Implementation of the Thin Layer Agar Method for Diagnosis of Smear-Negative Pulmonary Tuberculosis in a Setting with a High Prevalence of Human Immunodeficiency Virus Infection in Homa Bay, Kenya

Abstract

The objective of this study was to evaluate the performance of a low-cost method, the thin layer agar (TLA) method, for the diagnosis of smear-negative patients. This prospective study was performed in Homa Bay District Hospital in Kenya. Out of 1,584 smear-negative sputum samples, 212 (13.5%) were positive by culture in Löwenstein-Jensen medium (LJ) and 220 (14%) were positive by the TLA method. The sensitivities of LJ and TLA were 71% and 74%, respectively. TLA could become an affordable method for the diagnosis of smearnegative tuberculosis in resource-limited settings, with results available within 2 weeks.The prevalence of smear-negative pulmonary tuberculosis (TB) has been increasing in countries with a high incidence of human immunodeficiency virus (HIV), especially in low-income countries where many patients are coinfected with HIV and TB and where culture of Mycobacterium tuberculosis is often not available (3,4,5,6,13,15). The incidence of TB in Kenya is estimated at 384/100,000 inhabitants (23). Kenya ranks 13th on the World Health Organization list of 22 highburden countries for TB worldwide, and HIV prevalence in new TB cases is estimated at 52% (23). Culture is more sensitive than smear microscopy (1, 3, 7), but cultivation in Löwenstein-Jensen (LJ) medium is very slow. The thin layer agar (TLA) method has been described as a simple, rapid, and inexpensive method (11,12,18) allowing initial identification of M. tuberculosis based on colony morphology, visualized microscopically, and by incorporation of para-nitrobenzoic acid (PNB) in the medium (10,17,19). We performed a prospective study to evaluate the performance of the TLA method for detection of M. tuberculosis in smear-negative samples compared to cultivation in LJ medium.The study was conducted in Homa Bay District Hospital, Kenya. All smear-negative respiratory samples received between November 2007 and September 2008 from patients suspected of having TB were included. A total of 1,584 smearnegative samples were analyzed. Sputum was digested and decontaminated using the sodium hydroxide-N-acetyl-L-cysteine method (9). LJ cultures were examined twice weekly for up to 8 weeks (22). Cultures were considered positive for M. tuberculosis according to their morphological characteristics, positive acid-fast bacilli staining, and inhibition of growth by PNB on TLA (8). A culture was considered negative if no growth was observed after 8 weeks, and it was considered contaminated if there was growth but it was negative for acidfast bacilli. TLA plates were prepared as previously described (18) with small modifications. One hundred microliters of decontaminated sample was inoculated on a biplate petri dish of 100 mm by 15 mm (Becton Dickinson, Sparks, MD) containing 20 ml of 7H11 agar supplemented with 10% oleic acid-albumin-dextrose-catalase (Becton Dickinson) plus piperacillin, trimethoprim, and amphotericin B (Sigma Aldrich) at 0.05 g/ ml, 0.02 g/ml, and 0.02 g/ml, respectively. PNB at 500 g/ml was incorporated in one compartment. The plates were se...

Comparative performance of Thin Layer Agar and Löwenstein–Jensen culture for diagnosis of tuberculosis

“…The performance of both cultures was lower in smear negative samples, in particular for LJ. This is in agreement with other studies and was reported also for genetic testing and liquid culture .…”

Decontamination methods for samples preserved in cetylpyridinium chloride and cultured on thin-layer agar

“…6 Robledo et al, in a multicenter study, found rates of between 2.7% and 9.5%. 16 These higher rates may be due to the lower concentration of antibiotics added to the medium (0.02 ll/ml instead of the 4.0 ll/ml used in our study).…”

“…5 When used for smear-positive samples, the median time required for mycobacterial detection is 14 days, vs. 9.6 days with the liquid culture BACTECe MGITe 960 system (BD, Sparks, MD, USA) and 23 days using solid Löwenstein-Jensen (LJ) medium. [6][7][8] However, TLA culture can yield high contamination rates when inoculated with samples previously treated with Petroff or NALC-NaOH alone. 6,7 The combined application of NALC-NaOH and CPC has rarely been described, and combination of CPC with the Petroff method is discouraged.…”

“…[6][7][8] However, TLA culture can yield high contamination rates when inoculated with samples previously treated with Petroff or NALC-NaOH alone. 6,7 The combined application of NALC-NaOH and CPC has rarely been described, and combination of CPC with the Petroff method is discouraged. 9 The use of CPC is incompatible with agar-based media unless CPC is neutralised, limiting its application with TLA.…”

Rapid Direct Testing of Susceptibility of Mycobacterium tuberculosis to Isoniazid and Rifampin on Nutrient and Blood Agar in Resource-Starved Settings

“…However, this system is costly, its consumables are not easily available, and it requires technical expertise. Parallel to the progress in automated systems, much work has also been published on the development of rapid but economical methods which are equally effective and comparable to automated systems, especially for resource-limited countries (1,11,12,15,18). Drancourt et al were the first to report the incidental growth of M. tuberculosis colonies on blood agar and termed this finding the "end of dogma" (7).…”