Rapid Slide Latex Agglutination Test for Detection of Methicillin Resistance in Staphylococcus aureus

The MRSA screen test (Denka Seiken Co., Ltd.), a commercially available, rapid (20-min) slide latex agglutination test for the determination of methicillin resistance by detection of PBP 2a in Staphylococcus aureus, was compared with the oxacillin agar screen test and PCR detection of the mecA gene. A total of 563 S. aureus isolates were tested. Two hundred ninety-six of the isolates were methicillin-susceptible isolates from cultures of blood from consecutive patients. Also, 267 methicillin-resistant isolates that comprised 248 different phage types were tested. Methicillin resistance was defined as the presence of the mecA gene. Of the 267 mecA gene-positive isolates, 263 were positive by the MRSA screen test (sensitivity, 98.5%), and all the mecA-gene negative strains were negative by the MRSA screen test (specificity, 100%). The oxacillin agar screen test detected methicillin resistance in 250 of the mecA gene-positive isolates (sensitivity, 93.6%). The sensitivity of the MRSA screen test was statistically significantly higher than the sensitivity of the oxacillin agar screen test (P < 0. 05). The MRSA screen test is a highly sensitive and specific test for the detection of methicillin resistance. Also, it offers results within half an hour and is easy to perform, which makes this test a valuable tool in the ongoing battle against methicillin-resistant S. aureus.     

Bacteriophage-Based Latex Agglutination Test for Rapid Identification of Staphylococcus aureus

Rapid diagnosis is essential for the management of Staphylococcus aureus infections. A host recognition protein from S. aureus bacteriophage phiSLT was recombinantly produced and used to coat streptavidin latex beads to develop a latex agglutination test (LAT). The diagnostic accuracy of this bacteriophage-based test was compared with that of a conventional LAT, Pastorex Staph-Plus, by investigating a clinical collection of 86 S. aureus isolates and 128 coagulase-negative staphylococci (CoNS) from deep tissue infections. All of the clinical S. aureus isolates were correctly identified by the bacteriophage-based test. While most of the CoNS were correctly identified as non-S. aureus isolates, 7.9% of the CoNS caused agglutination. Thus, the sensitivity of the bacteriophage-based LAT for identification of S. aureus among clinical isolates was 100%, its specificity was 92.1%, its positive predictive value (PPV) was 89.6%, and its negative predictive value (NPV) was 100%. The sensitivity, specificity, PPV, and NPV of the Pastorex LAT for the identification of S. aureus were 100%, 99.2%, 98.9%, and 100%, respectively. Among the additionally tested 35 S. aureus and 91 non-S. aureus staphylococcal reference and type strains, 1 isolate was false negative by each system; 13 and 8 isolates were false positive by the bacteriophage-based and Pastorex LATs, respectively. The ability of the phiSLT protein to detect S. aureus was dependent on the presence of wall teichoic acid (WTA) and corresponded to the production of ribitol phosphate WTA, which is found in most S. aureus clones but only a small minority of CoNS. Bacteriophage-based LAT identification is a promising strategy for rapid pathogen identification. Finding more specific bacteriophage proteins would increase the specificity of this novel diagnostic approach.     

Direct Detection of Methicillin Resistance in Staphylococcus aureus in Blood Culture Broth by Use of a Penicillin Binding Protein 2a Latex Agglutination Test

We studied the utility of performing a penicillin binding protein 2a latex agglutination (PBP-LA) assay directly on Bactec blood culture broth samples containing Staphylococcus aureus to rapidly detect methicillin resistance. The sensitivity, specificity, positive predictive value, and negative predictive value of this method were 94.1%, 97.5%, 98%, and 92.9%, respectively.Staphylococcus aureus is the leading cause of bloodstream infections, with high levels of morbidity and mortality arising from complications (7, 9, 19). Complication rates increase with the duration of bacteremia and delay in appropriate therapy (5). Therefore, the early identification of S. aureus and determination of its susceptibility to methicillin are crucial to optimize outcomes. Classical culture-based identification and susceptibility testing of S. aureus bacteria isolated from blood cultures take at least 48 h. More rapid PCR methods have been developed for identification from culture broth samples (4, 15-17); however, PCR testing is expensive and often labor-intensive. The penicillin binding protein 2a latex agglutination (PBP-LA) assay (Denka Seiken Co., Japan/Oxoid Ltd., United Kingdom) is a rapid, simple, inexpensive, and FDA-approved test for the identification of methicillin resistance in S. aureus bacteria from culture plates (10, 12, 21). Here, we examined whether this test could be used directly on blood culture broth to expedite diagnosis. Previously, the application of this test on simulated specimens incubated in ESP blood culture broth showed excellent specificity (100%) but poor sensitivity (18%) (3). In contrast, other studies (1, 20) that examined a small number of samples with the BacT/Alert and Bactec blood culture systems claimed excellent sensitivities (96 to 100%) but low specificities (84 to 86%). We therefore sought to examine more definitively the utility of the PBP-LA method in clinical practice by testing a large set of S. aureus-positive Bactec 9240 Standard/10 Aerobic/F and Lytic/10 Anaerobic/F bottles from clinical blood cultures.In this study, positive blood cultures with Gram stain morphologies suggestive of Staphylococcus spp. were first tested by a direct tube coagulase (DTC) test to rapidly identify S. aureus (11). DTC test-positive cultures were then tested by the PBP-LA assay as follows. Briefly, 2 ml of blood culture broth was mixed with 3 or 2 ml of distilled water for aerobic and anaerobic cultures, respectively (see below for the rationale of different volumes). The tubes were centrifuged at 1,000 × g for 10 min, and the pellet was resuspended with 1.5 ml (aerobic culture) or 1 ml (anaerobic cultures) of 0.1 N NaOH. The suspensions were then centrifuged at 2,500 × g for 5 min in Microfuge tubes, and the pellet was then used as the sample for the PBP-LA assay according to the manufacturer''s instructions for testing of bacterial colonies. The identification of S. aureus was subsequently confirmed by slide latex agglutination (Staphaurex; Remel) or tube coagulase testing of subculture colonies. Methicillin susceptibility was determined by both Vitek-2 oxacillin MIC and mecA gene PCR (12).A total of 91 blood cultures positive for S. aureus were evaluated (51 methicillin-resistant S. aureus [MRSA] and 40 methicillin-susceptible S. aureus [MSSA] samples). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the PBP-LA test were 94.1%, 97.5%, 98%, and 92.9%, respectively, compared with mecA gene PCR (Table ​(Table1).1). In addition, the PBP-LA test showed identical performance characteristics with aerobic and anaerobic blood culture broth samples. When both aerobic and anaerobic bottles from the same set were positive for S. aureus (n = 40; 21 MRSA and 19 MSSA samples), the PBP-LA results from each bottle were concordant.

TABLE 1.

Overall performance of the PBP-LA test on positive blood culture broth

Evaluation of a Latex Agglutination Test (MRSA-Screen) for Detection of Oxacillin Resistance in Coagulase-Negative Staphylococci

The MRSA-Screen (Denka-Seiken, Tokyo, Japan) latex agglutination test was evaluated for its ability to detect PBP 2a from 200 clinical isolates of coagulase-negative staphylococci (CoNS; 84 mecA-positive strains and 116 mecA-negative strains) consisting of 108 Staphylococcus epidermidis, 37 S. saprophyticus, 15 S. haemolyticus, 11 S. hominis, 10 S. capitis, 10 S. warneri, and 3 S. lugdunensis species as well as 6 other species of CoNS. The assay was compared with susceptibility testing with an agar screen plate with oxacillin at 6 microg/ml (OXA6), by oxacillin disk diffusion (DD), by broth microdilution (BMDIL), by the E test, and with Vitek GPS-SV and Vitek GPS-107 susceptibility cards. PCR for the detection of the mecA gene was used as the "gold standard." The sensitivities and specificities for the methods evaluated were as follows: MRSA-Screen, 100 and 100%, respectively; OXA6, 100 and 99%, respectively; DD, 98 and 62%, respectively; BMDIL, 100 and 60%, respectively; E test, 100 and 51%, respectively; Vitek GPS-SV susceptibility card, 98 and 87%, respectively; and Vitek GPS-107 susceptibility card, 100 and 61%, respectively. The MRSA-Screen test accurately and rapidly detected oxacillin resistance in CoNS.     

Sensitivity and Specificity of an Improved Rapid Latex Agglutination Test for Identification of Methicillin-Sensitive and -Resistant Staphylococcus aureus Isolates

The performance of a second-generation rapid agglutination kit, Slidex Staph Plus (SSP; bioMérieux), was compared to those of the Slidex Staph (SS; bioMérieux), Staphaurex (SRX; Murex Diagnostics), and BBL Staphyloslide (BBL; Becton Dickinson) kits by using 508 clinical isolates composed of 150 methicillin-sensitive Staphylococcus aureus (MSSA) organisms, 154 methicillin-resistant S. aureus (MRSA) organisms, and 204 non-S. aureus Staphylococcus spp. Of the 508 isolates tested, 75% were fresh clinical isolates, with the remainder taken from five different freezer collections. All four agglutination tests had comparable sensitivities for MSSA and MRSA. However, the SS kit was significantly less specific (93.1%) than the three other tests (P > 0.05, McNemar test). These results demonstrate that the new rapid latex agglutination kit, SSP, was more specific for the identification of S. aureus than the previous version and performed comparably to the SRX and BBL kits.     

Rapid Latex Agglutination Test for Rubella Antibody

The latex agglutination card test (Rubascan) for the detection of rubella antibody was compared with the standard hemagglutination inhibition and enzyme-linked immunosorbent assay tests. There was complete agreement with sera which had hemagglutination inhibition titers of ≥16. Sera with low levels of antibody which were positive in the enzyme-linked immunosorbent assay, however, gave negative latex agglutination results approximately 25% of the time (false negatives), whereas sera which were negative in the enzyme-linked immunosorbent assay gave false-positive results in about 3% of the cases. The use of capillary “finger stick” plasma instead of venous sera resulted in additional false-negative latex agglutination tests among patients with very low antibody titers. Because of the simplicity of the method, it should be possible to use this test in physicians' offices and in large immunization campaigns. Care should be taken to become completely familiar with the procedures and reading of the agglutination patterns. Control sera should always be used. Interpretation of results should take into consideration the rates of false-negative and false-positive results noted above. These rates apply to sera with little or no antibody. In particular, negative tests should be confirmed with more specific methods in critical cases, such as pregnant women exposed to rubella or women of childbearing age who are being considered for immunization. There was no problem with the latex agglutination findings for sera with higher titers. Since results are available in 8 min, physicians should be able to counsel their patients rapidly and immunize, if necessary, while the patient is still present.     

Syphilis Fast Latex Agglutination Test, a Rapid Confirmatory Test

Using 255 serum samples with various reactivities, we evaluated the Syphilis Fast latex agglutination test (Syphilis Fast) against the Treponema pallidum particle agglutination test (TP-PA) for confirming a diagnosis of syphilis. We found 98.8% agreement between the Syphilis Fast and the TP-PA. The Syphilis Fast, however, had a couple of advantages over the TP-PA: the test takes only 8 min to perform and produces results that are easy to read. It appears to be a good confirmatory test for syphilis, especially for point-of-care clinics such as prenatal or sexually transmitted disease clinics.     

Evaluation of the BBL® CrystalTM MRSA ID System for Rapid Detection of Methicillin Resistance in Staphylococcus aureus

Twenty-four clinical isolates of Staphylococcus aureus collected from various geographic areas and four reference strains were studied by (i) agar diffusion with disks impregnated with 5 μg oxacillin and reading after incubation at 30°C for 24 hours, (ii) Southern hybridization with a probe specific for the mecA gene, and (iii) the BBL® Crystal^TM MRSA ID system. There was perfect correlation between the three methods: the BBL® Crystal^TM MRSA ID system detected methicillin resistance in the fifteen strains hybridizing with the mecA probe and classified as resistant by the oxacillin disk diffusion test; the thirteen remaining strains were susceptible by agar diffusion and by the BBL® test and did not hybridize with the mecA probe. The BBL® Crystal^TM MRSA ID System, therefore, appears to be an accurate method for rapid detection of Staphylococcus aureus exhibiting homogeneous resistance to methicillin.     

Multiplex PCR for Simultaneous Identification of Staphylococcus aureus and Detection of Methicillin and Mupirocin Resistance

In this work, we describe a multiplex PCR assay for the detection of clinically relevant antibiotic resistance genes harbored by some Staphylococcus aureus isolates and for the simultaneous identification of such isolates at the species level. Conditions were optimized for the simultaneous detection of the 310-, 456-, and 651-bp regions of the mecA (encoding high-level methicillin resistance), ileS-2 (encoding high-level mupirocin resistance), and femB (encoding a factor essential for methicillin resistance) genes, respectively, from a single colony in a single reaction tube. The femB PCR fragment allows the specific identification of S. aureus. Validation of the method was performed using 50 human isolates of methicillin-resistant S. aureus (MRSA) and the appropriate control strains. This assay offers a rapid, simple, feasible, specific, sensitive, and accurate identification of mupirocin-resistant MRSA clinical isolates and could be systematically applied as a diagnostic test in clinical microbiology laboratories, facilitating the design and use of antibiotic therapy.     

Comparison of the BD Phoenix System with the Cefoxitin Disk Diffusion Test for Detection of Methicillin Resistance in Staphylococcus aureus and Coagulase-Negative Staphylococci

The BD Phoenix system was compared to the cefoxitin disk diffusion test for detection of methicillin (meticillin) resistance in 1,066 Staphylococcus aureus and 1,121 coagulase-negative staphylococcus (CoNS) clinical isolates. The sensitivity for Phoenix was 100%. The specificities were 99.86% for S. aureus and 88.4% for CoNS.Infections caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) or coagulase-negative staphylococci (CoNS) are an increasing problem worldwide (1). Methicillin resistance is primarily due to the presence of a mecA gene which encodes penicillin binding protein 2a (PBP2a) (4).Detection of the mecA gene or PBP2a is considered the gold standard for detecting mecA-mediated methicillin resistance in staphylococci. Among available phenotypic methods, the Clinical and Laboratory Standards Institute (CLSI) has recently introduced the cefoxitin disk diffusion (DD) test for predicting the presence of mecA in S. aureus and CoNS (5, 6), which is preferred over the oxacillin DD test (21). Automated systems are widely used for species identification and susceptibility testing. The aim of the present study was to evaluate the performance of the BD Phoenix automated system (BD, Sparks, MD) in determining methicillin resistance in comparison to the cefoxitin DD test.The study was performed on 1,066 S. aureus isolates and 1,121 CoNS collected during the 2006-2007 routine clinical laboratory activity at the University Hospital of Perugia, Italy. Strains were isolated from the inpatient population of surgical and medical wards and, to a lesser extent, from the outpatient population. Some strains came from other laboratories, for which we are the reference center. Isolates obtained from consecutive cultures from the same patient were excluded. Identification of the isolates was done by conventional methods (colony pigmentation, hemolysis, coagulase production, and the clumping factor test), the Phoenix system (BD), and, in selected cases, the API Staph system (bioMérieux, Marcy l''Etoile, France). Isolates were tested with the cefoxitin DD test, which was performed using Mueller-Hinton agar plates (bioMérieux) and 30-μg cefoxitin disks (bioMérieux) and interpreted according to current CLSI breakpoints (6), and with PMIC/ID gram-positive Phoenix panels (BD), which were prepared from subcultures on Columbia sheep blood agar (BD) after isolation on primary plates, according to the manufacturer''s instructions. The agreement between both methods was considered the “consensus result.” PB2a expression, as detected by the latex agglutination test (Denka Seiken Co., Niigata, Japan), was used to resolve discrepancies. The test was carried out according to the manufacturer''s instructions on uninduced inocula for S. aureus (3, 22, 23) or inocula induced with a 1-μg oxacillin disk for CoNS (13, 14, 17). Methicillin susceptibility in PBP2a-negative CoNS strains was confirmed by mecA gene testing, performed with a LightCycler instrument (Roche Diagnostics, Indianapolis, IN) as described elsewhere (17). For S. aureus, PBP2a-positive isolates were considered methicillin resistant, while PBP2a-negative isolates were considered susceptible. For CoNS, PBP2a-positive isolates were considered methicillin resistant, while PBP2a-negative isolates were considered susceptible, when confirmed as negative for mecA. Categorical disagreements were classified as very major errors (VMEs; false identification of susceptibility by the Phoenix system) and major errors (MEs; false identification of resistance by the Phoenix system). VME and ME rates were calculated using the numbers of reference isolates confirmed as resistant and susceptible, respectively, as denominators. The Phoenix sensitivity rate was the number of strains identified as methicillin resistant by the Phoenix system over the total number of strains confirmed as resistant, and the Phoenix specificity rate was the number of strains identified as methicillin susceptible by the Phoenix system over the total number of strains confirmed as susceptible.Phoenix detection of MRSA is based on both oxacillin and cefoxitin MICs, interpreted according to CLSI breakpoints (for oxacillin, susceptible with MICs of ≤2 μg/ml and resistant with MICs of ≥4 μg/ml; for cefoxitin, susceptible with MICs of ≤4 μg/ml and resistant with MICs of ≥8 μg/ml) (7), in that if either oxacillin or cefoxitin MIC testing indicates that the isolate is resistant, the Phoenix final report is methicillin resistance. Among 1,066 S. aureus isolates, the cefoxitin DD test results and the Phoenix final reports were concordant for 718 methicillin-susceptible and 347 methicillin-resistant strains and discordant for only 1 strain, which was identified as susceptible by the cefoxitin DD test (diameter of 32 mm) and resistant by the Phoenix system (oxacillin MIC, >2 μg/ml; cefoxitin MIC, >4 μg/ml). This strain was negative for PBP2a. Moreover, it was identified as susceptible to oxacillin (MIC = 1 μg/ml) and cefoxitin (MIC = 4 μg/ml) by macrodilution testing according to the CLSI guidelines. Thus, it was finally referred to as methicillin susceptible (one ME; ME rate, 0.14%; 99.86% specificity). Indeed, considering separately the Phoenix oxacillin and cefoxitin MIC results, six S. aureus strains falsely identified as susceptible to oxacillin (six VMEs; VME rate, 1.73%) and one strain falsely identified as susceptible to cefoxitin (one VME; VME rate, 0.29%) were finally reported correctly by the Phoenix expert system, based on cefoxitin and oxacillin results, respectively. These strains were all identified as resistant by the cefoxitin DD test and were PBP2a positive. Thus, the Phoenix sensitivity was 100% and the specificity was 99.86%, provided that both oxacillin and cefoxitin MICs were determined in the panel.Unlike for S. aureus, oxacillin MIC alone is used by the Phoenix system to detect methicillin resistance in CoNS, interpreted according to CLSI breakpoints (for Staphylococcus lugdunensis, susceptible with MICs of ≤2 μg/ml and resistant with MICs of ≥4 μg/ml; for CoNS other than S. lugdunensis, susceptible with MICs of ≤0.25 μg/ml and resistant with MICs of ≥0.5 μg/ml). Moreover, the Phoenix expert system suggests user testing for PBP2a or mecA CoNS isolates (except Staphylococcus epidermidis) with MICs for oxacillin between 0.5 and 2 μg/ml, as recommended by the CLSI (7).The 1,121 CoNS studied included 629 S. epidermidis isolates, 169 Staphylococcus haemolyticus isolates, 101 S. hominis isolates, 66 S. capitis isolates, 63 CoNS not identified at the species level, 25 S. lugdunensis isolates, 22 S. simulans isolates, 17 S. warneri isolates, 15 S. saprophyticus isolates, 4 S. cohnii isolates, 4 S. sciuri isolates, 4 S. xylosus isolates, 1 S. caprae isolate, and 1 S. intermedius isolate. All CoNS not identified at the species level had been confirmed by a clumping factor test not to be S. lugdunensis, for which the cefoxitin DD and oxacillin MIC tests have different interpretation criteria.Among 25 S. lugdunensis isolates, concordant results were obtained for 24 susceptible isolates, while 1 isolate was discordant, being susceptible by the cefoxitin DD test (diameter 32 mm) and resistant by the Phoenix oxacillin test (MIC > 2 μg/ml). The result for the latex test for PBP2a was positive, and the isolate was finally identified as methicillin resistant, in accordance with the automated system. Among the other 1,096 CoNS, results were concordant for 304 methicillin-susceptible and 741 methicillin-resistant strains. Discrepant results were obtained for 51 strains, all identified as susceptible by the cefoxitin DD test and resistant by the Phoenix oxacillin test, with 47 having MICs between 0.5 and 2 μg/ml and 4 having MICs of >2 μg/ml. The latex test for PBP2a, performed after oxacillin induction, showed positive results for 8/51 strains, thus confirming them as methicillin resistant, and negative results for 43 strains. All 43 PBP2a-negative strains were tested for mecA by real-time PCR and were confirmed to be methicillin susceptible, being negative for the mecA gene (Table ​(Table1).1). Thus, among 1,121 CoNS (including S. lugdunensis), 43 MEs and no VMEs were recorded for the automated system, resulting in 88.4% specificity and 100% sensitivity.

TABLE 1.

Resolution of discrepancies among CoNS identified as methicillin susceptible by cefoxitin DD testing and methicillin resistant by Phoenix oxacillin MIC testing

Agglutination of Staphylococcus aureus by Rabbit Sera

Of 137 Staphylococcus aureus strains, 87 agglutinated in normal rabbit serum. The agglutination was shown to be caused by the Fc-part of immunoglobulin G (IgG). F(ab(1))(2)-fragments of IgG and immunoglobulin M (IgM) in corresponding concentrations were unreactive. The agglutinating strains had a high or moderate content of protein A. Strains with a low content of protein A and protein A-negative mutants did not agglutinate. The importance of the reaction between the Fc part of IgG and protein A for serotyping of S. aureus is demonstrated. Two alternative methods for serotyping S. aureus are suggested, using either F(ab(1))(2) fragments of IgG or intact IgM.     

Rapid Detection of Contagious Bovine Pleuropneumonia by a Mycoplasma mycoides subsp. mycoides SC Capsular Polysaccharide-Specific Antigen Detection Latex Agglutination Test

A latex agglutination test (LAT) has been developed for the diagnosis of contagious bovine pleuropneumonia (CBPP). The latex microspheres were coated with MmmSC polyclonal immunoglobulin G antiserum and detected MmmSC antigen in the serum of cattle infected with CBPP and in growth medium containing MmmSC. The specific antigen recognizsed by this test appeared to be the capsular polysaccharide (CPS). The LAT recognized all 23 strains of MmmSC examined in this study, with a sensitivity level of 2 ng of CPS, or the equivalent of 5 × 10³ CFU, in a reaction volume of 0.03 ml. Therefore, rapid identification of MmmSC cultures should be possible. Agglutination was also observed with the related goat pathogens and “Mycoplasma mycoides” cluster members Mycoplasma mycoides subsp. mycoides large colony biotype (four of six strains positive) and Mycoplasma mycoides subsp. capri (three of six strains positive), in agreement with the suggestion that these latter two mycoplasmas may in fact represent a single species (although collectively exhibiting two capsular serotypes). Comparisons in diagnosis with the complement fixation test (CFT) were made by using African field sera from CBPP-infected cattle. After 2 (or 3) min of incubation, the test detected 55% (or 61%) of CFT-positive sera and 29% (or 40%) of CFT-negative sera, with an overall correlation in diagnosis of 62% (or 61%). The rates for false-positive diagnoses made by using “known” CBPP-negative sera from the United Kingdom were 3 or 13% after 2 or 3 min of incubation, respectively. The data agree with previous findings that some CBPP CFT-negative misdiagnoses may occur due to “antibody eclipsing” by excess circulating antigen. The LAT combines low cost and high specificity with ease of application in the field, without the need for any specialist training or equipment.     

Evaluation of the MRSA-Screen Test in detecting oxacillin resistance in community and hospital isolates of Staphylococcus aureus

The MRSA-Screen Test (Denka Seiken Co., Japan), a latex agglutination test to detect penicillin-binding protein 2a, was compared with PCR for the detection of oxacillin resistance in Staphylococcus aureus. A total of 77 oxacillin-sensitive and 269 oxacillin-resistant (ORSA) isolates were evaluated. Of the ORSA isolates, 186 were non-multiresistant (NORSA), defined as being resistant to two or fewer antibiotics other than beta-lactams. Eighty-three were multiresistant ORSA (MORSA) strains. If PCR is considered the gold standard test, then the sensitivity, specificity, positive and negative predictive values of the MRSA-Screen Test were 100, 99, 99 and 100%, respectively. The endpoint was hard to read with NORSA strains that took longer than 60 s to react. MORSA strains took a median 12 s (range 5-60 s) to give a positive reaction with the MRSA-Screen Test, whereas NORSA strains took a median 30 s (range 5-180 s), a difference which was significantly different (P < 0.0001, two-tailed Mann-Whitney unpaired two sample test). NORSA strains had an MIC50 of 128 mg/l and MIC90 of 256mg/l, whereas MORSA strains had an MIC50 and MIC90 of >256mg/l. The time that the MRSA-Screen Test took to agglutinate with ORSA strains correlated weakly with the MIC (r2 = 0.26). Detection of methicillin resistance cost AUD$9 per isolate with the MRSA-Screen Test, compared with AUD$13 per isolate with mecA PCR. The MRSA-Screen Test gave excellent sensitivity and specificity, and was quicker and cheaper than PCR. The full 3 min should be allowed to elapse before calling a test negative. Organisms giving indeterminate reactions should be tested for the mecA gene by PCR.     

Development of a Latex Agglutination Test for Rapid Identification of Escherichia coli

 Escherichia coli, one of the most important human pathogens, is usually identified by a battery of biochemical tests that require overnight incubation. For rapid identification of Escherichia coli, a latex agglutination test (LAT) was developed. Rabbits were immunized with cell-surface antigens extracted from Escherichia coli CCRC 15481 with 4 M urea, and the affinity-purified antibodies were used to coat latex particles for the identification of the bacterium. The following gram-negative bacteria were used to evaluate the LAT: Escherichia coli (n=761), Enterobacteriaceae other than Escherichia coli (n=632), Aeromonas spp. (n=21), Pseudomonas spp. (n=75), Vibrio spp. (n=18), and other bacteria (n=64). The LAT had a sensitivity and specificity of 99.2 and 93.3%, respectively. If the LAT was used in conjunction with the tests of indole production or lactose fermentation, the specificity values for the identification of Escherichia coli increased from 93.3 to 98.8 and 98.7%, respectively. If the LAT, indole production, and lactose fermentation were used together for the identification of Escherichia coli, the sensitivity and specificity were 94 and 99.7%, respectively. Lactose fermentation could be detected by observing the colonies grown on selective media (e.g. MacConkey agar), and indole production could be analyzed simply by the spot indole test. Strains producing negative reactions (i.e. not identified as Escherichia coli) should be processed by the conventional procedures for identification. The present protocol integrating the LAT, indole production, and lactose fermentation for the identification of Escherichia coli offers considerable savings of time, manpower, and cost.     

Methicillin resistance in Staphylococcus aureus infections among patients colonized with methicillin-susceptible Staphylococcus aureus

Objectives

We have noticed that patients colonized with methicillin-susceptible Staphylococcus aureus (MSSA) rarely get methicillin-resistant S. aureus (MRSA) infections. The purpose of this study was to compare the odds of a Staphylococcus aureus (SA) infection being an MRSA infection in MSSA carriers, MRSA carriers and non-carriers of SA.