Evidence Based Practice on Typhoid Fever

TYPHOID FEVER

SALMATSalmonella Microagglutination

Technique

INTRODUCTION

• Salmonella genus is a complex bacterial group widely distributed in nature. In humans, Salmonella serotypes such as Typhi can cause typhoid fever, a life-threatening disease.

• During the late 1970s Ship and Rowe developed a mechanized technique for Salmonella serotyping that uses small volumes of commercial antisera (25 [micro]L). However, it depended on the purchase of specialized equipment.

• Other serotyping approaches are currently being developed, which also require acquisition of specialized equipment as well as expensive reagents like protein microarrays, and Luminex.

• Due to budget limitations and to the importance of maintaining Salmonella-laboratory-based surveillance, there has been a newly developed manual microtiter flagella test which is used regularly for Salmonella serotyping since 2005.

• This microtest requires smaller amounts of reagents than STAT and has allowed maintenance of Salmonella surveillance programs.

INTRODUCTION

ACTUAL STUDY

• The SALMATcor was developed and validated at the "Centro Nacional de Referencia en Bacteriologia from the Instituto Costarricense de Investigation y Ensenanza en Nutricion y Salud“

• The performance of this technique was compared with Standard Tube Agglutination Tecnique (STAT) as a reference method.

• For the intralaboratory validation, 141 Salmonella strains of 12 different serovars isolated from humans, animals, food, and environment were used

• In total, 1187 Salmonella H antigens were characterized by each method to compare concordance between STAT and SALMATcor.

ACTUAL STUDY

• For the intralaboratory statistical validation of the assay, the relative accuracy (AC), relative specificity (SP), and relative sensibility (SE) of the SALMATcor compared to the STAT.

ACTUAL STUDY

• AC - degree of correspondence between the response obtained by the STAT and the response obtained by the SALMATcor

• SE - ability of the SALMATcor to detect the antigen when it is detected by the STAT

• SP - ability of the SALMATcor not to detect the antigen when it is not detected by the STAT

• Cohen's kappa index was used to estimate the concordance between the results obtained by both analytical methods

ACTUAL STUDY

• Performance indicators

99.92% AC

100% SP

99.78% SE

ACTUAL STUDY

How to perform SALMAT

• FFA preparation for each Salmonella strain to be tested was prepared like it was prepared for STAT

• For each antigen to be evaluated, 100 [micro]L of FFA was placed into a well of a 96-well polystyrene microplate (round or flat bottom) and 20 [micro]L of diluted monovalent antiserum was added (or 100 [micro]L if using polyvalent antiserum)

• The plate was covered with a lid and then incubated in a water bath over a floating platform for 1 h at 50[degrees]C [+ or -] 1[degrees]C.

How to perform SALMAT

• After incubation, to avoid abrupt movements that could disrupt the aggregates, the wells were examined for agglutination under indirect white light, interpreting positive and negative reactions

• As it required for STAT, an autoagglutination control is always required. In this case, in one well of the plate 20 [micro]L of saline was added to 100 [micro]L of FFA, and the results were recorded and interpreted.

CONCLUSION

• SALMAT is an easy alternative procedure that reduces laboratory expenses and makes Salmonella serovar surveillance more accessible. This new method is as accurate and specific as the STAT, showing an excellent concordance in the intralaboratory validation assay and the WHO-GSS EQAS program.

CONCLUSION

• This microagglutination technique can be adopted even by low-income laboratories in developing countries, independently of antisera's brand/source, as long as they are designed for STAT applications.

• For those laboratories that already perform Salmonella serotyping by the tube technique or that are planning to implement it, adopting the SALMATcor is recommended.

• SALMATcor will contribute to improve Salmonella surveillance worldwide.

RECOMMENDATION

• SALMATcor is a newly developed method to determine the presence of the salmonella bacteria group. I greatly recommend the procedure because aside from it lessens the expenses in the salmonella antisera, it is easier to perform which makes it easier for the technicians to handle even a great number of samples at the same time. It is a great substitute to the usual and traditional agglutination method that most laboratories are using. It’s even more recommended because it can detect the presence of salmonella bacteria accurately.

PCR (Polymerase Chain Reaction)

method for

Salmonella

Introduction

Salmonella enterica serovar Typhi, the human-specific, causative

agent of typhoid fever, causes an estimated 21 million new cases and

216,000 deaths every year.

It is generally thought that Salmonella enterica serovar Typhi, an

enterically acquired invasive pathogen, penetrates the ileal epithelium and is

transported via underlying macrophages to spleen, liver, and other target

tissues during the normal disease course.

Natural clearance of the disease is thought to entail development

of specific humoral and cellular immune responses.

However, the precise mechanisms of Salmonella serovar Typhi

virulence and immune protection are not well understood, mainly due to the

absence of a suitable animal model.

We have been developing a human typhoid challenge model which

could increase understanding of the pathogenesis of typhoid fever, the

immunobiology of the disease and the correlates of protection, and provide a

model for evaluation of new vaccines.

In the case of Salmonella serovar Typhi, there is an urgent need for

new improved vaccines, as those currently available all have limitations.

Actual Study

As early diagnosis of the disease and prompt treatment are

essential for optimal management, especially in children, a rapid sensitive

detection method for typhoid fever is urgently needed. Although PCR is

sensitive and rapid, initial research indicated similar sensitivity to blood

culture and lower specificity.

They developed a fast and highly sensitive blood culture PCR

method for detection of Salmonella Typhi, allowing same-day initiation of

treatment after accurate diagnosis of typhoid. Methods an ox bile tryptone

soy broth was optimized for blood culture, which allows the complete lysis of

blood cells to release intracellular bacteria without inhibiting the growth of

Salmonella Typhi.

Using the optimised broth Salmonella Typhi bacteria in artificial blood samples

were enriched in blood culture and then detected by a PCR targeting the fliC-d

gene of Salmonella Typhi.

Several studies have been reported since the first evaluation of PCR

as a diagnostic tool for typhoid fever when Song et al. successfully amplified

the flagellin gene (fliC-d ) of Salmonella serovar Typhi in all cases of culture

proven typhoid fever and from none of the healthy controls.

These studies reported excellent sensitivity and specificity when

compared to positive (blood culture proven) and healthy controls.

The clinical utility of these PCR tests was inadequately evaluated though some studies

claimed that as few as 10 bacteria per millilitre of blood could be detected. The number

of Salmonella bacteria circulating in the blood of a patient with Salmonella bacteremia is

small.

One study found 0.5-22 bacteria per millilitre of blood in 15 patients with typhoid

fever, and another showed a median of 0.3 (IQR, 0.1-10) bacteria per millilitre of blood from

81 patients with typhoid fever. The very low ratio of bacterial to human DNA means that the

PCR template in clinical preparations is dominated by mammalian DNA and could cause

false-positive PCR signals due to the non-specific binding of primers and false-negative

results due to reduced sensitivity. In practice, the large excess of human DNA does indeed

cause problems for PCR-based pathogen detection in blood, particularly in samples with

low bacterial numbers.

Furthermore, small volumes of blood are often used for DNA

extraction or as template in the PCR, which will significantly lower the

sensitivity of these tests.

CONCLUSION

This novel blood culture PCR method is superior in speed and

sensitivity to both conventional blood culture and PCR assays. Its use in

clinical diagnosis may allow early detection of the causative organism and

facilitate initiation of prompt treatment among patients with typhoid fever.

Recommendation

Based on the research, I recommend the Polymerase Chain

Reaction (PCR) for early detection of Typhoid Fever . It’s a nice method to

use because it has a fast and highly sensitive blood culture method for

detection of Salmonella Typhi, that allowing same-day initiation of treatment

after accurate diagnosis of typhoid. PCR has an excellent sensitivity and

specificity when compared to positive (blood culture proven) and healthy

controls. It helps the patient to prevent the disease to become more

serious.