|Year : 2014 | Volume
| Issue : 3 | Page : 315-317
Detection of Mycobacterium tuberculosis growth by direct microscopic observation has a lower cost, higher sensitivity and higher diagnostic value than liquid and solid media cultures
S Olaru-Peter1, C Grigoriu2, LV Boiculese3, AC Gradinaru1, II Macovei4, D Diculencu1, B Grigoriu5
1 Departament of Bacteriology,Clinic of Pulmonary Diseases, Iasi, Romania
2 Department of Pathology, University of Medicine and Pharmacy "Gr.T.Popa", Iasi, Romania
3 Department of Medical Informatics and Biostatistics, University of Medicine and Pharmacy "Gr.T.Popa", Iasi, Romania
4 University of Agricultural Sciences and Veterinary Medicine, Iasi, Romania
5 Department of Pulmonary Diseases, University of Medicine and Pharmacy "Gr.T.Popa", Iasi, Romania
|Date of Submission||06-Mar-2013|
|Date of Acceptance||01-Jun-2014|
|Date of Web Publication||10-Jul-2014|
Department of Pulmonary Diseases, University of Medicine and Pharmacy "Gr.T.Popa", Iasi, Romania
Source of Support: CNCSIS: PN II-IDEI PCE 2011., Conflict of Interest: None
Background: Culture is needed to confirm tuberculosis but results are generally obtained after several weeks. Objectives: We compared a direct microscopic observation technique for detection of mycobacterial culture positivity (MODS) with the classic solid and MB/BacT cultures in terms of sensitivity, contamination rate, speed and cost on 488 samples. Results: The sensitivity of the MODS technique - 99,2% (162 positive samples) was higher than MB/BacT 78,4% (125 positive samples) and solid culture 69,6% (113 positive samples) P < 0.005 for all comparisons. The median times to positivity were 21, 13.3 and 3 days on solid media, B/BacT and MODS respectively. Conclusions: The MODS technique is faster and more sensitive than both solid media and MB/BacT culture.
Keywords: Bacteriological techniques, costs and cost analysis, diagnosis, liquid culture, sensitivity and specificity, tuberculosis
|How to cite this article:|
Olaru-Peter S, Grigoriu C, Boiculese L V, Gradinaru A C, Macovei I I, Diculencu D, Grigoriu B. Detection of Mycobacterium tuberculosis growth by direct microscopic observation has a lower cost, higher sensitivity and higher diagnostic value than liquid and solid media cultures. Indian J Med Microbiol 2014;32:315-7
|How to cite this URL:|
Olaru-Peter S, Grigoriu C, Boiculese L V, Gradinaru A C, Macovei I I, Diculencu D, Grigoriu B. Detection of Mycobacterium tuberculosis growth by direct microscopic observation has a lower cost, higher sensitivity and higher diagnostic value than liquid and solid media cultures. Indian J Med Microbiol [serial online] 2014 [cited 2019 Jul 22];32:315-7. Available from: http://www.ijmm.org/text.asp?2014/32/3/315/136585
| ~ Introduction|| |
Culture is the gold standard to confirm tuberculosis but the results are obtained after at least 10 days. We evaluated in a low resource setting, the MODS method for detection of mycobacterial culture positivity based on microscopical observation.
| ~ Materials and Methods|| |
MODS culture was implemented in the regional mycobacterial laboratory of the Iasi County, Romania, as described by Moore and Caviedes, on 24 wells cell-culture plates using 1 ml of the Middlebrook 7H9 media inoculated with 500 μl sample volume. Respiratory and gastric aspirate samples were decontaminated according to the sodium hydroxide protocol , while sterile samples were not. A microscopic examination (auramine-rhodamine confirmed by Ziehl-Nielsen staining) preceded the parallel inoculation on Lφwenstein-Jensen (LJ) (Sanimed, Romania) and MB/BacT system (BioMιrieux, Durham, USA) as instructed by the manufacturers. Culture media was the same for the MB/BacT and MODS. Positive, negative and inter-sample contamination controls were processed with each plate. Positive wells on MODS were confirmed by Ziehl-Neelsen staining  and subcultured on LJ. Statistical analyses were performed with SPSS 17.0 with two sided tests and significant P < 0.05.
| ~ Results|| |
Cultured samples (n = 488) originated from sputum n = 306, bronchial aspirates n = 116, morning gastric aspirate n = 13, biopsies n = 22, pleural fluid n = 30 and urine n = 1. A total of 401 samples were addressed for tuberculosis diagnosis while 87 were addressed for monitoring treatment response.
A total of 113 samples were positive for mycobacteria on LJ, 125 on MB/BacT and 162 with MODS. A number of cultures were also positive with other bacteria: 23 on LJ slants (4.7% of cultures) and 7 samples each on MB/BacT and MODS (1.4%). None of the control wells was contaminated.
Mycobacteria were isolated from 166 samples, sensitivities (95% confidence intervals) being 68.07% (60.4-75.08) for LJ, 75.3% (68.02-81.65) for MB/BacT and 97.59% (93.94-99.32) for MODS [Table 1]. Sensitivity was lower for the group of patients who were under treatment than those investigated for diagnosis 92.6% vs. 99.2% [Table 1].
Of the 21 cases positive with MODS and negative with LJ and MB/BacT, 3 came from scant smear positive patients, all of them receiving treatment, another one had a relapse with a scant positive sputum smear. Three additional samples were sputa from patients with a tuberculous pleurisy ( Adenosin deaminase (ADA)>100 UI/l). Four patients had radiological lesions suggestive of tuberculosis and at least one positive culture in the next 2 weeks.
The last 10 samples came from two patients with an undiagnosed exudative pleurisy (low ADA, negative pleural biopsy), two from a patient with an empyema and two from patients with lung cancer. Four patients had chronic cough with no diagnosis. Therefore the cross-contamination rate is less than 2%.
The performance of the three culture methods was similar but statistically the MODS method performed significantly better than the other two methods (P < 0.001) [Table 1].
The median time to positivity was 21 days on LJ, 13.3 days on MB/BacT and 3 days with MODS [Figure 1]a]. The difference was significant (P < 0.0001, Log Rank). The mean (± SD) time gain using MODS was 21 ± 7.65 days compared with LJ and 11 ± 6.2 days compared with MB/BacT.
|Figure 1: (a) Time to positive culture (b-d) Distribution of the time to a positive culture with all three culture methods Classification of positive Ziehl-Neelsen staining according to WHO Scanty 1-9 AFB/100 oil immersion fields, 1+ = 10-99 AFB/100 oil immersion fields, 2+ = 1-10 AFB/oil immersion field in at least 50 fields, 3+ = >10 AFB/oil immersion field in at least 20 fields, AFB=acid-fast bacilli|
Click here to view
The time to a positive culture was shorter in smear positive than in smear negative cases and was correlated with the bacterial load on microscopic examination [Figure 1]b-d]. It was shorter in cases addressed for the initial diagnosis vs. follow-up for both LJ (24.5 ± 6.3 vs. 28.44 ± 8.4 days, P = 0.013) and MB/BacT (14.2 ± 6.3 vs. 18.8 ± 9.8 days, P = 0.028) but not for MODS (4.9 ± 5 vs. 5.2 ± 3 days, P > 0.3) (Mann-Whitney test).
The costs related to sample processing was 3.3 €. The specific cost per sample were 1.1 € for MODS, 1.07 € for LJ and 10 € for MB/BacT.
| ~ Discussion|| |
Rapid confirmation of mycobacteria growth in clinical samples is essential and influences patient management. We demonstrate that implementation of the MODS technique is feasible in a low resource setting. MODS detected M. tuberculosis growth with greater sensitivity and faster than Lφwenstein-Jensen or MB/BacT cultures. The mean (±SD) time-gain was 2 weeks compared with LJ and 11 days compared with MB/BacT. These results are similar to previous investigations that demonstrated gains of 10-20 days compared with standard methods. ,,, MODS is also more sensitive than LJ and MB/BacT culture, which is a new finding. The improvement in sensitivity is possibly related to the stringent method used for decontamination, which is reflected in the low number of contaminated samples. These figures (4.7% for solid media and 1.4% for liquid cultures) are in the lower-end of recommended values and is similar to reports of Caviedes et al., (2%), Arias et al., (3.8%) and Michael et al., (4.8%). ,, The MODS method favours the growth of mycobacteria in samples with low bacterial burden and is able to "rescue" samples subjected to a very strict decontamination.
False positives from cross-contamination are concerning but rare as already reported by Moore et al. The majority of cases, which were positive with the MODS technique, are in fact negative samples at microscopy either secondary to treatment or from samples with a low number of mycobacteria.
The technique requires around 30 min of handling per plate but this is not an issue in low resource settings where personnel cost is low. A systematic review of MODS and an accompanying commentary in Lancet considered MODS to have a low cost effectiveness ratio due to the need of "specialised equipment and a high degree of technical expertise". , We demonstrate that this method can be implemented with good results in a low resource setting with proper training. Equipment costs are low and only a standard inverted microscope with a 40× phase contrast objective is necessary.
| ~ Conclusions|| |
We show that MODS is a sensitive and specific method and given its simplicity, speed and low cross-contamination rates, it is suitable for routine use in developing countries.
| ~ References|| |
|1.||World Health Organization, Laboratory services in Tuberculosis control Part III: Culture. Geneva, Switzerland 1998 |
|2.||Chakravorty S, Dudeja M, Hanif M, Tyagi JS. Utility of universal sample processing methodology, combining smear microscopy, culture, and PCR, for diagnosis of pulmonary tuberculosis. J Clin Microbiol 2005;43:2703-8. |
|3.||Arias M, Mello FC, Pavon A, Marsico AG, Alvarado-Galvez C, Rosales S, et al. Clinical evaluation of the microscopic-observation drug-susceptibility assay for detection of tuberculosis. Clin Infect Dis 2007;44:674-80. |
|4.||Caws M, Dang TM, Torok E, Campbell J, Do DA, Tran TH, et al. Evaluation of the MODS culture technique for the diagnosis of tuberculous meningitis. PLoS One 2007;2:e1173. |
|5.||Moore DA, Evans CA, Gilman RH, Caviedes L, Coronel J, Vivar A, et al. Microscopic-observation drug-susceptibility assay for the diagnosis of TB. N Engl J Med 2006;355:1539-50. |
|6.||Bwanga F, Haile M, Joloba ML, Ochom E, Hoffner S. Direct nitrate reductase assay versus microscopic observation drug susceptibility test for rapid detection of MDR-TB in Uganda. PLoS One 2011;6:e19565. |
|7.||Caviedes L, Lee TS, Gilman RH, Sheen P, Spellman E, Lee EH, et al. Rapid, efficient detection and drug susceptibility testing of Mycobacterium tuberculosis in sputum by microscopic observation of broth cultures. The Tuberculosis Working Group in Peru. J Clin Microbiol 2000;38:1203-8. |
|8.||Michael JS, Daley P, Kalaiselvan S, Latha A, Vijayakumar J, Mathai D, et al. Diagnostic accuracy of the microscopic observation drug susceptibility assay: A pilot study from India. Int J Tuberc Lung Dis 2010;14:482-8. |
|9.||Moore DA, Caviedes L, Gilman RH, Coronel J, Arenas F, LaChira D, et al. Infrequent MODS TB culture cross-contamination in a high-burden resource-poor setting. Diagn Microbiol Infect Dis 2006;56:35-43. |
|10.||Minion J, Leung E, Menzies D, Pai M. Microscopic- observation drug susceptibility and thin layer agar assays for the detection of drug resistant tuberculosis: A systematic review and meta-analysis. Lancet Infect Dis 2010;10:688-98. |
|11.||Banerjee Y, Taranikanti V, Bayoumi R. Assays for drug resistant tuberculosis in high burden countries. Lancet Infect Dis 2011;11:161-2. |