Selvakumar N

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

Evaluation of the Phenol Ammonium Sulfate Sedimentation Smear Microscopy Method for Diagnosis of Pulmonary Tuberculosis

ABSTRACT 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.

Lot Quality Assurance Sampling of Sputum Acid-Fast Bacillus Smears for Assessing Sputum Smear Microscopy Centers

ABSTRACT Assessment of 12 microscopy centers in a tuberculosis unit by blinded checking of eight sputum smears selected by using a lot quality assurance sampling (LQAS) method and by unblinded checking of all positive and five negative slides, among the slides examined in a month in a microscopy centre, revealed that the LQAS method can be implemented in the field to monitor the performance of acid-fast bacillus microscopy centers in national tuberculosis control programs.

Inefficiency of 0.3% Carbol Fuchsin in Ziehl-Neelsen Staining for Detecting Acid-Fast Bacilli

ABSTRACT 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.

High Rates of Ofloxacin Resistance in Mycobacterium tuberculosis among Both New and Previously Treated Patients in Tamil Nadu, South India

Periodic drug resistance surveillance provides useful information on trends of drug resistance and effectiveness of tuberculosis (TB) control measures. The present study determines the prevalence of drug resistance among new sputum smear positive (NSP) and previously treated (PT) pulmonary TB patients, diagnosed at public sector designated microscopy centers (DMCs) in the state of Tamil Nadu, India. In this single-stage cluster-sampling prevalence survey, 70 of 700 DMCs were randomly selected using a probability-proportional to size method. A cluster size of 24 for NSP and a varying size of 0 to 99 for PT cases were fixed for each selected DMC. Culture and drug susceptibility testing was done on Lowenstein-Jensen medium using the economic variant of proportion sensitivity test for isoniazid (INH), rifampicin (RMP), ofloxacin (OFX) and kanamycin (KAN). Human Immunodeficiency Virus (HIV) status was collected from patient records. From June 2011 to August 2012, 1524 NSP and 901 PT patients were enrolled. Any RMP resistance and any INH resistance were observed in 2.6% and 15.1%, and in 10.4% and 30% respectively in NSP and PT cases. Among PT patients, multi drug resistant TB (MDR-TB) was highest in the treatment failure (35%) group, followed by relapse (13%) and treatment after default (10%) groups. Extensively drug resistant TB (XDRTB) was seen in 4.3% of MDR-TB cases. Any OFX resistance was seen in 10.4% of NSP, 13.9% of PT and 29% of PT MDR-TB patients. The HIV status of the patient had no impact on drug resistance levels. RMP resistance was present in 2.6% of new and 15.1% of previously treated patients in Tamil Nadu. Rates of OFX resistance were high among NSP and PT patients, especially among those with MDR-TB, a matter of concern for development of new treatment regimens for TB.

Can LED fluorescence microscopy replace Ziehl-Neelsen microscopy in tuberculosis detection?

Ziehl-Neelsen (ZN) acid-fast bacilli (AFB) microscopy, which detects only about 50% of pulmonary tuberculosis patients in the community, is the cornerstone worldwide for the diagnosis of tuberculosis, as, apart from other constraints, it is infl uenced by the laboratory workload, stain quality and light microscope conditions in the fi eld. In comparison to ZN microscopy, conventional (mercury vapour lamp) fl uorescence microscopy (CFM), based on auramine phenol staining, can detect approximately 5–10% more AFB positive smears.1 However, CFM is used only in specialised laboratories and not in peripheral health institutions due to the requirement for expensive mercury vapour lamps and darkroom facilities. The light emitting-diode (LED) microscope, with its inexpensive, long-life LED lamps, is a boon to mycobacteriologists. The LED microscope can be housed and slides examined in an ordinary, well-lit room. Its major advantage is the ease of examination with 40×/20× objectives. Laboratory technicians (LTs) feel less fatigue on examining the slides, thus increasing the chances of AFB detection in paucibacillary samples. The World Health Organization recently recommended both the use of LED FM and replacing ZN microscopy in a phased manner. However, this gives rise to the following issues: 1) less experienced LTs are likely to commit false-positive errors,2 as impurities in auramine stains and artefacts such as blood3 in the sputum will fl uoresce like AFB; 2) detection of at least three AFB in a smear is likely to be highly confi rmative of culture-positive TB;4 3) AFB damaged by anti-tuberculosis drugs during treatment stain better with auramine than carbol fuchsin;5 4) positive smears are not checked before reporting the results in the fi eld; and 5) there is a paucity of knowledge about the adequacy of training on LED FM and on the quality of FM reporting in programmatic conditions: doubtful smears encountered with LED FM should be restained by ZN and verifi ed until the technicians are fully familiar with LED FM microscopy.

Processing Sputum Specimens in a Refrigerated Centrifuge Does Not Increase the Rate of Isolation of Mycobacterium tuberculosis

ABSTRACT A total of 1,047 sputum samples from pulmonary tuberculosis patients was collected in cetyl pyridinium chloride-sodium chloride solution. Each sample was divided into two parts and randomly allocated for the isolation of Mycobacterium tuberculosis, with one part to be processed by the standard method and the other by a modified method. In the standard method, the samples were processed by using nonrefrigerated centrifuges, while in the modified method, they were processed by using a refrigerated centrifuge. Fifty-seven samples that yielded contaminants were excluded, and the remaining 990 samples were taken up for analysis. The rates of isolation of M. tuberculosis with the standard and modified methods were 48.6 and 48.1%, respectively, and the difference was not statistically significant (McNemars test; P > 0.5). However, 51% of the positive cultures were isolated within 2 weeks with the modified method compared to 37% with the standard method (chi-square test; P < 0.001). The results of the study reveal that processing of sputum samples in a refrigerated centrifuge does not improve the rate of isolation but will result in rapid isolation of M. tuberculosis.

Characterization and phylogenetic analysis of antituberculous compound producing actinomycete strain D25 isolated from Thar Desert soil, Rajasthan.

During the course of the anti-infective drug discovery programme, actinomycete strain D25 was recovered from the Thar Desert soil, Rajasthan, India. Actinomycin type of compound isolated from the strain D25 showed promising activity against multi drug resistant and extensively drug resistant M. tuberculosis isolates. The present study reports the characteristics and phylogenetic status of the actinomycete strain D25. Phenotypic and cell wall characteristics revealed that the strain belongs to the genus Streptomyces. Further 16s rRNA analysis confined the genus Streptomyces with 97% similarity to the closely related species Streptomyces althioticus KCTC 9752. The 16s rRNA sequence was submitted to GenBank with the accession number JN604533.1. According to Bossard et al. (2003) strain D25 was found to be a novel species of the genus Streptomyces from Thar Desert soil, Rajasthan.

Retrieval of Mycobacterium tuberculosis cultures suspended in phosphate buffered saline

One hundred and twenty-seven of 130 isolates of Mycobacterium tuberculosis, suspended in phosphate buffered saline (PBS) and stored at ambient conditions in the laboratory for 14days, and another 55 of 60 cultures, suspended as above, transported from reference laboratories within 7days, were successfully retrieved on LJ medium. Considering the maximum retrieval of M. tuberculosis, use of PBS can be explored further for transportation of M. tuberculosis cultures across laboratories.

Quality indicators in a mycobacteriology laboratory supporting clinical trials for pulmonary tuberculosis

BACKGROUND Documentation of structured quality indicators for mycobacteriology laboratories supporting exclusively controlled clinical trials in pulmonary tuberculosis (PTB) is lacking. OBJECTIVE To document laboratory indicators for a solid (Lowenstein-Jensen medium) culture system in a mycobacteriology laboratory for a period of 4years (2007-2010). METHODS The sputum samples, collected from PTB suspects/patients enrolled in clinical trials, were subjected to fluorescence microscopy, culture and drug sensitivity testing (DST). Data was retrospectively collected from TB laboratory registers and computed using pre-formulated Microsoft Office Excel. Laboratory indicators were calculated and analyzed. RESULTS The number of samples processed in a calendar year varied from 6261 to 10,710. Of the samples processed in a calendar year, specimen contamination (4.8-6.9%), culture positives (78.4-85.1%) among smear positives, smear positives (71.8-79.0%) among culture positive samples, smear negatives among culture negative samples (95.2-96.7%), and average time to report DST results (76-97days) varied as shown in parentheses. CONCLUSION Values of quality indicators in mycobacteriology laboratories supporting exclusively clinical trials of PTB have to be defined and used for meaningful monitoring of laboratories.