JCM Accepts, published online ahead of print on 15 May 2013

J. Clin. Microbiol. doi:10.1128/JCM.00332-13

Copyright 2013, American Society for Microbiology. All Rights Reserved.

1 Detection of Mycobacterium tuberculosis in blood using the Xpert MTB/RIF assay

3 Padmapriya Banada, Ranie Koshy, and David Alland#

5 Center for Infectious Diseases, New Jersey Medical School UMDNJ, 185 South Orange

6 Avenue, Newark, NJ 07103

10

11

12

13 # Correspondent footnote:

14 Division of Infectious Disease, New Jersey Medical School UMDNJ, 185 South

15 Orange Avenue, MSB A920C, Newark, NJ 07103. Ph: (973) 972-2179. Fax: (973) 972-

16 0713. Email: allandda@umdnj.edu.

17

18 Key words: Blood, Tuberculosis, TB bacteremia, Xpert MTB/RIF assay, blood

19 processing

20

21 Running title: Detection of TB in blood

22

23

24
25 ABSTRACT

26 We have developed a novel blood lysis-centrifugation approach for highly-

27 sensitive Mycobacterium tuberculosis (MTB) detection in large volumes of blood using

28 the Xpert MTB/RIF assay. One through 20 ml of blood were spiked with 0.25 to 10

29 colony forming units (CFU)/ml of the MTB surrogate M. bovis BCG. Multiple replicates

30 of each sample were processed by a new lysis-centrifugation method, and tested with the

31 Xpert MTB/RIF assay. The assay was very sensitive at increased blood volumes. In the

32 20 ml samples, BCG was detected in blood spiked with 10, 5, 1, and 0.25 CFU/ml 100%,

33 100%, 83%, and 57% of the time, respectively, compared to 100%, 66%, 18% and 18%,

34 respectively in 1 ml blood samples. Assay sensitivity was influenced by the type of

35 anticoagulant used, with Acid Citrate Dextrose (ACD)-B providing the best results. Limit

36 of detection (LOD) of 10 CFU/ml was established with BCG spiked in ACD-B blood (1

37 to 50 CFU/ml in 5 ml) and 92%, 36% and 33% of samples with 5, 1 and 0.5 CFU/ml,

38 respectively, were assay positive. The lysis-buffer was stable both at room temperature

39 and 4C for two months. The assay could be tested with blood stored for 8 days without

40 change in sensitivity as measured by cycle threshold. This new assay format extends the

41 capability of the Xpert MTB/RIF test, enabling up to 20 ml of blood to be tested rapidly

42 for the presence of MTB. This approach may be a useful method to detect

43 extrapulmonary TB and mortality risk, in immunocompromized patients.

44

Detection of TB in blood- Banada et al., Page 2

45 INTRODUCTION

46 Tuberculosis (TB) is one of the leading causes of death from an infectious disease

47 worldwide (17). Diagnostic delays contribute significantly to mortality. In the case of

48 pulmonary TB (PTB), a diagnosis can often be made by testing expectorated sputum

49 samples. However, some patients are not able to expectorate sputum, especially if they

50 are very sick or young. Extra-pulmonary tuberculosis (EPTB) is common (18, 33),

51 especially in patients with human immunodeficiency virus (HIV) infection. Mortality in

52 subjects with dual HIV and Mycobacterium tuberculosis infection can be very high (15,

53 16). Extra-pulmonary tuberculosis is usually diagnosed by performing microscopic,

54 culture-based or nucleic acid amplification tests (NATs) on aspirated fluid or tissue

55 biopsy material, depending on the site that is infected (12, 38). Microscopy has a low

56 sensitivity for diagnosis of EPTB and reliance on culture-based detection methods can

57 lead to substantial delays in diagnosis (2, 41). Rapid tuberculosis NATs have not been

58 optimized for non-pulmonary samples and have had variable performance detecting TB

59 from tissue or fluids (19, 29, 41, 42). Thus, there is a real need for improved methods to

60 detect extra-pulmonary samples.

61 Blood is an attractive option for the detection of TB, especially in HIV infected

62 patients. Blood is easier and more reliable to sample than sputum, especially in patients

63 who are confused, obtunded or who do not have a productive cough. M. tuberculosis can

64 be cultured from 2% - 64% of blood samples depending on the study population (22). In

65 sub-Saharan Africa, M. tuberculosis is a common cause of bacteremia, accounting for

66 54% of all blood stream infections (BSI) especially among HIV positives (43).

67 Furthermore, disseminated M. tuberculosis infection resulting in BSI may be an

68 important predictor of mortality (15, 16).

Detection of TB in blood- Banada et al., Page 3
69 M. tuberculosis is rarely detected in blood tests despite the potential advantages of

70 this approach. TB blood culture usually takes several weeks to become positive; and

71 requires appropriately equipped facilities (30, 32). NATs such as the polymerase chain

72 reaction (PCR) have been investigated, but sensitivity in blood has been poor (20% to

73 55%) (1, 13, 15, 28, 34, 36). This could in part be due to the presence of PCR inhibitors

74 (3, 4). The poor sensitivity of blood-based NATs could also be due to the small volume

75 of blood (500 l to <5 ml) that is usually collected for these tests. These low volumes

76 could make it difficult or impossible to detect M. tuberculosis circulating in blood if the

77 bacteria are present at very low densities. However, 5 10 ml of blood is typically used

78 for blood cultures (20, 40). NATs for TB might have sensitivities equal or greater than

79 blood culture if similar volumes of blood could be tested by improved sample processing

80 methods.

81 The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) has greatly simplified

82 tuberculosis detection in sputum samples (9, 10, 26). This completely automated real

83 time PCR assay simultaneously detects the presence of M. tuberculosis and rifampicin

84 resistance (21). The assay is approximately 98.3% sensitive and 99% specific for

85 detecting M. tuberculosis in sputum samples{Boehme, 2010 #26;Helb, 2010 #29}.

86 Sensitivity for extra-pulmonary samples has varied from 25% to 100% depending on the

87 body site and study (1, 13, 28, 34, 39). The ability of the Xpert MTB/RIF assay to

88 detect M. tuberculosis in blood has not been investigated and the current sample

89 processing protocol used in the Xpert MTB/RIF assay has not been previously

90 optimized for blood. For example, the current Xpert MTB/RIF protocol uses 1 ml of

91 sputum, while a protocol for blood should arguably start from larger sample volumes.

Detection of TB in blood- Banada et al., Page 4

 92 Here, we present a novel lysis and centrifugation approach which permits

93 relatively large volumes of blood to be tested with the Xpert MTB/RIF assay. Analytic

94 results suggest that our new approach could be usefully applied to clinical testing of

95 blood samples from patients suspected of tuberculosis.

96 MATERIALS AND METHODS

97 Ethics statement. The study was approved by the University of Medicine and

98 Dentistry of New Jersey (UMDNJ)-Institutional review board (IRB) in protocol #

99 0120080060 (for use of discarded blood tests), #0120060015 (for use of discarded blood

100 bank blood), and #012010406 (for drawing and testing blood from healthy human

101 volunteers). Blood from human volunteers was obtained with informed consent.

102 Bacterial strain, culture conditions. The Mycobacterium bovis BCG Pasteur

103 used as a surrogate test strain for M. tuberculosis was a kind gift of William Jacobs, Jr.,

104 Albert Einstein College of Medicine, Bronx, NY. BCG was propagated as described

105 previously (5). Experiments used pre-quantitated BCG that was frozen in aliquots.

106 Aliquots were sonicated three times (Branson ultrasonic cleaner 1510, Branson

107 ultrasonics, Danbury, CT) for 30s each, before serial dilutions were made in Middlebrook

108 7H9 broth (BD, Franklin Lakes, NJ).

109 Blood sample collection and storage. Expired blood [with anticoagulant

110 Citrate-phosphate-dextrose-adenine (CPDA)] from the UMDNJ-University Hospital

111 (UH, Newark, NJ) blood bank that would normally have been discarded was collected

112 and stored at 4C until use. Hematocrit values were adjusted to 40% by diluting the

113 banked blood (hematocrit 60-80%) in phosphate buffered saline (PBS, 0.01M, pH 7.4) to

114 simulate to normal adult blood hematocrit (6, 7). Studies to examine the effect of

115 anticoagulants as well as limit of detection experiments were performed using fresh blood
Detection of TB in blood- Banada et al., Page 5
116 collected from healthy human volunteers. This blood was drawn directly into 8 ml BD

117 vacutainer tubes (BD, NJ) containing either Potassium Ethylenediaminetetraacetic acid

118 (K2EDTA), or Acid Citrate Dextrose (ACD-A, ACD-B) as anticoagulants and analyzed

119 immediately or stored at 4C for no longer than 24 h until use.

120 Blood sample processing. RBC lysis solution 2.2.1 was prepared to contain 10%

121 (w/v) sucrose, 0.5% (w/v) magnesium chloride, 5% (v/v) triton-X-100, in a 0.01M tris

122 HCl solution adjusted to pH 7.2. When processing blood sample volumes 5ml, RBC

123 lysing solution 2.2.1 was added at 15% v/v to blood, mixed well and incubated at room

124 temperature (20C-25C) for 10 min. The blood tubes were centrifuged for 30 min at

125 3000 xg. The supernatant was carefully decanted and the pellet was resuspended in 1 ml

126 of phosphate buffered saline (PBS, pH 7.2). Xpert MTB/RIF assay sample reagent (SR,

127 Cepheid) was added at 1:1 ratio to the resuspended sample, mixed well and the sample

128 was loaded into Xpert MTB/RIF assay cartridges [version G3, Research use only

129 (RUO)] after 15 min incubation at room temperature (Fig. 1). When processing 1 ml

130 blood volumes, the blood sample was mixed with 1 ml of the Xpert SR and incubated

131 for 15 min. The blood-SR mixture was then loaded into the sample loading chamber of

132 the Xpert MTB/RIF assay cartridge. Subsequent sample processing and PCR were then

133 performed as per the manufacturers recommendations for sputum samples (Cepheid,

134 CA).

135 Effect of blood volume on detection of very low levels of TB in blood. To

136 determine the effect of blood volume on sensitivity for M. tuberculosis, BCG cells were

137 spiked at 0.25, 1, 5 and 10 CFU/ml concentrations into various volumes of reconstituted

138 banked blood. Blood samples were processed as described above. All the samples were

139 run in replicates of seven.

Detection of TB in blood- Banada et al., Page 6
140 Limit of detection (LOD) of Mycobacteria in ACD-B treated blood. ACD-B

141 proved to be the best performing anticoagulant in our tests. Therefore the LOD was

142 established using the blood from healthy volunteers collected in ACD-B anticoagulant

143 tubes (BD). M. bovis BCG was spiked at concentrations of 0.5, 1, 5, 10 and 50 CFU/ml in

144 10 ml ACD-B blood. All the samples were processed following the lysis centrifugation

145 protocol. Replicates of 7 to 8 were run for each cell concentration in two separate

146 experiments or runs for a total of 15 replicates.

147 Stability of solution 2.2.1. RBC lysis solution 2.2.1 was prepared and stored at

148 both room temperature (RT) and at refrigerated conditions (4- 8C) for up to 3 months.

149 The aliquoted tubes were removed at days 1, 7, 15, followed by 1 month, 2 months, 3

150 months; and tested as follows. For each time point, reconstituted banked blood was

151 spiked with 10 CFU/ml of BCG per 10 ml of blood. RBC lysis solution 2.2.1 was added

152 at 15% v/v (1.5 ml per 10 ml) and the blood was processed as described above. Stability

153 was assessed by average cycle threshold (Ct) of the first rpoB probe measured in the

154 Xpert MTB/RIF assay, with increasing Ct number indicative of degraded performance.

155 Storage of BCG infected blood at refrigerated temperature. Tests were

156 performed to understand how long ACD-B anticoagulated blood could be stored in the

157 refrigerator before processing. BCG was spiked to 10 ml of ACD-B treated blood at 10

158 CFU/ml and stored in the refrigerator (2-8C) for up to 8 days. The sample was removed

159 and processed using solution 2.2.1 following the protocol described above. The effect of

160 storage time was evaluated by observing any change in the cycle threshold (Ct) or the end

161 point fluorescence (EPF) values in the first rpoB probe measured in the Xpert

162 MTB/RIF assay.

Detection of TB in blood- Banada et al., Page 7

163 Statistical analysis. Analysis (average, standard deviation and t-test) was

164 performed using Microsoft Excel 2000 for Windows. One-way Analysis of variance for

165 independent or correlated samples (ANOVA) was carried out to evaluate the P values

166 using the online ANOVA calculator http://faculty.vassar.edu/lowry/anova1u.html. Curve

167 fitting for LOD curve was done by sigmoid linear regression analysis using Sigmaplot

168 V8.0.

169

Detection of TB in blood- Banada et al., Page 8

170 RESULTS

171 Large volumes of blood detect and outperform small volumes. We developed

172 a protocol that enabled us to lyse up to 20 ml of blood, concentrate any M. tuberculosis

173 present in the sample by centrifugation, and re-suspend the resulting pellet in the Xpert

174 SR. The resulting sample was then suitable for testing using the standard sputum

175 protocol. We tested this approach by spiking between 0.25 CFU/ml and 10 CFU/ml of

176 BCG into different volumes of (blood-banked) blood ranging from 1 ml through 20 ml.

177 The 1 ml blood samples were processed without lysis/centrifugation by mixing them with

178 equal volumes of SR, and then loading the 2 ml mixtures into Xpert MTB/RIF assay

179 cartridges. The larger blood volume samples were processed using our novel

180 lysis/centrifugation protocol. As shown in Fig. 2, increasing the volume of blood that

181 was tested resulted in a substantially increased rate of positive results, especially for tests

182 performed on 0.25, 1, and 5 CFU/ml samples. Blood volumes of 20 ml that were spiked

183 with as few as 0.25 CFU/ml and 1 CFU/ml could be detected consistently 57% and >80%

184 of the times, respectively. However, lower blood volumes of 1 ml detected the same cell

185 concentrations <20% of the times. Thus, 1 ml blood samples needed to be spiked with 5-

186 10 times more M. tuberculosis CFU than 20 ml blood samples to achieve similar rates of

187 test positivity.

188 We considered the possibility that our lysis/centrifugation method might

189 concentrate PCR inhibitors at the same time that it concentrated M. tuberculosis bacilli.

190 The Xpert MTB/RIF assay includes a sample processing control assay for Bacillus

191 globigii, which can also be used to indicate the presence of PCR inhibitors (8). Inhibition

192 is detected by an increase in B. globigii cycle threshold (Ct). We did not observe any

Detection of TB in blood- Banada et al., Page 9

193 increase in B. globigii Ct with increasing blood volume, suggesting that our procedure

194 did not concentrate significant amounts of inhibitors to the Xpert MTB/RIF assay.

195 Role of anti-coagulants on TB detection in blood. Our processing protocol

196 requires anticoagulated blood samples. However, certain anticoagulants such as heparin

197 or EDTA can inhibit PCR assays. We tested the impact of various anticoagulants on

198 assay sensitivity after large volume lysis/centrifugation. We selected anticoagulants for

199 testing based on their use in commonly-available commercial blood collection tubes.

200 Blood was drawn from human volunteers into one of three collection tubes containing

201 either ACD-A, ACD-B or EDTA. BCG was spiked into 5 ml aliquots of each sample at

202 5 and 10 CFU/ml concentrations. The samples were then processed using our lysis

203 centrifugation protocol and tested with the Xpert MTB/RIF assay. As shown in Fig. 3,

204 the ACD anti-coagulated blood showed the best overall performance. M. tuberculosis

205 was detected in 100% of the test aliquots containing 10 CFU/ml that were anti-coagulated

206 with either ACD-A or ACD-B, and in 100% of the test aliquots containing 5 CFU/ml that

207 were anti-coagulated with ACD-B (ACD-A not tested at 5 CFU/ml). This is in contrast

208 to the EDTA anti-coagulated samples which had substantially fewer positive test results.

209 Comparing the rpoB values of the ACD-A and ACD-B anti-coagulated samples

210 containing 10 CFU/ml, we noted that the ACD-A anticoagulation resulted in a delayed

211 mean Ct (29.70.5 versus 28.11, p=0.015). This suggested that ACD-B anticoagulation

212 would provide the best assay sensitivity.

213 Limit of detection using ACD-B anticoagulated blood. We determined the

214 limit of detection of our lysis/centrifugation protocol using blood anticoagulated with

215 ACD-B, since this appeared to be the best candidate for eventual clinical studies. We

216 found that our approach identified M. tuberculosis 100% of the time in the aliquots
Detection of TB in blood- Banada et al., Page 10
217 containing, 50 CFU/ml and 10 CFU/ml, defining the limit of detection as 10 CFU/ml.

218 However the assay continued to perform well even at lower inoculums, detecting M.

219 tuberculosis in 91.6% of the blood samples spiked with 5 CFU/ml, 36% spiked with 1

220 CFU/ml and 33% spiked with 0.5 CFU/ml (Fig. 4). Overall, the performance of the

221 ACD-B anticoagulated blood samples was similar to that we observed with CPDA-

222 anticoagulated blood banked blood (Fig. 2), suggesting that blood banked blood can be

223 used for future analytic studies.

224 Stability studies. Our assay might be useful in clinics or laboratories that do not

225 have the capacity to easily make our lysis solution. Therefore, we performed stability

226 studies to determine how long the lysis solution could be stored without affecting assay

227 performance. The solution was stored at both room temperature (RT, 20C-28C) and at

228 4C for up to three months. Aliquots were removed periodically to process blood

229 samples spiked with known numbers of BCG CFU/ml. The Ct of the first rpoB probe

230 was used to asses assay performance (Fig. 5A). The storage temperature of the lysis

231 buffer did not appear to affect rpoB Ct value (95% CI, P=0.8).

232 Laboratories might find it convenient to batch test blood samples, or blood might

233 need to be transported to off-site locations. To study how long blood could be stored at

234 4C prior to processing, we spiked 10 CFU/ml of BCG into 10 ml aliquots of ACD-B

235 anticoagulated blood and then stored the samples at 4C for up to 8 days. Samples were

236 processed and analyzed at 0, 1, 2, 4 and 8 days by comparing number of positive samples

237 and assay Ct values (Fig. 5B). All assays were positive at all time points and no

238 difference in assay Ct values was noted even after the eight day time point. This study

239 suggests that M. tuberculosis is stable in human blood stored at 4C for at least eight

Detection of TB in blood- Banada et al., Page 11

240 days, and that large volume sample processing can be delayed for at least one week

241 without affecting assay performance.

242

Detection of TB in blood- Banada et al., Page 12

243 DISCUSSION

244 The major findings of this study were that a simple pre-processing step can be

245 used to sensitively detect M. tuberculosis in large volumes of blood using the Xpert

246 MTB/RIF assay. Important assay parameters affecting sensitivity of detection include

247 blood volumes and anticoagulants used.

248 Our proposed lysis centrifugation protocol has several notable advantages over

249 prior assays to detect M. tuberculosis in blood. First, our lysis buffer was concentrated

250 (used at 0.15:1 ratio with blood) compared to most blood lysis buffers reported, which

251 uses much higher ratios, usually between 1:1 to 10:1. The limited volume requirement of

252 the lysis buffer made it possible to lyse large blood volumes within the blood collection

253 tube or by transferring the collected blood sample into a simple test tube that was

254 prefilled with the lysis buffer. These procedures simplified the technical components of

255 the assay. Second, we showed that our lysis buffer completely removes inhibitors to

256 PCR, in the context of the Xpert MTB/RIF assay. This property made it possible for us

257 to test very large blood volumes, in this study up to 20 ml, without adding measurable

258 inhibitors into the PCR. The ability to test large blood volumes enabled us to achieve

259 substantial gains in sensitivity. Finally, we show that our assay has acceptable reagent

260 and sample stability.

261 Our goal was to develop the most sensitive blood PCR assay possible for M.

262 tuberculosis. This was based on the assumption that M. tuberculosis bacteria are present

263 in the blood of TB patients at very low concentrations, when they are present at all. We

264 are not aware of any studies that have formally examined this question in the case of M.

265 tuberculosis. However, for other microbial pathogens bacterial loads of <1 CFU/ml have

266 been reported among septic adult patients (25). Furthermore, the volume of blood tested
Detection of TB in blood- Banada et al., Page 13
267 by blood culture is considered to be the single most important factor for detecting

268 bacteria in the blood of adult patients (11, 35, 44). Each additional 1 ml of blood cultured

269 increases microbial recovery by up to 3% (11, 14, 24, 31). Thus, we expect that our large

270 volume approach will substantially improve M. tuberculosis detection.

271 It is unlikely that our method using Xpert MTB/RIF assay on blood will

272 diagnose all forms of TB with equal sensitivity. Rebello et al, (34) noted that the

273 sensitivity of PCR and culture of blood for TB was greatest in patients with disseminated

274 tuberculosis and those with HIV-confection. These are patient groups which frequently

275 do not have productive cough. Thus, it is likely that our approach will be especially

276 useful in a subset of TB cases which are particularly poorly served by current diagnostics.

277 Several anticoagulants that are used to collect blood from patients can be

278 inhibitory to PCR, and will affect assay sensitivity, if not properly removed from the

279 sample (37, 46). Heparin is a known PCR inhibitor (37, 45). EDTA can chelate the

280 Mg++ present in PCR buffer, thus affecting the PCR performance (23). Isolator tubes

281 recommended for lysis/centrifugation blood cultures contains high concentrations of

282 saponin and sodium polyanetholesulfonate (SPS) which have been shown to be inhibitory

283 to PCR based blood assays (27). In the current study, we examined blood collected in

284 EDTA, CPDA (for banked blood) and two different commercial ACD anticoagulated

285 tubes (ACD-A and ACD-B). In ACD (Acid citrate dextrose), citric acid prevents

286 coagulation by binding to calcium, and dextrose acts as a RBC nutrient and preservative

287 by maintaining RBC viability. It contains no known PCR inhibitors and thus was a good

288 candidate for our studies. Of the two different ACD formulations available in

289 commercial blood drawing tubes, ACD-A is more concentrated

290 (http://www.bd.com/vacutainer/pdfs/plus_plastic_tubes_wallchart_tubeguide_VS5229.pd
Detection of TB in blood- Banada et al., Page 14
291 f, http://www.pharmacopeia.cn/v29240/usp29nf24s0_m5100.html). It did not perform as

292 well as ACD-B in our hands, possibly because high concentrations of citrate in ACD-A

293 might have mildly inhibited the PCR.

294 In summary, we have shown that it is possible to test large volumes of blood for

295 the presence of MTB using the existing Xpert MTB/RIF platform. Our approach can

296 test samples as large as 20 ml of blood, and has a detection limit of five to ten CFU/ml.

297 Bacterial titers as low as 0.25 to one CFU/ml can also be routinely detected, although

298 there will be some false negative assays at the low end of these titers. The sample

299 processing protocol we developed takes approximately one hour. Combined with the

300 Xpert MTB/RIF on-machine time, the time to result of this assay can be as low as three

301 hours from the initial blood draw. This time frame compares very favorably to the

302 several week delay that is typical for M. tuberculosis blood cultures and may enable rapid

303 diagnosis in a patient group that will particularly benefit from rapid diagnosis and

304 initiation of treatment. The high sensitivity which we observed in our analytic study

305 suggests that our approach may have clinical utility. However, it will be necessary to

306 perform clinical trials in relevant patient populations before clinical sensitivity and

307 specificity can be established.

308

309

Detection of TB in blood- Banada et al., Page 15

310 ACKNOWLEDGEMENTS.

311 This work was supported by National Institutes of Health grants AI098713 and

312 AI080653. We wish to thank Dr. Fermina M. Mazzella, Department of Pathology and

313 Laboratory Medicine, New Jersey Medical School University of Medicine and

314 Dentistry of New Jersey for help with collecting the discarded blood tubes. We

315 acknowledge the blood bank staff at UMDNJ-university hospital, Newark, NJ, for

316 providing us with the expired blood.

317

318 COMPETING INTERESTS.

319 D.A. is one of a group of co-investigators who invented molecular beacon

320 technology and who receive income from licensees, including Cepheid which licenses the

321 molecular beacon technology in the Xpert MTB/RIF assay. To manage potential

322 conflicts of interest, Dr. Alland has irrevocably limited the fees that can accrue to him

323 from the Xpert MTB/RIF assay to $5000 per year.

324

Detection of TB in blood- Banada et al., Page 16

325 REFERENCES:

326 1. Ahmed, N., A. K. Mohanty, U. Mukhopadhyay, V. K. Batish, and S. Grover.

327 1998. PCR-based rapid detection of Mycobacterium tuberculosis in blood from

328 immunocompetent patients with pulmonary tuberculosis. J. Clin. Microbiol.

329 36:3094-3095.

330 2. Ait-Khaled, N., and D. A. Enarson. 2003. TUBERCULOSIS: A Manual for

331 Medical Students, vol. 99.272. World Health Organization.

332 3. Al-Soud, W. A., L. J. Jnsson, and P. Rdstrm. 2000. Identification and

333 Characterization of Immunoglobulin G in Blood as a Major Inhibitor of

334 Diagnostic PCR. J. Clin. Microbiol. 38:345-350.

335 4. Al-Soud, W. A., and P. Rdstrm. 2001. Purification and Characterization of

336 PCR-Inhibitory Components in Blood Cells. J. Clin. Microbiol. 39:485-493.

337 5. Banada, P. P., S. K. Sivasubramani, R. Blakemore, C. Boehme, M. D.

338 Perkins, K. Fennelly, and D. Alland. 2010. Containment of bioaerosol infection

339 risk by the Xpert MTB/RIF assay and its applicability to point-of-care settings. J.

340 Clin. Microbiol. 48:3551-3557.

341 6. Bensinger, T. A., and T. F. Zuck. 1976. Additional Studies Concerning the

342 Metabolism of Packed Erythrocytes in CPD Adenine. Transfusion 16:353-356.

343 7. Billett, H. H. 1990. Hemoglobin and Hematocrit p. 718-719. In H. W. Walker

344 HK, Hurst JW (ed.), Clinical Methods: The History, Physical, and Laboratory

345 Examinations, 3rd ed. Butterworth Publishers,, Boston.

346 8. Blakemore, R., E. Story, D. Helb, J. Kop, P. Banada, M. R. Owens, S.

347 Chakravorty, M. Jones, and D. Alland. 2010. Evaluation of the analytical

348 performance of the Xpert MTB/RIF assay. J Clin Microbiol 48:2495-2501.

Detection of TB in blood- Banada et al., Page 17
349 9. Boehme, C. C., P. Nabeta, D. Hillemann, M. P. Nicol, S. Shenai, F. Krapp, J.

350 Allen, R. Tahirli, R. Blakemore, R. Rustomjee, A. Milovic, M. Jones, S. M.

351 O'Brien, D. H. Persing, S. Ruesch-Gerdes, E. Gotuzzo, C. Rodrigues, D.

352 Alland, and M. D. Perkins. 2010. Rapid Molecular Detection of Tuberculosis

353 and Rifampin Resistance. N Engl. J. Med. 363:1005-1015.

354 10. Boehme, C. C., M. P. Nicol, P. Nabeta, J. S. Michael, E. Gotuzzo, R. Tahirli,

355 M. T. Gler, R. Blakemore, W. Worodria, C. Gray, L. Huang, T. Caceres, R.

356 Mehdiyev, L. Raymond, A. Whitelaw, K. Sagadevan, H. Alexander, H.

357 Albert, F. Cobelens, H. Cox, D. Alland, and M. D. Perkins. 2011. Feasibility,

358 diagnostic accuracy, and effectiveness of decentralised use of the Xpert MTB/RIF

359 test for diagnosis of tuberculosis and multidrug resistance: a multicentre

360 implementation study. The Lancet 377:1495-1505.

361 11. Bouza, E., D. Sousa, M. Rodrguez-Crixems, J. G. Lechuz, and P. Muoz.

362 2007. Is the Volume of Blood Cultured Still a Significant Factor in the Diagnosis

363 of Bloodstream Infections? J. Clin. Microbiol. 45:2765-2769.

364 12. Chakravorty, S., M. Dudeja, M. Hanif, and J. S. Tyagi. 2005. Utility of

365 universal sample processing methodology, combining smear microscopy, culture,

366 and PCR, for diagnosis of pulmonary tuberculosis. J. Clin. Microbiol. 43:2703-

367 2708.

368 13. Condos, R., A. McClune, W. N. Rom, and N. W. Schluger. 1996. Peripheral-

369 blood-based PCR assay to identify patients with active pulmonary tuberculosis.

370 The Lancet 347:1082-1085.

371 14. Dunne, J., F. Nolte, and M. Wilson. 1997. Blood cultures III. Cumitech 1B.

372 American Society for Microbiology,, Washington, DC.

Detection of TB in blood- Banada et al., Page 18
373 15. Folgueira, L., R. Delgado, E. Palenque, J. M. Aguado, and A. R. Noriega.

374 1996. Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR. J.

375 Clin. Microbiol. 34:512-515.

376 16. Fordham von Reyn, C. 1999. The significance of bacteremic tuberculosis among

377 persons with HIV infection in developing countries. AIDS 13:2193-2195.

378 17. Gandhi, N. R., A. Moll, A. W. Sturm, R. Pawinski, T. Govender, U. Lalloo,

379 K. Zeller, J. Andrews, and G. Friedland. 2006. Extensively drug-resistant

380 tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV

381 in a rural area of South Africa. The Lancet 368:1575-1580.

382 18. Golden, M. P., and H. R. Vikram. 2005. Extrapulmonary tuberculosis: an

383 overview. Am. Fam. Physician 72:1761-1768.

384 19. Gous, N., L. E. Scott, E. Wong, T. Omar, W. D. F. Venter, and W. Stevens.

385 2012. Performance of the Roche LightCycler Real-Time PCR Assay for

386 Diagnosing Extrapulmonary Tuberculosis. J. Clin. Microbiol 50:2100-2103.

387 20. Hanscheid, T., C. Monteiro, J. M. Cristino, L. M. Lito, and M. J. Salgado.

388 2005. Growth of Mycobacterium tuberculosis in Conventional BacT/ALERT FA

389 Blood Culture Bottles Allows Reliable Diagnosis of Mycobacteremia. J. Clin.

390 Microbiol. 43:890-891.

391 21. Helb, D., M. Jones, E. Story, C. Boehme, E. Wallace, K. Ho, J. Kop, M. R.

392 Owens, R. Rodgers, P. Banada, H. Safi, R. Blakemore, N. T. Lan, E. C.

393 Jones-Lopez, M. Levi, M. Burday, I. Ayakaka, R. D. Mugerwa, B. McMillan,

394 E. Winn-Deen, L. Christel, P. Dailey, M. D. Perkins, D. H. Persing, and D.

395 Alland. 2010. Rapid detection of Mycobacterium tuberculosis and rifampin

Detection of TB in blood- Banada et al., Page 19

396 resistance by use of on-demand, near-patient technology. J. Clin. Microbiol

397 48:229-237.

398 22. Heysell, S., T. Thomas, N. Gandhi, A. Moll, F. Eksteen, Y. Coovadia, L.

399 Roux, P. Babaria, U. Lalloo, G. Friedland, and S. Shah. 2010. Blood cultures

400 for the diagnosis of multidrug-resistant and extensively drug-resistant tuberculosis

401 among HIV-infected patients from rural South Africa: a cross-sectional study.

402 BMC Infect. Dis. 10:344.

403 23. Huggett, J. F., T. Novak, J. A. Garson, C. Green, S. D. Morris-Jones, R. F.

404 Miller, and A. Zumla. 2008. Differential susceptibility of PCR reactions to

405 inhibitors: an important and unrecognised phenomenon. BMC Res. Notes 1:70.

406 24. Ilstrup, D. M., and J. A. Washington Ii. 1983. The importance of volume of

407 blood cultured in the detection of bacteremia and fungemia. Diag. Microbiol.

408 Infect. Dis. 1:107-110.

409 25. Kellogg, J. A., J. P. Manzella, and J. H. McConville. 1984. Clinical laboratory

410 comparison of the 10-ml isolator blood culture system with BACTEC radiometric

411 blood culture media. J. Clin. Microbiol. 20:618-623.

412 26. Lawn, S. D., A. D. Kerkhoff, M. Vogt, Y. Ghebrekristos, A. Whitelaw, and R.

413 Wood. 2012. Characteristics and Early Outcomes of Patients With Xpert

414 MTB/RIF-Negative Pulmonary Tuberculosis Diagnosed During Screening Before

415 Antiretroviral Therapy. Clin. Infect. Dis. 54:1071-1079.

416 27. Levett, P. N., R. E. Morey, R. L. Galloway, D. E. Turner, A. G. Steigerwalt,

417 and L. W. Mayer. 2005. Detection of pathogenic leptospires by real-time

418 quantitative PCR. Journal of Medical Microbiology 54:45-49.

Detection of TB in blood- Banada et al., Page 20

419 28. Lima, J. F., L. M. Montenegro, A. Montenegro Rde, M. M. Cabral, A. S.

420 Lima, F. G. Abath, and H. C. Schindler. 2009. Performance of nested PCR in

421 the specific detection of Mycobacterium tuberculosis complex in blood samples

422 of pediatric patients. J. Bras. Pneumol. 35:690-697.

423 29. Mehta, P. K., A. Raj, N. Singh, and G. K. Khuller. 2012. Diagnosis of

424 extrapulmonary tuberculosis by PCR. FEMS Immunol. Med. Microbiol.:n/a-n/a.

425 30. Mendelson, M. 2007. Diagnosing tuberculosis in HIV-infected patients:

426 challenges and future prospects. Br. Med. Bull. 81-82:149-165.

427 31. Mermel, L. A., and D. G. Maki. 1993. Detection of bacteremia in adults:

428 consequences of culturing an inadequate volume of blood. Ann. Intern. Med.

429 119:270-272.

430 32. Perkins, M. D., and J. Cunningham. 2007. Facing the Crisis: Improving the

431 Diagnosis of Tuberculosis in the HIV Era. J. Infect. Dis. 196:S15-S27.

432 33. Peto, H. M., R. H. Pratt, T. A. Harrington, P. A. LoBue, and L. R.

433 Armstrong. 2009. Epidemiology of Extrapulmonary Tuberculosis in the United

434 States, 19932006. Clin. Infect. Dis. 49:1350-1357.

435 34. Rebollo, M. J., R. San Juan Garrido, D. Folgueira, E. Palenque, C. Daz-

436 Pedroche, C. Lumbreras, and J. M. Aguado. 2006. Blood and urine samples as

437 useful sources for the direct detection of tuberculosis by polymerase chain

438 reaction. Diag. Microbiol. Infect. Dis. 56:141-146.

439 35. Reimer, L. G., M. L. Wilson, and M. P. Weinstein. 1997. Update on detection

440 of bacteremia and fungemia. Clin. Microbiol. Rev. 10:444-465.

Detection of TB in blood- Banada et al., Page 21

441 36. Rolfs, A., J. Beige, U. Finckh, B. Kohler, T. Schaberg, J. Lokies, and H. Lode.

442 1995. Amplification of Mycobacterium tuberculosis from peripheral blood. J.

443 Clin. Microbiol. 33:3312-3314.

444 37. Satsangi, J., D. P. Jewell, K. Welsh, M. Bunce, and J. I. Bell. 1994. Effect of

445 heparin on polymerase chain reaction. The Lancet 343:1509-1510.

446 38. Singh, K. K., M. Muralidhar, A. Kumar, T. K. Chattopadhyaya, K. Kapila,

447 M. K. Singh, S. K. Sharma, N. K. Jain, and J. S. Tyagi. 2000. Comparison of

448 in house polymerase chain reaction with conventional techniques for the detection

449 of Mycobacterium tuberculosis DNA in granulomatous lymphadenopathy. J. Clin.

450 Path. 53:355-361.

451 39. Taci, N., A. S. Yurdakul, I. Ceyhan, M. B. Berktas, and M. Ogretensoy. 2003.

452 Detection of Mycobacterium tuberculosis DNA from peripheral blood in patients

453 with HIV-seronegative and new cases of smear-positive pulmonary tuberculosis

454 by polymerase chain reaction. Respir. Med. 97:676-681.

455 40. Tenney, J. H., L. B. Reller, S. Mirrett, W. L. Wang, and M. P. Weinstein.

456 1982. Controlled evaluation of the volume of blood cultured in detection of

457 bacteremia and fungemia. J. Clin. Microbiol. 15:558-561.

458 41. Tortoli, E., C. Russo, C. Piersimoni, E. Mazzola, M. P. Dal, M. Pascarella, E.

459 Borroni, A. Mondo, F. Piana, C. Scarparo, L. Coltella, G. Lombardi, and D.

460 M. Cirillo. 2012. Clinical validation of Xpert MTB/RIF for the diagnosis of

461 extrapulmonary tuberculosis. Eur. Respir. J. 40:442-447.

462 42. Tortoli, E., P. Urbano, F. Marcelli, M. T. Simonetti, and M. D. Cirillo. 2012.

463 Is real-time PCR better than conventional PCR for Mycobacterium tuberculosis

464 complex detection in clinical samples? J. Clin. Microbiol. 50:2810-2813.

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465 43. Varma, J. K., K. D. McCarthy, T. Tasaneeyapan, P. Monkongdee, M. E.

466 Kimerling, E. Buntheoun, D. Sculier, C. Keo, P. Phanuphak, N.

467 Teeratakulpisarn, N. Udomsantisuk, N. H. Dung, N. T. Lan, N. T. Yen, and

468 K. P. Cain. 2010. Bloodstream infections among HIV-infected outpatients,

469 Southeast Asia. Emerg. Infect. Dis. 16:1569-1575.

470 44. Weinstein, M. P., S. Mirrett, M. L. Wilson, L. G. Reimer, and L. B. Reller.

471 1994. Controlled evaluation of 5 versus 10 milliliters of blood cultured in aerobic

472 BacT/Alert blood culture bottles. J. Clin. Microbiol. 32:2103-2106.

473 45. Yokota, M., N. Tatsumi, O. Nathalang, T. Yamada, and I. Tsuda. 1999.

474 Effects of heparin on polymerase chain reaction for blood white cells. J. Clin. Lab

475 Anal. 13:133-140.

476 46. Zhang, Z., M. B. Kermekchiev, and W. M. Barnes. 2010. Direct DNA

477 Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and

478 Novel Mutants of Taq. J. Mol. Diagn. 12:152-161.

479

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484

485

486

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488
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489

490

491

492

493 Fig. 1. Flow chart describing the large volume blood sample processing

494 methodology.

495

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504
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505

506

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510

511 Fig. 2. Effect of blood volume on the probability of mycobacteria detection. BCG

512 was spiked at various CFU per ml into different volumes of blood. The proportion of

513 samples that were positive at each concentration for N = 7 per concentration are shown.

514

515

516

517

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518

519

520

521

522 Fig. 3. Comparison of various anticoagulants. Blood anticoagulated with ACD-A,

523 ACD-B and EDTA were spiked with 5 and 10 CFU/ml of BCG in 5 ml whole blood.

524 The proportion of samples that were positive (bars) and the Ct value of the rpoB assay

525 () are shown for each condition. N = the number of replicates run with each

526 anticoagulant type.

527

528

529

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531

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533

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534

535

536

537

538 Fig. 4. Limit of detection of mycobacteria in ACD-B anticoagulated blood. BCG was

539 spiked into 5 ml blood aliquots. The proportion of positive samples for each CFU

540 concentration out of 15 samples tested per concentration is shown. Each replicate is

541 indicative of a blood sample from one healthy individual.

542

543

544

545

546

547

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548

549

550 Fig. 5. Solution and sample stability. Stability studies were performed by running

551 Xpert MTB/RIF assays with (A) lysis solution stored between 0 and 90 days at room

552 temperature () or at refrigerated conditions (4-8C) (), (B) BCG-spiked blood samples

553 that had been stored for 0 to 8 days. Each time point shows the mean of N=5 test results.

554 An increase in cycle threshold (not seen) would have indicated that a stability storage

555 limit had been reached.

556

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