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Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of the tube coagulase test

Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of... Background: The ideal identification of Staphylococcus aureus clinical isolates requires a battery of tests and this is costly in resource limited settings. In many developing countries, the tube coagulase test is usually confirmatory for S. aureus and is routinely done using either human or sheep plasma. This study evaluated Mannitol salt agar and the deoxyribonuclease (DNase) test for improving the efficiency of the tube coagulase test in resource limited settings. The efficiency of human and sheep plasma with tube coagulase tests was also evaluated. Methods: One hundred and eighty Gram positive, Catalase positive cocci occurring in pairs, short chains or clusters were subjected to growth on Mannitol salt agar, deoxyribonuclease and tube coagulase tests. Of these, isolates that were positive for at least two of the three tests (n = 60) were used to evaluate the performance of the tube coagulase test for identification of S. aureus, using PCR-amplification of the nuc gene as a gold standard. Results: Human plasma was more sensitive than sheep plasma for the tube coagulase test (sensitivity of 91% vs. 81% respectively), but both plasmas had very low specificity (11% and 7% respectively). The sensitivity and specificity of the tube coagulase test (human plasma) was markedly improved when Mannitol salt agar and DNase were introduced as a tri-combination test for routine identification of Staphylococcus aureus (100% specificity and 75% sensitivity). The specificity and sensitivity of Mannitol salt agar/DNase/tube coagulase (sheep plasma) combination was 100% and 67%, respectively. Conclusion: The efficiency of the tube coagulase test can be markedly improved by sequel testing of the isolates with Mannitol salt agar, DNase and Tube coagulase. There is no single phenotypic test (including tube coagulase) that can guarantee reliable results in the identification of Staphylococcus aureus. Background are the mainstay in the diagnosis of staphylococcal Staphylococcus aureus is a ubiquitous commensal bac- infections, in which coagulase tests are usually confirma- terium on human skins and anterior nares, but fre- tory for S. aureus [4-8]. Coagulase testing is performed quently causes severe infections in humans [1]. Rapid using the slide coagulase (SCT) or the tube coagulase and direct identification of S. aureus is crucial for (TCT) methods [9]. Although these tests efficiently proper management of patients with skin infections, identify S. aureus, their performances vary from setting abscesses, septicemia/bacteremia, gastroenteritis, endo- to setting and need improvement [3,6]. carditis, toxic shock syndrome and certain food intoxi- Several laboratories in developing countries screen for cations [2,3]. In developing countries, phenotypic tests presumptive Staphylococcus aureus based on growth on Mannitol salt agar (MSA) and/or DNase tests and confir- mation is done with the TCT. In many settings, the use * Correspondence: najjuka2002@yahoo.com † Contributed equally of the TCT is curtailed by reliance on human plasma, Department of Medical Microbiology, School of Biomedical Sciences, since the recommended plasmas (from rabbit, horse [10]) Makerere University College of Health Sciences, Upper Mulago hill road, are either expensive or if locally available, are of poor Kampala, Uganda Full list of author information is available at the end of the article © 2010 Kateete et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Kateete et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:23 Page 2 of 7 http://www.ann-clinmicrob.com/content/9/1/23 quality. Human plasma is reported to give discordant Medical Microbiology, Makerere University College of results [6]; usually obtained from blood banks as outdated Health Sciences, from January through June, 2009. It materials, it contains variable amounts of CRF (Coagu- was a laboratory based study that involved frozen clini- lase-Reacting Factor) and anti-staphylococcal antibodies cal isolates from patients’ blood, cerebral spinal fluid, [9]. This type of plasma is not recommended for coagu- anterior nares, skin and wound swabs. lase tests [9]. Other factors which make human plasma inappropriate for the coagulase tests include; a high bur- Inclusion/exclusion criteria den of viral infections (such as HIV/AIDS, Hepatitis B One hundred and eighty Gram positive, Catalase posi- and C) in resource limited settings that can render the tive cocci occurring in pairs, short chains or clusters plasma risky to laboratory workers and prior to use, it were subjected to growth on MSA, tube coagulase and must be screened for safety. There are also ethical issues DNase tests (see below). Of these (N = 180), isolates accompanying the use of human specimens. Conversely, that were positive for at least two of the three tests the efficiency of other plasmas in many laboratory set- (n = 60) were used to evaluate the performance of the tings varies and has to be determined for proper identifi- tube coagulase test for identification of S. aureus,using cation of S. aureus with coagulase tests. PCR-amplification of the nuc gene (which is specific for Although coagulase tests are invaluable for identification S. aureus) as the gold standard. of Staphylococcus aureus, few studies have evaluated their use in routine practice [10]. In addition, diagnostic labora- Phenotypic identification of Staphylococcus aureus tories are occasionally faced with organisms with biochem- Isolates were incubated at 37°C for 24 hours on blood ical characteristics that do not fit into the patterns of a agar and then sub-cultured on TS (Tryptic Soya, Liofil- known genus and species [11]. Furthermore, there are chem, Italy) agar (Fisher, Leicestershire, UK). Single some problems associated with coagulase tests; firstly, colonies were tested with tube coagulase and DNase test some human CoNS (Coagulase Negative Staphylococci) andgrowthonMSA.Toevaluatethe performanceof produce clumping factor and may be falsely positive with the individual tests or a combination of tests, sequel the SCT [9,12]. Secondly, some staphylococci of animal testing of the isolates was done beginning with MSA, origin are clumping factor negative and tube coagulase followed by DNase and TCT. positive; these may be misidentified as S. aureus,unless To confirm fermentation of mannitol, growth of yel- the fermentation of Mannitol is utilized in addition [9]. low colonies on MSA (Oxoid, Cambridge, UK) sur- Whereas Mannitol salt agar was developed for the pre- rounded by yellow zones after 24 hours of incubation at sumptive isolation of S. aureus in a single step, which is 37°C indicated a positive result. DNase test was per- convenient for diagnostic laboratories, Mannitol positive formed by incubating the isolates for 24 hours at 37°C CoNS have been reported in nasal and clinical specimens on DNase agar (Scharlau, Barcelona, Spain), and pouring from Nigeria and Japan [13,14]. Furthermore, Mannitol an excess (~15 ml) of 1 N HCl. Excess acid was negative MRSA (Methicilin Resistant Staphylococcus aur- removed with a vacuum pipette and clear zones around eus) was reported from clinical specimens in Kwazulu the bacterial colonies indicated DNase positive colonies. Natal province, South Africa [15]. In view of the above, For tube coagulase tests, colonies of test isolates were the common identification methods for S. aureus were re-suspended in 2 ml of citrated sheep or human plasma evaluated, aiming at improving the diagnosis of S. aureus in sterile glass test-tubes. Since citrate is utilized by through a combination of available phenotypic methods. enterococci [16], pure colonies of Gram positive, Cata- This study reveals that there is no single test (includ- lase positive staphylococci (catalase tests preceded coa- ing the tube coagulase test) that can guarantee reliable gulase reactions) were used. Positive control tubes with results for the identification of S. aureus. However, citrated plasma and coagulase producing strain of improved diagnostic sensitivity and specificity of the S. aureus ATCC 25923 (which efficiently coagulates tube coagulase test were achieved upon simultaneous citrated plasma) were included. To rule out citrate utili- testing of the isolates with DNase and Mannital salt zation by other microorganisms, control TCTs contain- agar. In order to improve the identification of S. aureus ing citrated plasma with Staphylococcus epidermidis in resource limited settings, sequel testing of the isolates ATCC 12228 were included. In addition, negative con- with Mannitol salt agar, tube DNase and coagulase is trol tubes containing citrated plasma alone (with no cul- proposed. tures inoculated) were included. The tubes were incubated at 35°C for 4 hours and observed for clot for- Methods mation. Where clotting did not occur, the tubes were Study setting incubated at room temperature for an additional 18 This study was done in the Clinical Microbiology and hours [9]. Tubes were studied without agitation in order Molecular Biology laboratories of the Department of not to disrupt partially formed clots. Kateete et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:23 Page 3 of 7 http://www.ann-clinmicrob.com/content/9/1/23 Molecular assays Table 1 Deriving Sensitivity, Specificity, Positive/Negative Predictive Values for the common identification tests for PCR-amplification of the nuc gene was used as a baseline S. aureus test. Reaction mixes were done in a Cleanspot ultraviolet Test Staphylococcus Other Total workstation (COV Lab products, Michigan, USA). The results aureus staphylococci reactions contained 20 pmoles each of the nuc forward Positive a b a + b and reverse primers (5′-GCGATTGATGGTGATA Negative c d c + d CGGTT-3′ and 5′-AGCCAAGCCTTGACGAACTA Total a + c b + d (a + b) + (c + d) AAGC-3′, respectively, synthesized by Eurofins-MWG- =n Operon, Ebersberg, Germany), 1.5 units of Taq DNA Where, polymerase (Thermo-Fisher, Surry, UK) and 1 μlofcus- a = True positives tom PCR master mix (ThermoFisher, Surry, UK). In the b = False positives post-amplification room, ~100 ng of staphylococcal chro- c = False negatives mosomal DNA was added to the reactions as template. d = True negatives Diagnostic sensitivity = d/(b + d) Amplifications were done in a Peltier thermocycler (MJ Diagnostic specificity = a/(a + c) Research, Waterman, MA, USA) under the following Positive predictive value (PPV) = a/(a + b) conditions: initial denaturation at 94°C, 5 min, followed Negative predictive value (NPV) = d/(d + c) by 37 cycles each consisting of a denaturation at 94°C, n=60 1 min; primer annealing at 55°C, 0.5 min, and extension at 72°C, 1 min; followed by a final extension at 72°C, 7 minutes. After this, the amplicons were mixed with 5 μl production can be detected using either the slide coagu- of DNA loading buffer and electrophoresed in a 1% agar- lase test (SCT) or the tube coagulase test (TCT). Slide ose gel in TAE buffer (Tris, acetate and EDTA). Control coagulase detects bound coagulase (also called “clump- reactions included templates of Staphylococcus aureus ing factor”) [9], which reacts directly with fibrinogen in ATCC 25923 (positive control), Staphylococcus epidermi- plasma, causing rapid cell agglutination. Negative iso- dis ATCC 12228 and nuclease free water (negative lates following SCT require confirmation with the controls). superior TCT, since strains deficient in clumping factor usually produce free coagulase. Tube coagulase detects Quality control secreted extracellular free coagulase that reacts with a To minimize cross contamination, standard microbiolo- substance in plasma called “Coagulase-Reacting Factor” gical procedures were strictly followed. Positive and (CRF) to form a complex, which in turn reacts with negative controls were always included in the test reac- fibrinogen to form fibrin (the clot) [9]. Strains of coagu- tions. DNA extraction and PCR-amplifications were lase-positive-animal staphylococci have been isolated done in molecular laboratories that are separate from from human clinical specimens, yet some animal staphy- the clinical microbiology laboratory where cultures were lococcal isolates also ferment mannitol [13]. This study grown. The PCR laboratory has designated sections for evaluated the performance of TCTs, DNase and MSA, pre-amplification, amplification and post-amplification, the phenotypic methods commonly used in the identifi- with a unidirectional movement of staff. cation of Staphylococcus aureus. Data analysis Detection of Staphylococcus aureus with the available The data were analyzed using a 2 × 2 contingency table for phenotypic tests diagnostic specificity and sensitivity (table 1). Diagnostic PCR-amplification of the nuc gene, which was used as a sensitivities and specificities were calculated as follows: baseline test, detected 32 Staphylococcus aureus of the Sensitivity (%) = [True positive/(True Positive + False 60 staphylococcal isolates, see table 2. Of the 32 nuc- Positive)] × 100 positive Staphylococcus aureus isolates, MSA detected Specificity (%) = [True Negative/(True Negative + the highest number of isolates (30 of 32, 94%) while the False Negative)] × 100 TCT (human and sheep plasma respectively) detected The positive predictive value (PPV) (%) = [True Posi- 29 and 27 of the 32 isolates (91% and 84% respectively), tive/(True Positive + False Positive)] × 100 table 2. DNase detected the least number of isolates Thenegativepredictivevalue (NPV)(%) =[True (24 of 32, 75%). Negative/(True Negative + False Negative)] × 100. Nine of the 32 nuc-positive Staphylococcus aureus iso- lates (28%) were coagulase negative, implying that some Results and discussion isolates may be misidentified by the TCT as a sole test. Coagulasetesting is thesinglemostreliablemethodfor The coagulase negative Staphylococcus aureus may prob- identifying Staphylococcus aureus [9]. Coagulase ably be MRSA isolates which are reported to react weakly Kateete et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:23 Page 4 of 7 http://www.ann-clinmicrob.com/content/9/1/23 Table 2 Identification of S. aureus with the common tests in comparison to PCR-detection of the nuc gene Other tests nuc PCR Outcome Positive (% of 32) Negative (% of 28) Subtotal MSA Positive 30 (94) 6 (21) 36 Negative 2 (6) 22 (79) 24 DNase Positive 24 (75) 1 (4) 25 Negative 8 (25) 27 (96) 35 Human plasma Positive 29 (91) 25 (89) 54 Negative 3 (9) 3 (11) 6 Human plasma/MSA Positive 27 (84) 6 (21) 31 Negative 0 3 (11) 3 Human plasma/DNase Positive 21 (60) 1 (4) 22 Negative 0 3 (11) 3 Human plasma/MSA/DNase Positive 21 (60) 1 (4) 22 Negative 0 3 (11) 3 Sheep plasma Positive 26 (81) 26 (93) 52 Negative 6 (19) 2 (8) 8 Sheep plasma/MSA Positive 24 (75) 6 (21) 30 Negative 0 2 (8) 2 Sheep plasma/DNase Positive 18 (56) 1 (4) 19 Negative 0 1 (4) 1 Sheep plasma/MSA/DNase Positive 18 (56) 1 (4) 19 Negative 0 2 (8) 2 MSA/DNase Positive 24 (74) 1 (4) 25 Negative 2 (6) 22 (79) 24 : tube coagulase (human plasma) : tube coagulase (sheep plasma) MSA: Mannitol salt agar DNase: Deoxyribonuclease PCR: Polymerase Chain Reaction or negatively with TCTs [17], or rare Staphylococcus aur- aureus. Growth on MSA was the most sensitive (94% eus strains that are reported to be coagulase negative [9]. sensitivity), followed by the TCT (human and sheep Two Staphylococcus aureus isolates (6%) were also MSA plasma, 91% and 81% sensitivity respectively) while the negative. Shittu et al also reported mannitol negative Sta- DNase test was the least sensitive (75% sensitivity). Con- phylococcus aureus that was methicillin resistant [15]. versely, the DNase test was the most specific (96% speci- Furthermore, eight of the 32 nuc-positive Staphylococcus ficity) followed by MSA (79% specificity), while the TCT aureus isolates were DNase negative (25%). Rao et al (human plasma and sheep plasma) was the least specific reported DNase negative Staphylococcus aureus but with (11% and 7%, respectively), table 3. Overall, of the indi- no explanation for these findings [18]. Six staphylococci vidual tests studied, growth on MSA was the best at other than the nuc-confirmed Staphylococcus aureus iso- identifying Staphylococcus aureus (94% sensitivity and lates produced yellow colonies on MSA, and similar find- 79% specificity). Our results slightly differ from those of ings were reported by other investigators [19-24]. Han et al, who reported sensitivity and specificity of Another rare finding in this study was an isolate that was 76.5% and 99.6%, respectively, for MSA [25]. D’Souza DNasepositivebut MSA-negative andtubecoagulase- et al, also reported sensitivity of 71%, a little lower than negative (i.e. non-Staphylococcus aureus). We presume ours [26]. The high sensitivity of MSA in detecting Sta- this isolate could have been Staphylococcus schleiferi phylococcus aureus could be due to its ability to isolate subsp. coagulans, which according to the National Stan- pathogenic Staphylococcus aureus [27], differentiating dard Method is also DNase positive [9]. coagulase negative staphylococci from coagulase positive staphylococci. Single phenotypic tests are inefficient for the For tube coagulase, human plasma was more sensitive identification of Staphylococcus aureus than sheep plasma (91% vs. 81% sensitivity), implying We used sensitivity and specificity to evaluate the per- that using sheep plasma with TCTs may detect more formance of individual tests in detecting Staphylococcus false negative isolates. Thus, in this setting, it is unlikely Kateete et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:23 Page 5 of 7 http://www.ann-clinmicrob.com/content/9/1/23 Table 3 Sensitivity, Specificity, Negative/Positive staphylococci, such as Staphylococcus schleiferi subspe- Predictive values for the common diagnostic tests for cies coagulans, Staphylococcus delphini, Staphylococcus clinical S. aureus intermedius and Staphylococcus hyicus. Staphylococcus Test Sensitivity Specificity NPV PPV delphini and Staphylococcus intermedius are rare clinical (%) (%) (%) (%) isolates while Staphylococcus hyicus is indeterminate MSA 94 79 92 83 (with prevalence ranging from 11% to 89% [17]). The DNase 75 96 77 96 prevalence of these isolates may be high in certain set- Human plasma 91 11 33 54 tings. Although MSA and DNase had high specificities Sheep plasma 81 7 25 50 and sensitivities, as individual tests, their use in routine MSA/DNase 96 92 92 96 identification of Staphylococcus aureus is curtailed by Human plasma/MSA 33 100 100 82 their ability to detect other bacterial isolates [14], and Human plasma/DNase 75 100 100 95 are mostly used during initial screens [6]. Indeed, Man- Human plasma/DNase/ 75 100 100 94 nitol salt positive CoNS (Staphylococcus caprae, MSA S. hemolyticus and S. saprophyticus) have been reported Sheep plasma/MSA 25 100 100 80 in Nigeria and Japan [13,14]. Thus, in certain settings, if Sheep plasma/DNase 50 100 100 95 used individually to identify Staphylococcus aureus, Sheep plasma/MSA/ 67 100 100 95 common phenotypic tests may be insufficient; some iso- DNase lates will be misidentified, either as Staphylococcus aur- : Tube coagulase (human plasma) 2 eus or CoNS. : Tube coagulase (sheep plasma) MSA: Mannitol salt agar DNase: Deoxyribonuclease A combination of MSA and DNase improves the tube PCR: Polymerase Chain Reaction coagulase test We then evaluated the sensitivity and specificity of test that sheep plasma will replace human plasma for routine combinations (i.e. MSA/DNase/TCT) at detecting Sta- TCTs. Our data is in agreement with previous findings phylococcus aureus, aiming at improving the perfor- in which sensitivity of 94-100% was reported by other mance of the TCT. We achieved absolute specificity investigators [28-30]. However, in this study, the sensi- (100%) in detecting Staphylococcus aureus with test tivity for human and sheep plasma was higher than combinations, with the exception of the DNase/MSA what was reported in other settings, where values as low combination (92% specificity). Conversely, the sensitivity as 54.5% (human plasma) and 45.5% (sheep plasma) of test combinations varied, with the MSA/DNase/TCT were reported [28]. Therefore, with coagulase tests, (human plasma) being the most sensitive (75% sensitiv- plasma performance varies with setting and the choice ity), while MSA/DNase/TCT (sheep plasma) was the of plasma can influence the efficiency of the test. Also, least sensitive (25% sensitivity), table 3. the use of EDTA or citrate as anticoagulant influences A combination of MSA/DNase resulted in specificity the performance of the test. Noteworthy, citrate only and sensitivity of 92% and 96%, respectively, and this affects coagulase reactions of enterococci where it is uti- would be the combination of choice for identification of lized [16], but does not affect coagulase producing Staphylococcus aureus. However, since both tests are organisms such as Staphylococcus aureus [16]. not specific to Staphylococcus aureus and can detect The DNase test had a sensitivity of 75% and a specifi- other bacterial isolates, the dual combination can only city of 96%, which are comparable to other studies in be used to improve the TCT. Although other test com- which a sensitivity of 75% and a specificity of 100% were binations-MSA/DNase/TCT (human plasma) and MSA/ reported [31]. In this study, the DNase test was the TCT (human plasma) had specificity of 100%, they had most specific of all tests and had the least number of varying sensitivity (75% and 33%, respectively). In this false positive isolates (there was only one false positive). line, a combination of MSA/DNase/TCT (human This was in agreement with other reports, in which the plasma) is better at identifying Staphylococcus aureus DNase test was reported as superior to the TCT [28,31]. (100% specificity, 75% sensitivity) than the MSA/TCT Growth on MSA was also highly specific (79% specifi- (human plasma) combination (100% specificity, 33% sen- city), while the least specific test was the tube coagulase sitivity). Likewise, the DNase/TCT (sheep plasma) and (human and sheep plasma, 11% and 7% specificity, MSA/TCT (sheep plasma) and MSA/DNase/TCT respectively). (sheep plasma) combinations had an absolute specificity In contrast, other studies reported high specificity (100%) but with varying sensitivity (50%, 25% and 67%, (93.6%) for human and sheep plasma with TCTs [32]. respectively). The low specificity in our study may partly be due to Thus, the efficiency of the tube coagulase can be the non specific detection of other coagulase positive improved through simultaneous testing that includes Kateete et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:23 Page 6 of 7 http://www.ann-clinmicrob.com/content/9/1/23 both DNase and MSA. For higher sensitivity and specifi- DNase and Tube coagulase) in settings where rabbit or city, sequel identification of Staphylococcus aureus may horse plasmas are not readily available. Screening of commencewithMSA,DNase and then TCT.Note- plasmas from other species (such as rabbit, goat, pig, worthy, the improved specificity of the TCT did not sig- and bovine), which can be cheaper and safer, and nificantly alter the initially observed high sensitivity. The reported to be more efficient than human plasma with useofMSA priortoTCTs/DNaseishighlyrecom- TCTs, is recommended. In addition, genotyping of clini- mended due to the clumping factor negative and tube cal staphylococcal isolates is recommended to speciate coagulase positive staphylococci that are increasingly CoNS isolates, and to determine the prevalence of rare being recovered from human infections. These isolates Staphylococcus aureus and animal isolates in human also produce a heat stable DNase and can be misidenti- specimens in Uganda. fied as Staphylococcus aureus.However,these strains can be differentiated from Staphylococcus aureus by Conclusions their failure to produce acid from maltose, lactose and We have evaluated the performance of common labora- mannitol. Furthermore, rare strains of Staphylococcus tory tests used routinely in the identification of Staphy- aureus can be coagulase negative [9], yet animal isolates lococcus aureus infections in Uganda. The identification (S. intermidius, S. hyicus, S. delphini and S. schleiferi of clinical Staphylococcus aureus still largely relies on subsp. coagulans) can be tube coagulase positive the tube coagulase test, but it requires screening of the [9,33,34]; differentiation of these also requires growth isolates with two additional tests (MSA and DNase) on MSA. prior to TCTs, for improved efficiency. There is no sin- gle phenotypic test (including the tube coagulase test) Predictive values that can guarantee reliable results in the identification Growth on MSA had the highest negative predictive of Staphylococcus aureus. value (NPV) followed by DNase, while tube coagulase The specificity of sheep plasma was relatively low had the lowest NPV (which did not match the high sen- (even in combination with MSA/DNase), and may not sitivity initially observed, see table 3). Conversely, test be appropriate for the TCT in some settings. Thus, combinations gave absolute NPVs (100%) with the where rabbit or horse plasmas are unavailable, sheep exception of DNase/MSA (NPV of 92%), table 3. The plasma may not replace human plasma. Since human high NPVs of the test combinations, particularly those plasma is inappropriate or risky in some settings, the involving the TCT reveal that test combinations can be performance of plasmas from other animal species reliably used in routine identification of Staphylococcus should be investigated for a replacement of human aureus. DNase had the highest positive predictive value plasma in TCTs. (PPV), followed by MSA (96% and 83%, respectively). Conversely, tube coagulase alone resulted in average Abbreviations PPVs (54% and 50%), see table 3. Interestingly, test com- ATCC: American Type Culture Collection; CoNS: Coagulase Negative binations resulted in high PPVs, with those involving Staphylococci; CRF: Coagulase Reacting Factor; DNase: Deoxyribonuclease; EDTA: Ethylenediaminetetraacetic acid; HCl: Hydrochloric acid; HIV/AIDS: DNase having the highest PPVs (95%), see table 3. Thus, Human immunodeficiency virus/Acquired immunodeficiency syndrome; LB: as for NPVs, the specificity of the TCT can be improved Luria-Bertani; MRSA: Methicillin Resistant Staphylococcus aureus; MSA: by a simultaneous identification of the isolates with Mannital salt agar; nuc: thermonuclease gene; PCR: polymerase chain reaction; PPV: Positive Predictive Value; NPV: Negative Predictive Value; STC: DNase and MSA. Slide Coagulase Test; TCT: Tube Coagulase Test; TS: Tryptic soy; TAE: Tris Therefore, the ideal identification of Staphylococcus Acetate-EDTA aureus clinical isolates requires a battery of tests. Sta- Acknowledgements phylococcus aureus infections are more frequent than FCN and CNK thank the technical staff at the clinical microbiology laboratory those by other bacteria, particularly in settings with high (Makerere University College of Heath Sciences) for helping with microbial HIV/AIDS prevalence [35-37]. This warrants correct cultures, and Ms Geraldine Nalwadda for administrative assistance. This study obtained funding from Sida-Sarec (to FCN) through Makerere identification of the isolates to achieve better treatment University School of Graduate Studies. DPK was supported by the Fogarty outcomes. Since options for improving the sensitivity International Center training support (award #U2RTW006879) through and specificity are presented, these data will improve on ICHORTA-Clinical Operational & Health Services Research (COHRE) Training program at the Joint Clinical Research Center, Kampala, Uganda. the identification of Staphylococcal aureus in clinical specimens. Author details Department of Medical Microbiology, School of Biomedical Sciences, Makerere University College of Health Sciences, Upper Mulago hill road, Recommendations Kampala, Uganda. Department of Veterinary Parasitology and Microbiology, For routine identification of Staphylococcus aureus from Faculty of Veterinary Medicine, Makerere University, Kampala, Uganda. clinical specimens, we recommend simultaneous use of Current Address: P.O. BOX 25085, 00603, Nairobi, Kenya. all the three tests (beginning with growth on MSA, Kateete et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:23 Page 7 of 7 http://www.ann-clinmicrob.com/content/9/1/23 with a negative deoxyribonuclease (DNase) test-implications for Authors’ contributions laboratory diagnosis and infection control. J Hos Infec 2002, 51(3):238-239. FCN conceived and supervised the study. DPK wrote the manuscript and co- 19. Martinez OV, Cleary T, Baker M, Civetta J: Evaluation of a mannitol-salt- supervised the study. CNK performed the experiments in partial fulfillment oxacillin-tellurite medium for the isolation of methicillin-resistant for his BSC degree. The other authors provided intellectual support to CNK Staphylococcus aureus from contaminated sources. Diagn Microbiol Infec and contributed to the writing of the manuscript. All authors read and Dis 15(3):207-211. approved the final manuscript. 20. Merlino J, Gill R, Robertson GJ: Application of lipovitellin-salt-mannitol agar for screening, isolation, and presumptive identification of Competing interests Staphylococcus aureus in a teaching hospital. J Clin Microbiol 1996, The authors declare that they have no competing interests. 34(12):3012-3015. 21. 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Philadelphia: Lippincott- 31. Lagace-Wiens PRS, Alfa MJ, Manickam K, Karlowsky JA: Thermostable RavenKoneman EW , 5 1997, 551-576. DNase Is Superior to Tube Coagulase for Direct Detection of 10. McDonald CL, Chapin K: Rapid identification of Staphylococcus aureus Staphylococcus aureus in Positive Blood Cultures. J Clin Microbiol 2007, from blood culture bottles by a classic 2-hour tube coagulase test. J Clin 45(10):3478-3479. Microbiol 1995, 33(1):50-52. 32. Stevens M, Geary C: Comparative evaluation of a latex test for the 11. Woo PCY, Leung ASP, Leung KW, Yuen KY: Identification of slide identification of Staphylococcus aureus. Eur J Clin Microbiol Infec Dis 1989, coagulase positive, tube coagulase negative Staphylococcus aureus by 8(2):153-156. 16S ribosomal RNA gene sequencing. Mol Path 2001, 54:244-247. 33. Varaldo PE, Kilpper-Balz R, Biavasco F, Satta G, Schleifer KH: Staphylococcus 12. Freney J, Brun Y, Bes M, Meugnier H, Grimont F, Grimont PAD, Nervi C, delphini sp. nov., a Coagulase-Positive Species Isolated from Dolphins. Int Fleurette J: Staphylococcus lugdunensis sp. nov. and Staphylococcus J Syst Bacteriol 1988, 38(4):436-439. schleiferi sp. nov., Two Species from Human Clinical Specimens. Int J Syst 34. Vandenesch F, Lebeau C, Bes M, Lina G, Lina B, Greenland T, Benito Y, Bacteriol 1988, 38(2):168-172. Brun Y, Fleurette J, Etienne J: Clotting activity in Staphylococcus schleiferi 13. Kawamura Y, Hou X-G, Sultana F, Hirose K, Miyake M, Shu S-E, Ezaki T: subspecies from human patients. J Clin Microbiol 1994, 32(2):388-392. Distribution of Staphylococcus Species among Human Clinical 35. Nathoo KJ, Chigonde S, Nhembe M, Ali MH, Mason PR: Community- Specimens and Emended Description of Staphylococcus caprae. J Clin acquired Bacteremia in Human Immunodeficiency Virus-infected Microbiol 1998, 36(7):2038-2042. Children in Harare, Zimbabwe. Ped Infec Dis J 1996, 15(12):1092-1097. 14. Shittu A, Lin J, Morrison D, Kolawole D: Identification and molecular 36. Crum-Cianflone NF, Burgi AA, Hale BR: Increasing rates of community- characterization of mannitol salt positive, coagulase-negative acquired methicillin-resistant Staphylococcus aureus infections among staphylococci from nasal samples of medical personnel and students. J HIV-infected persons. Int J STD AIDS 2007, 18(8):521-526. Med Microbiol 2006, 55(Pt 3):317-324. 37. Nguyen MH, Kauffman CA, Goodman RP, Squier C, Arbeit RD, Singh N, M 15. Shittu A, Lin J, Morrison D: Molecular identification and characterization Wagener M, Yu VL: Nasal Carriage of and Infection with Staphylococcus of mannitol-negative methicillin-resistant Staphylococcus aureus. Diagn aureus in HIV-Infected Patients. Annal Internal Med 1999, 130(3):221-225. Microbiol Infec Dis 2007, 57(1):93-95. 16. Bayliss BG, Hall ER: Plasma Coagulation by Organisms Other Than doi:10.1186/1476-0711-9-23 Staphylococcus aureus. J Bacteriol 1965, 89(1):101-105. Cite this article as: Kateete et al.: Identification of Staphylococcus aureus: 17. National Standard Method Coagulase SOP, UK. [http://www.hpa- DNase and Mannitol salt agar improve the efficiency of the tube standardmethods.org.uk/]. coagulase test. Annals of Clinical Microbiology and Antimicrobials 2010 9:23. 18. Rao JG, Qamruddin AO, Hassan IA, Burnie JP, Ganner M: Cluster of clinical isolates of epidemic methicillin-resistant Staphylococcus aureus (EMRSA) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Clinical Microbiology and Antimicrobials Springer Journals

Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of the tube coagulase test

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Springer Journals
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Copyright © 2010 by Kateete et al; licensee BioMed Central Ltd.
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Biomedicine; Medical Microbiology; Infectious Diseases
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1476-0711
DOI
10.1186/1476-0711-9-23
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20707914
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Abstract

Background: The ideal identification of Staphylococcus aureus clinical isolates requires a battery of tests and this is costly in resource limited settings. In many developing countries, the tube coagulase test is usually confirmatory for S. aureus and is routinely done using either human or sheep plasma. This study evaluated Mannitol salt agar and the deoxyribonuclease (DNase) test for improving the efficiency of the tube coagulase test in resource limited settings. The efficiency of human and sheep plasma with tube coagulase tests was also evaluated. Methods: One hundred and eighty Gram positive, Catalase positive cocci occurring in pairs, short chains or clusters were subjected to growth on Mannitol salt agar, deoxyribonuclease and tube coagulase tests. Of these, isolates that were positive for at least two of the three tests (n = 60) were used to evaluate the performance of the tube coagulase test for identification of S. aureus, using PCR-amplification of the nuc gene as a gold standard. Results: Human plasma was more sensitive than sheep plasma for the tube coagulase test (sensitivity of 91% vs. 81% respectively), but both plasmas had very low specificity (11% and 7% respectively). The sensitivity and specificity of the tube coagulase test (human plasma) was markedly improved when Mannitol salt agar and DNase were introduced as a tri-combination test for routine identification of Staphylococcus aureus (100% specificity and 75% sensitivity). The specificity and sensitivity of Mannitol salt agar/DNase/tube coagulase (sheep plasma) combination was 100% and 67%, respectively. Conclusion: The efficiency of the tube coagulase test can be markedly improved by sequel testing of the isolates with Mannitol salt agar, DNase and Tube coagulase. There is no single phenotypic test (including tube coagulase) that can guarantee reliable results in the identification of Staphylococcus aureus. Background are the mainstay in the diagnosis of staphylococcal Staphylococcus aureus is a ubiquitous commensal bac- infections, in which coagulase tests are usually confirma- terium on human skins and anterior nares, but fre- tory for S. aureus [4-8]. Coagulase testing is performed quently causes severe infections in humans [1]. Rapid using the slide coagulase (SCT) or the tube coagulase and direct identification of S. aureus is crucial for (TCT) methods [9]. Although these tests efficiently proper management of patients with skin infections, identify S. aureus, their performances vary from setting abscesses, septicemia/bacteremia, gastroenteritis, endo- to setting and need improvement [3,6]. carditis, toxic shock syndrome and certain food intoxi- Several laboratories in developing countries screen for cations [2,3]. In developing countries, phenotypic tests presumptive Staphylococcus aureus based on growth on Mannitol salt agar (MSA) and/or DNase tests and confir- mation is done with the TCT. In many settings, the use * Correspondence: najjuka2002@yahoo.com † Contributed equally of the TCT is curtailed by reliance on human plasma, Department of Medical Microbiology, School of Biomedical Sciences, since the recommended plasmas (from rabbit, horse [10]) Makerere University College of Health Sciences, Upper Mulago hill road, are either expensive or if locally available, are of poor Kampala, Uganda Full list of author information is available at the end of the article © 2010 Kateete et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Kateete et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:23 Page 2 of 7 http://www.ann-clinmicrob.com/content/9/1/23 quality. Human plasma is reported to give discordant Medical Microbiology, Makerere University College of results [6]; usually obtained from blood banks as outdated Health Sciences, from January through June, 2009. It materials, it contains variable amounts of CRF (Coagu- was a laboratory based study that involved frozen clini- lase-Reacting Factor) and anti-staphylococcal antibodies cal isolates from patients’ blood, cerebral spinal fluid, [9]. This type of plasma is not recommended for coagu- anterior nares, skin and wound swabs. lase tests [9]. Other factors which make human plasma inappropriate for the coagulase tests include; a high bur- Inclusion/exclusion criteria den of viral infections (such as HIV/AIDS, Hepatitis B One hundred and eighty Gram positive, Catalase posi- and C) in resource limited settings that can render the tive cocci occurring in pairs, short chains or clusters plasma risky to laboratory workers and prior to use, it were subjected to growth on MSA, tube coagulase and must be screened for safety. There are also ethical issues DNase tests (see below). Of these (N = 180), isolates accompanying the use of human specimens. Conversely, that were positive for at least two of the three tests the efficiency of other plasmas in many laboratory set- (n = 60) were used to evaluate the performance of the tings varies and has to be determined for proper identifi- tube coagulase test for identification of S. aureus,using cation of S. aureus with coagulase tests. PCR-amplification of the nuc gene (which is specific for Although coagulase tests are invaluable for identification S. aureus) as the gold standard. of Staphylococcus aureus, few studies have evaluated their use in routine practice [10]. In addition, diagnostic labora- Phenotypic identification of Staphylococcus aureus tories are occasionally faced with organisms with biochem- Isolates were incubated at 37°C for 24 hours on blood ical characteristics that do not fit into the patterns of a agar and then sub-cultured on TS (Tryptic Soya, Liofil- known genus and species [11]. Furthermore, there are chem, Italy) agar (Fisher, Leicestershire, UK). Single some problems associated with coagulase tests; firstly, colonies were tested with tube coagulase and DNase test some human CoNS (Coagulase Negative Staphylococci) andgrowthonMSA.Toevaluatethe performanceof produce clumping factor and may be falsely positive with the individual tests or a combination of tests, sequel the SCT [9,12]. Secondly, some staphylococci of animal testing of the isolates was done beginning with MSA, origin are clumping factor negative and tube coagulase followed by DNase and TCT. positive; these may be misidentified as S. aureus,unless To confirm fermentation of mannitol, growth of yel- the fermentation of Mannitol is utilized in addition [9]. low colonies on MSA (Oxoid, Cambridge, UK) sur- Whereas Mannitol salt agar was developed for the pre- rounded by yellow zones after 24 hours of incubation at sumptive isolation of S. aureus in a single step, which is 37°C indicated a positive result. DNase test was per- convenient for diagnostic laboratories, Mannitol positive formed by incubating the isolates for 24 hours at 37°C CoNS have been reported in nasal and clinical specimens on DNase agar (Scharlau, Barcelona, Spain), and pouring from Nigeria and Japan [13,14]. Furthermore, Mannitol an excess (~15 ml) of 1 N HCl. Excess acid was negative MRSA (Methicilin Resistant Staphylococcus aur- removed with a vacuum pipette and clear zones around eus) was reported from clinical specimens in Kwazulu the bacterial colonies indicated DNase positive colonies. Natal province, South Africa [15]. In view of the above, For tube coagulase tests, colonies of test isolates were the common identification methods for S. aureus were re-suspended in 2 ml of citrated sheep or human plasma evaluated, aiming at improving the diagnosis of S. aureus in sterile glass test-tubes. Since citrate is utilized by through a combination of available phenotypic methods. enterococci [16], pure colonies of Gram positive, Cata- This study reveals that there is no single test (includ- lase positive staphylococci (catalase tests preceded coa- ing the tube coagulase test) that can guarantee reliable gulase reactions) were used. Positive control tubes with results for the identification of S. aureus. However, citrated plasma and coagulase producing strain of improved diagnostic sensitivity and specificity of the S. aureus ATCC 25923 (which efficiently coagulates tube coagulase test were achieved upon simultaneous citrated plasma) were included. To rule out citrate utili- testing of the isolates with DNase and Mannital salt zation by other microorganisms, control TCTs contain- agar. In order to improve the identification of S. aureus ing citrated plasma with Staphylococcus epidermidis in resource limited settings, sequel testing of the isolates ATCC 12228 were included. In addition, negative con- with Mannitol salt agar, tube DNase and coagulase is trol tubes containing citrated plasma alone (with no cul- proposed. tures inoculated) were included. The tubes were incubated at 35°C for 4 hours and observed for clot for- Methods mation. Where clotting did not occur, the tubes were Study setting incubated at room temperature for an additional 18 This study was done in the Clinical Microbiology and hours [9]. Tubes were studied without agitation in order Molecular Biology laboratories of the Department of not to disrupt partially formed clots. Kateete et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:23 Page 3 of 7 http://www.ann-clinmicrob.com/content/9/1/23 Molecular assays Table 1 Deriving Sensitivity, Specificity, Positive/Negative Predictive Values for the common identification tests for PCR-amplification of the nuc gene was used as a baseline S. aureus test. Reaction mixes were done in a Cleanspot ultraviolet Test Staphylococcus Other Total workstation (COV Lab products, Michigan, USA). The results aureus staphylococci reactions contained 20 pmoles each of the nuc forward Positive a b a + b and reverse primers (5′-GCGATTGATGGTGATA Negative c d c + d CGGTT-3′ and 5′-AGCCAAGCCTTGACGAACTA Total a + c b + d (a + b) + (c + d) AAGC-3′, respectively, synthesized by Eurofins-MWG- =n Operon, Ebersberg, Germany), 1.5 units of Taq DNA Where, polymerase (Thermo-Fisher, Surry, UK) and 1 μlofcus- a = True positives tom PCR master mix (ThermoFisher, Surry, UK). In the b = False positives post-amplification room, ~100 ng of staphylococcal chro- c = False negatives mosomal DNA was added to the reactions as template. d = True negatives Diagnostic sensitivity = d/(b + d) Amplifications were done in a Peltier thermocycler (MJ Diagnostic specificity = a/(a + c) Research, Waterman, MA, USA) under the following Positive predictive value (PPV) = a/(a + b) conditions: initial denaturation at 94°C, 5 min, followed Negative predictive value (NPV) = d/(d + c) by 37 cycles each consisting of a denaturation at 94°C, n=60 1 min; primer annealing at 55°C, 0.5 min, and extension at 72°C, 1 min; followed by a final extension at 72°C, 7 minutes. After this, the amplicons were mixed with 5 μl production can be detected using either the slide coagu- of DNA loading buffer and electrophoresed in a 1% agar- lase test (SCT) or the tube coagulase test (TCT). Slide ose gel in TAE buffer (Tris, acetate and EDTA). Control coagulase detects bound coagulase (also called “clump- reactions included templates of Staphylococcus aureus ing factor”) [9], which reacts directly with fibrinogen in ATCC 25923 (positive control), Staphylococcus epidermi- plasma, causing rapid cell agglutination. Negative iso- dis ATCC 12228 and nuclease free water (negative lates following SCT require confirmation with the controls). superior TCT, since strains deficient in clumping factor usually produce free coagulase. Tube coagulase detects Quality control secreted extracellular free coagulase that reacts with a To minimize cross contamination, standard microbiolo- substance in plasma called “Coagulase-Reacting Factor” gical procedures were strictly followed. Positive and (CRF) to form a complex, which in turn reacts with negative controls were always included in the test reac- fibrinogen to form fibrin (the clot) [9]. Strains of coagu- tions. DNA extraction and PCR-amplifications were lase-positive-animal staphylococci have been isolated done in molecular laboratories that are separate from from human clinical specimens, yet some animal staphy- the clinical microbiology laboratory where cultures were lococcal isolates also ferment mannitol [13]. This study grown. The PCR laboratory has designated sections for evaluated the performance of TCTs, DNase and MSA, pre-amplification, amplification and post-amplification, the phenotypic methods commonly used in the identifi- with a unidirectional movement of staff. cation of Staphylococcus aureus. Data analysis Detection of Staphylococcus aureus with the available The data were analyzed using a 2 × 2 contingency table for phenotypic tests diagnostic specificity and sensitivity (table 1). Diagnostic PCR-amplification of the nuc gene, which was used as a sensitivities and specificities were calculated as follows: baseline test, detected 32 Staphylococcus aureus of the Sensitivity (%) = [True positive/(True Positive + False 60 staphylococcal isolates, see table 2. Of the 32 nuc- Positive)] × 100 positive Staphylococcus aureus isolates, MSA detected Specificity (%) = [True Negative/(True Negative + the highest number of isolates (30 of 32, 94%) while the False Negative)] × 100 TCT (human and sheep plasma respectively) detected The positive predictive value (PPV) (%) = [True Posi- 29 and 27 of the 32 isolates (91% and 84% respectively), tive/(True Positive + False Positive)] × 100 table 2. DNase detected the least number of isolates Thenegativepredictivevalue (NPV)(%) =[True (24 of 32, 75%). Negative/(True Negative + False Negative)] × 100. Nine of the 32 nuc-positive Staphylococcus aureus iso- lates (28%) were coagulase negative, implying that some Results and discussion isolates may be misidentified by the TCT as a sole test. Coagulasetesting is thesinglemostreliablemethodfor The coagulase negative Staphylococcus aureus may prob- identifying Staphylococcus aureus [9]. Coagulase ably be MRSA isolates which are reported to react weakly Kateete et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:23 Page 4 of 7 http://www.ann-clinmicrob.com/content/9/1/23 Table 2 Identification of S. aureus with the common tests in comparison to PCR-detection of the nuc gene Other tests nuc PCR Outcome Positive (% of 32) Negative (% of 28) Subtotal MSA Positive 30 (94) 6 (21) 36 Negative 2 (6) 22 (79) 24 DNase Positive 24 (75) 1 (4) 25 Negative 8 (25) 27 (96) 35 Human plasma Positive 29 (91) 25 (89) 54 Negative 3 (9) 3 (11) 6 Human plasma/MSA Positive 27 (84) 6 (21) 31 Negative 0 3 (11) 3 Human plasma/DNase Positive 21 (60) 1 (4) 22 Negative 0 3 (11) 3 Human plasma/MSA/DNase Positive 21 (60) 1 (4) 22 Negative 0 3 (11) 3 Sheep plasma Positive 26 (81) 26 (93) 52 Negative 6 (19) 2 (8) 8 Sheep plasma/MSA Positive 24 (75) 6 (21) 30 Negative 0 2 (8) 2 Sheep plasma/DNase Positive 18 (56) 1 (4) 19 Negative 0 1 (4) 1 Sheep plasma/MSA/DNase Positive 18 (56) 1 (4) 19 Negative 0 2 (8) 2 MSA/DNase Positive 24 (74) 1 (4) 25 Negative 2 (6) 22 (79) 24 : tube coagulase (human plasma) : tube coagulase (sheep plasma) MSA: Mannitol salt agar DNase: Deoxyribonuclease PCR: Polymerase Chain Reaction or negatively with TCTs [17], or rare Staphylococcus aur- aureus. Growth on MSA was the most sensitive (94% eus strains that are reported to be coagulase negative [9]. sensitivity), followed by the TCT (human and sheep Two Staphylococcus aureus isolates (6%) were also MSA plasma, 91% and 81% sensitivity respectively) while the negative. Shittu et al also reported mannitol negative Sta- DNase test was the least sensitive (75% sensitivity). Con- phylococcus aureus that was methicillin resistant [15]. versely, the DNase test was the most specific (96% speci- Furthermore, eight of the 32 nuc-positive Staphylococcus ficity) followed by MSA (79% specificity), while the TCT aureus isolates were DNase negative (25%). Rao et al (human plasma and sheep plasma) was the least specific reported DNase negative Staphylococcus aureus but with (11% and 7%, respectively), table 3. Overall, of the indi- no explanation for these findings [18]. Six staphylococci vidual tests studied, growth on MSA was the best at other than the nuc-confirmed Staphylococcus aureus iso- identifying Staphylococcus aureus (94% sensitivity and lates produced yellow colonies on MSA, and similar find- 79% specificity). Our results slightly differ from those of ings were reported by other investigators [19-24]. Han et al, who reported sensitivity and specificity of Another rare finding in this study was an isolate that was 76.5% and 99.6%, respectively, for MSA [25]. D’Souza DNasepositivebut MSA-negative andtubecoagulase- et al, also reported sensitivity of 71%, a little lower than negative (i.e. non-Staphylococcus aureus). We presume ours [26]. The high sensitivity of MSA in detecting Sta- this isolate could have been Staphylococcus schleiferi phylococcus aureus could be due to its ability to isolate subsp. coagulans, which according to the National Stan- pathogenic Staphylococcus aureus [27], differentiating dard Method is also DNase positive [9]. coagulase negative staphylococci from coagulase positive staphylococci. Single phenotypic tests are inefficient for the For tube coagulase, human plasma was more sensitive identification of Staphylococcus aureus than sheep plasma (91% vs. 81% sensitivity), implying We used sensitivity and specificity to evaluate the per- that using sheep plasma with TCTs may detect more formance of individual tests in detecting Staphylococcus false negative isolates. Thus, in this setting, it is unlikely Kateete et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:23 Page 5 of 7 http://www.ann-clinmicrob.com/content/9/1/23 Table 3 Sensitivity, Specificity, Negative/Positive staphylococci, such as Staphylococcus schleiferi subspe- Predictive values for the common diagnostic tests for cies coagulans, Staphylococcus delphini, Staphylococcus clinical S. aureus intermedius and Staphylococcus hyicus. Staphylococcus Test Sensitivity Specificity NPV PPV delphini and Staphylococcus intermedius are rare clinical (%) (%) (%) (%) isolates while Staphylococcus hyicus is indeterminate MSA 94 79 92 83 (with prevalence ranging from 11% to 89% [17]). The DNase 75 96 77 96 prevalence of these isolates may be high in certain set- Human plasma 91 11 33 54 tings. Although MSA and DNase had high specificities Sheep plasma 81 7 25 50 and sensitivities, as individual tests, their use in routine MSA/DNase 96 92 92 96 identification of Staphylococcus aureus is curtailed by Human plasma/MSA 33 100 100 82 their ability to detect other bacterial isolates [14], and Human plasma/DNase 75 100 100 95 are mostly used during initial screens [6]. Indeed, Man- Human plasma/DNase/ 75 100 100 94 nitol salt positive CoNS (Staphylococcus caprae, MSA S. hemolyticus and S. saprophyticus) have been reported Sheep plasma/MSA 25 100 100 80 in Nigeria and Japan [13,14]. Thus, in certain settings, if Sheep plasma/DNase 50 100 100 95 used individually to identify Staphylococcus aureus, Sheep plasma/MSA/ 67 100 100 95 common phenotypic tests may be insufficient; some iso- DNase lates will be misidentified, either as Staphylococcus aur- : Tube coagulase (human plasma) 2 eus or CoNS. : Tube coagulase (sheep plasma) MSA: Mannitol salt agar DNase: Deoxyribonuclease A combination of MSA and DNase improves the tube PCR: Polymerase Chain Reaction coagulase test We then evaluated the sensitivity and specificity of test that sheep plasma will replace human plasma for routine combinations (i.e. MSA/DNase/TCT) at detecting Sta- TCTs. Our data is in agreement with previous findings phylococcus aureus, aiming at improving the perfor- in which sensitivity of 94-100% was reported by other mance of the TCT. We achieved absolute specificity investigators [28-30]. However, in this study, the sensi- (100%) in detecting Staphylococcus aureus with test tivity for human and sheep plasma was higher than combinations, with the exception of the DNase/MSA what was reported in other settings, where values as low combination (92% specificity). Conversely, the sensitivity as 54.5% (human plasma) and 45.5% (sheep plasma) of test combinations varied, with the MSA/DNase/TCT were reported [28]. Therefore, with coagulase tests, (human plasma) being the most sensitive (75% sensitiv- plasma performance varies with setting and the choice ity), while MSA/DNase/TCT (sheep plasma) was the of plasma can influence the efficiency of the test. Also, least sensitive (25% sensitivity), table 3. the use of EDTA or citrate as anticoagulant influences A combination of MSA/DNase resulted in specificity the performance of the test. Noteworthy, citrate only and sensitivity of 92% and 96%, respectively, and this affects coagulase reactions of enterococci where it is uti- would be the combination of choice for identification of lized [16], but does not affect coagulase producing Staphylococcus aureus. However, since both tests are organisms such as Staphylococcus aureus [16]. not specific to Staphylococcus aureus and can detect The DNase test had a sensitivity of 75% and a specifi- other bacterial isolates, the dual combination can only city of 96%, which are comparable to other studies in be used to improve the TCT. Although other test com- which a sensitivity of 75% and a specificity of 100% were binations-MSA/DNase/TCT (human plasma) and MSA/ reported [31]. In this study, the DNase test was the TCT (human plasma) had specificity of 100%, they had most specific of all tests and had the least number of varying sensitivity (75% and 33%, respectively). In this false positive isolates (there was only one false positive). line, a combination of MSA/DNase/TCT (human This was in agreement with other reports, in which the plasma) is better at identifying Staphylococcus aureus DNase test was reported as superior to the TCT [28,31]. (100% specificity, 75% sensitivity) than the MSA/TCT Growth on MSA was also highly specific (79% specifi- (human plasma) combination (100% specificity, 33% sen- city), while the least specific test was the tube coagulase sitivity). Likewise, the DNase/TCT (sheep plasma) and (human and sheep plasma, 11% and 7% specificity, MSA/TCT (sheep plasma) and MSA/DNase/TCT respectively). (sheep plasma) combinations had an absolute specificity In contrast, other studies reported high specificity (100%) but with varying sensitivity (50%, 25% and 67%, (93.6%) for human and sheep plasma with TCTs [32]. respectively). The low specificity in our study may partly be due to Thus, the efficiency of the tube coagulase can be the non specific detection of other coagulase positive improved through simultaneous testing that includes Kateete et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:23 Page 6 of 7 http://www.ann-clinmicrob.com/content/9/1/23 both DNase and MSA. For higher sensitivity and specifi- DNase and Tube coagulase) in settings where rabbit or city, sequel identification of Staphylococcus aureus may horse plasmas are not readily available. Screening of commencewithMSA,DNase and then TCT.Note- plasmas from other species (such as rabbit, goat, pig, worthy, the improved specificity of the TCT did not sig- and bovine), which can be cheaper and safer, and nificantly alter the initially observed high sensitivity. The reported to be more efficient than human plasma with useofMSA priortoTCTs/DNaseishighlyrecom- TCTs, is recommended. In addition, genotyping of clini- mended due to the clumping factor negative and tube cal staphylococcal isolates is recommended to speciate coagulase positive staphylococci that are increasingly CoNS isolates, and to determine the prevalence of rare being recovered from human infections. These isolates Staphylococcus aureus and animal isolates in human also produce a heat stable DNase and can be misidenti- specimens in Uganda. fied as Staphylococcus aureus.However,these strains can be differentiated from Staphylococcus aureus by Conclusions their failure to produce acid from maltose, lactose and We have evaluated the performance of common labora- mannitol. Furthermore, rare strains of Staphylococcus tory tests used routinely in the identification of Staphy- aureus can be coagulase negative [9], yet animal isolates lococcus aureus infections in Uganda. The identification (S. intermidius, S. hyicus, S. delphini and S. schleiferi of clinical Staphylococcus aureus still largely relies on subsp. coagulans) can be tube coagulase positive the tube coagulase test, but it requires screening of the [9,33,34]; differentiation of these also requires growth isolates with two additional tests (MSA and DNase) on MSA. prior to TCTs, for improved efficiency. There is no sin- gle phenotypic test (including the tube coagulase test) Predictive values that can guarantee reliable results in the identification Growth on MSA had the highest negative predictive of Staphylococcus aureus. value (NPV) followed by DNase, while tube coagulase The specificity of sheep plasma was relatively low had the lowest NPV (which did not match the high sen- (even in combination with MSA/DNase), and may not sitivity initially observed, see table 3). Conversely, test be appropriate for the TCT in some settings. Thus, combinations gave absolute NPVs (100%) with the where rabbit or horse plasmas are unavailable, sheep exception of DNase/MSA (NPV of 92%), table 3. The plasma may not replace human plasma. Since human high NPVs of the test combinations, particularly those plasma is inappropriate or risky in some settings, the involving the TCT reveal that test combinations can be performance of plasmas from other animal species reliably used in routine identification of Staphylococcus should be investigated for a replacement of human aureus. DNase had the highest positive predictive value plasma in TCTs. (PPV), followed by MSA (96% and 83%, respectively). Conversely, tube coagulase alone resulted in average Abbreviations PPVs (54% and 50%), see table 3. Interestingly, test com- ATCC: American Type Culture Collection; CoNS: Coagulase Negative binations resulted in high PPVs, with those involving Staphylococci; CRF: Coagulase Reacting Factor; DNase: Deoxyribonuclease; EDTA: Ethylenediaminetetraacetic acid; HCl: Hydrochloric acid; HIV/AIDS: DNase having the highest PPVs (95%), see table 3. Thus, Human immunodeficiency virus/Acquired immunodeficiency syndrome; LB: as for NPVs, the specificity of the TCT can be improved Luria-Bertani; MRSA: Methicillin Resistant Staphylococcus aureus; MSA: by a simultaneous identification of the isolates with Mannital salt agar; nuc: thermonuclease gene; PCR: polymerase chain reaction; PPV: Positive Predictive Value; NPV: Negative Predictive Value; STC: DNase and MSA. Slide Coagulase Test; TCT: Tube Coagulase Test; TS: Tryptic soy; TAE: Tris Therefore, the ideal identification of Staphylococcus Acetate-EDTA aureus clinical isolates requires a battery of tests. Sta- Acknowledgements phylococcus aureus infections are more frequent than FCN and CNK thank the technical staff at the clinical microbiology laboratory those by other bacteria, particularly in settings with high (Makerere University College of Heath Sciences) for helping with microbial HIV/AIDS prevalence [35-37]. This warrants correct cultures, and Ms Geraldine Nalwadda for administrative assistance. This study obtained funding from Sida-Sarec (to FCN) through Makerere identification of the isolates to achieve better treatment University School of Graduate Studies. DPK was supported by the Fogarty outcomes. Since options for improving the sensitivity International Center training support (award #U2RTW006879) through and specificity are presented, these data will improve on ICHORTA-Clinical Operational & Health Services Research (COHRE) Training program at the Joint Clinical Research Center, Kampala, Uganda. the identification of Staphylococcal aureus in clinical specimens. Author details Department of Medical Microbiology, School of Biomedical Sciences, Makerere University College of Health Sciences, Upper Mulago hill road, Recommendations Kampala, Uganda. Department of Veterinary Parasitology and Microbiology, For routine identification of Staphylococcus aureus from Faculty of Veterinary Medicine, Makerere University, Kampala, Uganda. clinical specimens, we recommend simultaneous use of Current Address: P.O. BOX 25085, 00603, Nairobi, Kenya. all the three tests (beginning with growth on MSA, Kateete et al. Annals of Clinical Microbiology and Antimicrobials 2010, 9:23 Page 7 of 7 http://www.ann-clinmicrob.com/content/9/1/23 with a negative deoxyribonuclease (DNase) test-implications for Authors’ contributions laboratory diagnosis and infection control. J Hos Infec 2002, 51(3):238-239. FCN conceived and supervised the study. DPK wrote the manuscript and co- 19. 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Published: Aug 13, 2010

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