Early diagnosis always leads to better treatment, which is why the point of care testing holds an essential place in the healthcare setting. This PPT includes different categories of RDT according to their turnaround time and technology.

1. MUBASHIR NAZIR
MSC. CLINICAL MICROBIOLOGY
SHERI KASHMIR INSTITUTE OF MEDICAL SCIENCES
SRINAGAR, INDIA

2. INTRODUCTION
 Also known as Rapid Point-of-Care Test
 Clinical specimen (serum, plasma, saliva, urine, stool, tissue, or body
fluids) is processed in a single step at the site where it is collected and a
qualitative or quantitative result is available within 20 minutes .
 RDT nowadays uses more than just a single step testing procedure.
 For certain RDTs, obtaining results within 18 hours instead of 4 days
(e.g. for MRSA) or 1 week instead of 6 weeks (e.g. tuberculosis) still
renders the test "rapid."

3. The Need for RDTs
 Demand for fast, easy-to-use and sensitive diagnostic tests for microbial
infections is on the rise.
 Disease epidemic requirements for biological confirmation at peripheral
level.
 Rapid diagnostics for improving the quality of care for patients with
suspected infections.
 Improve antibiotic targeting to only those who will benefit, thus reducing
overuse.
 Enhance surveillance of pathogens and infectious diseases.

4. FEATURES
Features of the ideal RDT are:
• High sensitivity and specificity;
• Relatively high negative and positive predictive values;
• Reproducible results;
• Rapid turnaround time;
• Availability and reporting of results to those who need them in
a timely manner;
• Affordable pricing.

5. CATEGORIES OF RDT
 RDT is generally grouped into the following categories:
1) Direct microscopy of specimens.
2) Rapid biochemical tests
3) Antigen/Antibody detection
4) Molecular detection
5) Mass spectrometry

6.  RDT for infectious diseases has been based largely on rapid microscopy.
 A Gram stain can confirm within
minutes the presence of
gram-positive diplococci
(eg,Streptococcus pneumoniae)
in a sputum smear, gram-negative
diplococcic (Neisseria gonorrhoea)
in a urethral smear, or gram-negative
rods in a spun specimen of urine.
 Mycobacteria can be identified
rapidly by microscopy of specimens
stained with Ziehl-Neelsen, Kinyoun,
or rhodamine - auramine stains

7. Contd……

 Staining with acridine orange, and
examining under a fluorescent microscope
reveals the presence of trypanosomes
that cause sleeping sickness (Trypanosoma
brucei) or Chagas disease (Trypanosoma
cruzi)
 Giardia lamblia and
Entamoeba histolytica
are readily identified by
microscopy of direct fecal
smears prepared with saline
or lugol’s iodine.

8. Contd……
Encapsulated cells of Cryptococcus
identified by india ink.
Treponema pallidum in dark-field microscopy

9. IMMUNOELECTRON MICROSCOPY
 Viral particles are mixed with
specific antisera and observed
under the electron microscope .
 Seen as clumps .
 Applied in some viruses such as
HAV, and viruses causing diarrhea.

10.  Screening for UTI
Nitrite
 Aromatic amine in reagent strip reacts with nitrite; producing a
diazonium salt.
 Diazonium salt reacts with sulfanilic acid and acetic acid to produce a
pink azo dye.
 UTIs caused by organisms that do not convert nitrates to nitrites are not
detected
• Staphylococcus
• Streptococcus

11. Contd…..
Leukocyte Esterase
 An enzyme present in granulocytes, hydrolyzes indoxylcarbonic acid to
indoxyl, which reacts with a diazonium salt to create a purple color
usually in 2 min.
 Following may cause a FALSE POSITIVE results
Trichomonas
Heavy vaginal discharge (mucus)
 Following may cause a FALSE NEGATIVE results
High level of protein
High level of vitamin C

12.  PRECPITATION
 AGGLUTINATION
 IMMUNOFLUORESENCE
 IMMUNOCHROMATOGRAPHY
 CHEMILUMENESCENCE ASSAY

13. PRECIPITATION REACTIONS
Definition :
 The reaction which occurs when specific antibody combines with
soluble antigen.
Principle :
 Multivalent antigens combine with bivalent antibodies;
precipitation occurs only when a large lattice is formed and this
is possible in the zone of equivalence.

14. Contd…
Precipitation reactions as RDT
 RING TEST : Antigen is layered over an antiserum in a narrow tube.
A precipitate ring appears at the junction of the two liquids e.g. CRP,
Lancefield's grouping of Streptococcus.
 FLOCCULATION TEST: When instead of sediment, the precipitate is
suspended as floccules e.g. VDRL
 ELECTROIMMUNODIFFUSION TESTS: are precipitation tests in gel (1
% agar) which can be speeded up by electrically driving the antigen
and antibody.

15. Contd…
 Methods of Electroimmunodiffusion:
o Counter immunoelectrophresis
Simultaneous electrophoresis of the
antigen and antibody in gel in opposite
directions.
o One dimensional single EID
mainly applied for quantization of
antigens; precipitation is formed in the
shape of cone like structures.

16. Contd………
o Two dimensional electrophoresis
Several antigens in a mixture can be
quantitated .
Applications
-screening tool for differentiation of
more than 30 serum proteins
-used for detection of :
Ig classes
Multiple myeloma

17. AGGLUTINATION
Definition
 It is an antigen-antibody reaction, in which a particulate antigen
combines with its antibody.
Principle
 Multivalent antigens combine with bivalent antibodies, agglutination
occurs only when a large lattice is formed and this is possible in the
zone of equivalence.
Uses
 Slide agglutination
 Used to identify bacterial strains isolated from clinical specimens .
 Used for blood grouping and cross- matching.
 Agglutination is more sensitive than precipitation for the detection of
antibodies.

18. Cotnd……
Passive agglutination test
 A precipitation reaction can be
converted into agglutination
 latex particles , bentonite and RBCs.
Latex agglutination test
• Polystyrene latex particles are
employed to adsorb several types of
antigens and antibodies.
• Convenient , rapid and specific.
• Detection of HBsAg, ASO, CRP, RF,
HCG, bacterial typing.

19. Contd……
Coagglutination
• Based on presence of protein A on
the surface of some strains of Staph.
aureus (cowan 1 strain).
•Specific IgG immunoglobulin is
coated on these cowan 1 strains of S.
aureus
• Detection of bacterial antigens in
blood, urine and CSF.
• N. gonorrhoeae
Strep. pyogenes
H. influenzae

20. IMMUNOFLUORESCENCE
 Light of a shorter wavelength incites the electrons of a molecule to a higher
energy state for a very short time; as the electrons return to the ground
state, the energy is released as light of a longer wavelength .
 Commonly used dyes are fluorescin isothiocyanate (blue green) and
lissamine rhodamine (orange red) .

21. Contd……
Direct immunoflouresence
 Specific antibodies tagged with fluorescent dye are
used for detection of unknown antigen in a specimen.
Uses
 Detection of T. pallidium, HSV, RSV, Influenza,
Cryptosporidium, Giardia , Legionella, etc.
 Sensitive method to diagnose rabies in brain tissue
smears.
Disadvantage
 Separate specific fluorescent labelled antibody has to
be prepared against each antigen to be tested .

22. Contd……
Indirect immunoflouresence
 Known antigen is fixed on a slide, the
unknown antibody (serum) is applied to
the slide.
 For detection of the Ag-Ab reaction ,
fluorescin-tagged antibody to human
globulin is added.
Uses
Detection of EBV, Toxoplasma gondii
Advantage
Single antihuman globulin fluorescent
conjugate can be employed for detection
of any Ab to any Ag.

23. IMMUNOCHROMATOGRAPHY
 Dye-labelled antibody, specific for target antigen, is present on the lower
end of nitrocellulose strip or in a plastic well provided with the strip.

24. Contd…….
 APPLICATIONS
DENGUE
RDT SENSITIVITY
IgM/IgG 70-80%
NS1 Ag 50-75%
IgM/IgG & NS1
Ag
> 90%

25. Contd….
INFLEUNZA
 Commercial influenza RDT can detect
Influenza virus antigens within 15
minutes of testing.
 Kits vary.
 None of the current commercial
influenza RDT assays are able to
identify any of the various influenza A
virus subtypes.
 Influenza RDTs have limited
sensitivity (11%-70%)

26. Contd…..
HIV
 For the majority of commercial diagnostic tests, the main serological
target for the detection of HIV infections is based on antibody reactivity
to the envelope transmembrane proteins:
gp41 for HIV-1 and gp36 for HIV-2

27. Contd……
HBsAg /Anti-HCV

28. Contd…….
MALARIA
 Some RDTs detect a single species
either P.falciparum (HRP-2) or
P.vivax (LDH).
 Some detect multiple spp. and
some further distinguish between
P.falciparum and non - falciparum.
 Falcivax (RDT kit) has a specificity
of 100% and sensitivity of 96.3%
and 94.7%.
 Blood for test is commonly
obtained from a finger prick and
results are available within 15-30
min.

29. Contd…….
SHYPHILIS
 Clinical diagnosis is of limited use
because chancres may heal, be
atypical, or patients may be
asymptomatic.
 p15, p17, p47
 Available within 30 minutes and do
not require a laboratory or other
instrumentation.

30. CHEMILUMINESCENCE
It is the emission of light as a result of
chemical reaction.
It involves excitation(absorption) and de-
excitation (emmision) of light
Light energy is detected by
chemiluminescence detector.
Applications
 Drug sensitivity of M.tuberculosis.
 Dx. of infectious diseases
CMV, Rubella ,Toxoplama ( IgG/IgM)
 Blood viruses
HBc-Ab, HBsAg, HCV-Ab, HIV
 Detection of tumour markers;
ovarian carcinoma : Ca-125.
 Hormonal analysis like T3, T4, TSH,
testosterone.

31. Nucleic acid probes
 ISH
 Hybrid capture
 Reverse hybridization
 Nucleic acid Amplification
 PCR
-RT- PCR
-Multiplex PCR
-Nested PCR
-Real time PCR
-Gene Xpert

32. Contd………
HYBRIDIZATION
 Process where two DNA or RNA single chains from different biological
sources, make the double stranded configuration, based on nucleotide
complementarity and of contingent sequence homology of the two
sources, resulting DNA-DNA, RNA-RNA or DNA-RNA hybrids.
 Basic steps in a hybridization assay:
1. Production and labeling of single-strand nucleic acid probe
2. Preparation of single-strand target nucleic acid
3. Mixture and hybridization of target and probe nucleic acids
4. Detection of hybridization

33. IN SITU HYBRIDIZATION
 Used in molecular pathology to detect chromosomal translocations, gene
amplifications, identification of infectious agents.
Fluorescent
ISH (FISH)
Chromog
enic ISH
(CISH)
Oligonucleotide
probe is labelled
with a
flourophore that
is detected by
direct
fluorescence
microscopy.
Olinucleotide
is labelled in
such a way
that an
enzymatic
reaction
generates a
color that can
be viewed
using a
traditional
microscope.

34. Applications
 rapid identification and differentiation of clinically important yeasts in
positive blood cultures e.g. C. albicans , C. glabrata, C. krusei ,C.
parapsolosis using four ISH probes.
 to detect and identify the bacteria likely to be present in the respiratory
specimens of patients with cystic fibrosis e.g. P . aeuroginosa,
Burkholderia cepacia, H. influenza , Staph. aureus.
 90% sensitivity and 100% specificity as compared to culture.
 96.5% of positive blood cultures could be identified within 2.5 hours
(in comparison to conventional culture ).
 study of H pylori in gastric biopsies ,Chlamydia, Legionella pneumophilia in
fixed respiratory specimens.

35. Contd…..
Peptide Nucleic Acid (PNA) Probes
 PNA probes are synthetic pieces of DNA in which the negatively charged
sugar-phosphate backbone of DNA is replaced by a neutral polyamide
backbone of repetitive units .
 PNA FISH is a novel FISH technique that uses PNA probes to target
species-specific ribosomal RNA (rRNA) sequences.
 Provide faster and more specific results than traditional DNA probes.
 AdvanDx (Woburn, Massachusetts) introduced in vitro diagnostic kits
(using PNA FISH); approved by FDA.
 These kits can be used to differentiate Enterococcus faecalis from other enterococci in
positive blood cultures (with high sensitivity and specificity).

36. Contd……..
MICROORGANISMS DETECTED
S. aureus
CoNS
Enterococcus faecalis
Enterococcus faecium,
Streptococcus pneumoniae
E. coli
Pseudomonas aeruginosa,
Candida albicans, C. parapsilosis, C. glabrata, C. krusei C. tropicalis

37. Contd……

38. HYBRID CAPTURE
 DNA is denatured
 Hybridized to RNA probe
 Captured by bound anti DNA/RNA antibodies
 Detected with multiple
antibodies conjugated
to alkaline phosphatase

39. Contd…..
Applications
 Detection of high risk human papillomavirus subtypes, N. gonorrhoeae,
Chlamydia tracomatis.
 Detection of CMV, HBV in blood . HBV test is qualitatative whereas CMV
test is quantitative.

40. REVERSE HYBRIDIZATION
APPLICATION
Line probe assay
 Alarming increase in MDR-TB, the emergence of extensively drug
resistant TB (XDR-TB), potential institutional transmission, and rapid
mortality of MDR-TB and XDR-TB patients with HIV co-infection, have
highlighted the urgency for rapid screening methods.
Steps
 DNA is extracted from M. tuberculosis isolates or directly from clinical
specimens.
 PCR amplification of the resistance-determining region of the gene under
question is performed using biotinylated primers.
 Labelled PCR products are hybridized with specific oligonucleotide probes
immobilized on a strip.
 If mutation is present in one of the target regions, the amplicon will not
hybridize with the relevant probe.

41. Contd….
 Mutations are detected by lack of binding to wild-type probes, as well as
by binding to specific probes for the most commonly occurring
mutations.
 The post-hybridization reaction leads to the development of colored
bands on the strip at the site of probe binding and is observed by eye.

42. NUCLEIC ACID AMPLIFICATION
PCR
 In-vitro technique for amplification of a region of DNA whose sequence is
known or which lies between two regions of known sequence.
 Highly sensitive and specific.
 Specificity may be improved by changing the reaction conditions such as:
Salt concentration or primer annealing temp.
Easiest of these to change is the primer temp , the specificity of the
binding of the primers to the target DNA molecule will increase as the
annealing temp used approaches the Tm of primers.

43. Contd……
40-60% GC preferred
for better annealing
0
500000
1000000
1500000
2000000
2500000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
DNAcopies
Cyclenumber
DNAcopies vsCycle number

44. Contd……
NESTED PCR
Involves the sequential use of two primer sets.
 The first set is used to amplify a target
sequence.
 The amplicon obtained is then used as the
target sequence for a second amplification
using primers internal to those of the first
amplicon.
 The advantage of this approach is extreme
sensitivity and confirmed specificity without the
need for using probes.

45. Contd......
Applications
 Used to improve the sensitivity of assay
 Used in detection of microorganisms that may be in low quantity
in the blood and tissues, such as Rickettsia, Bartonella
MTB
 IS6110- detection of M. tuberculosis and M. bovis BCG
 65 kDa protein gene- detect all mycobacterial strains
 Mpt40- distinguish between M. tuberculosis from M. bovis BCG

46. Contd……
REAL TIME PCR
 Quantitative PCR.
FEATURES
 Real-time detection of PCR products is made possible
by including in the reaction a fluorescent molecule
that reports an increase in the amount of DNA with a
proportional increase in fluorescent signal.
 The fluorescent chemistries employed for this purpose
include DNA-binding dyes and fluorescently labelled
sequence specific primers or probes.
 Specialized thermal cyclers equipped with fluorescence
detection modules are used to monitor the
fluorescence as amplification occurs.
 The measured fluorescence reflects the amount of
amplified product in each cycle.

47. Contd……
Advantages
 Main advantage of real-time PCR over conventional PCR is that real-time
PCR allows to determine the starting template copy number with
accuracy and high sensitivity over a wide dynamic range.
 Data can be evaluated without gel electrophoresis, resulting in reduced
experiment time and increased throughput.
 Reactions are run and data are evaluated in a closed-tube system,
opportunities for contamination are reduced and the need for post-
amplification manipulation is eliminated.

48. DNA Detection: SYBR Green I Dye
DENATURATION STEP: DNA + PRIMERS + DYE
WEAK BACKGROUND FLUORESCENCE
ANEALING STEP:DYE
BINDS dsDNA, EMITS LIGHT
EXTENSION STEP: MEASURE
LIGHT EMMISSION

49. Contd……
Hybridization probes
Tagman probes
 Light emission from the
reporter fluorophore (R) is
quenched because of its
proximity to the quencher (Q).
Cleavage by Taq polymerase
separates the reporter and
quencher allowing
fluorescence.
FRET probes
 probes are two separate
fluorescently labeled
oligonucleotides, one with a 5′
donor molecule and the other
with a 3′ acceptor molecule
attached

50. Contd…..
Applications
 Herpes viruses, adenoviruses, smallpox viruses, parvoviruses.
 Bartonella spp., differentiate HSV-1 from HSV-2,
 M. tuberculosis, staphylococcus, MRSA, Pseudomonas, Bacillus antracis.
 H.capsulatum, Coccidiodes immitis, Penicillium marneffi
 Toxopama gondii, Leishmania, Trypanosoma, Crytosporidium.
 18S rRNA gene has unique stretches that enable the identification of all
5 human infecting spp. of malaria – P. falciparum, P. vivax, P. ovale,
P. malaraie, P. knowlesi
Threshold
fluorescence level
Threshold cycles for each sample

51. GENE XPERT MTB/RIF
PRINCIPLE
 Nucleic acid amplification(NAA)
test which simultaneously detects
DNA of M tuberculosis complex
and resistance to rifampicin (i.e;
mutation of rpoB gene) in less
than 2 hours.
 Primers in the assay amplify a
portion of the rpoB gene
containing the 81 bp core region.
 The probes are able to
differentiate between the
conserved wild type sequence and
mutations in the core region that
are associated with rifampicin
resistance.

52. Contd……
Procedure
 Uses a disposable cartridge with the GeneXpert Instrument System.
 Sputum sample is collected from the patient with suspected TB.
 Sputum is mixed with the reagent that is provided with the assay, and a
cartridge containing this mixture is placed in the GeneXpert machine.
Advantages
 Results are available quickly
 Minimal technical training is required to run the test
 Assay can quickly identify possible multidrug-resistant TB (MDR TB)
• MDR TB is TB that is resistant to both isoniazid (INH) and RIF, two of the most
effective TB drugs.
• RIF resistance is a predictor of MDR TB because resistance to RIF, in most
instances, co-exists with resistance to INH.

53. INTERPRETATION OF RESULTS
RIF Resistance Detected
 Results that are positive for MTBC
and for RIF resistance mean that
the bacteria have a high
probability of resistance to RIF.
RIF Resistance Not Detected
 Results that are positive for MTBC,
but negative for RIF resistance
mean that the bacteria are
probably susceptible to RIF.
RIF Resistance Indeterminate
 Results that are positive for MTBC
and indeterminate for RIF
resistance mean that the test
could not accurately determine if
the bacteria are resistant to RIF.

54.  Create gas-phase ions
 Use the difference in mass-to-charge ratio (m/z) of ionized atoms or
molecules to separate them.
 Measure the quantity of ions of each mass-to-charge ratio and
provides structural information by the identification of distinctive
fragmentation patterns.
 Imaging mass spectrometry tools allow the two-dimensional visualization of the
distribution of trace metals, metabolites, surface lipids, peptides & proteins directly from
biological samples without the need for chemical tagging or antibodies.

55. Matrix-Assisted Laser Desorption/Ionization
Time Of Flight(MALDI)-TOF
PRINCIPLE
 Laser evaporation from a
crystallized sample/matrix
mixture. The matrix material must
have an absorption spectrum that
matches the laser wavelength of
energy.
 Matrix acts as a receptical for the
laser energy and facilitates
ionization while minimizing
ablation of the sample and analyte
ion that would otherwise occur
from direct laser desorption.

56. Contd……
 3 commonly used matrix molecules are
 3,5-dimethoxy-4-hydroxy cinnamic acid (sinapinic acid)
 alpha-cyano-4 hydroxycinnamic acid
 2,5-dihydroxybenzoic acid (DHB).
 Gram-negative organisms appear to be much easier to detect than Gram-positives,
 Fungi
 Brucella

57. Contd……
Surface-enhanced laser desorption/ionization-time of flight
 SELDI-TOF is an ionization method in mass spectrometry that is used
for the analysis of protein mixtures.
 The proteins are ionized with lasers and separated by size.
 It characterizes patterns of combinations of proteins or peptides in
blood or other body tissues that uniquely define a specific infectious
disease, rather than identifying only a single marker.
Applications
 SELDI-TOF include rapid diagnosis of sleeping sickness, invasive
Aspergillosis, tuberculosis, and Chagas' disease.