Roadmap to DILI research in Europe. A proposal from COST action ProEuroDILINet.

In the current article the aims for a constructive way forward in Drug-Induced Liver Injury (DILI) are to highlight the most important priorities in research and clinical science, therefore supporting a more informed, focused, and better funded future for European DILI research. This Roadmap aims to identify key challenges, define a shared vision across all stakeholders for the opportunities to overcome these challenges and propose a high-quality research program to achieve progress on the prediction, prevention, diagnosis and management of this condition and impact on healthcare practice in the field of DILI. This will involve 1. Creation of a database encompassing optimised case report form for prospectively identified DILI cases with well-characterised controls with competing diagnoses, biological samples, and imaging data; 2. Establishing of preclinical models to improve the assessment and prediction of hepatotoxicity in humans to guide future drug safety testing; 3. Emphasis on implementation science and 4. Enhanced collaboration between drug-developers, clinicians and regulatory scientists. This proposed operational framework will advance DILI research and may bring together basic, applied, translational and clinical research in DILI.

A biocompatible decellularized pulp scaffold for regenerative endodontics.

AIM: To investigate the feasibility of decellularizing the entire dental pulp using a mild treatment protocol to develop a decellularized biological extracellular matrix scaffold for use in regenerative endodontic procedures. METHODOLOGY: Decellularized human dental pulps were assessed using histological and immunohistochemical methods, scanning electron microscope and DNA quantification assay. Cytotoxicity assays to determine decellularized scaffold biocompatibility were also performed. Decellularized scaffolds were seeded with human dental pulp stem cells and cell viability assessed using Live/Dead® stain. Quantitative data were analysed statistically using Student's t-test and one-way analysis of variance to compare mean values between groups depending on group numbers. RESULTS: Assessment of decellularized tissues revealed an acellular matrix with preservation of native tissue histoarchitecture and composition. Decellularized tissues showed no evidence of cytotoxicity, with cell growth in direct contact with the scaffold and no reduction in cellular activity following extract incubation. Furthermore, the scaffold was able to support human dental pulp stem cell viability and attachment following recellularization. CONCLUSIONS: Promising results were observed in developing a decellularized biological scaffold derived from the dental pulp with the perseveration of extracellular structural components which are required for tissue-specific regeneration.

Clinical and epidemiological profile of women with breast cancer managed in a public referral hospital in northeastern Brazil.

AIM: To evaluate the clinical and epidemiological profile of women with breast cancer, managed in the Breast Service at Hospital Get6lio Vargas (HGV). MATERIALS AND METHODS: A descriptive, observational cross-sectional study, involving 174 breast cancer patients, managed at the Breast Service at HGV in Teresina, in the northeast of Brazil, from May 2011 to June 2014. Epidemiological variables included age group, age at menarche, age at first childbirth, and menopause. The tumor characteristics studied were histological type, TNM classification, and staging. The results were organized in distribution tables. RESULTS: Breast carcinoma was more common in patients aged 60 and over, in multiparous women, and in women who were menopausal before age 55. The most common histological type was ductal invasive carcinoma T2N1MO (Stage IIB) in 83 (48%) women. CONCLUSION: The current study shows that the majority of carcinomas were Stage II or advanced tumors, with clinically positive axillary lymph node status and weak correlation with reproductive risk factors.

Tumor necrosis factor-α and interleukin-6 gene polymorphism association with susceptibility to celiac disease in Italian patients.

The aim of this research was to study polymorphisms in the genes encoding cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in patients with celiac disease (CD) antigens DQ2 (DQ2-positive) or DQ8 (DQ8-positive). We compared the results with healthy controls to determine whether any of the polymorphisms have a role in susceptibility to CD. A case-control of 192 patients with CD (96 DQ2-positive and 96 DQ8-positive) and 96 healthy controls from northeast Italy were included in the study. Analysis of single nucleotide polymorphisms (SNPs) was carried out using the polymerase chain reaction-restriction fragment length polymorphism method. Significant differences for the TNF-α(-308 G>A) polymorphism were observed when we compared the flowing groups: DQ2-positive with controls [odds ratio (OR) = 0.45, P = 0.0002]; DQ8-positive with controls (OR = 3.55, P < 0.0001); and DQ2-positive with DQ8-positive (OR = 0.12, P < 0.0001). We did not observe a statistically significant association between IL-6 (-174 G>C) polymorphism and CD (P > 0.05). Our results suggest that TNF-α(-308 G>A) polymorphism may play a role in susceptibility to CD in Italian patients.

(1-3)-beta-D-glucan in association with lactate dehydrogenase as biomarkers of Pneumocystis pneumonia (PcP) in HIV-infected patients.

Pneumocystis pneumonia (PcP) is a major HIV-related illness caused by Pneumocystis jirovecii. Definitive diagnosis of PcP requires microscopic detection of P. jirovecii in pulmonary specimens. The objective of this study was to evaluate the usefulness of two serum markers in the diagnosis of PcP. Serum levels of (1-3)-beta-d-glucan (BG) and lactate dehydrogenase (LDH) were investigated in 100 HIV-positive adult patients and 50 healthy blood donors. PcP cases were confirmed using indirect immunofluorescence with monoclonal anti-Pneumocystis antibodies and nested-PCR to amplify the large subunit mitochondrial rRNA gene of P. jirovecii in pulmonary specimens. BG and LDH levels in serum were measured using quantitative microplate-based assays. BG and LDH positive sera were statistically associated with PcP cases (P ≤ 0.001). Sensitivity, specificity, positive/negative predictive values (PPV/NPV), and positive/negative likelihood ratios (PLR/NLR) were 91.3 %, 61.3 %, 85.1 %, 79.2 %, 2.359, and 0.142, respectively, for the BG kit assay, and 91.3 %, 35.5 %, 75.9 %, 64.7 %, 1.415 and 0.245, respectively, for the LDH test. Serologic markers levels combined with the clinical diagnostic criteria for PcP were evaluated for their usefulness in diagnosis of PcP. The most promising cutoff levels for diagnosis of PcP were determined to be 400 pg/ml of BG and 350 U/l of LDH, which combined with clinical data presented 92.8 % sensitivity, 83.9 % specificity, 92.8 % PPV, 83.9 % NPV, 5.764 PLR and 0.086 NLR (P < 0.001). This study confirmed that BG is a reliable indicator for detecting P. jirovecii infection. The combination between BG/LDH levels and clinical data is a promising alternative approach for PcP diagnosis.

Providing preoperative information for children undergoing surgery: a randomized study testing different types of educational material to reduce children's preoperative worries.

This study developed three types of educational preoperative materials and examined their efficacy in preparing children for surgery by analysing children's preoperative worries and parental anxiety. The sample was recruited from three hospitals in Lisbon and consisted of 125 children, aged 8-12 years, scheduled to undergo outpatient surgery. The participants were randomly assigned to one of the seven independent conditions that were combined into the following three main groups: an experimental group, which received educational materials with information about surgery and hospitalization (a board game, a video or a booklet); a comparison group, which received entertaining material with the same format type; and a control group, which did not receive any material. Children's preoperative worries and parental anxiety were evaluated after the experimental manipulation. Children who received educational materials were significantly less worried about surgery and hospital procedures than children in the comparison and the control groups, although no statistically differences were found between the type of materials within the experimental group, and no significant effect occurred on parental state anxiety. These results do however support the hypothesis that providing preoperative materials with educational information reduce children's preoperative worries.

Assessing cognitive decline in the aging brain: lessons from rodent and human studies.

As life expectancy continues to increase worldwide, age-related dysfunction will largely impact our societies in the future. Aging is well established to promote the deterioration of cognitive function and is the primary risk factor for the development of prevalent neurological disorders. Even in the absence of dementia, age-related cognitive decline impacts specific types of memories and brain structures in humans and animal models. Despite this, preclinical and clinical studies that investigate age-related changes in brain physiology often use largely different methods, which hinders the translational potential of findings. This review seeks to integrate what is known about age-related changes in the brain with analogue cognitive tests used in humans and rodent studies, ranging from "pen and paper" tests to virtual-reality-based paradigms. Finally, we draw parallels between the behavior paradigms used in research compared to the enrollment into clinical trials that aim to study age-related cognitive decline.

'Cloudbuster': a Python-based open source application for three-dimensional reconstruction and quantification of stacked biological imaging samples.

Three-dimensional (3D) spheroid cultures are generating increasing interest in cancer research, e.g. for the evaluation of pharmacological effects of novel small molecule inhibitors. This is mainly due to the fact that such 3D structures reflect physiological characteristics of tumours and the cellular microenvironments they reside in more faithfully than two-dimensional (2D) cell cultures; in addition, they allow the reduction of animal experiments while providing significantly relevant human-based models. Quantification of such organoid structures as well as the mainly slice-based acquisition and thus forced 2D representation of 3D spheroids provide a challenge for the interpretation of the associated generated data. Here, we provide a novel open-source workflow to reconstruct a 3D entity from slice-recorded microscopical images with or without treatment with anti-migratory small molecule inhibitors. This reconstruction produces distinct point clouds as basis for subsequent comparison of basic readout parameters using average computer processor, memory and graphics resources within an acceptable time frame. We were able to validate the usefulness of this workflow using 3D data generated by various imaging techniques, including z-stacks from confocal microscopy and histochemically labelled spheroid sectioning, and demonstrate the possibility to accurately characterize inhibitor effects in great detail.

Clinical relevance of multiple single-nucleotide polymorphisms in Pneumocystis jirovecii Pneumonia: development of a multiplex PCR-single-base-extension methodology.

Pneumocystis jirovecii pneumonia (PcP) is a major cause of respiratory illness in patients with AIDS. The identification of multiple single-nucleotide polymorphisms (SNPs) at three distinct P. jirovecii loci encoding dihydrofolate reductase (DHFR), mitochondrial large-subunit rRNA (mtLSU rRNA), and superoxide dismutase (SOD) was achieved using multiplex-PCR (MPCR) followed by direct sequencing and two single-base extension (SBE) techniques. Four SNPs (DHFR312, mt85, SOD215, and SOD110), correlated previously with parameters of disease, were amplified and genotyped simultaneously. The concordance of results between the standard sequencing technique (direct sequencing) and SBE analysis was 96.9% for the acrylamide gel electrophoresis and 98.4% for the capillary electrophoresis. The cross-genetic analysis established several statistical associations among the SNPs studied: mt85C-SOD110T, SOD110T-SOD215C, and SOD110C-SOD215T. These results were confirmed by cluster analysis. Data showed that among the isolates with low to moderate parasite burden, the highest percentages of DHFR312C, mt85C, SOD110T, and SOD215C were detected, whereas for high parasite burden cases the highest frequencies were observed among isolates with DHFR312T, mt85T, SOD110C, and SOD215T. The polymorphisms studied were shown to be suitable genetic targets potentially correlated with PcP clinical data that can be used as predictors of outcome in further studies to help clinical decision-making in the management of PcP. The MPCR/SBE protocol described for the first time in the present study was shown to be a rapid, highly accurate method for genotyping P. jirovecii SNPs encoded by different loci that could be used for epidemiological studies and as an additional procedure for the prognostic classification and diagnosis of PcP.

Epidemiology and clinical relevance of Pneumocystis jirovecii Frenkel, 1976 dihydropteroate synthase gene mutations.

A review was conducted to examine the published works that studied the prevalence of Pneumocystis jirovecii dihydropteroate synthase (DHPS) mutations in patients with P. jirovecii pneumonia (PcP), in develop and developing countries, and that focused the problem of the possible association of these mutations with exposure to sulpha or sulphone drugs and their influence in the PcP outcome. Studies conducted in United States of America presented higher P. jirovecii mutations rates, in comparison with European countries, and in developing countries, lower rates of DHPS mutations were reported, due to limited use of sulpha drugs. A significant association was reported between the use of sulpha or sulphone agents for PcP prophylaxis in HIV-infected patients and the presence of DHPS mutations. However these mutations were also detected in PcP patients who were not currently receiving sulpha or sulphone agents. The outcome and mortality of HIV-infected patients with PcP harbouring DHPS gene mutations were related primarily to the underlying severity of illness and the initial severity of PcP, more than to the presence of mutations.

Genetic characterization of the UCS and Kex1 loci of Pneumocystis jirovecii.

Nucleotide variation in the Pneumocystis jirovecii upstream conserved sequence (UCS) and kexin-like serine protease (Kex1) loci was studied in pulmonary specimens from Portuguese HIV-positive patients. DNA was extracted and used for specific molecular sequence analysis. The number of UCS tandem repeats detected in 13 successfully sequenced isolates ranged from three (9 isolates, 69%) to four (4 isolates, 31%). A novel tandem repeat pattern and two novel polymorphisms were detected in the UCS region. For the Kex1 gene, the wild-type (24 isolates, 86%) was the most frequent sequence detected among the 28 sequenced isolates. Nevertheless, a nonsynonymous (1 isolate, 3%) and three synonymous (3 isolates, 11%) polymorphisms were detected and are described here for the first time.

CrATP as a new inhibitor of ecto-ATPases of trypanosomatids.

Trypanosomatid protozoa include heteroxenic species some of them pathogenic for men, animals and plants. Parasite membrane contains ecto-enzymes whose active sites face the external medium rather than the cytoplasm. Herpetomonas sp. displayed a Mg2+-dependent ecto-ATPase activity, a Mg-independent ecto-ADPase and an ecto-phosphatase activity. Both, the ecto-ADPase and phosphatase activities were insensitive to CrATP (chromium(III) adenosine 5'-triphosphate complex). Ecto-ATPase activity was reversibly inhibited. At 2 mm ATP the apparent Ki was 4 x 7+/-1 x 0 microm but a fraction of about 40-50% was insensitive to CrATP. Remarkably, at low substrate concentration (0 x 2 mm) more than 90% of the ecto-ATPase was inhibited with Ki=0 x 33+/-0 x 10 microm. These parameter dependences are interpreted as the presence of 2 ecto-ATPases activities, one of them with high ATP apparent affinity and sensitivity to CrATP. DIDS (4,4 diisothiocyanatostilbene 2,2' disulfonic acid), suramin and ADP were also effective as inhibitors. Only ADP presented no additive inhibition with CrATP. The pattern of partial inhibition by CrATP was also observed for the ecto-ATPase activities of Leishmania amazonensis, Trypanosoma cruzi and Trypanosoma rangeli. CrATP emerges as a new inhibitor of ecto-ATPases and as a tool for a better understanding of properties and role of ecto-ATPases in the biology of parasites.

Pneumocystis jirovecii multilocus genotyping profiles in patients from Portugal and Spain.

Pneumonia caused by the opportunistic organism Pneumocystis jirovecii is a clinically important infection affecting AIDS and other immunocompromised patients. The present study aimed to compare and characterise the frequency pattern of DNA sequences from the P. jirovecii mitochondrial large-subunit rRNA (mtLSU rRNA) gene, the dihydropteroate synthase (DHPS) gene and the internal transcribed spacer (ITS) regions of the nuclear rRNA operon in specimens from Lisbon (Portugal) and Seville (Spain). Total DNA was extracted and used for specific molecular sequence analysis of the three loci. In both populations, mtLSU rRNA gene analysis revealed an overall prevalence of genotype 1. In the Portuguese population, genotype 2 was the second most common, followed by genotype 3. Inversely, in the Spanish population, genotype 3 was the second most common, followed by genotype 2. The DHPS wild-type sequence was the genotype observed most frequently in both populations, and the DHPS genotype frequency pattern was identical to distribution patterns revealed in other European studies. ITS types showed a significant diversity in both populations because of the high sequence variability in these genomic regions. The most prevalent ITS type in the Portuguese population was Eg, followed by Cg. In contrast to other European studies, Bi was the most common ITS type in the Spanish samples, followed by Eg. A statistically significant association between mtLSU rRNA genotype 1 and ITS type Eg was revealed.

Serologic evidence for Schmallenberg virus circulation at high altitude, Central Portugal, 2015-2016.

A few years after the initial 2011 large scale Schmallenberg virus (SBV) epidemic that affected Europe, a subsequent decrease in new SBV infections was observed presumably associated with natural substitution of previously exposed animals. In the present work, a 2-year prospective serosurvey was performed to evaluate SBV circulation in a population of sheep living at high altitude in the central region of Portugal and with restricted movement. Blood from a representative sample of 168 autochthonous sheep was collected in 2015 and again in 2016, and tested for the presence of anti-SBV IgG by ELISA. Of the 2015 sample collection, seven animals tested positive for anti-SBV IgG, corresponding to a seroprevalence of 4.2% while of the 2016 sample collection, 10 presented SBV antibodies, showing a seroprevalence of 6.0% (p = 0.619). Results show that SBV is endemic in sheep of central Portugal, even in herds at high altitude locations. When comparing anti-SBV seroprevalences of 2015/2016 found in this study, to one detected in 2014 in the same region, a steep decrease could be observed (p < 0.001). This is in accordance with what has been documented in Western European countries, where a decrease in the number of SBV-infected sheep has been found, a fact which may pose a new threat for SBV re-emergence.

Temporal and spatial fluctuations of phytoplankton in a tropical coastal lagoon, southeast Brazil.

Spatial and temporal variability of the phytoplankton community in the tropical coastal Imboassica lagoon, an environment naturally isolated from the ocean by a narrow sandbar, was analysed every two weeks for 19 months by sampling three sites. During this study, the lagoon received direct input of marine water three times, resulting in remarkable salinity, nutrient concentrations and phytoplankton biomass variations in both temporal and spatial aspects. The phytoplankton biomass presented relatively low values ranging, on average, from 0.54 mg x L(-1) in the station closest to the sea (station 1) to 1.34 mg x L(-1) in the station close to a macrophyte bank (station 3). Diatoms and cryptomonads dominated in stations 1 and 2 (located relatively close to station 1, yet receiving the runoff of domestic sewage), and euglenoids, cryptomonads and dinoflagellates at station 3. Stations 1 and 2 usually presented the same dominant species but station 2 presented a higher phytoplankton biomass. On the other hand, station 3 showed more similar results concerning phytoplankton biomass with station 2, however the dominant species were usually different. The high fluctuations of salinity and the reduced nutrient availability are pointed out as the main factors structuring the dynamics of the phytoplankton community at the Imboassica lagoon.

Environmental changes and zooplankton temporal and spatial variation in a disturbed Brazilian coastal lagoon.

The Imboassica lagoon, located in the Municipality of Macaé (RJ), is separated from the sea by a sand bar, and its surroundings are partially occupied by residential areas. This coastal lagoon has undergone environmental degradation due to sewage input and artificial sand bar openings. The temporal and spatial variation of environmental variables and zooplankton were studied monthly for four years. There were five artificial openings of the sand bar during the period of study, mostly in the rainy season. Besides osmotic changes, these events caused the drainage of the water of the lagoon into the sea, loss of total organic nitrogen, and an increase of total phosphorus. The zooplankton community of Imboassica lagoon included freshwater and marine taxa, holoplanktonic, meroplanktonic and nectobenthonic forms. Polychaeta, Bivalvia and Gastropoda larvae, and the taxa of Rotifera Hexarthra spp., Lecane bulla, Synchaeta bicornis, nauplii of Cyclopoida and Calanoida copepods were considered constant taxa. Distinct zooplankton assemblages were found during zooplankton spatial surveys in oligohaline and mesohaline conditions. The successful zooplankton populations were either favored by the disturbance of the sand bar opening, such as the veligers of the gastropod Heleobia australis, or capable of fast recovery after the closing of the sand bar, during the succession from a marine into an oligohaline environment, such as Hexarthra spp.. Such populations seemed well adapted to the stress conditions usually found in the lagoon due to osmotic changes, column mixing, nutrient input, and high fish predation pressure. Rare species in the community, such as Moina minuta, presented population increases all over the lagoon under oligohaline conditions.

Bacterial growth and DOC consumption in a tropical coastal lagoon.

The aims of this research were to determine the main limiting nutrient to bacterial growth in Imboassica lagoon, southeastern Brazil, to estimate the percentage of dissolved organic carbon (DOC) available for bacterial growth, and to determine the bacterial growth efficiency (BGE) of natural assemblages. Bacterial growth and DOC consumption were determined in batch culture experiments, in which water samples were supplemented with nitrogen and phosphorus together or separately, or incubated without nutrient additions. When added together, N and P stimulated higher bacterial growth rates and production, as well as higher DOC consumption. The BGEs and DOC consumption rates were strongly dependent on the method used to determine bacterial production. The BGE ranged from 11 to 72%. However, only a minor fraction of bulk DOC was consumed by the planktonic bacteria (from 0.7 to 3.4%). The results suggest that low availability of phosphorus and nitrogen coupled with excess organic carbon was the main factor responsible for the relatively low bacterial utilization of DOC in Imboassica lagoon.

Serological diagnosis of pneumocystosis: production of a synthetic recombinant antigen for immunodetection of Pneumocystis jirovecii.

Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. Thus, the development of a serological test is urgently needed as it will allow the diagnosis of PcP using blood, an inexpensive and non-invasive specimen. This study aims to combine the production of a multi-epitope synthetic recombinant antigen (RSA) and an ELISA test for detection of anti-P. jirovecii antibodies, in order to develop a new approach for PcP diagnosis. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein. This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii (patent-pending no. PT109078). Serum specimens from 88 patients previously categorized in distinct clinical subgroups and 17 blood donors, were analysed. The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. This innovative approach, provides good insights about what can be done in the future serum testing for PcP diagnosis.

Assessment of Corneal Epithelial Thickness in Asymmetric Keratoconic Eyes and Normal Eyes Using Fourier Domain Optical Coherence Tomography.

Purpose. To compare the characteristics of asymmetric keratoconic eyes and normal eyes by Fourier domain optical coherence tomography (OCT) corneal mapping. Methods. Retrospective corneal and epithelial thickness OCT data for 74 patients were compared in three groups of eyes: keratoconic (n = 22) and normal fellow eyes (n = 22) in patients with asymmetric keratoconus and normal eyes (n = 104) in healthy subjects. Areas under the curve (AUC) of receiver operator characteristic (ROC) curves for each variable were compared across groups to indicate their discrimination capacity. Results. Three variables were found to differ significantly between fellow eyes and normal eyes (all p < 0.05): minimum corneal thickness, thinnest corneal point, and central corneal thickness. These variables combined showed a high discrimination power to differentiate fellow eyes from normal eyes indicated by an AUC of 0.840 (95% CI: 0.762-0.918). Conclusions. Our findings indicate that topographically normal fellow eyes in patients with very asymmetric keratoconus differ from the eyes of healthy individuals in terms of their corneal epithelial and pachymetry maps. This type of information could be useful for an early diagnosis of keratoconus in topographically normal eyes.

Multicentre study highlighting clinical relevance of new high-throughput methodologies in molecular epidemiology of Pneumocystis jirovecii pneumonia.

Pneumocystis jirovecii causes severe interstitial pneumonia (PcP) in immunosuppressed patients. This multicentre study assessed the distribution frequencies of epidemiologically relevant genetic markers of P. jirovecii in different geographic populations from Portugal, the USA, Spain, Cuba and Mozambique, and the relationship between the molecular data and the geographical and clinical information, based on a multifactorial approach. The high-throughput typing strategy for P. jirovecii characterization consisted of DNA pooling using quantitative real-time PCR followed by multiplex-PCR/single base extension. The frequencies of relevant P. jirovecii single nucleotide polymorphisms (mt85, SOD110, SOD215, DHFR312, DHPS165 and DHPS171) encoded at four loci were estimated in ten DNA pooled samples representing a total of 182 individual samples. Putative multilocus genotypes of P. jirovecii were shown to be clustered due to geographic differences but were also dependent on clinical characteristics of the populations studied. The haplotype DHFR312T/SOD110C/SOD215T was associated with severe AIDS-related PcP and high P. jirovecii burdens. The frequencies of this genetic variant of P. jirovecii were significantly higher in patients with AIDS-related PcP from Portugal and the USA than in the colonized patients from Portugal, and Spain, and children infected with P. jirovecii from Cuba or Mozambique, highlighting the importance of this haplotype, apparently associated with the severity of the disease and specific clinical groups. Patients from the USA and Mozambique showed higher rates of DHPS mutants, which may suggest the circulation of P. jirovecii organisms potentially related with trimethoprim-sulfamethoxazole resistance in those geographical regions. This report assessed the worldwide distribution of P. jirovecii haplotypes and their epidemiological impact in distinct geographic and clinical populations.