TERM PAPER

ON
CORRELATION BETWEEN RAPID TEST AND AUTOMATED
METHODS IN DIAGNOSING INFECTIOUS DISEASE
LIKE TYPHOID

TABLE OF CONTENTS

TOPIC PAGES
INTRODUCTION 8

REVIEW OF LITERATURE
- Rapid and Automated tests
- Typhoid fever
- Machines and Tools required for diagnosis
- Diagnosis of Typhoid fever
Conclusion

References
ABSTRACT

Over the past several years, the development and application of molecular diagnostic techniques
has initiated a revolution in the diagnosis and monitoring of infectious diseases. The advances of
medical sciences and technology have brought us to a point where automation is the prime
method for diagnosing several diseases. Automated methods are considered advanced and highly
accurate for various diagnostic procedures. Although the gold standard for diagnosis of infectious
diseases still stands as microbial culture still cost effective and fast-paced rapid tests have been
developed for diagnosis of commonly occurring diseases. Different type of Rapid Diagnostic Tests
(RDT) is based on different principles like: lateral flow chromatographic assays that give fairly
accurate result with an incomparable pace. Though it cannot be concluded that they are the most
accurate or advance form of tests but their pace is their Unique Selling Proposition and when you
are in a profession of saving lives, time is of essence.
 INTRODUCTION

Automation was introduced to the clinical microbiology laboratories in the 1960s but it initially met
various challenges and criticism. Today, these instruments are an integral part of many clinical
laboratories and are used for microbial identification, susceptibility testing, and detection of
positive blood cultures and assaying levels of antimicrobial agents in body fluids. In many cases,
automated tests are more sensitive and specific than manual techniques. However, automated
testing is often more expensive and has a high possibility of mechanical failure than manual testing.
Automation continues to be an integral part of the clinical microbiology laboratories and with
advances in technology and medical sciences, it will definitely improve upon its quality and cost
effectiveness.

On the other hand, a new line of fast-paced diagnostic tests has been developed in recent times.
Rapid diagnostic tests (RDT) are the cost effective, fast paced solution to the diagnostic pothole that
exist in the developing parts of the world, they have allowed rapid diagnosis and elimination of the
subjective interpretation of various manual tests although they can never have a spot on accuracy
but their pros far outmatch their cons.

This report deals with comparison and analysis of automated and rapid diagnostic methods. The
focus of this report is on diagnosis of Typhoid fever.
 REVIEW OF LITERATURE

RAPID TESTS:
A rapid diagnostic test (RDT) is a medical diagnostic test that is quick and easy to perform. RDTs are suitable
for emergency medical screening and for use in hospitals with limited resources. They also allow testing in
primary care for things that formerly only a laboratory test could measure. They provide fast results within
20mins-2hrs. Nowadays, Expertise and scientists are using rapid tests for quick detection of disease in
emergency. Various features of RDTs are:

● High quality, easy-to-use tests.

● Tests are based on agglutination, immuno-dot, immuno-chromatographic and immuno-filtration
techniques.
● Quick and easy to perform, only takes 10 minutes to 2 hours – and require little or no additional
equipment.
● Are designed for use with individual or a extended number of samples, which make them more
reasonably prized then ELISAs in low throughput laboratories.
● Possibility to store at room temperature for long period of time.
● Same-day results provided for timely treatment interventions.

Some examples of RDTs are listed below:

● Rapid HIV test

● Rapid influenza diagnostic test
● Rapid malaria test
● Rapid plasma reagin
● Rapid strep test
● Rapid urease test
● Typhoid IgG/IgM
AUTOMATED TESTS

Automated tests are medical diagnostic test that are fairly accurate and sensitive. They are less labor
intensive but take time to give the result. Automated tests play a crucial role in diagnosis of a disease. In
automated methods, we use different types of machines for detection of various infectious agents that may
be causing the disease. These Tests are able to reduce or eliminate the high amount of labor work required
in manual testing.

In automated test, a machine follows a sequence of steps more quickly than a technician does, and it can
run the tests overnight to present the results in the morning.

Machines used and operated by us are:

 BACTEC 9120
 VITEC 2

BLOOD CULTURING

A blood culture is a routine test that checks for bacteria, yeast, and other microorganisms in the blood. The
sample retrieval is a relatively simple process for the patient and involves a simple blood draw.

After the blood draw, the blood is transferred into bottles with specific culture media. These bottles are then
incubated in automated machines for a period of time and then the machine checks for any microbial
growth to determine whether microorganisms have invaded the patient's bloodstream. The process is
completely automated and gives results in overnight.

The only risk of the test is when you give blood; though, blood draws are routine procedures and rarely
cause any serious side effects..
 TYPHOID FEVER

INTRODUCTION:
Typhoid fever is an acute illness associated with fever caused by the Salmonella typhi bacteria.

Typhoid fever is developed by drinking or eating contaminated food or water. People with acute illness
can contaminate the surrounding water through stool, which contains a high concentration of the
bacteria. Contamination of the water supply can, infect the food supply. The bacteria can survive for
weeks in water or dried sewage.

About 3%-5% of people become carriers of the bacteria after the acute illness. Others suffer a very mild
illness that goes unrecognized. These people may contain bacteria for long time -- even though they
have no symptoms -- and be the source of new outbreaks of typhoid fever for many years.

The incubation period for the bacteria is usually 1-2 weeks, and the duration of the illness is about 3-4
weeks. Symptoms include:

● Poor appetite

● Headaches

● Generalized aches and pains

● Fever as high as 104 degrees Fahrenheit

● Lethargy

● Diarrhea

● Chest congestion

● Abdominal pain and discomfort

Improvement may occur in the third and fourth week without complications. About 10% of people have
recurrent symptoms after feeling good for one to two weeks. Getting ill or worse again after few time is
actually more common in individuals treated with antibiotics, after some time the fever becomes
constant and the condition becomes worsened.
MACHINES USED FOR DIAGNOSIS

BACTEC 9120
Blood culture is one of the most important functions of a microbiology laboratory as clinicians rely on this, to help
in the diagnosis of bacteremia and fungemia. The BACTEC FX System builds on the established history of previous
BACTEC instrumentation; it goes much further with exciting innovations such as-Vial-activated workflow, advanced
ergonomics, remote alarms and blood culture observation, and customer-focused data management. The
instrument design provides optimal, easy-to-use workflow in a compact, modular design.

Bactec 9120 Blood Culture System is the world's most effective automated system for septicemia testing. It comes
with a unique feature of continuous monitoring, high performance results, and non-invasive design.

System has a separate data management capability to analyze the data provided and monitor the growth Kinetics
of every sample.
 Continuous Monitoring Technology

BACTEC 9000 Systems shows the distinctive BACTEC 9000 fluorescent sensor technology that
allows for fully automated, easy testing using a continuous-monitoring instrument that agitates and
incubates BACTEC/F blood culture bottles, resulting in earlier detection of positive vials.
The BACTEC instrument also provides advanced algorithms for individual bottle types, for cases such
as low blood volume, pediatric specimens, or to detect slow growing organisms such as Haemophilus.
These algorithms give rapid detection of pathogens in blood culture.

In the BACTEC 9000 instrument, systematic directions for bottle processing and test initiation are
provided by scanning the barcode test menu.
Patient statistic characterizing can be entered into the system using the BACTEC Barcode Scanner.
Also the information can be entered via keyboard, if desired.
Bottles are tested every 10 minutes. Positive results are picked out for quick processing. Negative
bottles can be scanned out of the system and unloaded at the end of protocol.

Test bottles with different media in which the samples are collected:

•BD BACTEC/F Blood Culture Media

The BACTEC/F line of blood culture media is used with the BACTEC 9000 Fluorescent Series
Systems. These media allow screening for bacteria, yeast and fungi present in the blood. This includes
standard broth media, resin media and media particularly designed for low blood volume inoculation.
•A perfect BACTEC resin media effectively neutralize a wide variety of antibiotics, permitting growth
of microorganisms, which will not grow in conventional media.
•BACTEC resin media achieve significantly higher isolation rates in antibiotic patients than systems
using only broth dilution. This greater recovery leads to more detailed diagnosis and effective
treatment, which can in turn lead to shorter hospital stays, lower patient costs, greater overall
laboratory and institutional efficiency.

BD BACTEC Plus Aerobic/F and Plus Anaerobic/F Medium

BACTEC plus Aerobic/F and Plus Anaerobic/F media are enriched soybean-casein digest broth media. These
media contain resins for neutralization of antibiotic, providing increased recovery as compared with
Standard media.

BACTEC Plus Aerobic/F Medium is capable of helping the growth of common aerobic and facultative
organisms;

BACTEC Plus Anaerobic/F Medium is capable of helping the growth of common anaerobic and facultative
organisms.

It consists of two types of bottles: Peds bottle and adult bottle

BD BACTEC Peds Plus™/F Medium

●BACTEC Peds Plus/F media are specialized media that contain small-volume samples (less than and equal
to 3 mL of blood), to optimize detection of common pediatric microorganisms. The medium is an improved
soybean-casein digest broth and contains resins for antibiotic neutralization.

BD BACTEC Adults plus™/F Medium

•BACTEC adults Plus/F media are media that contain large-volume samples (greater that and equal to 8 mL
of blood), to optimize detection of common adult’s pathogens/microorganisms. The medium is an
enriched soybean-casein digest broth and contains resins for neutralization of antibiotics.
BD BACTEC™ Blood Culture Procedural Tray
● Blood Culture Trays decrease the chances of contaminating a blood culture by having all of the
components required to draw blood for culture, in a convenient tray format. The trays standardize
procedures hospital-wide and increase compliance with physician requests.

●Using the Bactec Blood Culture Procedural Trays optimizes media performance for the best combination
of recovery and time-to-detection. The BACTEC Blood Culture Procedural Trays minimize needle-stick
potential and reduce inventory and supply costs by providing a capitated kit cost for blood culturing

Once the bottle in the BACTEC 9120 is positive, then it is cultured on agar plates and gram staining is done
in order to detect various diseases and types of bacterial growth in patient’s body.

Streak plate method

The streak plate method is a rapid qualitative isolation method. It is a techniques used for isolation of
discrete colonies, initially requires the number of organisms in the inoculums to reduced. This dilution
technique involves spreading a loop with culture over the surface of an agar plate. The resulting decrease in
the population size of bacteria ensures that, following colonies, individual cells will be sufficiently far apart
on the surface of the agar medium. Although different type of procedures are performed, the four ways or
quadrant streak is mostly done.
 Three-phase streaking
The three-phase streaking pattern, known as the T-Streak, is done by using a sterile tool, such as a cotton swab or
commonly an inoculation loop. In this, we will mark the plate in three parts The inoculation loop is sterilized by
passing it through a flame. When the loop is cool, we take the loop full of inoculum such as a broth or patient
specimen containing many species of bacteria and spread across the surface of the agar in a zigzag motion until
approximately 30% of the plate has been covered. We do the same for the next two part. Each time the loop have
fewer and fewer bacteria until it accumulate just single bacterial cells that can grow into a colony. The plate should
show massive growth in the first section. The second section will have less growth and a few isolated colonies,
while the final section will have the least growth and many isolated colonies.

Continuous streaking
Continuous streaking can be done on full or half plate but usually half plate streaking is done in most of
the places. Mark your agar plate into two halves. Flame your loop and take loop full of sample and streak
one of the half in a continuous back n-forth line. Now do the same on the other half.

Once the bacterial growth is obtained on the plates then it is processed in VITEK 2 for further
investigation and identification of species of bacteria and the types of antibiotics to be given.

VITEK 2

With increasing healthcare challenges like Multi-Drug Resistant Organisms (MDRO), microbiology labs play an critical
role. For fighting infectious organisms, microbial identification (ID) and antibiotic susceptibility tests (AST) providing
the right information for targeted clinical responses. Combining an advance, automated platform with the VITEK 2
microbial ID/AST testing system offers the confidence of fast, accurate results. Its design helps to ensure the better
laboratory workflow with fewer repetitive tasks, improved standardization, higher safety and rapid time-to-results and
reporting.

VITEK MS is an automated microbial identification system that uses an innovative mass spectrometry technology to
provide identification results in a few hours

VITEK2 uses unique ID and AST cards the size and shape of a playing card. Ready-to-use and low-cost, VITEK2
ID cards give a menu of available tests. Based on this advance innovation, VITEK2 can provide identification
and susceptibility results in as little as 5 hour
 How it works
After primary organism isolation, handling is minimized in a simple standardized inoculum. The inoculum is
placed into the VITEK2 Cassette at the Smart Carrier Station, where the VITEK® 2 Card gets in contact with
the sample. Once the Cassette is loaded, the incubation and reading of each card is managed by the system.

 
 DIAGNOSIS OF TYPHOID FEVER
After a person had eaten contaminated food or water, the Salmonella bacteria moves to the small intestine
and enter the blood temporarily. The bacteria are than carried by white blood cells in the liver, spleen and
bone marrow, where they reproduce or multiply and again re-enter the bloodstream. People develop
symptoms, including fever, at this point. Bacteria move to the gallbladder, biliary system, and the lymphatic
tissue of the bowel. Here, they multiply in high numbers. The bacteria pass through the intestinal tract and
can be identified in stool samples. If a test result is not clear, blood samples will be collected to do a
diagnosis.

Generally, typhoid fever can be detected by blood culturing. If a patient shows any infection in
his/her blood, then there may be chances of infection.
Typhoid fever can be detected by -
1. Slide Widal test
2. Tube Widal test
3. Salmonella typhi rapid test

Widal test
 Georges Ferdinand Widal developed Widal test in 1896.

• Widal Test is an agglutination test, which detects the presence of serum agglutinins (H and O) in patient's
serum with typhoid and paratyphoid fever.

• When facilities for culturing are not available, the Widal test is the reliable and can play a important role in
the diagnosis of typhoid fevers in endemic areas.

• The patient’s serum is tested for O and H agglutinins/antibodies against the following antigen suspensions
(usually stained suspensions)
 Principle of Widal test

Suspension that contain antigen will agglutinate on exposure to antibodies to Salmonella organisms.
Patients’ suffering from enteric fever will carry antibodies in their sera, which can react and agglutinate serial
doubling dilutions of killed, colored Salmonella antigens in a agglutination test.

The main principle of Widal test is that if homologous antibody is present in patient's serum, it will react
with respective antigen in the reagent and gives visible clumping on the test card and agglutination in the
tube. The antigens used in the test are “H” and “O” antigens of Salmonella Typhi and “H” antigen of
Salmonella Paratyphi. The paratyphoid “O” antigen is not used in this process as they cross react with
typhoid “O” antigen due to the sharing of factor 12. “O” antigen is a somatic antigen and “H” antigen is
flagellar antigen.
 SLIDE WIDAL TEST

Slide Widal test is a rapid test. It gives instant result approximately in 2 minutes. This test is useful for emergency
medical screening and for medical facilities with limited resources. They also allow testing in primary care for things
that formerly only a laboratory test could measure.

Procedure of Widal test:

1. Place one drop of positive control on one-reaction circles of the slide.

2. Pipette one drop of isotonic saline on the next reaction circle. (Negative Control).

3. Pipette one drop of the patient serum Tobe tested on the remaining four reaction circles.

4. Add one drop of Widal test antigen suspension ‘H’ to the first two reaction circles. Add one drop each of ‘O’,
‘H’, ‘AH’ and ‘BH’ antigens to the remaining four reaction circles.

5. Mix contents of each circle carefully over the entire circle using separate mixing sticks.

6. Rock the slide gently back and forth and observe for agglutination within one minute.

STANDARD TUBE TEST METHOD

In Widal tube Test, two types of tubes were originally used:

(1) Dreyer’s tube (narrow tube with conical bottom) for H agglutination and
(2) Felix tube (short round-bottomed tube) for O agglutination.

Now days 3 x 0.5 ml Kahn tubes is used for both types of agglutination.

PROCEDURE:
1. Take 4 sets of 6 Kahn tubes/test tubes and label them O, H, AH and BH.
2. Pipette 1.9 ml of isotonic saline into the tube No.1 of all sets.
3. To each of the remaining tubes add 1.0 ml of isotonic saline.
4. To the tube No.1 in each row add 0.1 ml of the sample, which needed to be tested and mix well.
5. Transfer 1.0 ml diluted serum from tube no.1 to tube no.2 and mix well.
6. Transfer 1.0 ml of the recent diluted sample from tube no.2 to tube no.3 and mix well. Continue this
serial dilution till tube no. 5 in each set.
7. Discard 1.0 ml diluted serum from tube No. 5 of each set.
8. Tube No. 6 acts as a saline control in all the sets. Now the dilution of the sample in each set is as
follows: Tube No. : 1 2 3 4 5 6 (control) Dilutions 1:20 1:40 1:80 1:160 1:320
9. To all the tubes of each set add one drop of the respective WIDAL TEST antigen (O, H, AH and BH)
from the reagent vials and mix well.
10. Incubate the tubes at 37° C overnight (approximately 18 hours).
11. Dislodge the sediment button gently and observe for agglutination

RAPID TEST FOR SALMONELLA TYPHI

OBJECTIVES:

As typhoid fever is endemic in India, there is a continuous search for a simple test, which can be easily
performed in small laboratories with fewer resources for an early and rapid diagnosis. Typhidot test is
evaluated for this purpose.

METHODS

Rapid test is used to detect presence of s. typhi in patient’s blood. In this test serum is extracted and one
drop is placed on the kit provides followed by one drop of buffer provided in the kit. The test is carried out
on coded sera according to the instructions written by manufacture.

RESULTS:

•If a red line is seen in IgM and IgG , then person is having infection.

•If a red line is only seen in IgM, then person is having acute infection.

•If a red line is only seen in C then person is not having any infection.

INTERPRETATION & CONCLUSION:

The Typhoid test is easy to perform, and requires no extra training of staff for explaining the results.
However, rapid test is not sufficient to conclude whether a person is suffering from typhoid fever or not so,
it is followed by other tests. Complementary test like blood culture and the Widal test are useful in the
diagnosis of typhoid fever.
 TREATMENT

Typhoid fever is treated with antibiotics, which kill the Salmonella bacteria. Prior to the use of
antibiotics, the fatality rate was 20%. Death occurred from overwhelming infection, pneumonia,
intestinal bleeding, or intestinal perforation. With antibiotics and supportive care, mortality has been
reduced to 1%-2%. With appropriate antibiotic therapy, there is usually improvement within one to
two days and recovery within seven to 10 days.
Several antibiotics are effective for the treatment of typhoid fever. Chloramphenicol was the original
drug of choice for many years. Because of rare serious side effects, other effective antibiotics have
replaced chloramphenicol. The choice of antibiotics is guided by identifying the geographic region
where the infection was contracted (certain strains from South America show a significant resistance
to some antibiotics). If relapses occur, patients are retreated with antibiotics.
Those who become chronically ill (about 3%-5% of those infected), can be treated with prolonged
antibiotics. Often, removal of the gallbladder, the site of chronic infection, will provide a cure.
.
 RAPID vs. AUTOMATED TESTS

Rapid test gives result within a short span of time but the percentage of accuracy is less whereas
automation takes time in giving the result but it gives an accurate result.

Therefore, it is always recommended to use automated machines in order to get accurate result and
detailed study of the infection and treatment to be given.

In case of VITEK2, we check the sensitivity easily and that too in short span of time whereas in other case
compared to automated machines it takes times as well as less accuracy.

Antibiotic sensitivity or antibiotic susceptibility is the susceptibility of bacteria to antibiotics. Because

susceptibility can differ within a species (with some strains being more resistant than others), antibiotic
susceptibility testing(AST) is done to determine which antibiotic will be most successful in treatment of
bacterial infection in vivo. Antibiotic sensitivity tests are often done by the Kirby-Bauer method.

Small discs containing antibiotics are placed onto a plate upon which bacteria are growing. If the bacteria are
sensitive to the antibiotic, a clear ring, or zone of inhibition, is seen around the disc indicating poor
growth. Other methods to test antimicrobial susceptibility include the Stokes method, Etest (also based on
antibiotic diffusion), Agar and Broth dilution methods for minimum inhibitory concentration (MIC)
determination. The results of the test are reported on the antibiogram.

The antibiotic susceptibility can be checked by measuring the diameter and the degree of lysis of the media.
 CONCLUSION
Diagnostic sciences are a very crucial part in treatment of any patient because without a proper
diagnosis one cannot randomly go on treating a disease. Efficiency, accuracy and speed these are the
3 main constituents that any good diagnostic method should have. With advancing technology and
increased understanding of nature, man is striving towards development of advanced methods for
microbial identification and eradication. Various types of diagnostic methods have been developed
which have their own unique pros and some cons. It’s important to understand that the world we
live in is diverse place some communities are more well off than other and vice versa, though
humanity at this time cannot provide everyone with the same resources but we can at least make
sure that everyone around the world is provided with the modern day basic necessities that includes
healthcare. While automation might be good enough for huge laboratories, rapid tests go a long way
in providing inexpensive and instant diagnosis

Automated and rapid methods both have their advantages and disadvantages, the automated method is
fairly accurate and less labor intensive but takes a handsome amount of time whereas rapid methods
though not that accurate but they produce almost instant results. One method cannot be overlooked in
presence of other and it's their coexistence that makes the diagnosis and treatment of the disease efficient
and viable.

Although blood culture will always maintain their gold standard rankings but the rapid tests and
automated methods are catching up fairly fast. That day is not very far when humans will develop
automated methods that have more than 99% accuracy and instant results.
 References

 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3920109/
 www.medicalnewstoday.com
 www.webmd.com
 www.who.int/bulletin/volumes
 www.atmph.org/article.asp
 www.tandfonline.com
 https://www.researchgate.net