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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 2  |  Page : 175-179

The importance of multiple samples in mycobacterial recovery: A 10-year retrospective study


1 Department of Clinical Pathology, São João Hospital Centre; Epidemiology Unit, Institute of Public Health, University of Porto, Porto, Portugal
2 Department of Clinical Pathology, São João Hospital Centre, Porto, Portugal
3 Department of Clinical Pathology, São João Hospital Centre; Epidemiology Unit, Institute of Public Health; Department of Biomedicine, Faculty of Medicine, University of Porto, Porto, Portugal

Date of Web Publication14-Jun-2019

Correspondence Address:
Angélica Ramos
Department of Clinical Pathology, São João Hospital Centre, Alameda Professor Hernâni Monteiro, 4200-319 Porto
Portugal
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_68_19

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  Abstract 


Background: Instead of the three samples previously recommended, the World Health Organization considered that two sputum samples are sufficient for the diagnosis of pulmonary tuberculosis. Our goal is to evaluate the value of multiple samples in the microscopic and cultural examination of Mycobacterium tuberculosis complex (MTBc) and nontuberculous mycobacteria (NTM). Methods: Through a 10-year retrospective study, including 38,887 sputum samples from 11,676 patients, the relative contribution of each sputum specimen for acid-fast bacilli detection and mycobacteria isolation was determined by calculating the yield of the first sample and the incremental yield of the second, third, and more than three samples. Results: With regard to patients with MTBc isolation, we found that 95.2% (435/457) of the cases could be detected in the first and second smear and 97.9% (659/673) in the first and second culture, with a very low increment of the third sample, corresponding to 3.1% (14/457) in smear and 1.3% (9/673) in culture. Among patients with an NTM strain, the percentage of cases detected in the first and second smear and culture was 69% (20/29) and 86.5% (316/365), respectively. In these patients, we found an increment yield of the third sample of 20.7% (6/29) for smear and 10.5% (38/365) for culture. The incremental yield of third and above-third sample for NTM detection was of 31% (9/29) in smear and 13.5% (49 of 365) in culture. Conclusions: We found an added value of multiple samples in NTM recovery. However, our findings suggest that only two samples are required for the diagnosis of pulmonary tuberculosis.

Keywords: Multiple samples, mycobacteria, nontuberculous mycobacteria, tuberculosis


How to cite this article:
Ramos A, Carvalho T, Guimarães JT. The importance of multiple samples in mycobacterial recovery: A 10-year retrospective study. Int J Mycobacteriol 2019;8:175-9

How to cite this URL:
Ramos A, Carvalho T, Guimarães JT. The importance of multiple samples in mycobacterial recovery: A 10-year retrospective study. Int J Mycobacteriol [serial online] 2019 [cited 2019 Sep 18];8:175-9. Available from: http://www.ijmyco.org/text.asp?2019/8/2/175/260389




  Introduction Top


Despite several advances in the early diagnosis of tuberculosis (TB), microscopic examination of the sputum smear remains a commonly used method in mycobacteriology laboratories. In developing countries, sputum smear microscopy is still the most commonly used diagnostic test for pulmonary TB. This is a simple technique that quickly identifies acid-fast bacilli (AFB) with a relatively low cost and high positive predictive value.[1] However, a definitive tuberculosis diagnosis always requires the isolation of the etiologic agent from clinical samples. The microbiologic criteria are equally important to make a diagnosis of lung diseases caused by nontuberculous mycobacteria (NTM).[2] Sputum is the specimen most commonly sent to the laboratory when pulmonary TB and NTM lung diseases are suspected.

In the decades of 1980 and 1990, most standard laboratory texts[3] and guidelines for mycobacteriology laboratories[3],[4] recommend that at least three sputum specimens, preferably collected on successive days, should be submitted to the laboratory for AFB smear and culture for patients suspected to have tuberculosis. From 2010 onward, the World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease also recommend the examination of three smears before declaring a person with suspected TB as smear-negative.[5],[6],[7] However, after several studies determined that two samples may detect >90% of the sputum smear and culture positive cases,[8],[9],[10],[11],[12] the WHO considered the collection of only two sputum samples as sufficient.[13]

In the last three decades, Portugal made great efforts in the fight against TB. In such a way that in 2014, and for the first time, it stepped into the low incidence group of countries, with an estimated incidence below 20 cases per 100,000 inhabitants.[14] In spite of all this improvement, Portugal still maintains a high incidence of disease in the European context. In addition, while the number of Mycobacterium tuberculosis is decreasing, the number of NTM and cases of disease caused by these species of mycobacteria have been increasing in various areas of the world.[15] In a recent study including a population attended at a Portuguese hospital, the percentage of NTM isolations corresponds to approximately 40% of the total of mycobacteria strains identified.[16]

The purpose of this work is thus, through a 10-year retrospective study, to assess the value of multiple samples in smear and culture examination for the diagnosis of TB. We only know one recent study that assesses the value of multiple samples in the TB diagnosis in Portuguese population.[17] As a second goal, the study evaluates the importance of the number of samples for the recovery of NTM.


  Methods Top


Study population

This 10-year retrospective study was conducted at the Clinical Pathology Department of São João Hospital Centre, a tertiary care university hospital located in north of Portugal. The study included 38,887 sputum samples from 11,676 patients analyzed between 2005 and 2014. The Institutional Review Board of the hospital approved this study.

Specimen processing, acid-fast bacilli smear, and culture

When they were received in the laboratory, the samples were kept at 4°C until processed. The samples were decontaminated according to the N-acetyl-L-cysteine-sodium hydroxide routine method.[18] AFB were detected microscopically in sputum decontaminated and concentrated, with a Kinyoun stain (performed in 270 of the 2763 samples) or auramine stain (performed in 2493 of the 2763 samples). The digested, decontaminated and concentrated samples were inoculated into MGIT™ 960 medium (Becton, Dickinson, and Company [BD], New Jersey, U.S.), supplemented with BBL™ MGIT™ OADC (BD) enrichment and BBL™ MGIT™ PANTA™ antibiotic mixture (BD) for 42 days at 37°C. The tubes were placed in the BACTEC™ MGIT™ 960 system (BD) where they were incubated and monitored continuously for an advanced fluorometric technology which permits highly accurate detection of O2 consumption. The positive cultures in BACTEC™ MGIT™ 960 system (manufacturer-set threshold: 75 Growth Units) were examined by smear microscopy by Kinyoun's stain to examine AFB.

Mycobacterial molecular identification

To determine the species of the isolates one or two of the following routine molecular approaches were used: AccuProbe M. tuberculosis complex culture identification test (GenProbe, San Diego, CA) and Genotype Mycobacterium CM, AS and MTBC kits (Hain Lifescience GmbH, Nehren, Germany).

Data and statistical analysis

The relative contribution of each sputum specimen for AFB detection and M. tuberculosis complex (MTBc) or NTM isolation was determined by calculating the yield of the first sample and the incremental yield of the second, third, and more than three samples. The incremental yield in smear was defined as a positive AFB result on the second, third, or more than three specimens when the previous specimens were negative; the incremental yield in culture was defined as a positive culture result on the second, third, or more than three specimens when the previous specimens were negative. SPSS (IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0., IBM Corp., Armoonk, NY, USA) was used on data analysis.


  Results Top


From the set of sputum samples received for mycobacteria research, 2763 samples, corresponding to 1038 patients, were positive for mycobacteria. Seven hundred and thirty-four (70.7%) of these patients were men and 304 (29.3%) women, with an average age of 53.9 and 54.1 years, respectively. The range of ages corresponds to 9–93 years. Five hundred and eighty-two (56.1%) were outpatients and 456 (43.9%) were inpatients.

An MTBc and an NTM strain were isolated in 673 (64.8%) and 365 (35.2%) of these patients, respectively. Our analysis included 457 patients with positive smear and 673 with a positive culture for MTBc and 29 patients with positive smear and 365 with a positive culture for NTM [Table 1]. In [Table 2], we present the distribution of patients with NTM and MTBc isolation by clinical departments where they were attended. Most patients with isolation of both MTBc and NTM strains came from the pulmonology department followed by the infectious diseases department.
Table 1: Distribution of nontuberculous mycobacteria species among patients with a nontuberculous mycobacteria positive smear and culture

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Table 2: Distribution of patients with nontuberculous mycobacteria and Mycobacterium tuberculosis complex isolation by clinical departments

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Results are presented in [Table 3]. Regarding patients with MTBc isolation, we observed that 95.2% (435/457) had the first positive AFB smear in the first and second sample. The percentage of MTBc patients who had the first positive result in the first and second culture was 97.9% (659/673).
Table 3: Relative contribution of each sputum specimen among patients with one, two, three, or more samples

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When we look at patients with NTM identification, we found that 69% (20/29) had the first positive AFB smear in the first and second sample. The incremental yield of third and more than three smears was of 31% (9/29). In turn, the percentage of NTM patients who had the first positive culture in the first and second sample was 86.5% (316/365), and the incremental yield of third and above three samples was 13.5% (49/365).


  Discussion Top


In this retrospective study, the most relevant finding we found was that about 13 in every 100 patients with NTM needed three or more samples for the isolation of these mycobacteria. With regard to NTMs, the incremental yield of the third and more than three smears was 20.7% and 10.3%, respectively. However, the number of NTM-patients with AFB positive smear included was too small, which is a limitation of the study. There are several reasons that may explain this high number of patients with negative smears in which an NTM strain is subsequently isolated. First, the decontamination method, namely that of N-acetyl-L-cysteine-sodium hydroxide, may render some NTMs strains unviable, which may also interfere with smear sensitivity. Second, some NTMs can be misidentified by the auramine stain. Third, many of these NTMs may correspond to colonizing strains and not true agents of infection, and according to the American Thoracic Society criteria, AFB smear, culture results, and clinical status suggest the close correlation between the three.[2]

Regarding culture, the percentage of patients who had their first positive culture in the first sample was 71.2% and the incremental yield of the second, third, and more than three samples was 15.3%, 10.5%, and 3.0%, respectively.

With the increase in the importance of NTM infections, due to an ever-growing susceptible population, we consider that our results are worth evaluating and should be considered.

Regarding MTBc, we estimated that >95% of the cases could be detected in the first and second smear and culture, with a very low increment of the third sample, corresponding to 3.1% in smear and 1.3% in culture. These results corroborate the findings of Mase et al. in a systematic review of 2007 that found that 85.8% of MTBc cases were positive in the first smear, that the incremental yield of the second sample was 11.9% and the increment yield of the third sample ranged from 2% to 5%.[8] The findings are also in line with the revision of Rieder et al. of 2005 that estimated an incremental yield of the second and third samples ranged from 4.5% to 26.9% and from 0.7% to 7.2%, respectively, as well as other recent publications.[8],[9],[10],[11] In addition, since 1998, Nelson et al. had described that 95% of positive cases were detected in the first sample and the increment yield of the third culture was 7%.[12]

Although molecular tests have improved TB diagnosis, in developing countries direct examination still has some advantages: it is easy to carry out and implement, inexpensive, and requires less equipment. In addition, the definite diagnosis of TB continues to depend on the agent isolation and culture is necessary for the subsequent realization of the susceptibility test. Here, we corroborate the previous findings of the number of smears and cultures that should be analyzed for MTBc examination.

As a result of its retrospective nature, the present study has some limitations related to the characterization of the study population. Although we know how many patients came from the infectious diseases department, we did not access the number of HIV-positive patient, for whom in general, the sputum smear microscopy has lower yields. Furthermore, we did not access other underlying diseases of the patients studied. Aspects related to the quality of the samples, such as the timing of the sputum collection, time intervals for sample collection, conditions of conservation of samples before being received in the laboratory, visual appearance, and volume of sputum samples, were also not controlled in the present study. The differences in yield of the detection and isolation of mycobacteria between the months of summer and winter were also not evaluated here.

It is important to note also some aspects of the staining methods as well as the culture media used in the study. The smear of about 90% of the samples included in the study was stained by auramine, considered more sensitive than the carbol fuchsin methods such as the Ziehl-Neelsen and Kinyoun stainings.[1],[19] The smears of the remaining samples were stained by Kinyoun and corresponded to samples from the 1st year of the study, in which the auramine methodology was not yet implemented in our hospital. Regarding the culture media, only the liquid medium results of MGIT were considered since over the 10 years of the study we only found one sputum sample where a mycobacterium was isolated in the solid medium after the liquid medium has not been successful in the recovery of this same mycobacteria. In addition to the decrease in turnaround time, several studies have shown that the BACTEC MGIT liquid culture system has better MTBc recovery rates compared to conventional Löwenstein-Jensen method.[20],[21],[22]

Our study also presents some advantages compared to previous studies. On the one hand, the majority of studies evaluate only the AFB microscopic examination,[9],[10],[11],[23] and here, we evaluated also the mycobacterial culture. On the other hand, all of the studies to which we had access refer only to the MTBc and here, we also accessed the value of multiple samples for NTM identification.


  Conclusions Top


In this study, we found an added value of multiple samples in NTM recovery, which prevalence is increasing worldwide, representing a new challenge for clinical mycobacteriology and the diagnosis of nontuberculosis lung disease. If there is a suspicion of NTM lung disease, the analysis of more than two sputum cultures is recommended. However, since the diagnostic yield of the third sputum is low, our findings in a Portuguese population are in line with current WHO recommendations for submission of only two sputum specimens for the diagnosis of pulmonary tuberculosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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