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Primary cultivation: factors affecting contamination and Mycobacterium ulcerans growth after long turnover time of clinical specimens.

Bratschi MW, Bolz M, Grize L, Kerber S, Minyem JC, Um Boock A, Yeboah-Manu D, Ruf MT, Pluschke G - BMC Infect. Dis. (2014)

Bottom Line: The analysis showed: i) that the use of moist transport media significantly increased the recovery rate of M. ulcerans compared to samples kept dry; ii) that the choice of the decontamination method had no significant effect on the chance of M. ulcerans isolation; and iii) that Löwenstein-Jensen supplemented with antibiotics as inoculation medium yielded the best results.We further found that, ten extra days between sampling and inoculation lead to a relative decrease in the isolation rate of M. ulcerans by nearly 20%.Finally, collection and processing of multiple samples per patient was found to significantly increase the M. ulcerans isolation rate.

View Article: PubMed Central - PubMed

Affiliation: Swiss Tropical and Public Health Institute, Basel, Switzerland. martin.bratschi@unibas.ch.

ABSTRACT

Background: While cultivation of pathogens represents a foundational diagnostic approach in the study of infectious diseases, its value for the confirmation of clinical diagnosis of Buruli ulcer is limited by the fact that colonies of Mycobacterium ulcerans appear only after about eight weeks of incubation at 30°C. However, for molecular epidemiological and drug sensitivity studies, primary isolation of M. ulcerans remains an essential tool. Since for most of the remote Buruli ulcer endemic regions of Africa cultivation laboratories are not easily accessible, samples from lesions often have to be stored for extended periods of time prior to processing. The objective of the current study therefore was to determine which transport medium, decontamination method or other factors decrease the contamination rate and increase the chance of primary isolation of M. ulcerans bacilli after long turnover time.

Methods: Swab and fine needle aspirate (FNA) samples for the primary cultivation were collected from clinically confirmed Buruli ulcer patients in the Mapé Basin of Cameroon. The samples were either stored in the semi-solid transport media 7H9 or Amies or dry for extended period of time prior to processing. In the laboratory, four decontamination methods and two inoculation media were evaluated and statistical methods applied to identify factors that decrease culture contamination and factors that increase the probability of M. ulcerans recovery.

Results: The analysis showed: i) that the use of moist transport media significantly increased the recovery rate of M. ulcerans compared to samples kept dry; ii) that the choice of the decontamination method had no significant effect on the chance of M. ulcerans isolation; and iii) that Löwenstein-Jensen supplemented with antibiotics as inoculation medium yielded the best results. We further found that, ten extra days between sampling and inoculation lead to a relative decrease in the isolation rate of M. ulcerans by nearly 20%. Finally, collection and processing of multiple samples per patient was found to significantly increase the M. ulcerans isolation rate.

Conclusions: Based on our analysis we suggest a procedure suitable for the primary isolation of M. ulcerans strains from patients following long delay between sample collection and processing to establish a M. ulcerans strain collection for research purposes.

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Related in: MedlinePlus

Predicted probabilities forM. ulceransgrowth. Based on the M. ulcerans growth vs. no growth or contamination model the probability of M. ulcerans growth was predicted as a function of transport time for samples transported in Amies medium, decontaminated with NaOH for 10 minutes, inoculated onto LJ medium supplemented with PANTA and if the Ct value of the qPCR was 27.8. Mean predicted probability of the M. ulcerans growth rate and 95% confidence intervals are shown.
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Fig2: Predicted probabilities forM. ulceransgrowth. Based on the M. ulcerans growth vs. no growth or contamination model the probability of M. ulcerans growth was predicted as a function of transport time for samples transported in Amies medium, decontaminated with NaOH for 10 minutes, inoculated onto LJ medium supplemented with PANTA and if the Ct value of the qPCR was 27.8. Mean predicted probability of the M. ulcerans growth rate and 95% confidence intervals are shown.

Mentions: For the identification of factors that affect M. ulcerans recovery in a scenario where some cultures are contaminated, 700 inoculated cultures from 302 qPCR positive swabs (average qPCR Ct value of 28.09) taken from 72 qPCR positive patients were analyzed. As shown in Table 1, 88 inoculations collected from 31 patients of the total 700 inoculations resulted in M. ulcerans growth. This corresponded to a per patient positivity rate of 43.1%. Further, 37.1% (260/700) of the inoculations were contaminated and 50.3% (352/700) did not result in any growth. Analysis of these inoculations in a multivariate analysis showed, that transport medium had a significant effect (p-value: 0.019) on the recovery of M. ulcerans (Table 4). Specifically, swabs transported in Amies medium showed the best recovery rate of M. ulcerans, although not significantly better than 7H9 medium (95% CI of OR: 0.269 - 1.415). As also seen in the analysis for non-contamination (Table 2) and M. ulcerans growth (Table 3), the decontamination methods evaluated here did not significantly differ in their effect on the chance of M. ulcerans recovery (p-value: 0.295; Table 4). On the other hand the use of the inoculated media had a significant (p-value: 0.003) impact on the chance of M. ulcerans recovery, with the use of LJ as compared to LJ_PANTA reducing the probability of M. ulcerans recovery by 65.5%. As further shown in Table 4, a one unit increase in the qPCR Ct value away from the mean qPCR Ct value of the inoculated swabs decreased the chance of M. ulcerans recovery by 10.8% (p-value: 0.011). Furthermore, consistent with the analysis of factors affecting M. ulcerans growth (Table 3), the time span from sampling to inoculation significantly (p-value: 0.006) reduced the chance of M. ulcerans isolation, with a decrease of 19.1% for every 10 extra days of storage compared to the mean storage time of 80.2 days (Table 4, Figure 2). As shown in Figure 2, the predicted probability of M. ulcerans recovery decreased from 61.2% at zero days of storage to 26.8% within 70 days of storage. Specifically for swabs with a qPCR Ct value of 27.8, that were transported in Amies medium, decontaminated using NaOH for 10 minutes and inoculated onto LJ with PANTA, a probability of M. ulcerans growth of 61.2% can be expected if samples were stored for zero days. If the samples are stored for 50 or 100 days, the predicted probability decreases to 35.3% and 15.9% respectively.Table 4


Primary cultivation: factors affecting contamination and Mycobacterium ulcerans growth after long turnover time of clinical specimens.

Bratschi MW, Bolz M, Grize L, Kerber S, Minyem JC, Um Boock A, Yeboah-Manu D, Ruf MT, Pluschke G - BMC Infect. Dis. (2014)

Predicted probabilities forM. ulceransgrowth. Based on the M. ulcerans growth vs. no growth or contamination model the probability of M. ulcerans growth was predicted as a function of transport time for samples transported in Amies medium, decontaminated with NaOH for 10 minutes, inoculated onto LJ medium supplemented with PANTA and if the Ct value of the qPCR was 27.8. Mean predicted probability of the M. ulcerans growth rate and 95% confidence intervals are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4264541&req=5

Fig2: Predicted probabilities forM. ulceransgrowth. Based on the M. ulcerans growth vs. no growth or contamination model the probability of M. ulcerans growth was predicted as a function of transport time for samples transported in Amies medium, decontaminated with NaOH for 10 minutes, inoculated onto LJ medium supplemented with PANTA and if the Ct value of the qPCR was 27.8. Mean predicted probability of the M. ulcerans growth rate and 95% confidence intervals are shown.
Mentions: For the identification of factors that affect M. ulcerans recovery in a scenario where some cultures are contaminated, 700 inoculated cultures from 302 qPCR positive swabs (average qPCR Ct value of 28.09) taken from 72 qPCR positive patients were analyzed. As shown in Table 1, 88 inoculations collected from 31 patients of the total 700 inoculations resulted in M. ulcerans growth. This corresponded to a per patient positivity rate of 43.1%. Further, 37.1% (260/700) of the inoculations were contaminated and 50.3% (352/700) did not result in any growth. Analysis of these inoculations in a multivariate analysis showed, that transport medium had a significant effect (p-value: 0.019) on the recovery of M. ulcerans (Table 4). Specifically, swabs transported in Amies medium showed the best recovery rate of M. ulcerans, although not significantly better than 7H9 medium (95% CI of OR: 0.269 - 1.415). As also seen in the analysis for non-contamination (Table 2) and M. ulcerans growth (Table 3), the decontamination methods evaluated here did not significantly differ in their effect on the chance of M. ulcerans recovery (p-value: 0.295; Table 4). On the other hand the use of the inoculated media had a significant (p-value: 0.003) impact on the chance of M. ulcerans recovery, with the use of LJ as compared to LJ_PANTA reducing the probability of M. ulcerans recovery by 65.5%. As further shown in Table 4, a one unit increase in the qPCR Ct value away from the mean qPCR Ct value of the inoculated swabs decreased the chance of M. ulcerans recovery by 10.8% (p-value: 0.011). Furthermore, consistent with the analysis of factors affecting M. ulcerans growth (Table 3), the time span from sampling to inoculation significantly (p-value: 0.006) reduced the chance of M. ulcerans isolation, with a decrease of 19.1% for every 10 extra days of storage compared to the mean storage time of 80.2 days (Table 4, Figure 2). As shown in Figure 2, the predicted probability of M. ulcerans recovery decreased from 61.2% at zero days of storage to 26.8% within 70 days of storage. Specifically for swabs with a qPCR Ct value of 27.8, that were transported in Amies medium, decontaminated using NaOH for 10 minutes and inoculated onto LJ with PANTA, a probability of M. ulcerans growth of 61.2% can be expected if samples were stored for zero days. If the samples are stored for 50 or 100 days, the predicted probability decreases to 35.3% and 15.9% respectively.Table 4

Bottom Line: The analysis showed: i) that the use of moist transport media significantly increased the recovery rate of M. ulcerans compared to samples kept dry; ii) that the choice of the decontamination method had no significant effect on the chance of M. ulcerans isolation; and iii) that Löwenstein-Jensen supplemented with antibiotics as inoculation medium yielded the best results.We further found that, ten extra days between sampling and inoculation lead to a relative decrease in the isolation rate of M. ulcerans by nearly 20%.Finally, collection and processing of multiple samples per patient was found to significantly increase the M. ulcerans isolation rate.

View Article: PubMed Central - PubMed

Affiliation: Swiss Tropical and Public Health Institute, Basel, Switzerland. martin.bratschi@unibas.ch.

ABSTRACT

Background: While cultivation of pathogens represents a foundational diagnostic approach in the study of infectious diseases, its value for the confirmation of clinical diagnosis of Buruli ulcer is limited by the fact that colonies of Mycobacterium ulcerans appear only after about eight weeks of incubation at 30°C. However, for molecular epidemiological and drug sensitivity studies, primary isolation of M. ulcerans remains an essential tool. Since for most of the remote Buruli ulcer endemic regions of Africa cultivation laboratories are not easily accessible, samples from lesions often have to be stored for extended periods of time prior to processing. The objective of the current study therefore was to determine which transport medium, decontamination method or other factors decrease the contamination rate and increase the chance of primary isolation of M. ulcerans bacilli after long turnover time.

Methods: Swab and fine needle aspirate (FNA) samples for the primary cultivation were collected from clinically confirmed Buruli ulcer patients in the Mapé Basin of Cameroon. The samples were either stored in the semi-solid transport media 7H9 or Amies or dry for extended period of time prior to processing. In the laboratory, four decontamination methods and two inoculation media were evaluated and statistical methods applied to identify factors that decrease culture contamination and factors that increase the probability of M. ulcerans recovery.

Results: The analysis showed: i) that the use of moist transport media significantly increased the recovery rate of M. ulcerans compared to samples kept dry; ii) that the choice of the decontamination method had no significant effect on the chance of M. ulcerans isolation; and iii) that Löwenstein-Jensen supplemented with antibiotics as inoculation medium yielded the best results. We further found that, ten extra days between sampling and inoculation lead to a relative decrease in the isolation rate of M. ulcerans by nearly 20%. Finally, collection and processing of multiple samples per patient was found to significantly increase the M. ulcerans isolation rate.

Conclusions: Based on our analysis we suggest a procedure suitable for the primary isolation of M. ulcerans strains from patients following long delay between sample collection and processing to establish a M. ulcerans strain collection for research purposes.

Show MeSH
Related in: MedlinePlus