Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
letter
. 2020 Aug 24;58(9):e00278-20. doi: 10.1128/JCM.00278-20

Discriminative Potential of the Vitek MS In Vitro Diagnostic Device Regarding Haemophilus influenzae and Haemophilus haemolyticus

Sebastian Nürnberg a, Heike Claus a, Manuel Krone a, Ulrich Vogel a, Thiên-Trí Lâm a,✉
Editor: Yi-Wei Tangb
PMCID: PMC7448641  PMID: 32404483

LETTER

Haemophilus influenzae can cause respiratory and invasive infections (1), whereas H. haemolyticus (Hh) is generally considered apathogenic (2) and is rarely associated with disease (3). Since both species colonize the upper respiratory tract, their distinction is crucial in routine diagnostics (4).

Difficulties in discriminating H. influenzae and H. haemolyticus using the MALDI Biotyper system (Bruker Daltonics, Bremen, Germany) (5) were solved by integrating additional H. haemolyticus spectra into the database (5, 6). Other studies hint at similar problems with Vitek MS (bioMérieux, Marcy-l’Etoile, France) but were carried out with small numbers of Haemophilus spp., and species diagnosis was not sufficiently assured (7–9). Our objective was to verify the Vitek MS IVD V3 identification of H. influenzae and H. haemolyticus as available in a medical laboratory by using a significant number of well-defined isolates. The IVD database is CE certified and, therefore, is suitable for routine diagnostics in accredited laboratories. It contains 210 H. influenzae and 26 H. haemolyticus spectra for the differentiation of these two species.

The 286 strains of this study included 236 invasive clinical H. influenzae isolates from blood and cerebrospinal fluid and 50 clinical H. haemolyticus strains. All strains were characterized by colony morphology, Gram staining, oxidase test, and factor-dependent growth using BBL Hemo ID Quad (BD, Heidelberg, Germany). H. influenzae isolates additionally were serotyped by slide agglutination and PCR. The species-specific genes fucK (10) and ompP2 (11, 12) were amplified to confirm H. influenzae. If both genes were missing, ompP6 was sequenced to diagnose H. haemolyticus (13). Multilocus sequence typing (MLST) was performed on 108 H. influenzae and 43 H. haemolyticus isolates by concatenating the sequences of the six loci adk, atpG, frdB, mdh, pgi, and recA (14–16). The resulting contigs were aligned with four reference strains from each species (17) using MEGA-X software 10.0.5 (18). A neighbor-joining net, created with SplitsTree4 14.15.1 software (19), separated H. haemolyticus and H. influenzae strains into two distinct genogroups with diversity within the two groups (Fig. 1). Therefore, MLST not only confirmed the species assignment (15) but also showed high genetic diversity. Comparison of the MLST sequences to the Vitek MS results did not show remarkable differences between the sequence types of strains with correct and incorrect/ambiguous identification by Vitek MS.

FIG 1.

FIG 1

Open in a new tab

Neighbor-net representation of the concatenated H. influenzae and H. haemolyticus MLST sequences using Splitstree4 software. H. influenzae and H. haemolyticus strains constitute two distinct clusters (boxes A and B). The strains within each cluster are genetically diverse. Strain numbers are marked with Hh for H. haemolyticus, Hi for H. influenzae, and Hp for H. parainfluenzae.

All strains were tested in duplicate with Vitek MS according to the manufacturer’s instructions. Therefore, each strain was cultured on chocolate agar plates supplemented with hemin and NAD (chocolate agar PolyViteX; bioMérieux) and incubated at 35°C ± 1°C with 5% CO2 for 24 h. We applied colony material of one strain onto a target spot. For the duplicate measurement, a similar amount of material from different colonies of the same isolate was applied to another spot. According to the manufacturer, there is a “certain identification” if the confidence value ranges between 60 and 99.9 and an “uncertain identification” if the confidence value is below 60. When there were various results in the duplicate measurements, the result with the higher confidence value was taken. According to these criteria, the Vitek MS showed a certain identification for 277 out of 286 tested isolates and an uncertain identification for 9 isolates.

The Vitek MS showed a concordance of 100% for H. influenzae with certain and correct identification of all H. influenzae isolates. Correct results were found for 19 out of 50 H. haemolyticus isolates. However, in 22 H. haemolyticus isolates the Vitek MS results did not correspond to the reference method: 21 were indicated as H. influenzae and one as H. parainfluenzae. Nine H. haemolyticus isolates showed uncertain identification between H. influenzae and H. haemolyticus (Tables 1 and 2). As a result, 42% of H. haemolyticus isolates were misidentified as H. influenzae. As a consequence, a strain identified as H. influenzae using Vitek MS may not necessarily be H. influenzae but potentially is H. haemolyticus. This reduces the positive predictive value for the Vitek MS diagnosis of H. influenzae and might lead to false or unnecessary therapy. We are the first to examine this topic using a large number of unambiguously defined isolates of these two species, and the findings are in accordance with those of other reports (5, 7–9). Most diagnostic studies have been carried out using the Bruker MALDI Biotyper and showed a sensitivity ranging between 76.9% and 100% for the identification of H. influenzae but a low specificity for H. haemolyticus. Some investigators included more reference strains in the database for the MALDI Biotyper, which resulted in improved accuracy (5, 6). We assume that this also could be achieved with the Vitek MS IVD, which would make it a more precise diagnostic technique to distinguish the two Haemophilus spp.

TABLE 1.

Characteristics of all H. haemolyticus strains tested by Vitek MS IVDa

Isolate no. Clinical source Molecular tests for:
 ID from reference method Vitek MS results (confidence value and ID) for spot:
ompP2 fucK ompP6 1 2
H2172 Throat swab N N H. haemolyticus H. haemolyticus 99.9 H. haemolyticus 99.9 H. haemolyticus
H2518 Throat swab N N H. haemolyticus H. haemolyticus 99.9 H. haemolyticus 99.9 H. haemolyticus
H2757 Sputum N N H. haemolyticus H. haemolyticus 99.9 H. haemolyticus 99.9 H. haemolyticus
H2761 Throat swab N N H. haemolyticus H. haemolyticus 99.9 H. haemolyticus 99.9 H. haemolyticus
H2778 Tracheal secretion N N H. haemolyticus H. haemolyticus 99.9 H. haemolyticus 99.9 H. haemolyticus
H2883 Throat swab N N H. haemolyticus H. haemolyticus 99.9 H. haemolyticus 99.9 H. haemolyticus
H2922 Throat swab N N H. haemolyticus H. haemolyticus 99.9 H. haemolyticus 99.9 H. haemolyticus
H1148 Blood N N H. haemolyticus H. haemolyticus 99.9 H. haemolyticus No ID
H1530 Blood N N H. haemolyticus H. haemolyticus 99.9 H. haemolyticus 52.8 H. haemolyticus; 47.1 H. influenzae
H2263 Throat swab N N H. haemolyticus H. haemolyticus 99.9 H. haemolyticus 57.5 H. haemolyticus; 42.4 H. influenzae
H2433 Throat swab N N H. haemolyticus H. haemolyticus 52.8 H. haemolyticus; 47.1 H. influenzae 99.9 H. haemolyticus
H2588 Throat swab N N H. haemolyticus H. haemolyticus 53.4 H. haemolyticus; 46.5 H. influenzae 99.9 H. haemolyticus
H2589 Tracheal secretion N N H. haemolyticus H. haemolyticus 50.7 H. haemolyticus; 49.2 H. influenzae 99.9 H. haemolyticus
H2872 Throat swab N N H. haemolyticus H. haemolyticus 99.9 H. haemolyticus 99.0 H. influenzae
H2934 Throat swab N N H. haemolyticus H. haemolyticus No ID 99.9 H. haemolyticus
H3035 Throat swab N N H. haemolyticus H. haemolyticus 99.4 H. influenzae 99.9 H. haemolyticus
H3045 Throat swab N N H. haemolyticus H. haemolyticus 99.9 H. haemolyticus 50.2 H. haemolyticus; 49.7 H. influenzae
H3470 Sputum N N H. haemolyticus H. haemolyticus 99.9 H. haemolyticus 53.5 H. haemolyticus; 46.4 H. influenzae
LNP30497 Vaginal swab N N H. haemolyticus H. haemolyticus 45.5 H. influenzae; 54.4 H. haemolyticus 99.9 H. haemolyticus
H1495 Throat swab N N H. haemolyticus H. haemolyticus 50.3 H. haemolyticus; 49.6 H. influenzae 53.6 H. haemolyticus; 46.6 H. influenzae
H1613 Blood N N H. haemolyticus H. haemolyticus 50.0 H. haemolyticus, 49.9 H. influenzae 56.6 H. haemolyticus; 43.3 H. influenzae
H2121 Sputum N N H. haemolyticus H. haemolyticus 50.1 H. haemolyticus; 49.8 H. influenzae 50.0 H. haemolyticus; 49.9 H. influenzae
H2129 Throat swab N N H. haemolyticus H. haemolyticus 51.2 H. haemolyticus; 48.7 H. influenzae 50.0 H. haemolyticus; 49.9 H. influenzae
H2735 Sputum N N H. haemolyticus H. haemolyticus 50.0 H. influenzae; 49.9 H. haemolyticus 50.0 H. influenzae; 49.9 H. haemolyticus
H2943 Throat swab N N H. haemolyticus H. haemolyticus 52.8 H. haemolyticus; 47.1 H. influenzae 54.9 H. haemolyticus; 45.0 H. influenzae
H2947 Tracheal secretion N N H. haemolyticus H. haemolyticus 50.0 H. haemolyticus; 50.0 H. influenzae 50.0 H. haemolyticus; 49.9 H. influenzae
H3050 Throat swab N N H. haemolyticus H. haemolyticus 50.0 H. haemolyticus; 49.9 H. influenzae 50.0 H. influenzae; 49.9 H. haemolyticus
H3659 Throat swab N N H. haemolyticus H. haemolyticus 50.0 H. influenzae; 50.0 H. haemolyticus 49.9 H. influenzae; 50.0 H. haemolyticus
H2288 Blood N N H. haemolyticus H. haemolyticus 99.9 H. influenzae 99.9 H. influenzae
H2297 Sputum N N H. haemolyticus H. haemolyticus 96.7 H. influenzae 99.9 H. influenzae
H2299 Sputum N N H. haemolyticus H. haemolyticus 61.5 H. influenzae No ID
H2300 Throat swab N N H. haemolyticus H. haemolyticus 99.9 H. influenzae 99.9 H. influenzae
H2311 Sputum N N H. haemolyticus H. haemolyticus No ID 99.9 H. influenzae
H2317 Sputum N N H. haemolyticus H. haemolyticus 99.9 H. parainfluenzae 99.9 H. parainfluenzae
H2331 Throat swab N N H. haemolyticus H. haemolyticus 49.9 H. haemolyticus; 50.0 H. influenzae 99.9 H. influenzae
H2347 Sputum N N H. haemolyticus H. haemolyticus 94.8 H. influenzae 99.9 H. influenzae
H2498 Blood N N H. haemolyticus H. haemolyticus 96.0 H. influenzae No ID
H2645 Throat swab N N H. haemolyticus H. haemolyticus 49.6 H. influenzae; 50.3 H. haemolyticus 99.9 H. influenzae
H2722 Sputum N N H. haemolyticus H. haemolyticus 50.0 H. haemolyticus; 49.9 H. influenzae 99.9 H. influenzae
H2822 Sputum N N H. haemolyticus H. haemolyticus 45.1 H. influenzae; 54.8 H. haemolyticus 99.6 H. influenzae
H2939 Tracheal secretion N N H. haemolyticus H. haemolyticus 99.9 H. influenzae 99.9 H. influenzae
H2944 Throat swab N N H. haemolyticus H. haemolyticus 99.9 H. influenzae 99.9 H. influenzae
H2946 Throat swab N N H. haemolyticus H. haemolyticus 50.3 H. haemolyticus; 49.6 H. influenzae 99.4 H. influenzae
H2961 Tracheal secretion N N H. haemolyticus H. haemolyticus 99.9 H. influenzae 99.9 H. influenzae
H3386 Throat swab N N H. haemolyticus H. haemolyticus 99.9 H. influenzae 99.9 H. influenzae
H3622 Throat swab N N H. haemolyticus H. haemolyticus 50.0 H. haemolyticus; 50.0 H. influenzae 99.7 H. influenzae
LNP29052 Eye swab N N H. haemolyticus H. haemolyticus 91.0 H. influenzae 60.9 H. influenzae
LNP29248 Bronchoalveolar lavage N N H. haemolyticus H. haemolyticus 97.0 H. influenzae No ID
LNP29731 Eye swab N N H. haemolyticus H. haemolyticus No ID 83.8 H. influenzae
LNP30199 Eye swab N N H. haemolyticus H. haemolyticus 99.9 H. influenzae 99.9 H. influenzae
Open in a new tab
a

Most isolates showed ambiguous species identification (ID) or incorrect results in at least one of the measurements using Vitek MS. Strains with correct identification that matched the reference methods are listed first. Strains with unambiguous results (uncertain identification) and strains with no correct identification (ID not in concordance with the reference methods) are found in the lower sections. P, positive; N, negative; ND, not determined; ID, identification; no ID, no identification.

TABLE 2.

Summary of Vitek MS results and the concordance with the reference method

ID by reference method Identification (no.)
Certain Uncertain Concordance with the reference method
H. influenzae (n = 236) 236 0 236
H. haemolyticus (n = 50) 41 9 19
Total (n = 286) 277 9 255
Open in a new tab

ACKNOWLEDGMENTS

We are thankful to all laboratories that submitted isolates to the NRZMHi and to the cooperating German local health authorities. We thank Muhamed-Kheir Taha (Institut Pasteur, Paris) for contributing H. haemolyticus strains. Furthermore, we thank Sabrina Hebling, Alexandra Sikora, Beate Stirzel, Theresa Höferth, and Lena Zubrod for their excellent technical assistance.

The NRZMHi is supported by the Robert Koch-Institute with funds of the Federal Ministry of Health (funding code 1369-237).

REFERENCES

  • 1.Turk DC. 1984. The pathogenicity of Haemophilus influenzae. J Med Microbiol 18:1–16. doi: 10.1099/00222615-18-1-1. [DOI] [PubMed] [Google Scholar]
  • 2.Mukundan D, Ecevit Z, Patel M, Marrs CF, Gilsdorf JR. 2007. Pharyngeal colonization dynamics of Haemophilus influenzae and Haemophilus haemolyticus in healthy adult carriers. J Clin Microbiol 45:3207–3217. doi: 10.1128/JCM.00492-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Anderson R, Wang X, Briere EC, Katz LS, Cohn AC, Clark TA, Messonnier NE, Mayer LW. 2012. Haemophilus haemolyticus isolates causing clinical disease. J Clin Microbiol 50:2462–2465. doi: 10.1128/JCM.06575-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hinz R, Zautner AE, Hagen RM, Frickmann H. 2015. Difficult identification of Haemophilus influenzae, a typical cause of upper respiratory tract infections, in the microbiological diagnostic routine. Eur J Microbiol Immunol 5:62–67. doi: 10.1556/EUJMI-D-14-00033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Chen JHK, Cheng VCC, Wong CP, Wong SCY, Yam WC, Yuen KY. 2017. Rapid Differentiation of Haemophilus influenzae and Haemophilus haemolyticus by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry with ClinProTools mass spectrum analysis. J Clin Microbiol 55:2679–2685. doi: 10.1128/JCM.00267-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Bruin JP, Kostrzewa M, van der Ende A, Badoux P, Jansen R, Boers SA, Diederen BM. 2014. Identification of Haemophilus influenzae and Haemophilus haemolyticus by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Eur J Clin Microbiol Infect Dis 33:279–284. doi: 10.1007/s10096-013-1958-x. [DOI] [PubMed] [Google Scholar]
  • 7.Broyer P, Perrot N, Rostaing H, Blaze J, Pinston F, Gervasi G, Charles MH, Dachaud F, Dachaud J, Moulin F, Cordier S, Dauwalder O, Meugnier H, Vandenesch F. 2018. An automated sample preparation instrument to accelerate positive blood cultures microbial identification by MALDI-TOF mass spectrometry (Vitek MS). Front Microbiol 9:911. doi: 10.3389/fmicb.2018.00911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Powell EA, Blecker-Shelly D, Montgomery S, Mortensen JE. 2013. Application of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of the fastidious pediatric pathogens Aggregatibacter, Eikenella, Haemophilus, and Kingella. J Clin Microbiol 51:3862–3864. doi: 10.1128/JCM.02233-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.López-Fabal MF, Gómez-Garcés JL, López-Hontangas JL, Sanz N, Muñoz C, Regodón M. 2015. Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry for identifying respiratory bacterial pathogens: a fast and efficient method. Rev Esp Quimioter 28:242–246. [PubMed] [Google Scholar]
  • 10.Norskov-Lauritsen N. 2009. Detection of cryptic genospecies misidentified as Haemophilus influenzae in routine clinical samples by assessment of marker genes fucK, hap, and sodC. J Clin Microbiol 47:2590–2592. doi: 10.1128/JCM.00013-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Binks MJ, Temple B, Kirkham LA, Wiertsema SP, Dunne EM, Richmond PC, Marsh RL, Leach AJ, Smith-Vaughan HC. 2012. Molecular surveillance of true nontypeable Haemophilus influenzae: an evaluation of PCR screening assays. PLoS One 7:e34083. doi: 10.1371/journal.pone.0034083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Hobson RP, Williams A, Rawal K, Pennington TH, Forbes KJ. 1995. Incidence and spread of Haemophilus influenzae on an Antarctic base determined using the polymerase chain reaction. Epidemiol Infect 114:93–103. doi: 10.1017/s0950268800051943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Murphy TF, Brauer AL, Sethi S, Kilian M, Cai X, Lesse AJ. 2007. Haemophilus haemolyticus: a human respiratory tract commensal to be distinguished from Haemophilus influenzae. J Infect Dis 195:81–89. doi: 10.1086/509824. [DOI] [PubMed] [Google Scholar]
  • 14.Meats E, Feil EJ, Stringer S, Cody AJ, Goldstein R, Kroll JS, Popovic T, Spratt BG. 2003. Characterization of encapsulated and noncapsulated Haemophilus influenzae and determination of phylogenetic relationships by multilocus sequence typing. J Clin Microbiol 41:1623–1636. doi: 10.1128/jcm.41.4.1623-1636.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.McCrea KW, Xie J, LaCross N, Patel M, Mukundan D, Murphy TF, Marrs CF, Gilsdorf JR. 2008. Relationships of nontypeable Haemophilus influenzae strains to hemolytic and nonhemolytic Haemophilus haemolyticus strains. J Clin Microbiol 46:406–416. doi: 10.1128/JCM.01832-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Norskov-Lauritsen N, Overballe MD, Kilian M. 2009. Delineation of the species Haemophilus influenzae by phenotype, multilocus sequence phylogeny, and detection of marker genes. J Bacteriol 191:822–831. doi: 10.1128/JB.00782-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.National Center for Biotechnology Information. 1988. Nucleotide. National Library of Medicine, National Center for Biotechnology Information, Bethesda, MD: https://www.ncbi.nlm.nih.gov/nucleotide/. Accessed 28 October 2019. [Google Scholar]
  • 18.Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. doi: 10.1093/molbev/msy096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Huson DH, Bryant D. 2006. Application of phylogenetic networks in evolutionary studies. Mol Biol Evol 23:254–267. doi: 10.1093/molbev/msj030. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES