Sputum smear microscopy in the Xpert® MTB/RIF era.

A balanced perspective is advocated for the assessment and application of the most recent and the oldest diagnostic methods for pulmonary tuberculosis (TB)-the molecular Xpert® MTB/RIF assay and microscopy for acid-fast bacilli. We discuss their respective merits and shortcomings and identify threats that may hamper their use in TB control. Neither test on its own provides all the information needed for diagnosis and treatment monitoring. Considering all aspects important for both individual patient care and disease control, neither seems 'better' than the other. The required advancement of microscopy had already been hampered before the introduction of the GeneXpert technology by unsuccessful and probably misguided attempts to decentralise culture-based diagnosis and drug susceptibility testing. It seems evident that systematic replacement of microscopy by Xpert is not a viable option for the foreseeable future. Instead, the two methods should complement each other to arrive at a comprehensive, accessible and continuous service for a maximum number of patients. This will intrinsically prioritise targeting the most potent transmitters with the worst prognosis, simultaneously offering optimised prospects for efficient TB control. New microscopy and Xpert applications are expected to ultimately make control programmes independent of culture-based methods in diagnosis, treatment monitoring and outcome assessment.

Evaluation of new external quality assessment guidelines involving random blinded rechecking of acid-fast bacilli smears in a pilot project setting in Mexico.

SETTING: Laboratories in Mexico that support the national tuberculosis (TB) control program have been involved in an acid-fast bacilli (AFB) microscopy external quality assurance program which includes rechecking 100% of smears identified as AFB-positive by the local laboratories and 10% of smears identified as AFB-negative. Very few errors have been detected in Mexico using non-random selection and unblinded rechecking of the slides. OBJECTIVE: To evaluate the results from a 1-year pilot program involving blinded rechecking of randomly selected AFB slides from local TB laboratories in two Mexican states and determine its feasibility for future implementation. DESIGN: To reduce potential bias, laboratory staff from the National TB Laboratory, Institute for Epidemiological Diagnosis and Reference (InDRE), performed quarterly statistical sampling of AFB smears and on-site evaluations in local laboratories in each state. AFB smears were rechecked at the respective state laboratories with discordant results resolved at InDRE. RESULTS: A significantly greater percentage of errors was detected on the randomly selected, blinded AFB smears than on the non-randomly selected, unblinded smears. CONCLUSION: Random blinded rechecking provides more accurate estimates of AFB microscopy results, resulting in improved diagnosis and monitoring of treatment response.

Implementation of proficiency testing in conjunction with a rechecking system for external quality assurance in tuberculosis laboratories in Mexico.

SETTING: In developing countries, tuberculosis is diagnosed by identification of acid-fast bacilli (AFB) on sputum smears. OBJECTIVE: To evaluate the quality of AFB microscopy, the Mexican Secretary of Health National Reference Laboratory implemented proficiency testing for its network of 637 laboratories. DESIGN: A total of 586 (92%) laboratories were inspected and 430 technicians evaluated by proficiency testing consisting of 10 slides with known numbers of AFB. Results were compared with those of slide rechecking and with proficiency testing performed 2 years later. RESULTS: Of the 430 technicians evaluated by proficiency testing in 1998, 196 (46%) scored less than 80% and received intensive training in 1999. From a previous mean score of 65% their results increased to 90% (P < 0.0001). In 2001, they again underwent proficiency testing, and the mean score was 83%. The main factors affecting proficiency testing results were the type of laboratory in which the microscopists worked and the number of low-positive slides (1-9/100) in the test. Laboratories whose work was rechecked had better scores (P = 0.002). Proficiency testing scores and the estimated sensitivity of the microscopist's laboratory were associated (P = 0.01). CONCLUSION: External quality assessment and training improve diagnostic performance. Rechecking and proficiency testing are both viable measures of laboratory performance.

Quality assurance in the mycobacteriology laboratory. Quality control, quality improvement, and proficiency testing.

In conclusion, the components of QA (QC, QI, and PT) in the mycobacteriology laboratory address not only the accuracy of testing but also provide a measure for the laboratory practices that are necessary to effectively diagnose and control tuberculosis in the community. Given the extremely important role of laboratory testing in the control of tuberculosis, providing rapid diagnosis and determination of susceptibility test results, laboratories should use the recommendations concerning turnaround times as either achievable goals or a measure of whether the laboratory should be performing mycobacteriology tests. We strongly urge laboratories to include their turnaround times for test results and the timeliness of reporting those results in their annual QA program.

Fusion of inclusions following superinfection of HeLa cells by two serovars of Chlamydia trachomatis.

We used a double-label immunofluorescence assay to examine the ability of Chlamydia trachomatis serovar F to infect and develop within HeLa 229 cells previously infected with serovar E. No exclusion to superinfection occurred for up to 24 h following infection by serovar E. The percentage of HeLa cells infected in cultures inoculated with both strains was identical to that of cells in cultures inoculated with one strain as a control. Organisms of both serovars were located within the same intracellular inclusion in 88 to 95% of HeLa cells infected with both serovars. The proportion of superinfected HeLa cells containing both strains in separate inclusions increased when there was exposure to inhibitors of cytoskeletal structure and transport. We used this inhibition to demonstrate that fusion of C. trachomatis phagosomes occurs throughout the developmental cycle.

Two confirmatory tests for identification of Neisseria gonorrhoeae from primary culture.

We compared a fluorescent monoclonal antibody and a DNA probe for identification of Neisseria gonorrhoeae (GC) from primary genital cultures of presumptive GC and selected bacterial isolates other than GC. The monoclonal antibody was sensitive (94%) and specific (100%) enough to identify GC in selective primary genital culture. The DNA probe was sensitive (95%) but not adequately specific (65%) to function as a confirmatory test.

Multicenter evaluation of ethambutol susceptibility testing of mycobacterium tuberculosis by agar proportion and radiometric methods.

Reproducibility of ethambutol (EMB) susceptibility test results for Mycobacterium tuberculosis has always been difficult for a variety of reasons, including the narrow range between the critical breakpoint for EMB resistance and the MIC for susceptible strains, borderline results obtained with the BACTEC 460TB method, the presence of microcolonies determined using the agar proportion (AP) method, and a lack of agreement between these two testing methods. To assess the frequency of these problems, M. tuberculosis drug susceptibility data were collected in a multicenter study involving four laboratories. Resistant, borderline, and susceptible isolates were shared among the laboratories to measure interlaboratory test agreement. Half of isolates determined by BACTEC 460TB to be resistant were determined to be susceptible by the AP method. Isolates determined to be resistant to EMB by both BACTEC 460TB and AP methods were almost always resistant to isoniazid. Results from isolates tested by the BACTEC 460TB method with an EMB concentration of 3.75 micro g/ml in addition to the standard 2.5 micro g/ml did not show improved agreement by the AP method. While these results do not indicate that the AP method is more accurate than the BACTEC 460TB method, laboratories should not report EMB monoresistance based on BACTEC 460TB results alone. Monoresistance to EMB should only be reported following confirmation by the AP method. Microcolonies could not be confirmed as resistant by the BACTEC 460TB method or by repeat testing with the AP method and do not appear to be indicative of resistance.

Chronic tenosynovitis of the hand due to Mycobacterium nonchromogenicum: use of high-performance liquid chromatography for identification of isolates.

Six cases of chronic tenosynovitis of the hand due to the Mycobacterium terrae complex were identified. All isolates from the six cases were identified as Mycobacterium nonchromogenicum by high-performance liquid chromatography and by testing for susceptibility to ofloxacin and to 5% NaCl. Ethambutol, sulfonamides (or trimethoprim-sulfamethoxazole), erythromycin, and streptomycin are the drugs most active against isolates of the M. terrae complex, and therapy with some combination of these agents plus surgical debridement offers the best current treatment of this disease. This study supports the contention arising from previous case reports of pulmonary disease that M. nonchromogenicum is the pathogenic member of the M. terrae complex.

Multicenter evaluation of a nonweekend reading schedule for radiometric pyrazinamide susceptibility testing of Mycobacterium tuberculosis.

Pyrazinamide (PZA) is an integral component of the short-course chemotherapy regimen for tuberculosis. The BACTEC 460TB PZA susceptibility test for Mycobacterium tuberculosis with a daily (D) reading schedule has been available for more than 10 years, but weekend laboratory staffing is necessary. A nonweekend (NW) reading schedule has not been validated in a multicenter study. This prospective multicenter study compares the interlaboratory reproducibility of PZA susceptibility results by following both the D and NW schedules. A total of 181 cultures were shared among four laboratories. Isolates were selected based on resistance or borderline resistance to at least one streptomycin-isoniazid-rifampin-ethambutol drug or PZA. One laboratory used a D reading schedule, and three laboratories used a NW schedule. Both reading schedules are based on the standard BACTEC 460TB PZA protocol. With the NW schedule, the growth index (GI) is not available for test interpretation on Saturday, Sunday, and Monday. Of the 181 shared cultures, 154 were found to be susceptible by all laboratories, 19 were found to be resistant, and 8 had discordant results. The overall pairwise interlaboratory agreement was 97.7%. The discrepancies were not associated with the type of reading schedule used. However, the median control GI was significantly higher for the NW schedule (321) than for the D schedule (259) (P < 0.0001) although results were available on average in about 7 days from setup for both schedules. These results show that the NW schedule is a suitable alternative for laboratories that do not read and interpret PZA susceptibility tests on weekends.

Laboratory diagnosis of sexually transmitted diseases in facilities within the United States. Results of a national survey.

BACKGROUND AND OBJECTIVES: The diagnosis of many sexually transmitted diseases (STD) requires laboratory testing. The authors assessed the effects of the introduction of new tests and regulations on STD testing. STUDY DESIGN: A questionnaire survey was mailed to a random sample of facilities listed in the STD Referral Database inquiring about tests offered, changes in testing, and reasons for changes. RESULTS: Responses from 405 facilities were analyzed. Most responding facilities collected specimens for nontreponemal tests for syphilis (352 of 405 [86.9%]). Since each facility's information was last updated, the number reporting testing for Chlamydia trachomatis rose from 160 of 405 (39.5%) to 288 of 405 (71.1%), but testing for gonorrhea and chancroid decreased (365 of 405 [90.1%] to 328 of 405 [81%], and 182 of 405 [44.9%] to 32 of 405 [7.9%], respectively). Of 364 responses to a question on changes in tests performed in the last 2 years, 249 (68.4%) reported no change, 81 (22.3%) reported an increase, and 37 (10.2%) reported a decrease. The most frequently added tests were nonculture tests for C. trachomatis (34 of 81 [42%]) and the most frequent reason for adding tests was targeted funding (25 of 81 [30.9%]). The most frequently discontinued tests were cultures and gram stains for gonorrhea (15 of 37 [40.5%]) and other in-house tests (9 of 37 [24.3%]). Most facilities that discontinued testing cited the Clinical Laboratory Improvement Act as the reason (34 of 37 [91.9%]; 95% confidence interval = 78.1%, 98.3%). CONCLUSIONS: The number of facilities testing for C. trachomatis has increased with funding and with the availability of nonculture tests, but the number of those testing for chancroid and gonorrhea has decreased. Implementation of the Clinical Laboratory Improvement Act may be associated with a decrease in the number of facilities performing tests for STD.