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. 2022 Apr 20;60(4):e0211121.
doi: 10.1128/jcm.02111-21. Epub 2022 Mar 21.

Direct Real-Time PCR for the Detection and Serotyping of Haemophilus influenzae without DNA Extraction

Daya Marasini  1 , Melissa J Whaley  1 , Laurel T Jenkins  1 , Fang Hu  2 , Wenxin Jiang  3 , Nadav Topaz  4 , Alex Chen  1 , Susanna Schmink  1 , Jennifer Dolan Thomas  1 , Brian H Harcourt  1 , Henju Marjuki  1 , Xin Wang  1
Affiliations

Direct Real-Time PCR for the Detection and Serotyping of Haemophilus influenzae without DNA Extraction

Daya Marasini et al. J Clin Microbiol. .

Abstract

To monitor the burden and changes in Haemophilus influenzae (Hi) disease, direct real-time PCR (drt-PCR) assays have been developed for Hi detection in monoplex form and its six serotypes in triplex form, directly from cerebrospinal fluid (CSF) specimens. These assays target the phoB gene for the species detection (Hi-phoB) and serotype-specific genes in region II of the capsule biosynthesis locus (Hi-abf and Hi-cde), identified through comparative analysis of Hi and non-Hi whole-genome sequences. The lower limit of detection (LLD) is 293 CFU/mL for the Hi-phoB assay and ranged from 11 to 130 CFU/mL for the triplex serotyping assays. Using culture as a reference method, the sensitivity and specificity of Hi-phoB, Hi-abf, and Hi-cde were 100%. Triplex serotyping assays also showed 100% agreement for each serotype compared to their corresponding monoplex serotyping assay. These highly sensitive and specific drt-PCR assays do not require DNA extraction and thereby reduce the time, cost, and handling required to process CSF specimens. Furthermore, triplex drt-PCR assays combine the detection of three serotypes in a single reaction, further improving testing efficiency, which is critical for laboratories that process high volumes of Hi specimens for surveillance and diagnostic purposes.

Keywords: Haemophilus influenzae; Hi-phoB; direct real-time polymerase chain reaction; serotyping; triplex.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Sequence alignment for phoB assay primer/probe design. The figure shows the segment of the phoB gene with the consensus to the primer and probes sequences used for the assay. The alignment was constructed using 65 representative Hi genomes.
FIG 2
FIG 2
Bland-Altman plots resulting from the pairwise comparison of Ct values between triplex and monoplex direct real-time PCR assays by H. influenzae serotypes. (a) Hia-acs3. (b) Hib-bsc3. (c) Hic-ccs4. (d) Hid-dcs5. (e) Hie-ecs8. (f) Hif-fcs3. The level of agreement between the two assays by serogroup (Ct triplex – Ct monoplex), also known as observed mean difference (lines of short dashes), is presented as a function of the average Ct values of the two assays ([Ct triplex + Ct monoplex]/2). Reference lines (solid lines) indicate the ideal zero difference. Upper and lower limits of agreement (lines of long dashes) defined by mean Ct difference of ±1.96 SD.
See this image and copyright information in PMC

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