Jose C. Palomares

Direct Detection of Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis in Auramine-Rhodamine-Positive Sputum Specimens by Real-Time PCR

ABSTRACT Our objective was to evaluate the feasibility of a molecular assay based on a real-time PCR technique, carried out with a LightCycler instrument (Roche Biochemicals), to identify Mycobacterium tuberculosis bacilli and to detect rifampin and isoniazid resistance in DNA extracts from sputum samples. We studied three genes: rpoB, which is associated with rifampin resistance, and katG and inhA, which are associated with isoniazid resistance. A total of 205 sputum samples collected from 108 patients diagnosed with pulmonary tuberculosis with positive auramine-rhodamine-staining (AR) sputum samples, were tested. The sensitivities of the LightCycler PCR assay for the positive AR specimens was 97.5% (200 of 205) for rpoB and inhA genes and 96.5% (198 of 205) for the katG gene. For the total number of patients tested, the sensitivity was 100% (108 of 108 patients) for rifampin, whereas the sensitivity was 98.1% (106 of 108 patients) for isoniazid. Full agreement was found with the Bactec MGIT 960 method and the genotype inferred from the LightCycler data for rifampin. The phenotypic method for isoniazid reported 13 resistant strains (≥0.1 μg/ml). In seven (53.8%) strains there was a concordance between both methods, but we found that six (46.2%) strains reported as resistant by the phenotypic method were determined to be susceptible by real-time PCR. For the 75 strains reported as susceptible by the phenotypic method, the concordance with the LightCycler data was 100%. Our results demonstrate that rifampin-resistant M. tuberculosis could be detected in DNA extracted from auramine-rhodamine-positive sputum samples in a single-tube assay that took less than 3 h to perform for a collection of auramine-rhodamine-positive specimens obtained from patients with culture-documented pulmonary tuberculosis. Similarly, this occurs in half of the isoniazid-resistant M. tuberculosis DNA extracted from auramine-rhodamine-positive specimens.

Improved real-time PCR for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates

In this study we designed two pairs of probes for the detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis with real-time PCR procedures. One pair of probes spans the region between codon 510 and 528 of the rpoB gene, and the other one screens for mutation at the regulatory region of the inhA gene. We have evaluated these probes in combination with two other pairs of probes previously described to detect mutations in 20 susceptible and 53 unique resistant M. tuberculosis clinical isolates. We were able to detect nine different mutations affecting five codons of the rpoB gene, two different mutations at codon 315 of the katG gene and a nucleotide substitution (C209T) in the regulatory region of the inhA gene within two hours turnaround.

Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the detection and identification of organisms of clinical and epidemiologic importance. Staphylococcus aureus, one of the most frequent causes of human infections, was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. One hundred clinical isolates of S. aureus, verified by biochemical reactions and latex agglutination and 90 negative control clinical isolates were screened in the assay. Moreover, fifty blood broth samples from blood culture bottles showing Gram-positive cocci in clusters on direct Grams stain and 25 showing Gram-negative bacilli were screened. The probes, constructed from the nuc gene, correctly detected all S. aureus genomes present without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from other types of clinical specimens.

Activities of new quinoline derivatives against genital pathogens.

The in vitro activities of four quinoline carboxylic acids against 48 strains of Neisseria gonorrhoeae, 10 of Chlamydia trachomatis, and 32 of Ureaplasma urealyticum were compared. Ciprofloxacin was the most active against N. gonorrhoeae and C. trachomatis but had poor bactericidal activity against U. urealyticum, whereas ofloxacin showed the most bactericidal activity against U. urealyticum but was less active than ciprofloxacin against the two former pathogens. Norfloxacin and enoxacin were less active against all the studied pathogens.

Nosocomial transmission of tuberculosis infection in pediatrics wards

Analysis of restriction fragment length polymorphisms is a well‐established method of “DNA fingerprinting” that has been used to trace the transmission of particular strains of Mycobacterium tuberculosis during investigations of outbreaks. This report describe the use of restriction fragment length polymorphisms and arbitrarily primed polymerase chain reaction analysis to investigate two outbreaks of tuberculosis that affected six children who attended two pediatric wards in our hospital. In both outbreaks a history of household exposure to an adult with M. tuberculosis was obtained and suspected tuberculous contacts were identified. We have demonstrated unequivocally the strain relationship among the isolates in all the cases by restriction fragment length polymorphisms and arbitrarily primed polymerase chain reaction analysis. These techniques are very useful for performing epidemiologic studies of tuberculosis in children where natural history of tuberculosis infection is different from that in adults in that it is almost always primary infection rather than reactivation.

HPV Testing by cobas HPV Test in a Population from Catalonia

Background HPV testing in cervical cancer screening has been proposed as an alternative or complementary to cytology in women older than 30 years. However, adequate clinical sensitivity and specificity are crucial for a new test to be implemented. Hybrid Capture 2 (HC2) has proved good clinical performance in selecting women at risk for high-grade intraepithelial lesions with a high sensitivity and specificity. cobas HPV Test has been recently launched and its performance in different clinical settings needs to be determined. Objectives The aim of this study was to evaluate the cobas HPV Test for the detection of cervical HPV infection in a population of women in Catalonia (Spain) using HC2 as a reference. Materials and Methods Cervical liquid cytology samples from 958 women have been studied. Sensitivity was analyzed in 60 samples from patients with a high-grade intraepithelial lesion (≥CIN2) on histology and specificity was determined in 898 samples from women with no ≥CIN2. All cases had HC2 and cobas HPV Test performed. Statistical analyses of sensitivity, specificity and comparison between HC2 and cobas HPV Test by a non-inferiority test were applied. Results Sensitivity of HC2 and cobas HPV Test for detecting ≥CIN2 proved identical (98.3%) while specificity was 85.3% and 86.2% respectively. The non-inferiority test demonstrated that cobas HPV Test surpassed 90% sensitivity and 98% specificity of HC2. Conclusion The cobas HPV Test results fulfilled sensitivity and specificity requirements for HPV based cervical cancer screening and for the triage of minor cytological abnormalities, allowing its introduction in clinical settings.

Molecular detection and identification of Aspergillus spp. from clinical samples using real-time PCR.

The definite and rapid diagnosis of invasive aspergillosis is necessary because of the high mortality caused. The objective of this study was to evaluate a real‐time PCR assay to detect Aspergillus spp. in clinical samples, based on the Light Cycler technology. Specificity was assessed by using DNA extracted from pathogenic and non‐pathogenic bacteria/fungi from Spanish Collection including: two Aspergillus flavus, four Aspergillus fumigatus, two Aspergillus nidulans, two Aspergillus niger and two Aspergillus terreus isolates. The analytical sensitivity was evaluated with different inocula (101–105 conidia ml−1), and serially diluted DNA of A. fumigatus. To assess clinical applicability, samples from patients at risk were analysed. Species identification was determined by analysing the melting curves. Reactions using genomic DNA from other species of different genera than Aspergillus were negative (specificity: 100%). Analytical sensitivity was 60 fg using DNA and 5–20 conidia using conidial suspensions. The linear range was from 60 to 6 × 107 fg. The Tm ranged from 67.34 to 70.7 °C for the different Aspergillus spp. studied. Nine hundred and forty‐eight consecutive blood samples from 127 patients were processed. In total, 10 (1%) of 948 samples from blood samples were PCR‐positive. The real‐time PCR assay provides a high sensitivity and specificity for detection of fungal DNA and rapidly identifies most of clinically relevant Aspergillus species.

Comparison of the Cobas 4800 Human Papillomavirus test against a combination of the Amplicor Human Papillomavirus and the Linear Array tests for detection of HPV types 16 and 18 in cervical samples.

The greater prevalence of human papillomavirus (HPV) types 16 and 18 compared to the other high-risk HPV types of cervical cancer led to the development of clinical tests that detect both types separately from other genotypes. One method is the Roche Cobas 4800 HPV test, which is based on a real-time PCR. The aim of this study was to evaluate the performance of the Cobas 4800 HPV test for detecting genotypes 16 and 18 by comparing the results with those obtained in a combination of the Roche Amplicor HPV assay and the Roche Linear Array (LA) HPV genotyping assay. Excellent concordance was found between both methods (92.7%, kappa value=0.872). The Cobas 4800 HPV test could be used as a single test for identifying HPV types 16 and 18 directly from clinical specimens.

Evaluation of the cobas 4800 CT/NG Test for detecting Chlamydia trachomatis and Neisseria gonorrhoeae DNA in urogenital swabs and urine specimens

We have evaluated 696 samples (488 swabs and 208 urine specimens) with the cobas 4800 (c4800) CT/NG Test for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae DNA in swab and urine specimens. c4800 results were compared with those obtained from COBAS AMPLICOR (CAM) CT/NG Test. Discordant results were reanalyzed with the MultiNA system and compared with clinical data. For C. trachomatis detection by both methods, we obtained 93.8%, 100%, 100%, and 99.1% for sensitivity, specificity, and positive and negative predictive values, respectively. For urine specimens analyzed in c4800, our results were 96.6%, 100%, 100%, and 99.4%, respectively. For N. gonorrhoeae detection, swab results were:88.0%, 100%, 100%, and 99.4%. For urine specimen, results obtained were 100%, 100%, 100%, and 100%. Reanalyses were all concordant between both methods. c4800 results were comparable with those obtained with the CAM system. We had an excellent correlation between swab and urine specimens analyzed by c4800.

Comparison of urine and cervical samples for detecting human papillomavirus (HPV) with the Cobas 4800 HPV test

BACKGROUND Human papillomavirus (HPV) is the main cause of cervical cancer. The development of non-invasive self-sample collection methods would have the potential advantage of increasing the acceptance of the screening procedures. OBJECTIVES To compare human papillomavirus (HPV) DNA detection and genotyping with the Cobas 4800 HPV test (Roche Diagnostic, Spain) on paired cervical and first voided urine. STUDY DESIGN Paired urine and cervical samples were collected from 125 women referred for evaluation of abnormal Pap smear results. RESULTS The overall percent agreement between HPV detection in urine and cervical samples was 88%. A substantial concordance rate of HPV DNA detection in both samples was observed (κ=0.76; 95% IC: 64-87). In this high prevalence population the sensitivity, specificity, NPV and PPV for detection of HPV DNA from urine versus cervical samples were 90.5% (95% IC: 80-95%), 85%, (95% IC: 74-92%), 89.8% (95% IC: 79.5-95.3) and 86.4% (95% IC: 76.1-92.7) respectively. Compared to histologically confirmed CIN 2/3 disease, the clinical sensitivity and specificity for the detection of high-risk HPV in urine samples were 95% (95% IC: 76-97%) and 52.4% (95% IC: 40-64%) respectively. CONCLUSIONS These results suggest that urine samples processed with Cobas 4800 HPV test may be useful for clinical management of HPV infection.