Urinalysis

METHODS OF URINE COLLECTION

1. Random collection taken at any time of day with no precautions regarding contamination. The sample may be dilute, isotonic, or hypertonic and may contain white cells, bacteria, and squamous epithelium as contaminants. In females, the specimen may cont contain vaginal contaminants such as trichomonads, yeast, and during menses, red cells.
2. Early morning collection of the sample before ingestion of any fluid. This is usually hypertonic and reflects the ability of the kidney to concentrate urine during dehydration which occurs overnight. If all fluid ingestion has been avoided since 6 p.m. the previous day, the specific gravity usually exceeds 1.022 in healthy individuals.
3. Clean-catch, midstream urine specimen collected after cleansing the external urethral meatus. A cotton sponge soaked with benzalkonium hydrochloride is useful and non-irritating for this purpose. A midstream urine is one in which the first half of the bladder urine is discarded and the collection vessel is introduced into the urinary stream to catch the last half. The first half of the stream serves to flush contaminating cells and microbes from the outer urethra prior to collection. This sounds easy, but it isn't (try it yourself before criticizing the patient).
4. Catherization of the bladder through the urethra for urine collection is carried out only in special circumstances, i.e., in a comatose or confused patient. This procedure risks introducing infection and traumatizing the urethra and bladder, thus producing iatrogenic infection or hematuria.
5. Suprapubic transabdominal needle aspiration of the bladder. When done under ideal conditions, this provides the purest sampling of bladder urine. This is a good method for infants and small children.

Full details here

Read more...

Clinically Relevant Microbiology Starts at the Source

Clinically Relevant Microbiology Starts at the Source
By: Mike Costello, PhD, MT(ASCP)
ACL Laboratories
Mary Dikeman, MT (ASCP)
Affinity Health System

Program Objectives
* Emphasize that obtaining sensitive and specific microbiology results begins with the patient and not at the door of the microbiology laboratory.
* Accentuate the importance of proper collection and transport of specimens in both local and referral environments
* Stress the importance of timely communication between the Microbiology laboratory and those collecting specimens
* Describe common pitfalls in specimen collection and transport
* Discuss What rules or principles must be followed in order to collect microbiology specimens which will accurately reflect the pathogenesis of the microbiological agent. (Church D. The Seven Principles of Accurate Microbiology Specimen Collection. . Calgary Laboratory Services Microbiology Newsletter. Volume 6, 2005)

Introduction
The practice of sensitive, specific and cost effective clinical microbiology is intimately tied to the submission and proper handling of optimal specimens for analysis. Unfortunately, these aspects of clinical microbiology are not as critically controlled as our laboratory assays. It is our responsibility to educate and notify our healthcare colleagues when specimens arrive at the laboratory that will yield inferior results.

Quality assurance of specimen collection and transport is a never ending battle and requires long term commitment of your time and resources, but the end results are better patient care and a more rewarding experience for those of us who work in the microbiology laboratory.

Principle #1: The specimen must be collected with a minimum of contamination as close to
site of infection as possible

Urine Culture Contamination Rates

* Urine Culture contamination rates (>2 bacteria at >100,000 CFU) should be <20%
o CAP Q-Probe study (Valenstein P Meier F. Urine culture contamination: a College of American Pathologists Q-Probes study of contaminated urine cultures in 906 institutions. Arch Pathol Lab Med. 1998;122:123-129)..
+ 630 participants collected information of 155,037 urine culture specimens; 20.1% were considered contaminated (>2 organisms at >105 CFU)
+ The top 10% of institutions reported a rate of 5.6%. Bottom 10% of institutions reported a contamination rate of 36.8%
+ Males have a lower contamination rate than females (11.2% Vs. 22.8%)
+ ER departments had a contamination rate of 17.8%, sites adjacent to lab had rates of 19.5%, and other sites had rates of 22.1%

Blood Culture
* Two sets of blood cultures should be drawn. Number of sets positive correlates with true sepsis (except for coagulase negative Staph?) (Clin Microbiol. Rev 19:788-802, 2006)
* Catheter drawn blood cultures
o Catheter drawn blood cultures are equally likely to be truly positive (associated with sepsis), but more likely to be colonized (J Clin Microbiol 38:3393, 2001.)
+ One drawn through catheter and other though vein PPV 0f 96%
+ Both drawn from catheter PPV 0f 50%
+ Both drawn through vein PPV of 98%
o Study of positive coagulase negative Staphylococcus cultures and sepsis (Clin Infect Dis. 39:333, 2004.)

Blood Culture Contamination Rate
By Service Drawing Culture
What is an “Acceptable” Blood Culture Contamination Rate for Your Lab??
Blood Culture Contamination in Pediatric Patients
Young Children and Young Doctors
Inexperienced physician-young child
Inexperienced physician-older child
Experienced physician-younger child
Experienced physician-older child
Predicative Value of a Positive Result
False Positive
True Positive
Variable
Ped Infect Dis. 2006, 25:611-614.

Inexperienced Physicians = Interns and residents in 1st half of training
Experience Physicians = Residents in 2nd half of training and senior physicians
What is an “Acceptable” Blood Culture Contamination Rate for Your Lab??

What is an “acceptable” blood culture contamination rate*?
Berkeris LG, JA Toworek, MK Walsh, PN Valenstein. Trends in Blood Culture Contamination.
Arch Pathol Lab Med 129:1222-1294, 2005

Respiratory Cultures
* Community Acquired Pneumonia – Sputum rejection rate and culture correlation with gram stain
o 54% of all samples were judged to be of good quality.
o Presence of a (predominant morphology) PM on Gram stain was predictive of whether the sputum culture could demonstrate a pathologic organism. In the presence of a positive PM, 86% of cultures yielded a pathologic organism, while a positive culture was obtained in 19.5% of Gram stains without a predominant organism. S. pneumoniae was the most common infection, growing in 55.7% of positive sputum cultures.
o The sensitivity and specificity of finding Gram-positive diplococci for a positive culture of S. pneumoniae were 60% and 97.6%, respectively (Arch Intern Med. 2004;164:1725-1727, 1807-1811)
* Ventilator associated pneumonia (VAP) – appropriate specimen
o Blood cultures highly specific but not sensitive (positive in <10% of VAP)
o Quantitative cultures of lower respiratory tract specimens show a closer clinical correlation than sputum subcultures (Clinical Microbiol. Rev. 19:637-657, 2006.)

Viral Respiratory Cultures – Collect Sample From Site of Infection
How do you know that an adequate
Specimen was submitted for rapid
EIA assays???
Throat swabs are even worse!
Samples for Diagnosis of Viral Respiratory Infections
Lung biopsy
Bronchial alveolar
lavage/wash/brush
Nasopharygeal secretion
Nasopharygeal wash
Induced sputum
Nasopharygeal swab
Nasal wash


Throat swab (adenovirus only)
Saliva
Blood?
Sputum
DFA/EIA
OIA
Culture
LRTC*present
LRTC cells absent
Reagent Cost

Skin and Soft Tissue (Wound) Cultures
* Collect with steel (needle aspirate or scalpel)
* Discourage the use of swabs
* If infection NOT suspected, DON’T culture
* Get infected tissue or body fluid [ discourage swabs! ]
* -use something sharp ( syringe, scalpel, etc )
* -close doesn’t count
* *Don’t culture the surface / get deep infected sample*
* Remove needles / send capped syringe with aspirate
* Share specimen: Microbiology-Surgical Path-Cytology
* ** Label specimen and site accurately
* ** Give appropriate history
(Matkoski C. Sharp SE, Kiska DL. Evaluation of the Q Score and Q234 Systems for cost-effective and clinically relevant interpretation of wound cultures. J Clin Microbiol 2006;44:1869-1872)

Principle #2: A specimen must be collected at the optimal time(s) in order to recover the pathogen(s) of interest
Principle #3: A sufficient quantity of the specimen must be obtained to perform the requested tests
Blood Cultures
* Volume of blood drawn is the single most important factor influencing sensitivity. A single set for an adult blood culture consists of one aerobic and one anaerobic bottle. Optimally 10 mL of blood should be inoculated into each bottle. Volume of blood for a pediatric culture can be related to the infants weight
* Solitary blood cultures should be less than 5% (Arch Pathol Lab Med. 2001 125:1290-1294)
* If only enough blood can be drawn for one bottle, inoculate the aerobic bottle.
o 644 positive blood cultures, 59.8% from both bottles, 29.8% from aerobic bottle only and 10.4% from anaerobic bottle only (J Infect Chemother 9:227, 2003).
Pediatric Blood Cultures - Volume
Surgical Specimens (Other Shared Specimens)
TISSUE
FLUID
Specimen size of pea or larger
Divide
Anaerobic transport tube
Hold
upright,
uncap,
insert specimen and recap
Anaerobic
Culture
and stain

COLLECTION AND HANDLING OF OPERATING ROOM SPECIMENS FOR MICROBIOLOGY
Acceptable Specimens For Anaerobic Culture

Principle #4: Appropriate collection devices and specimen containers must be used to ensure recovery of all organisms
Recovery of Anaerobic Bacteria Placed in in Aerobic/Anaerobic Transport Media
CVP = Copan Vi-Pak Amies Agar Gel collection and transport swabs
SSS = Starplex StarSwab II,
PAC = BBL Port-A-Cult

How Does Transport Time Affect Yields?
J Clin Microbiol. 2001:39 377-380

Suggested Transport Media – General Comments

Principle #5: Collect all microbiology test samples prior to the institution of antibiotics
Principle #6: The specimen container must be properly labeled and sealed prior to transport
Principle #7: Minimize transport time or maximize transport media. There is always some loss of viability during transport
Minimize transport time and maximize use of transport media as much as possible
Environmentally Fragile Organisms
QA monitor??
Principle #8: Special handling/Collection instruction must be followed
* First, communicate with those that are doing collections.
* Collection instructions are written and available.
* Get involved with nursing orientation/education days and ask to have the instructions given out; poster board learning; quiz or competencies.
* Talk to providers when there are problems with specimen collection; they sometimes do not know they could do it better.


Principle #9: Improper specimen Collected for Ordered Test

Criteria For Rejection of Microbiological Specimens
* Criteria for rejection must be readily available and laboratory specific
* Unlabeled or improperly labeled specimen
* Prolonged storage or transport
* Improper or damaged container
* Specimen received in fixative
* Oropharyngeal contaminated sputum
* Duplicate specimens stools, sputum) within a 24 hour period. Exceptions cleared by the laboratory
* Specimens unsuitable for culture request (anaerobic culture from not acceptable source, urine from Foley catheter)
* Dry Swab
* 24-hr collection of urine or sputum for AFB or fungal culture
* Other criteria specific to your laboratory

Cultures That Should Include a Gram Stain
* CSF or sterile body fluid (cytospin)
* Eye
* Purulent discharge
* Sputum or transtracheal aspirate
* All surgical specimens
* Tissue
* Urethral exudates (male only, intracellular gonococcus))
* Vaginal specimens
* Wounds

Summary
* Publish specific rules for specimen collection
o There will be exceptions!
+ Make physician or healthcare provider aware of implications of culturing suboptimal specimens
* Communicate, communicate, communicate!
o Real time feedback
o Contact the health care worker who collected the suboptimal specimen

References

* Clinical Microbiology Procedures Handbook. 2nd Edition. . HD Isenberg ed. ASM. Cumitechs. ASM Press. Wash. DC.
* Manual of Clinical Microbiology, 9th Edition. ASM Press. Wash. DC. 2007.Miller MJ.
* A Guide To Specimen Management in Clinical Microbiology. ASM Press. Wash. DC. 1999.

Clinically Relevant Microbiology Starts at the Source.ppt

Read more...

Specimen Collection and Laboratory Diagnosis of Lower Respiratory Infections

Specimen Collection and Laboratory Diagnosis of Lower Respiratory Infections

By:Mohammad Rahbar (PhD)
Department Of Microbiology Reference Laboratory of Iran

Anatomy of Respiratory Tract

“ 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
o Aging population
o Increase in patients with immunocompromising conditions
* Overtreatment has lead to resistance
o Multidrug resistant Streptococcus pneumoniae
o Resistance among hospital acquired pathogens such as Acinetobacter, Pseudomonas aeruginosa E.coli K.pneumonia (ESBLs) MRSA and others
* Major sections
o Clinical aspects of diseases of LRT
o Specimen collection
o Specimen processing
o Interpretation of bacterial cultures
o Most common pathogens
o Methods for implementing change
o Guidelines for frequency of testing
o Public health issues
o 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
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
o Food should not have been ingested for 1-2 h prior to expectoration
o The mouth should be rinsed with saline or water
o Patient should breathe and cough deeply
o Patient should expectorate into a sterile container
* Transport container immediately to lab
* Perform Gram stain and plant specimen as soon as possible

Sputum collection
* Sputum of less than 2ml should not be processed unless obviously purulent
* Only 1 sputum per 24hr .submitted
* Some scoring system should be used to reject specimen that re oral contamination.
* Transportation in <2 hr is recommended with refrigeration if delays anticipated.
* Handle all samples using universal precautions.
* Perform Gram stain and plant specimen as soon as possible

Induced sputum
Patients who are unable to produce sputum may be assisted by respiratory therapy technician. Aerosol induced specimen are collected by allowing the patient to breath aerosolized droplets of a solution of 15% sodium chloride and 10% glycerin for approximately 10 minute . obtaining such specimen may avoid the need for a more invasive procedures ,such as bronchoscopy or needle aspiration, in many cases.

Gastric aspiration
* The gastric aspiration is used exclusively for isolation of acid-fast bacilli and may be collected from patients who are unable to produce sputum, particularly young children. The relative resistance of mycobacteria allows them to remain viable for a short period. Gastric lavage must be delivered to the lab immediately so that the acidity can be neutralized. Specimen can be first neutralized and then transported if immediate delivery is not possible.

Sputum Gram Stain Unacceptable
Sputum Gram Stain Good Quality
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)
o Semiquantitate and report organism with descriptive
o If no predominant organism is present, report “mixed gram positive and gram negative flora”

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
* Sputum collected under supervision of nurse or resident
o Samples were processed immediately
o Screened for epithelial cells
o Screened for predominant morphotype (> 75% of the organisms seen)
o 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
o Moderate neutrophils
o Moderate Gram positive diplococci suggestive of Streptococcus pneumoniae
o 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
o < normal flora, quantify and limit ID; no susceptibility
o Add comment that organism not predominant on stain
* ID mould, Mycobacteria or Nocardia spp.

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

ATS Guidelines Diagnostic Tests for CAP
* Empiric therapy for outpatients
o Macrolide or tetracycline
* Hospitalized patients with CAP
o 2 sets of pre-treatment blood cultures
o Pleural fluid Gram stain/culture when appropriate
o Studies for Legionella, Mtb, fungi in select patients
o Sputum Gram stain/culture only if resistant or unusual pathogen is suspected
o 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 (%)
o Gram positive cocci
+ S. aureus 17
+ S. pneumoniae 2-20

AGENTS OF HAP
* Aerobic gram-neg bacilli 60
o Pseudomonas aeruginosa
o Enterobacter sp.
o Klebsiella pneumoniae
o Acinetobacter
o Legionella
o Anaerobes 10-20
o 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
o Bronchial brushings
o Bronchial washes
o Protected specimen brushing
o Bronchoalveolar lavage specimens (BAL)
o Transbronchial biopsy

Respiratory Specimens
* Protected Brush Specimen
o To procure uncontaminated lower airway secretions
o Brush within 2 catheters
* Bronchoalveolar Lavage (BAL)
o Samples large area of the lung
o Performed using a bronchoscope
o 100 to 250 ml of saline injected
o Injected saline along with secretions is collected by aspiration
* Transthoracic Aspiration
o Involves percutaneous introduction of a needle directly into the infiltrate

Bronchoalveolar Lavage (BAL) Specimen Acceptability
* Microscopic examination of Gram-stained smear
o Acceptable
+ <1% of cells present are squamous epithelial cells
o 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
o Aerobic bacterial culture and Gram stain
o Anaerobic bacterial culture
o Limited volume
* Bronchoscopy brush, unprotected
o No anaerobic culture
o Limited volume
* Bronchial washings
o Useful only for pneumonia caused by strict pathogens
o Reasonable requests: Mtb, Fungi, Legionella, Pneumocystis
* Bronchoalveolar lavage
o No anaerobe culture
o Amenable to extensive testing for all opportunistic pathogens

Interpretation of Quantitative PSB/BAL
* Dilution Method
o Quantify each morphotype present and express as CFU/ml
* Calibrated Loop Method
o Quantify each morphotype present and express as log10 colony count ranges
* Thresholds for significance
o PSB > 103 CFU/ml
o BAL > 104 CFU/ml

Bronchoscopy Samples Quantitative Methods
Routine culture
* Most of the commonly sought etiologic agents of lower respiratory tract infection will isolated on routinely used media : 5% sheep blood agar ,MacConkey agar for isolation and differentiation of gram-negative bacilli ,and chocolate agar for Neisseria spp and Haemophilus
* Because of contaminating oral flora ,sputum specimens ,specimens obtained by bronchial washing, and lavage trachestomy, or endotracheal tube aspirates are not inoculated to enriched broth or incubated anaerobically. Only specimens obtained by percutaneous aspiration (including transtracheal aspiration )and by protected bronchial brush are suitable for anaerobic culture: he latter must be done quantitatively for proper interpretation.
* Transtracheal and percutaneous lung aspiration material may be inoculated to enriched thioglycollate ,as well as to solid media. For suspected cases of legionnaires disease buffered charcoal –yeast extract (BCYE) agar and selective BCYE are inoculated.

* Sputum specimens from patients known to have cystic fibrosis should be inoculated to selective agar ,such as manitol salt agar for recovery of S .aureus and selective horse blood-bacitracin ,incubated anaerobically and aerobically ,for recovery of H,influenzae that may be obscured by the mucoid P,aeroginosa on routine media. The use of selective medium for B.cepacia ,such as PC or OFPBL agar ,is also recommended

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

Specimen Collection and Laboratory Diagnosis of Lower Respiratory Infections.ppt

Read more...