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Bacteriology Program
Modules: Throat, Skin, AFB stain, Difficult/Blood culture and
Mycobacteriology culture.
Generic Report

2019, Survey 4

Report prepared by

RCPAQAP Microbiology

Version 1: Initial Publication

Copyright

This material is copyright and may not be used in any form for advertising, sales promotion or publicity. The material may not be reproduced in whole or in part for any
purpose whatsoever (including presentations at meetings and conferences), without the prior written permission of the RCPA Quality Assurance Programs Pty
Limited. Permission must be sought in writing from the Program but will not be unreasonably refused.

Confidentiality

RCPA Quality Assurance Programs Pty Limited keeps all participant details confidential. No information related to any of the participants will be divulged to a third party,
unless required by legislation, without the express written consent of the participant. General information may be discussed at meetings or presented as papers to journals.

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Assurance Programs
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St Leonards NSW 2065
Phone +61 2 9045 6000 © 2019 RCPA Quality Assurance Programs Pty Ltd. All rights reserved
Fax +61 2 9356 2003 Report Issued Friday, 2 August 2019
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Accredited for compliance with ISO/IEC 17043
Accreditation Number: 14863

Report ID: MIBP2019040000000000Gener01

Report issued: 2/08/2019 Page 2 of 8

FINAL RESULTS AND COMMENTS Survey 4, 2019

Item 1 Urine for microscopy, bacterial count, identification and susceptibilities

SPECIAL ATTENTION:

Result entry for the Bacteriology Urine program – Survey 2 (dispatch 4) was captured separately on the
new myQAP platform. As a result, this module will no longer be included in the Bacteriology survey or
generic report.

Participants in the Bacteriology Urine program will now be issued with a separate survey report, which
includes the generic report.

The RCPAQAP is harmonising the approach to the assessment of survey results for all qualitative pro-
grams. The assessment process for survey results for this program will change from the current scoring
system, to one that utilises ‘terms’ such as Concordant or Discordant to assess qualitative responses
against a known target.

It is expected that this report may be delayed by a few weeks for this survey due to extensive testing and
validation required.

ITEM 2019:4:2 THROAT SWAB

ITEM 2019:4:2 Throat swab

Quality Control
This item contained no pathogens. Staphylococcus epidermidis, and an α-haemolytic Streptococcus represented normal flora. This
item passed all homogeneity and stability testing with the normal flora counts remaining above 5.0 x 10 9 CFU/L throughout the
survey period.

Culture and identification results

Two hundred and seventeen participants returned results for this item with 88% correctly reporting “No likely pathogen” or “No
a.
pathogens isolated”. Nine participants reported a pathogen. Four reported Streptococcus group C, two S. pneumoniae, two S.
pyogenes and one S. aureus. Seventeen reported one of the normal flora isolates as the final result while two reported mixed flora.

a.

ITEM 2019:4:2 CLERICAL ERROR

Specimen label: Kate CONNER U/N 622856 Table 1: Clerical errors reported.

Clerical Errors reported in Item 2019:4:2 Number labs (217)

Paperwork: Kate CONNER U/N 568226 No errors reported 12

Error Unit number only 205 (94%)

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ITEM 2019:4:3 SKIN SWAB

Streptococcus pyogenes

Streptococcus pyogene s 163 163 75.8

45 20.9
Streptococcus group A 45

1 0.5

Beta-h aemolytic Stre p. Spe cie s 1

2 0.9

Staphylococcus species 2

2 0.9

Staphylococcus epid ermidis 2

1 0.5

Staphylococcus aure us 1

1 0.5

MRSA isol ate d 1

0 20 40 60 80 100 120 140 160 180

ITEM 2019:4:3
Quality Control

This item contained the pathogen, Streptococcus pyogenes with Staphylococcus epidermidis representing normal skin flora.
Homogeneity and stability testing were satisfactory and viability counts by the end of survey were > 1.0 X 10 10 CFU/L for the
pathogen.

Identification– Streptococcus pyogenes

Two hundred and fifteen participants returned results. All but six participants reported a β-haemolytic Streptococcus species. One
participant reported MRSA. Reporting MRSA could have repercussions for the patient with an incorrect alert to infection control.
Participants used various methods alone or in combination to identify as S. pyogenes/ Streptococcus group A. Results were
submitted for bacitracin susceptibility (n=42), Lancefield grouping (n=124) and commercial kits/systems (n=140). MALDI-ToF-MS
was used by 84 with the next popular kit/system being Vitek2 GP with 40 users.

Susceptibility testing - Streptococcus pyogenes

One hundred and ninety three (90%) participants submitted susceptibility results. Susceptibility testing was performed well.
Susceptibility results reported are tabulated in Table 2.

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Table 2: Streptococcus pyogenes Table 3. Susceptibility methods used. 2019:4:3 Streptococcus pyogenes

Streptococcus Susceptibility method used n

pyogenes
CDS 21
DISK DIFFUSION (CLSI) 83
susceptibilities DISK DIFFUSION (EUCAST) 40
(all methods) Etest 1
Kirby Bauer (CLSI) 11
MicroScan (CLSI) 2
Antibiotic S I/DS R
MicroScan (EUCAST) 1
Amoxycillin-clavulanate 6 - - Not performed 22
Ampicillin/Amoxycillin 35 - - Phoenix (CLSI) 2
Cefazolin 8 - - Vitek 2 (CLSI) 23
Cefotaxime 14 - - Vitek 2 (EUCAST) 9
Ceftriaxone 36 - -
Cefuroxime 5 - -
Cefuroxime (IV) 1 - -
Cephalexin 19 - -
Cephalothin 4 - -
Chloramphenicol 15 1 -
Clindamycin 124 - 1
Doxycycline 10 - -
Erythromycin 153 - 3
Levofloxacin 25 - -
Linezolid 15 - -
Penicillin 180 - -
Tetracycline 73 1 -
Trimethoprim-sulfamethoxazole 33 - 8
Vancomycin 63 - -

Table 4: Streptococcus pyogenes. CDS results

2019:4:3 Streptococcus pyogenes

Skin swab
Antibiotic Disc Content Exception to Interpretation MIC (mg/L)
standard Susceptible
Interpretation strains

Ampicillin/amoxycillin 5 4mm S1,2 ≤2

Benzylpenicillin 0.5 u S1,2 ≤ 0.125
Cefotaxime 0.5 4mm S ≤ 0.5
Clindamycin 2 S3 ≤ 0.5
Erythromycin 5 S3 ≤ 0.5
Tetracycline 10 S ≤4
Vancomycin 5 2mm S ≤4

CDS notes
1. For streptococci groups A, B, C, G and Streptococcus milleri group, the susceptibility to benzylpenicillin, ampicillin, amoxycillin and
cephalosporin antibiotics (except Ceftazidime) is extrapolated from the testing of benzylpenicillin 0.5 u.
2. If the organism is resistant to benzylpenicillin 0.5 u and susceptible to ampicillin 5 µg, report as having a decreased susceptibility.
3. No inducible clindamycin resistance (ICR) detected. Report clindamycin as susceptible.

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ITEM 2019:4:4A,B,C,D CULTURE AND MICROSCOPY FOR AFB

Reference laboratory report Microscopy Results
Slides were prepared in this survey using sputum that was
Table 5: 4A-D expected results examined using a Ziehl-Neelsen stain and tested for M.
tuberculosis and MAC by molecular methods and found to be
Test A B negative. AFB positive smears were seeded with Mycobacterium
AFB Ziehl 10-99/100 HPF (1+) ; 1 None tuberculosis H37Ra.
stain Neelsen -10 /HPF (2+)
Smear
C D Three hundred and nine participants returned results.
Culture Bactec Negative (normal flora M.chelonae Both smears passed homogeneity testing.
MGIT only)
960 Item 4A
(BD)/L-J Smear 4A was positive and contained 10-99/100 HPF (1+).
Some smears contained clumps in some areas which may
account for the higher counts [1-10 /HPF (2+)]. Four reported none
Table 6: Participant smear results (two a possible transposition of results) and two participants
reported “none” for both 4A and 4B. All four used only the Ziehl-
Participant smear results A B Neelsen stain.

>10 /HPF (3+) 7 - Item 4B

1-10 /HPF (2+)
10-99/100 HPF (1+)
1-9/100 HPF (Scanty)
None
Table 7: Participant culture results
Culture results
Mycobacterium species isolated
Mycobacterium species not isolated
Table 8: Results for media/methods used for specimen 4D
136 - Smear 4B was negative. Five participants reported a false
positive smear. Two of these appear to have transposed results.
143 2 The remaining three reported both smears positive.
19 3
Culture Results
4 304 Sixty four laboratories participated and returned a culture result
for at least one of these items.
Item 4C was negative, containing only normal flora. All
participants returned a correct “not isolated” result.
Item 4D was positive, containing Mycobacterium chelonae and
normal flora (S.epidermidis and an α-hemolytic streptococcus).
C D The sample passed all homogeneity and stability testing.
For the positive culture, 38 (60%) used both a manual and
automated method with 95% of these participants reporting at
- 60 least one method positive. Over 93% of all participants reported
64 4 “isolated” by at least one method. Twenty participants use only
an automated method while six use a standalone non-
automated method.
Two participants used two manual methods but no automated
method. Their overall result was correct.

Molecular Diagnostics Results

Media^ Total no. Number Thirteen laboratories also performed a molecular method for
Inoculated positive detection of M. tuberculosis. Where tested, all returned a correct,
4D negative result for Item 4C and 4D. Ten used a commercial kit
BD BBL MGIT (manual) 1 1 while the remaining used an Inhouse PCR. Please note that no
Brown and Buckle Slope 3 3 scoring is applied to molecular results from these items. To be
Gerloff with NVAP 2 2
assessed on molecular testing of Mycobacterium tuberculosis/
avium, the Molecular MTB/avium (now includes RIF resistance)
LJ 27 23 module is available for enrolment.
LJ Glycerol 2 1
LJ Pyruvate 8 7
Table 9. Combinations of AFB stains reported.
LJ Pyruvate Glycerol 4 4
Ogawa 4 4 Stain 1 Stain 2 n
B83 agar 1 1
Middlebrook 7H11 Agar 1 1 Ziehl-Neelsen - 162
Kirchner broth 1 1 Auramine O - 95
BacT/ALERT 3D 2 1 Kinyoun - 23
BD BACTEC MGIT 960 System 53 45
Auramine O Ziehl-Neelsen 15
BD BACTEC MGIT 320 System 3 2
AO + Rhodamine B - 6

^more than one medium may have been used by a participant. AO + Rhodamine B Ziehl-Neelsen 5
Auramine O Kinyoun 3
Acridine Orange Ziehl-Neelsen 1

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ITEM 2019:4:5 ISOLATE FOR IDENTIFICATION

%

Abio tro phia defectiva* 123 123 65.4

Abio tro phia sp ecies* 5 5 2.7

11 5.9
Gra nulicatella adiacens* 11
2 1.1
Gra nulicatella sp .* 2
3 1.6
Gemell a morb illorum 3
1 0.5
Gemell a h aemolysa ns 1
2 1.1
Nutritio nally varia nt Streptococcus* 2
2 1.1
Arcano bacter ium ha emolyticum 2
1 0.5
Gra m positive cocco bacilli 1
1 0.5
Gra m positive ba cillu s 1
1 0.5
Gra m neg ative co ccoba cillu s 1
1 0.5
Microbacte rium sp. 1
1 0.5
Leu con ostoc spe cie s 1
1 0.5
Glo bica tel la sang uinis 1
1 0.5
Facklamia lan guida* 1
1 0.5
Erysipelothrix rhusiopathiae * 1
1 0.5
Bacillu s sp eci es (?contaminan t) 1
1 0.5
Ana erococcus sp eci es 1
1 0.5
Aer oco ccus u rinae 1
1 0.5
Acti nobaculum scha alii* 1
1 0.5
Streptococcus vir idans* 1

No growth 1 1 0.5

Unable to Ide ntify - Refe r 25 25 13.3

0 20 40 60 80 100 120 140

* includes presumptive identification(s)

ITEM 2019:4:5
Isolate for Identification– Abiotrophia defectiva

QA Report
This item contained a pure growth of Abiotrophia defectiva and passed homogeneity
testing and counts remained above 4.0 x 10 9 CFU/L throughout the survey period. One
out of three vials failed stability testing so the participant who reported no growth has
not been scored.

Identification
A. defectiva was originally referred to as nutritionally variant streptococci (NVS)
because of its fastidious nutritional requirement for thiol compounds (pyroxidol/ Figure 5.1: Gram stain, Kingella kingae © ICPMR

cysteine).1 Two distinct groups of NVS exist, A. defectiva and A. adiacens. In 1995, 16S rRNA studies in Japan revealed that these
fastidious organisms were not related to the genus Streptococcus at all. They were transferred to the new genus, Abiotrophia. Of the
four species of Abiotrophia, three could be distinguished from A. defectiva and so were placed in another genus called Granulicatella
in 2000, leaving A. defectiva as the sole species in the genus.1,2,3,4
Abiotrophia and Granulicatella are catalase-negative, pleomorphic gram-positive cocci which exhibit satelliting behavior because of
their requirement for pyridoxal (vitamin B6). The presence of α– and β-galactosidase and the absence of β-glucuronidase in A.
defectiva distinguishes it from the closely related Granulicatella species. A. defectiva and Granulicatella are LAP-positive like the
streptococci, but unlike streptococci are PYR-positive.1

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SUMMARY OF PHENOTYPIC AND BIOCHEMICAL RESULTS

Phenotypic characteristics

• Gram stain: gram-positive –mainly cocci but may be pleomorphic with bulbous forms. Maybe Gram variable. Colonial appearance:
viridans-like (α-haemolytic) colonies.
• Catalase: negative
• Oxidase: negative
• Motility: negative
• Vancomycin: susceptible

Growth conditions

growth conditions may vary according to the strain isolated and the degree of B6
requirement.
• Growth on HBA @ 35°C in O2 : +
• Growth on HBA @ 35°C in CO2 : +++
• Growth on HBA @ 35°C in ANO2: +
• Growth on CHOC @ 35°C in CO2 : +++
• Growth on MacConkey agar: no growth

Identification was confirmed by 16S rRNA sequencing.

Supplementary tests Figure 5.2 Abiotrophia defectiva. 48 hours incubation,

CO2. © 2011 RCPA Microbiology QAP

• PYR: positive
• LAP: positive
• Arginine dihydrolase: negative
• α-Galactosidase: positive
• ß-Glucuronidase: negative
• Trehalose: fermented.
• Growth in 6.5% NaCl: negative3
• Satellitism: positive3

Participant results

Excluding the laboratory unable to grow this isolate, 187 participants returned a result with 128/187(68%) reporting Abiotrophia species.
Thirteen percent could not identify in this survey.
As discussed earlier Granulicatella species reported by 13 participants, although pyridoxyl-dependent and closely related to A. defectiva
are distinguished by the α– and β-galactosidase tests and the β-glucuronidase test (A. defectiva is pos/pos/neg and Granulicatella are
neg/neg/pos or neg depending on species). All 13 participants who incorrectly reported Granulicatella species used Vitek2 GP.
Another misidentification reported was Gemella species by four laboratories. Two had used Phoenix, another Vitek2 GP and the
remaining did not report using a kit. Like Abiotrophia, Gemella species (other than G. haemolysans and G. asacchararolyticus) are PYR
and LAP positive, will not grow in 6.5% NaCl and produce slow-growing, α-haemolytic streptococcus-like colonies on blood agar. The test
for satellitism will distinguish Gemella (negative) from the NVS (positive). The isolate is streaked for confluent growth onto a blood agar
plate and a single cross streak of Staphylococcus aureus or S. epidermidis is applied. The plate is incubated at 35°C in CO2 and
examined for enhanced growth of the isolate colonies surrounding the staphylococcal streak. Alternatively, media supplemented with
pyridoxyl HCl (final concentration of 0.001%) or commercial pyridoxyl discs may be used to show vitamin B6 dependency. 3
The pleomorphism of NVS may also have caused confusion. Most laboratories (176/184) reported Gram reactions as gram-positive
cocci/diplococci/coccobacilli. Six reported gram-variable cocci/coccobacilli/rods. Two laboratories reported gram-negative rods/cocci/
coccobacilli with both unable to identify. It is worth noting that pyridoxal HCl added to media may also help convert NVS to a more
Streptococcus-like Gram stain morphology and gram-positivity.4 Aerococcus species reported by one lab have a more staphylococcus-
like Gram reaction. All 93 reporting a catalase result (negative) reported correctly.

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One hundred and forty nine participants used a commercial kit/system to identify this isolate. Below shows the percentage correct for an
identification of A. defectiva/Abiotrophia species for the most commonly used. Sixteen used a second kit or method to confirm
identification.

• API Strep: 7/10 (70%). Aerococcus urinae 1); Facklamia languida (1); NVS (1).
• API Coryne: 0/2. Arcanobacterium haemolyticum (2).
• MALDI-TOF Bruker: 55/55 (100%).
• MALDI-TOF VITEK MS: 32/32 (100%).
• Sequencing 16S rRNA: 3/3 (100%).
• VITEK2 GP (Biomerieux): 21/40 (53%). Gemella morbillorum (1); Granulicatella adiacens (11); Granulicatella species (2);
Globicatella sanguinis (1); NVS (1); Leuconostoc species (1); Unable to Identify - Refer (2).

Abiotrophia are part of the normal flora of the upper respiratory tract and are a known cause of endocarditis and septicaemia. The gram-
positive cocci in the Gram stain of blood cultures may look ‘antibiotic affected’ and laboratories will often fail to isolate any organism. The
frequency of infection and colonisation of cardiac valves by Abiotrophia strongly suggests an affinity for avascular tissue. 5 The clinical
course of endocarditis with NVS is often more severe and difficult to treat than with the viridans streptococci or enterococci and relapse
rates have been documented as being relatively high. 3,4 A high prevalence of β-lactam and macrolide resistance among the NVS has
been reported.6 Predisposing factors for NVS infection among neutropenic patients include chemotherapy-induced neutropenia and oral
mucositis.6

References Item 5
1. The Gram-Positive Cocci: in Koneman's Colour Atlas and Textbook of Diagnostic Microbiology. Winn and Akllen et al. 2016, 7th Ed.
Lippincott Williams and Wilkins, Philadelphia. p774.
2. Collins M.D, Lawson P.A. The genus Abiotrophia (Kawamura et al.) is not monophyletic: proposal of Granulicatella gen. nov., Granulicatella
adiacens comb. nov., Granulicatella elegans comb. nov. and Granulicatella balaenopterae comb. nov. Int J Syst Evol Microbiol 2000;50: 365-
9. http://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/00207713-50-1-365
3. Christensen, JJ and Ruoff, KL. Aerococcus, Abiotrophia, and other aerobic, catalase-negative, gram-positive cocci: In Manual of Clinical
Microbiology, Editors in chief J.H. Jorgensen and M.A Pfaller. 11th Edition. ASM Press: Chapter 24.
4. Christensen, J. and Facklam R. Granulicatella and Abiotrophia species from human clinical specimens. J Clin Microbiol 2001;39: 3520-3523.
http://www.ncbi.nlm.nih.gov/pubmed/11574566
5. Ince, A et al. Total knee arthroplasty infection due to Abiotrophia defectiva. J Med Microbiol. 2002. 51, 899-902.
6. Senn, L et al. Bloodstream and endovascular infections due to Abiotrophia defectiva and Granulicatella species. BMC Infectious Diseases
2006. 6: 9. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1360077/pdf/1471-2334-6-9.pdf

Other references:
7. Rhodes, H.M., Hirigoyen, D., Shabnam, L., Williams, D.N., Hansen, G.T. Infective endocarditis due to Abiotrophia defectiva and
Granulicatella spp. complicated by infectious intracranial cerebral aneurysms: a report of three cases and review of the literature. J Med
Microbiol. 2016;65: 493. https://jmm.microbiologyresearch.org/content/journal/jmm/10.1099/jmm.0.000260#tab1
8. Escarcega, E., Trovato, C., Idahosa, O., Gillard, J., Stankewicz, H. Abiotrophia defectiva endocarditis: an easy miss. Clin Pract Cases Emerg
Med. 2017;1: 229 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5965177/
9. Chowdhury,S., and German, M.L, Rare but not infrequent: infective endocarditis caused by Abiotrophia defectiva, Case Reports in Infectious
Diseases, vol. 2018, Article ID 5186520, 3 pages, 2018. https://www.hindawi.com/journals/criid/2018/5186520/cta/
10. Ratcliffe, P., Fang, H., Thidholm, E., Boräng, S., Westling, K., Özenci, V. Comparison of MALDI-TOF MS and VITEK 2 system for laboratory
diagnosis of Granulicatella and Abiotrophia species causing invasive infections. Diagn Microbiol Infect Dis. 2018;77: 216. https://
www.ncbi.nlm.nih.gov/pubmed/24034902

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