Mycobacterium and Aerobic actinomycetes Culture

22 Pages·2016·0.39 MB·English

JCM Accepted Manuscript Posted Online 10 February 2016
J. Clin. Microbiol. doi:10.1128/JCM.02838-15
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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3 Mycobacterium and Aerobic actinomycetes Culture:
4 Are Two Medium Types and Extended Incubation Times Necessary?
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6 Patricia J. Simner, Kelly A. Doerr, Lory K. Steinmetz and Nancy L. Wengenack
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8 Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology,
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9 Mayo Clinic, Rochester, MN

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12 Running Title: Mycobacterium Culture Incubation Time s
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15 Corresponding Author: a
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16 Nancy L. Wengenack, Ph.D. 2
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17 Division of Clinical Microbiology 2
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18 Department of Laboratory Medicine and Pathology
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19 Mayo Clinic g
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20 200 First Street t
21 Rochester, MN 55905
22 E-mail: [email protected]
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24 ABSTRACT:
25 Mycobacterial cultures are historically performed using a liquid medium and a solid agar
26 medium with an incubation period of up to 60 days. We performed a retrospective
27 analysis of 21,494 mycobacterial and aerobic actinomycetes cultures performed over 10
28 months to determine whether two medium types remain necessary and to investigate
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29 whether culture incubation length could be shortened. Specimens were cultured to
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30 BACTEC™ MGIT™ liquid medium and Middlebrook 7H11/S7H11 solid medium with a
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31 incubation for 42 and 60 days, respectively. Time-to-positivity and the identity of isolates
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32 recovered from each medium were evaluated. 1205/21,494 cultures (6%) were positive

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33 on at least one medium. Of the 1353 isolates recovered, 1110 (82%) were p
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34 nontuberculous mycobacteria, 145 (11%) were aerobic actinomycetes and 98 (7%) were m
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35 Mycobacterium tuberculosis complex. Assessing medium types, 1121 isolates were s
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36 recovered from solid medium cultures, 922 isolates were recovered from liquid medium r
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37 and 690 isolates were recovered on both media. Liquid cultures were positive an average n

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38 of 10 days before solid cultures when both medium types were positive (p<0.0001). a
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39 Isolates detected on solid medium after 6 weeks of incubation included 65 (5%) 2
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40 nontuberculous mycobacteria, 4 (0.3%) aerobic actinomycetes and 2 (0.2%) M. 2
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41 tuberculosis complex. Medical chart review suggested most of these later-growing
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42 isolates were insignificant as the diagnosis was already known or they were considered g
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43 colonizers/contaminants. t
44 This study reaffirms the need for both liquid and solid medium for mycobacterial
45 and aerobic actinomycetes culture and demonstrates that solid medium incubation times
46 may be reduced to 6 weeks without significantly impacting sensitivity.
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48 INTRODUCTION
49 Traditionally, mycobacterial and aerobic actinomycetes cultures are performed
50 using both liquid and solid medium with extended incubation times of up to 60 days
51 recommended for optimal recovery (1). The use of two types of medium is
52 recommended by the College of American Pathologists (CAP) accreditation program
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53 (MIC.32250) and the Clinical and Laboratory Standards Institute (CLSI) (1, 2).
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54 Many of the studies supporting these recommendations were performed 10-20 a
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55 years ago (3-6). Since that time much has changed in mycobacteriology including the
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56 transition of many laboratories from use of radiometric liquid medium to Mycobacteria

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57 Growth Indicator Tubes (MGIT, Becton Dickinson, Franklin Lakes, NJ) or VersaTREK p
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58 (TREK Diagnostics/Thermo Fisher, Oakwood Village, Ohio) medium and the description m
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59 of many new species of mycobacteria (7, 8). s
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60 As laboratories enter into an era with a significant emphasis on cost-effective r
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61 health care, it becomes increasingly important to examine traditional laboratory practices n

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62 to ensure they meet the needs of a changing health-care environment and to look for cost- a
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63 savings opportunities. The purpose of this study was to determine whether one type of 2
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64 medium could be eliminated or if incubation times could be shortened for mycobacteria 2
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65 and aerobic actinomycetes cultures, including Nocardia spp., as a cost-saving measure
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66 without impacting the quality of patient care. A retrospective study was performed by g
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67 examining results for 21,494 mycobacterial and aerobic actinomycetes cultures in liquid t
68 medium and on solid medium with an incubation period of up to 8 weeks.
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70 MATERIALS AND METHODS
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71 Study Design
72 A retrospective study was performed over 10 months examining culture results
73 and time to positivity for 21,494 mycobacterial and aerobic actinomycetes cultures.
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75 Mycobacterial and Aerobic actinomycetes Culture:
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76 Specimens submitted for mycobacterial and aerobic actinomycetes culture were
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77 cultured to BACTEC™ MGIT™ liquid medium with incubation for 42 days and to a
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78 Middlebrook 7H11/S7H11 solid medium biplates which were incubated for 60 days.
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79 Biplate cultures were incubated at 37°C in an atmosphere of 5-7% CO and were
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80 examined weekly for growth. In addition, any specimen submitted from the extremities p
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81 (e.g., skin, arm, leg) were inoculated on a solid 7H10 media with a heme supplement m
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82 (i.e., X-factor disk) and incubated at 30ºC for 60 days. Specimens from non-sterile s
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83 sources (e.g., respiratory specimens) were processed using the BBL MycoPrep Specimen r
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84 Digestion/Decontamination Kit (Becton, Dickinson and Company (BD), Franklin Lakes, n

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85 NJ) which included a 15 min exposure to N-acetyl-cysteine/2% NaOH and resuspension a
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86 of the pellet in 3mL of MycoPrep Phosphate Buffer (BD). Inoculation of medium was 2
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87 done by adding 0.5mL of sample to MGIT tube broth and to each side of a Middlebrook 2
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88 7H11/S7H11 agar plate. Negative MGIT tube broth cultures were visually checked
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89 following 42 days of incubation for growth (clumps/flecks) in the medium before g
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90 discarding and if any were noted, the broth was subcultured to a Middlebrook agar plate t
91 and a blood agar plate to look for organism(s).
92 When a MGIT liquid medium tube signaled positive or when growth was visually
93 observed on solid medium, a Kinyoun acid fast stain or modified acid fast stain was
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94 performed to confirm the presence of a mycobacterium or an aerobic actinomycetes,
95 respectively. Broth from a positive MGIT tube was mixed and then subcultured to a
96 Middlebrook medium plate and streaked for isolation of colonies and to a blood agar
97 plate to look for potential contamination by bacteria or yeasts. Isolates were identified
98 from positive MGIT tubes or from isolates on Middlebrook agar using either the
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99 AccuProbe® nucleic acid hybridization probes for M. tuberculosis complex, M. avium w
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100 complex and M. gordonae (Hologic GenProbe, San Diego, CA) or using 500bp partial a
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101 16S rRNA gene sequencing as previously described (9).
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102 Analysis of Data: h
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103 The time-to-positivity (TTP) and the identity of isolates recovered from liquid and //
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104 solid medium were compared. Statistical analysis was performed using a one-tailed .
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105 Student’s T test.
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107 Chart Review: M
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108 For isolates demonstrating growth on solid medium after 6 weeks of incubation, a c
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109 chart review was performed to determine whether the attending physician chose to treat 6
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110 the patient as a result of the delayed growth of organism or whether they regarded the 1
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111 isolate as a potential contaminant or colonizer that did not require therapy. Some isolates y

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112 growing after 6 weeks on solid medium were from specimens submitted to our reference e
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113 laboratory and therefore medical records were not available for review. The Mayo
114 Clinic Institutional Review Board (IRB) approved this study.
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117 RESULTS:
118 During the study period, 1205/21,494 cultures (6%) were positive for
119 mycobacteria or aerobic actinomycetes on at least one medium type. From the 1205
120 positive cultures, 1353 mycobacteria or aerobic actinomycetes were identified of which
121 1110 (82%) were nontuberculous mycobacteria (NTM), 145 (11%) were aerobic
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122 actinomycetes and 98 (7%) belonged to the Mycobacterium tuberculosis complex. The
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123 most common mycobacterial species and aerobic actinomycetes genera and species a
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124 isolated are summarized in Tables 1 and 2. Of the 1205 positive cultures, 175 “mixed”
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125 cultures (15%) were observed with 86/175 cultures containing multiple organisms while

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126 89/175 mixed cultures actually contained multiple morphotypes (e.g., a rough and a p
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127 smooth morphology or a dry and a moist morphology) of the same organism. The most m
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128 common organisms with multiple morphotypes observed were M. avium complex (MAC, s
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129 58/89; 65%), M. abscessus complex (18/89; 20%) and M. fortuitum (3/89; 3%). M. r
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130 abscessus complex includes M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, n

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131 and M. abscessus subsp. massiliense. A total of 45 different combinations of organisms a
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132 were observed among truly mixed cultures. The most common combinations from mixed 2
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133 cultures were MAC and M. gordonae (13; 15%), MAC and M. abscessus complex (11; 2
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134 13%), MAC and M. fortuitum (7; 8%) and M. abscessus complex and M. gordonae (5;
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135 6%). g
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136 Of the 1353 total organisms, 1121 grew on solid medium (83%), 922 (68%) grew t
137 in liquid medium, and 690 (51%) grew on/in both media types. Solid medium was
138 positive without corresponding liquid medium positivity for 431 isolates from 395
139 cultures (33% of positive cultures). Previous literature reports comparing the recovery of
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140 mycobacteria in broth and on solid medium often Negative liquid medium cultures in
141 the face of a positive solid medium culture occurred most often when low quantities of
142 organism was recovered (1-3 colonies of growth for 288 cultures), when a mixed culture
143 was identified (69 cultures), or when the MGIT liquid medium was contaminated with
144 bacteria or fungi (55 cultures). Of the 431 isolates that grew only on solid medium, 348
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145 were NTM, 70 were aerobic actinomycetes and 13 were M. tuberculosis complex. Liquid
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146 medium was positive without corresponding solid medium positivity in 232 isolates from a
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147 230 cultures (19%). These included 189 NTM, 34 aerobic actinomycetes and 9
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148 Mycobacterium tuberculosis complex isolates. Of a total of 20 isolates that grew on the

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149 extremity plate, 7 NTM isolates grew strictly on the extremity plate at 30ºC with a X- p
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150 factor disk, including 2 M. haemophilum isolates and 1 isolate each of M. marinum, M. m
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151 chelonae, M. monacense, M. peregrinum/M. septicum and a Mycobacterium species s
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152 which could not be further identified. Interestingly, 2 additional M. haemophilum r
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153 isolates grew on solid medium on the standard 7H11/S7H11 biplate from 2 blood n

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154 specimens without the addition of an X-factor disk or lower incubation temperatures. a
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155 The time-to-positivity (TTP) for mycobacterial and aerobic actinomycetes 2
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156 cultures from liquid medium and solid medium are summarized in Tables 3 and 4, 2
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157 respectively. A comparison of TTP for isolates with both positive MGIT liquid and solid
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158 cultures is summarized in Table 5. Overall, identification of mycobacterial isolates was g
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159 significantly quicker by an average of 10 days with liquid medium cultures compared to t
160 solid medium cultures when both media types were positive (p<0.0001). Greater than
161 90% of all liquid medium cultures were positive after 4 weeks of incubation (92% of
162 NTM, 93% of M. tuberculosis complex and 89% of aerobic actinomycetes) and 6 weeks
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163 of incubation for solid medium (92% of NTM, 98% of M. tuberculosis complex and 96%
164 of aerobic actinomycetes). The greatest differences in TTP were observed among NTM
165 (liquid medium TTP = 10 days vs solid medium TTP = 20 days; difference of 10 days; p-
166 value <0.0001) and M. tuberculosis complex isolates (liquid medium TTP = 14 days vs
167 solid medium TTP = 23 days; difference of 9 days; p-value <0.0001). A difference in
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168 TTP of 1 day was observed when comparing liquid medium (10 days) to solid medium
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169 (11 days) for aerobic actinomycetes (p-value = 0.1538). Not surprisingly, the TTP was a
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170 faster if the AFB smear was positive from specimen (data not shown).
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171 A chart review was performed where possible to determine the clinical

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172 significance of isolates that grew on solid medium after more than 6 weeks of incubation. p
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173 Overall, 65 NTM, 4 aerobic actinomycetes and 2 M. tuberculosis complex isolates were m
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174 positive on solid medium after 42 days of incubation. Many of the NTM isolates (45/65; s
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175 71%) were present in low quantities (1-3 colonies). Of the 65 NTM, 42 isolates were r
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176 identified as MAC, 5 were M. xenopi, 7 were M. gordonae, 4 were M. lentiflavum, 3 were n

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177 Mycobacterium species that could not be further identified and there was one isolate each a
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178 for M. abscessus, M. chelonae, M. kansasii and M. paraffinicum. Of the MAC isolates, 2
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179 21/42 (50%) were already identified from a liquid culture of the same specimen. A chart 2
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180 review was able to be performed on 9 of the remaining 21 MAC cultures. The cultures
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181 were from either a patient previously known to have a MAC pulmonary infection who g
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182 was already on antimycobacterial therapy or they were deemed not significant by the t
183 clinical attending as a contaminant or colonizer since only 1 or 2 colonies were isolated
184 from a single culture. Interestingly from a total of 11 M. xenopi isolates from this study,
185 5 were positive by solid medium only after 42 days of incubation. This may be due to
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186 the fact that they were not incubated at the optimal growth temperature for M. xenopi
187 which is 42ºC and this may have resulted in a slower growth rate. Three of the 5 isolates
188 were from an arm biopsy specimen that had grown M. xenopi from liquid medium within
189 42 days on a separate culture. Also of 7 total M. lentiflavum isolates from this study 4
190 (57%) were positive after 6 weeks on solid medium. This may be due to its slower
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191 growth rate (“lentus” is from the Latin for “slow”) with the range for TTP of 22 to 56
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192 days. Unfortunately, all the M. xenopi and M. lentiflavum cultures positive after 42 days a
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193 were from reference laboratory clients and no charts were available for review to
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194 determine significance. The remaining NTM isolated after 42 days of incubation were

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195 deemed not significant after chart review as most only had one or two colonies isolated p
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196 and the physician elected not to treat with antimycobacterial agents. Of the 5 aerobic m
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197 actinomycetes that grew after 42 days of incubation, 4 were identified as Rhodococcus s
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198 equi (1 colony up to “few” colonies isolated) and 1 isolate was identified as Gordonia r
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199 bronchialis (1 colony isolated). None of the 5 isolates were recovered in liquid culture. n

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200 Importantly, the 2 M. tuberculosis complex isolates that were positive after 6 weeks on a
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201 solid medium were previously positive in liquid medium culture and were identified after 2
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202 15 days and 16 days of incubation in liquid medium, respectively. 2
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204 DISCUSSION g
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205 As the laboratory evolves into an era of increased emphasis on cost-effective t
206 health care and reduced reimbursement, evaluating traditional practices for continued
207 effectiveness is important. An example is the suggested use of inexpensive and readily
208 available 5% blood agar plates as a replacement to specialized egg-based medium (LJ) or
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209 agar based medium (Middlebrook) for the recovery of mycobacteria (10). The purpose of
210 our study was to challenge the dogma of requiring both solid and liquid medium as well
211 as extended incubation periods for isolation of mycobacteria and aerobic actinomycetes.
212 Several studies have been performed previously to examine this issue but they
213 encompassed a smaller number of cultures, evaluated the recovery of M. tuberculosis
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214 complex only or were performed 15-20 years ago when the recognized diversity of
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215 Mycobacterium species and aerobic actinomycetes genera and species was much smaller a
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216 and the available culture platforms were different (11-15). Thus, we performed a large
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217 retrospective study over 10 months examining 21,494 mycobacterial and aerobic

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218 actinomycetes cultures using both liquid and solid medium. p
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219 This study confirms the continued necessity for both liquid medium and solid m
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220 medium to optimize the culture recovery of mycobacteria and aerobic actinomycetes. s
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221 Our results are somewhat in contrast to a study by Sharp et al that concluded that r
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222 Lowenstein-Jensen (LJ) solid medium was no longer necessary with the implementation n

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223 of the BACTEC MB9000 broth system for mycobacterial culture (14). The Sharp et al a
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224 study was published in 2000 which was prior to the recognition of many species of 2
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225 nontuberculous mycobacteria. Isolates in that study were identified as M. tuberculosis 2
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226 complex, M. avium complex, M. fortuitum-chelonae complex, "pigmented
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227 Mycobacterium species" and "Mycobacterium species" based on the available methods at g
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228 the time. In that study, 72 of 331 isolates (22%) were recovered only on solid LJ medium t
229 which is similar to our results but they were only able to be identified as "pigmented
230 Mycobacterium species" and "Mycobacterium species" at that time. These isolates were
231 deemed to be non-pathogens in that study and so their recovery on solid medium alone
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