Monday, August 11, 2014

DIAGNOSTIC MEDICAL MICROBIOLOGY AND CLINICAL CORRELATIONS

PRINCIPLES OF DIAGNOSTIC MEDICAL MICROBIOLOGY CONTD...

LABORATORY AIDS IN THE SELECTION OF ANTIMICROBIAL THERAPY
The antimicrobial drug used initially in the treatment of an infection is chosen on the basis of clinical impression after the physician is convinced that an infection exists and has made a tentative etiologic diagnosis on clinical grounds.  On the basis of this "best guess," a probable drug of choice can be selected.  Before this drug is administered,  specimens are obtained for laboratory isolation of the causative agent.  The results of these examinations may necessitate selection of a different drug.  The identification of certain microorganisms that are uniformly drug-susceptible eliminates the necessity for further testing and permits the selection of optimally effective drugs solely on the basis of experience.  Under other circumstances,  tests for drug susceptibility of isolated microorganisms may be helpful. 
The commonly performed disk diffusion susceptibility test must be used judiciously and interpreted with
restraint.  In general,  only one member of each major class of drugs is represented.  For staphylococci,  penicillin G,  oxacillin,  cefazolin,  erythromycin,  gentamicin,  and vancomycin are used.  For gram-negative rods,  ampicillin,cefazolin and second- and third-generation cephalosporins,  piperacillin and other "antipseudomonal penicillins,"carbapenems,  trimethoprim-sulfamethoxazole,  fluoroquinolones,  and the aminoglycosides (amikacin,tobramycin,  gentamicin) are included.  For urinary tract infections with gram-negative rods,  nitrofurantoin,quinolones,  and trimethoprim may be added.  The choice of drugs to be included in a routine susceptibility test battery should be based on the susceptibility patterns of isolates in the laboratory ,  the type of infection (community-acquired or nosocomial),  the source of the infection,  and cost efficacy analysis for the patient population. 
The sizes of zones of growth inhibition vary with the molecular characteristics of different drugs.  Thus,  the zone size of one drug cannot be compared to the zone size of another drug acting on the same organism.  However,for any one drug the zone size can be compared to a standard,  provided that media,  inoculum size, and other conditions are carefully regulated.  This makes it possible to define for each drug a minimum diameter of inhibition zone that denotes "susceptibility" of an isolate by the disk diffusion technique.
The disk test measures the ability of drugs to inhibit the growth of bacteria.  The results correlate reasonably
well with therapeutic response in those disease processes where body defenses can frequently eliminate
infectious microorganisms.In a few types of human infections,  the results of disk tests are of little assistance (and may be misleading) because a bactericidal drug effect is required for cure.  Outstanding examples are infective endocarditis,  acute osteomyelitis,  and severe infections in a host whose antibacterial defenses are inadequate,  eg,  persons with neoplastic diseases that have been treated with radiation and anti-neoplastic chemotherapy ,  or persons who are being given corticosteroids in high dosage and are immunosuppressed. Instead of the disk test,  a semi quantitative minimum inhibitory concentration (MIC) test procedure can be used.  It measures more exactly the concentration of an antibiotic necessary to inhibit growth of a standardized inoculum under defined conditions.  A semi automated microdilution method is used in which defined amounts of drug are dissolved in a measured small volume of broth and inoculated with a standardized number of microorganisms.  The end point,  or minimum inhibitory concentration,  is considered the last broth cup (lowest concentration of drug) remaining clear ,  ie,  free from microbial growth.  The minimum inhibitory concentration provides a better estimate of the probable amount of drug necessary to inhibit growth in vivo and thus helps in gauging the dosage regimen necessary for the patient.

Clinical microbiology laboratories perform disk diffusion tests and tests based upon determining the MIC and
interpret their results using guidelines established by the Clinical Laboratory and Standards Institute (CLSI)
located in Wayne,  Pennsylvania.  In addition,  to help guide empiric therapy choices before the results of
antimicrobial susceptibility tests are available,  it is recommended by CLSI that laboratories publish an
antibiogram annually that contains the results of susceptibility testing in aggregate for particular organism–drug
combinations.  For example,  it may be important to know the most active  -lactam antimicrobial agent targeted against Pseudomonas aeruginosa among ICU patients in a particular hospital so that agent can be used when a patient develops an infection while in that unit. There are other methods for assessing the efficacy of antimicrobial treatment.  Bactericidal effects can be estimated by subculturing the clear broth onto antibiotic-free solid media.  The result,  eg,  a reduction of colony forming units by 99.9% below that of the control,  is called the minimal bactericidal concentration (MBC). The selection of a bactericidal drug or drug combination for each patient can be guided by specialized laboratory tests.  Such tests measure either the rate of killing (time-kill assay) or the proportion of the microbial population that is killed in a fixed time (serum bactericidal testing). In urinary tract infections,  the antibacterial activity of urine is far more important than that of serum.

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.

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 and locate a fixed vein by touch.  Release the tourniquet while the skin is being
prepared.
3.   Prepare the skin for venipuncture by cleansing it vigorously with 70–95% isopropyl alcohol.  Using 2%
tincture of iodine or 2% chlorhexidine,  start at the venipuncture 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,  and 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.
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,  three 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 pneumoniaeP 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.
Microbiology Student At PBPC, Nayabazzar-9, Pokhara.


































































































































































Tuesday, August 5, 2014

DIAGNOSTIC MEDICAL MICROBIOLOGY

PRINCIPLES OF DIAGNOSTIC MEDICAL MICROBIOLOGY CONTD...
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,  C immitis, Histoplasma capsulatum,  and others. Portions of the 16S rRNA are conserved across many species of microorganisms.  Amplifying the 16S rRNA using primers to these conserved regions allows isolation and sequencing of the variable regions of the molecules. These variable sequences are genus- or species-specific markers that allow identification of microorganisms.  Pathogens that are difficult or impossible to culture in the laboratory have been identified using this technique.One example is Tropheryma whipplei,  the cause of Whipple disease. Molecular diagnostic assays that use amplification of nucleic acid have become widely used and are evolving rapidly .  These amplification systems fall into several basic categories as outlined below.
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.  Conventional PCR utilizes three sequential reactions—denaturation,  annealing,  and primer extension—as follows.  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 10^5 - 10^6 fold. 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 a broad range of bacterial and viral pathogens such as Chlamydia trachomatis,  N gonorrhoeae,  M 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 B 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.

PROBE AMPLIFICATION SYSTEMS
The ligase chain reaction (LCR) is an amplification system different from PCR.  LCR uses thermostable DNA polymerase and thermostable DNA ligase.  LCR uses four oligonucleotide probes of 20–24 bases each.  Each pair of oligonucleotides is designed to bind to the denatured target DNA only a few bases apart.  The oligonucleotides are mixed with extracted target DNA from the specimen and other reagents and then heated to denature the target DNA.  The reaction is then cooled to allow binding of the oligonucleotide probes to the target DNA.  The short gap between the two probes is filled in by the DNA polymerase and linked by the DNA ligase,  yielding double-stranded DNA molecules 40–50 bp in length.  The cycle is repeated 30–40 times,  yielding a large number of DNA molecules.  This commercially available system includes automated detection of the amplified DNA.  It can be used to detect C trachomatis and N gonorrhoeae.  It is available only outside of the United States.

SIGNAL AMPLIFICATION TECHNIQUES
These assays strengthen the signal by amplifying the label (eg,  fluorochromes,  enzymes) that is attached to the target nucleic acid.  The branched DNA (bDNA) system has a series of primary probes and a branched
secondary probe labeled with enzyme.  Multiple oligonucleotide probes specific for the target RNA (or DNA) are fixed to a solid surface such as a microdilution tray .  These are the capture probes.  The prepared specimen is added,  and the RNA molecules are attached to the capture probes on the microdilution tray .  Additional target probes bind to the target but not to the tray .  The enzyme-labeled bDNA amplifier probes are added and attach to the target probes.  A chemiluminescent substrate is added,  and light emitted is measured to quantitate the amount of target RNA present.  Examples of the use of this type of assay include the quantitative measurement of HIV -1,  hepatitis C virus,  and hepatitis B virus.

AMPLIFICATION METHODS: NON–PCR-BASED
The transcription-mediated amplification (TMA) and the nucleic acid sequence-based amplification
(NASBA) systems amplify large quantities of RNA in isothermal assays that coordinately use the enzymes
reverse transcriptase,  RNase H,  and RNA polymerase.  An oligonucleotide primer containing the RNA
polymerase promoter is allowed to bind to the RNA target.  The reverse transcriptase makes a single-stranded cDNA copy of the RNA.  The RNase H destroys the RNA of the RNA -cDNA hybrid,  and a second primer anneals to the segment of cDNA.  The DNA -dependent DNA polymerase activity of reverse transcriptase extends the DNA from the second primer ,  producing a double-stranded DNA copy ,  with intact RNA polymerase.  The RNA polymerase then produces many copies of the single-stranded RNA.  Detection of C trachomatis,  N gonorrhoeae,and M tuberculosis and quantitation of HIV -1 load are examples of the use of these types of assays. Strand displacement assays (SDA) are isothermal amplification assays that employ use of restrictive endonuclease and DNA polymerase.

REAL-TIME PCR
Technologic advances,  which have lead to "real-time amplification," have streamlined nucleic acid amplification platforms,improved the sensitivity of amplification tests,  and have drastically reduced the potential for contamination.  Real-time instruments have replaced the solid block used in conventional thermocyclers with fans that allow more rapid PCR cycling.  Dramatic improvements in the chemistry of nucleic acid amplification reactions have resulted in homogeneous reaction mixtures in which fluorogenic compounds are present in the same reaction tube in which the amplification occurs.  A variety of fluorogenic molecules are used.  These include nonspecific dyes such as SYBR green,  which binds to the minor groove of double-stranded DNA,  and amplicon specific detection methods using fluorescently labeled oligonucleotide probes,  which fall into three categories:
TaqMan or hydrolysis probes; fluorescence energy transfer (FRET) probes; and molecular beacons. All of the methods allow for measurement of fluorescence with each amplification cycle,  that is,  "real-time" assessment of results.  Since the reaction tube does not need to be opened to analyze the PCR products on a gel,  there is much less risk of amplicon carry-over to the next reaction.

THE IMPORTANCE OF NORMAL BACTERIAL & FUNGAL FLORA
Organisms such as M tuberculosis,  Salmonella typhi,  and Brucella species are considered pathogens whenever they are found in patients.  However ,  many infections are caused by organisms that are permanent or transient members of the normal flora. For example,  Escherichia coli is part of the normal gastrointestinal flora and is  also the most common cause of urinary tract infections. Similarly ,  the vast majority of mixed bacterial infections with anaerobes are caused by organisms that are members of the normal flora. The relative numbers of specific organisms found in a culture are important when members of the normal flora are the cause of infection.When numerous gram-negative rods of species such as Klebsiella pneumoniae are found mixed with a few normal nasopharyngeal bacteria in a sputum culture,  the gram-negative rods are strongly suspect as the cause of pneumonia because large numbers of gram-negative rods are not normally found in sputum or in the nasopharyngeal flora; the organisms should be identified and reported.  In contrast,abdominal abscesses commonly contain a normal distribution of aerobic,  facultatively anaerobic,  and obligately anaerobic organisms representative of the gastrointestinal flora.  In such cases,  identification of all species present is not warranted; instead,  it is appropriate to report "normal gastrointestinal flora." Yeasts in small numbers are commonly part of the normal microbial flora.  However ,  other fungi are not normally present and therefore should be identified and reported.  Viruses usually are not part of the normal flora as detected in diagnostic microbiology laboratories.  However ,  some latent viruses,  eg,  herpes simplex, or live vaccine viruses such as poliovirus occasionally appear in cultures for viruses.  In some parts of the world,stool specimens commonly yield evidence of parasitic infection.  In such cases,  it is the relative number of parasites correlated with the clinical presentation that is important.

CITED BY ANIL BHUJEL
BSC MICROBIOLOGY, TU.
MICROBIOLOGY STUDENT AT PBPC, NAYABAZZAR-9, POKHARA.

SOME SUGGESTED REFERENCES:
www.ncbi.nlm.nih.gov › ... › J Clin Microbiol › v.45(9); Sep 2007
en.wikipedia.org/wiki/16S_ribosomal_RNA
biotech.bio5.org/sites/default/files/pdf/16s%20rDNA%20SeqAnal.pdf
link.springer.com/chapter/10.1007%2F0-387-32892-0_20
amrita.vlab.co.in/?sub=3&brch=76&sim=1421&cnt=1
www.austincc.edu/.../mdfund_unit9Chapter7NucleicAcidAmplificationT...
www.genengnews.com/.../targeted-amplification-system.../81245623/?...
www.ncbi.nlm.nih.gov/pubmed/1370001
https://www.inkling.com/read/.../signal-amplification-methods
en.wikipedia.org/wiki/Polymerase_chain_reaction.
books.google.com.np/books?isbn=1402062419
www.mdpi.com/2079-6374/3/1/18/pdf























   















































































Sunday, August 3, 2014

DIAGNOSTIC MEDICAL MICROBIOLOGY

PRINCIPLES OF DIAGNOSTIC MICROBIOLOGY...

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 specimens and culture media used to diagnose the more common bacterial infections. 
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 and are
discussed in separate sections in this blog on the diagnosis of bacterial,  chlamydial,  and viral infections.
EIAs,  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.  An example of this type of assay is the Binax NOW Streptococcus pneumoniae urinary antigen test.  In some EIAs,  the initial antibody is not necessary ,  because the antigen will bind directly to the plastic of the wells. EIAs are used to detect viral,  bacterial,  chlamydial,  protozoan,  and fungal antigens in a variety of specimen types such as stool,  cerebrospinal fluid,  urine,  and respiratory samples.
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 (Cowan I strain) 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 such as S pneumoniae,  Neisseria meningitidis,  N gonorrhoeae,  and Beta-hemolytic streptococci.  Latex agglutination tests are primarily directed at the detection of carbohydrate antigens of encapsulated microorganisms.  Antigen detection is used most often in the diagnosis of group A streptococcal pharyngitis. Detection of cryptococcal antigen is useful in the diagnosis of cryptococcal meningitis in patients with AIDS or other immunosuppressive diseases. The sensitivity of latex agglutination tests in the diagnosis of bacterial meningitis may not be better than that of Gram stain,  which is approximately 100,000 bacteria per milliliter .  For  that reason,  the latex agglutination test is not recommended for direct specimen testing.

Western Blot Immunoassays
These assays are usually performed to detect antibodies against specific antigens of a particular organism.  This method is based upon the electrophoretic separation of major proteins of the organism in question in a
 twodimensional agarose gel.  Organisms are mechanically or chemically disrupted and resultant solubilized antigen of the organism is placed in a polyacrylamide gel.   An electric current is applied and major proteins are separated out on the basis of size (smaller proteins travel faster).  The protein bands are transferred to strips of nitrocellulose paper .  Following incubation of the strips with a patient's specimen containing antibody (usually serum),  the antibodies bind to the proteins on the strip and are detected enzymatically in a fashion similar to the EIA methods described above.  Western blot tests are used as 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.  F or 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.  The Gen-Probe test is an example of a hybridization test format in which the probe and target are in solution.  Most of the applications in use in clinical microbiology laboratories make use of solution hybridization formats.  In situ hybridization involves the use of labeled DNA probes or labeled RNA probes to detect complementary nucleic acids in formalin-fixed paraffin-embedded tissues,  frozen tissues,  or cytologic preparations mounted on slides.  Technically,  this can be difficult and is usually performed in histology laboratories and not clinical microbiology laboratories.  However,this technique has increased the knowledge of the biology of many infectious diseases,  especially the hepatitides and oncogenic viruses,  and is still useful in infectious diseases diagnosis.A novel technique that is somewhat of a modification of in situ hybridization makes use of peptide nucleic acid probes.  Peptide nucleic acid probes are synthesized pieces of DNA in which the sugar phosphate backbone of DNA (normally negatively charged) is replaced by a polyamide of repetitive units (neutral charge).  Individual nucleotide bases can be attached to the now neutral backbone,  which allows for faster and more specific hybridization to complementary nucleic acids.  Because the probes are synthetic,  they are not subject to degradation by nucleases and other enzymes.  A commercial company (AdvanDx,  Woburn MA) has a number of FDA cleared assays for confirmation of Staphylococcus aureus,  enterococci,  certain Candida sp.  and some gram-negative bacilli in positive blood culture bottles.  The probe hybridization is detected by fluorescence and is called Peptide Nucleic Acid-Fluorescence In Situ Hybridization (PNA -FISH).

Cited By Anil Bhujel
Bsc Microbiology, TU.
Microbiology Student At Pokhara Bigyan Tatha Prabidhi Campus(PBPC), Nayabazzar-9, Pokhara.

SOME SUGGESTED REFERENCES:






















  























  

Thursday, July 31, 2014

DIAGNOSTIC MEDICAL MICROBIOLOGY

PRINCIPLES 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,  enzyme immunoassay
[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.  Although physicians should be competent to perform a few simple,  crucial microbiologic tests—make and stain a smear ,  examine it microscopically ,  and streak a culture plate—technical details of the more involved procedures are usually left to the bacteriologist or virologist and the technicians on the staff .   Physicians who deal with infectious processes must know when and how to take specimens, what laboratory  examinations to request, and how to interpret the results.
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.  F or each type of specimen,  suggestions for optimal handling are given in the following paragraphs and in the section on diagnosis by anatomic site,  below. Recovery of bacteria and fungi is most significant if the agent is isolated from a site normally devoid of microorganisms (a normally sterile area).  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 normal microbiota  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 microbiota of each particular site.  Microbiologic data must be correlated with clinical information in order to arrive at a meaningful interpretation of 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 .
The type of specimen to be examined is determined by the presenting clinical picture.  If symptoms or signs
point to involvement of one organ system,  specimens are obtained from that source.  In the absence of
localizing signs or symptoms,  repeated blood samples for culturing are taken first,  and specimens from other
sites are then considered in sequence,  depending in part upon the likelihood of involvement of a given organ

system in a given patient and in part upon the ease of obtaining specimens.

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 10^5 organisms per milliliter before it is likely that organisms will be seen on a smear .  Liquid medium containing 10^5 organisms per milliliter does not appear turbid to the eye. Specimens containing 10^2 to 10^3 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. 
Gram & Acid-Fast Staining Methods
Gram stain
(1) Fix smear by heat or using methanol.
(2) Cover with crystal violet.
(3) Wash with water .  Do not blot.
(4) Cover with Gram's iodine.
(5) Wash with water .  Do not blot.
(6) Decolorize for 10–30 seconds with gentle agitation in acetone (30 mL) and alcohol (70 mL).
(7) Wash with water .  Do not blot.
(8) Cover for 10–30 seconds with safranin (2.5% solution in 95% alcohol).
(9) Wash with water and let dry . Ziehl-Neelsen acid-fast stain
(1) Fix smear by heat.
(2) Cover with carbol fuchsin,  steam gently for 5 minutes over direct flame (or for 20 minutes over a water
bath).  Do not permit slides to boil or dry out.
(3) Wash with deionized water .
(4) Decolorize in 3.0% acid-alcohol (95% ethanol and 3.0% hydrochloric acid) until only a faint pink color
remains.
(5) Wash with water .
(6) Counterstain for 1 minute with Loeffler's methylene blue.
(7) Wash with deionized water and let dry .
Kinyoun carbolfuchsin acid-fast stain
(1) Formula: 4 g basic fuchsin,  8 g phenol,  20 mL 95% alcohol,  100 mL distilled water .
(2) Stain fixed smear for 3 minutes (no heat necessary) and continue as with Ziehl-Neelsen stain.
Specimens submitted for examination for mycobacteria should be stained for acid-fast organisms,  using either Ziehl-Neelsen stain or Kinyoun stain.  An alternative fluorescent stain for mycobacteria,
auramine-rhodamine stain,  is more sensitive than other stains for acid-fast organisms but requires fluorescence microscopy and,  if results are positive,  confirmation of morphology with an acid-fast stain.
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.

Cited By Anil Bhujel.
Bsc Microbiology, TU.
Microbiology Student At PBPC, Nayabazzar-9, Pokhara.

SOME SUGGESTED REFERENCES:
www.ncbi.nlm.nih.gov › NCBI › Literature › Bookshelf
https://sites.google.com/.../allmicrobiologysite/diagnostic-medical-microb...
www.slideshare.net/chhaya30/diagnostic-microbiology
biomedicgen.files.wordpress.com/.../diagnostic-medical-microbiology-cli...
accessmedicine.mhmedical.com/content.aspx?bookid=504&sectionid...
en.wikipedia.org/wiki/Medical_microbiology
cc.shsmu.edu.cn/G2S/Template/ShowNoMeau.aspx?type=3...pdf...
tainguyenso.vnu.edu.vn/xmlui/bitstream/handle/.../01071000101.pdf?...
en.wikipedia.org/wiki/Staining
https://www.microscopeworld.com/t-microscope_slide_staining.aspx
www.microscopemaster.com/cell-staining-microscopy.html