Abstract
Diagnostic misidentifications of commensalic Haemophilus haemolyticus as pathogenic Haemophilus influenzae are frequent. This pilot study evaluates whether isolations of H. haemolyticus are frequent enough in Germany to cause a relevant diagnostic problem, considering the fact that even H. influenzae is a mere colonizer in about 30% of isolations.
In microbiological laboratories of two hospitals located in Northern and Southern Germany, the distribution of Haemophilus spp. was analyzed during a six-month-period. Site of infection, sex, and age of the patients was taken into consideration.
A total of 77 Haemophilus spp. isolates was acquired and discriminated on species level, comprising: 48 H. influenzae, 25 Haemophilus parainfluenzae, 3 H. haemolyticus, and 1 Haemophilus parahaemolyticus. The proportion of H. haemolyticus was calculated to range between 1.2% and 16.2 % within the 95% confidence limits. Commensalic Haemophilus spp. were isolated from oropharynx-associated sites only. H. influenzae, in contrast, was detected in clinically relevant materials like lower respiratory materials and conjunctiva swabs.
Altogether, there was a low proportion of clinical H. haemolyticus isolates. Accordingly, the problem of unnecessary antibiotic therapies due to misidentifications of H. haemolyticus as H. influenzae is quantitatively negligible compared with the risk of confusing H. influenzae colonizations with infections.
Keywords: antibiotic therapy, epidemiology, Germany, Haemophilus influenza, misidentification, upper respiratory tract infection
Introduction
Most upper respiratory tract infections (URTI) are caused by viral agents [1–7]. Among the bacterial infections, the Gram-negative rod-shaped bacterium Haemophilus influenzae is one of the quantitatively most important pathogens [8, 9]. As shown in our previous study [10], H. influenzae is frequently isolated from adult patients with recurrent tonsillitis and retropharyngeal abscesses [10–12], particularly in case of fine-needle aspiration [13]. Combined infections due to Staphylococcus aureus and H. influenzae were frequently observed [10, 14]. H. influenzae type b conjugate vaccines allow for an efficient protection against severe infections by this serotype in case of boostered vaccination [15]; however, there is no general cross-protection at the species level.
In spite of the pathogenic potential of H. influenzae, asymptomatic colonization of the upper respiratory tract (URT) by this microbial species occurs frequently [16, 17]. Colonization rates of the respiratory tract are particularly high in children and decrease with increase in age [17]. Altogether, a total of 30% of healthy volunteers was found to be colonized by H. influenzae in the respiratory tract [16].
Other Haemophilus spp. that are occasionally isolated from the URT include: Haemophilus parainfluenzae, Haemophilus haemolyticus, and Haemophilus parahaemolyticus. These species are phylogenetically related but virtually apathogenic. Altogether, Haemophilus spp. account for about 10% of the culturable bacterial flora of the URT [17]. H. parainfluenzae shows saprophytic growth in the URT but hardly ever causes local infections; however, infrequent cases of infectious endocarditis due to H. parainfluenzae have been described [18]. Although recent studies suggest occasional etiological relevance of H. haemolyticus [19], most studies describe this species as a commensal [20–22] without clinical importance.
In case of URTI, a reliable discrimination of H. influenzae from other Haemophilus spp. in the diagnostic routine is crucial due to the species-depending etiological relevance. However, as recently shown, traditional biochemical differentiation systems like api-NH (bioMérieux, Nürtingen, Germany) or VITEK-NH cards (bioMérieux) fail in 1–10% of the analyses [23–26]. Modern routine-compatible approaches like matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI–TOF–MS) or fluorescence in situ hybridization (FISH) do not score better in the discrimination of H. influenzae from other Haemophilus spp. as recently shown in our study [27].
The differentiation between H. influenzae and H. haemolyticus is particularly difficult. Most H. haemolyticus isolates lead to an alpha-hemolysis on horse, cow, or rabbit blood agar [28–30]. However, this phenomenon is of little practical relevance because sheep blood agar, where H. haemolyticus usually grows without hemolysis, is usually used in microbiological routine diagnostics. To make things even more complicated, nonhemolytic H. haemolyticus isolates are frequently cultured in microbiological routine diagnostics [22, 31].
Biochemical identification of Haemophilus spp. is frequently used in diagnostic routine conditions. Growth factor-based methods make use of the fact that H. influenzae lacks the enzymatic capacity to convert d-aminolevulinic acid (ALA) to protoporphyrin, hence, depends on factor X (heme) for growth [24, 32]. However, these approaches fail to discriminate H. haemolyticus from H. influenzae [27]. Sophisticated, labor-consuming, and expensive molecular approaches like ribosomal DNA sequence analysis, multilocus sequence analysis, DNA–DNA hybridization, and P6 gene sequencing reliably allow for such discrimination [22, 33]. MALDI–TOF–MS also allows for discrimination but on condition that a new H. haemolyticus mass spectrum is manually implemented as we previously showed [27]. However, the mass spectra of both species are quite similar. Accordingly, H. haemolyticus isolates will usually be misidentified as H. influenzae under routine conditions by a large proportion of diagnostic laboratories.
Consequently, in patients with URTI, confusions of H. haemolyticus to be H. influenzae will lead to unnecessary antibiotic therapies while the relevant, usually viral causative agent remains undetected. Retrospective analyses suggest that 10–40% presumed H. influenzae isolates were indeed H. haemolyticus [22, 33]. The present study tries to estimate the quantitative dimension and relevance of this problem in Germany to allow for a substantiated discussion about the need for more sophisticated approaches to discriminate within the Haemophilus genus in routine diagnostics: Are isolations of H. haemolyticus frequent enough in Germany to cause a relevant diagnostic problem? Or is there a higher risk of confusing mere H. influenzae colonization with real H. influenzae infections?
Materials and methods
Isolate collection and differentiation
During a six-month sampling period between June and November 2011, clinical Haemophilus spp. isolates were collected at the Institutes for Microbiology, Virology, and Hygiene of the University Hospitals Rostock, Germany and Ulm, Germany.
To become included into the analysis, reliable differentiation of the isolates on species level had to be guaranteed as follows. All included isolates were analyzed by two diagnostic MALDI–TOF–MS approaches at the Departments of Microbiology, Virology, and Hygiene of the University Hospitals Hamburg and Rostock, Germany, as well as by FISH at the Bernhard Nocht Institute for Tropical Medicine Hamburg, Germany. In case of contradicting results, partial 16S rRNA gene sequencing was used as the diagnostic gold standard. The test comparison describing the reliability of the different approaches has been previously published, and sequence information was deposited in FASTA format in the supplementary data [27]. Isolates for which individual test results were missing were not included into the study to assure standardized quality.
Isolate- and patient-related data
Isolate-specific information was documented for each included isolate. This information comprised the site of the presumed infection, age, and sex of the patients. Afterwards, patient data on each isolate were anonymized. The clinicians that sent the clinical samples provided all assessed information. Further inquiries on the wards regarding severity or other details of the disease were not performed.
Statistics
Regarding the parameter patient age, mean and median values were calculated for each Haemophilus sp.
With respect to the samples sizes, the distribution of the different Haemophilus sp. regarding site of infections, patient age, and patient sex was descriptively assessed.
Based on the assessed species distribution, the true proportion of H. haemolyticus among the subgroup of H. haemolyticus/H. influenzae isolates was estimated by calculating the 95% confidence interval using the Clopper–Pearson method [34] that leads to conservative only positive confidence limits [35].
Ethical approval
Ethical clearance for the analysis was obtained from Ethics Committee of the University Hospital Rostock, Germany.
Results
Distribution of Haemophilus spp. in relation to the sites of infection
In total, 60 clinical Haemophilus isolates from the University Hospital Rostock and 17 isolates from the University Hospital Ulm were included into the study [27] (Table 1). Isolates from Rostock comprised: 35 H. influenzae, 23 H. parainfluenzae, 1 H. haemolyticus, and 1 H. parahaemolyticus. Isolates from Ulm included: 13 H. influenzae, 2 H. parainfluenzae, and 2 H. haemolyticus.
Table 1.
Summarized presentation of the clinical Haemophilus isolates with details on clinical material, patient age, patient gender, and origin
| Species | Clinical material | Patient age (in years) | Patient gender | Origin |
|---|---|---|---|---|
| H. haemolyticus | Lower airways | 65 | Male | Ulm |
| H. haemolyticus | Lower airways | 61 | Female | Ulm |
| H. haemolyticus | Oropharynx | 9 | Female | Rostock |
| H. influenzae | Lower airways | 70 | Female | Rostock |
| H. influenzae | Lower airways | 55 | Male | Rostock |
| H. influenzae | Lower airways | 34 | Male | Rostock |
| H. influenzae | Lower airways | 67 | Male | Rostock |
| H. influenzae | Nasopharynx | 9 | Female | Rostock |
| H. influenzae | Nasopharynx | 0.67 | Female | Rostock |
| H. influenzae | Nasopharynx | 1 | Female | Rostock |
| H. influenzae | Nasopharynx | 1 | Male | Rostock |
| H. influenzae | Lower airways | 54 | Male | Rostock |
| H. influenzae | Conjunctiva | 6 | Male | Ulm |
| H. influenzae | Conjunctiva | 6 | Male | Ulm |
| H. influenzae | Lower airways | 44 | Male | Ulm |
| H. influenzae | Oropharynx | 14 | Male | Ulm |
| H. influenzae | Oropharynx | 23 | Female | Ulm |
| H. influenzae | Oropharynx | 14 | Male | Ulm |
| H. influenzae | Conjunctiva | 65 | Female | Ulm |
| H. influenzae | Lower airways | 45 | Male | Ulm |
| H. influenzae | Nasopharynx | 3 | Male | Rostock |
| H. influenzae | Lower airways | 51 | Male | Rostock |
| H. influenzae | Lower airways | 60 | Male | Rostock |
| H. influenzae | Nasopharynx | 2 | Male | Rostock |
| H. influenzae | Oropharynx | 2 | Male | Rostock |
| H. influenzae | Lower airways | 17 | Male | Rostock |
| H. influenzae | Nasopharynx | 1 | Female | Rostock |
| H. influenzae | Conjunctiva | 0.07 | Female | Rostock |
| H. influenzae | Lower airways | 48 | Female | Rostock |
| H. influenzae | Lower airways | 2 | Female | Rostock |
| H. influenzae | Nasopharynx | 10 | Male | Rostock |
| H. influenzae | Nasopharynx | 2 | Male | Rostock |
| H. influenzae | External ear | 1 | Male | Rostock |
| H. influenzae | Lower airways | 62 | Female | Rostock |
| H. influenzae | Nasopharynx | 2 | Male | Rostock |
| H. influenzae | Lower airways | 32 | Male | Rostock |
| H. influenzae | Lower airways | 52 | Female | Rostock |
| H. influenzae | Lower airways | 59 | Male | Rostock |
| H. influenzae | Lower airways | 48 | Male | Rostock |
| H. influenzae | Nasopharynx | 0.83 | Male | Rostock |
| H. influenzae | Lower airways | 59 | Female | Ulm |
| H. influenzae | Conjunctiva | 5 | Male | Ulm |
| H. influenzae | Nasopharynx | 0.06 | Female | Ulm |
| H. influenzae | Lower airways | 54 | Male | Ulm |
| H. influenzae | Conjunctiva | 37 | Male | Ulm |
| H. influenzae | Nasopharynx | 2 | Female | Rostock |
| H. influenzae | Paranasal sinus | 64 | Male | Rostock |
| H. influenzae | Lower airways | 22 | Female | Rostock |
| H. influenzae | External ear | 1 | Female | Rostock |
| H. influenzae | Nasopharynx | 2 | Female | Rostock |
| H. influenzae | Lower airways | 62 | Male | Rostock |
| H. parahaemolyticus | Oropharynx | 6 | Male | Rostock |
| H. parainfluenzae | Lower airways | 30 | Female | Rostock |
| H. parainfluenzae | Lower airways | 77 | Female | Rostock |
| H. parainfluenzae | Lower airways | 73 | Male | Rostock |
| H. parainfluenzae | Lower airways | 29 | Male | Rostock |
| H. parainfluenzae | Middle ear | Not assessed | Not assessed | Rostock |
| H. parainfluenzae | Lower airways | 58 | Male | Rostock |
| H. parainfluenzae | Lower airways | 67 | Female | Rostock |
| H. parainfluenzae | Lower airways | 25 | Male | Rostock |
| H. parainfluenzae | Lower airways | 28 | Male | Rostock |
| H. parainfluenzae | Lower airways | 2 | Female | Rostock |
| H. parainfluenzae | Lower airways | 61 | Male | Rostock |
| H. parainfluenzae | Lower airways | 61 | Male | Rostock |
| H. parainfluenzae | Lower airways | 61 | Male | Rostock |
| H. parainfluenzae | Lower airways | 82 | Female | Rostock |
| H. parainfluenzae | Oropharynx | 46 | Male | Rostock |
| H. parainfluenzae | Lower airways | 74 | Male | Rostock |
| H. parainfluenzae | Lower airways | 76 | Male | Ulm |
| H. parainfluenzae | Oropharynx | 38 | Female | Ulm |
| H. parainfluenzae | Oropharynx | 13 | Female | Rostock |
| H. parainfluenzae | Lower airways | 80 | Male | Rostock |
| H. parainfluenzae | Lower airways | 71 | Male | Rostock |
| H. parainfluenzae | Lower airways | 71 | Male | Rostock |
| H. parainfluenzae | Lower airways | 60 | Male | Rostock |
| H. parainfluenzae | Lower airways | 60 | Male | Rostock |
| H. parainfluenzae | Lower airways | 60 | Male | Rostock |
The presumed sites of infection of these 77 Haemophilus spp. were as follows: the lower airways (44 samples), the oropharynx (nine samples, three of them from known mucoviscidosis patients), the nasopharynx (14 samples), the conjunctiva (six samples), the external ear (two samples), the middle ear (one sample), and the paranasal sinus (one sample) (Table 2).
Table 2.
Distribution of Haemophilus isolates regarding clinical material, patient age, and patient sex
| Haemophilus spp. | H. influenzae | H. parainfluenzae | H. haemolyticus | H. parahaemolyticus | |
|---|---|---|---|---|---|
| Distribution of presumed sites of
infection | |||||
| Lower airways | 44 | 21 (48%) | 21 (48%) | 2 (5%) | – |
| Nasopharynx | 14 | 14 (100%) | – | – | – |
| Oropharynx | 9 | 4 (including 3/3 mucoviscidosis patients) (44%) | 3 (33%) | 1 (11%) | 1 (11%) |
| Conjunctiva | 6 | 6 (100%) | – | – | – |
| External ear | 2 | 2 (100%) | – | – | – |
| Middle ear | 1 | – | 1 (100%) | – | – |
| Paranasal sinus | 1 | 1 (100%) | – | – | – |
| Total | 77 (100%) | 48 (62%) | 25 (32%) | 3 (4%) | 1 (1%) |
| Distribution of patient
age | |||||
| Median age (years) | 37.5 (0.058–82) | 15.5 (0.058–70) | 60.5 (2–82) | 61 (9–65) | 6 (–) |
| Mean age (years) (± standard deviation) | 35.7 (± 27.7) | 26.5 (± 25.5) | 54.3 (± 22.5) | 45 (± 31.2) | 6 |
| Distribution of patient
sex | |||||
| Male | 49 (64%) | 30 (62.5%) | 17 (70.8%) | 1 (33.3%) | 1 (100%) |
| Female | 27 (36%) | 18 (37.5%) | 7 (29.2%) | 2 (66.6%) | 0 (0%) |
The chances of isolating H. influenzae or other Haemophilus sp. strains with low pathogenic potential from the lower airways and other directly oropharynx-associated sites including the middle ear were nearly equal. All four H. haemolyticus or H. parahaemolyticus isolates were isolated either from the lower airways or the oropharynx. In contrast, only H. influenzae was isolated from the nasopharynx, the paranasal sinus, the external ear, and the conjunctiva.
Distribution of Haemophilus spp. in relation to sex and age
In all instances except one, information on patients’ age and sex was extractable from the accompanying forms. The remaining single isolate, a H. parainfluenzae isolate, was collected at the University of Rostock from an anonymous patient who visited the University of Rostock hospital complaining of an URT infection in the course of the study [36].
The 76 patients comprised 49 men and 27 women. 62.5% (30/48) of all H. influenzae isolates originate from male and, therefore, 37.5% (18/48) from female patients. A comparable male–female ratio was observed for H. parainfluenzae. There, 70.8% (17/25) of the isolates were cultured from men and 29.2% (7/25) from women. Thus, the sex-specific distributions of H. influenzae and H. parainfluenzae were similar to the overall sex distribution of the study participants. The three cultured H. haemolyticus isolates were from two men and one woman, while the single H. parahaemolyticus isolate was obtained from a man. Table 2 shows a summary of the distribution of Haemophilus isolates from men and women.
The median age of the participating patients was 37.5 (0.058–82) years, and the mean age was 35.7 (±27.7) years (standard deviation (SD) in brackets). H. haemolyticus was isolated from a nine-year-old child and two patients aged 61 and 65 years; H. parahaemolyticus was isolated from a six-year-old child. H. influenzae strains were predominantly isolated from young patients: median age of 15.5 (0.058–70 years), whereas H. parainfluenzae isolates were more frequent in the elderly: median age of 60.5 (2–82) years (Table 2).
Proportion of H. haemolyticus among the subgroup of H. haemolyticus/H. influenzae isolates
Three H. haemolyticus isolates and 48 H. influenzae isolates were cultured. H. haemolyticus isolates were identified in a proportion of three out of 51 (5.88%) of the H. haemolyticus/H. influenzae subgroup. With respect to the total number of samples, the 95% confidence interval was 5.9% ± 6.4%. The application of the Clopper–Pearson method identified a range between 1.2% and 16.2 % within the 95% confidence limits.
Discussion
In this study, we have used an expensive but reliable technique to identify clinical Haemophilus spp. isolates from two German University Hospitals and to determine their distribution on species level. As expected, due to the sample size, no obvious age and sex distribution patterns in the isolation frequency were observed with the exception of H. influenzae, which was frequently isolated from young patients and H. parainfluenzae that was frequently isolated from the elderly. This result is in line with the previously observed phenomenon of decreasing colonization rates of the upper respiratory tract with H. influenzae with increasing age [17]. However, the practical relevance regarding therapeutic decisions is low because the discrimination of H. influenzae and H. parainfluenzae is highly reliable in routine diagnostics [27].
H. haemolyticus and H. parahaemolyticus were too infrequently isolated to draw any conclusions regarding their distribution in the population. This can be attributed to the sample size and/or the geographical regions of the study. Importantly, the results that we obtained during this study reveal two very interesting observations. Firstly, the observation that all H. parainfluenzae, H. haemolyticus, and H. parahaemolyticus were isolated from materials directly associated to the oropharynx. Secondly, the observation that H. influenza was isolated from the nasopharynx, the paranasal sinus, the external ear, and the conjunctiva-associated materials, indicates its clinical relevance. Our data is in line with previous investigations that H. influenzae accounts for less than 2% of the Haemophilus burden in the pharynx [17, 37]. However, we strongly suggest further studies to be performed from larger population sizes probably nationwide to confirm or exclude site-specific differences in the colonization frequency with different Haemophilus spp. and to confine colonization with species of low pathogenic potential on sites in the oropharynx. Our pilot study about 6 months delivers preliminary data only.
Nevertheless, the data suggest that colonization or infection due to H. haemolyticus is rare at least in severely ill patients who are treated in German University Hospitals. Previously published rates of 10–40% from other geographic regions [22, 33] were not confirmed by our data that suggested a ratio of 5.9% ± 6.4%. In contrast, a previous analysis of healthy volunteers suggested a colonization rate of 30% with the facultative pathogenic species H. influenzae [16]. Even when considering that the H. haemolyticus proportion among those isolates from healthy individuals could be as high as 10%, there is a considerable and at least two- to threefold higher risk of misidentifying an H. influenzae colonization as an H. influenzae infection than an H. haemolyticus colonization as an H. influenzae infection in German URTI patients. In contrast, a reliable discrimination based on sophisticated, expensive, and laborious molecular procedures like ribosomal DNA sequence analysis, multilocus sequence analysis, DNA–DNA hybridization, and P6 gene sequencing [22, 33] is hardly realistic in the microbiological routine laboratory at present due to its labor efforts and costs.
Accordingly, the diagnosis of H. influenzae in case of URTI has to be interpreted with care, both due to the risk of misidentifying H. haemolyticus as H. influenzae [22, 27, 33] and due to the high overall colonization rate with H. influenzae [16]. Therefore, this microbiological diagnosis can only be considered as one piece of information for narrowing the differential diagnosis of URTI and, consecutively, finding reasons for the administration of antibiotic drugs.
The lately developed and readily available H. haemolyticus mass spectrum [27] that is based on data from the reference strain H. haemolyticus ATCC 33390 is recently the most reliable to discriminate H. haemolyticus and H. influenzae. However, the mass spectrum differences between these species are marginal [27] and more evaluations with higher numbers of well-characterized isolates are needed to estimate the diagnostic accuracy of this procedure.
Acknowledgements
This publication was funded by the Open Access support program of the Deutsche Forschungsgemeinschaft and the publication fund of the Georg August Universität Göttingen. I am grateful to Wycliffe O. Masanta for revising the text of the article.
Footnotes
Potential conflicts of interest: All authors report no potential conflicts of interest.
Contributor Information
H. Frickmann, 1 Fachbereich Tropenmedizin am Bernhard-Nocht Institut, Bundeswehrkrankenhaus Hamburg, Hamburg, Germany; 2 Institut für Mikrobiologie, Virologie und Hygiene, Universitätsmedizin Rostock, Rostock, Germany.
A. Podbielski, 2 Institut für Mikrobiologie, Virologie und Hygiene, Universitätsmedizin Rostock, Rostock, Germany.
A. Essig, 3 Institut für Mikrobiologie, Virologie und Hygiene, Universitätsklinikum Ulm, Ulm, Germany.
N. G. Schwarz, 4 AG Infektionsepidemiologie, Bernhard-Nocht Institut für Tropenmedizin Hamburg, Hamburg, Germany.
A. E. Zautner, 2 Institut für Mikrobiologie, Virologie und Hygiene, Universitätsmedizin Rostock, Rostock, Germany; 5 UMG-Labor – Institut für Klinische Chemie/Zentrallabor, Universitätsmedizin Göttingen, Göttingen, Germany; 6 Institut für Medizinische Mikrobiologie, Universitätsmedizin Göttingen, Göttingen, Germany.
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