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Diagnosis of partially treated culture-negative bacterial meningitis using 16S rRNA universal primers and restriction endonuclease digestion

¹Department of Neurology, KS Hegde Medical Academy, Deralakatte, Mangalore, India ²Department of Fisheries Microbiology, University of Agricultural Sciences, Mangalore, India

Correspondence Iddya Karunasagar mircen{at}sancharnet.in

Received January 15, 2004
Accepted February 5, 2005

Cerebrospinal fluid (CSF) obtained from patients with partially treated and culture-negative meningitis was subjected to PCR using 16S rDNA universal primers followed by restriction endonuclease digestion. In all, 43 patients and 7 controls were enrolled in this study. Twenty-one meningitic samples were positive by PCR. Mycobacterium tuberculosis was the causative agent in seven cases followed by Haemophilus influenzae (four), Streptococcus pneumoniae (two), Listeria monocytogenes (one), Escherichia coli (one), Pseudomonas aeruginosa (one) and Staphylococcus aureus (one). Only two meningitic CSF samples were culture-positive. In this study, PCR using bacterial 16S rDNA specific universal primers was found to be superior to conventional methods in the diagnosis of partially treated meningitis.

	INTRODUCTION

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Acute bacterial meningitis constitutes a serious neurological disorder associated with significant morbidity and mortality in developing countries. The common practice of antibiotic therapy prior to cerebrospinal fluid (CSF) evaluation coupled with inconsistent laboratory support in developing countries makes aetiological diagnosis extremely difficult (Cartwright et al., 1992). Prior antibiotic therapy 12 h or more before lumbar puncture can sterilize the CSF (Solbrig et al., 2000). In addition, tuberculous meningitis, which resembles partially treated pyogenic meningitis in many respects, creates further diagnostic difficulties.

Diagnosis of bacterial meningitis based on direct microscopy is quick but lacking in sensitivity. Culture of CSF and blood takes at least 24 h and is very often negative due to prior treatment with antibiotics. Various laboratory investigations of the CSF have been developed for the rapid diagnosis of acute bacterial meningitis. Nevertheless, none of these tests alone or in combination are dependable because of low sensitivity and specificity (Nato et al., 1991).

In recent years, PCR techniques have been increasingly used to amplify and detect microbial DNA in CSF. PCR assay has been used for identification of Streptococcus pneumoniae (Pozzi et al., 1989; Cherian et al., 1998) and for simultaneous detection of Neisseria meningitidis, Haemophilus influenzae and streptococci (Hassan-King et al., 1996; Corless et al., 2001) as aetiological agents of bacterial meningitis. Use of broad-range bacterial primers in the diagnosis of bacterial meningitis has been reported in earlier studies (Greisen et al., 1994; Radstrom et al., 1994; Kotilainen et al., 1998; Backman et al., 1999). In these studies, the diagnosis of meningitis, especially in the culture-negative cases, is not supported by clinical details and CSF cytochemistry. Response to treatment is also not included. The present study incorporates clinical and CSF data and response to treatment in all cases subjected to PCR. PCR was performed using 16S rRNA primers followed by restriction endonuclease digestion as described previously by Lu et al. (2000).

	METHODS

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Clinical specimens.

Patients diagnosed with meningitis by clinical and cytochemical criteria were selected for the study. All patients had received empirical therapy with broad-spectrum antibiotics, anti-tuberculous therapy or both, prior to entry into the study. The decision to treat as pyogenic or tuberculous meningitis, in culture-negative patients, was based on several criteria which included clinical presentation, CSF cytochemistry, and presence or absence of extra central nervous system tuberculosis as in previous studies (Narayanan et al., 2001). Parenteral antibiotic therapy 24 h or more prior to diagnostic lumbar puncture was used to define partially treated meningitis. From January to October 2002, 43 CSF samples were obtained from patients aged between 2 and 65 years. Samples were stored in sterile containers in ice, transported to the laboratory and stored at –20 °C. Direct examination was done by Gram's staining and Ziehl–Neelsen staining methods and all the samples were subjected to bacteriological culturing. Clinical details including treatment received and final outcome were recorded. An additional seven samples from patients who underwent diagnostic lumbar puncture for conditions other than central nervous system infections, such as peripheral neuropathy and normal pressure hydrocephalus, were included as controls. Clinical and laboratory parameters of patients and controls are shown in Tables 1 and 2.

Bacterial strains and preparation of crude lysate.

The strains used in this study included H. influenzae, Strep. pneumoniae, N. meningitidis, Escherichia coli, Salmonella typhi and Listeria monocytogenes. The test strains were grown in Brain Heart Infusion broth (BHI) (Hi-Media) overnight at 37 °C. A 1 : 10 dilution of the culture was prepared by adding 50 µl of the bacterial culture to 450 µl TE (10 mM Tris, pH 8.0; 1 mM EDTA) buffer. The cell lysate was prepared by boiling at 95 °C for 10 min and centrifuged at 10 000 g for 10 min. A 5 µl quantity of the supernatant was used for PCR.

Extraction of DNA from CSF samples.

DNA from CSF samples was extracted by the CTAB–phenol–chloroform–isoamyl alcohol method. Briefly, 1 ml CSF was centrifuged at 14 000 g for 15 min, the supernatant was discarded and the pellet was resuspended in 567 µl TE (10 mM Tris, 1 mM EDTA; pH 8.0) buffer, CTAB and NaCl. This was followed by extraction once each with phenol–chloroform (24 : 1) and phenol–chloroform–isoamyl alcohol (25 : 24 : 1). The DNA was precipitated using 2-propanol, washed with 70 % ethanol and resuspended in 20 µl sterile TE buffer. Five microlitres of this was used as template for PCR. Sterile water was subjected to the same procedure every time the DNA was extracted from CSF, and this acted as a negative control for PCR.

PCR conditions.

For PCR, primers specific for eubacterial 16S rDNA were used to generate a 996 bp product (Lu et al., 2000). The amplification was performed in a 50 µl PCR mixture consisting of 1 x PCR buffer (10 mM Tris/HCl, pH 9.0; 50 mM KCl; 1.5 mM MgCl₂; 0.01 % gelatin), 0.5 µM of each primer, 200 µM of each of the four dNTPs and 1.25 U Taq polymerase (Bangalore Genei). The cycling conditions were as follows. After an initial denaturation for 3 min at 94 °C, the next 35 cycles consisted of denaturation at 94 °C for 1 min, primer annealing at 55 °C for 1 min and primer extension at 72 °C for 2 min. The post-amplification of the flush ends was performed at 72 °C for 5 min. For specific detection of Mycobacterium tuberculosis, the primer pair was used as previously described (Renz et al., 1991). All PCR reactions were performed in a PTC-100 Thermal cycler (M. J. Research). The products of PCR were separated on a 1.6 % agarose gel, stained with ethidium bromide (0.5 µg ml^–1) and photographed using a gel documentation unit (Hero lab).

Restriction endonuclease digestion.

Ten microlitres of the PCR product was digested with HaeIII restriction enzyme (Bangalore Genei). One product was in addition digested with MnlI. The digestion products were resolved on a 2 % agarose gel, stained with ethidium bromide and photographed.

	RESULTS AND DISCUSSION

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A total of 50 CSF specimens were used for PCR, which included 43 cases of meningitis and 7 controls. The universal primers amplified bacterial 16S rDNA from 21 out of 43 meningitic samples (48.8 %). The PCR products generated thus were digested with HaeIII restriction enzyme and the digestion patterns were analysed as described previously by Lu et al. (2000). In this study, the 996 bp amplicon from known strains of bacteria was digested and the patterns generated were used as reference for subsequent studies using CSF specimens. From these studies, the bacterial DNA from CSF specimens was determined to be from M. tuberculosis (seven cases), H. influenzae (four), Strep. pneumoniae (two), L. monocytogenes (one), E. coli (one), Pseudomonas aeruginosa (one) and Staphylococcus aureus (one). Restriction digestion with MnlI helped to ascertain the aetiology of the Staph. aureus-positive sample. In cases where tuberculous meningitis was diagnosed, an additional Mycobacterium-specific PCR (Renz et al., 1991) was performed. All seven samples which yielded restriction patterns typical of M. tuberculosis were also positive by this PCR. Two CSF samples yielded faintly positive PCR signals that were too weak to be restriction digested. In another two samples, the digestion patterns were not recognizable as belonging to any common bacterial pathogen. Two samples were culture-positive for Strep. pneumoniae and P. aeruginosa, respectively, and the restriction digestion patterns of the PCR products from these samples resembled those of cultured organisms.

In this study, 41 of 43 meningitic CSF specimens tested were culture-negative, unlike most previous studies which have used culture-positive samples for PCR analysis. All PCR-positive cases were supported clinically and by routine cytochemistry. Eleven cases were treated as tuberculous meningitis, all of which responded to the treatment. PCR was negative in four of these cases, two of which also had pulmonary tuberculosis. A negative PCR in the presence of disseminated tuberculosis has also been observed in previous studies (Nato et al., 1991). A paucibacillary CSF or alternatively prior treatment with anti-tuberculous treatment have been conjectured to be the reason for PCR negativity. Two patients with pyogenic meningitis expired (Tables 1 and 2); both cases were caused by uncommon meningitis pathogens. They were empirically treated with broad-spectrum antibiotics and anti-tuberculous drugs prior to entry into the study. One patient with PCR-proven tuberculous meningitis expired. Prior to entry into the study, this patient had received broad-spectrum antibiotics based on a presumptive diagnosis of pyogenic meningitis.

In two samples, the restriction enzyme digestion results did not match any of those described by Lu et al. (2000) or the reference strains used in our study. It is possible that these were caused by pathogens whose restriction digestion patterns are not known to us. Alternatively, strain variations in common pathogens may have been responsible. In 22 meningitis samples, PCR was negative. It is possible that in some cases PCR inhibition was responsible for the negative results. Sufficient volume of specimen was not available for a spike-back experiment to prove this hypothesis.

We used the CTAB method for the extraction of DNA from CSF specimens. This method of DNA extraction enhanced PCR detection in the majority of the samples that were PCR-negative when a simple boiling method was used to prepare DNA from CSF for PCR (data not shown). In order to avoid contamination by bacterial DNA during extraction, we co-extracted TE buffer each time DNA was extracted from CSF specimens by the CTAB method and used it as a negative control. To keep costs down, commercial DNA extraction kits were not employed. Moreover, they often require the presence of 10³ c.f.u. organisms ml^–1 for detection (Pozzi et al., 1989; Davis & Fuller, 1991). In our study, we opted to perform restriction digestion after the preliminary PCR, once again to cut costs and duration of the test.

The results of this preliminary study, done for the first time in India, indicate that PCR-based assays are useful adjuncts to conventional bacterial culture and antigen detection methods in establishing the bacterial aetiology in meningitis in settings where substantial numbers of specimens are culture-negative. The aim of the present study was to evaluate the usefulness of the PCR method in detecting bacteria in CSF from patients with clinically diagnosed bacterial meningitis. This work corroborates the previously published work of Lu et al. (2000). Molecular diagnostic methods, though expensive, are justified in developing countries like India. Culture negativity in CSF results in empiric treatment of meningitis often with combinations of antibiotics and/or anti-tuberculous therapy. This often results in high morbidity and mortality, frequent need for intensive care therapy and prolonged hospital stay, all of which singly or in combination make treatment expensive and unaffordable for the majority of patients.

	REFERENCES

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