BACTERIOLOGY AND PARASITOLOGY SECTION

The Bacteriology and Parasitology section performs a wide range of diagnostic testing. Our specimens are received from both public and private agencies, hospitals, clinics and doctor’s offices. Specimens may be human or animal in origin, from the environment or foods. We are able to accommodate testing during outbreaks or increased incidence. We provide telephone consultation and referral of unusual requests to the CDC. The following is a brief description of the services that we provide.

REFERENCE BACTERIOLOGY: If possible, submit a pure isolate of the unknown bacteria for identification. Please provide prior laboratory results, i.e. gram stain, growth requirements, organism suspected etc. This serves to insure proper handling by this Laboratory upon receipt of the unknown. Serological typing for Haemophilus influenzae and grouping for Neisseria meningitidis is performed at no charge providing the specimen is from a normally sterile site, i.e. blood or spinal fluid. Submit isolates on appropriate growth supporting media in screw capped tubes. Avoid sending plates.

BORDETELLA PERTUSSIS: Nasopharyngeal swabs are the specimen of choice. Transport media, FA slides, swabs and specimen collection instructions are provided by the Health and Environmental Testing Laboratory. There is an estimated 95 % likelihood of a positive isolation in the first week of disease, dropping to less than 50 % in the fourth week.

DIPHTHERIA: Pure isolates for identification are accepted. Clinical material submitted in a suitable transport system, i.e. culturette, is also acceptable. Please call prior to the submission of specimens.

DNA PROBE TESTING FOR NEISSERIA GONORRHOEAE AND CHLAMYDIA TRACHOMATIS: There are two chlamydia DNA probes available. The amplified probe is offered for use on routine screening of endocervical and urethral swab specimens as well as male and female urine specimens.

The nonamplified DNA probe is used to test conjunctival specimens. A specimen collection kit different from the kits used for the amplified probe must be requested.

PARASITIC DISEASES: This service is no longer routinely offered by the laboratory. Please call for assistance during outbreaks or in identifying unusual parasites. Stools are examined for helminths and their ova, protozoa and cryptosporidium. Stools should be preserved using a two bottle transport system, one with 10% formalin and the other with PVA. Recovered helminths should be placed in a preservative solution such as formalin for shipment to the Laboratory. Intermittent passage of parasites by the host may require multiple specimens taken over a period of several days. Blood parasites are referred.

ENTERIC BACTERIOLOGY: Isolation, identification, serogrouping and serotyping of enteric species from stool or reference culture. There in no charge to confirm or perform serologic testing of isolates for Salmonella species and E. coli O157:H7. All E. coli O157:H7 isolates are also strain typed using Pulsed Field Gel Electrophoresis.

VIROLOGY SECTION

In the Virology section the major serologic test is for the diagnosis of human immunodeficiency virus (HIV-1 and HIV-2 screen), the virus known to cause acquired immunodeficiency or AIDS. The screening test, enzyme immunoassay test (EIA) is very sensitive and is used for detecting reactive specimens. The western blot is very specific and is used for confirmation of true positives to HIV-1

The Virology section also performs a non-invasive test procedure for HIV-1 using oral mucosal transudate which is secreted between the teeth instead of serum or plasma normally required. This is convenient for sexually transmitted (STD) clinics which do not have a qualified phlebotomist to draw blood on a routine basis or when the patient is reluctant to give a blood sample.

Other serologic tests include rubella (German measles), varicella zoster (Chicken Pox), rubeola (old fashion measles), mumps, cytomegalovirus, cryptococcus, toxoplasmosis, herpes simplex, Lyme Disease, syphilis, hepatitis B and C.

The isolation and identification of viruses is an important aspect of viral diagnosis. Since many viruses are readily transmissible, all clinical specimens must be handled in such a way as to minimize the risk of laboratory infection. Personnel can be immunized against hepatitis B, influenza and rabies if necessary to perform a task which subjects them to these agents. Using the procedure of universal precautions, personnel use lab coats, face shields, gloves, and safety biological cabinets. All equipment and discarded material are sterilized, disinfected or incinerated. During the year viruses, which cause respiratory, gastrointestinal, central nervous system infections and also rashes, are routinely cultured and identified using cell lines from human or monkey origin. They include herpes simplex virus, cytomegalovirus, respiratory syncytial virus, adenovirus, and enterovirus. Many nursing homes dealing with susceptible senior citizens are anxious to know when influenza A has infected their patients in order to institute effective chemotherapy. Recently we have been routinely offering a rapid antigen test that can be used for presumptive results. The cell culture isolation system is the most sensitive test but may take several days to detect influenza virus. Since the rapid test is less sensitive it must be backed up by the cell culture method. The viral isolates can subsequently be strain-identified by Hemagglutination inhibition and the results are sent to the influenza program at the Centers for Disease Control and Prevention program in Atlanta, Georgia. In this way, the most suitable vaccine strains can be used for the following influenza season for maximum protection of the immunized population.

Molecular Strain Typing
Pulsed Field Gel Electrophoresis

Strain typing is an invaluable tool for determining relatedness between isolates in a suspected outbreak. Each species of microorganisms has an almost limitless number of strains due to normal random mutations that occur in nature such as single base pair substitutions, deletion of individual genes, or even acquisition of DNA from other microbial species. Phenotypic characteristics such as bacterial biotyping, antibiograms, serotyping, and bacteriophage typing has been used to type strains. Unfortunately, these methods are limited by inconsistencies in their discriminating ability, their labor intensity, and their lack of reproducibility. Pulsed field gel electrophoresis (PFGE) is a molecular subtying method based on genotypic characteristics of an organism. It produces clearly interpretable results that are reproducible and have a highly discriminatory capability. It involves the separation of specific DNA fragments obtained from the whole DNA of an individual isolate by a unique electrophoresis technique capable of separating large DNA fragments, ³ 1000 Kb, as well as small fragments. The DNA "fingerprint" produced can be compared to that of other isolates to determine their probable relatedness.

There are three primary applications for strain typing:

1. Assessing relatedness among multiple isolates from an individual patient can help differentiate the true cause of an acute infection from independent contaminants.

2. Differentiate a relapse of an old infection from a new infection on a given patient.

3. Distinguish a true outbreak of a single strain of organism and linking the outbreak to a given source. Identification of the reservoir and mode of transmission of a pathogen is a critical part of recognizing and controlling disease outbreaks, nosocomial or the public community. Differentiating a true outbreak from random coincidental occurrences prevents costly, time consuming investigations. This powerful tool can also link multiple outbreaks that would otherwise not have been connected.

We now offer this technology to assist hospital infection control programs and to aid in diagnostic patient care. Typing of Staphylococcus aureus isolates, particularly methicillin resistant strains, is available at no charge and we hope to offer PFGE typing for other nosocomial organisms as well.

Maine is currently an active participant of PulseNET. PulseNET is a national network of public health laboratories that perform DNA ‘fingerprinting" on foodborne bacteria. The network permits rapid comparison of these "fingerprint" patterns through an electronic database at the Centers for Disease Control and prevention (CDC). The foodborne bacterial pathogens currently being DNA subtyped by the HETL are E. coli O157:H7, Salmonella typhimurium, and other Salmonella serotypes. Other foodborne pathogens for which we have the capacity to type by PFGE are Listeria and Yersinia.

For more information on submitting specimens for molecular subtyping by PFGE, please contact the Pulsed Field Laboratory at the following address, fax, or telephone.

Health and Environmental Testing Laboratory
Donna Wrigley, Microbiologist
PFGE Laboratory
221 State St., Station 12
Augusta, ME 04330
Tel: (207) 287-2727  FAX: (207) 287-6832