88
Pathology (2015), 47(1), January
CORRESPONDENCE
Number of cases
30
Serotype 1b 2a
20
2b 3a
surveillance of emerging antibiotic resistance in Shigella species is warranted, and clinical laboratories should be aware of possible azithromycin-resistant Shigella species locally acquired in Australia when providing advice to clinicians regarding empiric therapy.
6
10
Other
20 0 20 0 0 20 1 0 20 2 0 20 3 0 20 4 0 20 5 0 20 6 0 20 7 0 20 8 0 20 9 1 20 0 1 20 1 1 20 2 1 20 3 14 *
0
Year Fig. 1 Number of cases of Shigella flexneri serotypes in Victoria tested at MDU PHL, from 2000. *Data for 2014 is January to June inclusive.
minimum inhibitory concentration (MIC) 16 mg/mL is considered susceptible in the normal wild-type distribution.3,4 The Microbiological Diagnostic Unit Public Health Laboratory (MDU PHL) routinely performs identification, serotyping and antimicrobial susceptibility testing of Shigella isolates from clinical cases in Victoria and selected isolates from NSW.5 Recent reports of increasing antibiotic resistance in Shigella species, including a cases of infection where high-level resistance to azithromycin was demonstrated,3,6 and an increase in local laboratory confirmed cases of Shigella flexneri, prompted our investigation for local azithromycin resistance. Between 1 May 2013 and 30 June 2014, the number of Shigella flexneri 3a cases reported in Melbourne and Sydney was 29 and 16, respectively, a significant increase compared to previous years, with the major increase seen in 2014. Figure 1 summarises the serotype prevalence data for cases notified in Victoria from 2000 and where isolates were characterised at MDU PHL, demonstrating the significant increase in Shigella flexneri 3a cases in the first half of 2014. The number of infections caused by other Shigella flexneri serotypes was unchanged over the same time period. Thirty-eight of 45 isolates (14 from 2013, 12 males; 24 from 2014, all males) were submitted for azithromycin susceptibility testing, using azithromycin Etest according to the manufacturer’s instructions (bioMerieux, France). Susceptibility to azithromycin (MIC 16 mg/mL) was only seen in four 2013 isolates from two males and two females. Of the 34 remaining azithromycinresistant isolates (89% of isolates tested), 33 were resistant to azithromycin (MIC >256 mg/mL), ampicillin, and tetracycline, while one isolate was resistant to azithromycin (MIC 128 mg/ mL), ampicillin, tetracycline and exhibited decreased susceptibility to ciprofloxacin (0.25 mg/mL). Approval to include de-identified demographic data summarising risk factors for Shigella infection was granted by Department of Health, Victoria. The median age for the azithromycin resistant cases was 40 years (range 21–65 years), with most cases reported as occurring in MSM without overseas travel, indicating local acquisition in the MSM population in Melbourne and Sydney. These data are the result of passive surveillance of notifiable infections undertaken during this period, and not active surveillance in this risk group. Emerging azithromycin resistance in Shigella species further highlights the growing burden of antibiotic resistant bacterial infections, and has significant implications for the empiric treatment of severe bacterial gastrointestinal disease. Particularly in the MSM community, possible cases of shigellosis should not be empirically treated with azithromycin. Active
Conflicts of interest and sources of funding: The authors state that there are no conflicts of interest to disclose. Mary Valcanis1 Jeremy D. Brown2 Briony Hazelton2 Matthew V. O’Sullivan2,3 Alex Kuzevski1 Courtney R. Lane4,5 Benjamin P. Howden1,6 1
Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, The University of Melbourne at The Doherty Institute for Infection and Immunity, Melbourne, Vic, 2Centre for Infectious Disease and Microbiological Laboratory Services, Institute for Clinical Pathology and Medical Research, Westmead Hospital, Sydney, NSW, 3Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Sydney, NSW, 4Victorian Department of Health, Communicable Disease Epidemiology and Surveillance, Health Protection Branch, Melbourne, Vic, 5National Centre for Epidemiology and Population Health, Australian National University, Canberra, ACT, and 6Infectious Diseases Department, Austin Health, Heidelberg, Vic, Australia Contact Professor Benjamin Howden. E-mail: [email protected] 1. Rowe SL, Radwan S, Lalor K, et al. An outbreak of shigellosis among men who have sex with men, Victoria, 2008. Vic Infect Dis Bull 2010; 13: 119–23. 2. Gastrointestinal Expert Group. Therapeutic Guidelines: Gastrointestinal. Version 5. Melbourne: Therapeutic Guidelines Limited, 2011. 3. Hassing RJ, Melles DC, Goessens WHF, Rijnders BJA. Case of Shigella flexneri infection with treatment failure due to azithromycin resistance in an HIV-positive patient. Infection 2014; 42: 789–90. 4. European Committee on Antimicrobial Susceptibility Testing. Breakpoint Tables for Interpretation of MICs and Zone Diameters, Version 4, 2014. Cited 20 Jul 2014. http://www.eucast.org/clinical_breakpoints 5. Valcanis M. Laboratory testing of Shigella. Vic Infect Dis Bull 2010; 13: 114–8. 6. Heiman KE, Karlsson M, Grass J, et al. Notes from the field: Shigella with decreased susceptibility to azithromycin among men who have sex with men – United States, 2002-2013. MMWR Morb Mortal Wkly Rep 2014; 63: 132–3.
DOI: 10.1097/PAT.0000000000000207
Direct identification of bacteria from positive BD-Bactec blood culture bottles on the Vitek MS Sir, Timely and the appropriate choice of antimicrobial therapy is associated with lower mortality in patients with bacterial sepsis.1 Unfortunately there is often a delay of days before
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
CORRESPONDENCE
bacteria are isolated from blood cultures, identified and antimicrobial susceptibilities are determined, during which time patients may remain on inappropriate empirical antimicrobial therapy. The recent widespread implementation of matrix assisted laser desorption and ionisation time of flight mass spectrometry (MALDI-TOF MS) into diagnostic laboratories offers a rapid and reliable alternative method for direct identification of isolates from blood cultures. Identification of the isolate may allow tailoring of antibiotic therapy based on local sensitivity data. It may also be used in combination with molecular techniques such as the Xpert MRSA/SA BC2 (Cepheid, USA), or inoculation of appropriate antimicrobial susceptibility cards on automated systems3 such as the Vitek-2 (bioMe´rieux, France). In this study we evaluated the performance of a modified extraction protocol previously reported for the BD-Bruker Maldi Biotyper4 (Becton Dickinson, USA), on the Vitek-MS (bioMe´rieux), including the relative contribution of protocol steps to yield. LabPlus serves as the diagnostic laboratory for a 1000 bed university-affiliated tertiary care hospital. Blood cultures are processed 24 h a day. All positive blood cultures have a Gram stain performed; the results of which determine the media that the broth is subcultured onto. Bacterial isolates are identified using the Vitek MS. Direct identification using the MS was performed on positive blood cultures prior to growth on subculture. Fisher’s exact test was used for comparing unmatched, and McNemar’s test for comparing matched data. For the basic protocol extraction method: 3.5 mL of broth was removed from the blood culture bottles and injected into a 3.5 mL serum separator tube (SST) (Becton Dickinson). This was centrifuged at 3300 rpm for 10 min. The supernatant was removed, and pellet re-suspended in 1.0 mL of sterile water. This was manually agitated prior to transfer to a 2 mL tube. After centrifugation at 13,000 rpm for 1 min, the supernatant was removed and resulting bacterial pellet spotted in quadruplicate onto a 48-well Vitek MS-DS disposable target slide. Sample wells were overlaid with 1 mL alpha-cyano-4-hydroxycinnamic acid (HCCA) matrix (bioMe´rieux), and run on the Vitek MS IVD system. For the additional centrifugation step, following centrifugation of the SST, the pellet was re-suspended in 1.5 mL of sterile water, transferred to a 2 mL tube, and centrifuged at 2100 rpm for 1 min. Then 1 mL of the supernatant was transferred to a 2 mL tube and centrifuged at 13,000 rpm for 1 min. The pellet was processed as for the basic protocol. For evaluation of formic acid extraction, a subset of samples were overlaid with 0.5 mL formic acid (bioMe´rieux) on two sample wells of the target slide. These were then overlaid with matrix and run as per the basic protocol. A total of 128 BD Bactec Plus Aerobic/F, Plus Anaerobic/F and Peds Plus (Becton Dickinson) blood culture bottles from 86 patients (73 adults and 13 children) collected between January and October 2013, were processed using either the basic method, or with the additional centrifugation step. Of these, 105 (82%) bottles were positive by Gram stain and had a single isolate grown on subculture. Fifteen (12.5%) bottles had growth of two or more isolates on subculture. Two bottles contained yeasts, and six bottles had flagged positive but had negative Gram stains (false positive).
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For blood cultures with a single isolate, the overall correct bacterial identification to species level by Vitek MS software was achieved for 57 of 105 (54%) of isolates; 47 of 61 (77%) Gram negative organisms and 10 of 44 (23%) Gram positive organisms using either method. No identification was given for 40 of 105 (38%) of isolates. Incorrect identification occurred in eight of 105 (7.6%) single isolate cultures; two of 61 (3.3%) Gram negative organisms, and six of 44 (14%) Gram positive organisms (Table 1). For polymicrobial samples, correct identification for one organism was achieved in 13 of 15 (87%) of samples. In one of the six blood culture bottles which had flagged falsely positive, direct identification gave an erroneous identification of Vibrio fluvialis with high confidence (98.7%). The blood cultures remained negative after 5 days incubation. The method did not identify yeasts. Using the basic method, 15 of 24 (63%) of Gram negative organisms, six of 26 (23%) of Gram positive organisms, and 21 of 50 (42%) of all isolates were correctly identified. With the additional centrifugation step, 32 of 37 (86%) of Gram negative organisms, four of 18 (22%) of Gram positive organisms, and 36 of 55 (65%) of all bacteria were correctly identified. Formic acid extraction gave correct identification for 31 of 52 (60%) isolates compared with 30 of 52 (58%) ( p ¼ 0.262) of those processed without this step. We found this method to be reliable for the identification of Gram negatives, and were able to improve on an initial correct identification rate of 63% to 86% ( p ¼ 0.059) for Gram negatives (42% to 65%, p ¼ 0.019 for all bacteria) with an additional centrifugation step to remove remaining cellular debris, which may interfere with MS spectral recognition.5 Our results compare favourably both with the findings of Moussaoui et al. (91%),4 and those of other recent reports for the identification of Gram negatives.6–8 Identification of Gram positive organisms (23%) by this method was poor, compared with that reported by Moussaoui et al. (89%).4 The yield did not substantially improve with an additional centrifugation step. A significant (14%) risk of false positive identifications contributes to rendering this method unsuitable for testing Gram positive organisms on the Vitek MS. The performance of individual organisms using either the basic method, or with the additional centrifugation step is presented in Table 1. Issues with identification of Salmonella, Campylobacter, Rhizobium, and Fusobacterium have been reported elsewhere.4,5 The relatively small number of samples in this study restricted our ability to assess the performance of other less commonly isolated organisms. The sensitivity of this method for identification of Staphylococcus aureus was particularly poor (18%), and inferior to the sensitivity of the two hour tube coagulase test (68%) performed at our laboratory. Rapid identification of S. aureus isolates when paired with genotypic identification of methicillin resistance2 can be used to guide antibiotic choice. Coagulase negative staphylococci, which represented the largest group of Gram positive organisms, were also infrequently identified to species level (21%). Reliable identification of these organisms may provide valuable information that a positive blood culture is likely to be a contaminant, avoiding unnecessary escalation of antibiotics. The differences observed for identification of Gram positive organisms compared with Moussaoui et al. may be accounted for in part by differences in the performance of the Vitek MS,
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
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Pathology (2015), 47(1), January
CORRESPONDENCE
Table 1
Direct MALDI-TOF identification compared with conventional subculture MALDI-TOF identification result for all isolates tested Correct identification Species level
Organism Gram negatives E. coli Enterobacter spp Klebsiella spp P. mirabilis Salmonella spp S. marcescens Y. enterocolitica C. jejuni H. influenzae K. kingae P. aeruginosa R. radiobacter Bacteroides spp F. necrophorum Subtotal (%) Gram positives CONS S. aureus Micrococcus spp Gemella sputi S. agalactiae Viridans streptococci Enterococcus spp L. monocytogenes Rhodococcus equi C. perfringens Subtotal (%) Total
All samples
Monomicrobial
Polymicrobial
35/38 4/5 6/9 0/1 3/8 3/3 1/1 0/1 1/1 0/1 1/1 0/1 3/3 0/2 60/75 (80%)
27/30 3/4 6/6 0/0 3/8 3/3 1/1 0/1 1/1 0/1 1/1 0/1 2/2 0/2 47/61 (77%)
8/8 1/1 0/3 0/1
7/34 2/12 0/1 0/1 1/2 0/3 2/3 1/1 0/1 1/2 14/60 (23%) 71/135 (53%)
6/24 2/11 0/1 0/1 1/2 0/3 0/0 1/1 0/1 0/0 10/44 (23%) 57/105 (54%)
Genus
Incorrect
4/8
1/1 1/1 1/1 10/14 (71%) 1/10 0/1
2/61 (3%) 10/34 4/12
1/2 2/3 1/1 1/2 4/16 (25%) 14/30 (47%)
6/44 (14%) 8/105 (8%)
CONS, coagulase negative staphylococcus.
and BD-Bruker MS for direct blood culture identification for this group of organisms.9 Others have also reported difficulties with Gram positive organism identification using centrifugation based methods on the BD-Bruker MS,6 possibly due to loss of bacteria in multiple centrifugation steps, as the number of bacteria present in positively flagged cultures with Gram positive organisms is generally lower than for Gram negative organisms.10 Therefore, a different approach for Gram positive organisms may be required on the Vitek MS platform. Two alternative methods, both evaluated on the Vitek MS have reported higher Gram positive organism identification rates, using lysis-centrifugation8 (92%) and lysis-filtration7 (75%). A known limitation of direct identification is the inability of Vitek MS software to detect polymicrobial infections, which mandates that the MS result be interpreted in conjunction with the Gram stain. The minimum time to results for samples was 35 min, with a median of 110 min in routine circumstances. This fitted well with hospital laboratory and clinical routines. We found that formic acid extraction made no difference to performance, allowing a time saving of 5–10 min. Direct identification was usually achieved 24 h before conventional methods, although we did not assess the use of the MS for identification of early growth from subcultures outside routine laboratory workflow, which may offer a labour saving compromise to this technique.11 The reagent cost for this method was NZ$1.16, comparing favourably with the Foster method (NZ$2.69) and the
Sepsityper (Becton Dickinson) (NZ$6.20). No additional equipment is required, and steps were simple to perform. The impact of additional labour time on laboratory functioning was not assessed, a factor likely to be a major limitation on the implementation of direct identification in laboratories. Several studies have shown that the routine identification of bacteria directly from blood cultures using MALDI-TOF MS has a positive impact on outcomes of patients with bacterial sepsis, increasing early institution of appropriate antibiotics and reducing cost and length of hospital stay,6,12 although the contribution of direct identification alone to these improved outcomes has been difficult to determine. This method is rapid, sensitive and reliable for Gram negative organism identification, simple to perform and requires only readily available and inexpensive consumables. Although it performs poorly for Gram positive organisms, laboratories may find routine application of this protocol useful for Gram negative organisms, as improvements in the early institution of appropriate antimicrobial therapy have been reported with these bacteria alone.12 Conflicts of interest and sources of funding: The authors state that there are no conflicts of interest to disclose. Gary N. McAuliffe Mary Bilkey Sally A. Roberts
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
CORRESPONDENCE
Department of Microbiology, LabPlus, Auckland, New Zealand Contact Dr Gary McAuliffe. E-mail: [email protected] 1. Ferrer R, Martin-Loeches I, Phillips G, et al. Empiric antibiotic treatment reduces mortality in severe sepsis and septic shock from the first hour: results from a guideline-based performance improvement program. Crit Care Med 2014; 42: 1749–55. 2. Clerc O, Prod’hom G, Senn L, et al. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR-based rapid diagnosis of Staphylococcus aureus bacteraemia. Clin Microbiol Infect 2014; 20: 355–60. 3. Machen A, Drake T, Wang YF. Same day identification and full panel antimicrobial susceptibility testing of bacteria from positive blood culture bottles made possible by a combined lysis-filtration method with MALDITOF VITEK mass spectrometry and the VITEK2 system. PLoS One 2014; 9: e87870. 4. Moussaoui W, Jaulhac B, Hoffmann AM, et al. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directly from blood culture vials. Clin Microbiol Infect 2010; 16: 1631–8. 5. Stevenson LG, Drake SK, Murray PR. Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionizationtime of flight mass spectrometry. J Clin Microbiol 2010; 48: 444–7. 6. Vlek AL, Bonten MJ, Boel CH. Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry improves appropriateness of antibiotic treatment of bacteremia. PLoS One 2012; 7: e32589. 7. Fothergill A, Kasinathan V, Hyman J, Walsh J, Drake T, Wang YF. Rapid identification of bacteria and yeasts from positive-blood-culture bottles by using a lysis-filtration method and matrix-assisted laser desorption ionization-time of flight mass spectrum analysis with the SARAMIS database. J Clin Microbiol 2013; 51: 805–9. 8. Foster AG. Rapid Identification of microbes in positive blood cultures by use of the vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system. J Clin Microbiol 2013; 51: 3717–9. 9. Chen JH, Ho PL, Kwan GS, et al. Direct bacterial identification in positive blood cultures by use of two commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry systems. J Clin Microbiol 2013; 51: 1733–9. 10. Ferreira L, Sa´nchez-Juanes F, Mun˜oz-Bellido JL, Gonza´lez-Buitrago JM. Rapid method for direct identification of bacteria in urine and blood culture samples by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: intact cell vs. extraction method. Clin Microbiol Infect 2011; 17: 1007–12. 11. Idelevich EA, Schu¨le I, Gru¨nastel B, Wu¨llenweber J, Peters G, Becker K. Rapid identification of microorganisms from positive blood cultures by MALDI-TOF mass spectrometry subsequent to very short-term incubation on solid medium. Clin Microbiol Infect 2014; Apr 3;. (Epub ahead of print). 12. Clerc O, Prod’hom G, Vogne C, Bizzini A, Calandra T, Greub G. Impact of matrix- assisted laser desorption ionization time-of-flight mass spectrometry on the clinical management of patients with Gram-negative bacteremia: a prospective observational study. Clin Infect Dis 2013; 56: 1101–7.
DOI: 10.1097/PAT.0000000000000206
Evaluation of HemoCue white blood cell differential counter at a remote health centre in Australia’s Northern Territory Sir, The total white blood cell (WBC) count and differential is a frequently ordered pathology test to detect infection, inflammation or as part of a full blood count or complete blood count in a routine health assessment.1 Differentiation of the number of white blood cells into subtypes (neutrophils, lymphocytes, monocytes, eosinophils and basophils) provides additional clinical information; for example, to distinguish between a potential bacterial, viral or parasitic infection.2
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Traditionally the differential WBC count is measured by automated laboratory cell counters. The ability to measure a differential WBC count by point-of-care testing (POCT) would be useful in extra-laboratory (e.g., emergency departments and outpatient clinics) and primary care (e.g., general practices and Aboriginal Medical Services) settings where the convenience, accessibility, portability and immediacy of the POCT result would be advantageous.3 In 2008, HemoCue AB (Sweden) developed a POCT device for total WBC count. In 2012, this device was further refined to additionally provide a 5-part differential count. In this study we examined the analytical performance characteristics and usefulness of the HemoCue WBC Diff counter in a remote Aboriginal medical service, where access to pathology services is difficult due to the geographical isolation from the nearest laboratory and the high burden of infection among its Aboriginal community members. The study was implemented in a remote Indigenous community in Australia’s Northern Territory, located over 600 km from the nearest major town and laboratory service (Mt Isa, Queensland). The community has a population between 500 and 800 people, of which 94% are Aboriginal.4 The Remote Health Centre is serviced by two Remote Area Nurses (RAN) and a visiting medical practitioner. During the wet season the community is frequently cut off via road due to flooding. Ethics registration for this project was obtained in March 2013 from the Menzies School of Medicine Ethics Committee. Informed verbal consent was obtained from each patient routinely presenting to clinic with symptoms indicating a WBC count was needed. Patient participation in the study was voluntary. A 10 mL sample of capillary or venous blood is loaded into a HemoCue WBC Diff microcuvette. The red blood cells are lysed and the WBCs stained with methylene blue. Thirty seven images are then taken of the stained WBCs using microphotographs, images are focused and mathematical algorithms classify and count cell types. Total WBC and a 5-part differential count are displayed in less than 5 min. The measuring range for the total WBC count is 0.3–30 109/L. The analyser is small, lightweight and portable (size 190 160 160 mm, weight 1.3 kg). The analyser operates by batteries or by AC power adaptor and has external connectivity capability. Venous whole blood samples sent to the laboratory were measured on a Sysmex XE-2100 analyser (Sysmex America, USA). Fluorescent labelling is used to measure the nucleusplasma ratio of each individually stained cell, enabling differentiation and reporting of six WBC populations. The XE-Series utilises an adaptive cluster analysis system (ACAS) to separate cell populations into well-defined three-dimensional clusters. This study comprised 62 adult patients routinely presenting to clinic with symptoms indicating a WBC differential count was needed. Each patient result was recorded using a unique code number to maintain confidentiality in the study. The Flinders University International Centre for Point-of-Care Testing (iPOCT) developed a training resource package and delivered on-site training to the clinical team at the health service. A RAN collected the venous sample to be sent to the laboratory and tested a small aliquot of the venous sample on the WBC Diff prior to despatch to the laboratory. A fingerpick capillary sample was also taken at the same time from each patient and tested on the WBC Diff on-site. The venous sample was then sent via Mount Isa to the nearest accredited pathology laboratory in Brisbane some 2000 km from the remote community. The
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
Pathology (2015), 47(1), January
CORRESPONDENCE
Number of cases
30
Serotype 1b 2a
20
2b 3a
surveillance of emerging antibiotic resistance in Shigella species is warranted, and clinical laboratories should be aware of possible azithromycin-resistant Shigella species locally acquired in Australia when providing advice to clinicians regarding empiric therapy.
6
10
Other
20 0 20 0 0 20 1 0 20 2 0 20 3 0 20 4 0 20 5 0 20 6 0 20 7 0 20 8 0 20 9 1 20 0 1 20 1 1 20 2 1 20 3 14 *
0
Year Fig. 1 Number of cases of Shigella flexneri serotypes in Victoria tested at MDU PHL, from 2000. *Data for 2014 is January to June inclusive.
minimum inhibitory concentration (MIC) 16 mg/mL is considered susceptible in the normal wild-type distribution.3,4 The Microbiological Diagnostic Unit Public Health Laboratory (MDU PHL) routinely performs identification, serotyping and antimicrobial susceptibility testing of Shigella isolates from clinical cases in Victoria and selected isolates from NSW.5 Recent reports of increasing antibiotic resistance in Shigella species, including a cases of infection where high-level resistance to azithromycin was demonstrated,3,6 and an increase in local laboratory confirmed cases of Shigella flexneri, prompted our investigation for local azithromycin resistance. Between 1 May 2013 and 30 June 2014, the number of Shigella flexneri 3a cases reported in Melbourne and Sydney was 29 and 16, respectively, a significant increase compared to previous years, with the major increase seen in 2014. Figure 1 summarises the serotype prevalence data for cases notified in Victoria from 2000 and where isolates were characterised at MDU PHL, demonstrating the significant increase in Shigella flexneri 3a cases in the first half of 2014. The number of infections caused by other Shigella flexneri serotypes was unchanged over the same time period. Thirty-eight of 45 isolates (14 from 2013, 12 males; 24 from 2014, all males) were submitted for azithromycin susceptibility testing, using azithromycin Etest according to the manufacturer’s instructions (bioMerieux, France). Susceptibility to azithromycin (MIC 16 mg/mL) was only seen in four 2013 isolates from two males and two females. Of the 34 remaining azithromycinresistant isolates (89% of isolates tested), 33 were resistant to azithromycin (MIC >256 mg/mL), ampicillin, and tetracycline, while one isolate was resistant to azithromycin (MIC 128 mg/ mL), ampicillin, tetracycline and exhibited decreased susceptibility to ciprofloxacin (0.25 mg/mL). Approval to include de-identified demographic data summarising risk factors for Shigella infection was granted by Department of Health, Victoria. The median age for the azithromycin resistant cases was 40 years (range 21–65 years), with most cases reported as occurring in MSM without overseas travel, indicating local acquisition in the MSM population in Melbourne and Sydney. These data are the result of passive surveillance of notifiable infections undertaken during this period, and not active surveillance in this risk group. Emerging azithromycin resistance in Shigella species further highlights the growing burden of antibiotic resistant bacterial infections, and has significant implications for the empiric treatment of severe bacterial gastrointestinal disease. Particularly in the MSM community, possible cases of shigellosis should not be empirically treated with azithromycin. Active
Conflicts of interest and sources of funding: The authors state that there are no conflicts of interest to disclose. Mary Valcanis1 Jeremy D. Brown2 Briony Hazelton2 Matthew V. O’Sullivan2,3 Alex Kuzevski1 Courtney R. Lane4,5 Benjamin P. Howden1,6 1
Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, The University of Melbourne at The Doherty Institute for Infection and Immunity, Melbourne, Vic, 2Centre for Infectious Disease and Microbiological Laboratory Services, Institute for Clinical Pathology and Medical Research, Westmead Hospital, Sydney, NSW, 3Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Sydney, NSW, 4Victorian Department of Health, Communicable Disease Epidemiology and Surveillance, Health Protection Branch, Melbourne, Vic, 5National Centre for Epidemiology and Population Health, Australian National University, Canberra, ACT, and 6Infectious Diseases Department, Austin Health, Heidelberg, Vic, Australia Contact Professor Benjamin Howden. E-mail: [email protected] 1. Rowe SL, Radwan S, Lalor K, et al. An outbreak of shigellosis among men who have sex with men, Victoria, 2008. Vic Infect Dis Bull 2010; 13: 119–23. 2. Gastrointestinal Expert Group. Therapeutic Guidelines: Gastrointestinal. Version 5. Melbourne: Therapeutic Guidelines Limited, 2011. 3. Hassing RJ, Melles DC, Goessens WHF, Rijnders BJA. Case of Shigella flexneri infection with treatment failure due to azithromycin resistance in an HIV-positive patient. Infection 2014; 42: 789–90. 4. European Committee on Antimicrobial Susceptibility Testing. Breakpoint Tables for Interpretation of MICs and Zone Diameters, Version 4, 2014. Cited 20 Jul 2014. http://www.eucast.org/clinical_breakpoints 5. Valcanis M. Laboratory testing of Shigella. Vic Infect Dis Bull 2010; 13: 114–8. 6. Heiman KE, Karlsson M, Grass J, et al. Notes from the field: Shigella with decreased susceptibility to azithromycin among men who have sex with men – United States, 2002-2013. MMWR Morb Mortal Wkly Rep 2014; 63: 132–3.
DOI: 10.1097/PAT.0000000000000207
Direct identification of bacteria from positive BD-Bactec blood culture bottles on the Vitek MS Sir, Timely and the appropriate choice of antimicrobial therapy is associated with lower mortality in patients with bacterial sepsis.1 Unfortunately there is often a delay of days before
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
CORRESPONDENCE
bacteria are isolated from blood cultures, identified and antimicrobial susceptibilities are determined, during which time patients may remain on inappropriate empirical antimicrobial therapy. The recent widespread implementation of matrix assisted laser desorption and ionisation time of flight mass spectrometry (MALDI-TOF MS) into diagnostic laboratories offers a rapid and reliable alternative method for direct identification of isolates from blood cultures. Identification of the isolate may allow tailoring of antibiotic therapy based on local sensitivity data. It may also be used in combination with molecular techniques such as the Xpert MRSA/SA BC2 (Cepheid, USA), or inoculation of appropriate antimicrobial susceptibility cards on automated systems3 such as the Vitek-2 (bioMe´rieux, France). In this study we evaluated the performance of a modified extraction protocol previously reported for the BD-Bruker Maldi Biotyper4 (Becton Dickinson, USA), on the Vitek-MS (bioMe´rieux), including the relative contribution of protocol steps to yield. LabPlus serves as the diagnostic laboratory for a 1000 bed university-affiliated tertiary care hospital. Blood cultures are processed 24 h a day. All positive blood cultures have a Gram stain performed; the results of which determine the media that the broth is subcultured onto. Bacterial isolates are identified using the Vitek MS. Direct identification using the MS was performed on positive blood cultures prior to growth on subculture. Fisher’s exact test was used for comparing unmatched, and McNemar’s test for comparing matched data. For the basic protocol extraction method: 3.5 mL of broth was removed from the blood culture bottles and injected into a 3.5 mL serum separator tube (SST) (Becton Dickinson). This was centrifuged at 3300 rpm for 10 min. The supernatant was removed, and pellet re-suspended in 1.0 mL of sterile water. This was manually agitated prior to transfer to a 2 mL tube. After centrifugation at 13,000 rpm for 1 min, the supernatant was removed and resulting bacterial pellet spotted in quadruplicate onto a 48-well Vitek MS-DS disposable target slide. Sample wells were overlaid with 1 mL alpha-cyano-4-hydroxycinnamic acid (HCCA) matrix (bioMe´rieux), and run on the Vitek MS IVD system. For the additional centrifugation step, following centrifugation of the SST, the pellet was re-suspended in 1.5 mL of sterile water, transferred to a 2 mL tube, and centrifuged at 2100 rpm for 1 min. Then 1 mL of the supernatant was transferred to a 2 mL tube and centrifuged at 13,000 rpm for 1 min. The pellet was processed as for the basic protocol. For evaluation of formic acid extraction, a subset of samples were overlaid with 0.5 mL formic acid (bioMe´rieux) on two sample wells of the target slide. These were then overlaid with matrix and run as per the basic protocol. A total of 128 BD Bactec Plus Aerobic/F, Plus Anaerobic/F and Peds Plus (Becton Dickinson) blood culture bottles from 86 patients (73 adults and 13 children) collected between January and October 2013, were processed using either the basic method, or with the additional centrifugation step. Of these, 105 (82%) bottles were positive by Gram stain and had a single isolate grown on subculture. Fifteen (12.5%) bottles had growth of two or more isolates on subculture. Two bottles contained yeasts, and six bottles had flagged positive but had negative Gram stains (false positive).
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For blood cultures with a single isolate, the overall correct bacterial identification to species level by Vitek MS software was achieved for 57 of 105 (54%) of isolates; 47 of 61 (77%) Gram negative organisms and 10 of 44 (23%) Gram positive organisms using either method. No identification was given for 40 of 105 (38%) of isolates. Incorrect identification occurred in eight of 105 (7.6%) single isolate cultures; two of 61 (3.3%) Gram negative organisms, and six of 44 (14%) Gram positive organisms (Table 1). For polymicrobial samples, correct identification for one organism was achieved in 13 of 15 (87%) of samples. In one of the six blood culture bottles which had flagged falsely positive, direct identification gave an erroneous identification of Vibrio fluvialis with high confidence (98.7%). The blood cultures remained negative after 5 days incubation. The method did not identify yeasts. Using the basic method, 15 of 24 (63%) of Gram negative organisms, six of 26 (23%) of Gram positive organisms, and 21 of 50 (42%) of all isolates were correctly identified. With the additional centrifugation step, 32 of 37 (86%) of Gram negative organisms, four of 18 (22%) of Gram positive organisms, and 36 of 55 (65%) of all bacteria were correctly identified. Formic acid extraction gave correct identification for 31 of 52 (60%) isolates compared with 30 of 52 (58%) ( p ¼ 0.262) of those processed without this step. We found this method to be reliable for the identification of Gram negatives, and were able to improve on an initial correct identification rate of 63% to 86% ( p ¼ 0.059) for Gram negatives (42% to 65%, p ¼ 0.019 for all bacteria) with an additional centrifugation step to remove remaining cellular debris, which may interfere with MS spectral recognition.5 Our results compare favourably both with the findings of Moussaoui et al. (91%),4 and those of other recent reports for the identification of Gram negatives.6–8 Identification of Gram positive organisms (23%) by this method was poor, compared with that reported by Moussaoui et al. (89%).4 The yield did not substantially improve with an additional centrifugation step. A significant (14%) risk of false positive identifications contributes to rendering this method unsuitable for testing Gram positive organisms on the Vitek MS. The performance of individual organisms using either the basic method, or with the additional centrifugation step is presented in Table 1. Issues with identification of Salmonella, Campylobacter, Rhizobium, and Fusobacterium have been reported elsewhere.4,5 The relatively small number of samples in this study restricted our ability to assess the performance of other less commonly isolated organisms. The sensitivity of this method for identification of Staphylococcus aureus was particularly poor (18%), and inferior to the sensitivity of the two hour tube coagulase test (68%) performed at our laboratory. Rapid identification of S. aureus isolates when paired with genotypic identification of methicillin resistance2 can be used to guide antibiotic choice. Coagulase negative staphylococci, which represented the largest group of Gram positive organisms, were also infrequently identified to species level (21%). Reliable identification of these organisms may provide valuable information that a positive blood culture is likely to be a contaminant, avoiding unnecessary escalation of antibiotics. The differences observed for identification of Gram positive organisms compared with Moussaoui et al. may be accounted for in part by differences in the performance of the Vitek MS,
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Pathology (2015), 47(1), January
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Table 1
Direct MALDI-TOF identification compared with conventional subculture MALDI-TOF identification result for all isolates tested Correct identification Species level
Organism Gram negatives E. coli Enterobacter spp Klebsiella spp P. mirabilis Salmonella spp S. marcescens Y. enterocolitica C. jejuni H. influenzae K. kingae P. aeruginosa R. radiobacter Bacteroides spp F. necrophorum Subtotal (%) Gram positives CONS S. aureus Micrococcus spp Gemella sputi S. agalactiae Viridans streptococci Enterococcus spp L. monocytogenes Rhodococcus equi C. perfringens Subtotal (%) Total
All samples
Monomicrobial
Polymicrobial
35/38 4/5 6/9 0/1 3/8 3/3 1/1 0/1 1/1 0/1 1/1 0/1 3/3 0/2 60/75 (80%)
27/30 3/4 6/6 0/0 3/8 3/3 1/1 0/1 1/1 0/1 1/1 0/1 2/2 0/2 47/61 (77%)
8/8 1/1 0/3 0/1
7/34 2/12 0/1 0/1 1/2 0/3 2/3 1/1 0/1 1/2 14/60 (23%) 71/135 (53%)
6/24 2/11 0/1 0/1 1/2 0/3 0/0 1/1 0/1 0/0 10/44 (23%) 57/105 (54%)
Genus
Incorrect
4/8
1/1 1/1 1/1 10/14 (71%) 1/10 0/1
2/61 (3%) 10/34 4/12
1/2 2/3 1/1 1/2 4/16 (25%) 14/30 (47%)
6/44 (14%) 8/105 (8%)
CONS, coagulase negative staphylococcus.
and BD-Bruker MS for direct blood culture identification for this group of organisms.9 Others have also reported difficulties with Gram positive organism identification using centrifugation based methods on the BD-Bruker MS,6 possibly due to loss of bacteria in multiple centrifugation steps, as the number of bacteria present in positively flagged cultures with Gram positive organisms is generally lower than for Gram negative organisms.10 Therefore, a different approach for Gram positive organisms may be required on the Vitek MS platform. Two alternative methods, both evaluated on the Vitek MS have reported higher Gram positive organism identification rates, using lysis-centrifugation8 (92%) and lysis-filtration7 (75%). A known limitation of direct identification is the inability of Vitek MS software to detect polymicrobial infections, which mandates that the MS result be interpreted in conjunction with the Gram stain. The minimum time to results for samples was 35 min, with a median of 110 min in routine circumstances. This fitted well with hospital laboratory and clinical routines. We found that formic acid extraction made no difference to performance, allowing a time saving of 5–10 min. Direct identification was usually achieved 24 h before conventional methods, although we did not assess the use of the MS for identification of early growth from subcultures outside routine laboratory workflow, which may offer a labour saving compromise to this technique.11 The reagent cost for this method was NZ$1.16, comparing favourably with the Foster method (NZ$2.69) and the
Sepsityper (Becton Dickinson) (NZ$6.20). No additional equipment is required, and steps were simple to perform. The impact of additional labour time on laboratory functioning was not assessed, a factor likely to be a major limitation on the implementation of direct identification in laboratories. Several studies have shown that the routine identification of bacteria directly from blood cultures using MALDI-TOF MS has a positive impact on outcomes of patients with bacterial sepsis, increasing early institution of appropriate antibiotics and reducing cost and length of hospital stay,6,12 although the contribution of direct identification alone to these improved outcomes has been difficult to determine. This method is rapid, sensitive and reliable for Gram negative organism identification, simple to perform and requires only readily available and inexpensive consumables. Although it performs poorly for Gram positive organisms, laboratories may find routine application of this protocol useful for Gram negative organisms, as improvements in the early institution of appropriate antimicrobial therapy have been reported with these bacteria alone.12 Conflicts of interest and sources of funding: The authors state that there are no conflicts of interest to disclose. Gary N. McAuliffe Mary Bilkey Sally A. Roberts
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CORRESPONDENCE
Department of Microbiology, LabPlus, Auckland, New Zealand Contact Dr Gary McAuliffe. E-mail: [email protected] 1. Ferrer R, Martin-Loeches I, Phillips G, et al. Empiric antibiotic treatment reduces mortality in severe sepsis and septic shock from the first hour: results from a guideline-based performance improvement program. Crit Care Med 2014; 42: 1749–55. 2. Clerc O, Prod’hom G, Senn L, et al. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR-based rapid diagnosis of Staphylococcus aureus bacteraemia. Clin Microbiol Infect 2014; 20: 355–60. 3. Machen A, Drake T, Wang YF. Same day identification and full panel antimicrobial susceptibility testing of bacteria from positive blood culture bottles made possible by a combined lysis-filtration method with MALDITOF VITEK mass spectrometry and the VITEK2 system. PLoS One 2014; 9: e87870. 4. Moussaoui W, Jaulhac B, Hoffmann AM, et al. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directly from blood culture vials. Clin Microbiol Infect 2010; 16: 1631–8. 5. Stevenson LG, Drake SK, Murray PR. Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionizationtime of flight mass spectrometry. J Clin Microbiol 2010; 48: 444–7. 6. Vlek AL, Bonten MJ, Boel CH. Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry improves appropriateness of antibiotic treatment of bacteremia. PLoS One 2012; 7: e32589. 7. Fothergill A, Kasinathan V, Hyman J, Walsh J, Drake T, Wang YF. Rapid identification of bacteria and yeasts from positive-blood-culture bottles by using a lysis-filtration method and matrix-assisted laser desorption ionization-time of flight mass spectrum analysis with the SARAMIS database. J Clin Microbiol 2013; 51: 805–9. 8. Foster AG. Rapid Identification of microbes in positive blood cultures by use of the vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system. J Clin Microbiol 2013; 51: 3717–9. 9. Chen JH, Ho PL, Kwan GS, et al. Direct bacterial identification in positive blood cultures by use of two commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry systems. J Clin Microbiol 2013; 51: 1733–9. 10. Ferreira L, Sa´nchez-Juanes F, Mun˜oz-Bellido JL, Gonza´lez-Buitrago JM. Rapid method for direct identification of bacteria in urine and blood culture samples by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: intact cell vs. extraction method. Clin Microbiol Infect 2011; 17: 1007–12. 11. Idelevich EA, Schu¨le I, Gru¨nastel B, Wu¨llenweber J, Peters G, Becker K. Rapid identification of microorganisms from positive blood cultures by MALDI-TOF mass spectrometry subsequent to very short-term incubation on solid medium. Clin Microbiol Infect 2014; Apr 3;. (Epub ahead of print). 12. Clerc O, Prod’hom G, Vogne C, Bizzini A, Calandra T, Greub G. Impact of matrix- assisted laser desorption ionization time-of-flight mass spectrometry on the clinical management of patients with Gram-negative bacteremia: a prospective observational study. Clin Infect Dis 2013; 56: 1101–7.
DOI: 10.1097/PAT.0000000000000206
Evaluation of HemoCue white blood cell differential counter at a remote health centre in Australia’s Northern Territory Sir, The total white blood cell (WBC) count and differential is a frequently ordered pathology test to detect infection, inflammation or as part of a full blood count or complete blood count in a routine health assessment.1 Differentiation of the number of white blood cells into subtypes (neutrophils, lymphocytes, monocytes, eosinophils and basophils) provides additional clinical information; for example, to distinguish between a potential bacterial, viral or parasitic infection.2
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Traditionally the differential WBC count is measured by automated laboratory cell counters. The ability to measure a differential WBC count by point-of-care testing (POCT) would be useful in extra-laboratory (e.g., emergency departments and outpatient clinics) and primary care (e.g., general practices and Aboriginal Medical Services) settings where the convenience, accessibility, portability and immediacy of the POCT result would be advantageous.3 In 2008, HemoCue AB (Sweden) developed a POCT device for total WBC count. In 2012, this device was further refined to additionally provide a 5-part differential count. In this study we examined the analytical performance characteristics and usefulness of the HemoCue WBC Diff counter in a remote Aboriginal medical service, where access to pathology services is difficult due to the geographical isolation from the nearest laboratory and the high burden of infection among its Aboriginal community members. The study was implemented in a remote Indigenous community in Australia’s Northern Territory, located over 600 km from the nearest major town and laboratory service (Mt Isa, Queensland). The community has a population between 500 and 800 people, of which 94% are Aboriginal.4 The Remote Health Centre is serviced by two Remote Area Nurses (RAN) and a visiting medical practitioner. During the wet season the community is frequently cut off via road due to flooding. Ethics registration for this project was obtained in March 2013 from the Menzies School of Medicine Ethics Committee. Informed verbal consent was obtained from each patient routinely presenting to clinic with symptoms indicating a WBC count was needed. Patient participation in the study was voluntary. A 10 mL sample of capillary or venous blood is loaded into a HemoCue WBC Diff microcuvette. The red blood cells are lysed and the WBCs stained with methylene blue. Thirty seven images are then taken of the stained WBCs using microphotographs, images are focused and mathematical algorithms classify and count cell types. Total WBC and a 5-part differential count are displayed in less than 5 min. The measuring range for the total WBC count is 0.3–30 109/L. The analyser is small, lightweight and portable (size 190 160 160 mm, weight 1.3 kg). The analyser operates by batteries or by AC power adaptor and has external connectivity capability. Venous whole blood samples sent to the laboratory were measured on a Sysmex XE-2100 analyser (Sysmex America, USA). Fluorescent labelling is used to measure the nucleusplasma ratio of each individually stained cell, enabling differentiation and reporting of six WBC populations. The XE-Series utilises an adaptive cluster analysis system (ACAS) to separate cell populations into well-defined three-dimensional clusters. This study comprised 62 adult patients routinely presenting to clinic with symptoms indicating a WBC differential count was needed. Each patient result was recorded using a unique code number to maintain confidentiality in the study. The Flinders University International Centre for Point-of-Care Testing (iPOCT) developed a training resource package and delivered on-site training to the clinical team at the health service. A RAN collected the venous sample to be sent to the laboratory and tested a small aliquot of the venous sample on the WBC Diff prior to despatch to the laboratory. A fingerpick capillary sample was also taken at the same time from each patient and tested on the WBC Diff on-site. The venous sample was then sent via Mount Isa to the nearest accredited pathology laboratory in Brisbane some 2000 km from the remote community. The
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