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Rapid and Fatal Meningococcal Disease Due to a Strain of Neisseria meningitidis Containing the Capsule Null Locus

  1. Linda M. N. Hoang1,
  2. Eva Thomas1,2,
  3. Shaun Tyler5,
  4. Andrew J. Pollard6,
  5. Gwen Stephens3,
  6. Larry Gustafson4,
  7. Alan McNabb3,
  8. Ingrid Pocock3,
  9. Raymond Tsang5, and
  10. Rusung Tan1,2
  1. 1Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver
  2. 2Children's & Women's Health Centre of British Columbia, Vancouver
  3. 3BC Centre for Disease Control, Vancouver
  4. 4Fraser Health, Surrey, British Columbia, Manitoba, Canada
  5. 5National Microbiology Laboratory, Health Canada, Winnipeg, Manitoba, Canada
  6. 6Department of Paediatrics, University of Oxford, United Kingdom
  1. Reprints or correspondence: Dr. Rusung Tan, Dept. of Pathology and Laboratory Medicine, British Columbia's Children's Hospital, 4480 Oak St., Rm. 2G5, Vancouver, BC V6H 3V4 (roo{at}interchange.ubc.ca).
 
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Abstract

Background. Neisseria meningitidis continues to be an important cause of invasive bacterial disease among children and young adults worldwide. In Canada, N. meningitidis strains that bear serogroups B and C polysaccharide capsules predominate. We report the first documented case of invasive meningococcal disease in an immunocompetent host caused by an acapsular strain of N. meningitidis containing the capsule null locus (cnl).

Methods. Analysis of the isolate was performed with use of serological and molecular methods, including multilocus sequence typing and cnl gene identification. Analysis of 16S ribosomal RNA (rRNA) and porA genes was also performed to confirm the identity of the bacterium.

Results. The patient was a healthy, immunocompetent 13-year-old child, and N. meningitidis was recovered from a sample of her cerebrospinal fluid before death. The isolate was nontypeable by both conventional antisera and indirect whole-cell enzyme-linked immuosorbent assay methods using antibodies to serogroups B, C, Y, and W135. The isolate was further identified as a cnl strain, serotype 15 (ST-198). N. meningitidis–specific DNA was identified in the isolate and in the pre- and postmortem specimens by 16S rRNA and porA gene analysis.

Conclusions. This is the first reported case of fatal meningococcal disease caused by an acapsular cnl strain of N. meningitidis that was isolated from an immunocompetent host. Routine molecular diagnostic methods targeted at the cnl locus failed to detect this organism, indicating a need to determine the incidence of infection with cnl strains among patients with culture-negative invasive disease.

Invasive meningococcal disease is a rapidly progressive and often fatal illness that mainly affects young adults and children, and it continues to be a significant public health concern. In 2002, ∼200 persons in Canada were reported to have invasive meningococcemia, an incidence of 0.6 cases per 100,000 population [1]. In British Columbia, there was a total of 32 cases, for an incidence of meningococcal disease of 0.8 cases per 100,000 population, with the highest incidence among children aged <1 year and the bulk of the cases (n = 26) occurring among people aged >14 years [1]. The etiologic agent, Neisseria meningitidis, colonizes the human naso-oropharyngeal mucosa in 10% of individuals and is transferred from person to person by direct contact or droplets [2, 3]. Invasive disease begins with breech of the oropharyngeal mucosa followed by systemic dissemination of the bacteria, septicemia, and/or acute meningitis [4].

The meningococcal capsule is an important virulence factor critical for the pathogenesis of meningococcal disease, and the invasiveness of the organism is thought to be highly dependent on the presence of the capsular gene complex that encodes factors necessary for serogroup expression [5–7]. Invasive disease is primarily caused by the serogroups A, B, C, W135, and Y. The genes involved in capsular polysaccharide synthesis (cps and sia) and transport (ctr) operon are siaA, siaB, siaC, ctrA, ctrB, ctrC, and ctrD. In 2002, Claus et al. [8] demonstrated that, in strains that lacked the cps operon, the region is replaced by a noncoding region, termed the “capsule null locus” (cnl). These unencapsulated strains of N. meningitidis have continued to be associated with a carrier state and have been found as human commensals. Several studies have examined the carriage of nontypeable N. meningitidis. During an outbreak of serogroup C disease in British Columbia, in 2003, Patrick et al. [9] evaluated N. meningitidis carriage in patients aged 13–29 years and found that 33% of isolates obtained from 153 individuals with N. meningitidis carriage were nontypeable by PCR. Sadler et al. [10] reported that 50% of meningococcal carrier isolates were nontypeable by conventional antisera studies. Of these, 47% were either ctrA gene negative or an unknown type.

Recently, a case of cnl meningococcemia in a severely immunocompromised patient was described. The authors suggested that the isolate, although pathogenic in an immunocompromised host, was not pathogenic for immunocompetent individuals [11]. We present, to our knowledge, the first documented case of rapid and fatal meningococcemia in an immunocompetent patient due to a cnl strain of N. meningitidis.

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Case Report

A previously healthy 13-year-old girl presented to her local hospital at 2 A.M. feeling unwell and with a widespread, purpuric rash. She had returned home from school early the previous day with flulike symptoms, back pain, and nausea, and she developed a fever that night. There was no history of recurrent, unusual, or frequent infections; no recent travel history; and no personal or family history suggestive of immunodeficiency. She was HIV and hepatitis B virus seronegative. Other family members were healthy. She had not received immunization against any meningococcal infection.

At presentation, the patient was drowsy and had vital signs consistent with mild septic shock. Mild neck stiffness and purpura fulminans were noted during clinical examination. Laboratory investigations revealed thrombocytopenia, coagulopathy, and marked neutropenia. The patient started receiving ceftriaxone, and within 15 min after drug administration, blood and CSF samples were obtained and submitted for culture. The CSF had a mononuclear cell count of 13 × 106 cells/L, an RBC count of 1925 × 106 cells/L, a glucose level of 3.9 mmol/L, and a protein level of 228 mg/L. She became severely hypotensive and was initially stabilized with volume resuscitation and dopamine infusion. The patient was transferred to the British Columbia Children's Hospital (BCCH) intensive care unit but died of cardiorespiratory failure ∼5 h from the time of initial hospital presentation.

On autopsy, there were no signs of immunological disease. There were no structural anomalies noted, and the reticuloendothelial system was fully developed. A nontypeable strain of N. meningitidis was isolated from the CSF culture. All cultures of brain, kidney, lung, heart, and liver specimens obtained at autopsy were negative for bacteria. Molecular detection of meningococci from the patient's pre- and postmortem specimens was required to confirm that this organism was the pathogen.

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Methods

Bacterial isolation. A volume of the patient's CSF sample was inoculated directly onto a chocolate blood agar plate with 5% sheep blood (PML Microbiologicals), onto a Columbia blood agar plate with 5% sheep blood (PML Microbiologicals), and into brain heart infusion (BHI) broth (PML Microbiologicals) with standard laboratory methods, and these samples were incubated in an atmosphere of 5% CO2 at 35°C for 5 days. A volume of the BHI broth was subsequently subcultured onto chocolate blood agar plates, which were incubated in an atmosphere of 5% CO2 for an additional 3 days.

Samples of the patient's blood were collected into 1 aerobic and 1 anaerobic BacT Alert 3D bottle (Organon Teknika) for culture shortly after antibiotics had been administered, and the cultures were monitored for growth. Both blood culture bottles were discarded, because there was no growth after 5 days of incubation. Autopsy specimens were directly inoculated onto a chocolate blood agar plate with 5% sheep blood and a Columbia blood agar plate with 5% sheep blood (PML Microbiologicals) and were incubated in an atmosphere of 5% CO2 at 35°C for 5 days.

Conventional serotype studies of isolate. Conventional serotyping of the meningococcal isolate was performed with rabbit antisera to serogroups A, B, C, W135, X, Y, Z, and 29E and with indirect whole-cell ELISA for detection of serogroups B, C, Y, and W135.

Molecular serogroup studies. Routine PCR detection of ctrA and siaD gene targets of meningococci was performed with the patient's serum and autopsy specimens as described elsewhere [12]. Further PCR studies were performed on the isolate to specifically target the genes for the ctr operon (ctrA, ctrB, ctrC, and ctrD) and the sialic acid synthesis genes (siaA, siaB, and siaC and serogroup B-, C-, Y-, and W135-specific siaD). In addition, Southern hybridization was performed using full-length probes for the ctr and sia operons. PCR and sequencing was also performed for cnl, as described elsewhere [8]. Multilocus sequence typing was performed as previously described by Maiden et al. [13], with the use of modifications recommended on the MLST Web site (http://www.mlst.net).

porA and 16S rRNA detection and sequencing studies. The specimens from the patient that were available for testing were CSF samples obtained at hospital admission, an EDTA blood specimen drawn just before the patient's death, and specimens of heart blood, brain, and pericardial fluid obtained at autopsy. Premortem specimens (CSF and EDTA blood) and postmortem specimens (pericardial fluid, brain tissue, and heart tissue) that were culture-negative for bacteria were subjected to porA gene and 16S rRNA detection and sequencing studies. The N. meningitidis isolated from the patient's CSF specimen was also evaluated by porA gene and 16S rRNA studies.

Tissue specimens were digested using a QIAamp Tissue Kit (Qiagen), and DNA was extracted with a QIAamp DNA Mini Kit (Qiagen) in accordance with the manufacturer's instructions. The DNA from body fluid specimens was extracted directly from the samples by use of a QIAamp DNA Mini Kit (Qiagen). Negative control samples were included to detect contamination during the tissue digestion and nucleic-acid extraction steps. DNA amplification was performed using a nested PCR reaction. The nested PCR assay targeting the porA gene, which encodes the outer membrane protein that is the subtype determinant antigen for N. meningitidis, used primers that were described elsewhere [14, 15]. The PCR product was visualized on an ethidium bromide—stained agarose gel. In the nested PCR assay that targeted the 16S rRNA, the first round of PCR amplification was performed using primers 27f and 1525r, as described elsewhere [16]. The secondary PCR amplification used the primers 27f and 519r and cycling conditions described elsewhere [16]. The PCR product of appropriate size was determined by gel electrophoresis in the presence of ethidium bromide. Cycle sequencing of the amplicons was performed using primers 27f and 519r and Big Dye Terminator chemistry, version 3.1 (Applied Biosystems), in accordance with the manufacturer's instructions. Sequences of the fragments were determined on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Sequences generated by the paired primers were transferred to, assembled, and edited using SeqManTM software (DNAStar). Consensus sequences were compared to sequences in GenBank using a BLAST search.

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Results

Bacterial cultures. Cultures of blood samples that had been collected at the time of admission to the local hospital were negative for bacteria after 5 days of incubation and were discarded. However, a CSF culture yielded a single gray colony on the inoculum site on both the chocolate agar plate and the blood agar plate after 2 days of incubation. Gram stains of the colony revealed gram-negative diplococci consistent with Neisseria species. The isolate was oxidase positive and was later identified as N. meningitidis by the API NH system (bioMériuex). All specimens obtained at autopsy and submitted for bacterial culture were negative for bacterial growth. In many cases of meningococcal sepsis, N. meningitidis is recovered in only ∼50% of blood cultures [17, 18]. Factors that may contribute to the negative blood culture results include the presence of sodium polyanethol sulfonate, which is present in the blood culture system and may be sufficient to suppress growth [19, 20], and the administration of antibiotics before collection of patient specimens, which may inhibit bacterial growth in artificial media.

Molecular analysis. To rule out the possibility that the isolates were contaminated from an environmental source, direct molecular detection from tissue specimens was performed using 16S rRNA and porA PCR followed by nucleotide sequencing. Amplicons of the correct size were amplified from the EDTA premortem blood specimen and postmortem heart blood and pericardial fluid specimens obtained at autopsy in the primary 16S rRNA PCR. Amplicons of the correct size in the second round of PCR were also obtained from these 3 specimens. BLAST searches in GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) of the sequences obtained from the 3 amplicons matched a nonserogroupable N. meningitidis strain M7892 (AF382303) with 100% similarity. The porA gene was detected in the premortem CSF and blood specimens, as well as in postmortem heart and pericardial specimens; the latter 3 were also positive for the 16S rRNA gene (table 1). The sequence of the porA gene product from the isolated N. meningitidis organism was identical to those found in both pre- and postmortem specimens, confirming the presence of N. meningitidis in the patient's clinical samples.

Table 1
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Table 1

Neisseria meningitidis detection in pre- and postmortem specimens by conventional culture, 16S rRNA gene PCR, and porA gene PCR methods.

N. meningitidis typing studies. Serological typing (for serogroups A, B, C, 135W, X, Y, Z, and 29E) that was performed and repeated at a second laboratory showed nonagglutination. Serogrouping by indirect whole-cell ELISA with monoclonal antibodies to detect serogroups B, C, Y, and W135 bacteria also yielded negative results.

PCR diagnostic assays failed to detect the ctrA gene or siaD genes. Similarly, the results of PCR for N. meningitidis with primers specific for the ctrA and siaD gene were also negative for all autopsy specimens. Additional PCR investigations to elucidate the serogroup of this N. meningitidis isolate also failed to detect the capsule synthesis genes—specifically, the capsule transport genes ctrA, ctrB, ctrC, and ctrD. The sialic acid synthesis genes, siaA, siaB, and siaC and serogroup B-, C-, Y-, and W135-specific siaD were also absent, as was the serogroup A–specific orf2 gene. Southern hybridization also failed to detect ctr- or sia-related gene sequences. PCR for cnl yielded an amplicon of the expected size, and subsequent sequencing typed the strain as cnl-2. These findings suggest that the isolate lacked genes within the capsular synthesis and transport operon, thus explaining the autoagglutination phenomenon observed. The possibility of a novel capsule structure, however, cannot be excluded.

Antibiotic therapy was initiated within 15 min before a CSF specimen was obtained from which the nonencapsulated strain grew. Therefore, it is unlikely that the antibiotic would have influenced the genotypic or phenotypic characteristics of the isolate. In addition, the N. meningitidis 16S rRNA sequence was identical to that obtained from the postmortem tissues, and this sequence corresponds to a nonserogroupable strain, as shown in figure 1.

Figure 1
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Figure 1

Dendogram of 16S rRNA sequence of the Neisseria meningitidis strain isolated from the CSF of a previously healthy, immunocompetent 13-year-old girl. The sequence matched a nonserogroupable N. meningitidis strain (AF382303) on BLAST searches in GenBank with 100% similarity.

The isolate was typed as serotype 15, serosubtype P1 (nonserotypeable), and ST-198 by multilocus sequence typing. This serotype has previously been identified as a commensal strain isolated from a group of healthy children and young adults in Bavaria, Germany, and it contained the cnl locus [8]. Serotype 15 has previously been isolated from a CSF specimen from a patient with invasive meningococcal disease in Canada [21].

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Discussion

Currently, rapid laboratory diagnosis of N. meningitidis disease is important not only for assisting in the management of the illness but also for determining the necessity of subsequent prophylaxis and vaccination strategies. In British Columbia, PCR studies are routinely performed on CSF and blood specimens for the identification of N. meningitidis. The PCR strategy used targets the ctrA gene, a highly conserved segment of the N. meningitidis genome encoding an outer-membrane protein involved in the transport of the capsular polysaccharide [22]. The gene is a frequent target of many PCR assays, generally allowing for rapid and reliable diagnosis of infections [12]. This case, however, provides evidence that an acapsular strain of N. meningitidis undetectable by ctrA PCR assays retains the capacity to cause rapid and invasive meningococcal disease. A proportion of cases of presumed meningococcal disease are culture and PCR negative and may be due to cnl strains. Should such strains of acapsular, virulent meningococci increase in prevalence, conventional PCR diagnosis may be unreliable, and assays targeting other conserved sequences of the organism will be necessary. Additional studies are needed to determine the incidence of infection with capsule null strains among cases of culture-negative invasive disease.

In this particular case, laboratory confirmation of meningococcemia would have been missed without the isolated colonies from the CSF culture. Because less than one-half of the cases of invasive meningococcal disease yield positive culture results, and because PCR diagnosis has become a “gold standard” for diagnosis [14, 15], these findings have implications for the interpretation of PCR results, as well as for the design of future assays. Similarly, vaccines based on the capsular antigens may not provide adequate protection and might drive evolution of hyperinvasive acapsular strains of N. meningitidis.

The inability to readily identify the isolate in this case might have had significant public health implications, particularly with regard to close-contact prophylaxis and cluster surveillance, were it not for the high level of suspicion of meningococcal disease and the prompt reporting by the local emergency physician. In accordance with hospital infection-control policy and emergency department protocol, prophylaxis of household contacts was initiated by the local hospital, and the public health on-call physician for the regional health authority was notified immediately. Extensive contact tracing was initiated, with chemoprophylaxis provided to household and other close contacts. Conjugate meningococcal C vaccine was also provided to the contacts for whom chemoprophylaxis was indicated on the basis of local epidemiology of invasive meningococcal disease in adolescents and young adults.

In contrast to a recent report by Vogel et al. [11], which described a case of invasive meningococcal disease caused by a commensal cnl strain of N. meningitidis, this report involved a healthy, presumably immunocompetent 13-year-old child with no obvious predisposition for invasive meningococcal disease. This case underscores the importance of antibiotic prophylaxis for persons in close contact with such patients, regardless of the capsulation status of the isolate. With emerging strains of virulent bacteria, which may not be identifiable through conventional methods, a high clinical index of suspicion in combination with multiple modalities of diagnostic microbiology tools for rapid and reliable identification of the offending etiology is becoming the mainstream standard in clinical microbiology and infectious diseases.

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Acknowledgments

Potential conflicts of interest. All authors: no conflicts.

  • Received September 27, 2004.
  • Accepted November 8, 2004.
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