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Basis for treatment of tuberculosis among HIV-infected patients in Tanzania: the role of chest x-ray and sputum culture
© Bakari et al; licensee BioMed Central Ltd. 2008
Received: 21 June 2007
Accepted: 06 March 2008
Published: 06 March 2008
Active tuberculosis (TB) is common among HIV-infected persons living in tuberculosis endemic countries, and screening for tuberculosis (TB) is recommended routinely. We sought to determine the role of chest x-ray and sputum culture in the decision to treat for presumptive TB using active case finding in a large cohort of HIV-infected patients.
Ambulatory HIV-positive subjects with CD4 counts ≥ 200/mm3 entering a Phase III TB vaccine study in Tanzania were screened for TB with a physical examination, standard interview, CD4 count, chest x-ray (CXR), blood culture for TB, and three sputum samples for acid fast bacillus (AFB) smear and culture.
Among 1176 subjects 136 (12%) were treated for presumptive TB. These patients were more frequently male than those without treatment (34% vs. 25%, respectively; p = 0.049) and had lower median CD4 counts (319/μL vs. 425/μL, respectively; p < .0001). Among the 136 patients treated for TB, 38 (28%) had microbiologic confirmation, including 13 (10%) who had a normal CXR and no symptoms. There were 58 (43%) treated patients in whom the only positive finding was an abnormal CXR. Blood cultures were negative in all patients.
Many ambulatory HIV-infected patients with CD4 counts ≥ 200/mm3 are treated for presumptive TB. Our data suggest that optimal detection requires comprehensive evaluation, including CXR and sputum culture on both symptomatic and asymptomatic subjects.
Tuberculosis (TB) is the major cause of death from AIDS in most areas of the developing world [1, 2]. The high mortality of HIV-associated TB reflects multiple factors, including lack of access to care, delayed or missed diagnosis of TB and acceleration of HIV infection [2–5]. Active TB is considered an indication for anti-retroviral therapy (ART) in HIV-infected persons with CD4 counts <350, but treatment of co-infection may be complicated by drug interactions and by immune reconstitution syndrome [6, 7]. For these reasons screening for active TB is recommended for patients with HIV living in TB-endemic regions. However, only limited data are available on the relative sensitivity of different methods of screening for active TB among HIV-infected patients with CD4 counts ≥ 200. The World Health Organization (WHO) recommends screening for TB prior to ART based on symptoms followed by sputum acid fast bacillus (AFB) smear; chest x-ray (CXR) and sputum culture are not routine in their method [6, 8].
This report describes our experience applying a comprehensive screening program (including both CXR and sputum culture) to both symptomatic and asymptomatic ambulatory HIV-infected subjects with CD4 counts ≥ 200 being evaluated for a TB vaccine trial in Tanzania. With this approach, 12% of those screened were treated for presumptive TB based on the local standards of care. Fewer than half of these cases would have been detected by symptoms and AFB smear alone; many were only detected by CXR and some cases only by sputum culture. These observations have major implications for HIV programs.
Study subjects and protocol
Subjects are screened for HIV and tuberculosis as part of the DarDar (Dartmouth Medical School-Dar es Salaam) Study, a Phase III trial of a prime-boost vaccine strategy for the prevention of HIV-associated TB (prime = childhood BCG, boost = multiple dose whole cell inactivated mycobacterial vaccine)  being conducted in Tanzania. Ambulatory HIV-infected subjects age ≥ 18 years in apparent good health are referred from HIV voluntary counseling and testing (VCT) centers in Dar es Salaam or by other study subjects. Informed consent was obtained from all subjects.
Eligibility requirements for the vaccine study include two positive ELISA antibody tests for HIV, CD4 count ≥ 200/mm3 and BCG scar. All subjects meeting these criteria were evaluated for active TB by symptoms, chest x-ray and microbiology. The latter involved collecting three expectorated sputum samples for AFB smear and culture. Patients were instructed to cough deeply and were encouraged to produce one spot sample and bring two subsequent first morning samples; sputum induction was not used. A single 10 mL mycobacterial blood culture was processed using the MB/BacT automated system (bioMerieux, Inc, Durham, NC, USA). A tuberculin skin test (TST) was performed with 0.1 mL intradermal RT-23 (State Serum Institute, Copenhagen) and read at 48–72 hrs as mm induration in the transverse diameter.
For the purposes of this report symptoms of TB were defined as either cough or fever for ≥ 2 weeks or both. Chest x-rays were read by the principal radiologist at the university teaching hospital, Muhimbili University College of Health Sciences (MUCHS); the requisition indicated whether the patient has suspect TB. An abnormal x-ray is defined as the presence of a focal infiltrate, cavity formation, hilar adenopathy, or a miliary pattern. The decision to treat for suspect TB was made by study physicians in collaboration with physicians from the Tanzania National Tuberculosis and Leprosy Program (NTLP) based on clinical, laboratory and/or radiologic features consistent with TB. CXR and AFB smear results were available for these decisions and patients were typically assessed for radiologic response to a 10 day course of antibiotic therapy for community acquired pneumonia before the decision to treat for TB. Other than blood culture, there was no detailed testing for extrapulmonary TB among the subjects described in the present report. Follow-up data on whether the subject started treatment as recommended was obtained from the NTLP.
Definitions of TB for the DarDar Study
1. One or more sputum cultures positive for Mycobacterium tuberculosis (MTB) with ≥ 10 colony forming units (CFU); or,
2. Two or more sputum cultures with 1–9 CFU of MTB (indeterminate MTB culture); or,
3. Two or more positive sputum smears for acid fast bacilli (AFB) *; or,
4. One or more cultures for MTB from the blood or other sterile body site.
1. Positive chest x-ray plus either
a. one positive sputum AFB smear, or,
b. one indeterminate MTB culture result; or,
2. Clinical symptoms/signs plus either
a. one positive sputum AFB smear, or,
b. an indeterminate MTB culture result; or,
3. Clinical symptoms/signs and a positive x-ray plus a response to anti-TB therapy; or,
4. One positive sputum AFB smear from a sterile site plus clinical symptoms/signs of tuberculosis; or,
5. Caseous necrosis on a tissue biopsy.
The research protocol was approved by the Ethics Committee of the Muhimbili University College of Health Sciences and the Dartmouth Committee for the Protection of Human Subjects.
Serum samples are tested using 2 different HIV ELISA methods: Vironostika HIV Uni-Form II Ag/Ab (BioMerieux, Boxtel, The Netherlands) and Vironostika HIV Uni-Form II Plus O (BioMerieux, Boxtel, The Netherlands). Subjects reactive on both assays are considered HIV-infected. Blood is collected in EDTA tubes for enumeration of CD4 cells by FASCount (BD Biosciences, Franklin Lakes, NJ) after staining with monoclonal antibodies .
Sputum samples are examined by direct microscopy using auramine-rhodamine staining with confirmation of positives by Ziehl Neelsen AFB staining. Sputum samples are also decontaminated in 2% NaOH for 30 minutes, concentrated by centrifugation at 3000 g, examined again by microscopy and cultured on Lowenstein Jensen slants for 10 weeks at 37°C. Sputum culture contamination rates were approximately 1% during the period of this report.
Blood is cultured using the MB/BacT method according to manufacturer's instructions (Bio-Merieux, Lyon, France). Characteristic slow-growing mycobacteria are presumptively identified as M. tuberculosis. Putative M. tuberculosis isolates are shipped to Dartmouth to confirm M. tuberculosis complex by DNA probe testing (AccuProbe, Gen-Probe, San Diego, CA)
Standard descriptive statistical methods are used to determine median, interquartile range and the percent of subjects with particular characteristics. All p-values are based on Fisher's exact test or Wilcoxon test, as appropriate, and are 2-sided. A p-value < 0.05 is considered statistically significant.
Characteristics of HIV-infected patients with CD4 ≥ 200 with and without TB treatment
TB treatment (N = 136)
No TB treatment (N = 1040)
Age, median (interquartile range)
Female N (%)
CD4, median (interquartile range)
History of TB N (%)*
Tuberculin skin test ≥ 5 mm N (%)*
Tuberculin skin test ≥ 10 mm*
Basis for TB treatment among 1176 HIV-infected subjects with CD4 ≥ 200/mm3
AFB positive only
Culture positive only
Symptoms (fever or cough ≥ 2 wks)
Abnormal chest x-ray
Symptoms and chest x-ray
Among 124 subjects who produced sputum samples 106 were submitted for AFB stain and culture and 18 for AFB stain only. A total of 38 (31%) of 124 tested subjects had a positive AFB smear or mycobacterial culture; these subjects meet a study definition of definite or probable TB (Table 1). Positive sputum microbiology was present in 14 (32%) of 44 subjects tested with both symptoms and an abnormal chest x-ray and in 1 (20%) of 5 subjects tested with symptoms only (p = 0.7). DNA probes were positive for M. tuberculosis complex on all 36 isolates tested. Blood cultures were negative on all subjects.
In 13 (10%) of 136 cases of TB the diagnosis was established by sputum microbiology alone in patients with no symptoms and negative chest x-rays. Ten of these cases of "subclinical TB" were reported previously . These patients represent 34% (13 of 38) of all subjects with positive sputum microbiology.
We have shown that 12% of ambulatory HIV-infected patients in Tanzania undergoing comprehensive screening are treated for presumptive and previously undiagnosed active TB. Our results suggest that the optimal diagnosis of TB in this setting requires assessment of both symptomatic and asymptomatic HIV-positive patients using chest x-ray and sputum culture, an observation that differs from consensus international recommendations and has obvious implications for the resources needed to conduct effective and optimal treatment of persons with HIV infection. Confirmation of this preliminary finding will be available when our expert panel completes classification of TB endpoints among the eligible subjects in the trial, including independent review of chest x-rays and response to treatment.
An abnormal chest x-ray in the absence of symptoms was the most common basis for the suspicion of TB. This finding differs from an initial report from Botswana in which an abnormal chest x-ray was very rare among HIV- infected subjects with no symptoms of TB  and a study in South Africa where symptom-based screening was effective . However, our results and those of other recent surveys [13, 14] including a more recent report from Botswana  suggest that the chest x-ray is important in screening and that the standard recommendation to screen for TB based on symptoms alone may be unreliable and insensitive in sub-Saharan Africa . Based on our data, if AFB smear had only been performed on patients with symptoms, 55 patients would have had an AFB smear and only 8 of these (6% of all treated cases) would have been positive and been treated.
Prevalence rates for TB depend on the patient population, screening methods and TB case definitions employed. The 12% point prevalence of TB treatment in our study compares to rates of 3–12% in other studies among HIV- infected persons living in TB endemic countries [12, 13, 17–22]. A recent study from South Africa using chest x-ray, sputum culture and a clinical definition category for TB found that 11% of subjects in an ART program had previously undiagnosed TB at baseline and that rates of incident TB in those on ART for 3 years continued to be 5–10 fold higher than those for HIV-uninfected persons living in the same region .
Microbiologic studies confirmed the diagnosis of TB in 31% of those with suspect disease. Our relatively low rate of positive sputum cultures for TB might have been influenced by overly vigorous decontamination of sputum specimens as suggested by our low sputum culture contamination rate of 1%. However, by routinely performing sputum microbiology in this population, including in those without symptoms, we were able to identify a unique group of subjects with "subclinical TB", i.e., active, culture-positive disease without symptoms (including study definition symptoms as well as absence of additional symptoms such as weight loss or fever <2 weeks) or x-ray findings. A previous report from our study focused on 10 such patients whom we encountered among the initial 500 subjects screened; that report also presented detailed data to eliminate the possibility of false positive cultures and follow-up data to indicate that the early treatment was associated with a more favorable prognosis than typically seen with HIV-associated tuberculosis . The 13 patients in the present series comprise 9% of all patients treated for TB. Rates of subclinical TB might be found to be even higher if more sensitive liquid based media had been used for sputum culture . Investigators in Uganda, India, New York and London have also described HIV-infected subjects with subclinical forms of TB [7, 24–26]; in some of these studies "subclinical" includes any active disease without symptoms, regardless of chest x-ray findings.
Sputum culture not only makes an important contribution to the diagnosis of both clinical and subclinical TB in patients with HIV [20, 25]. It will also be an increasingly important TB diagnostic tool for another reason: the detection and spread of XDR TB in sub-Sarharan Africa . As we showed previously in Kenya  and others have confirmed , blood culture for M. tuberculosis may make an additional contribution in hospitalized patients with advanced and symptomatic HIV infection but did not contribute to detection of TB in ambulatory subjects with earlier stage HIV infection.
Our overall study is focused on ambulatory HIV-infected persons with CD4 = 200 residing in an area with endemic tuberculosis. We believe our subjects are generally representative of such patients, although there are potential sources of bias. Conceivably, persons with subtle or unreported symptoms of TB might have been preferentially drawn to a TB vaccine trial. Conversely, because all subjects had received BCG at birth, this could have reduced the rate of active TB. In general populations, BCG immunization does not typically affect adult TB rates  and among HIV-infected persons limited retrospective data is conflicting [30, 31].
Our goal in this report was to describe the frequency and characteristics of subjects who were judged by TB clinicians to require treatment for presumptive TB at the time of screening. Our evaluations were conducted by trained, experienced local physicians, supported by expert radiologic interpretation, albeit by a single radiologist aware of the suspicion of TB. In a study from the United States expert radiologists agreed on chest x-ray suspicion of tuberculosis in 139 subjects (HIV status not stated) approximately 70% of the time, and this suspicion appeared to accurately identify smear negative tuberculosis in 48% of cases . We cannot confirm that all patients treated for TB without microbiologic confirmation had active disease because they were ineligible for our vaccine study and did not have follow-up to assess response to therapy. Current microbiologic methods often fail to confirm disease in subjects who have classic TB symptoms with x-ray changes and subsequent radiologic and clinical response to therapy for TB. In other published series of HIV-associated TB 19–66% of subjects have met a "clinical" definition of TB with negative microbiology [21, 33, 34].
Our findings reflect the realities of a presumptive TB diagnosis in regions endemic for both TB and HIV infection. If confirmed in the eligible subjects in our trial whose TB endpoints are classified according to our strict case definitions our findings will have important implications for ART programs in such environments. First, chest x-ray and sputum microbiology will identify a significant number of cases of presumptive TB even among those who deny fever or cough; treatment of these subclinical cases may reduce mortality from HIV-associated TB; thus, an effort should be made to expand availability of these tests in resource poor regions . Second, with rigorous TB screening as many as 12% of ambulatory HIV-infected subjects with CD4 counts = 200/mm3 will qualify for ART based on the decision to treat for TB; early ART in these subjects may reduce mortality . Third, a substantial proportion of ART eligible subjects will require deferral of single drug preventive therapy for latent TB while active TB is being excluded by culture. Finally, these data emphasize the importance of integrating TB and HIV programs in countries with high rates of co-infection, of educating clinicians about the diverse manifestations of TB in HIV-infected patients, and of providing optimal methods for TB diagnosis.
We thank Wendy Wieland-Alter, Theckla Kazimoto, and Godfrey Marwa for laboratory support, Emily Wroe for assistance with data collection, and Nancy Wray (Dartmouth College) for administrative support. Support: National Institutes of Health, DAIDS, AI 45407 and Fogarty International Center, D43-TW006807.
- Frieden T, Sterling TR, Munsliff SS, Watt CJ, Dye C: Tuberculosis. Lancet. 2003, 362: 887-99. 10.1016/S0140-6736(03)14333-4.View ArticlePubMedGoogle Scholar
- Harries AD, Hargreaves NJ, Kemp J, et al: Deaths from tuberculosis in sub-Saharan African countries with a high prevalence of HIV-1. Lancet. 2001, 357: 1519-1523. 10.1016/S0140-6736(00)04639-0.View ArticlePubMedGoogle Scholar
- Mtei L, Matee M, Herfort O, et al: High rates of clinical and subclinical tuberculosis among ambulatory HIV-positive subjects in Tanzania. Clin Infect Dis. 2005, 40: 1500-7. 10.1086/429825.View ArticlePubMedGoogle Scholar
- Whalen C, Horsburgh CR, Hom D, Lahart C, Simberkoff M, Ellner J: Accelerated course of human immunodeficiency virus infection after tuberculosis. Am J Respir Crit Care Med. 1995, 151: 129-35.View ArticlePubMedGoogle Scholar
- Talbot EA, Hay Burgess DC, Hone NM, et al: Tuberculosis serodiagnosis in a predominantly HIV-infected population of hospitalized patients with cough, Botswana, 2002. Clin Infect Dis. 2004, 39: e1-7. 10.1086/421388.View ArticlePubMedGoogle Scholar
- WHO: Antiretroviral therapy for HIV infection in adults and adolescents in resource limited settings: towards universal access: recommendations for a public health approach (2006 revision). 2006, Geneva, Switzerland: WHO, 1-140.Google Scholar
- Breen RA, Smith CJ, Cropley I, Johnson MA, Lipman MC: Does immune reconstitution syndrome promote active tuberculosis in patients receiving highly active antiretroviral therapy?. Aids. 2005, 19: 1201-6.View ArticlePubMedGoogle Scholar
- Organization WH: Scaling up antiretroviral therapy in resource limited settings: treatment guidelines for a public health approach. Geneva, Switzerland. 2004, 1-67.Google Scholar
- Vuola J, Ristola M, Cole B, et al: Immunogenicity of an inactivated mycobacterial vaccine for the prevention of HIV-associated tuberculosis: a randomized, controlled trial. AIDS. 2003, 17: 2351-2355. 10.1097/00002030-200311070-00010.View ArticlePubMedGoogle Scholar
- Landay A, Ohlsson-Wilhelm B, Giorgi JV: Application of flow cytometry to the study of HIV infection. Aids. 1990, 4: 479-97. 10.1097/00002030-199006000-00001.View ArticlePubMedGoogle Scholar
- Mosimaneotsile B, Talbot EA, Moeti TL, et al: Value of chest radiography in a tuberculosis prevention programme for HIV-infected people, Botswana. Lancet. 2003, 362: 1551-2. 10.1016/S0140-6736(03)14745-9.View ArticlePubMedGoogle Scholar
- Mohammed A, Ehrlich R, Wood R, Cilliers F, Maartens G: Screening for tuberculosis in adults with advanced HIV infection prior to preventive therapy. Int J Tuberc Lung Dis. 2004, 8: 792-5.PubMedGoogle Scholar
- Wood R, Middelkoop K, Myer L, et al: Undiagnosed tuberculosis in a community with high HIV prevalence: implications for tuberculosis control. Am J Respir Crit Care Med. 2007, 175: 87-93. 10.1164/rccm.200606-759OC.View ArticlePubMedGoogle Scholar
- den Boon S, White NW, van Lill SW, et al: An evaluation of symptom and chest radiographic screening in tuberculosis prevalence surveys. Int J Tuberc Lung Dis. 2006, 10: 876-82.PubMedGoogle Scholar
- Samandari T, Agizew T, Arwady A, et al: Asymptomatic pulmonary TB among HIV-infected adults screened for the Botswana isoniazid preventive therapy clinical trial, 2004–2006 [abstract 862]. 14th Conference on Retroviruses and Opportunistic Infections. Los Angeles, CA. 2007Google Scholar
- Corbett EL, Marston B, Churchyard GJ, De Cock KM: Tuberculosis in sub-Saharan Africa: opportunities, challenges, and change in the era of antiretroviral treatment. Lancet. 2006, 367: 926-37. 10.1016/S0140-6736(06)68383-9.View ArticlePubMedGoogle Scholar
- Nachega J, Coetzee J, Adendorff T, et al: Tuberculosis active case-finding in a mother-to-child HIV transmission prevention programme in Soweto, South Africa. AIDS. 2003, 17 (9): 1398-1400. 10.1097/00002030-200306130-00018.View ArticlePubMedGoogle Scholar
- Kimerling ME, Schucter J, Chanthol E, et al: Prevalence of pulmonary tuberculosis among HIV-infected persons in a home care program in Phnom Penh, Cambodia. Int J Tuberc Lung Dis. 2002, 6 (11): 988-94.PubMedGoogle Scholar
- Desormeaux J, Johnson MP, Coberly JS, et al: Widespread HIV counseling and testing linked to a community-based tuberculosis control program in a high-risk population. Bulletin of the Pan American Health Organization. 1996, 30: 1-8.PubMedGoogle Scholar
- Corbett EL, Charalambous S, Moloi VM, et al: Human immunodeficiency virus and the prevalence of undiagnosed tuberculosis in African gold miners. Am J Respir Crit Care Med. 2004, 170: 673-9. 10.1164/rccm.200405-590OC.View ArticlePubMedGoogle Scholar
- Day JH, Charalambous S, Fielding KL, Hayes RJ, Churchyard GJ, Grant AD: Screening for tuberculosis prior to isoniazid preventive therapy among HIV-infected gold miners in South Africa. Int J Tuberc Lung Dis. 2006, 10: 523-9.PubMedGoogle Scholar
- Lawn SD, Myer L, Bekker LG, Wood R: Burden of tuberculosis in an antiretroviral treatment programme in sub-Saharan Africa: impact on treatment outcomes and implications for tuberculosis control. Aids. 2006, 20: 1605-12.View ArticlePubMedGoogle Scholar
- Abe C, Hosojima S, Fukasawa Y, et al: Comparison of MB-Check, BACTEC, and egg-based media for recovery of mycobacteria. J Clin Micro. 1992, 30 (4): 878-881.Google Scholar
- Guwatudde D, Nakakeeto M, Jones-Lopez EC, et al: Tuberculosis in household contacts of infectious cases in Kampala, Uganda. Am J Epidemiol. 2003, 158: 887-98. 10.1093/aje/kwg227.View ArticlePubMedPubMed CentralGoogle Scholar
- Swaminathan S, Paramasivan CN, Kumar SR, Mohan V, Venkatesan P: Unrecognised tuberculosis in HIV-infected patients: sputum culture is a useful tool. Int J Tuberc Lung Dis. 2004, 8: 896-8.PubMedGoogle Scholar
- Singh KK, Dong Y, Belisle JT, Harder J, Arora VK, Laal S: Antigens of Mycobacterium tuberculosis recognized by antibodies during incipient, subclinical tuberculosis. Clin Diagn Lab Immunol. 2005, 12: 354-8. 10.1128/CDLI.12.2.354-358.2005.PubMedPubMed CentralGoogle Scholar
- Gandhi NR, Moll A, Sturm AW, et al: Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet. 2006, 368: 1575-80. 10.1016/S0140-6736(06)69573-1.View ArticlePubMedGoogle Scholar
- Gilks CF, Brindle RJ, Mwachari C, et al: Disseminated Mycobacterium avium infection among HIV-infected patients in Kenya. J AIDS. 1995, 8 (2): 195-8.Google Scholar
- von Reyn CF, Vuola J: New vaccines for the prevention of tuberculosis. Clin Infect Dis. 2002, 35: 465-74. 10.1086/341901.View ArticlePubMedGoogle Scholar
- Marsh BJ, von Reyn CF, Edwards J, et al: The risks and benefits of childhood bacille Calmette-Guerin immunization among adults with AIDS. AIDS. 1997, 11: 669-72. 10.1097/00002030-199705000-00015.View ArticlePubMedGoogle Scholar
- Waddell RD, Lishimpi K, von Reyn CF, et al: Bacteremia due to Mycobacterium tuberculosis or M. bovis, Bacille Calmette-Guerin (BCG) among HIV-positive children and adults in children. AIDS. 2001, 15: 55-60. 10.1097/00002030-200101050-00009.View ArticlePubMedGoogle Scholar
- Gordin FM, Slutkin G, Schecter G, Goodman PC, Hopewell PC: Presumptive diagnosis and treatment of pulmonary tuberculosis based on radiographic findings. Am Rev Respir Dis. 1989, 139: 1090-1093.View ArticlePubMedGoogle Scholar
- Whalen CC, Johnson JL, Okwera A, et al: A trial of three regimens to prevent tuberculosis in Ugandan adults infected with the human immunodeficiency virus. N Engl J Med. 1997, 337: 801-8. 10.1056/NEJM199709183371201.View ArticlePubMedGoogle Scholar
- Apers L, Wijarajah C, Mutsvangwa J, Chigara N, Mason P, van der Stuyft P: Accuracy of routine diagnosis of pulmonary tuberculosis in an area of high HIV prevalence. Int J Tuberc Lung Dis. 2004, 8: 945-51.PubMedGoogle Scholar
- De Cock K, Marston B: The sound of one hand clapping. Am J Respir Crit Care Med. 2005, 172: 3-4. 10.1164/rccm.2504003.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/8/32/prepub
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