sodC-Based Real-Time PCR for Detection of Neisseria meningitidis

Real-time PCR (rt-PCR) is a widely used molecular method for detection of Neisseria meningitidis (Nm). Several rt-PCR assays for Nm target the capsule transport gene, ctrA. However, over 16% of meningococcal carriage isolates lack ctrA, rendering this target gene ineffective at identification of this sub-population of meningococcal isolates. The Cu-Zn superoxide dismutase gene, sodC, is found in Nm but not in other Neisseria species. To better identify Nm, regardless of capsule genotype or expression status, a sodC-based TaqMan rt-PCR assay was developed and validated. Standard curves revealed an average lower limit of detection of 73 genomes per reaction at cycle threshold (Ct) value of 35, with 100% average reaction efficiency and an average R2 of 0.9925. 99.7% (624/626) of Nm isolates tested were sodC-positive, with a range of average Ct values from 13.0 to 29.5. The mean sodC Ct value of these Nm isolates was 17.6±2.2 (±SD). Of the 626 Nm tested, 178 were nongroupable (NG) ctrA-negative Nm isolates, and 98.9% (176/178) of these were detected by sodC rt-PCR. The assay was 100% specific, with all 244 non-Nm isolates testing negative. Of 157 clinical specimens tested, sodC detected 25/157 Nm or 4 additional specimens compared to ctrA and 24 more than culture. Among 582 carriage specimens, sodC detected Nm in 1 more than ctrA and in 4 more than culture. This sodC rt-PCR assay is a highly sensitive and specific method for detection of Nm, especially in carriage studies where many meningococcal isolates lack capsule genes

Characterization of Streptococcus-Pneumoniae From Human Immunodeficiency Virus-Seropositive Patients with Acute and Recurrent Pneumonia

Thirty-two isolates of clinically significant Streptococcus pneumoniae from 11 human immunodeficiency virus (HIV)-seropositive patients with single or multiple episodes of pneumonia were characterized by antibiotic susceptibility testing, serotyping, ribotyping, and repetitive extragenic palindromic polymerase chain reaction (REP-PCR). The isolates comprised 10 serotypes, 12 ribotyping patterns, and 12 REP-PCR patterns. There was close but not absolute correlation between techniques. By combining these characterization methods, 14 strains were identified. Five strains were found in = 1 patient, suggesting their frequent occurrence in this population. Two isolates of different serotype from 1 patient were highly related by ribotyping and REP-PCR, suggesting possible in vivo serotype change. Acute infection was associated with single strains or coinfection by distinct strains. Recurrent pneumonia was identified as relapse with the same strain or reinfection with new strains. The molecular characterization of pneumococci from HIV-seropositive persons refines our understanding of pneumococcal infection in these patients

Population-based study of non-typable Haemophilus influenzae invasive disease in children and neonates.

The extent of non-capsulate, non-serotypable Haemophilus influenzae (NST) as a cause of serious invasive disease in children has not been fully defined. We describe the epidemiology of these childhood infections from cases identified during a continuing prospective survey of invasive H influenzae disease in the Oxford region, UK. 408 strains of H influenzae were isolated from cases of invasive disease. 383 (94%) were H influenzae type b (Hib), 24 (6%) were NST strains, and 1 was a type f strain. 3 of the NST strains were non-capsulate type b mutants (b-), but the remaining 21 strains were from the phylogenetically distinct and heterogeneous population of non-capsulate H influenzae (NC). 10 of the NC strains were isolated from neonates with sepsis; crude mortality rate was 40%, with an incidence of 4.6 cases per 100,000 livebirths. 11 NC strains were isolated from children after the neonatal period and under 10 years of age, 4 (36%) of which had severe, unrelated, predisposing conditions. The incidence of NC invasive diseases in these children was 0.5 per 100,000 per year. The attributable mortality for these infections was 10%. Infections due to these H influenzae strains are, after the implementation of Hib vaccines, likely to persist and represent a substantial proportion of the serious infections caused by this species

Nontypeable Haemophilus influenzae strains with the capsule-associated insertion element IS1016 may mimic encapsulated strains

With the elimination of Haemophilus influenzae type b through vaccination, it has been suggested that other types of H. influenzae strains might acquire virulence traits and emerge as important pathogens. The gene sequence IS1016 has been associated with an increased capacity to cause severe infections. It is usually present in encapsulated strains but is sometimes harbored by nontypeable H. influenzae strains. To explore this further, 118 H. influenzae isolates, collected from both patients and healthy carriers, were investigated with PCR with reference to this gene sequence. Isolates positive for the insertion element were bio- and serotyped. The presence of hmw genes for adherence, the genetic profile, and the ability to form biofilm in vitro were investigated. A total of 15 isolates were IS1016-positive, whereof 12 were nontypeable. All 12 nontypeable isolates were obtained from healthy carriers, and 92% of the isolates were biotype I. They cross-reacted to some extent with type-specific antisera or exhibited a restricted genetic diversity like encapsulated strains. Furthermore, they lacked hmw-genes, and their ability to form biofilms was comparable with a capsule-deficient type b strain. Although this subset of strains mimicked traits usually exhibited by encapsulated strains, the isolation frequency did not seem to have been affected by vaccination