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Optimization of Molecular Approaches to Genogroup Neisseria meningitidis Carriage Isolates and Implications for Monitoring the Impact of New Serogroup B Vaccines.

Rojas E, Hoyos J, Oldfield NJ, Lee P, Flint M, Jones CH, Ala'Aldeen DA, Jansen KU, Anderson AS - PLoS ONE (2015)

Bottom Line: Isolates were typed for the Nm vaccine antigen factor H binding protein (fHbp), and were found to represent the known diversity of this antigen.Thirty seven NGG isolates evidenced a disrupted capsular polysaccharide operon judged by a ctrA negative result.Only 28.6% (67/234) of the isolates were serogrouped by slide agglutination (SASG), highlighting the reduced capability of carriage strains to express capsular polysaccharide.

View Article: PubMed Central - PubMed

Affiliation: Vaccine Research and Development, Pfizer Inc, Pearl River, New York, United States of America.

ABSTRACT
The reservoir for Neisseria meningitidis (Nm) is the human oropharynx. Implementation of Nm serogroup C (NmC) glycoconjugate vaccines directly reduced NmC carriage. Prophylactic vaccines are now available to prevent disease caused by the five major Nm disease causing serogroups (ABCWY). Nm serogroup B (NmB) vaccines are composed of antigens that are conserved across Nm serogroups and therefore have the potential to impact all Nm carriage. To assess the effect of these vaccines on carriage, standardized approaches to identify and group Nm are required. Real-time PCR (rt-PCR) capsule grouping assays that were internally controlled to confirm Nm species were developed for eight serogroups associated with carriage (A, B, C, E, W, X, Y and Z). The grouping scheme was validated using diverse bacterial species associated with carriage and then used to evaluate a collection of diverse Nm carriage isolates (n=234). A scheme that also included porA and ctrA probes was able to speciate the isolates, while ctrA also provided insights on the integrity of the polysaccharide loci. Isolates were typed for the Nm vaccine antigen factor H binding protein (fHbp), and were found to represent the known diversity of this antigen. The porA rt-PCR yielded positive results with all 234 of the Nm carriage isolates. Genogrouping assays classified 76.5% (179/234) of these isolates to a group, categorized 53 as nongenogroupable (NGG) and two as mixed results. Thirty seven NGG isolates evidenced a disrupted capsular polysaccharide operon judged by a ctrA negative result. Only 28.6% (67/234) of the isolates were serogrouped by slide agglutination (SASG), highlighting the reduced capability of carriage strains to express capsular polysaccharide. These rt-PCR assays provide a comprehensive means to identify and genogroup N. meningitidis in carriage studies used to guide vaccination strategies and to assess the impact of novel fHbp containing vaccines on meningococcal carriage.

No MeSH data available.


Related in: MedlinePlus

Location of serogroup-specific PCR amplicons in capsular biosynthesis genes of N. meningitidis serogroups A, B, C, W, Y, E, Z, and X.Genes ctrA-D encode capsule transport proteins conserved among all eight serogroups. Genes cssA-C are responsible for the sialic acid capsular component of serogroups B, C, W, and Y, while csb-c-w-y encode the sialyltransferases that produce the serogroup-specific linkages of sialic acid with glucose or galactose. Capsule biosynthesis genes csaA-D, csxA-C, cseA-G, and cszA-D, are unique to A, X, E, and Z serogroups, respectively. Polysaccharide transport and biosynthesis genes are flanked by tex and galE (black arrows). Genes at the distal end of the biosynthetic clusters in serogroups B, C, W, and Y are shown as dotted arrows, and annotated as described previously [35]. The green arrows indicate the location of the ctrA primers used to detect all eight serogroups. Light blue arrows represent the initial primers designed to identify NmE and NmZ, based on sequence at the 5’ end of the ctrA gene. The red arrows represent the position of the final primers designed for the eight serogroup-specific assays. The blow-up at the bottom illustrates the position of ctrA primers and probes in greater detail.
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pone.0132140.g001: Location of serogroup-specific PCR amplicons in capsular biosynthesis genes of N. meningitidis serogroups A, B, C, W, Y, E, Z, and X.Genes ctrA-D encode capsule transport proteins conserved among all eight serogroups. Genes cssA-C are responsible for the sialic acid capsular component of serogroups B, C, W, and Y, while csb-c-w-y encode the sialyltransferases that produce the serogroup-specific linkages of sialic acid with glucose or galactose. Capsule biosynthesis genes csaA-D, csxA-C, cseA-G, and cszA-D, are unique to A, X, E, and Z serogroups, respectively. Polysaccharide transport and biosynthesis genes are flanked by tex and galE (black arrows). Genes at the distal end of the biosynthetic clusters in serogroups B, C, W, and Y are shown as dotted arrows, and annotated as described previously [35]. The green arrows indicate the location of the ctrA primers used to detect all eight serogroups. Light blue arrows represent the initial primers designed to identify NmE and NmZ, based on sequence at the 5’ end of the ctrA gene. The red arrows represent the position of the final primers designed for the eight serogroup-specific assays. The blow-up at the bottom illustrates the position of ctrA primers and probes in greater detail.

Mentions: PCR grouping of meningococci relies on the accurate detection of the gene(s) responsible for determining the capsular type. The enzymes that synthesize and translocate capsular polysaccharides are encoded by an operon that is composed of between seven and eleven genes flanked by tex and galE (Fig 1) [34,35]. Thus, rt-PCR assays for genotypic grouping of Nm based on the detection of serogroup-specific genes csaB (NmA), csb (NmB), csc (NmC), csw (NmW), csxB (NmX) and csy (NmY) have been described [36,37]. However, no rt-PCR assays for genogrouping NmE and NmZ have been reported [29,37,38]. We therefore sought to develop assays to identify and genogroup carriage isolates, while trying to address the shortcomings present in current assay designs. We developed optimized porA and ctrA rt-PCR assays that identified Nm and assays that grouped isolates from the eight major serogroups (A, B, C, E, W, X, Y and Z) to assist in surveying the current state of meningococcal carriage. Carriage isolates were also typed for fHbp to evaluate its distribution across the Nm carriage isolates.


Optimization of Molecular Approaches to Genogroup Neisseria meningitidis Carriage Isolates and Implications for Monitoring the Impact of New Serogroup B Vaccines.

Rojas E, Hoyos J, Oldfield NJ, Lee P, Flint M, Jones CH, Ala'Aldeen DA, Jansen KU, Anderson AS - PLoS ONE (2015)

Location of serogroup-specific PCR amplicons in capsular biosynthesis genes of N. meningitidis serogroups A, B, C, W, Y, E, Z, and X.Genes ctrA-D encode capsule transport proteins conserved among all eight serogroups. Genes cssA-C are responsible for the sialic acid capsular component of serogroups B, C, W, and Y, while csb-c-w-y encode the sialyltransferases that produce the serogroup-specific linkages of sialic acid with glucose or galactose. Capsule biosynthesis genes csaA-D, csxA-C, cseA-G, and cszA-D, are unique to A, X, E, and Z serogroups, respectively. Polysaccharide transport and biosynthesis genes are flanked by tex and galE (black arrows). Genes at the distal end of the biosynthetic clusters in serogroups B, C, W, and Y are shown as dotted arrows, and annotated as described previously [35]. The green arrows indicate the location of the ctrA primers used to detect all eight serogroups. Light blue arrows represent the initial primers designed to identify NmE and NmZ, based on sequence at the 5’ end of the ctrA gene. The red arrows represent the position of the final primers designed for the eight serogroup-specific assays. The blow-up at the bottom illustrates the position of ctrA primers and probes in greater detail.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493136&req=5

pone.0132140.g001: Location of serogroup-specific PCR amplicons in capsular biosynthesis genes of N. meningitidis serogroups A, B, C, W, Y, E, Z, and X.Genes ctrA-D encode capsule transport proteins conserved among all eight serogroups. Genes cssA-C are responsible for the sialic acid capsular component of serogroups B, C, W, and Y, while csb-c-w-y encode the sialyltransferases that produce the serogroup-specific linkages of sialic acid with glucose or galactose. Capsule biosynthesis genes csaA-D, csxA-C, cseA-G, and cszA-D, are unique to A, X, E, and Z serogroups, respectively. Polysaccharide transport and biosynthesis genes are flanked by tex and galE (black arrows). Genes at the distal end of the biosynthetic clusters in serogroups B, C, W, and Y are shown as dotted arrows, and annotated as described previously [35]. The green arrows indicate the location of the ctrA primers used to detect all eight serogroups. Light blue arrows represent the initial primers designed to identify NmE and NmZ, based on sequence at the 5’ end of the ctrA gene. The red arrows represent the position of the final primers designed for the eight serogroup-specific assays. The blow-up at the bottom illustrates the position of ctrA primers and probes in greater detail.
Mentions: PCR grouping of meningococci relies on the accurate detection of the gene(s) responsible for determining the capsular type. The enzymes that synthesize and translocate capsular polysaccharides are encoded by an operon that is composed of between seven and eleven genes flanked by tex and galE (Fig 1) [34,35]. Thus, rt-PCR assays for genotypic grouping of Nm based on the detection of serogroup-specific genes csaB (NmA), csb (NmB), csc (NmC), csw (NmW), csxB (NmX) and csy (NmY) have been described [36,37]. However, no rt-PCR assays for genogrouping NmE and NmZ have been reported [29,37,38]. We therefore sought to develop assays to identify and genogroup carriage isolates, while trying to address the shortcomings present in current assay designs. We developed optimized porA and ctrA rt-PCR assays that identified Nm and assays that grouped isolates from the eight major serogroups (A, B, C, E, W, X, Y and Z) to assist in surveying the current state of meningococcal carriage. Carriage isolates were also typed for fHbp to evaluate its distribution across the Nm carriage isolates.

Bottom Line: Isolates were typed for the Nm vaccine antigen factor H binding protein (fHbp), and were found to represent the known diversity of this antigen.Thirty seven NGG isolates evidenced a disrupted capsular polysaccharide operon judged by a ctrA negative result.Only 28.6% (67/234) of the isolates were serogrouped by slide agglutination (SASG), highlighting the reduced capability of carriage strains to express capsular polysaccharide.

View Article: PubMed Central - PubMed

Affiliation: Vaccine Research and Development, Pfizer Inc, Pearl River, New York, United States of America.

ABSTRACT
The reservoir for Neisseria meningitidis (Nm) is the human oropharynx. Implementation of Nm serogroup C (NmC) glycoconjugate vaccines directly reduced NmC carriage. Prophylactic vaccines are now available to prevent disease caused by the five major Nm disease causing serogroups (ABCWY). Nm serogroup B (NmB) vaccines are composed of antigens that are conserved across Nm serogroups and therefore have the potential to impact all Nm carriage. To assess the effect of these vaccines on carriage, standardized approaches to identify and group Nm are required. Real-time PCR (rt-PCR) capsule grouping assays that were internally controlled to confirm Nm species were developed for eight serogroups associated with carriage (A, B, C, E, W, X, Y and Z). The grouping scheme was validated using diverse bacterial species associated with carriage and then used to evaluate a collection of diverse Nm carriage isolates (n=234). A scheme that also included porA and ctrA probes was able to speciate the isolates, while ctrA also provided insights on the integrity of the polysaccharide loci. Isolates were typed for the Nm vaccine antigen factor H binding protein (fHbp), and were found to represent the known diversity of this antigen. The porA rt-PCR yielded positive results with all 234 of the Nm carriage isolates. Genogrouping assays classified 76.5% (179/234) of these isolates to a group, categorized 53 as nongenogroupable (NGG) and two as mixed results. Thirty seven NGG isolates evidenced a disrupted capsular polysaccharide operon judged by a ctrA negative result. Only 28.6% (67/234) of the isolates were serogrouped by slide agglutination (SASG), highlighting the reduced capability of carriage strains to express capsular polysaccharide. These rt-PCR assays provide a comprehensive means to identify and genogroup N. meningitidis in carriage studies used to guide vaccination strategies and to assess the impact of novel fHbp containing vaccines on meningococcal carriage.

No MeSH data available.


Related in: MedlinePlus