Laboratory diagnosis of pertussis infections: the role of PCR and serology

Abstract

This study reports on practical laboratory aspects of pertussis diagnosis. PCR assays were applied to respiratory specimens obtained during a large study of infants (less than 5 months old) admitted to paediatric intensive care units (n ¼ 122), children (less than 15 years old) admitted to paediatric wards (n ¼ 16) and their household contacts (n ¼ 320). Estimation of antibodies to pertussis toxin and culture for Bordetella pertussis were attempted on specimens from the same patients, where available, and the overall utility of the diagnostic PCR assays was assessed by comparison to these results. A PCR assay for the human mitochondrial cytochrome oxidase (HMCO) gene was used for quality control of the extracted samples and an internal process control (IPC) was included in each sample to test for PCR inhibition. Four of 458 samples were considered unsuitable (three HMCO negative, one IPC negative) and excluded from further analyses. Positive PCR results were considered valid if they were either (i) positive for both of two B. pertussis gene targets (pertussis toxin S1 promoter and the insertion element IS481), i.e. consensus PCR positive, or (ii) repeatably positive in only one assay. Using these criteria, 52 of 454 (11 . 5 %) samples were considered as PCR positive for B. pertussis. Six of 356 samples were culture-positive for B. pertussis, 1/88 infants, 3/14 children and 2/254 contacts, giving an overall isolation rate of 1 . 7 %. Using these data, PCR gave an almost fivefold increase in diagnostic yield compared with culture (McNemar's test; P , 0 . 0001). Sera from 9/111 infants, 5/10 children and 14/210 contacts were positive. Serology and PCR results showed a high level of agreement (113/121) for infants and children. PCR demonstrated a significant improvement in diagnostic yield over culture. Serological testing also resulted in a significant increase in diagnostic yield compared to culture alone. PCR is a useful technique, but validity of results must be assured by careful control. Rapid diagnosis of B. pertussis infection particularly in infants by PCR, together with serological assays, can enhance surveillance systems for pertussis in all age groups.

Nucleic Acid Amplification Tests for Diagnosis of Bordetella Infections

“…Although the pertussis toxin operon is present in B. pertussis, B. parapertussis, and B. bronchiseptica, the pertussis toxin promoter (ptxA-Pr) is a target for B. pertussis-specific assays using a real-time format (5,14). It was, however, consistently less sensitive than IS481 (Caro et al, unpublished).…”

“…The sensitivity of PCR appears to be highest in newborns and young infants (5,6). In adolescents and adults, the sensitivity depends on the time between cough onset and specimen collection and on the bacterial load but was found to be always lower than 50% (23).…”

Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Diagnosis of Bordetella pertussis Infection

“…Cross-reactivity with B. bronchiseptica in IS481-based PCR was also observed in the external quality assessment program (20). Therefore, it should be noted that IS481-based PCR provided the only evidence for the presence of Bordetella species in clinical specimens, as described by Fry et al (7).…”

Prevalence and Sequence Variants of IS 481 in Bordetella bronchiseptica : Implications for IS 481 -Based Detection of Bordetella pertussis

“…On the basis of a comparison of the B. bronchiseptica IS481 sequences with those of IS481 primers used for conventional PCR identification of B. pertussis (2,3,9,10,12,13,16,18,20,21,23,25,37,45,46,47,53), it seems likely that misidentification of IS481-containing strains of B. bronchiseptica would occur. Many of the suggested primers are 100% identical to the B. bronchiseptica sequence reported here, while others have a few base substitutions which may still permit amplification.…”