Methicillin-Resistant Staphylococcus aureus vs. traditional culture methods

Is the cepheid GeneXpert a specific and sensitive method to identify Methicillin-Resistant Staphylococcus aureus (MRSA) vs. traditional culture methods?

Abstract

Methicillin-Resistant Staphylococcus Aureus (MRSA) is a bacterial disease opposed to antibiotic methicillin. Since it was first reported in the 1960s, reported incidence on its transmission and infections has continued to rise. Statistics from disease control centers show that by the year 2000, the number of sick persons in intensive care units who had been infected with MRSA had risen to 54.5% in most cases this has consequently increased the length of admission in hospitals, high cost of treatment and even in some cases mortality. Reports have shown that in most infections, organisms resistant to remedy such as MRSA have institutions as the epicenter of the spread. Equipments, hands and apparels of health workers have been known to be responsible for the spread of these organisms. Reliable and early detection of MRSA agents is important for the prompt implementation of seclusion of infected patients.

 A characteristic feature of methicillin resistance is its varied nature, this varies in according to the culture method used, and the salt concentration in the media as well as the temperature of incubation but only a minute percentage of MRSA shows resistance at any given time. Specific methods are used in the detection of MRSA most of these methods have been known to be successful in the detection of the organism. The American society of microbiology recommends an approach that combines blood agar plate with mannitol-salt agar and media salt that has trypticase soy broth. It has been noted that the use of the PCR real time based IDI-MRSA assay coupled with the smart cycler rapid DNA system or the geneXpert system, significantly improves the detection time for MRSA (Cepheid, 2009). It is also seen to be sensitive and specific, the test is based on the detection of the sequence of the staphylococcal organism with its chromosomal make up as well the chromosomal make up of orfx. At the moment, tests involving MRSA are from nasal and groin specimens. The use of the combined specimens in the use of IDI-MRSA has shown to be sensitive and particular for detecting MRSA. An assessment of the test when both specimens were processed separately and when combined was carried out this was to establish the sensitivity and the specifity of the IDI-MRSA. The results were then compared with those done using the routine culture (Johnson et al., 2006).

Introduction

Staphylococcus aureus is a type of bacterium that is commonly found on the skin and noses of healthy individuals. Although on most cases it is seen to be harmless, it may at times enter the body through cuts, wounds causing infections that may be mild or serious. MRSA is mostly opposed to antibacterial agents i.e. of methicilin and other related antibiotics this is because; MRSA creates a substance around it called lactamase that destroys the antibacterial properties of methicilin.

In the recent past MRSA infections has been on the increase especially in hospitals. Some patients hide the bacteria in their noses and sometimes in the groins without the threat of getting any infections. These patients however, may get infected if the bacterium infects other parts of the body such as open wounds. Patients who are colonized with the bacteria also serve as an agent of the bacteria which spreads to other patients. This is possible if hospital staffs or attendants become colonized with the bacterium themselves and spread the bacteria to other patients whom they come into contact with.

 The spreading of infection rate has continued to be noted across the world especially in the USA, however further investigations have revealed that the strain of the hospital MRSA is different with that found in the community at large which has the capacity to cause more serious infection in individuals. This is mostly due to the high level of toxins in this type of bacterium which increases its ability to cause illness (DHQP, 2008).

 But the early detection and containment of MRSA has been proven to reduce its prevalence. Studies in DNA sequencing have shown that the staphylococcal chromosome mec integrates with the s.aureus chromosome. The SCCmec carries the resistant mecA. To detect the MRSA the use of assays that are designed to target sequences in the sccmec is implemented while other subsequent tests focus on orfx.

 This assay is capable of differentiating between the methicilin susceptible aureus and methicilin resistant staphylococci. As tests have progressed a new model that targets DNA sequences in the chromosomal orfx-sccmec called the Xpert MRSA has become available. This is used together with the GeneXpert PCR platform (Diagn, 2007).

 Using the expert MRSA assay, the DNA samples are extracted and detected in cultures of different test chambers. These chambers contain frozen beads that have reagents required to process in real time. Minimal time in processing ensures that more batches can be tested (Johnson et al., 2006).

Methods/Materials

This study was conducted at a public teaching and referral hospital in Melbourne Australia. Previous screenings had indicated that there was a high colonization of Methicillin-Resistant Staphylococcus aureus (MRSA). Patients had been relocated to other ward blocks following redevelopments in their initial wards. But prior to this and within the 48 and 96 hours the patients aged above 18 had to undergo a screening process for nose and cutaneous groin MRSA. Consent had to be sort first from the patients; the hospitals human ethics team subsequently approved the procedure. The first procedure involved the collection of swabs from patients with emphasis on the cutaneous areas of the groin and nasal area. This was aided by the use of Copan trasystem liquid Stuart swabs with the processing of the samples, commencing within twelve hours.  The second step involved the testing of the samples through the comparisons of the IDI-MRSA assay. This involved conducting separate tests of both swabs and a sample a combined site. This method was seen to be appropriate inorder to avoid sampling variations. Using each swab collected, DNA lysates were prepared using the reagents that had been supplied with the MRSA assay kit. (Infecto diagnostic inc.) also provided more buffer tubes in order to carry out the combined nose-groin DNA lysate specimen.4ug/ml oxacilin was used to inoculate nose-groin swabs from each patient this was then placed into different buffer tubes containing 1ml sample buffer. Each of the groin nose samples measuring 0.5ml were then transferred into different lysis tubes. This was after vortexing for 1min. a combination of the samples measuring 0.5 from each groin and nose samples were then placed into a third lysis tube bringing a total to three of the DNA lysis tubes for each patient i.e. 0.5ml for each nose and groin samples and 0.25 for the combined nose and groin samples. The first two samples were then sent for MRSA culture. The DNA lysate preparation was then completed for the three lysis tubes. This was as prescribed by the manufacturer. The lysis tube were set up at 21000*g for a period of five minutes at room temperature, they were later removed and a fresh sample of 50ul added to the lysis tubes. The lysis tube were then vortexed for a further period of five minutes at room temperature and centrifuged for a period of 30seconds at 21000*g. this procedure was then followed with heating for a period of 2minutes with subsequent storage in ice prior to the commencing of the IDI- MRSA procedure (Nasia et al., 2003).

The IDI-MRSA assay procedure was carried out on all the three samples provided as prescribed by the manufacturer. The procedure was as follows; 2.8ul of DNA lysate sample was added to 25ul of master mix which was then moved to a smart cycler tube. The tubes were then inserted into an I-CORE module of the cycler which was then run using the IDI-MRSA. A positive and negative control DNA was then included in each cycle. The tests were repeated using the DNA lysate that had been kept in ice if they demonstrated inhibition. A recurrence in inhibition led to the exclusion of samples upon retesting.

            Culture methods: the specimens from the patients which consisted of the residual sample buffer and swabs, contained 1ml of typtone Soya broth, with an addition of 6.5% sodium chloride. These broths were cultured for 48hours at 350c this was then sub cultured onto MSO before storage at 40c. The MSO cultures were then incubated for 48 hours at 350c this was followed by the sub culturing of colonies of s. aureus onto DNase agar and blood agar. The MRSA isolates were then confirmed using regular testing methods. Antibiotic susceptibility was then evaluated using CLSI methods by agar dilution.

Additional investigations were conducted if it was found that the results of IDI-MRSA assay on any specimen showed discordance. For PCR positives where the culture was negative specimen, both culture broths were subcultured in manitol salt agar that was antibiotic free in order to ascertain the presence of MRSA. The results in this procedure were however not considered in the analysis of specificity since the culture methodology here was more detailed. Patient samples that had PCR positive but culture negative specimens underwent a review of their laboratory records to ascertain infection of MRSA. However the IDI-MRSA PCR assay that was negative but with positive assay culture, were tested using gel based PCR for the presence of MecA and nuc genes.

This data was further subjected to analysis where the DNA specimens obtained from the groin and nose or the combination specimen was compared to MRSA results that were arrived at using the culture method. Furthermore, the specificity and sensitivity of each IDI-MRSA were put into comparison with that of traditional culture assay (Nasia et al., 2003).

TABLE 1. Comparison of the IDI-MRSA assay using individual and combined nose-groin

DNA specimens with culture of swabs for detection of MRSA

IDI-MRSA assay

sample (n _ 192)

No. of

positive

samples

No. of

negative

samples

Sensitivity

(%)

Specificity

(%)

PPVc

(%)

NPVd

(%)

No. of

positive

samples

No. of

negative

samples

Sensitivity

(%)

Specificity

(%)

PPVc

(%)

NPVd

(%)

Nose

+ve

-ve

18

2

14

158

90

91..7

56

98.8

23

3

9

157

88.5

94.6

72

98.1
Groin

+ve

_ve

15

3

17

157

83

90.2

46

98

18

4

14

156

81.8

91.8

56

97.5
Combi

+ve

_ve

22

3

14

153

88

91.6

61.

98

27

4

9

152

87.1

94.4

75

97

TABLE 3. Sensitivity, specificity, and positive and negative predictive values of the Xpert MRSA kit compared with those from direct and enrichment cultures from nose, throat, and

groin/perineum specimens

Reference method and and analysis group
Site
Sensitivity
Specifity
Ppv
Npv
Direct culture
All
88
92
53
99
Enrichment culture
All

Nose

Throat

groin
79

84

75

97
94

92

98

94
71

67

83

69
96

97

93

99
Amended results all patients
All

Nose

Throat

groin
90

95

75

97
97

98

98

96
86

90

88

80
98

99

96

99
Previously positive patients
All

Nose

Throat

groin
87

93

73

95
100

100

100

100
100

100

100

100
87

88

77

96

Results

Generally, all the specimens from the patients were assessed by IDI-MRSA assay and culture in total, 211 patients specimens were tested. Results from some patients totaling 19 were cancelled this was due to either failure in IDI-MRSA assay external control or constant PCR inhibition in the specimens. A number of patients totaling 18 had discordant results where one or more specimens were inhabited on the PCR assay but faced no inhibition on the assay of the stored frozen lysate specimen. The analysis though was relatively successful with 91% of the tests getting valid results using the various methods.16% of patients were colonized using the MRSA while 13% were colonized  on MRSA based on the agar culture assay with the options of groin alone 2.6%, nose alone3.6% or a combination of the two 6.8%. Six patients were found to be positive by broth culture alone. Groin culture in comparison to nose culture alone identified a further five patients that would have been locked out had agar culture alone been performed. The sensitivity and specifity of IDI-MRSA assay in comparison to agar culture assay on nose and groin specimen were compared to those performed on the combined specimen of nose and groin the specifity was found to be 91%, 90% and 91.6% respectively. It was also found that the incorporation of the broth culture resulted in slight difference in terms of specifity and sensitivity. Of the 200 patients, 19 tested positive by the PCR when swabs from the groin and nose were assessed separately. 36 of the patients tested positive by the PCR when the combination of nose and groin swabs was used. Twenty patients with a total of 32 specimens had the IDI-MRSA testing positive but tested negative in the agar and broth cultures. five patients though, tested positive for MRSA upon the subculture of sodium chloride. The remaining eleven patients 5 had MRSA cultured during the specific month when the procedure was carried out. In essence the probability of the study returning a false result on the infection of MRSA by patients was significantly low. Although having registered a positive MRSA, four patients had negative IDI-MRSA assay results for all the three studies carried out. A positive result was also found when the IDI-MRSA was repeated using the DNA lysate for the four patients. Tests for mecA and nuc for the four patients also returned positive results (Emma et al., 2006).

All the procedure was carried out using the IDI-MRSA assay and smart cycler systems with each group of samples taking two and half hours to process.

Discussion

This study was to ascertain the performance of the IDI-MRSA assay in the detection of nose and groin MRSA infection using separate groin and nose samples and combined samples. In comparison with the culture analysis, it was found that the use of IDI-MRSA was effective and responsive in identifying the presence of MRSA of the nose. Other studies performed before also recorded the same findings. It was also evident that IDI MRSA sensitivity and specifity 91% and 96% respectively, was high according to prior studies. IDI-MRSA assay was also found to be sensitive and specific, 83% and 90% respectively in detecting the presence of MRSA in the groin. We have also been able to establish that the IDI-MRSA of a single DNA lysate combination sample was found to be sensitive at 88% and specific 91.6% in comparison when the samples were prepared separately. Nose cultures identified only 80% percent of colonized samples. Given this observations it was therefore conclusive to say that a combined assay was enough to give conclusive and accurate results (gene xpert). This method was also found to be cost effective since a significantly low number of IDI MRSA as well as personnel were required to carry out the tests. When the IDI-MRSA was used on fresh specimen, a low percentage of the samples were found to be inhibited. However it continues to be evident that the level of inhibition continued to be lower when the specimens of the groin and nose were combined. The issue of inhibition was significantly addressed by the use of frozen DNA lysates; this resolved the issue of inhibition in 23 out of 33 samples. It was also found that it required a maximum of two and half hours to complete a test of a batch of 14 specimens. In light of this it is possible to test samples of a large number of patients and have them concluded within the shortest amount of time compared with the culture assays which would take relatively longer. Despite these advantages, we found that the studies had some shortcomings. For example the entire specimens sampled were of adults. So it is not easy to establish if the procedure would perform in the same way with children. Secondly it was required that half the samples be used in the preparation of the DNA lysates. This might have compromised the results compared with that obtained from swabs with the recommended sample buffer volumes but since the IDI-MRSA using this methods had some similarities in sensitivity and specifity with prior studies using other methods showed that there was little effect. Speedy detection of the MRSA colonization is very important so as to provide for effective control mechanisms. The use of combined specimens was found to be the most appropriate since it provided results faster and in a more clinically useful time compared to the culture methods (Emma et al., 2006).

Summary

It should be noted that anyone can carry the MRSA bacteria effects can range from mild to very serious recent studies on the bacteria have tended to reveal that its epidemiology is changing. Risk factors in the infection of MRSA may include admissions to a health facility especially in the intensive care unit, exposure to infected patients or contact to health workers who have been colonized. Young children are also bound to have a high colonization rate. When this case has been identified in the community, studies normally reveal a history of long-term hospitalization, close contact with someone who had been diagnosed with MRSA, previous antimicrobial use and other risk factors. Generally , transmission in most cases has been narrowed down to touching of the infected skin of someone who has MRSA, sharing of personal effects like towel and face cloths with the infected, touching of public objects and gadgets such as phones and being in crowded places. a person may also increase his chances of getting MRSA by abusing antibiotics through a disregard of prescriptions . Studies have also shown that patients who are getting better from surgery and burns are also susceptible to the bacteria. MRSA symptoms may vary depending on the style of infection. This may include reddening of the infected area incase of a wound, fever back pain incase of a urinary tract infection or a severe cough incase of pneumonia. Due to its resistance; effective treatment of MRSA has become almost impossible. However few strains of staphylococcus aureus have developed resistance to a new antibiotic called vancomycin. General hygiene and careful hand washing is advised as the single most trusted way of controlling the infection of MRSA. Health care providers are advised to use protective wear when handling patients who are known to have an MRSA infection. Patients should also practice good hygiene to avoid spreading the bacteria to others (Johnson et al., 2006).

References

Emma J. Bishop,Elizabeth A. Grabsch, Susan A. Ballard, Barrie Mayall, Shirley Xie, Rhea        Martin, and M. Lindsay Grayson. ( 2006). Infectious Diseases,1 Microbiology,2 and         Infection Control3 Departments, Austin Health, Heidelberg, Victoria, Australia;Department of Epidemiology and Preventive Medicine, Monash University,      Victoria, Australia4; and Department of Medicine, University of Melbourne, Australia:             Victoria.

Nasia, Safdar,Leah Narans, Barbara Gordon, and Dennis G. Maki. (2003). Section of Infectious             Diseases, Department of Medicine, University of Wisconsin Medical School.  Wisconsin:             Madison.

Division of Healthcare Quality Promotion (DHQP). (2008). Center for Disease Control and       Prevention. Retrieved February 8, 2009, from     http://www.cdc.gov/ncidod/dhqp/ar_MRSA.html

Mayo Clinic Staff. (2008).MRSA infection. Retrieved February 8, 2009, from             http://www.mayoclinic.com/health/mrsa/DS00735

Diagn, J Mol. (2007).Evaluation of the Cepheid GeneXpert BCR-ABL Assay. Pubmed Central,           9(2), 220–227. Retrieved February 8, 2009, from             http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1867453

Cepheid. (2009).GeneXpert System. Retrieved February 8, 2009, from    http://www.cepheid.com/systems-and-software/genexpert-system/

Johnson et al. (2006).Evaluation of BacLite Rapid MRSA, a rapid culture based screening test for the detection of ciprofloxacin and methicillin resistant S. aureus (MRSA) from          screening swabs. BMC Microbiology. 6, (83), 1471-2180. Retrieved February 8, 2009,      from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1592303

 

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