To investigate the usefulness of meibomian gland (MG) dropout rate in the evaluation of MG morphological change associated with the use of prostaglandin for glaucoma treatment through the association between MG and the ocular surface parameters and medication duration and presence of preservative.This cross-sectional study was conducted on 88 eyes of 88 patients who were diagnosed with glaucoma and used only Tafluprost as treatment. The patients were divided into four “user” groups: 1) 23 patients used preservative-free (PF) Tafluprost for 6 months; 2) 21 patients used preservative-containing (PC) Tafluprost for 6 months; 3) 23 patients used PF-Tafluprost for 24 months; 4) 21 patients used PC-Tafluprost for 24 months. Ocular surface parameters and the MG condition, including MG dropout rate and meiboscale, were evaluated. Multiple regression was used to identify associations.There were significant differences in age (p = 0.003), tear breakup time (p = 0.016), lid margin abnormality (p = 0.016), expressibility (p = 0.039), meiboscale (p<0.001), and MG dropout rate (p<0.001) among the 4 groups. MG dropout rate and meiboscale showed significant differences in all post hoc analyses, except for the comparison between the PF-Tafluprost and PC-Tafluprost 6-month user groups. Medication duration, preservative status, and meiboscale were significantly correlated with MG dropout rate . In the 6-month user group, preservative status significantly correlated with MG dropout rate (p = 0.015). However, in the 24-month user group, meiboscale was the only parameter significantly associated with MG dropout rate (p<0.001).MG dropout rate in patients using Tafluprost showed a significant correlation with medication duration and preservative status. This result indicates MG dropout rate reflects MG morphologic change associated with prostaglandin. Glaucoma is an irreversible progressive disorder of the optic nerve, which can cause blindness if not properly treated. Treatment of glaucoma comprises reduction of intraocular pressure (IOP), which can be achieved with eye drops or laser or surgical methods; however, the primary treatment method is the use of anti-glaucoma eye drops. Continuous use of topical anti-glaucoma medications is necessary to maintain the IOP-lowering effect. A notable limitation in this approach is that long-term use of topical anti-glaucoma medications increases the risk of developing ocular surface diseases –5. SeverMeibomian gland (MG) condition in glaucoma patients is related to the behaviors involved in medication use, such as frequency and duration, as well as the type of preservative and active ingredient in the medication ,9,10,12.MG dropout rate is an objective parameter that reflects morphologic changes of the MG, indicating loss of the gland. Unlike hyperkeratinization of the MG duct, MG dropout is more closely associated with MG atrophy, suggesting underlying MGD ,18. SeveTherefore, we conducted this study to evaluate the usefulness of the MG dropout rate for assessing MG status in glaucoma patients using topical PGAs. We analyzed the correlation between medication factors including medication duration and presence or absence of preservative and parameters for ocular surface and MG status, including MG dropout rate. Among topical PGAs, we only used preservative-containing (PC) and preservative-free (PF) Tafluprost  to minimize the effect of medication differences.This cross-sectional study was conducted in the Department of Ophthalmology, Severance Hospital, Yonsei University School of Medicine , and followed the tenets of the Declaration of Helsinki. Ethical approval was obtained from the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine, Seoul, Korea; written informed consent was obtained from all subjects who enrolled in the study. By retrospectively reviewing medical records from the period between January 2015 and December 2017, we identified 88 patients who met the inclusion criteria. The patients were diagnosed with glaucoma for the first time in our glaucoma clinic and began using topical PC- or PF-Tafluprost, which has been used as one of standard treatment methods to treat glaucoma patients in our institution. To the included patients who had no change or addition of topical anti-glaucoma medications during the follow-up period, ophthalmic examinations were performed cross-sectionally at the 6- or 24-month follow-up visit. Patients with a history of ocular surgery, ocular injury, or disease affecting the ocular surface and MG such as allergic conjunctivitis or demodex on eyelashes, as well as patients using other topical agents  were excluded in the process of reviewing the medical records of patients. This was confirmed again directly with the patients who visited the outpatient clinic at 6 or 24 months after topical Tafluprost use. In addition, ocular surface and lid margin status were also evaluated at the time of visiting the outpatient clinic to exclude patients having conditions affecting the ocular surface and MG. Among the included patients, we evaluated the ocular surface and MG status in patients who used the medication for 6 months and those who used the medication for 24 months. Included patients did not have abnormal findings regarding the MG or ocular surface conditions in the medical records of their first visit.Forty-four eyes of 44 glaucoma patients who used topical Tafluprost for 6 months and 44 eyes of 44 glaucoma patients who used topical Tafluprost for 24 months were included in the present study. In each group, 23 eyes of 23 patients used PF-Tafluprost and the remaining 21 eyes of 21 patients used PC-Tafluprost.As a control group, we enrolled 64 eyes of 64 subjects who visited our hospital for regular check-up or preoperative examination associated with refractive surgery or cataract surgery by reviewing medical records retrospectively. For these subjects, the same examinations for ocular surface and MG conditions were conducted.Ocular surface and MG conditions were evaluated using the same methods as described in previous studies published by ophthalmologists from our institution ,19,21. TThrough direct observation using slit-lamp microscopy, assessments of lid margin abnormality, meibum quality, and meibum expressibility were conducted. Lid margin abnormality was scored from 0 to 4 on the basis of four factors: vascular engorgement, plugging of the MG orifice, anterior or posterior displacement of the mucocutaneous junction, and irregularity of the lid margin. Eight lower lid glands were assessed using a scale of 0 to 3 points at each gland for meibum quality . Expressibility of MG was scored by applying digital pressure on five glands of the central one-third of the lower lid, with a score range from 0 to 3 .Tear break up time (TBUT) using a fluorescein strip, type I Schirmer test, and ocular surface staining score (Oxford score) were used to assess ocular surface condition. The ocular surface disease index (OSDI) was used to evaluate subjective ocular discomfort. As described previously , the exaFor comparison of continuous and categorical parameters between the groups, the independent two-sample t-test, analyses of variance, and chi-squared tests were performed. A multivariate regression analysis was used to investigate correlations among parameters showing significant differences in group comparisons, including age, sex, medication duration, and presence or absence of preservative in the medication (preservative status). In addition, a multivariate regression analysis was used to identify factors correlated with MG dropout rate on the basis of medication duration and preservative status. All statistical analyses were performed using SAS version 9.4 software . Statistical significance was defined as p<0.05.We compared age, sex ratio, ocular surface parameters, and MG parameters among four groups: 1) 6-month users of PF-Tafluprost; 2) 6-month users of PC-Tafluprost; 3) 24-month users of PF-Tafluprost; and 4) 24-month users of PC-Tafluprost. There were significant differences in age (p = 0.003), TBUT (p = 0.016), lid margin abnormality (p = 0.016), expressibility (p = 0.039), meiboscale (p<0.001), and MG dropout rate (p<0.001) among the four groups . ResultsTBUT, lid margin abnormality, expressibility, meiboscale, and MG dropout rate showed significant differences in comparisons among the groups. Correlations among these parameters were identified by multiple regression analyses including age, sex, medication duration, and preservative status . TBUT waMG dropout rate was the only parameter in the multiple regression analyses that was correlated with both medication duration and preservative status. Because of their ease of use (once per day) and strong IOP-lowering effects, PGAs have been used as a first-choice medication for treatment of glaucoma ,23. TherIn the present study, MG dropout rate was significantly correlated with medication duration and preservative status, when the data were adjusted for age and sex. Notably, MG dropout rate was significantly correlated with preservative status in 6-month user group; however, there was no significant correlation between preservative status and MG dropout rate in the 24-month user group. Considering the results of another subgroup analysis conducted under a different preservative status in the present study, which showed a significant correlation between MG dropout rate and medication duration only in the PF-Tafluprost user group, MG dropout rate may represent morphologic changes in the MG that are affected by preservative status and medication duration. Meiboscale, another parameter indicative of morphologic changes of the MG, showed correlation results that were similar to those exemplified by MG dropout rate. However, multivariate analyses showed that preservative status was not correlated with meiboscale; thus, the effect of preservative on MG may not be accurately reflected by meiboscale. Several previous studies indicated that MG loss was affected by age and sex –28. HoweA number of previous studies have reported that PF anti-glaucoma topical medications have advantages in treatment of ocular surface disease –32. TearThe MG dropout rate and meiboscale among 24-month users of PF-Tafluprost were higher than those among 6-month users of PC-Tafluprost. In addition, the MG dropout rate was significantly correlated with the presence of preservative only in patients who used Tafluprost for 6 months. These results support the possibility that PF-PGAs may exhibit a limited prevention effect on the development and progression of MGD. However, this limited prevention effect is not an indication that PF-PGAs have no clinical use. Rather, it suggests that PF-PGAs should be the primary consideration when using PGAs in glaucoma patients who have already begun to exhibit atrophic changes in the MG. According to our results, MG dropout rate can be used to identify the glaucoma patients who should use PF-PGAs preferentially by measuring objective status of MG. Further studies are needed to define the cut-off value of MG dropout rate for determining whether PF-PGAs are required.To investigate the effect of topical anti-glaucoma medication on the ocular surface and MG condition, behavior regarding medication use  as well as the type or concentration of active ingredient and preservative, should be well-controlled. However, it is difficult to conduct research in which these conditions are well-controlled in clinical practice. Therefore, specific formulas, such as burden of anti-glaucoma score, were used to assess the effects of different anti-glaucoma medications in a previous study . We condThis study has several limitations. First, we could not confirm the longitudinal change of the MG dropout rate because this study used a cross-sectional design. By performing longitudinal follow-up of the same patients included in this study, patterns of change in MG dropout rate might be identified. Second, we could not evaluate primary conditions of MG and ocular surface in included patients, although we excluded patients whose medical records described abnormal MG findings. Lastly, only Korean patients were included in this study. Because the reported MGD prevalence in the Asian population is higher than that in the Western population ,38, addiIn conclusion, MG dropout rate in patients using Tafluprost was significantly correlated with medication duration and preservative status. Considering the changing pattern and correlation of MG dropout rate with preservative status and medication duration, MG dropout rate could be used to assess MG status in users of PC-Tafluprost or PF-Tafluprost. This result could be applied to users of other PGAs in addition to Tafluprost.S1 Dataset(XLSX)Click here for additional data file.


Background: A solid pseudopapillary tumor (SPT) of the pancreas is a rare neoplasm with low malignancy, constituting about 2% of pancreatic tumors, which mainly occurs in young women.Case Presentation: We herein report a case of a small SPT arising from the head of the pancreas in an asymptomatic 50-year-old man. This patient was admitted to our department at Henan Provincial People's Hospital for the evaluation of a pancreatic mass and a pancreatic resection was performed. Histology revealed the lesion to be an SPT of the pancreas, with the characteristic pseudopapilla formation, central degeneration, and capsule formation. The tumor was positive for vimentin, CD10, α1-antichymotrypsin, α1-antitrypsin, β-catenin, neuron-specific enolase, synaptophysin, and progesterone receptor.Conclusion: We diagnosed an SPT in the patient based on these histological findings and immunophenotype. Pathological examination reveals that SPTs are usually a large, encapsulated mass composed of a mixture of cystic, solid, and hemorrhagic components. Both a capsule and intratumoral hemorrhage are important clues to the diagnosis because these features are rarely found in other pancreatic neoplasms. SPTs generally occur in young women and are rarely reported in middle-aged or elder males. In this article, we report a case of a 50-year-old man who presented with a small SPT in the area of the pancreatic head with clinical information, computed tomography (CT) findings, pathological features, and a literature review.Solid pseudopapillary tumors (SPTs) of the pancreas, first described by Virginia Frantz in 1959,A previously healthy 50-year-old man was admitted to Henan Provincial People's Hospital with a month's history of unspecific epigastric abdominal discomfort. This patient had no significant history of medical or alcohol abuse and hospitalization. Physical examination and laboratory tests were unremarkable. Abdominal ultrasonography (USG) and CT scan showed an enlarged pancreatic head containing an uneven mixed solid and cystic composition about 3.7 × 4.0 cm . No surr2) from various areas of the tumor. On immunohistochemical analysis, the tumor cells were positive for vimentin, CD10, α-1-antichymotrypsin (AACT), α-1-antitrypsin (AAT), β-catenin, neuron-specific enolase (NSE), progesterone receptor (PR), and synaptophysin (Syn) in approximately 30% of the cells, whereas cells were positive for Ki-67 antigen (a proliferation marker) less than 1% of the neoplastic cells . and PRs , all of 10 Some support exists for both hypotheses. Many researchers have demonstrated that SPTs have a complex immunophenotype that is inconsistent with that of any of the pancreatic cell types and that a pancreatic origin is unlikely. On the basis of some similarities between SPTs and ovarian surface cells and the proximity between genital ridges and the pancreas anlage during early embryogenesis, SPTs might originate from the genital ridge-related cells that were incorporated into the pancreas during organogenesis.10 Although nearly all studies show no evidence of estrogen receptors, progesterone receptors are present in most cases.8 Furthermore, Morales et al.11 described the temporal relationship between a fast-growing SPT and pregnancy in a young woman. Hence, sex hormones may play a pivotal role in the pathogenesis or growth of SPTs. In addition, the low Ki67 expression in SPTs may be associated with a good clinical outcome of these tumors.The exact cause of SPTs of the pancreas is unknown. Different hypotheses of histogenesis of SPTs have been postulated. Many investigators support the theory that SPTs originate from multipotent primordial cells, whereas others suggest that SPTs have an extra-pancreatic origin from genital ridge angle-related cells that were incorporated into the pancreas during organogenesis.12 After intravenous contrast material administration, enhancing solid areas are typically noted peripherally, whereas cystic spaces are usually more centrally located.13 MR imaging typically shows a well-defined lesion with heterogeneous signal intensity on T1- and T2-weighted images, which reflects the complex nature of the mass. Areas of high signal intensity on T1-weighted images and low or inhomogeneous signal intensity on T2-weighted images can help identify blood products and may also help differentiate SPTs from other pancreatic tumors. Although recent advances of these imaging techniques have significantly improved pancreatic imaging, preoperative diagnosis of SPTs is still difficult. Accurate diagnosis of the special type of pancreatic tumor is obviously important. Fine needle aspiration or biopsy and histological analysis are needed for definitive diagnosis in certain cases.A variety of imaging techniques define the imaging features of SPTs as hypervascular, well-encapsulated round mass with combined cystic and solid components. USG findings of SPTs have been described as well-encapsulated cystic and solid masses. CT scan usually shows a well-encapsulated lesion with varying solid and cystic components owing to hemorrhagic degeneration.15 The accumulation recognition and diagnosis of SPTs in males remains challenging.In conclusion, SPTs of the pancreas are a rare neoplasm comprising a wide range of differing underlying pathologies from completely benign through premalignant to frankly malignant. Surgical resection is the treatment of choice even in the case of distant hepatic metastasis, and complete resection is usually curative. The efficacy of chemotherapy or radiation therapy for SPTs has yet to be shown, although some cases were reported to be resectable after preoperative chemotherapy.


Co-circulation of dengue virus (DENV) and chikungunya virus (CHIKV) is increasing worldwide but information on the viral dynamics and immune response to DENV-CHIKV co-infection, particularly in young infants, is scant.Blood samples were collected from 24 patients, aged 2 months to 82 years, during a CHIKV outbreak in Mexico. DENV and CHIKV were identified by RT-PCR; ELISA was used to detect IgM and IgG antibodies. CHIKV PCR products were cloned, sequenced and subjected to BLAST analysis. To address serological findings, HMEC-1 and Vero cells were inoculated with DENV-1, DENV-2 and CHIKV alone and in combination (DENV-2-CHIKV and DENV-1-CHIKV); viral titers were measured at 24, 48 and 72 h.Nine patients (38%) presented co-infection, of who eight were children. None of the patients presented severe illness. Sequence analysis showed that the circulating CHIKV virus belonged to the Asian lineage. Seroconversion to both viruses was only observed in the four patients five years or older, while the five infants under two years of age only seroconverted to CHIKV. Viral titers in the CHIKV mono-infected cells were greater than in the DENV-1 and DENV-2 mono-infected cells. Furthermore, we observed significantly increased CHIKV progeny and reduction of DENV progeny in the co-infected cells.in vitro assay findings strongly suggest that the lack of DENV IgG conversion in the co-infected infants is due to suppression of DENV replication by the Asian lineage CHIKV. The presence of maternal antibody and immature immune responses in the young infants may also play a role.In our population, DENV-CHIKV co-infection was not associated with increased clinical severity. Our The online version of this article (10.1186/s13071-018-2942-1) contains supplementary material, which is available to authorized users. Over the last few decades, dengue virus (DENV) has spread throughout most tropical and subtropical areas of the world where it has become endemic , 2. SincAedes mosquitoes. The broad geographical distribution of the Aedes aegypti and Aedes albopictus vectors has allowed for the widespread transmission of CHIKV in DENV endemic areas [DENV and CHIKV are RNA viruses transmitted to humans through the bite of ic areas . Vector ic areas . A recenic areas .Both sporadic and outbreak-associated cases of virologically confirmed co-infections have been reported worldwide . The preSeveral reports have documented a more severe clinical outcome for DENV-CHIKV co-infected patients when compared to mono-infected patients. Notably, all these studies involve the East/Central/South African (ECSA) CHIKV genotype .DENV is highly endemic in southern Mexico, where it has caused major outbreaks since the 1980s . During Although co-infections and viral-viral interactions have been widely described in nature, few reports address the role of viral co-infections on the course of human illness . The dynFlavivirus/Alphavirus co-infections have yielded conflicting results; two studies have demonstrated interference of CHIKV by DENV in mosquitoes and mosquito cell lines [Viral interference and genetic re-assortments are the most common form of interactions among arboviral co-infections . Studiesll lines , 15. In ll lines  found thInformation on viral interactions and the immune response in the human co-infected host is scant . Previouin vitro assays to support our assumption. This paper presents the first detailed description of DENV-CHIKV co-infections in children from Yucatan, Mexico, and show that CHIKV-DENV co-infection impairs host responses, which induce memory antibody responses to CHIKV alone.During the outbreak, we were interested in determining whether DENV-CHIKV co-infection was occurring in our patients, and its potential impact on their severity of illness. We unexpectedly found that co-infection in young infants resulted in positive IgG seroconversion against CHIKV but not against DENV. We postulated that viral interference could plausibly explain these results and performed g; serum samples were aliquoted and stored at -20 °C until use.The patients included in this study were enrolled at the Hospital General O’Horan Emergency Department during a two-week period in September 2015, at the height of the CHIKV outbreak in Yucatan. Patients presented fever greater than 38 °C and two or more of the following symptoms: rash, arthralgia, myalgia and headache. After obtaining written informed consent, clinical data was registered on a standardized questionnaire and blood samples were collected during acute illness on admission or the first medical visit. Whole blood was collected in Trizol tubes (0.5 ml) and serum clot activator tubes (1–8 ml depending on age) on Day 1 to 7, hereby referred to as the acute sample. Hospitalized patients were followed prospectively until discharge. Patients returned for a follow-up visit 4–6 weeks after onset of illness for a full physical examination and collection of convalescent sera . Trizol tubes were stored at -70 °C until RNA extraction. Serum activator tubes were centrifuged for 15 min at 2000× The following viral isolates were used for the cell assays: A CHIKV strain from a clinically ill patient from the state of Yucatan in 2015 (GenBank: MF407264); a DENV-2 strain with high nucleotide sequence homology to the New Guinea strain , obtaine2 tissue culture flasks at 75–85% confluence.Virus titers were determined by plaque assays. Briefly, 24-well plates were seeded with Vero cells in duplicate, and when they reached 80–90% confluence; serial 10-fold dilutions were made of supernatants collected from the different experimental conditions . Infected Vero cells were then completely overlaid with DMEM containing methylcellulose and maintained for 4 days at 37 °C. The overlays were removed with gentle washes and fixed with methanol at 80% and the monolayers were blocked with 5% PBS-milk. After washing, one plate was incubated with mouse IgG antibody 4G2 at 1:2000 (anti DENV E protein) and the second plate was incubated with mouse IgG at 1:500 (anti E2 CHIKV). Subsequently, anti-mouse IgG (H+L) was added to the assay at a 1:2000 dilution with horseradish peroxidase (HRP) and incubated for 1 h. Plates were washed and True Blue was added to reveal the plaques. Stock viruses were prepared by infecting a C6/36 cell monolayer in 75 cmInfected Vero cells (0.25 MOI) were analyzed at 48 h post-infection. Cells were detached with trypsin solution and treated with trypsin-neutralizing solution . Once in suspension, cells were treated with fixing/permeabilising solution  at 4 °C for 1 h with gentle agitation, washed twice with PBS/0.5% albumin, and treated for 30 min with an unrelated immunoglobulin solution (10% goat serum in PBS). Cells were then incubated with rat anti-NS5 monoclonal antibody for DENV  and withThe detection of anti-DENV and anti-CHIKV IgM and IgG was performed with commercial ELISA kits ; all assays were run in duplicate. The optical density (OD) was measured at 450 nm and the units of antibody concentration and cut-off values were calculated as described by the manufacturer. A positive IgG seroconversion was defined as the change from a negative IgG serological value in the acute sample to a positive IgG value in the convalescent serum sample.Trizol was used for extraction of viral RNA from the acute samples  according to the manufacturer’s instructions. Retrotranscription of cDNA was performed with a Superscript III reverse transcriptase kit (Invitrogen). DENV PCR was performed according to the methods described by Lanciotti et al.  . Phylogenetic trees were constructed by using the neighbour-joining algorithm as implemented in PHYLIP  [The CHIKV PCR fragment was cloned using the PCR-XL TOPO vector according to the manufacturer’s instructions (Invitrogen). The Topo vector containing a 1032 bp fragment of CHIKV was sequenced in the forward and reverse directions, and sequences were submitted to the GenBank database. The viral sequences were aligned, analysed and subjected to homology search by BLAST analysis. Analysis of nucleotide sequences and deduced amino acid sequences were performed by using EXPASY Tools  and Clusstalw2/) .in vitro by using Vero and HMEC-1 cells infected with CHIKV, DENV-1 and DENV-2 strains obtained from Mexican subjects during the current study (DENV-1 and CHIKV) or previous studies conducted by our team (DENV2). DENV-1 was selected because it was the predominant serotype in Yucatan during 2015 and DENV-2 was selected, as it was the most widespread serotype in Mexico during recent years. Vero cells (6 × 104), highly permissive for both DENV and CHIKV, were seeded on glass coverslips  in 24-well plates and incubated overnight. The monolayers of Vero cells were infected at MOI of 0.25 for DENV-1/2 and CHIKV and the monolayers of HMEC-1 cells were infected at MOI of 2.5 for DENV-1/-2 and CHIKV; both were placed in a shaking incubator for 2 h at 37 °C. Upon removal, cells were washed with PBS and supplemented with complete medium. Cells were subsequently incubated at 12, 24, and 48 h at 37 °C, stained by immunofluorescence, and viral titers were measured from the supernatants of Vero cells at each time point [Because the young co-infected infants underwent IgG seroconversion to CHIKV but not to DENV, we performed co-infection experiments in both HMEC-1 and Vero cells to assess whether this could be due to DENV suppression by CHIKV. Co-infection assays were performed me point . Mock-inme point . After wWe included 24 patients aged 2 months to 82 years. More than half of them (63%) were children, six of whom (25%) were less than one year of age. Among the 24 patients, 14 (58%) had positive PCR results. Three patients were positive for DENV only, five for CHIKV only and six for both viruses . We chose three colonies from each patient for sequencing analysis. We then performed a phylogenetic analysis of the CHIKV E2 region comparing our sequences to others available in the GenBank database. The nucleotide sequences of our two isolates possessed 100% identity to each other. Furthermore, the nucleotide sequence of one isolate (GenBank: MF407264) was aligned with a CHIKV sequences previously reported for an isolate from Chiapas, Mexico . The phylogenetic tree showed that our isolates belonged to the Asian genotype , and 25.6 % positive cells (detection of NS5 protein) was observed in DENV-2 mono-infected cells. In contrast, when Vero cells were co-infected with DENV-2-CHIKV we observed that the percent of detectable DENV-2 infected cells (NS5 fluorescent cells) dropped to 2.3% while 34.0% of cells were positive for CHIKV  was remarkably high. Given the small number of patients and the short time period studied, it is not possible to determine whether this reflected the true prevalence of co-infection in the population of Yucatan during the 2015 dengue season. Our findings, however, do support evidence from other countries that DENV-CHIKV co-infections occur frequently during outbreaks % was rem. Co-infeOur CHIKV isolates had a 100% nucleotide identity and a 100% amino acid similarity with the first CHIKV isolated in Mexico, which belonged to the Asian genotype . MoreoveThe preferential IgG seroconversion of young infants to CHIKV but not to DENV is an interesting and unexpected finding that may have several explanations: (i) CHIKV replicates and disseminates faster than DENV; as a consequence, CHIKV may trigger and simultaneously control the innate immune response through different evasion mechanisms before DENV can induce the innate immune response; (ii) the presence of circulating maternal anti-DENV but not anti-CHIKV antibodies in young infants; IgG may resolve the DENV infection in the infants before a proper adaptive immune response can be initiated; and (iii) intrinsic differences in the immune response of young infants compared to older children and adults.Several studies  have shoVaccine studies have shown that antibodies of maternal origin bind to vaccine antigens and prevent infant B cells from accessing immunodominant vaccine epitopes. Furthermore, these maternal antibodies decrease the antigen load in such a manner that plasma cell differentiation is impaired in young infants , 40. It Our data also show, however, that the presence of maternal virus-specific antibodies in the young infants alone cannot explain the lack of DENV-specific IgG seroconversion, as two other co-infected infants, one four months of age (#147) and an older toddler, 21 months of age (#155) lacked IgG in the acute sample. Another possibility is that some of these children were sequentially infected, with CHIKV being the first virus introduced into the host. This would induce a proper host immune response for CHIKV, but would interfere with a subsequent infection by DENV. A possible mechanism would involve the first virus taking over the cellular factors required for viral replication or interfering with the replication of a secondary viral infection. The interference of one virus by another during co-infection could differentially induce an antiviral state mediated by IFNs. Each virus possesses their own signature, and an effective IgG response by the host depends on which virus first infected the host. Additionally, the efficiency of the nuclear translocation of IRF3- and IRF7 type I IFN-dependent antiviral responses which is the main protagonist to control viral replication and dissemination and also the IFN stimulated response elements (ISRG) located in promoter regions of different ISGs, may be differential depending on the virus involved.Potiwat et al.  studied In the co-infection assays that we performed for this study, we observed that CHIKV replication in mono- and co-infected cells exceeded that of DENV-1 and DENV-2 by more than 200-fold. In DENV-CHIKV co-infected cells we detected low DENV-1 viral particles in the supernatant but DENV-2 was still capable of low-level replication. This suggests that in natural conditions the circulating Asian genotype has higher replication rates than DENV, which might lead to the interspecific competition of DENV in co-infected mosquitoes and in human hosts. Recently, investigators observed that DENV was rapidly outcompeted by CHIKV in a cell culture from a DENV-CHIKV co-infected patient from Yucatan, and was attributed to the slower replication rate of DENV . The conAge-dependent differences in the host-immune response may also partially explain some of our findings. There is ample evidence that young infants have decreased expression of cell surface receptors in naïve B cells and poor IgG responses to protein and polysaccharide antigens. Thus, it is plausible that our older co-infected children have a more finely tuned immune system and are capable of detecting both antigens even at very low levels, thus generating an adaptive response with memory IgG antibodies.While our study results strongly suggest that the Asian lineage CHIKV suppresses DENV replication, it is important to mention its limitations. The number of co-infected patients we studied was small and it was not possible to determine whether the co-infections were simultaneous or sequential, a factor that may be determinant for viral replication. Future research studies should attempt to include a larger number of subjects from different age groups and to quantify the viral load in ill patients by real-time PCR.vs superinfection). These findings have important implications for the development of polyvalent vaccines that include multiple DENV serotypes or multiple arboviruses such as dengue, Zika and chikungunya. Future vaccine research should elucidate whether suppression of viral replication occurs in young children and whether this plays a critical role in effective vaccine response.In conclusion, our study suggests that DENV-CHIKV co-infection occurred in Yucatan, Mexico during 2015 and that these co-infections were not associated with increased clinical severity. Among co-infected patients, older children and adults presented specific IgG that demonstrate an immune response to both viruses, while young infants seroconverted to CHIKV alone. Our findings could be explained by a myriad of factors such as the presence of DENV-reacting maternal antibodies and the immune responses in young infants, the viral competence of the circulating DENV and CHIKV strains, and the timing of the viral infections in the host  and infected cells with CHIKV anti-E2 (Y). (TIFF 917 kb)


M. tuberculosis-specific CD4 T cells. It was performed at time-points T0 (10 days), T1 (1 month), T2 (6 months), and T3 (12 months) post-treatment initiation. All markers were able to detect active tuberculosis (aTB) within this patient at T0 and reverted to a healthy/LTBI phenotype at the end of treatment. Tantalizingly, there was a clear trend toward the healthy/LTBI phenotype for the markers at T1 and T2, indicating a potential role in monitoring anti-TB treatment in the future. This assay may therefore contribute to improved TB diagnostic algorithms and TB treatment monitoring, potentially allowing for individualization of TB treatment duration in the future.Tuberculosis (TB) epidemiology is changing in Western and Central Europe due to the rise in immigration and refugees fleeing high-TB-burden areas of war and devastation. The change in local demography and the lack of sensitive and specific TB diagnostic and monitoring tools, especially for cases of childhood TB, leads to either missed cases or over-treatment of this group. Here we present a promising new diagnostic approach, the T cell activation marker (TAM)-TB assay, and its performance in a case of extra-pulmonary TB occurring in a 16 year old refugee from Afghanistan. This assay is based on the characterization of 3 activation markers  and one maturation marker (CD27) on  Mycobacterium tuberculosis (MTB) infection, however their inability to differentiate between aTB, LTBI and successfully treated TB makes them an unsatisfactory tool for diagnosis of active TB. Only two diagnostic methods in use form the current “gold standard,” the liquid culture method, and the PCR technique  both of which involve the detection of MTB in sputum or other clinical samples. However, the reliance on sputum samples makes the liquid culture susceptible to contamination , work-up for suspected tuberculosis of the hip was initiated. IGRA testing  was positive; AFBs were seen on microscopy from joint fluid aspirate, subsequently confirmed as M. tuberculosis (MTB) using PCR  and culture. Since we were able to diagnose this patient on his joint fluid aspirate (microscopy results available on the same day), we deliberately refrained from a synovial biopsy, a more invasive diagnostic approach, generally recommended to obtain the gold standard specimen if joint involvement is suspected in tuberculosis. An HIV test was negative. The patient reported that he had never received any treatment for TB previously. He was started on isoniazid, rifampicin, pyrazinamide, and ethambutol for 2 months, followed by an additional 10 months period on rifampicin and isoniazid. Drug resistance testing showed a fully sensitive MTB isolate. Treatment was well tolerated and during the 12 months course, the patient showed continuous clinical improvement with regular ibuprofen required until month 3 of treatment. During this phase, treatment was supplemented by pantoprazole. Follow-up clinical visits revealed no signs of side effects and radiological assessment documented gradual improvement on MRI imaging but also unavoidable long-term damage to the left hip joint on conventional x-ray in terms of severe narrowing of the joint space, osteophytes and severe deformity of the femoral head was evident , normal FBC and chemistry. Conventional radiography and a subsequent MRI scan of the left hip showed blurring of cortical margins of the femoral head and acetabulum, hyper intensity of the left hip and acetabulum (oedema of the bone marrow) and marked joint effusion on the T2 weighted image with fat suppression as well as synovial thickening and enhancement on the post-contrast T1 weighted image –C. A che evident . Though +, HLA-DR+, and Ki67+), CD27low effector memory MTB-specific CD4 T cells is indicative of active TB. Furthermore, results using this approach have been shown to correlate with disease severity and lung tissue destruction   -TB assay. The TAM-TB assay has been shown to accurately differentiate between active pulmonary TB (aTB) and latent TB infection (LTBI) in different age groups by determination of the phenotypic and functional characteristics of MTB-specific CD4 T cells via flow cytometry –12; detetruction . Sequentescribed , the assere used . Remarkaonary TB . For ESAonary TB . Unlike ent (T3) . A subst2 and T3 . It is p2 and T3 –H. Two oent (T3) –H.LTBI or cure” (data not shown).Future studies will be needed to determine whether TAM-TB assay results could have guided earlier discontinuation of anti-TB therapy in patients before the defined 12 months period. Of note, an additional TAM-TB result obtained from the patient 2 months after discontinuation of anti-TB treatment (14M) was still consistent with “Central and Western Europe, Germany in particular, has seen an increase in TB incidence rate in recent years changing from 5.2/100.000 (2012) to 7.2/100.000 2015) ( (15). ThWith this perspective we aim to highlight that the TAM-TB assay is a promising new tool with great potential to improve TB diagnostics, particularly in difficult-to-diagnose cases of extra-pulmonary TB or children and adolescents in whom conventional tests are frequently unsuccessful. Immunological biomarkers have already demonstrated to be very promising to indirectly diagnose TB; and they are highly likely to play an even more prominent role in TB diagnostics in the years and decades to come , 17, 18.® with a heparin-binding hemagglutinin antigen (HBHA)-based IGRA helped to differentiate Quantiferon-positive children with LTBI from those with aTB  technique is currently not feasible. However, several studies have shown that flow-cytometry-based assays can be further simplified by using whole blood rather than PBMC, thus reducing the blood volume required and, in consequence, making them more feasible to use, especially in infants and younger children , 27Nevertheless, our results provide a first important proof-of-concept that the TAM-TB assay might also be a useful and powerful tool to monitor clinical response to treatment in cases of extra-pulmonary TB. Though the results of this case look promising, they only represent the treatment course of a single patient and one needs to be aware that the respective values of the four TAM-TB markers were referenced to cut-off values obtained from a pulmonary TB cohort. It is likely that since extra-pulmonary TB is a different clinical entity than the pulmonary form of TB, adjusted cut-offs may be required for extra-pulmonary TB cases to allow more accurate interpretation of the results. We do not propose using the TAM-TB assay as a new stand-alone technique for diagnosing tuberculosis, but instead we rather suggest to develop a diagnostic algorithm involving all currently available tools in order to be able to eventually tailor TB treatment duration for individual patients. Still, we are confident that this new assay holds a great potential for the challenging area of TB care and larger prospective clinical trials are currently underway to further validate its performance in the field.MA, UvB, and CG designed and conducted the diagnostic work-up. MA and UvB wrote the manuscript. ID, CZ, and UvB took clinical care of the patient and collected TAM TB samples. MA, KH, and CG ran and analyzed TAM TB assay. JL-Z reported on imaging results and created respective figure for image display.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.


We identified 36 miRNAs that were differentially expressed between newborns and 7-year-olds after controlling for blood cell composition. Using pathway analysis, we found that differentially expressed miRNAs targeted genes enriched for processes related to post-translational modifications, metabolism, and immune response. Our study found that unlike adults, where miRNA expression levels in peripheral blood may decrease with age, expression levels of most miRNAs increased from birth to mid-childhood. This may be reflective of the role miRNAs may play in the highly coordinated mechanisms regulating genes involved in children’s development. Furthermore, it will be important to adjust for both age and blood cell composition in future pediatric studies of miRNA expression in blood.Epigenetic mechanisms have emerged as an important pathway through which environmental exposures can affect health through the regulation of gene expression without changes in DNA sequence: microRNAs (miRNAs) are short non-coding RNAs that target protein-coding mRNAs, leading to post-transcriptional repression. They are involved in important physiologic processes, but little is known about how miRNA expression may change with age in children. We used an nCounter miRNA assay to assess the expression of 43 miRNAs in buffy coat samples collected from newborns ( Epigenetics has emerged as an important biological mechanism through which environmental exposures can affect health ,2,3. EpiGrowing evidence has demonstrated that non-coding RNAs also play a major role in important cellular processes and disease etiology : miRNAs n = 89) and found 94 miRNAs differentially expressed by sex in cord blood: The mRNA targets of these differentially expressed miRNAs were significantly enriched in pathways related to neurodevelopment, RNA metabolism, and post-transcriptional gene silencing [Host factors including sex and age can also influence interindividual differences in epigenetic profiles. Yet little is known about sex- and age-specific differences in human miRNA expression, particularly in children. In adult human plasma, 7 out of 179 candidate circulating miRNAs have been associated with sex . We receilencing .Age-related changes in miRNA expression may be one mechanism regulating gene expression at different developmental stages. Only a few studies have examined age-associated differences in miRNA expression in children. Over 100 liver miRNAs (out of 533) were differentially expressed among fetal, pediatric, and adult subjects, with the largest differences observed between fetal and pediatric liver samples . Among aWhile studies on the relationship between age and sex and miRNA expression are still limited, the existing data suggest that consideration of important host factors is critical for epidemiological studies of miRNA expression. In the present study, we sought to better understand age-related differences in miRNA expression among newborns and 7-year-old children.n = 121) and/or a blood sample from age 7 years (n = 147) available for analysis: 79 children had samples assessed at both time points. Children included in this study did not differ significantly from all children in the cohort by demographic and exposure variables . Furthermore, children with measurements available at both ages compared to those with measurements at only one time point did not differ by demographic and exposure variables, with the exception of being overweight at age 7 years. There was a suggestive trend of a higher incidence of being overweight and obesity among the children with only a single time point (p = 0.05), but this association did not remain statistically significant after adjustment for multiple testing.Children and mothers in this study were participants of the Center for Health Assessment of Mothers and Children of Salinas (CHAMACOS) birth cohort, a longitudinal study of farmworker families from the agricultural region of Salinas Valley, California . PregnanStudy protocols were approved by the University of California, Berkeley, Committee for the Protection of Human Subjects (2010-03-949). Written informed consent was obtained from all mothers, and children provided verbal assent at age 7.® , were centrifuged, separated into aliquots of plasma, buffy coats, and red blood cells, and then stored at −80 °C at the School of Public Health Biorepository, University of California, Berkeley.Blood specimens were collected from CHAMACOS children at birth  and at approximately 7 years of age (by venipuncture). We piloted the protocol for cord blood collection in CHAMACOS participants using two assays, fluorescent in situ hybridization (FISH) analysis for sex chromosomes and polymerase chain reaction (PCR) analysis using human leukocyte antigen (HLA) markers, to assure that cord blood samples were not contaminated by maternal blood. Of the 30 samples randomly selected from CHAMACOS boys, none of the assays indicated any contamination with maternal blood. Heparinized whole blood, collected in BD vacutainersIn our pilot studies, we compared RNA yields from clot, serum, and buffy coat fractions  of blood and found that the highest RNA yields came from the buffy coat fractions. Based on these data and previously published research utilizing miRNA expression data from buffy coat fractions in human populations ,21,22,23In our previous study, we used an miRNAome wide approach, identifying >500 miRNAs with measurable expression in cord blood buffy coat samples using an EdgeSeq miRNA whole transcriptome assay . Here, wminfi [Cell type proportions were calculated in the R-package minfi  based onminfi ,26. For minfi . For blominfi .2 mean miRNA expression with log2 mean SD of miRNA expression at each time point. To model relationships of miRNA expression with white blood cell counts, we constructed separate linear regression models for each miRNA (outcome) at each time point (birth and age 7 years), including blood cell type proportions as the independent variables in the model. To determine whether the miRNAs were differentially expressed by sex at each age, we used the R/Bioconductor package limma, controlling for white blood cell composition.Raw miRNA counts measured by nCounter were imported for quality control and normalization using proprietary software from NanoString, nSolver 3.0 . Raw miRNA expression counts were normalized using the geometric mean of all miRNAs with mean raw counts >50. To determine whether variability of miRNA expression increases as miRNA expression increases, we calculated the Pearson’s correlation coefficient of logn = 79). All other analyses included all children (121 newborns and 142 7-year-olds). To determine whether children’s miRNA expression levels cluster by age, we applied principal components analysis (PCA) to log2 normalized miRNA expression counts using the princomp command in R. We also used the pca3d package in R to visualize the results by age group. To determine which miRNAs were differentially expressed between the two time points, while controlling for blood cell composition, we also utilized the limma package.To determine correlations of miRNAs across time points, we also calculated the partial correlation of each miRNA at birth and age 7 years, controlling for cell composition, among children with miRNAs assessed at both ages  were used to visualize and categorize GO terms via semantic similarity dimension reduction [Homo sapiens GO term database.To determine the potential regulatory function of miRNAs differentially expressed between newborns and 7-year-old children, we used DIANA miRpath v3.0, a web-based computational tool  to condueduction . Allowedp-values <0.05 were considered significant. However, to account for multiple testing, we also used the Benjamini–Hochberg (BH) method for false discovery rate (FDR) with an FDR q-value < 0.05 threshold for significance [Unadjusted ificance . StatistWe measured miRNA expression in a set of 43 miRNAs that had measurable miRNA expression in our previous study  with a rminfi in newborns and 7-year-old children are shown in p-value < 0.05) with blood cell proportions at either age. Among the 43 miRNAs assessed, we found 8 miRNAs that had suggestive associations (2 positive and 6 negative), with blood cell proportions in cord blood .Blood cell proportions estimated by rd blood  indicatird blood , was alsp-values were all >0.05).To examine whether the 43 miRNAs assessed in cord and 7-year-old blood samples differed by sex, we performed differential expression (DE) analysis for sex. We observed a very consistent trend of lower expression in girls compared to boys among the majority of cord blood miRNAs (32 out of 43), as indicated by the negative fold-change values shown in For each miRNA, we calculated the partial correlations between newborn and 7-year-old expression, controlling for cell composition among the 79 children with measurements at both ages . CorrelaWe performed principal components analysis on all miRNAs assessed in newborns and 7-year-old children. The first four principal components accounted for >60% of the variability of miRNA expression levels . Figure limma to identify miRNAs differentially expressed between newborns and 7-year-old children, adjusting for white blood cell proportions. p-values < 0.05). For example, miR-1260b was 6.8-fold lower in newborns compared to 7-year-old children . There were also a few miRNAs (8 out of 43) with positive fold-change values, indicating a small numbers of miRNAs with higher expression in newborns compared to 7-year-old children. In addition to PCA, we also used the R/Bioconductor package p-value < 0.05, We applied DIANA-miRPath in miRNAs differentially expressed between newborns and 7-year-old children. Using the union of predicted genes targeted by differentially expressed miRNAs, we found 130 gene ontology (GO) terms that were significantly enriched , the predicted mRNA targets and their associated pathways highlighted processes important in biological development as children get older.


Changes in different states of MDD, SCZ or BD were also observed in various comparisons. Studies comparing first-episode SCZ to controls may have more reproducible findings than those comparing pre- and post-treatment results. Longitudinal, system-wide studies of inflammation regulation that can differentiate trait- and state-specific changes will be needed to establish valuable biomarkers.Inflammation is a natural defence response of the immune system against environmental insult, stress and injury, but hyper- and hypo-inflammatory responses can trigger diseases. Accumulating evidence suggests that inflammation is involved in multiple psychiatric disorders. Using inflammation-related factors as biomarkers of psychiatric disorders requires the proof of reproducibility and specificity of the changes in different disorders, which remains to be established. We performed a cross-disorder study by systematically evaluating the meta-analysis results of inflammation-related factors in eight major psychiatric disorders, including schizophrenia (SCZ), bipolar disorder (BD), autism spectrum disorder (ASD), major depression disorder (MDD), post-trauma stress disorder (PTSD), sleeping disorder (SD), obsessive–compulsive disorder (OCD) and suicide. A total of 43 meta-analyses involving 704 publications on 44 inflammation-related factors were included in the study. We calculated the effect size and statistical power for every inflammation-related factor in each disorder. Our analyses showed that well-powered case–control studies provided more consistent results than underpowered studies when one factor was meta-analysed by different researchers. After removing underpowered studies, 30 of the 44 inflammation-related factors showed significant alterations in at least one disorder based on well-powered meta-analyses. Eleven of them changed in patients of more than two disorders when compared with the controls. A few inflammation-related factors showed unique changes in specific disorders . MDD had the largest number of changes while SD has the least. Clustering analysis showed that closely related disorders share similar patterns of inflammatory changes, as genome-wide genetic studies have found. According to the effect size obtained from the meta-analyses, 13 inflammation-related factors would need <50 cases and 50 controls to achieve 80% power to show significant differences ( Furthermore, pharmacology studies suggest that inflammation may have a causal effect in the development of psychiatric disorders. On one hand, drugs that target IRFs have been proposed for treating psychiatric disorders, including major depression (MDD)12, schizophrenia (SCZ)13 and bipolar disorder (BD)14, particularly in a subset of patients with chronic inflammation10; a recent meta-analysis of anti-cytokine treatment showed significant antidepressant effects10. On the other hand, some current psychotropic drugs, including antipsychotics and antidepressants, have anti-inflammatory effects, though with inconsistent findings19.Inflammation is a regulated process that occurs in response to injury or stress. Inflammation is also implicated in psychiatric disorders. Triggered by various internal and external stressors, inflammation is moderated by the immune system through a series of signalling pathways, particularly proteins like pro- and anti-inflammatory factors. Changes of some inflammation-related factors (IRFs) have been intensively studied and reviewed in relation to psychiatric disorders20 as well as the treatment response21. Measurable in peripheral blood, IRFs have the advantage of accessibility. Currently, diagnosis of functional psychiatric disorders is based mainly on subjective interview. The addition of trait biomarkers associated with disease diagnoses can only improve the accuracy of diagnosis. Furthermore, state markers associated with specific symptoms or stages of disease can help to monitor treatment. Accordingly, it is crucial to explore whether IRFs can be used as trait and/or state markers of psychiatric disorders. IRF changes need to be associated with diseases, symptoms or treatment effects, and more importantly, such associations must be specific and reproducible to turn IRFs into biomarkers. Although dozens of meta-analyses have been published on IRFs in psychiatric disorders, the reproducibility of those results has not been thoroughly evaluated. Without comparing the studies from different disorders, the specificity of IRFs cannot be determined.The relevance of inflammation to psychiatric disorders suggests its potential as a suitable biomarker. A biomarker is defined as “any substance, structure, or process that can be measured in the body,” which can “influence or predict the incidence of outcome or disease”Here, we systematically reviewed meta-analysis studies of IRF changes in major psychiatric disorders. Through the evaluation, we found that meta-analyses with sufficient statistical power produced stable results. After filtering out the underpowered data, we identified IRF changes that were shared across disorders or unique to specific disorders as potential trait markers. We identified 30 IRFs showing alterations in at least one disorder. We also identified IRFs as potential state markers for specific types of patients with MDD, SCZ or BD in various comparisons. We discussed the missing aspects of existing studies and recommended future study designs needed to translate IRFs into biomarkers.22, VEGF23, NGF22, FGF24 and TNF24. The inclusion criteria were the following: 1) published in English before March 2018; 2) assessed circulating cytokine/chemokine/inflammation-related factor levels in plasma or serum samples or the central nervous system; 3) meta-analysis only. The studies involving patients of different treatment stages or symptom states (state-related) were analysed separately from the case–control studies (trait-related). We discarded two studies testing for the correlation between the IRF levels with depressive symptoms27 since their measurements were not compatible with the other differential studies. The literature selection followed the standard by Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)28  OR ((chemokine) OR (Interleukin) OR (interferon) OR (tumour necrosis factor) OR (colony-stimulating factor) OR (growth factor) OR inflammat%))) AND (#DISORDER#) AND meta-analysis. #DISORDER# is one of the major psychiatric disorders: SCZ, BD, autism spectrum disorder (ASD), MDD, post-traumatic stress disorder (PTSD), anxiety, suicide, sleeping disorder (SD) or obsessive–compulsive disorder (OCD). For growth factors, we selected only those known to regulate inflammation, such as IGFn = 11), MDD (n = 16), BD (n = 6), OCD (n = 1), suicide (n = 2), ASD (n = 1), PTSD (n = 1), SD (n = 1), and cross-disorder studies (n = 4). Anxiety did not meet all our criteria for inclusion. One publication can contain meta-analyses of several IRFs in multiple disorders targeting different comparisons; therefore, we partitioned the data to serve two different analyses: trait marker analysis and state marker analysis 31. The most frequently studied disorders are MDD, BD, suicide and SCZ. These four disorders were meta-analysed numerous times, giving us an opportunity to assess the stability of the meta-analysis results over time.We collected 43 meta-analysis publications on 44 IRFs in eight major psychiatric disorders. Data from 43 meta-analysis studies included SCZ  produced relatively stable findings. Therefore, we used statistical power to filter all meta-analyses and identified inflammatory changes that are likely to be reproducible.34. Some significant IRF changes only appeared in recent studies as more data became available. For example, the difference of IFN-gamma was not significant between MDD and controls in a study of 238 subjects35, but a later meta-analysis that includes a total of 1470 subjects found IFN-gamma to be significantly decreased in MDD patients36. IL-8 was reported to have no significant changes in MDD by a study of 560 subjects37, but it was found to be increased in MDD in a later study of 3788 subjects38. These conflicting results suggest that those changes can only be detected in larger sample sizes.By using the stability of the results to assess the reproducibility for the IRFs changed in BD, SCZ, suicide and MDD, we found that some findings in these meta-analyses were stable while others were not. For example, sIL-2R and sTNF-R1 were significantly elevated consistently in BD39 targeting p < 0.05. Totally, 26% of the meta-analyses did not have enough power (<80%).We hypothesised that the inconsistent results of the same IRF within the same disorder may result from underpowered studies. To test this hypothesis, we extracted the effect size measured as a standardised mean difference of every IRF in each disorder from those meta-analyses, and calculated their statistical powers by using G*powerFor the IRF meta-analysed multiple times, 42% (16/38) of the tests showed inconsistent results in case–control comparisons. Totally, 67% (24/36) of the tests showed inconsistent results in state-related meta-analyses. After filtering out the tests with insufficient statistical power, only 16% (5/31) of case–control-related tests still showed inconsistent results. Totally, 48%  of the state-related tests remained inconsistent. Filter by statistical power clearly improved the consistency of the results by about 20% or more for both trait and state-related analyses. If two studies were completely independent, as each result has three possible states: increase, decrease and no change, the chance of two results having the same type of state is 11% (1/3 × 1/3). The observed consistency here (>33%) is much higher than 11% even in the underpowered meta-analyses. This high consistency can be partially caused by the relatedness of data used in the two meta-analyses. But the overall pattern of consistency, particularly that improved consistency associated with filter by statistical power is very encouraging. All consistencies are higher than 50% after filtering. Case–control trait-related findings have 84% consistency. The results of SCZ and BD case–control comparisons showed 100% consistency. Trait-related findings are generally more reliable than state-related tests, which have consistencies ranging between 16% and 60%.We looked into the inclusion and exclusion criteria of all the meta-analyses and noticed large variations: 37% of the meta-analyses required diagnosis with DSM/ICD; 20% of the studies required specific age ranges; 70% of the studies removed data without controls; only 20% of the studies removed duplicated data; 11% of the studies excluded participants with other comorbidities. Very few used identical criteria. The inconsistent use of inclusion/exclusion criteria could potentially increase the inconsistencies for the results of meta-analyses. But our analyses showed sufficient consistency for those well-powered data (84% consistency for the results of case–control comparisons). The inconsistent inclusion/exclusion criteria used by different studies did not destroy the findings.Based on the stability evaluation, we decided to analyse only the data from the well-powered studies because they were more likely to yield reproducible findings. We removed the data with statistical power lower than 0.8. We applied this filter to all data, assuming that all data including those tested only once will have better reproducibility. The single meta-analysis of OCD was filtered out because of the insufficient statistical power. Thirty-eight IRFs survived the filtering. Thirty of the 38 IRFs showed significant alterations in at least one disorder Table . All wel29. Five IRFs were significantly changed in SCZ patients based on CSF samples. IL-6 was consistent with the changes detected in blood while IL-1beta was inconsistent. Three others  were not studied in blood meta-analyses.We also examined the impact of tissue types on the reproducibility of findings. The single study on IRFs from the brain was under-poweredTo evaluate the specificity of each IRF in disorders, we investigated whether the change of one IRF occurs universally in all psychiatric disorders or only in some disorders by using only well-powered data. Whenever an IRF only shows a change in one or several disorders but not every disorder, we consider that it merits disease specificity. An IRF change is considered to be non-specific when all the disorders carry the same change.We used the IRFs that changed for multiple disorders patients compared with controls to assess their degree of specificity. Thirteen of them were analysed in more than half (4) the disorders, including TGF-beta, IL-10, IL-1RA, IL-1beta, IL-2, IL-4, IL-6, IL-8, sIL-2R, sIL-6R, TNF-alpha, IFN-gamma and C-reactive protein (CRP). All these IRFs changed in some disorders but had no change or even opposite changes in other disorders. Among them, IL-2 is uniquely significantly low in suicide; IL-4 is significantly low in suicide, but high in BD; sIL-6R is uniquely significantly high in BD; sIL-2R is uniquely not changed in PTSD. IL-6 and CRP are the two most commonly increased IRFs changed in four disorders. The most commonly decreased IRF is the nerve growth factor (NGF) in MDD and SCZ.40.The specificity of IRFs across disorders can reflect a similarity in the aetiology or pathology of disorders. Unsupervised clustering analysis on the presence of the changes of the 30 IRFs in the seven disorders showed an interesting pattern—BD and SCZ were closer to each other than the others, while suicide clustered with PTSD and SD Fig. . This paIRFs that change at different states of disorders were also examined in the well-powered studies. Two types of studies have been used in state-related analyses, including ten studies that compared BD, SCZ and MDD patients at different clinical states with controls, and twelve studies that compared pre- and post-treatment patients. Quantitative differences in IRFs among states of diseases were observed.42, but was reported to have consistent changes in several different states of SCZ: higher in acutely ill, chronically ill and first-episode SCZ patients than in controls. CRP is the only IRF that was reported to change in all three BD  states. But the depression state has conflicting results44. Studies comparing patients with first-episode SCZ patients with controls contain the most consistent results as reported in the two meta-analyses by Miller and collaborators45, many were also replicated by Upthegrove et al.46 except for IL-4 and IFN-gamma.Out of the sufficiently powered meta-analyses, the SCZ data have the same consistency  as BD  data, and are better than MDD  data when IRFs were meta-analysed multiple times. The pre- and post-treatment comparison is one of the major sources of inconsistency (3/6 in SCZ and 5/5 in MDD). For example, IL-6 has inconsistent findings in SCZ pre- and post-treatment comparisonThe state-related data carry more uncertainty than trait-related data. We are particularly concerned by the frequent inconsistencies observed in the pre- and post-treatment comparison studies. Among these state-related studies, findings from first-episode SCZ studies are likely more reliable. All the results are shown in Supplementary Table It should be noted that our study was built upon meta-analysis results, which represented the consensus of multiple individual studies, less influenced than original individual studies by biological or technical noises or variations from individual studies. At the same time, meta-analyses cannot recover signals that may be missed by original studies due to technical limitations. Meta-analyses can be misled by systematic bias in individual data. Therefore, systematic investigation with better technologies and better experimental design has the potential to uncover IRF changes that we missed, or to disapprove a finding claimed here.More research is necessary before IRFs can be used as clinically useful biomarkers. We noticed some major pieces missing in previous studies, mostly due to the limitations of technology and costs. Most of those studies focused on candidate genes, which do not provide system-wide information. Statistical procedures were commonly liberal, leaving more space for false findings. Since longitudinal studies are still rare, we can neither differentiate acute and chronic changes nor can we discern trait and state markers. Lastly, blood–brain comparisons may not be necessary for developing biomarkers, but such comparisons are important for establishing the mechanisms of psychiatric disorders.Candidate gene studies contributed to answering specific questions in biology, but they frequently missed the big picture, without assessing the impact of the genes that changed the most. Candidate genes are generally not the top signals in genome-wide or system-wide studies. The bigger differences between cases and controls with better disease specificity are hidden in the systems and remain to be discovered. Therefore, the current candidate genes, including all the IRFs reviewed here, may not be the best genes for biomarker development. The other genes discovered from system-wide hunting may be better biomarker candidates.47. One GWAS of inflammation quantitative trait locus (QTL) identified some potential genetic regulators of inflammation48. The omics-based approaches can help reveal more about these regulatory processes. Genomic and epigenetic methods have been applied to adipose tissue49 and blood leucocytes50, leading to the discovery of large inflammatory response networks. These studies have only scratched the surface of this complex regulatory system. More remains to be learned regarding the regulation of inflammation in response to both acute and chronic stressors.The regulatory system that modulates inflammation remains poorly understood. Regulation of inflammatory responses occurs at various levels including genetic, transcriptional, translational, protein modification and protein interaction, and involves many signalling pathways51. It has been used in studies of alcoholism52, depression53 and autism54, with up to 41 IRFs examined simultaneously53. The rapidly evolving mass spectrometry-based proteomics will soon provide more comprehensive coverage. The advanced technology will make system-wide studies of inflammation feasible in the near future.Hundreds of genes or proteins are involved in immunity and inflammation regulation and should be studied in relation to psychiatric disorders. Only a limited number of them have been investigated in psychiatric disorders due to the constraints of methodology and resources. Previous studies were completed by using the enzyme-linked immunosorbant assay (ELISA) or multiplex protein assays. Multiplex protein assay is a technology getting more attention recently39 on the 427 meta-analyses of 44 IRFs done in the eight disorders, 318 of them (74%) have sufficient power ( ≥ 0.8), while the rest were under-powered. Sample size, one of the major drivers of statistical power, is a limiting factor for many studies. The good news from this study is that the sample size required to detect the differences in IRFs between patients and controls might be relatively small for many IRFs. Based on the median effect size summarised in Table p < 0.0016. At that p value, 13 IRFs would only need <100 subjects (50 cases and 50 controls) to achieve 80% power to detect a significant difference  and site effects (data collected from different machines may not be measured consistently). Ignoring the covariates for inflammation such as blood counts, BMI, sex and chronic stress levels can lead to false findings. The inappropriate management of covariates can misdirect meta-analyses. As shown in Supplementary Table p ≤ 0.05 to be significant even when multiple IRFs were tested. With that, publication bias for the positive results likely occurred55. In Supplementary Table Strict statistical criteria for defining significance should be consistently applied across studies. Many publications considered 56, acute ethanol intoxication57, long-term smoking59, binge drinking60, psychological stress63, diet64, air pollution65 and infection all can induce inflammation. The inflammation levels likely also fluctuate temporally due to environmental and circadian rhythm changes regardless of health status. Single-time- point measures could be misleading. Therefore, longitudinal studies with multiple time-point measures of inflammation in peripheral tissues are necessary to accurately assess the inflammatory changes. The economical and convenient methods of data collection need to be developed for longitudinal studies.Acute and chronic inflammation can have very different impacts and consequences in aetiology and pathology. Many physical and psychological stressors, environmental insults including acute smoking66, as well as cancer68. Therefore, much remains to be learned about the specificity of each individual IRF as related to various psychiatric and non-psychiatric diseases.Biomarkers will be more informative if they can differentiate traits and states to facilitate diagnosis and differential diagnosis and monitor prognosis. In this study, we only had data to compare eight psychiatric disorders. Changes of IRFs are not limited to neuropsychiatric disorders; they are also reported in patients with cardiovascular and metabolic diseases69. The clinical symptoms can change through the disease course while the diagnosis remains unchanged. To differentiate trait and state markers, we will need to examine patients of different episodes, illness or symptom stages, instead of lumping all patients into one case group.State markers represent the status of clinical manifestations in patientsA few IRFs were presented as potential state and trait markers. Our consistency analysis based on sufficiently powered meta-analyses suggests that trait-related findings might be more reliable than state-related findings so far. The state-related analyses have more inconsistent results than the trait-related analyses. More work remains to be done to accurately identify the state-related changes.46; however, IL-12 significantly increased in the blood of patients with SCZ45. We can reasonably hypothesise that inflammation could be tissue-specific. Tissue specificity may differentiate some disorders. The central nervous system may have a different inflammatory response than the peripheral tissue depending on the source of the insult. This may be particularly true in relation to the blood–brain barrier, which separates the central nervous system from peripheral circulation and responds to inflammation under its own regulation71. A systematic evaluation of the similarity and dissimilarity of inflammation across tissues in different disorders is needed. Peripheral blood data are useful for developing clinically informative biomarkers, but the brain is the ultimate affected tissue of psychiatric disorders. Therefore, building the blood–brain relationship will be critical in proving the biological relevance and illustrating the biological mechanisms. Postmortem brain data are valuable, and the PsychENCODE project72 brought more insights into the inflammation status of brains affected by SCZ, BD and ASD. However, resolving the causal relationships in the study of inflammation using postmortem brains will be challenging. Animal and cellular models will hold great value in understanding the regulation of inflammation75.Biomarkers of psychiatric disorders need to have biological relevance based on solid mechanistic understanding that will ultimately link blood and brain function. In our data, the difference of IL-12 levels in CSF between SCZ and controls was not significantIn summary, this systematic evaluation of inflammatory changes across multiple psychiatric disorders showed us the intriguing possibility of differentiating psychiatric disorders by using inflammatory biomarkers. Moreover, inflammation may have a large enough effect size that these biomarkers could be detected in relatively small sample sizes. We propose that a system-wide longitudinal study using strict analytical procedures could render effective and useful biomarkers.List of Supplementary MaterialsSupplementary Fig.1 Flowchart of Data Selection Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)Supplementary Table S1 -original ExcelSupplementary Table S2 -original ExcelSupplementary Table S3 -original ExcelSupplementary Table S4 -original ExcelSupplementary Table S5 -original Excel


Preventing unintended pregnancy is critical for women living with HIV (WLWH) to safely achieve their reproductive goals. Family planning services should support WLWH at risk of repeat unintended pregnancies. We examined the relationship between unintended pregnancy and subsequent contraception use among WLWH in Uganda.This was a retrospective analysis of data from a longitudinal cohort of individuals initiating antiretroviral therapy (ART), restricted to women with pregnancy (confirmed via urine β-hcg testing) between 2011–2013. The exposure of interest was intended vs unintended pregnancy, and the outcome was self-report of modern contraceptive use  at 12 (range 6–18) months post-partum. A log-binomial model was used to estimate relative risks of modern contraceptive use post-partum based on intent of the index pregnancy, adjusted for age, socioeconomic status, education, relationship and HIV status of pregnancy partner, contraceptive use prior to pregnancy, years since HIV diagnosis, ART regimen, and CD4 cell count.3, and were living with HIV for 3.8 years. Fifty pregnancies (45%) were reported as unintended and 60 (55%) as intended. Postpartum, 64% of women with unintended and 51% with intended pregnancy reported modern contraception (p = 0.24). In adjusted models, there was no association between pregnancy intent and post-partum contraception. However, contraceptive use prior to the referent pregnancy was positively associated with post-partum contraceptive use , while higher baseline CD4 cell count was associated with lower post-partum contraceptive use .Among 455 women, 110 women reported 110 incident pregnancies with report on intent. Women had a baseline median age of 29 years, baseline CD4 count 403 cells/mmAlmost half of incident pregnancies among WLWH in this cohort were unintended. Experiencing an unintended pregnancy was not associated with post-partum contraceptive use. Creative strategies to support contraceptive uptake for birth spacing and prevention of unintended pregnancies in the post-partum period are needed. Individuals’ freedom to decide on the number and timing of their children is central to the attainment of sexual and reproductive health and rights worldwide , an inteDespite these well-publicized goals, millions of women worldwide continue to have unmet family planning needs, with rates in some parts of the world, such as sub-Saharan Africa, stagnant from 1970–2010 , with thWomen in Uganda, with one of the highest fertility rates in the world at 5.4 births per woman , and a nUnmet need refers both to need for limiting (for women who do not desire more children) and spacing  pregnancies. In bothStudy participants were enrolled in the Uganda AIDS Rural Treatment Outcomes (UARTO) cohort study, which ran from 2005 to 2015. Clients initiating ART at an HIV clinic in Southwestern Uganda, at least 18 years old, and living within 60 kilometers of the clinic were eligible to enroll. Contraceptive services were not directly available at the HIV clinic site, but were available, free of charge, through the family planning clinic at the same hospital campus. Participants completed baseline and approximately quarterly interviews and phlebotomy. Interviewer-administered questionnaires detailed socio-demographics, sexual behavior, and partner dynamics, including partner HIV status. Laboratory data included CD4 cell count measurements and HIV-RNA, and clinical records indicated ART regimen.This analysis utilized data from the Reproductive Health Component of this cohort, initiated in October 2011 with follow-up through April 2014. This annual questionnaire assessed sexual and reproductive health and history, fertility desire, contraceptive use, partner HIV status, and partner fertility desire; the questionnaire was also administered with incident pregnancy (up to quarterly). Among women of reproductive age, pregnancy was assessed quarterly with urine β-Hcg testing.All women of reproductive age (18–49 years old) with a positive pregnancy test between 2011–2013  and who completed at least one post-partum reproductive health survey were eligible for inclusion in this analysis. Women who were not tested with urine β-Hcg but self-reported pregnancy were considered pregnant. Women who reported pregnancy but had a negative urine β-Hcg were not considered pregnant.The exposure of interest was the intent of the index pregnancy, determined using questions derived from the Pregnancy Risk Assessment Monitoring System (PRAMS) developed and validated by the U.S. Centers for Disease Control (CDC) , measureThe primary outcome was self-reported use of modern contraception at 12 months post-partum . Since most women did not have a visit at exactly 12 months post-partum, we considered visits within 6–18 months after birth outcome, based on extrapolation to a 9-month pregnancy (dates of pregnancy outcome were missing for most participants). Sensitivity analyses expanded this window to range from 6 to 36 months from referent pregnancy.Modern contraceptive use was derived at this post-partum visit from self-report of a modern contraceptive method , educatA log-binomial model was used to obtain relative risks of modern contraceptive use post-partum based on intention of the index pregnancy. This model was separately adjusted for the covariates of interest. Covariates with a significant effect on outcome at a level of p<0.05 were then incorporated into serial adjusted models. Given the relatively small sample size and inconsistent missingness across variables, several smaller models were evaluated rather than one large model.This study was approved by the ethics review boards of Partners Healthcare, the Mbarara University of Science and Technology, and Simon Fraser University. Administrative approvals were secured from the President’s Office and the Ugandan National Council for Science and Technology.3), and were more likely to report that their primary partner either “did not want the current pregnancy then or at any time” (26% vs 3%) or “didn’t care or don’t know” (26% vs 5%).Among 455 women of reproductive age who completed the Reproductive Health Component of the study, there were 120 incident pregnancies during 1,161 woman-years of follow-up. Pregnancy intent was reported for 110 of these pregnancies; 50 (45%) reported as unintended, and 60 (55%) as intended. Of the 110 pregnancies, 26 resulted in a live birth, 5 in miscarriage, 0 in stillbirth, 2 had other pregnancy outcomes, and 78 were missing data on pregnancy outcome. At the post-partum visit, 85% of women with unintended and 67% of women with intended referent pregnancy reported not wanting any more children in the future . 62% of . At the time of the post-partum visit, 10 women had a repeat positive pregnancy test, among whom 8 reported contraceptive use .Data on the primary outcome, contraceptive use 6–18 months post-partum, was available for 82 participants. The post-partum visit occurred a median of 11.2 months (range 6–18) post-partum. Of these, 47 (57%) reported modern contraceptive use postpartum, the proportion was 64% among women with an unintended and 51% with a referent intended pregnancy . Of the . At the 3 increase , p = 0.24, increase , when adModels did not show an association of post-partum contraceptive use with participants’ age, socioeconomic status, education level, years since HIV diagnosis, efavirenz-containing ART regimen, or participant’s relationship with or HIV status of the pregnancy partner. Self-reported personal or partner fertility desire at the time of the post-partum visit was similarly not associated with post-partum contraceptive use.Sensitivity analysis classifying 10 women with repeat pregnancies at the post-partum visit as not using effective contraception did not alter the results of the primary analysis: intent of referent pregnancy was not associated with post-partum contraceptive use . Similarly, expanding the post-partum visit window to include all visits within 6–36 months after the initial pregnancy visit did not meaningfully alter the results.In this cohort of WLWH in rural Uganda, 45% of pregnancies were unintended. Intent of referent pregnancy was not associated with effective post-partum contraception use. Overall, 57% of participants reported modern contraception at 12 months post-partum, despite 75% of women not wanting to have children at that time. Among those using contraception, over half were using injectable hormonal methods.The reported rate of unintended pregnancy in our cohort (45%) is similar to that reported nationally for Uganda in 2011 (44%). StudiesIn this study, 64% of women with referent unintended and 51% with intended pregnancy reported modern contraceptive use at 12 months post-partum, with an overall post-partum contraceptive use rate of 57%. These results are somewhat higher than overall contraceptive use reported among WLWH at this site ,38, and Referent pregnancy intent was not associated with contraception use. It is possible that there is a true lack of association between pregnancy intent and post-partum contraceptive use in this cohort. A similar absence of correlation was noted in a study of adolescent girls living with HIV in Kenya, in whicPost-partum contraceptive use was not associated with participant fertility desire in the post-partum period, in contrast to a previous study from this same site examining contraceptive use. Three qhttps://clinicaltrials.gov/ct2/show/study/NCT02964169).These data highlight the importance of efforts to focus on strengthening contraceptive counseling and utilization in the postpartum period. This was a quantitative study that did not explore women’s self-reported reasons for not using contraception, therefore, comprehensive recommendations regarding increased utilization of post-partum contraception in this population is beyond the scope of this study. However, the findings shed light on several areas in which contraceptive care provision for this population could be improved. Integration of HIV care delivery and contraceptive services is an evidence-based approach to increasing access to contraception for women living with and without HIV in sub-Saharan Africa ,42. InteContraceptive use prior to index pregnancy and baseline CD4 count were the only significant determinants of post-partum contraceptive use. The association with prior contraceptive use suggests that women who were more likely to be using contraception prior to conception, due to either better access or better education, were again more likely to be using contraception post-partum, irrespective of whether their intervening pregnancy was intended or not. While the numbers in this study are small, this finding suggests that WLWH with incident pregnancies should be assessed for contraceptive use in the period prior to pregnancy. Those reporting no contraceptive use in the pre-conception period should be focused on for additional counseling regarding post-partum contraceptive uptake. The reasons for the statistically significant  association between baseline CD4 cell count and post-partum contraceptive use are not clear. One potential reason may be that women entering the cohort with lower CD4 cell counts were more frequently engaged in care due to advanced disease, thus they were exposed to a greater amount of counseling and health education, and may have therefore been more likely to use contraception in the post-partum period.In sum, data from this cohort of WLWH in Uganda suggests that eliciting intent of an index pregnancy using currently-available tools does not predict effective post-partum contraceptive use. A stronger emphasis in counseling on post-partum contraceptive use and prevention of repeat unintended pregnancies should be incorporated into care for WLWH. WLWH who are pregnant should be assessed for contraceptive use in the pre-conception period, and those who report not using contraceptive methods prior to their current pregnancy should be supported to access post-partum contraception commensurate with their reproductive goals. Access to long-acting and injectable methods of contraception should be ensured in this setting. The development of a validated tool to assess pregnancy intent among women living with HIV in sub-Saharan Africa should be further explored.There are several limitations to this study. Determination of pregnancy intent is challenging without a current, culturally-appropriate, validated questionnaire, and women may be misreporting pregnancy intent or future fertility desire due to social desirability bias in the healthcare setting. There is potential for reporting bias leading to the underestimation of rates of unintended pregnancy, since women may be less likely to report a pregnancy as unintended once they are pregnant. Data on outcomes and covariates were not available for all study participants, leading to decreased power to detect significant differences in contraceptive use by pregnancy intent. Some covariates (e.g. pregnancy outcome) were not included in models due to missing data. Data on post-partum contraceptive use were not available for all incident pregnancies, however, rates of missing data were similar for women with referent unintended and intended pregnancies.Understanding contraceptive use and fertility desire after unintended pregnancies can provide an opportunity for focused interventions to address unmet family planning needs among women of reproductive age with HIV in Uganda. This exploratory study, with the objective of better understanding the relationship between unintended pregnancy and subsequent contraceptive use, aims to inform targeted contraceptive counseling for post-partum women living with HIV in sub-Saharan Africa.


An overlooked sequela of HIV risk is trauma exposure, yet few HIV interventions address trauma exposure, mental health, and substance misuse. In a two-arm randomized controlled trial 73 Native American women were randomized to a culturally-adapted Cognitive Processing Therapy (CPT) or 6-weeks waitlist. Outcomes assessed: PTSD symptom severity, alcohol use frequency, substance abuse or dependence diagnosis, and high-risk sexual behavior defined as vaginal/anal intercourse (a) under the influence of alcohol and/or illicit substances, (b) with a partner who was concurrently sexually active with someone else, and/or (c) with more than one partner in the past 6 weeks. Among immediate intervention participants, compared to waitlist participants, there were large reductions in PTSD symptom severity, high-risk sexual behavior, and a medium-to-large reduction in the frequency of alcohol use. CPT appears to improve mental health and risk behaviors, suggesting that addressing PTSD may be one way of improving HIV-risk related outcomes. An important but overlooked sequela of trauma exposure is risk for HIV . Trauma Exposure to trauma over the past several centuries has led to devastating consequences for AIAN health and wellness . AIAN exTrauma exposure and its impact on substance misuse and PTSD have received special attention from many AIAN communities . HistoriCulturally-tailored interventions are critical for this population, as tailored interventions may reduce high attrition of racial and ethnic minorities from programs developed for non-minority communities , 29. CogThis study’s primary objective was to assess the feasibility and effectiveness of CPT for AIAN woman with PTSD symptoms, high-risk sexual behavior, and substance use. Our study hypotheses were: (1) as compared to waitlist, improvements in PTSD, substance use, and HIV sexual-risk behavior will be greater at post intervention for those receiving immediate CPT; and (2) at 3 months post intervention, improvements in PTSD, substance use, and HIV sexual-risk behavior compared to pre-intervention will be greater for those receiving CPT compared to waitlist.The study was conducted at two rural Pacific Northwest behavioral health clinics: a tribally-operated clinic located on the reservation and a privately-operated nonprofit clinic located in a town adjacent to the reservation. Our community assessment shown that 39.5% of women between the ages of 15 and 35 had high rates of mental health diagnoses  , 38. RecAll participants completed 45-min audio computer-administered assessments at baseline, immediate post-, and 3-month post-intervention. Following baseline assessment, participants were randomly assigned to either receive immediate CPT or to a 6-week waitlist. At the end of the waitlist period, participants completed a post-waitlist assessment. Random assignment to condition was computer-generated with block sizes ranging from 2 to 8 to reduce the detection of a pattern and prepared by an external statistician. Allocation concealment involved the use of sequentially numbered, opaque, sealed envelopes containing the group assignment, which the research manager opened at the moment of randomization. Due to the nature of the intervention, participants and the study team could not be blinded to condition. Participants were compensated for each interview: baseline ($30), waitlist , post-intervention ($40 approximately 10 weeks after first session), and three-month post-intervention ($50). Travel was reimbursed up to $20.00 for each intervention session .Based on community feedback we used CPT without the trauma narrative . In CPT Map of Adaptation Process, which is a systematic approach for adapting evidence-based behavioral interventions (EBIs) ) and a 0.4-point greater reduction in alcohol problems .Across the 6 outcomes, there were two statistically significant dose–response effects. Each additional CPT session attended was associated with a 1.3-point greater reduction in PTSD symptoms  of clients never receiving any CPT. The modal number of therapy sessions in clinical settings is one . This maThis study has several limitations. With over 567 federal and 200 state-recognized tribes as well as multiple unique urban AIAN communities, our Pacific Northwest regional focus may not fully capture AIAN retention and response to CPT. In addition, we did not utilize independent, blinded interviewer administered assessments, which are the recommended method of assessing treatment response . HoweverOverall, results of this study suggest that providing a short-term, structured, culturally adapted intervention for PTSD can decrease PTSD, substance use, and HIV risk behaviors in a sample of AIAN women. Given the high rates of trauma exposure and associated negative outcomes in AIAN communities this study offers a potential efficacious option for addressing an important public health disparity. Future studies should expand on this work by exploring briefer therapy protocols to help improve retention and engagement. Addressing trauma is a community driven and crucial priority for the health and prosperity of AIAN communities and continues to be an area of great importance.