# Presentation: 839551

## Advanced Specimen Collectionand Culture Workup

- Karen C. Carroll, M.D.Associate Professor PathologyThe Johns Hopkins University School of MedicineDirector, Division Medical MicrobiologyThe Johns Hopkins Hospital, Baltimore, MD
- _[kcarrol7@jhmi.edu](mailto:kcarrol7@jhmi.edu)_
- Punam Verma, Ph.D., D (ABMM), MT(ASCP)
- Assistant Professor, Rush Medical College
- Assistant Director, Clinical Microbiology
- Rush University Medical Center, Chicago, IL
- _[Punam_Verma@rush.edu](mailto:Punam_Verma@rush.edu)_

## Objectives

- At the completion of this program the participants will be able to:
- Review collection and transport procedures for blood, urine, CSF, and other sterile fluid specimens submitted for microbiological culture.
- Summarize appropriate algorithms for culture workup of blood, urine, CSF, and other sterile fluid specimens.
- Correlate culture types with clinical relevance

## Role of the Microbiology Laboratory in Patient Care

- To analyze and manage specimens
- To communicate effectively with healthcare professionals involved, both before and after specimen analysis

- J. M. Miller, 1998 MLO 6: 28-34

## The Total Testing Process 

**Pre-analysis**

**The Total Testing Process**

## Specimen Selection

- The specimen must be material from actual infection site and must be collected with a minimum of contamination from adjacent tissues, organs, or secretions
- Optimal times for specimen collection must be established for the best chance of recovery of causative microorganisms.

## Specimen Collection

- A sufficient quantity of specimen must be obtained to perform the culture techniques requested.
- Appropriate collection devices, specimen containers, and culture media must be used to ensure optimal recovery of microorganisms
- Cultures must be obtained prior to the administration of antibiotics
- The culture container must be properly labeled
- Provide complete information on specimen requisition forms

## Specimen Transport Systems

- Sterile screw-cap cups, petri dishes, tubes
- Swabs
- Swab Transport system
- Calcium Alginate Swabs
- Cotton Swabs
- Dacron Swabs
- Nasopharyngeal-urethrogenital swabs
*N. gonorrhoeae* transport systems
- Proprietary swab systems for molecular testing for GC/CT
- Anaerobic Transport Systems
- Viral Transport Systems

- Perry, J.L. 1997. Assessment of swab transport systems for aerobic and anaerobic organism recovery. J. Clin. Microbiol 35:1269-1271

- PAC - Port-A-Cul (Becton Dickinson, Cockeysville, Md.)
- EZ - Culturette EZ (Becton Dickinson, Cockeysville, Md.)
- CAG – Copan Venturi Transystem Amies gel without charcoal (Copan Diagnostics, Carona, CA

## Quality Control Of Microbiological Transport Systems

- NCCLS document – M40-A
- Describes criteria to consider when choosing a microbiological transport device
- Presents quality control guidelines for both the manufacturer and the testing laboratory
- Provides a method by which laboratories can validate the manufacturer’s claims and compare devices

## CAP CHECKLIST

- Question MIC.11030	PHASE: II
- Is there a documented procedure describing methods for patient identification, patient preparation, specimen collection and labeling, specimen preservation, and conditions for transportation, and storage before testing, consistent with good laboratory practice?

- User guides
- Virtual user guides or procedure manuals

## Web-based User’s Guide

- Benefits
- Users can access information with less effort.
- More accessible information venues (internet and intranet)
- Providing accurate and up-to-date information saves time, effort and money in communicating changes and information to the staff

## Slide 13

## Slide 14

## Slide 15

## Slide 16

## Slide 17

## Slide 18

## Slide 19

## Blood Cultures

## Clinical Significance of Bacteremia

- 200,000 cases of bloodstream infections per year in the US
- Associated with mortality rates from 20% to 50%
- Nosocomial episodes >50% in some hospitals
- Prolonged hospitalizations
- Increased mortality compared to community-acquired episodes

- Magadia RR and Weinstein MP. 2001. Laboratory diagnosis of bacteremia
- and fungemia.  *Infect Dis Clinics N Amer* 15:1009

## Bacteremias

- Blood cultures are the gold standard for diagnosing a bacteremia
- Detecting the presence of bacteria in the blood is one of the most important functions in the clinical microbiology laboratory

**Notes:**
Because bacteremias are highly prevalent and are associated with a high mortality, detecting the presence of bacteria in the blood is one of the most important functions in the clinical micro lab.

## Diagnostic and Prognostic Importance of Positive Blood Cultures

- Diagnostic
- Establishes infectious etiology for patient’s illness
- Provides organism for susceptibility testing and optimization of antimicrobial therapy
- Prognostic
- Provides evidence of failure of host defenses to contain infection at primary site
- Provides evidence of failure of physician to remove, drain, or otherwise adequately treat primary infection

## Blood Cultures: Key Elements

- Timing
- Skin Antisepsis
- Collection
- Number
- Volume
- Choice of blood culture media
- Duration of Incubation
- Special pathogens
- Quality assurance

## Timing of Blood Cultures

- Optimal time
- Just before the onset of a shaking chill
- Fever detected
- General rule
- Collect 2 BC simultaneously
- Li et. al. (JCM 1994; 32:2829-2831) found that the interval between blood cultures was not clinically important

## Common Antiseptic Agents

|  | Mechanism of Action | Rapidity of Action | Residual Effect | Affected by Organic Matter | Primary Use |
| --- | --- | --- | --- | --- | --- |
| 2% chlorhexidine gluconate/70% isopropyl alcohol | Denature protein & disrupt cell membrane | Rapid | Excellent | Efficacy not affected by organic matter | Skin prep |
| Iodophors | Substitution by free iodine | Intermediate | Minimal | Diminished efficacy by organic matter | Surgical hand scrub, handwash & skin prep |
| Alcohol | Denature proteins | Rapid | None | No data | Surgical hand scrub, handwash & skin prep |
| Tincture of Iodine (2%) | Denature proteins & substitution by free iodine | Rapid | Minimal | No data | Skin prep |

## Comparison of Antiseptic Agents

- Mimoz, O et. al.** **1999. Chlorhexidine compared with povidone-iodine as skin preparation before blood culture. A randomized, controlled trial. *Ann Intern Med.* Dec 7;131(11):834-7

## Skin Antisepsis

- After palpitation, scrub the venipuncture site with 70% alcohol for a minimum of 30 s.
- Apply antiseptic agent in concentric circles away from the puncture site covering a circular area 1.5 to 2 in. in diameter

## Blood Collection

- Methods
- Needle and syringe
- Butterfly draw
- Direct draw
- Vacutainer-type
- Needle transfer devices
- Aspiration from IV catheters
- Increasing use
- Increased contamination rates
- If done, also obtain peripheral BC to validate

## BLOOD CULTURES

- Definition
- Blood obtained from one venipuncture site defines one blood culture, regardless of the number of bottles filled
- Highly dependent on the collection technique for its sensitivity and specificity

## Optimal Blood Volume

- Most important variable for improving detection of bacteremia and fungemia
- Number of microorganisms present in blood
- Adults - <1 to 10 CFU/ml
- Pediatric – 100 to 1000 CFU/ml
- Recommended volume for adults
- 20 to 30 ml per venipuncture

- Magadia RR and Weinstein MP. 2001. Laboratory diagnosis of bacteremia and fungemia.  *Infect Dis Clinics N Amer* 15:1009

## Rate of positivity increases (up to a point) as more cultures are obtained
Detection of etiologic agent
1st BC – 91.5%
2nd BC – 99.3%
3rd BC – 99.6%

- Detection of etiologic agent
- 1st BC – 91.5%
- 2nd BC – 99.3%
- 3rd BC – 99.6%

- Number of Blood Cultures

- Weinstein, M.P., et. al. 1983. The clinical significance of positive blood cultures: a comprehensive analysis of 500 episodes of bacteremia and fungemia in adults. I. Laboratory and epidemiologic observations. *Rev. Infect. Dis.* 5:35-53

## Diagnostic Importance of Separate Blood Cultures

- Weinstein, M.P., et. al. 1983. The clinical significance of positive blood cultures: a comprehensive analysis of 500 episodes of bacteremia and fungemia in adults. I. Laboratory and epidemiologic observations. *Rev. Infect. Dis.* 5:35-53

## True Bacteremia vs. Contamination

| Clinical significance of Staphylococcus epidermidis isolates from blood cultures |  |  |  |  |
| --- | --- | --- | --- | --- |
| # of sets positive | # of sets obtained | % Significant | % Contaminate | % Indeterminate |
| 1 | 1 | 0 | 97 | 3 |
| 1 | 2 | 2 | 95 | 3 |
| 2 | 2 | 60 | 3 | 37 |
| 1 | 3 | 0 | 100 | 0 |
| 2 | 3 | 75 | 0 | 25 |
| 3 | 3 | 100 | 0 | 0 |

- Weinstein, M.P.,et. al.1997. The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults. *Clin. Infect. Dis.* 24:584-602

## Volume of Blood Sampled

- Kaditis, A.G.,et. al. 1996.** **Yield of positive blood cultures in pediatric oncology patients by a new method of blood culture collection. *Pediatr. Infect. Dis. J.* 15:615-620

## CAP CHECKLIST

- Question MIC.22630	PHASE: II
- Are sterile techniques for drawing and handling of blood cultures defined, made available to individuals responsible for specimen collection, and practiced?
- Question MIC.22640	PHASE: I
- Are adequate volumes of blood collected for detection of sepsis?

## Choice of Blood Culture Media

- Several commercially available blood culture systems
- Two-bottle blood culture sets
- Aerobic and anaerobic
- Blood-broth ratio (1:5)
- Inactivation or binding of antimicrobials
- Resin, activated charcoal
- Detects more contaminants, more expensive

## Duration of Incubation

- Continuous-monitoring blood culture systems
- Five-6 day protocols are acceptable for majority of pathogens (BacTAlert and BACTEC series)1
- Four day protocols suggested for ESP system, BACTEC 92402-4
- Terminal subcultures are not required for any system
- Prolonged incubation periods (or alternative systems) may be required for fastidious pathogens
- 1  Hardy DJ, et. al. 1992. Time to detection of positive BacT/Alert blood cultures and lack of need for routine subculture of 5-to-7-day negative cultures. *J Clin Microbiol* 30:2743.
- 2 Doern GV, et. al. 1997. Four-day incubation period for blood culture bottles processed with the Difco ESP blood culture system.  *J Clin Microbiol* 35:1290.
- 3 Han XY and Truant AL. 1999. The detection of positive blood cultures by the AccuMed ESP-384 System: The clinical significance of three-day testing. *Diagn Micro Infect Dis* 33:1.
- 4 Johnson AS, et. al. 2000. Four day incubation for detection of bacteremia using the BACTEC 9240.  *Diagn Microbiol Infect* *Dis* 38: 195-9

## Extended Incubation for Presumptive Endocarditis

- Many labs are doing this
- Limited data on utility
- Rush unpublished data---no meaningful information
- Not needed for HACEK group detection

## Multicenter Study of Extended Incubation for HACEK Organisms

| 
Study site | 
Total positive 
blood cultures | Number of cultures positive for HACEK  |  HACEK Species 
Isolated |  Time to detection (days) |
| --- | --- | --- | --- | --- |
| 
JHH | 
6519 | 
7 | A. actinomycetemcomitans (1)                                    
E. corrodens (1)
C. hominis (2)*
H. parainfluenzae (3) | 5
4
3                 
3-5              |
| ARUP |  2301 | 1 |  H. parainfluenzae (1) | 4                                            |
| RWJUH | 1462 | 1 |  H. parainfluenzae (1) | 4 |
| TOTAL | 10,282 | 9 (0.08%) |   | 4 days (mean duration) |

- Bhally HS, et. al. 2004. Abstracts of the 42nd Annual Meeting of IDSA, Boston, MA, #453, p.120

## Quality Assurance

- Monitor contamination rates
- Workup and reporting leads to over-utilization of antibiotics and other associated costs
- Monitor volume
- Low volumes may decrease sensitivity
- When < 10 ml available fill aerobic bottle only
- Monitor positivity rates
- Infection control issues
- Contamination issues
- Monitor solitary blood cultures
- May compromise care due to poor sensitivity

## Blood Culture Contamination

- Definition
- Typical skin pathogens
- viridans strep, *Corynebacterium* sp., *Bacillus *sp., coagulase neg staph, *P. acnes*
- Number of positive and negative cultures in an episode
- Results of concurrent microbiology tests
- Compatibility of clinical features with typical features of infection
- Rates should be _< _3.0%
- Weinstein MP, 1997.  The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults.  *Clin Infect Dis* 24:585-602

## Cerebrospinal Fluid Specimens

## Acute Meningitis: Etiologic Agents

*Streptococcus pneumoniae*
*Neisseria meningitidis*
*Listeria monocytogenes*
*Streptococcus agalactiae*
*Haemophilus influenzae*
*Staphylococcus aureus*
- Gram negative bacilli
- Anaerobes
- Amoeba
- Thomson RB, Bertram H. 2001. Laboratory diagnosis of central nervous system infections.  *Infect Dis Clinics N America* 15:1047.

## Slide 45

## Acute Meningitis: Shunt Related

- Coagulase-negative staphylococci
*Staphylococcus aureus*
*Proprionibacterium acnes*
- Gram negative enteric bacilli
- Non-fermenting gram negative bacilli

## CSF Collection

## CSF Collection and Processing

- Major Pitfalls and Controversies
- Failure to properly decontaminate
- VOLUME
- Timely transport
- Pre-treatment with antibiotics
- Bacterial antigen detection tests
- Media
- Duration of incubation

## CSF Antigen Detection Revisited

- First and second generation “BAD” tests
- Poor sensitivity and specificity
- Results do not alter therapy
- Use selectively based upon lab practice
- New assay for *S. pneumoniae*
- NOW *S. pneumoniae* Urinary Antigen Test (Binax, Inc. Portland, ME)
- Immunochromatographic membrane assay—detects C polysaccharide cell wall antigen

## NOW Streptococcus pneumoniae Assay for CSF

- Samra Z, et. al. 2003. *Diagn Microbiol* *Infect Dis* 45:237.
- Study performed in 900 bed children’s hospital in Israel
- 519 pts. with suspected meningitis were enrolled
- CSF and blood obtained concomitantly
- CSF and urine samples obtained for NOW testing
- Results
- Pos CSF antigen from 21/22 pts. with pneumococcal meningitis (sens 95.4%); pos urine antigen 12/22 pts.; Gram stain 68.2% sensitive
- Neg CSF antigen from all 27 pts with other pathogens; 5 false pos urine antigens
- All 470 pts with no pathogens recovered were CSF antigen neg; 63 were urine antigen pos

## CSF Specimens: Processing 

- Process within 1-2 hr of collection
- Centrifuge volumes > 0.5 ml to concentrate pathogens
- Treat as “stat” for Gram stain performance and interpretation
- Cytocentrifugation
- Pos stain: microbiology “critical value”
- Inoculate sediment to:
- 5% sheep blood agar, chocolate agar incubate in 5-10% CO2 x 72 h
- Include broth for patients with shunts and infection adjacent to subarachnoid space; incubate x 5-7 days

## Acute Bacterial MeningitisUse of Gram Stain

- Dunbar SA, et. al. 1998. *J. Clin. Microbiol* 36: 1617
- 2653 adult CSF specimens
- 56 positive for *C. neoformans*, *S. pneumoniae*, or *N. meningitidis*
- 88% had a positive stain result
- If patients with prior antibiotic therapy excluded
- CSF Gram stain  - 92% sensitive

## CSF Broth Cultures:Evidence Against Routine Use

- Lessing MP, et.al. 1996. Eur J Clin Microbiol Infect Dis 15;79.
- Morris AJ, et. al. 1995. *J Clin Microbiol* 33:161.
- Only 2/88 broth only isolates were clinically significant
- Continued use of broth recommended only for shunt infections
- Sturgis CD , et. al. 1997. *Am J Clin Pathol* 108:217.
- Dunbar SA, et. al. 1998. *J Clin Microbiol* 36: 1617
- 82% of pathogens recovered on both solid media and in broth
- 220 contaminants; 55% broth only
- Exclusion of broth would have missed no acute bacterial meningitis cases, but 25% of shunt assoc cases

## Sterile Body Fluids

##  Inoculation of Sterile Body Fluids to Blood Culture Bottles

- Inoculate blood culture bottles at bedside when volume is sufficient
- Minimum of 1 mL of specimen/bottle
- Collect aerobic and anaerobic bottle set
- BacT/Alert FAN bottles outperformed conventional media and standard bottles in several studies
- Send additional fluid in sterile container for immediate Gram’s stain and culture for Mycobacteria, fungus or “special” pathogens
- Most useful for synovial fluids and peritoneal fluid/CAPD; literature mixed for pleural fluid

## Inoculation of Sterile Body Fluids to Blood Culture Bottles

- Advantages
- Simplifies specimen processing
- Shortens time to detection
- Enhances recovery of fastidious pathogens
- Disadvantages
- Contamination rates increase
- Simor AE, et. Al. 2000. Evaluation of the BacT/Alert microbial detection system with FAN aerobic and FAN anaerobic bottles for culturing normally sterile body fluids other than blood. *Diagn Microbiol Infect Dis* 37:5.
- Bourbeau P, et. Al. 1998. Use of the BacT/Alert blood culture system for culture of sterile body fluids other than blood. *J Clin Microbiol* 36: 3273.

## Inoculation of Synovial Fluid to Blood Culture Bottles

- Enhances recovery of fastidious and slow-growing pathogens eg. *Kingella kingae*
- Yagupsky P. 1992. *J Clin Microbiol* 30:1278
- Host B. 2000. *Eur J Clin Microbiol* Infect Dis 19:608-11
- Improves recovery of organisms from patients on antibiotics at time of specimen collection
- Von Essen R. 1997. *Scand J Rheumatol* 26:293

## Urine Specimens

## Urinary Tract Infections

- Urinary tract infections- most common bacterial infectious disease
- Urines-- most common sample type for culture
- Microbiologic diagnosis of UTIs has been impacted by several factors:
- Changes in criteria for defining significant bacteriuria
- Laboratory consolidation
- Emergence of resistant organisms
- Increases in numbers of immunocompromised pts.
- Changes in technological advancements

## UTI:Definitions

- UTI: presence of microorganisms in urine
- Symptomatic UTI: presence of clinical features
- Asymptomatic UTI:absence of symptoms in setting of critical numbers of potential uropathogens; local host response usually present

## Criteria for Defining Significant Bacteriuria

- Symptomatic women
- _>_ 102 coliforms/mL or _>_105 non-coliforms/mL
- Symptomatic men
- _>_ 103 CFU bacteria/mL
- Asymptomatic individuals
- _>_105 CFU bacteria/mL
- Catheterized patients
- _>_103CFU bacteria/mL
- Any growth on suprapubic aspirate or intraoperatively obtained sample

## Etiology of Urinary Tract Infections

**Figure 1 Etiology of UTI in Men**
**Figure 2 Etiology of UTI in Women**

## Complicated/Hospital Acquired UTIs: Etiology

*E. coli*
*Klebsiella*
*Proteus*
*Providencia*
*Serratia*
*Enterobacter*
*Acinetobacter*
*Pseduomonas*

- Coagulase negative staphylococci
- Enterococci
*Corynebacterium urealyticum*
- Yeasts

## Urine Cultures: Indications

- Culture is not always necessary in women with dysuria, pyuria
- Cultures _are_ indicated in the following situations:
- complicated or uncertain clinical features
- UTI in past 3 weeks indicating possible relapse
- symptoms for more than 7 days
- recent hospitalization or catheterization indicating possible nosocomial infection
- pregnancy
- diabetes

## Urine Culture Specimen Collection

- Routine midstream clean catch urine specimen
- Appropriate instructions must be provided to females, specimen should collected after thoroughly cleansing the urethral opening with soap
- Cleansing  not necessary in males
- Specimen should be a clean caught, mid-stream, early morning specimen
- Transport urine specimens in _leakproof _ containers within an one hour of collection
- Refrigerate or use boric acid transport system if it cannot be transported and plated within an hour
- Include pertinent patient information

## Urine Culture Specimen Collection

## Urine Culture Specimen Collection

- Straight catheterization or suprapubic aspiration
- performed under strict aseptic conditions
- to determine the significance of borderline counts in repeated clean catch midstream specimen
- to diagnose anaerobic bacteriuria

## Urine Culture Specimen Collection

- Chronic bladder catheterization
- clean collection port of catheter tubing carefully with 70% alcohol
- aspirate specimen using a sterile syringe and dispense in a sterile container

## Urine CultureSpecimen Rejection Criteria

- Urine collected from bedpan or urinal or collection bag
- Frozen specimen
- 24 h. collections for microbiologic culture
- A Foley catheter tip

## Urine Culture Algorithms:Women

- Leukocyte esterase pos.		     Leukocyte esterase neg.;
- 			                                                  Screen for asymptomatic
- Plate 0.001 mL SBA/MAC			bacteriuria
- Plate 0.01 mL SBA					
- 				   		    Plate 0.001 mL SBA/MAC			       				
- Work up any quantity of potential              Work up potential pathogens
- pathogens up to 3 org.			>50,000 up to 3 org.
- 	                 				               
-     Gram ID > 4 org. 			       Gram ID potential pathogens
- 						          < 50,000 up to 3 org.      						                             
-  Recollect if > 3 saprophytic org.	  Recollect if > 3 saprophytic indicating contamination   		     org. indicating contamination

## Urine Culture Algorithms: Men

- Plate 0.001 mL loop SB/MAC
- 
- 		Work up any quantity of potential
- pathogens up to 3 organisms
- 
-      Gram ID > 3 potential pathogens

## Urine Culture AlgorithmsIndwelling Catheters

- Leukocyte esterase pos.
- 
- Plate 0.001 mL SBA/MAC/CNA
- 
- Work up potential pathogens > 50,000 up to 3 org.
- 
- Gram ID potential pathogens < 50,000 up to 3 org.
- 
- > 4 org., call physician
- consider changing catheter
- recollect urine at time of catheter change

## Media, Inoculum and Incubation

- Media
- 5% sheep blood agar
- MacConkey agar or EMB
- Colistin-nalidixic acid agar (CNA)
- Inoculate using calibrated loop method
- Incubate at 35-37o C for 18-24h; 48 h for the following organisms
- Yeasts
*Corynebacterium urealyticum*
- Other coryneforms
*P. aeruginosa*

## Differential Media

**Dip inoculum methods**
**Chromogenic media**** ****	**
**Potential advantages**
**Ease in differentiating mixed flora**
**Eliminate need for subculture when performing susceptibility testing**
**Control of swarming *****Proteus***
**Limitations**
**Few studies have performed in depth cost-analysis  **
**Accuracy for detection of non-enterococcal, Gram-positive cocci and yeast is variable and needs more study**

**CHROMagar TM Orientation  **

## Questions?