INTRODUCTION
Access to reliable diagnostic microbiologic testing is limited in austere environments, leading to potential misdiagnosis, inadequate treatment, increased mortality, and the inability to determine the prevalence and dynamics of disease etiology.1 A systematic review of community-acquired bloodstream infections in sub-Saharan Africa yielded mortality rates of 19% and 39% for sepsis and severe sepsis, respectively.2 Sepsis can lead to lifelong disability in many survivors, and the cost and complexity required for effective microbiologic testing can be a deterrent to implementation.3–6 Exacerbating these challenges in austere environments is the increasing degree of analytical specificity demanded by ever-increasing bacterial taxonomic refinements and technological sophistication.7 Laboratory analyses fall across a spectrum, from rapid testing that provides presumptive evidence for clinical decision making, to advanced analytical reference testing for confirmation and the advancement of medical sciences. There is a growing inequality between the emerging literature in clinical bacteriology, generated with technologically advanced instruments, and what can be implemented sustainably in austere environments. More research is needed to enable laboratories-in-development to provide quality microbiologic testing that has clinical utility and supports public health.
Most positive blood culture bottles require 1 to 3 days of incubation before microbial growth is detected using manual or automated continuous-monitoring instruments.8,9 Thereafter, current standard laboratory practice requires the recovery and preparation of a pure bacterial isolate at a standardized inoculum density for subsequent species-level identification and antimicrobial susceptibility testing (AST)— adding an additional 1 to 2 days of analysis. Such delays in turnaround time, together with the direct costs associated with blood cultures and analytical methods, create a barrier to implementation of blood culture testing at laboratories-in-development (personal observations).
Advanced technologies for identification and AST directly from positive blood culture bottles include matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, nucleic acid amplification tests, microfluidics, microcalorimetry, and next-generation sequencing.10 However, in addition to interpretive and analytical challenges, cost and infrastructure requirements prevent sustainable implementation. Rapid, potentially more sustainable, phenotypic culture-based approaches remain an attractive alternative. In the late 1970s and early 1980s, promising results were reported with the direct use of positive blood culture bottle broth for disk diffusion AST (direct disk diffusion [dDD]) as compared with standard disk diffusion (DD) using isolated colonies.11–15 More recently, dDD was found to compare favorably with newly introduced automated microbroth dilution instruments.16 Although concern about regulatory restrictions may have hampered widespread use of dDD in the absence of consensus guidelines, more recent studies confirming accuracy and reproducibility call for a reinvigoration of dDD studies.10,17–19
DD zone size interpretations of the Clinical Laboratory Standards Institute (CLSI) and the European Society Clinical Microbiology and Infectious Diseases identifies only seven groups of non-fastidious organisms for zone size interpretation: the Enterobacterales, Pseudomonas aeruginosa, Staphylococcus, Enterococcus, Acinetobacter, the Burkholderia cepacia complex, and Stenotrophomonas maltophilia. Within these groups, differentiating the coagulase-negative staphylococci from Staphylococcus aureus, and Salmonella and Shigella within the Enterobacterales is currently required for result interpretation.20,21 Globally and in sub-Saharan Africa, the most common bacterial causes of sepsis are Escherichia coli, Klebsiella, Salmonella, P. aeruginosa, Acinetobacter, S. aureus, beta-hemolytic Streptococcus, and Streptococcus pneumoniae.2,22,23 Limiting the identification of isolates to CLSI or European Society Clinical Microbiology and Infectious Diseases interpretive groups provides an opportunity for laboratories-in-development to introduce rapid abbreviated blood culture testing with meaningful clinical utility.
Chromogenic agar is an accurate, rapid, and cost-effective alternative media to traditional bacteriological culture media for isolation and differentiation of urinary tract pathogens E. coli, Klebsiella–Enterobacter–Serratia group, the Proteus–Providencia–Morganella group, P. aeruginosa, Acinetobacter, Staphylococcus, Enterococcus, and beta-hemolytic Streptococcus.24–29 In addition, Singh and Bhunia30 used optical laser technology for label-free, real-time, on-plate colony screening to differentiate clinically relevant pathogens accurately without the need for additional tests. The chromogens incorporated into chromogenic bacteriological media are typically analogs of naturally occurring di- and oligosaccharides, peptides, or esters. Such chromogens are comprised of two parts: the first is recognized by a transport system and a hydrolytic enzyme, often capable of operon induction; and a second molecule, the chromophore, when hydrolyzed leads to dimerization, chelation of metal ions, or electronic rearrangement, resulting in a shift in light absorption seen as a color change.31,32 Chromatic MH agar (Liofilchem, Roseto degli Abruzzi, Italy) is a non-selective, Mueller–Hinton-based agar incorporating a proprietary blend of chromogenic substrates that, when cleaved by microbial enzymes, result in color changes that correlate to clinically relevant pathogens. Chromatic MH agar has been used as substitute for traditional Mueller–Hinton agar for DD and gradient strip AST and performs well for the simultaneous isolation and direct AST of non-fastidious pathogens in urine,33 respiratory specimens,34 and vascular prosthetic graft specimens.35 Combined, these characteristics raise the possibility of using Chromatic MH as a solid agar medium for isolation, identification, and dDD of positive blood culture bottle broth with reduced turnaround time and a minimum of reagents, media, labor, and complexity, rendering this an attractive option in resource-limited settings.
The Austere Environments Consortium for Enhanced Sepsis Outcomes is engaged in observational studies of sepsis at Komfo Anokye Teaching Hospital (KATH), Kumasi, Ghana, where our collaboration is seeking to enable sustainable laboratory approaches that may be implemented in resource-constrained environments. To this end, we assessed the performance of Chromatic MH agar for dDD from contrived positive blood culture bottles and compared findings with current standard practice using subcultured isolates and the CLSI method. Furthermore, we characterized the growth of 24 different bacterial and 3 yeast species on Chromatic MH agar with the aid of rapid spot tests for same-day identification.
MATERIALS AND METHODS
Chromogenic culture characterization.
Twenty-four bacterial and three yeast reference strains were obtained from the American Type Culture Collection (ATCC) and are detailed in Table 1. Lyophilized strains were revived and sub-cultured serially twice before inoculation onto Chromatic MH agar plates. Plates were incubated at 37°C in room air or a candle jar (S. pneumoniae and Haemophilus influenzae) and evaluated for chromogenic reactions and colonial growth characteristics daily for up to 72 hours. For Erysipelothrix rhusiopathiae, Yersinia enterocolitica, and Pseudomonas fluorescens, plates were transferred from 37°C to room-temperature incubation after the initial 24 hours. Photos of agar plates were taken using a mobile phone. Rapid identification reagents were used on colonies taken directly from Chromatic MH to aid differentiation: catalase (3% hydrogen peroxide; LabChem Inc., Zelienople, PA), slide coagulase (CoaguStaph; Hardy Diagnostics, Santa Maria, CA); and the following in-house prepared reagents (Sigma-Aldrich, St. Louis, MO): rapid spot indole filter paper method using 1% 4-dimethylaminocinnamaldehyde, spot oxidase filter paper using Kovác’s 1% tetramethyl-p-phenylenediamine dihydrochloride solution, 0.02% L-pyrrolidonyl-β-naphthylamide visualized with 4-dimethylaminocinnamaldehyde, spot filter paper with 0.02% esculin visualized with 1% ferric ammonium citrate, and sulfur–indole–motility medium (Criterion; Hardy Diagnostics, Santa Maria, CA).36,37 Testing and quality control (QC) were performed according to York et al.38
Strains and isolates
| Number | Strains and isolates |
|---|---|
| 1 | Enterococcus faecalis ATCC 29212 |
| 2 | Staphylococcus aureus subsp. aureus ATCC 25923 |
| 3 | Staphylococcus epidermidis ATCC 12228 |
| 4 | Streptococcus agalactiae ATCC 12386 |
| 5 | Streptococcus pneumoniae ATCC 6305 |
| 6 | Streptococcus pyogenes ATCC 19615 |
| 7 | Erysipelothrix rhusiopathiae ATCC 19414 |
| 8 | Listeria monocytogenes ATCC 19115 |
| 9 | Citrobacter freundii ATCC 8090 |
| 10 | Cronobacter sakazakii ATCC 29544 |
| 11 | Escherichia coli ATCC 35218 |
| 12 | E. coli ATCC 25922 |
| 13 | Haemophilus influenzae ATCC 10211 |
| 14 | Klebsiella pneumoniae subsp. pneumoniae ATCC 13883 |
| 15 | Proteus mirabilis ATCC 12453 |
| 16 | Salmonella enterica Enteritidis ATCC 13076 |
| 17 | Salmonella enterica Typhimurium ATCC 14028 |
| 18 | Serratia marcescens ATCC 43861 |
| 19 | Yersinia enterocolitica subsp. enterocolitica ATCC 9610 |
| 20 | Acinetobacter baumannii ATCC 19606 |
| 21 | Burkholderia cepacia ATCC 25416 |
| 22 | Pseudomonas aeruginosa ATCC 27853 |
| 23 | Pseudomonas fluorescens ATCC 13525 |
| 24 | Stenotrophomonas maltophilia ATCC 13637 |
| 25 | Cryptococcus neoformans ATCC 32045 |
| 26 | Candida krusei ATCC 34135 |
| 27 | Candida albicans ATCC 10231 |
| Challenge isolates* | |
| 1 | S. aureus |
| 2 | Klebsiella aerogenes |
| 3, 4, 5 | K. pneumoniae (3 isolates) |
| 6 | Salmonella enteritidis |
| 7 | A. baumannii |
| 8, 9 | P. aeruginosa (2 isolates) |
| 10 | Vibrio parahaemolyticus |
ATCC = American Type Culture Collection.
Challenge isolates consisted of clinical isolates from Komfo Anokye Teaching Hospital and isolates from external proficiency surveys.
Standard AST.
We performed DD QC testing on Chromatic MH, Mueller–Hinton Agar (Thermo Scientific Oxoid; Thermo Fisher Scientific, Waltham, MA), and antimicrobial disks as described by CLSI using S. aureus ATCC 25923, E. coli ATCC 25922, E. coli ATCC 35218, and P. aeruginosa ATCC 27853.20 In addition, we subjected 10 challenge isolates (Table 1) to DD on both agar media.
Contrived blood culture-positive sample preparation.
Blood culture bottles (Bactec Plus Aerobic/F 30 mL; BD Diagnostics, Sparks, MD) were inoculated with approximately 15 colony forming units (CFU) of each of the 4 QC strains and the 10 challenge isolates. Briefly, a bacterial suspension approximating a 0.5 McFarland standard (1.5 × 108 CFU/mL) was prepared in normal saline from an overnight solid agar culture and diluted 1:100 by transferring 100 μL into 9.9 mL trypticase soy broth (BD Bacto; Becton, Dickinson and Company, Franklin Lakes, NJ). A further 2 × 1:100 serial dilution in trypticase soy broth was made to yield a final concentration of approximately 1.5 × 102 CFU/mL. From this, 0.1 mL was inoculated (approximately 15 CFU) with a tuberculin syringe into a blood culture bottle. A total of 8 mL of expired human blood collected in citrate–phosphate–dextrose–adenine obtained from the KATH blood bank was added to each bottle and incubated in either a Bactec 9240 and/or FX40 instrument (BD Diagnostics, Sparks, MD). Replicates were prepared on different days using two lots of blood culture bottles: 4 replicates of S. aureus ATCC 25923, 8 replicates of each of E. coli ATCC 25922 and E. coli ATCC 35218, and 10 replicates of P. aeruginosa ATCC 27853. A single preparation was made of each of the 10 challenge isolates. Blood bottles were removed and processed for Gram stain and culture within 6 hours of the instrument positive signal.
Direct disk diffusion susceptibility testing.
Antimicrobial susceptibility disks used (Table 2) were obtained from two manufacturers (BBL Sensi-Disc, BD Diagnostics, Sparks, MD; and Thermo Scientific Oxoid, Thermo Fisher Scientific, Waltham, MA). Briefly, 4 mL of broth was transferred from a positive blood culture bottle into a sterile Bijou bottle. From the Bijou bottle, Chromatic MH agar plates were inoculated using a sterile cotton-tipped swab rubbed in three directions while rotating the swab before antimicrobial disks were placed. Antimicrobial susceptibility agar plates were incubated in ambient air at 37°C for 18 to 20 hours before inhibition zone size measurements were taken with a clear ruler, against a black background with reflected light, from the top of the agar plate with lids removed. For the QC strains, the zone size diameters were compared with the acceptable QC ranges established for each specific antimicrobial disk and QC strain combination.20 Zone size measurements obtained from dDD onto Chromatic MH agar were compared with those obtained from the standard CLSI DD method using Mueller–Hinton agar.20
Antimicrobial susceptibility disk
| Antimicrobial agent and disk content | Abbreviation |
|---|---|
| Amoxicillin–clavulanate 20/10 μg | AMC |
| Ampicillin 10 μg | AMP |
| Azithromycin 15 μg | AZM |
| Cefazolin 30 μg | CZ |
| Cefoxitin 30 μg | FOX |
| Cefpodoxime 10 μg | CPD |
| Ceftazidime 30 μg | CAZ |
| Ceftriaxone 30 μg | CRO |
| Cefuroxime 30 μg | CXM |
| Chloramphenicol 30 μg | C |
| Ciprofloxacin 5 μg | CIP |
| Clindamycin 2 μg | CC |
| Erythromycin 15 μg | E |
| Gentamicin 10 μg | GM |
| Imipenem 10 μg | IMP |
| Meropenem 10 μg | MEM |
| Penicillin 10 U | P |
| Rifampicin 5 μg | RA |
| Tetracycline 30 μg | TE |
| Trimethoprim–sulfamethoxazole 1.25/23.75 μg | SXT |
| Vancomycin 30 μg | VA |
| Nitrofurantoin 300 μg | NIT |
Statistical analysis.
The reproducibility of dDD on Chromatic MH agar was assessed by comparing the mean, SD, coefficient of variation (CV), range, and 95% CI (Student’s t-test for small sample size, rounded to the nearest whole millimeter) of zone sizes obtained. The precision (reproducibility) categorical agreement was calculated as the number of categorical result matches/total number of results for all organisms and drugs combined.39 The categorical agreement (CA), very major error (VME) rate, major error rate, and minor error rate for dDD was determined by comparison to standard DD for the 10 challenge isolates and the 4 QC strains combined against all drugs tested. VME (false-susceptible) rates were calculated as number of results resistant by DD but susceptible by dDD/total number of resistant results. Major error (false-resistant) rates were calculated as number susceptible by DD but resistant by dDD/number of susceptible isolates. Minor errors were defined as one result yielding an intermediate category, and the other either a susceptible or resistant result.39
RESULTS
Chromogenic culture characterization.
To assess the utility of Chromatic MH agar for the isolation and differential identification of pathogens commonly encountered in blood cultures, we evaluated 24 bacteria and 3 yeasts (Table 3). Plates with insufficient growth for characterization were reincubated and inspected daily for an additional 48 hours. No growth was obtained for S. pneumoniae and H. influenzae. All other organisms yielded sufficient growth on Chromatic MH agar after overnight incubation for characterization except for E. rhusiopathiae, Y. enterocolitica, P. fluorescens, and Cryptococcus neoformans, which required 72 hour for faint, clear growth of tiny colonies. Photos of chromogenic cultures are shown in Supplemental Figures S1 through S24. Rapid tests were used to differentiate further between species with the same color when possible (Table 3).
Chromogenic culture and differentiation
| Culture result | Rapid test | Organism |
|---|---|---|
| Gram positive | ||
| Cream/white | Cat+, Coag+ | Staphylococcus aureus |
| Cat+, Coag– | Staphylococcus epidermidis | |
| Cat– | Streptococcus pyogenes | |
| Turquoise | Cat–, Esc+, PYR+ | Enterococcus faecalis |
| Cat–, Esc–, PYR– | Streptococcus agalactiae | |
| Cat+, Esc+, PYR– | Listeria monocytogenes | |
| Clear faint | S+, Ind–, M+ | Erysipelothrix rhusiopathiae |
| Gram negative | ||
| Mauve | PYR–, Ind+ | Escherichia coli |
| PYR–, Ind– | Shigella sonnei | |
| PYR+, Ind– | Citrobacter freundii | |
| Dark blue | – | Klebsiella pneumoniae |
| – | Cronobacter sakazakii | |
| – | Klebsiella aerogenes | |
| Turquoise | Ox–, PYR+ | Serratia marcescens |
| Ox–, PYR– | Stenotrophomonas maltophilia | |
| Ox+, PYR– | Vibrio parahaemolyticus | |
| Green | – | Pseudomonas aeruginosa |
| Clear | Ox+, PYR– | Burkholderia cepacian |
| Ox–, PYR– | Acinetobacter baumannii | |
| Ox–, PYR– | Salmonella enterica | |
| Ox–, PYR+, purple at RT | Yersinia enterocolitica | |
| Clear | Faint at 72h at RT | Pseudomonas fluorescens |
| Brown precipitate | – | Proteus mirabilis |
| Yeast | ||
| Cream/white | – | Candida albicans |
| – | Candida krusei | |
| Clear, faint at 72 hours | – | Cryptococcus neoformans |
Cat = catalase; Coag = coagulase; Esc = esculin; Ind = indole; M = motility; Ox = Oxidase; PYR = pyrrolidonyl arylamidase; RT = room temperature; S = sulfur.
Standard AST.
Prior to examining the potential of dDD on Chromatic MH agar, we first performed QC testing of type strains using standard DD methods described elsewhere.20 QC of standard DD of antimicrobial disks using Mueller–Hinton agar yielded seven instances of zone sizes outside of the acceptable range: S. aureus ATCC 25923 against cefoxitin 30 μg (FOX; +1 mm), meropenem 10 μg (MEM; +1 mm), and tetracycline 30 μg (+4 mm); E. coli ATCC 25922 against chloramphenicol (C; +1 mm), rifampicin 5 μg (–1 mm); and P. aeruginosa ATCC 27853 against gentamicin 10 μg (GM; +3 mm) and MEM (+1 mm). Upon retesting, all QC failures were resolved. In contrast, the same strains and disks yielded no zone sizes out of range with Chromatic MH (data not shown).
Direct disk diffusion susceptibility testing.
Having established that standard DD performs favorably well on Chromatic MH agar, we next sought to compare categorical agreement between dDD from spiked positive blood culture bottles with standard DD. For the 10 challenge isolates (Supplemental Table S1), the categorical agreement with 113 data points was 79.6%. No VMEs were seen. Three major errors (3 of 61, 4.9%) were seen: one for each of two K. pneumoniae isolates imipenem 10 μg (IMP) was found resistant by dDD on Chromatic MH (both 19 mm) versus susceptible by DD on Mueller-Hinton agar (MHA) (29 and 30 mm); and for the third K. pneumoniae isolate, FOX was resistant (14 mm) versus susceptible (21 mm). The minor error rate was 17.7% (20 of 113).
To assess reproducibility of dDD on Chromatic MH agar we prepared 4 to 10 replicates of the 4 QC strains in contrived blood culture bottles and subjected them to testing: S. aureus ATCC 25923 tested against 21 antimicrobial agents in 4 replicates yielded 4 of 84 zone sizes outside acceptable QC ranges (Supplemental Table S2): one instance of vancomycin 30 μg; E. coli ATCC 25922 tested against 17 agents in 8 replicates yielded zone sizes outside the acceptable QC range for cefazolin 30 μg (CZ; v5 replicates), ciprofloxacin 5 μg (4 replicates), amoxicillin–clavulanate 20/10 μg (AMC; 3 replicates), MEM (3 replicates), trimethoprim–sulfamethoxazole 1.25/23.75 μg (3 replicates), nitrofurantoin 300 μg (3 replicates), ceftriaxone 30 μg (2 replicates), cefpodoxime 10 μg (1 replicate), cefuroxime 30 μg (1 replicate), C (1 replicate), GM (1 replicate), IMP (1 replicate), rifampicin 5 μg (1 replicate), and tetracycline 30 μg (1 replicate); E. coli ATCC 35218 tested against 2 agents in 8 replicates yielded zone sizes outside the QC range for AMC (2 replicates); P. aeruginosa tested against 6 agents in 10 replicates yielded zone sizes outside the QC range for GM (2 replicates) and ceftriaxone 30 μg (1 replicate). The imprecision of replicate zone sizes measured for 238 data points ranged from 0% to 16.3% CV. CA interpretations showed no VMEs. However, six instances of minor errors were observed for E. coli ATCC 259223: three of eight AMC replicates yielded intermediate results (expected susceptible), one of eight CZ yielded resistant (expected susceptible or intermediate), and two of eight C yielded intermediate results (expected susceptible). The precision (reproducibility) CA yielded 97.5% (232 of 238) categorical result matches. During analysis for CA calculations, it was noted that the acceptable CLSI zone sizes in particular instances encompassed two categories: for S. aureus ATCC 25923 the acceptable erythromycin zone size range is 22 to 30 mm, which encompass the breakpoints susceptible ≥ 23 mm, and intermediate 14 to 22 mm. Similarly, for E. coli ATCC 25922, the AMC and CZ acceptable QC ranges yield susceptible or intermediate categories, and for E. coli ATCC 35218 against AMC, either susceptible or intermediate is acceptable.20 This raises a question about the appropriateness of the type strains recommended for QC, and the meaning of measurement variations as currently defined.
DISCUSSION
Of the 24 bacteria cultured onto Chromatic MH agar, 19 yielded sufficient growth for identification after overnight incubation. Erysipelothrix rhusiopathiae, P. fluorescens, and Y. enterocolitica required re-incubation for an additional 24 to 48 hours at room temperature for sufficient growth, consistent with culture on routinely used non-selective clinical bacteriological media. As expected, S. pneumoniae and H. influenzae failed to grow, because no blood, hemoglobin, or X and V factors were added to the medium. With simple rapid spot identification reagents, organisms were readily assigned to the appropriate CLSI interpretive categories. Candida was readily recovered on Chromatic MH agar and can be differentiated with the germ tube test. Cryptococcus growth was scant.
QC testing of media and disks showed that Chromatic MH agar used with the standard CLSI DD method yielded no QC failures, whereas traditional MHA yielded seven instances requiring retesting. This may be a result of enhanced readability of zone size edges with a chromogenic color reaction as opposed to traditional MHA. Ease of zone size readability is beneficial for initial skills development.
The performance of dDD onto Chromatic MH agar was satisfactory in our study. The overall CA between standard dDD and DD was 91.7%, which satisfies the Cumitech-recommended 90%.39 However, we did observe a lower CA (79.6%) in the subset of 10 challenge isolates. Of note, three major errors were seen with K. pneumoniae affecting IMP and FOX results, possibly a result of an inoculum effect. The precision (CV, 0–16%) and reproducibility CA (97.5%) of replicate measures zone size measures were high.
There have been at least eight larger previous studies evaluating dDD on positive blood culture bottles since 1976, comprising a total of 3,949 blood culture isolates and 28,126 organism–antibiotic combinations. From these studies, CA ranged from 93.9% to 97.7%, of which VMEs comprised 0% to 1.6%—and major errors, 0.03% to 2.1%—supporting the direct use of positive blood culture broth for disk diffusion susceptibility testing.10,11,13,14,16–19 Our study suggests that the use of Mueller–Hinton-based chromogenic media for dDD of positive blood culture bottles shows potential for rapid, clinically useful presumptive overnight simultaneous identification and AST, but requires further evaluation with routine blood culture samples from patients. There is, however, a need to optimize the medium formulation to allow the recovery of S. pneumoniae and H. influenzae, and to assess the potential and limitations for identifying Neisseria meningitidis and/or ruling out select agents of concern for laboratory exposure.
Supplemental Materials
ACKNOWLEDGMENTS
We thank the laboratory technicians and scientists at KATH microbiology laboratory for support and workflow accommodations. We also thank the anonymous reviewer for useful suggestions included in the supplemental identification algorithm.
REFERENCES
- 1.↑
Petti CA , Polage CR , Quinn TC , Ronald AR , Sande MA , 2006. Laboratory medicine in Africa: a barrier to effective health care. Clin Infect Dis 42: 377–382.
- 2.↑
Lewis JM , Feasey NA , Rylance J , 2019. Aetiology and outcomes of sepsis in adults in sub-Saharan Africa: a systematic review and meta-analysis. Crit Care 23: 1–11.
- 3.↑
Fleischmann C , Scherag A , Adhikari N , Hartog C , Tsaganos T , Schlattmann P , Angus D , Reinhart K , 2015. Global burden of sepsis: a systematic review. Crit Care 19 (Suppl 1) (abstract): 21.
- 4.↑
Iwashyna TJ , Ely EW , Smith DM , Langa KM , 2010. Long-term cognitive impairment and functional disability among survivors of severe sepsis. JAMA 304: 1787–1794.
- 5.↑
Al Khalaf MS , Al Ehnidi FH , Al-Dorzi HM , Tamim HM , Abd-Aziz N , Tangiisuran B , Hassan Y , Arabi YM , 2015. Determinants of functional status among survivors of severe sepsis and septic shock: one-year follow-up. Ann Thorac Med 10: 132.
- 7.↑
Munson E , Carroll KC , 2019. Whither extensive genomic-based microbial taxonomic revision? Clin Chem 65: 1343.
- 8.↑
Ahmad A , Iram S , Hussain S , Yusuf NW , 2017. Diagnosis of paediatric sepsis by automated blood culture system and conventional blood culture. J Pak Med Assoc 67: 192–195.
- 9.↑
Butler-Laporte G et al., 2020. Real-world time to positivity of 2 widely used commercial blood culture systems in patients with severe manifestations of sepsis: an analysis of the FABLED study. Open Forum Infect Dis 7: 1–5.
- 10.↑
Périllaud C , Pilmis B , Diep J , Péan de Ponfilly G , Vidal B , Couzigou C , Mizrahi A , Lourtet-Hascoët J , le Monnier A , Nguyen Van JC , 2019. Prospective evaluation of rapid antimicrobial susceptibility testing by disk diffusion on Mueller–Hinton rapid-SIR directly on blood cultures. Diagn Microbiol Infect Dis 93: 14–21.
- 11.↑
Mirrett S , Reller LB , 1979. Comparison of direct and standard antimicrobial disk susceptibility testing for bacteria isolated from blood. J Clin Microbiol 10: 482–487.
- 12.↑
Wegner DL , Mathis CR , Neblett TR , 1976. Direct method to determine the antibiotic susceptibility of rapidly growing blood pathogens. Antimicrob Agents Chemother 9: 861–862.
- 13.↑
Fay D , Oldfather JE , 1979. Standardization of direct susceptibility test for blood cultures. J Clin Microbiol 9: 347–350.
- 14.↑
Doern GV , Scott DR , Rashad AL , Kim KS , 1981. Evaluation of a direct blood culture disk diffusion antimicrobial susceptibility test. Antimicrob Agents Chemother 20: 696–698.
- 15.↑
Coyle MB , McGonagle LA , Plorde JJ , Clausen CR , Schoenknecht FD , 1984. Rapid antimicrobial susceptibility testing of isolates from blood cultures by direct inoculation and early reading of disk diffusion tests. J Clin Microbiol 20: 473–477.
- 16.↑
Edelmann A , Pietzcker T , Wellinghausen N , 2007. Comparison of direct disk diffusion and standard microtitre broth dilution susceptibility testing of blood culture isolates. J Med Microbiol 56: 202–207.
- 17.↑
Tan TY , Ng LSY , Kwang LL , 2008. Evaluation of disc susceptibility tests performed directly from positive blood cultures. J Clin Pathol 61: 343–346.
- 18.↑
Stokkou S , Geginat G , Schlüter D , Tammer I , 2015. Direct disk diffusion test using European Clinical Antimicrobial Susceptibility Testing breakpoints provides reliable results compared with the standard method. Eur J Microbiol Immunol (Bp) 5: 103–111.
- 19.↑
Rajshekar D et al., 2019. Evaluation of performance of direct disk diffusion test from positively flagged blood culture broth: a large scale study from South India. J Lab Physicians 11: 154–160.
- 20.↑
Clinical and Laboratory Standards Institute , 2020. Performance Standards for Antimicrobial Susceptibility Testing. 30th edition. Wayne, PA: Clinical and Laboratory Standards Institute.
- 21.↑
The European Committee on Antimicrobial Susceptibility Testing , 2020. Breakpoint Tables for Interpretation of MICs and Zone Diameters. Version 10. The European Committee on Antimicrobial Susceptibility Testing.
- 22.↑
Waters D , Jawad I , Ahmad A , Lukšić I , Nair H , Zgaga L , Theodoratou E , Rudan I , Zaidi AKM , Campbell H , 2011. Aetiology of community-acquired neonatal sepsis in low- and middle-income countries. J Glob Health 1: 154–170.
- 23.↑
Downie L , Armiento R , Subhi R , Kelly J , Clifford V , Duke T , 2013. Community-acquired neonatal and infant sepsis in developing countries: efficacy of WHO’s currently recommended antibiotics: systematic review and meta-analysis. Arch Dis Child 98: 146–154.
- 24.↑
Samra Z , Heifetz M , Talmor J , Bain E , Bahar J , 1998. Evaluation of use of a new chromogenic agar in detection of urinary tract pathogens. J Clin Microbiol 36: 990–994.
- 25.↑
Sánchez-Calvo JM , de Francisco JL , Torres-Martos E , Alados Arboledas JC , López Prieto MD , 2017. A cost-saving strategy for processing isolated uropathogens in community-acquired urinary tract infections. J Microbiol Methods 139: 130–134.
- 26.↑
Carricajo A , Boiste S , Thore J , Aubert G , Gille Y , Freydiere AM , 1999. Comparative evaluation of five chromogenic media for detection, enumeration and identification of urinary tract pathogens. Eur J Clin Microbiol Infect Dis 18: 796–803.
- 27.↑
Croxatto A , Marcelpoil R , Orny C , Morel D , Prod’hom G , Greub G , 2017. Towards automated detection, semi-quantification and identification of microbial growth in clinical bacteriology: a proof of concept. Biomed J 40: 317–328.
- 28.↑
Hakalehto E , Nyholm O , Bonkoungou IJO , Kagambega A , Rissanen K , Heitto A , Barro N , Haukka K , 2014. Development of microbiological field methodology for water and food-chain hygiene analysis of Campylobacter spp. and Yersinia spp. in Burkina Faso, West Africa. Pathophysiology 21: 219–229.
- 29.↑
Merlino J , Siarakas S , Robertson GJ , Funnell GR , Gottlieb T , Bradbury R , 1996. Evaluation of CHROMagar orientation for differentiation and presumptive identification of Gram-negative bacilli and Enterococcus species. J Clin Microbiol 34: 1788–1793.
- 30.↑
Singh AK , Bhunia AK , 2016. Optical scatter patterns facilitate rapid differentiation of Enterobacteriaceae on CHROMagarTM orientation medium. Microb Biotechnol 9: 127–135.
- 31.↑
Druggan P , 2012. Chromogens, fluorogens, Trojan horses and their use in culture media. Correy JEL , Gordon DW , Baird RM , eds. Handbook of Culture Media for Food and Water Microbiology, 3rd edition. London, United Kingdom: RSC Publishing, 623–624.
- 32.↑
Manafi M , Kneifel W , Bascomb S , 1991. Fluorogenic and chromogenic substrated used in bacterial diagnostics. Microbiol Rev 55: 335–348.
- 33.↑
Strumbelj I , 2013. New broth – chromogenic Mueller Hinton agar procedure for urine samples – next-day result of Enterobacteriaceae antimicrobial susceptibility testing. 23rd European Congress of Clinical Microbiology and Infectious Diseases. April 27–30, 2013, Berlin, Germany.
- 34.↑
d’Humières C , Belhiba M , Brocco F , Andremont A , Grall N . Direct determination of antimicrobial susceptibility of Gram negative bacilli from respiratory samples on chromogenic agar plates with gradient antibiotic strips. 24th European Congress of Clinical Microbiology and Infectious Diseases. May 1–13, 2014, Barcelona, Spain.
- 35.↑
M’Zali FH , Stecken L , Berard X , Quinart A , Sztark F . A novel, direct susceptibility testing method of sonicated vascular prosthetic graft samples by combination of Liofilchem MIC test strip and chromogenic agar plates. 23rd European Congress of Clinical Microbiology and Infectious Diseases. April 27–30, 2013, Berlin, Germany.
- 36.↑
Morgan JW , 1987. Evaluation of a rapid method for the determination of L-pyrrolidonyl-B-naphthylamide hydrolysis. Lab Med 18: 682–683.
- 37.↑
Edberg SC , Gam K , Bottenbley CJ , Singer JM , 1976. Rapid spot test for the determination of esculin hydrolysis. J Clin Microbiol 4: 180–184.
- 38.↑
York MK , Traylor MM , Hardy J , Henry M , 2004. Biochemical tests for the identification of aerobic bacteria. Isenberg HD , ed. Clinical Microbiology Procedures Handbook. 2nd edition. Washington, DC: ASM Press.
- 39.↑
Clark RB , Lewinski MA , Loeffelholz MJ , Tibbetts RJ , 2009. Cumitech 31A, Verification and Validation of Procedures in the Clinical Microbiology Laboratory. Washington, DC: ASM Press.