# Presentation: 108585

## Advanced Specimen Collectionand Culture Work-up

- 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

- Review collection and transport procedures for wound, stool, respiratory, and genitourinary specimens submitted for microbiological culture.
- Summarize appropriate algorithms for culture workup of wound, stool, respiratory, and genitourinary specimens submitted for microbiology culture.
- Discuss the importance of the clinician-laboratory interface

## Routine Stool Culture

- Q-Probe Study of 601 institutions
- Majority of laboratories (99.3%) included *Salmonella* and *Shigella* in the routine stool workup
- 96% routinely included *Campylobacter*
- 30-60% of laboratories surveyed also included other organisms such as *Aeromonas*, *Plesiomonas*, *Yersinia*, *Escherichia coli* O157, & *Vibrio*

- Valenstein, P, M. Pfaller, and M. Yungbluth. 1996. The use and abuse of routine stool microbiology. *Arch Pathol Lab Med*. 120:206-211.

## Role of Rapid Nonculture Screening Tests

- Fecal Leukocyte Test
- Fecal Lactoferrin Assay
- Gram stain

## Fecal Leukocyte Test

- Hines, J and I. Nachamkin. 1996. Effective use of the clinical microbiology laboratory for diagnosing diarrheal diseases. Clin Infect Dis. 23:1292–1301.

## Fecal Lactoferrin Assay

- To detect *Salmonella*, *Shigella* and *Campylobacter* spp.
- The sensitivity ranged from 83 to 93%, and specificity ranged from 61% to 100% 1
- 85% sensitive and 79% specific when compared to culture 2
- As a marker for inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) 3
- Significantly higher lactoferrin levels in patients with active and inactive IBD (85.9% sensitivity and 100% specific)
- Elevated fecal lactoferrin levels – 100% specific in ruling out IBS

- Choi et. al. 1996. To culture or not to culture: fecal lactoferrin screening for inflammatory diarrhea. *J. Clin. Microbiol*. 34:928-932.
- Silletti, R. P. et. al. 1996. Role of stool screening tests in diagnosis of inflammatory bacterial enteritis in selection of specimens likely to yeild invasive enteric pathogens.  J. Clin. Microbiol. 34:1161-1165.
- Kane, S. V., et. al. 2003. Fecal lactoferrin is a sensitive and specific marker in identifying intestinal inflammation.  Am. J. Gastroenterol. 98:1309-1314.

## Gram Stain

- The only role of Gram stain is in the diagnosis of *Campylobacter* sp.
- The sensitivity ranges from 66 to 94% with high specificity
- No value for detecting other enteric pathogens

- Hines, J and I. Nachamkin. 1996. Effective use of the clinical microbiology laboratory for diagnosing diarrheal diseases. Clin Infect Dis. 23:1292–1301.

## Number of Specimens Required to Identify a Pathogen

| Number of Specimens Collected per patient  | Cumulative Percent of Infected Patients Detected |  |
| --- | --- | --- |
|  | Bacteria/Fungi
(n = 3349) | Parasites
(n = 1159) |
| 1 | 96.9 | 91.9 |
| 2 | 99.0 | 97.6 |
| 3 | 99.3 | 99.8 |
| 4 | 99.4 | 99.9 |

- Valenstein, P, M. Pfaller, and M. Yungbluth. 1996. The use and abuse of routine stool microbiology. *Arch Pathol Lab Med*. 120:206-211.

## Bacteriology Results on Inpatients by Day of Hospital Stay

| Result  | Hospital Day  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | 1 | 2 | 3 | 4 | >4 |
| Total number of specimens collected | 7924 | 6886 | 3369 | 2022 | 10009 |
| Total number of 1st positives, with or without previous specimens | 374 | 155 | 35 | 19 | 49 |
| Percentage of total specimens that were patient's 1st positive specimen | 4.7 | 2.3 | 1.0 | 0.9 | 0.5 |
| Number of 1st  positive specimens from patients for whom previous negative specimens were collected
 | 6 | 0 | 1 | 0 | 1 |

- Valenstein, P, M. Pfaller, and M. Yungbluth. 1996. The use and abuse of routine stool microbiology. *Arch Pathol Lab Med*. 120:206-211.

## CAP CHECKLIST

- Question MIC.22440 	PHASE: I
- Does the laboratory have guidelines (developed with clinicians) for the number and/or timing of collection of stool specimens submitted for routine bacterial testing?
- Question MIC.22336  	PHASE: I
- Does the final report for routine bacterial stool cultures list the organisms for which the specimen was cultured (*e.g.,* Salmonella, Shigella, Vibrio, *etc.*)?

## Acceptable Specimens

- Fecal sample
- Fresh
- received within 1 to 2 h of passage
- Preserved
- Buffered Glycerol Saline
- recommended for *Salmonella* & *Shigella* but not for *Campylobacter* or *Vibrio* sp., unless enriched with CaCl2
- Modified Carey-Blair
- good overall transport media
- inappropriate for *C. difficile*

## Acceptable Specimens

- Rectal swabs
- Duodenal, colostomy or ileostomy contents
- stool transport vials
- Rectal biopsy samples
- sterile container with a small amount of sterile water

## Unacceptable Specimens

- Unpreserved stool samples > 2h old
- Dry rectal swabs or biopsy specimens
- Multiple specimens received on the same day
- Specimens received from inpatients after the third hospital day, without prior consultation

## Workup Guidelines for Salmonella, Shigella, Aeromonas & Plesiomonas species

- Media
**Non-selective**
- BAP
*Aeromonas* sp., *Plesiomonas shigelloides*, Yeasts, *Staphylococcus aureus*, *Pseudomonas aeruginosa*
**Differential enteric agar**
- MacConkey agar, Eosin Methylene Blue (EMB)
- differentiates lactose-fermenting from non-lactose fermenting colonies
**Moderately selective**
- Hektoen Agar, Xylose Lysine Deoxycholate Agar (XLD), or Salmonella Shigella Agar
- allows growth of *Salmonella* & *Shigella* sp. while suppressing the growth of most members of family *Enterobacteriaceae*
**Enrichment broth**
- GN Broth, Selenite F
- increases chances of detecting low numbers of pathogens

## Workup Guidelines for Salmonella, Shigella, Aeromonas & Plesiomonas species

- Enteric Screening Procedure
- Conventional
- TSI or KIA, LIA, and Urea
- Commercial kits
- latex agglutination
- Full ID of suggestive screening results
- Serological identification of *Salmonella* and *Shigella* sp.

## Campylobacter sp.

**Most frequently isolated**
**Selective media**
**Campy-Thio (enrichment broth)**
**Campy-BAP**
**Skirrow medium**
**Campylobacter-cefoperazone-vancomycin-amphotericin (CVA)**
**Identification**
**growth at 42°C, oxidase and catalase positive, Hippurate positive**
**Nalidixic Acid susceptible, Cephalothin resistant**
**Latex agglutination test**

## Campylobacter sp.

- ProSpecT® *Campylobacter* Microplate Assay
- detects *Campylobacter* specific antigens in stool (fresh or in transport medium)
- utilizes polyclonal anti- *Campylobacter* specific antigens capture antibody
- can be read visually or spectrophometrically
- Evaluated in three studies
- Sensitivities of 80, 89 and 96%
- Specificities of 99%
- Flexiable, easy to use
- reduces cost, reduce turnaround time
- Cross-reactivity with *C. upsaliensis*, *C. hyointestinalis*, or *C. helveticus* unknown

- Endtz, H. P., et. al. 2000. Evaluation of a New Commercial Immunoassay for Rapid Detection of Campylobacter jejuni in Stool Samples. *Eur. J. Clin. Microbiol. Infect. Dis.* 19:794-797.
- Hindiyeh, M. et. al. 2000.  Rapid Detection of *Campylobacter jejuni* in Stool Specimens by an Enzyme Immunoassay and Surveillance for *Campylobacter upsaliensis* in the Greater Salt Lake City Area**.** *J. Clin. Microbiol*. 38: 3076-3079.
- Tolcin, R. et. al. 2000. Evaluation of the Alexon-Trend ProSpecT Campylobacter Microplate Assay. *J. Clin. Microbiol*. 38: 3853-3855.

## Genus Vibrio

- Laboratory Diagnosis
- Media
- TCBS (thiosulfate citrate bile salts sucrose) agar
- sucrose-fermenting (Yellow colonies)
*V. cholerae, V. alginolyticus*, & *V. fluvialis*
- non-sucrose-fermenting (**green** colonies)
*V. parahaemolyticus, V. vulnificus *(Lactose Fermentor)
- Susceptible to 150 g of vibriostatic agent (O/129)

## Escherichia coli

- Three paradigms by which diarrhea is produced:
- enterotoxin production
- Enterotoxigenic E. coli (ETEC)
- Enteroaggregative E. coli (EAEC)
- invasion
- Enteroinvasive E. coli (EIEC)
- intimate adherence with membrane signaling
- Enteropathogenic E. coli (EPEC)
- Enterohemorrhagic E. coli (EHEC/ STEC)

## Enterohemorrhagic E. coli

- Aka Shiga toxin-producing *E. coli* (STEC)
- There are at least 100 serotypes of STEC
- Only one serotype, namely *E. coli* O157:H7 can be detected in clinical laboratories.
- Selective media: sorbitol-MacConkey agar
- confirm by latex agglutination
- Varied geographic distribution - evaluate prevalence for the need of routine workup
- Availability of EIA for detection of STEC

## Yersinia enterocolitica

- Detection based on conventional methods
- Selective media - CIN agar
- dark red “bull’s eye” with a transparent border

## Clostridium difficile Testing

- Acceptable specimen
- Unformed stool specimen unless ileus due to *C. difficile* is suspected.
- Rejection criteria
- Specimens that are not liquid or soft
- Specimens from infants under 1 year old should be discouraged
- Specimen more than 24 hours old.
- Rectal swab specimens
- “Test for cure” or testing from asymptomatic individuals

- Gerding, D.N. et. al. 1995. Clostridium difficile-associated diarrhea and colitis.Infect. Control. Hosp. Epidemol. 16:459-477.
- Johnson, S. and D. N. Gerding. 1998. Clostridium difficile associated diarrhea: a review. Clin. Infect. Dis. 26:1027-1034.

## Clostridium difficile Testing

- Culture – most sensitive
- Selective medium - Cycloserine-cefoxitin-fructose agar
- Characteristic horse-dung smell
- typical yellow-green fluorescence under UV light
- Limitations
- does not distinguish  between toxigenic and non-toxigenigic strains
- delayed turn-around time
- Use of latex agglutination test that detects glutamte dehydrogenase is discouraged

## Clostridium difficile Testing

- Cell Culture Cytotoxicity Assay – most specific
- detects Toxin B
- Limitations
- Requires 24 to 48 hours
- Tedious
-  Non-commercial versions are not standardized
- EIAs for toxin A or toxins A and B
- Rapid
- Less sensitive than cell cyotoxicity assay
- Tests that detect only toxin A may miss isolates that are toxin A-B+

## Wounds

## Wound Cultures: Controversies

- Is sampling a wound for culture relevant?
- When and how should wounds be sampled?
- How should samples be transported?
- What analysis should be requested?
- Gram stain only? Culture only? Susceptibility testing?
- Quantitative cultures?

## Wounds:Classification

- Acute
- Caused by external damage to intact skin
- Types
- Surgical
- Bites
- Burns
- Minor cuts
- Abrasions
- Severe traumatic

- Chronic
- Precipitated by predisposing conditions that lead to compromise of dermal/epidermal tissue
- Types
- Impaired venous drainage
- Impaired arterial supply
- Metabolic diseases eg. diabetes

- Bowler PG, et. al. 2001. Wound microbiology and associated approaches to wound management. *Clin Microbiol Rev* 14: 244.

## Wound Infections:Etiology

- Surgical wounds
- Aerobes: *S. aureus*, coagulase negative staphylococci, *Enterococcus *spp. *E. coli*, *P*. *aeruginosa*, *Enterobacter *spp.
- Anaerobes: *Bacteroides *spp., *Peptostreptococcus*, *Clostridium *spp.
- Acute soft tissue infections
*Staph aureus* _only _organism in 30%
- 30-50% mixed aerobes/anaerobes
- 20-30% other eg. Group A streptococci, *Clostridium spp.*
- Bite wounds
- Special pathogens: *Pasteurella multocida*, *Capnocytophaga canimorsus*, *Bartonella henselae*, *Eikenella corrodens*
- Other mixed aerobes and anaerobes

## Wound Infections:Etiology

- Burn wounds
- Primarily aerobic organisms: *P. aeruginosa*, *Staphylococcus aureus*, *E. coli*, *Klebsiella *spp. *Enterococcus *spp. and *Candida *spp.
- Diabetic foot ulcers
- Aerobes: *Staph aureus*, *Streptococcus *spp. *P*. *aeruginosa*, *Enterococcus* spp., enterics
- Anaerobes: *Peptostreptococcus*, *Bacteroides *spp*.*, *Prevotella *spp.
- Decubitus ulcers
- Mixed aerobic and anaerobic bacteria

## Wound Cultures

- For open wounds
- Clean the wound margins with surgical soap or 70% ethyl or isopropyl alcohol
- Aspirate from the depth of the wound using a sterile syringe and needle
- Aspirated fluid should be sent to the laboratory in an appropriate transport system
- Alternatively, a curette may be used to obtain tissue from base of the wound
- Swabs are strongly discouraged

## Wound Cultures

- For closed wounds
- Prepare site as described for obtaining blood culture
- Aspirate as much purulent material as possible
- Transport in aerobic/anaerobic transport system

## Wound Cultures: Gram stain

- Pros
- Useful in estimating organism load from tissue biopsies
- Presence of microorganisms on smear from swabs correlates with _>_ 106 organisms (burns)
- Facilitates identification of etiologic agent of wound infection following clean surgery

- Cons
- Poor correlation seen between Gram stain and culture results from biopsy of diabetic foot infections
- In mixed infections, little value although presence of leukocytes indicates infection

## Wound Specimens: Algorithms

- Three approaches
- PMN predominance
- Q-Score
- Q-2-3-4 system

## Wound Specimens: Algorithms

- Modified from Sharp SE. *Clin Micro Newsletter* 21:14, 1999

## Wound Cultures

- Culture for aerobic and anaerobic bacteria if appropriately collected
- Gram stain results suggest adequate collection or presence of inflammation
- Tissues or aspirates vs. swabs
- Primary plating media: 5% SBA, Choc agar, MacConkey agar; anaerobic plates and thio if appropriately collected
- Identify anaerobes to Genus level only
- Perform susceptibility testing of predominant organisms only

## Wound Cultures: Extent of Workup

- Possible approaches
- Use Gram stain result
- Work up organisms seen on stain only
- List others
- Work up any potential pathogens to maximum of three, list others present by morphology
- Work up any quantity *S. aureus*, *P. aeruginosa*, beta hemolytic streptococci, enterics and gram-negative anaerobes

## Wound Cultures: Examples

- Gram stain results: (Acceptable)
- Many neutrophils, no epithelial cells
- Many gram positive cocci in clusters
- Many gram negative bacilli
- Few morphotypes resembling skin flora
- Work up (identify and perform susceptibility testing): Gram positive cocci in clusters and gram neg bacilli
- Culture report: Many *S. aureus*, many *Klebsiella* *pneumoniae*, light aerobic bacteria resembling skin flora

## Wound Cultures: Examples

- Gram stain: many neutrophils, few epithelial cells, Gram positive cocci in clusters, Gram positive cocci in chains,
- Culture grows: many *S. aureus*, many Group A streptococci, few enteric bacilli
- Work up: *S. aureus*, Group A streptococcus: limited ID and no susceptibility on enteric bacilli; susceptibility testing on Group A strep not  required

## Wound Cultures: Examples

- Gram stain: Many neutrophils, few epithelial cells, multiple morphotypes
- Culture grows: more than 3 potential pathogens
- Consider source
- Tissue or aspirate ?
- Contamination likely ?
- Type of patient
- May need to consult with clinician or Infectious Diseases service

## Q-Score

|  |  |  |  | Squamous Epithelial Cells |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  | No Cells | 1-9/lpf | 10-24/lpf
 | >25/lpf |
|  |  |  | Score | 0 | -1 | -2 | -3 |
|  | Neutrophils | No Cells | 0 | 3 | 0 | 0 | 0 |
|  |  | 1-9/lpf | +1 | 3 | 0 | 0 | 0 |
|  |  | 10-24/lpf | +2 | 3 | 1 | 0 | 0 |
|  |  | >25/lpf | +3 | 3 | 2 | 1 | 0 |

- Q-Score = # of potential pathogens (PP)
- 	   to work up

## Workup of Wound Cultures

- Q-Score System
- Good quality specimen (Q3)
- Up to 3 organisms can be considered as potential pathogens and worked up (ID/AST)
- Lower quality specimen (Q2, Q1)
- More SEC
- Fewer organisms are worked up

## Workup of Wound Cultures

- Q-Score System
- If the Q-score is greater than or equals the PP in culture
- Workup all potential pathogens
- If Q-Score is less than the PP in culture
- Look at the Gram stain
- Workup all PP that are seen on GS
- Morphologically ID others
- If all PP present on GS then only Morph ID all

## Workup of Wound Cultures

- Q/2-3-4 System
- Culture workup is based on the # of PP present
- 2PP – ID/AST
- 3PP
- Look at the Gram stain
- Workup two PP if they are seen on GS
- If all 3 present on GS then Morph ID
- 4PP
- Morph ID only

## Wound Cultures: Example

- Gram stain: many neutrophils, few epithelial cells, Gram positive cocci in clusters, Gram positive cocci in chains,
- Culture grows: many *S. aureus*, many Group A streptococci, few enteric bacilli
- Q score = 2  [PMN (+3), few epi (-1)]
- Q/2-3-4 = 3 PP
- look at gram stain
- Work up: *S. aureus*, Group A streptococcus, Morph ID and no susceptibility on enteric bacilli

## Respiratory Specimens

## Respiratory Specimens

- Upper respiratory tract specimens
- Throat
- detection of streptococcal pharyngitis

## Respiratory Specimens

- Upper respiratory tract specimens
- Nose
- detection of MRSA carriers
- Nasopharyngeal swabs
- diagnosis of *Bordetella pertussis*
- Nasopharyngeal swabs and washings
- diagnosis of viral disease

## Lower Respiratory Tract Infections

## “ The culture of lower respiratory specimens may result in more unnecessary microbiologic effort than any other type of specimen.”Raymond C Bartlett

## Lower Respiratory Tract InfectionsEpidemiology

- Pneumonia is the sixth leading cause of death in US
- Increasing numbers of patients at risk
- Aging population
- Increase in patients with immunocompromising conditions
- Overtreatment has lead to resistance
- Multidrug resistant *Streptococcus pneumoniae*
- Resistance among hospital acquired pathogens such as *Acinetobacter*, *Pseudomonas aeruginosa *and others

## Cumitech 7B:2003 Lower Respiratory Tract Infections

- 6 contributing authors
- Major sections
- Clinical aspects of diseases of LRT
- Specimen collection
- Specimen processing
- Interpretation of bacterial cultures
- Most common pathogens
- Methods for implementing change
- Guidelines for frequency of testing
- Public health issues
- Reimbursement codes

## Categories of Lower Respiratory Tract Infections

- Acute bronchitis
- Community acquired pneumonia
- Hospital acquired pneumonia
- Pneumonia in the immunocompromised host

## Community Acquired PneumoniaEtiologic Agents

| Pathogen | Frequency (%) |
| --- | --- |
| Streptococcus pneumoniae | 66 |
| Haemophilus influenzae | 1-12 |
| M catarrhalis | 10 |
| Legionella species | 2-15 |
| Mycoplasma pneumoniae | 2-14 |
| Klebsiella species | 3-14 |
| Enteric gram negative bacilli | 6-9 |
| Staphylococcus aureus | 3-14 |
| Chlamydia species | 5-15 |
| Influenza viruses | 5-12 |
| Other viruses | <1-12 |
| Unknown | 23-49 |

- Carroll KC. 2002. *J Clin Microbiol* 40:3115-3120.         Sharp SE, et.al. Cumitech 2003

## Community Acquired PneumoniaDiagnosis

- Available Test Methodologies
- Sputum Gram stain and culture
- Blood cultures
- Serologic studies
- Antigen detection tests
- Nucleic acid amplification tests

## Sputum Gram Stain and Culture

- Proponents
- Demonstration of predominant morphotype on Gram stain guides therapy
- Accuracy is good when strict criteria are used
- Cheap, so why not?

- Antagonists
- Poor specimen collection
- Intralaboratory variability (Gram stain interpretation)
- Low sensitivity and specificity
- Empiric treatment guidelines
- Not cost effective

## Sputum Collection

- Proper patient instruction
- Food should not have been ingested for 1-2 h prior to expectoration
- The mouth should be rinsed with saline or water
- Patient should breathe and cough deeply
- Patient should expectorate into a sterile container
- Transport container immediately to lab
- Perform Gram stain and plant specimen as soon as possible

## Sputum Gram Stain

- Screen for acceptability
- Examine specimen under low power (x 10 objective)
- Examine 10 representative fields
- Specimens that show few squamous epithelial cells (< 10/lpf) and many PMNs (> 25/lpf) are acceptable
- Notify physician of unacceptable samples

## Sputum Gram StainUnacceptable

## Sputum Gram Stain Good Quality

## Sputum Gram Stain Good Quality

## Sputum Gram Stain

- Good quality specimens
- Quantify number and types of inflammatory cells
- Note presence of bronchial epithelial cells
- Concentrate on areas with WBCs when looking for organisms
- Determine if there is a predominant organism (> 10 per oil immersion field)
- Semiquantitate and report organism with descriptive
- If no predominant organism is present, report “mixed gram positive and gram negative flora”

## Slide 62

## Utility of the Gram Stain in Diagnosis of Pneumonia

**  ****Roson, B, et. al. 2000. *****Clin Infect Dis***** 31:869-74**.
- Prospective study
- Non immunocompromised patients hospitalized with CAP
- 1,000 bed hospital in Spain
- ER physicians instructed on sputum collection for Gram stain and culture
- Sputum collected _under supervision_ of nurse or resident
- Samples were processed immediately
- Screened for epithelial cells
- Screened for predominant morphotype (> 75% of the organisms seen)
- Sputum planted to blood agar, chocolate agar and MacConkey agar
- Strictly defined clinical and diagnostic parameters

##  Utility of the Gram Stain in Diagnosis of Pneumonia 

**Roson, B, et. al. 2000. *****Clin Infect Dis***** 31:869-74**
- Results
- 190/533 (35.6%) patients had no sputum sample submitted (these patients were included in the calculations)
- 133/533 (25%) patients had a poor quality specimen
- 210/533 (39.4%) patients had a good quality specimen
- Overall sensitivity and specificity for pneumococcal pneumonia: 57% and  97%
- Overall sensitivity and specificity for *H. influenzae* pneumonia: 82 % and 99%
- Gram stain gave presumptive diagnosis in 80% of patients who had a good specimen submitted
- > 95% of patients in whom a predominant morphotype was seen on Gram stain received monotherapy

## Gram Stain Reports

- Be as descriptive as possible
- Moderate neutrophils
- Moderate Gram positive diplococci suggestive of *Streptococcus pneumoniae*
- Few bacteria suggestive of oral flora
- Keep report short—avoid line listing of all morphotypes present

## Sputum and Endotracheal SuctionCulture Evaluation

- Identify and perform susceptibility testing on 2-3 potential pathogens seen as predominant on Gram stain
- Alpha strep—rule out *S. pneumoniae*
- Yeast—rule out *Cryptococcus neoformans* only
*S. aureus*, Gram negative bacilli
- < normal flora, quantify and limit ID; no susceptibility
- Add comment that organism not predominant on stain
- ID mould, Mycobacteria or *Nocardia spp*.
- Modified from Sharp SE, et. Al. 2003. *Cumitech* 7B. ASM Press.

## IDSA Practice GuidelinesDiagnostic Tests for CAP

- Outpatients
- Empiric therapy with a macrolide, doxycycline, or a fluoroquinolone
- Hospitalized patients with CAP
- Gram stain and culture of sputum
- 2 pretreatment blood cultures
- Studies for Mtb, Legionella in select patients
- Rationale
- To improve patient care
- Advance knowledge of epidemiologically important organisms
- Prevent antibiotic abuse
- Reduce antibiotic expense
- Bartlett JG. 2000. *Clin Infect Dis* 31:347-82.

## ATS GuidelinesDiagnostic Tests for CAP

- Empiric therapy for outpatients
- Macrolide or tetracycline
- Hospitalized patients with CAP
- 2 sets of pre-treatment blood cultures
- Pleural fluid Gram stain/culture when appropriate
- Studies for Legionella, Mtb, fungi in select patients
- Sputum Gram stain/culture only if resistant or unusual pathogen is suspected
- Avoid extensive testing
- ATS. 2001. *Am J Respir Crit Care Med* 163: 1730-1754.

## Hospital Acquired Pneumonia

- Most frequent nosocomial infection (30-33% of cases) among combined medical surgical intensive care units
- 83% are ventilator associated
- Etiologic agents				Frequency (%)
*Gram positive cocci*
*S. aureus*  				17
*S. pneumoniae*				2-20
- Aerobic gram-neg bacilli	 		60
*Pseudomonas aeruginosa*
*Enterobacter sp.*
*Klebsiella pneumoniae*
*Acinetobacter*
*Legionella*
- Anaerobes				10-20
- Fungi					0-10
- Modified from: Carroll KC. 2002.* J Clin Microbiol *40: 3115-3120.

## Hospital Acquired PneumoniaDiagnosis

- American College of Chest Physicians: Clinical findings are not sufficient for definitive diagnosis
- Qualitative culture or endotracheal sputum has poor predictive value
- Bronchoscopy is recommended by many pulmonologists
- Bronchial brushings
- Bronchial washes
- Protected specimen brushing
- Bronchoalveolar lavage specimens (BAL)
- Transbronchial biopsy

## Respiratory Specimens

- Protected Brush Specimen
- To procure uncontaminated lower airway secretions
- Brush within 2 catheters

## Respiratory Specimens

- Bronchoalveolar Lavage (BAL)
- Samples large area of the lung
- Performed using a bronchoscope
- 100 to 250 ml of saline injected
- Injected saline along with secretions is collected by aspiration
- Transthoracic Aspiration
- Involves percutaneous introduction of a needle directly into the infiltrate

## Bronchoalveolar Lavage (BAL) Specimen Acceptability

- Microscopic examination of Gram-stained smear
- Acceptable
- <1% of cells present are squamous epithelial cells
- Unacceptable
- >1% of cells present are squamous epithelial cells

- Thorpe JE et. al.  1987. Bronchoalveolar lavage for diagnosing acute bacterial
- pneumonia. *J. Infect. Dis.* 155:855-861

## Processing Bronchoscopy Specimens

- Bronchoscopy brush protected
- Aerobic bacterial culture and Gram stain
- Anaerobic bacterial culture
- Limited volume
- Bronchoscopy brush, unprotected
- No anaerobic culture
- Limited volume
- Bronchial washings
- Useful only for pneumonia caused by strict pathogens
- Reasonable requests: Mtb, Fungi, *Legionella*, *Pneumocystis*
- Bronchoalveolar lavage
- No anaerobe culture
- Amenable to extensive testing for all opportunistic pathogens

## Interpretation of Quantitative PSB/BAL

- Dilution Method
- Quantify each morphotype present and express as CFU/ml
- Calibrated Loop Method
- Quantify each morphotype present and express as log10 colony count ranges
- Thresholds for significance
- PSB > 103 CFU/ml
- BAL > 104 CFU/ml
**Baselski and Wunderink. 1994. *****Clin***** *****Micro Rev***** 7:547**

## Bronchoscopy SamplesQuantitative Methods

- PSB or BAL	**Baselski and Wunderink. 1994. *****Clin***** *****Micro Rev***** 7:546.**  		       vortex 30-60 s		Final dilutions

- Plate 0.1 ml

- Chocolate, blood                        1:10

- Dilute
- 0.1 ml to 9.9 ml saline

- Plate 0.1 ml

- Chocolate           1:1000
- blood

- Dilute 0.1 ml to 9.9 ml saline

- Plate 0.1 ml

- Chocolate        1:100,000
- blood

## Bronchoscopy SamplesQuantitative Methods

- Calibrated loop method
-  **Baselski and Wunderink. 1994. *****Clin***** *****Micro Rev***** 7:547**
-                PSB	                                   vortex 30-60 s	BAL

- Plate 0.1 ml

- Chocolate		      Chocolate			              Chocolate
- 			          **Final Dilutions**
- 1:10				1:100				1:1000

-        Plate 0.01 ml

- Plate 0.001 ml

## Immunocompromised PatientsSuggested BAL Protocol

- Aerobic Gram stain quantitative bacterial culture
- Fungal stain and culture
- Mycobacterial stain and culture
- Viral culture/Respiratory DFA
- Pneumocystis DFA
- Legionella culture

## Genital Specimens

## GENITAL TRACT SPECIMENS

**Patients in high risk situations:**
- Patients known to have gonorrhea
- Male patients with NGU, PGU, epididymitis, and Reiter's Syndrome
- Females with mucopurulent cervicitis, urethral syndrome, endometriosis, and salpingitis
- Neonates born to infected mothers
- Infertility investigations

## GENITAL TRACT SPECIMENS

**Sexually active asymptomatic females who:**
- Are age 25 years or younger
- Are pregnant
- Have evidence of purulent or mucopurulent cervical discharge
- Exhibit endocervical bleeding, induced by swabbing on examination
- Have had a new sex partner in the preceding 2 months
- Use no contraceptives or a non-barrier method for contraception

## GENITAL TRACT SPECIMENS

- For Females
- Cervical specimens should be collected after removing excess mucous from the cervical os and surrounding mucosa
- Use a second swab to collect specimen by rotating the swab for 10 to 30 secs. in the endocervical canal
- Collect vaginal specimens using a speculum without any lubricant

## GENITAL TRACT SPECIMENS

- For males
- Urethral specimens are collected by inserting a swab 2 to 4 cm. into the urethra and rotating the swab for 2 to 3 seconds

## GENITAL TRACT SPECIMENS

- For HSV lesions
- Fluid from lesions should be aspirated using a syringe
- Swab can be used to collect vesicle fluid or cellular material from the base of the lesion before crusting and healing have begun

## Genital Specimens

| Specimen source | Potential 
Pathogens | Primary plating media | Special considerations |
| --- | --- | --- | --- |
| Cervix | Chlamydia; GC; herpes | SBA, Choc, TM; viral transport media for herpes | NAT testing recommended for GC, CT |
| Cul-de-sac | Anaerobes, GC, CT, enterics  | SBA, choc, TM,
Mac, ana, thio | Collect aspirate;
Anaerobe transport |
| Endometrium | Mixed aerobes /anaerobes | SBA, choc, TM,
Mac, ana, thio
 | Surgical biopsy or sheathed catheter |
| Vagina | Group B strep;
Mixed aerobes anaerobes BV | SBA; LIM or other special broth | Culture for Group B strep only; do not culture for BV |

## LABORATORY DETECTION OF BV

- Clue Cells: vaginal epithelial cells studded with coccobacilli
- wet mount
- pH > 4.5
- Whiff test + (10-20% KOH)
- Scored gram stain
- Culture = NO
*G. vaginalis* isolated in > 92% women with BV and 70% asymptomatic woman
- Probe: AFFIRM (Becton Dickinson)
- agrees well with high count *G. vaginalis*

## Trichomonas vaginalis

- Common sexually transmitted disease
- Disease associations and adverse outcomes
- Vaginitis
- Urethritis—men and women
- Outcomes
- Adverse pregnancy events
- Associated with increased HIV shedding

## Trichomonas vaginalisDiagnosis

- Culture- “gold standard”
- Diamond’s media
- InPouch TV; BioMed Diagnostics, San Jose CA)
- Barenfanger J, et. al. 2002. *J Clin Microbiol* 40:1387.
- Wet mount—insensitive (~ 50%)
- Rapid tests
- XenoStrip-Tv (GenzymeDiagnostics, Inc. San Antonio, Tex.)
- more sensitive than wet prep
- less sensitive than culture
- useful as a POC test
- Pillay A, et. al. 2004. *J Clin Microbiol* 42:3853.
- Kurth A, et. al. 2004. *J Clin Microbiol* 42:2940.

## Lab detection (cont.)

- GLC—no longer used
- Detection of sialidases (neuraminidases that remove sialic acid from sialogly-coconjugates)
- In BV, associated with *Prevotella* and *Bacteroides *sp.
- Colorimetric test BVBlue System (Gryphus Diagnostics—91.7% sensitive; 97.8% specific
- Myziuk L, et. al. 2003. BVBlue test for diagnosis of bacterial vaginosis. *J Clin Microbiol* 41:1925.

## Slide 90

## Clue Cell of BV

## BV Scored Gram Stain ( from Nugent RP 1991;29:297)

| TYPE | Number seen/OIF |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | None | <1 | 1-5 | 6-30 | >30 |
| Lacto | 4 | 3 | 2 | 1 | 0 |
| Gard/Bact | 0 | 1 | 2 | 3 | 4 |
| Curved GNR | 0 | 1 | 2 | 3 | 4 |

## Interpretation of Scored Gram Stain

- 0-3 = Normal
- 4-6 = Intermediate
- may indicate trichomoniasis, GC or CT
- abnormal gram stain, but not consistent with BV
- 7-10 = Consistent with Bacterial Vaginosis
- Significance of results unknown in pre-menarchal girls or post-menopausal women

## Slide 94

## Who Should be Screened for BV?

- Women with vaginal symptoms
- esp. if failed therapy
- Pregnant women at high risk of preterm birth
- Pregnant women with genital symptoms
- rule out trichomoniasis as well
- Women with gynecologic surgery

## Thank You

- Questions ?????