Chlamydia trachomatisdiversity viewed as a tissue-specific coevolutionary arms race
© Nunes et al.; licensee BioMed Central Ltd. 2008
Received: 28 July 2008
Accepted: 23 October 2008
Published: 23 October 2008
The genomes of pathogens are thought to have evolved under selective pressure provided by the host in a coevolutionary arms race (the 'Red Queen's Hypothesis'). Traditionally, adaptation by pathogens is thought to rely not on whole chromosome dynamics but on gain/loss of specific genes, yielding differential abilities to infect distinct tissues. Thus, it is not known whether distinct host organs differently shape the genome of the same pathogen. We tested this hypothesis using Chlamydia trachomatis as model species, looking at 15 serovars that infect different organs: eyes, genitalia and lymph nodes.
We analyzed over 51,000 base pairs from all serovars using various phylogenetic approaches and a non-phylogenetic indel-based algorithm to study the evolution of individual and concatenated loci. This survey comprised about 33% of all single nucleotide polymorphisms in C. trachomatis chromosomes. We present a model in which genome evolution indeed correlates with the cell type (epithelial versus lymph cells) and organ (eyes versus genitalia) that a serovar infects, illustrating an adaptation to physiologically distinct niches, and discarding genetic drift as the dominant evolutionary driving force. We show that radiation of serovars occurred primarily by accumulation of single nucleotide polymorphisms in intergenomic regions, housekeeping genes, and genes encoding hypothetical and cell envelope proteins. Furthermore, serovar evolution also correlates with ecological success, as the two most successful serovars showed a parallel evolution.
We identified a single nucleotide polymorphism-based tissue-specific arms race for strains in the same species, reflecting global chromosomal dynamics. Studying such tissue-specific arms race scenarios is crucial for understanding pathogen-host interactions during the course of infectious diseases, in order to dissect pathogen biology and develop preventive and therapeutic strategies.
When two species interact with each other, such as a pathogen and human, a never-ending reciprocal and dynamic adaptation process takes place. Whereas the 'goal' of the human being is to try to avoid, solve or minimize the infection, the 'goal' of the pathogen is to deal with this constant host environmental and immune pressure, through genomic evolutionary changes, in order to win this arms race [1–4]. Typically, genome evolution within same-species strains of a pathogen has been studied mainly in the light of horizontal gene transfer (HGT) at specific chromosome loci [5, 6], as for Escherichia coli [7, 8], Staphylococcus aureus , Streptococcus pyogenes , Salmonella enterica , Shigella flexneri , and Pseudomonas syringae . An extreme example is provided by the well-studied E. coli, where strains K-12 and O157 differ by more than 1 million base pairs , and same-serovar strains were found to present profound differences in gene content [13, 14]. Globally, these targeted HGT events reflect different pathoadaptation processes for microrganisms with reversible genome size-plasticity; depending on the transitory 'cassette-genes' carried at any specific time, the pathogenecity or ability of these microrganisms to infect different tissues may vary . Thus, generally, these processes rely on gain/loss of virulence/colonization factors rather than reflect whole chromosomal dynamics, the evaluation of which remains complex. Indeed, assessment of tissue-specific adaptive evolution at the whole genome level demands that same-species strains of a pathogen specifically and non-transitorily infect different tissues. Therefore, on behalf of the arms race theory assumed by the evolutionary Red Queen's Hypothesis [15, 16], one question arises: do distinct host organs differently shape the genome of the same pathogen? No microrganism is more suitable than Chlamydia trachomatis, the most prevalent sexually transmitted bacterial pathogen worldwide, to test this hypothesis, as the species comprises several serovars with a wide range of specific human tissue tropism. This pathogen is mainly classified into 15 serovars based on the differential immunoreactivity of the major outer membrane protein (MOMP), constituting three disease groups : serovars A-C and Ba are commonly associated with ocular trachoma; serovars D-K infect the epithelial cells of genitalia and are normally found in non-invasive sexually transmitted infections (where serovar E represents about one-third of all infections, and together with serovar F constitute up to 50% of them); serovars L1-L3 are also sexually transmitted but are invasive and disseminate into the local lymph nodes causing lymphogranuloma venereum (LGV). However, in the context of this classification system, the evaluation of adaptive evolution becomes enigmatic because there is no correlation between it and C. trachomatis tropism nor with the ecological success of the different serovars, as strains with different organ specificities are placed within the same classification group.
As occurred for Mycobacterium leprae , Rickettsia prowazekii , and the aphid endosymbiont Buchnera aphidicola , the first stages of Chlamydia evolution consisted of a massive genome reduction upon becoming an obligate intracellular parasite [21, 22]. However, comparative genomics over the few currently fully sequenced C. trachomatis genomes [20, 23–25] revealed that gene decay is not involved in the more recent evolutionary stages. Indeed, contrary to most pathogens, the core- and the pan-genome  of this microrganism are near identical, indicating that the factors involved in the differential organ specificity among serovars are not acquired by gene transfer .
To evaluate if distinct arms races occur between different infected human organs and this pathogen's serovars, we performed high-scale concatenation-based phylogenomics, using about one-third of all chromosome single nucleotide polymorphisms (SNPs). So far, in contrast to the ocular group, only one strain from the epithelial-genital and LGV groups has been fully sequenced [20, 23–25], making our multiple-loci scrutiny of all 15 serovars the ideal tool to track the evolutionary diversity of a microrganism characterized for its distinct infection niches. Here, we show a matchless model of SNP-based adaptive evolution of same-species strains to each infected cell-type and organ that relies on whole chromosome evolutionary dynamics, unlike previous reports for other pathogens focused on specific gene gain/loss.
Evaluation of the degree of polymorphism for the selected loci
Additionally, a global overview of GC content revealed a mean value for all loci categories (data not shown) that is similar to the total mean GC content of approximately 41% observed for the fully sequenced genomes [21, 23–25] with a standard deviation of 2.9%, which is not indicative of any putative HGT event.
Correlation of individual loci with tissue-specific strain radiation
Genomic analysis of the concatenated loci
Genetic polymorphism for the concatenated sequences
Parsimony informative sites†
Overall mean distance (nucleotides)
65.8 (SE 4.7)
96.4 (SE 5.3)
168.1 (SE 7.6)
701.9 (SE 15.6)
1,032.1 (SE 17.2)
Overall mean p-distance (nucleotides)
0.0172 (SE 0.0013)
0.0121 (SE 0.0007)
0.0430 (SE 0.0020)
0.0199 (SE 0.0004)
0.0202 (SE 0.0003)
Evolutionary history of C. trachomatis
Overall mean genetic distances within and between disease groups
Genetic distance (%)*
133 (SE 10)
0.27 (SE 0.02)
Genital (without E/F)
309 (SE 14)
0.63 (SE 0.03)
Genital (with E/F)
460 (SE 13)
0.93 (SE 0.03)
69 (SE 8)
0.14 (SE 0.02)
Ocular/Genital (without E/F)
942 (SE 21)
1.91 (SE 0.04)
Ocular/Genital (with E/F)
983 (SE 20)
2.00 (SE 0.04)
1,484 (SE 42)
3.02 (SE 0.08)
LGV/Genital (without E/F)
1,241 (SE 36)
2.52 (SE 0.07)
LGV/Genital (with E/F)
1,226 (SE 34)
2.49 (SE 0.07)
673 (SE 16)
1.37 (SE 0.03)
We also highlight the loci that most contribute to the final tree topology (Figure 4), as they may be relevant for the evolutionary adaptation to each specific niche. Among these loci, we have found either highly conserved or polymorphic loci for strains infecting the same cell-type/organ. The former may represent a step forward in the evolutionary process by revealing the final stages  of this tissue-specific adaptive evolution, while the latter may also be involved in pathogenic differences between strains infecting the same tissue . The most extreme case is given by the CEP pmpF, where all the strains that infect the lymph nodes are 100% similar but show a mean distance of 312 and 421 SNPs to strains infecting the epithelial-genital and ocular tissues, respectively. In contrast, the epithelial-genital strains reveal up to 129 SNPs among them (data not shown). Although less markedly, CT049 is polymorphic among the LGV strains but near 100% identical among the ocular strains.
Polymorphic loci among strains that infect the same biological niche
Impact of small insertions/deletions (indels) on tissue-specific strain radiation
In order to have a more complete picture of the evolution of the serovars, we studied the chromosomal occurrence of small insertion/deletion (indel) events, which are non-phylogenetic parameters. We observed 84 small indel events (from 1-43 bp) inside the global concatenated loci for all strains, which mainly occurred within the IGR and CEP categories (Additional data file 4). None of these events was found to disrupt the coding sequence of the respective loci, indicating the absence of gene decay in the studied regions.
Evolutionary inferences on the ecological success
Analysis of the global phylogenetic tree (Figure 4) also shows that the two most prevalent genital serovars worldwide, E and F, are closely related and separated from the other epithelial-genital strains. This segregation is observed for the majority of loci, with the exception of the HPs (Figure 2a). From all these loci, 70% of CEPs show an amino acid replacement for >50% of SNPs, compared to only 20% of HKs (Figure 2b). Curiously, the most remarkable segregation of E and F was seen for two IGRs (rs2/ompA and yfh0_1/parB) and three HKs (karG, tsf and rs2) (Figure 4). Furthermore, for the still unclassified protein gene CT622 and for the IGR rs2/ompA, we observed a non-random distribution of SNPs that are present in serovars E and F but not in the other epithelial-genital strains (Figure 3c,d). Finally, the mean γ-distance from any epithelial-genital strain to serovar E or F was from 3.4-fold (between G and E/F) to 4.7-fold (between I and E/F) higher than the distance between E and F (Figure 5b), which supports this close relationship between the two most ecologically successful serovars.
We have hypothesized that distinct arms races may occur inside the same host when the same pathogen is able to infect different organs. In contrast to free living bacteria, where HGT is strongly associated with a pathogen's adaptive evolution [3, 5–11], Chlamydia has been characterized by genetic isolation and, while cumulative studies suggest that HGT has almost certainly occurred in Chlamydiaceae [35–37], there is no report to date of transferable mobile elements in C. trachomatis. Here, we demonstrate that C. trachomatis strains that preferentially infect the eyes, the epithelial-genital cells or the lymph nodes present a distinct evolutionary pattern likely illustrating a SNP-based tissue-specific arms race.
In order to develop a more compelling argument for a causal link between genome profile and cell/organ appetence, the use of genetic modification and especially the use of animal models are appealing approaches. However, C. trachomatis is genetically non-tractable and, except for the cynomolgus monkey (accurate for studying the trachoma pathology) , no suitable animal model exists for the three types of C. trachomatis disease. Also, there is no in vitro model, such as cell culture, that mirrors the chlamydial infection in vivo, and it has been previously demonstrated that intensive serial passaging of chlamydial strains yielded no mutations on the most variable chlamydial gene (ompA) . Furthermore, it would be inconceivable that these approaches could represent millions of years of chlamydial evolution.
It is believed that the LGV biovar was the first to diverge from a common C. trachomatis ancestor when new primate hosts evolved after the dinosaur extinction, whereas separation of genital and ocular serovars might have occurred with the appearance of early humanoid primate hosts . The skill to colonize different organs and cell-types likely developed through indel events and SNP accumulation on virulence/colonization factors. So far, chlamydial putative virulence factors, such as the type III effector tarp , the cytotoxin gene , and especially the tryptophan operon [40, 41], are the best candidates for providing that skill. In particular, while the first of these factors differentiates the LGV strains from the other groups, the other two differentiate the strains colonizing the genitalia from the strains colonizing other niches. For example, it was clearly demonstrated that only strains possessing a functional trpBA operon are able to colonize the genital tract . With respect to type III effectors, although their role in C. trachomatis tropism is not clear, it was shown that evolutionary genetic diversification of the type III effector HopZ family, via horizontal transfer, had clear implications for Pseudomonas syringae host specificity . However, none of the chlamydial putative virulence factors fully explain the existence of the three major tropism groups made up from the different serovars. Also, the putative emergence of tissue-specific adhesins cannot be discarded.
With regard to our results (Figure 4), strain radiation within each disease group likely occurred because of accumulation of mutations throughout the chromosome caused by environmental and immune pressure in each niche, giving rise to the contemporary serovars. Within the genitalia, the higher serovar multiplicity and radiation of epithelial-genital strains compared to the LGV strains would be unexpected in the light of the earlier evolutionary divergence of the latter . However, besides the different host immune responses in those niches, the epithelial-genitalia environment presents pH and hormonal fluctuations that are variable among individuals, and also an abundant nutrient-competing flora, which could have strongly influenced the evolutionary pathway of the infecting strains. In support of this, nutrient-competing flora were shown to be a major factor in the successful pathoadaptation of Salmonella enterica serovar Typhimurium to the intestinal tract, as the inflammatory process induced by this pathogen was shown to make a negative impact on mainly the other colonizing microrganisms and, thus, a positive impact on its arms race with the host .
Globally, we have observed that the loci that most contribute to strain segregation by cell-type/organ are spread throughout the chromosome (Figure 1) and belong to different functional categories, suggesting that this dynamic evolutionary adaptation is a general trait of the entire genome. Whereas the contribution of CEPs is likely associated with putative structural, antigenic or host-adhesion roles, no assumption can be made for the HPs. However, we found that HPs were the most variable among the serovars, with an overall polymorphism 2.2-fold higher than the CEPs (Table 1), which suggests a higher involvement in chromosomal dynamics. With respect to IGRs, we speculate that their contribution to strain segregation may be associated with recombination events that may promote genetic variability, as we recently described . Nevertheless, the high variability of IGRs was surprising, as they commonly involve regulatory regions that are expected to be conserved; thus, the existence of random genetic drift may also be considered for IGRs. Finally, although the HKs are involved in strain segregation, the vast majority of them showed <50% non-synonymous mutations (Figure 2b), which is consistent with their role in essential biological functions.
It is known that in populations without HGT and with bottlenecks, as is the case for C. trachomatis, random genetic drift can play a major role in evolution, being responsible for the fixation of unfavorable mutations . However, our results suggest that chlamydial strain segregation according to tropism properties occurred mainly through an adaptive evolutionary process and not through dominant genetic drift. Several arguments point in this direction: the statistical association found between most polymorphic loci (number of SNPs/loci and p-distance/loci) and the strain clustering according to their tissue specificity; Chlamydiae presents a relatively high ratio of non-synonymous to synonymous changes when compared, for example, to E. coli and Buchnera , further supported by our findings where the majority of HPs and CEPs involved in the segregation of the LGV strains showed >50% non-synonymous SNPs (Figure 2b); for at least eight loci (CT049, CT144, CT622, pmpE, pmpF, pmpH, rs2/ompA IGR and ompA/pbpB IGR), we observed a non-random fixation of SNPs exclusive of same niche-infecting strains (Figure 3), corresponding to specific clusters of amino acid changes in coding sequences; the extremely robust global phylogenetic tree with maximum bootstrap support (99-100%) in the branch nodes where strains are separated by their cell-type/organ specificity (Figure 4); 20 out of the 22 loci that contribute to the segregation of strains that preferentially infect the eyes are also involved in the segregation of strains that colonize the lymph nodes (Figure 4) by presenting a dissimilar and specific SNP pattern; and finally, the well-known differences in environmental and immune pressure as well as competing flora and physiological specificities between ocular, epithelial-genital and lymph node tissues.
Within all the loci that are more likely to be involved in the adaptive evolution to each specific niche, we have found either highly conserved or polymorphic loci among strains infecting the same cell-type/organ (Figure 4), where the most remarkable examples are pmpF and CT049 (see Results). We hypothesize that pmpF and CT049 may be good representatives of a final stage of the adaptive evolution to the lymph nodes and the eyes, respectively, considering their extreme conservation among the corresponding strains. On the other hand, these genes may be responsible for pathogenic differences among epithelial-genital and LGV strains, respectively, based on their strong polymorphism among the corresponding strains. While PmpF has been implicated as a potential target for the host immune response, as it contains several putative major histocompatibility epitopes , biological information for CT049 is lacking.
Additionally, we found several loci that are polymorphic among strains infecting the same cell-type/organ that seem not to have been involved in the adaptation to each niche, but which may have been involved in the pathogenesis of trachoma, genital infections or LGV disease (Table 3). Indeed, 4 of these loci (CT622, tsf, rs2 and pbpB) belong to a pool of 22 genes that are responsible for profound differences in virulence among two C. trachomatis ocular strains in nonhuman primates .
Interestingly, we also observed a clear evolutionary co-segregation of the two most ecologically successful serovars (E and F). This is intriguing as there is a 15% difference between them in the gene coding for the major antigen (the major outer membrane protein (MOMP)), which constitutes about 60% of the membrane dry-weight  and is a putative cytoadhesin . Although it is not known why serovars E and F are the most prevalent worldwide, their ecological success seems not to be associated with intracellular multiplication rate , indicating that it is likely defined at the host cell adhesion and entry steps. However, the existence of E/F specific virulence factors or adhesins cannot be addressed in this study. Even so, tarp is the unique virulence factor that distinguishes serovar E from the other epithelial-genital serovars (including F), as it presents fewer repeat motifs in the 5' region , but its phenotypic consequences are not known. Moreover, a more successful host immune evasion could also be speculated for serovars E and F considering the well-known different antigenic profile among epithelial-genital serovars .
Regarding the loci that most markedly contribute to the segregation of serovars E and F, we highlight the IGRs tsf, rs2 and rs2/ompA (Figure 4). The first two of these may be involved in hypothetical differences in strain growth , while the last involves the regulatory region of rs2. This IGR includes specific domains where most SNPs are exclusive of strains E and F (Figure 3d), suggesting a potential impact on the rs2 regulation and, thus, on strain growth. Also, the IGR rs2/ompA is a recombination hotspot for the generation of mosaic structures within chlamydial strains , suggesting that recombination may contribute to the ecological success of the two serovars. However, as most SNPs of the CEPs involved in the E/F segregation confer amino acid replacements (Figure 2b), we suggest that the positive selection for the membrane proteins may also be a driving force for the E/F evolutionary divergence, likely through antigenic variability.
It is not surprising that bacterial populations that evolved in different ecological niches have different profiles of genetic variability. However, contrary to all previous reports for other pathogens focused on HGT events and gene decay, we present evidence of SNP-based, tissue-specific evolutionary adaptation relying on whole chromosome dynamics, as a consequence of the occurrence of dissimilar arms races between the pathogen and diverse host organs. Answering the proverbial question of 'which came first' (tropism or SNPs), the scenario presented here suggests that while some SNPs, on very few and specific loci, are likely responsible for tropism differences, the vast majority of SNPs throughout the chromosome are a consequence of different tissue tropisms and are expected to be involved in maintaining organ appetence, as per the Red Queen's Hypothesis. Mirroring bacterial virulence , we present evidence that a 'one size fits all' approach cannot be applied to adaptive evolution. This phenomenon is illustrated by a pathogen believed to infect 140 million people, where the incidence rate can be as high as 30% among adolescent females . We believe that grasping a pathogen's genetic trends with regard to its interaction with the host will be an essential tool in deciphering the molecular genetic aspects of infectious diseases.
Materials and methods
Culture of C. trachomatisreference strains
We used the most common reference strains representing the 15 C. trachomatis serovars: A/Har13, B/TW5, Ba/Apache2, C/TW3, D/UW3, E/Bour, F/IC-Cal3, G/UW57, H/UW4, I/UW12, J/UW36, K/UW31, L1/440, L2/434 and L3/404. McCoy cell culture of all strains plated in T-25 cm2 flasks was performed as previously described . At 48-72 h post-infection, elementary bodies were harvested, and DNA was extracted using QIAamp® DNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Serovar confirmation of each reference strain was performed using ompA genotyping with BLAST comparison of the available GenBank sequences.
Selection of loci
A GenBank search was performed to look for genomic regions that had been sequenced for at least one C. trachomatis reference strain from each of the three disease groups. Up to 93 loci were found, comprising about 84,000 bp of the chromosome, and involving IGRs, HKs, HPs and CEPs. Only non-constant loci were selected (51 of the 93; Figure 1; Additional data file 2) for sequencing the other reference strains if their sequences were not available yet. Automated sequencing was performed as previously described . The DNA sequence data have been deposited in a public database ([GenBank: EU239694-EU239702], [GenBank:EU239705-EU239712], and [GenBank:EU247618-EU247753]). Primer sequences are given in Additional data file 5. For all strains, five types of concatenated sequences were created in a head-to-tail fashion: one for each loci category (IGRs, HKs, HPs and CEPs) and a global concatenated sequence involving all loci (approximately 50,000 bp for each taxon).
Contingency table for estimating polymorphism significance
For all individual loci and concatenated sequences, alignments of all strains were generated using LaserGene (DNASTAR, Madison, WI, USA) and MEGA 3.1 . MEGA 3.1 was also used to create matrices of pairwise comparisons and to estimate the number of variable sites, the number of parsimony informative sites and overall mean genetic distances. The pairwise-deletion option was chosen to remove all sites containing missing data or alignment gaps from all distance estimations, only when the need arose and not prior to the analysis.
In order to search for distinct regions that may be associated with strains belonging to a specific disease group, SimPlot 3.5.1  was used on all 51 loci. For each similarity plot, serovars were grouped according to the cell-type/organ that they infect, and nucleotide pairwise distances were calculated using the K2P method (gaps excluded; ts/tv of 2.0) in a sliding window size of 160 bp moved across the alignment in a step size of 10 bp. Additionally, for all loci where serovars E and F clustered apart from the other epithelial-genital serovars was observed, a SimPlot analysis was also performed to evaluate if the E/F nucleotide differences compared to the other genital serovars were clustered in specific domains of each locus.
Prior to the phylogenetic reconstructions, and in order to select the appropriate evolutionary models, we evaluated the homogeneity of substitution patterns between sequences by calculating the Monte Carlo test-based Disparity Index per site . This gives the probability of rejecting the null hypothesis that sequences have evolved with the same pattern of substitution. The NJ method  was used with K2P , Jukes-Cantor  and Tamura-Nei  models to generate phylogenies. For the concatenated sequences, in order to examine the accuracy of the major conclusions reached from the NJ analysis, trees were also constructed under the maximum parsimony criterion , using the max-min branch-and-bound algorithm.
Considering that recombination disturbs a phylogenetic signal since the two parts of the recombined region may have different evolutionary histories [61, 62], one locus (ompA) was excluded from the phylogenetic concatenated analysis, as its highly recombinant nature has already been demonstrated [48, 63]. The use of outgroup sequences was discarded in the present study because no rooted trees were needed to achieve the objectives defined above. Also, the most suitable strain for use as an outgroup, C. muridarum (MoPn strain), has several loci that vary greatly in size and diverge from those in the C. trachomatis strains, which would entail the removal of a huge portion of the sequences being analyzed.
We used a non-phylogenetic method for estimating the evolutionary distance between each pair of homologous DNA sequences, which is given by the parameter γ :
γ = -2logeP
P = n xy /√n x n y
where n xy is the number of nucleotides shared by the two sequences, and n x and n y are the number of nucleotides of each sequence. For comparative purposes, we used the same set of loci as for the phylogenetic concatenated analyses, that is, we excluded the recombinant ompA gene. The γ variability was estimated by Monte Carlo using the alignments of each individual locus through the statistical platform R 2.5.1 . Each time, 20 loci were randomly selected with replacement and γ-distances were calculated by repeating this procedure 50 times.
The statistical association between genetic and phylogenetic variables was performed using the ANOVA test by comparing groups' population means. We considered as genetic variables the overall mean values of percent GC content, p-distance and absolute SNPs obtained for each of the selected loci. The phylogenetic variables were: clustering of strains according to tropism properties; co-segregation of E/F strains; segregation of a LGV cluster or an ocular cluster; and the 'weight' of each locus in the final concatenated tree. The homogeneity of variances was tested using the Levene's test. Whenever the hypothesis of homogeneity of variances was rejected, the non-parametric Mann-Whitney test was used to compare distributions among groups. A P-value of 0.05 or less was considered significant.
Additional data files
The following additional data are available with the online version of this paper. Additional data file 1 is a figure showing the overall mean genetic distances among all 15 C. trachomatis serovars for the 51 loci. Additional data file 2 is a table listing the cellular roles of the 51 loci. Additional data file 3 is a figure showing C. trachomatis's evolutionary history by loci category. Additional data file 4 is a table listing the indel events found among the 15 C. trachomatis serovars for all loci. Additional data file 5 is a table listing the primers used for PCR and sequencing of selected loci.
cell envelope protein gene
horizontal gene transfer
hypothetical or unclassified protein gene
single nucleotide polymorphism.
We would like to thank Dr Brendan Wren and Dr Bush for useful comments, critical discussions and reading of the manuscript prior to publication. This work was supported by grants from Fundação para a Ciência e a Tecnologia (FCT) (PTDC/BIA-BCM/71117/2006) and Comissão de Fomento da Investigação em Cuidados de Saúde (n°112/2007) to JPG. AN is the recipient of a PhD Grant (SFRH/BD/25651/2005) from FCT.
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