Tech.Sight. Molecular testing for infectious disease.
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Automation↗
Biomedical subjects
D Jungkind
Publications and source records attributed to D Jungkind.
Automation of laboratory testing for infectious diseases using the polymerase chain reaction-- our past, our present, our future.
While it is an extremely powerful and versatile assay method, polymerase chain reaction (PCR) can be a labor-intensive process. Since the advent of commercial test kits from Roche and the semi-automated microwell Amplicor system, PCR has become an increasingly useful and widespread clinical tool. However, more widespread acceptance of molecular testing will depend upon automation that allows molecular assays to enter the routine clinical laboratory. The forces driving the need for automated PCR are the requirements for diagnosis and treatment of chronic viral diseases, economic pressures to develop more automated and less expensive test procedures similar to those in the clinical chemistry laboratories, and a shortage in many areas of qualified laboratory personnel trained in the types of manual procedures used in past decades. The automated Roche COBAS AMPLICOR system has automated the amplification and detection process. Specimen preparation remains the most labor-intensive part of the PCR testing process, accounting for the majority of the hands-on-time in most of the assays. A new automated specimen preparation system, the COBAS AmpliPrep, was evaluated. The system automatically releases the target nucleic acid, captures the target with specific oligonucleotide probes, which become attached to magnetic beads via a biotin-streptavidin binding reaction. Once attached to the beads, the target is purified and concentrated automatically. Results of 298 qualitative and 57 quantitative samples representing a wide range of virus concentrations analyzed after the COBAS AmpliPrep and manual specimen preparation methods, showed that there was no significant difference in qualitative or quantitative hepatitis C virus (HCV) assay performance, respectively. The AmpliPrep instrument decreased the time required to prepare serum or plasma samples for HCV PCR to under 1 min per sample. This was a decrease of 76% compared to the manual specimen preparation method. Systems that can analyze more samples with higher throughput and that can answer more questions about the nature of the microbes that we can presently only detect and quantitate will be needed in the future.
Hepacivirus↗
Enhancing the specificity of the COBAS AMPLICOR CT/NG test for Neisseria gonorrhoeae by retesting specimens with equivocal results.
The COBAS AMPLICOR CT/NG test for Neisseria gonorrhoeae cross-reacts with certain strains of nonpathogenic Neisseria species. In some strains, the target sequence is identical to that of N. gonorrhoeae, whereas other strains have a small number of mismatches within the regions recognized by the primers or probe used in the COBAS AMPLICOR NG test. These cross-reactive strains are occasionally present in urogenital specimens, causing false-positive results in the COBAS AMPLICOR NG test. Analysis of the data generated in a large multicenter clinical trial showed that 2.9% of the specimens gave signals between A(660)s of 0.2 and 3.5 but that one-half of these equivocal specimens did not contain N. gonorrhoeae. Most of these equivocal specimens were correctly classified as true positive or true negative by retesting in duplicate and defining a PCR-positive result as two of three results with an A(660) of > or =2.0. If specimens had been classified as positive or negative based on a single test result using a cutoff of an A(660) of 0.2, specificity would have ranged from 96.2 to 98.9% depending on specimen type, sex, and presence of symptoms. By employing the equivocal zone-retesting algorithm, specificity increased to 98.6 to 99.9% with little effect (0.1 to 4.9% decrease) on sensitivity in most specimen types, enabling the test to achieve a positive predictive value of at least 90% in populations with a prevalence of 4% or higher. In lower-prevalence populations, the test could be used to screen for presumptive infections that would have to be confirmed by an independent test.
Adult↗
A receptor-specific peptide for imaging infection and inflammation.
The chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP), when radiolabelled, continues to be an attractive agent for imaging infection or inflammation. Previously, several analogues of fMLP have been prepared and radiolabelled using a bifunctional chelating agent conjugation procedure that was relatively long and complex. We have prepared a new analogue of fMLP, TP765, by the addition of 4-aminobutyric acid (4-ABA) and a group of four amino acids, Gly-Gly-d-Ala-Gly, to the carboxy terminus (i.e. to the phenylalanine) of fMLP. The adduct -(4-ABA)-Gly-Gly-d-Ala-Gly- serves as a chelating moiety for strong chelation with 99Tcm. The use of a peptide as a chelating moiety greatly simplified the synthetic procedure and rendered the analogue ready for instant chelation with 99Tcm. HPLC analysis revealed that 99Tcm-TP765 was a single chemical entity that retained biological activity and neutrophil specificity. 99Tcm-TP765 was stable when challenged with strong chelating agents in vitro and had rapid but biphasic blood clearance (alphat1/2 = 7 min, betat1/2 = 45 min). Approximately 90% of the radioactivity had cleared from circulation within 45 min post-injection and the agent had accumulated in experimental bacterial or sterile abscesses in significantly (P<0.05) higher quantities than the analogues evaluated previously. Generally, the biodistribution pattern of 99Tcm-TP765 was similar to that of other analogues examined and its abscess uptake was independent of the abscess age. In conclusion, a new analogue of fMLP, 99Tcm-TP765, was prepared by a simple procedure. This new analogue has properties similar to those of previously examined analogues used as agents for imaging infection or inflammation.
Animals↗
Multicenter evaluation of AMPLICOR and automated COBAS AMPLICOR CT/NG tests for Neisseria gonorrhoeae.
The fully automated COBAS AMPLICOR CT/NG and semiautomated AMPLICOR CT/NG tests were evaluated in a multicenter trial for their ability to detect Neisseria gonorrhoeae infections. Test performance compared to that of culturing was evaluated for 2,192 matched endocervical swab and urine specimens obtained from women and for 1, 981 matched urethral swab and urine specimens obtained from men. Culture-negative, PCR-positive specimens that tested positive in a confirmatory PCR test for an alternative target sequence within the N. gonorrhoeae 16S rRNA gene were considered to be true positives. The overall prevalences of gonorrhea were 6.6% in women and 20.1% in men. The COBAS AMPLICOR and AMPLICOR formats yielded concordant results for 98.8% of the specimens and exhibited virtually identical sensitivities and specificities. The results that follow are for the COBAS AMPLICOR format. With the infected patient as the reference standard, the resolved sensitivities of PCR were 92.4% for endocervical swab specimens and 64.8% for female urine specimens. There were no significant differences in these rates between women with and without symptoms. Among symptomatic men, COBAS AMPLICOR sensitivities were 94.1% for urine and 98.1% for urethral swabs; for asymptomatic men, the results were 42.3 and 73.1%, respectively. In comparison, the sensitivities of culturing were 84.8% for endocervical specimens, 92.7% for symptomatic male urethral specimens, and only 46.2% for urethral specimens obtained from asymptomatic men. When PCR results were analyzed as if only a single test had been performed on a single specimen type, the resolved sensitivity was always higher. The resolved specificities of PCR were 99.5% for endocervical swab specimens, 99.8% for female urine specimens, 98.9% for male urethral swab specimens, and 99.9% for male urine specimens. The internal control revealed that 2.1% of specimens were inhibitory when initially tested. Nevertheless, valid results were obtained for 99.2% of specimens because 60.0% of the inhibitory specimens were not inhibitory when a second aliquot was tested. The COBAS AMPLICOR CT/NG test for N. gonorrhoeae exhibited high sensitivity and specificity with urethral swab and urine specimens from men and endocervical swab specimens from women and thus is well suited for diagnosing and screening for N. gonorrhoeae infection.
Animals↗
Multicenter evaluation of the AMPLICOR and automated COBAS AMPLICOR CT/NG tests for detection of Chlamydia trachomatis.
The fully automated COBAS AMPLICOR CT/NG and semiautomated AMPLICOR CT/NG tests were evaluated in a multicenter trial for the ability to detect Chlamydia trachomatis infections. Test performance compared to that of culture was evaluated for 2,236 matched endocervical swab and urine specimens obtained from women and for 1,940 matched urethral swab and urine specimens obtained from men. Culture-negative, PCR-positive specimens that tested positive in a direct fluorescent-antibody test or in a confirmatory PCR test for an alternative target sequence were resolved as true positives. The overall prevalences of chlamydia were 2.4% in women and 7.2% in men. The COBAS AMPLICOR and AMPLICOR formats yielded concordant results for 98.1% of the specimens. With the infected patient as the reference standard, the resolved sensitivities of COBAS AMPLICOR were 89.7% for endocervical swab specimens, 89.2% for female urine specimens, 88.6% for male urethral swab specimens, and 90.3% for male urine specimens. When results were analyzed as if only a single test had been performed on a single specimen type, the resolved sensitivity was always higher. The resolved specificities of PCR were 99.4% for endocervical swab specimens, 99.0% for female urine specimens, 98.7% for male urethral swab specimens, and 98.4% for male urine specimens. The internal control revealed that 2.4% of the specimens were inhibitory when initially tested. Nevertheless, valid results were obtained for 98.6% of the specimens because 59.1% of the inhibitory specimens were not inhibitory when a second aliquot was tested. The COBAS AMPLICOR and AMPLICOR CT/NG tests for C. trachomatis exhibited equally high sensitivity and specificity with both urogenital swab and urine specimens and thus are well suited for screening for C. trachomatis infection.
Automation↗
Some characteristics of platelet concentrates contaminated with Staphylococcus epidermidis.
The study investigates platelet concentrates (PCs) contaminated with Staphylococcus epidermidis. The work was conducted using 12 pairs of PCs. Light transmission through the PCs was observed over 7 days. Immediately after preparation, one PC was inoculated with Staphylococcus epidermidis (17-43 colony forming units (CFU)/ ml) and the other served as a non-contaminated control. In the contaminated PCs the CFU/ml and swirling were determined on days 1, 3 and 7. In all PCs determination of pH and the extent of shape change (ESC) was carried out on days 3 and 7. All contaminated PCs had visible swirling on day 3 but they contained 2.0 X 10 4 to 9.0 X 10 8 CFU/ml. pH (P=0.05) and ESC (P<0.05) then were slightly lower. On day 7, eight of 12 of the contaminated PCs failed to display transmission changes despite deranged analytical results and impaired swirling. During early storage the contaminated units had unimpaired swirling and only slightly impaired in vitro quality. They appear to be difficult to identify in the routine.
Journal Article↗
Evaluation of AMPLILINK software for the COBAS AMPLICOR system.
The use of AMPLILINK version 1.0 software was evaluated for the operation and control of one COBAS AMPLICOR instrument and for two COBAS AMPLICOR instruments run simultaneously to perform and detect nucleic acid amplification reactions. A total of 3,384 results were analyzed. The initial accuracy of the results was 99.91%. Three errors of omission of transfer of data from the COBAS AMPLICOR to the AMPLILINK system were observed. Two of these errors were from a single specimen, where both the analyte and internal control results were not transmitted. These errors did not interfere with the correctness of any other data. There were no interruptions of runs, and no data were mixed. AMPLILINK increased convenience, saved labor, and was found to be a very useful addition for clinical laboratories performing molecular-diagnostic procedures with the COBAS AMPLICOR system.
Evaluation Studies as Topic↗
Viral burden and disease progression in HIV-1-infected patients with sickle cell anemia.
The spleen and lymph nodes are major sites of human immunodeficiency virus type 1 (HIV-1) replication, mutation, and genetic variation in vivo. If a major portion of the lymphatic tissue, such as the spleen, is removed or otherwise is unavailable for invasion by the HIV-1 virus, will the course of the infection be altered, resulting in a prolonged symptom-free interval or even increased survival? The spleen of most adults with sickle cell anemia (SS) is nonfunctional due to recurrent episodes of microinfarction. If autosplenectomized SS patients are exposed to HIV-1, they may be ideal candidates to examine the question of whether absence of splenic function at the time of infection will positively alter the course of HIV-1-related disease. All SS patients with a diagnosis of HIV-1 infection at five university sickle cell centers were included in the patient cohort. Patients in active treatment or in follow-up (group A, n = 11) underwent a series of quantitative viral studies to determine their HIV-1 viral burden. The studies included the branched-DNA signal amplification assay, quantitative DNA-polymerase chain reaction (PCR), quantitative reverse transcription (RT)-initiated-PCR, and in situ PCR. All patients who died of the complications of the acquired immunodeficiency syndrome (AIDS) or of SS, lost to follow-up, or were otherwise unavailable for study (Group B: n = 7) were included in the total patient group. None of the patients in group B underwent quantitative viral studies. In addition, a control population (group C, n = 36) of HIV-1-infected African Americans without SS, of similar age and gender to the SS patients, were compared with the study population for outcomes. In eight of 11 active patients (group A), the CD4+ T-lymphocyte counts were normal and viral burdens were low for an average of 10.25 years following diagnosis. These eight patients all from group A were the only long-term nonprogressors (44%) among a total of 18 SS patients (groups A and B). In group C (control), only five patients of 36 were long-term nonprogressors (13.9%). Five patients (28%) of the total SS group (groups A and B) succumbed to AIDS. One of the five was from Group A. The evaluation of a limited number of adult individuals suggests that a significant proportion of HIV-1-seropositive SS patients (44%) may be asymptomatic long-term nonprogressors. In these patients, the CD4+ T-lymphocyte counts remained high and their viral burdens were remarkably lower than in non-SS HIV-1-seropositive individuals. Whereas this study does not prove an "autosplenectomy" hypothesis, it suggests that in patients with both SS and HIV-1 infection, the retroviral disease may be ameliorated by host factors of which absence of splenic function prior to HIV-1 infection may be one.
Adolescent↗
Evaluation of automated COBAS AMPLICOR PCR system for detection of several infectious agents and its impact on laboratory management.
We evaluated the COBAS AMPLICOR (CA) PCR system (Roche Diagnostic Systems) designed for automated PCR amplification and detection of nucleic acids from infectious agents in clinical samples. The Roche AMPLICOR microwell plate (MWP) PCR was the reference method. CA amplifies target nucleic acid, captures the biotinylated amplification products by using magnetic particles coated with specific oligonucleotide probes, and detects the bound products colorimetrically. For Mycobacterium tuberculosis, the correlation of the results of CA tests with those of MWP tests was 100% with 230 samples, including 20 culture-positive samples. For hepatitis C virus, the correlation was 100% with 214 samples, including 60 positive samples. MultiPlex CA analysis of 199 cervical specimens for Chlamydia trachomatis, Neisseria gonorrhoeae, and the internal control gave 100% concordance. These samples included 19 C. trachomatis and 3 N. gonorrhoeae culture-positive samples. Overall, the agreement between PCR methods for all 842 comparisons was 100%. Compared with culture, the sensitivities of the assays for C. trachomatis and M tuberculosis were > or = 95%. After spiking alternating amplification tubes in the CA system with 10(14) copies of the Chlamydia amplicon per ml, we were unable to demonstrate any carryover cross-contamination of negative samples. Using the criteria of the College of American Pathologists workload recording method, we found that the total hands-on time to produce CA PCR results was 4.4, 7.9, and 3.3 min for M. tuberculosis, hepatis C virus, and the MultiPlexed assay for chlamydia plus gonorrhea and an internal control, respectively. The CA system brings true PCR automation to laboratories. In addition to the accuracy of automated results, the CA system provides labor savings, provides containment of the amplification and detection components of PCR, and supports both MultiPlex amplification and sequential algorithm (ReFlex) detection of analytes.
Automation↗
Procedure for evaluation of neutralizing antibody to cytomegalovirus in commercial intravenous gamma globulin preparations.
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Antibodies, Viral↗
Technetium-99m-labeled proteins for imaging inflammatory foci.
Polyclonal human IgG (IgG), antinuclear antibody (TNT-1), and human serum albumin (HSA), were labeled with 99mTc by a method recently developed in our laboratory, and administered i.v., each to a separate group of five mice, bearing inflammatory foci induced by an i.m. injection of 40 microL turpentine or 5 x 10(8) E. coli and 5 x 10(8) Entercocci. TNT-1 labeled with 125I served as a control and 67Ga-citrate as a "gold standard". At 4 or 24 h post injection, animals were imaged and sacrificed for tissue distribution studies. At 4 h in the turpentine group, the abscess-to-muscle ratios were: 67Ga, 4.8 +/- 2.1, 125I-TNT-1, 4.3 +/- 1; 99mTc-TNT-1, 3.5 +/- 1.8; 99mTc-IgG, 3.9 +/- 0.6; and 99mTc-HSA, 4.3 +/- 1. In the microorganism group, these ratios were 2.6 +/- 0.6, 3.3 +/- 0.5, 3.4 +/- 0.08, 3 +/- 1.1 and 4.1 +/- 0.6, respectively. Autoradiographic examination of infected tissues indicated that leakage of labeled proteins into interstitial space due to increased capillary permeability may be one of the major mechanisms of uptake.
Animals↗
Detection of borderline oxacillin-resistant Staphylococcus aureus and differentiation from methicillin-resistant strains.
Eighty-eight Staphylococcus aureus clinical isolates meeting criteria for borderline oxacillin resistance (intermediate susceptibility or resistance to oxacillin but susceptibility to amoxicillin/clavulanic acid upon disk diffusion testing) were studied to determine optimal test techniques and conditions for differentiating borderline oxacillin-resistant Staphylococcus aureus (BORSA) from methicillin-resistant Staphylococcus aureus (MRSA). Further testing revealed three distinct resistance patterns: 61 strains (69%) consistently met BORSA criteria and had average beta-lactamase levels five- to six-fold higher than oxacillin-susceptible controls; 11 strains (13%) were markedly heteroresistant MRSA with delayed appearance of resistant colonies leading to spurious susceptibility to amoxicillin/clavulanic acid; 16 strains (18%) appeared to be oxacillin-susceptible on repetitive testing. Under conditions used to elicit intrinsic methicillin resistance in Staphylococcus aureus, a large percentage of BORSA appeared resistant to amoxicillin/clavulanic acid. This clearly shows that BORSA may be misidentified as MRSA while heteroresistant MRSA may appear to be BORSA. It is concluded that amoxicillin/clavulanic acid zone sizes should be measured after a full 24 hours of incubation, that susceptibility testing of Staphylococcus aureus under certain environmental conditions should be interpreted with caution, and that MIC testing is the most reliable technique for differentiating these two resistance patterns in Staphylococcus aureus.
Amoxicillin↗
Chlamydial antibody, as determined with an enzyme-linked immunosorbent assay, in tubal factor infertility.
Serum chlamydial antibody (CA), as determined with an enzyme-linked immunosorbent assay (ELISA), was evaluated as a predictor for the presence or absence of tubal factor infertility. Two hundred fifty-eight infertile women had CA drawn at the initial visit of an infertility workup. Of them, 46.3% were CA positive (CA+). One hundred forty-five patients underwent laparoscopy (LPY). Tubal factor was diagnosed in 87.2% of CA+ patients and 13.6% of CA negative (CA-) ones (P less than .001), with a rising frequency by CA positivity. CA correctly predicted the presence or absence of tubal factor in 86.9% of patients. The frequency of abnormal hysterosalpingograms (HSG) was higher in CA+ patients. The predictive values for tubal factor with low, mid and high CA+ were 62.5%, 97.5% and 95.8%, respectively, and for no tubal factor with CA- was 72.3%. Combining HSG with CA- increased that value. Agreement between the LPY and HSG findings by the CA result showed a high correlation. A history of pelvic inflammatory disease (PID) or intrauterine device use was more common in CA+ patients, but only 25.3% of patients with tubal factor had a history of PID. The frequency of positive cervical chlamydial cultures was 0.8%. CA determined with ELISA appears to be an accurate screening test for tubal factor infertility and can be used to reliably select the procedure of choice for tubal evaluation.
Antibodies, Bacterial↗
Comparison of two rapid latex agglutination methods for detection of group A streptococcal pharyngitis.
Throat swabs from 404 patients with suspected pharyngitis were collected using duplicate swabs. Both swabs were used to inoculate 5% sheep blood agar plates, which were incubated in an anaerobic atmosphere for the isolation of Group A streptococci. The throat swabs were tested for the presence of Group A antigen using the Culturette Brand 10-Minute Group A Strep ID kit (Marion Scientific, Kansas City, MO), and the Direct Antigen Identification D.A.I. Strep A Test (Difco Laboratories, Inc., Detroit, MI). We found that 77 of the 404 specimens were culture positive for Group A streptococci. The Strep ID kit had a sensitivity of 83.7% and a specificity of 91.6%. The positive and negative predictive values were 72% and 95.6%, respectively. The D.A.I. test had a sensitivity of 80.2% and a specificity of 100%. The positive and negative predictive values were 100% and 94.5%, respectively. There was not a significant difference in the sensitivity of the two kits (P less than 0.1), but there was a significant difference in the specificity (P less than 0.01).
Humans↗
Clinical comparison of a new automated infrared blood culture system with the BACTEC 460 system.
A new blood culture instrument, the BACTEC (Johnston Laboratories, Inc., Towson, Md.) NR-660, which utilizes infrared detection of carbon dioxide from microbial metabolism, was compared with the radiometric BACTEC 460 system. There were 1,554 isolates from 18,785 paired aerobic blood cultures. Of these isolates, 1,303 were isolated from the radiometric 6B medium, and 1,259 were isolated from the NR6A medium (P = 0.06). Analysis of the data indicated no significant differences in recovery when any individual species was considered. When organisms were considered as groups, there were no significant detection differences for gram-negative bacilli, yeasts, or anaerobes. For gram-positive cocci in aerobic medium, the BACTEC 460 detected 84.3% of the total isolates, and the BACTEC NR-660 detected 79.7% (P = 0.04). There were 891 isolates from 13,983 paired anaerobic blood cultures. Of these isolates, 725 were recovered from the radiometric 7D BACTEC medium, and 723 were recovered from the NR7A BACTEC medium (P greater than 0.9). In the anaerobic media there was no significant difference in detection of any organism group, including the gram-positive cocci. When the results of the aerobic and anaerobic media were combined, there was equivalence between the two systems for the detection of gram-positive cocci (P greater than 0.2) and other organism groups. When the ability to detect septic episodes was compared, there was no significant difference for any organism group (P = 0.12). For aerobic media, the mean times for detection were 30.5 and 29.5 h for the BACTEC 460 and NR-660, respectively. For anaerobic media, the mean times for detection were 39.8 and 41.6 h for the BACTEC 460 NR-660, respectively. Compared with the BACTEC 460, the BACTEC NR-660 system had a greater ease of operation, faster test cycle, computerized data base, and equally rapid detection of positive cultures.
Bacteria↗
Chlamydial infections in pregnancy.
Chlamydia trachomatis is recognized as a common sexually transmitted cervical pathogen that may be transmitted to the neonate at delivery. Neonatal infection may be manifested as conjunctivitis, pneumonitis or both. Additionally, cervical infection may be related to premature rupture of the membranes and premature delivery. Women registering for prenatal care in the first half of pregnancy were cultured for Chlamydia to further define its role as a cause of perinatal complications and to evaluate a method of preventing transmission to the neonate. Positive cultures were obtained in 33 of 221 women evaluated (14.9%). No significant differences were found in pregnancy or neonatal complications between the groups with positive and negative cultures. Women with positive cultures were treated at 36 weeks' gestation with erythromycin; 18 received an adequate course of therapy. Of these 18, 16 returned with their infants four to eight weeks postpartum. No symptoms of conjunctivitis or pneumonitis were noted at that time, and cultures of the conjunctiva and nasopharynx were negative in all the infants. Thus, surveillance for Chlamydia and treatment late in pregnancy appear to have no adverse impact on pregnancy and effectively block vertical transmission of the organism.
Chlamydia Infections↗
Frequency of Chlamydia trachomatis as the cause of pharyngitis.
We evaluated the incidence of Chlamydia trachomatis as the etiologic agent of uncomplicated pharyngitis by the cell culture procedure recommended by the Centers for Disease Control, Atlanta, Ga., and by the MicroTrak direct immunofluorescent stain (Syva Co., Palo Alto, Calif.) for elementary bodies on throat swabs collected from 126 symptomatic patients. Of the 126 cultures, 8% were positive for group A beta-hemolytic streptococci. Of 126 chlamydia cultures, none was positive. The MicroTrak test gave one borderline positive result. In contrast to a previously published report that C. trachomatis is the most frequent nonviral cause of adult pharyngitis (A. L. Komaroff, M. D. Aronson, T. M. Pass, C. T. Ervin, and W. T. Branch, Jr., Science 222:927-929, 1983), our data indicated an infection rate of less than 1%.
Adolescent↗