REPORT OF RECENT MICROBIAL TECHNIQUES DEVELOPED IN DIAGNOSING SOME COMMON DISEASES

1. REPORT OF RECENT MICROBIAL TECHNIQES DEVELOPED IN
DIAGNOSING SOME COMMON DISEASES
PRESENTING BY:
RATNAKAR
PHARM-D (2ND-YEAR)
SGRRITS, UTTARAKHAND

2. SOME COMMON DISEASE AND DIAGNOSIS
1.TYPHOID AND DIAGNOSIS
2.TUBERCULOSIS
3.MALARIA
4.CHOLERA
5.HEPATITIS
6.MENINGITITS
7.SYPHILIS
8.GONORRHEA
9.HIV/AIDS

3. TYPHOID CONTI.....
• Typhoid
Typhoid is a bacterial infection that can lead to a high fever, diarrhea, and
vomiting. It can be fatal. It is caused by the bacteria Salmonella typhi.
Causes
Bacterium Salmonella Typhi .
Salmonellae typhi
 Salmonellae typhae are gram – ve rods, facultative aerobic, Motile
with peritrichate flagella, non-spore-forming
 1-3μm ×0.5μm in size
DIAGNOSIS: according to week
First Week
 Blood Culture, Bone Marrow Culture

4. Typhoid conti..
Second Week
 Antibody Detection (Widal Test)
Widal Test is an agglutination test which detects the presence of serum
agglutinins (H and O) in patients serum with typhoid and paratyphoid fever.
" A test involving agglutination of typhoid bacilli when they are mixed with serum
containing typhoid antibodies from an individual having typhoid fever; used to
detect the presence of Salmonella typhi and S.paratyphi."
Third Week
 Stool Culture
Fourth Week
 Urine Culture

5. Typhoid conti...
NEW DIAGNOSTIC TESTS
• IDL Tubex detects IgM09 antibodies with in few minutes
• Typhidot test that detects presence of IgM and IgG in one hour
(sensitivity>95%, Specificity 75%)
• Typhidot-M, that detects IgM only (sensitivity 90% and specificity 93%)
• Typhidot rapid (sensitivity 85% and Specificity 99%) is a rapid 15 minute
immunochromatographic test to detect IgM.
2.Tuberculosis
Tuberculosis is an infectious disease that usually affects the lungs. Compared
with other diseases caused by a single infectious agent, tuberculosis is the
second biggest killer, globally.

6. Tuberculosis conti...
cause
Mycobacterium Tuberculosis Discoverer by Robert Koch so that disease is also
called koch disease.
Mycobacteria
• Obligate aerobes growing most successfully in tissues with a high oxygen
content, such as the lungs.
Diagnosis
Acid-fast staining
The material to be examined on a glass slide and staining with carbol-fuchsin
by means of either the Ziehl-Neelsen or Kinyoun technique.
Fluorochrome staining :
Sensitivity of detection of acid-fast organisms is increased by a fluorochrome
staining procedure with auramine O, a fluorescent stain.

7. Tuberculosis conti...
• This procedure requires use of a fluorescent microscope but is faster than
acid-fast staining because the intensity of the fluorescent signal enables
slides to be scanned at lower magnification.
Cultures
• Cultures of mycobacteria require only 10 to 100 organisms to detect M.
tuberculosis.
• The sensitivity of culture is excellent, ranging from 80% to 93%.
• The specificity is quite high, at 98%.
Radiometric and Colorimetric Detection Systems
• Radiometric culture systems (the BACTEC) incorporate 14 14C-labeled
palmitic acid into a liquid culture medium.

8. Tuberculosis conti...
• Growth of mycobacteria results in liberation of CO2 that can be measured by
the detection device.
• The increased sensitivity of the system enables growth to be detected
sooner, usually in 10 to 14 days.
Chromatography
• High-performance liquid chromatography (HPLC) is a rapid and highly
specific method for detecting the unique pattern of mycolic acids for
identifying mycobacterial species.
• Tuberculostearic acid, a component of M. tuberculosis, can be easily
detected even in infinitesimal (femtomole) quantities by gas liquid
chromatography (GLC).
Nucleic Acid Amplification Tests
• In each test, either DNA or ribonucleic acid (RNA) is amplified to detectable
levels.

9. Tuberculosis conti...
• Nucleic acid amplification tests include those that involve PCR amplification,
transcriptionmediated amplification, strand-displacement amplification, ligase
chain reaction, and Q Beta replicase amplification.
• NAA assays are also quite specific for M. tuberculosis, with specificities in the
range of 98% to 99%.
Polymerase chain reaction (PCR)
• PCR targets DNA, rRNA, insertion and repetitive elements, and various
proteinencoding genes.
• The PCR amplification process can be completed in 2–4 h after obtaining the
• processed clinical sample. The detection assay requires an additional 2–24 h
but it is very costly

10. Tuberculosis conti...
Transcription-mediated amplification (TMA) “MTD test”
Isothermal, target-based amplification system is based on amplification of
ribosomal RNA (rRNA) unlike PCR, which is based on amplification of DNA.
3.Malaria
Malaria is a life-threatening mosquito-borne blood disease caused by a
Plasmodium parasite.
It is transmitted to humans through the bite of the Anopheles mosquito.
Once an infected mosquito bites a human, the parasites multiply in the host's
liver before infecting and destroying red blood cells.
Diagnosis
• Light microscopy

11. Malaria conti...
• Antigen detection - Rapid malaria diagnosis tests (RDTs)
• Serology
• PCR
CULTURE OF THE PARASITE:
• Trager and Jensen’s method of petridish culture employed with a candle jar
to provide an atmosphere of 3% oxygen and 10% carbondioxide and a
relatively simple culture medium supplemented with human, rabbit or calf
serum to maintain infected erythrocytes. Fresh red cells were added
periodically for continuation and growth of Plasmodium.
• Rapid diagnostic tests (RDTs) are also employed for easy detection and take
around 2 to 15 minutes. These tests detect parasite antigens. These can be
used by relatively untrained staff.

12. Cholera conti..
4.Cholera
Cholera is an acute epidemic infectious disease. It is characterized by watery
diarrhea, extreme loss of fluid and electrolytes, and severe dehydration. It can be
fatal.
It is caused by the bacterium Vibrio cholera (V. cholera).
Diagnosis
• Rapid diagnosis of cholera by coagglutination test
A simple, rapid, and reliable method to detect Vibrio cholerae in fecal specimens
would assist in the management of cases of severe diarrhea, especially since
most such cases occur in areas with minimal laboratory facilities. A
coagglutination test was used to detect V. cholerae antigen in bile-peptone broth
incubated with feces.
• PCR test
• Bipolar microorganismus by bluedomethylene staining

13. HEPATITIS Conti..
VIRAL HEPATITIS
• The term viral hepatitis is used to describe infection of thenliver caused by
hepatotropic viruses. Currently there are 5 main varieties of these viruses
and a sixth poorlycharacterised virus, causing distinct types of viral hepatitis:
• Hepatitis A virus (HAV), causing a faecally-spread selflimiting disease.
• Hepatitis B virus (HBV), causing a parenterally transmitted disease that may
become chronic.
• Hepatitis C virus (HCV), previously termed non-A, non-B (NANB) hepatitis
virus involved chiefly in transfusionrelated hepatitis.
• Hepatitis delta virus (HDV) which is sometimes associated as superinfection
with hepatitis B infection.
• Hepatitis E virus (HEV), causing water-borne infection.

14. HEPATITIS Conti..
• Hepatitis G virus (HGV), is a recently discovered transfusion-transmitted
hepatotropic virus but is not known to cause hepatitis.
All these human hepatitis viruses are RNA viruses except HBV which is a DNA
virus.
Diagnosis
Liver fuction test
PCR test
ELISA
Detection HBs(ab)
6.Meningitis
Meningitis is an inflammation of the meninges. The meninges are the three
membranes that cover the brain and spinal cord. Meningitis can occur when
fluid surrounding the meninges becomes infected..

15. Meningitis conti..
The causative organisms
1. Escherichia coli infection is common in neonates with neural tube defects.
2. Haemophilus influenzae is commonly responsible for infection in infants and
children.
3. Neisseria meningitidis causes meningitis in adolescent and young adults and
is causative for epidemic meningitis.
4. Streptococcus pneumoniae is causative for infection at extremes of age and
following trauma.
Diagnosis

16. SYPHILIS Conti...
7.SYPHILIS
Syphilis is a venereal (sexually-transmitted) disease caused by spirochaetes,
Treponema pallidum. Other treponemal diseases are yaws, pinta and bejel. The
word ‘syphilis’ is
derived from the name of the mythological handsome boy,Syphilus, who was
cursed by Greek god Apollo with the disease.
Causative Organism
T. pallidum is a coiled spiral filament 10 μm long that moves actively in fresh
preparations. The organism cannot be stained by the usual methods and can
be demonstrated in the exudates and tissues by.
Diagnosis
1. Dark ground illumination (DGI) in fresh preparation;
2. Fluorescent antibody technique

17. SYPHILIS Conti...
3. silver impregnation techniques; and
4. PCR as a research method.
The organism has not been cultivated in any culture media but experimental
infection can be produced in rabbits and chimpanzees. The organism is rapidly
destroyed by cold, heat, and antiseptics.
8.Gonorrhea
Gonorrhea is a sexually transmitted disease (STD). It’s caused by infection with
the bacterium Neisseria gonorrhoeae.
Diagnosis
Microscopy of a direct smear of the discharge stained with Gram stain reveals
Gram-negative diplococci within polymorphonuclear leucocytes. The sensitivity
of the microscopy is highest in urethral samples in men, reaching 90–95%,
whereas for endocervical smears sensitivity drops to 30–50%. In asymptomatic
patients sensitivity is extremely low (20%).

18. Gonorrhea conti...
• Culture of N. gonorrhoeae requires the use of agar enriched with blood or
hemoglobin and several agents such as glucose, amino acids and antibiotics
(colistin, vancomycin, nystatin, etc.), to suppress the growth of commensal
neisseriae, and Gram-positive and Gram-negative bacteria and fungi.
• Enzyme immunoassays (EIAs) can be used for quick identification of
gonococcal urethritis with good sensitivity and specificity. These tests lack
sensitivity and specificity when used for cervical, pharyngeal and rectal
samples.
• Nucleic acid detection methods enable the detection of N. gonorrhoeae from
clinical samples without the requirement of bacterial viability. These are the
nucleic acid amplification tests (NAATs) that are used in pharyngeal and
rectal samples, with greater sensitivity than cultures.

19. HIV/ AIDS conti..
9.HIV/ AIDS
HIV is a virus that damages the immune system. The immune system helps the
body fight off infections. Untreated HIV infects and kills CD4 cells, which are a
type of immune cell called T cells.
Diagnosis
ELISA
An enzyme-linked immunosorbent assay, also called ELISA or EIA, is a test that
detects and measures antibodies in your blood. This test can be used to
determine if you have antibodies related to certain infectious conditions.
PROCEDURE
The ELISA test is simple and straightforward. You’ll probably need to sign a
consent form, and your doctor should explain the reason for doing the test.
The ELISA test involves taking a sample of your blood.

20. HIV/ AIDS conti..
• If your blood contains antibodies to the antigen, the two will bind together.
Western blot
• Western blot is often used in research to separate and identify proteins. In
this technique a mixture of proteins is separated based on molecular weight,
and thus by type, through gel electrophoresis.
The procedure involves:
• Separation of the proteins on polyacrylamide gel.
• Transfer (blotting) of proteins from the gel to a membrane (nitrocellulose or
nylon) and identification of the protein with a specific Ab.
• The method is sensitive for detecting proteins in complex mixtures.
• Immunoblot is used as a confirmatory test for HIV

21. THANK YOU.....