DIAGNOSTIC MEDICAL MICROBIOLOGY

PRINCIPLES OF DIAGNOSTIC MEDICAL MICROBIOLOGY CONTD....
DIAGNOSIS OF INFECTION BY ANATOMIC SITE

Wounds,  Tissues,  Bones,  Abscesses,  & Fluids:
Microscopic study of smears and culture of specimens from wounds or abscesses often gives early and
important indications of the nature of the infecting organism and thus helps in the choice of antimicrobial drugs. Specimens from diagnostic tissue biopsies should be submitted for bacteriologic as well as histologic
examination.  Such specimens for bacteriologic examination are kept away from fixatives and disinfectants,minced,  and cultured by a variety of methods.
The pus in closed,  undrained soft tissue abscesses frequently contains only one organism as the infecting agent;most commonly staphylococci,  streptococci,  or enteric gram-negative rods.  The same is true in acute
osteomyelitis,  where the organisms can often be cultured from blood before the infection has become chronic.Multiple microorganisms are frequently encountered in abdominal abscesses and abscesses contiguous with mucosal surfaces as well as in open wounds.  When deep suppurating lesions,  such as chronic osteomyelitis,drain onto exterior surfaces through a sinus or fistula,  the flora of the surface through which the lesion drains must not be mistaken for that of the deep lesion.  Instead,  specimens should be aspirated from the primary infection through uninfected tissue. Bacteriologic examination of pus from closed or deep lesions must include culture by anaerobic methods. Anaerobic bacteria (Bacteroides,  peptostreptococci) sometimes play an essential causative role,  and mixtures of anaerobes are often present.
The methods used for cultures must be suitable for the semi-quantitative recovery of common bacteria and also for recovery of specialized microorganisms,  including mycobacteria and fungi.  Eroded skin and mucous
membranes are frequently the sites of yeast or fungus infections.  Candida,  Aspergillus,  and other yeasts or
fungi can be seen microscopically in smears or scrapings from suspicious areas and can be grown in cultures.
 Treatment of a specimen with KOH and calcofluor white greatly enhances the observation of yeasts and molds in the specimen. Exudates that have collected in the pleural,  peritoneal,  pericardial,  or synovial spaces must be aspirated with aseptic technique.  If the material is frankly purulent,  smears and cultures are made directly .  If the fluid is clear ,it can be centrifuged at high speed for 10 minutes and the sediment used for stained smears and cultures. 
 The culture method used must be suitable for the growth of organisms suspected on clinical grounds—eg,
mycobacteria,  anaerobic organisms—as well as the commonly encountered pyogenic bacteria.  Some fluid
specimens clot,  and culture of an anticoagulated specimen may be necessary .  The following chemistry and
hematology results are suggestive of infection: specific gravity > 1.018,  protein content >3 g/dL (often
resulting in clotting),  and cell counts >500–1000/ L.  Polymorphonuclear leukocytes predominate in acute
untreated pyogenic infections; lymphocytes or monocytes predominate in chronic infections.  Transudates 
resulting from neoplastic growth may grossly resemble infectious exudates by appearing bloody or purulent and by clotting on standing.  Cytologic study of smears or of sections of centrifuged cells may prove the neoplastic nature of the process. 
1) BLOOD:  
Since bacteremia frequently portends life-threatening illness,  its early detection is essential.  Blood culture is the single most important procedure to detect systemic infection due to bacteria.  It provides valuable information for the management of febrile,  acutely ill patients with or without localizing symptoms and signs and is essential in any patient in whom infective endocarditis is suspected even if the patient does not appear acutely or severely ill.  In addition to its diagnostic significance,  recovery of an infectious agent from the blood provides invaluable aid in determining antimicrobial therapy .  Every effort should therefore be made to isolate the causative organisms in bacteremia. In healthy persons,  properly obtained blood specimens are sterile.  Although microorganisms from the normal respiratory and gastrointestinal flora occasionally enter the blood,  they are rapidly removed by the reticuloendothelial system.  These transients rarely affect the interpretation of blood culture results.  If a blood culture yields microorganisms,  this fact is of great clinical significance provided that contamination can be excluded.  Contamination of blood cultures with normal skin flora is most commonly due to errors in the blood collection procedure.  Therefore,  proper technique in performing a blood culture is essential. The following rules,  rigidly applied,  yield reliable results:
  1. Use strict aseptic technique.  Wear gloves—they do not have to be sterile.
  2. Apply a tourniquet & locate a fixed vein by touch.Release tourniquet while the skin is being prepared.
  3. Prepare skin for vein puncture by cleansing vigorously with 70%-95% isopropyl alcohol.Using 2% tincture of iodine or 2% chlorhexidine, start a vein puncture site and cleanse the skin in concentric circles of increasing diameter .  Allow the antiseptic preparation to dry for at least 30 seconds.  Do not touch the skin after it has been prepared.
  4. Reapply the tourniquet,  perform venipuncture,  and (for adults) withdraw approximately 20 mL of blood..
  5. Add the blood to labeled aerobic and anaerobic blood culture bottles.
  6.  Take specimens to the laboratory promptly ,  or place them in an incubator at 37°C.

Several factors determine whether blood cultures will yield positive results: the volume of blood cultured,  the
dilution of blood in the culture medium,  the use of both aerobic and anaerobic culture media,  and the duration of incubation.  F or adults,  20 to 30 mL per culture is usually obtained,  and half is placed in an aerobic blood culture bottle and half in an anaerobic one,  with one pair of bottles comprising a single blood culture.  However, different volumes of blood may be required for the many different blood culture systems that exist.  One widely used blood culture system uses bottles that hold 5 mL rather than 10 mL of blood.  An optimal dilution of blood in a liquid culture medium is 1:300–1:150; this minimizes the effects of the antibody,complement,& white blood cell antibacterial systems that are present.  Because such large dilutions are impractical in blood cultures,most such media contain 0.05% sodium polyanetholesulfonate (SPS),  which inhibits the antibacterial systems. However ,  SPS also inhibits growth of some neisseriae and anaerobic gram-positive cocci and of Gardnerella vaginalis.  If any of these organisms are suspected,  alternative blood culture systems without SPS should be used.
Blood cultures are incubated for 5–7 days.  Automated blood culture systems use a variety of methods to detect positive cultures.  These automated methods allow frequent monitoring of the cultures—as often as every few minutes—and earlier detection of positive ones.  The media in the automated blood culture systems are so enriched and the detection systems so sensitive that blood cultures using the automated systems do not need to be processed for more than 5 days.  In general,  subcultures are indicated only when the machine indicates that the culture is positive.  Manual blood culture systems are obsolete and are likely to be used only in laboratories in developing countries that lack the resources to purchase automated blood culturing systems.
 In manual systems,  the blood culture bottles are examined two or three times a day for the first 2 days and daily thereafter for 1 week.  In the manual method,  blind subcultures of all the blood culture bottles on days 2 and 7 may be necessary. The number of blood specimens that should be drawn for cultures and the period of time over which this is done depend in part upon the severity of the clinical illness.  In hyperacute infections,  eg,  gram-negative sepsis with shock or staphylococcal sepsis,  it is appropriate to culture two blood specimens obtained from different anatomic sites over a period of 5–10 minutes.  In other bacteremic infections,  eg,  subacute endocarditis,3 blood specimens should be obtained over 24 hours.  A total of three blood cultures yields the infecting bacteria in more than 95% of bacteremic patients.  If the initial three cultures are negative and occult abscess,  fever of unexplained origin,  or some other obscure infection is suspected,  additional blood specimens should be cultured when possible before antimicrobial therapy is started.Several types of blood culture bottles are available that contain resins or other substances that absorb most antimicrobial drugs and some antimicrobial host factors as well.  Indications for the use of the resin-containing bottles include the following: a clinically septic patient receiving antimicrobial therapy who already had negative sets of blood cultures; a patient with clinical evidence of endocarditis and negative blood cultures and who is receiving antimicrobial therapy; and a patient admitted to the hospital with sepsis who had been given antimicrobial therapy prior to admission.  The resin-containing bottles should not be used to follow the effectiveness of therapy because the resin may absorb antimicrobials in the specimen and allow the culture to turn positive in spite of clinically efficacious therapy. It is necessary to determine the significance of a positive blood culture.
 The following criteria may be helpful in differentiating "true positives" from contaminated specimens:
1.   Growth of the same organism in repeated cultures obtained at different times from separate anatomic sites strongly suggests true bacteremia.
2.   Growth of different organisms in different culture bottles suggests contamination but occasionally may
follow clinical problems such as enterovascular fistulas.
3.   Growth of normal skin flora,  eg,  coagulase-negative staphylococci,  diphtheroids (corynebacteria and
propionibacteria),  or anaerobic gram-positive cocci,  in only one of several cultures suggests contamination.
Growth of such organisms in more than one culture or from specimens from a patient with a vascular
prosthesis or central venous catheter enhances the likelihood that clinically significant bacteremia exists.

4.   Organisms such as viridans streptococci or enterococci are likely to grow in blood cultures from patients
suspected to have endocarditis,  and gram-negative rods such as E coli in blood cultures from patients with
clinical gram-negative sepsis.  Therefore,  when such "expected" organisms are found,  they are more apt to be etiologically significant.
The following are the bacterial species most commonly recovered in positive blood cultures: staphylococci,
including S aureus; viridans streptococci; enterococci,  including Enterococcus faecalis; gram-negative enteric bacteria,  including E coli and K pneumoniae; P aeruginosa; pneumococci; and H influenzae.  Candida species,other yeasts,  and some dimorphic fungi such as H capsulatum grow in blood cultures,  but many fungi are rarely ,  if ever ,  isolated from blood.  Cytomegalovirus and herpes simplex virus can occasionally be cultured from blood,  but most viruses and rickettsiae and chlamydiae are not cultured from blood.  Parasitic protozoa and helminths do not grow in blood cultures.
In most types of bacteremia,  examination of direct blood smears is not useful.  Diligent examination of Gram stained smears of the buffy coat from anticoagulated blood will occasionally show bacteria in patients with S
aureus infection,  clostridial sepsis,  or relapsing fever .  In some microbial infections (eg,  anthrax,  plague,
relapsing fever ,  rickettsiosis,  leptospirosis,  spirillosis,  psittacosis),  inoculation of blood into animals may give positive results more readily than does culture.  In practicality ,  this is almost never done.

CITED BY ANIL BHUJEL
Bsc Microbiology,TU.
Pokhara Bigyan Tatha Prabidhi Campus, Nayabazar-9, Pokhara.

REFERENCES:
Forbes BA,  Sahm DF ,  Weissfeld AS (editors): Bailey and Scott's Diagnostic Microbiology,  12th ed.  ASM Press, Washington,  DC,  2007.
Murray PR et al (editors): Manual of Clinical Microbiology,  9th ed.  ASM Press,  2007. Persing D et al (editors): Molecular Microbiology: Diagnostic Principles and Practice,  2nd edition,  ASM Press,  (In
Press).
Winn W et al (editors): Koneman's Color Atlas and Textbook of Diagnostic Microbiology 6th ed.  Lippincott Williams and Wilkins,  2006.









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