Preparation of mycobacteria-containing artificial sputum for TB panel testing and microscopy of sputum smears

Panel testing, blinded cross rechecking and on-site evaluation are the three methods for external quality assessment (EQA) of acid-fast bacilli (AFB) smear microscopy. Panel testing can provide data on laboratory capabilities prior to implementing a rechecking programme, assess the current status of laboratory performance and detect problems associated with diagnostic performance. Thus far, two methods for preparing panel test slides have been reported: these use real AFB-positive and -negative sputum treated with sodium hydroxide (NaOH) or N-acetyl-L-cysteine (NALC).

To evaluate the above methods and to develop a new method to prepare panel test slides with artificial sputum.

Panel test slides were prepared using the NaOH and NALC methods. New artificial sputum preparation methods were developed and examined using a cultured monocyte cell line, cultured avirulent mycobacteria and methylcellulose or polyacrylamide gel as substrate. Smears prepared by the four methods were compared.

Panel test slides prepared with NaOH and NALC methods are not macroscopically or microscopically similar to real smears. Our new artificial sputum is similar to real sputum in viscosity and macroscopic and microscopic appearance; it is also consistent in panel positivity grades.

The artificial sputum described here could contribute to the EQA and training in tuberculosis laboratories or microscopy centres.