Evaluation of detection of M. tuberculosis in clinical specimens of tuberculosis by DNA amplification

The sensitivity of detection of M. tuberculosis genomic DNA were 1pg or 10-100 bacterial cell by PCR. Only M. tuberculosis, M. bovis and BCG were positive with 165 b.p band, but all other 14 mycobacterium and 10 bacteria of non-mycobacterial tested, were negative. Of 75 sputum specimens of pulmonary tuberculosis, the positive rate of PCR were 53.3%, culture method showed only 21.3%, fast-acid staining were 25.3%. 17 non-tuberculosis lung disease were negative in three methods. Of 58 tuberculosis meningitis, the positive rate of PCR, the fast-acid staining and culture in cerebrospinal fluid were 51.7%, 8.6%, 1.7% respectively. 30 non-tuberculosis meningitis were negative in three methods. The results showed that DNA amplification is a superior method with high degree of sensitivity and specificity for rapid diagnosis of pulmonary tuberculosis and tuberculosis meningitis.     

Quality evaluation of sputum specimens for mycobacterial culture

The evaluation of sputum specimen quality before bacterial culture is an accepted practice. Traditionally, sputum received for mycobacterial culture has been processed regardless of specimen quality. In view of the increasing emphasis placed on the primary acid-fast smear, the effect of specimen quality and the contribution of the presence of neutrophils on subsequent smear and culture positivity for mycobacteria was assessed. A total of 873 sputa were evaluated and initially assigned a quality (Q) score of 1 (many squamous cells relative to neutrophils) to 3 (no squamous cells) based on criteria for specimen acceptability for routine bacterial culture. The percentage of specimens that were Q1, Q2, and Q3 were 46.8, 35.3, and 17.9, respectively. Most of the specimens received were Q1, and these specimens demonstrated the highest number of positive smears and cultures compared to Q2 or Q3 specimens. Thus, routine bacterial culture criteria were not helpful in assessing specimen quality for mycobacterial culture. The contribution of the presence of neutrophils to smear and culture positivity was assessed. A total of 724 sputa were evaluated for cellular composition: 665 (91.9%) specimens contained neutrophils, and 59 (8.1 %) did not contain neutrophils. A total of 51 (7.0%) primary smears and 121 (16.7%) cultures were positive for mycobacteria; 92.2% of the positive smears; and 90.1 % of the positive cultures were from specimens that contained neutrophils. Thus, screening sputum specimens for the presence of neutrophils provides an effective method to evaluate the acceptability of sputum for mycobacterial smear and culture, especially from patients in respiratory isolation.     

Rapid detection and typing of herpes simplex virus DNA in clinical specimens by the hybrid capture II signal amplification probe test

A second-generation signal amplification, nucleic acid-based test for the rapid detection and typing of herpes simplex virus (HSV) DNA was developed and evaluated with artificial and clinical specimens. The analytical sensitivity of the Hybrid Capture II (HC II) HSV DNA assay was determined by testing either cloned HSV DNA or total genomic HSV DNA titrations and resulted in detection thresholds of between 5 x 10(3) and 1 x 10(4) copies per assay. Specificity was assessed by testing a panel of bacteria and viruses commonly found in the female genital tract. Sensitivity was assessed by testing 112 ulcerative genital lesions by the HC II assay and comparing the results to those obtained by routine cell culture. Discrepant results were resolved by PCR testing. After resolution of the discrepant results, the sensitivity of the HC II assay compared to the consensus result (the results of two of three tests, the HC II assay, culture, and PCR, were in agreement) was 93.2% (41 of 44 specimens), and the specificity was 100% (60 of 60 specimens). Culture gave a sensitivity of 84.1% (37 of 44 specimens) and a specificity of 100% (60 of 60 specimens) compared to the consensus result. The results of HSV typing by the HC II assay and culture agreed in all cases. The HC II assay is a rapid and accurate assay for detecting and typing HSV types 1 and 2, with a sensitivity comparable to that of culture and greater ease of use than culture.     

Sequence analysis of mitochondrial DNA hypervariable regions using infrared fluorescence detection

The non-coding region of the mitochondrial genome provides an attractive target for human forensic identification studies. Two hypervariable (HV) regions, each approximately 250-350 bp in length, contain the majority of mitochondrial DNA (mtDNA) sequence variability among different individuals. Various approaches to determine mtDNA sequence were evaluated utilizing highly sensitive infrared (IR) fluorescence detection. HV regions were amplified either together or separately and cycle-sequenced using a Thermo Sequenase protocol. An M13 universal primer sequence tail covalently attached to the 5' terminus of an amplification primer facilitated electrophoretic analysis and direct sequencing of the amplification products using IR detection.     

Rapid enzymatic analysis for human immunodeficiency virus type 1 DNA in clinical specimens

A clinical procedure for rapid detection of human immunodeficiency virus type 1 (HIV-1) by DNA amplification is demonstrated. The rapid procedure reduces handling requirements and amplification time and eliminates use of radioactivity for the detection of the amplification product. Total leukocyte lysates are the amplification substrates. Two conserved regions in the HIV-1 genome are amplified by 45 cycles of a two-temperature thermal cycle and the amplification products are detected by ultraviolet light after electrophoresis on agarose gels. Twenty-four specimens clinically diagnosed by detection of antibody (IgG) to HIV-1 were confirmed by the rapid DNA amplification procedure. In a blind study, 56 samples positive for HIV-1 DNA were detected in 503 individuals by the current classical polymerase chain reaction method; the same 56 positive samples were also detected by the rapid amplification protocol. No false-positive or false-negative results were obtained. The turnaround time for analysis has been reduced to < 24 h without compromising test results.     

Effect of centrifuging shell vials at 3,500 x g on detection of viruses in clinical specimens

An increase in shell vial centrifugation force to 3,500 x g and a concomitant reduction in spin time to 15 min did not decrease the sensitivity of detecting viruses in clinical specimens compared with the accepted practice of using 700 x g for 40 min. No damage to the cell monolayer (ML) at the higher g force was observed. Toxicity to the ML is decreased with the shorter spin, probably because of reduced time of contact between the specimen and the ML.     

Direct genotypic detection of Mycobacterium tuberculosis rifampin resistance in clinical specimens by using single-tube heminested PCR

Recent analysis of the gene encoding the beta subunit of Mycobacterium tuberculosis RNA polymerase (rpoB) has demonstrated a small region that harbors the mutations most frequently associated with rifampin resistance. Earlier reports have described a high degree of sequence conservation of rpoB among mycobacteria other than M. tuberculosis and other GC-rich bacteria that can lead to false-positive amplification when applied directly to clinical specimens. We developed reagents for PCR amplification that are based on signature nucleotides discovered by comparative sequence analysis of the rpoB genes of organisms phylogenetically related to M. tuberculosis. The specificities of the reagents were challenged with 20 isolates of multiple-drug-resistant M. tuberculosis and more than 20 species of mycobacteria other than M. tuberculosis and other GC-rich organisms. A single-tube heminested PCR protocol was devised to obtain sensitivity equal to those of an IS6110-based PCR assay and culture in spiked sputum experiments. The assay correctly identified 21 of 24 (87.5%) culture-positive specimens, 13 of which were acid-fast smear-negative, in a panel of 51 clinical specimens. Three specimens that were false-positive initially were negative upon repeat testing when the assay was modified to eliminate the potential for aerosol carryover of the first-round amplification product during the open-tube addition of the second set of reaction reagents. This assay is the most sensitive and specific test to date for the direct detection of M. tuberculosis rpoB in clinical specimens. This rapid PCR-based assay can be used for the simultaneous identification of M. tuberculosis and its rifampin susceptibility genotype.     

Fingerprinting of mycobacterial DNA in the molecular epidemiology of tuberculosis]

Immunologic status was studied in 86 patients with type-I diabetes mellitus before and after treatment with decarise, thymaline and T-activin. With the aid of ECG, phonocardiogramme, apex cardiogramme, oscillogramme, there has been calculated index of vitality of the myocardium reflecting the subendocardial bloodflow. The principal parameters characterising bodily immune status (absolute number of T- and B-lymphocytes), T-suppressors counts as well as those of circulating immune complexes (CIC) were compared to the values for subendocardial bloodflow (end-diastolic pressure, index of vitality of the myocardium). There has been established strong inverse relation between the absolute numbers of T-, B-lymphocytes, and CIC, end-diastolic pressure and index of vitality of the myocardium, this being regarded as evidence in confirmation of the guarding role of the former and damaging one of the latter (CIC) under vascular pathology in diabetes mellitus.     

Antiserum-agar plate method for simultaneous detection and direct isolation of Legionella species in clinical and environmental specimens

Colonies of Legionella pneumophila serotypes 1 through 6, L. micdadei, L. bozemanii, L. dumoffii, and L. gormanii, which were developed on filtered yeast extract agar containing polyvalent antiserum, were surrounded by distinct, specific precipitin rings.     

Rapid detection of different RNA respiratory virus species by multiplex RT-PCR: application to clinical specimens

Inconsistent findings from recent mortality studies of workers exposed to magnetic fields have led to calls for more detailed understanding of exposure distributions and metrics in various industries. The authors undertook personal monitoring at an automobile transmission plant to (a) learn if magnetic field exposure differences were present, (b) make assignments for a brain cancer study, and (c) compare two exposure indices. A wide range of average exposures occurred (i.e., 0.016-4.6 microtesla). Within-day variability was also large, and it reached 4 orders of magnitude for some workers. Unexpectedly, demagnetizers were found among the strong sources that contributed to elevated exposures. The authors used conventional summary measures to assign job groups to exposure categories, and they used a new index of exposure irregularity to make alternative assignments. These new assignments appeared to differ from the original ones with respect to work time in each exposure group (i.e., 54% of the work time fell into different exposure categories).     

A trial of clinical application of sparfloxacin for treating mycobacterial infections

Sparfloxacin seems to be a good candidate for antimycobacterial treatment. However, there have been no clinical studies. We experienced 2 SPFX-treated cases, who could not use other antimycobacterial agents because of side effect, we tried SPFX-treatment on these cases. Good results were obtained, however, for long time use to prevent side effects, we tried SPFX every second day and monitored the serum levels of SPFX. SPFX-every second day treatment gave good clinical results and adequate serum levels of SPFX were observed.     

The clinical use of fluoroquinolones for the treatment of mycobacterial diseases

Mycobacterial diseases often require prolonged therapy with multidrug regimens. Fluoroquinolones have excellent bactericidal activity against many mycobacteria; achieve effective serum, tissue, and intracellular levels following oral administration; and produce few adverse effects. These properties have led to the increasing use of fluoroquinolones for the treatment of mycobacterial infections. We reviewed clinical studies and reports involving the use of fluoroquinolones for mycobacterial diseases. Ofloxacin, ciprofloxacin, sparfloxacin, and pefloxacin exhibit clinical efficacy against mycobacterial diseases, especially tuberculosis and leprosy. Fluoroquinolones have generally been administered in regimens that include other agents. However, when a fluoroquinolone has been found to be the sole active agent in a multidrug regimen, the ready emergence of resistance to fluoroquinolones has been recognized, just as when they have been used as monotherapy. Therefore, to forestall the emergence of resistance to fluoroquinolones during the treatment of mycobacterial diseases, these drugs should always be used in combination with at least one other active agent, and they should be used only when effective alternative drugs are not available.     

Qualitative and quantitative detection of cytomegalovirus DNA in sera by PCR as a clinical marker

The amount of human cytomegalovirus (CMV) DNA in sera is considered to be a direct marker for CMV infection. We established conditions for nested PCR that detected one copy of CMV DNA, and for competitive PCR, which detected five or more copies of CMV DNA quantitatively. We tested 50 microl each of 16 freeze-stored and 5 fresh sera from patients, for CMV DNA. In sera obtained from the same patient at different time points, small amounts of CMV DNA were detected before the onset of CMV pneumonia. In sera from certain CMV-infected patients who were treated with the anti-CMV agent, ganciclovir, CMV DNA was not detected. Quantitative PCR detection of CMV DNA seems to be suitable for predicting early recurrent CMV infection and monitoring the efficacy of antiviral therapy. The qualitative nested PCR examination of CMV DNA in 40 cord blood plasma samples was carried out for the purpose of preventing CMV infection by cord blood stem cell transplantation, and they were all negative for CMV DNA.     

Presence of Streptococcus DNA sequence in surgical specimens of gastric cancer

In Southern blot analysis using Mycoplasma 16S ribosomal DNA (rDNA) as a probe, positive signals were detected in DNA samples from surgical specimens of gastric cancers. The DNA that hybridized to Mycoplasma 16S rDNA was eluted from the gel, cloned and sequenced. The cloned sequence was identical to 16S rDNA of Streptococcus anginosus. In Southern blot analysis with the S. anginosus 16S rDNA fragment as a new probe, positive signals were detected in 9 (20%) out of 43 cases of gastric cancer.     

Isolation and characterisation of nocardia from clinical specimens

Sixteen isolations of nocardia of which 12 were from pulmonary infections, one from wound infection, one from mycetoma and 2 from eye infections were studied from June, 1989 to May, 1990. The importance of Gram's stain findings of primary smear is being highlighted. The nocardia species were identified utilising the morphological characters including acid fastness and cultural and biochemical characters. Notable among the isolates were Nocardia brasiliensis, one each from mycetoma and pulmonary infection, which are rare in South India and Nocardia asteroides from a case of endophthalmitis probably of endogenous origin.     

Sequence polymorphism of mitochondrial DNA control region in Japanese

Sequence polymorphisms of the mitochondrial DNA (mtDNA) control region, hypervariable regions I and II, from 100 unrelated Japanese were determined by PCR amplification and direct sequencing. Sequences of 404 nucleotides for hypervariable region I and 379 nucleotides for region II were obtained. Variable sites (85 and 45) were revealed in region I and region II, respectively, as compared to the reference sequence, and a total of 96 different genetic patterns from both regions I and II were determined. A point mutation heteroplasmy was observed at the ratio of approximately 50:50 from one individual at the sequence position 151 showing a nucleotide transition from C to T. The probability of identity was estimated as 2.3% for region I, 3.9% for region II, and 1.1% combined for both regions. These results suggest that sequence polymorphism of mtDNA control region would be very useful in forensic practice as a marker for individual identification.     

Multiplex PCR-based assay for detection of Bordetella pertussis in nasopharyngeal swab specimens

A multiplex PCR-based assay was developed for the detection of Bordetella pertussis in nasopharyngeal swab specimens. The assay simultaneously amplified two separate DNA targets (153 and 203 bp) within a B. pertussis repetitive element and a 438-bp target within the beta-actin gene of human DNA (PCR amplification control). PCR products were detected by a sensitive and specific liquid hybridization gel retardation assay. A total of 496 paired nasopharyngeal swab specimens were tested by both the PCR-based assay and culture. Although 30 (6%) of the specimens inhibited the amplification of the beta-actin target, in all 29 specimens studied, the inhibition disappeared on repeat testing or was easily overcome with a 1:8 dilution or less of specimen digest. Of the 495 specimen pairs yielding a final evaluable result by the PCR-based assay, 19.0% were positive by the PCR-based assay, whereas 13.9% were positive by culture (P < 0.0001). After resolving the PCR-positive, culture-negative results by testing an additional aliquot from these specimens by the multiplex PCR-based assay, the PCR-based assay had a sensitivity and specificity of 98.9 and 99.7%, respectively, compared with values of 73.4 and 100%, respectively, for culture. In comparison with patients with culture-confirmed pertussis, those with PCR-positive, culture-negative results were older and more likely to have had prolonged cough, immunization with pertussis vaccine, or treatment with erythromycin. This multiplex PCR-based assay is substantially more sensitive than culture and identifies specimens that contain inhibitors of PCR.