Association Between Mycoplasma and Ureaplasma Airway Positivity, Ammonia Levels, and Outcomes Post–Lung Transplantation: a Prospective Surveillance Study

Received: 13 April 2020 | Revised: 1 November 2020 | Accepted: 2 November 2020 DOI: 10.1111/ajt.16394

ORIGINAL ARTICLE

Association between Mycoplasma and Ureaplasma airway positivity, ammonia levels, and outcomes post–lung transplantation: A prospective surveillance study

1Transplant Infectious Diseases, Division of Infectious Diseases, University of Abstract Alberta, Edmonton, AB, Canada Hyperammonemia syndrome (HS) is a rare complication with high mortality described 2Lung Transplant Program, Division of Respiratory Medicine, University of after lung transplantation. Its pathophysiology is still unclear, but previous studies, Alberta, Edmonton, AB, Canada including murine models, have linked the identification of Mycoplasmataceae in air- 3 Division of Cardiac Surgery, University of way specimens with HS occurrence. This study explores the association between Alberta, Edmonton, AB, Canada Mycoplasmataceae polymerase chain reaction (PCR) positivity, ammonia levels, HS, and 4Department of Critical Care Medicine, University of Alberta, Edmonton, AB, mortality post–lung transplant. Adults who underwent lung transplantation between Canada July 2017 and August 2019 had prospective surveillance testing for Mycoplasma and 5Division of Laboratory Medicine and Pathology, University of Alberta, Ureaplasma using PCR on post-operative bronchoscopy samples. One hundred and Edmonton, AB, Canada fifty-nine patients underwent lung transplantation during the study period. Mean age

Correspondence was 54 (±13) years; baseline diseases were predominantly pulmonary fibrosis (37.7%) Wendy I. Sligl, Transplant Infectious and chronic obstructive pulmonary disease (35.8%). Mycoplasma and/or Ureaplasma Diseases (Division of Infectious Diseases, Department of Medicine) and Department airway positivity was found in 42 (26.4%) of tested patients, represented mostly by M. of Critical Care Medicine, University of salivarium (26/43; 60.4%), U. parvum (7/43; 16.2%), and U. urealyticum (5/43; 11.6%). Alberta, Edmonton, AB, Canada. Email: [email protected] Median peak ammonia levels were higher in those with Ureaplasma colonization compared to uncolonized patients (p = .04), however, only three patients developed HS. Recipient airway Ureaplasma positivity was independently associated with younger (aOR 0.94, 95% CI 0.88–0.99, p = .04) and female donors (aOR 4.29; 95% CI 1.01–18.2, p = .05).

KEYWORDS clinical research/practice, donors and donation: donor-derived infections, infection and infectious agents, infection and infectious agents – bacterial, infectious disease, lung disease: infectious, lung transplantation/pulmonology, organ procurement and allocation

Abbreviations: CSA-IRD, Canadian Standards Association increased-risk donor; HS, hyperammonemia syndrome; IQR, medians and interquartile ranges; NAAT, nucleic acid amplification testing; NML, National Microbiology Laboratory; OPO, organ procurement organization; PCR, polymerase chain reaction; SDs, standard deviations; SOFA, Sequential Organ Failure Assessment.

amjtransplant.com | 1 Am J Transplant. 2020;00:1–9. 2 | BUZO et al.

1 | INTRODUCTION universal surveillance post–lung transplantation is not standardized and in vivo relationships between airway mollicute identification and Hyperammonemia syndrome (HS) is a rare complication of im- the occurrence of HS are not well understood. munocompromised hosts described in both adults and children Treatment is generally multi-faceted and varies by center. It gen- following hematological malignancy, stem cell or solid organ trans- erally includes antimicrobial therapy but may also include reduced plantation, particularly in lung transplants.1-6 It was firstly de- protein intake, nitrogen scavenger agents, and renal replacement scribed in lung transplant patients by Lichtenstein and Kotloff in therapy. Some authors have extrapolated data from cirrhotic pa- 1993.7 This syndrome is characterized by elevated serum ammonia tients and have used rifaximin and lactulose, aiming to reduce am- levels associated with worsening sensorium (i.e., lethargy, confu- monia-producing colonic bacteria and promote ammonia excretion sion, and agitation), seizures, and normal liver enzymes. Cerebral from the gastrointestinal tract, respectively.15,18 edema may develop, leading to death in some cases.8-10 Previous Even though HS has been associated with Ureaplasma spp and M. studies have demonstrated a relationship between Ureaplasma hominis airway positivity, there are few prospective studies correlat- urealyticum, Ureaplasma parvum, and Mycoplasma hominis identifi- ing airway positivity and ammonia levels in asymptomatic patients. cation, mostly but not limited to airway specimens, and the devel- This relatively large study aims to describe airway positivity among opment of HS.4-6,8,11,12,13 An immunocompromised murine model post–lung transplant patients using institutional Mycoplasma and also demonstrated a relationship specifically between Ureaplasma Ureaplasma active surveillance data, and correlate these data with urealyticum infection and HS.14 donor characteristics, ammonia levels, hyperammonemia syndrome, The occurrence of HS in lung transplant patients is rare, esti- and mortality. mated between 1 and 4%, but mortality rates range from 69% to 75%. The syndrome typically presents within 3 weeks posttransplant, with a median time of onset between days 6 and 9.15-17 There 2 | MATERIAL AND METHODS is still no consensus on ammonia thresholds to guide clinical inter- ventions, but most reported HS cases have ammonia levels twice Adult patients (≥18 years) who underwent lung or heart-lung the upper limit of normal,3 with ammonia peaks ranging from transplantation from July 2017 to August 2019 underwent pro- 5,17,18 193 μmol/L to 1000 μmol/L. Hyperammonemia pathogenesis spective surveillance for hyperammonemia and mollicute positiv- is thought to be related to the overgrowth of urea-splitting organ- ity posttransplant. Our surveillance protocol includes testing for isms leading to increased urea hydrolysis and subsequent elevation Mycoplasmataceae on the first bronchoalveolar lavage fluid obtained of serum ammonia.4 between days 1 and 3 posttransplant, as well as daily serum ammo- The reasons for overgrowth and predilection of Mycoplasmataceae nia testing for 7 days post-transplant (normal range 20–50 μmol/L) in transplanted lungs are still unknown, however, it has been hypoth- (Figure 1). The testing protocol includes Mycoplasmataceae family esized by many authors to be donor derived.12,19 Others believe that 16S rRNA gene detection using PCR, with final species identifica- local immunosuppression and the use of broad spectrum antimicro- tion performed on positive samples by sequencing at the National bials peri-transplant might play a role in their proliferation.14,20 A Microbiology Laboratory (NML), Winnipeg, Manitoba. Multiple sam- recent prospective study identified Ureaplasma positivity in 14.2% ples from individual patients were pooled for testing. of lung-transplant donor-recipient matched pairs, and associated Baseline variables including recipient gender, age at time of Ureaplasma positivity with donors who had multiple sexual partners transplant, baseline pulmonary disease, Acute Physiology and and aspiration events prior to brain death.13 Chronic Health Evaluation (APACHE II) and Sequential Organ Failure Previous studies have tested bronchoalveolar samples for Assessment (SOFA) scores at the time of transplant were col- Mycoplasma and Ureaplasma using nucleic acid amplification test- lected. Antimicrobial use was also collected during the first 14 days ing (NAAT) and Mycoplasma culture in cases of suspected HS,4 but posttransplant.

Dailyserum ammonialevelsuntil day7post-transplant

Lung transplantation

Intraoperative swabs Post-operative 24-72h bronchoscopy • Bacterialculture; • Anastomosischeck; • Fungal culture; • Bacterialculture; • Mycobacteria culture; • Fungal culture; • Respiratoryviruses NAAT; • Mycoplasma/Ureaplasma NAAT; Sampledfromdonorsand recipients

FIGURE 1 Surveillance protocol for Mycoplasma/Ureaplasma and ammonia post-lung transplant BUZO et al. | 3

Compliance to the protocol was calculated if both PCR testing performed using the Chi-square or Fisher's Exact tests to assess and daily ammonia serum level (for 7 days posttransplant) were for correlations between recipient/donor risk factors and mollicute collected. Hyperammonemia syndrome was defined as any new identification, incidence of hyperammonemia syndrome and 30- neurologic abnormality (agitation, decreased level of conscious- day mortality. Multivariable logistic regression was subsequently ness, seizures, or cerebral edema), without an alternative diagno- performed to assess for associations between donor variables sis, with serum ammonia levels above 50 μmol/L and normal liver and PCR-positivity status. Clinically relevant variables (e.g., donor enzymes. Our criteria were revised using previously published age and sex) were included in the model regardless of significance criteria21,22 to include ruling out any urea cycle or metabolism on univariate analysis. Variables identified as being significant disorders. (p ≤ .10) on univariate screen were also included. Separate models Donor data were retrieved from local organ procurement orga- were constructed to identify associations with Mycoplasma and/or nization (OPO) records. Donor variables of interest included gen- Ureaplasma positivity and Ureaplasma positivity alone. C-statistic der, age, date of transplant, province of procurement, race, cause values were reported as measures of goodness of fit. All p-values of death, increased risk donor status, any active and frequent were two tailed with statistically significant defined by a p < .05. use of tobacco and or marijuana preceding organ procurement, All statistical analyses were performed using IBM SPSS Statistics, any use of intravenous drugs previous to organ procurement, Version 26.0 (IBM Corp). and high-risk sexual behaviors (as per 2012 Canadian Standards Association increased-risk donor [CSA-IRD] criteria). Also, the use of antimicrobials in the 72 h prior to organ procurement was 3 | RESULTS retrieved. Demographic variables were reported as numbers and percent- 3.1 | Study population and baseline diseases ages for categorical variables and means with standard deviations (SD) or medians and inter-quartile ranges (IQR), as appropriate One hundred and fifty-nine adults underwent lung transplantation for continuous variables. Median peak ammonia levels by molli- during the study period. Most patients were male (n = 106, 66.6%) cute PCR positivity and outcome categories were compared using and mean age was 54.0 years (±13.2). Most common baseline dis- the Independent Samples Median Test. Univariate analyses were eases pretransplant included pulmonary fibrosis (n = 60, 37.7%),

TABLE 1 Recipient baseline Total PCR positive PCR negative p- characteristics and univariate analyses for n = 159 (100%) n = 42 (29%) n = 105 (71%) value correlations with Mycoplasma/Ureaplasma airway PCR positivity Male gender, n (%) 106 (66.6) 33 (79) 62 (59) .025 Age (years), mean (SD) 54.2 (13.20) 53.5 (13.0) 53.7 (13.7) .92 Type of transplant, n (%) Bilateral sequential 154 (96.8) 42 (100) 100 (95.2) .97 lung transplant Heart-lung transplant 3 (1.8) 0 (0) 3 (2.8) Single-lung transplant 2 (1.2) 0 (0) 2 (1.9) Baseline disease, n (%) Pulmonary fibrosis 60 (37.7) 23 (55) 34 (32) .15 Chronic pulmonary 57 (35.8) 10 (24) 42 (40) obstructive disease Cystic fibrosis 21 (13.2) 4 (10) 15 (14) Pulmonary 15 (9.4) 4 (10) 10 (10) hypertension Alpha-1 antitrypsin 6 (3.7) 1 (2.3) 4 (3.8) deficiency APACHE II, at ICU 19.7 (5.8) 21.6 (6.8) 19.0 (5.4) .018 admission, mean (SD) SOFA, at ICU admission, 7.9 (3.1) 8.2 (3.7) 7.8 (2.8) .53 mean (SD)

Abbreviations: APACHE, Acute Physiology and Chronic Health Evaluation; PCR, polymerase chain reaction; SOFA, sequential organ failure score. 4 | BUZO et al. chronic obstructive pulmonary disease (n = 57, 35.8%), and cystic sub-speciation was not possible. Two patients (4.7%) had more than fibrosis (n = 21, 13.2%). Baseline APACHE II and SOFA scores were one mollicute species identified (Table 2). 19.7 (±5.8) and 7.9 (±3.1) at the time of ICU admission posttransplant, respectively (Table 1). 3.3 | Ammonia levels posttransplant

3.2 | Protocol compliance and Median peak ammonia levels were 45 μmol/L (IQR 36–58) in PCR- microbiologic findings negative patients, 52 μmol/L (IQR 39–60) in those who tested positive for Mycoplasma spp., and 61 μmol/L (IQR 46–75) in those Compliance to the protocol was 92.4% (147/159) for who tested positive for Ureaplasma spp (Figure 2). Median peak Mycoplasma/Ureaplasma PCR in the first post-operative bronchos- ammonia levels varied significantly among the groups (p = .03); copy specimen. After excluding five early deaths (within 7 days and were specifically higher in those with Ureaplasma-positivity of transplant) 95.4% (147/154) of patients had at least one serum compared with PCR-negative patients (p = .04), but not when ammonia level collected and 67.5% (104/154) had complete 7-day compared with Mycoplasma-positive patients (p = .46). Those who ammonia testing posttransplant. Forty-two (26.4%) patients had tested positive for Mycoplasma did not have significantly higher Ureaplasma or Mycoplasma airway PCR positivity. M. salivarium ammonia levels when compared with PCR-negative recipients was the most common isolated species (n = 26, 59.0%), followed by (p = .20). Median day of ammonia peak posttransplant also varied U. parvum (n = 7, 15.9%), U. urealyticum (n = 5, 11.3%), M. hominis among groups (p = .03); 4 days (IQR 2–6) in Mycoplasma-positive (n = 3, 6.8%), and M. pneumoniae (n = 2, 4.5%). There was one patient recipients, 4 days (IQR 1–5) in Ureaplasma-positive recipients, and with Mycoplasma non-pneumoniae identified (2.2%), but further 2 days (IQR 2–4) in PCR-negative recipients (Figure 3). Pairwise comparisons only demonstrated a difference in median day of

TABLE 2 Ureaplasma/Mycoplasma airway positivity by species ammonia peak between PCR-negative recipients and those who (42 patients; 44 isolates) tested positive for Mycoplasma (p = .02). The median peak ammonia level in patients with HS (n = 3) was 79 μmol/L (IQR 67.5–110.5) Total of compared to 47.5 mol/L (IQR 36–59.5) in patients without HS isolates (%) μ (p = .19) (Figure 4). Mycoplasma salivarium 26 (59.0) Ureaplasma parvum 7 (15.9) Ureaplasma urealyticum 5 (11.3) 3.4 | Donor characteristics Mycoplasma hominis 3 (6.8) Mycoplasma pneumoniae 2 (4.5) Donors were mostly male (n = 84, 52.8%) and Caucasian (n = 120, Mycoplasma non-pneumoniae (not further speciated) 1 (2.2) 75.4%), and the mean age was 37.7 years (±14.8). Increased risk Co-identification of species 2/42 (4.7) sexual behavior (n = 60, 37.7%) and increased risk donor status

FIGURE 2 Median peak ammonia levels posttransplant among airway positive vs. negative patients. Box plot denotes median values (dark lines in the middle of boxes), interquartile ranges (boxes), T-bars or inner fences (1.5 times the height of the box or minimum/maximum values), and outliers (small circles and asterisks; the latter which are extreme outliers). p-value for differences in medians = .031 BUZO et al. | 5

FIGURE 3 Median day of peak ammonia level posttransplant among airway positive vs. negative patients. Box plot denotes median values (dark lines in the middle of boxes), interquartile ranges (boxes), T-bars or inner fences (1.5 times the height of the box or minimum/ maximum values), and outliers (small circles and asterisks; the latter which are extreme outliers). p-value for differences in medians = .028

FIGURE 4 Median peak ammonia levels posttransplant among patients with and without hyperammonemia syndrome. Box plot denotes median values (dark lines in the middle of boxes), interquartile ranges (boxes), T-bars or inner fences (1.5 times the height of the box or minimum/maximum values), and outliers (small circles and asterisks; the latter which are extreme outliers). p-value for differences in medians = .19

(n = 77, 48.4%) were common. The most common cause of death The only donor characteristic associated with Mycoplasma and/ was hemorrhagic cerebrovascular accident (n = 46, 28.9%), fol- or Ureaplasma positivity on multivariable logistic regression was lowed by opioid overdose (n = 18, 11.3%) and traumatic brain injury marijuana use (aOR 2.30, 95% CI 1.03–5.14, p = .04) (C-statistic (n = 14, 8.8%). 0.70). Multivariable analysis for Ureaplasma positivity alone demonstrated independent associations with younger donor age (aOR 0.94, 95% CI 0.88–0.99, p = .04) and female donors (aOR 4.29; 95% CI 3.5 | Characteristics and outcomes in patients with 1.01–18.2, p = .05) (C-statistic 0.84) (Tables 4 and 5). mollicute PCR-positive airway samples Of 159 donors, 125 (78.6%) were treated with antimicrobials prior to organ procurement, however, only one received antimicro- Recipients who tested positive for Ureaplasma or Mycoplasma had bial therapy active against Mycoplasma/Ureaplasma spp. younger donors, with increased risk sexual behavior and increased Hyperammonemia syndrome was identified in nine recipients frequency of marijuana use (Table 3). Ureaplasma/Mycoplasma- (1.8%), all of whom underwent bilateral sequential lung transplant, positive recipients were mostly male and had higher APACHE II one of whom died. All cases had elevated ammonia levels and neu- scores, but not higher SOFA scores (Table 1). rologic symptoms (including confusion, agitation, decreased level 6 | BUZO et al.

TABLE 3 Donor characteristics and Total PCR positive PCR negative univariate analyses for correlations with n = 159 n = 42 (29%) n = 105 (71%) p-value Mycoplasma/Ureaplasma airway positivity Age, years, mean (SD) 36 (14.8) 32.9 (13.9) 40.3 (14.7) .006 Male gender, n (%) 84 (52.8) 26 (62) 48 (46) .076 Donor classification, n (%) NDD 135 (84.9) 35 (83) 90 (86) .54 DCD 22 (13.8) 7 (17) 13 (12) MAID 2 (1.2) 0 (0) 2 (2) Donor ethnicity, n (%) Caucasian 119 (74.8) 31 (74) 80 (76) .89 Indigenous 22 (13.8) 6 (14) 14 (13) Asian 15 (9.4) 5 (12) 10 (10) Others 3 (1.8) 1 (2) 2 (2) Group characteristics, n (%) IRD 77 (48.4) 25 (60) 44 (42) .053 Tobacco use 95 (59.7) 29 (69) 57 (54) .1 Marijuana use 70 (44.0) 25 (60) 37 (32) .007 IDU 28 (17.6) 7 (17) 17 (16) .94 High-risk sexual 37 (23.2) 16 (38) 17 (16) .014 behaviora Steroid use 84 (52.8) 19 (42) 60 (57) .19 pretransplant

Abbreviations: DCD, donation after cardiac death; IRD, increased risk donor, IDU, injection drug use; MAID, medical assistance in dying; NDD, neurologic determination of death; PCR, polymerase chain reaction; SD, standard deviation. aMissing data (22 patients).

TABLE 4 Multivariable logistic regression examining donor TABLE 5 Multivariable logistic regression examining donor characteristics associated with Mycoplasma and/or Ureaplasma characteristics associated with Ureaplasma positivity alone (n = 147) positivity (n = 147a) Variable aOR (95%CI) p-value Variable aOR (95%CI) p-value Age 0.94 (0.88–0.99) .046 Age 0.97 (0.94–1.00) .062 Female gender 4.30 (1.01–18.26) .048 Female gender 0.53 (0.25–1.16) .11 Increased risk donor 2.12 (0.39–11.60) .39 Increased risk donor 1.09 (0.40–3.00) .87 Marijuana use 5.67 (0.99–32.5) .052 Marijuana use 2.30 (1.03–5.14) .042 High-risk sexual behavior 0.59 (0.20–1.78) .35 High-risk sexual behavior 1.02 (0.55–1.91) .95 Abbreviations: 95% CI, 95% confidence interval; aOR, adjusted odds Abbreviations: 95% CI, 95% confidence interval; aOR, adjusted odds ratio. ratio. a12 patients did not have Mycoplasma/Ureaplasma PCR testing. a12 patients did not have Mycoplasma/Ureaplasma PCR testing.

nitrogen scavenger agents, and renal replacement therapy. On of consciousness, or diffuse cerebral edema) with no other eti- univariate analysis, Ureaplasma infection was associated with HS ology. The median day of HS diagnosis was 5.0 days (±2.8) post- (p < .05). Further analyses were not conducted due to inadequate transplant and median ammonia level at time of diagnosis was statistical power. 69.0 (±9.0) μmol/L. Of these three patients, one was positive for With regard to specific treatments for Mycoplasmataceae air- Ureaplasma urealyticum and two patients had both Ureaplasma way positivity, 24 recipients (15.1%) received active antibacterial parvum and Mycoplasma hominis identified. All three cases were therapy, of which 16 (10.0%) received antimicrobials specifically negative for urea cycle disorders. In addition, there was no history for hyperammonemia. Of those treated, 14 (58.3%) were treated of metabolic diseases in any of the donors. Survivors were treated with doxycycline, 8 (33.0%) with ciprofloxacin, 1 (4.2%) with with active antimicrobial therapy (one with doxycycline, the levofloxacin and 1 (4.2%) with minocycline. The mean duration other with doxycycline and ciprofloxacin), dietary modifications, of therapy was 8 (±6) days. The use of active antimicrobials was BUZO et al. | 7 not associated with decreased 30-day mortality (p = 1.00) in our likely. In particular, increased donor sexual risk behavior can facil- analysis. It is important to mention that, as a send-out test, most itate donor airway positivity with these genitourinary pathogens clinicians did not have Mycoplasmataceae PCR results at the time and subsequent recipient transmission. The association with female of HS diagnosis. As such, antimicrobial therapy would have been gender is novel but may relate to the high prevalence of Mollicutes started empirically. genitourinary colonization (specifically Ureaplasma spp.) in child- The median ICU length of stay in this cohort was 6.09 (3.9– bearing age women.26 14.03) days, and median hospital length of stay was 27 (17–47) days. Subgroup analysis did not show different positivity rates for There were no differences in ICU or hospital lengths between Mycoplasma and/or Ureaplasma spp among various pulmonary diseases. Mycoplasma-positive or Ureaplasma-positive versus PCR-negative Regarding the prevalence by species, Mycoplasma salivarium was the recipients (p-values .42 and .18, respectively). The overall 30-day most identified species, which has not been widely reported in the liter- mortality rate was 4.4% (7/159) in this cohort. ature. We suspect this species is not the same in terms of pathobiology compared to other mollicute species, given its nature as an upper airway and oral cavity contaminant. In support of this, most cases of hyperam- 4 | DISCUSSION monemia syndrome reported in the literature have been associated with M. hominis or Ureaplasma sp. positivity, not with M. salivarium. The high In summary, hyperammonemia syndrome was rare in our cohort of prevalence of M. salivarium may be the result of our screening method, lung transplant recipients, but it was diagnosed early and treated 16S rRNA PCR, which has a high sensitivity for detection. aggressively. We report high Mycoplasma and Ureaplasma positiv- Unfortunately, our surveillance testing requires sending specimens ity rates (28.5%) immediately post–lung transplant and a significant to a reference laboratory as we do not have in-house testing available, association between Ureaplasma-positivity and elevated ammonia which results in delayed turnaround times (TAT). In our cohort, the mean levels. This association, however, cannot be considered causative TAT of the 159 sent specimens was 14.9 (±7.7) days. Development of at this time. on-site, rapid, real-time PCR testing for Ureaplasma spp and Mycoplasma We believe the higher ammonia levels seen in recipients with hominis may be justified in large transplant centers to ensure early diag- Ureaplasma-positive airway samples may be explained by the dif- nosis and therapy, and avoidance of empiric therapies. ferent metabolic pathways used by these bacteria; Mycoplasma Despite the previous association between Ureaplasma infec- species are able to metabolize carbohydrates and amino acids to tion and high ammonia levels in vivo,4,11,14,24,25 the role of anti- produce energy alternatively to urea, while Ureaplasma species microbial therapy in these cases is still unclear but has been are non-fermentative mollicutes—relying strictly on urea for their associated with favorable outcomes.13 As Mollicutes lack a cell growth as evidence by in vitro models.23 However, the occurrence wall, empiric therapy with ß-lactams (usual empiric choices for of hyperammonemia syndrome has also been linked to host factors. post-operative prophylaxis, including at our site) is ineffective. Rueda et al hypothesize that glutamine synthetase deficiency might Previous case reports have used tetracyclines, fluoroquinolones, increase the likelihood of ammonia accumulation, justifying why and azithromycin as empiric options, however, independent asso- low protein intake may be a therapeutic option.2 Another potential ciations with outcomes are lacking. In our center, we use doxycy- hypothesis is that the lungs are known for being highly metabolic cline empirically, given gyrA and gyrB fluoroquinolones-resistance post–lung transplantation and could be overwhelmed by specific mutations are increasingly common and M. hominis is highly resis- bacterial co-signaling, leading to elevated ammonia production.21 tant to macrolides.27-29 Indeed, ammonia has been previously reported as a marker for This study highlights the potential value of prospective sur- inflammatory lung diseases and hyperammonemia syndrome has veillance of serum ammonia and Mollicutes airway positivity post- been well described in lung and hematopoietic stem cell transplants transplant as we believe that prompt (and directed) initiation of with mollicute-positive airway samples.24,25 therapy can lead to better outcomes. Ideally, point-of-care testing It has been hypothesized that Mycoplasmataceae infection post- for Ureaplasma would be available in all large transplant centers transplant is donor derived. Wright et al. described three donor to facilitate prompt diagnosis of hyperammonemia. Although not bronchoalveolar lavage samples with M. hominis identified by PCR supported by high-quality data, we suggest treating hyperam- in two of three matched recipient samples, one of which developed monemia syndrome not only with antimicrobials, but also with HS.19 Similarly, Fernandez et al. systematically tested recipients dietary modifications, nitrogen scavenger agents and renal re- and donors for mollicutes colonization. None of the recipients in placement therapy. The length of treatment is not established Fernandez's study had a positive bronchoalveolar lavage fluid for but varies between 10 and 21 days in previous reports with good Ureaplasma or Mycoplasma, whereas four donors (14%) had posi- outcomes.6,11,12,25 tive samples for Mollicutes testing. Fernandez also demonstrated an Despite our findings, our study has several limitations. First, it association with younger donor age and high-risk sexual activity,13 is a single-center study and thus may not be generalizable. Second, as we have reported in this study. We were unable to microbiologi- compliance with the surveillance protocol was not 100%, leading to cally confirm whether PCR positivity was donor derived as we only missing data (12 patients [7.6%] did not have any Mycoplasma nor tested recipients posttransplant, however, we believe this is most Ureaplasma testing, and ammonia levels were not systematically 8 | BUZO et al.

3. Navaneethan UVP. Idiopathic hyperammonemia in a patient tested daily for 7 days in 55 [35.6%] patients). Third, given that both with total pancreatectomy and islet cell transplantatione. JOP. mortality and hyperammonemia syndrome were rare conditions post- 2010;11(6):620-624. transplant, the study was underpowered to examine these outcomes. 4. Bharat A, Cunningham SA, Budinger GRS, et al. Disseminated Fourth, given that we defined HS as any elevated serum ammonia Ureaplasma infection as a cause of fatal hyperammonemia in humans. Sci Transl Med. 2015;7(284):284ra59. level associated with neurologic dysfunction not attributable to an- 5. Somerville L, Sligl W, Zelyas N, Lien D, Preiksaitis J. Surveillance for other cause, we risked misclassifying patients as previously reported Mycoplasma/Ureaplasma Infection in Lung Transplant Recipients cases of HS are usually associated with marked elevated ammonia (LTRs). https://atcmeetingabstracts.com/abstract/surveillance-for- levels. However, given that hyperammonemia can develop very rap- mycoplasma-ureaplasma-infection-in-lung-transplant-recipients- ltrs/. Accessed April 11, 2020. idly, we believe this definition best identifies patients early, avoiding 6. Nowbakht C, Edwards AR, Rodriguez-Buritica DF, et al. Two cases late diagnosis and poor outcomes. With only three cases of HS, mis- of fatal hyperammonemia syndrome due to mycoplasma homi- classification could not be substantial. Last, both donor and recipient nis and ureaplasma urealyticum in immunocompromised pa- testing would be required to determine if recipient airway positivity tients outside lung transplant recipients. Open Forum Infect Dis. 2019;6(3):1-3. is truly donor derived, however, only post-operative recipient testing 7. Kotloff RM, Lichtenstein GR, Berry GT, et al. Idiopathic hyper- was possible in our population. ammonemia following lung transplantation. Am Rev Respir Dis. In conclusion, we have demonstrated that patients positive for 1993;4:A603. Ureaplasma from airway specimens post–lung transplant have higher 8. Watson AJ, Karp JE, Gordon Walker W, Chambers T, Risch VR, Brusilow SW. Transient idiopathic hyperammonaemia in adults. ammonia levels and may be at risk for hyperammonemia syndrome. Lancet. 1985;326(8467):1271-1274. In fact, all three patients with hyperammonemia syndrome in this 9. Yoshida EM, Ostrow DN, Erb SR, Fradet G. Hyperammonemia after cohort were positive for Ureaplasma spp (one with Ureaplasma urea- heart-lung transplantation. Gastroenterology. 1997;112(6):2162. lyticum and two with positive samples for Ureaplasma parvum in the 10. Wylam ME, Kennedy CC, Hernandez NM, et al. Fatal hyperammonaemia caused by Mycoplasma hominis. Lancet. context of Mycoplasma hominis co-colonization). Ureaplasma identi- 2013;382(9908):1956. fication was independently associated with younger, female donors. 11. Smith M, Crews JD, Cheek N, Srivastava R, Appachi E. Based on these data, surveillance and/or pre-emptive therapy for Hyperammonemic encephalopathy due to ureaplasma par- Ureaplasma in selected groups may improve outcomes post–lung vum infection in an immunocompromised child. Pediatrics. transplantation. 2019;144(2):e20190601. 12. Fernandez R, Ratliff A, Crabb D, Waites KB, Bharat A. Ureaplasma transmitted from donor lungs is pathogenic after lung transplanta- ACKNOWLEDGMENTS tion. Ann Thorac Surg. 2017;103(2):670-671. We would like to acknowledge the Provincial Laboratory for 13. Fernandez R, Chi M, Ison M, et al. Sequelae of donor-derived mol- Public Health (ProvLab) in Alberta and the National Microbiology licute transmission in lung recipients. Am J Respir Crit Care Med. 2017;195(5):687-689. Laboratory (Winnipeg, MB), for performing PCR surveillance test- 14. Wang X, Karau MJ, Greenwood-Quaintance KE, et al. Ureaplasma ing. Also, TRACER program for retrieving supplementary data about urealyticum causes hyperammonemia in an experimental immuno- critical care scores and Alberta Health Services to allow us to access compromised murine model. PLoS One. 2016;11(8):1-8. its database to develop this study. 15. Lichtenstein GR, Kaiser LR, Tuchman M, et al. Fatal hyperammonemia following orthotopic lung transplantation. Gastroenterology. 1997;112(1):236-240. DISCLOSURE 16. Chen C, Bain KB, Iuppa JA, et al. Hyperammonemia syndrome after The authors of this manuscript have no conflicts of interest to dis- lung transplantation: a single center experience. Transplantation. close as described by the American Journal of Transplantation. 2016;100(3):678-684. 17. Krutsinger D, Pezzulo ABA, et al. Idiopathic hyperammonemia after solid organ transplantation: primarily a lung problem? A single-cen- ORCID ter experience and systematic review. Clin Transplant. 2017;31(5). Bruno F. 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