Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology

Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay for Mycobacterium tuberculosis DNA in order to rapidly diagnose tuberculous meningitis. This technique combined the high sensitivity of nested PCR with the accurate quantification of real-time PCR. In this study, we attempted to improve the original QNRT-PCR assay and newly developed the wide-range QNRT-PCR (WR-QNRT-PCR) assay, which is more accurate and has a wider detection range. For use as an internal-control "calibrator" to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. It had artificial random nucleotides in five regions annealing specific primers and probes. The NM-plasmid demonstrated statistically uniform amplifications (F = 1.086, P = 0.774) against a range (1 to 10(5)) of copy numbers of mimic M. tuberculosis DNA and was regarded as appropriate for use as a new internal control in the WR-QNRT-PSR assay. In addition, by the optimization of assay conditions in WR-QNRT-PCR, two-step amplification of target DNA was completely consistent with the standard curve of this assay. Due to the development of the NM-plasmid as the new internal control, significantly improved quantitative accuracy and a wider detection range were realized with the WR-QNRT-PCR assay. In the next study, we will try to use this novel assay method with actual clinical samples and examine its clinical usefulness.     

Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: clinical application for diagnosis of tuberculous meningitis

Although the "gold standard" for diagnosis of tuberculous meningitis (TBM) is bacterial isolation of Mycobacterium tuberculosis, there are still several complex issues. Recently, we developed an internally controlled novel wide-range quantitative nested real-time PCR (WR-QNRT-PCR) assay for M. tuberculosis DNA in order to rapidly diagnose TBM. For use as an internal control calibrator to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. Due to the development of the NM-plasmid, the WR-QNRT-PCR assay demonstrated statistically significant accuracy over a wide detection range (1 to 10(5) copies). In clinical applications, the WR-QNRT-PCR assay revealed sufficiently high sensitivity (95.8%) and specificity (100%) for 24 clinically suspected TBM patients. In conditional logistic regression analysis, a copy number of M. tuberculosis DNA (per 1 ml of cerebrospinal fluid) of >8,000 was an independent risk factor for poor prognosis for TBM (i.e., death) (odds ratio, 16.142; 95% confidence interval, 1.191 to 218.79; P value, 0.0365). In addition, the copy numbers demonstrated by analysis of variance statistically significant alterations (P < 0.01) during the clinical treatment course for 10 suspected TBM patients. In simple regression analysis, the significant correlation (R(2) = 0.597; P < 0.0001) was demonstrated between copy number and clinical stage of TBM. We consider the WR-QNRT-PCR assay to be a useful and advanced assay technique for assessing the clinical treatment course of TBM.     

Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) for detection of bovine ephemeral fever virus

A rapid, sensitive, and specific assay, RAPID-BAP assay, was developed to detect and quantify the G protein-encoding gene of bovine ephemeral fever virus (BEFV). This new technique uses a nested PCR and magnetic bead-based DNA probing assay. The optimal conditions for the assay were examined. By applying a nested PCR, a minimum of 1 copy/mul of the BEFV plasmid DNA could be detected by the assay. The optimal hybridization conditions at 50 degrees C in 5x SSC and 0.5% SDS with a 20-min incubation allowed clear discrimination between negative and positive controls. The assay was also highly specific as all negative controls failed to show any positive detection. The diagnostic sensitivity of the RAPID-BAP assay, real-time RT-PCR, and conventional RT-PCR in the detection of 34 clinical blood samples suspected to have BEFV infections were 72.73, 36.36, and 18.18%, respectively. The results indicated that the RAPID-BAP assay developed in this study was more sensitive than the conventional RT-PCR and real-time RT-PCR assays for the detection of BEFV. The novel RAPID-BAP assay is an excellent diagnostic tool with high sensitivity, specificity, and fast turnaround time.     

Loop-mediated isothermal amplification for rapid and reliable diagnosis of tuberculous meningitis

Diagnosis of tuberculous meningitis (TBM) is often difficult. A reliable, simple, and rapid diagnostic test that can be performed in any standard laboratory could be helpful in TBM diagnosis. In this study, a loop-mediated isothermal amplification assay (LAMP) was evaluated to rapidly detect and diagnose TBM infection and was compared to the performance of nested PCR. Six specific primers were used to recognize the IS6110 genomic sequence from Mycobacterium tuberculosis, which included one forward outer primer, one reverse outer primer, two respective inner primers, and two loop primers. The optimum reaction temperature and time were 63°C and 60 min, respectively. Nested PCR was performed targeting the IS6110 region from M. tuberculosis using a commercial kit. The LAMP method yielded a sensitivity of 88.23% and a specificity of 80%, compared to the nested-PCR assay, which yielded a sensitivity of 52.9% and a specificity of 90% for TBM diagnosis. Comparative experiments showed that the LAMP assay is a rapid, sensitive, and specific method to detect TBM infection and that it is superior to the nested-PCR assay. LAMP is very simple, and it can be performed in any laboratory and in rural settings.     

Comparative evaluation of early diagnosis of tuberculous meningitis by different assays

Cerebrospinal fluid (CSF) and peripheral blood (PBL) were sampled multiple times from 25 patients with a clinical diagnosis of tuberculous meningitis (TBM) and 49 controls, including 27 patients with other infectious diseases of the central nervous system and 22 patients with other noninfectious neurological diseases. We used an enzyme-linked immunospot assay (ELISPOT) to detect anti-Mycobacterium bovis BCG antibody-secreting cells in CSF and PBL, PCR to detect a repeated insertion sequence (IS6110) specific for Mycobacterium tuberculosis in CSF, and an enzyme-linked immunosorbent assay (ELISA) to detect anti-BCG antibodies in CSF and PBL. In the meantime, culture of CSF from every TBM and control patient was done on Lowenstein-Jensen medium. ELISPOT proved to be the most valuable test, with a sensitivity of 84.0% and a specificity of 91.8%, and showed a sensitivity of 100.0% with the CSF specimens obtained within 4 weeks after the onset of TBM. The numbers of CSF anti-BCG immunoglobulin-secreting cells tested by ELISPOT were even higher in the early phase of TBM and declined while the disease was going on (P = 0.008), which allowed an early diagnosis to be made. The sensitivities of PCR and ELISA were only 75.0% and 52.3%, respectively; and the specificities were 93.7% and 91.6%, respectively. Culture of CSF on Lowenstein-Jensen medium was the least sensitive (16%) compared to the sensitivities of the other three assays. Our results demonstrate that the ELISPOT technique is worthy for routine use in the laboratory to support the clinical diagnosis of TBM.     

Occurrence of overlooked zoonotic tuberculosis: detection of Mycobacterium bovis in human cerebrospinal fluid

The paucibacillary nature of the cerebrospinal fluid (CSF) has been a major obstacle in the diagnosis of human tuberculous meningitis (TBM). This study shows that with molecular techniques direct precise determination to the species level of mycobacterial pathogens can be made. The present report describes the utility of a nested PCR (N-PCR) assay (A. Mishra, A. Singhal, D. S. Chauhan, V. M. Katoch, K. Srivastava, S. S. Thakral, S. S. Bharadwaj, V. Sreenivas, and H. K. Prasad, J. Clin. Microbiol. 43:5670-5678, 2005) in detecting M. tuberculosis and M. bovis in human CSF. In 2.8% (6/212) of the samples, M. tuberculosis was detected, and in 17% (36/212), M. bovis was detected. Mixed infection was observed in 22 samples. Comparative analysis of clinical diagnosis, smear microscopy, and N-PCR in 69 patients (TBM, 25; non-TBM, 44) showed that the sensitivity of N-PCR (61.5%) was greater than that of smear microscopy (38.4%). Determination to the species level is important from the viewpoint of determining the prevalence of these mycobacteria in a community and would influence strategies currently adopted for the prevention of tuberculosis.     

Utility of the polymerase chain reaction in the diagnosis of tuberculous meningitis

Due to inconsistent clinical presentations and the lack of a rapid, sensitive and specific test, tuberculous meningitis (TBM) is particularly difficult to diagnose. The present study was carried out to determine the utility of the polymerase chain reaction (PCR) using INS primers in the diagnosis of TBM and to compare the efficacy of two different DNA extraction protocols. Fifty-seven cerebrospinal fluid (CSF) samples from suspected cases of meningitis -- 30 definitive/possible TBM and 27 non-TBM -- were processed for microscopy, culture and PCR. Results of computer tomographic (CT) scan findings were noted. The results of smear, culture and PCR were compared using culture and/or clinical response to treatment as the gold standard. The sensitivity of microscopy, culture, CT scan and PCR was 3.3%, 26.7%, 60.0% and 66.7%, respectively. PCR following QIAmp DNA extraction had a sensitivity of 66.7% compared to PCR following a DNA extraction protocol based on the use of cetyl trimethyl ammonium bromide (CTAB) (50%). PCR was positive in all culture-positive CSF samples using either extraction method. PCR is a rapid and sensitive technique; above all, it can diagnose tuberculous meningitis at a very early stage.     

Rapid detection of herpes simplex virus DNA in cerebrospinal fluid: comparison between loop-mediated isothermal amplification and real-time PCR

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency, and speed under isothermal conditions. To evaluate the usefulness of LAMP for diagnosing central nervous system infection with herpes simplex virus (HSV), we compared the LAMP method with real-time PCR, using samples that were previously tested by nested PCR. We examined 69 cerebrospinal fluid (CSF) samples from patients suspected of having HSV infection of the central nervous system. The results of the real-time PCR analysis and nested PCR assay were in complete accord. When nested PCR was regarded as standard, the sensitivity of LAMP was 81%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 90%. Although further improvement is necessary for the wide spread use, the LAMP method might be applicable to diagnosis of HSV infection of the central nervous system.     

Improved detection of JC virus DNA in cerebrospinal fluid for diagnosis of AIDS-related progressive multifocal leukoencephalopathy.

Several methods to increase the sensitivity of JC virus (JCV) DNA detection in cerebrospinal fluid (CSF) for a noninvasive diagnosis of AIDS-related progressive multifocal leukoencephalopathy (PML) were investigated. When CSF collected at clinical presentation was tested, JCV DNA was detected in 8 of 19 patients with PML by standard PCR (sensitivity, 42%; 95% confidence interval [CI], 21 to 66%) and in 14 of 19 by nested PCR (sensitivity, 74% [95% CI, 49 to 90%]; P = 0.014 [McNemar's test]. For multiple serial CSF samples, standard PCR yielded JCV DNA for 11 of 19 PML patients (sensitivity, 58% [95% CI, 34 to 79%]) and nested PCR yielded JCV DNA for 17 of 19 patients (sensitivity, 90% [95% CI, 66 to 98%]; P = 0.014). The majority of the false-negative samples were found to contain PCR inhibitors. Standard PCR did not detect JCV DNA in CSF from any of the 83 AIDS patients with other diagnosis (100% specificity [95% CI, 95 to 100%]); JCV DNA was found in CSF from one control patient by nested PCR (99% specificity [95% CI, 93 to 100%]).     

Evaluation of IS6110 as amplification target for direct tuberculosis diagnosis

We describe in the present study an evaluation of the IS6110 repetitive element in the rapid diagnosis of pulmonary and extrapulmonary tuberculosis by polymerase chain reaction (PCR). A pair of oligonucleotide primers was designed to amplify a 201-bp DNA fragment of IS6110. The amplified DNA was detected by ethidium bromide stained agarose gel electrophoresis and confirmed by Sal I digestion and Southern blot hybridization with a 32P-labeled probe. To detect the presence of amplification inhibitors, an internal control DNA that used the same primers as for the target sequence was added to each PCR reaction. PCR results were compared with the results of acid fast stained smears, cultures, and clinical data in 102 sputum and 41 extrapulmonary specimens. With the exception of four samples, M. tuberculosis was detected by PCR in all smear- and culture-positive cases and in all smear-negative, culture positive cases. Additionally, PCR was able to detect 6 cases that were smear and culture negative but clinically strongly suspected of tuberculosis. The final PCR sensitivity and specificity were 93.1% and 95.18%, respectively. One M. tuberculosis strain isolated from a sputum was found to lack IS6110. This study shows that (1) PCR diagnosis based on IS6110 reached the best sensitivity and specificity but must be considered carefully since some M. tuberculosis strains lack IS6110; and (2) PCR must be interpreted in conjunction with clinical and radiological data when it is discordant with conventional methods results.     

Comparative evaluation of BACTEC 460TB system and Lowenstein-Jensen medium for the isolation of M. tuberculosis from cerebrospinal fluid samples of tuberculous meningitis patients

PURPOSE: To evaluate the role of the radiometric BACTEC 460TB system and the conventional Lowenstein-Jensen (LJ) medium for isolation of M. tuberculosis from cerebrospinal fluid (CSF) samples of tuberculous meningitis (TBM) patients. METHODS: CSF specimens (n=2325) from suspected TBM patients were processed for isolation of mycobacteria by inoculating BACTEC 12B medium and the LJ medium. The isolation of mycobacteria in both media was confirmed by microscopy and biochemical identification. Drug sensitivity testing for the anti-TB drugs was carried out by BACTEC radiometric method. RESULTS: Among the total 2325 CSF specimens processed by both methods, M. tuberculosis was isolated from 256 specimens. The isolation rates were 93% and 39% for the BACTEC system and LJ medium respectively. Both the media supported growth in 32% of the culture-positive specimens. BACTEC system alone yielded growth in 61% and LJ alone in 7%, of the culture-positive specimens. Among 205 isolates tested for drug susceptibility 81% were sensitive to all the drugs tested and 19% were resistant. CONCLUSIONS: The BACTEC 460TB system provides a highly sensitive and rapid tool for the isolation and drug susceptibility testing of M. tuberculosis, from CSF of TBM patients. Use of a solid medium in conjunction with the BACTEC 12B medium is essential for optimal recovery for M. tuberculosis from CSF specimens.     

Simple and rapid detection of Mycobacterium tuberculosis complex organisms in bovine tissue samples by PCR.

Mycobacterium bovis is a slowly growing microorganism, and confirmation of the diagnosis by conventional culture is a lengthy process. A simple, rapid method for the extraction of DNA from bovine tissue samples was developed and used in a PCR designed for the diagnosis of tuberculosis. Tissues from 81 cattle from tuberculosis-infected herds (group 1) and 19 cattle from tuberculosis-free herds (group 2) were tested in this PCR, and the results were compared with those of conventional culture. The PCR assay detected 71.4% of the culture-positive animals from group 1. Tissue from all animals in group 2 were negative in the PCR assay and by culture. The described method could be used as a rapid screening technique which would be complementary to culture of tissue specimens for the routine diagnosis of bovine tuberculosis. The PCR technique is much faster than culture and reduces the time for diagnosis from several months to 2 days. It also provides for the detection of M. bovis when rapidly growing Mycobacterium spp. are present in the sample and may be able to detect the presence of M. bovis in samples even when organisms have become nonviable.     

A rapid and highly reliable field-based LAMP assay of canine parvovirus

Loop-mediated isothermal amplification (LAMP) is known as a rapid and reliable alternative to conventional single-step or nested PCR for detection of genomic DNA of various pathogens in clinical samples. In this study, LAMP assay was developed for canine parvovirus (CPV) and compared with single-step and nested PCR assays. Out of 50 fecal samples from dogs clinically suspected for CPV infections, 19 were found positive by single-step PCR, 22 by nested PCR and 26 by LAMP. LAMP products were subjected to restriction analysis and sequencing to check their specificity. LAMP assay turned out to be a rapid and fairly reproducible method, did not amplify other common canine pathogens and was more sensitive than nested PCR assay. Therefore, it can be regarded as a highly reliable method for routine field diagnosis of CPV infection. Keywords: canine parvovirus; nested polymerase chain reaction; loop-mediated isothermal amplification; sensitivity; specificity.     

Correlation between culture of Mycobacterium tuberculosis and detection of mycobacterial antigens in cerebrospinal fluid of patients with tuberculous meningitis

A retrospective study was done to correlate culture of Mycobacterium tuberculosis and detection of mycobacterial antigen in cerebrospinal fluid (CSF) by an inhibition enzyme-linked immunosorbent assay (ELISA). M. tuberculosis was cultured from CSF of 14 out of 70 patients with a clinical diagnosis of tuberculous meningitis (TBM). Mycobacterial antigens were demonstrated in CSF specimens by inhibition ELISA in all 14 culture-positive patients with antigen concentrations of 14.5-295 ng/ml (mean 158.8 ng/ml). Thus there was positive correlation between the detection of mycobacterial antigen and isolation of M. tuberculosis. Based on this observation, 56 CSF specimens from culture-negative patients with clinically diagnosed TBM were examined for mycobacterial antigen and the data were compared with those from culture positive patients. ELISA gave positive results in 38 specimens, with antigen levels of 12.5-280 ng/ml (mean 152.6 ng/ml). In 70 CSF specimens from patients with non-tuberculous neurological disease (control group), ELISA results were negative. Thus, detection of mycobacterial antigen in CSF specimens by inhibition ELISA had a specificity of 100% and a sensitivity of 67.8% for the diagnosis of TBM and is of potential value in the laboratory diagnosis of TBM.     

Improved diagnosis of mycobacterial infections in formalin-fixed and paraffin-embedded sections with nested polymerase chain reaction

Traditional histological diagnosis of mycobacterial infection in formalin-fixed and paraffin-embedded (FFPE) tissues is insensitive and poorly specific. To improve this, we developed nested polymerase chain reaction (PCR) protocols for detecting a Mycobacterium genus-specific 65-kDa heat shock protein (HSP65) sequence and the M. tuberculosis complex-specific insertion sequence IS6110 in FFPE sections. Protocols were optimized on tissues from 20 patients with a final clinical diagnosis of mycobacterial infection. Amplicons were controlled by sequencing and restriction endonuclease digestion. PCR could detect as few as three mycobacterial genomes per reaction. Assays showed 100% sensitivity and specificity for both M. tuberculosis complex and M. avium complex infection. Paraffin blocks from a second group of 26 patients with histological evidence of necrotizing granulomas of unknown etiology were then analyzed as a surrogate group to test the assay under conditions similar to those applying during routine diagnosis. Twenty-three of these blocks contained amplifiable DNA; nine were positive for M. tuberculosis complex DNA and four for other types of mycobacterial DNA. Furthermore, digestion of HSP65 amplicons with NarI could distinguish M. tuberculosis from M. avium complex. In conclusion, our nested PCR assays can be used as reliable tools for the detection of mycobacterial infections in FFPE tissues. The assays are simple and rapid to perform and show improved sensitivity and specificity compared to previously reported protocols.     

Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus

One-step SYBR Green I-based real-time RT-PCR assay for rapid detection as well as quantitation of Japanese encephalitis virus (JEV) in acute-phase patient CSF samples by targeting the NS3 gene was developed. The assay developed in this study was found to be more sensitive as compared to conventional RT-PCR. The specificity of the reported assay system was established through melting curve analysis as well as by cross-reactivity studies with related members of Flavivirus. The applicability of Real-time PCR assay for clinical diagnosis was validated with 32 suspected acute-phase CSF samples of Gorakhpur epidemic, India, 2005. The improved sensitivity of real-time RT-PCR was reflected by picking up 10 additional samples with low copy number of template in comparison to conventional RT-PCR. The quantitation of the viral load in acute-phase CSF samples was done using a standard curve obtained by plotting cycle threshold (C(t)) values versus copy numbers of the RNA template. This is the first report on the application of real-time RT-PCR for detection as well as quantitation of JEV from patient CSF samples. These findings demonstrate the potential clinical application of the reported assay as a sensitive diagnostic test for rapid and real-time detection and quantitation of JEV in acute-phase clinical samples.     

Detection of Aspergillus DNA in cerebrospinal fluid from patients with cerebral aspergillosis by a nested PCR assay

Invasive aspergillosis (IA), a complication with high mortality rates, especially in disseminated IA with cerebral involvement, is difficult to diagnose. Biopsy of cerebral lesions is often not feasible, and culture of Aspergillus spp. from cerebrospinal fluid (CSF) is frequently negative. New molecular methods have emerged for diagnosing IA. So far, there are only few reports of Aspergillus DNA detection in CSF. After modifying the DNA extraction protocol, we detected Aspergillus DNA in CSF samples by a previously described nested PCR assay. In six patients with hematologic malignancy and cerebral aspergillosis, CSF samples were investigated for Aspergillus DNA. IA was classified according to the EORTC/MSG 2002 criteria. Two patients each had proven, probable, and possible IA. Thirty-five CSF samples were investigated for Aspergillus DNA by nested PCR. Samples with positive results in the nested PCR assay were quantified by LightCycler PCR assay. Fourteen CSF samples showed positive results in the nested PCR assay. Of these, six samples gave positive results in real-time PCR. The range of CFU per ml was 2,154 to 63,100,000. The highest number of CFU per ml was found in a CSF sample of a patient with acute lymphocytic leukemia and probable cerebral aspergillosis. Detection of Aspergillus DNA in CSF samples is thus possible and has the potential to improve diagnosis of cerebral aspergillosis. Further prospective studies with larger numbers of patients must be performed to evaluate the clinical significance of Aspergillus PCR with CSF samples.     

Evaluation of a PCR-immunoassay technique for detection of Neisseria meningitidis in cerebrospinal fluid and peripheral blood

A multiplex PCR-immunoassay for the rapid diagnosis of bacterial meningitis from cerebrospinal fluid (CSF) or peripheral blood was compared with conventional microbiological techniques used in the routine diagnostic laboratory. The multiplex PCR was designed to detect simultaneously a universal conserved sequence coding for bacterial 16S rRNA and the Neisseria meningitidis porA gene. The PCR-immunoassay had a detection limit of (3-5) x 10(2) cfu/ml (equivalent to 1-3 cfu/PCR) with spiked CSF or blood samples, compared with (3-5) x 10(3) cfu/ml for PCR followed by conventional agarose gel electrophoresis for detection of PCR products. Of 294 CSF samples from patients suspected on clinical grounds of suffering from meningitis, the PCR-immunoassay detected bacterial DNA in 29 CSF samples, 15 of which were also positive for N. meningitidis DNA. The 29 positive CSF samples comprised 25 samples that were also reported positive and four that were reported negative by conventional methodology; the latter four were all positive for N. meningitidis by the PCR-immunoassay. Of 173 peripheral blood samples examined, the PCR-immunoassay detected bacterial DNA in 18 samples, 14 of which were also positive for N. meningitidis DNA. In comparison, only 10 samples were reported positive for N. meningitidis by conventional methodology. All negative PCR-immunoassay results correlated with those obtained by conventional methodology for both CSF and blood samples. The sensitivity and speed of the PCR-immunoassay system indicated that it could be used as a routine diagnostic test for meningococcal meningitis, enabling a diagnosis to be made within 4 h of receipt of the specimen.     

聚合酶链反应检测结核分枝杆菌DNA诊断关节结核的临床评价

目的评价聚合酶链反应(polymerase chain reaction,PCR)检测关节结核标本结核分枝杆菌脱氧核糖核酸(deoxyribonucleic acid,DNA)诊断关节结核的临床价值。方法对20份标准标本(5份结核分枝杆菌标准菌株、5份卡介苗和10份其它细菌标本)分别应用PCR盲法检测结核分枝杆菌DNA。对95例关节结核标本和98例非关节结核标本分别应用PCR检测结核分枝杆菌DNA。应用疾病诊断方法的临床评价原则对PCR诊断关节结核的敏感性、特异性和准确性进行评价。结果 (1)20份标准标本PCR检测中,结核分枝杆菌和卡介苗均为阳性,而其它细菌均为阴性;(2)95例关节结核标本中,PCR阳性78例、阴性17例;98例非关节结核标本中,PCR阳性9例、阴性89例;(3)PCR检测关节结核标本结核分枝杆菌DNA的敏感性为82.11%、特异性为90.81%、准确性为86.60%、阳性预测值为89.80%、阴性预测值为84.00%;(4)PCR整个检测过程自动化控制,可在3~6h内完成。结论 PCR是一种敏感、特异、快速、简便、无创、标本微量的关节结核标本结核分枝杆菌检测方法,对于关节结核的早期、快速诊断与鉴别诊断具有极其重要的临床价值。     

早期诊断结核性脑膜炎的新方法:检测浓缩脑脊液中CFP-10和ESAT-6

目的:应用酶联免疫吸附试验检测浓缩的脑脊液中结核分枝杆菌特异性抗原培养滤液蛋白10(CFP10)和6000早期分泌性抗原靶(ESAT-6),评价其在结核性脑膜炎(TBM)早期诊断中的价值。方法:应用ELISA测定46例临床诊断为TBM和56例非TBM患者脑脊液原液及经透析袋浓缩10倍的脑脊液浓缩液中CFP10和ESAT-6。结果:TBM患者脑脊液原液CFP10检测敏感度为13.04%、特异度为100.00%,ESAT-6的敏感度为13.04%、特异度为100.00%;而浓缩液CFP10的敏感度为78.26%、特异度为96.42%,ESAT-6的敏感度为76.09%、特异度为98.18%。结论:应用反透析方法浓缩脑脊液,使用抗CFP10和ESAT-6 ELISA检测脑脊液CFP10和ESAT-6能显著提高其敏感度,是一种简单、快速早期诊断结核性脑膜炎的有效辅助方法。