Uk standards for Microbiology Investigations Acknowledgments

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+ = positive, - = Negative, (+) = 80-89% positive, d= 11-89% positive, V= variable result

Growth requirement for X and V factors

This is used to distinguish Haemophilus species (TP 38 - X and V factor test or TP 29 - Porphyrin synthesis (ala) test).

Summary of X and V test results


X factor

V factor

X + V factor


H. influenzaea

No growth

No growth



H. haemolyticusb

No growth

No growth



H. parainfluenzae

No growth




H. pittmaniae

No growth




H. parahaemolyticus

No growth




H. paraphrohaemolyticus

No growth




Haemophilus ducreyi


No growth



Haemophilus sputorum

No growth




aH. aegyptius is indistinguishable from H. influenzae biotype III in normal laboratory tests.

b-haemolytic on horse blood agar.

Serotyping H. influenzae with commercial type-specific antisera

Commercial identification kit

Several commercial identification systems that use biochemical or enzymatic substrates are available for identification of Haemophilus species. The manufacturer’s instructions should be followed precisely when using these kits. In many cases, the commercial identification kit may not reflect recent changes in taxonomy.

3.5 Further Identification

Rapid Molecular Methods

Molecular methods have had an enormous impact on the taxonomy of Haemophilus. Analysis of gene sequences has increased understanding of the phylogenetic relationships of Haemophilus species and related organisms and has resulted in the recognition of numerous new species. Molecular techniques have made identification of many species more rapid and precise than is possible with phenotypic techniques.

A variety of rapid identification and sensitivity methods have been developed for isolates from clinical samples; these include molecular techniques such as Real-time Polymerase Chain reaction (PCR), Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST), Ribotyping, 16S rRNA gene sequencing, and Matrix Assisted Laser Desorption Ionisation Time-of-Flight (MALDI-TOF) Mass Spectrometry. All of these approaches enable subtyping of unrelated strains, but do so with different accuracy, discriminatory power, and reproducibility.

However, these methods remain accessible to reference laboratories only and are difficult to implement for routine bacterial identification in a clinical laboratory.

16S rRNA gene sequencing

A genotypic identification method, 16S rRNA gene sequencing is used for phylogenetic studies and has subsequently been found to be capable of re-classifying bacteria into completely new species, or even genera. It has also been used to describe new species that have never been successfully cultured.

This has been used for better discrimination of closely related species such as C. hominis and C. valvarum17,50.

Real-time Polymerase Chain reaction (PCR)

PCR has been used to identify H.ducreyi in clinical specimens. Orle et al.51 reported on the development of a commercial multiplex PCR assay that permits the simultaneous amplification of DNA targets from H. ducreyi, T. pallidum, and Herpes Simplex Virus types 1 and 2 directly from genital ulcer specimens.

16s rRNA PCR assay followed by sequencing and analysis has been used for rapid identification of difficult and serious infections due to fastidious microorganisms – Cardiobacterium hominis52. In addition, this method can also be used to discriminate C. hominis from C. valvarum, which has recently been found to be responsible for endocarditis.

H. haemolyticus and H. influenzae differ from other Haemophilus species because they require haemin (X factor) and NAD (V factor) for growth. H. haemolyticus can easily be distinguished from encapsulated H. influenzae because H. influenzae isolates produce one of the six structurally distinct capsules that can be easily determined by slide agglutination assay, whereas H. haemolyticus has never been shown to produce a capsule. However, due to the high similarity in morphology, biochemistry, and genetics between H. haemolyticus and non-encapsulated or nontypeable H. influenzae, distinguishing the two by standard microbiology methods has been challenging. A new PCR assay has been developed and this has proved to be a superior method for discrimination of non-typeable Haemophilus influenzae from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. It has also been suggested it would be suitable for routine non-typeable Haemophilus influenzae surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease53. The hpd- and iga- based PCR assays can be used in combination with standard microbiological methods to improve the identification of H. haemolyticus from non-typeable Haemophilus influenzae54.


Ribotyping is based on restriction fragment length polymorphisms of rRNA genes, which are highly conserved and are usually present in multiple copies on the genome. Ribotyping does however present some disadvantages; it is labour intensive and requires costly enzymes and materials. Nevertheless, ribotyping provides a highly reproducible and reliable reference typing system.

This has been used to identify and characterise H. ducreyi and it was found to be highly reproducible and that it discriminated among strains of H. ducreyi55,56. It may be used to study the epidemiology of H. ducreyi and chancroid.

This has also been used successfully in the identification of H. influenzae57 and may help to understand the molecular characteristics of outbreaks, endemicity and value of vaccination.

Pulsed Field Gel Electrophoresis (PFGE)

PFGE detects genetic variation between strains using rare-cutting restriction endonucleases, followed by separation of the resulting large genomic fragments on an agarose gel. PFGE is known to be highly discriminatory and a frequently used technique for outbreak investigations and has gained broad application in characterizing epidemiologically related isolates. However, the stability of PFGE may be insufficient for reliable application in long-term epidemiological studies. However, due to its time-consuming nature (30hr or longer to perform) and its requirement for special equipment, PFGE is not used widely outside the reference laboratories.

This has been used successfully to identify and discriminate between strains of non-typeable Haemophilus influenzae58.

Multilocus Sequence Typing (MLST)

Multilocus sequence typing (MLST) is a tool that is widely used for phylogenetic typing of bacteria. MLST is based on PCR amplification and sequencing of internal fragments of a number (usually 6 or 7) of essential or housekeeping genes spread around the bacterial chromosome. MLST has been extensively used as the one of the main typing methods for analysing the genetic relationships within the genus Haemophilus population.

This has been used to describe new specie, H. pittmaniae9 and to also separate H. haemolyticus and H. influenzae into distinct clusters using concatenated sequences of multiple genes, including the 16S rRNA gene, adk (adenylate kinase gene), pgi (glucose-6-phosphate isomerase gene), recA (recombination protein gene), and infB (translation initiation factor 2 gene)59.

Matrix-Assisted Laser Desorption/Ionisation - Time of Flight (MALDI-TOF) Mass Spectrometry

This has been shown to be a rapid and powerful tool because of its reproducibility, speed and sensitivity of analysis. The advantage of MALDI-TOF as compared with other identification methods is that the results of the analysis are available within a few hours rather than several days. The speed and the simplicity of sample preparation and result acquisition associated with minimal consumable costs make this method well suited for routine and high-throughput use.

This has been used to describe and characterise new specie, Haemophilus sputorum3. Members of the species yield a unique MALDI-TOF mass spectrum distinct from other related Haemophilus species.

MALDI- TOF MS can be used to accurately identify the HACEK organisms despite their fastidious nature60. This technique can also be used for rapid discrimination of Haemophilus influenzae, H. parainfluenzae and H. haemolyticus, although, there are suggestions of misidentifications of commensal H. haemolyticus as H. influenzae61. This could be resolved by the addition of a suitable H. haemolyticus reference spectrum to the system’s database as well as alternative tests being applied in case of ambiguous test results on isolates from seriously ill patients.

This has also been used to rapidly distinguish between C. hominis and C. valvarum62.

3.6 Storage and Referral

If required, save pure isolate on a chocolate agar slope for referral to the Reference Laboratory.

4a Identification of Haemophilus species

This flowchart is for guidance only

4b Identification of HACEK group

This flowchart is for guidance only
5 Reporting

5.1 Presumptive Identification

If appropriate growth characteristics, colonial appearance and Gram stain of the culture are demonstrated.

5.2 Confirmation of Identification

Following serotyping of H. influenzae, appropriate X and V and/or commercial identification kit results and/or the Reference Laboratory report.

5.3 Medical Microbiologist

Inform the medical microbiologist of all positive cultures from normally sterile sites.

According to local protocols, the medical microbiologist should also be informed of presumptive or confirmed Haemophilus species or other member of the HACEK group of organisms when the request card bears relevant information eg:

  • Meningitis or brain abscess

  • Facial cellulitis

  • Septic arthritis

  • Osteomyelitis

  • Epiglottitis, pneumonia, mastoiditis or empyema thoracis

  • Septicaemia or endocarditis

Follow local protocols for reporting to clinician.

5.4 CCDC

Refer to local Memorandum of Understanding.

5.5 Public Health England63

Refer to current guidelines on CDSC and COSURV reporting.

5.6 Infection Control Team

6 Referrals

6.1 Reference Laboratory

For information on the tests offered, turnaround times, transport procedure and the other requirements of the reference laboratory refer to:

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