Xem mẫu

M E T H O D S I N M O L E C U L A R M E D I C I N ETM Meningococcal Disease Methods and Protocols Edited by Andrew J. Pollard, MD, PhD Martin C. J. Maiden, PhD Humana Press Microbiology and Laboratory Diagnosis 1 1 Microbiology and Laboratory Diagnosis Keith Cartwright 1. Introduction 1.1. Historical Background In 1887, Anton Weichselbaum, a Viennese doctor, was the first to report the isolation of meningococci from patients with meningitis (1). Shortly after, came the first description of lumbar puncture in living patients (2), leading to the iso-lation of meningococci from acute cases of meningitis. Three years later, Kiefer grew meningococci from the nasopharynx of cases of meningococcal disease, and from their contacts (3), a finding of immense significance in advancing understanding of the epidemiology and pathogenesis of the disease. Early serological typing systems demonstrated that there were important differences between meningococci in terms of their virulence (4). 1.2. Meningococcal Carriage and Disease It is believed that meningococci only occur in humans. They have never been isolated from other animals, possibly owing to their inability to acquire iron from any other than human sources (transferrin and lactoferrin). Their fastidious nature makes it most unlikely that there are any important environ-mental reservoirs. Meningococci form part of the normal commensal flora and can be isolated from the nasopharynx of approx 10% of individuals overall. Nasopharyngeal carriage is age-dependent, peaking in late teenage and early adulthood at 20–30% or more, but with low prevalence in the young and in the elderly. It is not clear whether acquisition of a new meningococcus in the nasopharynx results in respiratory illness. Meningococci may also be isolated from the urethra and from the rectum from time to time and appear to be capable of causing urethritis. From: Methods in Molecular Medicine, vol. 67: Meningococcal Disease: Methods and Protocols Edited by: A. J. Pollard and M. C. J. Maiden © Humana Press Inc., Totowa, NJ 1 2 Cartwright Invasion is a rare phenomenon, though probably more frequent than would be suggested by the measured rates of disease. It is well-recognized that a small pro-portion of young children may present in hospital with a mild febrile illness that resolves rapidly without antibiotic treatment and from whom a meningococcus is subsequently isolated from blood cultures. For both ethical and logistic reasons, blood culture studies of febrile (but otherwise healthy) children in the community are difficult to mount. Were they to be undertaken with large numbers of partici-pants, it seems likely that they would identify at least a small number of febrile children from whose bloodstream a meningococcus could be isolated. Is it important to confirm the diagnosis in cases of suspected meningococcal infection? The answer must be in the affirmative, both for the optimal management of the patient and his or her contacts, and also for the epidemiological added value. Though meningococci are almost invariably sensitive to penicillin, the exclusion of other causes of meningitis and septicemia remains a key rationale for the full microbiological investigation of both these conditions. Without a detailed under-standing of the range of meningococci causing human disease, and the age groups affected, development of effective vaccines is impossible. 1.3. The Changing Pattern of Meningococcal Disease Diagnosis Diagnostic algorithms in suspected meningococcal infection have changed considerably in the UK over the last 10 years. The drivers have been changes in clinical management and changes in disease epidemiology, allied to techni-cal advances in the laboratory. In the UK and in other countries where most patients with suspected meningo-coccal disease present first to a primary care medical practitioner, a substantial and increasing proportion of patients are being treated with a dose of parenteral benzyl-penicillin. To date, all but one of the published studies (together with unpublished data) support the efficacy of this early management step. Though beneficial, administration of benzylpenicillin prior to the patient’s admission to hospital normally renders blood cultures sterile. It has been suggested that general practitioners administering benzylpenicil-lin to patients with suspected meningococcal disease should take blood cul-tures prior to administering the antibiotic, sending them in to hospital with the patient. This diagnostic step is theoretically possible, but would present a num-ber of logistic difficulties. It is probably not warranted now that good nonculture diagnostic techniques are available (see Subheading 2.1.). 1.4. Microbiological Investigation as Part of the Early Management of Meningococcal Infection There is strong evidence to support the view that delay in the active manage-ment of meningococcal infection is a major factor increasing the risk of a poor Microbiology and Laboratory Diagnosis 3 outcome. Studies in various countries have documented some of the reasons for delay in treatment. One of the most frequent reasons for failing to institute prompt treatment is the fear that initiation of antibiotic treatment may adversely affect the microbiological investigations. As a consequence, a patient may arrive in hospital, be subjected to initial clinical evaluation and may be sus-pected of having meningococcal meningitis. A lumbar puncture may be ordered, and antibiotics withheld pending the results of the lumbar puncture. In a busy pediatric or adult medical unit, this may take an hour or two, or some-times longer, to arrange. This is unacceptable. As soon as meningococcal infection is suspected, blood cultures should be drawn, a drip set up, and intra-venous antibiotics commenced. A lumbar puncture (if deemed appropriate) can then be carried out at the earliest available opportunity. Because it takes at least an hour for antibiotics to begin to arrive in the sub-arachnoid space (even when given by the intravenous route), the chances of isolating a meningococ-cus (or other bacterium) from the cerebrospinal fluid (CSF) are still high. Even if CSF cultures are negative, the diagnosis may be confirmed by microscopic examination of CSF, by latex agglutination tests, or by amplification of micro-bial DNA by polymerase chain reaction (PCR). It is also not widely appreciated that meningococcal DNA is cleared only slowly from the CSF in meningococcal meningitis. If the patient is too unwell or too unstable for lumbar puncture to be contemplated at the time of admis-sion to hospital, and if the diagnosis has not been established within the first 24–48 h, a lumbar puncture is still likely to give a positive PCR result even on d 3 or d 4 of inpatient management. Such a late lumbar puncture will only be needed rarely, but the possibility should be borne in mind. 1.5. Changing Perceptions of Lumbar Puncture Lumbar puncture is now used less frequently, especially by pediatricians (5). This change in clinical practice has arisen from a combination of concern over its perceived dangers, together with a sense of its lack of contributory value in some situations. Coning, frequently fatal, may occur in about 1% of cases of meningo-coccal meningitis where lumbar puncture is undertaken, and lumbar puncture may exacerbate hemodynamic instability in a patient verging on the brink of shock. There is also an increasing understanding that analysis of CSF may provide little additional information relevant to the management of the acutely ill patient (espe-cially if fever and a vasculitic rash are present and a diagnosis of meningococcal infection is overwhelmingly likely). Add to this the fact that the results of all initial examinations (protein, glucose, cell count, and Gram-stained smear) may be nega-tive and yet a meningococcus may be grown on the following day from 5–10% of patients(5), and the exercise of caution over the use of lumbar puncture in children is very understandable. 4 Cartwright The same is not true in adults with symptoms and signs of meningitis. Here, the epidemiology of bacterial meningitis is very different (6). A wider range of pathogens is possible, including the pneumococcus, and other more arcane bac-teria such as Listeria monocytogenes. A few patients with pneumococcal men-ingitis may have a vasculitic rash and their infection may be confused on clinical grounds with meningococcal meningitis or septicemia. The overriding importance of accurate diagnosis of meningitis in adults is the risk (small as yet in the UK, but substantial in countries such as Spain, France, and South Africa) of true penicillin, or penicillin- and cephalosporin-resistant infection. Lumbar puncture is still the most important investigation in adult patients with suspected bacterial meningitis (7). 2. Specific Clinical Issues Impacting on Microbiological Diagnosis 2.1. Effect of Early Parenteral Antibiotic Treatment on Diagnostic Investigations In the 1980s, the great majority of patients in the UK with suspected menin-gococcal meningitis were not treated with benzylpenicillin prior to hospital admission. In such patients (both adults and children), blood cultures were posi-tive in about 50%, and if meningitis was present and a lumbar puncture was undertaken, the CSF would either yield Gram-negative diplococci on the stained smear, or a meningococcus would be isolated on culture in more than 90% of cases. Alternative diagnostic methods had to be devised to cope with patients with negative blood cultures, and in whom lumbar puncture was contraindicated. Throat swabs have proved of great value in this situation, giving a positive result in up to 50% of patients, a proportion that is largely unaffected by prior benzylpenicillin treatment (7). Per-oral swabs give a better yield than per-nasal swabs. If the intention is to isolate a meningococcus, the swab must be plated out as soon as it is obtained. A swab taken in the middle of the night cannot be left to be cultured in the morning. If a skin rash is present, aspiration of an affected area of skin may yield diplococci on a Giemsa-stained smear, or in a somewhat smaller proportion of cases, a positive culture. Agglutination of latex particles coated with meningo-coccal serogroup-specific antibodies by meningococci of the homologous serogroup can be made more sensitive by inducing better agglutination by means of ultrasound enhancement. Demonstration of a rising antimeningococcal antibody titer between acute and convalescent serum samples may also be helpful for epidemiological rea-sons, though it does not provide information at the time that it is needed for the acute management of the patient. ... - tailieumienphi.vn
nguon tai.lieu . vn