Revisiting the susceptibility testing of Mycobacterium tuberculosis to ethionamide in solid culture medium.

BACKGROUND & OBJECTIVES: Increase in the isolation of drug resistant phenotypes of Mycobacterium tuberculosis necessitates accuracy in the testing methodology. Critical concentration defining resistance for ethionamide (ETO), needs re-evaluation in accordance with the current scenario. Thus, re-evaluation of conventional minimum inhibitory concentration (MIC) and proportion sensitivity testing (PST) methods for ETO was done to identify the ideal breakpoint concentration defining resistance. METHODS: Isolates of M. tuberculosis (n=235) from new and treated patients were subjected to conventional MIC and PST methods for ETO following standard operating procedures. RESULTS: With breakpoint concentration set at 114 and 156 µg/ml, an increase in specificity was observed whereas sensitivity was high with 80 µg/ml as breakpoint concentration. Errors due to false resistant and susceptible isolates were least at 80 µg/ml concentration. INTERPRETATION & CONCLUSIONS: Performance parameters at 80 µg/ml breakpoint concentration indicated significant association between PST and MIC methods.

Modification of proportion sensitivity testing method for ethionamide.

Standardized methodology for drug susceptibility testing of second line drugs is vital for treatment of multi/extensively drug resistant tuberculosis. Discrepancy between laboratory methods and clinical interpretation is well established for bacteriostatic drugs such as ethionamide. Optimization of the standard proportion sensitivity testing (PST) method for ethionamide was under taken in 235 Mycobacterium tuberculosis isolates from new and previously treated pulmonary tuberculosis patients. An additional higher concentration of 57 µg/ml was evaluated against at the standard 40 µg/ml concentration in PST method. Performance parameters and agreement between the two drug concentrations was higher indicating the efficiency of PST method at its present format at 40 µg/ml and additional higher concentration of 57 µg/ml as an alternative when required.

Consistency of standard laboratory strain Mycobacterium tuberculosis H 37 Rv with ethionamide susceptibility testing.

Drug susceptibility pattern of standard Mycobacterium tuberculosis strain H 37 Rv showed discrepancy in minimum inhibitory concentration method for ethionamide and consistent results were obtained for the other second line drugs namely, kanamycin and ofloxacin. It is, therefore, necessary to revisit the susceptibility testing method for ethionamide for effective clinical management of patients with drug resistant tuberculosis.

Radiological staging of rectal cancer in a resource limited setting.

OBJECTIVE: Current guidelines on rectal cancer (RC) management recommend pre-operative MRI for loco-regional staging and CT for staging of metastases. This allows appropriate selection of patients for chemo-radiotherapy (CRT). However, MRI is not freely available in many low-income countries. We assessed the status of pre-operative imaging for RC in Sri Lanka and evaluated the performance of CT in RC staging. RESULTS: A pre-tested interview-administered questionnaire was used to assess the pre-operative use of MRI and CT in RC. CT findings from 37 RC patients were then compared with histopathology findings. Of the 64 surgeons interviewed, 57 (89.1%) did not request an MRI for their RC patients. Reasons cited included limited availability and long waiting times due to competing health needs. A CT was requested by all. In RC, the overall accuracy of CT for T staging was 43.2% and 29.7% of T1-T2 tumours were over-staged as T3. The overall accuracy of CT for regional lymph node staging was 70.3%. In summary, CT alone is not suitable for RC staging in any setting. It leads to over-staging and patients may thus receive unnecessary CRT. Steps must be taken to improve access to pre-operative MRI among Sri Lankan RC patients.

Sputum conversion at the end of intensive phase of Category-1 regimen in the treatment of pulmonary tuberculosis patients with diabetes mellitus or HIV infection: An analysis of risk factors.

BACKGROUND & OBJECTIVE: New smear-positive pulmonary tuberculosis (PTB) patients in the Revised National Tuberculosis Control Programme (RNTCP) are treated with a 6-month short-course chemotherapy (SCC) regimen irrespective of co-morbid conditions. We undertook this retrospective analysis to compare sputum conversion rates (smear, culture) at the end of intensive phase (IP) of Category-1 regimen among patients admitted to concurrent controlled clinical trials: pulmonary tuberculosis alone (PTB) or with type 2 diabetes mellitus (DM-TB) or HIV infection (HIV-TB), and to identify the risk factors influencing sputum conversion. METHODS: In this retrospective analysis sputum conversion rates at the end of intensive phase (IP) in three concurrent studies undertaken among PTB, DM-TB and HIV-TB patients, during 1998 - 2002 at the Tuberculosis Research Centre (TRC), Chennai, were compared. Sputum smears were examined by fluorescent microscopy. HIV infected patients did not receive anti-retroviral treatment (ART). Patients with DM were treated with oral hypoglycaemic drugs or insulin (sc). RESULTS: The study population included 98, 92 and 88 patients in the PTB, DM-TB and HIV-TB studies. At the end of IP the smear conversion (58, 61, and 62%) and culture conversion (86, 88 and 92%) rates were similar in the three groups respectively. The variables associated with lack of sputum smear or culture conversion were age >45 yr, higher pre-treatment smear and culture grading, and extent of the radiographic involvement. INTERPRETATION & CONCLUSION: Our findings confirm that the current policy of the control programme to treat all pulmonary TB patients with or with out co-morbid conditions with Category-I regimen appears to be appropriate.

Sensitivity of Ziehl-Neelsen method for centrifuged deposit smears of sputum samples transported in cetyl-pyridinium chloride.

BACKGROUND & OBJECTIVES: Sensitivity of Ziehl-Neelsen (ZN) method is known to be low for liquefied sputum smears. Information on the ZN sensitivity for centrifuged deposit smears is not known. This study was carried out to determine the sensitivity of ZN method for acid fast bacilli (AFB) in centrifuged deposit smears and liquefied sputum smears made from sputum samples transported in cetyl-pyridinium chloride (CPC) solution. METHODS: Liquefied sputum smears and the corresponding centrifuged deposit smears from each of the 607 consecutive sputum samples collected from tuberculosis patients admitted to receive treatment transported in CPC were read by the same readers and their results compared with culture results. RESULTS: A significantly (P<0.001) higher proportion of samples were positive in centrifuged deposit smears (40%) compared to liquefied sputum smears (30%). The results of 341 culture-positive specimens revealed that the sensitivity of ZN method was 47 per cent using liquefied sputum smears and 63 per cent using centrifuged deposit smears (P<0.001). INTERPRETATION & CONCLUSION: Our study demonstrated that the sensitivity of ZN method for AFB in centrifuged deposit smears and liquefied sputum smears was reduced if sputum samples are transported in CPC solution.

Stratifying low level Isoniazid resistance using additional intermediate drug concentration.

Isoniazid (INH) susceptibility testing for 100 Mycobacterium tuberculosis performed by conventional minimum inhibitory concentration (MIC) method was stratified using additional drug concentrations. Introduction of additional drug concentrations did not greatly improve the discriminatory capacity, but can be used in specialized studies pertaining to cross resistance between structural analogues of INH.

Randomized clinical trial of thrice-weekly 4-month moxifloxacin or gatifloxacin containing regimens in the treatment of new sputum positive pulmonary tuberculosis patients.

BACKGROUND: Shortening tuberculosis (TB) treatment duration is a research priority. This paper presents data from a prematurely terminated randomized clinical trial, of 4-month moxifloxacin or gatifloxacin regimens, in South India. METHODS: Newly diagnosed, sputum-positive HIV-negative pulmonary TB patients were randomly allocated to receive gatifloxacin or moxifloxacin, along with isoniazid and rifampicin for 4 months with pyrazinamide for first 2 months (G or M) or isoniazid and rifampicin for 6 months with ethambutol and pyrazinamide for first 2 months (C). All regimens were administered thrice-weekly. Clinical and bacteriological assessments were done monthly during treatment and for 24 months post-treatment. The Data and Safety Monitoring Board recommended termination of the trial due to high TB recurrence rates in the G and M regimens. RESULTS: Of 416 patients in intent-to-treat analysis, 6 (5%) of 124, 2 (2%) of 110 and 2 (2%) of 137 patients with drug-susceptible TB in the G, M and C arms respectively had unfavorable response at the end of treatment; during the next 24 months, 17 (15%) of 115, 11 (11%) of 104 and 8 (6%) of 132 patients respectively, had TB recurrence. Of 38 drug-resistant patients 1 of 8 and 3 of 26 in the G and C arms respectively had unfavourable response at the end of treatment; and TB recurrence occurred in 2 of 7 and 2 of 23 patients, respectively. The differences in TB recurrence rates between the G and C arms was statistically significant (p = 0.02). Gastro-intestinal symptoms occurred in 23%, 22% and 9% of patients in the G, M and C arms respectively, but most reactions were mild and manageable with symptomatic measures; 1% required regimen modification. CONCLUSIONS: 4-month thrice-weekly regimens of gatifloxacin or moxifloxacin with isoniazid, rifampicin and pyrazinamide, were inferior to standard 6-month treatment, in patients with newly diagnosed sputum positive pulmonary TB. TRIAL REGISTRATION: Clinical Trials Registry of India CTRI/2012/10/003060.

Assessment of long term status of sputum positive pulmonary TB patients successfully treated with short course chemotherapy.

BACKGROUND: Long term status of pulmonary tuberculosis (PTB) patients treated with short course chemotherapy (SCC) regimens remains unknown. OBJECTIVE: To assess the clinical, bacteriological, radiological status and health related quality of life (HRQoL) of PTB patients 14-18 years after successful treatment with SCC. METHODOLOGY: In a cross-sectional study, cured PTB patients treated during 1986-1990 at the Tuberculosis Research Centre (TRC) were investigated for their current health status including pulmonary function tests (PFT). The St Georges respiratory questionnaire (SGRQ) was used to assess the HRQoL. RESULTS: The mean period after treatment completion for the 363 eligible participants was 16.5 yrs (range 14-18 yrs., 84% coverage); 25 (7%) had been re-treated and 52 (14%) died. Among the investigated, 58 (29%) had persistent respiratory symptoms; 170 (86%) had radiological sequelae but none had active disease. Abnormal PFT was observed in 96 (65%) with predominantly restrictive type of disease in 66 (45%). The SGRQ scores for activity and impact were high implying impairment in HRQoL. CONCLUSION: Assessment of long term status of cured PTB patients showed an impairment of lung functions and HRQoL highlighting the need to address these issues in the management of TB that may provide added value to patient care.

Simple direct drug susceptibility tests on sputum samples for early detection of resistance in tubercle bacilli.

BACKGROUND: Direct sensitivity test either by sputum concentrate (DS) or swab method (DSM) set up along with the primary culture would avoid the delay of four or more weeks required for the indirect test. A comparison of these two methods against the standard indirect sensitivity method under routine laboratory conditions is necessary to prove their merit. METHOD: Smear positive sputum samples were aliquoted and sensitivity tests were set up by both the direct methods as also an indirect test set up from the primary culture of the same sample. RESULTS: The agreement with the indirect test results for isoniazid (INH) ranged from 97-98% for the DS method and 93-97% for the DSM method. The corresponding figures were 96-98% by the DS and 94-99% by the DSM method for rifampicin (R). The agreement was less satisfactory for ethambutol (Emb). CONCLUSION: This study showed that direct sensitivity tests such as DS and DSM methods can detect most of the cultures resistant to INH and R (MDR) from the time growth appears on the primary culture, even as early as the second week of setting up the tests.

Inefficiency of 0.3% carbol fuchsin in ziehl-neelsen staining for detecting acid-fast bacilli.

We compared the sensitivity and specificity of a modified Ziehl-Neelsen (modified-ZN) staining method for acid-fast bacilli (AFB) with that of the standard Ziehl-Neelsen (standard-ZN) staining method, using culture results with Mycobacterium tuberculosis as the "gold standard." The sensitivity (72%; 101 of 140) of the modified-ZN staining method, which uses 0.3% carbol fuchsin, was significantly lower than that of the standard-ZN staining method (84%; 117 of 140); the modified-ZN method missed 21% of cases detected by the standard-ZN method and 11% more of culture-positive samples than the standard-ZN method. The World Health Organization recommendation of 0.3% carbol fuchsin in the ZN method for staining AFB needs to be reconsidered.

Evaluation of the phenol ammonium sulfate sedimentation smear microscopy method for diagnosis of pulmonary tuberculosis.

We compared the sensitivity and specificity of the phenol ammonium sulfate (PhAS) sediment smear microscopy method for detection of acid-fast bacilli with those of direct smear microscopy, using culture results for Mycobacterium tuberculosis as the "gold standard." The sensitivities of the PhAS and direct smear methods were 85% (465 of 547) and 83% (454 of 547), respectively, and the specificity of each method was 97%. The PhAS method was better accepted by the laboratory technicians and safer but necessitates an overnight sedimentation, which delays reporting of results until 1 day after sputum collection.