Principle of diagnostic medical microbiology

Introduction

Diagnostic medical microbiology is concerned with the etiologic diagnosis of infection. Laboratory procedures used in the diagnosis of infectious disease in humans include the following:

(1)  Morphologic identification of the agent in stains of specimens or sections of tissues (light and electron microscopy).

(2)  Culture isolation and identification of the agent.

(3)  Detection of antigen from the agent by immunologic assay (latex agglutination, EIA, etc) or by fluorescein-labeled (or peroxidase-labeled) antibody stains.

(4)  DNA-DNA or DNA-RNA hybridization to detect pathogen-specific genes in patients'  specimens.

(5)  Detection and amplification of organism nucleic acid in patients' specimens.

(6)  Demonstration of meaningful antibody or cell-mediated immune responses to an infectious agent.

In the field of infectious diseases, laboratory test results depend largely on the quality of the specimen, the timing and the care with which it is collected, and the technical proficiency and experience of laboratory personnel. 

Communication between Physician & Laboratory

Diagnostic microbiology encompasses the characterization of thousands of agents that cause or  are  associated  with  infectious  diseases.  The  techniques  used  to  characterize  infectious agents  vary  greatly  depending  upon  the  clinical  syndrome  and  the  type  of  agent  being considered,  be  it  virus,  bacterium,  fungus,  or  other  parasite.  Because  no  single  test  will permit  isolation  or  characterization  of  all  potential  pathogens,  clinical  information  is  much more important  for  diagnostic  microbiology than it is  for  clinical chemistry  or  hematology. The  clinician  must  make  a  tentative  diagnosis  rather  than  wait  until  laboratory  results  are available.  When  tests  are  requested,  the  physician  should  inform  the  laboratory  staff  of  the tentative  diagnosis  (type  of  infection  or  infectious  agent  suspected).  Proper  labeling  of specimens  includes  such  clinical  data  as  well  as  the  patient's  identifying  data  (at  least  two methods  of  definitive  identification)  and  the  requesting  physician's  name  and  pertinent contact information. Many  pathogenic  microorganisms grow  slowly, and  days or  even  weeks  may elapse  before they are isolated and identified. Treatment cannot be deferred until this process is complete. After  obtaining  the  proper  specimens  and  informing  the  laboratory  of  the  tentative  clinical diagnosis, the physician should begin treatment with drugs aimed at the organism thought to be responsible  for  the patient's illness.  As the  laboratory  staff  begins to  obtain results, they inform the physician, who can then reevaluate the diagnosis and clinical course of the patient and perhaps make changes in the therapeutic program. This "feedback" information from the laboratory  consists  of  preliminary  reports  of  the  results  of  individual  steps  in  the  isolation and identification of the causative agent.  

Diagnosis of Bacterial & Fungal Infections

Specimens

Laboratory  examination  usually  includes  microscopic  study  of  fresh  unstained  and  stained materials and preparation of cultures with conditions suitable for growth of a wide variety of microorganisms, including the type of organism most likely to be causative based on clinical evidence.  If  a  microorganism  is  isolated,  complete  identification  may  then  be  pursued. Isolated  microorganisms  may  be  tested  for  susceptibility  to  antimicrobial  drugs.  When significant pathogens are isolated before treatment, follow-up laboratory examinations during and after treatment may be appropriate.

A  properly  collected  specimen  is  the  single  most  important  step  in  the  diagnosis  of  an infection,  because  the  results  of  diagnostic  tests  for  infectious  diseases  depend  upon  the selection,  timing,  and  method  of  collection  of  specimens.  Bacteria  and  fungi  grow  and  die, are  susceptible  to  many  chemicals,  and  can  be  found  at  different  anatomic  sites  and  in different body fluids and tissues during the course of infectious diseases. Because isolation of the  agent  is  so  important  in  the  formulation  of  a  diagnosis,  the  specimen  must  be  obtained from  the  site  most  likely  to  yield  the  agent  at  that  particular  stage  of  illness  and  must  be handled  in  such  a  way  as  to  favor  the  agent's  survival  and  growth..  Any  type  of microorganism cultured from blood, cerebrospinal fluid, joint fluid, or the pleural cavity is a significant diagnostic  finding. Conversely,  many  parts of the  body  have a  normal  microbial flora  that  may  be  altered  by  endogenous  or  exogenous  influences.  Recovery  of  potential pathogens  from  the  respiratory,  gastrointestinal,  or  genitourinary  tracts;  from  wounds;  or from  the  skin  must  be  considered  in  the  context  of  the  normal  flora  of  each  particular  site.  Microbiologic  data  must  be  correlated  with  clinical  information  in  order  to  arrive  at  a meaningful interpretation of the results.

 A few general rules apply to all specimens:

 (1) The quantity of material must be adequate.

(2)  The sample should be representative of the infectious process (eg, sputum, not saliva; pus from the underlying lesion, not from its sinus tract; a swab from the depth of the wound, not from its surface).

 (3) Contamination  of  the  specimen  must  be  avoided  by  using  only  sterile  equipment  and aseptic precautions.

(4) The specimen must be taken to the laboratory and examined promptly. Special transport media may be helpful.

 (5) Meaningful specimens to diagnose bacterial and fungal infections must be secured before antimicrobial  drugs are administered. If antimicrobial drugs are  given  before  specimens are taken  for  microbiologic  study,  drug  therapy  may  have  to  be  stopped  and  repeat  specimens obtained several days later.

Microscopy & Stains

Microscopic  examination  of  stained  or  unstained  specimens  is  a  relatively  simple  and inexpensive  but  much  less  sensitive  method  than  culture  for  detection  of  small  numbers  of bacteria. A specimen must contain at least 10⁵organisms per milliliter before it is likely that organisms will  be  seen  on  a  smear.  Liquid  medium  containing  10⁵ organisms  per  milliliter does  not  appear  turbid  to  the  eye.  Specimens containing  10²-10³ organisms  per  milliliter produce growth on solid media, and those containing ten or fewer bacteria per milliliter may produce growth in liquid media.

Gram  staining  is  a  very  useful  procedure  in  diagnostic  microbiology.  Most  specimens submitted  when  bacterial  infection  is  suspected  should  be  smeared  on  glass  slides,  Gram-stained,  and  examined  microscopically.  On  microscopic  examination,  the  Gram  reaction (purple-blue indicates gram-positive organisms; red, gram-negative) and morphology (shape: cocci, rods, fusiform, or other) of bacteria should be noted. The appearance of bacteria on Gram-stained smears does not permit identification of species. Reports of gram-positive cocci in chains are suggestive of, but not definitive for, streptococcal species; gram-positive cocci in clusters suggest a staphylococcal species. Gram-negative rods can be large, small,  or  even  coccobacillary.  Some  nonviable  gram-positive  bacteria  can  stain  gram-negatively.  Typically,  bacterial  morphology  has  been  defined  using  organisms  grown  on agar. However, bacteria in body fluids or tissue can have highly variable morphology.

Immunofluorescent  antibody  (IF)  staining  is  useful  in  the  identification  of  many microorganisms.  Such  procedures  are  more  specific  than  other  staining  techniques  but  also more  cumbersome  to perform. The  fluorescein-labeled antibodies in  common use are  made from  antisera  produced  by  injecting  animals  with  whole  organisms  or  complex  antigen mixtures.  The  resultant  polyclonal  antibodies  may  react  with  multiple  antigens  on  the organism that  was injected and  may also  cross-react  with antigens  of other microorganisms or  possibly  with  human  cells  in  the  specimen.  Quality  control  is  important  to  minimize nonspecific  IF  staining.  Use  of  monoclonal  antibodies  may  circumvent  the  problem  of nonspecific  staining.  IF  staining  is  most  useful  in  confirming  the  presence  of  specific organisms  such  as  Bordetella  pertussis  or  Legionella  pneumophila  in  colonies  isolated  on culture media. The use of direct IF staining on specimens from patients is more difficult and less specific.

Stains  such  as  calcofluor  white,  methenamine  silver,  and  occasionally  periodic  acid-Schiff (PAS) and  others are used for tissues and other specimens in  which  fungi  or other parasites are  present.  Such  stains  are  not  specific  for  given  microorganisms,  but  they  may  define structure  so  that  morphologic  criteria  can  be  used  for  identification.  Specimens  to  be examined  for  fungi  can  be  examined  unstained  after  treatment  with  a  solution  of  10% potassium hydroxide, which breaks down the tissue surrounding the fungal mycelia to allow a  better  view  of  the  hyphal  forms.  Phase  contrast  microscopy  is  sometimes  useful  in unstained specimens. Darkfield microscopy is used to detect  Treponema pallidum in material from primary or secondary syphilitic lesions.

Culture Systems

For diagnostic bacteriology, it is necessary to use several types of media for routine culture, particularly  when  the  possible  organisms  include  aerobic,  facultatively  anaerobic,  and obligately  anaerobic  bacteria.  The  standard  medium  for  specimens  is  blood  agar,  usually made with 5% sheep blood. Most aerobic and facultatively anaerobic organisms will grow on blood agar. Chocolate agar, a medium containing heated blood with or without supplements, is  a  second  necessary  medium;  some  organisms  that  do  not  grow  on  blood  agar,  including pathogenic neisseria and haemophilus, will grow on chocolate agar. A selective medium for enteric gram-negative rods (either MacConkey agar or eosin-methylene blue [EMB] agar) is a third type of medium used routinely. Specimens to be cultured for obligate anaerobes must be  plated  on  at  least  two  additional  types  of  media,  including  a  highly  supplemented  agar such  as  brucella  agar  with  hemin  and  vitamin  K  and  a  selective  medium  containing substances that inhibit the growth  of  enteric gram-negative rods and  facultatively anaerobic or anaerobic gram-positive cocci.

 Antigen Detection

Immunologic  systems  designed  to  detect  antigens  of  microorganisms  can  be  used  in  the diagnosis  of  specific  infections.  IF  tests  (direct  and  indirect  fluorescent  antibody  tests)  are one form of antigen detection .

Enzyme immunoassays (EIA), including enzyme-linked immunosorbent assays (ELISA) and  agglutination  tests  are  used  to  detect  antigens  of  infectious  agents  present  in  clinical specimens. The principles of these tests are reviewed briefly here.

There are many variations of EIAs to detect antigens. One commonly used format is to bind a capture  antibody,  specific  for  the  antigen  in  question,  to  the  wells  of  plastic  microdilution trays. The specimen containing the antigen is incubated in the wells followed by washing of the  wells.  A  second  antibody  for  the  antigen,  labeled  with  enzyme,  is  used  to  detect  the antigen.  Addition  of  the  substrate  for  the  enzyme  allows  detection  of  the  bound  antigen  by colorimetric  reaction.  A  significant  modification  of  EIAs  is  the  development  of immunochromatographic  membrane  formats  for  antigen  detection.  In  this  format,  a nitrocellulose membrane  is used to absorb the antigen  from a  specimen.  A colored reaction appears  directly  on  the  membrane  with  sequential  addition  of  conjugate  followed  by substrate.  In  some  formats,  the  antigen  is  captured  by  bound  antibody  directed  against  the antigen. These assays have the advantage of being rapid and also frequently include a built -in positive  control.  In  latex  agglutination  tests,  an  antigen-specific  antibody  (either  polyclonal or monoclonal) is fixed to latex beads. When the clinical specimen is added to a suspension of the latex beads, the antibodies bind to the antigens on the microorganism forming a lattice structure,  and  agglutination  of  the  beads  occurs.  Coagglutination  is  similar  to  latex agglutination  except  that staphylococci rich in  protein  A are used  instead  of latex  particles; coagglutination  is less useful  for antigen  detection compared  with  latex agglutination  but is helpful  when  applied  to  identification  of  bacteria  in  cultures.Latex  agglutination  tests  are primarily directed at the detection of carbohydrate antigens of encapsulated microorganisms. Another  form  of  EIA,  to  detect  antibody,  is  immunoblotting  ("Western  blot"),  whereby defined antigens are placed  on strips  of  nitrocellulose  paper.  Following incubation  with the test  antibody-containing  specimen,  the  strip  is  further  treated  with  an  enzyme-labeled antibody, usually from another animal, against the test antibody. Addition of the substrate for the enzyme allows detection of the antigen-specific bound antibody by colorimetric reaction. Western  blot  tests  are  used  as  the  specific  tests  for  antibodies  in  HIV  infection  and  Lyme disease.

Molecular Diagnostics

The  principle  behind  early  molecular  assays  is  the  hybridization  of  a  characterized  nucleic acid  probe  to  a  specific  nucleic  acid  sequence  in  a  test  specimen  followed  by  detection  of the  paired  hybrid.  For  example,  single-stranded  probe  DNA  (or  RNA)  is  used  to  detect complementary RNA or denatured DNA in a test specimen. The nucleic acid probe typically is labeled with enzymes, antigenic substrates, chemiluminescent molecules, or radioisotopes to  facilitate  detection  of  the  hybridization  product.  By  carefully  selecting  the  probe  or making a specific oligonucleotide and performing the hybridization under conditions of high stringency, detection of the nucleic acid in the test specimen can be extremely specific. Such assays  are  currently  used  primarily  for  rapid  confirmation  of  a  pathogen  once  growth  is detected, eg, the identification of Mycobacterium tuberculosis in culture using the Gen-Probe Inc. (San Diego, CA) DNA-probe.

Identifying Bacteria Using 16S rRNA

The 16S rRNA  of  each  species  of  bacteria has  stable  (conserved)  portions of  the  sequence. Many  copies  are  present  in  each  organism.  Labeled  probes  specific  for  the  16S  rRNA  of  a species are added, and the amount of label on the  double-stranded hybrid is  measured. This technique  is  widely  used  for  the  rapid  identification  of  many  organisms.  Examples include the most common and important  Mycobacterium species, Coccidioides immitis, Histoplasma capsulatum, and others. Molecular diagnostic assays that use amplification techniques have become widely used and are evolving rapidly. 

Target Amplification Systems

In  these  assays,  the  target  DNA  or  RNA  is  amplified  many  times.  The  polymerase  chain reaction  (PCR)  is  used  to  amplify  extremely  small  amounts  of  specific  DNA  present  in  a clinical  specimen,  making  it  possible  to  detect  what  were  initially  minute  amounts  of  the DNA.  PCR  uses  a  thermostable  DNA  polymerase  to  produce  a  twofold  amplification  of target  DNA  with  each  temperature  cycle.  The  DNA  extracted  from  the  clinical  specimen along  with  sequence-specific  oligonucleotide  primers,  nucleotides,  thermostable  DNA polymerase, and buffer are heated to 90–95 °C to denature (separate) the two strands of the target DNA. The temperature in the reaction is lowered, usually to 45–60 °C depending upon the  primers,  to  allow  annealing  of  the  primers  to  the  target  DNA.  Each  primer  is  then extended by the thermostable DNA polymerase by adding nucleotides complementary to the target  DNA  yielding  the  twofold  amplification.  The  cycle  is  then  repeated  30–40  times  to yield amplification of the target DNA segment by as much as 10⁵  to 10⁶fold. The amplified segment  often  can  be  seen  in  an  electrophoretic  gel  or  detected  by  Southern  blot  analysis using labeled DNA probes specific for the segment or by a variety of proprietary commercial techniques.

PCR  can  also  be  performed  on  RNA  targets,  which  is  called  reverse  transcriptase  PCR.

The  enzyme  reverse  transcriptase  is  used  to  transcribe  the  RNA  into  complementary  DNA for amplification.

PCR  assays  are  available  commercially  for  identification  of  Chlamydia  trachomatis, Neisseria  gonorrhoeae,  Mycobacterium  tuberculosis,  cytomegalovirus,  enteroviruses,  and many  others.  An  assay  is  available  for  HIV-1  viral  load  testing  also.  There  are  many  other "in-house" PCRs that have been developed by individual laboratories to diagnose infections. Such assays are the tests of choice to diagnose many infections—especially when traditional culture  and  antigen  detection  techniques  do  not  work  well.  Examples  include  testing  of cerebrospinal  fluid  for  herpes  simplex  virus  to  diagnose  herpes  encephalitis  and  testing  of nasopharyngeal wash fluid to diagnose Bordetella pertussis infection (whooping cough).

A  major  consideration for laboratories that perform PCR assays is to  prevent contamination of  reagents  or  specimens  with  target  DNA  from  the  environment,  which  can  obscure  the distinction  between  truly  positive  results  and  falsely  positive  ones  because  of  the contamination.