Comparative analysis of clinical presentation of community-acquired pneumonia induced by Chlamydia psittaci and Legionella Diagnosed through Metagenomic Next-Generation Sequencing

Legionella and Chlamydia psittaci cause atypical community-acquired pneumonia, which mimic each other. Our aim was to compare the clinical characteristics of Legionella pneumonia (LP) and psittacosis and assess whether metagenomic next-generation sequencing (mNGS) is an effective method for early diagnosis. We conducted a retrospective study and compared seventeen patients with Chlamydia psittaci pneumonia and nine patients with LP, diagnosed by mNGS. This study was carried out in the First Aliated Hospital of Wenzhou Medical University, China, from July 2018 to May 2020. mNGS was carried out from bronchial alveolar lavage uid (BALF) and/or lung tissues.

inhalation of contaminated aerosols originating from the excretions of infected birds. Psittacosis can be asymptomatic or acute symptomatic infection characterized by mild in uenza-like illness to severe pneumonia and extra-pulmonary systemic disease, which can be fatal. Chlamydia psittaci leads to CAP in rare cases (1.03%, range: 0-6.7%) [4]. Because of the low disease awareness, nonspeci c clinical manifestations, and lack of rapid and accurate diagnostic methods, psittacosis is easy to be underdiagnosed. Metagenomic next generation sequencing (mNGS) has been increasingly applied to diagnose many infectious diseases especially when traditional diagnostic methods have limitations [5].
With the widespread application of mNGS in clinical settings, psittacosis cases are getting reported frequently in recent years [6][7][8].
Legionella spp. are gram-negative intracellular bacteria that frequently leads to severe CAP (SCAP) requiring admission in intensive care unit (ICU). Legionella pneumonia (LP) is most likely caused by Legionella pneumophila. The most common bacterial mimics of legionnaire's disease (LD) are Streptococcus pneumoniae and psittacosis [9]. Studies have been conducted to determine whether LP can be distinguished from pneumonia caused by S. pneumoniae [10]. However, few studies describe the problem of differentiating LP from C. psittaci pneumonia in clinical settings.
We performed a retrospective study of 17 cases of C. psittaci pneumonia diagnosed by mNGS and compared them with 9 cases of LP. This comparison was carried out to nd whether the cases differ in clinical manifestations and to demonstrate whether mNGS is an effective diagnostic method. We present the following article in accordance with the STROBE reporting checklist.

Study design
This was a retrospective study. The study was conducted in accordance with the Declaration of Helsinki.
The study was approved by the Ethics Committee of the First A liated Hospital of Wenzhou Medical University. The requirement for informed consent was waived due to the retrospective nature of the study.

Diagnostic criteria
Cases of C. psittaci pneumonia met the following three criteria: (1) Ful lled the diagnostic criteria for CAP [11]; (2) A speci c DNA fragment of C. psittaci identi ed by mNGS; and (3) Negative routine etiological tests, including blood, sputum, and bronchial alveolar lavage uid (BALF) culture.
Cases of LP ful lled the criteria for CAP and met at least one of the following criteria: (1) Legionella successfully isolated from a respiratory sample or lung tissue sample and cultured on buffered charcoal yeast extract (BCYE) media; (2) Positive urine test for Legionella antigen; (3) A speci c DNA fragment of Legionella identi ed by mNGS and negative routine etiological tests.

Study participants
A total of seventeen cases with C. psittaci pneumonia and nine cases with LP who were admitted to the First A liated Hospital of Wenzhou Medical University, a tertiary hospital in Zhejiang, China, between July 2018 and May 2020. For each case, we extracted the demographic data, comorbidities, illness severity, clinical manifestations, laboratory and imaging data, treatment, and outcomes from electronic medical records.

mNGS
The procedures such as sample processing, DNA extraction, construction of DNA libraries, sequencing, and bioinformatic analysis were completed by BGI-Huada Genomics Institute (Shenzhen, China), according to the previously described procedures by Miao et al. [12].

Statistical analysis
Continuous variables were expressed as means ± standard deviation (SD), or medians (25th, 75th percentiles), depending on whether the variables were normally distributed or not. Categorical variables were expressed as percentages. T-test or the Mann-Whitney U-test were used to compare continuous variables. Categorical variables were compared using chi-square or Fisher's exact test. A p value of less than 0.05 was considered as statistically signi cant. All analyses were performed using the SPSS software (version 26.0, SPSS Inc., Chicago, Illinois).

Psittacosis and LP diagnosis
During the study period, 17 patients were diagnosed with psittacosis, and 9 patients with LP. C. psittaci was detected in BALF samples of 17 psittacosis patients by mNGS. Blood and BALF cultures were negative for all the 17 patients. We detected Legionella pneumophila in BALF samples of 8 LP patients using mNGS and Legionella bozemanii in lung tissue sample through percutaneous lung biopsy of 1 patient using mNGS. Blood and BALF cultures were conducted for all LP patients; BALF samples of 3 out of 8 patients were found positive for Legionella pneumophila. Tissue culture conducted in 1 patient through percutaneous lung biopsy revealed Legionella bozemanii growth. The mean time when the culture turned positive was 9.5 days. Urine Legionella antigen test were found positive in 3 out of 9 patients.

General characteristics
The demographic and general characteristics of the patients are shown in Table 1. 13 of the 17 patients of psittacosis group (76.5%) and no patient of the LP group (0%) had a history of avian or poultry exposure (p < 0.001). The median time from onset of illness to admission was 5 days in both the groups.
Psittacosis patients were admitted at all seasons while no LP patients were admitted in the winter. 41.2% of the psittacosis patients (7/17) were admitted in the spring, while 44.4% of the LP patients (4/9) were admitted in the autumn.

Clinical characteristics
All the patients infected with psittacosis and LP were febrile with a mean temperature of 39.8 °C. Majority of the patients in both the groups had similar symptoms (no signi cant difference): non-speci c rigors, fatigue, cough, sputum, and dyspnoea. Also, many extra-pulmonary manifestations were similar between both the two groups: relative bradycardia, neurological symptoms (such as headache, dizziness), and gastrointestinal symptoms (vomiting and diarrhea). Meanwhile, 5 patients in the psittacosis group and 1 in the LP group were complicated with rhabdomyolysis. The diagnosis of rhabdomyolysis was con rmed when the level of serum creatine kinase (CK) was found higher than 1000 U/L [13]. Clinical manifestations of the patients are shown in Table 2.
The laboratory and radiological tests results are shown in Table 3. The psittacosis patients had a mean white blood cell counts, neutrophils and lymphocyte of 10.5 × 10 9 /L, 9.4 × 10 9 /L, and 0.7 × 10 9 /L, respectively, while the corresponding counts in LP patients were 9.5 × 10 9 /L, 8.3 × 10 9 /L, and 0.6 × 10 9 /L respectively. Similarly, C-reactive protein (CRP) and procalcitonin (PCT) levels were 205.1 mg/L and 0.8 ng/mL, respectively, in psittacosis patients whereas the corresponding levels in the LP patients were 234.9 mg/L and 5.1 ng/mL. As compared with LP patients, psittacosis patients had signi cantly higher levels of hemoglobin, serum sodium, and higher proportion of elevated aspartate aminotransferase (AST). Both the groups had elevated levels of IL-6. Moreover, consolidation, in ammatory exudation, and pleural effusions were frequently observed in computed tomography (CT) scans in both psittacosis and LP patients ( Fig. 1, 2).

Treatment and outcomes
All antimicrobial treatments received by the patients are listed in Table 4. 23.5% of psittacosis and 44.4% of LP patients did not receive active antibiotics against C. psittaci or Legionella on admission. Reporting of the mNGS results took 48-72 h from the time of receipt of the samples. One psittacosis patient refused hemodialysis and one LP patient refused invasive mechanical ventilation; both the patients received carbapenem as initial empirical antibiotic and died before receiving their mNGS reports. Quinolone (moxi oxacin, 0.4 g intravenously q.d.) was frequently administered to patients of both the groups. The median fever clearance time in psittacosis patients (6 days) did not signi cantly differ from that in LP patients (5 days). 58.8% (10/17) of psittacosis and 44.4% (4/9) of LP patients ful lled the criteria for SCAP. The nal prognosis was similar in both the groups; hospital mortality rate was 11.8% (2/17) in psittacosis patients and 22.2% (2/9) in LP patients (p = 0.591).

Discussion
Legionella accounts for approximately 1 to 10% of CAP and is the second most common cause of SCAP, which requires ICU admission [14]. Although C. psittaci is documented as an uncommon cause of CAP, CAP cases induced by C. psittaci may be underdiagnosed. A French study on distinctive features of pneumonia caused by C. psittaci and L. pneumophila showed that severe psittacosis and severe legionellosis share many common characteristics in ICU patients [15]. However, the recommended rst line antibiotics are different for psittacosis and LP. For Legionella species, uoroquinolone and azithromycin are the preferred antimicrobials, while for C. psittaci tetracycline is recommended [11,16]. Consequently, to an optimal initial administration of empirical antibiotics, the differentiation of psittacosis from LP is frequently concerned in clinical practice.
Our results suggest that psittacosis and LP indeed share many similarities consistent with reported ndings[6, 15,17]. They both manifest as hyperpyrexia with a mean temperature of 39.8 °C. In addition, they have similar extra-pulmonary symptoms: neurological symptoms, gastrointestinal symptoms, relative bradycardia and rhabdomyolysis. Legionella-associated rhabdomyolysis has been frequently reported in literature [18,19]. Rhabdomyolysis is not usually caused by pathogens, but most pathogeninduced rhabdomyolysis in the patients is due to an infection by Legionella species [18]. The exact mechanism is unclear, but it involves either the release of endotoxin in the circulation, resulting in muscle and kidney damage, or the direct invasion of Legionella into the muscle tissues [20]. Meanwhile, case reports suggest that rhabdomyolysis also occurs in psittacosis [21,22]. Our study reveals that psittacosisassociated rhabdomyolysis is not rare, and even more common than Legionella (no signi cant difference). But the mechanism behind the complication of psittacosis by rhabdomyolysis is unknown.
The laboratory ndings of psittacosis and LP groups generally showed normal or slightly increased white blood cell counts, neutrophils, and PCT level, highly elevated CRP, and decreased lymphocytes. Gacouin et al. reported that the outcome of patients with psittacosis is similar to that of LP patients [15]. Our study also reveals that psittacosis and LP groups share similar proportion of SCAP and outcomes.
However, there are some differences between psittacosis and LP. We observed that 76.5% of the patients with psittacosis and 0% of the patients with LP had a contact with birds. Studies report that about 70% of psittacosis cases have a known source of infection as a result of contact with birds [23]. A study reported that 100% of the psittacosis cases had a bird exposure history while the corresponding percentage for LP cases is only 5.9% [15]. Therefore, a history of avian or poultry contact is an important diagnostic feature of psittacosis.
This study also shows that psittacosis patients are admitted at all seasons, and LP patients are not admitted in the winter season. Moreover, 41.2% of the psittacosis patients were admitted in the spring, while 44.4% of the LP patients were admitted in the autumn. In a Britain study, no seasonal variation was observed in psittacosis [24]. Furthermore, a higher portion of pigeons were found positive for C. psittaci IgG antibodies in the spring season. This observation suggests that pigeons may act as a signi cant source of human infection during the spring season [25]. Furthermore, raising poultry in all the seasons is quite common in China, which adds to the problem. Legionnaires' disease is more common in the late summer and early autumn, seasons that are associated with wet and humid weather [26]. In our study, 44.4% of LP patients were admitted in the autumn and 0% in the winter season. This trend might be due to the fact that the weather in Wenzhou, China is wet throughout the year except winters. However, due to a limited sample size, the correlation between the admission of psittacosis or LP patients and the seasons could not be elucidated.
In Legionella CAP and psittacosis, hepatic involvement is common and generally manifested by mildly increased serum transaminases [9,23]. It is reported that abnormal liver function tests occurs in some cases of psittacosis, and occurs in many cases in LP [17]. However, in our study, a signi cant number of patients with psittacosis had a higher proportion of elevated AST. We hypothesize that this observation might be attributed to the co-involvement of rhabdomyolysis. In addition, compared to psittacosis patients, LP patients have lower levels of serum sodium and hemoglobin. LP is frequently associated with hyponatremia [23], which may in turn be associated with the syndrome of inappropriate antidiuretic hormone secretion [27]. There is some anecdotal evidence associating hemolytic anemia to legionellosis, but the underlying mechanism is unknown [28,29]. However, in our study, the bilirubin levels in both LP and psittacosis patients were found to be in the normal range, suggesting no evidence of hemolytic anemia. 2 LP patients out of 9 had comorbidities of chronic renal failure; another 2 patients had hematologic diseases (1 patient had myeloproliferative neoplastic disease and the other one had leukemia) (no signi cant difference as compared with psittacosis, data not shown). The comorbidities may be the cause of the lower hemoglobin levels in LP patients. As compared with severe legionellosis patients, patients with severe psittacosis are reported to be younger and less frequent smokers, having fewer chronic diseases and longer duration of pre-hospital symptoms [15]. However, in our study both legionellosis and psittacosis patients shared similar such features. The differences between our study and the reported studies might be due to variations in study population.
Although certain clinical features may suggest Legionella or C. psittaci infection, microbiological tests are required to con rm the diagnosis. Available diagnostic methods for C. psittaci infection are not ideal. Culture methods are time consuming and requires at least biosafety level III facility [30]. Serological tests are suitable for retrospective diagnosis as sera are required in both acute and convalescent phases [31]. Also, cross-reactivity with other Chlamydia spp. also poses problems [3]. Polymerase chain reaction (PCR) based testing is a more speci c and rapid detecting method but has high sensitivity only in the acute stage [32]. For diagnosis of legionnaire's disease, in vitro culture is the gold standard, requiring a speci c BCYE agar media. However, the prolonged incubation time of ≥ 3 days limits its utility for early diagnosis [3]. Also, prior antibiotic exposure in the patient may decrease the culture positivity. The urinary antigen test, of good sensitivity and speci city [33], can detect the pathogen early and the antigen persists for several weeks despite the antibacterial therapy [3]. But such tests can only detect L. pneumophila serogroup I. PCR assays can rapidly detect most Legionella species, but its sensitivity and speci city varies [34,35]. Moreover, PCR is available only in a few hospitals in China. mNGS has emerged as a high-throughput method for pathogen identi cation and is superior to the currently available microbiological diagnostic methods for the identi cation of hard-to-culture pathogens. Because of its potential to use non-speci c primers and a short turnaround time, mNGS can detect etiologic pathogens rapidly, leading to early diagnosis and a better prognosis [36]. Furthermore, mNGS is less affected by prior antibiotic exposure [12]. Several studies have discussed the potential of mNGS in diagnosing psittacosis or legionnaire's disease [6][7][8]37]. In our study, all 17 psittacosis patients and 9 LP patients were diagnosed with the help of mNGS. Only 44.4% (4/9) patients tested positive for Legionella using culture methods, with a mean time to positivity as 9.5 days. Urine Legionella antigen test was positive only in 33.3% (3/9) patients. Therefore, mNGS may be a promising and rapid method for diagnosing psittacosis or legionnaire's disease.
The tetracyclines are the preferred antibiotics for the treatment of psittacosis [11,16]. Macrolides are considered as alternatives for patients who are contraindicated for tetracyclines [16]. Studies are needed to establish the utility of quinolones in treating these diseases. A few uoroquinolones were found to be active against C. psittaci in experimental models [17]. In the present study, quinolones were administered to 10 psittacosis patients, and on one out of 10 died, indicating that quinolones may be effective against C. psittaci. The unavailability of doxycycline in the hospital might be a reason why quinolones were frequently administered in our study. Besides, the use of tigecycline is restricted because of the high price and high incidence of hepatic side effects.
This study has several limitations. Sample size of this study is limited, and the diagnosis of psittacosis was made solely through mNGS, with no con rmation by other methods. However, clinical manifestations are consistent with literature and BALF in all cases showed C. psittaci reads with mNGS. Further, direct comparison between psittacosis and LP provides important information on the differential diagnosis of the two diseases based on clinical parameters.

Conclusions
There are many similarities between psittacosis and LP, including extra-pulmonary manifestations, biological features, and prognosis. Psittacosis is a vital differential diagnosis of LP, especially in patients with prior avian exposure. mNGS is a sensitive tool and has potential for the early detection of C. psittaci and Legionella. These preliminary ndings should be correlated with diagnostic trials in the future studies.

Availability of data and materials
The dataset used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of the First A liated Hospital of Wenzhou Medical University. The requirement for informed consent was waived due to the retrospective nature of the study. The study was conducted in accordance with the Declaration of Helsinki.

Consent for publication
Not Applicable.

Competing interests
The authors have no con icts of interest to declare.
Author details 1