MALDI-TOF MS Distinctly Differentiates Nontypable Haemophilus influenzae from Haemophilus haemolyticus

Zhu, Bingqing; Xiao, Di; Zhang, Huifang; Zhang, Yongchan; Gao, Yuan; Xu, Li; Lv, Jing; Wang, Yingtong; Zhang, Jianzhong; Shao, Zhujun

doi:10.1371/journal.pone.0056139

Cited by 40 publications

(30 citation statements)

References 27 publications

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“…However, it may not be able to provide sufficient resolution for subtyping of bacterial pathogens, which is often performed in public health microbiology laboratories for disease surveillance and other large epidemiological surveys. The power of MALDI-TOF for identification of H. haemolyticus and H. influenzae is highly dependent on a well-defined reference spectrum for H. haemolyticus in species databases, which varies between laboratories (35,36). Its utility in discriminating between H. haemolyticus and H. influenzae remains to be further validated.…”

Section: Discussionmentioning

confidence: 99%

Comparative Genomic Analysis of Haemophilus haemolyticus and Nontypeable Haemophilus influenzae and a New Testing Scheme for Their Discrimination

Rishishwar

Sivadas

et al. 2016

J Clin Microbiol

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g Haemophilus haemolyticus has been recently discovered to have the potential to cause invasive disease. It is closely related to nontypeable Haemophilus influenzae (NT H. influenzae). NT H. influenzae and H. haemolyticus are often misidentified because none of the existing tests targeting the known phenotypes of H. haemolyticus are able to specifically identify H. haemolyticus. Through comparative genomic analysis of H. haemolyticus and NT H. influenzae, we identified genes unique to H. haemolyticus that can be used as targets for the identification of H. haemolyticus. A real-time PCR targeting purT (encoding phosphoribosylglycinamide formyltransferase 2 in the purine synthesis pathway) was developed and evaluated. The lower limit of detection was 40 genomes/PCR; the sensitivity and specificity in detecting H. haemolyticus were 98.9% and 97%, respectively. To improve the discrimination of H. haemolyticus and NT H. influenzae, a testing scheme combining two targets (H. haemolyticus purT and H. influenzae hpd, encoding protein D lipoprotein) was also evaluated and showed 96.7% sensitivity and 98.2% specificity for the identification of H. haemolyticus and 92.8% sensitivity and 100% specificity for the identification of H. influenzae, respectively. The dual-target testing scheme can be used for the diagnosis and surveillance of infection and disease caused by H. haemolyticus and NT H. influenzae. Haemophilus haemolyticus is a human commensal that colonizes the respiratory tract. It shares high similarities in morphology, biochemistry, and genetics with nontypeable Haemophilus influenzae (NT H. influenzae) (1-3). NT H. influenzae has emerged as the major cause of invasive H. influenzae disease since the implementation of H. influenzae serotype b (H. influenzae b) vaccine (4). NT H. influenzae infections such as childhood otitis media and respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD) result in an enormous social and economic burden to societies (5). H. haemolyticus was recently reported to cause invasive disease (1). These cases were previously misidentified as NT H. influenzae due to the lack of proper identification methods to discriminate between the two species (6). Reevaluation of NT H. influenzae cases from previous years revealed that about 2% (7/374) of the NT H. influenzae invasive cases reported from Active Bacterial Core surveillance (ABCs) were in fact caused by H. haemolyticus (1).Misidentification of H. haemolyticus as NT H. influenzae has been repeatedly reported by multiple research groups, with up to 40% of isolates being misidentified as NT H. influenzae using classical phenotypic methods in clinical microbiology laboratories (1, 7-11). The misidentification of H. haemolyticus has a potential impact on accurate assessment of the prevalence of antibioticresistant NT H. influenzae (12). Therefore, a rapid and accurate method is needed to distinguish H. haemolyticus from NT H. influenzae and to better understand the epidemiology of H. haemolyticus infections....

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Section: Discussionmentioning

confidence: 99%

Comparative Genomic Analysis of Haemophilus haemolyticus and Nontypeable Haemophilus influenzae and a New Testing Scheme for Their Discrimination

Rishishwar

Sivadas

et al. 2016

J Clin Microbiol

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“…This is rather different from the observation of Sethi et al 1 , who reported 145 cases of new H. influenzae acquisition versus 1503 cases of no new strain acquisition (8.7%). Incorrect identification of H. haemolyticus as H. influenzae cannot be the cause of this difference, because we used the advanced version of database for MALDI-TOF MS identification 10 . However, in the study of Sethi et al 1 , COPD patients were requested to come for screening for bacterial presence in sputum at regular monthly intervals regardless of their health status, unlike in our study, where sputum was sampled from patients who sought medical care or were treated during hospital stay.…”

Section: Discussionmentioning

confidence: 99%

McRAPD unlike MALDI-TOF MS is a suitable candidate for routine discrimination of new Haemophilus influenzae strain acquisition in chronic obstructive pulmonary disease (COPD) and cystic fibrosis

Raclavsky¹,

Stromerova

Šafářová

et al. 2016

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub

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a Background and Aims. Haemophilus influenzae new strain acquisition has been demonstrated to increase the relative risk of acute exacerbation fourfold in contrast to colonisation or chronic infection by the same strain in chronic obstructive pulmonary disease (COPD). Unfortunately, molecular typing techniques are not suitable for routine use due to cost, labour-intensity and need for special expertise. We tested two techniques potentially useful for routine typing, namely the newly available MALDI-TOF MS and the modified McRAPD compared to MLST as the gold standard. Methods. In 10 patients (10.8%) suffering from COPD or cystic fibrosis, H. influenzae isolates were recovered repeatedly at different timepoints from the same patient during the study period. This allowed for thirteen pairwise comparisons of typing results in isolates recovered consecutively from the same patient to test the ability of the techniques to uncover new strain acquisition. Results. MLST detected 9 cases of new strain acquisition among the 13 pairwise comparisons. However, MALDI-TOF MS reported all 13 pairs as different and thus new. In contrast, McRAPD was able to differentiate all the new strain acquisitions from pre-existing ones, both by visual inspection of melting profiles and by Relative Significant Difference values. Conclusions. Unlike MALDI-TOF MS, McRAPD appears to be a suitable candidate for routine discrimination of new strain acquisitions because of its accuracy and, rapid, easy and economic performance.

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“…If this technology is available and suitable reference spectra are included in the database, MALDI-TOF-MS has the potential to provide diagnostic results with sufficient reliability for the diagnostic routine situation [40][41][42]. For less well-equipped laboratories, FISH might be a cost-effi cient and easy-to-perform alternative but will not provide conclusive results in all instances [40].…”

Section: Discussionmentioning

confidence: 99%

“…Suitability of MALDI-TOF-MS for the discrimination of H. infl uenzae from H. haemolyticus has been described in 2013 [40,41] and confi rmed in 2014 [42]. Nevertheless, the availability of a well-defi ned reference spectrum for H. haemolyticus in the applied database is an important prerequisite for this.…”

Section: Matrix-assisted Laser-desorption-ionization Time-of-flight Mmentioning

confidence: 99%

Difficult identification ofHaemophilus influenzae, a typical cause of upper respiratory tract infections, in the microbiological diagnostic routine

Hinz¹,

Zautner²,

Hagen³

et al. 2015

European Journal of Microbiology and Immunology

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Haemophilus influenzae is a key pathogen of upper respiratory tract infections. Its reliable discrimination from nonpathogenic Haemophilus spp. is necessary because merely colonizing bacteria are frequent at primarily unsterile sites. Due to close phylogenetic relationship, it is not easy to discriminate H. influenzae from the colonizer Haemophilus haemolyticus. The frequency of H. haemolyticus isolations depends on factors like sampling site, patient condition, and geographic region.Biochemical discrimination has been shown to be nonreliable. Multiplex PCR including marker genes like sodC, fucK, and hpd or sequencing of the 16S rRNA gene, the P6 gene, or multilocus-sequence-typing is more promising. For the diagnostic routine, such techniques are too expensive and laborious. If available, matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry is a routine-compatible option and should be used in the first line. However, the used database should contain well-defined reference spectra, and the spectral difference between H. influenzae and H. haemolyticus is small. Fluorescence in-situ hybridization is an option for less well-equipped laboratories, but the available protocol will not lead to conclusive results in all instances. It can be used as a second line approach. Occasional ambiguous results have to be resolved by alternative molecular methods like 16S rRNA gene sequencing.

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MALDI-TOF MS Distinctly Differentiates Nontypable Haemophilus influenzae from Haemophilus haemolyticus

Cited by 40 publications

References 27 publications

Comparative Genomic Analysis of Haemophilus haemolyticus and Nontypeable Haemophilus influenzae and a New Testing Scheme for Their Discrimination

Comparative Genomic Analysis of Haemophilus haemolyticus and Nontypeable Haemophilus influenzae and a New Testing Scheme for Their Discrimination

McRAPD unlike MALDI-TOF MS is a suitable candidate for routine discrimination of new Haemophilus influenzae strain acquisition in chronic obstructive pulmonary disease (COPD) and cystic fibrosis

Difficult identification ofHaemophilus influenzae, a typical cause of upper respiratory tract infections, in the microbiological diagnostic routine

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