CORRESPONDENCE Presence of Tropheryma whipplei in Different Body Sites in a Cohort of Healthy Subjects
Table 1. Characteristics of the Cohort with and without Tropheryma whipplei in Bronchoalveolar Lavage
To the Editor: Characteristic
The organism Tropheryma whipplei causes Whipple’s disease, a systemic infectious disease that primarily involves the gastrointestinal tract (1). Recent examination of the lung microbiome using culture-independent techniques has shown that the organism is present in the lungs of healthy individuals, but the origin of the organism is unknown. The Lung HIV Microbiome Project discovered that T. whipplei is more common in the lungs of HIV-infected individuals and that antiretroviral therapy significantly reduces its relative abundance (2, 3). T. whipplei has been previously detected in saliva at low prevalence (0.2%), and more commonly in gastric samples (11.4%), from individuals without Whipple’s disease (4, 5), but whether T. whipplei in the lung results from aspiration of gastric contents or from some other mechanism is unknown. T. whipplei hsp65–specific polymerase chain reaction assay is based on the T. whipplei hsp65 gene sequence and has very high sensitivity and specificity, particularly in individuals without active disease (6). To investigate potential sources for T. whipplei detected in the lung, we performed polymerase chain reaction and sequencing for T. whipplei in simultaneous bronchoalveolar lavages (BALs), oral wash samples, and gastric aspirate samples in a cohort of HIVuninfected, healthy subjects. Some of the results of this study have been previously reported in the form of an abstract at the 2015 International Conference of the American Thoracic Society (7).
T. whipplei Detected (n = 5)
Age, mean yr (SD) Male sex, n (%) White, n (%) Smoker, n (%) Post-BD FEV1, mean % predicted (SD) Post-BD FEV1/FVC, mean % (SD) DLCO, mean % predicted (SD)
42.4 3 4 3 98.7
(17.4) (60) (80) (60) (21.6)
T. whipplei Not Detected (n = 32) 41.1 8 27 11 102.6
(16.0) (25) (84) (34) (15.5)
82.6 (10.7)
83.6 (6.0)
104.2 (30.1)
100.5 (32.0)
Definition of abbreviations: BD = bronchodilator; DLCO = diffusing capacity of the lung for carbon monoxide. No significant differences between groups on any measure.
T. whipplei hsp65–specific nested polymerase chain reaction (6). The amplified products were purified using Agencourt AMPure XP PCR Purification kit (Beckman Coulter, Brea, CA) and then were sequenced using specific primers by the Genomics and Proteomics Core laboratories at the University of Pittsburgh. CLC Main Workbench 6.5 and the Molecular Evolutionary Genetics Analysis version 6 (MEGA6) software packages (12) were used for analyses of T. whipplei hsp65 partial gene sequences. Clinical characteristics of individuals with and without T. whipplei were compared using Stata 13 (StataCorps, College Station, TX).
Methods
Participants were a subset of the Michigan site of the Lung HIV Microbiome Project cohort. Informed consent was obtained from each subject, and the study protocol was approved by institutional review boards at the University of Michigan, the Ann Arbor Veterans Affairs Medical Center, and the University of Pittsburgh. The study was registered with ClinicalTrials.gov (NCT02392182). The subjects were clinically well and could not have received antibacterials or corticosteroids in the 3 months before sampling. Demographic data were collected, and pulmonary function testing was performed according to American Thoracic Society guidelines (8, 9) (Table 1). BAL and paired oral wash samples were collected from 37 healthy subjects, as per protocol (10). Gastric aspirate samples were obtained from 29 individuals simultaneously, as previously described (11). After 18 months, 27 individuals had second visits with repeat BAL and oral wash. DNA was isolated using the PowerSoil DNA isolation kit (MoBio, Carlsbad, CA). T. whipplei was detected using
Results
Two of 37 (5.4%) individuals had T. whipplei detected in BAL on visit 1 and 5 of 27 (18.5%) on visit 2. Clinical characteristics did not differ between individuals with and without T. whipplei in BAL (Table 1). T. whipplei was not detected in any oral wash samples. Two (6.9%) of 29 gastric aspirates had detectable T. whipplei (Table 2). Individuals with a positive BAL were more likely to have positive gastric aspirates (P , 0.001) (Table 2). T. whipplei also persisted, as both individuals with a positive BAL on the first visit also had a positive BAL on the second visit (P = 0.002). To compare the genetic Table 2. Comparison of Partial hsp65 Gene Sequence of Tropheryma whipplei in Bronchoalveolar Lavage and Gastric Aspirate Samples Subject 11002 11020
This work was supported by National Institutes of Health grants U01 HL098962 (A.M.), K24 HL023342 (A.M.), and U01 HL098961 (J.M.B. and J.L.C.), Merit Review Award BX001389, and the Department of Veterans Affairs (C.M.F.). Author Contributions: Conceived and designed the experiments: S.Q., A.M., J.M.B., and J.L.C.; collected the samples: C.M.F. and J.L.C.; performed the experiments: S.Q., L.L., and H.M.; analyzed the data and statistics: S.Q., A.M., and E.C.; and wrote the manuscript: S.Q. and A.M. All authors revised the manuscript before submission.
Correspondence
11024 11031 11036
BAL1
OW1
GA1
BAL2
2 Mutation (4 nt) 2 Wild type 2
2 2
2 Mutation (4 nt) 2 Wild type N/A
Wild type Mutation (4 nt) Wild type Wild type Wild type
2 2 2
OW2 2 2 2 2 2
Definition of abbreviations: 2, negative polymerase chain reaction products; 4 nt = 4 nucleotides different from reference sequence; BAL = bronchoalveolar lavage; GA = gastric aspirate; N/A = sample was not available; OW = oral wash.
243
CORRESPONDENCE T. whipplei hsp65
T CTC T GC C GG GGA T CC GC A GA T A GG G G A T A T T A T TG C C C A A G C A C T C G A G A A G G TTGGC A A GG A A G GC GT T G T C A CT G
11020 BAL1 11020 GA1 11020 BAL2 T. whipplei hsp65
T CGA GGA GT C A A A T A CT T T C GGG A C C G A A C T C G AGA T A A CC G A GGG CA T G C G T T TT G A T A A A GG C T A C C T T T C G G C T T A
11020 BAL1 11020 GA1
A
11020 BAL2
A
T. whipplei hsp65
A T TT T GT A A C C G A T GC G GA G C GG C A G GA A A C GG T TT T T G A G A A C C C T T A C A T T C TT A T T T GT G A T A GC A A A A T A T C G A
11020 BAL1
C
G
A
11020 GA1
C
G
A
11020 BAL2
C
G
A
T. whipplei hsp65
G T GT T AA A GA T CT G C T C C CG GT T G T T G A C A A GG T TA T CC A G T C T G G T A A G C A A C TT CT T A T C A T T G C T G A A G A T G T
11020 BAL1 11020 GA1 11020 BAL2
Figure 1. Alignment of partial hsp65 gene sequence of Tropheryma whipplei in one individual’s bronchoalveolar lavage and gastric aspirate. BAL = bronchoalveolar lavage; GA = gastric aspirate; T. whipplei hsp65 = partial T. whipplei reference sequence.
identities of T. whipplei from the lung and gastric aspirate in the same subject, we applied standard DNA sequencing to amplified products of partial T. whipplei hsp65 gene. Compared with T. whipplei hsp65 gene reference sequence, one subject (11031) had wild-type T. whipplei detected in gastric aspirate on visit 1 and both BALs. Another subject (11020) had the same mutant of T. whipplei detected in both BALs and the gastric aspirate, and the other three subjects had wild-type T. whipplei detected in BALs on visit 2 (Table 2 and Figure 1). These results demonstrate that the isolates of T. whipplei in the BAL and gastric aspirate in the same subject shared genetic identity. Discussion
In this study, we detected T. whipplei in BAL and gastric aspirate samples, but not oral washes, from a cohort of healthy subjects. This study is the first description of T. whipplei colonization in lung and stomach using T. whipplei hsp65–specific nested polymerase chain reaction and sequencing approaches in a cohort of HIV-uninfected individuals without clinical diseases. Recent examination of the lung microbiome has shown that carriage of T. whipplei in the lung is found in about 16.7–26.0% of healthy individuals and 53.7% of HIV-infected individuals, using 16S rRNA gene sequencing (2, 3, 13). In our study, using specific nested polymerase chain reaction and sequencing, which may be more sensitive than 16S rRNA sequencing, T. whipplei was detectable in the lungs of 5% of HIV-uninfected, healthy individuals, with 19% of individuals demonstrating detectable T. whipplei at a subsequent bronchoscopy. Similar to previous work, T. whipplei was not detected in any oral wash samples in this cohort (2, 3). Our results 244
and others indicate that asymptomatic carriage of T. whipplei in the mouth may occur, but the prevalence is likely quite low (4). Individuals with T. whipplei in the lung were more likely to have T. whipplei detected in the stomach and were also more likely to be persistently colonized with T. whipplei. There was strong genetic identity of T. whipplei detected in the BAL and gastric aspirate samples from the same subjects or in the lungs of the same individuals over time. These results indicate that T. whipplei in the lung may come from aspiration of gastric contents or could translocate from the gastrointestinal tract to the lung. We cannot prove the mode of colonization in the lung, but the lack of detection of T. whipplei in oral samples, despite its presence in the lung and stomach, suggests that aspiration of oral or gastric contents into the lung or swallowing of lung organisms is unlikely or quite transient. The detection of genetically similar T. whipplei in repeated BAL samples also suggests T. whipplei colonization is persistent. Additional longitudinal work could help elucidate the epidemiology of T. whipplei colonization in healthy individuals. n Author disclosures are available with the text of this letter at www.atsjournals.org. Shulin Qin, M.D., Ph.D. Emily Clausen, M.D. Lorrie Lucht, B.S. Heather Michael, B.S. University of Pittsburgh Pittsburgh, Pennsylvania James M. Beck, M.D. University of Colorado School of Medicine Aurora, Colorado
American Journal of Respiratory and Critical Care Medicine Volume 194 Number 2 | July 15 2016
CORRESPONDENCE and Veterans Affairs Eastern Colorado Health Care System Denver, Colorado
The Methodology of Assessing Long-Term Mortality and Cardiovascular Risks in Survivors of Sepsis
Jeffrey L. Curtis, M.D. Christine M. Freeman, Ph.D. University of Michigan Ann Arbor, Michigan and VA Healthcare System Ann Arbor, Michigan
To the Editor:
Alison Morris, M.D., M.S. University of Pittsburgh Pittsburgh, Pennsylvania
References 1. Desnues B, Al Moussawi K, Fenollar F. New insights into Whipple’s disease and Tropheryma whipplei infections. Microbes Infect 2010;12: 1102–1110. 2. Morris A, Beck JM, Schloss PD, Campbell TB, Crothers K, Curtis JL, Flores SC, Fontenot AP, Ghedin E, Huang L, et al.; Lung HIV Microbiome Project. Comparison of the respiratory microbiome in healthy nonsmokers and smokers. Am J Respir Crit Care Med 2013; 187:1067–1075. 3. Lozupone C, Cota-Gomez A, Palmer BE, Linderman DJ, Charlson ES, Sodergren E, Mitreva M, Abubucker S, Martin J, Yao G, et al.; Lung HIV Microbiome Project. Widespread colonization of the lung by Tropheryma whipplei in HIV infection. Am J Respir Crit Care Med 2013;187:1110–1117. 4. Fenollar F, Laouira S, Lepidi H, Rolain JM, Raoult D. Value of Tropheryma whipplei quantitative polymerase chain reaction assay for the diagnosis of Whipple disease: usefulness of saliva and stool specimens for firstline screening. Clin Infect Dis 2008;47:659–667. 5. Ehrbar HU, Bauerfeind P, Dutly F, Koelz HR, Altwegg M. PCR-positive tests for Tropheryma whippelii in patients without Whipple’s disease. Lancet 1999;353:2214. 6. Morgenegg S, Dutly F, Altwegg M. Cloning and sequencing of a part of the heat shock protein 65 gene (hsp65) of “Tropheryma whippelii” and its use for detection of “T. whippelii” in clinical specimens by PCR. J Clin Microbiol 2000;38:2248–2253. 7. Clausen ES, Freeman CM, Curtis JL, Morris AM, Qin SL. Presence of Tropheryma whipplei in the lung and gastrum [abstract]. Am J Respir Crit Care Med 2015;191:A4723. 8. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, Crapo R, Enright P, van der Grinten CP, Gustafsson P, et al.; ATS/ERS Task Force. Standardisation of spirometry. Eur Respir J 2005;26:319–338. 9. Macintyre N, Crapo RO, Viegi G, Johnson DC, van der Grinten CP, Brusasco V, Burgos F, Casaburi R, Coates A, Enright P, et al. Standardisation of the single-breath determination of carbon monoxide uptake in the lung. Eur Respir J 2005;26:720–735. 10. Beck JM, Schloss PD, Venkataraman A, Twigg H III, Jablonski KA, Bushman FD, Campbell TB, Charlson ES, Collman RG, Crothers K, et al.; Lung HIV Microbiome Project. Multicenter comparison of lung and oral microbiomes of HIV-infected and HIV-uninfected individuals. Am J Respir Crit Care Med 2015;192:1335–1344. 11. Bassis CM, Erb-Downward JR, Dickson RP, Freeman CM, Schmidt TM, Young VB, Beck JM, Curtis JL, Huffnagle GB. Analysis of the upper respiratory tract microbiotas as the source of the lung and gastric microbiotas in healthy individuals. MBio 2015;6:e00037. 12. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. Mega6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013;30:2725–2729. 13. Charlson ES, Bittinger K, Haas AR, Fitzgerald AS, Frank I, Yadav A, Bushman FD, Collman RG. Topographical continuity of bacterial populations in the healthy human respiratory tract. Am J Respir Crit Care Med 2011;184:957–963.
Copyright © 2016 by the American Thoracic Society
Correspondence
We read with great interest the study by Ou and colleagues on the long-term risk for cardiovascular events among patients discharged with a diagnosis of sepsis (1). On the basis of discharge diagnoses of septicemia (International Classification of Diseases, Ninth Revision, 038.x), the authors demonstrate an increased risk for cardiovascular events among such patients compared with two different control populations: a matched hospital population and a control population. Although we appreciate the efforts made by the authors to address a difficult issue in health care, we believe the study methodology and results raise several issues of concern that compromise the external and internal validity of the article. The validity of the discharge diagnosis is crucial to justify the overall approach. Although an acceptable positive predictive value of 92% is documented (2), the sensitivity of the exact diagnosis is only 9% (3). The limitations of an approach using the discharge diagnosis in this context have been subject to much debate, and the so-called Angus algorithm, although not flawless, is generally accepted as the gold standard of sepsis identification in administrative cohorts (3–5). We acknowledge that the authors included a recording of antibiotic treatment but do not believe this would contribute to an already very specific diagnosis. In addition, the authors’ approach renders meaningful comparisons with other studies difficult. In the propensity score–matched analysis, the authors find similar hazard ratios for cardiovascular events and all-cause mortality when comparing the patients with sepsis with each control group. These results are counterintuitive and very difficult to comprehend. However, from their Table 1, it appears that population control subjects have more comorbidity and are older than nonsepsis hospitalized control subjects. Using the authordefined definition of comorbidity, an individual has to be admitted (or seen in an outpatient clinic) to be assigned a diagnosis code for calculation of the Charlson Comorbidity Index. We would appreciate an author clarification on this point. It would be of interest to see the distribution curves of the propensity distributions of both matchings, which may clarify some of these features. From the all-cause mortality Kaplan-Meier curves (the authors’ Figures 1A and 1B), it is apparent that almost one-third of all persons in the population control group are dead after 10 years and that the hospital control subjects have a lower 10-year mortality rate than the population control subjects. These observations are quite astounding and warrant an in-depth discussion and clarification. The observation window for an all-cause mortality event to appear is widely defined and could potentially approach 10 years (e.g., an event occurring 9 d from the index date is weighted equally to one occurring 9 yr from the index date in the hazard ratio analysis). We believe that “late events” a priori mitigate biological
Author Contributions: conception and design: D.P.H., P.D., J.R.D., and C.B.L.; analysis and interpretation: D.P.H., P.D., J.R.D., and C.B.L.; drafting the manuscript for important intellectual content: D.P.H., P.D., J.R.D., and C.B.L.
245
Table 1. Characteristics of the Cohort with and without Tropheryma whipplei in Bronchoalveolar Lavage
To the Editor: Characteristic
The organism Tropheryma whipplei causes Whipple’s disease, a systemic infectious disease that primarily involves the gastrointestinal tract (1). Recent examination of the lung microbiome using culture-independent techniques has shown that the organism is present in the lungs of healthy individuals, but the origin of the organism is unknown. The Lung HIV Microbiome Project discovered that T. whipplei is more common in the lungs of HIV-infected individuals and that antiretroviral therapy significantly reduces its relative abundance (2, 3). T. whipplei has been previously detected in saliva at low prevalence (0.2%), and more commonly in gastric samples (11.4%), from individuals without Whipple’s disease (4, 5), but whether T. whipplei in the lung results from aspiration of gastric contents or from some other mechanism is unknown. T. whipplei hsp65–specific polymerase chain reaction assay is based on the T. whipplei hsp65 gene sequence and has very high sensitivity and specificity, particularly in individuals without active disease (6). To investigate potential sources for T. whipplei detected in the lung, we performed polymerase chain reaction and sequencing for T. whipplei in simultaneous bronchoalveolar lavages (BALs), oral wash samples, and gastric aspirate samples in a cohort of HIVuninfected, healthy subjects. Some of the results of this study have been previously reported in the form of an abstract at the 2015 International Conference of the American Thoracic Society (7).
T. whipplei Detected (n = 5)
Age, mean yr (SD) Male sex, n (%) White, n (%) Smoker, n (%) Post-BD FEV1, mean % predicted (SD) Post-BD FEV1/FVC, mean % (SD) DLCO, mean % predicted (SD)
42.4 3 4 3 98.7
(17.4) (60) (80) (60) (21.6)
T. whipplei Not Detected (n = 32) 41.1 8 27 11 102.6
(16.0) (25) (84) (34) (15.5)
82.6 (10.7)
83.6 (6.0)
104.2 (30.1)
100.5 (32.0)
Definition of abbreviations: BD = bronchodilator; DLCO = diffusing capacity of the lung for carbon monoxide. No significant differences between groups on any measure.
T. whipplei hsp65–specific nested polymerase chain reaction (6). The amplified products were purified using Agencourt AMPure XP PCR Purification kit (Beckman Coulter, Brea, CA) and then were sequenced using specific primers by the Genomics and Proteomics Core laboratories at the University of Pittsburgh. CLC Main Workbench 6.5 and the Molecular Evolutionary Genetics Analysis version 6 (MEGA6) software packages (12) were used for analyses of T. whipplei hsp65 partial gene sequences. Clinical characteristics of individuals with and without T. whipplei were compared using Stata 13 (StataCorps, College Station, TX).
Methods
Participants were a subset of the Michigan site of the Lung HIV Microbiome Project cohort. Informed consent was obtained from each subject, and the study protocol was approved by institutional review boards at the University of Michigan, the Ann Arbor Veterans Affairs Medical Center, and the University of Pittsburgh. The study was registered with ClinicalTrials.gov (NCT02392182). The subjects were clinically well and could not have received antibacterials or corticosteroids in the 3 months before sampling. Demographic data were collected, and pulmonary function testing was performed according to American Thoracic Society guidelines (8, 9) (Table 1). BAL and paired oral wash samples were collected from 37 healthy subjects, as per protocol (10). Gastric aspirate samples were obtained from 29 individuals simultaneously, as previously described (11). After 18 months, 27 individuals had second visits with repeat BAL and oral wash. DNA was isolated using the PowerSoil DNA isolation kit (MoBio, Carlsbad, CA). T. whipplei was detected using
Results
Two of 37 (5.4%) individuals had T. whipplei detected in BAL on visit 1 and 5 of 27 (18.5%) on visit 2. Clinical characteristics did not differ between individuals with and without T. whipplei in BAL (Table 1). T. whipplei was not detected in any oral wash samples. Two (6.9%) of 29 gastric aspirates had detectable T. whipplei (Table 2). Individuals with a positive BAL were more likely to have positive gastric aspirates (P , 0.001) (Table 2). T. whipplei also persisted, as both individuals with a positive BAL on the first visit also had a positive BAL on the second visit (P = 0.002). To compare the genetic Table 2. Comparison of Partial hsp65 Gene Sequence of Tropheryma whipplei in Bronchoalveolar Lavage and Gastric Aspirate Samples Subject 11002 11020
This work was supported by National Institutes of Health grants U01 HL098962 (A.M.), K24 HL023342 (A.M.), and U01 HL098961 (J.M.B. and J.L.C.), Merit Review Award BX001389, and the Department of Veterans Affairs (C.M.F.). Author Contributions: Conceived and designed the experiments: S.Q., A.M., J.M.B., and J.L.C.; collected the samples: C.M.F. and J.L.C.; performed the experiments: S.Q., L.L., and H.M.; analyzed the data and statistics: S.Q., A.M., and E.C.; and wrote the manuscript: S.Q. and A.M. All authors revised the manuscript before submission.
Correspondence
11024 11031 11036
BAL1
OW1
GA1
BAL2
2 Mutation (4 nt) 2 Wild type 2
2 2
2 Mutation (4 nt) 2 Wild type N/A
Wild type Mutation (4 nt) Wild type Wild type Wild type
2 2 2
OW2 2 2 2 2 2
Definition of abbreviations: 2, negative polymerase chain reaction products; 4 nt = 4 nucleotides different from reference sequence; BAL = bronchoalveolar lavage; GA = gastric aspirate; N/A = sample was not available; OW = oral wash.
243
CORRESPONDENCE T. whipplei hsp65
T CTC T GC C GG GGA T CC GC A GA T A GG G G A T A T T A T TG C C C A A G C A C T C G A G A A G G TTGGC A A GG A A G GC GT T G T C A CT G
11020 BAL1 11020 GA1 11020 BAL2 T. whipplei hsp65
T CGA GGA GT C A A A T A CT T T C GGG A C C G A A C T C G AGA T A A CC G A GGG CA T G C G T T TT G A T A A A GG C T A C C T T T C G G C T T A
11020 BAL1 11020 GA1
A
11020 BAL2
A
T. whipplei hsp65
A T TT T GT A A C C G A T GC G GA G C GG C A G GA A A C GG T TT T T G A G A A C C C T T A C A T T C TT A T T T GT G A T A GC A A A A T A T C G A
11020 BAL1
C
G
A
11020 GA1
C
G
A
11020 BAL2
C
G
A
T. whipplei hsp65
G T GT T AA A GA T CT G C T C C CG GT T G T T G A C A A GG T TA T CC A G T C T G G T A A G C A A C TT CT T A T C A T T G C T G A A G A T G T
11020 BAL1 11020 GA1 11020 BAL2
Figure 1. Alignment of partial hsp65 gene sequence of Tropheryma whipplei in one individual’s bronchoalveolar lavage and gastric aspirate. BAL = bronchoalveolar lavage; GA = gastric aspirate; T. whipplei hsp65 = partial T. whipplei reference sequence.
identities of T. whipplei from the lung and gastric aspirate in the same subject, we applied standard DNA sequencing to amplified products of partial T. whipplei hsp65 gene. Compared with T. whipplei hsp65 gene reference sequence, one subject (11031) had wild-type T. whipplei detected in gastric aspirate on visit 1 and both BALs. Another subject (11020) had the same mutant of T. whipplei detected in both BALs and the gastric aspirate, and the other three subjects had wild-type T. whipplei detected in BALs on visit 2 (Table 2 and Figure 1). These results demonstrate that the isolates of T. whipplei in the BAL and gastric aspirate in the same subject shared genetic identity. Discussion
In this study, we detected T. whipplei in BAL and gastric aspirate samples, but not oral washes, from a cohort of healthy subjects. This study is the first description of T. whipplei colonization in lung and stomach using T. whipplei hsp65–specific nested polymerase chain reaction and sequencing approaches in a cohort of HIV-uninfected individuals without clinical diseases. Recent examination of the lung microbiome has shown that carriage of T. whipplei in the lung is found in about 16.7–26.0% of healthy individuals and 53.7% of HIV-infected individuals, using 16S rRNA gene sequencing (2, 3, 13). In our study, using specific nested polymerase chain reaction and sequencing, which may be more sensitive than 16S rRNA sequencing, T. whipplei was detectable in the lungs of 5% of HIV-uninfected, healthy individuals, with 19% of individuals demonstrating detectable T. whipplei at a subsequent bronchoscopy. Similar to previous work, T. whipplei was not detected in any oral wash samples in this cohort (2, 3). Our results 244
and others indicate that asymptomatic carriage of T. whipplei in the mouth may occur, but the prevalence is likely quite low (4). Individuals with T. whipplei in the lung were more likely to have T. whipplei detected in the stomach and were also more likely to be persistently colonized with T. whipplei. There was strong genetic identity of T. whipplei detected in the BAL and gastric aspirate samples from the same subjects or in the lungs of the same individuals over time. These results indicate that T. whipplei in the lung may come from aspiration of gastric contents or could translocate from the gastrointestinal tract to the lung. We cannot prove the mode of colonization in the lung, but the lack of detection of T. whipplei in oral samples, despite its presence in the lung and stomach, suggests that aspiration of oral or gastric contents into the lung or swallowing of lung organisms is unlikely or quite transient. The detection of genetically similar T. whipplei in repeated BAL samples also suggests T. whipplei colonization is persistent. Additional longitudinal work could help elucidate the epidemiology of T. whipplei colonization in healthy individuals. n Author disclosures are available with the text of this letter at www.atsjournals.org. Shulin Qin, M.D., Ph.D. Emily Clausen, M.D. Lorrie Lucht, B.S. Heather Michael, B.S. University of Pittsburgh Pittsburgh, Pennsylvania James M. Beck, M.D. University of Colorado School of Medicine Aurora, Colorado
American Journal of Respiratory and Critical Care Medicine Volume 194 Number 2 | July 15 2016
CORRESPONDENCE and Veterans Affairs Eastern Colorado Health Care System Denver, Colorado
The Methodology of Assessing Long-Term Mortality and Cardiovascular Risks in Survivors of Sepsis
Jeffrey L. Curtis, M.D. Christine M. Freeman, Ph.D. University of Michigan Ann Arbor, Michigan and VA Healthcare System Ann Arbor, Michigan
To the Editor:
Alison Morris, M.D., M.S. University of Pittsburgh Pittsburgh, Pennsylvania
References 1. Desnues B, Al Moussawi K, Fenollar F. New insights into Whipple’s disease and Tropheryma whipplei infections. Microbes Infect 2010;12: 1102–1110. 2. Morris A, Beck JM, Schloss PD, Campbell TB, Crothers K, Curtis JL, Flores SC, Fontenot AP, Ghedin E, Huang L, et al.; Lung HIV Microbiome Project. Comparison of the respiratory microbiome in healthy nonsmokers and smokers. Am J Respir Crit Care Med 2013; 187:1067–1075. 3. Lozupone C, Cota-Gomez A, Palmer BE, Linderman DJ, Charlson ES, Sodergren E, Mitreva M, Abubucker S, Martin J, Yao G, et al.; Lung HIV Microbiome Project. Widespread colonization of the lung by Tropheryma whipplei in HIV infection. Am J Respir Crit Care Med 2013;187:1110–1117. 4. Fenollar F, Laouira S, Lepidi H, Rolain JM, Raoult D. Value of Tropheryma whipplei quantitative polymerase chain reaction assay for the diagnosis of Whipple disease: usefulness of saliva and stool specimens for firstline screening. Clin Infect Dis 2008;47:659–667. 5. Ehrbar HU, Bauerfeind P, Dutly F, Koelz HR, Altwegg M. PCR-positive tests for Tropheryma whippelii in patients without Whipple’s disease. Lancet 1999;353:2214. 6. Morgenegg S, Dutly F, Altwegg M. Cloning and sequencing of a part of the heat shock protein 65 gene (hsp65) of “Tropheryma whippelii” and its use for detection of “T. whippelii” in clinical specimens by PCR. J Clin Microbiol 2000;38:2248–2253. 7. Clausen ES, Freeman CM, Curtis JL, Morris AM, Qin SL. Presence of Tropheryma whipplei in the lung and gastrum [abstract]. Am J Respir Crit Care Med 2015;191:A4723. 8. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, Crapo R, Enright P, van der Grinten CP, Gustafsson P, et al.; ATS/ERS Task Force. Standardisation of spirometry. Eur Respir J 2005;26:319–338. 9. Macintyre N, Crapo RO, Viegi G, Johnson DC, van der Grinten CP, Brusasco V, Burgos F, Casaburi R, Coates A, Enright P, et al. Standardisation of the single-breath determination of carbon monoxide uptake in the lung. Eur Respir J 2005;26:720–735. 10. Beck JM, Schloss PD, Venkataraman A, Twigg H III, Jablonski KA, Bushman FD, Campbell TB, Charlson ES, Collman RG, Crothers K, et al.; Lung HIV Microbiome Project. Multicenter comparison of lung and oral microbiomes of HIV-infected and HIV-uninfected individuals. Am J Respir Crit Care Med 2015;192:1335–1344. 11. Bassis CM, Erb-Downward JR, Dickson RP, Freeman CM, Schmidt TM, Young VB, Beck JM, Curtis JL, Huffnagle GB. Analysis of the upper respiratory tract microbiotas as the source of the lung and gastric microbiotas in healthy individuals. MBio 2015;6:e00037. 12. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. Mega6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013;30:2725–2729. 13. Charlson ES, Bittinger K, Haas AR, Fitzgerald AS, Frank I, Yadav A, Bushman FD, Collman RG. Topographical continuity of bacterial populations in the healthy human respiratory tract. Am J Respir Crit Care Med 2011;184:957–963.
Copyright © 2016 by the American Thoracic Society
Correspondence
We read with great interest the study by Ou and colleagues on the long-term risk for cardiovascular events among patients discharged with a diagnosis of sepsis (1). On the basis of discharge diagnoses of septicemia (International Classification of Diseases, Ninth Revision, 038.x), the authors demonstrate an increased risk for cardiovascular events among such patients compared with two different control populations: a matched hospital population and a control population. Although we appreciate the efforts made by the authors to address a difficult issue in health care, we believe the study methodology and results raise several issues of concern that compromise the external and internal validity of the article. The validity of the discharge diagnosis is crucial to justify the overall approach. Although an acceptable positive predictive value of 92% is documented (2), the sensitivity of the exact diagnosis is only 9% (3). The limitations of an approach using the discharge diagnosis in this context have been subject to much debate, and the so-called Angus algorithm, although not flawless, is generally accepted as the gold standard of sepsis identification in administrative cohorts (3–5). We acknowledge that the authors included a recording of antibiotic treatment but do not believe this would contribute to an already very specific diagnosis. In addition, the authors’ approach renders meaningful comparisons with other studies difficult. In the propensity score–matched analysis, the authors find similar hazard ratios for cardiovascular events and all-cause mortality when comparing the patients with sepsis with each control group. These results are counterintuitive and very difficult to comprehend. However, from their Table 1, it appears that population control subjects have more comorbidity and are older than nonsepsis hospitalized control subjects. Using the authordefined definition of comorbidity, an individual has to be admitted (or seen in an outpatient clinic) to be assigned a diagnosis code for calculation of the Charlson Comorbidity Index. We would appreciate an author clarification on this point. It would be of interest to see the distribution curves of the propensity distributions of both matchings, which may clarify some of these features. From the all-cause mortality Kaplan-Meier curves (the authors’ Figures 1A and 1B), it is apparent that almost one-third of all persons in the population control group are dead after 10 years and that the hospital control subjects have a lower 10-year mortality rate than the population control subjects. These observations are quite astounding and warrant an in-depth discussion and clarification. The observation window for an all-cause mortality event to appear is widely defined and could potentially approach 10 years (e.g., an event occurring 9 d from the index date is weighted equally to one occurring 9 yr from the index date in the hazard ratio analysis). We believe that “late events” a priori mitigate biological
Author Contributions: conception and design: D.P.H., P.D., J.R.D., and C.B.L.; analysis and interpretation: D.P.H., P.D., J.R.D., and C.B.L.; drafting the manuscript for important intellectual content: D.P.H., P.D., J.R.D., and C.B.L.
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