Use of a multiplex PCR-based reverse line blot (mPCR/RLB) hybridisation assay for the rapid identification of bacterial pathogens.

The aim of this study was to develop a sensitive and reliable method for the molecular identification of pathogenic bacteria. A multiplex PCR-based reverse line blot (mPCR/RLB) hybridisation assay was developed and evaluated for the rapid identification of 24 systemic and respiratory bacterial pathogens in routine diagnosis. All species-specific probes designed for the RLB hybridised with amplified DNA only from the corresponding species. Sensitivity limits of the mPCR/RLB assay varied among the 24 target organisms from 0.05 pg to 0.5 ng of genomic DNA. The sensitivity of the assay was 2 x 10(2) CFU/mL for Streptococcus pneumoniae and 6 x 10(2) CFU/mL for Escherichia coli. The specificity of each probe was tested against 24 species. There were no cross-reactions among any of the 43 probes. The mPCR/RLB assay appeared to be a useful alternative tool for the molecular identification of common pathogens.     

Development and evaluation of a line probe assay for rapid identification of pncA mutations in pyrazinamide-resistant mycobacterium tuberculosis strains

Resistance of Mycobacterium tuberculosis to pyrazinamide (PZA) derives mainly from mutations in the pncA gene. We developed a reverse hybridization-based line probe assay with oligonucleotide probes designed to detect mutations in pncA. The detection of PZA resistance was evaluated in 258 clinical isolates of M. tuberculosis. The sensitivity and specificity of PZA resistance obtained by this new assay were both 100%, consistent with the results of conventional PZA susceptibility testing. This assay can be used with sputa from tuberculosis patients. It appears to be reliable and widely applicable and, given its simplicity and rapid performance, will be a valuable tool for diagnostic use.     

Development of a DNA microarray for detection and identification of fungal pathogens involved in invasive mycoses

Invasive fungal infections have emerged as a major cause of morbidity and mortality in immunocompromised patients. Conventional identification of pathogenic fungi in clinical microbiology laboratories is time-consuming and, therefore, often imperfect for the early initiation of an adequate antifungal therapy. We developed a diagnostic microarray for the rapid and simultaneous identification of the 12 most common pathogenic Candida and Aspergillus species. Oligonucleotide probes were designed by exploiting the sequence variations of the internal transcribed spacer (ITS) regions of the rRNA gene cassette to identify Candida albicans, Candida dubliniensis, Candida krusei, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida lusitaniae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus. By using universal fungal primers (ITS 1 and ITS 4) directed toward conserved regions of the 18S and 28S rRNA genes, respectively, the fungal ITS target regions could be simultaneously amplified and fluorescently labeled. To establish the system, 12 pre-characterized fungal strains were analyzed; and the method was validated by using 21 clinical isolates as blinded samples. As the microarray was able to detect and clearly identify the fungal pathogens within 4 h after DNA extraction, this system offers an interesting potential for clinical microbiology laboratories.     

Evaluation of a multiplex PCR-based reverse line blot-hybridization assay for identification of serotype and surface protein antigens of Streptococcus agalactiae

A 33-primer multiplex PCR-based reverse line blot (mPCR/RLB) assay was developed to identify Streptococcus agalactiae serotypes and surface protein antigen genes simultaneously. It was evaluated by using 551 clinical isolates. The mPCR/RLB assay was more sensitive than conventional serotyping, especially for protein antigen typing, but otherwise the results correlated well.     

Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.

Human papillomavirus (HPV) can be detected by amplification of viral DNA. A novel PCR primer set generating a short PCR fragment (SPF PCR) was used for amplification of a fragment of only 65 bp from the L1 region and permitted ultrasensitive detection of a broad spectrum of HPV genotypes. The intra- and intertypic sequence variations of the 22-bp interprimer region of this amplimer were studied. Among 238 HPV sequences from GenBank and clinical specimens, HPV genotypes were correctly identified based on the 22-bp sequence in 232 cases (97.2%). Genotype-specific probes for HPV genotypes 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, 51 to 54, 56, 58, 59, 66, 68, 70, and 74 were selected, and a reverse hybridization line probe assay (LiPA) (the INNO-LiPA HPV prototype research assay) was developed. This LiPA permits the use of amplimers generated by the SPF as well as the MY 09/11 primers. The assay was evaluated with a total of 1, 354 clinical specimens, comprising cervical scrapes (classifications ranging from normal cytology to severe dyskaryosis) and formalin-fixed, paraffin-embedded cervical carcinoma samples. LiPA results were highly concordant with sequence analysis of the SPF amplimer, genotype-specific PCR, and sequence analysis of amplimers generated by MY 09/11 primers. The sensitivity of the SPF primers was higher than that of the GP5(+)/6(+) primers over a broad range of HPV types, especially when multiple HPV genotypes were present. In conclusion, the SPF LiPA method allows extremely sensitive detection of HPV DNA as well as reliable identification of HPV genotypes in both cervical smears and paraffin-embedded materials.     

Use of a commercial PCR-based line blot method for identification of bacterial pathogens and the mecA and van genes from BacTAlert blood culture bottles

In this study, the PCR-based DNA strip assay GenoType BC for the identification of bacteria and the resistance genes mecA, vanA, vanB, vanC1, and vanC2/3 directly from positive BacTAlert blood culture bottles was evaluated in a multicenter study. Of a total of 511 positive blood cultures, correct identification percentages for Gram-negative bacteria, Gram-positive bacteria, and the mecA gene were 96.1%, 89.9%, and 92.9%, respectively. Results were available 4 h after growth detection.     

Evaluation of a commercial DNA probe assay for the identification ofMycobacterium kansasii

A commercially available DNA probe (the Accu-ProbeMycobacterium kansasii culture identification test, Gen-Probe, USA) for the identification ofMycobacterium kansasii was tested on a panel of 143 fully characterized mycobacterial strains. The isolates included 70Mycobacterium kansasii and 73 mycobacteria other thankansasii. The specificity was 100 % while the sensitivity was 72.8 %. This sensitivity is unusually low in comparison with that of commercial DNA probes for other mycobacteria and confirms a previous study that found genetic heterogeneity within the speciesMycobacterium kansasii. Strains that do not hybridize with the AccuProbe are particularly prevalent in Italy and perhaps elsewhere in Europe.     

Evaluation of a chemiluminescent probe assay for identification of Histoplasma capsulatum isolates.

Fifty-three of 54 isolates of fungi were correctly identified with an acridinium ester-labelled probe for Histoplasma capsulatum (Accuprobe; Gen-Probe, San Diego, Calif.). One isolate of Aspergillus niger was incorrectly identified as H. capsulatum. Age of the culture, medium for isolation, and morphologic state did not affect the results.     

Use of a panfungal PCR assay for detection of fungal pathogens in a commercial blood culture system

A panfungal PCR assay was used to evaluate the ability of the ESP blood culture system to detect fungemia. The results showed that the ESP system is reliable for the detection of fungi and showed the applicability of using a molecular-based assay as a potential rapid and reliable method for the identification of fungi.     

Evaluation of a commercial exoantigen test system for the rapid identification of systemic fungal pathogens

Seventy-nine mycelial-form stock cultures of Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, and morphologically similar fungi were extracted and tested by using commercial macroimmunodiffusion exoantigen test kits and the Centers for Disease Control (CDC) reference system for identifying fungal isolates. Results showed 100% correlation between the two systems. Specific exoantigens of C. immitis and H. capsulatum extracted from agar slant cultures (slant extraction method) readily were identified. In eight of 26 cultures of B. dermatitidis, broth culture filtrates (broth-shake-flask method) were required to demonstrate the specific bands of identity. No false-negative reactions or cross-reactivity among the pathogens and other fungi were observed. The commercial test kits provided a rapid and specific method for identifying or confirming suspected fungal pathogens.     

Comprehensive multicenter evaluation of a new line probe assay kit for identification of Mycobacterium species and detection of drug-resistant Mycobacterium tuberculosis

We evaluated a new line probe assay (LiPA) kit to identify Mycobacterium species and to detect mutations related to drug resistance in Mycobacterium tuberculosis. A total of 554 clinical isolates of Mycobacterium tuberculosis (n = 316), Mycobacterium avium (n = 71), Mycobacterium intracellulare (n = 51), Mycobacterium kansasii (n = 54), and other Mycobacterium species (n = 62) were tested with the LiPA kit in six hospitals. The LiPA kit was also used to directly test 163 sputum specimens. The results of LiPA identification of Mycobacterium species in clinical isolates were almost identical to those of conventional methods. Compared with standard drug susceptibility testing results for the clinical isolates, LiPA showed a sensitivity and specificity of 98.9% and 97.3%, respectively, for detecting rifampin (RIF)-resistant clinical isolates; 90.6% and 100%, respectively, for isoniazid (INH) resistance; 89.7% and 96.0%, respectively, for pyrazinamide (PZA) resistance; and 93.0% and 100%, respectively, for levofloxacin (LVX) resistance. The LiPA kit could detect target species directly in sputum specimens, with a sensitivity of 85.6%. Its sensitivity and specificity for detecting RIF-, PZA-, and LVX-resistant isolates in the sputum specimens were both 100%, and those for detecting INH-resistant isolates were 75.0% and 92.9%, respectively. The kit was able to identify mycobacterial bacilli at the species level, as well as drug-resistant phenotypes, with a high sensitivity and specificity.     

Evaluation of a line probe assay for identification of hepatitis B virus precore variants in serum from chronic hepatitis B carriers

A prototype line probe assay (LiPA) for identifying hepatitis B virus (HBV) precore variants (INNO-LiPA HBV precore) was evaluated using a panel of 50 sera from 46 patients with HBV infection. The assay detected sequence variations detected commonly in the precore promoter region and in amino acid codons 28 and 29 of the precore gene. There was strong agreement between INNO-LiPA HBV precore results and those of a codon 28 point mutation assay (PMA), with identical results obtained in 40 of 43 sera (93%) typeable by both assays (kappa coefficient (kappa)=0.90). In addition, the precore codon 29 sequence identified by the INNO-LiPA HBV precore was confirmed by nucleotide sequencing in all seven samples analysed. However, the INNO-LiPA HBV precore identified precore promoter sequences much less efficiently. The prototype assay could identify codon 28/29 sequences from as little as 10 HBV genome equivalents in 10 microl serum, and in experiments using artificially prepared mixtures of variants could identify a minor component constituting 2.5% of the total viral DNA population. The INNO-LiPA HBV precore was also straightforward technically and rapid, and is therefore likely to be useful for epidemiological investigations into the prevalence, distribution and clinical significance of HBV precore variants.     

Second-generation line probe assay for hepatitis C virus genotyping.

Because of the enormous variability of hepatitis C virus (HCV), the development of reliable genotyping assays is a formidable challenge. The optimal genotyping region appears to be the 5' untranslated region (UR) because of high conservation within, but considerable variability between, genotypes. In this study, 21 probes dispersed over seven variable 5' UR areas were applied to a line probe assay (LiPA) and used to analyze 506 HCV-infected sera from different geographical regions representing a multitude of subtypes. At least 31 different reactivity patterns emerged, with 404 (80%) of 506 distributed over 11 prototype patterns, in general corresponding to subtypes 1a, 1b, 2a/2c, 2b, 3a, 5a, and 6a and several type 4 subtypes. Subtyping specificity ranged from 97% in Hong Kong to 90% in Europe but was only 11% in West Africa, while typing specificity was always 100% when samples from Vietnam were excluded. In a second evaluation, the subtype prediction by LiPA of 448 GenBank 5' UR HCV sequences was scored. Of the 58 theoretically predicted patterns, 321 sequences (72%) were covered by the 11 prototype patterns. We concluded that (i) the selected probes detected the corresponding signature motifs in the seven variable regions with 100% reliability; (ii) these motifs allowed correct type interpretation of samples collected worldwide, with the exclusion of Vietnam, Thailand, or Vietnamese patients residing in European hospitals; and (iii) subtyping specificities vary according to geographical region, with 11 prototype subtyping patterns identifying the majority of samples from Europe and the Americas. These results indicate that the LiPA is a reliable assay applicable to routine typing and subtyping of HCV specimens.     

Evaluation of a novel PCR-based assay for detection and identification of Chlamydia trachomatis serovars in cervical specimens

The aims of this study were to compare a novel PCR-based Chlamydia trachomatis detection and genotyping (Ct-DT) assay with the FDA-approved, commercially available C. trachomatis detection Hybrid Capture 2 (HC2) assay and to investigate the C. trachomatis serovar distribution among young women in a rural Costa Rican study population. A total of 5,828 sexually active women participating in a community-based trial in Costa Rica were tested for C. trachomatis by HC2. A sample of 1,229 specimens consisting of 100% HC2 C. trachomatis-positive specimens (n = 827) and a random sample of 8% HC2 C. trachomatis-negative specimens (n = 402) were tested with the Ct-DT assay. Agreement between the two assays was determined by the unweighted kappa statistic. Discrepant specimens were tested with a second commercially available test (COBAS TaqMan). The Ct-DT-positive specimens were further analyzed with the Ct-DT genotyping step to investigate the distribution of 14 different C. trachomatis serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3). After accounting for the sampling fraction selected for Ct-DT testing, crude agreement with the HC2 assay was 98% and the kappa was 0.92 (95% confidence interval [CI], 0.89 to 0.97). The 33 discordant samples that were further analyzed with the COBAS TaqMan test showed better agreement with the Ct-DT assay (31/33, P < 0.001). Among the 806 Ct-DT-positive samples, serovar E was the most common serovar (31%), followed by serovars F and D (both 21%) and serovar I (15%). In conclusion, the novel Ct-DT assay permits reliable detection and identification of C. trachomatis serovars.     

基因芯片技术检测鉴定临床常见致病真菌的初步研究

目的为了快速、简便、高通量地鉴定临床常见致病真菌,建立了一种采用基因芯片技术对临床常见的致病真菌鉴定的分子生物学方法。方法以5.8S rDNA与28S rDNA间的内转录间区2(internal transcribed spacer-2,ITS-2)为靶标,针对待检的临床常见致病真菌设计合成一系列寡核苷酸探针,制成寡核苷酸芯片。待检真菌DNA经通用引物扩增标记后,与芯片杂交,对杂交图谱分析归纳,得到一套种特异性的典型杂交图谱。待检的样品菌与基因芯片杂交,得到的杂交结果与典型图谱比对即可判断出样品的种类。结果以涉及8个属20个种的标准致病真菌菌株对芯片的特异性、重复性、灵敏度进行考察,结果表明,该研究建立的基因芯片技术可以有效地区分20种临床常见致病真菌,特异性良好,重复性良好(信噪比CV<10%),灵敏度为15 pg/ml真菌DNA。收集从临床分离的84株致病真菌菌株对基因芯片进行试用,结果显示基因芯片的鉴定结果与常规鉴定方法的鉴定结果一致。结论这项技术的建立可以稳定、特异性地实现临床常见致病真菌的高通量鉴定,为进一步检测研究奠定了基础。     

Evaluation of multiplexed PCR and liquid-phase array for identification of respiratory fungal pathogens

Invasive fungal infections are the cause of serious morbidity and high mortality in immunocompromised patients. Early laboratory diagnostic options remain limited; however, rapid detection and accurate identification may improve outcome. Herein, multiplexed PCR followed by liquid-phase array was evaluated for detection and identification of common respiratory fungal pathogens, including Aspergillus fumigatus, Rhizopus microsporus, Scedosporium apiospermum and Fusarium solani. The limit of detection ranged 0.1-1 ng of DNA, depending on the fungus being tested. Primer cross-reactivity was seen for some fungi: Aspergillus flavus primers detected Aspergillus oryzae; Scedosporium apiospermum primers detected Paecilomyces lilacinus, and Aspergillus terreus primers detected S. apiospermum. PCR followed by liquid-phase array is potentially useful for the identification of clinically relevant fungal pathogens.     

Performance assessment of new multiplex probe assay for identification of mycobacteria

A new DNA probe assay (INNO LiPA Mycobacteria; Innogenetics, Ghent, Belgium) for the simultaneous identification, by means of reverse hybridization and line-probe technology, of Mycobacterium tuberculosis complex, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium gordonae, the species of the Mycobacterium avium complex (MAC), Mycobacterium scrofulaceum, and Mycobacterium chelonae was evaluated on a panel of 238 strains including, besides representatives of all the taxa identifiable by the system, a number of other mycobacteria, some of which are known to be problematic with the only other commercial DNA probe system (AccuProbe; Gen-Probe, San Diego, Calif.), and two nocardiae. The new kit, which includes a control probe reacting with the whole genus Mycobacterium, correctly identified 99.6% of the strains tested; the one discrepancy, which remained unresolved, concerned an isolate identified as MAC intermediate by INNO LiPA Mycobacteria and as Mycobacterium intracellulare by AccuProbe. In five cases, because of an imperfect checking of hybridization temperature, a very slight, nonspecific, line was visible which was no longer evident when the test was repeated. Two strains whose DNA failed amplification at the first attempt were regularly identified when the test was repeated. Interestingly, the novel kit dodged all the pitfalls presented by the strains giving anomalous reactions with AccuProbe. A unique feature of INNO LiPA Mycobacteria is its ability to recognize different subgroups within the species M. kansasii and M. chelonae, while the declared overlapping reactivity of probe 4 with some M. kansasii and Mycobacterium gastri organisms and of probe 9 with MAC, Mycobacterium haemophilum, and Mycobacterium malmoense, may furnish a useful aid for their identification. The turnaround time of the method is approximately 6 h, including a preliminary PCR amplification.     

Development of PCR-based hybridization protocol for identification of streptococcal species.

16S rRNA of Streptococcus agalactiae, S. uberis, and S. parauberis was bound to streptavidin-coated magnetic beads by using a biotinylated oligonucleotide probe complementary to a highly conserved region of the molecule. In-solution hybridization of radiolabelled oligonucleotide probes to immobilized 16S rRNA allowed the specific identification of S. agalactiae and S. parauberis but not S. uberis. PCR was used to amplify a species-specific region of the 16S rRNA gene from these species. One of the PCR primers was biotinylated at the 5' end to allow purification of the amplified product on streptavidin-coated magnetic beads and subsequent denaturation to yield immobilized single-stranded DNA. Radiolabelled oligonucleotide probes were hybridized in solution to the single-stranded target molecule and enabled species-specific identification of the target organism. This protocol overcame problems associated with hybridization of the S. uberis-specific probe to 16S rRNA in solution. A similar procedure may enable the specific detection of other streptococci which exhibit a species-specific sequence in this region of the gene.