Microbiology and Molecular Diagnostics - Infectious Diseases

Faculty Director:                         Melinda Poulter, PhD

Scientific Director:                       Larry Silverman, PhD, D (ABCC),(ABMG),F.A.C.M.G

MANAGER:                                   Susan Spanos, MS ,BS M(ASCP)

TECHNICAL SPECIALIST:             Jim Bowden, CLSp (MB)

SUPERVISORS:

Accession                                    Frankie Brewster, MT (ASCP)

Bacteriology                                Joanne Carroll, MT(ASCP)

Mycobacteriology & Mycology      Michelle Sheurenbrand MT (ASCP)

                                                   Sharon Dudley MT (ASCP)

Virology                                       Shirley Kyger, I (ASCP)

TELEPHONE:                               924-LABS (924-5227)

DIRECTOR-ON-CALL:                   PIC 1221

HOURS:                                       Open 24 hours/day

General Guidelines and Policies for Microbiology

The most important contribution to the effectiveness of the microbiology laboratory is the specimen that is appropriately selected, collected and transported. Since specimens for microbiological analysis are likely to contain living organisms, specimen collection, handling and transport should be accomplished with the following factors in mind:

1. Select the correct anatomic site from which to obtain the specimen and communicate this to the laboratory via the appropriate request mechanism (see Section I General Policies).

2. Collect the specimen using the proper technique and supplies (refer to the individual test listings for this information).

3. Package the specimen in a container designed to promote survival of the causative organism and to eliminate leakage. Label completely (refer to Section I General Policies B).

4. Transport the specimen to the laboratory expeditiously or make sure that, if it must be stored, the storage conditions are appropriate for the suspected organism.

It is also important to note that the laboratory needs specific and critical information from the physician regarding the patient and the specimen.  Please refer to Section I for detailed information about requesting laboratory analysis. If a particular agent/organism is suspected, please communicate this fact to the laboratory when making requests for analysis.

If a specimen is determined unacceptable for culture, it will be retained until the requesting physician is notified.

The report of "No Significant Change" indicates that the same organism/s was isolated from the same site within the last 72 hours. After 72 hours, cultures submitted from the same site will be repeated with full identification and susceptibility if appropriate.  The laboratory will evaluate culture results and perform identification and susceptibility on potential pathogens for the clinical site. An additional charge will be generated for each identification and susceptibility performed. Reports will be updated daily via the laboratory and hospital computer system.

Bacteriology Specimen Submission Guidelines

Anaerobic- cultures should be performed on the following specimens when  the specimens are submitted in an approved transport container, appropriately labeled and ordered, and meet the requirements for anaerobic culture:

Aspirates

Sterile Body Fluids

Bone Tissues

Tissue/Biopsy

Specimens for anaerobic culture require special collection and transport techniques to prevent loss of anaerobic conditions. Culturettes are unacceptable for anaerobic cultures.  Aspirates or biopsies are the preferred specimens.  Sterile body fluids should be submitted in a sterile cup or a sterile black top tube. Sterile fluids should not be submitted in blood culture bottles because no gram stain or quantification of organisms can be made. Place tissues in a sterile cup. Tissue specimens should be no larger than pea size. All specimens for anaerobic culture should be submitted to the laboratory within one hour after collection.  In instances where the above specimens are not ordered for anaerobic culture, but are appropriately collected and submitted, the laboratory will add anaerobic media to detect anaerobic bacteria that may be present in the specimen.  An additional charge will be assessed.

Samples from superficial sites or sites that have anaerobic normal flora are not suitable for anaerobic culture. If unacceptable specimens are received with requests for anaerobic testing, physicians or units will be notified by computer.

Aerobic (but not anaerobic) cultures are performed on virtually any body site provided the specimen is submitted in an approved transport container, appropriately labeled and ordered. See individual test listings for specific specimen requirements. All swabs should be submitted using the Culturette II system available from the hospital storeroom. Blood Cultures: A two bottle CO₂ detection system is employed for the recovery of aerobic and anaerobic bacteria and yeast. Bottles are available on nursing units or from the hospital storeroom. A peptide nucleic acid fluorescent in situ hybridization test (PNA FISH) will be used within several hours on smears from positive blood cultures. The test will detect Staph aureus, Enterococcus faecalis or Candida albicans within several hours of the positive blood culture detection. If routine methods of culture fail to provide a microbiological diagnosis, the physician is encouraged to consult with the senior microbiology staff.  If unusual organisms are suspected, the laboratory must be notified by phone and the suspected agent should be noted on the requisition

Reporting of Results: All inpatient results will be reported through the hospital information system. Preliminary reports will be available by computer immediately after the completion of a screening test or the first reading. All cultures, as appropriate, are evaluated each day. Reports will be updated daily or whenever new information becomes available. The nursing staff in the intensive care units, the physician, or if appropriate, the chief resident of the service will be notified by telephone of positive blood, tissue and sterile body fluid cultures as well as reportable and/or communicable disease isolates.

Refer to appendix VII for definitions of reports.

Antimicrobial Susceptibility Testing and Reporting: Automated Minimum Inhibitory Concentrations (MIC) results are reported in ug/mL with interpretations and are performed routinely on most clinically significant organisms.

S,I,R assignments are not possible for some drug / microbe combinations due to insufficient validation data.

Standardized disk diffusion tests are performed on some isolates and are reported as:

Susceptible: The isolate appears to be susceptible to ordinarily achievable blood levels.

Intermediate: Susceptibility of the isolate is indeterminate; some strains may respond to concentrations achieved by high dosage or in areas of the body where the drug is concentrated (e.g.urine).

Resistant: The isolate is not completely inhibited by drug concentrations within the usual therapeutic range.

Streptococcus pneumoniae has become more resistant to antibiotics, approximately 56% of these isolates were resistant to penicillin in  2006.

Testing  will be done to confirm the presence of extended spectrum beta lactamases in the organisms E. col, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis according to NCCLS guidelines for 2007. A comment will be attached to the report to indicate the presence of these extended spectrum beta lactamases. Physicians should consult Infectious Diseases for treatment options. Enterococcus sp. will be tested for high level aminoglycosides, penicillin and vancomycin resistance. Vancomycin resistant Enterococcus faecium is frequently seen in our hospital. The physician is advised to consult Infectious Diseases for treatment of infections with resistant enterococci.

Mycobacteriology Specimen Submission Guidelines

Guidelines for obtaining specimens:

For cases of suspected pulmonary tuberculosis:

See test listing for Culture, AFB for optimal specimen types and volumes.

For outpatients, the three sputa can be collected at home in sterile containers, refrigerated, and all brought to the laboratory the same day.

Only 1 sputum and/or bronchoscopy specimen is accepted per day; three sputa and/or bronchoscopy specimens are accepted per week.  If the smears from these specimens are negative, another set of three specimens is accepted after one week if the suspicion of tuberculosis remains.

Once the diagnosis of tuberculosis is established from the initial series of three sputa, only one sputum is accepted every other week for AFB smear. An AFB culture is done from an acceptable sputum every other week. If more than one sputum is received by the laboratory within a week, the physician will be called to determine if the additional sputa are being submitted to discontinue isolation. Exception: to discontinue isolation requires 3 consecutive negative smears from sputa obtained at least 24 hours apart.

Three consecutive negative (AFB smears) from different days are required for release from isolation for Tuberculosis.

For cases of suspected non-pulmonary tuberculosis:

Swabs are not an appropriate collection device for most specimens submitted for mycobacterial culture.  If swabs must be submitted, however, a separate swab must be included for the AFB culture. The clinician should be aware that the sample on a swab is suboptimal for the isolation of a mycobacteria species.

For urogenital tuberculosis, collect 3 early-morning, voided, midstream or catheter urines on successive days.  For outpatients, the three urines can be collected at home in sterile containers, refrigerated, and all brought to the laboratory the same day.

All other specimens are collected as you would for a bacterial culture.

Identification/Reporting:

Molecular probes are used to identify M. tuberculosis, M. avium, and M. gordonae. Because the probe used for M. tuberulosis cannot distinguish between M. tuberculosis, M. bovis, BCG, M. African, M. microti, and M. canetti, a positive result is reported as M. tuberculosis complex.

The probe for M. avium cannot distinguish between M. avium, and M. intracellulare, therefore a positive result is reported as M. avium complex.

Mycobacteria not identified by probe are usually identified by 16S ribosomal DNA sequencing with results available within one to two weeks.

Susceptibility Testing:

M. tuberculosis complex is tested against isoniazid, streptomycin, ethambutol, rifampin and pyrazinamide (if the isolate is resistant to isoniazid). Between 1999 and 2006, 43 M. tuberculosis complex patient isolates were tested for susceptibility to INH.  6.9% of the isolates were INH resistant.

Mycology Specimen Submission Guidelines

Specimen requirements

See test listing Culture, Fungus

Suspicion of infection with the systemic fungal pathogens, Histoplasma capsulatum, Blastomyces dermatitidis, or Coccidioides immitis should be indicated on the test request.

Suspicion of infection with the unusual dermatophytes, Trichophyton verrucosum or Trichophyton schoenleinii should be indicated on the test request so the media can be incubated at the appropriate temperatures. If Malassezia species is suspected, please note this on the test request.

Susceptibility Testing

Candida species can be tested in house for susceptibility to flucytosine, fluconazole, and itraconazole. Additional susceptibilities and susceptibility testing on Nocardia species requires consultation with the director-on-call and must be accompanied by a patient history. These tests are performed by an accredited referral laboratory.

Virology Specimen Submission Guidelines

Culture and Direct Antigen Detection:

See individual test listings for Virus Cultures

Transport Media:

Viral transport medium (VTM) is available in the Clinical Microbiology Laboratory.  VTM should not directly contact patients. Very acidic (yellow) or alkaline (purple) media should not be used. Observe the expiration date on the label. The medium can not be used for bacterial culture specimens as it contains antibiotics.

Specimen Collection for virus isolation:

The collection of a proper specimen and its correct handling are critical steps in virus isolation. Specimens for virus isolation must be collected and processed in a different manner than those for routine microbiologic studies. The following guidelines should be followed in submitting specimens for virus isolation:

1. Only two specimens for viral culture from the same site will be accepted within a seven day period per patient. These two specimens must be at least 48 hours apart.

2. The amount of virus is usually maximal at or just after the appearance of symptoms, so that specimens should be collected as early as possible in the course of illness.

3.Most viruses are inactivated by adverse environmental conditions and/or delays in specimen processing.

a. Whenever possible, specimens for isolation should be submitted fresh in the morning. Specimens should be transported to the laboratory immediately after collection (keep all specimens except blood refrigerated until transport). Blood should be kept at room temperature.

b. For unavoidable delays, specimens may be held at 4^oC (refrigerator) overnight, but they should not be frozen.

c. Fluctuations in temperature, especially freezing and thawing, should be avoided. Certain viruses, such as respiratory syncytial, cytomegalovirus, and varicella-zoster virus, are particularly labile, and specimens suspected of containing these agents should be collected at times when they can be processed promptly without freezing.

d. Containers for virus transport should be made of glass or plastic and have airtight lids.

e. Swabs and other samples that could dry out in transport should be placed in vials containing viral transport medium (VTM). Dry swabs are not acceptable specimens. Culturette II swabs are not acceptable specimen systems for recovery of viruses.

f. Only cotton or dacron swabs with aluminum or plastic shafts should be used for collection. Calciumalginate swabs are not acceptable for collecting virus samples nor are swabs with wooden shafts.

4. Bacterial overgrowth can seriously hinder efforts at virus isolation. Specimens should be collected aseptically with attention to minimize contamination by microbial flora and should be processed promptly.

5. When a virus is initially isolated, from blood or sterile body fluids or from known immunocompromised patients, the physician will be notified by telephone.

Susceptibility Testing:

Susceptibility testing on isolates of CMV, HSV and VZV is performed by special request at an accredited referral laboratory.

Viral Serology:

Serological testing for viral antibodies should be carried out on acute and convalescent sera. Individual tests are listed in the index. Questions concerning testing for viral antibodies should be directed to the Clinical Immunology (Davis) Laboratory (924-5179).

Chart saved on legal landscape to be inserted at this location.

^aViruses which cannot be detected in our laboratory are not listed.

*For rapid testing for Influenza A and B virus, nasal washings give the best results.

Molecular Diagnositic Submission Guidelines

Molecular testing is available for several infectious diseases including:

Herpes Simplex Virus types I and II

Enterovirus

Bordetella pertussis

Bordetella parapertussis

Methcillin Resistant Staph aureus

Influenza A & B

HIV viral load and genotyping

HCV viral load and genotyping

Cytomegalovirus (viral load)

Chlamydia trachomatis

Neisseria gonorrhoeae

16 S ribosomal sequencing for various bacterial and fungal isolates

See individual test listing for the particular organism of interest.