Processing postmortem specimens with C18-carboxypropylbetaine and analysis by PCR to develop an antemortem test for Mycobacterium avium infections in ducks.

Mycobacterium avium is the causative agent of the avian mycobacteriosis commonly known as avian tuberculosis (ATB). This infection causes disseminated disease, is difficult to diagnose, and is of serious concern because it causes significant mortality in birds. A new method was developed for processing specimens for an antemortem screening test for ATB. This novel method uses the zwitterionic detergent C18-carboxypropylbetaine (CB-18). Blood, bone marrow, bursa, and fecal specimens from 28 ducks and swabs of 20 lesions were processed with CB-18 for analysis by smear, culture, and polymerase chain reaction (PCR). Postmortem examination confirmed nine of these birds as either positive or highly suspect for disseminated disease. The sensitivities of smear, culture, and PCR, relative to postmortem analysis and independent of specimen type, were 44.4%, 88.9%, and 100%, respectively, and the specificities were 84.2%, 57.9%, and 15.8%, respectively. Reductions in specificity were due primarily to results among fecal specimens. However, these results were clustered among a subset of birds, suggesting that these tests actually identified birds in early stages of the disease. Restriction fragment length polymorphism mapping identified one strain of M. avium (serotype 1) that was isolated from lesions, bursa, bone marrow, blood, and feces of all but three of the culture-positive birds. In birds with confirmed disease, blood had the lowest sensitivity and the highest specificity by all diagnostic methods. Swabs of lesions provided the highest sensitivity by smear and culture (33.3% and 77.8%, respectively), whereas fecal specimens had the highest sensitivity by PCR (77.8%). The results of this study indicate that processing fecal specimens with CB-18, followed by PCR analysis, may provide a valuable first step for monitoring the presence of ATB in birds.

Evaluation of a Treponema pallidum enzyme immunoassay as a screening test for syphilis.

The CAPTIA Syphilis-G enzyme immunoassay for the detection of antibodies to Treponema pallidum was evaluated as a screening test for syphilis in comparison with the standard rapid plasma reagin (RPR) test. One thousand samples were tested, and the standard fluorescent treponemal antibody absorption test and the standard microhemmaglutination test were used to confirm the presence of treponemal antibodies. Diagnosis of syphilis was based on traditional standard serology results. Clinical data used in the diagnosis of patients whose samples yielded conflicting results were provided by physicians. Initially, 7 patients whose samples were reactive in the RPR test and 14 patients whose samples yielded positive or equivocal results in the CAPTIA Syphilis-G test were diagnosed as not being infected. After discrepancies due to technical problems were reconciled, samples from six patients remained reactive in the RPR test and that from one patient remained positive in the CAPTIA Syphilis-G test. In addition, seven patients whose samples were nonreactive in the RPR test and two patients whose samples were negative in the CAPTIA Syphilis-G test were diagnosed as having untreated syphilis. After discrepancies were reconciled, samples from five patients remained nonreactive in the RPR test and none remained negative in the CAPTIA Syphilis-G test. Final results indicate that the specificities are 99.4 and 99.9%, respectively. In addition to the improved sensitivity and specificity of the CAPTIA Syphilis-G screen, other potential benefits of this assay lead us to believe that this method could serve as a better screening tool than the RPR test.

In vitro comparison of NALC-NaOH, tween 80, and C18-carboxypropylbetaine for processing of specimens for recovery of mycobacteria.

A recent article (C. G. Thornton et al., J. Clin. Microbiol. 36:1996-2003, 1998) reported a new specimen-processing method for improved recovery of mycobacteria. This method used C18-carboxypropylbetaine (CB-18) and increased both smear and culture sensitivity. The companion article (C. G. Thornton et al., J. Clin. Microbiol. 36:2004-2013, 1998) described initial improvements to this method. Additional significant parameters of the CB-18 processing method are identified herein. First, eliminating the incubation step was shown to further improve culture sensitivity. Subsequently, recovery of several mycobacterial isolates by the CB-18 method was compared to a contemporary processing method that combines NALC and NaOH (NALC-NaOH) and a Tween 80-based method. Recovery of the tuberculous isolates following NALC-NaOH processing averaged 20% and ranged from 1.6 to 45%, whereas recovery of the nontuberculous isolates averaged 11% and ranged from 0.1 to 55%. Recovery of the tuberculous and nontuberculous isolates by the Tween 80-based method ranged from 22 to 92% and 27 to 93%, respectively, with averages of 58 and 65%, respectively. Recovery of the tuberculous and nontuberculous mycobacteria following CB-18 processing averaged 86 and 73%, respectively, with ranges from 61 to over 100% and from 43 to over 100%, respectively. Other parameters of the CB-18 method were also examined, including recovery versus CB-18 concentration and the relationship between CB-18 concentration and the tuberculocidal effect. The tuberculocidal effect was time dependent but independent of concentration, whereas recovery was directly proportional to concentration. Increasing the CB-18 concentration to 4 mM provided quantitative recovery on solid medium; however, higher concentrations of CB-18 were not compatible with liquid culture. Examination of the relationship between increasing CB-18 and lecithin concentrations suggested that lecithin could not overcome the deleterious effects of CB-18 in liquid culture at these higher concentrations.

Processing respiratory specimens with C18-carboxypropylbetaine: development of a sediment resuspension buffer that contains lytic enzymes to reduce the contamination rate and lecithin to alleviate toxicity.

The C18-carboxypropylbetaine (CB-18) procedure for processing respiratory specimens for the detection of mycobacteria was shown to provide significant increases in sensitivity by smear and culture. However, the procedure also produced increased contamination, a loss in liquid culture sensitivity, and a reduction in smear specificity. Because of these observations, the toxicity of CB-18 and the nature of the contamination were characterized. Preincubation in 1 mM CB-18 impacted viability in a time-dependent fashion, but the magnitude of the loss was species and isolate dependent. Mycobacterium tuberculosis isolates were the most susceptible, losing 20 to 30% of the CFU within 30 min and 30 to 60% after 3 h, whereas Mycobacterium avium and Mycobacterium fortuitum isolates were unaffected by CB-18. In liquid culture, when the concentration of CB-18 exceeded 5 microg/ml, there was an impact on growth characteristics for the most susceptible M. tuberculosis isolate. In contrast, M. fortuitum isolates were able to grow in 100 microg of CB-18 per ml. In liquid culture, the deleterious effects of CB-18 were enhanced in the presence of antibiotics, whereas growth on solid media was not similarly affected. Supplementation of the resuspension buffer with 0.15% lecithin alleviated toxicity. Initial attempts to modify the CB-18 procedure to control contamination incorporated acids or alkalis; however, losses in culture sensitivity occurred. Studies to identify these contaminants led to the development of a sediment resuspension buffer that contained lytic enzymes to combat contamination and lecithin to alleviate toxicity. This formulation included lysozyme, zymolyase, and Cytophaga and Trichoderma extracts and was seen to reduce contamination to acceptable levels (<5%).

Novel method for processing respiratory specimens for detection of mycobacteria by using C18-carboxypropylbetaine: blinded study.

A novel method for processing respiratory specimens to improve culture and acid-fast staining of mycobacteria is introduced. This new method utilized N,N-dimethyl-N-(n-octadecyl)-N-(3-carboxypropyl)ammonium inner salt (Chemical Abstract Service no. 78195-27-4), also known as C18-carboxypropylbetaine (CB-18). In a blinded, five-center study, CB-18-based processing was compared to the standard method combining NALC and NaOH (NALC/NaOH). A total of 573 respiratory specimens were tested. Individual specimens were split approximately equally; the host institutions processed half of each specimen by the NALC/NaOH method, while the other half was processed with CB-18 at Quest Diagnostics--Baltimore. A total of 106 specimens were culture positive for acid-fast bacilli (AFB). Replacement of the primary decontamination agent with CB-18 caused changes in all diagnostic parameters. Aggregate culture sensitivity improved by approximately 43% (P < 0.01), and smear sensitivity improved by approximately 58% (P < 0.01). The sensitivity of smear relative to that of M. tuberculosis isolates exceeded 93% (P < 0.01) when specimens were processed with CB-18. The average times to a positive result were reduced by 7.3 days in liquid culture (P < 0.01) and 5.3 days on solid media (P < 0.05); however, the CB-18 method had a 20.8% contamination rate in liquid culture versus a rate of approximately 7.5% with NALC/NaOH processing. There were also unusual reductions in liquid culture sensitivity and smear specificity among CB-18-processed specimens. The characteristics of the latter parameters suggested that refinement of the CB-18 processing method should allow further improvements in culture sensitivity. This study showed that the CB-18 method has the potential to improve both smear and culture detection for these important human pathogens.