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Journal of Clinical Microbiology, June 1999, p. 1797-1801, Vol. 37, No. 6
PHLS Meningococcal Reference Unit,
Received 29 September 1998/Returned for modification 2 December
1998/Accepted 12 March 1999
Preadmission administration of antibiotics to patients with
suspected meningococcal infection has decreased the likelihood of
obtaining an isolate and has stimulated development of rapid and
reliable non-culture-based diagnostic methods. The sensitivity of the
conventional test card latex agglutination test (TCLAT) for detection
of capsular polysaccharide has been reported to be suboptimal. In the
United Kingdom meningococcal DNA detection by PCR has become readily
available and is now used as a first-line investigation. Recently, the
performance of latex antigen detection has been markedly improved by
ultrasound enhancement. Three tests for laboratory confirmation of
meningococcal infection, (i) PCR assays, (ii) TCLAT, and (iii)
ultrasound-enhanced latex agglutination test (USELAT), were
compared in a retrospective study of 125 specimens (serum, plasma, and
cerebrospinal fluid specimens) from 90 patients in whom meningococcal
disease was suspected on clinical grounds. Samples were from patients
with (i) culture-confirmed meningococcal disease, (ii) culture-negative
but PCR-confirmed meningococcal disease, and (iii) clinically suspected
but non-laboratory-confirmed meningococcal disease. USELAT
was found to be nearly five times more sensitive than TCLAT. Serogroup
characterization was obtained by both PCR and USELAT for 44 samples; all results were concordant and agreed with the serogroups
determined for the isolates when the serogroups were available. For 12 samples negative by USELAT, the serogroup was determined by
PCR; however, for 12 other specimens for which PCR had failed to
indicate the serogroup, USELAT gave a result.
USELAT is a rapid, low-cost method which can confirm a
diagnosis, identify serogroups, and guide appropriate management of
meningococcal disease contacts. A complementary non-culture-based confirmation strategy of USELAT for local use supported by a
centralized PCR assay service for detection of meningococci would give
the benefits of timely information and improved epidemiological data.
Growth of Neisseria
meningitidis from a normally sterile site has long been the
definitive test for the diagnosis of meningococcal disease, although
microscopy of Gram-stained films of cerebrospinal fluid (CSF) can
demonstrate the presence of diplococci and strongly supports the
diagnosis. The recent trend in the United Kingdom to preadmission
administration of antibiotics to patients with suspected cases of
meningococcal disease coupled with a greater circumspection about
obtaining CSF from patients in whom the clinical picture is typical has
decreased the likelihood of culture confirmation (6). Rapid,
non-culture-based methods for determination of the causative
meningococcal serogroup have been developed to improve epidemiological
information and to guide contact management by chemoprophylaxis and
vaccination. PCR-based DNA detection (9, 21, 23, 24, 25, 26)
represents an improvement in non-culture-based serogroup confirmation
over that currently available through capsular polysaccharide detection
by the conventional test card latex agglutination test (TCLAT).
The Public Health Laboratory Service (PHLS) Meningococcal Reference
Unit (MRU) for England and Wales is screening more than 10,000 samples
per annum using a sensitive and specific PCR assay (ctrA
assay) to detect meningococcal DNA (20). The
ctrA-reactive specimens are further tested by the
siaD serogroup B and C PCR assays (5). The
siaD assay is inherently less sensitive than the
ctrA assay, with the result that only 65 to 70% of
ctrA assay-positive samples provide a serogroup result
(20).
Commercial TCLATs can be used by nonspecialist laboratories for the
rapid detection of serogroup-specific meningococcal antigen in CSF or
serum with minimal preprocessing and modest expenditure, but poor
sensitivity has been reported (12). Recently, improved performance of antigen detection has been described by performing assays in an ultrasonic standing wave. Latex particles suspended in the
wave are subjected to physical forces that promote formation of
aggregates (8) by increasing particle-particle contact. Enhanced agglutination between antibody-coated microparticles in the
presence of antigen occurs, giving substantial increases in sensitivity
of antigen detection compared to that of TCLAT (14, 16, 17,
27). Application of the ultrasound-enhanced latex agglutination
test (USELAT) for detection of capsular polysaccharide antigen from N. meningitidis serogroup A, B, C, Y, and W135
strains has demonstrated sensitivity enhancements with clinical samples without nonspecific reactivity (2, 18).
The aim of this study was to compare PCR and latex agglutination
tests (LATs) for the non-culture-based diagnosis of meningococcal infection and also to determine whether USELAT testing of
clinical samples could be a practical first-line investigation in
general microbiology laboratories.
(This work has been presented in part at the 38th Interscience
Conference on Antimicrobial Agents and Chemotherapy, San Diego, Calif.,
24 to 27 September 1998, and the 11th Pathogenic
Neisseria Conference, Nice, France, November 1998.)
Patients and samples.
One hundred twenty-five specimens from
90 patients were studied (Table 1). All
patients had clinical features consistent with meningococcal infection
(meningococcal meningitis and/or septicemia) as the most probable
diagnosis. Samples had been submitted to the MRU specifically for
testing by PCR assays for the detection of meningococci. Specimens were
retrieved following storage at Isolate confirmation and characterization.
Isolates of
N. meningitidis are received at the PHLS MRU from nearly all
culture-positive patients identified by clinical microbiology
laboratories throughout England and Wales. Strains are confirmed to be
meningococci and are characterized with a panel of antisera (1,
10, 13, 28) to define the serogroup, serotype, and serosubtype.
Molecular detection.
Clinical samples for DNA-based
non-culture-based diagnosis of meningococcal infection are submitted to
MRU and are stored at 4°C prior to testing. Template DNA is extracted
from 100 µl of the clinical specimen (CSF, serum, or plasma) with a
commercial kit (DNAzol: Gibco BRL, Paisley, Scotland) to produce 50 µl (final volume) in sterile water (sterile injectible), of which 2 µl is used in each PCR assay.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Ultrasound-Enhanced Latex Immunoagglutination and
PCR as Complementary Methods for Non-Culture-Based Confirmation of
Meningococcal Disease
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
80°C for examination by TCLAT and
USELAT. Primary culture and other investigations for the
detection of bacterial pathogens were performed at the laboratories
that submitted the specimens. Apart from one patient in whom
Streptococcus pneumoniae was subsequently identified as the
causative organism, no etiology other than meningococci was
definitively identified. Samples negative by the meningococcus-specific PCR assays and latex tests were not investigated further. The study
samples were selected to include (i) PCR-positive specimens from
patients from whom meningococci had been cultured from normally sterile
sites, (ii) specimens which were positive by PCR but which were culture
negative, (iii) specimens which were positive by culture but which were
PCR negative, and (iv) PCR- and culture-negative samples.
TABLE 1.
Samples used in the study
Latex agglutination reagents. Commercially available latex agglutination reagents (Wellcogen kit; Murex Diagnostics, Dartford, United Kingdom) for the detection of meningococcal polysaccharide were used in the TCLAT and USELAT formats. The N. meningitidis ACYW135 reagent detects four serogroups (serogroups A, C, Y, and W135). The N. meningitidis B-Escherichia coli K1 monoclonal antibody latex reagent exploits the cross-reactivity of E. coli K1 and N. meningitidis serogroup B polysaccharide and, as such, is not strictly specific.
Ultrasonic equipment. The ultrasound apparatus has been described in detail elsewhere (15, 16). Essentially, a tubular ultrasonic transducer (PCA4; Morgan Matroc, Wrexham, United Kingdom) with a fundamental thickness resonance frequency of 1.5 MHz was mounted as shown schematically in Fig. 1. The transducer was driven at 4.5 MHz by the 20 Vp-p (volts peak-peak) output of an RF amplifier (model 240L; ENI, Rochester, N.Y.). The amplifier input came from a Hewlett-Packard 33120A frequency synthesizer.
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Conventional agglutination and ultrasonic test procedure. Specimens (serum, plasma, or CSF specimens) were prepared for processing, in accordance with Wellcogen kit instructions, which included centrifugation and subsequent filtration to remove protein (29) with 0.2 µm-pore-size Millex-GS (Sigma Chemical Co., Poole, United Kingdom) membrane filters when the sample volume permitted. Each patient specimen was tested with N. meningitidis B-E. coli K1 and N. meningitidis ACYW135 test latexes by using the appropriate negative controls supplied with the kits. The manufacturer's instructions were followed for performance and interpretation of TCLAT.
In the USELATs, 25 µl of test sample was mixed on a nonadsorbing surface with 25 µl of diluted test or control latex suspension prediluted 1 in 8 in phosphate-buffered saline (pH 7.4). With a syringe, the reaction droplet was immediately drawn into a 2-mm (internal diameter) glass capillary (Fisher Scientific, Loughborough, United Kingdom) which was positioned on the transducer axis and sonicated for 60 s in the axial high-acoustic-pressure region of the sound field (15). On expulsion from the capillary onto a test card, the droplet was stirred four times to break up nonspecific aggregates formed as a result of ultrasonic concentration. Droplets were loaded into microslides (Camlab, Cambridge, United Kingdom) with a 200-µm-path-length cross section by capillarity and were examined for agglutination by microscopy (×10 objective). Samples were scored as positive when particle agglutination clearly exceeded any slight residual particle aggregation in control samples. The investigators who performed the LATs were unaware of PCR or culture results.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Detection of meningococci.
Table
2 demonstrates the benefit of
non-culture-based detection by comparing the number of samples positive
by the PCR screening assay (ctrA assay) and USELAT
for culture-confirmed and culture-unconfirmed samples. The total
numbers positive by the ctrA PCR assay and USELAT
were 77 (62%) and 61 (49%), respectively, whereas 42 (34%) samples
were positive by culture. Forty-five ctrA PCR assay-positive and 32 USELAT-positive samples were found among 83 samples
from culture-negative patients.
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Serogrouping of N. meningitidis infection by PCR
and USELAT.
There were 56 siaD PCR-positive
samples (32 serogroup B-positive samples and 24 serogroup C-positive
samples). USELAT indicated the serogroup in 44 (79%)
specimens for which the siaD PCR assay result was positive
(23 N. meningitidis B-E. coli K1-positive samples
and 21 ACYW135-positive samples). There was complete concordance between the USELAT and PCR serogroup identifications for 44 samples (Table 3). When considering all
results, USELAT and PCR were in agreement for 99 (79.2%) of
the 125 samples overall.
|
Comparison of ultrasound-enhanced detection with the
standard test card method.
Overall, 61 (49%) specimens were
positive by USELAT, whereas 13 (10%) were positive
by TCLAT. Table 4 shows that 84%
(27 of 32) of the total culture-positive and PCR-positive samples (Table 2) were positive by USELAT, whereas 22% (7 of 32)
were positive by the commercial test card procedure. No discrepant serogroup results were found by either latex agglutination method. There were no TCLAT-positive results among the PCR-negative samples, whereas USELAT was positive for five PCR-negative samples.
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Sensitivity of meningococcal detection. By using the clinical diagnosis of meningococcal disease as a standard, the relative sensitivities of culture, USELAT, TCLAT, the ctrA PCR assay, and the siaD PCR assay were 28% (26 of 90), 50% (45 of 90), 10% (9 of 90), 67% (60 of 90), and 48% (43 of 90), respectively. Results for 26 patients with culture-confirmed meningococcal disease and for whom the specimen from the site that was culture positive was available for PCR and LAT gave USELAT, TCLAT, ctrA PCR assay, and siaD PCR assay sensitivities of 76% (20 of 26), 19% (5 of 26), 88% (23 of 26), and 69% (18 of 26), respectively. There were no discrepancies in the serogroups identified in 15 patients who were positive by culture, the siaD PCR assay, and USELAT.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Rapid laboratory confirmation of meningococcal infection can help ensure the most appropriate patient care and contact management to prevent secondary cases of infection. Nonculture-based methods, specifically, PCR assays, have greatly improved the rate of confirmation of infection in the United Kingdom and have resulted in closer approximation of the number of patients notified by clinicians to the office of National Statistics (United Kingdom) on the basis of clinical diagnosis and the total number of laboratory-proven cases of infection (7, 20). Increased confidence in the available epidemiological information is especially important prior to the anticipated introduction of serogroup C conjugate meningococcal vaccines to the immunization schedule in the United Kingdom within the next 2 years (11) so that the effect of the new vaccine can be accurately monitored.
The comparison of USELAT and a PCR test for the detection of meningococci demonstrated that there was good concordance between the results of the two assays. Overall, the ctrA PCR screening assay yielded 13% more positive results than USELAT. It is probably more appropriate, however, to compare USELAT with the siaD PCR since by determining the serogroup they provide equivalent epidemiological information.
The siaD PCR assay detected 4% fewer positive results; moreover, the results for small numbers of specimens could be available more rapidly by USELAT than by the PCR. A single USELAT result would be possible within 10 min following boiling and filtration, which take about 20 min; each additional test would take another 8 to 10 min. This compares with about 2.5 h for a single PCR assay. PCR assays are, however, more efficient with large numbers of samples; results from 80 assays can be available within 5 h, which includes the time required for specimen processing.
When both USELAT and the siaD PCR assay were positive, there were no discrepancies in the meningococcal serogroups identified. USELAT provided serogroup characterization in instances in which the PCR assays could only confirm meningococcal infection and in five (4%) specimens identified meningococcal antigen when the ctrA PCR screening assay had been negative. The 12 specimens positive by USELAT (7 N. meningitidis B-E. coli K1-positive specimens and 5 ACYW135-positive specimens and negative by siaD PCR are balanced by the 12 USELAT-negative samples positive by the siaD PCR assay (9 serogroup B-infected specimens and three serogroup C-infected specimens).
In this study of 90 patients with clinical meningococcal disease, the most sensitive test for meningococcal detection was the ctrA PCR assay. USELAT was more sensitive than the siaD PCR assay for serogroup confirmation, and both assays were superior to standard LAT (with Murex reagents) and culture. The relative positions of the sensitivities of the USELAT, TCLAT, and the two PCR assays remained the same when the sensitivities were determined for the subset of samples from patients with culture-confirmed cases of infection. Previous studies of USELAT (2) and meningococcal PCR (5) estimate 100% specificity. A single sample from a patient with an S. pneumoniae infection and three samples from one patient infected with meningococcal serogroup 29E were nonreactive by both USELAT and PCR.
The likelihood of serogroup determination by USELAT or the siaD PCR will be affected by the amount of target present. PCR techniques detect unique N. meningitidis gene sequences present as single copies on each genome, giving a lower limit of detection (250 organisms/ml of fluid tested). In contrast, USELAT detects extracellular capsular polysaccharide, which is produced in excess and which blebs off the organism into the surrounding medium. LAT was performed with 25 µl of a 1-in-4-diluted plasma or serum sample, representing approximately 6 µl of the original specimen, while the 2 µl of the DNA extract tested by PCR was from 4 µl of plasma or serum from the original sample. The volume of the specimen from which target for the respective tests was obtained was therefore equivalent.
One of the five USELAT (ACYW135)- and ctrA PCR-positive samples was found to be a culture-proven serogroup Y infection not detected by the siaD PCR assay used. It is possible that any of the other four samples could have been from patients with serogroup Y or W135 infection, but it is most unlikely that any were from patients with serogroup A infections, since only two cases of infection with these globally important strains have been identified in the United Kingdom over the last 3 years. An alternative explanation is that the samples contained small numbers of genome copies that were detectable by the ctrA PCR assay but not by the siaD PCR assay because the currently available assays exhibit different sensitivities (20).
The sensitivities of the PCR assays may be improved by better DNA extraction and by redesigning the TaqMan assay to include probes for the less common serogroups that cause infection in England and Wales. The current TaqMan ctrA PCR assay detects serogroups B, C, W135, and Y. Serogroups A, X, 29E, and Z are not detected by the current TaqMan ctrA PCR assay but can be detected by a ctrA PCR and enzyme-linked immunosorbent assay (20). The siaD primers described previously (5) were redesigned for the TaqMan assay for increased sensitivity. Serogroup Y- and W135-specific (siaD) PCR assays have recently been developed in a PCR and enzyme-linked immunosorbent assay format (4) and will, in future, be used with specimens that are positive by the ctrA PCR assay but that are nonreactive by the serogroup B- or C-specific siaD PCR assay.
Because of developments in the molecular characterization of strains by the sequencing of alleles of housekeeping or outer membrane protein-encoding genes, such assays are now able to furnish more information than can be provided by any serologically based method (22). The facility of translating sequencing techniques into a non-culture-based diagnostic format is being explored. Data generated in this way could provide important additional information for the management of clusters of infections and outbreaks.
All the LAT investigations were initially performed 2 months after the PCR assays, demonstrating that polysaccharide can persist in stored specimens. In performing the tests independently at the two study sites (Cardiff and Manchester), the technique was found to be portable between laboratories. Undoubted advantages of USELAT over PCR are the lower costs in terms of labor, staff training, equipment, and reagents. A compact voltage-generating device with the essential characteristics of the test equipment described in Materials and Methods has recently been developed (Electro-Medical Supplies, Wantage, United Kingdom). The performance of the device is under evaluation at six hospital locations in the British Isles. If trials are successful a device could become commercially available for less than $4,000. The possibility of using the ultrasound equipment for other latex agglutination-based assays (14, 16) would make any investment even more cost-effective. To develop and maintain confidence on the part of both operators and end users as to the validity of the test results, it would be necessary to develop an external quality assurance scheme (perhaps comprising a panel of negative material and stringently quantified meningococcal polysaccharide samples weighted to include a high proportion of samples containing amounts close to detection thresholds).
Ultrasound assays with individual serogrouping latex reagents for serogroups A, C, Y, and W135 (currently commercially available from Sanofi and bioMerieux) are under evaluation to see if it is possible to provide specific information on the serogroup. Performance of the test with the serogroup-specific latex agglutination reagent after demonstration of reactivity with the pooled reagent would be efficient and cost-effective. Knowledge of the local serogroup incidence would dictate the sequence in which such specific assays were performed. A DNA-based detection strategy would require five separate PCR assays (meningococcal DNA detection, followed by detection of DNAs of serogroups B, C, Y, and W135), and the cost would be considerably greater than those of the LATs.
Although the increased overall sensitivity of the ctrA PCR assay over that of USELAT was clearly shown in this study, the latter would be an extremely useful method of case confirmation in those countries where rapid PCR diagnosis is unavailable. USELAT could notably improve the epidemiological data from areas of the world where routine culture is often impracticable.
USELAT could be used as a first-line investigation in local microbiology laboratories (19) for rapid non-culture-based diagnosis with relevant clinical samples collected as soon after admission as possible. Potentially helpful additional information can be obtained from PCR-based testing schemes, and this will increase over the next few years. When meningococcal PCR assays are available, as is currently the case at reference units in the United Kingdom and the Republic of Ireland, early specimens from all patients with suspected meningococcal infection should be sent for PCR, regardless of any other techniques that are available locally.
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ACKNOWLEDGMENTS |
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The Meningitis Research Foundation, Bristol, United Kingdom, and the United Kingdom Biotechnology and Biological Sciences Research Council (BBSRC) Analytical Biotechnology Initiative have provided support for this study.
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FOOTNOTES |
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* Corresponding author. Mailing address: PHLS Meningococcal Reference Unit, Manchester PHL, Manchester, M20 2LR, United Kingdom. Phone: 44 (0)1612 914628. Fax: 44 (0)1614 462180. E-mail: ed{at}manphl.demon.co.uk.
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Introduction
Materials and Methods
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Discussion
References
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Journal of Clinical Microbiology, June 1999, p. 1797-1801, Vol. 37, No. 6
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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