FINAL RESULTS AND COMMENTS 2013-5 BACTERIOLOGY …dataentry.rcpaqap.com.au/micro/reports/uploadedfiles/Fnl2013-5.pdfAeromonas species may also grow yellow colonies on TCBS. Results

FINAL RESULTS AND COMMENTS

2013-5

BACTERIOLOGY PROGRAM

PARASITOLOGY

_________________________________________________________________________________________

NATA Accredited Proficiency Testing Scheme Provider Number: 14863 This Facility is accredited by the National Association of Testing Authorities, Australia and complies with the Requirements of ISO/IEC 17043:2010

13th September 2013

Suite 201, Level 2 8 Herbert Street, St Leonards NSW 2065 Australia PH: +61 2 9045 6060 Fax: 1300 78 29 21 (Australia) +61 2 9356 2003 (International) [email protected] www.rcpaqap.com.au/microbiology

AUTHORISED BY CHAIR CHAIR: DR ARTHUR MORRIS PROGRAM MANAGER: ELIZABETH HAREMZA Report prepared by D.Walker and M.Bullivant No information related to any of the participants will be divulged to a third party, unless required by legislation, without the prior written consent of the participant. General information may be discussed at meetings or presented as papers to journals.

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©2013 RCPA Quality Assurance Programs Pty Limited 1 Date of issue: 13th September 2013

FINAL RESULTS AND COMMENTS D5/2013 ITEM 2013:5:1A,B FAECES FOR CULTURE

%

62 30.4

75 36.8

3 1.5

1 0.5

5 2.5

1 0.5

2 1.0

49 24.0

2 1.0

1 0.5

1 0.5

2 1.0

Pathogens reported 1A

QA Report Item 1A contained Campylobacter jejuni, with Escherichia coli, Enterococcus faecalis and Citrobacter freundii representing bowel flora. Viability of the pathogen and homogeneity and stability testing were satisfactory with Campylobacter counts of greater than 2 x 108 CFU/L throughout the survey period. Isolation and Identification of Pathogens Item 1A: The results of this survey compare poorly with previous surveys (Table 1). Of 204 laboratories returning a result only 141 (69% (or 70% including transcription errors)) reported Campylobacter species. Of the 63 participants not reporting the pathogen 31 (49%) did not appear to culture for it. Campylobacter jejuni isolation requires the use of selective agar such as a moist CCDA plate incubated at 42°C in a microaerobic atmosphere (5% O2, 10% CO2, and 85% N2)

1 for 48 to 72 hours.2 It is essential to meet these conditions in order to isolate. Since Campylobacter is one of the most common causes of bacterial enteritis1 all laboratories culturing for faecal pathogens should routinely investigate for Campylobacter species. Motility and Gram stain should reveal rapidly darting rods that have a gull‐winged appearance and stain faintly gram‐negative. Oxidase is positive. Hippurate hydrolysis is positive for C. jejuni and this result was confirmed by at least 20 laboratories. The laboratory reporting C. coli reported hippurate test as negative. The two reports of Vibrio parahaemolyticus were transcription errors. Appropriate commercial systems/kits used and success rates in the hands of the users were: API Campy—Biomerieux: 4/4 C. jejuni (100% correct) MALDI‐TOF: 39/42 C. jejuni/species (92% correct)

* NLP/NPI = no likely pathogen/no pathogens isolated

Survey 2012:1:1A 2010:3:1A 2009:7:1B

% reporting Campylobacter species 82 80 82

2008:7:1A

84

2006:3:1B

88

Table 1 : Past survey results for Campylobacter species

2

1

1

2

49

2

1

5

1

3

75

62

0 10 20 30 40 50 60 70 80

Vibrio parahaemolyticus

Unable to Ident ify ‐ Refer

Salmonella species

No Salmonella Campylobacter Shigella or Yersinia

*NLP/NPI

Escherichia coli

Enterococcus faecium

Citrobacter freundii

Campylobacter coli

Campylobacter jejuni/coli

Campylobacter species

Campylobacter jejuni

References Item 1A: 1. Fitzgerald, C., and Nachamkin, I., Campylobacter and Arcobacter: In Manual of Clinical Microbiology, Versalovic and Carroll et al. 10th

Edition. ASM Press: Chapter 53. 2. Curved gram‐negative bacilli and oxidase‐positive fermenters: Campylobacteriaceae and Vibrionaceae : in Koneman’s Color Atlas and

Textbook of Diagnostic Microbiology, Winn and Allen et al. 2005. 6th Ed. Lippincott Williams and Wilkins, Philadelphia; Chapter 8.


Pathogens reported 1B %

182 88.8

2 1.0

3 1.5

2 1.0

1 0.5

1 0.5

1 0.5

2 1.0

1 0.5

1 0.5

5 2.4

1 0.5

1 0.5

1 0.5

1 0.5

1

1

1

1

5

1

1

2

1

1

1

2

3

2

182

0 20 40 60 80 100 120 140 160 180 200

Salmonella species

Pseudomonas stutzeri

Plesiomonas shigelloides

No salmonella, shigella or campylobacter isolated

No likely pathogen

Escherichia coli

Citrobacter freundii

Campylobacter jejuni

Aeromonas species

Aeromonas salmonicida

Aeromonas hydrophila/caviae

Aeromonas hydrophila

Vibrio vulnificus

Vibrio species

Vibrio parahaemolyt icus

QA Report Item 1B contained Vibrio parahaemolyticus, with Enterococcus faecalis, Escherichia coli and Citrobacter freundii representing faecal flora. Colony counts for V. parahaemolyticus remained above 7.5 x 109 CFU/L during survey period. The sample also satisfactorily passed homogeneity and stability testing. Isolation and Identification of Pathogens Item 1B: Two hundred and five laboratories returned results for this survey with 187 (91% (or 92% counting transcription errors)) reporting Vibrio species. Any oxidase‐positve organism in a stool sample could be Vibrio, Plesiomonas or Aeromonas and these must be distinguished from one another and from Pseudomonas species. A TSI/KIA slope will eliminate Pseudomonas as this is an oxidative organism and the others are fermentative. There was one false report of Pseudomonas stutzeri (a yellow‐pigmented, wrinkled organism). Pseudomonas is usually not β‐haemolytic or indole‐positive as are the other three pathogenic species. Six laboratories failed to distinguish between them with reports of various Aeromonas species (5) and Plesiomonas shigelloides (1). Most laboratories (4/5) reporting Aeromonas species used API 20 NE – Biomerieux and appeared to over‐read the esculin hydrolysis reaction, calling it positive and tipping the scales in favour of an Aeromonas identification. The most common profile reported by other users of this kit was 7077744 (i.e. esculin reported as negative) confirming a very good identification (%ID = 99.1) for V. parahaemolyticus after 48 hours. P. shigelloides is positive for arginine dihydrolase (ADH), and ornithine (ODC) and lysine (LDC) decarboxylases, inhibited by 4% salt and DNAase‐negative. Vibrio species are usually tolerant to 4% salt and are either negative for ADH or for ODC and LDC. V. vulnificus reported by one laboratory are usually lactose‐positive. They are also sensitive to O/129 disks (as are V. cholerae and V. mimicus), whereas V. parahaemolyticus (and V. alginolyticus, V. fluvialis and Aeromonas species) are resistant. V. cholerae, V. alginolyticus and V. fluvialis all grow yellow colonies on TCBS agar due to their ability to utilize sucrose. Aeromonas species may also grow yellow colonies on TCBS. Results of the most commonly used commercial kits/systems in the hands of the users are shown in Table 3.

Despatch 2010:7:1A 2008:5:1B 2006:3:1A

% reporting Vibrio species

89 86 69

Table 2: Despatch comparison of results

1B Most common kit/Identification systems used

Number of users

% of users reporting

Vibrio species

API 20 E—Biomerieux 36 94

API 20 NE—Biomerieux 36 89

BBL Crystal—BD Enteric/Non Fermenter Kit

8 100

Phoenix 4 100

VITEK2 GN‐Biomerieux 64 95

MALDI‐TOF 44 98

Table 3: Kit performance

Reference Item 1B: 1. Clinical Microbiology Procedures handbook. Lynne S.

Garcia, Editor in Chief. Vol.1, Section 3 (3.17.36. 1‐3 and 3.18.2.17).


ITEM 2013:5:2A,B SPUTUM FOR CULTURE

Pathogens reported 2A Item 2013:5:2A,B Sputum for Culture

QA Report Item 2A contained a pure growth of Moraxella catarrhalis. Throughout the three week despatch period, the bacterial count remained above 1.0 x 1010 CFU/L. Stability and homogeneity tests were satisfactory. Isolation, identification and susceptibility testing of pathogens One participant transposed 2A and 2B results.

%

239 94.1

6 2.4

2 0.8

1 0.4

1 0.4

1 0.4

1 0.4

3 1.2

Item 2A: Identification Item 2A contained the pathogen Moraxella catarrhalis. The graph above illustrates the identifications reported. Two hundred and fifty four participants returned results for item 2A. Ninety five percent of participants identified the pathogen as Moraxella catarrhalis or Branhamella catarrhalis. Moraxella catarrhalis is now the accepted name for this organism.1 Three participants reported “no likely pathogen”. One hundred and thirty five participants used a commercial kit/system to identify this pathogen. All kits performed well. MALDI‐TOF MS (46), API NH (41 users) and Vitek 2 NH (20) were the most popular commercial kit/systems used. A variety of methods used either alone or in combination were used to identify this isolate. The main identification tests used were Gram stain, oxidase, “push test” (pushed along the agar like a hockey puck), Catarrhalis disk/tributyrin hydrolysis and DNAse.2 One participant transposed results with item 2B. Susceptibility Testing: Moraxella catarrhalis Like most strains of M.catarrhalis, this strain was a β‐lactamase producer. CLSI does not recommend routine susceptibility testing, however interpretative criteria for broth microdilution susceptibility testing is available if required.3 β‐lactamase can be performed using chromogenic cephalosporin methods, e.g. nitrocefin. One hundred and sixteen participants reported this strain as a β‐lactamase producer and only one reported this strain as β‐lactamase negative. The participant reporting β‐lactamase negative did not report ampicillin/amoxycillin. Of those 17 participants who reported ampicillin/amoxycillin susceptible, all used CLSI. However three of these participants reported the β‐lactamase as positive. References Item 2A

1. Acinetobacter, Chryseobacterium, Moraxella, and other Nonfermentative gram‐negative rods: in Manual of Clinical Microbiology, 10th Edition. Editor in chief James Versalovic. ASM Press; Chapter 42. p720‐22.

2. Speeleveld.E, Fossépré J.M, Gordts B, and Van Landuyt H W. Comparison of three rapid methods, tributyrine, 4‐methylumbelliferyl butyrate, and indoxyl acetate, for rapid identification of Moraxella catarrhalis. J. Clin. Microbiol. 1994;32: 1362‐1363.

http://jcm.asm.org/cgi/reprint/32/5/1362 3. Clinical and Laboratory Standards Institute. Methods for Antimicrobial

Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline. CLSI document M45‐A2 Clinical and Laboratory Standards Institute, Pennsylvania USA. 2010

Susceptibilities (all methods) 2A M.catarrhalis S I R

Ampicillin/Amoxycillin 17 ‐ 73

Ampicillin/sulbactam 1 ‐ ‐

Augmentin 136 ‐ 1

Azithromycin 25 ‐ ‐

Cefaclor 42 ‐ ‐

Cefoperazone 1 ‐ ‐

Cefotaxime 20 ‐ 1

Ceftazidime 11 ‐ ‐

Ceftriaxone 25 ‐ ‐

Cefuroxime 44 ‐ ‐

Chloramphenicol 13 ‐ ‐

Ciprofloxacin 54 ‐ ‐

Clarithromycin 6 ‐ ‐

Cotrimoxazole 97 1 2

Doxycycline 4 ‐ 1

Erythromycin 91 ‐ 1

Gentamicin 7 1 1

Imipenem 6 ‐ 1

Levofloxacin 10 ‐ ‐

Meropenem 8 ‐ ‐

Penicillin 3 ‐ 56

Tetracycline 101 1 2

Table 4: 2A Moraxella catarrhalis susceptibilities reported– all methods.

3

1

1

1

1

2

6

239

0 50 100 150 200 250 300

No likely pathogen

Micrococcus species

Staphylococcus aureus

Acinetobacter baumannii

Branhamella species

Branhamella catarrhalis

Moraxella species

Moraxella catarrhalis


Item 2B: Acinetobacter baumannii 2B contained Acinetobacter baumannii with an α‐haemolytic streptococcus species, Neisseria subflava and Staphylococcus epidermidis present as normal flora. Throughout the three week despatch period, the bacterial count remained above 4.5 x 109

CFU/L for the pathogen and 3.5 x 109 CFU/L for the normal flora. Stability and homogeneity tests were satisfactory. Identification Acinetobacter species are oxidase negative, nitrate negative, non‐motile gram‐negative short rods. They utilise carbohydrates oxidatively. A.baumannii may be differentiated from other Acinetobacter species by growth at 44oC (+), glucose oxidation (+), hydrolysis of gelatin (‐), haemolysis on SBA and the ability to

Pathogens reported 2B

Susceptibilities (all methods)

2B A.baumannii S I R Amikacin 68 ‐ ‐


Ampicillin/Sulbactam 16 ‐ ‐

Augmentin 44 1 46

Cefazolin ‐ ‐ 28 Cefepime 18 ‐ ‐

Cefotaxime 5 7 32

Ceftazidime 92 1 10

Ceftriaxone 6 15 47

Cefuroxime 2 4 10

Cephalexin ‐ ‐ 12

Cephalothin ‐ 1 18

Ciprofloxacin 208 ‐ ‐

Colistin 7 ‐ ‐

Cotrimoxazole 135 ‐ 2

Gentamicin 224 2 1

Imipenem 89 ‐ ‐


Meropenem 120 1 ‐

Piperacillin 10 2 2

Piperacillin/tazobactam 71 ‐ ‐

Polymyxin B 14 ‐ ‐

Tetracycline 18 1 2

Timentin 36 ‐ 1

Tobramycin 26 ‐ ‐

Trimethoprim 4 ‐ 17

Unasyn 9 ‐ ‐

utilise various carbon substrates. Table 7‐22 and 7‐23 in Koneman outlines a practical approach to the identification of non‐fermenters.1 Two hundred and thirty eight participants used a commercial kit/system. All kits performed well. Forty eight participants used MALDI ‐TOF– all reporting A.baumannii /A.baumannii complex . The only erroneous identification (besides the transposed result) was P.luteola. This participant used API 20E but unlike all the other API 20E users they reported the ADH positive. Susceptibility testing Acinetobacter baumannii: Susceptibility testing A.baumannii is often responsible for hospital‐acquired infections and its ability to acquire multi‐antibiotic resistance is causing concern. This isolate was not multi‐resistant. For participants’ susceptibility testing results please refer to Table 5.

Reference Item 2B

1. The nonfermentative gram‐negative bacilli in Koneman's Colour Atlas and Textbook of Diagnostic Microbiology. Winn and Akllen et al. 2005 6th Ed. Lippincott Williams and Wilkins, Philadelphia; p360‐361.

Table 5: Acinetobacter baumannii susceptibilities reported– all methods.

1

1

9

4

29

70

140

0 20 40 60 80 100 120 140 160


Pseudomonas luteola

Acinetobacter species

Acinetobacter calcoaceticus

Acinetobacter calcoaceticus/baumannii

Acinetobacter baumannii complex

Acinetobacter baumannii

%

140 55.1

70 27.6

29 11.4

4 1.6

9 3.5

1 0.4

1 0.4


ITEM 2013:5:3 VAGINAL SWAB

% 182 76.2

3 1.3

12 5.0

9 3.8

18 7.5

1 0.4

1 0.4

1 0.4

1 0.4

1 0.4

1 0.4

1 0.4

1 0.4

1 0.4

4 1.7

2 0.8

*NLP/NPI = no likely pathogen/no pathogens iolated **includes presumptive identifications

Table 7: Streptococcus species (various)

QA Report This sample contained a mixture of commensals including Neisseria cinerea, Staphylococcus epidermidis and α‐haemolytic streptococci to represent oral/urogenital tract and skin flora. This item passed homogeneity and stability testing with counts above 4.0x 109 CFU/L for the N. cinerea and above 3.0 x 109 CFU/L for the other organisms throughout the survey period. Isolation and Identification of Pathogens Neisseria cinerea is a Neisseria gonorrhoeae‐like isolate which is a commensal of the naso– and oropharynx and less commonly, genital sites. There have been documented cases where N. cinerea has been misreported as N. gonorrhoeae because of its similarities in colony appearance and biochemical tests.1 In this survey 77% reported no likely pathogen or similar with 5% reporting N. cinerea. The last time this organism was issued was in survey 2011:5:3. Other Neisseria species reported are listed above. N. cinerea is glucose‐negative but rapidly overoxidizes this acid to CO2 so that the carbonic acid produced may result in a weakly positive test depending on the buffering capacity of the medium used.2 This can result in false reports of N. gonorrhoeae if only a narrow range of tests are performed. In this survey 18 laboratories (7.5%) reported N. gonorrhoeae. This is an improvement on the 2011 survey where 21% reported N. gonorrhoeae but is still unacceptably high. Out of 8 laboratories reporting glucose as positive, 6 concluded that it was N. gonorrhoeae. Reference 2 below suggests a number of other supplementary tests to perform in order to distinguish similar Neisseria species from N. gonorrhoeae. Serological/immunological/molecular methods are important confirmatory follow‐up tests when N. gonorrhoeae is suspected. Some N. cinerea isolates have reacted in gonococcal coagglutination serology tests.2 A simple test is that N. cinerea will grow on basic agars such as blood agar, trypticase soy agar and Mueller‐Hinton agar which N. gonorrhoaea will not.1 Caution should be exercised when identifying suspected gonococcal isolates that exhibit atypical reactions. References Item 3: 1. Dossett, JH., Applebaum, PC., Knapp, JS., and Totten, PA. Proctitis associated with Neisseria cinerea misidentified as Neisseria gonorrhoeae in a child.

J Clin Microbiol. 1985; 21: 575‐7. http://www.ncbi.nlm.nih.gov/pubmed/3921562 2. http://www.cdc.gov/std/Gonorrhea/lab/Ncin.htm

Streptococcus sanguis 1

Streptococcus mitis 1

Streptococcus group B 1

Streptococcus canis 1

Table 6: Other Neisseria species

2

4

1

1

1

1

1

1

1

1

1

18

9

12

3

182

0 20 40 60 80 100 120 140 160 180 200

Unable to Identify ‐ Refer

Streptococcus species (various)

Shigella sonnei


Gram positive coccus

Escherichia coli

Enterococcus fae calis

Aeromonas hydrophila group

?Ureaplasma urealyticum

?Staphylococcus epidermidis

?Gram negative diplococci

**Neisseria gonorrhoeae

**Other Neisseria species

**Neisseria cinerea

No Neisseria gonorrhoeae isolated

*NLP/NPI

Neisseria species 7

Neisseria elongata 1

?Neisseria elongata 1


ITEM 2013:5:4 WOUND SWAB

Item 2013:5:4 Wound Swab QA Report This item contained Staphylococcus aureus and the anaerobe, Clostridium septicum. Homogeneity and stability testing were satisfactory and viability counts by the end of despatch were > 1.0 X 1010 CFU/L for both pathogens. Culture The challenge was to isolate and identify an aerobic and anaerobic pathogen from this item. Six participants did not culture for anaerobes. Of those 232 performing anaerobic culture, 25 participants (11%) failed to report the anaerobe.

Aerobic isolate Staphylococcus aureus %

235 98.7

1 0.4

1 0.4

1 0.4

Identification: Staphylococcus aureus All participants isolated the staphylococcus. One participant, even though the slide and tube coagulase were both positive did not report S.aureus but chose to report “Staphylococcus coag. positive”. The participant reporting Staphylococcus cohnii reported both the slide and tube coagulase negative. Seventy seven used a commercial kit/system to identify this pathogen. All kits performed well. Susceptibility Testing: Staphylococcus aureus Susceptibility testing was performed well. One participant incorrectly reported that the isolate was MRSA which could have treatment and infection control implications.

Susceptibilities (all methods) Item 4 Staphylococcus

aureus S I R


Augmentin 30 ‐ 1 Cefazolin 16 ‐ ‐ Cefoxitin 127 ‐ 1

Cefuroxime 7 ‐ ‐

Cephalexin 34 ‐ ‐

Cephalothin 19 ‐ 1

Chloramphenicol 5 ‐ ‐

Ciprofloxacin 63 ‐ 2

Clindamycin 152 1 1

Cotrimoxazole 140 ‐ 1

Doxycycline 7 ‐ ‐

Erythromycin 215 2 1

Flucloxacillin 13 ‐ 1

Fusidic Acid 60 ‐ ‐

Gentamicin 72 ‐ ‐


Linezolid 24 ‐ ‐

Methicillin 33 ‐ ‐

Oxacillin 69 ‐ 1

Penicillin 1 ‐ 202

Rifampicin 61 ‐ ‐

Teicoplanin 13 ‐ ‐

Tetracycline 91 ‐ ‐

Trimethoprim 9 ‐ ‐

Vancomycin 73 ‐ ‐

Table 8: Staphylococcus aureus susceptibilities reported– all methods.

1

1

1

235

0 50 100 150 200 250

Staphylococcus cohnii

Staphylococcus coag. positive

Staphylococcus aureus (MRSA)

Staphylococcus aureus

ITEM 2013:5:4 CLERICAL ERROR

Clerical Errors reported in Item 2013:5:4 Number labs (238)

No error reported 11

Error unit number 227 (95%)

Table 9: Clerical errors reported.

Specimen label: Rubi BURNS U/N 314699 Paperwork: Rubi BURNS U/N 781013


Anaerobic isolate Clostridium septicum

%

129 55.6

32 13.8

9 3.9

1 0.4

1 0.4

1 0.4

4 1.7

3 1.3

1 0.4

1 0.4

1 0.4

1 0.4

13 5.6

2 0.9

6 2.6

1 0.4

1 0.4

25 10.8

*includes presumptive identifications

Anaerobic Isolate ‐ Clostridium septicum Two hundred and thirty two participants cultured for anaerobes with 89% isolating an anaerobe and 78% reporting a Clostridium species. C.septicum is a rapidly growing large filamentous gram‐positive rod. Spores, often rare, are subterminal “lemon” forms. It produces a thick swarming growth. The key features of C.septicum include negative reactions for: catalase, lecithinase, lipase, sucrose and indole while positive eactions are recorded for gelatin, esculin and DNAse. One hundred and forty participants used a commercial kit/system. For those isolating and reporting an anaerobe most popular kits with identifications in the hands of the users were as follows (=Clostridium septicum): API 20 A: 13/18 (72%). Clostridium species (2); C.paraputrificum (1); Actinomyces naeslundii (1); Fusobacterium species (1). RapiD 32 A: 8/10 (80%). C.perfringens (2). Rapid ANA II : 27/35 (77%). C.perfringens (4); C.tertium (2); C.hastiforme (1); Clostridium species (1). VITEK 2 ANC: 23/31 (74%).Clostridium species (4); C.sporogenes (1); C. bifermentans (1); C. tertium (1).C.perfringens (1). MALDI‐TOF MS: 43/45 (96%). C.tertium (1= Bruker); Clostridium species (1= manufacturer not specified).

Reference Item 4

1. The Anaerobic Bacteria: in Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Winn and Allen et al. 2005. 6th Ed. Lippincott Williams and Wilkins, Philadelphia; page 913

25

1

1

6

2

13

1

1

1

1

3

4

1

1

1

9

32

129

0 20 40 60 80 100 120 140

No anaerobe isolated (culture performed)


Anaerobic species

Anaerobic Gram negative rod*

Anaerobic Gram positive coccus

Anaerobic Gram positive bacillus*

Fusobacterium species

Peptococcus species

Actinomyces naeslundii

Clostridium bifermentans

Clostridium tetani

Clostridium tertium

Clostridium hastiforme

Clostridium paraputrificum

Clostridium sporogenes

Clostridium perfringens*

Clostridium species*

Clostridium septicum*

Fugure 4.1 C.septicum swarming over S.aureus colonies. Growth on HBA @ 35°C in ANO2. © Microbiology QAP 2009.


ITEM 2013:5:5 ISOLATE FOR IDENTIFICATION

%

94 42.7

17 7.7

1 0.5

38 17.3

1 0.5

1 0.5

1 0.5

1 0.5

4 1.8

4 1.8

1 0.5

1 0.5

7 3.2

1 0.5

9 4.1

34 15.5

5 2.3

* includes presumptive identification(s)

Isolate for Identification– Actinomyces meyeri QA Report This item contained a pure growth of Actinomyces meyeri and passed all homogeneity and stability testing with counts remaining above >1.0 x 1010 CFU/L throughout the despatch period. Identification Actinomyces are anaerobic to micoaerophilic, non‐sporulating gram‐positive rods more typically found in polymicrobial infections than alone.1 Actinomycosis is a chronic, granulomatous infection of which A. Israelii is the most common cause.1,2 A. meyeri is described in the literature as an obligate anaerobe3 but is reported in one paper on a case of

Other identifications No. Labs

Shigella sonnei 1

Pasteurella multocida 1

Gardnerella vaginalis 1

Gemella morbillorum 1

Staphylococcus aureus 1

Corynebacterium species* 1

Erysipelothrix rhusiopathiae* 1

Arcanobacterium haemolyticum 1

Arcanobacterium pyogenes 1

Figure 5.1. A. meyeri Gram stain X100 a. from colony, b. from broth. © 2013 RCPAQAP/microbiology

Table 10. Other identifications reported

5

34

9

1

7

1

1

4

4

1

1

1

1

38

1

17

94

0 10 20 30 40 50 60 70 80 90 100

No growth


Other identifications (incorrect)

Gram positive rod (branching)*

Anaerobic Gram positive bacillus*

Bacteroides ureolyticus

Propionibacterium propionicum

Propionibacterium species*

Bifidobacterium species*

Eggerthella lenta*

Clostridium botulinum

Clostridium perfringens

Clostridium species

Actinomyces species*

Actinomyces neuii spp neuii

Actinomyces turicensis*

Actinomyces meyeri*

pulmonary actinomycosis as demonstrating growth aerobically as well as anaerobically.2 Our isolate grows best anaerobically and at a much slower rate and more feebly aerobically. Actinomyces are predominantly found in the dental cavity and have been associated with thoracic, brain, cervico‐facial and abdomino‐pelvic disease.1 A. meyeri has been isolated from abscesses, particularly of the brain and from pleural fluid and bites. 3 It is an uncommon cause of actinomycosis. Periodontal disease and alcoholism appear to be risk factors for infection with A. meyeri which has a predilection for disseminated disease.2 ,4

A. meyeri cells are irregular, beaded rods with rudimentary branching. Colonies on blood agar are non‐haemolytic, translucent, whitish‐grey, raised, shiny, entire with no pigment. Biochemically they are catalase‐negative, do not generally hydrolyse esculin and nitrate reduction is negative. Urease is usually negative and the CAMP test is positive. Acid is produced from glucose, maltose, sucrose and xylose, whereas mannitol, trehalose, raffinose and mannose are not fermented.1,5

5.1a 5.1b


SUMMARY OF PHENOTYPIC AND BIOCHEMICAL RESULTS3,6

Phenotypic characteristics Gram stain: Gram‐positive rod. Cells are irregular and beaded in appearance with rudimentary branching. Motility @ RT (BHI broth) : non‐motile Colonial appearance: 0.5mm – 1.0mm translucent whitish grey, raised,

shiny, entire colonies. Pigment: None Haemolysis: None Catalase: Negative Oxidase: Not applicable KIA slope: Not applicable Growth conditions Growth on HBA @ 35°C in O2 : No growth Growth on HBA @ 35°C in CO2 : ++ Growth on HBA @ 35°C in ANO2: +++ Growth on CHOC @ 35°C in CO2 : ++ Biochemical reactions Kit Name: ID 32 A Identification: Numerical Profile: 0504473705 ID: 99.9% Actinomyces meyeri T value: 0.62 Second choice: ID: 0.1% Bifidobacterium spp. T value = 0.0 Kit Name: Remel RapID ANA II Identification: Actinomyces meyeri Numerical Profile: 030671 Probability: 91.44% Bioscore: 1/19

Analysis of whole cell long chain fatty acids was performed using the Microbial Identification System (MIS), MIDI Corporation, which consists of an Agilent technologies 7890A Gas Chromatograph Unit coupled to a computer system which runs the Sherlock® software version 6.1. MIDI identified this isolate as Actinomyces meyeri (SI value 0.647). SI values of 0.5 or above are generally correct to the species level for a typical strain. On cluster analysis the isolate clustered with a group of organisms including various Actinomyces spp. Identification of Bacterial Isolates by DNA Sequence Analysis Accurate identification of bacterial isolates is achieved by analysis of the DNA sequence of specific genes such as the 16S rRNA gene that is found in all bacteria. The 16S rRNA gene is just over 1500bp long and is made up of regions of highly conserved sequences. Interspersed within the conserved areas are other regions that have been found to be highly variable between bacteria. Identification is obtained by analysis of the variable regions within the gene by DNA sequencing. The resulting sequence is compared against a large database of known bacterial sequences for an exact match. Amplification of the 16S rRNA gene was achieved using polymerase chain reaction (PCR). The 16S rRNA gene sequence analysis identified the isolate as an Actinomyces meyeri with 99% match to the GenBank library entry number NR_029286.1. It is important to note that the library entries are not peer reviewed before being added to the database; however, a published entry holds greater validity.

Figure 5.2. A. meyeri . © RCPAQAP/microbiology 2013

Nitrate broth (NO3) Negative

Urease Negative

PAL Negative

Aesculin Negative α‐Fucosidase Negative

α‐Glucosidase Positive

β‐N‐Acetylglucosaminidase Negative

β‐Galactosidase Positive

Hippurate Negative

Arabinose Negative

Maltose Positive

Mannitol Negative

Raffinose Negative

Rhamnose Negative

Sucrose Positive

Xylose Positive

Trehalose Negative

Distinguishing features3 A. meyeri can be distinguished from A. israelii and A. naeslundii by not fermenting raffinose, from A. viscosus by not producing catalase, from A. odontolyticus by not producing pink colonies on blood agar and by not reducing nitrate, from A. bovis by fermenting ribose and xylose, and from Actinomyces sp. strain WVU 963 by not hydrolysing esculin.


Participants’ results Two hundred and twenty participants submitted results for this item. One hundred and fifty (68%) identified the isolate to the genus level. Commercial kit/system performance in the hands of the users (=Actinomyces species): API 20A: 13/19 (68%). Clostridium perfringens (1); Gram positive bacillus (1); Bifidobacterium species (2); Propionibacterium species (2). API Coryne: 18/21 (86%). Unable to identify (1); Gardnerella vaginalis (1); Arcanobacterium haemolyticum (1). MALDI‐TOF: 57/57 (100%). RapiD 32 A: 10/10 (100%). Rapid ANAII: 19/21 (90%). Bacteroides ureolyticus (1); Eggerthella lenta (1). VITEK2 ANC: 43/43 (100%). Sequencing 16S rRNA: 4/4 (100%). References Item 5: 1. Wade, WG., and Kononem, E. Propionibacterium, Lactobacillus, Actinomyces, and other non‐spore‐forming anaerobic gram‐positive rods: In Manual

of Clinical Microbiology, Versalovic and Carroll et al. 10th Edition. ASM Press: Chapter 49. 2. Fazili, T., Blair, D., Riddell, S., Kiska, D. and Nagra, S. Actinomyces meyeri infection: Case report and review of the literature. J inf. 2012;65: 357‐361.

http://www.sciencedirect.com/science/article/pii/S0163445312000461 3. Aerobic and facultative gram‐positive bacilli: in Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Winn and Allen et al. 2005. 6th Ed.

Lippincott Williams and Wilkins, Philadelphia; Chapter 14. 4. Apotheloz, C., and Regamey, C. Dissemated infection due to Actinomyces meyeri: case report and review. Clin Infect Dis. 1996;22: 621‐5. http://www.ncbi.nlm.nih.gov/pubmed/8729199 5. Cato, EP., Moore, WEC., Nygaard, G., and Holdeman, LV. Actinomyces meyeri sp. nov. specific epithet rev. Int J Sys Bac 1984;34: 487‐9. http://ijs.sgmjournals.org/content/34/4/487.full.pdf 6. Identification Reference Laboratory Centre for Infectious Diseases and Microbiology, ICPMR, Westmead Hospital. Other reference: Sarkonen, N., Könönen, E., Summanen, P., Knöönen M., and Jousimies‐Somer, H. Phenotypic identification of Actinomyces and related species isolated from human sources. J. Clin. Microbiol. 2001; 39: 3955‐3961. http://jcm.asm.org/content/39/11/3955.full.pdf+html


ITEM 2013:5:8A,B PARASITOLOGY

2013:5:8A Entamoeba coli, Blastocystis hominis and Entamoeba histolytica/dispar/moshkovskii were seen by all four referral laboratories in sufficient numbers to score. Entamoeba hartmanni and Endolimax nana were also seen but not by all referral laboratories and therefore have not been scored. Entamoeba histolytica and Entamoeba dispar are morphologically identical species. In bright‐field microscopy, E. histolytica/E. dispar cysts are spherical and usually measure 12 to 15 μm (range may be 10 to 20 μm). A mature cyst has four nuclei while an immature cyst may contain only one to three nuclei. Peripheral chromatin is fine, uniform and evenly distributed. Elongated, chromatoid bodies with bluntly rounded ends may sometimes be found. Glycogen can be diffuse or absent in mature cysts while clumped in immature cysts.1 The 22 participants reporting E.histolytica have been scored incorrect as reporting E.histolytica based upon the microscopic morphology of cysts alone or trophozoites without ingested red blood cells, is incorrect and clinically misleading (final report 2008‐1).Table 11 lists other results reported by participants. More than one parasite may have been reported by a participant.

8A Entamoeba coli, Blastocystis hominis and Entamoeba histolytica/dispar/moshkovskii

8A ‐ Other parasite No. Labs

Ascaris lumbricoides 1

Balantidium coli 1

Chilomastix mesnili 1

Cryptosporidium parvum 1

Cryptosporidium species 1

Giardia intestinalis 1

Taenia species 1

Entamoeba polecki 2

Entamoeba species 2

Giardia lamblia 3

Dientamoeba fragilis 5

Iodamoeba butschlii 12

Entamoeba histolytica 22

Table 11. other parasites reported

1

49

52

31

56

4

94

112

0 20 40 60 80 100 120

No parasites seen

Endolimax nana

Entamoeba hartmanni

Entamoeba histolytica/dispar/moshkovskii

Entamoeba histolytica/dispar

Blastocystis species

Blastocystis hominis

Entamoeba coli

%

112 84.8

94 71.2

4 3.0

56 42.4

31 23.5

52 39.4

49 37.1

1 0.8

References Item 8A. 1. http://www.dpd.cdc.gov/dpdx/HTML/PDF_Files/Entamoeba_benchaid.pdf 2. http://www.dpd.cdc.gov/dpdx/HTML/MorphologyTables.htm


8B Virtual slide: Cryptosporidium species

8B ‐ Parasites No. Labs

Cryptosporidium species 50

Cryptosporidium parvum 38

Dientamoeba fragilis 44

Blastocystis hominis 14

Endolimax nana 13

Entamoeba coli 10

Giardia intestinalis 5

Entamoeba histolytica 4

Entamoeba hartmanni 3

Entamoeba histolytica/dispar 3

Giardia lamblia 3

Giardia species 3

Taenia species 3

Ascaris lumbricoides 2

Hymenolepis species 2

Strongyloides species 2

Strongyloides stercoralis 2

?Unidentified piece of fluke or worm 1

Ascaris species 1

Cyclospora cayetanensis 1

Cyclospora species 1

Dientamoeba species 1

Giardia duodenalis 1

Gnathostoma spinigerum 1

Iodamoeba butschlii 1

Myxobolus plectrophiles 1

Table 12. parasites reported

Figure 8B (i). Cryptosporidium oocysts (4‐6 µm) stain light pink to dark red.

Figure 8B (ii). Arrows indicate trophozoites resembling D.fragilis.

References Item 8B: 1. http://www.dpd.cdc.gov/dpdx/HTML/PDF_Files/Crypto_benchaid.pdf 2. http://www.dpd.cdc.gov/dpdx/HTML/MorphologyTables.htm Figure 3

This virtual slide could be accessed at http://images.rcpaqap.com.au/RCPAQAP/Microbiology/2013-5-8B.svs (type link) , although a number of participants (n=32) were unable to open this link either due to RCPAQAP server problems, possible firewall restrictions by their institutions or did not have internet available at the time. This was educational only and has not been scored, being a pilot for the suitability of virtual microscopy of parasites. It is hoped that in the future we will be able to demonstrate rarer parasites we are unable to get adequate volumes of samples to distribute to our participants.

The specimen was collected in SAF, concentrated and stained using a modified Iron‐ Haematoxylin stain. The modification to the stain was to incorporate the acid fast stain as part of the process. The below images, Figures 8B (i) and (ii) are snapshots from the virtual image. Of the 100 participants submitting results on this item, 88% reported Cryptosporidium. In the Modified Acid stain, oocysts (4‐6 µm) stain light pink to dark red. There were a few trophozoites of possibly D.fragilis but as most participants commented, the limitations of the software did not allow adequate focussing to determine internal structure.

Figure 8B (iii). Protozoa found in stool specimens of humans: Coccidia and Blastocystis.2

Documents

FINAL RESULTS AND COMMENTS 2013-5 BACTERIOLOGY …dataentry.rcpaqap.com.au/micro/reports/uploadedfiles/Fnl2013-5.pdfAeromonas species may also grow yellow colonies on TCBS. Results