Introduction of an automated service for the laboratory confirmation of meningococcal disease in Scotland

The Scottish Meningococcus and Pneumococcus Reference Laboratory provides a national service for the laboratory confirmation of meningococcal and pneumococcal disease in Scotland. The main tests used for the laboratory confirmation of meningococcal disease are culture, the polymerase chain reaction (PCR), antibody testing, and more recently DNA sequencing. This paper describes the automation of PCR for the laboratory confirmation of meningococcal disease and the typing of meningococcal isolates using DNA sequencing. Both methods have been automated using a robotic liquid handler and automated DNA sequencer. These methods, along with standard culture phenotyping and antibody testing, provide Scotland with an excellent service for the confirmation of meningococcal disease.     

Meningococcal disease due to serogroup Y in Scotland, 1992-1999

Meningococcal disease is an important cause of morbidity and mortality. A retrospective analysis was performed of all cases of invasive group-Y disease that were laboratory-confirmed in Scotland between 1992 and 1999. A total of 1881 meningococcal isolates were characterised, 78 of which were serogroup Y. The incidence of non-invasive group-Y disease remained level between 1992 and 1999. Only 12 isolates were from invasive disease, comprising five strain types. Invasive group-Y disease was associated mostly with the young or old. Serogroup-Y meningococcal disease was uncommon and a rare cause of invasive disease in Scotland between 1992 and 1999; however, it is essential that microbiologists are aware of its potential for increasing in incidence due to the recent introduction of the MenC vaccine, and its increased incidence in the USA.     

Evaluation of a diagnostic polymerase chain reaction assay for Neisseria meningitidis in North America and field experience during an outbreak

CONTEXT: Meningococcal infection has a high public profile because of its dramatic presentation, high fatality rate, and propensity to occur in outbreaks and clusters of cases. Use of a diagnostic polymerase chain reaction (PCR) assay could enhance laboratory confirmation of cases and guide the public health response in North America. OBJECTIVE: To assess the performance of a PCR assay for the diagnosis of meningococcal disease after its implementation in a North American setting and to evaluate sensitivity and specificity of the assay for the detection of prevalent bacterial isolates. DESIGN: Laboratory evaluation of the sensitivity and specificity of a PCR assay for Neisseria meningitidis and observational study of a series of cases comparing molecular diagnosis against the criterion standard of conventional laboratory diagnostic tests. SETTING: A Canadian province with a population of 4 million people. PATIENTS: Children and adults presenting with suspected meningococcal disease in British Columbia. MAIN OUTCOME MEASURES: The sensitivity and specificity of the PCR assay when compared against standard laboratory methods. RESULTS: The PCR assay correctly identified all of 38 Canadian isolates of Neisseria meningitidis and correctly assigned the serogroup to each isolate. None of 57 other gram-positive or gram-negative bacteria or yeasts were detected by the PCR assay. In a clinical evaluation, for diagnosis of meningococcal disease, the PCR assay had a sensitivity and specificity of 91% and 76%, respectively, against conventional methods of diagnosis. Use of the PCR assay increased the laboratory confirmation of clinically suspected cases by 36%. During an outbreak, the PCR assay allowed serogroup determination in 3 of 7 cases, aiding in the public health decision to launch an immunization campaign. CONCLUSIONS: The PCR assay is more sensitive than conventional methods for the diagnosis of meningococcal disease, and enhanced surveillance may help direct the public health response to the changing epidemiology of disease in North America.     

Automated non-culture-based sequence typing of meningococci from body fluids.

In recent years, the polymerase chain reaction has been used for the non-culture diagnosis of meningococcal disease, and sequence-based typing takes this further by providing the full characterisation normally only available by culture. In this study, porA gene sequencing was used to perform non-culture-based sequence typing of Neisseria meningitidis strains direct from body fluids. Non-culture porA gene sequencing provided the serosubtype of the infecting organism, and proved to be a useful method as N. meningitidis was not isolated from any of the patients in this study. In conclusion, porA gene sequencing is a very useful tool for the non-culture characterisation of meningococci and provides important information for public health management of cases and contacts.     

Semiautomation of multilocus sequence typing for the characterization of clinical isolates of Neisseria meningitidis

The Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) provides a national service for the laboratory confirmation of meningococcal and pneumococcal disease in Scotland. Part of this service includes the serogrouping of meningococcal isolates followed by typing and subtyping. The procedures for this are labor-intensive but important for the identification of linked cases and the surveillance of disease so that effective public health measures can be taken. However, different strains of meningococci, such as those within the electrophoretic type 37 complex, occurring during case clusters of disease are now indistinguishable by current methods. The SMPRL has started using multilocus sequence typing (MLST) as a routine method for the characterization of isolates of Neisseria meningitidis. MLST produces nucleotide sequence data of seven housekeeping genes providing results that are useful for public health management. However, the method is laborious and time-consuming and therefore lends itself towards automation. The SMPRL therefore developed a semiautomated method for MLST using a 96-well format liquid handler and an automated DNA sequencer. Semiautomated MLST is now provided as a reference service for Scotland. This work describes the methodology required for the characterization of N. meningitidis and highlights its usefulness for public health intervention.     

Ultrasound-enhanced latex immunoagglutination test (USELAT) for detection of capsular polysaccharide antigen of Neisseria meningitidis from CSF and plasma

AIMS: An ultrasonic instrument, the Immunosonic, was used to evaluate ultrasound-enhanced latex immunoagglutination testing (USELAT) for detection and serogroup determination of Neisseria meningitidis in clinical specimens. METHODS: Eighty-two CSF and EDTA blood specimens from patients with suspected meningococcal disease (MD) were tested by USELAT. Results were compared with routine laboratory tests for confirmation of MD and discrepant results were resolved by analysis of further laboratory and clinical data. RESULTS: Using the Wellcogen Bacterial Antigen Kit, USELAT was positive in 20 (24%) specimens. The resolved sensitivity of USELAT was 49% compared with 67% for PCR. There were no discrepancies between serogroups indicated by USELAT and serogroups confirmed by PCR or culture grouping. CONCLUSIONS: Although USELAT could be performed in laboratories without facilities for PCR testing, a specific ultrasonic instrument is necessary and some experience is required in interpreting results. The lower resolved sensitivity makes USELAT unsuitable as a stand-alone rapid test, and it added little value to standard laboratory culture with PCR testing.     

Fatal outcome from meningococcal disease--an association with meningococcal phenotype but not with reduced susceptibility to benzylpenicillin

Penicillin has been the mainstay of treatment for meningococcal disease. Isolates of Neisseria meningitidis that are less susceptible to penicillin have been reported in several countries and in recent years have become more common. The clinical significance of this reduced susceptibility has not been investigated on a large scale. Hence, N. meningitidis isolates from culture-confirmed cases of meningococcal disease in England and Wales, between 1993 and 2000, were routinely serogrouped, serotyped and tested for susceptibility to penicillin. These data were linked to death registrations and analysed retrospectively. The changing trends in susceptibility were described and multivariate logistic regression was used to examine associations between strain characteristics and fatal outcome. The frequency of N. meningitidis isolates less susceptible to penicillin increased from < 6% in 1993 to > 18% in 2000. In particular, isolates expressing serogroup C with serotype 2b and serogroup W135 had a higher frequency of reduced penicillin susceptibility (49% and 55%, respectively). There was no evidence of an association between fatal outcome and infection with a less penicillin-susceptible isolate. Fatal outcome was associated with serogroup and serotype, with the odds of death for cases infected with C:2a and B:2a strains three-fold higher when compared with the baseline. For this large dataset the serogroup and serotype of the infecting strain influenced mortality from meningococcal disease and may be markers for hypervirulence. No association was found between reduced penicillin susceptibility and fatal outcome, but the increasing frequency of isolates less susceptible to penicillin highlights the need for continued surveillance.     

PCR of peripheral blood for diagnosis of meningococcal disease.

Meningococcal disease is normally suspected on clinical grounds and is confirmed by isolation of Neisseria meningitidis bacteria from blood or cerebrospinal fluid or, more recently, by serology or PCR of cerebrospinal fluid. Achieving confirmation of a clinical diagnosis of meningococcal disease has become more difficult in the last few years. The pre-hospitalization administration of parenteral benzylpenicillin normally renders blood cultures sterile, and lumbar puncture is undertaken less frequently, especially in young children. We evaluated PCR for the detection of meningococcal DNA in 80 blood samples taken from patients with known or suspected meningococcal disease or from patients with other diagnoses (negative controls). Both the sensitivity and the specificity of the test were 100% for patients with confirmed cases of meningococcal disease when the blood buffy coat was used (83 to 100% sensitivity and 87 to 100% specificity with 95% confidence limits). Positive PCR results could be obtained from both blood buffy coat and serum samples. Sensitivity was unaffected by prior antibiotic treatment. PCR is a rapid, sensitive test that may be used to confirm a diagnosis of meningococcal disease by using peripheral blood samples. Introduction of this test into clinical laboratories may in some cases obviate the need for lumbar puncture to be performed on patients with suspected meningococcal disease. Our results demonstrate that a substantial number of cases of meningococcal disease are not confirmed by conventional techniques and remain undiagnosed. If the PCR test described here was widely applied, the number of cases of meningococcal disease ascertained might rise by as much as 60% greater than that recognized at present. It is likely that we are in a prevaccination era for meningococcal disease. Better case ascertainment is urgently required to assess the need for vaccines, to determine their costs and benefits, and to monitor their efficacies.     

Confirmation of invasive meningococcal disease by single point estimation of IgM antibody to outer membrane protein of Neisseria meningitidis

OBJECTIVES: The sensitivity of laboratory confirmation of invasive meningococcal disease (IMD) by culture or PCR is affected by prior antibiotic treatment and decreasing use of early lumbar puncture. Serological diagnosis of IMD is not widely used because of reliance on paired serum samples. The application of single point estimations of IgM antibodies in the diagnosis of IMD was explored. DESIGN: Outer membrane proteins from a mix of commonly encountered meningococcal serotypes were partially purified and used as an antigen in an enzyme immunoassay for the detection of IgM antibody. The cut-off for the assay was derived using sera from blood bank donors and the accuracy then evaluated with sera from patients with culture-confirmed IMD, other bacterial infections and culture-proven nasopharyngeal colonisation with Neisseria meningitidis. RESULTS: The coefficient of variability of the assay was < 10% in negative, mid- and high-range positive sera and the specificity of the assay was at least 93%. In sera collected from 49 adult patients at various times after positive blood or CSF culture-confirmed IMD, the assay had a sensitivity of 100% in specimens collected between 5 and 18 days. At the time of isolation of meningococci from either blood or CSF, eight of 29 sera were IgM-positive, but beyond 70 days no positive results were detected. No differences were seen in the IgM responses in patients from whom different serogroups of N. meningitidis were recovered. CONCLUSIONS: Serological examination by single point IgM enzyme immunoassay (EIA) offers the possibility of an expanded laboratory confirmation of IMD in adults for samples taken between 5 and 18 days after onset.     

Ultrasound-Enhanced Latex Immunoagglutination and PCR as Complementary Methods for Non-Culture-Based Confirmation of Meningococcal Disease

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.     

Prospective study of a real-time PCR that is highly sensitive, specific, and clinically useful for diagnosis of meningococcal disease in children

Due to the early administration of antibiotics, meningococcal disease is increasingly difficult to diagnose by culturing. Laboratory studies have shown PCR to be sensitive and specific, but there have been few clinical studies. The objectives of this study were to determine the diagnostic accuracy and clinical usefulness of meningococcal PCR through a prospective comparison of real-time PCR, nested PCR, and standard culturing of blood and cerebrospinal fluid (CSF). The setting was a tertiary-care pediatric hospital in Australia, and the participants were 118 children admitted with possible septicemia or meningitis. The main outcome measures-sensitivity, specificity, and positive and negative predictive values-were compared to a "gold standard " fulfilling clinical and laboratory criteria. For 24 cases of meningococcal disease diagnosed by the gold standard, culturing of blood or CSF was positive for 15 (63%), nested PCR was positive for 21 (88%), and real-time PCR was positive for 23 (96%). The sensitivity, specificity, and positive and negative predictive values of real-time PCR (the most sensitive test) for all specimens were, respectively, 96% (95% confidence interval, 79 to 99%), 100% (95% confidence interval, 96 to 100%), 100% (95% confidence interval, 85 to 100%), and 99% (95% confidence interval, 94 to 100%). Of 54 patients with suspected meningococcal disease at admission, 23 had positive PCR results. Only one PCR specimen was positive in a patient thought unlikely to have meningococcal disease at admission. Blood PCR remained positive for 33% of patients tested at up to 72 h. Real-time PCR has high positive and negative predictive values in this clinical setting, with better confirmation of cases than nested PCR. Targeting patients for PCR based on admission criteria appears to be practical, and the test may remain useful for several days after the start of antibiotic administration.     

Invasive meningococcal disease in Scotland, 1994 to 1999, with emphasis on group B meningococcal disease

A review was carried out on 774 invasive meningococcal isolates reported to the active meningococcal surveillance system in Scotland from 1994 to 1999. This showed that serogroups B (51.7%) and C (39.2%) caused the majority of disease. The six common PorB proteins (4, 1, 15, 2B, 12, and 21) and PorA proteins (serosubtypes) (P1.4, P1.15, P1.9, P1.14, P1.7, and P1.16) accounted for 50 and 51% of all group B isolates, respectively, during the study period.     

Characterization ofNeisseria meningitidis isolates and clinical features of meningococcal conjunctivitis in ten patients

Cases of meningococcal conjunctivitis occurring in Denmark in the period 1982–1991 were reviewed. In a survey of laboratory reports, ten cases were identified. The meningococcal strains were characterized by serological grouping, typing and subtyping, and by antimicrobial susceptibility testing. Five cases were caused by serogroup B meningococci (B:15:P1.16, B:15:P1.6, B:4:P1.15) and five cases by serogroup C meningococci (C:2a:P1.2 (4 strains), C:14:NST). The median age of the patients was 12.5 months (range 7 days to 9 years). Signs of conjunctivitis were predominant; in addition, five of the patients had fever and general malaise. In one patient the same strain was recovered from blood and eye secretions. None of the patients had signs of meningitis. All meningococcal strains isolated from patients with meningococcal conjunctivitis were assumed to be virulent and had the same characteristics as strains causing meningococcal disease in Denmark within the same period.     

From genomics to surveillance, prevention and control: new challenges for the African meningitis belt

An international conference was held in Niamey, Niger, in November 2005. It aimed at reviewing the current situation in the meningitis belt. This region stretches from Senegal to Ethiopia and is characterized by high levels of seasonal endemicity with large epidemics of meningococcal meningitis occurring cyclically, generally caused by N. meningiditis serogroup A. WHO currently recommends a reactive strategy based on rapid detection of epidemics, intervention with antibiotics to treat cases and mass vaccination with a meningococcal polysaccharide vaccine to halt the outbreak. Epidemiological patterns of the disease in Africa have been changing with the occurrence of outbreaks outside the meningitis belt and with the emergence of serogroup W135, which first caused an epidemic among Hajj pilgrims in 2000 and then a large-scale meningitis outbreak in Burkina Faso in 2002. Consequently enhanced laboratory surveillance and confirmation of the strain responsible for the outbreak are required. New rapid dipstick tests have been developed through a collaboration between Institut Pasteur and CERMES. They are designed for bedside diagnosis and detect meningococcal antigens present in CSF using immunochromatography. The treatment of meningococcal meningitis during epidemics is based on short-course, long-acting oily chloramphenicol. An alternative is the use of ceftriaxone, which is equally effective and can be used in pregnant women and infants. A low-cost, monovalent serogroup A meningococcal conjugate vaccine for large-scale use in Africa is under development. In spite of the emergence of W135 strains in the meningitis belt, N. meningiditis A continues to be the principal strain isolated during the epidemic seasons and elimination of outbreaks of N. meningiditis serogroup A can still be considered as the primary objective of a preventive vaccination strategy.     

Confirmation of meningococcal disease by urinary antigen testing

The meningococcus is an important cause of morbidity and mortality and a rapid laboratory diagnosis is required through accurate, non-culture-based methods. Body fluids that are easily obtainable are preferred for this route of diagnosis and urine is the specimen of choice as it can be obtained non-invasively. Urine samples were tested from patients with suspected meningococcal disease and tested by latex agglutination and PCR. It was shown that urinary PCR is not useful for the laboratory confirmation of MD but latex agglutination testing may be useful in certain settings prior to confirmatory testing by a reference laboratory.     

The epidemiology of liver disease in Tayside database: a population-based record-linkage study

BACKGROUND: The true incidence and prevalence of liver disease is difficult to ascertain because there are few, if any, population-based registers of liver disease available to ensure proper case and comparator selection. The epidemiology of liver disease in Tayside (ELDIT) is a specially built register of liver disease for a well-defined geographical area of Scotland. AIMS: This paper describes the electronic linkage of multiple data sources to form ELDIT and provides initial results from the database. PATIENTS: All subjects resident in Tayside and registered with a general practitioner in the study period 1980-1999, approximately 400,000 people. METHODS: Electronic record-linkage techniques were employed to include anonymised data from primary and secondary sources. Hospital admissions, dispensed medication, and laboratory results from immunology, virology, and biochemistry were used to identify cases of liver disease. Diagnostic algorithms were used to verify and classify subjects with liver disease. A validation of the algorithms against the clinical diagnosis was used to determine the measure of agreement (true positive rate) of ELDIT. RESULTS: At present approximately 10,000 subjects have been identified with liver disease or abnormal liver function. The data set is nearing completion with cases of rarer liver disease being identified last. Incidence densities for the population were calculated. From the validation study, agreement between electronic and clinical diagnosis was 0.98 and positive predictive value was 0.83 showing electronic diagnostic algorithms are sensitive enough to identify liver disease using para-clinical data. CONCLUSIONS: ELDIT demonstrates how clinical information can be harnessed electronically to provide a better understanding of liver disease in a population.     

Detection of Meningococcal Carriage by Culture and PCR of Throat Swabs and Mouth Gargles

The standard method for detecting meningococcal carriage is culture of throat swabs on selective media, but the levels of carriage determined depend heavily on the skills of the individuals taking the swab and interpreting the cultures. This study aimed to determine the most sensitive detection method for meningococcal carriage. Throat swabs and saline mouth gargles, obtained from 89 university students, were processed in parallel by conventional culture and TaqMan ctrA PCR. Carriage of meningococci, as detected by the combined methods, was 20%. The sensitivities of throat swab culture, throat swab PCR, gargle culture, and gargle PCR were 72, 56, 56, and 50%, respectively, and the probabilities that these techniques would correctly identify the absence of carriage (negative predictive value [NPV]) were 93.4, 89.9, 89.9, and 88.8%. Culturing both throat swabs and gargles increased the NPV to 98.6%. The further addition of throat swab PCR increased this to 100%. Testing gargles by both culture and PCR was as sensitive as testing throat swabs by both methods, suggesting that gargles may be a suitable alternative for large-scale screening studies when throat swabs are difficult to obtain, although they required more lengthy laboratory processing. PCR was a useful adjunct to culture for detecting nasopharyngeal carriage, but it failed to detect some nongroupable strains. For maximum sensitivity, a combination of techniques was required. This study indicates the confidence with which health care professionals involved in meningococcal screening can regard laboratory results.     

The Scotland and Newcastle epidemiological study of Hodgkin's disease: impact of histopathological review and EBV status on incidence estimates

AIMS: The epidemiological and pathological features of Hodgkin lymphoma (HL) are complex. The Epstein-Barr virus (EBV) is consistently associated with a proportion of cases, and these cases are thought to represent a distinct aetiological subgroup of HL. The aim of the present analysis was to determine the age and sex specific incidence of EBV associated and non-associated HL, analysed separately, using data derived from a population based study-the Scotland and Newcastle epidemiological study of Hodgkin's disease (SNEHD). This study also provided a unique opportunity to evaluate accuracy in the current diagnosis and classification of HL. METHODS: SNEHD analysed consecutive cases of HL diagnosed in the study area between 1993 and 1997. Diagnostic biopsy material was retrieved, EBV status of tumours was determined, and histological review was performed. RESULTS: In total, 622 cases were eligible for the study, and EBV studies and histopathological review were performed on biopsy material from 537 and 549 cases, respectively. Accuracy in the overall diagnosis of HL and classification of nodular sclerosis HL was good, but diagnosis of HL in the elderly and classification of other subtypes was less reliable. One third of classic HL cases were EBV associated, and age specific incidence curves for EBV associated and non-associated cases were distinct. CONCLUSIONS: Comparison of age specific incidence curves for EBV associated and non-associated HL supports the hypothesis that these are two distinct aetiological entities. Accuracy in the diagnosis of HL is generally good, but certain subgroups of cases continue to present diagnostic difficulties.     

Assessing the risk of laboratory-acquired meningococcal disease

Neisseria meningitidis is infrequently reported as a laboratory-acquired infection. Prompted by two cases in the United States in 2000, we assessed this risk among laboratorians. We identified cases of meningococcal disease that were possibly acquired or suspected of being acquired in a laboratory by placing an information request on e-mail discussion groups of infectious disease, microbiology, and infection control professional organizations. A probable case of laboratory-acquired meningococcal disease was defined as illness meeting the case definition for meningococcal disease in a laboratorian who had occupational exposure to an N. meningitidis isolate of the same serogroup within 14 days of illness onset. Sixteen cases of probable laboratory-acquired meningococcal disease occurring worldwide between 1985 and 2001 were identified, including six U.S. cases between 1996 and 2000. Nine cases (56%) were serogroup B; seven (44%) were serogroup C. Eight cases (50%) were fatal. All cases occurred among clinical microbiologists. In 15 cases (94%), isolate manipulation was performed without respiratory protection. We estimated that an average of three microbiologists are exposed to the 3,000 meningococcal isolates seen in U.S. laboratories yearly and calculated an attack rate of 13/100,000 microbiologists between 1996 and 2001, compared to 0.2/100,000 among U.S. adults in general. The rate and case/fatality ratio of meningococcal disease among microbiologists are higher than those in the general U.S. population. Specific risk factors for laboratory-acquired infection are likely associated with exposure to droplets or aerosols containing N. meningitidis. Prevention should focus on the implementation of class II biological safety cabinets or additional respiratory protection during manipulation of suspected meningococcal isolates.     

Detection and serogroup determination of Neisseria meningitidis in CSF by polymerase chain reaction (PCR)

A PCR protocol for the detection and serogroup determination of Neisseria meningitidis in CSF from 85 cases of suspected meningitis was evaluated. Screening assays for both IS1106 and the ctrA gene were used to detect meningococcal DNA, and a further two assays using the siaD gene were performed to determine the serogroup. PCR results were compared with results of bacteriological culture and discrepant results resolved by analysis of clinical data and further laboratory test results. The resolved sensitivity and specificity of the PCR screening assay were 89 and 100%, and those of bacteriological culture were 37 and 100%, respectively. The siaD B/C PCR assay was able to determine a serogroup in 85% of cases positive by the PCR screening assay compared with 50% of cases where a serogroup was determined by traditional methods. PCR is a useful tool for diagnosis of meningococcal meningitis when Gram stain and culture tests are negative, a situation that may arise when antibiotic treatment has commenced prior to lumbar puncture.