Genotyping of resistant strains isolated from patients treated with the different antimalarial drug combinations

<p><b>Copyright information:</b></p><p>Taken from "Efficacy and tolerability of four antimalarial combinations in the treatment of uncomplicated malaria in Senegal"</p><p>http://www.malariajournal.com/content/6/1/80</p><p>Malaria Journal 2007;6():80-80.</p><p>Published online 14 Jun 2007</p><p>PMCID:PMC1919387.</p><p></p

The endonuclease activity of ZAM integrase correlates with the presence of a 388 bp fragment from the <i>white</i> locus.

<p>A) Schematic representation of the ZAM integrase “IN” and a carboxy-terminal deleted integrase “ΔIN” used in the <i>in vitro</i> DNA binding assay. The three main domains: the zing finger “HHCC”, the catalytic domain “DDE” and a predictive DNA binding domain “BD” are represented. Nucleotide numbers according to ZAM sequence are indicated below. The hatched box indicated the region deleted to generate the ΔIN protein. The full length and the truncated integrases were expressed in bacteria as GST fusion proteins and fixed on agarose beads. IN and ΔIN purified proteins were analysed on SDS-PAGE electrophoresis followed by coomassie staining (right panel). The molecular masses of proteins are indicated in kilodalton. B) Circular representation of the 2686 bp pUC18 plasmid. Palindromic sequences CGCGCG present in pUC18 are indicated. The Drosophila genomic locus known to be the target of ZAM integration and located 3 kb upstream of the <i>white</i> gene is presented above. The <i>white</i> fragment (from positions 4278 to 4666 according to the drosophila sequence) was cloned in the pUC18 plasmid and is represented by the grey box. The black dot at position 4314 indicates the CGCGCG integration site of ZAM. C) <i>In vitro</i> endonuclease activity of ZAM integrase: pUC, pUC/white and pUC/white1mut plasmids were incubated without (−IN) or with (+IN) purified ZAM-IN. Positions of the supercoiled, nicked (circle) and linear (bar) DNAs are indicated.</p

LTR binding property of ZAM integrase.

<p>A) ZAM LTR fragments. The grey box represents the full length LTR of ZAM and the solid bars represent the KpnI “K” and HindIII “H” restriction sites at the position 21 and 82, respectively. A full length LTR and truncated PCR product deleted of the first 40 bp of ZAM LTR called “ΔLTR” are presented below. A double stranded oligonucleotide spanning from position 28 to 40 and called “BS” was also used in these experiments. B) <i>In vitro</i> DNA binding assays with ZAM-IN on the LTR fragments. <u>Left panel:</u> The full length “LTR” and the two truncated LTR fragments digested by KpnI “K” or HindIII “H” were tested as indicated above each lane. <u>Middle panel:</u> the full length LTR and a truncated PCR product “ΔLTR” were used in these assays. <u>Right panel: </u><i>In vitro</i> binding assays with ΔIN on ZAM LTR fragments: the full length “LTR” and the two truncated LTR fragments digested by KpnI “K” or HindIII “H” were tested as indicated above each lane. C) <i>In vitro</i> DNA binding assays performed with a double stranded oligonucleotide from base 28 to 40 according to ZAM sequence. The 13 bp fragment is retained by ZAM-IN. DNA fragments sizes are indicated as “L”. DNA fragments sizes are indicated for each panel. In B and C, bound and unbound fractions are presented in upper and lower panels respectively. The percentage of bound and unbound fractions is presented below each panel in B. In C, 100% of the 274 bp fragment was recovered in the unbound fraction whereas 100% of the 66 and 48 bp fragments were recovered in the bound fraction.</p

ZAM Integrase interacts with specific genomic DNA sequences.

<p>A) Diagram of the <i>white</i> DNA fragment from nucleotide positions 4278 to 4666. Two PCRs products used in this experiment and called “w4278” and “w4392” are represented underneath. The two AluI restriction sites and the resulting DNA fragment sizes are presented above. The palindromic cleavage site CGCGCG is indicated by a white box. B) <i>In vitro</i> binding assays with ZAM-IN protein performed on the <i>white</i> PCR products “w4278” and “w4392”. C) Assays performed with the <i>white</i> PCR product w4278 digested by AluI. The percentage of bound (upper panels) and unbound (lower panels) fractions is presented below each panel. DNA fragments sizes are indicated for each panel.</p

ZAM integrase binds two specific genomic DNA sites.

<p>A) Sequence of the Drosophila <i>white</i> locus from base 4278 to 4326. The oligonucleotides w0, w1, w1mut, w2, w3, Δw3 and w3mut used in the experiments are represented under the sequence. The integration site CGCGCG, the sequence of the mutated integration site of the w1mut oligonucleotide and the nucleotides mutated to generate the w3mut oligonucleotide are indicated by boxes. B) Left panel: <i>In vitro</i> binding assays were performed with ZAM integrase “IN” and the double stranded oligonucleotides w0, w1, w1mut, w2, w3, Δw3 and w3mut. w1 and w3 are the only two oligonucleotides retained by ZAM integrase. Right panel: <i>In vitro</i> endonuclease activity of ZAM integrase: pUC/white and pUC/white3mut plasmids were incubated without (−IN) or with (+IN) purified ZAM-IN. Positions of the supercoiled, nicked (circle) and linear (bar) DNAs are indicated. C) Alignment of a conserved motif detected in the ZAM LTR and w3. The first 60 nucleotides of the LTR sequence are presented as the upper sequence. The binding site of w3 is presented below.</p

Ecosystems and sand fly captures.

<p>Odds ratios are computed by multiple logistic regression analysis (all species included). <i>P. duboscqi</i> (PD), <i>S. antennata</i> (SA), <i>S. schwetzi</i> (SS), <i>S. dubia</i> (SD), <i>S. magna</i> (SM), <i>S. clydei</i> (SC), <i>S</i>. <i>adleri</i> (SA.1), <i>S. buxtoni.</i> The indicated <i>P</i>-values result from likelihood ratio tests. Stars denote the degree of significance (* ≤5%, ** <1%, *** <0.1%).</p

Discriminant analysis showing the relations between sand fly species and the three ecosystems.

<p>Each dot represents one village. Villages were classified in the ecosystem (LG, S and SC) they belong to. Sand fly captures were used to compute the coordinates of the dots (dimensionless units). Confidence ellipses materialized the variability of capture data across ecosystems. The Ndiaye Bopp (NB) village was excluded from this analysis because it is the only village with an intermediate ecosystem between S and SC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014773#pone-0014773-g001" target="_blank">Figure 1</a>).</p

Distribution of sand fly species and leishmaniasis seropositive dogs in the rural community of Mont Rolland.

<p>The map shows the rural community of Mont Rolland (the villages are represented by their initials), the distribution of the different species of phlebotomine sand flies in each village and, in between brackets, the number of seropositive dogs out of the total number of tested dogs in each village <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014773#pone.0014773-Faye1" target="_blank">[7]</a>.</p

Sand fly species caught in the Mont Rolland rural community.

<p>This table presents the nine species of sand flies caught in the Mont Rolland rural community, the number of females and males and their percentage in the whole population.</p><p>*Differently from females, the distinction between males of <i>S. dubia</i> and males of <i>S. antennata</i> was difficult. Therefore we calculated the ratio between <i>S. dubia</i> and <i>S. antennata</i> females (2.19) and then we used the same ratio to estimate the number of males in each of the two species.</p

Ecosystems, number of traps and number of sand flies in each trap in the different villages.

<p>The table presents the number of phlebotomine sand flies collected in each of the 16 villages:</p><p>Guidieur (G), Khaye Diagal (KaD), Fouloum (Fm), Sambaye Karang (SK), Twin Djassa (TD), Ndiaye Bopp (NB), Loukhouss (L), Pallo Dial (PD), Pallo Youga (PY), Paham Kouye (PK), Kémaye (K), Fouloune (Fne), Keur Daouda (KeD), Colobane Thiombane (CT), Keur Lat Diop (KLD) and Nguith Fall (NF); the different traps used [light traps (CDC), sticky traps (ST) and indoor residual spraying (IRS)]; the number of traps (No.T) and in bracket the number of sand flies caught <b>(</b>No.flies) in each village with that type of trap; the mean number of sand flies/trap (Mean No.flies/T). Eco, Ecosystem; SC, sandy clay soil; S, sandy soil; LG, lateritic gravel soil.</p><p>*The Ndiaye Bopp village presents intermediate ecological features between S and SC.</p