relates not only to replacement infection but also inadequate coverage rates with 23vPPV.Where governments can afford to use both vaccines for the elderly (as the US has done), better disease prevention may be achieved. Most importantly, vaccines with a broader coverage and duration of protection are needed.Most national advisory bodies have recommended continuation of a polysaccharide program for older persons without the addition of a conjugated vaccine to their program. Monitoring of the burden of disease in different countries over the next few years will be important in providing better data for decision making.


Chromosome conformation capture (3C)-based techniques have revolutionized the field of nuclear organization, partly replacing DNA FISH as the method of choice for studying three-dimensional chromosome architecture. Although DNA FISH is commonly used for confirming 3C-based findings, the two techniques are conceptually and technically different and comparing their results is not trivial. Here, we discuss both 3C-based techniques and DNA FISH approaches to highlight their similarities and differences. We then describe the technical biases that affect each approach, and review the available reports that address their compatibility. Finally, we propose an experimental scheme for comparison of 3C and DNA FISH results. Understanding the three-dimensional 3D) structure of chromosomes is a central focus in modern molecular biology. Besides characterizing how chromosome folding is achieved in order to package meters of DNA inside a cell nucleus that is only a few micrometers in diameter, it is important to understand how regulatory sequences interact with each other in 3D nuclear space , 2. MoreD structuTwo experimental approaches that have been extensively used to investigate chromosome structure in eukaryotes are DNA fluorescent in situ hybridization (DNA FISH)  and chroThe recent advent of 3C-based approaches (such as circularized chromosome conformation capture (4C), chromosome conformation capture carbon copy (5C), and Hi-C ) has revIn fact, DNA FISH and 3C are very different techniques that provide intrinsically different (and complementary) types of information. 3C-based approaches detect the cell population-averaged crosslinking probabilities of the chromatin fiber. The most recent versions of these techniques enable genome-wide, high-resolution measurements of the spatial proximity between genomic elements to be obtained. DNA FISH, on the other hand, enables the measurement of 3D distances between a limited number of genomic loci; it also enables the distribution of these distances within a cell population to be determined. This information is not accessible in 3C-based experiments. Moreover, the two techniques are affected by common, as well as specific, potential sources of experimental error. Nevertheless, with appropriate precautions and carefully designed experiments, the two methods can be powerfully combined to bring comprehensive insights into the folding of the genome over a wide range of length scales.Several reviews have already covered recent studies that have explored the different scales of chromosome folding , 9, 16. DNA FISH usually involves fixation and permeabilization of cells, followed by hybridization of fluorescently labeled DNA probes (either single-stranded oligos or denatured double-stranded DNA) to specific loci on the chromatin fiber. Prior to hybridization, the sample must be slightly denatured to allow base-pairing to occur between the probe and the double-stranded target DNA. The target locus can then be directly visualized using fluorescence microscopy, enabling its localization to be assessed in the context of the overall nuclear architecture and/or with respect to other genomic loci . Dependithree-dimensional distance (rab) between two targeted loci a and b inside single cells. Consequently, the associated probability distribution P(rab) that measures the variation of rab across the cell population can be extracted , as welCab between loci a and b in Hi-C and 5C experiments can, in principle, be thought of as being proportional to the number of cells in which the two loci were located closer than a certain range R within which crosslinking occurs  by the following relationship [α is a proportionality constant, rmin is the minimum distance between loci a and b that can be reached due to steric repulsion between the two parts of the fiber, and R is the crosslinking range . In support of this, 3C- and DNA FISH appear to be highly correlated when comparing 3C-based counts with colocalization of corresponding FISH probes, but only when defining colocalization radii that are much smaller (five- to tenfold) than the maximal distances measured by FISH [a and b , it is already clear that relating 3C-based counts with 3D distances measured by FISH is not a trivial task. Notably, the 3C signal is dominated by the low-distance portion of the distance distributions  to infer the full distributions of 3D distances P(rab), for all loci a and b considered in the experiment. In order to do this quantitatively, it is possible to use computational strategies based on coarse-grained physical models of the chromatin fiber, which try to find the 3D fiber conformations that are compatible with the set of restraints provided by the 3C results. To convert 3C counts into distances, all these models assume some sort of functional relationship between 3C count and the 3D distance of loci. Some models assume that the 3C count is inversely proportional to the mean 3D distance and produce an average conformation of a chromosomal region  to be deconvolved out of the 3C data and to be compared with high-resolution DNA FISH experiments [As we have seen, relating the 3C count to the full distribution of distances through Eq. 1 is a complicated “inverse problem” that requires using data of reduced dimensionality ; oth. 1 see , 34). Al. AlCab)eriments , 34.P(rab) between two loci a and b (Eq. 1).Both 3C and DNA FISH experiments are subject to a range of technical issues. Some of these impact the two techniques in a comparable manner, which facilitates comparison of the results obtained, whereas other technical issues are specific to one or the other or affect the two approaches differently. One fundamental similarity (and common bias) is that both DNA FISH and 3C are based on chemical crosslinking with formaldehyde. Formaldehyde crosslinking favors DNA–protein  and, to A second similarity is that both DNA FISH and 3C require that nuclei are permeabilized in order to allow fluorescent probes or restriction enzymes to access the chromatin. Permeabilization is usually achieved by treatment with detergents: Triton X-100 (in FISH) or Igepal CA-630 (in 3C). Although this step might be thought to lead to perturbations in 3D nuclear structure, both electron and high-resolution optical microscopies have shown that nuclear ultrastructure is well conserved, at least upon Triton permeabilization , 41. AltAside from these similarities, the 3C and DNA FISH techniques also have specific technical issues that need to be evaluated accurately and controlled in order to ensure that the results can be correctly interpreted. In 3D DNA FISH, crosslinked chromatin must be mildly denatured in order to allow base-pairing between the probe and the target DNA. This is achieved by heating the sample in the presence of formamide, which reduces the melting temperature of double-stranded DNA and allows DNA denaturation at lower temperatures than in aqueous solution. Heat denaturation is, of course, a major source of concern in DNA FISH experiments because it may lead to changes in nuclear and chromatin organization. Indeed, electron microscopy observations  have shoP(rab) in Eq. 1 is correctly sampled. This is especially important for the small distances that dominate the 3C signal. The amount of aberrations present in a microscopy setup can be easily estimated by using calibration beads labeled in multiple fluorophores and then measuring the shift in the positions of bead centers when beads are imaged in the various colors. Correction of such aberrations can be performed by elastic registration of DNA FISH images. Various methods that can be used to achieve this have been implemented in user-friendly computer programs such as ImageJ . Such aberrations have to be taken into account and can be corrected for computationally  to ensurple, see , 48).The 3C-based techniques are also characterized by certain distinct technical issues that can affect the experimental procedure and impact on how the data are analyzed, especially when they are to be compared to DNA FISH data. Such biases include the efficiency of chromatin digestion, the choice of restriction enzyme used, the efficiency of ligation, and other biases  DNA FISH measurements.Ring1B−/−) cells, the HoxD locus remains associated with high levels of PRC2 proteins, whereas in PRC2 (Eed−/−) mutant cells, the PRC2 coating is lost. Williamson et al. [Eed−/−) cells, where both techniques pointed to chromatin unfolding, when compared to wild-type cells. However, in PRC1 (Ring1B−/−) cells, discordant results were observed; here, strong 5C interactions  were contradicted by decompaction observed in FISH. The authors suggested that the observed 5C versus FISH discrepancy could result from differential crosslinking efficiencies when large quantities of PRC2 are lost (Eed−/−) or maintained (Ring1b−/−) at the HoxD locus. Thus, it is possible that, in certain specific cases, where the chromatin composition changes in a major way between experimental samples, the FISH and 3C-based results may diverge. Another study on the same locus, the physical compaction at the HoxD locus was measured in vivo by DNA FISH and super-resolution microscopy [Although negative (or discordant) 3C versus FISH correlations may be underrepresented in the literature because of an obvious publication bias towards positive reports, the majority of studies suggest that 3C-based techniques and 3D DNA FISH give concordant results over a wide range of genomic and spatial ranges , 52–54. n et al.  reportedcroscopy . HoweverR dominate the 3C count (Eq. 1), methods that detect physical proximity rather than mean 3D distances should be more suited for comparisons between 3C and DNA FISH results. In fact, colocalization methods that are based on correlations of pixel intensities [An important aspect that needs to be taken into consideration when comparing DNA FISH and 3C results is the choice of the criteria that have to be used to quantify DNA FISH distance distributions. Since short-range distances below the crosslinking range ensities , 27 haveOn the other hand, measuring 3D distances is clearly the method of choice for either assessing difference in structure over large genomic distances  or testiTo minimize the effect of potential fixation biases, fixation conditions for 3C and FISH should be as similar as possible. Possibly, a fraction of the same cells that were fixed for 3C should be separated from the main batch and used immediately to prepare coverslips for DNA FISH . Alternatively, another batch of cells grown on coverslips should be fixed with the reagents used for 3C.When comparing different conditions in DNA FISH (such as different differentiation stages or mutants), the various samples should be processed in parallel in order to minimize differences in denaturation time and temperature. Ideally, all samples should be dispensed on the same coverslip to ensure maximal uniformity of conditions.Optical aberrations should be limited as much as possible during image acquisition. Mechanical stage drifts, which can result in image distortions, should be minimized . Chromatic aberrations should be reduced by preferring microscopes that mount filter wheels in combination with multiband-pass dichroic mirrors, instead of rotating filter cubes .Despite all precautions and even in stable and well-aligned microscopes, residual aberrations can be of the order of a few hundreds of nanometers, and thus comparable to the distances that dominate the 3C signal. These residual aberrations need to be measured using fluorescent beads and computationally corrected after image acquisition using elastic channel registration  in orderReplicate 3C-based experiments should be performed in parallel to account for technical and biological variability correctly .3C counts should not be exclusively compared to mean 3D distances measured by DNA FISH. We suggest that different methods should be used in parallel to compare the two techniques, notably those that measure spatial proximity .In conclusion, we propose an experimental scheme for the careful comparison of 3C and DNA FISH results to ensure that the validity of the comparison is maximized:Although it is impossible to rule out confounding effects resulting from the specific bias of each technique completely, these expedients should at least ensure that the comparison between 3C-based results and DNA FISH measurements is fair.Accurately designed combined 3C–DNA FISH experiments represent a powerful tool that can be used to determine the 3D conformation of chromosomes, including its cell-to-cell variability and its relevance in the context of fundamental biological functions such as transcription. Further, the possibility of combining DNA FISH with super-resolution microscopy , 55, 57 Live-cell imaging is likely to remain a low-throughput technique that allows the investigation of a small number of genomic loci in tightly controlled experimental systems, such as clonal culture cell lines expressing controlled levels of fluorescent proteins. In developmental or disease-related contexts , 3C-based approaches complemented with DNA FISH will probably remain the methods of choice for the investigation of chromatin structure. It will therefore be crucial to implement accurately controlled 3C–FISH experiments to compare the results of these two techniques, notably by adopting the procedures we have proposed in this review. In addition, novel approaches to 3C and FISH that do not require crosslinking, such as native 3C  and CASFFinally, thanks to the greatly improved ability to modify mammalian genomes with CRISPR/Cas9 or TALEN approaches, we are entering an era when the structural and functional models of chromatin organization that can be built on the basis of 3C and FISH results can be tested genetically. On the one hand, this will require carefully controlled quantitative experiments, but on the other hand, it will provide an unprecedented opportunity to obtain a mechanistic description of chromosome conformation and its function in the context of transcription and other important biological processes.


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 ocularChlamydia 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 theCt 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<0.0001) but not severe (p=0.149) scars increased in prevalence with age.Conclusions: Neither conjunctival scars nor lymphoid follicles were associated with antibodies toCt, suggesting that they are unlikely to be a direct result of ocularCt infection. Clinical signs of trachoma were prevalent in this population but were not indicative of the underlying rates ofCt infection. The current World Health Organization guidelines for trachoma elimination indicated that this population should receive intervention with mass distribution of antibiotics, but the data presented here suggest that this may not have been appropriate. International partners have committed to elimination of trachoma as a public health problem by the year 2020 and the global elimination strategy is guided by the clinical signs trachomatous trichiasis (TT) and trachomatous inflammation–follicular (TF). The World Health Organization (WHO) recommends at least three years of mass drug administration (MDA) with azithromycin in districts with ≥10% TF prevalence in 1–9 year-olds to treat the causative agentChlamydia trachomatis (Ct)2.Trachoma is responsible for approximately 1.9 million cases of visual impairment or blindness globally3. In response to this and in accordance with WHO guidelines, MDA took place throughout the Solomon Islands in 2014 and the national program administered approximately 24,000 doses of azithromycin . Data from the 2013 PBPS suggested that whilst TF was prevalent, TT (0.1% of those ≥15 years), trachomatous inflammation—intense , and ocular infection withCt (1.3% of 1–9 year-olds) were all rare3. Our recent survey of Kiritimati Island, Kiribati4, used the same tools and estimated more typically-matched values of TF andCt infection prevalence (among children) at 28% and 24%, respectively. We therefore questioned the underlying biology of the TF signs that were observed in the Solomon Islands.A 2013 population-based prevalence survey (PBPS) covering two provinces (Temotu and Rennell & Bellona) of the Solomon Islands showed that the proportion of 1–9-year-old children with TF was moderately high (26.1%)Ct exposure. We set out to investigate this hypothesis by returning to the same two provinces of the Solomon Islands six months after MDA took place. This study used tests for two different persistent markers of previousCt infection. The first was an enzyme-linked immunosorbent assay (ELISA) that measured antibodies against theCt antigen Pgp36. This tool has been used to assess transmission of both urogenital7 and ocular8 infections, including in the study on Kiritimati Island, where we showed that there were strong associations betweenCt infection, TF and anti-Pgp3 antibody levels. We also observed a rapid increase in age-specific Pgp3 seroprevalence throughout childhood years in that population4.We hypothesised that the clinical signs in the Solomon Islands were not consistent with repeatedCt seropositivity, is generally considered to be a persistent marker of previous ocularCt infection. In trachoma, it is characterised by a gradual accumulation of scar tissue in the tarsal conjunctivae9, which typically begins to develop to the point of being visible in late childhood. Scarring is more commonly found in those who have experienced prolonged, severe inflammation and infection11. Very few young children in trachoma-endemic communities have signs of scarring, but as many as 10–30% of older children may do so13. Scarring progresses throughout a lifetime and, in severe cases, is the underlying cause of TT11. Assuming that trachoma was an endemic problem in this population, we would expect to observe an age-dependent accumulation of scarring, with an increasing proportional representation of severe scars with advancing age.This study also assessed trachomatous scarring. Scarring, caused by immuno-pathological responses to repeated cycles of infection, is an irreversible process that, likeThe methods used in this study adhered to the tenets of the Declaration of Helsinki. Ethical approval for the study was granted by the London School of Hygiene & Tropical Medicine  and Solomon Islands National Health Research Ethics Committee (HRC15/03). Subjects aged 18 years or older gave written, informed consent to participate. A parent or guardian provided written, informed consent on behalf of those aged under 18 years.To enable comparison to pre-MDA data, only villages in Temotu and Rennell & Bellona provinces where baseline mapping had been conducted were eligible for inclusion. Due to their small respective populations , the two provinces were combined into one evaluation unit during baseline mapping. The survey took place in June–July 2015, six months after a single round of azithromycin MDA had been delivered by the Solomon Islands National Trachoma Elimination Program.3. We included numbers of villages in each province to reflect the proportion of the total population of the two provinces combined . The proportions of active trachoma and infection cases in study villages before MDA were extracted from the full baseline dataset and are presented here for comparison.Thirteen villages were selected in which more than 10% of the community  had previously had signs of TFCt antibody seropositivity in children aged 1–9 years. Based on the low prevalence of ocularCt infection prior to MDA (1.3%), we expected the seroprevalence to be approximately 10%, in line with other communities with lowCt prevalence14. To estimate seroprevalence with ±5% precision at the 95% confidence level assuming a design effect of 2.65 (as utilised in the baseline study) at least 367 children were required15. In our pre-MDA PBPS survey, we examined a mean of 1.1 children per household and therefore estimated that 25 households in each of 13 clusters were needed to reach our sample size. All residents aged 1 year or above living in households drawn at random from a list of all households in a study cluster were eligible to participate.This survey was powered to estimate the prevalence of anti-16. Clinical grading of TF, TI and TT is a routine part of trachoma surveys and prevalence estimates of TF and TT are the basis for programmatic decision making. These signs have, therefore, been the focus of programmatic scale-up of mapping activities whereas similar standardisation does not exist for scar grading. Clinical grading according to the WHO simplified system17 was therefore used for TF, TI and TT, whilst photo-grading was used for scarring.Clinical TF, TI and TT grading was performed in the field by two Global Trachoma Mapping Project (GTMP)-certified graders wearing 2.5× binocular magnifying loupes. Graders were trained according to a training scheme developed under the GTMP which was designed to be as standardisable as possible between countries. To become certified, graders were required to achieve a kappa score of ≥0.7 compared to expert consensus on a set of 50 photographs, then a kappa of ≥0.7 compared to a highly experienced grader on 50 schoolchildren’s eyes18. Photographs were graded by two photo-graders who had previously achieved kappa scores for inter-grader agreement of >0.7 for F (follicles), >0.7 for P (papillae) and >0.7 for C  grades, compared to a highly-experienced trachoma grader. Photographs were graded for F, P and C. The F grades were used to retrospectively check the accuracy of the TF field grading, although it should be noted the two grading systems are not entirely concordant. Photograph grading was undertaken masked to field grading, laboratory results and the other photograph grader’s assessment. Discrepant grades were arbitrated by a third highly experienced grader. Grading was performed using “FPC_Grader”, an open source software tool based on R.High-resolution digital photographs of the right tarsal conjunctivae were graded for scarring using the modified WHO trachoma grading systemDried blood spots were collected for assessment of anti-Pgp3 antibody level. Participants’ fingers were cleaned and then pricked with new, sterile lancets, and blood was collected onto filter paper calibrated to absorb 10 µL . Filter wheels were air-dried for 4–12 hours before being sealed in plastic bags with desiccant sachets. These were refrigerated for up to one week and then stored at -20°C before shipping at ambient temperature to LSHTM, London, UK, where they were again stored at -20°C.Swabs were passed three times (with a 120°-turn between each pass) over the right conjunctiva of children aged 1–9 years. The examiner and specimen manager took precautions to avoid cross contamination between participants or swabs in the field. In each village, one clean swab was passed within 20 cm of a seated participant and then processed identically to participant swabs to test whether cross contamination between swabs took place in the field. Swabs were refrigerated for up to one week and then stored at -20°C before shipping to LSHTM on dry ice for processing.19. Optical density (OD) at 450 nm was measured using SpectraMax M3 photometric plate reader  then normalised to a 20% dilution of high-titre (presumed positive) serum in low-titre (presumed negative) serum.Anti-Pgp3 antibody level was assessed using ELISA, as described elsewhereHomo sapiens ribonuclease subunit  and open reading frame 2 of theCt plasmid (diagnostic target) using a previously evaluated droplet digital PCR assay20 with minor modifications21.DNA was extracted from swabs with the QIAamp DNA mini kit . Samples were tested for22. Pre- and post-MDA proportions were compared using Wilcoxon’s rank sum test. Fleiss’ Kappa scores were calculated using the ‘irr’ package in R. ddPCR tests for current ocularCt infection were classified into negative and positive populations according to methods described previously20. Anti-Pgp3 antibody titre was divided into two populations using an expectation-maximisation finite mixture model6, with individuals classified seropositive if their normalised OD was more than three standard deviations above the mean of the presumed-negative population. Using this method, the threshold normalised OD value for positivity was 0.7997. Data from comparable studies in Bijagos Islands, Guinea-Bissau23, and Kiritimati Island, Kiribati4, are shown inCC-BY 4.0) andCC BY 3.0 IGO licenses, respectively, to illustrate how patterns of antibodies and scars look in hyper-endemic settings.All data analyses were conducted using R 3.2.324.1511 people  aged 1 year and over were examined in 382 households from the 13 selected study villages. By comparison, the pre-MDA survey of the same villages yielded 1534 people (490 1–9-year-olds) in 394 households. Data on non-participation were not collected in the June 2015 study, but the number enrolled was similar to that for the pre-MDA survey, suggesting a similar participation rate of around 90% on both occasions. In this study, there was a mean of 4 people per household aged 1 year and over, and a mean of 1.2 children per household aged 1–9 years. After accounting for non-participants, this is similar to the means in the 2009 Solomon Islands National Census 3. We observed 66/466 (14.2%) cases of TF and no cases of TI in either eye after MDA, representing a decrease in TF of 58% (p<0.0001). Following MDA, 65% of TF cases were bilateral. Retrospective comparison of field grade of TF to photograph grade of F2–3 indicated moderate agreement between field and photograph grade (kappa = 0.52).Prior to MDA, there were 167/490 (34.0%) cases of TF and 1/490 (0.2%) case of TI in either eye in those aged 1–9 years in study villagesIn the two villages enrolled in Rennell & Bellona, a slight increase in the prevalence of TF in either eye in those aged 1–9 years following MDA was noted, but it was not statistically significant . In contrast, in the 11 enrolled villages of Temotu, a substantial decrease in TF  was observed .No cases of TT were identified during this study.Of the right eye photographs that were collected, 1440/1511 (95.3%) were suitable for grading conjunctival scarring. 42% of photograph grades did not match on either F, P or C grade and so were adjudicated (the majority of these discrepancies were due to disagreements in P grade). 188/1440 (13.1%) photographs were graded as having visible scars (C>0), of which 127 were C1 (mild), 53 were C2 (moderate) and eight were C3 (severe). Four out of eight cases of C3 were found in children aged 1–9 years, these photographs are shown inCt infections were common (22% of 1–9 year olds had detectableCt infection)23. These data are included to demonstrate the contrast between photo-grading data sets from the Solomon Islands and Guinea-Bissau, the latter of which reflects the typical patterns of scar accumulation that would be expected in a setting where ocularCt infection is hyperendemic , but the proportion of people with more severe scarring (C2 or C3) did not (logistic regression p=0.149). There was also no significant association between having C>0 and gender (chi-squared test p=0.80). In Rennell & Bellona, 25/225 (11.1%) of photos were graded C>0, whereas in Temotu, 163/1215 (13.4%) of photos were graded C>0; the difference in scarring between provinces was not significant (chi-squared test p = 0.404).Ct infection was much higher4. Among children aged 1–9 years, the rate and dynamics of accumulation of seropositivity differed substantially between the Solomon Islands and Kiritimati (Dried blood spots were collected from 1499/1511 (99.2%) people aged ≥1 year during the post-MDA survey; the other 12 people declined finger-prick. The distribution of normalised OD for all individuals, grouped into five-year age brackets, is shown initimati .The largest increase in seroprevalence was observed between those aged 16–20 years and 21–25 years where seroprevalence rose significantly from 30.4% to 71.6% (chi-squared test p<0.0001). Of those aged over 25 years, 67.4% were seropositive. In the 16–20-year-old age group, the prevalence of seropositivity amongst females was higher than in males . The seroprevalence among children in Rennell & Bellona was significantly higher than that in Temotu .25) there was no association between C grade and anti-Pgp3 OD (linear regression adjusted for age and gender p=0.453) or anti-Pgp3 positivity (logistic regression adjusted for age and gender p=0.472).80.3% of Solomon Islands children with TF were seronegative. 82.3% percent of children without TF were seronegative. There was no association between seropositivity and signs of TF in children aged 1–9 years (logistic regression adjusted for age and gender p=0.616). In those who were younger than the population median self-reported age of sexual debut  of the 61 children with TF also hadCt infection. We previously showed that, of 462 swabs from the pre-MDA study which passed quality control, 5/462 (1.1%) had infection. All five infection cases came from children with active trachoma in the right eye . The median pre-MDA load ofCt infections in those villages was 14,260 plasmid copies/swab3. Neither the difference between the pre- and post-MDACt prevalence nor the pre- and post-MDACt load were statistically significant . The relationship betweenCt infection, signs of trachoma and seropositivity was examined in children aged 1–9 years and is summarised inCt infections identified during this study.Positive endogenous control tests were obtained from 457/466 swabs from children aged 1–9 years. The median load of the endogenous human RPP30 target was 33,500 copies, equivalent to over 15,000 conjunctival cells. In this study, 8/457 (1.8%) children had evidence ofCt infection and late-stage disease (TT) are rare3. If the village-level findings of the current study were replicated throughout the district, then TF would still be sufficiently prevalent to warrant continued intervention. The conjunctival scarring and serological data presented here, combined with previousCt infection data, suggest that ocularCt is scarce and is not being widely transmitted. TF is not concurrent with an appreciable burden of infection, severe scarring or TT in this population. Our most significant finding, which is that 80% of TF cases occur in people who are seronegative for antibodies againstCt, questions whether further rounds of MDA are warranted in this population.Based on moderate estimates of province-level prevalence of TF, the Solomon Islands has  been identified as having endemic trachoma. Whilst measures for trachoma elimination have already been deployed in Temotu and Rennell & Bellona, we have previously noted that TI, ocularCt infection. More important still is that in Kiritimati Island, children with TF were far more likely to be seropositive than those without TF. In the Solomon Islands, however, we not only found that most TF cases were seronegative, but that individuals with TF were no more likely to be seroreactive to Pgp3 than their peers without TF. We can rule out the possibility that Solomon Islanders are collectively non-responsive to Pgp3  because the majority of the adult population do have antibodies against Pgp3. The most parsimonious explanation of our findings is that TF in this population is caused by a factor other thanCt.In Kiritimati Island, we found that just 20.3% (23/119) of children with TF were seronegative according to the same ELISA test that was used here. We were not surprised to find some individuals with TF were seronegative because (1) a proportion of individuals who have primary infections will not yet have seroconverted and (2) anti-Pgp3 antibody responses may not be the same in all people due to natural variability in host responses. However, the fact that 80% of TF cases in Solomon Islands were seronegative suggests that many cases are not caused byCt infections. This is concordant with our previous data, which suggested that although ocularCt strains are present in the Solomon Islands, they are rare3. This contrasts with the data from Kiritimati Island, where we saw that there was a substantial year-on-year increase in age-specific seropositivity  and older children (6–10 years), which suggests that children here occasionally do encounteritivity . The inc26 after clearance of infection. It is, in any case, hard to imagine a biologically plausible situation in which seroreversion would fully account for the contrasts between serological data from Kiritimati and data from the Solomon Islands.While seroreversion due to clearance of infection by MDA is a possible explanation for the low seroprevalence and absence of association of anti-Pgp3 antibodies with TF in the Solomon Islands, there is currently no evidence for complete seroreversion for Pgp3-specific antibodies13, no eyelid-distorting scars were found in 311 adults aged above 40 years. Some cases of severe scarring we observed in children were not typical of trachoma and were found in children who lacked Pgp3 reactivity . This was considered to be acceptable as this is a post-treatment scenario where there are likely to be more borderline cases where disagreement may be more common. Also, the discrepancy between F2 and TF grades  may lead to further disagreement. The most common type of disagreement was field TF+ / photograph F≤1, as we might expect given the discrepancies between the FPC and the simplified system. There is also a possibility that the grader had overcalled some milder cases of follicular inflammation as TF. When a discrepancy occurs, we consider the field grade to be more likely to be accurate as the field grader can achieve a three-dimensional real-time view of the eyelid.Ct in children with TF, which actually increased slightly . From interventions in other settings, we might expect TF prevalence to have approximately halved six months after a single round of MDA, given 80% population coverage31. Azithromycin has anti-inflammatory and broad-spectrum antibiotic effects, which may help explain the observed decrease in clinical disease, but would of course only be effective in controlling a subset of bacterial genera. Given the method of village selection, regression to the mean would be another potential explanation for the fall in prevalence.The 13 communities included here were the most highly endemic of those surveyed in Temotu and Rennell & Bellona during the GTMP, with at least 15% of children aged 1–9 years living in selected villages having TF before MDA. In this study, we showed that the burden of TF in many of these villages dropped significantly following a single round of MDA, but still remained above the threshold for continued intervention. The drop in clinical disease was not reflected by a simultaneous drop in ocularWe observed regional variation across the study villages. Compared to Temotu, we noted that MDA did not have as significant an impact on TF prevalence in Rennell & Bellona, that there were more children there who were seropositive and that there were more children with TF who also had infection. Our survey was not prospectively designed to assess these differences, and the subgroup size in Rennell & Bellona precluded more detailed analysis. Temotu is more similar to the rest of the Solomon Islands in terms of the geology and geography of the islands as well as in relation to the lifestyle and ethnicity of the majority of the inhabitants. Further studies on the localisation of trachoma in the islands are warranted.32. Data from cross-sectional surveillance tools used in isolation can be hard to interpret, especially given the prolonged persistence of TF after clearance of infection33. Some features of conjunctivitis in the Solomon Islands resemble trachoma, particularly the prevalent follicular inflammation and some of the severe conjunctival scarring. Crucially, we found that these clinical features were not co-endemic with TT at a prevalence that indicated an ongoing public health problem. In this setting, we believe that tests for infection gave a better indication of the public health threat from trachoma than TF. A combined approach in which various age-specific markers of trachoma are assessed together across the complete age range of the population may prove useful for prioritising areas for intervention where the prevalence of TF alone does not coherently reflect trachoma’s public health importance.The complex, multistage nature of trachoma makes it difficult to predict the outcome of any given interventionCt34 and yaws35), further rounds of azithromycin MDA do not appear to be indicated for the purposes of trachoma elimination. As the positive predictive value of TF for ocularCt infection decreases globally in response to declining prevalence, it is likely that other regions and countries will be identified where a high prevalence of TF is not reflective of threat to vision and may not require MDA. WHO recommendations for implementation of MDA and the SAFE strategy should be reviewed in the light of this evidence.Contrary to the WHO recommendation for treatment based solely on prevalence of TF, our data suggest that trachoma is not a public health problem in these villages. Whilst there have been substantial collateral benefits to local residents from having received MDA .Data are available under the terms of thehttps://github.com/chrissyhroberts/FPC_GRADER/FPC_GRADER is available from:http://doi.org/10.5281/zenodo.111679937Archived source code as at time of publication:License: GNU This manuscript describes trachoma prevalence in the Solomon Islands over an almost 2 year period. The data highlight the discrepancy between the presence of high TF rates, which would indicate that trachoma is a public health problem in this area, while the rates of other signs of trachoma  are very low to none. The study is well designed and the manuscript well written. The concern raised by the authors that the high TF rates seen may not actually reflect ongoing trachoma infection and thus MDA may not be warranted are well supported by the data as is their suggestion that consideration be given to using multiple markers of trachoma to determine whether or not intervention is needed. I have some comments for the authors which I list below:What the inter observer agreement for grading scarring alone was in this paper?What percent of grades had to be adjudicated?Where else this Dawson et. al scarring grading scheme applied to photographs has been used/validated?In the Methods section the authors state that grading of TF, TI, and TT was done clinically and that only scarring was graded by photographs. However, when they describe the inter observer agreement for the grading they refer to the grading of F, P, and C . Could the authors please report the following:The authors followed the grading system of conjunctival scarring described by Dawson et al.; the grade descriptions do not have clear cut offs. How did the authors modify this grading system for the photographic graders? How did they determine the cut off between C1 and C2? How did they determine shortening/distortion for C3 from photographs alone? Did they include looking for signs of lid margin conjunctivilization as they state in their C3 example photograph? These are areas that could lead to a great deal of disagreement between graders...In the comparison population whose scarring data is presented in Figure 2 B, was the grading done in the same way as in this paper, through photographs with two graders and an adjudicator? If so, what was the inter-observer agreement? This information was not readily available in the paper cited. TSSince the authors mention the kappa of F, P, and C, I wonder if they happened to grade follicles and papillae photographically? If so, how does this compare to the field grades? Given the discrepancy between TF and all other signs of trachoma, having a way to ensure accurate field grading of TF would be reassuring.Do the authors have any hypotheses as to why the prevalence of TF might be so high in this community if it is not due to trachoma ?   TF1.In the discussion the authors state that there is no evidence for complete seroreversion. Although I agree with the overall conclusion that seroreversion cannot account for the differences in the data that were observed, the authors may want to consider looking at/adding the following reference which does support the presence of seroreversion: West et al. (2018) PGP3 AntibodyI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We are grateful to Dr Wolle for her feedback on our article. We have taken her comments into account as we prepared the second version of this manuscript, and believe they have enhanced the quality and transparency of the article. We hope the following additional information addresses some of her comments directly. The photograph graders had to achieve a kappa score of >0.7 in C grading, a kappa score of >0.7 in F grading and a kappa score of >0.7 in P scoring when compared to a highly experienced grader before they were considered to be ‘validated’ for photograding. It is hard to calculate a kappa score between graders in this particular manuscript because those where the primary graders did not reach consensus were arbitrated. Where the first two grades agreed, the grade was accepted as the final grade. Where the first two grades disagreed on F, P or C, an independent expert adjudicated. There are, therefore, a subset of photographs where both primary graders have called the same C grade, and the arbitrator has also graded C. In these cases, the kappa agreement was 0.76 between primary and arbitrator grades. 42% of participant photographs did not agree on F or P or C score, so were arbitrated. The majority of these were due to disagreement in P score.et al. Immunofibrogenic Gene Expression Patterns in Tanzanian Children with Ocular Chlamydia trachomatis Infection, Active Trachoma and Scarring: Baseline Results of a 4-Year Longitudinal Study.Front. Cell. Infect. Microbiol., 2017Ramadhaniet al. The conjunctival microbiome in health and trachomatous disease: A case control study.Genome Medicine, 2014.Zhouet al., Conjunctival Scarring in Trachoma Is Associated with the HLA-C Ligand of KIR and Is Exacerbated by Heterozygosity at KIR2DL2/KIR2DL3.PLoS NTDs, 2014.Roberts While we are aware of other grading systems developed specifically for grading scarring in photographs, we considered this grading system to be the best balance between the relatively simplistic tool of the simplified system and the highly complex tools described by others. We felt the experience our group had using the FPC system would lead to more reproducible grading which would outweigh the benefit incurred by using more specialised methods. We acknowledge that the difference between borderline cases of C0 and C1 or between C1 and C2 may be difficult to arbitrate, although felt that was a problem common to all trachoma grading, for example with regards to interpretation of what constitutes a follicle >0.5mm or the difference between different levels of inflammation. Regarding the question of distortion of the tarsus, we believe in the photographs where C3 was called (examples of which are displayed in figure 1) the distortion of the tarsus is clear, however, it is possible that some cases of C3 were missed. We believe at the population level, this is unlikely to lead to gross shifts in results, especially in the context of this population where over 85% of people show no scarring at all. Finally, the comparator data we present in figure 2b demonstrate that the same grading scheme used in an area with hyperendemic infection does identify cases of moderate and severe scarring. We have presented data in the article from a comparable study in Guinea Bissau where the same grading process was used, and a starkly different picture emerges. In addition, we have listed below some examples of recent studies using the FPC system. In the comparator population from Guinea Bissau, the photographs were reviewed by two experienced graders who met to discuss the outcome of discrepant results. Further information about this process was not recorded, so it is not possible to determine what proportion needed to be discussed. The kappa agreement score between photograde of F2–3 and field grade of TF is 0.52 and additional information surrounding this comparison is included in response to Dr Nash’s comment.Ct infection is not the same as that in other countries, so does not result in the antibody and cicatrizing responses seen in other endemic areas. We consider the former to be more likely, and mention that in the final sentence of the second paragraph in the discussion. The two hypotheses that have emerged as a result of this set of work are (1) that an as-yet uncharacterised, non-bacterial cause of follicular conjunctivitis is inflating estimates of TF or (2) that the host response of residents of the Solomon Islands to circulatinget al.. Our interpretation of the data in that paper is that the small proportion of people classed as sero-reverters at one year actually have a distinguishably lower antibody level at baseline than those who were positive at both timepoints, and a distinguishably higher antibody level at follow up than those classed as negative at both timepoints, therefore we believe there is no evidence for ‘complete’ seroreversion, and the statement should stand. Thank you for directing us to the paper from West The manuscript describes the results of a trachoma prevalence survey which measured clinical signs, chlamydial infection, as well as anti-Pgp3 seropositivity among a population from the Solomon providences of Temotu, and Rennell & Bellona. This report is one of a number of recent reports which have examined this population in great detail, including through the use of conjunctival transcriptome profiling and measures of microbial diversity. The manuscript is well written and the laboratory and analytic techniques were appropriate for the main aims of the survey. Within this population, at both baseline, and at the survey time-point described in this manuscript, the clinical signs of trachoma were high enough to warrant interventions with antibiotics per WHO guidelines. However, other markers of trachoma, such as low infection and seropositivitiy, as well as the very low prevalence of trichiasis, suggested the possibility of alternative etiologies for the clinical signs (TF) of trachoma in these providences. The findings of this study are important, as is the author’s call for the use multiple age-specific markers of trachoma to help in prioritizing areas for intervention.Given that a major finding here is that TF remained above 10%, while other trachoma indicators suggested that trachoma was not a public health problem in these providences, it would strengthen the manuscript if the authors provided some more information on whether or not they took additional steps to ensure accurate grading of TF in the field. For example:The authors state that one of two GTMP-certified graders did the grading for TF, TI, and TT. Please state the month and year that this individual was certified for this particular survey, and what criteria were used for certification. The kappas achieved were reported for the photograph grading, please report for the field grading.Second, it appears that this study used only one grader to capture field grades. Is there a concern that the single chosen grader may have been a person who systematically overcalls TF ?Were there any quality control steps taken to ensure the quality of the field TF grading? Given that the discrepancy between TF and other markers of trachoma were discovered in these providences at an earlier timepoint , and that this current survey for these providences was already more thorough than standard trachoma surveys, the authors could have taken some steps to ensure the quality of field grading. For example, was it possible to recheck a certain percentage of TF cases while still in the field; how did the photo graders compare to the field graders for the TF grade within this particular survey?   Major CommentsUnder trachoma grading in the methods section, the authors discuss standardization of clinical signs but only cite Thylefors in the section. That paper shows comparable inter- and intra-observer variation for TF, TI and scarring (TS). Is there a different reference that can be used to support the assertion that TF, TI, and TT are more “standardized” than scar grading? Minor commentsI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We appreciate the time Dr Nash has taken to appraise this manuscript. We include a point-by-point response to his comments below. We believe his feedback has improved the article, and hope he agrees. Graders were trained according to the GTMP training protocol as described elsewhere. The training was robust; trainees attended a taught course on trachoma, were required to achieve a kappa score of ≥0.7 when grading a set of 50 photographs in which the trachoma grade had been agreed by several experts and were required to achieve a kappa score of ≥0.7 against a highly experienced grader trainer in the assessment of 50 children aged 1–9 years in the field. Both graders working on this study had met those criteria on the first attempt during a GTMP training in the Solomon Islands in September 2013 (unfortunately we do not have their specific kappa scores available). The grading in Temotu (where 11 of the 13 study villages were located) was undertaken by a single grader who had also successfully completed a GTMP training course in Ethiopia prior to the Solomon Islands course. A second grader conducted the grading in Rennell & Bellona (2 of the 13 study villages were in this province). We believe by training in this manner, we have ensured our data is as comparable as possible to data generated by the GTMP, and by impact and pre-surveillance surveys in the majority of trachoma-endemic areas. Data generated by graders trained in this manner are used to guide treatment decisions around the world. However, to support our research programme alongside the Solomon Islands National Trachoma Control programme we have collected photographs to enable us to retrospectively check grading. In a sample of photographs from a previous survey, the kappa score between field grade of TF and photograde of TF was 0.88. In this survey the photographs were graded with the FPC system rather than the simplified system. The kappa agreement between a photograph grade of F2/F3 and field grade of TF was 0.52, indicating moderate agreement. This is lower than in previous studies in the Solomon Islands, potentially because of more borderline cases in a post-treatment community or because F2/F3 is not exactly comparable to TF. We have expanded the methods section in version 2 to give a fuller account of the training process, we have added the F2/F3 vs TF kappa score to the results, and have added a section in the discussion regarding this process. Two graders were involved in this study. Both had met the predefined criteria for successful grading and were therefore accredited to grade trachoma to the quality required to guide treatment decisions. The kappa agreement between field and photograph grades along with caveats about expectations for that agreement has been included in the manuscript in line with the previous comment. The most common type of disagreement was field+ / photograph-, therefore there is a possibility that that grader had overcalled some milder cases of follicular inflammation as TF. For the reasons described above, we expect a certain number of discrepancies. Also, when a discrepancy occurs, we believe the field grade to be more likely to be accurate as the field grader can achieve a three-dimensional real-time view of the eyelid. It is worth noting that the key finding of this paper that the majority of TF cases do not have antibodies to Pgp3 holds true if we define the clinical phenotype using the photograph grades . It wasn't possible to recheck grading in the field, however, we have presented the comparisons between field and photograph grade above and in the paper. Because specific interventions at the programmes are guided by TF, TI and TT grades but not TS grades, scale up of mapping activities has focussed on those signs. Therefore, grading for TF, TI and TT has been repeatedly replicated in thousands of districts round the world, whereas TS grading is relatively uncommon. Therefore, while graders may be able to be trained to assess TS reliably, training for TF, TI and TT is ‘standardised’. We have amended the terminology in the methods to reflect this, and added a reference where this is also discussed.


Sarcomas are rare, heterogeneous tumors for which prognosis remains dismal in patients with advanced disease. Pazopanib, a vascular endothelial growth factor receptor inhibitor, has shown modest efficacy in patients with soft tissue sarcoma who fail cytotoxic chemotherapy. The cytotoxic agent temozolomide has also demonstrated activity in patients with advanced sarcoma.We performed a retrospective case series to evaluate the feasibility of adding temozolomide to pazopanib in advanced sarcoma patients following single-agent pazopanib failure.Patients with recurrent, metastatic sarcomas who had progressed on single-agent pazopanib and continued on pazopanib with the addition of temozolomide were included in this retrospective analysis to examine the tolerability and responses associated with the treatment combination.Nine patients with a range of sarcoma subtypes were identified . All patients received combination therapy. One patient was recently started on therapy and was excluded from the analysis . Median PFS for single-agent pazopanib was 7.5 months (range 2–19). For the eight evaluable patients , best response at 4 months with pazopanib plus temozolomide was partial response (n = 1), stable disease (n = 3) and progressive disease (n = 4), with a median PFS of 3.5 months (range 0–15). Median PFS with combination treatment in patients with stable disease or response was 8 months (range 5–15). All four patients who achieved clinical benefit remain on therapy and are tolerating the combination therapy with expected but manageable side effects.In heavily pretreated patients with advanced sarcoma, the addition of temozolomide to pazopanib was found to be tolerable. Future prospective trials are required to deduce whether temozolomide extends the clinical benefit of pazopanib. Sarcomas are a rare and heterogeneous set of diseases that have a mesenchymal origin in bone or soft tissue. Soft tissue sarcomas (STS) are composed of more than 70 different subtypes and account for approximately 1% of all tumors . In 2017Treatment of metastatic disease remains unsatisfactory due to limited active chemotherapy options . TraditiPazopanib is a multi-targeted tyrosine kinase inhibitor (TKI) that inhibits vascular endothelial growth factor receptor (VEGFR)-1, -2, and -3; PDGFR-α and -β, and stem cell factor receptor (c-Kit), B-Raf and others ,21. In tTo this end, temozolomide is a novel alkylating agent that exerts its effects through the formation of 5-3-methyl-1-triazenolimidazole-4 carboxamide, which is the putative active metabolite of dacarbazine . TemozolWe thus hypothesize that during progression on pazopanib, the tumor cells have begun developing escape pathways around the tyrosine kinase inhibition. However, the addition of temozolomide after progression on pazopanib may re-induce a tumor response by inducing cellular damage that necessitates the upregulation of the PI3K/Akt pathway. Despite the upregulation of escape pathways, the tumor now becomes increasingly dependent on PI3K/Akt activation; however, this activation remains downregulated by pazopanib , leadingWe conducted a retrospective analysis of data from patients with advanced sarcoma who received pazopanib plus temozolomide at our institution from January 1, 2014 to June 30, 2016, and who were evaluable for toxicity and tumor response. The study was approved by the Ohio State University Institutional Review Board (OSU: 2016C0022).Patients were eligible for inclusion in the study if they were older than 18 years of age with a pathologically-confirmed diagnosis of soft tissue or bone sarcoma, had received therapy with a single-agent VEGFRi (pazopanib) in the recurrent or metastatic setting and were subsequently treated with pazopanib in combination with temozolomide. Patients were required to have received at least one cycle of pazopanib-based therapy prior to combination with temozolomide.Data collection  was performed by a thorough retrospective review of each patient’s medical record.Adverse events were evaluated using Common Terminology Criteria for Adverse Events version 4.0.PFS, defined as the time from the date of clinic visit prior to starting temozolomide plus pazopanib to disease progression or death, whichever occurred first. Patients were censored if there was no progression at the time of last follow up. All patients were evaluated with computed tomography scans every 8 weeks. Response to therapy was defined according to RECIST version 1.1 criteria and evaluated by the treating physician.Demographics, patient characteristics, toxicities, and clinical response were summarized using descriptive statistics . Survival curves were estimated using the Kaplan-Meier method.Nine patients were identified and eight patients with a range of sarcoma subtypes were evaluated in this analysis . At the 2, 7 days on with 7 days off) on a 28-day cycle  until progression. Median PFS for single-agent pazopanib was 7.5 months (range 2–19) with a best response of stable disease (SD) for most patients . At the ay cycle .The combination therapy was tolerated, with only one patient having a grade 4 toxicity of thrombocytopenia . ProteinThe combination of pazopanib plus temozolomide resulted in disease stabilization as best response in three of eight patients at 4 months . The cliIn addition, we also evaluated next-generation sequencing for all patients to identify if there are any key genomic alternations that could be used as a possible predictive biomarker. We did not find any similarities among the patients .Metastatic STS are difficult to treat and typically resistant to cytotoxic chemotherapy, with a poor overall prognosis ,4. With 2 for 7 days on, 7 days off) was selected based on previous studies using the combination of temozolomide and the VEGFi bevacizumab in the treatment of solitary fibrous tumors [This is the first case series, to our knowledge, evaluating the feasibility of the combination of pazopanib and temozolomide in patients with advanced sarcoma. Our data, although retrospective, comprising a small sample size, and a range of sarcoma subtypes, tentatively suggest that this combination is tolerable and that further investigation of the efficacy of this regimen in a subset of patients with refractory sarcoma may be worth consideration. The ability of this combination regimen to help overcome tolerance to single-agent pazopanib cannot be deduced from this case series and would need to be assessed in a prospective randomized clinical trial vs a group receiving temozolomide monotherapy. At our institution, the starting dose of pazopanib (400 mg/day) administered in combination with temozolomide was lower than the approved dose of 800 mg/day  and was s tumors .The median PFS for patients receiving combination treatment in the present study was 3.5 months with a range from 0–15. There were three patients in our cohort who were started on combination therapy and who were already experiencing rapid progression of disease to the point of requiring an ICU admission. The median PFS for these three patients was 1 month compared to 8 months for the remainder of the patients. This suggests that there could be two distinct populations of patients. One that achieves no benefit from the combination therapy and is generally not likely to tolerate even one cycle, and a second population that may achieve slight benefit from the combination, but where potentially neither population may be considered a true responder to combination therapy.In this population, patients had a median time of 7.5 months (range 2–19) on pazopanib monotherapy. The median time on pazopanib monotherapy in this case series appears to be longer than that in the PALETTE trial, wherein the median duration of pazopanib treatment was 16.4 weeks (range 0–79) ,34,35. TLimitations of this study include the retrospective nature, range of sarcomas included, and small sample size. Adverse event data may not have been collected in a uniform fashion compared to those participating in a clinical trial. Next-generation sequencing performed on these patients as part of routine clinical practice failed to identify any key genomic alterations that could be used as a possible predictive biomarker.In conclusion, our retrospective case series evaluated the feasibility of the addition of temozolomide to pazopanib in heavily pretreated patients with advanced sarcoma, who had predominantly begun developing slow disease progression. The combination of temozolomide with pazopanib was found to be tolerable. In order to determine whether temozolomide extends the clinical benefit of pazopanib vs temozolomide monotherapy, a large, randomized, prospective study would need to be completed. Next generation sequencing could aid in the identification of sarcoma subtype-specific alterations and help elucidate key predictive biomarkers.


To the Editor: We read with interest the article by Chochua et al. from the Centers for Disease Control and Prevention  156 was the major contributor to antimicrobial resistance among serotype 35B isolates, as stated by Chochua et al. We reported a predominance (69.2%) of 35B sequence type (ST) 558 among invasive isolates across the entire study period ; 95% of the ST558 isolates were penicillin nonsusceptible. We noted that clonal expansion of ST558 was the major contributor to the increase in prevalence of serotype 35B, as did Chochua et al. Furthermore, we observed the emergence of 35B-CC156 after introduction of PCV13 and noted that 35B-CC156 isolates were multidrug-resistant , similar to previous observations of CC156 associated with other pneumococcal polysaccharide capsules . Thus, the increase in multidrug resistance among serotype 35B isolates in the post-PCV13 era was strongly associated with the emergence of CC156.Our conclusion that both clonal expansion and diversification had occurred in the post-PCV13 era is validated by the results of Chochua et al. During 2015–2016, we observed no further increase in serotype 35B. We agree with Chochua et al. that the emergence of serotype 35B is of concern and the development of a new generation pneumococcal vaccine is necessary. We will continue to monitor and report data regarding ongoing changes in pneumococci; although our study is not population based, we believe it provides reliable data that are useful for clinical, epidemiologic, and vaccine-related considerations.


In the budding yeast S. cerevisiae, septins are found through most of the cell cycle in an hourglass organization at the mother-bud neck until cytokinesis when the collar splits into two rings that disassemble prior to the next cell cycle. Experiments using polarized fluorescence microscopy have suggested that septins are arranged in ordered, paired filaments in the hourglass and undergo a coordinated 90° reorientation during splitting at cytokinesis. This apparent reorganization could be due to two orthogonal populations of filaments disassembling and reassembling or being preferentially retained at cytokinesis. In support of this idea, we report a decrease in septin concentration at the mother-bud neck during cytokinesis consistent with other reports and the timing of the decrease depends on known septin regulators including the Gin4 kinase. We took a candidate-based approach to examine what factors control reorientation during splitting and used polarized fluorescence microscopy to screen mutant yeast strains deficient in septin interacting proteins. Using this method, we have linked known septin regulators to different aspects of the assembly, stability, and reorganization of septin assemblies. The data support that ring splitting requires Gin4 activity and an anillin-like protein Bud4, and normal accumulation of septins at the ring requires phosphorylation of Shs1. We found distinct regulatory requirements for septin organization in the hourglass compared to split rings. We propose that septin subpopulations can vary in their localization and assembly/disassembly behavior in a cell-cycle dependent manner at cytokinesis.Septins are conserved filament-forming proteins that act in diverse cellular processes. They closely associate with membranes and, in some systems, components of the cytoskeleton. It is not well understood how filaments assemble into higher-order structures  Saccharomyces cerevisiae, septins assemble into a ring at the new bud site and this ring transitions into an hourglass which splits into two rings at cytokinesis  while either Cdc11 or Shs1 occupies the terminal position  a concerted rotation of the entire higher-order assembly; (2) the selective departure of a majority population of filaments that then “reveals” a second, orthogonal population; and (3) a triggered disassembly and reassembly of filaments into a new ensemble that is perpendicular to the starting state  based on rationale from previous work  was generated by amplifying ScCDC12 from S. cerevisiae genomic DNA using the oligonucleotides AGO1181 and AGO1182. The PCR product was ligated into the pRS416 plasmid (AGB441) and verified by test digestions using PspXI and SlaI and sequencing . Next, the plasmid AGB459 (pScCdc12-GFP) was generated by first amplifying the GFP insert from the plasmid AGB005 using the oligonucleotides AGO1187 and AGO1188 and ligating the product into the digested AGB455 plasmid. Again this plasmid was verified with test digestions and sequencing . Constraining of GFP to Cdc12 was completed using PCR to amplify the entire AGB459 using the oligonucleotides AGO1203 and AGO1204 which contain homology over the CDC12-GFP linker and this amplification removed nucleotides encoding 4 amino acids from the N-terminus of GFP and 3 amino acids from the C-terminus of Cdc12. AGB467 (pRS416-ScCDC12-conGFP) was verified by sequencing . Once verified, AGB467 was transformed into wild-type lab strain (AGY000), and the following yeast deletion strains: bnr1Δ, bni1Δ, bud4Δ, cla4Δ, gin4Δ, rts1Δ, and shs1Δ . Doug Kellogg generously supplied us with shs1 phosphomutant strains  was integrated into the same strains as the AGB467 plasmid generating the strains AGY131-AGY137, and AGY323-AGY327. All strain, plasmid, and oligonucleotide information is present in Tables We generated S. cerevisiae cells were grown overnight in appropriate media with proper selection for the specific plasmids, collected by centrifugation, and resuspended in 2x low fluorescence minimal media (LFMM). Cells were mounted on gel pads of 1.4% agarose and LFMM on glass depression slide, covered with a coverslip (no. 1.5), sealed with Valap, and imaged.For imaging, Time-lapse recordings for estimating septin intensity of mutant strains through the cell cycle were acquired with a Nikon Eclipse Ti-E inverted wide-field microscope equipped with a 60x (1.4 N.A.) plan-apochromat oil objective and a Andor Zyla 4.2 sCMOS camera. A Chroma DAPI/FITC/TRITC/Cy5 quad filter set was used for fluorescent imaging of GFP. The fluorescent light source was a Spectra LED lamphead and images were acquired with 12% laser power, 100 ms exposure, and 90 s time intervals.t0) was manually determined. This t0 represented the maximum septin intensity value close (not more than 4–5 time points) to the visible ring split. In most cases the quantitatively determined t0 was not more than two time points (3 min) away from the visible split. This quantitative approach was taken to account for different visible split times depending on how a given image was automatically contrasted by FIJI. t0 therefore represented the quantitative onset of ring splitting. Since the primary focus of this study was the septin concentration change during ring splitting, all following analyses were limited to 24 min before and after t0. Percent change over the entire 24 min was determined and reported in Table t = 0 to t = 24 min after splitting; Phase 2 is the rate of disassembly from the last time point of Phase 1 to t = 24. After disassembly rates were determined, the fold change compared to wild-type was calculated.Using FIJI, individual cells were cropped from the timelapse recordings and the mean background-subtracted septin intensity at the mother-bud neck were determined for every time point for a given cell using MATLAB. These septin intensity values were saved and plotted in an Excel file. For every cell, a time zero (shs1-phosphomutant strains) were acquired using the MF-PolScope  using the OpenPolScope software (www.openpolscope.org).All polarization fluorescence images (except the shs1-phosphomutant strains and bud4Δ splitting events were acquired with the Zeiss Axio Imager.M1 wide-field microscope with five linear polarized filters (Chroma 21033a) in a filter wheel  and UV blocker 420 nm LP emission filter 32 nm . The fluorescent light source used was an EXFO X-Cite 120 lamp. Images were acquired through Micro-manager (www.micro-manager.org) using the OpenPolScope software (www.openpolscope.org).Polarized fluorescence measurements of the Current Protocols in Cell Biology . Using two different Shs1-GFP strains we observed the accumulation of Shs1 in the septin hourglass is only ~50% of the level of either Cdc3 or Cdc11 . These data indicate that the difference between the septin populations that stay associated with the cortex and that are released into the cytoplasm is not simply the presence or absence of Cdc11 or Shs1.We hypothesized that whether a given septin filament is released or stays localized at the neck could be explained by the composition of septin heteromeric rods, specifically the terminal subunit, which is occupied by either Cdc11 and/or Shs1. If Cdc11 or Shs1 specified which complexes were retained or exited from the neck, we predicted that Cdc11 and Shs1 would display different rates of change in concentration at splitting. To assess this, we monitored the abundance of 3 different septins at the mother-bud neck over the course of septin ring splitting. We used widefield fluorescence microscopy to image GFP-Cdc3, Shs1-GFP (in two different strain backgrounds), and Cdc11-GFP every 90 s to ensure sufficient time resolution for assessing intensity differences and kinetics . This rapid disassembly from the bud neck is consistent with previous data that Bud4 is required for stability of split septin rings during and after cytokinesis , but the net orientation remained ~90° with some decrease in anisotropy , and similar to what we observed in gin4Δ mutant cells, a monophasic disassembly rate of septins from the bud neck slower than either observed phase in wild-type cells , ps2 (CDK sites), and ps4 (Pho85/CDK sites)  Figure . Septin y Figure . In contshs1 phosphorylation-deficient mutants were comparably highly disorganized and showed a wide distribution of orientations and cell-to-cell variability in both hourglass and asymmetric split ring structures ; (2) Increased rate of disassembly (bud4Δ); (3) Monophasic rate of disassembly ; (4) Misorganized hourglass but organized split rings (gin4Δ); (5) Organized hourglass but misorganized split rings (bud4Δ); (6) Misorganized hourglass and split rings ; and (7) correctly oriented but less ordered (low anisotropy) assemblies . Of note, many regulators (Class1), including those influencing F-actin have an appreciable impact of the abundance of septins in the hourglass structure. It is unclear whether this is due to some altered membrane composition in these mutants or direct impacts on septins that may lower their affinity for the membrane.We hoped that through careful analysis of splitting kinetics and organization states that we would be able to dissect the molecular control of the transition. We identified several categories of defects in both the kinetics of splitting and septin organization by combining timelapse imaging with polarized fluorescence analysis Table : (1) Decper se. It is likely that this next phase of screening will require identifying partial loss-of-function or separation-of-function alleles because it is probable that whatever responds to or executes the signal for reorientation also contributes to overall septin organization.In this study, we had aimed to identify mutants that would have specific defects in reorientation. However, the only cases where we see definite defects in reorientation is in scenarios where the ring fails to split or the split rings are unstable such as what happens in cells lacking Bud4. A clear challenge that emerged from this study is that perturbing many of the factors that we suspect might contribute to the process leads to defects in the starting higher-order structure of the septin hourglass. In many cases these defects were not detectable until analysis with polarized light. It is difficult to disentangle the degree to which defects arise due to poor construction as opposed to an inability to respond appropriately to cell cycle triggers of the transition. Future work will need to expand this screen to the vast array of septin regulators and interacting proteins that are known to exist with the hope of still identifying a regulator that might be responsible for this transition without contributing to the organization Despite the constellation of phenotypes, there are a few key aspects of the data that point to potential mechanisms relevant to the splitting transition. In particular, consistent with previous observations, Bud4 is required for a proper split ring state  and see that non-phosphorylatable mutants altered septin levels in the hourglass and monophasic disassembly. Unfortunately, uncertainty about the background of these mutants with regard to the BUD4 locus makes it challenging to interpret the rate of disassembly in these strains because the disassembly was more rapid than expected in controls. Analysis of an shs1 mutant that lacks the entire C-terminus was illuminating in that it also has a major defect in the recruitment of septins to the hourglass however it shows a biphasic disassembly that is not slowed compared to wildtype, unlike the shs1 null. This mutant still retains a phosphorylation site at T6 that is missing in the PS alleles, pointing to this residue as a potentially important site for regulating contact with the membrane for both assembly of the hourglass and release of septins at splitting. Overall, the data point to Gin4 and Shs1 as modulators of the rate of change in septin dynamics and orientation at late stages of the cell cycle. It is likely that this process involves phosphorylation-dependent changes in membrane composition and/or septin-membrane binding that change the affinity of septins for the membrane and possibly change the preference of septins to bind membranes of specific curvatures.Another clue emerges from the similar behavior of A real power of using polarized fluorescence microscopy is the ability to detect aspects of macromolecular organization not readily apparent in standard total fluorescence measurements and to assess the degree of order in an ensemble of molecules. In our previous work we have seen that no matter how we view the hourglass or rings, the average dipole orientation was the same and we interpreted this to mean that a majority of septins are symmetric relative to the membrane supporting paired filaments. Our motivation for using the MF-PolScope was to be able to simultaneously capture multiple focal planes in the panel of mutants in hopes of detecting defects in pairing that might inform the mechanisms of reorientation. To our surprise, we could see no evidence of systematic differences in orientation that were dependent on focal plane suggesting that pairing may not be impacted in these diverse mutant backgrounds. In summary, this work set out to analyze the requirements for septin rearrangement at cytokinesis in budding yeast. The findings point to a function for Shs1, Gin4, and Bud4 in this process but there is clearly much more work to be done to understand how the dramatic cytoskeletal reorientation occurs in this small window of time and space.MM performed experiments, assembled figures, analyzed data and wrote manuscript, MJ performed experiments and analyzed data, SM and AV assisted in data analysis, RO contributed instrumentation and expertise in analysis, AG designed the study and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.