Clinical Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Neisseria meningitidis in Cerebrospinal Fluid

BackgroundNeisseria meningitidis (Nm) is a leading causative agent of bacterial meningitis in humans. Traditionally, meningococcal meningitis has been diagnosed by bacterial culture. However, isolation of bacteria from patients’ cerebrospinal fluid (CSF) is time consuming and sometimes yields negative results. Recently, polymerase chain reaction (PCR)-based diagnostic methods of detecting Nm have been considered the gold standard because of their superior sensitivity and specificity compared with culture. In this study, we developed a loop-mediated isothermal amplification (LAMP) method and evaluated its ability to detect Nm in cerebrospinal fluid (CSF).Conclusions/SignificanceCompared to PCR, LAMP detected Nm with higher analytical and clinical sensitivity. This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.     

False positive diagnosis of meningococcal infection by the IS1106 PCR ELISA

At a time when optimal case ascertainment for meningococcal infection is a high priority, the need for non-culture case confirmation, in particular by DNA amplification, is seen as being of vital importance to assist contact management and cluster recognition. A solution hybridisation assay with colorimetric microtitre plate detection (polymerase chain reaction-enzyme-linked immunosorbent assay (PCR ELISA)) has been developed using the multicopy insertion sequence IS1106 which had reportedly achieved a specificity of 100% and was described as being meningococcal specific. This PCR ELISA assay was evaluated on specimens from over 5000 patients at the national Meningococcal Reference Unit (MRU) between late 1995 and early 1997 and was found to be highly sensitive. Insertion sequences, however, are genetically mobile with the ability to spread between species and even genera. During the evaluation period of the IS1106 PCR ELISA a number of false positives proved to be caused by organisms other than N. meningitidis were recorded resulting in the withdrawal of this assay as a front line screening assay for routine confirmation of meningococcal infection.     

A Multi-Target Real-Time PCR Assay for Rapid Identification of Meningitis-Associated Microorganisms

A central nervous system (CNS) infection, such as meningitis, is a serious and life-threatening condition. Bacterial meningitis can be severe and may result in brain damage, disability or even death. Rapid diagnosis of CNS infections and identification of the pathogenic microorganisms are needed to improve the patient outcome. Bacterial culture of a patient??s cerebrospinal fluid (CSF) is currently considered the ??gold standard?? for diagnosing bacterial meningitis. From the CSF cultures researchers can assess the in vitro susceptibility of the causative microorganism to determine the best antibiotic treatment. However, many of the culture assays, such as microscopy and the latex agglutination test are not sensitive. To enhance pathogen detection in CSF samples we developed a multi-target real-time PCR assay that can rapidly identify six different microorganisms: Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae, Listeria monocytogenes and Cryptococcus neoformans. In this study we applied this PCR analysis to 296 CSF samples from patients who were suspected of having meningitis. Of the 296 samples that were examined, 59 samples were positive according to the CSF culture and/or molecular assays. Forty-six CSF samples were positive for both the CSF culture and our real-time PCR assay, while 13 samples were positive for the real-time PCR but negative for the traditional assays. This discrepancy may have been caused by the fact that these samples were collected from 23 patients who were treated with antimicrobials before CSF sampling.     

Meningococcal disease: A paradigm of type‐IV pilus dependent pathogenesis

Neisseria meningitidis (meningococcus) is a Gram‐negative bacterium responsible for two devastating forms of invasive diseases: purpura fulminans and meningitis. Interaction with both peripheral and cerebral microvascular endothelial cells is at the heart of meningococcal pathogenesis. During the last two decades, an essential role for meningococcal type IV pili in vascular colonisation and disease progression has been unravelled. This review summarises 20 years of research on meningococcal type IV pilus‐dependent virulence mechanisms, up to the identification of promising anti‐virulence compounds that target type IV pili.     

Quick Diagnosis of Human Brain Meningitis Using Omp85 Gene Amplicon as a Genetic Marker

The usual diagnosis of life-threatening human brain bacterial meningitis are expensive, time consuming or non-confirmatory. A quick PCR based diagnosis of meningitis in cerebrospinal fluids (CSF) using specific primers of virulent Omp85 gene of Neisseria meningitidis can detect as low as 1.0 ng of genomic DNA (G-DNA) in 80 min for confirmation of bacterial meningitis caused by N. meningitidis infection. The 257 bp amplicon of Omp85 gene does not show homology with other suspected pathogens in CSF and can be used as a specific genetic marker for diagnosis of the disease.     

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.     

Evaluation of non-culture diagnosis of invasive meningococcal disease by polymerase chain reaction (PCR)

Antibiotic treatment prior to transport or admission to hospital has reduced the proportion of cases of invasive meningococcal disease (IMD) from which Neisseria meningitidis can be isolated by standard microbiological techniques. Identification of meningococci by polymerase chain reaction (PCR) was assessed in relation to microbiological diagnosis for cases over a 4-year period between 1998 and 2001. A screening assay for the IS1106 gene was used to detect meningococcal DNA and five additional assays for siaD and orf-2 genes were performed to determine the serogroup. PCR results were compared with results of bacteriological culture, other laboratory test results and clinical data. The sensitivity of the PCR assay for culture-confirmed cases was 98.5%. The specificity of the assay was 96% based on test results for patients from whom other bacteria were isolated, children with viral meningitis and afebrile negative controls. The siaD B/C/W-135 and Y as well as the orf-2 gene for serogroup A PCR assays were able to determine the serogroup for 75.2% of cases that were positive by PCR screening assay. When isolates from patients with IMD were tested by both agglutination and PCR, the results agreed in all cases. PCR is a useful tool for diagnosis of IMD when Gram stain and culture tests are negative due to antibiotic treatment prior to collection of samples for microbiological analyses.     

Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>, <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> and <Emphasis Type="Italic">Neisseria meningitidis</Emphasis>

Background  

Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients.     

Diagnosis of Streptococcus pneumoniae meningitis by polymerase chain reaction amplification of the gene for pneumolysin

Diagnosis of bacterial meningitis has long been based on classical methods of Gram stain, serological tests, and culture of cerebrospinal fluid (CSF). The performance of these methods, especially culture and direct smear is thwarted by failure to detect bacteria following administration of antimicrobial agents and reluctance to performance lumbar punctures at admission. Indeed, patients with meningitis frequently receive antibiotics orally or by injection before the diagnosis is suspected or established Thus an alternative method has become necessary to help clinicians and epidemiologists to management and control of bacterial meningitis. We evaluate the application of a polymerase chain reaction-based (PCR) assay for amplification of pneumolysin gene (ply) to diagnosis of Streptococcus pneumoniae meningitis. The PCR assay sensitivity for CSF was 96% (95% confidence interval, CI, 90-99%) compared to a sensitivity of 59% for culture (95% CI 49-69%), 66% for Gram stain (95% CI 56-74%), and 78% for latex agglutination test (95% CI 69-86%); PCR specificity was 100% (95% CI 83-100%). PCR results were available within 4 h of the start of the assay. This molecular approach proved to be reliable and useful to identify this bacterium compared with other classical laboratory methods for identification of bacterial meningitis pathogens.     

Comparison of Real Time IS6110-PCR,Microscopy, and Culture for Diagnosis of Tuberculous Meningitis in a Cohort of Adult Patients in Indonesia

Background

Bacteriological confirmation of tuberculous (TB) meningitis is difficult. Culture is slow and microscopy has insufficient sensitivity. We evaluated real time PCR targeting insertion sequence IS6110 among 230 consecutive adult patients with subacute meningitis in a referral hospital in Indonesia.

Methods

Cerebrospinal fluid (CSF) samples were examined using microscopy, solid and liquid culture, and real time IS6110-PCR with a fluorescence-labeled probe using DNA extracted from CSF. CSF samples from 40 non-infectious neurology patients were used as negative controls. IS6110-PCR results were linked with clinical and CSF characteristics.

Results

Most patients presented with subacute meningitis, after a median of 14 days of symptoms (range 7–30). After exclusion of cryptococcal and bacterial meningitis, 207 patients were classified as definite or probable TB meningitis; 17.9% with HIV infection. Among this group IS6110-PCR gave the highest positivity rate (68%, 95% CI 62–74%) compared with microscopy of ZN-stained slides (11%, 95% CI 7–15%), and mycobacterial culture using solid (36%, 95% CI 29–42%) and liquid (44%, 95% CI 37–51%) media. IS6110-PCR was positive in 92% of patients with culture-positive and 42% of patients with culture-negative probable TB meningitis. Among culture-negative patients, a positive PCR was associated with a history of TB treatment, a longer duration of illness, a higher CSF cell count and protein, and a lower CSF glucose. IS6110-PCR was negative in all CSF samples from non-meningitis control patients.

Conclusions

Real time IS6110-PCR is a quick, sensitive, and specific test for diagnosing of TB meningitis in this setting. Its performance in other (less-developed) settings needs further study.     

Assessment of a two-step nucleic acid amplification assay for detection of Neisseria meningitidis followed by capsular genogrouping

Immediate prevention of meningococcal disease relies in part on the prompt treatment with antibiotics of household and other close contacts of cases; however intervention with effective vaccination relies on identification of serogroup-causing strains. Parenteral antibiotic for patient with suspected meningococcal disease before hospital admission is currently recommended. Laboratory standard methods are hindered by failure to detect bacteria by this medical approach to improve patient prognosis. We assessed two polymerase chain reaction (PCR) assays to detect (crgA) and define the serogroups (siaD, orf-2, and ctrA) of Neisseria meningitidis in 120 cerebrospinal fluid (CSF) samples from positive cases (culture or antigen detection or direct smear). The PCR sensitivity for the identification of N. meningitidis was 100% (95% confidence interval, CI, 96-100%) compared to a sensitivity of 46% for culture (95% CI 37-55%), 61% for latex agglutination test (95% CI 52-70%), and 68% for Gram stain (95% CI 59-76%); PCR specificity was 97% (95% CI 82-100%). PCR correctly identified the serogroups A, B, C, W135, Y, and X in CSF samples with a sensitivity of 88% (95% CI 80-93%); the primer sets were 100% specific. The introduction of PCR-based assays shall increase laboratory confirmed cases, consequently enhancing surveillance of meningococcal disease.     

Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients

Cerebrospinal fluid (CSF) samples from clinically diagnosed patients with detectable Angiostrongylus canto-nensis-specific antibodies (n = 10), patients with clinically suspected cases that tested negative for A. cantonensis-an-tibodies (n = 5) and patients with cerebral gnathostomiasis (n = 2) and neurocysticercosis (n = 2) were examined by a single-step polymerase chain reaction (PCR) method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.     

The class 1 outer membrane protein of Neisseria meningitidis produced in Bacillus subtilis can give rise to protective immunity

The class 1 outer membrane protein of Neisseria meningitidis B:15:P1.7, 16 was expressed in Bacillus subtilis in high yield as intracellular aggregates. These were easy to isolate and the protein (called BacP1) could be solubilized under denaturing conditions. Sera of mice immunized with thus-solubilized BacP1 contained high titres of antibodies that reacted with the class 1 protein of the meningococcal envelope in immunoblots but did not react with native meningococcal envelope in enzyme immunoassays (EIA) or with intact meningococci in bactericidal assays. However, when the BacP1 protein was complexed with heterologous (Salmonella) lipopolysaccharide, the ensuing sera reacted with meningococcal envelope preparations in both EIA and immunoblots, showed subtype-specific bactericidal activity, and were protective in an infant rat meningitis model.     

Employment of broad range 16S rDNA PCR and sequencing in the detection of aetiological agents of meningitis

To employ partial 16S rDNA PCR and automated sequencing technique to identify non-culturable causal agents of bacterial meningitis, 73 peripheral blood samples and 413 culture-negative and eight culture-positive CSF clinical specimens from patients with suspected acute meningitis were examined for the presence of bacterial genomic DNA employing broad range 16S rDNA PCR followed by sequencing of the amplicons. In blood samples, 63/73 specimens were PCR positive (86.3%) and after direct sequencing of the PCR amplicons, only 12.7% (8/63) gave clear sequencing results and 55/63 (87.3%) were mixed with more than one organism detected. The mixed PCR amplicons were separated by using PAGE and mixed amplicons from 29/55 (52.7%) specimens were successfully identified through sequencing. Of the CSF samples, 8/8 culture-positive samples were also PCR positive and 45/413 (10.9%) of culture-negative gave a strong PCR signal and 88/413 (21.3%) specimens yielded a weak PCR signal. The remaining 280 culture-negative specimens were also PCR negative. Nested PCR was set up for the 88 weak positive samples and yielded 72/88 (81.8%) strong positives, with the remainder failing to amplify 133/413 (32.2%) culture-negative samples were PCR positive. In this study, the most common bacteria identified from blood specimens were Neisseria meningitidis, 13/63 (20.6%); Streptococcus spp, 5/63 (7.9%); Acinetobacter spp and Pseudomonas spp 4/63 (6.3%). From culture-negative CSF, the pattern was different in that Staphylococcus spp (13/58, 22.4%), Neisseria meningitidis (9/58, 15.52%) and Pseudomonas spp (8/58, 14.79%), were the most frequent. Overall, 16S rRNA broad-range PCR combined with direct DNA sequencing is a valuable molecular tool to aid with the detection as well as identification of non-culturable aetiological agents of acute bacterial meningitis and can augment cultural methods in the diagnosis of central nervous system infections in patients who have been treated with antibiotics. However, this study demonstrates that contamination is an important complication of the molecular assay, which should be attempted to be eliminated through careful laboratory controls. Hence there should be careful interpretation of any molecular finding, in tandem with other laboratory findings, such as culture, immunological and biochemical markers, and the clinical scenario of the patient.     

Evaluation of the MicroSeq 500 16S rDNA-based gene sequencing for the diagnosis of culture-negative bacterial meningitis

Gene amplification using 16S rDNA primers has been proposed as a strategy for the diagnosis of bacterial meningitis. The aim of this study was to evaluate the performance of the MicroSeq 500 16S ribosomal DNA test (Applied Biosystems) from patients with suspected bacterial meningitis and CSF negative-culture in comparison to traditional methods. Twelve purulent culture-negative CSF samples were collected between January 2005 and January 2007. For DNA extraction, 500 microl of CSF samples were treated using the QIAamp mini kit (QIAGEN). The extracted DNA was examined amplifying 500 bp at the 5' end of 16S rRNA gene using MicroSeq500 16S rDNA Bacterial Identification PCR kit and the sequencing reactions were performed with the MicroSeq500 16S rDNA Bacterial Identification Sequencing kit (Applied Biosystems). The sequences were compared with those available in GenBank. For the culture-negative CSF samples the MicroSeq 500 16S rDNA yielded a positive result in 9 cases (75.0%): three samples were identified as Streptococcus. pneumoniae, three as Neisseria meningitidis, and the remaining 3 as Haemophilus influenzae, Abiotrophia defectiva and Porphyromonas gingivalis. The MicroSeq 500 16S ribosomal DNA test may improve the microbiological diagnosis of bacterial meningitis, especially when spinal fluid samples are obtained after the administration of antimicrobial therapy.     

Simultaneous amplification of multiple DNA fragments by polymerase chain reaction in the analysis of transgenic plants and their progeny

We describe the simultaneous amplification of different segments of foreign DNA in transgenic plants using the polymerase chain reaction (PCR). We used PCR to simultaneously amplify different regions of transformed T-DNA in order to assay the integrity of transformed constructions in primary tomato transformants. We also used simultaneous PCR amplification to examine the segregation of transformed sequences in progeny of primary transformants. A tomato transformant containing the maize transposable elementAc was crossed to transformants containing the non-autonomousDs1 element flanked by maizeAdh1 sequences. We then ran PCR reactions on DNA from F1 progeny using two sets of primers, one set homologous toAc and one set homologous toAdh1 sequences on either side ofDs1. Because theAc andAdh1 primers resulted in amplification of fragments of different sizes, it was possible to monitor the inheritance ofAc and theDs1 containingAdh1 genein a single reaction. Additionally, it was possible to identify F1 plants in whichDs1 had excised by the amplification of a fragment the size predicted for an empty donor site. In order to run these reactions, we have constructed a simple and inexpensive thermal cycler which, when used in conjunction with the rapid miniscreen plant DNA isolation procedure described, allows the processing of a large number of samples in a single day. Therefore, we have shown that PCR can be a useful tool to monitor the integrity of foreign genes in transgenic plants, to follow the segregation of foreign DNA in progeny, and to assay for the excision of transposable elements.     

Characterization of<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> var.<Emphasis Type="Italic">neoformans</Emphasis> serotype A and A/D in samples from Egypt

The cryptococcal polysaccharide antigen was detected in 10 cerebrospinal fluid (CSF) and 23 serum samples from cryptococcal meningitis and intestinal cryptococcosis by the cryptococcal antigen latex agglutination system (CALAS). CALAS titers in CSF and serum samples of cryptococcal meningitis ranged over 8-2048 and 32-2048, respectively, while in cases of intestinal cryptococcosis, serum titers ranged over 8-2048. The isolates of yeast Cryptococcus neoformans were determined to be of serotype A or of the A/D pair. The total leukocyte count and biochemical parameters in CSF were significantly increased as indicators of microbial infection. Furthermore, the in vitro change of the teleomorph (sexual state) to the anamorph (asexual state) was also detected and the teleomorph state changed in vivo to the encapsulated anamoph state which is more virulent during infection in vivo than the yeast-like noncapsulated form. Two primers for internal transcribed spacer (ITS) regions of ribosomal DNA were used for molecular detection of C. neoformans. After PCR amplification, a DNA band of 415 bp, visualized on agarose gel, indicated the presence of C. neoformans cells in the tested CSF and serum samples. The primer sensitivity was also characterized using purified yeast chromosomal DNA as template; it was about 20 pg or more chromosomal DNA which represents about 10 cells of C. neoformans. The primers were also specific for ITS regions of C. neoformans and gave negative results with Candida albicans and E. coli chromosomal DNA templates.     

Incorporation of Real-Time PCR into Routine Public Health Surveillance of Culture Negative Bacterial Meningitis in S?o Paulo,Brazil

Real-time (RT)-PCR increases diagnostic yield for bacterial meningitis and is ideal for incorporation into routine surveillance in a developing country. We validated a multiplex RT-PCR assay for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae in Brazil. Risk factors for being culture-negative, RT-PCR positive were determined. The sensitivity of RT-PCR in cerebrospinal fluid (CSF) was 100% (95% confidence limits, 96.0%–100%) for N. meningitidis, 97.8% (85.5%–99.9%) for S. pneumoniae, and 66.7% (9.4%–99.2%) for H. influenzae. Specificity ranged from 98.9% to 100%. Addition of RT-PCR to routine microbiologic methods increased the yield for detection of S. pneumoniae, N. meningitidis, and H. influenzae cases by 52%, 85%, and 20%, respectively. The main risk factor for being culture negative and RT-PCR positive was presence of antibiotic in CSF (odds ratio 12.2, 95% CI 5.9-25.0). RT-PCR using CSF was highly sensitive and specific and substantially added to measures of meningitis disease burden when incorporated into routine public health surveillance in Brazil.     

DNA probes for the Y-chromosome ofSilene latifolia,a dioecious angiosperm

Summary In order to obtain markers for the Y chromosome ofSilene latifolia, we pooled equal weights of leaf tissue from 18 female siblings into one sample and repeated the process with 18 male siblings. Pooling was intended to provide a common genetic background for each sample, leaving the absence or presence of the Y chromosome as the primary difference between the two samples. DNA was extracted from each sample and subjected to polymerase chain reaction (PCR) amplification with arbitrary 10 bp primers. Four of 60 primers used gave an amplification with the male DNA not found among those from the female DNA. Each of these was subsequently shown to provide a reliable marker for the Y chromosome.     

Galectin‐3 binds Neisseria meningitidis and increases interaction with phagocytic cells

Galectin‐3 is expressed and secreted by immune cells and has been implicated in multiple aspects of the inflammatory response. It is a glycan binding protein which can exert its functions within cells or exogenously by binding cell surface ligands, acting as a molecular bridge or activating signalling pathways. In addition, this lectin has been shown to bind to microorganisms. In this study we investigated the interaction between galectin‐3 and Neisseria meningitidis, an important extracellular human pathogen, which is a leading cause of septicaemia and meningitis. Immunohistochemical analysis indicated that galectin‐3 is expressed during meningococcal disease and colocalizes with bacterial colonies in infected tissues from patients. We show that galectin‐3 binds to N. meningitidis and we demonstrate that this interaction requiresfull‐length, intact lipopolysaccharide molecules. We found that neither exogenous nor endogenous galectin‐3 contributes to phagocytosis of N. meningitidis; instead exogenous galectin‐3 increases adhesion to monocytes and macrophages but not epithelial cells. Finally we used galectin‐3 deficient (Gal‐3^?/?) mice to evaluate the contribution of galectin‐3 to meningococcal bacteraemia. We found that Gal‐3^?/? mice had significantly lower levels of bacteraemia compared with wild‐type mice after challenge with live bacteria, indicating that galectin‐3 confers an advantage to N. meningitidis during systemic infection.