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






















   

































  





  
  







Monday, July 28, 2014

CONTROL OF MICROBIAL INFECTIONS

ANTIVIRAL DRUG RESISTANCE:
When an antibacterial or antiviral agent acts at a single target site then resistance can develop through 
mutations at that site. The high mutation rates in RNA viruses will mean that such problems will occur 
more readily than with DNA-based bacteria and this is witnessed in HIV therapies. Again, as with 
antibiotic policies, certain manoeuvres can be employed to minimise resistance developing. These 
include the use of combinations of antiviral compounds, switching between two unrelated agents 
(although sequential use of antivirals has been criticised for simply promoting resistance rather than 
preventing it) and the use of high concentrations to prevent any viral replication (‘knock out’). This, 
theoretically, prevents any viable mutants being released. 
Resistance to antiviral agents will result from mutations in the viral genome. The modified proteins may 
act on cell events that occur upstream of the active compound (e.g. mutations in viral thymidine kinase 
will reduce its affinity of binding to the nucleoside) or mutations may occur in the target protein itself (e.
g. retroviral reverse transcriptase). Whilst mutation is a frequent event in RNA viruses, and resistance 
develops frequently during the course of an infection, the spontaneous mutation events in a DNA virus 
like Herpes simplex will be of much lower frequency. Note that latent Herpes simplex virus does not 
express TK or DNA polymerase, hence the virus remains unaffected by the drug. 
The laboratory testing for resistance to antiviral agents has a number of problems. Direct testing of the 
inhibitory concentrations of antiviral agent (phenotypic testing) gives a concentration that is active, at 
least under laboratory conditions. Phenotypic testing requires that the organism has been cultured and 
this is not possible for those viruses that have not yet been successfully cultured in vitro. Molecular 
mechanisms avoid this problem. Genotype testing can, in theory, be carried out on clinical samples as 
well as cultured virus. Polymerase chain detection for resistant gene sequences are increasingly available 
and are less technically demanding than phenotypic tests. The interpretation of both types of testing is 
somewhat arbitrary and, like antibiotic susceptibility testing in bacteria, will only indicate possible outcomes or provide frames of reference.

SURVEILLANCE:
In the latter half of the 1980s, the incidence of tuberculosis had increased to ‘epidemic’ proportions in a 
developed city, New York, in the most prosperous country in the world. How could such a well recognised infectious disease against which an effective vaccine and a range of antibiotics are available 
spread unnoticed in such a city? The reasons illustrate how good public health infrastructure can be 
undermined if unsupported. Famously associated with poverty, tuberculosis had steadily increased in a 
place where HIV was endemic amongst people living in overcrowded, poor housing. The treatment of 
tuberculosis uses three antibiotics over 6 months and patients with pulmonary disease who are the infectious sources quickly become non-infectious following antibiotic treatment. If patients fail to complete the treatment, they will relapse into infectious cases and 
antibiotic resistance may result. Of the measures taken, DOT (direct observation of therapy) was 
crucial to the management of the epidemic. DOT targets the source by effecting a reduction of the 
proportion of infectious cases. One of the reasons for the return of tuberculosis was the reduced funding 
to public health departments responsible for monitoring (surveillance) and treating the disease. Partly 
this was a consequence of years of success in reducing the incidence of tuberculosis such that 
complacency had set in. Not surprisingly the cost of bringing the epidemic under control was more than 
the savings made in the first place. 
The value of quarantine measures to screen imported animals for infectious diseases needs 
consideration. The ancient practice of retaining ships in port for 40 days (‘quarantina’) 
applied to all trade, not just to animals, and was driven mostly by a need to prevent the spread 
of plague. With the spatial isolation of an island, quarantine can prevent the introduction of a 
disease. With increasing international travel by air, the principle has been undermined. The 
building of a tunnel under the English channel to connect the United Kingdom with the rest of 
Europe means the isolation is breached again. 



FUTURE EPIDEMICS
Epidemics have been a regular feature of human existence throughout history, so it is easy to state with 
confidence that they will continue to be so. The reasons for such a gloomy outlook are not difficult to find.  As populations have increased in size, particularly in cities, so has the incidence of 
infectious disease. The proportion of people infected by certain infectious diseases (tuberculosis is again 
an example) may fall, but the absolute numbers of people infected will still increase. The present 
population forecasts indicate increasing numbers of large (greater than 10 million people) cities, mostly 
in ports and concentrated in developing countries. Other than providing a critical population threshold to 
sustain diseases like measles, sexually transmitted diseases and the common cold, large cities need 
global commerce to support them, as well as attracting migration, which both increase the inward and 
outward flux of people, goods and microbes. The public health infrastructure needs to be appropriately 
sized and funded to prevent the resurgence of old infections that, although once under control, will 
readily return without continued control. The expansion of the areas of known infectious disease is a 
result of expanding urban slums into the country such that the associated risks of deforestation and 
monoculture of crops all result in disturbing zoonotic cycles. It is also depressing to note the regular 
appearance of epidemics that accompany war, a habit that, far from diminishing, seems to be on the 
increase and risks employing biological weapons. Although poverty is famously linked with poor health, 
notably tuberculosis, wealthy nations are not excluded from risk as advances in medicine keep more people alive with increasing opportunities for infections. 

                               EXAMPLES OF CHANGING PATTERNS IN INFECTIOUS DISEASE 
•  Previously unrecognised diseases: Lyme disease (Borrelia burgdoferi), Helicobacter 
pylori gastritis
•  Increasing zone of disease: increasing malaria and Dengue due to the spread of 
mosquito vectors into new areas. 
•  Resurgence of ‘old’ infections: epidemic wave of diphtheria in Europe affecting the 
old communist bloc. 
•  Apparent synergy in tuberculosis with HIV in AIDS patients. 
•  Increasing incidence of drug-resistant tuberculosis. 
•  Food-borne infections increasing with globalisation of food products.

International surveillance will become increasingly important as the world shrinks through globalisation 
in order that infectious diseases can be tracked. The bioterrorist threat, exemplified by the crippling cost  of sending a few samples of anthrax spores through the post, is a further call to adequate microbiological infrastructure and expertise. The factors that are important in protecting the public from biological weapons are similar to those that are considered in all microbiology laboratories. The need for microbiologists is obvious but should that idea need reinforcement then it is worth noting 
that the cost of eradication of poliomyelitis in the USA has paid for itself every 26 days since 1977. The 
financial losses to a country incurred through infectious disease every year may yet make governments 
take notice. 
Having described the human activities that promote infectious disease, what are the features of the 
organisms that will continue to create problems? RNA viruses are the organisms with the highest 
mutation rates. It can be expected that such organisms will continue to cause epidemics, just as HIV has 
been the most frightening of recent times because of the genetic variation that occurs through inaccurate 
transcription and genetic reassortment. HIV has demonstrated the potential impact that an RNA virus 
can have if it is able to cross the species barrier (from apes to humans). The risk of mutational events 
resulting in greater virulence in bacteria and fungi is less than for RNA viruses, but the increasing 
recognition that toxins and pathogenicity islands are coded for on mobile genetic elements means that 
bacteria will continue to present threats. The increase in antibiotic resistance genes through horizontal 
gene transfer over the last 50 years reminds us that we have not yet overcome the existing microbial 
threats.   HAZARD GROUPS
In order to protect the staff in microbiology laboratories as well as the public and the environment, 
micro-organisms have been categorised into four Hazard Groups. The groups, in increasing order of 
hazard, are as follows: 
Group 1:  Unlikely to cause human disease. 
Group 2:  Can cause disease and may be a hazard to employees. 
Unlikely to spread to the community. 
Effective prophylaxis or treatment available. 
Group 3:  Can cause human disease. 
Risk of spread to the community. 
Effective prophylaxis or treatment available. 
Group 4:  Can cause severe human disease. 
Likely risk of spread into the community. 
No effective prophylaxis and treatment. 
The micro-organisms considered the most effective biological weapons are chosen because 
they: 
•  are easily disseminated into the public or are highly infectious (readily transmitted 
from person to person). 
•  cause high mortality. 
•  require special action for public health control
The organisms mostly fall into Hazard Group 4. Viruses such as Variola major virus 
(smallpox) and Ebola and Marburg viruses are not easily treated with antiviral agents but 
there are effective vaccines available (although not routinely used). Bacillus anthracis and Yersinia pestis(the aetiological agent of plague) can be treated with antibiotics.





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Friday, July 18, 2014

CONTROL OF MICROBIAL INFECTIONS

ANTIBIOTIC RESISTANCE(BACTERIA):
The most pressing problem facing medicine is the growing number of micro-organisms that are now 
resistant to a wide range of antibiotics. The term antibiotic resistance describes the condition where 
bacteria have developed or acquired means to overcome the inhibitory effect of one or more antibiotics. A chilling example is multidrug-resistant Mycobacterium tuberculosis. Without the development of new antibiotics, thoracotomy (removal of the affected lobes of the lung) appears to be
the only treatment option. The mechanisms by which bacteria develops resistance to antibiotics are either due to mutation or acquisition of resistance genes from other organisms.  
MUTATION
Clinically, mutation leading to resistance has not been a major problem compared with the acquisition of 
resistance genes. In general, the effect of mutation will be to modify the target protein such that the 
binding affinity of the antibiotic is reduced. The protein will tolerate a certain loss of efficiency due to 
mutations, but constraints will limit the number and frequency of viable mutations in the active site. The 
affinity of binding will fall, but not such that the protein function is lost completely. If the binding site 
for the antibiotic is distinct from the active site of the protein, then there will be more scope for mutation 
to occur without significant loss of function. For example, the binding of an antibiotic on a ribosome 
might sterically hinder the correct binding of the tRNA and lead to misreading. If the antibiotic 
competed with the tRNA for the same binding site, then mutation of the tRNA binding site will limit the 
extent of mutation. Mutational events reduce the potency of the antibiotic for the target site and this is observed by small increases in the concentration of antibiotic needed to inhibit the growth of the organism (minimum inhibitory concentration, MIC); for example, the MIC of Staphylococcus aureus to penicillin may increase from 0.01 µg/ml to 0.05 µg/mL. By comparison, acquisition of 
resistance genes usually results in large increases in the MIC (e.g. from 1 µg/ml to >64 µg/mL). 
An example of mutational resistance of clinical importance is the resistance of Mycobacterium 
tuberculosis to the aminoglycoside streptomycin. During the treatment of patients with tuberculosis, the 
proportion of bacteria that are resistant to the effects of streptomycin increases steadily with time due to 
mutations in the ribosomal binding site for streptomycin. The frequency of mutation of Mycobacterium 
tuberculosis is approximately 1 in 10^7 , meaning that one in every 10 million cells undergoes a 
mutational event that affects streptomycin activity. The mutation rate is fixed and therefore occurs 
irrespective of the presence of streptomycin. Thus, in patients taking streptomycin, the selective pressure 
results in the resistant mutants continuing to grow (unlike the susceptible cells), displacing the falling 
numbers of streptomycin susceptible cells until eventually all the bacteria will be resistant to 
streptomycin. If the antibiotic is withdrawn then the selective pressure vanishes and the susceptible cells 
will regain dominance because of the cost of streptomycin resistance in terms of reduced efficiency of 
ribosomal function. The key feature here is the constant rate of mutation; all that changes is the selective 
pressure on the organism by exposing the organisms to streptomycin. 
Mutational resistance that causes problems for the treatment of a bacterial infection is largely restricted 
to Mycobacterium tuberculosis.Why? 
The organism has a long generation time (2–4 hours) compared with ‘fast’ growing organisms such as 
Esch. coli with a generation time of 20–30 minutes and Escherichia coli has multiple copies of the 
rRNA gene whereas Mycobacterium tuberculosis has only one. Any mutation in one of the appropriate 
genes in Escherichia coli will be recessive to the unaffected gene copies whereas mutations in the single 
copy of the gene in Mycobacterium tuberculosis will be expressed.

ACQUISITION OF RESISTANCE GENES (HORIZONTAL GENE TRANSFER):
Resistance genes are thought to exist in populations of bacteria that will be exposed to antibiotics in their 
natural environment. Bacteria in the soil will encounter antibiotics produced by fungi and Streptomyces 
spp. The strong selection pressures exerted by antibiotic use in agriculture (animal feeds, crop spraying, 
etc.), hospitals and the home (in countries where antibiotics can be bought freely at the pharmacy 
without prescription) has resulted in bacterial pathogens acquiring resistance genes from other 
organisms. The horizontal gene exchange of transmissible plasmids containing resistance genes means 
that effective antibiotic resistance is acquired in one step rather than waiting for mutational events to 
develop. The resistance genes have become widespread throughout different pathogens across the globe. 
Their mobility is conferred by becoming incorporated in transposons that, in turn, accumulate as gene 
cassettes in mobile plasmids called multidrug resistance plasmids(often abbreviated to R plasmids). They resemble the pattern observed with pathogenicity islands, clusters of genes all coding for related functions. 
Mutational events may be the only option for organisms that do not readily exchange genes horizontally. 
Again, Mycobacterium tuberculosis is a good example. The cell wall structure of Mycobacterium 
tuberculosis has the extra mycolic acid/arabinogalactan layers that may prevent transformation, 
conjugation and transduction.
RESISTANCE TO ANTIBIOTICS: CELLULAR MECHANISMS:
Enzymatic inactivation of the antibiotic:
A good example is the production of β-lactamase, an enzyme that hydrolyses the β-lactam nucleus of β-lactam antibiotics (penicillins). These enzymes may be found on the chromosome or on resistance 
plasmids and are expressed constitutively or as an inducible enzyme, only when β-lactams are present. 
The strategy adopted is exemplified in the Gram positive organism Staphylococcus aureus which 
produces inducible β-lactamase to be released extracellularly. In contrast the Gram negative Escherichia 
coli constitutively produces much smaller amounts of β-lactamase but sites it in the periplasmic space. 
In this way Escherichia coli chooses to only degrade β-lactam antibiotic that permeates through the 
porins in the outer membrane. Inactivating enzymes are also found that attack aminoglycosides and 

chloramphenicol. 
Active efflux of antibiotic:
The bacterium expends energy to actively extrude antibiotic from within the bacterial cell. The efflux 
pumps responsible may be able to pump more than one type of antibiotic (multidrug resistance efflux 

pumps). 
Reduced uptake:
An alternative to removing antibiotic once it has reached the cytosol is to mutate the mechanisms that 
are responsible for the uptake of the antibiotic in the first place. Such a strategy relies on active, i.e. 

energy expending, mechanisms of uptake. Passive diffusion will not apply.
Modification of drug target:
Mutations will result in alterations of the binding sites in antibiotic target proteins. Penicillin binding 
proteins, for example, can reduce their binding affinity for -lactam antibiotics. Alterations in the amino 
acid sequences of the ribosomal proteins can also result in reduced binding of the antibiotics that act on 

protein synthesis. 
Overproduction of target:
With antibiotics such as the sulphonamides and trimethoprim, competitive inhibitors of enzymes 
involved in the biosynthesis of folic acid, it is logical to see how bacteria have developed resistance to 

these drugs by simply overproducing enzymes. In the case of trimethoprim, the organism produces excess dihydrofolate reductase in order to overcome the competition from the drug. 
Some of these mechanisms are produced by mutation, others are simply acquired through horizontal 
gene transfer. The origins of the resistance mechanisms lie in other organisms, presumably those that 
produce antibiotics themselves. When an organism is under prolonged pressure through exposure to 
antibiotics, in time, the appropriate resistance mechanism will eventually make itself available. 
It is important to realise that bacteria can be ‘resistant’ to the inhibitory effects of antibiotics through 
mechanisms that are neither mutational events nor resulting from acquisition of exogenous resistance 
genes. 
•  Antibiotics have a particular spectrum of activity. Those organisms that are unaffected by a 
particular antibiotic may lack the target site or the mechanisms by which the antibiotic enters the 
bacterium.  
•  Even though several antibiotics are bactericidal (e.g. β-lactams), they can only exert this effect on 
actively growing bacteria; the organisms in stationary phase are not killed. This is a general 
phenomenon and applies not just to antibiotics but to disinfectants and other antibacterial 
conditions (starvation, drought). Endospores represent the extreme example of this strategy: 
completely inert structures that dominate in unfavourable conditions that are effectively unaffected 
by antibiotics. 
•  Dense collections of bacteria as biofilms offer protection partially through physical restriction and 

binding of the antibiotic so as to reduce the active concentration that can penetrate the biofilm.

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

SOME SUGGESTED REFERENCES:
en.wikipedia.org/wiki/Antibiotic_resistance
www.sciencedaily.com/articles/a/antibiotic_resistance.htm
textbookofbacteriology.net/resantimicrobial.html
www.who.int/mediacentre/factsheets/fs194/en/
www.rxlist.com/antibiotic_resistance-page3/drugs-condition.htm
textbookofbacteriology.net/resantimicrobial_3.html
www.ncbi.nlm.nih.gov/pubmed/21822035
www.life.umd.edu/classroom/.../MechanismsofAntibioticResistance.htm
www.sciencedirect.com/science/article/pii/S009286740700311X