Abstract
With the advent of effective vaccination strategies, life-threatening Haemophilus influenzae infections are now rare in developed countries (1). However, H. influenzae can still cause infection at a variety of anatomical sites, especially the upper and the lower airway. For this reason, adequate diagnosis of H. influenzae infections remains important. Classical microbiological diagnosis relies on growth on blood-based agar media, including chocolate agar or brain heart infusion (BHI) agar supplemented with Levinthals blood, hemoglobin, or hemin. In addition, H. influenzae can be distinguished from its closest relatives because it is incapable of producing porphyrin (2). Modern developments allow for testing without a cultivation step. The most direct and simple way of detection and identification without culture is provided by commercially available latex agglutination tests (2). Antibody-coated latex particles are mixed directly with clinical samples and agglutinate if H. influenzae is present. Sophisticated means for direct H. influenzae detection and identification are now available in the medical microbiology laboratory, including immunological and molecular methods. They include specific DNA probing and ultrasensitive nucleic acid amplification. This chapter surveys several of the more frequently applied culture-based, immunological and molecular approaches to diagnosis.
References
Urwin G., Krohn J. A., Deaver-Robinson K., Wenger J. D., Farley M. M., and the Haemophilus influenzae Study Group. (1996) Invasive disease due to Haemophilus influenzae serotype f: clinical and epidemiological characteristics in the H. influenzae serotype b vaccine era. Clin. Infect. Dis. 22, 1069ā1076.
Isenberg H.D. (1998) Essential procedures for clinical microbiology, American Society for Microbiology, Washington, DC.
Groeneveld K., van Alphen L., van Ketel R. J., Geelen van den Broek N. J., Eijk P. P., and Zanen H. C. (1989) Nonculture detection of Haemophilus influenzae in sputum with monoclonal antibodies specific for outer-membrane lipoprotein P6. J. Clin. Microbiol. 27, 2263ā2267.
Altschul S., Gish W., Miller W., Meyers E. W., and Lipman.D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403ā410.
Doern G., and Brogden-Torres B. (1992) Optimum use of selective plated media in primary processing of respiratory tract specimens from patients with cystic fibrosis. J. Clin. Microbiol. 30, 2740ā2742.
Mƶller L.V.M., van Alphen L., Grasselier H., and Dankert J. (1993) N-Acetyl-D-glucosamine medium improves recovery of Haemophilus influenzae from sputa of patients with cystic fibrosis. J. Clin. Microbiol. 31, 1952ā1954.
Smith A., and Baker M. (1997). Cefsulodin chocolate agar: a selective medium for the recovery of Haemophilus influenzae from the respiratory secretions of patients with cystic fibrosis. J. Med. Microbiol. 46, 883ā885.
Boom R., Sol C. J. A., Salimans M. M. M., Jansen C. L., Wertheim-van Dillen P. M. E., and van der Noordaa J. (1990) Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28, 495ā503.
Van Ketel R.J., de Wever B., and van Alphen L. (1990) Detection of Haemophilus influenzae in cerebrospinal fluids by polymerase chain reaction DNA amplification. J. Med. Microbiol. 33, 271ā276.
Mƶller L.V.M., Ruijs G. J., Heijerman H. G. M., Dankert J., and van Alphen L. (1992) Haemophilus influenzae is frequently detected with monoclonal antibody 8BD9 in sputum samples from patients with cystic fibrosis. J. Clin. Microbiol. 30, 2495ā2497.
Ueyama T., Gu X. X., Tsai C. M., Karpas A. B., and Lim D. J. (1999) Identification of common lipooligosaccharide types in isolates from patients with otitis media by monoclonal antibodies against nontypeable Haemophilus influenzae 9274. Clin. Diagn. Lab. Immunol. 6, 96ā100.
Becton Dickinson (2000) Difco manual: Haemophilus influenzae antisera, pp. 646ā648.
Hamel J., Brodeur B. R., Belmaaza A., Montplaisir S., Musser J. M., and Selander R. K. (1987) Identification of Haemophilus influenzae type b by a monoclonal antibody coagglutination assay. J. Clin. Microbiol. 25, 2434ā2436.
Landgraf I.M., and Vieira M. F. P. (1993) Biotypes and serotypes of Haemophilus influenzae from patients with meningitis in the city of Sao Paulo, Brazil. J. Clin. Microbiol. 31, 743ā745.
Daly J.A., Clifton N. L., Seskin K. C., and Gooch W. M. (1991) Use of rapid non-radioactive DNA probes in culture confirmation tests to detect Streptococcus agalactiae, Haemophilus influenzae, and Enterococcus spp. from paediatric patients with significant infections. J. Clin. Microbiol. 29, 80ā82.
Davis T.E., and Fuller D. D. (1991) Direct identification of bacterial isolates in blood cultures by using a DNA probe. J. Clin. Microbiol. 29, 2193ā2196.
Malouin F., and Bryan L. E. (1987) DNA probe technology for detection of Haemophilus influenzae. Mol. Cell. Probes 1, 221ā232.
Malouin F., Bryan L. E., Shewciw P., Douglas J., Li D., van den Elzen H., and Lapointe J. R. (1988) DNA probe technology for rapid detection of Haemophilus influenzae in clinical specimens. J. Clin. Microbiol. 26, 2132ā2138.
Terpstra W.J., ter Schegget J., and Schoone G. J. (1990) Detection of Leptospira, Haemophilus and Campylobacter using DNA probes, in: Gene Probes for Bacteria, (Macario A. J. L, and Conway de Macario E. eds.), Academic, New York, pp. 295ā322.
St. Geme J.W., Kumar V. V., Cutter D., and Barenkamp S. J. (1998) Prevalence and distribution of the hmw and hia genes and the HMW and Hia adhesins among genetically diverse strains of nontypeable Haemophilus influenzae. Infect. Immun. 66, 364ā3
Hogardt M., Trebesius K., Geiger A. M., Hornef M., Rosenecker J., and Heeseman J. (2000) Specific and rapid detection by fluorescent in situ hybridization of bacteria in clinical samples obtained from cystic fibrosis patients. J. Clin. Microbiol. 38, 818ā825.
Kroll J. S., Ely S., and Moxon E. R. (1991) Capsular genotyping of Haemophilus influenzae with a DNA probe. Mol. Cell. Probes. 5, 375ā379.
Muehlemann K., Balz M., Aebi S., and Schopfer K. (1996) Molecular characteristics of Haemophilus influenzae causing invasive disease during the period of vaccination in Switzerland: analysis of strains isolated between 1986 and 1993. J. Clin. Microbiol. 34, 560ā563.
Casin I., Grimont F., and Grimont P. A. (1986) Deoxyribonucleic acid relatedness between Haemophilus aegyptius and Haemophilus influenzae. Ann. Inst. Pasteur Microbiol. 137B, 155ā163.
Dewhirst F. E., Paster B. J., Olsen I., and Fraser G. J. (1992) Phylogeny of 54 representative strains of species in the family Pasteurellaceae as determined by comparison of 16S rRNA sequences. J. Bacteriol. 174, 2002ā2013.
Quentin R., Ruimy R., Rosenau A., Musser J. M., and Christen R. (1996) Genetic identification of cryptic genospecies of Haemophilus causing urogenital and neonatal infections by PCR using specific primers targeting genes coding for 16S rRNA. J. Clin. Microbiol. 34, 1380ā1385.
Tenover F. C., Huang M. B., Rasheed J. K., and Persing D. H. (1994) Development of PCR assays to detect ampicillin resistance genes in cerebrospinal fluid samples containing Haemophilus influenzae. J. Clin. Microbiol. 32, 2729ā2737.
Hassan-King M., Adegbola R., Baldeh I., Mulholland K., Omosigho C., Oparaugo A., et al. (1998) A polymerase chain reaction for the diagnosis of Haemophilus influenzae type b disease in children and its evaluation during a vaccine trial. Pediatr. Infect. Dis. 17, 309ā312.
Falla T. J., Crook D. W. M., Brophy L. N., Maskell D., Kroll J. S., and Moxon E. R. (1994) PCR for capsular typing of Haemophilus influenzae. J. Clin. Microbiol. 32, 2382ā2386.
Ueyama T., Kurono Y., Shirabe K., Takeshita M., and Mogi G. (1995). High incidence of Haemophilus influenzae in nasopharyngeal secretions and middle ear effusions as detected by PCR. J. Clin. Microbiol. 33, 1835ā1838.
Sakamoto N., Kurono Y., Ueyama T., and Mori G. (1996) Detection of Haemophilus influenzae in adenoids and nasopharyngeal secretions by polymerase reaction. Acta Otolaryngol. Suppl. 523, 145ā146.
Hassan-King M., Baldeh I., Adegbola R., Omosigho C., Usen S. O., Oparaugo A., and Greenwood B. M. (1996) Detection of Haemophilus influenzae and Streptococcus pneumoniae DNA in blood cultures by a single PCR assay. J. Clin. Microbiol. 34, 2030ā2032.
Post J.C., White G. J., Aul J. J., Zavoral T., Zadowsky R. M., Zhang Y., et al. (1996). Development and validation of a multiplex PCR-based assay for the upper respiratory tract bacterial pathogens Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis. Mol. Diagn. 1, 29ā39.
Bakaletz L.O., White G. I., Post J. C., and Ehrlich G. D. (1998) Blinded multiplex PCR analyses of middle ear and nasopharyngeal fluids from chinchilla models of single and mixed-pathogen-induced otitis media. Clin. Diagn. Lab. Immunol. 5, 219ā224.
Hendolin P.H., Paulin L., and Ylikoski J. (2000) Clinically applicable multiplex PCR for four middle ear pathogens. J. Clin. Microbiol. 38, 125ā132.
Backman A., Lantz P., Radstrom P., and Olcen P. (1999) Evaluation of an extended diagnostic PCR assay for detection and verification of the common causes of bacterial meningitis in CSF and other biological samples. Mol. Cell. Probes 13, 49ā60.
Van Belkum A., Renders N. H. M., Smith S., Overbeek S. E., and Verbrugh H. A. (2000) Comparison of conventional and molecular methods for the detection of bacterial pathogens in sputum samples from cystic fibrosis patients. FEMS Immunol. Med. Microbiol. 27, 51ā57.
Greisen K., Loeffelholz M., Purohit A., and Leong D. (1994) PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria including bacteria found in cerebrospinal fluid. J. Clin. Microbiol. 32, 335ā351.
Dicuonzo G., Lorino G., Lilli D., et al. (1999) Use of oligoprobes on amplified DNA in the diagnosis of bacterial meningitis. Eur. J. Clin. Microbiol. Infect. Dis. 18, 352ā357.
Rantakokko-Jalava K., Nikkari S., Jalava J., et al. (2000) Direct amplification of rRNA genes in diagnosis of bacterial infections. J. Clin. Microbiol. 38, 32ā39.
Turenne C.Y., Witwicki E., Hoban D. J., Karlowsky J. A., and Kabani A. M. (2000) Rapid identification of bacteria from positive blood cultures by fluorescence-based PCR-single āstrand conformation polymorphism analysis of the 16S rRNA gene. J. Clin. Microbiol. 38, 513ā520.
Anthony R.M., Brown T. J., and French G. L. (2000) Rapid diagnosis of bacteremia by universal amplification of 23S ribosomal DNA followed by hybridization to an oligonucleotide array. J. Clin. Microbiol. 38, 781ā788.
Fleischmann R. D., Adams M. D., White O., et al. (1995). Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 496ā512.
Karlin S., Mrazek J., and Campbell A. M. (1996) Frequent oligonucleotides and peptides of the Haemophilus influenzae genome. Nucleic Acids Res. 21, 4263ā4272.
Gilsdorf J. R. (1998) Antigenic diversity and gene polymorphisms in Haemophilus influenzae. Infect. Immun. 66, 5053ā5059.
van Belkum A., Scherer S., van Leeuwen W., Willemse D., van Alphen L., and Verbrugh H. (1997) Variable number of tandem repeats in clinical strains of Haemophilus influenzae. Infect. Immun. 65, 5017ā5027.
Hood D.W., Deadman M. E., Jennings M. P., Bisercic M., Fleischmann R. D., Venter J. C., and Moxon E. R. (1996) DNA repeats identify novel virulence genes in Haemophilus influenzae. Proc. Natl. Acad. Sci. USA 93, 11,121ā11,125.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
Ā© 2003 Humana Press Inc.
About this protocol
Cite this protocol
van Belkum, A., van Alphen, L. (2003). Diagnosis of Infection. In: Herbert, M.A., Hood, D.W., Moxon, E.R. (eds) Haemophilus influenzae Protocols. Methods in Molecular Medicineā¢, vol 71. Humana Press. https://doi.org/10.1385/1-59259-321-6:71
Download citation
DOI: https://doi.org/10.1385/1-59259-321-6:71
Publisher Name: Humana Press
Print ISBN: 978-0-89603-928-5
Online ISBN: 978-1-59259-321-7
eBook Packages: Springer Protocols