Evaluation of a novel endoluminal stapling procedure to restrict the lower esophageal sphincter and reduce reflux.

BACKGROUND AND STUDY AIMS: An effective, safe, and long-lasting endoluminal treatment for gastroesophageal reflux disease (GERD) would be an attractive prospect. We developed an endoluminal technique to restrict and tighten the lower esophageal sphincter (LES), by using a transoral endoscopic stapling device in a porcine model. PATIENTS AND METHODS: Pre-interventional evaluation comprised endoscopy, manometry, and 48-hour pH measurement of the distal esophagus using the catheterless BRAVO pH capsule. By placing the endoluminal stapling device at the LES and firing a 2.5-cm staple line, a vertical plication was created. In five pilot pigs (phase 1), plications were placed in various locations at the LES. In another five pigs (phase 2), plications were placed uniformly at the mid level of the LES on the lesser curvature side. Measurements were repeated 2 weeks after the procedure. Necropsy and histological analysis were performed. RESULTS: Endoluminal stapling was successfully completed in all animals. In phase 2, the median procedure time was 15 minutes (range 10-55 minutes). LES pressure increased from 10.5 mmHg (+/- 2.5 mmHg) to 14.3 mmHg (+/- 3.8 mmHg) (P = 0.038). Median percentage of time with pH below 4 decreased from 6.6% (range 2.9%-48.8%) to 2.2% (range 0%-10.4%) (P = 0.043). Histology showed the staple line involving the muscular layer in all pigs. A gap was present in the central part of the staple line in three pigs resulting in a mucosa-muscular bridge of tissue. This bridge did not influence the results. CONCLUSION: This novel endoluminal technique is feasible and safe in a porcine model over 2 weeks. It is appealing due to its simplicity and ease of application. Further studies aimed at eliminating the gap in the staple line and investigating more animals over longer survival periods are needed.

Sialoblastoma and hepatoblastoma in a neonate.

We report a case of salivary gland neoplasm and associated hepatoblastoma. The sialoblastoma was diagnosed by prenatal sonography; however, the hepatoblastoma was imaged post-operatively. Prior knowledge could have prevented a subsequent operation, including the additional risks of repeat anesthesia. We suggest that if a sialoblastoma is in the differential diagnosis, then additional imaging may be indicated because of the possibility of associated lesions.

Growth inhibition of Mycobacterium tuberculosis by polyoxyethylene stearate present in the BACTEC pyrazinamide susceptibility test.

We have previously found that approximately 3.5% of 428 clinical isolates of Mycobacterium tuberculosis yield uninterpretable results in the BACTEC pyrazinamide (PZA) susceptibility test system, because of inadequate growth. We tested the hypothesis that polyoxyethylene stearate (POES), the ingredient of the reconstituting fluid for the test, was the cause of this growth inhibition. A total of 15 isolates known for their previously uninterpretable results and 100 randomly chosen clinical isolates were tested in parallel both with and without POES. Repeat testing of the isolates with previously uninterpretable results yielded results in the presence of POES in only seven (47%). In the absence of POES, all gave interpretable results but one such result showed false resistance. For the other 100 clinical isolates, interpretable results were obtained with and without POES, but growth was enhanced in the absence of POES, especially in the PZA-susceptible strains. This was evidenced by a decreased time to attain a growth index of 200 in the control vial (4.9 days without POES versus 5.8 days with POES; P < 0.001) and a higher mean growth index ratio on the day of interpretation of the test (7.4% without POES versus 2.2% with POES; P < 0.001). However, the enhanced growth without POES led to 20 susceptible strains being misinterpreted as either resistant or borderline. We suggest that isolates of M. tuberculosis which yield uninterpretable results in the BACTEC PZA test system should be retested both with and without POES. If interpretable results indicating PZA resistance are obtained only in the absence of POES, the result should be confirmed by a pyrazinamidase assay or by the conventional proportion method. Routine omission of POES from the BACTEC test for all clinical strains is discouraged because of the unacceptably high false-resistance rates.

Testing of susceptibility of Mycobacterium tuberculosis to pyrazinamide: comparison of Bactec method with pyrazinamidase assay.

The susceptibility of 428 clinical isolates of Mycobacterium tuberculosis to pyrazinamide was assessed by the Bactec method and the Wayne pyrazinamidase assay. The correlation between the two tests was 98.2 and 100% for susceptible and resistant strains, respectively. False resistance was seen in four (0.8%) strains with the Bactec test, and false-susceptible results occurred in two (0.5%) pyrazinamidase assays. The Bactec test is rapid and reliable, and the Bactec results correlate well with the pyrazinamidase test results, although some strains did not grow well in the test medium.

Rapid broth macrodilution method for determination of MICs for Mycobacterium avium isolates.

A multicenter study was done to investigate the accuracy and reproducibility of a method for determining the MICs of antimicrobial agents against the Mycobacterium avium complex in 7H12 broth with the BACTEC system. In phase I, with eight drugs and 10 strains, intralaboratory reproducibility was 95.7 to 100%, allowing a 1-dilution difference upon repeat testing. The results of phase II testing with 41 additional strains were consistent with those obtained in phase I, with good interlaboratory reproducibility. The radiometric method was validated by sampling and plating of the same broth cultures and determining, by the number of CFU per milliliter, the lowest drug concentration that inhibited more than 99% of the initial bacterial population. Three test concentrations of each drug and the tentative interpretation of results are proposed. Radiometric MIC determination has the potential to become the method of choice for clinical microbiology laboratories and evaluation of new agents for the treatment of M. avium infections, both pulmonary and disseminated.

Bacteriologic investigations of unusual mycobacteria isolated from immunocompromised patients.

Mycobacterial isolates from blood and other extrapulmonary sites of six patients with AIDS were investigated because the isolates grew only in liquid media and failed to grow on solid culture media even on subculturing. Our investigations indicated that these mycobacteria possess common, but unusual, characteristics and probably belong to an unrecognized species recently reported as "Mycobacterium genavense."

Role of solid media when used in conjunction with the BACTEC system for mycobacterial isolation and identification.

This study evaluated the necessity and the contribution of solid media when used in conjunction with radiometric Middlebrook 7H12 (BACTEC 12B; Becton Dickinson, Towson, Md.) medium for recovery and complete identification of mycobacteria. Each of 1,184 digested, decontaminated respiratory specimens was inoculated into one BACTEC 12B vial, one 7H11 plate, and two Lowenstein-Jensen (LJ) slants. When the 12B vial was smear positive for acid-fast bacilli, the organisms were subcultured onto LJ slants and the BACTEC p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test system was inoculated with the organisms. Niacin tests were performed by using the growth from the original LJ slants and organisms from the LJ slants subcultured from 12B or 7H11 medium. The times to achieve definitive NAP and niacin test results were recorded. Recovery of all 143 isolates found in this study could not be achieved with a single medium. Among the three media, the highest percentage (92.8%) of Mycobacterium tuberculosis isolate recovered was with BACTEC 12B. The use of either 7H11 medium or LJ slants along with a 12B vial increased by 4 to 6% the total percentage of M. tuberculosis organisms that were isolated. Isolation of the M. tuberculosis complex and NAP differentiation in 12B medium were completed in an average of 17 days. On average, isolation and definitive niacin test results for M. tuberculosis cultures were obtained in 39.3 days by a conventional procedure and in 36.3 days when 12B subcultures were used. These results support the conclusion that LJ slants contribute 4 to 6% increased recovery of M. tuberculosis when used in conjunction with 12B medium. Additionally, a subculture onto LJ slants from 12B medium yielded sufficient growth for niacin testing earlier than an original LJ slant did.

Drug resistance of Mycobacterium tuberculosis isolated from treated patients in Pakistan.

An investigation was carried out to establish the extent of drug resistance among treated patients. A sample population of patients living in Lahore, Pakistan, which is a high prevalence area for tuberculosis, was studied. The total of 256 culture-positive cases in this study were divided into three groups according to the length of previous treatment. There was no significant difference in the antituberculosis treatment regimens or the drug resistance pattern among the three groups. All the patients had had at least three drugs for more than 6 months, and streptomycin and isoniazid were always included in the regimen. About one-third of the patients showed resistance to one or more drug, with the highest resistance being to streptomycin and INH. Resistance to rifampicin, which was introduced fairly recently in this area, was a little more than 5%, which is an increase from the last report.

Evaluation of a new blood culture medium for mycobacteria.

This study evaluates the sensitivity and detection time of a new radiometric broth (BACTEC 13A medium, Johnston Laboratories, Towson, MD) designed for direct blood culturing for Mycobacteria. A total of 1,848 blood specimens were cultured in parallel by a new method in which blood was directly inoculated into 13A medium and a comparative method involving lysis-centrifugation and inoculation of 7H11 agar and 7H12 broth media. There were 65 blood cultures with positive results (64 Mycobacterium avium-intracellulare and 1 Mycobacterium tuberculosis) from 43 patients. For the direct inoculation and lysis-centrifugation methods, the recovery rates were 96.9% and 98.5%, respectively, and the mean times to detection were essentially the same (23.6 and 23.8 days, respectively). Thus, the direct-inoculation technique using 13A medium showed performance equivalent to the lysis-centrifugation method. Also, the direct method has the advantage of elimination of the time-consuming manual manipulations that are required by lysis-centrifugation methods.

Comparison of BACTEC 13A medium and Du Pont isolator for detection of mycobacteremia.

BACTEC 13A medium (Johnston Laboratories, Towson, Md.) was compared with Isolator (Du Pont Co., Wilmington, Del.) concentrate for sensitivity, speed, and technical ease of isolation of mycobacteria from paired patient blood samples. Of 72 positive cultures, 63 were positive by both systems. Five positive cultures were detected by BACTEC 13A medium alone, and four were detected by Isolator alone. The median numbers of days to positivity were 12 for BACTEC 13A medium and 14 for Isolator concentrate. BACTEC 13A medium has an advantage over the Isolator in requiring less laboratory manipulation of the specimen but has the disadvantages of not providing isolated colonies or quantitation of organisms. Some technical problems with contamination in both systems are also discussed.

Identification of Mycobacterium tuberculosis in sputum by direct inoculation of the BACTEC NAP vial.

The Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, and M. africanum) can be differentiated from mycobacteria other than M. tuberculosis (MOTT bacilli) with the BACTEC NAP test (Johnston Laboratories, Becton, Dickinson & Co., Towson, MD), by selectively inhibiting their growth with p-nitro-alpha-acetyl-amino-beta-hydroxypropriophenone (NAP). The BACTEC NAP test is recommended for isolated mycobacterial cultures. In this report, a direct NAP test is performed by immediate inoculation of BACTEC NAP vials with processed sputum specimens that stain acid-fast positive. Seventy-six of 80 M. tuberculosis were correctly identified and all of the MOTT bacilli (nine M. avium complex and one M. kanasii) were correctly classified. The average time required for identification of M. tuberculosis with the direct BACTEC NAP test described here is more convenient than the recommended indirect test, and it is an accurate and rapid method to differentiate the M. tuberculosis complex from MOTT bacilli.

Direct antimicrobial drug susceptibility testing of Mycobacterium tuberculosis by the radiometric method.

Direct-drug-susceptibility tests were performed on clinical specimens positive for acid-fast bacilli by either Ziehl-Neelsen or fluorochrome staining. The results of conventional agar dilution (Vestal, 1975) and a modified radiometric (BACTEC) method were compared. A total of 580 smear-positive specimens were tested by the BACTEC method at three separate sites. Three hundred and seventy-seven of these were culture positive for M. tuberculosis, and 343 (91%) yielded acceptable direct-susceptibility-test results. We used the conventional method to determine that 343 of 519 smear-positive specimens were culture positive for M. tuberculosis, and 212 (62%) produced acceptable results within 3 wks. Conventional results were reported in 3-4 wks, while the time required to obtain results with the BACTEC method ranged from 5 to 21 days (average 11.5 days). Results indicate that the radiometric method provides reportable results more frequently with time savings as compared to the conventional method.

Interlaboratory drug susceptibility testing of Mycobacterium tuberculosis by a radiometric procedure and two conventional methods.

A total of 224 recent isolates of Mycobacterium tuberculosis from 163 patients selected to have multidrug resistance were tested against streptomycin (SM), isoniazid, rifampin, and ethambutol (EMB) by the rapid radiometric BACTEC method and two conventional proportion methods: the World Health Organization (WHO) method, using Lowenstein-Jensen medium; and the Veterans Administration reference laboratory for mycobacteria (VA) method, using Middlebrook 7H10 agar medium. The results were compared, focusing on the concentrations of the drugs in all three methods. Among the four drugs tested, most of the discrepancies in measured activity were observed with SM and EMB, generally because of differences in the drug concentrations used by the three methods. A 4-micrograms amount of SM in the BACTEC method was found to be slightly less active than 10 micrograms in the VA method and significantly more active than 4 micrograms of dihydrostreptomycin in the WHO method. With EMB, 2.5 micrograms in BACTEC was similar to 5 micrograms in the VA method and 2 micrograms in the WHO method, while 10 micrograms in the BACTEC method was found to be more active than 10 and 2 micrograms in the VA and WHO methods, respectively. To attain close agreement, drug concentrations used in the BACTEC method should be carefully selected when a comparison is to be made with any conventional method employed in a laboratory. Standardization of in vitro susceptibility testing is greatly needed to achieve uniformity among the test methods used to evaluate tuberculosis therapeutics.

Rapid radiometric methods to detect and differentiate Mycobacterium tuberculosis/M. bovis from other mycobacterial species.

Rapid methods for the differentiation of Mycobacterium tuberculosis/M. bovis (TB complex) from other mycobacteria (MOTT bacilli) were developed and evaluated in a three-phase study. In the first phase, techniques for identification of Mycobacterium species were developed by using radiometric technology and BACTEC Middlebrook 7H12 liquid medium. Based on 14CO2 evolution, characteristic growth patterns were established for 13 commonly encountered mycobacterial species. Mycobacteria belonging to the TB complex were differentiated from other mycobacteria by cellular morphology and rate of 14CO2 evolution. For further differentiation, radiometric tests for niacin production and inhibition by Q-nitro-alpha-acetyl amino-beta-hydroxy-propiophenone (NAP) were developed. In the second phase, 100 coded specimens on Lowenstein-Jensen medium were identified as members of the TB complex, MOTT bacilli, bacteria other than mycobacteria, or "no viable organisms" within 3 to 12 (average 6.4) days of receipt from the Centers for Disease Control. Isolation and identification of mycobacteria from 20 simulated sputum specimens were carried out in phase III. Out of 20 sputum specimens, 16 contained culturable mycobacteria, and all of the positives were detected by the BACTEC method in an average of 7.3 days. The positive mycobacterial cultures were isolated and identified as TB complex or MOTT bacilli in an average of 12.8 days. The radiometric NAP test was found to be highly sensitive and specific for a rapid identification of TB complex, whereas the radiometric niacin test was found to have some inherent problems. Radiometric BACTEC and conventional methodologies were in complete agreement in Phase II as well as in Phase III.

Evaluation of a rapid radiometric differentiation test for the Mycobacterium tuberculosis complex by selective inhibition with p-nitro-alpha-acetylamino-beta-hydroxypropiophenone.

This study is an evaluation of a rapid technique for the differentiation of the Mycobacterium tuberculosis complex from other mycobacteria, using p-nitro-alpha-acetylamino-beta- hydroxypropiophenone (NAP) as a selective inhibitory agent. A total of 416 coded cultures, 234 cultures belonging to the M. tuberculosis complex and 182 cultures belonging to 35 other mycobacterial species, were tested in two laboratories for p-nitro-alpha-acetylamino-beta- hydroxypropiophenone inhibition to concentrations of 5 and 10 micrograms of NAP per ml in Middlebrook 7H12 liquid medium. Two testing modes were compared: the indirect, in which a large bacterial inoculum was used from an isolated culture on a solid medium, and the direct, which used a small inoculum from 7H12 medium. A decrease or no increase in daily 14CO2 output as measured by a BACTEC system was considered evidence of inhibition. The data presented show that a concentration of 5 micrograms of NAP per ml can effectively separate the M. tuberculosis complex from other mycobacterial species in 4 to 6 days. The direct test data show that, unlike other conventional biochemical tests, it does not require a heavy inoculum of mycobacteria and can therefore be performed soon after growth is detected by the radiometric method.

Evaluation of the BACTEC radiometric method for recovery of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis from acid-fast smear-positive specimens.

A total of 463 respiratory specimens, all smear positive for acid-fast bacteria, were inoculated onto routine solid media and into BACTEC 7H12 Middlebrook medium for detection of mycobacterial growth. Conventional drug susceptibility testing (1% proportion method) was performed on Middlebrook 7H10/7H11 medium, and radiometric susceptibility testing was performed on BACTEC 7H12 medium. The average detection times for BACTEC-positive cultures were 8.3 days for Mycobacterium tuberculosis and 5.2 days for mycobacteria other than tuberculosis; by conventional methods, they were 19.4 and 17.8 days, respectively. These detection times do not include time required for identification, which was done by the conventional method only. There was an excellent correlation in the recovery rates of mycobacteria by the two methods. Drug susceptibility test results of M. tuberculosis isolates by the two methods showed 95.1 to 100% overall agreement. The average reporting time for drug susceptibility results ranged from 4.2 to 6.9 days for the BACTEC method and 13.7 to 21 days for the conventional methods. An average of 18 days was required by the BACTEC method for complete recovery and drug susceptibility testing of M. tuberculosis, as compared with 38.5 days for the conventional methods.

Resistance to rifampicin and isoniazid in strains of Mycobacterium tuberculosis.

Drug susceptibility studies on strains of Mycobacterium tuberculosis isolated from widely different populations of patients and tested by two different techniques indicated that all 55 strains resistant to rifampicin were also resistant to isoniazid, while many strains resistant to isoniazid were found to be susceptible to rifampicin. This observation, which has as yet unknown laboratory and clinical significance, may be particularly useful in management of patients. Further studies are called for to establish this relation.

Evaluation of rapid radiometric method for drug susceptibility testing of Mycobacterium tuberculosis.

A total of 106 isolates of Mycobacterium tuberculosis were tested for drug susceptibility by the conventional 7H11 plate method and by a new rapid radiometric method using special 7H12 liquid medium with 14C-labeled substrate. Results obtained by the two methods were compared for rapidity, sensitivity, and specificity of the new test method. There was 98% overall agreement between the results obtained by the two methods. Of a total of 424 drug tests, only 8 drug results did not agree, mostly in the case of streptomycin. This new procedure was found to be rapid, with 87% of the tests results reportable within 4 days and 98% reportable within 5 days as compared to the usual 3 weeks required with the conventional indirect susceptibility test method. The results of this preliminary study indicate that the rapid radiometric method seems to have the potential for routine laboratory use and merits further investigations.