Haemophilus influenzae serotype a as a cause of serious invasive infections

Review

Haemophilus influenzae serotype a as a cause of serious invasive infections Marina Ulanova, Raymond S W Tsang Lancet Infect Dis 2014; 14: 70–82 Published Online November 20, 2013 http://dx.doi.org/10.1016/ S1473-3099(13)70170-1 Northern Ontario School of Medicine, Lakehead University, Thunder Bay, ON, Canada (M Ulanova MD); and National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada (R S W Tsang PhD) Correspondence to: Dr Raymond S W Tsang, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB R3E 3R2, Canada raymond.tsang@phac-aspc. gc.ca

Haemophilus influenzae, particularly H influenzae serotype b (Hib), is an important pathogen that causes serious diseases like meningitis and septicaemia. Since the introduction of Hib conjugate vaccines in the 1990s, the epidemiology of invasive H influenzae disease has changed substantially, with most infections now caused by nonHib strains. We discuss the importance of H influenzae serotype a (Hia) as a cause of serious morbidity and mortality and its global epidemiology, clinical presentation, microbiology, immunology, prevention, and control. Much like Hib, the capsule of Hia is an important virulence factor contributing to the development of invasive disease. Molecular typing of Hia has identified distinct clonal groups, with some linked to severe disease and high case-fatality rates. Similarities between Hia and Hib capsules, their clinical presentation, and immunology of infection suggest that a bivalent Hia–Hib capsular polysaccharide-protein conjugate vaccine could offer protection against these two important serotypes of H influenzae.

Introduction Haemophilus influenzae is an important human pathogen that causes severe infections including meningitis, sepsis, and bacteraemic pneumonia, mostly affecting young children. The genus was named for its requirement for blood to grow and the species epithet given in the erroneous belief that these bacteria were the cause of influenza. H influenzae is a non-motile, non-acid-fast, and non-spore-forming Gram-negative coccobacillus. It belongs to the Pasteurellaceae family—most members of which are commensals but some are important human and animal pathogens.1 Although most people regard H influenzae as a strictly human parasite, infections in non-human primates have also been reported.2,3 In human beings, H influenzae is seen in mainly the upper respiratory tract (oropharynx and nasopharynx), although colonisation of the urinary and genital mucosa has also been described and can lead to urinary tract, neonatal, and obstetric infections.4–6 Some H influenzae strains are covered by a carbohydrate capsule, which forms the basis of a serotyping scheme— six serotypes (a–f) have been identified.7 Nonencapsulated strains are termed non-typeable. In-vitro growth of H influenzae needs two factors found in blood: haemin (the so-called X factor) and nicotinamide adenine dinucleotide (known as V factor). Presumptive identification is based on the growth requirement for both X and V factors, characteristic colony morphology on a chocolate blood agar plate, and Gram-stain

Region I

Region II

Region III

Region I

Region II

Region III

Characteristics of Hia

bexA*-D

acs1-4

hcsA-B

bexA-D

acs1-4

hcsA-B

The capsule of serotype a is a polymer of glucose and ribitol connected by phosphodiester linkages.19 The polysaccharide capsule serves as a physical barrier to block binding of complement components to the bacterial surface that can lead to the activation of the complement system and deposition of the complement membrane attack complex. Findings from animal studies

IS1016†

IS1016

IS1016

Figure 1: Organisation of the Haemophilus influenzae serotype a capsule synthesis genes *Partial deletion of the bexA gene. †Partial deletion of the IS1016. Image adapted from Kroll and colleagues30 and Follens and colleagues.31

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morphology.8 On the basis of their reactions in three biochemical tests (indole, urease, and ornithine decarboxylase), H influenzae can be divided into eight biotypes.9 Definitive identification of H influenzae, especially of non-typeable strains, needs 16S rRNA sequencing or other genetic methods.10 Using the technique of multilocus enzyme electrophoresis (MLEE), H influenzae serotype a (Hia) and serotype b (Hib) can be divided into different genetic lineages, whereas serotypes c, d, e, and f form monophyletic groups.11,12 By contrast with these serotypes, non-encapsulated H influenzae strains seem to be non-clonal and distinct from the encapsulated strains, and they are genetically diverse.13 Before effective vaccines were available, invasive H influenzae disease was dominated by Hib strains, which were a major cause of childhood meningitis.14 Hib conjugate vaccines were introduced initially in the late 1980s for children aged 12 months or older, and later in the early 1990s for infants younger than 6 months old. Near elimination of Hib disease in children occurred in countries that implemented paediatric Hib immunisation programmes.15 Even before the introduction of Hib conjugate vaccines, invasive disease caused by Hia had been described in indigenous populations in Australia, Papua New Guinea, The Gambia, and the USA.16–18 Findings from some reports suggest an increase in invasive Hia disease in some populations. In this Review, we discuss Hia biology, bacterial population genetics, global epidemiology, immunology, clinical presentation of invasive Hia disease, and potential infection control and prevention measures.

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Review

with isogenic mutants that differ from each other only by the type of capsule expressed on their surface have shown that Hib is the most virulent type, followed by Hia, and then other capsular types.20 The structure of the Hib capsule is polyribose ribitol phosphate linked by phosphodiester bonds.21 The genetic basis of capsule synthesis and expression in H influenzae has been investigated in much detail for Hib,22–25 and subsequently for the other serotypes.26–29 The genes involved in the synthesis and expression of the Hib capsule can be categorised into three functional regions. Region I contains four genes, bexA–D, which encode proteins associated with transport or export of the capsule to the cell surface. Region III contains two genes, hcsA and hcsB, which are associated with post-translational modification and expression of the capsule. Between regions I and III, region II contains genes associated with synthesis of the serotype-specific carbohydrate unit. In most Hib strains, regions I and III are flanked by insertion elements IS1016. The capsule polysaccharide synthesis (cps) genes (regions I–III) and the flanking elements exist in duplicates in a tandem orientation, but in one of the duplicate copies a partial deletion exists in the bexA gene and the insertion element (figure 1).30,31 Whereas most serotype a, c, d, e, and f strains have only one copy of the cps gene packaged as in Hib, some Hia strains have two copies of the cps gene with one of these copies having the partial deletion.26 This unique arrangement of the cps genes allows for recombination events to occur that can result in the loss of capsule expression. Such an arrangement has been described in

the case of the Hib– strain, whereby the bexA gene was lost and, therefore, the serotype b capsule was not expressed despite the presence of an intact region II.32,33 In other instances, recombination can also result in strains containing up to six copies of the cps gene.34,35 Also, the amount of capsular material produced is proportional to the number of cps genes present in a strain.36 Although the capsule is the best described virulence factor in H influenzae, other non-capsular components might also contribute to virulence. Because H influenzae normally colonises human mucosal surfaces where IgA antibodies are present, it—much like other mucosal pathogens such as Neisseria meningitidis, Neisseria gonorrhoeae, and Streptococcus pneumoniae—produces proteases that specifically break down human IgA.37 Three distinct types of H influenzae IgA proteases, each attacking a different peptide bond, have been described.38 Three of four Hia isolates analysed produced the type 1 protease, which cleaves the proline–serine bond at position 231–232 of the IgA molecule.38 Another molecule associated with H influenzae virulence is the lipo-oligosaccharide.39 15 Hia strains analysed with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) had lipo-oligosaccharide molecules of apparently uniform molecular weights that produced homogeneous bands of identical mobility.40

Typing of Hia Various typing methods with both phenotypic and genetic approaches have been described for H influenzae, but only some have been applied to study Hia (table 1).7,9,11–13,41–48

Principles behind test

Comment

Reference

Serotyping

Bacterial agglutination (usually done on slide) with specific rabbit anti- Very useful in defining the importance of each serotype associated with invasive disease capsular antiserum; expression of capsules measured by bacterial Serotyping discrepancies have been reported agglutination; PCR with primers specific for each serotype to detect serotype-specific genes

Pittman (1931);7 Falla et al (1994);41 LaClaire et al (2002)42

Biotyping

Based on three biochemical reactions: indole, urease, and ornithine decarboxylase phenotypic method

Eight biotypes identified; restricted use; most Hia were biotype II

Kilian (1976);9 Tsang et al (2006)43

OMP subtyping

Based on the electrophoretic mobility of a strain’s major OMPs in a polyacrylamide gel on electrophoresis under denaturing conditions

Restricted sensitivity in typing of strains

Barenkamp et al (1981)44

Capsular typing

Based on Southern blot hybridisation with a DNA probe; can identify six serotypes, including b- variants

Replaced largely by PCR

Kroll et al (1991)45

MLEE

Based on electrophoretic mobilities of 17 housekeeping enzymes, each given an allelic number; each strain is characterised by its allelic profile described as the electrophoretic type

For understanding the population biology of Hia; good for studying evolution of strains over time; not useful for comparing strains over short period of time—eg, in the identification of relatedness of strains during an outbreak

Musser et al (1988)11,12

MLST

Based on nucleotide sequences of internal fragments of seven housekeeping enzyme genes; each unique housekeeping gene sequence is given an allelic number and each strain is characterised by its allelic profile described as sequence type

Application and limitations similar to MLEE

Meats et al (2003)13

PFGE

Fingerprinting by restricting genomic bacterial DNA into a few large DNA fragments and separation of the DNA fragments into patterns called pulse types

Highly sensitive; especially if more than one restriction enzyme is used to generate different profiles (one profile per enzyme)

Millar et al (2005);46 Hammitt et al (2005);47 Tsang et al (2006)43

Antibiogram

Based on susceptibility pattern to a panel of antibiotics (including Useful for detection of resistance markers—eg, ampicillin-resistant those commonly prescribed for treatment of the infection in question) strains; otherwise restricted use

Sill et al (2007)48

Hia=Haemophilus influenzae serotype a. OMP=outer membrane protein. MLEE=multilocus enzyme electrophoresis. MLST=multilocus sequence typing. PFGE=pulsed-field gel electrophoresis.

Table 1: Typing methods for study of Haemophilus influenzae serotype a

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Clonal division I

Multilocus enzyme electrophoresis11,12

Multilocus sequence typing13

Lineage B2 Lineage B4

ST-5, ST-23, ST-56 ST-4

Clonal division II Lineage H1 Lineage I1

ST-21, ST-25, ST-30, ST-59, ST-60 ST-20, ST-62

Hia=Haemophilus influenzae serotype a. ST=sequence type.

Table 2: Population genetics of Hia studied by multi-locus enzyme electrophoresis and multilocus sequence typing

Housekeeping genes allelic profile adk

atpG

frdB

Countries

fucK

mdh

pgi

recA

ST-23 related ST-23

13

16

5

2

3

11

7

Malaysia

ST-56

13

16

5

18*

3

11

7

Papua New Guinea

ST-397

13

16

5

2

3

11

85*

Canada

ST-405

13

5

2

3

11

7

Canada

7*

ST-557

13

16

5

2

30*

11

7

Canada

ST-833

13

16

5

2

3

11

110*

Norway

ST-1035

13

16

5

90*

3

11

7

Canada

ST-576

13

16

5

18*

3

11

36*

Canada

ST-403

13

11*

5

18*

3

11

36*

Canada

ST-929

13

16

ST-5

12*

ST-529

5*

5

2

30*

11

121*

5

2

3

11

7

USA

Canada

18*

3

135*

7

Canada

108*

16

5

ST-4

4

17

4

1

2

9

6

The Gambia and Kenya

ST-1139

4

17

4

5*

2

9

6

Portugal

ST-62

21

12

9

7

19

24

19

Dominican Republic

ST-20

21

13*

9

7

19

24

19

UK

ST-775

21

12

122*

7

19

24

19

New Mexico, USA

ST-21

20

12

1

7

20

23

19

UK

ST-30

20

12

25*

7

20

23

19

UK

ST-59

20

12

1

8*

20

23

19

UK

ST-25

20

12

2*

1*

21*

23

19

UK

ST-372

20

12

9*

14*

21*

23

19

USA UK

ST-4 related

ST-62 related

ST-21 related

Unrelated to above groups or each other ST-60

20

12

21

20

34

30

19

ST-723

26

20

36

1

21

49

64

Nepal

ST-747

22

19

11

11

35

18

5

Nepal

ST-858

20

12

9

14

21

3

34

Israel

Sequence types (ST) are grouped by their relatedness, with the founding ST at the top. *Allele numbers vary from other sequence types in group. Data are from Haemophilus influenzae multilocus sequence typing database (accessed on Aug 14, 2012).

Table 3: Allelic differences of 26 sequence types of Haemophilus influenzae serotype a

For the H influenzae MLST database see http:// haemophilus.mlst.net/

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Serotyping is traditionally done by slide agglutination with specific rabbit antiserum. Detection of the capsular serotype-specific genes with PCR has also been described,41 and this method can be used to resolve any discrepancies of bacterial agglutination results between

laboratories.42 The PCR technique can also identify strains (eg, the b strains) that have the bex gene deletion.33 Biotyping of Hia has given little information about either the epidemiology or genetic background of strains. In Canada, most invasive Hia isolates are biotype II and are related to the multilocus sequence type (ST)-23.48–50 By contrast with Hia isolates, most invasive Hib isolates are biotype I.51–53 However, two Hia strains with sequence types unrelated to ST-23 were biotype I.54 In Brazil, four of 13 invasive Hia isolates were biotype I and the remaining nine isolates were either biotype II or biotype III.55,56 Analysis of outer membrane proteins by SDS-PAGE has allowed Hib– strains to be typed according to the mobility of their major outer membrane proteins.44 Analysis of 54 Hia isolates from different geographical regions identified seven protein subtypes, which seemed to be associated with their capsular genotypes.57 Capsular typing by hybridisation with a pU038 DNA probe has divided Hia isolates into three genotypes (aM, aN, and aT).45 The capsular genotype aN is associated with Hia strains, showing a partial deletion in one copy of the IS1016-bexA.26,58,59 These strains are associated with more serious disease. The population biology of encapsulated H influenzae was first studied with MLEE.11,12 With this powerful technique, encapsulated H influenzae can be divided into two clonal divisions: I and II. Clusters of electrophoretic types are seen within each clonal division. In a study of 52 Hia isolates from different countries, 22 (42%) isolates belonged to two clusters (B2 and B4) within the clonal division I, and 30 isolates were assigned to two clusters (H1 and I1) within the clonal division II.11,12 When the population genetics of H influenzae was studied by multi-locus sequence typing (MLST),13 a comparison of the MLEE and MLST data confirmed the existence of two phylogenetic groups, clonal divisions I and II.11,13 Furthermore, Hia isolates can be separated into four clusters by use of either method (table 2). We searched an online H influenzae MLST database and detected 44 Hia isolates, from 12 different countries, grouped into 26 different STs. The 26 types can be divided into four groups together with four others that seem to be unrelated to one another or to the four groups of related STs (table 3). In three of the four groups of related types, ST-23, ST-62, and ST-21 were identified by eBURST (version 3) analysis to be the founding STs in their respective groups. In the fourth group, only two STs exist and, therefore, not enough information is available to infer which is the founder. In nine studies,43,46,47,49,54,55,58–60 researchers used pulsedfield gel electrophoresis (PFGE) to analyse Hia isolates. Although all these studies analysed SmaI-restricted genomic DNA, comparison of the DNA fingerprints was not possible because no standardised method is available. www.thelancet.com/infection Vol 14 January 2014

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Nevertheless, PFGE analysis has given useful information about the clonal nature of Hia isolates assessed in the different studies. For example, Hia detected in the Navajo and White Mountain Apache children in southwest USA seemed to be clonal, with two PFGE patterns noted, both sharing an overall similarity of 90% or more.46 In a study of two Hia isolates from two infants admitted to hospital in Georgia, USA, PFGE results confirmed that they were identical and both contained the IS1016-bexA partial deletion.59 In Brazil, 13 Hia invasive isolates had two closely related PFGE

patterns.55 In another study in Brazil,60 28 invasive isolates had two PFGE patterns: A (corresponding to the MLST group ST-4) and B (ST-23). Strains with PFGE pattern A had the IS1016-bexA partial deletion and were associated with severe disease and higher case-fatality rate, whereas strains of PFGE pattern B did not have the IS1016-bexA partial deletion and were associated with lower case-fatality.60 In Canada, most Hia isolates were ST-23 and had related PFGE patterns,43,49 whereas two Hia isolates with sequence types unrelated to ST-23 had very different PFGE patterns.54

Geographical location; years

Characteristics of the population

Prevalence of Hia in Haemophilus influenzae invasive isolates

Annual incidence

Study type

Ladhani et al (2010)69

14 European countries (Austria, England, Greece, Finland, Iceland, Ireland, Italy, Malta, Netherlands, Norway, Portugal, Scotland, Slovenia, and Wales); 1996–2006

Annual population of 150 million; all ages

26 Hia of 10 081 H influenzae isolates

0·12 per 1 000 000 children younger than 5 years

European Union invasive bacterial infection surveillance (EU-IBIS)

Mudhune et al (2009)70

East African region (Kenya, Uganda, Tanzania, Ethiopia); 2003–07

Children between 2 months and 5 years of age

16 Hia of 119 H influenzae Not reported isolates

Network for surveillance of pneumococcal disease in the east African region

Millar et al (2005)46

Southwestern USA; 1988–2003

Navajo and White Mountain Apache children younger than 5 years

76 Hia of 378 H influenzae 20·2 per 100 000 children isolates

Population-based, active laboratory surveillance

Bruce et al (2008)71

Alaska, USA, and northern Canada; 2000–05

Alaska: population of 655 435 (19% Indigenous); northern Canada: 132 956 (59% Indigenous); all ages

42 Hia of 88 typeable H influenzae isolates

19·7 per 100 000 children younger than 2 years; 52·6 per 100 000 Indigenous children younger than 2 years

Population-based surveillance

Bender et al (2010)72

Utah, USA; 1998–2008

Children younger than 18 years

23 Hia of 91 H influenzae isolates

0·5 per 100 000 children (<5 years, 1998); 2·6 per 100 000 children (2008)

Retrospective study: populationbased surveillance

Rotondo et al (2013)73

Northern Canada; 2000–10

All ages; 58% Indigenous population

72 Hia of 142 H influenzae isolates

4·6 per 100 000 people (all ages); 87·5 per 100 000 (children younger than 2 years); 6·9 per 100 000 people (Indigenous population)

Population-based surveillance

Brown et al (2006)74

Northwestern Ontario, Canada; 2002–08

Population of 235 000; 13 Hia of 31 H influenzae all ages; 20% indigenous isolates with serotype information

7·7–23·2 per 100 000 per year in children younger than 5 years

Population-based surveillance

Adam et al (2010)75

Ontario, Canada; 1989–2007

All ages

2·1% of 1455 invasive H influenzae isolates

Not reported

Population-based surveillance

McConnell et al (2007)76

Canada; 1996–2001

3 million children admitted to paediatric tertiary care centres

26 Hia of 166 H influenzae Keewatin Region: 418·8 per 100 000 Inuit children younger than 5 years in 2001; isolates British Columbia, Alberta, Manitoba, and Saskatchewan: 3·7 per 100 000 Indigenous children younger than 5 years in 1996–2001; 2·3 per 100 000 non-Indigenous children younger than 5 years, in 1996–2001

Rubach et al (2007)77

Utah, USA; 1998–2008

Adults

15 Hia of 101 H influenzae isolates

0·08 per 100 000 adults

Population-based surveillance

Zanella et al (2008)78

Brazil; 1990–2008

Children

1990–99: 24 Hia of 3050 H influenzae isolates from meningitis cases; 2000–08: 118 Hia of 860 isolates

Incidence per 100 000 children younger than 1 year: 0·31 in 2000–02; 0·90 in 2003–05; 1·48 in 2006–08

Passive retrospective laboratory surveillance

Menzies et al (2013)79

Northern Territory, Australia; 1985–88 and 2001–11

Aboriginal children younger than 5 years

Not reported

14·7 per 100 000 children before introduction Retrospective study: populationbased surveillance of the Haemophilus influenzae type b (Hib) vaccine (1985–88); 11 per 100 000 children after introduction of the Hib vaccine (2001–11)

Population-based surveillance encompassing 12 Canadian paediatric tertiary care centres

Hia=Haemophilus influenzae serotype a.

Table 4: Prevalence and incidence rates of invasive Haemophilus influenzae serotype a disease worldwide

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Alaska, USA47,71,80 British Columbia, Canada50

Canadian Arctic73,76,81

Utah, USA72,77 Arizona, USA18,46 New Mexico, USA*

Manitoba, Canada43,82 Northern Ontario, Canada49,74 Quebec, Canada54

Ethiopia70

Papua New Guinea16,86,87

Kenya70 Uganda70

The Gambia14,17 Brazil55,56,60,78,83

Tanzania70 Australia88–90 South Africa84,85

Key Where either systematic laboratory surveillance has been done or where many cases have been detected Where either one or few cases have been reported

For the multilocus sequence typing database see http:// www.mlst.net

Figure 2: Geographical distribution of invasive Haemophilus influenzae serotype a disease *Data from the Haemophilus influenzae multilocus sequence typing database.

Antibiotic susceptibility The most commonly described antibiotic resistance in H influenzae is resistance to ampicillin or amoxicillin, first reported in 1972.61 Resistance of H influenzae to ampicillin is usually a result of the TEM-1 β-lactamase,62 and in some cases the ROB-1 enzyme.63 Besides production of β-lactamases, some H influenzae are resistant to ampicillin because of changes in their penicillin-binding proteins, leading to decreased binding of β-lactam antibiotics.64,65 In Canada, although resistance to ampicillin is common in invasive Hib isolates,48 ampicillin resistance in Hia is rare.48,49,66 Canadian Hia isolates show no resistance to other antibiotics, even though resistance to co-trimoxazole is common worldwide in non-typeable H influenzae.66 We identified only a few studies that provided antibiotic susceptibility data for Hia. Of the 15 Hia isolates for which susceptibility results were mentioned,47,49,55,58,67,68 two were described as β-lactamase-positive and resistant to ampicillin, with the rest reported to be ampicillin sensitive. Resistance to other antibiotics was not reported.

Global epidemiology Before the introduction of paediatric Hib conjugate vaccines, 99% of cases of invasive H influenzae disease in high-income countries were caused by Hib; invasive Hia disease was rarely reported.14 In the post-Hib vaccine era, the presence of invasive Hia disease has been reported in the North American Arctic, western Canada, northwestern 74

Ontario, Canada, and southwestern parts of the USA (table 4). This disease has also been detected in Brazil and Australia (figure 2).14,16–18,43,46,47,49,50,54–56,60,70–74,76–78,–80–90 Although Hia seems to be rare in the rest of the world, the scarcity of systematic reporting of invasive H influenzae disease or serotyping data from most low-income countries could lead to underestimation of the presence of this infection worldwide. More details about the global epidemiology of invasive Hia disease are available elsewhere.91 In 21 European countries where the surveillance of invasive H influenzae infection has been done since the 1990s, the incidence of invasive Hia disease is very low.69,92–98 The incidence of invasive Hia disease in Asia and Africa are unknown. Systematic studies of invasive H influenzae disease or serotype information are missing from most Asian and African countries. In Asia, some studies reported that Hia was either not detected or detected in isolated cases. For example, in a prospective surveillance of invasive H influenzae disease in six International Clinical Epidemiological Network-affiliated medical colleges in India (1993–97), only one Hia was identified in 125 H influenzae isolates by use of slide agglutination method.99 In Africa, invasive Hia disease has been reported from South Africa and The Gambia before the introduction of Hib vaccines.17,84 In The Gambia, between December, 1982, and January, 1984, Hia was isolated from four of 55 cases of H influenzae meningitis and three of 20 cases of bacteraemic pneumonia.26 A national surveillance study during the first 5 years of introduction of conjugate Hib vaccine in www.thelancet.com/infection Vol 14 January 2014

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South Africa identified ten Hia isolates in 712 invasive H influenzae specimens (the study used PCR to confirm serotype).85 Some cases of invasive Hia disease have also been reported in east Africa.70 In the USA, high incidence of invasive Hia disease were detected in Navajo and White Mountain Apache populations in southwestern USA, as well as in Alaska and Utah.18,46,80,71,72,77 In Utah, the incidence of invasive Hia disease in children younger than 5 years increased between 1998 and 2008.72 In Canada, high incidences of invasive Hia disease were reported from the Northern Territories and western provinces (Manitoba, Saskatchewan, and British Columbia) where most Indigenous Canadian people live.43,50,73,76,81,82 High incidences of invasive Hia disease have been seen in northwestern Ontario, where Indigenous people comprise 20% of the population49,74—this finding is in clear contrast with the rest of this population of the province in whom invasive Hia disease is rare.75 Findings from a large population-based study encompassing 12 Canadian paediatric tertiary care centres showed in 2001 an incidence rate of 418·8 per 100 000 Inuit children younger than 5 years who lived in the Keewatin Region.76 Surveillance data from 2000 to 2010 substantiate the high incidences of invasive Hia disease in young Indigenous children in the Canadian Arctic.73 Hia is uncommon in the general US population—Hia accounts for about 2% of all invasive H influenzae isolates.100,101 In Brazil, an increase in incidence rates and emergence of severe cases of invasive Hia disease have been reported in the post-Hib vaccine era.55,56,60,83 Although data from the rest of South America are incomplete, findings from a 2000–05 study showed Hia in 6·1% of all invasive H influenzae isolates from 19 Latin American and four Caribbean countries.102 In Papua New Guinea, findings from studies done in the 1980s showed high prevalence of Hia as a cause of bacterial meningitis and other invasive disease in young children,16,86 and of pneumonia in adults (based on blood or lung aspirate culture).87 In one study of children younger than 5 years with meningitis, Hia accounted for nine (12%) of 73 H influenzae isolates from cerebrospinal fluid (CSF) and six (7%) of 92 H influenzae isolates from blood cultures.86 In Australia, Hia was detected in Aboriginal children with invasive disease both before and after introduction of Hib vaccines—in 1985–93, this serotype caused 6–8% of invasive H influenzae infections in children.79,88–90 No data are available from systematic population-based studies of invasive Hia disease in New Zealand or Papua New Guinea. The reasons for an increased susceptibility of the indigenous populations to Hia infection are unclear, but might include the population genetics and various environmental and socioeconomic factors that could contribute to enhanced carriage and high transmission rates of Hia in specific areas and in specific population groups. www.thelancet.com/infection Vol 14 January 2014

The challenges in studying the global epidemiology of invasive Hia disease are very similar to those encountered in the surveillance of other invasive bacterial diseases. In countries with restricted resources, surveillance might be based on clinical cases only, without or with minimum laboratory investigation, thereby not providing details such as serotype information. Furthermore, true cases treated with antibiotics before bacteriological cultures can be obtained might be missed unless molecular methods such as PCR are available to detect H influenzae in clinical specimens and subsequently identify them to the serotype level. Because almost all invasive H influenzae disease has been historically due to Hib, and Hia is only recently recognised as an important pathogen, some laboratories might not have reagents or methods capable of detecting Hia. Furthermore, a shortage of experience in the serotyping technique and the availability of high quality serological reagents might also contribute to Hia cases being missed or misidentified. All these factors can therefore contribute towards potential discrepancies in the rates of invasive Hia infections around the world and complicate our understanding of the global epidemiology.

Clinical presentations Much like Hib, Hia can be carried in the nasopharynx without any signs of local or systemic infection, and Hia carriage has been seen in otherwise healthy children and patients with respiratory infections (table 5).47,86,103–112 Clinical presentations of invasive Hia disease are analogous to those caused by Hib, including meningitis, bacteraemic pneumonia (often with pleural involvement), septic arthritis, osteomyelitis, and bacteraemia without localised disease;46,49,55,71,80 by contrast with Hib, no cases of epiglottitis caused by Hia, have been recorded. A summary of cases of invasive Hia disease reported during the past 30 years show typical characteristics of this infection (table 6).26,47,49,56,58,59,67,68,107,113–117 The invasive disease mostly affects children between 6 months and 2 years of age.58 For example, Active Bacterial Core surveillance data obtained from nine participating centres in the USA show that, in a population of about 35 million, almost 40% of invasive Hia disease occurred in children younger than 2 years.59 By comparison with this finding, before introduction of the Hib vaccine, about 80% of invasive Hib cases occurred in children younger than 2 years.118 The median age of 76 Navajo and White Mountain Apache children affected by invasive Hia disease during 1988–2003 was 12 months—38 (50%) of these 76 children had meningitis, making it the most common clinical presentation followed by pneumonia (in 21 [30%] of children).46 Among 42 cases of invasive Hia disease in the North American Arctic in 2000–05, 30 were in children aged 2–5 years, with meningitis and pneumonia being the most common clinical presentations, followed by septic arthritis.71 Of the 25 paediatric cases of invasive 75

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Geographical location; year

Population

Study design

Gratten et al (1984)103

Papua New Guinea; March, 1980, to January, 1983

Children aged between 2 weeks and 5 years (healthy or with pneumonia); healthy adults

Laboratory study of H influenzae isolates from 43 Hia of 505 H influenzae isolates from the the upper respiratory tract of healthy children, nose or nasopharynx children with pneumonia, and healthy adults

Findings

Gratten et al (1991)86

Eastern Highlands, Papua New Guinea; 1978–88

Children younger than 5 years

Laboratory-based surveillance

16 Hia (8%) of 209 H influenzae isolates from the nose

Gratten et al (1994)104

Central Australia; May, 1990, to August, 1991

Aboriginal children aged between 1 month and 12 years

Study of carriage in Indigenous children admitted to hospital with acute lower respiratory tract infection in Alice Springs, NT

Six Hia (2%) of 254 H influenzae isolates from nasopharyngeal aspirates

Kwak et al (2000)105

Seoul, South Korea; January, 1992, to December, 1997

Children admitted to a tertiary care Prospective laboratory-based study; and referring centre H influenzae isolated from various clinical specimens

Four Hia of 29 H influenzae isolates from nonsterile body fluids

Lai et al (2002)106

Fuzhou, China; September, 1998, to September, 1999

Healthy children attending kindergartens

Collection of oropharyngeal samples from healthy preschool children

Six Hia of 283 H influenzae isolates obtained from 603 children

Hammitt et al (2005)47

Alaska; 2003

Indigenous

Investigation of an outbreak of invasive Hia disease; collecting oropharyngeal samples from close contacts of patients

Positive Hia cultures in five (16%) of 31 close contacts of two patients

Hammitt et al (2006)107

Alaska; 2005

Indigenous

Collection of oropharyngeal samples from close contacts of one patient with invasive Hia disease and from non-close contacts residents of the same community

Positive Hia cultures in three (43%) of seven close contacts and in none of 20 comparison participants

Bae et al (2010)108

Seoul, South Korea; 2002–04

Patients admitted to hospital with respiratory tract infections

Laboratory-based study of H influenzae isolates from various non-sterile body sites

Two Hia of 58 H influenzae strains isolated from sputum

Solorzano-Santos et al (2011)109

Mexico City (year not indicated)

Children between 2 months and 5 years of age

Cross-sectional survey; collecting nasopharyngeal samples

Three Hia of 88 H influenzae isolates obtained from 573 children (1% carriage rate)

De Carvalho et al (2011)110

Goiania, Brazil; August to September, 2005

Children aged 2–59 months

Collection of nasopharyngeal swabs

Hia in 2% of 1192 children, more common in infants (4–11 months)

Naranjo et al (2012)111

Caracas, Venezuela; December, 2008, to December, 2009

Children aged between 3 months and 5 years

Prospective epidemiological study; collecting Two Hia of 35 H influenzae isolates obtained middle-ear fluid samples from children from 87 children attending medical clinics for acute otitis media

Setchanova et al (2013)112

Sofia, Bulgaria; 1994–2011

Children younger than 14 years with otitis media

Laboratory-based study of isolates from middle-ear fluid

One Hia of 40 H influenzae isolates from middle-ear fluid

Hia=Haemophilus influenzae serotype a.

Table 5: Reports of non-invasive isolates of Haemophilus influenzae serotype a

Hia disease reported in Canada during 1996–2001, 20 were in children younger than 1 year (median age 8·5 months); meningitis was the most common presentation (in 13 children), followed by pneumonia (in five children).76 73% of 72 cases of invasive Hia disease reported in northern Canada between 2000 and 2010 were in children younger than 2 years; the most common clinical presentation was meningitis (33%), followed by bacteraemia (30%) and pneumonia (23%).73 Although most cases in young children occurred without any apparent predisposing medical disorder,56,59,68,76 some studies identified disorders that could have potentially decreased children’s immunity (table 6).47,49 Most children who developed invasive Hia disease had been vaccinated against Hib, although some had not received the full vaccine schedule.58,71,76 Findings from both clinical observations and laboratory studies have confirmed that the immunity given by Hib vaccination is serotypespecific and that Hib vaccination does not offer protection to Hia. Several cases of invasive Hia disease have been reported in adults with specific underlying disorders such as alcoholism, diabetes, or chronic obstructive pulmonary disease.49,67 On the basis of findings in northern Ontario in 2004–08 and in the 76

North American Arctic in 2000–05, adults with invasive Hia disease most commonly presented with pneumonia rather than meningitis.49,71 Severe cases of invasive Hia disease are similar to invasive Hib disease in the prevaccine era.55,58,59 Very severe cases presenting as meningitis or septic arthritis with serious complications have been reportedly caused by highly virulent Hia strains, which had the IS1016bexA deletion in the encapsulation (cap) locus of Hia; such cases occurred in young children without any apparent predisposing disorders.26,58–60 The case-fatality rate of invasive Hia disease was reported to be 14% in children with Hia meningitis in Brazil60 and 16% in Canadian paediatric cases.76 In the North American Arctic, during the past decade, the case-fatality rate was about 6%.71,73 The apparent disparity in reported casefatality rates might be related to Hia clones in different series of cases. The highest case-fatality rate was identified for meningitis caused by Hia strains that had the IS1016-bexA deletion—33% compared with 5% for cases caused by Hia strains without the IS1016-bexA deletion.60 By comparison, the case-fatality rate of invasive Hib disease before introduction of vaccine was between 2% and 5% in high-income countries and www.thelancet.com/infection Vol 14 January 2014

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between 26% and 57% in resource-poor countries.14 Invasive Hia disease can be recurrent—eg, during an outbreak of invasive Hia disease in Alaska Native infants, two cases of recurrent disease occurred within 3–4 months after full recovery from the first episode of invasive Hia infection.47 Findings from this study suggested that re-exposure to infection was the most possible reason for disease recurrence.47

Immunology The most important antigen of Hia is the capsular polysaccharide. Because Hia and Hib capsular polysaccharides are similar in structure,19,21 and both serotypes cause analogous clinical presentations, immunological responses to both infections are possibly

similar. However, the immune response to Hia infection is poorly studied. The immune response to Hib capsular polysaccharide, polyribosylribitol phosphate (PRP) has been extensively studied and the circulating anti-PRP antibodies have been established to be the major host defence mechanism against this infection. In immune individuals, anti-PRP IgG and IgM effectively protect against invasive disease through activating the classic complement pathway, resulting in complement-dependent bacteriolysis.119 Bacterial capsular polysaccharides are T-cell independent antigens and stimulate antibody production through direct activation of B cells.120 A subpopulation of spleen marginal zone B cells, the main contributors to anticapsular polysaccharide humoral immune responses, does not

Geographical Age; sex (ethnic origin [if location available])

Clinical presentation

Outcome

Underlying disorders

Source of isolation and characteristics of the pathogen

LeBlank and Hagarty (1983)113

New York, USA

Meningitis

Recovered

Skull fracture

Blood, cerebrospinal fluid (CSF); β-lactamase-negative

Rutherford and Wilfert (1984)67

North 1) 2·5 years; female Carolina, USA 2) 45 years; male

1) Pneumonia with empyema 2) Sepsis

1) Recovered with a left lower lobe cyst 1) None 2) Alcoholism that needed surgical removal 2) Acute renal failure, acute respiratory distress syndrome, death

1) Pleural effusion; β-lactamasepositive 2) Blood

Kroll et al (1994)26

The Gambia

1) Meningitis, septicaemia 2) Pneumonia

Not reported

Not reported

1) Blood, CSF IS1016-bexA deletion in the encapsulation (cap) locus of Hia in isolates from three children 2) Blood, lung aspirate

4 years; female

1) four children aged from 5 weeks to 8 months 2) one infant aged 9 months

Adderson Utah, USA et al (2001)58

1) 6 months; female (white) 1–4) Meningitis and bacteraemia 2) 1 year; female (white) 5) Pneumonia 3) 7 months; female 4) 13 months; male 5) 4 years; male

1) Renal failure, purpura fulminans, brain abscess, subdural empyema, softtissue necrosis that needed surgery. Outcome: hearing loss, development delay 2) Aseptic necrosis of hip, subdural empyema, lengthy fever. Outcome: hearing loss, development delay 3) Subdural effusion, lengthy fever; recovered 4) Recovered 5) Recovered

None

1–4) Blood, CSF 5) Pure culture of Hia from endotracheal secretions IS1016-bexA deletion in the encapsulation (cap) locus in isolates from children 1, 2, and 5

Mulder et al (2002)114

6 months, female

Meningitis

Recovered

None

Blood and CSF

Hammitt Western et al (2005)47 Alaska, USA

1) 6 months; female 2) 8 months; male 3) 4 months; male (all Alaska Native)

1) Pneumonia 2) Septic arthritis 3) Bilateral pneumonia

1) Recovered, but 4 months later developed a separate episode of Hia meningitis 2) After successful treatment of septic arthritis of left leg with ceftriaxone, 3 months later developed a recurrent episode of septic arthritis of left leg and left arm 3) Recovered without complications

1) History of gastrooesophageal reflux, reactive airway disease, frequent respiratory infections 2) History of pyelonephritis 3) History of neurodegenerative disease

1) Blood; β-lactamase-negative 2) Joint fluid and blood 3) Blood All (1–3) isolates were negative for IS1016-bexA deletion

Hammitt Alaska, USA et al (2006)107

6 months; male

Pneumonia

Recovered

Not reported

Blood; β-lactamase-negative

Kapogiannis Georgia, USA et al (2005)59

1) 14 months; male (Middle Eastern descent) 2) 30 months, male (black)

1) Infection of right hand, bacteraemia 2) Meningitis and septic arthritis

1) Recovered 2) Recovered, but had substantial cognitive and motor developmental deficits and hearing loss

None

1) Blood; β-lactamase-negative 2) Blood, CSF, synovial fluid; β-lactamase-negative Both cases caused by strains showing IS1016-bexA deletion

De Padua et al (2009)68

5 months; female (white)

Meningitis

Recovered after lengthy admission to hospital (57 days of antibiotic treatment)

None

CSF; β-lactamase-negative

Netherlands

Southern Brazil

(Continues on next page)

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Geographical Age; sex (ethnic origin [if location available])

Clinical presentation

Outcome

Underlying disorders

Source of isolation and characteristics of the pathogen

Septic arthritis

Recovered

None

Joint biopsy material; β-lactamase-positive, ampicillin resistant

(Continued from previous page) de Almeida et al (2008)56

Rio de 3 years; female Janeiro, Brazil

Kelly et al (2011)49

Northern Ontario, Canada

1) 15 months; male 2) 33 months; male 3) 4 years; male 4) 34 years; female 5) 65 years; female 6) 6·5 years; female 7) 47 years; male (all Indigenous)

1) Pneumonia and empyema 2) Septic arthritis 3) Pneumonia 4) Pneumonia 5) Septic arthritis 6) Tonsillitis and bacteraemia 7) Pneumonia

All recovered

1) Not reported 2) None 3) Morbid obesity 4) Diabetes, reactive airway disease, hypercholesterolaemia, smoking 5) History of tuberculosis, congestive heart failure, chronic obstructive pulmonary disease 6) History of surgical correction of congenital heart defect; iron deficiency anaemia 7) History of rheumatic fever

All isolates from blood; β-lactamase-negative and susceptible to antibiotics used for treatment; no IS1016-bexA deletion

Bezuhly and Fish (2012)115

Halifax, Canada

13 months; female (white)

Hand abscess, septicaemia

Recovered

None

Blood and wound

SadeghiAval et al (2013)116

Northern Ontario, Canada

1) 18 months; male 2) 8 months; female 3) 29 months; female 4) 15 years; male

1) Meningitis, pneumonia 2) Meningitis, pneumonia 3) Pneumonia 4) Pneumonia

All recovered

1) Not reported 2) Multicystic dysplastic kidney 3) Not reported 4) Not reported

1 and 2) Blood and CSF 3 and 4) Blood. All isolates: β-lactamase-negative and susceptible to antibiotics used for treatment; no IS1016-bexA deletion

Francis et al (2013)117

Western Australia

10 months; male

Sepsis

Septic shock and death

None

Clotted blood from a vascular catheter

Hia=Haemophilus influenzae serotype a.

Table 6: Clinical characteristics of invasive Haemophilus influenzae serotype a disease described in case studies published during the past 30 years

completely mature until children are aged 2–5 years.120 As a result, young children are unable to develop protective antibodies against such antigens and are highly susceptible to infections caused by encapsulated H influenzae. Newborn babies and infants during the first months of life are protected by maternal IgG antibodies acquired through the transplacental transfer, but later become susceptible to invasive Hib disease after a decrease in maternal IgG.121 In Hib-protein conjugated vaccines, PRP is covalently linked to a protein carrier, resulting in T-cell dependent immune response that develops at an early age.118 In unvaccinated individuals, natural immunity develops after exposure to Hib during its carriage or to some common nonpathogenic bacteria cross-reacting with PRP—eg, Escherichia coli K100.122 Natural immunity against invasive Hib disease is highly efficient, and increased serum titres of bactericidal antibody with age are strongly associated with a decrease in the incidence of disease.118 The natural history of Hia infection is very similar to that of Hib in the prevaccine era. Young infants seem to be protected against invasive Hia disease by maternal antibody because the disease does not typically affect children younger than 6 months. Bactericidal IgG antibodies against Hia capsular polysaccharide have been detected in cord blood serum samples from Mexican and Chilean mothers and in serum samples from healthy adult donors.123,124 Healthy individuals might acquire natural 78

immunity against Hia as a result of their exposure to either Hia or to some cross-reactive bacteria with shared epitopes.124 We have detected serum bactericidal activity against Hia in most adults (Tsang SW, Ulanova M, unpublished). After the decrease of maternal antibody concentrations, infants become susceptible to invasive Hia disease and, if infected, are unable to fully develop an antibody response to the pathogen. Hammitt and colleagues107 described a case of a 6-month-old infant who developed invasive Hia disease, but detected no IgG specific to the Hia capsular polysaccharide in acute-phase or convalescent-phase serum samples. Findings from earlier studies showed similar characteristics of anti-Hib immunity in young Alaska Native infants who often did not mount an antibody response to invasive Hib disease.125 This absence of response could be the reason that infants in this population developed recurrent episodes of invasive Hib disease when they were re-exposed to the pathogen.125 In line with similarities between Hib and Hia, a recurrence of invasive Hia disease has also been reported during an outbreak of invasive Hia disease in Alaska Native infants.47 Whether any immunological factors bring about an increased susceptibility of specific populations to invasive Hia disease is unknown. The fact that immune response to bacterial capsular polysaccharides involves few genes that encode the immunoglobulin variable domains might explain genetic predisposition to infections caused www.thelancet.com/infection Vol 14 January 2014

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by encapsulated bacteria.126 Indeed, a defective gene allele encoding the κ light chain of anti-PRP immunoglobulin variable part was detected in Navajos, with researchers suggesting that this absence could be the reason for an increased susceptibility to invasive Hib disease in this population.127

Prevention and control The tremendous success of the Hib conjugate vaccine and similarities between Hia and Hib capsules and the disease caused by these two serotypes provide rational background to suggest that a potential Hia conjugate vaccine will elicit protective antibodies in infants and will offer protection against Hia disease. Various issues related to the development and use of an Hia conjugate vaccine have been reviewed elsewhere.128 A bivalent Hia– Hib conjugate vaccine for infant immunisation will not only protect the most vulnerable population through induction of serum bactericidal antibodies to these two invasive serotypes, but will probably decrease carriage of these organisms and thereby further interrupt pathogen transmission through population-level herd immunity. Analogous to the recommendation of using chemoprophylaxis to prevent secondary Hib infections,129 chemoprophylaxis might prevent secondary Hia infections, although no data are available for drug choice, dosage, and effectiveness in prevention of development of disease and carriage.

Conclusions In the post-Hib vaccine era, two encapsulated serotypes of H influenzae (a and f) have emerged to replace Hib as important causes of invasive disease. The relative importance of each serotype varies with geographical location—eg, in Manitoba, Canada, serotype f caused only 4% of all invasive H influenzae disease but Hia was seven times more common, causing nearly 30% of the cases.43 In Europe, H influenzae serotype f caused only 5% of all invasive H influenzae disease,69 whereas in the USA, Hif was more common, accounting for 18% of all invasive H influenzae isolates serotyped.100,101 A clone of

Search strategy and selection criteria We systematically searched PubMed for articles published from June 1, 1970, to April 30, 2013, using the terms “Haemophilus influenzae”, “Haemophilus influenzae serotype a”, “Haemophilus influenzae invasive”, and “bacterial meningitis”. We also searched relevant references cited by retrieved papers. We used the same search terms to search Google Scholar to identify research reports, working papers, government documents, and abstracts. We included articles published in English, French, German, Dutch, Spanish, Portuguese, and Chinese. We also searched the Haemophilus influenzae multilocus sequence typing website for information about Hia on April 30, 2013.

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Hia with genetic organisation of the capsule synthesis genes similar to Hib has been reported to be associated with severe disease, with severity similar to Hib in the pre-Hib vaccine era. Specific reasons behind an increased incidence of invasive Hia disease in indigenous populations are unknown. Surveillance of Hia needs identification of expression of the Hia capsule by both phenotypic and genetic methods. Global distribution and potential migration of virulent clones that might be associated with severe disease, such as the ST-4, should be tracked by standardised techniques such as MLST, PFGE, or both to allow laboratories to compare their results. Knowledge of the type of immune response to Hia infection will be needed for vaccine development. A bivalent Hia–Hib conjugate vaccine could protect vulnerable individuals, such as indigenous populations, against invasive Hia and Hib. Cost–benefit analysis and the effect and acceptance of adding another vaccine against Hia to the present immunisation arsenal are needed for an evidence-based decision. Finally, while a vaccine against Hia is unavailable, the potential benefit of prophylaxis for close contacts of individuals with confirmed Hia disease in settings where secondary transmission is deemed likely should be studied. Contributors MU and RSWT had the idea for the Review, searched the available studies, wrote the Review, reviewed the different revisions, and approved the final version. Conflicts of interest We declare that we have no conflicts of interest. Acknowledgments We thank Luis Barreto for reading an early draft of this paper and for providing helpful discussion. This publication made use of data from the multilocus sequence typing website at Imperial College London, developed by David Aanensen and funded by Wellcome Trust. References 1 Kuhnert P, Christensen H. Pasteurellaceae: biology, genomics and molecular aspects. Norfolk, UK: Caister Academic Press, 2008. 2 Dochez AR, Mills KC, Kneeland Y. Variation of H influenzae during acute respiratory infection in the chimpanzee. Proc Soc Exp Biol N Y 1932; 30: 314–16. 3 Good RC, May BD. Respiratory pathogens in monkeys. Infect Immun 1971; 3: 87–93. 4 Gabre-Kidan T, Lipsky BA, Plorde JJ. Hemophilus influenzae as a cause of urinary tract infections in men. Arch Intern Med 1984; 144: 1623–27. 5 Quentin R, Ruimy R, Rosenau A, Musser JM, Christen R. 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 1996; 34: 1380–85. 6 Wallace RJ Jr, Baker CJ, Quinones FJ, Hollis DG, Weaver RE, Wiss K. Nontypable Haemophilus influenzae (biotype 4) as a neonatal, maternal, and genital pathogen. Rev Infect Dis 1983; 5: 123–36. 7 Pittman M. Variation and type specificity in the bacterial species Haemophilus influenzae. J Exp Med 1931; 53: 471–92. 8 Slack MPE. Haemophilus. In Topley & Wilson’s microbiology & microbial infections. 10th edition, volume 2 Bacteriology, pp. 1692–1718. Edited by S. Peter Borriello, Patrick R. Murray, Guido Funke. London, UK Edward Arnold Ltd, 2006. 9 Kilian M. A taxonomic study of the genus Haemophilus, with the proposal of a new species. J Gen Microbiol 1976; 93: 9–62.

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