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CLINICAL MICROBIOLOGY PROFICIENCY TESTING: CLINICAL BACTERIOLOGY

AUGUST 2008

M082-2 Knee joint fluid: Haemophilus aphrophilus / Aggregatibacter aphrophilus

HISTORY This sample was a joint fluid collected from a 30year old athlete with a swollen knee. The sample was sent to category A and B laboratories to set up and report as per their laboratory protocol. CMPT QA Internal quality testing yielded 3+ pure growth of Haemophilus aphrophilus/Aggregatibacter aphrophilus, viable for 11 days. The isolate was beta-lactamase negative and susceptible to ampicillin, amoxicillin, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, trimethoprimsulfamethoxazole, ciprofloxacin, and imipenem/meropenem. GRADING (grade=4) Consensus for grading was achieved with 93% (14/15) reference laboratories reporting Haemophilus aphrophilus or Aggregatibacter aphrophilus; one reference laboratory reported Pasteurella species. Results received from participants varied so greatly by laboratory category that results are shown in two tables, Table 1 for category A and Table 2 for category B laboratories (page 3). NOMENCLATURE CHANGE & IDENTIFICATION Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans, and Haemophilus segnis have recently been transferred to a new genus Aggregatibacter 1,2 in the family Pasteurellaceae. The species of the genus Aggregatibacter are independent of X factor and variably dependent on V factor for growth in vitro. Originally, H. aphrophilus was so named because it requires a high carbon dioxide atmosphere for growth, and "aphrophilus" means "foam-loving". Noted in the original descriptions of GRADING (maximum grade = 8)

IDENTIFICATION: 84% (58/69) category A laboratories and only 23% (5/22) of category B laboratories received a grade of 4/4 or 3/4. AST: see Table 3. NOTES A. aphrophilus has been implicated in serious infections: endocarditis, brain abscess, spinal epidural abscesses, meningitis, osteomyelitis, arthritis, lymphadenitis, and soft tissue infections following trauma (teeth manipulation, lacerations, bites, and postoperative wounds) particularly in absence of antibiotic prophylaxis.

`Bacterium actinomycetecomitans' (Klinger, 1912) and Haemophilus aphrophilus (Khairat, 1940) were the conspicuous growth in broth of small granules adhering to the walls of the test tube. Accordingly, Norskov-Lauritsen and Kilian (2006) proposed Aggregatibacter gen. nov. as the generic name for the group Aggregatibacter (L. v. aggregare to come together, aggregate, N.L. masc. n. bacter bacterial rod, N.L. masc. n. Aggregatibacter rod-shaped bacterium that aggregates with others) 2,3. In addition, several investigators now consider Haemophilus paraphrophilus and H. aphrophilus as a single species--Aggregatibacter aphrophilus 1. Aggregatibacter aphrophilus is a small gram-negative coccobacillus that is oxidase­positive. Colonies of A. aphrophilus on chocolate agar incubated in air supplemented with 5­10% CO2 are high convex, granular, yellowish, and opaque and

(Continued on page 2)

Table 1. M082-2 Results received from category A laboratories and grades assigned. Identifications submitted Haemophilus aphrophilus (44) / Aggregatibacter/Haemophilus aphrophilus, presumptive, refer (22/44); Haemophilus aphrophilus/ paraphrophilus (4), refer (1/4) Haemophilus species, not H. influenzae, refer Gram-negative coccobacilli, refer for ID & sens, possible HACEK group organism Gram-negative coccobacilli, refer for ID & sens CNS & gram-negative coccobacilli, refer Pasteurella species, +/- presumptive, refer (2); Eikenella corrodens, presumptive, refer (1) Sphingomonas paucimobilis +/- refer (2); unable to ID, possible Kingella sp., refer (1) Joint fluids not routinely processed (3); sample leaked in transit-CMPT unable to send a replacement sample (1) Total A

(% out of 69)

Methods

classical (8) & API NH (23) & Vitek2 (4); RapID NH, classical (10) & Vitek2 (2); Vitek2, classical (6); API 20 NH, classical (1); API 20 NH, Haem ID Quad (1); BD Phoenix, RapID NH, classical (1); MicroScan, classical (2) classical classical classical Vitek, classical API 20E, API 20NE, MicroScan, classical (1), API 20E, classical (1); Vitek, classical (1); Vitek2 compact, classical (1), Vitek2, classical (1); classical (2)

Grade 4

58 (84%)

2 1 1 1 6

3 3 1 0 0

4

/

ungraded

73

M082-2 Joint fluid: A. aphrophilus (continued from page 1)

2 CLINICAL SIGNIFICANCE A. aphrophilus is a fastidious gram-negative bacillus belonging to the HACEK group 6,7. It is usually present as a normal commensal in oral cavity and upper respiratory tract of human beings and animals such as dogs 6. It was first reported by Khairat et al. in 1940 from a fatal case of endocarditis (reported in reference 8). In addition to endocarditis, it has been implicated in other serious infections (brain abscess, spinal epidural abscesses, meningitis, osteomyelitis, arthritis, lymphadenitis, and soft tissue infections) following trauma (teeth manipulation, lacerations, bites, and postoperative wounds) particularly in absence of antibiotic prophylaxis 6, 8­18 . It causes human infections particularly in middle-aged and old people but only rarely in pediatrics population. Involvement of brain by this specific organism in pediatrics without cardiac predisposition is very rare. It is of interest that A. aphrophilus does not appear to be associated with respiratory infections although it is commensal in the upper respiratory tract. ANTIMICROBIAL SUSCEPTIBILITY & TREATMENT Unlike H. influenzae, A. aphrophilus usually behaves as a wild type Haemophilus species. Isolates are usually betalactamase negative, and are susceptible to ampicillin, betalactam/beta-lactam inhibitor combinations, cephalosporins, sulphonamides, tetracyclines, macrolides, and chloramphenicol. There is very little data on acquired resistance to antimicrobial agents in species other than H. influenzae 1,4. Appropriate antimicrobial therapy, such as a beta-lactam/ beta-lactamase inhibitor, ceftriaxone or cefotaxime or a fluoroquinolone, can lead to a favorable clinical outcome 4. As A.. aphrophilus is more commonly associated with infections of joints, bones, soft-tissue or brain, more prolonged parenteral treatment may be required than for respiratory infections with common Haemophilus species. REFERENCES

reach a diameter of 1.0­1.5 mm within 24 h. When plates are incubated without extra CO2, the growth is characteristically stunted, with very small colonies interspersed with a few larger colonies 2. Growth in broth is granular, with heavy sediment on the bottom of the tube; adhering colonies on the walls are difficult to remove. The presence of hemin may enhance growth, but porphyrins are synthesized from d-aminolaevulinic acid and X factor is not required. Some A. aphrophilus isolates are capable of synthesizing NMN from nicotinamide and 5phosphoribosyl pyrophosphate, while others (formerly Haemophilus paraphrophilus) lack a functional nicotinamide phosphoribosyltransferase and are dependent on V factor. A V-factor requiring (NAD) positive isolate may be summarily assumed to be H. parainfluenzae. Fermentation of lactose and trehalose (A. aphrophilus, positive; H. parainfluenzae, negative) are key tests that can be used to discrimination between these isolates. Organisms recovered from sites other than the respiratory tract should be investigated further to determine species 4. A.aphrophilus reactions for other tests include acid produced from glucose, fructose, maltose, mannose, and sucrose, whereas arabinose, cellobiose, mannitol, melibiose, melezitose, salicin, sorbose, sorbitol and xylose are not fermented. Variable fermentation is observed with galactose and raffinose. The catalase test is negative; ONPG is positive. Differentiation from other related microorganisms such as Pasteurella, Eikenella, Actinobacillus (now called Aggregatibacter actinomycetemcomitans 2), Cardiobacterium and Suttonella may also be difficult. Laboratories should consult appropriate manuals and reference information in this regard for review 1,4-6. An overview with flow charts and identification tables may be found in reference 7, see page 12 (URL provided) . ANTIMICROBIAL SUSCEPTIBILITY TESTING (grade=4) Consensus between the reference laboratories was attained for beta-lactamase testing and ampicillin, amoxicillin or amoxicillin-clavulanic acid reporting. Eighty percent 12/15 reference laboratories reported the isolate as beta-lactamase negative, 1 laboratory stated refer, and 2 did not report. Eighty percent (12/15) reference laboratories also reported susceptible results for ampicillin, amoxicillin or amoxicillin-clavulanic acid. One laboratory referred susceptibilities, one laboratory only submitted a comment "Usually susceptible to ampicillin, amoxicillin and 2nd generation cephalosporins." and one did not report. Results received and grades assigned are shown in Table 3.

1. Kilian M. 2007. Haemophilus. p. 636­648. In PR Murray et al. (eds.), Manual of Clinical Microbiology, 9th ed., American Society for Microbiology, Washington, D.C. 2. Norskov-Lauritsen, N., Kilian, M. 2006. Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V Table 3. M082-2 AST results received, methods reported, and grades assigned. factor-independent isolates). Int J Syst ampicillin, amoxilillin or Grade Beta-lactamase Evol Microbiol. 56: 2135-2146. amoxicillin-clavulanic acid 3. German Collection of Microorgannegative susceptible isms and Cell Cultures http:// 4 51 (45A, 6B) 40 (37A, 3B) www.dsmz.de/index.htm cefinase (26), nitrocefin (4), chromoge E-test (16), KB (15), KB, Etest 4. Murphy T. 2004. Haemophilus infections. p. 2667-2668. In GL Mannic cephalosporin (2), HNID panel (1); (5), no report (2), MS (1) dell et al (eds.), Principles and Pracoxoid (1); no method reported (17) tices of Infectious Diseases, 6th ed., 1 1 weakly positive (cefinase) / Elsevier, Churchill, Livingstone, PhilaUngraded No report, refer No report, refer, comment only delphia, PA.

(Continued on page 3)

M082-2 Joint fluid: A. aphrophilus (continued from page 2)

3

14. Clapper WE, Smith EA. 1971. H. aphrophilus in brain abscess: report of case. Am J Clin Pathol. 55: 726-728. 15. O'Driscoll JC, Keene GS, Weinbren MJ, Johnson AP, Palepou MF, George RC. 1995. Haemophilus aphrophilus discitis and vertebral osteomyelitis . Scand J Infect Dis. 27: 291-293. 16. Kao PT, Tseng HK, Su SC, Lee CM. 2002. Haemophilus aphrophilus brain abscess: a case report. J Microbiol Immunol Infect. 35: 184-186. 17. Bieger RC, Brewer NS, Washington JA 2nd. 1978. Haemophilus aphrophilus: a microbiologic and clinical review and report of 42 cases. Medicine. 57: 345-355. 18. Sharma BS, Gupta SK, Khosla VK. 2000. Current concepts in the management of pyogenic brain abscess. Neurol India. 48: 105-111. See nomenclature changes at these URLs: http://www.bacterio.cict.fr/ http://www.dsmz.de/bactnom/genera1.htm Recommended reading: Vandamme PAR. 2007. Taxonomy and classification of bacteria. P. 275-290.In PR Murray et al. (eds.), Manual of Clinical Microbiology, 9th ed., American Society for Microbiology, Washington, D.C.

5. Albritton, W.L. 1986. Species identification in Haemophilus infection. Rev Infect Dis. 10:1226. 6. Koneman EM, Allen SD, Janda WM, Schreckenberger PC, Jr Winn WC. 1997. p. 395-398. Miscellaneous fastidious gram-negative bacilli. Color Atlas and Textbook of Diagnostic Microbiology. Lippincott, PA. 7. Identification of Haemophilus species and the HACEK group. Standards Unit, Evaluations and Standards Laboratory. "under review" http://www.hpa-standardmethods.org.uk/documents/bsopid/pdf/ bsopid12.pdf 8. Webb CH, Hogg GM. 1990. Haemophilus. aphrophilus endocarditis. Br J Clin Pract. 44 :329-331. 9. Tsung, HS., Chun, LH, Yao, LN, Hung, LK, Chien KW. 2005. Clinical characteristics of invasive Haemophilus aphrophilus infections. J Microbiol Immunol Infect. 38: 271-276. 10. Yamashita J, Bone FJ, Hitchcock E. 1972. Brain abscess due to Haemophilus aphrophilus: case report. J Neurol Neurosurg Psychiatry. 35: 909-911. 11. Abla AA, Maroon JC, Slifkin M. 1986. Brain abscess due to Haemophilus aphrophilus: possible canine transmission. Neurosurgery. 19: 123-124. 12. De Louvis J, Gortval P, Hurley R. 1977. Bacteriology of abscesses of the central nervous system: a multicentre prospective study. Br Med J. 2: 981-984. 13. Isom JB, Gordy PD, Selner JC, Brown LJ, Willis M. 1964. Brain abscess due to H. aphrophilus. N Engl J Med. 271:1059-1061.

Table 2. M082-2 Results received from category B laboratories and grades assigned. Identifications submitted Haemophilus aphrophilus (1) presumptive, refer (1); Haemophilus aphrophilus /paraphrophilus (1) Haemophilus sp., not H. influenzae, refer; Haemophilus species, refer Haemophilus influenzae and CNS Sphingomonas paucimobilis, presumptive, refer Pasteurella pneumotropica/Mannheimia haemolytica, +/- refer Acinetobacter lwoffii/junii, presumptive, refer non-fermentative gram-negative coccobacillus (possible Acinetobacter sp), refer (1); aerobic gram-negative coccobacilli, refer (1); small gramnegative coccobacilli, refer (1); gram-negative bacilli, refer for ID and susceptibilities (5) non-hemolytic streptococci, refer gram-negative cocci, refer no report

Joint fluid samples not routinely processed (5); sample leaked in transit-CMPT unable to send a replacement sample (5); sample not labelled, unable to analyze. Please recollect and resubmit a new sample (1); no aerobic/anaerobic growth after 72 hours incubation, CMPT unable to send 2nd sample (1)

B 3 2 1 2 2 1 8

Methods API NH (1) & classical (2)

Classical & API 20E (1) or API NH, RapIDNH (1)

Grade 4 3 0 0 0 0 1

API NH, classical Vitek2 API 20E, classical Vitek, classical classical (1) & API 20E (2) or Vitek2 (2) or BBL crystal (1); refer (1); Vitek, classical (1)

1 1 1 12

classical refer n/a n/a

0 0 0 ungraded

Total

34

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