Azlocillin

Study objective: The purpose of this study was to determine whether the time to detection (TTD) of Mycobacterium tuberculosis in sputum culture correlates with the response to antituberculous treatment in patients with pulmonary tuberculosis.

Study design: Twenty-six consecutive patients were studied who bad active pulmonary tuberculosis and sufficient sputum cultures and clinical follow-up to allow adequate assessment.

Results: Following initiation of antituberculous therapy, 13 patients (group 1, responders) had a complete response to treatment, and the TTD of M tuberculosis using the mycobacterial growth indicator tube increased steadily. The remaining 13 patients (group 2, nonresponders) had persistent evidence of active disease and demonstrated little or no increase in the TTD with treatment unless an additional therapeutic intervention was implemented (surgery, improved compliance with medications, or a change in medications). The presence of HIV infection, intravenous drug use, multidrug resistance, treatment with second-line therapy, extensive radiographic involvement, and cavitary disease were associated with a delayed increase in the TTD.

Conclusions: The TTD was superior to clinical, radiographic, or conventional bacteriologic evaluation in determining treatment outcome. The TTD closely correlates with the overall response to treatment for pulmonary tuberculosis and may represent a useful adjunct to predict outcome in these patients. (CHEST 1998; 113:379-86)

Key words: Mycobacterium tuberculosis: outcome; treatment

Abbreviations: AFB=acid-fast bacilli; PCR=polymerase chain reaction; TTD=time to detection

Consequent to the recent resurgence of tuberculosis, the rapid diagnosis of patients with active disease has become a focus of interest.[1,2] Of equal importance to the control of tuberculosis is the assurance of effective treatment,[3] yet over the past two decades, there has been little progress in the ability to predict a patient's response to antituberculous therapy. Assessment or prediction of treatment response is especially important in patients at high risk for treatment failure (such as those with multi-drug-resistant tuberculosis or HIV infection or both) but remains essentially limited to clinical, radiographic, and conventional bacteriologic evaluation despite the inherent inaccuracies of these methods.[2,4] For example, although improving clinical status may indicate a response to treatment, patient heterogeneity and the presence of other concurrent illnesses reduce the certainty of clinical evaluation. Radiographic resolution as a marker of treatment response is inadequate because it lags significantly behind clinical improvement.[5,6] Various serologic markers have been evaluated as indicators of disease activity but their clinical usefulness remains uncertain.[7-9] The most widely accepted measure of treatment response in patients with pulmonary tuberculosis is the disappearance of acid-fast bacilli (AFB) from sputum, as assessed by microscopic examination and culture.[6] Unfortunately, smear sensitivity depends on the patient, the type and degree of pulmonary parenchymal involvement, and the skill of the microscopist, and it ranges from 22 to 80%.[10] Specificity is reduced by the presence of nontuberculous mycobacteria or dead organisms.[2,4] Sputum culture is superior to direct examination in detecting AFB, but because Mycobacterium tuberculosis is relatively slow growing, serial sputum cultivation is an inefficient means to assess treatment response.[4]

In 1993, a preclinical study of the mycobacterial growth indicator tube (MGIT; Becton-Dickinson; Cockeysville, Md) was conducted and found that this system can accurately detect the presence of M tuberculosis in as short a time as 2 days.[11] This study observed that the time to culture detection (TTD) of M tuberculosis in serial samples steadily increased in most patients receiving treatment for pulmonary tuberculosis. Another observation was that in those patients in whom the TTD did not increase, there was a poor response to antituberculous therapy.[12] In order to further evaluate the relationship between the TTD of M tuberculosis in the sputum of patients with active pulmonary tuberculosis and their response to treatment, a systematic analysis was executed on patients who had received treatment for pulmonary tuberculosis and a comparison of their TTD data was made between these and clinical, radiographic, and microbiologic characteristics.

Methods

Patient Study Group

A review of data was done on all patients with a diagnosis of pulmonary tuberculosis who were admitted to the Chest Service of Bellevue Hospital Center in New York City between January 1, 1995, and June 30, 1995. Patients were included in this study if they had adequate clinical, radiographic, and microbiologic records; 6 or more positive sputum cultures over a period of at least 2 weeks (to allow for adequate data analysis); and adequate follow-up data. Medical records were reviewed to obtain the following information: age, gender, race, usage of tobacco, alcohol and illicit drug usage, domiciliary status, and HIV serostatus.

Clinical presentation, symptoms, treatment regimens, and patient compliance were reviewed. Microbiologic data included AFB smear analysis, drug susceptibility patterns, and the TTD of M tuberculosis in culture using the MGIT system (described later). Chest radiographs were examined to' determine initial radiographic manifestations and changes with treatment.

Response to treatment was determined (blinded to TTD results) by review of inpatient and outpatient medical records of Bellevue Hospital Center and the New York City Department of Health. Clinical improvement was defined as defervescence, weight gain, decreased cough or hemoptysis, and increased appetite. Bacteriologic improvement (culture conversion) was defined as three or more consecutive, culture-negative specimens with no subsequent positive cultures. Clinical, bacteriologic, and radiographic follow-up was obtained for at least 6 months after hospital admission.

Specimen Processing for MGIT Cultures

Sputum induction was performed by having patients rinse their mouths with sterile water and then inhale nebulized 3% saline solution for 10 to 15 min or until the onset of sputum expectoration. All patient material was collected in sterile containers for processing. All specimens were digested and decontaminated with 2% NaOH, 2.9% sodium citrate, and 5% n-acetylcysteine for 15 min, and the reaction was terminated by addition of an equal volume of 0.9% saline solution. Specimens were centrifuged at 5,000 g for 15 min at 18 [degrees] C and decanted, and the sediment was used to inoculate culture media and prepare smears using an auramine acid-fast stain. All cultures were inoculated to solid media and to a MGIT system. The MGIT is a tube containing Middlebrook and Cohn 7H9 broth supplemented with oleic acid, albumin, dextrose, catalase, polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin sodium. An indicator at the bottom of the tube fluoresces as the level of oxygen decreases, signaling microbial growth. Tubes were examined daily on an ultraviolet transilluminator to detect growth, and a positive result was verified by examining Kinyoun- and Gram-stained smears. Specimens without growth after 60 days in culture were considered negative. If present, mycobacteria were then identified by DNA-RNA hybridization with the M tuberculosis culture confirmation system (AccuProbe Culture Confirmation System; Gen-Probe; San Diego, Calif). Susceptibility testing was performed using a modification of the method of proportions.[13] M tuberculosis isolates were tested for susceptibility to isoniazid, rifampin, pyrazinamide, ethambutol hydrochloride, streptomycin, kanamycin sulfate, ethionamide, rifabutin, ciprofloxacin hydrochloride, and amikacin sulfate.

Statistical Analysis

Fisher's Exact Test was used to compare patient characteristics. The relationship between the TTD and treatment duration was studied using a linear mixed-effects model. Two such models were considered: the first (model 1) had a random baseline value of TTD for each patient and a fixed effect for treatment duration, ie, a common rate of change (slope) of TTD. The second model (model 2) included a mixed term formed from a group indicator and treatment duration, ie, distinct slopes of TTD for the two groups. To establish whether the change in TTD as a linear function of treatment duration differed by treatment response group, a likelihood ratio test of model 2 against model 1 was performed.

Results

Demographic, Clinical, Bacteriologic, and Radiographic Characteristics

During the study period, 65 patients were admitted to the Bellevue Chest Service with active pulmonary tuberculosis. Twenty-six of these patients had 6 or more positive sputum cultures processed by the MGIT system during a period of at least 2 weeks and had sufficient clinical, radiographic, and bacteriologic follow-up to assess outcome. The remaining patients were excluded from this study.

Characteristics of the study population are summarized in Tables 1 and 2; groups 1 and 2 represent patients who were differentiated by their response to treatment and are further described later. At the time of hospitalization, 81% of patients had constitutional symptoms (fever, sweats, weight loss, malaise) and 88% had respiratory symptoms (cough, hemoptysis, dyspnea, pleuritic pain). In 12 patients, signs and symptoms of tuberculosis were either absent or indiscernible from other concomitant illnesses, which included anemia, end-stage HIV infection, psychosis, COPD, hepatitis, lung cancer, and chronic pancreatitis.

(*) Constitutional symptoms include fever, sweats, weight loss, and malaise. Respiratory symptoms include cough, hemoptysis, dyspnea, and pleuritic pain. [dagger] Pan-s=pan-susceptible; R=resistant; MDR=multidrug-resistant. [double dagger] First-line therapy included both isoniazid and rifampin; second-line therapy did not include both isoniazid and rifampin.

Clinical Outcome and Time to Detection

Sixteen patients received standard therapy, which included both isoniazid and rifampin in addition to pyrazinamide for the first 2 months of treatment. Ethambutol also was given until drug susceptibilities were known. Two patients were not initially treated with both isoniazid and rifampin because of abnormal liver function. An additional eight patients received a second-line regimen that did not contain both isoniazid and rifampin because of drug resistance.

Following initiation of antituberculous therapy, 13 patients (group 1, responders) had clinical improvement (defervescence, weight gain, decreased cough or hemoptysis, and increased appetite) and a progressive rise in the TTD with treatment, while the remaining 13 patients (group 2, nonresponders) had persistent, active disease and demonstrated little or no increase in TTD during treatment (Fig 1). Tables 1 and 2 compare characteristics between patients in groups 1 and 2. No statistically significant differences (between groups) were noted for any of the characteristics listed.

[FIGURE 1 GRAPH OMITTED]

In a test of model 2 (change in TTD related to clinical improvement) against model 1 (change in TTD unrelated to clinical improvement), the resulting change in (restricted maximal) log likelihood was 70.5. Comparing this with a [Chi.sup.2] distribution with 1 degree of freedom gives a probability value [is less than] 0.001. Thus, change in TTD as a linear function of treatment duration was significantly different for the two groups. For group 1, the estimate of this change was 0.71 with a 95% confidence interval (0.62,0.80). For group 2, the estimate of this change was 0.14 with a 95% confidence interval (0.05,0.24).

Seven of 13 patients in group 2 received a therapeutic intervention (surgery, improved compliance, or a change in medications) to augment their treatment. At the time of intervention, treatment had been given for an average of 85 days (range, 40 to 200 days), and the mean TTD was 16 days (range, 12 to 28 days). Following intervention, all patients manifested both clinical improvement and an increase in TTD. The remaining 6 patients in group 2 failed to improve with treatment. At last follow-up, one patient with advanced AIDS and multidrug-resistant tuberculosis died of hepatorenal failure, and the other five patients had persistent, active disease.

Bacteriologic Outcome and Time to Detection

The time from treatment initiation until culture conversion averaged 45 days (range, 15 to 75 days) for patients in group 1. For patients in group 2 who received an intervention, the mean time from intervention until culture conversion was 31 days (range, 7 to 107 days) and the total treatment time until culture conversion averaged 115 days (range, 54 to 246 days). At last follow-up, group 2 patients not undergoing an intervention had received an average of 201 days (range, 117 to 450 days) of treatment, and the mean TTD of their most recent culture was 18 days.

At the time of treatment initiation, all patients in both groups had one or more cultures with a TTD fewer than 20 days (TTD [is less than] 20 [Fig 2]). By treatment day 30, a TTD [is less than] 20 was present in 1 of 13 patients (8%) in group 1 and in all 13 patients in group 2. After 40 days of treatment, no patient in group 1 but all 13 patients in group 2 had a TTD of [is less than] 20.

Figure 3 shows the results of sputum AFB smears during the course of treatment. Five of 13 patients in group 1 (38%) and all patients in group 2 (not receiving an intervention) had at least 1 positive smear after 60 days of treatment. Five of 13 group 1 patients (38%) had at least 1 positive sputum smear after their last positive culture. Four of 6 group 2 patients who did not receive intervention had a negative smear associated with their most recent positive culture, despite a mean TTD of 18 days. Analysis of sputum smear sensitivity is shown in Table 3.

[FIGURE 3 GRAPH OMITTED]

(*) Prior to intervention. [dagger] Percentages in each column refer to the numbers of patients from Tables 1 and 2.

Discussion

Although the prompt identification of patients with tuberculosis has become a subject of increasing interest,[2] a simple, objective measure to predict treatment outcome remains elusive. The effectiveness of antituberculous therapy is determined by a variety of factors, including underlying immune status, compliance with and bioavailability of medications, drug susceptibility, and burden of infection. Hence, the assessment of a patient's response to antituberculous therapy often is difficult and imprecise.[4]

The analysis in this study demonstrates that the relationship between the TTD of M tuberculosis and the duration of antituberculous therapy was significantly different among patients who responded to treatment and those who did not. This difference in TTD was apparent after just a few weeks of treatment and increased progressively thereafter. The prognostic utility of TTD analysis is further illustrated by the dramatic change in TTD in group 2 patients who had been failing treatment and who then underwent a therapeutic intervention. The persistently low TTD in group 2 patients (not receiving an intervention) clearly reflected treatment failure; their most recent mean TTD (18 days) was similar to that of untreated patients with active disease.

In comparison with sputum smear evaluation, the TTD [is less than] 20 was a better prognostic indicator, decreasing in a sigmoidal fashion as a function of treatment response. After just 20 days of treatment, less than half of the patients in group 1 had a TTD [is less than] 20, and after 40 days, no group 1 patient had a TTD [is less than] 20. In contrast, 100% of patients in group 2 bad a TTD [is less than] 20 after 40 days of treatment, and over 80% of those not receiving an intervention had a TTD [is less than] 20 after 60 days of treatment.

Current recommendations for determining the response to antituberculous treatment emphasize sputum evaluation by direct examination and culture.[6,14] During the first 2 weeks of antituberculous therapy, a greater decrease in the number of colony-forming units of M tuberculosis occurs during the first 2 days than in the remaining 12 days.[15] Thus, in patients receiving a short-course regimen for tuberculosis, culture conversion occurs in approximately 50% after 1 month and in 95% after 4 months of treatment.[16] Cavitary disease and substance abuse have been associated with failure to convert sputum cultures,[16] which is in agreement with the findings from this study.

Overall acid-fast smear sensitivity in the patients in this study was comparable to that previously reported.[4] Sputum smear conversion during first-line antituberculous therapy typically occurs in 75 to 85% of patients after 2 months and in 96% after 6 months of therapy.[14,16] Smear results at 2 months have been reported to correlate with initial smear positivity and disease extent and may predict culture results at 3 months.[17] Despite the universal acceptance of smear evaluation as the best marker of treatment response, we found that the TTD-20 was superior in differentiating between group 1 and group 2 patients. For example, 5 of 13 patients (38%) in group 1 had false-positive smears following their last positive culture, and 9 of 26 patients (35%) failed to manifest a quantitative reduction in smear positivity during the course of treatment.

The chest radiograph may not indicate improvement until the patient has received several months of antituberculous treatment. Because radiographic assessment is inferior to clinical and bacteriologic findings as a marker of treatment response or relapse, it has little role in assessing the response to therapy, except when diagnosis and treatment of tuberculosis are presumptive.[5]

The duration of clinical signs and symptoms, especially fever, has been the subject of investigation as a potential marker of treatment response. In a pre-HIV era study, 79% of tuberculosis patients were febrile at the time of diagnosis.[18] Treatment with isoniazid and ethambutol, with or without rifampin or streptomycin, resulted in defervescence in 64% of patients after 2 weeks. In the HIV era, fever was reported in two thirds of patients presenting with tuberculosis.[19] Following treatment with isoniazid, rifampin, and pyrazinamide, 93% of patients became afebrile within 2 weeks. A more recent study found that in patients receiving a 4-drug regimen, defervescence occurred within 2 weeks in 9% of patients with multidrug-resistant tuberculosis compared with 78% of those with drug-susceptible organisms.[20] The demographic and clinical profile of patients in this study was representative of the population with tuberculosis served by Bellevue Hospital Center, as was the incidence of constitutional and respiratory Symptoms.[4,21] However, it is of note that many of the patients in this study were referred by other institutions or the New York City Department of Health due to management difficulty, drug resistance or toxicity, or treatment noncompliance. In this group of patients, the clinical assessment of response to treatment can be especially challenging, as demonstrated by the fact that signs and symptoms, including fever, often were indiscernible from other concomitant diseases. Despite this spectrum of patient complexity, after just several weeks of therapy, the TTD correlated well with treatment outcome.

Other methods devised to predict treatment outcome in tuberculosis patients include clinical scoring systems, serologic measures, and the use of polymerase chain reaction (PCR). Barnes et al[22] developed a system to determine short-term outcome and to predict the need for hospitalization based on patients' clinical features on admission. Absolute lymphocyte count, age, alcoholism, and extrapulmonary disease were associated with the need for hospitalization, while cavitary disease was actually associated with a better short-term outcome. However, the long-term prognostic value of these or other clinical factors is unknown.

Several studies have correlated the serologic levels of various acute-phase reactants with the response to antituberculous treatment. C-reactive protein is elevated in children with tuberculosis, and serum levels have been found to correlate with disease activity and treatment response.[7] A study of adults with tuberculosis found that those who had a poor outcome with treatment had persistently elevated C-reactive protein levels.[8] However, a more recent study of Japanese adults with tuberculosis reported a correlation between disease activity and serum levels of orosomucoid [Alpha.sub.1]-acid glycoprotein), haptoglobin, [Alpha.sub.1]-antitrypsin, and sialic acid, but not C-reactive protein.[9] Thus, the clinical utility of serologic measurements as an indicator of disease activity requires further investigation.

PCR, which is exquisitely sensitive in detecting the presence of M tuberculosis, was studied by Kennedy et al[23] as a potential indicator of treatment response. An evaluation of serial sputum samples from ten patients found that the identification of M tuberculosis by PCR continued following culture conversion, and persistent detection correlated with disease relapse. Unfortunately, PCR cannot differentiate viable from nonviable organisms; thus, its clinical applicability as a prognostic measure is uncertain.

The MGIT is a highly accurate system for the detection of mycobacteria.[11] However, it is the rapidity with which MGIT detects AFB and the ease of reading the cultures daily that facilitates the usefulness of the TTD measurement. Factors that determine an individual TTD value include the size of the inoculum, organism viability, the bacillary burden of the patient, and the quality of the sputum sample. Detection of MGIT fluorescence also is subject to observer variability, but the recent introduction of an automated reader should obviate this factor. The change in TTD with treatment most likely reflects a decline in the number and in the growth rate of bacilli as a consequence of the antituberculous medications. Intuitively, a close correlation between smear positivity and TTD would be expected, and experimentally, the detectability of tuberculous bacilli correlates with the size of the inoculum.[24] Surprisingly, this study found that a rapid TTD did not correlate with smear positivity, as exemplified by 4 of 6 group 2 patients who were failing treatment and had a short TTD despite negative sputum smears.

This study is limited primarily because it was a retrospective analysis. Hence, sputum samples, chest radiographs, clinical follow-up, and therapeutic interventions were all performed at the discretion of the treating physicians, thus limiting the number of patients eligible for inclusion in the study.

In conclusion, the TTD of M tuberculosis in serial sputum samples, as determined by the MGIT system, correlated closely with the overall response to therapy in patients with active pulmonary tuberculosis. TTD analysis was superior to clinical, radiographic, and conventional bacteriologic evaluation and may represent a novel method and useful adjunct to predict outcome in these patients.

References

[1] Barnes PF, Barrows SA. Tuberculosis in the 1990s. Ann Intern Med 1993; 119:400-10

[2] Schluger NW, Rom WN. Current approaches to the diagnosis of active pulmonary tuberculosis. Am J Respir Crit Care Med 1994; 149:264-67

[3] American Thoracic Society. Control of tuberculosis in the United States. Am Rev Respir Dis 1992; 146:23-33

[4] Garay SM. Pulmonary tuberculosis. In Rom WN, Garay SM, eds. Tuberculosis. Boston; Little, Brown, 1996

[5] Albert RK, Iseman M, Sbarbaro JA, et al. Monitoring patients with tuberculosis for failure during and after treatment. Am Rev Respir Dis 1976; 114:1051-60

[6] Core curriculum on tuberculosis. 2nd ed. Bethesda, Md; US Dept of Health and Human Services, April 1991

[7] Bajaj G, Tattan A, Ahmad P. Prognostic value of "C" reactive protein in tuberculosis. Indian Pediatr 1989; 26:1010-13

[8] Scott GM, Murphy RG, Gemidjioglu ME. Predicting deterioration of treated tuberculosis by corticosteroid reserve and C-reactive protein. J Infect 1990; 21:61-69

[9] Suzuki K, Takashima Y, Yamada T, et al. The sequential changes of serum acute phase reactants in response to antituberculous chemotherapy. Kekkaku 1992; 67:303-11

[10] Nolte FS, Metchock B. In: Murray PR, Barron EJ, Pfaller MA, eds. Manual of clinical microbiology. American Society of Microbiology, 1995;400-37

[11] Hanna BA, Walters SB, Kodsi SE, et al. Detection of Mycobacterium tuberculosis directly from patient specimens with the mycobacterial growth indicator tube: a new rapid method [abstract C112]. Presented at the American Society of Microbiology meeting, Las Vegas, Nev, May 23-27, 1994

[12] Hanna BA, Walters SB, Heller PA, et al. Time to culture detection of Mycobacterium tuberculosis as a possible index of patient progress and outcome [abstract D44]. Presented at the Interscience Conference on Antimicrobial Agents & Chemotherapy, San Francisco, September 17-20, 1995

[13] Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory. Atlanta: Centers for Disease Control, 1985; 159-184. US Public Health Service

[14] Treatment of tuberculosis and tuberculosis infection in adults and children. Am J Respir Crit Care Med 1994; 149:1359-74

[15] Jindani A, Aber VR, Edwards EA, et al. The early bactericidal activity of drugs in patients with pulmonary tuberculosis. Am Rev Respir Dis 1980; 121:939-49

[16] Combs DL, O'Brien RJ, Geiter LJ. USPHS tuberculosis short-course chemotherapy trial 21: effectiveness, toxicity and acceptability. Ann Intern Med 1990; 112:397-406

[17] Reider HL. Sputum smear conversion during directly observed treatment for tuberculosis. Tuber Lung Dis 1996; 77:124-29

[18] Kiblawi SSO, Jay SS, Stonehill RB, et al. Fever response of patients on therapy for pulmonary tuberculosis. Am Rev Respir Dis 1981; 123:20-24

[19] Barnes PF, Chan LS, Wong SF. The course of fever during treatment of pulmonary tuberculosis. Tubercle 1987; 68: 255-60

[20] Salomon N, Perlman DC, Friedmann P, et al. Predictors and outcome of multidrug-resistant tuberculosis. Clin Infect Dis 1995; 21:1245-52

[21] Neville K, Bromberg A, Bromberg R, et al. The third epidemic -- multidrug-resistant tuberculosis. Chest 1994; 105: 45-48

[22] Barnes PF, Leedom JM, Chan LS, et al. Predictors of short-term prognosis in patients with pulmonary tuberculosis. J Infect Dis 1988; 158:366-71

[23] Kennedy N, Gillespie SH, Saruni AOS, et al. Polymerase chain reaction for assessing treatment response in patients with pulmonary tuberculosis. J Infect Dis 1994; 170:713-16

[24] Pfyffer GE, Kissling P, Jahn EMI, et al. Diagnostic performance of amplified Mycobacterium tuberculosis direct test with cerebrospinal fluid, other nonrespiratory, and respiratory specimens. J Clin Microbiol 1996; 34:834-41

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