Case Report Severe pneumonia caused by Chlamydia psittaci : report of two cases and literature review

Introduction: Chlamydia psittaci pneumonia is a zoonotic infectious disease caused by Chlamydia psittaci . Its clinical manifestations are nonspecific. Diagnosis of the disease is difficult. In recent years, next-generation sequencing has played an important role in pathogen detection. We report two cases with severe Chlamydia psittaci pneumonia confirmed by next-generation sequencing. Case Study: The first case is that of a 50-year old man who presented with high fever for four days and cough with sputum for two days. The second case is that of a 57-year-old man who was admitted with high fever for one week, dyspnea and cough with sputum for four days. The second man worked at a chicken farm in the last two months. In both cases, the usual laboratory examination for pathogens detection was negative, and the initial anti-infectious therapy had limited effect. The bronchoalveolar lavage fluid of case 1 and the blood and sputum of case 2 were sent for next-generation sequencing which resulted in sequence reads of Chlamydia psittaci . Antibiotics were adjusted according to the diagnosis. Results: The diagnosis of the two cases was confirmed by next-generation sequencing detecting Chlamydia psittaci , and the patients had positive results after treatment. Conclusions: The two cases suggest that next-generation sequencing could be used in early diagnosis of Chlamydia psittaci infection to initiate specific anti-infection therapy in time.


Introduction
Chlamydia psittaci (C. psittaci) pneumonia is a zoonotic infectious disease caused by C. psittaci, and is estimated to cause approximately 1% of cases of community-acquired pneumonia [1]. C. psittaci infection is associated with various clinical manifestations from asymptomatic infection to severe atypical pneumonia and systemic disease. At present, the diagnosis of C. psittaci infection mainly depends on laboratory examination, which makes early diagnosis difficult. The next-generation sequencing (NGS) technique enables rapid screening of pathogens and helps with early identification of pathogens to initiate specific anti-infective therapy [2]. In this article, we reported two cases of severe Chlamydia psittaci pneumonia, C. psittaci was detected in blood, sputum, and bronchoalveolar lavage fluid (BALF) by NGS and was successfully rescued. We have also reviewed relevant literature on C. psittaci infection which include a total of 36 cases of infection.

Case 1
A 50-year-old male was admitted to the hospital on December 2, 2020. The patient had high fever for 4 days, cough with sputum and dyspnea for 2 days. He was an office worker. Four days earlier, the patient had fever after travelling, with a peak body temperature of 40 ℃, along with fatigue, muscle pain, and chills. There was no improvement after self-administered paracetamol and tylenol. Two days later he visited our fever clinic. His blood routine showed the white blood cell (WBC) count was 9.21(*10 9 / L), the neutrophil percentage was 82.9%. The chest CT ( Figure 1) showed variegated high-density shadow and abrasive glass density shadow in the upper lobe and middle lobe of the right lung. Considering that the patient was diagnosed with pulmonary infection, he was given moxifloxacin and imipenem and cilastatin sodium, but his condition deteriorated. One day before admission, his oxygenation index fell to 83 and he was sent to the intensive care unit (ICU). Emergency tracheal intubation and ventilator assisted ventilation was applied.  The chest CT showed that the upper lobe and middle lobe of the right lung were stained with high-density shadow and ground glass density shadow, and the bronchus inflation sign was found. The trachea and main bronchi are unobstructed.
The patient had hypertension for more than four years, and he was undergoing treatment with oral nifedipine and benazepril hydrochloride, and he did not monitor blood pressure regularly. The patient had been living abroad recently (Zhucheng to Gaomi, Shandong Province, China).
His body temperature was 38.2 °C, pulse was 128 bpm, blood pressure was 140/88 mmHg, and his respiratory rate was 20 breaths per minute at the time of admission. Following physical examination, the patient was kept under sedation in tracheal intubation and ventilator assisted ventilation. His Glasgow score was 3 and he could not cooperate with physical examination. The skin was stained and the skin temperature of both hands and feet was low. Wet rales could be heard and scattered in both lungs, especially in the right lung. The After active anti-infection treatment, the overall trend of chest image changes is the gradual absorption and reduction of inflammation in both lungs. Among them, chest image (2021-12-19) contrast with 2020-12-15 showed that the double lung texture increased, visible flake shadow can be found, especially in the right lung;. The inflammation in both lungs gradually reduced after adjusting the plan of antibiotics. patient's laboratory test results for blood routine tests and procalcitonin (PCT) are shown in Figure 2. Other laboratory test results can be found in Table 1.
After admission, diagnosis of type I respiratory failure and severe pneumonia was clear. Based on tracheal intubation assisted ventilation, his oxygen saturation was maintained at about 90% and oxygenation index was 73. The emergency extracorporeal membrane oxygenation (ECMO) was administrated. When using ECMO, simultaneous sedation and analgesia were required, and the use of heparin was adjusted based on the ACT (120-160s) or APTT (60-80s). The parameters of ECMO were adjusted according to the patient's condition. At the same time, the patient was treated with levofloxacin, imipenem and cilastatin sodium, and oseltamivir. Supportive treatment was also given. To identify the pathogen, bronchoalveolar lavage (BAL) was conducted on the second day and bronchoalveolar lavage fluid (BALF) was sent for testing with NGS. On the third day of arrival to our hospital, NGS of BALF and blood reported sequence reads of Chlamydia psittaci. Therefore, the antibiotics were adjusted to minocycline, azithromycin, and imipenem and cilastatin sodium. After treatment, the patient was in a stable condition and the lung inflammation was absorbed ( Figure 3). Finally, the patient was discharged the twenty sixth day after admission. The patient went for reexamination after 1 week (January 1, 2021) and was in good condition, his chest imaging ( Figure 3) showed the double lung inflammation was reduced.

Case 2
A 57-year-old male was admitted to the hospital on October 17, 2020 after suffering from a high fever for 1 week, and cough with sputum and dyspnea for 4 days. His peak body temperature reached 38.9 ℃, accompanied by fatigue, chills, and poor appetite, and there was no improvement after self-administered cephalosporin antibiotics. Four days prior to hospitalization, he started to have cough with white sticky phlegm accompanied with dyspnea. His poor appetite and fatigue were aggravated progressively. Three days prior to hospitalization he went to the local hospital, where his blood pressure was 68/40mmHg; his chest CT ( Figure 4) showed double lung inflammation and lung occupation. Considering the diagnosis of severe pneumonia, septic shock, and type I respiratory failure, he was treated with piperacillin sulbactam and oxygen inhalation. He was transferred to the ICU, and the chest CT indicated the progression of pneumonia. Oxygen storage mask and non-invasive ventilation (NIV) were given for assisted breathing. However hypoxemia was difficult to ameliorate and he was treated with emergency tracheal intubation and ventilator assisted ventilation. After the onset of the disease, his mental condition and sleep were poor, stool was normal, urine was dark brown and reduced, and he lost two kilograms in weight.
The patient had a history of splenic tumor resection which happened 40 years ago, and esophageal venous sclerosis treatment owing to cirrhosis and gastrointestinal bleeding 32 years ago. He was diagnosed with "portal vein congenital malformation, cirrhosis" and treated through a surgery. In the previous two months, he worked for a chicken farm. He had a 30year smoking history and no history of hereditary diseases in the family.
His body temperature was 36.8 °C, pulse 83 bpm, blood pressure 132/85 mmHg, respiratory rate 29 breaths per minute, and his PaO 2 was 75mmHg at the time of admission. Upon physical examination, the patient was kept in tracheal intubation and ventilator assisted ventilation. He appeared acutely ill and and in pain. He was under sedation and could not cooperate. There were liver palms and spider nevus. Wet rales could be heard in both lungs. The heart rate was 94 beats/min, the rhythm was absolutely uneven, and the intensity of the first heart sound was different. The patient's laboratory test results of blood routine and PCT after admission are shown in Figure 5. Other laboratory test results are listed in Table 1. His phlegm was tested negative for tuberculosis and fungus.
After admission, he was diagnosised with type I respiratory failure, severe pneumonia, and adult respiratory distress syndrome (ARDS). Therefore, imipenm, cilastatn sodium and moxifloxacin were used as anti-infective therapy. He was also treated with physical cooling, plasma transfusion, diuretic treatment and other symptomatic therapy. After admission, his blood and sputum were sent for testing for pathogens by using NGS and sequence reads of Chlamydia psittaci were reported. This result was in combination with his recent history of poultry exposure. The the antibiotics were adjusted to minocycline, azithromycin, imipenm and cilastatn sodium. The use of antibiotics and parameters of invasive ventilator were adjusted based on parameters such as hospital infection and changes in chest imaging ( Figure 6 and Figure 7). As a result of the treatment, the patient was in stable condition, the lung inflammation was absorbed, tracheal intubation was removed on the 17th day after admission.
He was also treated for atrial fibrillation during hospitalization. Owing to high levels of BUN, Scr, liver function, and bilirubin after admission, the protection of essential organs and maintaining water and electrolyte balance were required. Finally, the patient was discharged the 25th day after admission. The patient returned for reexamination after 1 week (November 18, 2020) and was in good condition, his chest imaging showed the double lung inflammation reduced and a small amount of pleural effusion in the left.

Results
The two cases were diagnosed with Chlamydia psittaci though NGS. Antibiotics and other therapies were adjusted according to the result and the patients' condition. The patients improved after treatment and were discharged on the 26th and 25th day, respectively. Reexamination showed good clinical and radiological prognosis.   The chest images of 10-17 showed bilateral lung inflammation and left pleural effusion. The chest images of 10-19 and 10-21 showed that the inflammation of both lungs was more advanced than before; after adjusting antibiotics, the inflammation of both lungs was absorbed and less than that of 10-21; 11-03 compared with 10-25, the inflammation of both lungs was advanced, hence the use of antibiotics was adjusted.

Discussion
Chlamydia psittaci is an obligatory intracellular Gram-negative bacterium, which can be found in the tissues, blood, and fecal material of parrots and other birds. C. psittaci mainly infects birds, but can occasionally cause psittacosis in humans when contaminated aerosols from infected birds are inhaled. Based on sequencing of the major outer protein gene (ompA), C. psittaci can be classified into 10 genotypes, namely A~G, WC, E/B, and M56 [15]. Genotypes A and E can infect humans. The disease usually occurs sporadically, but outbreaks have been described [16,17,18].
C. psittaci pneumonia mainly occurs in young and middle-aged people, and more than half of the patients have a history of contact with birds. As we reviewed in Table 2, most cases (77.8%, 28/36) had a history of contact with birds. The clinical manifestation of C. psittaci pneumonia is unspecific. It can appear as high fever, cold, headache, myalgia, cough, pulmonary infiltration, etc. In severe cases, patients can develop severe pneumonia, and even have a poor prognosis [3,19]. As summarized in Table 2 previous studies have identified the symptoms as fever (97.2%, 35/36), cough (75.0%, 27/36), myalgia (36.1%, 13/36), headache (33.3%, 12/36), malaise (25%, 9/36); some cases developed to ARDS; most of the cases were cured, only five cases (13.9%, 5/36) dead. In addition to the respiratory tract, infection with C. psittaci has been reported to affect organ systems, resulting in conditions including endocarditis, myocarditis, hepatitis, arthritis, keratoconjunctivitis, encephalitis, and ocular adnexal lymphoma [20]. Some patients may have rhabdomyolysis. Laboratory test results suggest that the WBC count is normal in most patients. If systemic diseases exist, patients may develop liver function abnormalities, hyponatremia, and elevated BUN and Scr. High-level C-reaction protein and low-level ALB can also occur. In the two cases we report here, there were fever, cough, respiratory failure, ARDS, and abnormalities of liver function and ALB; moreover, case 2 presented with abnormal renal function and coagulation.
There is also a lack of specificity in imaging of C. psittaci pneumonia. In the review (Table 2), chest imaging of most patients showed consolidation of infiltration, mainly in the lower lobe and unilateral lung, although some involved bilateral lung [21]; hilar lymphadenopathy and pleural effusion (13.9%, 5/36) are rare. In these cases, only two patients had normal chest imaging. High-resolution CT shows that some lesion areas are solid nodular and ground glass [22]. Among the two cases we have reported, there were solid or ground glass pulmonary shadows, and hilar lymphadenopathy and pleural effusion in case 2. Combined with respiratory failure and chest imaging The overall trend of chest CT was decreased inflammation and pleural effusion. findings, severe pneumonia could be diagnosed, but not C. psittaci infection.
The diagnosis of C. psittaci infection mainly depends on laboratory examination, including cell culture, serological tests, and polymerase chain reaction (PCR). Among them, cell culture is only performed in specialized laboratories (P3 facility), and C. psittaci is highly infectious when cultured; C. psittaci serological tests show cross-reaction with other chlamydial species, they do not allow source tracing, and the early diagnosis value is low [20,21]. PCR, especially real-time PCR (RT-PCR) is a faster, more sensitive, and more specific way for identification [23]. However, PCR for C. psittaci is unavailable in most hospitals in China, including many tertiary hospitals; it is only performed if clinicians have a high suspicion for C. psittaci infection. Most of the literature we reviewed were diagnosed by PCR or serological tests.
In recent years, NGS has shown some advantages in pathogen detection. NGS can directly obtain pathogen information from diseased tissues and body fluids [24,25], shorten the detection period significantly, and the high throughput sequencing technology can detect more gene sequences of pathogens simultaneously. NGS is theoretically based on specific nucleic acid sequences, and can realize the unique identification of almost all pathogens, which has obvious advantages for the detection of rare, new and complex pathogens. There are also some limitations of NGS, such as the lack of recognized interpretation standards, unclear relationship between sequencing results and treatment, possible contamination, and high costs, and the results need to be verified by combining with PCR or RT-PCR [26]; However, it plays a role in the diagnosis and treatment of patients with unknown pneumonia. The two cases in this paper were negative for the pathogen detection test which our hospital can perform; their final diagnosis was to detect C. psittaci in sputum, blood, and BALF by NGS.
Clinicians need to pay attention to the identification of other infections during treatment. Differential diagnosis is broad and includes infection with Coxiella burnetii (Q fever), Legionella, Chlamydophila pneumoniae, Mycoplasma pneumoniae, and respiratory viruses such as influenza. In the context of the novel coronavirus disease 2019 (COVID-19) epidemic since 2020, more attention should be paid to the differential diagnosis between C. psittaci pneumonia and COVID-19.
Tetracycline is the first choice in the treatment of C. psittaci infection [20]. In Table 2, tetracycline was used on 23 patients and 21 cases were cured, indicating that the efficacy of tetracycline was positive. Macrolides and quinolones could also be considered. The treatment of C. psittaci infection must last at least 10-14 days. The two patients in this paper adjusted the antibiotics to tetracycline (minocycline) after identifying C. psittaci infection. Their condition improved and they eventually recovered after the adjustment of antibiotics, thus confirming the efficacy of tetracycline in treating C. psittaci infection, as reviewed in Table 2.   [13] 64 (44-83)/3F and 6M 7 (77.8%) patients had a history of exposure to, or close contact with birds or poultry.
Fever and chills (9/9), cough and hypodynamia (9/9), headache (7/9), myalgia (7/9) Lesion began in superior lobe of lung (8/9), consolidation with air bronchograms (9/9), pleural effusions ( Moreover, both of the patients in this paper had severe pneumonia, respiratory failure, and ARDS, needed respiratory support in treatment to maintain oxygenation saturation and intensive care; and case 1 used ECMO. ECMO is a modified cardiopulmonary bypass circuit that serves as an artificial membrane lung and blood pump to provide gas exchange and systemic perfusion for patient, is a rapidly evolving alternative treatment modality for lung injury in severe acute respiratory distress syndrome and can improve the prognosis [27]. While using ECMO, ECMO operation, oxygenation saturation monitoring, anticoagulant management, pipeline safety, and complication prevention are also important.

Conclusions
Owing to the unspecific clinical features and imaging findings, the diagnosis of C. psittaci pneumonia is difficult and it can develop into severe pneumonia.
This indicates that relevant epidemiological history and timely pathogen detection are important for the diagnosis. NGS can shorten the time needed for diagnosis and enable earlier initiation of targeted antibiotic therapy, it could be used in early diagnosis of Chlamydia psittaci infection. Furthermore, ECMO also showed efficacy in the treatment of severe pneumonia.