Principal coordinates analysis of sequence diversity between chronic Chagas Disease patient TcGP63I antigenic repertoires.

<p>Genetic distances are based on a weighted unifrac metric. Plot A shows diversity comparisons among Go-as asymptomatic (asympt) and symptomatic (sympt) clinical cases, as well as one acute case. Plot B shows Goias cases with symptoms categorised as acute, card (cardiopathy), card + mega (cardiopathy as well as megacolon and / or megaesophagous), mega (megacolon and / or megaesophagous) or asympt (asymptomatic). Plot C shows comparisons among Cochabamba clinical cases (not including congenital cases) classified as either asymptomatic (asympt) and symptomatic (sympt). The dashed circle on plot C indicates samples unambiguously defined as TcI at the ND5 locus. Pairs of sequential isolates from the same patient are labelled x and y respectively.</p

Alpha diversity indices for TcGP63I amplicon diversity derived from pairs of congenital Chagas disease cases.

<p>Diversity indices were derived from STs defined at 99% sequence similarity. Bar plot and associated <i>x</i>-axis on the right hand side shows the Shannon diversity index calculated in Mothur [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003458#pntd.0003458.ref034" target="_blank">34</a>], with error bars defining upper and lower 95% confidence intervals.</p

Samples provenance and symptoms.

<p>^a Samples from Goias congenital case</p><p>^x Samples from the same patient taken >12 months apart</p><p>^y Samples from the same patient taken < 6 months apart</p><p>^z Samples taken from the same patient >12 months apart</p><p>Samples provenance and symptoms.</p

Yang and Neilson estimates for positive selection within and among abundant 97% STs identified in this study.

<p>^a Numbers in brackets represent the number of 99% STs define within each cluster from which estimates were generated.</p><p>^b P values are give for Fisher’s exact tests for deviation from the neutral expectation of Ka/Ks = 0.</p><p>Yang and Neilson estimates for positive selection within and among abundant 97% STs identified in this study.</p

Bar plot showing sequence type identity and abundance defined at 97% similarity for the ND5 locus across all samples.

<p>A—Goias cohort chronic/intermediate cases; B—Cochabamba chronic/intermediate cases; C—Cochabamba congenital cases. Y axes show log transformed abundance (read counts). X axes show clustered bars for individual samples. Sequence type identities are given in the legend. Stars denote congenital pair from Goias. Labels x (6416 / 6452), y (6401 / 6536) and z (6379 / 6445) sample pairs from the same patient at different time points (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003458#pntd.0003458.t001" target="_blank">Table 1</a>).</p

Geographical distribution of antibody responses to lineage-specific synthetic peptides, as determined by ELISA.

<p>EHC =  Endemic healthy controls (* a further 17 Colombian sera that were serologically negative with the lysate were included in the peptide ELISAs as additional controls); ND =  not determined.</p>a<p>these 98 comprised 1 sample from each of 90 patients, plus 2 paired samples from each of 4 patients. All eight paired samples reacted with TSSApep-II/V/VI, and are included within the 67 Brazilian reactors to this peptide. 1 set of these pairs also reacted with TSSApep-V/VI.</p>b<p>same sample, which did not react with TSSApep-II/V/VI, TSSApep-V/VI or chimeras.</p>c<p>these 9 samples also reacted with chimera TSSA-II/-I peptide.</p>d<p>same sample, which did not react with TSSApep-II/V/VI, TSSApep-V/VI or chimeras.</p>e<p>same sample.</p>f<p>same sample, which did not react with TSSApep-I, TSSApep-II/V/VI, TSSApep-V/VI or chimera TSSApep-II/-I.</p>g<p>non-specific binding; see text.</p>h<p>in each case the same sample reacted with TSSApep-III and TSSApep-IV.</p

TSSA provides potential epitopes that are <i>T. cruzi</i> lineage-specific.

<p>[A] Components of the peptides synthesised: N-terminal biotinylation; PEG spacer; Gly; the lineage-specific sequence; C-terminal Cys. [B] Amino acid sequences of the <i>T. cruzi</i> lineage-specific TSSA potential epitopes in the synthetic peptides (TSSApep-), with polymorphic residues underlined; for the two chimeric peptides the TSSA-II residues are shown in bold.</p

<i>T. cruzi</i> strains used here for comparative analysis of the ORF containing the reported TcI-applicable peptide (GenBank accession numbers refer to sequences determined here).

a.<p> =  no amino acid change.</p

Chagasic sera recognise TSSA lineage-specific peptides.

<p>Lineage-specific peptides or lysate were added to rows of the ELISA plate as indicated. ELISA plates showing: recognition of TSSApep-II/V/VI and TSSApep-V/VI, among Brazilian, Ecuadorean and Argentine sera; recognition of TSSApep-I and TSSApep-IV by an Argentine serum and a Venezuelan serum; recognition of chimera TSSApep-II/I by Argentine sera. All patients were seropositive with <i>T. cruzi</i> lysate. NEHC =  non-endemic healthy control.</p

Computer-predicted antigenicity score is much higher for TSSA-II/V/VI sequence than TSSA-I.

<p>Polymorphic sequences of [A] TSSA-I and [B] TSSA-II/V/VI showing regions of high antigenicity in red, and low antigenicity in blue. A few amino acid replacements in TSSA-I lead to disappearance of the immunogenic epitope that is present in TSSA-II/V/VI sequence.</p