literature review current from email final

8/14/2019 Literature Review Current From Email Final

1/21

Samantha Baillie

Infection and Immunity BSc

The role of MALDI TOF MS in the diagnosis and treatment of

Cystic Fibrosis

Abstract

Cystic Fibrosis (CF) is the most common inherited lethal disorder in

Caucasian patients. Despite extensive research into the condition, sufferers

still have an average life expectancy of less than 40 years compared to the 79

predicted for non-sufferers (1).

The most common cause of mortality and morbidity in these patients is

bacterial lung infections. The correct identification of infecting bacteria is

paramount to providing effective treatment, yet current diagnostic techniques

have levels of correct identification as low as 17% (2). Particularly inaccurate

diagnosis is shown for abnormal Pseudomonas aeruginosa (2) and

Burkholderia species (3), two of the most significant and severe infections in

CF.

New technology could change this. The Matrix-Assisted Laser-Desorption

Ionisation time-of-flight mass spectrometer (MALDI TOF MS) provides a rapid,

accurate and cheap bacterial identification. Correct diagnosis of samples

increases to between 79 and 100% when using MALDI TOF MS (4, 5).

Furthermore, the use of MALDI TOF MS in CF research has allowed

investigation into the proteomics of infecting bacteria (6, 7). This will allowbetter understanding of the infections and provide new targets for treatment.

Use of MALDI TOF MS could open the door to the desperately needed

improvements in CF management.

1


2/21

Samantha Baillie


Introduction

Cystic Fibrosis

Over 8,000 people in the UK have the inherited disorder CF, and one in 25

Caucasians carry the gene for it (8).

In 1989 it was discovered that CF is caused by a mutation to a gene located

on the long arm of chromosome seven. More specifically, this mutation affects

the transmembrane conductance regulator (CFTR) protein encoded at this

region (9). This protein functions as a chloride channel in the epithelium of the

lungs, pancreas, gastrointestinal system and skin. The defective chloride

channel causes a decreased chloride secretion into the airways and a

subsequent increase in sodium absorption. The critical consequence of this is

that the airways become dehydrated and thick mucous secretions can

accumulate.

The warm, damp conditions of these mucous secretions provide an ideal

environment for bacteria to colonise and breed in. The additional protection

against the immune system, provided by the sticky secretions and immobility

of the mucociliary escalator, allows colonisation by rare opportunistic bacteria

not found in the respiratory tract of unaffected individuals.

Lung infections are the primary cause of morbidity and mortality in CF patients

and up to 90% of deaths are due to infection induced respiratory failure (10).

In the infant CF patient, the most common bacteria isolated from the

respiratory tract are Staphylococcus aureus, but in adolescence

Pseudomonas aeruginosa infection overtakes. Another group of clinically

relevant bacteria affecting CF sufferers are those of the Burkholderia cepacia

complex (BCC) which significantly increase morbidity and mortality (11). Other

infections affecting CF patients includeAchromobacter xylosoxidans

Haemophilus influenza and Stenotrophomonas maltophilia. More recent and

2


3/21

Samantha Baillie


novel bacteria include Ralstonia mannitolilytica, Pandoraea apista, and

Inquilinus limosus (2, 4).

(1)

Pseudomonas and Burkholderia infections are the most significant infections

and are frequently referred to in the papers and research included in this

review.

3

Figure 1. CF lung infections vs. age.P. aeruginosa becomes the most commonly isolated pathogen of theairways in the adolescent years. Eventually up to 80% of patientsbecome infected. Although rates ofB. cepacia complex infectionremain low this is still significant due to the mortality associated. (1)


4/21

Samantha Baillie


Pseudomonas aeruginosa

This is the pathogen most commonly found in the respiratory tract of CF

patients. P. aeruginosa infection results in a more rapid deterioration of lung

function, especially in the case of antibiotic resistant Pseudomonas

aeruginosa (7).

Once Pseudomonas aeruginosa infection becomes chronic it is considered

almost impossible to eradicate and can increase mortality over an 8 year

period by 2.6 times (13)

The wide diversity in P. aeruginosa strains affects their virulence, their

aggressiveness and their response to antibiotics (12). The variation ofP.

aeruginosa is so great that there can be altered antibiotic sensitivity even

among various colonies of the same strain.

In CF centres the spread of resistant strains of bacteria between vulnerable

patients has caused controversy and unnecessary deaths. The most notable

case to date is the emergence of the Liverpool strain ofPseudomonas

aeruginosa which has now spread within and between CF centres nationwide.

This strain is especially aggressive and resistant and has been known to

cause pneumonia in non CF relations of CF patients. (12). As a result,

patients infected with such virulent or resistant strains are often kept in

isolation. This has subsequent effect on the psychological state of the patient

and is especially problematic in teenagers, many of whom already haveexisting compliance issues.

Burkholderia cepacia complex (BCC)

Although BCC is somewhat rarer than P. aeruginosa, found only in 3% of

patients (1), it has a more dramatic clinical picture and is therefore consideredof equal importance. 20 to 30% of patients infected with BCC develop cepacia

4


5/21

Samantha Baillie


syndrome in which they experience accelerated pulmonary deterioration,

fulminant necrotizing pneumonia and rapidly fatal bacteraemia (14).

Like P. aeruginosa, BCC is resistant to many antimicrobials and infection with

certain strains of the BCC can be a contra indication to lung transplant (4).

Again like P. aeruginosa, isolation may be necessary in BCC infection to

reduce the spread to other susceptible CF patients. This is considered so

important that patients with BCC infection are banned from attending any CF

events. If diagnosis is incorrect then this isolation may not commence earlyenough to prevent cross infection (15) or conversely, isolation in the case of

false positives can cause unnecessary distress for the patient.

Despite the importance of accurate BCC diagnosis there is often

misidentification within the species or confusion with Pandoraea spp.,

Ralstonia spp., orAchromobacterspp..

Biofilms

Multiple bacteria invading the airways of CF patients have the ability to form a

biofilm. Biofilms are communities of bacteria enclosed in a matrix of proteins

and nucleic acids (16). The biofilm provides the bacteria up to a thousand fold

resistance against host defences and antimicrobial agents compared to the

planktonic form (17).

Resistance has been attributed to the physical barrier formed by the biofilm

which prevents access of drugs, immune cells and immune products.

However, recently it has been suggested that in the conversion from

planktonic cells to those forming biofilms, genes coding for additional

protective factors are switched on. These factors are periplasmic glucans

which are believed to bind to antibiotics and interfere with their passage intocells (18)

5


6/21

Samantha Baillie


Biofilm formation enables bacteria to use quorum sensing to enhance their

chances of survival. In quorum sensing the bacteria wait until their numbers

are high enough to overcome the host immune system before 'turning on'

pathogenic virulence factors. The bacteria secrete N-acyl homoserine lactone

(AHL) which acts as a signalling molecule. Once AHL levels reach a critical

point, genes are activated via secondary signalling mechanisms. These genes

encode proteins that will assist the bacteria, including proteins necessary for

chronic infection and resistance (6).

Although this process is known to increase the virulence of bacteria, the

mechanism is still not fully understood. Arevalo-Ferro et al. used 2D gel

electrophoresis (2DE) to isolate those proteins up-regulated by quorum

sensing, followed by MALDI TOF mass spectrometry to identify them. By

identifying which proteins increase Pseudomonas aeruginosa pathogenicity

and encourage chronic infection, it may be possible to find targets for future

drug development. The ability of these bacteria to form biofilms and therefore

use quorum sensing may be the defining factor that allows Pseudomonas

aeruginosa to chronically affect the airways in immunocompromised patients.

The diversity of pathogens, their antimicrobial resistance, and their ability to

form biofilms all allow these bacteria to drastically affect the lifestyle and life

span of CF patients

Therefore, a rapid and accurate tool of diagnosis is required to ensure that

the correct treatment is given as soon as possible to prevent chronic infection

and the development of resistance. Unfortunately the current methods of

diagnosis are slow, expensive and often inaccurate. The new Matrix

associated laser desorption ionising time of flight mass spectrometer (MALTI-

TOF MS) could potentially revolutionise the way that infections are diagnosed.

6


7/21

Samantha Baillie


MALDI TOF MS

The MALDI TOF MS is a revolutionary new technology that allows rapid

identification of bacteria from their protein profile. Isolates of bacteria from

blood, sputum, urine, stool, cerebro-spinal fluid, swabs or biopsies are

cultured to form individual colonies.

It is important to note that methods of preparing the samples for MALDITOF

MS vary between laboratories. Most laboratories suspend the culture in a

sterile container with 20-300 l of water (4, 5, 19). In some cases the sampleis then centrifuged up to 6 times (19). The supernatant is used to inoculate

two wells of the 96 well chip to be inserted into the MALDITOF MS with 0.5-

1.0 l in each. This is allowed to dry in air before being covered with matrix

and allowed to dry again in order to crystallise the matrix (20). This sample is

now ready for analysis. It is disputed whether adding ethanol to the sample at

this point produces better spectra (27, 28).

A laser is fired in bursts at each well, providing energy to the matrix which is

turn ionises the particles in the sample. The ionised particles are released

from the sample and travel down the tunnel of the MALDI TOF MS (See

Figure 2). The time taken to reach a detector at the end of this tunnel and the

charge on the ion are combined to give a molecular mass/charge ratio. The

mass/charge ratios of all ions omitted are combined to give a spectrum.

7

.

.


8/21

Samantha Baillie


The spectrum produced can be modified using MALDI TOF MS software and

compared to existing databases. Software can perform tasks to refine the

data such as excluding peaks that are less than a certain amplitude, thereby

removing any ions present in too small a proportion to be significant. This

removes the background noise and allows the more relevant peaks to be

focused on. Database software performs the important task of comparing the

spectrum of a given sample to reference strains previously examined and

stored on the database. The spectra are compared to all reference strains and

the best match is given as the diagnosis. This match is graded according to

how close the match is, and also how close the match is to the second best

diagnosis. Ideally the spectra would have a very high level of similarity to a

8

Figure 2. MALDI TOF ionisation1. The laser fires at the sample (S) and matrix (M) in bursts in order to

ionise the particles.MH+ + S M + SH+

2. The ionised particles travel down the tunnel towards the detector.3. The time taken and molecular mass of the particles are combined to givethe mass/charge ratio.


9/21

Samantha Baillie


reference strain yet be highly differentiated from the second best match in

order to give confident confirmation of a given diagnosis.

Discussion

Current diagnosis of lung and airway infections in CF sufferers is based on

phenotypic and molecular analysis of sputum samples. Phenotypic methods

are becoming unpopular due to their time consuming nature and the

inaccuracy of the results produced.

Phenotypic testing

CF lung infections are frequently caused by atypical, opportunistic pathogens

not found in the normal respiratory tract. Furthermore, the invading pathogens

also have an atypical appearance and altered metabolic behaviour due to the

abnormal conditions of the CF airways (5). Current standard commercial kits

such as API 20E and the VITEK 2 cannot be relied upon torecognise the

pathogens as they are targeted towards more common infections, and

therefore often produce no diagnosis or incorrect diagnosis (10).

Additional difficulty arises in the case of biofilm formation, as this can prevent

test reagents from accessing the bacteria. This leads to incorrect results even

for those bacteria that the systems can recognise.

Diagnosis by MALDI TOF is a relatively new technique and therefore there

are few studies directly comparing its potential to current methods. Many

studies compare molecular techniques to older biochemical testing so these

9


10/21

Samantha Baillie


can be combined with those comparing molecular to MALDI TOF MS to gain

an overall picture.

Levels of misdiagnosis were found to be 11-36% in a collection of 1051 CF

sputum isolates collected from 115 treatment centres in the US (3). The

sputum samples were collected with the diagnoses given by current

techniques. Further testing with a combination of selective media, sodium

dodecyl sulfate polyacrylamide gel electrophoresis and DNA sequencing was

carried out on the samples. Results showed that 11% of isolates described as

BCC were not BCC and 36% of those described as non-BCC were in fact

BCC. Of the test centres involved, 70 used only commercial testing systems.

As described earlier, the correct diagnosis of BCC is paramount to the

patients physical and psychological health; therefore these findings highlight

the importance of more accurate diagnoses.

The performance of commercial biochemical kits in diagnosis of gram

negative rods showed lower levels of accuracy with correct diagnoses ranging

from only 17% identification to 56% (2). The highest levels of inaccuracy were

shown using the API 20E kit for identifying abnormal strains ofPseudomonas,

such as those often found in CF patients. The 17% that were correctly

identified were only matched to 16S rRNA sequencing when the result

accuracy was described as exceptional according to manufacturer guideline.

The very good and good results were often unreliable (67% and 84%

misidentification respectively) (2). Better results were shown for the API 20 E

when identifying a range of gram negative bacteria with levels of recognition

at 54%- similar to the alternative kit being compared (VITEK 2) which

achieved recognition levels of 56% (21). This shows that the kits do vary in

their ability to diagnose relevant bacterial strains; however, neither set of

results show levels as high as would be expected when the importance of

their accuracy is considered.

An especially common error in the commercial kits is the inability to identify

between BCC isolates and non BCC isolates (10, 14, 23). Burkholderia

10


11/21

Samantha Baillie


species are one of the most significant infections in CF patients, yet using the

BD phoenix and VITEK 2 systems, misdiagnosis were 56 and 55%

respectively (14). Similar results were given in a comparison of four

commercial systems including RapID NF plus, API Rapid NFT, VITEK Auto

Microbic system and Uni-N/F Tek. The Vitek sytem consistently confused

BCC with Burkholderia pickettiiand the API rapid NFT labelled 7 out of 15 non

BCC isolates as BCC. The Remel and RapID NF were declared to have the

most accurate results with the Remel database detecting 53 out of 58

Burkholderia species. The Remel database shows more promising results but

the low accuracy in the other systems highlight the importance of focusing on

differentiation between Burkholderia species.

Molecular testing

So far these studies have shown the shortcomings of phenotypic techniques,

especially the commonly used commercial kit systems. Now the improved

molecular techniques, specifically 16s rRNA gene sequencing can be

compared to the MALDI TOF mass spectrometer.

A sample of 80 blind-coded, clinically relevant, non-fermenting gram negative

bacteria were examined using both the 16s rRNA sequencing method and

MALDI TOF MS. While the sequencing method accurately identified 57 out of

80 non fermenting isolates, the MALDI TOF MS recognised 67 of 78 (5).Although two isolates were removed between the 16s rRNA typing and the

MALDI TOF MS typing, the difference in accuracy would still be significant

enough to show the improvement in diagnosis by MALDI TOF MS. The most

notable improvement is the correct identification of the Burkholderia species

by MALDI TOF MS. 16s rRNA sequencing could not discriminate between

Burkholderia cepaciaDSM 7288, Burkholderia cepacia LMG 2161 and

Burkholderia stabilis nor could it distinguish between the three strains

Burkholderia stabilisLMG 14294, Burkholderia pyrrocinia LMG 14191 and

11


12/21

Samantha Baillie


Burkholderia ambifaria LMG 11351. In contrast the database of the MALDI

TOF MS could distinguish between both sets ofBurkholderia strains. This is

an improvement on both the 16s rRNA sequencing technique and the results

from the phenotypic diagnostic tests described earlier.

When MALDI TOF MS was used to focus specifically on strains of non

fermenting bacteria isolated from CF patients, 549 of 559 strains were

correctly identified. 9 of the incorrectly identified strains belonged to

Burkholderia species and one to the Ralstonia genus (4). Comparing these

results to those described earlier for other phenotypic methods, with levels of

identification at 56%, it is clear that there is a huge improvement in the level of

diagnosis. Furthermore, an additional database was created with refined

diagnosis for the BCC and Ralstonia mannitolilytica and as a result the

identification improved from 83% to 98% for the BCC and from 94% to 100%

for the Ralstonia genus (4).

The use of MALDI TOF MS in combination with other diagnostic techniques

Thus far the MALDI TOF mass spectrometer has been compared to current

methods of diagnosis, but its use alongside 16S rDNA has also proved

beneficial. Von Wintzingerode et al. describe how MALDI TOF can be used to

analyse fragments of bacterial DNA created by PCR (23). 16s rDNA was

converted to 16s rRNA by PCR amplification in the presence of dUTP insteadof dTTP. The sequences were then fragmented using uracil-DNA-glycocylase,

followed by further amplification to create DNA signature sequences. MALDI

TOF was then used to analyse these fragments. The MALDI TOF MS can

discriminate between single nucleotide differences in the fragments allowing

reliable and accurate identification when the fragments were compared to a

16s rDNA database.

12


13/21

Samantha Baillie


The advantages of combining these methods are speed and reproducibility of

results. The rapid detection suggests that this method could potentially be

used for high-throughput identification with subsequent pharmaceutical

applications.

Similarly 2 dimensional gel electrophoresis (2DE) and MALDI-TOF were

combined to investigate the periplasmic proteome ofP. aeruginosa. This

research found the function of periplasmic proteins to be in 'transport, cellenvelope integrity and protein folding control' (24). In this process the proteins

were first obtained from the periplasmic material by centrifugation, then 2DE

was carried out on them. The 495 spots produced were excised, digested

using trypsin and analysed by MALDI TOF mass spectrometry. The database

MASCOT was used to confirm which proteins were present in the periplasmic

space. Thus, the combination of 2DE and mass spectrometry allowed the

identification of the periplasmic proteins in P. aeruginosa. Prior to this

research, only the bacterial membrane had been studied in such a way and

not the periplasm itself. Investigating which periplasmic proteins are present

or absent in various environmental conditions could help in the understanding

ofP. aeruginosa infection and its response to antibiotics.

13

1) Bacteria arecultured

2) 2DElectrophoresis

separates theproteins accordingto size

3) MALDI TOFcreates spectra for

individual proteins
http://img.directindustry.com/images_di/photo-g/maldi-tof-mass-spectrometer-57008.jpghttp://images.google.com/imgres?imgurl=http://www.braile.net/Newsletters/2008NYK/culture%2520dish.jpg&imgrefurl=http://www.hiddenhealthchiro.com/index.php%3Ffile%3D/nyk/templates20/shared/article.html%26aid%3D1752&usg=__wuAn4aaXW1fMd3iCfPFniS2g4qU=&h=272&w=316&sz=18&hl=en&start=1&um=1&tbnid=FM-1e98u6oCVGM:&tbnh=101&tbnw=117&prev=/images%3Fq%3Dculture%2Bdish%26hl%3Den%26rls%3Dcom.microsoft:en-us%26sa%3DN%26um%3D1


14/21

Samantha Baillie


A combination of MALDI TOF MS and 2DE was also used to investigate

quorum sensing in P. aeruginosa. 2DE was used to separate the proteins that

were present when the P. aeruginosa was using quorum sensing and those

present when the P. aeruginosa was not. This was followed by MALDI TOF

mass spectrometry to determine which proteins these were in order to gain a

better understanding of the change in protein activity and the role of quorum

sensing (25). Quorum sensing and biofilm production are closely linked and

are believed to play a central part in the chronic infection of bacteria such as

P. aeruginosa. Studying the proteins and mechanisms involved in quorum

sensing is vital if it is to be understood why P. aeruginosa can become

impossible to eradicate in CF patients. In this case almost 25% of proteins

had their levels altered by at least 2.5 fold highlighting the significance of this

process.

MALDI TOF and CF related proteomics

Some of the research described above investigated the proteomics of CF

infections. MALDI TOF MS has proved to be a useful tool, not only in looking

at the intact cell but also the interaction of proteins that occurs in the cells and

in biofilms.

Patrauchan et al used MALDI TOF mass spectrometry to examine the

proteomics ofP. aeruginosa and the differences in protein expressionbetween the planktonic and biofilm form. The biofilm form ofP. aeruginosa is

associated with longer and more chronic infections such as those in CF. The

effect of variations in calcium levels on the formation of biofilms was also

investigated; as calcium levels are higher in the CF airways. Results showed

that between 40-60% of proteins, representing 146 proteins, were altered by

these two factors. The affected proteins were thought to be involved in

metabolic processes such as iron acquisition, nitrogen metabolism and

oxidative stress responses, all of which aid bacterias survival in the

14


15/21

Samantha Baillie


respiratory tract. This discovery will aid the understanding of processes that

lead to P. aeruginosa infection becoming chronic and why this can occur in

CF airways. It also provides targets for future pharmaceutical research to find

methods of preventing the establishment of chronic P. aeruginosa infection.

A potential new therapeutic technique for CF treatment uses chicken

antibodies targeted towards antigens on P. aeruginosa. These are thought to

attach and prevent entry to cells and may provide prophylactic treatment for

CF patients to prevent P. aeruginosa infection (7). To further investigate the

mechanism of this process, Nilson et al. used MALDI TOF MS to determine

which bacterial proteins were targeted by the antibodies. Again 2DE was used

to isolate the proteins, and these were processed by the MALDI TOF MS,

matching the spectra given to those in the ProFound database. Prior to using

MALDI TOF MS it was known that these antibodies provided prophylactic

protection against P. aeruginosa infection, but not what the target was. MALDI

TOF MS allowed analysis to the level of amino acid sequence and identified

the target as Flagellin. This protein is not only involved in bacterial invasion

but can also cause an inflammatory response. Thus the research had a role in

confirming the target, and also in drawing attention to other advantages of the

chicken antibodies because of their ability to reduce inflammation.

15

Figure 3.Bacteria with FlagellinTargetting the Flagellin proteinwith antibodies prevents cellmotility and reduces inflammation.(7)


16/21

Samantha Baillie


Problems with MALDI TOF MS

Databases

The evidence given so far highlights the advantages of MALDI TOF MS in the

diagnosis of bacteria infecting patients with CF. However, as with all new

technology, there are questions that must first be answered in order for its use

to become widespread.

The first of these is whether the dependency on the database and software

used with the mass spectrometer will affect results. If the database does not

have a wide variety of reference bacteria or proteins then the identification will

not be specific or sensitive. As the MALDI TOF becomes more widely used,

the reference strains recognised by the MALDI TOF will become more diverse

and accurate. As described above the CF lung infections are especially

diverse and so databases must be taught to recognise these species and

strains specifically.

Degand et al. created their own database using 58 reference strains of non

fermenting gram negative bacilli including those frequently found in CF

infections. These were then used to identify 559 clinical isolates of which 549

were correctly identified. A separate database was created for the incorrectly

identified Burkholderia and Ralstonia species, with several reference strains

for each genospecies. As a result the identification ofRalstonia species

increased to 100% and only one strain ofBurkholderia species remained

incorrectly identified (4).

Equally good results were seen in a database created for non-fermenting

bacterial strains. 248 strains were used to make a database which then

identified 85.9% of isolates correctly to the level of species. (5)

16


17/21

Samantha Baillie


Different laboratories vary in the equipment and databases that they use

when performing MALDI TOF mass spectrometry analysis. Investigations

examining specific ion identification by various laboratories showed that there

were slight variations in the ions that were best recognised. Nine of the ions,

produced by MALDI TOF mass spectrometry carried out on an Escherichia

Coliwere recognised by all three labs, while more unique ions were

recognised by different laboratories. Interestingly when the results from all

three labs were combined they gave 100% identification record for various

ions possibly suggesting the advantage of using more than laboratory when in

investigating samples (26).

It is true that there is variation in accuracy between the described databases

set up by individual research teams, however the level of identification

remains high for all. There does not seem to be a correlation between the

number of strain used to set up the databases but this is hard to compare as

the types of bacteria also vary. Continuing the work to refine the databases for

undiagnosed or misdiagnosed bacteria is important and the remaining error in

Burkholderia identification shows that further work is especially needed in this

area.

Variables affecting spectrum quality

It has been suggested that variables, including the culture medium, cultureage, the matrix applied to the sample and the solvents used, can affect the

spectra produced by the MALDI-TOF MS (27).

Valentine et al. found that variations in the growth medium affected the visible

appearance of the spectra produced for the same strain of bacteria. However,

it was also found that while there was a noticeable difference in the spectra,

there were sufficient conserved peaks to recognise the pathogen. The

bacteria were cultured in different conditions and therefore produced varied

17


18/21

Samantha Baillie


proteins. However, the bacteria express a certain number of essential

housekeeping genes, therefore certain proteins are conserved and the

bacteria give an identifiable fingerprint spectrum. The growth mediums

investigated were minimal medium M9, rich media, tryptic soy broth, Luria-

Bertani broth and blood agar. The results are reassuring and illustrate that,

while the growth medium affects the phenotypic appearance of the cell and

therefore the visible spectra, the software itself can be relied on to identify

more subtle similarities.(28)

Likewise, when 10 randomly selected strains from a large sample were

removed and cultured in different ways by Mellman et al., the results were

identical. These strains were all grown on Mueller-Hinton agar in identical

conditions, then analysed by three different MALDI TOF mass spectrometers.

The results were reproducible with all isolates being correctly identified. (5)

Figure 2. shows the similarity between the spectra produced.

18

Figure 2. Mass spectra of a single strain by three MALDITOF mass sectrometers.

The similarity in peaks and troughs is noticeable forautoflex, microflex and ultraflex MALDI TOF MS. (5)


19/21

Samantha Baillie


The ten strains were then grown on 4 different media including Columbia

blood agar, Chocolate agar, Mueller-Hinton agar, and tryptic soy agar. All

isolates were identified correctly with high levels of confidence. Finally, three

of the strains were grown on Columbia blood agar media in identical

conditions before being left for 2, 5 or 7 days at room temperature. These

were then analysed and, once again, were all identified correctly.

These results are reassuring and imply that the growth medium will not affectthe results given. Additionally the machine used did not significantly affect the

results, or the lab used. Using ethanol in the preparation of samples

consistently showed improvement so this is something to be considered when

using MALDI TOF MS (27).

Conclusion

Respiratory infections in CF patients present a complex problem for clinicians

and researchers alike. Despite variations in pathogenicity, ranging from mild

chest infection to severe and often fatal cepacia syndrome, the invading

pathogens remain hard to differentiate between by commonly used diagnostic

techniques. The particularly severe Burkholderia species have proven to be

especially hard to accurately diagnose (11).

The currently high levels of misdiagnoses lead to delayed and inaccurate

treatment with subsequent detrimental effects on patients mental and

physical health. With emerging epidemics of resistant and virulent bacterial

strains in CF patients, accurate treatment is more important than ever (24).

19


20/21

Samantha Baillie


Much remains unknown about the behaviour of pathogens invading the CF

respiratory tract and why chronic infection can be established, unlike in the

normal airways. To fully understand the processes involved, better tools for

diagnosing and investigating infections are required. Factors specific to CF

infections that need to be overcome by investigative techniques include the

abnormality of the pathogens found, the difficulty in accessing them through

biofilms and the difficulty in differentiating between closely related bacterial

species or strains. 16s rRNA gene sequencing and other molecular methods

are a useful tool in providing this information but despite this these techniques

remain unsuitable as they are expensive, time consuming and require

expertise.

Rapid and cheap analysis of the molecular structure of pathogens must

therefore come from more modern techniques, the most successful of which

currently is the MALDI TOF mass spectrometer. Using mass spectrometry to

analyse the protein make up of a cell provides a wealth of information not only

about the cells individually, (14, 21, 23, 4, 11) but also about their protein

activity (6, 24, 25, 27).

Studies using MALDI TOF MS have assisted research into new CF treatment

strategies that are already underway (such as those using chicken antibodies

to Pseudomonas aeruginosa) (7). New potential targets for future treatment

that have been identified by MALDI TOF MS include quorum sensing proteins

(29), periplasmic proteins (24) and calcium regulation (6).

Costing 22-32% of the price of current identification methods, and taking only

6 minutes per isolate (20) it seems inevitable that MALDI TOF MS will

eventually become a primary tool of identification. In order for MALDI TOF MS

to be relied on clinically it must first be proven that the databases are accurate

and diverse enough to identify even rare and unusual forms of pathogens.

Additionally, direct comparisons between MALDI TOF MS and current

methods of diagnosis must be carried out to prove the superiority of

identification. It can be suggested that MALDI TOF MS provides a cheaper,

20


21/21

Samantha Baillie


faster and more accurate identification than phenotypic methods from

identification of similar samples. However, in order to indisputably prove that

MALDI TOF MS should replace current diagnostic techniques direct

comparisons must be performed on clinical samples with results that show a

higher level of identification. In this way price, speed of diagnosis and

technical difficulty can also be compared.

Further Study

Samples taken from CF patients and identified by regular methods should be

analysed by MALDI TOF MS to confirm that the accuracy of diagnosis is

improved or at least equal. It should be ensured that Burkholderia species are

included in these samples as they are rarer but of great importance. Due to

the high degree of error shown in identification by phenotypic methods (14,

21, 3) a further reliable reference method must run in parallel with the

phenotypic and MALDI TOF MS analyses. This will give unambiguous results

as to which of the two techniques provides better identification.

Evidence from this study will allow the MALDI TOF MS to become a more

widely used method of identification in both the research and clinical setting.

This potentiates immediate and future improvements in the diagnosis and

management of CF patients in order to extend and improve the lives of CF

sufferers.

literature review current from email final

Documents