meningococcal disease, methods and protocols

Meningococcal Disease Meningococcal Disease Humana Press Edited by Andrew J. Pollard, MD, PhD Martin C. J. Maiden, PhD Methods and Protocols Humana Press Edited by Andrew J. Pollard, MD, PhD Martin C. J. Maiden, PhD Methods and Protocols 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 E TM Microbiology and Laboratory Diagnosis 1 1 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 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. 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. Microbiology and Laboratory Diagnosis 5 However, the test that has emerged from the status of a research tool into one of fundamental utility is the detection of meningococcal DNA following its amplification by PCR. In the UK, the Public Health Laboratory Service (PHLS) Meningococcal Reference Unit (MRU) located at the Manchester Public Health Laboratory provides this test. From an initial experimental clini- cal service in 1996, the service has grown such that there were more than 16,000 requests for meningococcal PCR in 1999. The PCR test can be carried out on peripheral blood or on CSF, and is thought to be specific for meningo- coccal DNA. 2.2. CSF with Polymorphs but no Organisms Seen or Grown Another common clinical situation is that in which a febrile child or adult is subjected to lumbar puncture to exclude the possibility of meningitis, and tur- bid CSF is obtained, in which neutrophils are observed on microscopy and from which no bacteria are grown. Though neutrophils may occasionally pre- dominate in viral meningitis, the presumption is that most such patients have bacterial meningitis. Most will be treated empirically with a third-generation cephalosporin such as cefotaxime or ceftriaxone, but the need to establish (if possible) a more accurate diagnosis lies in the possibility that pneumococci (with the small attendant risk of treatment failure with either penicillin or with cephalosporins) may be the cause of the meningitis. Antigen-detection tests may be of value here and their sensitivity may be enhanced considerably by the use of ultrasound. Agglutination tests are prob- ably inherently less sensitive than PCR tests. Meningococcal PCR testing of CSF is now widely used in the UK and multiplex PCR tests that will detect DNA from meningococci, pneumococci and from Haemophilus influenzae type b are now being evaluated. 2.3. Unusual Presentations of Meningococcal Infection Patients with meningococcal infection may occasionally present with syn- dromes other than meningitis or septicemia. Urethritis, conjunctivitis, and pneumonia are all possibilities, as are septic arthritis, endophthalmitis, peri- carditis, and other infections of deep, normally sterile, tissues. Isolation of a meningococcus from a normally sterile site is diagnostic, but more difficulty arises in the interpretation of the significance of a meningococcus isolated from a superficial site. Clinical and microbiological judgement may be required, but if there is doubt, and particularly if the meningococcus is present in substantial numbers, and is well-endowed with capsular polysaccharide, the isolate should be treated with a high degree of suspicion. For example, primary meningococ- cal conjunctivitis should be treated aggressively, because there is a high risk of 6 Cartwright invasive disease if this is not done. In the US in recent years, there has been an increase in meningãíoccal infections owing to serogroup Y strains, and these may have a particular predilection for the respiratory tract. Chronic meningococcemia is now very rare, accounting for about 1% of all cases. It is normally diagnosed clinically at first. Blood cultures may need to be repeated frequently before a positive culture is obtained. Meningococcal blood PCR will probably be positive in the periods immediately after live meningo- cocci have been cleared from the bloodstream. 2.3. Clusters When a sporadic case of suspected meningococcal disease occurs, it is, of course, impossible to say if it will be followed rapidly by another. For this reason (as well as for clinical reasons), all suspected cases of meningococcal disease should be investigated as fully as possible. There is an ever-present risk that a sporadic case of meningococcal infection may be followed by others in the same family, school, or community. The public-health management of clusters of cases is made much more difficult when the diagnosis is uncertain in one or more of the cases. A typical situation with which public health-medicine specialists have to cope is that in which one or more suspected but unconfirmed cases in a school or other defined commu- nity is followed by a confirmed case (or vice versa). Trying to manage the possible cluster in such circumstances is extremely difficult. If one or more of the cases has died, pressure from the community for intervention may be intense, but might not be justified on epidemiological grounds, were good diagnostic information to be available from all cases. Having an accurate knowledge of the characteristics of the responsible strains is of fundamental value in guiding the management. For example, two or more cases of serogroup C disease occurring within a few days of each other within a defined small community would warrant consideration of the use of vaccine in addition to chemoprophylaxis. This would not apply if the cases were caused by serogroup B strains, or to a mixture of capsular serogroups. 2.4. Postmortem Diagnosis Because of the aggressive and rapid nature of the infection, some patients with suspected meningococcal infection will die before, or very shortly after arrival in hospital, and before there has been a chance to carry out any investi- gations. It is the author’s experience that a microbiologist is rarely involved in the investigation of such cases, only getting to hear of them many hours, or even days later, by which time chances of a positive culture are remote. Requests for autopsy are often declined by grieving relatives. Blood and/or CSF PCR tests should be of great value in this situation, though they are as yet Microbiology and Laboratory Diagnosis 7 formally untested. Aspiration, microscopy, culture, and PCR testing of any areas of skin rash are also worth considering. 2.5. The Impact of Changing Epidemiology on Diagnosis When a patient is suspected of having meningitis, and when there is no microbiological diagnosis, knowledge of the local epidemiology can be of great help in guiding management of both case and contacts. For example, in the UK, the introduction of conjugated Hib vaccines in 1992 has almost eliminated invasive Hib infections in all age groups, and not just in children. The intro- duction of conjugated meningococcal group C vaccines in November 1999 will result in a rapid fall in the incidence of meningococcal disease owing to this serogroup. Consequently, the relative (though not the absolute) risk of a case of meningitis of unknown etiology proving to be owing to a pneumococcus, with the attendant possibility of penicillin resistance, will rise. 3. The Future 3.1. Nonculture Detection of Meningococci from Throat Swabs Though it is believed that most, if not almost all invasive meningococcal disease follows initial colonization of the upper respiratory tract, meningo- cocci can only be cultured from throat swabs in about 50% of cases. PCR test- ing for detection of meningococci in throat swabs is currently under development at the PHLS Meningococcal Reference Unit. It may prove a use- ful addition to the available range of diagnostic techniques. 3.2. PCR Tests for Penicillin Resistance Clinical isolates of meningococci remain sensitive to penicillin, despite a small decrease in sensitivity in strains submitted to the England and Wales reference laboratory over the last few years. To date, there have been only a handful of reports of `-lactamase producing meningococci from clinical cases (and no cases of treatment failure owing to this cause) and none of these strains has survived for detailed examination today. Nevertheless, the risk of penicil- lin resistance remains, with the potential for treatment failure. There would be some value in having available a molecular method for detection of penicillin resistance, and in particular, the capacity to identify `-lactamase producing strains. Such a test could be carried out in conjunction with screening and serogroup-specific PCRs. 3.3. DNA Chips The pace of development of molecular diagnostics makes it seem increas- ingly likely that DNA chips for the diagnosis of meningococcal disease (and for a wide range of other meningitis pathogens) will become available within 8 Cartwright the next few years. As with meningococcal vaccines, their use in developing countries is likely to be restricted by cost factors. References 1. Weichselbaum, A. (1887) Ueber die aetiologie der akuten meningitis cerebro- spinalis. Fortschr. Med. 5, 573–583, 620–626. 2. Quincke, H. I. (1893) Ueber meningitis serosa. Samml. Klin. Vort. (Leipzig) 67, 655–694. 3. Kiefer, F. (1896) Zur differentialdiagnose des erregers der epidemischen cerebrospinalmeningitis und der gonorrhoe. Berl. Klin. Woch. 33, 628–630. 4. Gordon, M. H. and Murray, E. G. (1915) Identification of the meningococcus. J. R. Army Med. Corps. 25, 411–423. 5. Wylie, P. A. L., Stevens, D. S., Drake III, W., Stuart, J. M., and Cartwright, K. (1997) Epidemiology and clinical management of meningococcal disease in Gloucestershire: retrospective population-based study. BMJ 315, 774–779. 6. Begg, N., Cartwright, K. A. V., Cohen, J., Kaczmarski, E. B., Innes, J. A., Leen, C. L. S., et al. (1999) Consensus statement on diagnosis, investigation, treat- ment and prevention of acute bacterial meningitis in immunocompetent adults. J. Infect. 39, 1–15. 7. Cartwright, K., Reilly, S., White, D., and Stuart, J. (1992) Early treatment with parenteral penicillin in meningococcal disease. BMJ 305, 143–147. Meningococci from Clinical Specimens 9 9 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 2 Isolation, Culture, and Identification of Meningococci from Clinical Specimens Per Olcén and Hans Fredlund 1. Introduction Humans are the only natural reservoir for meningococci. The appropriate specimens that should be taken for isolation of meningococci are dependent on the clinical question. The most appropriate specimen and/or laboratory tech- niques for microbiological diagnosis in an acutely sick patient with suspected invasive disease like meningitis/septicemia (1) may be quite different from those required for diagnosis of the cause of a local infection in eye, upper res- piratory tract, lower respiratory tract, or urogenital tract, or for the study of the carrier state of healthy persons. Culture still forms the backbone of diagnosis in spite of major improve- ments in nonculture diagnostic methods (see Chapters 3–5), the latter being especially valuable when cultures are “falsely” negative. This can occur for a number of reasons, most often owing to antibiotic treatment before culture, but might also be related to transport media and isolation media. Necropsy tissues and fluids are also particularly difficult (2,3). Culture is very important because the availability of an isolate growing in the laboratory will allow species designation, antibiotic-susceptibility testing (see Chapter 6), and characterization of an isolate for public-health and epidemiological purposes (see Chapters 8–22). An evident factor of importance is also that almost every microbiological laboratory can perform cultures for meningococci. 2. Materials (for Diagnostic Sampling Procedures) In patients with suspected invasive meningococcal disease, it is logical to take cultures from the suspected primary site of infection (throat/nasophar- [...]... samples must first be carried out Protocols for the optimal extraction from cerebrospinal fluid (CSF), ethelyne diamine tetraacetic acid (EDTA) whole blood, plasma, and serum are described here Evaluation of the Qiagen and Gentra capture column systems described From: Methods in Molecular Medicine, vol 67: Meningococcal Disease: Methods and Protocols Edited by: A J Pollard and M C J Maiden © Humana Press... sistent suspicion of meningococcal disease Some of these methods are described in other chapters of this book and comprise antigen-detection methods including latex- and co-agglutination techniques (10); direct immunofluorescence with specific conjugates (3,10); enzyme immunoassays (34,35); and DNA amplification methods like PCR for different target sequences like the 16S rRNA gene (36–38) and the ctrA gene... M., and Fox, A J (1998) siaD PCR ELISA for confirmation and identification of serogroup Y and W135 meningococcal infections FEMS Microbiol Lett 159, 209–214 34 Salih, M A M., Ahmed, H S., Hofvander, Y., Danielsson, D., and Olcén, P (1989) Rapid diagnosis of bacterial meningitis by an enzyme immunoassay of cerebrospinal fluid Epidemiol Infect 103, 301–310 35 Salih, M A M., Ahmed, A A., Ahmed, H S., and. .. (nonselective) and selective medium including antibiotics and have a distinctive smell Suspected colonies are tested for fast oxidase activity and those giving positive results subjected to Gram staining and microscopy for Gram-negative diplococci Colonies suspected to be meningococci are subcultured and biochemically tested for degradation of glucose and maltose without degradation of fructose and lactose... Master mix contains AmpliTaq gold, 5 mM MgCl 2 , Uracil DNA glycosylase and dNTPs.) 2.4.3 Primers and Probes Taqman probes require HPLC purification Primer and probe for the ctrA assay are the same as those used for agarose and PCR ELISA detection described in Subheadings 2.2.3 and 2.3.2 Primers and probe sequences for 30 Guiver and Borrow Table 3 Primers for Use with the PE-ABI TaqMan System Gene Target... Olcén, P., Kjellander, J., Danielsson, D., and Linquist, B L (1979) Culture diagnosis of meningococcal carriers J Clin Path 32, 1222–1225 13 Abramson, J S and Spika, J S (1985) Persistence of Neisseria meningitidis in the upper respiratory tract after intravenous antibiotic therapy for systemic meningococcal disease J Infect Dis 151, 370–371 14 Cartwright, K., Reilly, S., White, D., and Stuart, J (1992)... parenteral penicillin in meningococcal disease BMJ 305, 143–147 15 Gästrin, B., Kallings, L O., and Marcetic, A (1968) The survival time for different bacteria in various transport media Acta Pathol Microbiol Scand 74, 371–380 20 Olcén and Fredlund 16 Kellog, J A and Manzella, J P (1986) Detection of group A streptococci in the laboratory or physician’s office Culture vs antibody methods JAMA 255, 2638–2642... normally non sterile sites and when mixed infections can be suspected This includes necropsy material Meningococci from Clinical Specimens 19 References 1 van Deuren, M., Brandtzaeg, P., and van der Meer, J W M (2000) Update on meningococcal disease with emphasis on pathogenesis and clinical management Clin Microbiol Rev 13, 144–166 2 Danielsson, D., Nathorst-Windahl, G., and Saldén, T (1971) Use of... Rådström, P., and Olcén, P (1999) Evaluation of an extended diagnostic PCR assay for rapid detection and verification of bacterial meningitis in CSF and other biological samples Mol Cell Probes 13, 49–60 39 Danielsson, D and Johannisson, G (1973) Culture diagnosis of gonorrhoea A comparison of the yield with selective and non-selective gonococcal culture media inoculated in the clinic and after transport... identification of Haemophilus influenzae and Neisseria meningitidis in postmortem human tissue Ann NY Acad Sci 177, 23–31 3 Danielsson, D and Forsum, U (1975) Diagnosis of Neisseria infections by defined immunofluorescence Methodologic aspects and applications Ann NY Acad Sci 254, 334–349 4 Weinstein, M P (1996) Current blood culture methods and systems: clinical concepts, technology, and interpretation of results . Meningococcal Disease Meningococcal Disease Humana Press Edited by Andrew J. Pollard, MD, PhD Martin C. J. Maiden, PhD Methods and Protocols Humana Press Edited by Andrew J. Pollard,. PhD Methods and Protocols 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 E TM Microbiology and Laboratory Diagnosis 1 1 From: Methods in Molecular Medicine, vol. 67: Meningococcal Disease: Methods. penicillin in meningococcal disease. BMJ 305, 143–147. Meningococci from Clinical Specimens 9 9 From: Methods in Molecular Medicine, vol. 67: Meningococcal Disease: Methods and Protocols Edited