Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization

A polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary tuberculosis was developed by using oligonucleotide primers to amplify a fragment of IS6110, an insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis. Sediment obtained from sputa processed by the N-acetyl-L-cysteine-NaOH method was suspended in a simple lysis buffer and was heated at 100 degrees C for 30 min prior to amplification. A dUTP-uracil N-glycosylase PCR protocol was used to prevent false-positive test results because of the carryover of products from previous amplification reactions. The 317-bp amplicon was detected by direct gel analysis and Southern blotting and then hybridization with a biotin-labeled internal probe. Hybrid molecules were detected by using a commercially available avidin-alkaline phosphatase-chemiluminescent substrate system (Tropix, Inc., Bedford, Mass.). The analytical sensitivity of the assay was 10 fg of purified mycobacterial DNA. The limits of detection by culture (Middlebrook 7H11 agar and Lowenstein-Jensen medium) and by PCR were equivalent in terminal dilution experiments for organism suspensions and positive sputa. An internal control was used to detect the presence of amplification inhibitors in each negative reaction mixture. DNA was purified from inhibitory specimens by phenol-chloroform extraction and ethanol precipitation. PCR results were compared with results of microscopy and conventional culture for the detection of M. tuberculosis in 313 sputum specimens. There were 124 specimens that were positive for M. tuberculosis by conventional methods and 113 (91%) that were positive by PCR. PCR detected 105 of 110 (95%) of the smear-positive and 8 of 14 (57%) of the smear-negative specimens. There were no false-positive results by PCR (specificity, 100%). This PCR assay innovations that make application of this new technology feasible in clinical microbiology laboratories.     

Direct in situ reverse transcriptase polymerase chain reaction for detection of measles virus

Behavioral contexts can evoke a variety of autonomic modes of response, characterized by reciprocal, coactive, or independent changes in the autonomic divisions. The present study investigated the modes of autonomic response to visual illusion and mental arithmetic tasks, by the use of noninvasive measures of sympathetic (pre-ejection period; PEP) and parasympathetic (respiratory sinus arrhythmia; RSA) cardiac control. As previously demonstrated, mental arithmetic was associated with a reciprocal pattern of sympathetic activation and vagal withdrawal. The illusion task, however, yielded a distinct mode of vagal activation in the absence of sympathetic change. Responses within tasks were reliable. In contrast to the general intertask consistency reported for stress tasks that yield similar autonomic modes of response, however, neither PEP nor RSA responses were correlated across the illusion and arithmetic tasks. This may be attributable to the dissimilar modes of autonomic control evoked by these tasks. The distinct modes of autonomic response to arithmetic and illusions emphasize the importance of a bivariate model of autonomic control, and may offer important experimental tools for psychophysiological studies of autonomic control.     

GBV-RNA detection by polymerase chain reaction with several primer pairs

The identification of specific genomic sequences of GB viruses (GBV) has made it possible to utilize the polymerase chain reaction (PCR) for evaluation of the viraemia. Several studies have demonstrated the RNA-GBV presence in sera from different patients amplifying several portions of the virus. In this investigation the PCR results when different regions of GBV (NS3, UTR and putative CORE and E1) were amplified in the same sample. In 245 samples studied there were two (0.8%) discordant results and the NS3 primer showed the greatest sensitivity. The lowest percentage of positivity was obtained with CORE-E1 primers. These results could be because the nucleocapside/E1 region was extremely variable in length and sequences, although degenerated primers and probes were used. Discordances were attributable to laboratory errors, variability in the viral genome, the presence of primer inhibitors in samples or a low viral load.     

Effect of long-term therapies with penicillin and sulfadiazine on Streptococcus mutans and lactobaccilli in dental plaque

Plaque samples were obtained from 13 children receiving long-term therapy with benzathine penicillin for the prevention of rheumatic fever recurrences, 31 children receiving oral sulfadiazine for the same purpose, and 29 untreated siblings. The therapies were found to have no effect upon the proportions of Streptococcus mutans or lactobacilli in dental plaque, upon the percentage of children harboring the organisms, nor upon the susceptibility of the organisms to penicillin and sulfadiazine. Of the S. mutans strains tested, 97% had a minimal inhibitory concentration of penicillin G of less than 48 ng/ml and, of the lactobacillus strains tested, 96.8% had a minimal inhibitory concentration of less than 1,600 ng/ml. All strains of both organisms were profoundly resistant to sulfadiazine.     

Direct detection of Mycobacterium tuberculosis using polymerase chain reaction assay among patients with hepatic granuloma

BACKGROUND: In liver tuberculosis, demonstration of acid bacilli by conventional methods remains futile. Since the definitive diagnosis of liver tuberculosis is based on the histologic evidence of granulomatous process with caseation necrosis, seen in only a third of cases, the diagnosis is made retrospectively by response to empirical anti-tuberculous drug therapy. AIMS: Our objective is to establish a polymerase chain reaction assay for detection of Mycobacterium tuberculosis affecting the liver using the paraffin-embedded liver biopsy specimens obtained from patients with hepatic granulomas. METHODS: As positive control, patients having either "definitive" (n=8) or "presumptive" (n=9) tuberculosis on the basis of clinical, microbiological, histologic data and their positive response to empirical treatment of anti-tuberculous drugs were used. Patients with hepatic granulomas secondary to schistosomiasis (n=6), sarcoidosis (n=2) and other liver diseases (n=10) were used as negative control. RESULTS: Of those patients who were diagnosed as having "definitive" and "presumptive" liver tuberculosis, positivity by one-step polymerase chain reaction was 100% and 44%, respectively. Using the nested polymerase chain reaction, positivity increased to 78% with "presumptive" liver tuberculosis. In contrast, the polymerase chain reaction assays were negative among all patients with hepatic granuloma due to non-tuberculous-in-origin and other liver diseases. CONCLUSIONS: The overall positivity of this polymerase chain reaction assay (88%) compares favorably with that of other conventional methods (12%). Thus, this polymerase chain reaction assay may be a reliable diagnostic tool for liver tuberculosis in a patient population in which the prevalence of diseases associated with hepatic granuloma is common.     

Mycoplasma pneumoniae detection from throat swab by two-step polymerase chain reaction

Two-step polymerase chain reaction (PCR) with primers designated against 16S rRNA gene of Mycoplasma pneumoniae for diagnosis of infection was evaluated in comparison with the conventional single-step PCR and culture methods. The two-step PCR method showed specific amplification of M. pneumoniae DNA and higher sensitivity (1.5 fg/assay) than the single-step PCR method. With the two-step PCR method, 76 of 322 throat swabs (23.6%) from patients with acute respiratory complaints gave positive results whereas 20.2% were positive in the culture method. Seven of 13 samples which were negative in the single-step PCR method but positive in either serological or the culture method showed positive results by the two-step PCR method. In addition, 5 samples which were weakly positive in the single-step PCR method showed distinctly positive results in the two-step PCR. These results indicate that the two-step PCR method is a useful tool for detection of M. pneumoniae in clinical specimens, although it requires a relatively sophisticated in technique.     

Rapid detection and typing of human papillomaviruses by consensus polymerase chain reaction and enzyme-linked immunosorbent assay

This long-term prospective study evaluates the clinical results of subsequent laminectomy in 103 consecutive patients who initially underwent chemonucleolysis (CNL) or laminectomy for lumbar disc herniation. Between 1981 and 1994, 53 patients who had received CNL initially and then underwent laminectomy and 50 patients treated initially with laminectomy underwent a repeat laminectomy. Clinical assessment at 6 weeks showed a success rate of 80.8% for post-CNL laminectomy and 78% for repeat laminectomy. At 6 months, the success rate for patients treated with CNL was 86% versus 78.7% for laminectomy. At 12 months, the overall success rate for the CNL group was 80.4% versus 83.3% for the laminectomy group, but in patients who had not obtained relief from the first procedure the success rate for the second procedure was higher for the post-CNL patients. A questionnaire was sent to all patients for 1- to 13-year follow-up review. The average follow-up period was 6.6 years for post-CNL laminectomy and 5.2 years for repeat laminectomy. The long-term success rate (81.8%) was higher in the post-CNL group compared to 64.4% in the repeat laminectomy group. Seven patients in the post-CNL group and nine in the repeat laminectomy group had undergone a third operation. When these originally successfully treated patients were reassigned after unsuccessful outcomes, the success rate for the CNL groups was 72.7%, versus 51.1% in the laminectomy group (p = 0.049). Employment rates were 80% for patients with CNL (21.8% changed jobs) and 76.3% for patients undergoing laminectomy (48.3% changed jobs) (p = 0.036). In conclusion, patients who underwent laminectomies after receiving CNL had significantly better long-term results than those who had repeat laminectomies.     

Paleomicrobiological study in dental calculus: Streptococcus mutans

Morphological types of bacterial remains preserved in ancient tartar of teeth from extinct human groups, which included some communities of coastal gatherers, fishermen, hunters, and farmers, and those practicing a mixed economy, were analyzed. Previous studies have shown the presence of bacteria in ancient tartar. The aim of this work was to determine whether Streptococcus mutans was present in ancient populations (500-12,000 years old). Teeth samples were from ancient skulls obtained from different anthropological collections: the north and south of Chile (before the Spanish conquest), Palencia, Spain, and an eastern Mediterranean region (Levant). Optical microscopy showed Gram positive and Gram negative bacteria. Scanning electron microscopy identified morphological types of bacteria. Transmission electron microscopy enabled categorization of bacterial structures. Fluorescence microscopy helped label and identify S. mutans, using polyclonal antibodies. Bacterial morphotypes were related to different subsistence patterns. Hunters, fishermen, and gatherers had a less diverse flora with bacillary and coccal morphotypes. Agricultural groups showed greater diversity with additional filamentous and spiral morphotypes. The best preserved ultrastructural feature was the cell wall. The existence and colonization capacity of the mutans-like streptococci preserved in tartar was established for the ancient populations studied, with the exception of Cerro Sotta (south of Chile). Hence, their occurrence could not be related to diet or subsistence pattern.     

Detection of pathogenic fungi in human blood by the polymerase chain reaction

The ability of the polymerase chain reaction (PCR) to detect pathogenic fungi in human blood was investigated. A DNA fragment of about 300 bp from the 18S rDNA, highly conserved in all fungi, was amplified with target DNA from 18 different species of fungi commonly isolated from clinical samples. The presence of PCR products was confirmed by hybridization with a fluorescein-labelled internal probe (21-mer). The PCR assay described is sensitive enough to detect 125 fg of purified Candida albicans DNA and 10 to 100 yeast cells per millilitre of blood.     

Analyte detection with DNA-labeled antibodies and polymerase chain reaction

We demonstrate immuno-polymerase chain reaction (PCR) assays for two clinical analytes--human thyroid-stimulating hormone and chorionic gonadotropin (hTSH, hCG)--using DNA-labeled antibodies and PCR for amplification of assay response. DNA-antibody conjugates were synthesized by using heterobifunctional cross-linker chemistries to covalently attach single- or double-stranded DNA labels through amine or sulfhydryl groups on the analyte antibodies. These approaches yielded molecular chimeras possessing both analyte-specific antibody binding and nucleic acid amplification functionalities. Dose-response relationships were demonstrated for immuno-PCR assays of both analytes in a microtiter plate-based, two-antibody sandwich assay format. Detection limits for hTSH (1 x 10(-19) mol, < 1.4 mIU/L) and hCG (5 x 10(-18) mol, 0.025 IU/L) exceeded those of conventional enzyme immunoassays by 2-3 orders of magnitude. We also evaluated various DNA design factors influencing label amplification and assay performance, such as primer sequence, strand number, and DNA length. Our findings, in concert with previous reports, suggest that this hybrid technology could provide the basis for a new generation of ultra-sensitive immunoassays offering multianalyte capabilities.     

Comparison of polymerase chain reaction and culture methods for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in subgingival plaque samples

In a formerly drug-dependent patient of 35 years of age suffering from an advanced HIV infection there was a development within a period of a few months of rapid weight loss amounting to 12 kg, persistent subfebrile temperatures and progressive dyspnoea on exercise. The histological pattern obtained via bronchoscopy revealed not only pneumocystis carinii pneumonia, which had already been suspected clinically, but also a not very differentiated adenocarcinoma of the lung with lymphangiosis carcinomatosa. The patient died three months after tis diagnosis was established, which had been followed by the usual pneumocystosis therapy and palliative treatment with glucocorticoids.     

The in situ polymerase chain reaction for detection of chlamydia trachomatis

The in situ polymerase chain reaction (PCR) is a technique that has important applications in the diagnosis of viral and bacterial diseases. This study investigated an in situ PCR assay established to detect the presence of Chlamydia trachomatis in endocervical swabs. In addition, histological sections of endocervical squamous cell carcinoma were analyzed because previous studies had revealed a significant association with C. trachomatis. A total of 20 cervical neoplasms (squamous cell carcinoma in situ; n = 10; invasive squamous cell carcinoma; n = 10) and endocervical smears taken from five patients with and without inflammatory changes were analyzed by conventional PCR. Chlamydial DNA was found in 10 histological samples (six carcinomas in situ, four invasive carcinomas) and in one endocervical swab from a patient with known C. trachomatis infection. Positive specimens were used for establishing an in situ PCR assay (IS-PCR). After IS-PCR, these samples showed dense cytoplasmic staining of endocervical cells (smears) and non-neoplastic epithelial cells (cervical neoplasms). The other tumor samples and smears did not demonstrate positive PCR reaction. The results indicate that in situ PCR is an effective technique for localizing C. trachomatis in target cells because IS-PCR detection of chlamydial DNA correlated with histological and cytological features.     

Rapid detection of aneuploidies by microsatellite and the quantitative fluorescent polymerase chain reaction

Several studies have been performed to assess the diagnostic value of using small tandem repeat (STR) markers and quantitative fluorescent polymerase chain reaction (QF-PCR) assays for the rapid detection of aneuploidies involving chromosomes 21, 18, 13 (Mansfield, 1993; Pertl et al., 1994, 1996; Adinolfi et al., 1995a). The results of these investigations have documented the diagnostic advantages of this approach to perform prenatal tests using amniotic and chorionic samples, or fetal nucleated cells retrieved from peripheral maternal blood or endocervical samples. The use of two or more STR markers for each autosome facilitates the diagnosis of aneuploidies, while avoiding the need to employ internal non-polymorphic markers. Multiplex quantitative fluorescent analyses can be performed in about six hours from the collection of the samples and, although targeted to specific abnormalities, they can exclude the presence of the most frequent chromosomal disorders. QF-PCR can be exploited to analyse DNA present in single or clumps of cells and thus to perform prenatal diagnoses on maternal peripheral blood or transcervical cell samples and on preimplantation embryos.     

Bordetella pertussis diagnosed by polymerase chain reaction

The object of this work was to test the polymerase chain reaction (PCR) for demonstration of Bordetella pertussis (BP) in nasopharyngeal secretions. The method was applied to patients with recently diagnosed pertussis, as verified by BP culture. In order to test the sensitivity and specificity of PCR for the diagnosis of BP, we used known concentrations of BP, Bordetella parapertussis and Bordetella bronchiseptica in aqueous solutions. PCR was furthermore carried out on species of bacteria that might be isolated from the nasopharynx. The applicability of PCR to patient specimens was tested in 25 patients in whose nasopharyngeal secretions BP had been demonstrated after 4-7 days of culture. The detection limit of PCR in aqueous solution was 1-2 BP bacteria per reaction tube. PCR was 100% specific for BP, showing no response with other Bordetella species or other bacteria known to colonize the nasopharynx. Of 25 patient specimens, 16 were PCR-positive 4-7 days after the positive primary culture had been established; only 5 out of 13 patient specimens were positive by repeated conventional nasopharyngeal culture at that time. We conclude that PCR is a possible alternative to culture for the demonstration of BP, as PCR is considerably faster than culture and might be more sensitive.     

Detection of Leishmania DNA by polymerase chain reaction in scars of treated human patients

Leishmaniasis is an endemic disease in developing countries. The efficacy of therapy is usually evaluated through clinical parameters. To define the parasitologic cure, 20 patients were biopsied before and 1 month to 8 years after treatment. Paraffin-embedded tissue was used for DNA isolation. All patients had a positive polymerase chain reaction (PCR) result before therapy, except 1, for whom no histopathologic material was available. The causative agent was identified as belonging to the Leishmania (Viannia) subgenus by hybridization. Despite clinical healing and absence of reactivation or development of mucosal lesions, PCR was positive in scars of 16 patients (80%). The results suggest that parasites persist in the skin for many years despite treatment. Depending on specific pathogenetic features of the parasite and the immune status of the host, this phenomenon might result in mucosal lesions. Alternatively, it could have a role in the maintenance of immunologic memory in patients living in areas in which leishmaniasis is endemic.