Putative genes in the <i>E</i>. <i>coli</i> O5 O-antigen gene cluster.

<p>^aaa, amino acid.</p><p>Putative genes in the <i>E</i>. <i>coli</i> O5 O-antigen gene cluster.</p

<i>E</i>. <i>coli</i> O5 and O76 O-antigen gene clusters.

<p>All genes are transcribed in a direction from the JUMPstart sequence to <i>gnd</i>.</p

Putative genes in the <i>E</i>. <i>coli</i> O76 O-antigen gene cluster.

<p>^a aa, amino acid.</p><p>Putative genes in the <i>E</i>. <i>coli</i> O76 O-antigen gene cluster.</p

Primers and concentrations used in the serogroup-specific multiplex PCR assays.

<p>^aGenBank accession numbers for each of the gene targets: O5<i>wzx</i> (KM881565); O91<i>wzy</i> (AY035396); O103<i>wzx</i> (AP010958); O55<i>wzx</i> (NC_013941); O128<i>wzx</i> (AY217096); O113<i>wzx</i> (AF172324); O146<i>wzx</i> (DQ465249); O76<i>wzx</i> (KM881564); O45<i>wzx</i> (AY771223); O177<i>wzx</i> (DQ008593); O157<i>wzx</i> (AE005174); O15<i>wzx</i> (AY647261); O104<i>wzx</i> (AF361371); O118<i>wzx</i> (DQ990684); O123<i>wzx</i> (DQ676933); O165<i>wzx</i> (GU068045); O172<i>wzx</i> (AY545992).</p><p>^bCross reaction with <i>wzx</i> from <i>E</i>. <i>coli</i> O9.</p><p>^cCross reaction with <i>wzx</i> from <i>E</i>. <i>coli</i> O151.</p><p>Primers and concentrations used in the serogroup-specific multiplex PCR assays.</p

Development of Three Multiplex PCR Assays Targeting the 21 Most Clinically Relevant Serogroups Associated with Shiga Toxin-Producing <i>E. coli</i> Infection in Humans

<div><p><i>Escherichia coli</i> serogroups O5, O15, O26, O45, O55, O76, O91, O103, O104, O111, O113, O118, O121, O123, O128, O145, O146, O157, O165, O172, and O177 are the O-antigen forms of the most clinically relevant Shiga toxin-producing <i>E. coli</i> (STEC) serotypes. In this study, three multiplex PCR assays able to specifically detect these 21 serogroups were developed and validated. For this purpose, the O-antigen gene clusters of <i>E. coli</i> O5 and O76 were fully sequenced, their associated genes were identified on the basis of homology, and serogroup-specific primers were designed. After preliminary evaluation, these two primer pairs were proven to be highly specific and suitable for the development of PCR assays for O5 and O76 serogroup identification. Specific primers were also designed for serogroups O15, O45, O55, O91, O104, O113, O118, O123, O128, O146, O157, O165, O172, and O177 based on previously published sequences, and previously published specific primers for serogroups O26, O103, O111, O121, and O145 were also included. These 21 primer pairs were shown to be specific for their target serogroup when tested against <i>E. coli</i> type strains representing 169 known O-antigen forms of <i>E. coli</i> and <i>Shigella</i> and therefore suitable for being used in PCR assays for serogroup identification. In order to validate the three multiplex PCR assays, 22 <i>E. coli</i> strains belonging to the 21 covered serogroups and 18 <i>E. coli</i> strains belonging to other serogroups were screened in a double-blind test and their sensitivity was determined as 1 ng chromosomal DNA. The PCR assays developed in this study could be a faster, simpler, and less expensive strategy for serotyping of the most clinically relevant STEC strains in both clinical microbiology and public health laboratories, and so their development could benefit for clinical diagnosis, epidemiological investigations, surveillance, and control of STEC infections.</p></div

Agarose gel electrophoresis of the PCR products obtained from <i>E</i>. <i>coli</i> type strains belonging to the 21 covered serogroups by using the three multiplex PCR assays.

<p><b>(a) Multiplex 1</b>: lanes 1 and 9, 100 bp DNA ladder; lane 2, O5; lane 3, O91; lane 4, O26; lane 5, O103; lane 6, O145; lane 7, O121; lane 8, O111. <b>(b) Multiplex 2</b>: lanes 1 and 9, 100 bp DNA ladder; lane 2, O55; lane 3, O128; lane 4, O113; lane 5, O146; lane 6, O76; lane 7, O45; lane 8, O177. <b>(c) Multiplex 3</b>: lanes 1 and 9, 100 bp DNA ladder; lane 2, O157; lane 3, O15; lane 4, O104; lane 5, O118; lane 6, O123; lane 7, O165; lane 8, O172.</p

Weekly notification of suspected cholera cases.

<p>The Disease Surveillance Service of the Ghana Health Service reports 20,120 cholera cases according to the WHO case definition between May 2014 and December 2014 with a peak number of 2,853 cases in the 35^th calendar week (25–31 August).</p

Spatial and temporal location of suspected cholera cases (Mai 2014-December 2014; n = 20,120).

<p>As notified to the Disease Surveillance Service of the Ghana Health Service according to the WHO case definition suspected cholera cases are plotted by district by 5-week period panels. The figure was produced with Arc GIS 10.0 (ESRI: ArcGis Desktop: Release 10.2011).</p

Antimicrobial resistance for each antibiotic (A) and resistance profile (B) of <i>Vibrio cholerae</i> isolates, by year of disease onset (n = 92).

<p>Antimicrobial resistance for each antibiotic (A) and resistance profile (B) of <i>Vibrio cholerae</i> isolates, by year of disease onset (n = 92).</p

Minimum spanning tree of multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) for <i>Vibrio cholerae</i> isolates (n = 45) by year of disease onset.

<p>Clonal complexes (CC 1, CC 2, CC 3) were defined as isolates connected through a chain of single-locus variants. Grey figures indicate the number of different alleles. Three-digit codes present the laboratory isolate number.</p