New Detection and Treatment for Chlamydia Psittaci: A Case Report

Background: The presentation of psittacosis can vary from subclinical infection to fatal pneumonia with a high mortality rate. Chlamydia psittaci infection during pregnancy is rare and might result in placental involvement, premature delivery or miscarriage. Herein, we report a case of severe pneumonia in a pregnant woman caused by C. psittaci. Case presentation: A 27-year-old female with a pregnancy at 22 weeks was admitted with hyperpyrexia, dry cough and dyspnea. Laboratory tests showed increased white blood cell count and slightly-elevated procalcitonin. Chest computed tomography scan revealed consolidation in the lingual and inferior lobe of the left lung. She suffered rapidly progressing respiratory failure and required mechanical ventilation and extracorporeal membrane oxygenation (ECMO). Her sample of bronchoalveolar lavage uid (BALF) was collected, from which C. psittaci was detected using next-generation sequencing (NGS). The pathogen was further conrmed by polymerase chain reaction (PCR) with the primers specic for the microorganism. Moreover, paired serum tests showed elevated C. psittaci group titer in both IgG and IgM. She was treated with azithromycin and tigecycline. After 24-day hospitalization, the patient was clinically well and discharged home. Conclusion: We report a case of severe pneumonia in a pregnant woman caused by C. psittaci, which diagnosed by NGS, and was cured without adverse effects on infant with tigecycline. The introduction of NGS and other effective assays might increase the detection rate. C. psittaci infection in pregnant women is very rare, but critical. Early diagnosis and correct treatment may save the mother and fetus. New tetracycline agent, tigecycline, may also be an effective alternative and should be further evaluated in psittacosis. pneumonia with high morbidity and appreciable mortality[3]. The main imaging features are single lobe exudation or consolidation, and the lower lobes are more commonly observed[4]. Denitive diagnosis can be established by culture, serology test, or polymerase chain reaction (PCR) specically targeting at C. psittaci. Infections during pregnancy by C. psittaci are rare, but might result in placental involvement, premature delivery or miscarriage[5, 6]. Herein, we report a case of severe pneumonia and respiratory failure due to the microorganism in a pregnant woman, requiring mechanical ventilation and extracorporeal membrane oxygenation (ECMO). Next-generation sequencing (NGS) of bronchoalveolar lavage uid (BALF) was used and C. psittaci was identied, and later conrmed by PCR and sera test. To the best of our knowledge, this is the rst report of C. psittaci as the causative agent of severe community acquired pneumonia (CAP) in pregnant woman which was diagnosed by NGS in China.


Background
Chlamydia psittaci, an intracellular bacterium, can infect birds and other animals, such as livestock, and can cause zoonotic infection in human psittacosis [1]. The symptoms of pneumonia due to C. psittaci were usually mild, including fever, headache, and general discomfort [2]. However, the organism can also lead to fatal pneumonia with high morbidity and appreciable mortality [3]. The main imaging features are single lobe exudation or consolidation, and the lower lobes are more commonly observed [4]. De nitive diagnosis can be established by culture, serology test, or polymerase chain reaction (PCR) speci cally targeting at C. psittaci. Infections during pregnancy by C. psittaci are rare, but might result in placental involvement, premature delivery or miscarriage [5,6]. Herein, we report a case of severe pneumonia and respiratory failure due to the microorganism in a pregnant woman, requiring mechanical ventilation and extracorporeal membrane oxygenation (ECMO). Next-generation sequencing (NGS) of bronchoalveolar lavage uid (BALF) was used and C. psittaci was identi ed, and later con rmed by PCR and sera test. To the best of our knowledge, this is the rst report of C. psittaci as the causative agent of severe community acquired pneumonia (CAP) in pregnant woman which was diagnosed by NGS in China.

Case Presentation
A 27-year-old female was admitted to China-Japan Friendship hospital on 7th October 2018, with hyperpyrexia, dry cough and dyspnea. On admission, she was fully conscious. On examination, the patient was febrile (39.5℃), with a respiratory rate of 25 breaths/minute, a pulse rate of 110 beats/minute and a blood pressure of 101/55 mmHg. Her breathing sound of the left lower lung was low. In addition, history taking revealed that she was pregnant at 22 weeks and physically t. Laboratory tests showed a white blood cell count of 7.69 × 10 9 /L, comprising 90.7% neutrophils, 5.6% lymphocytes and 2.2% monocytes, a hemoglobin of 70 g/L and a platelet count of 138 × 10 9 /L. Besides, hyponatremia and hypokalemia were found, and no abnormalities in liver or renal function tests were observed. Blood gas analysis suggested type I respiratory failure. Chest computed tomography (CT) scan revealed consolidation in lingual and inferior lobe of her left lung ( Fig. 1). Hypoxic dyspnea progressed rapidly after admission and she was transferred to the intensive care unit (ICU) after tracheal intubation on the same day. With the support of invasive ventilation, her oxygenation index stilled remained below 70 mmHg, therefore, the venousvenous extracorporeal membrane oxygenation (VV-ECMO) was given as adjuvant supportive therapy. The severe circulatory failure occurred rapidly after VV-ECMO was established. Echocardiography showed that ejection fraction dropped from 71% on the previous day to 30%, and the apex expanded globally and contraction weakened. Stress cardiomyopathy was considered.
She was empirically treated with azithromycin, ceftriaxone and oseltamivir as a case of severe community-acquired pneumonia. Tracheoscopy revealed congestion and edema of bilateral bronchial mucosa, with white and thin secretions in each lumen. Furthermore, her urine was negative for Legionella pneumophila serogroup 1 and pneumococcal antigens, and her sputum and BALF were negative for respiratory viruses and atypical pathogens identi ed by PCR, too. Moreover, the culture of bacteria and fungi remained negative after 2-7 day incubation. Then, we got another BALF and NGS was used to identify the possible pathogen. BALF was lysed and subjected to nucleic acid extraction with nucleic acid mini kit (Qiagen) following the manufacturer's instruction. The concentration of DNA was determined by Qubit 4 (Thermo). Then library construction was completed by DNA library construction kit (Vision medicals), which including fragmentation, the synthesis of 1st and 2nd strand, end repairment and adaptor ligation. The ampli ed libraries of each sample were pooled together and sequenced on an Illumina Nextseq sequencer for clinical metagenomic analysis. A minimum of 10M single-end 75 bp reads was obtained per sample. Sequence analysis was performed through Vision Medicals' IDseq™ commercial bioinformatic pipeline. Brie y, low-quality and short (length < 35 bp) reads and reads that mapped to human genome and plasmids were removed. The remaining reads were taxonomically classi ed by aligning curated microbial database consisting of viruses, bacteria, fungi, and parasites. The taxonomic references were downloaded from National Center Biotechnology Information (NCBI). Upon identi cation of critical pathogen, the identi ed species-speci c sequences were further con rmed by Blastn for further accuracy validation. At last, 18 reads of C. paittaci were detected (1 read is equal to approximately 1 fragment of 75 ~ 150 bp). The antibiotics were changed to azithromycin and tigecycline. Four days later, her pulmonary imaging improved (Fig. 2), and cardiac function gradually restored after one week. After 11 days, the ventilator and ECMO were both evacuated. Paired serum drawn tow weeks apart showed rising C. psittaci group titer both in IgG and IgM (Table 1). PCR for C. psittaci with the primers speci c the microorganism by Cp2-F, Cp2-R, Cp-1, Cp-2 and Cp-3 [7] was also positive in her BALF. The region of gene ompA was targeted. And a 131-bp speci c sequence was ampli ed and sequenced with the primers (3'-TGTGATTCACAAACCAAGAGGCTATA − 5' and 3'-CGAGGCCTACTTGCCATTCA-5'). Re-examination of medical history revealed that the woman once played with the parrots for about 15 minutes ve days before the onset of psittacosis.  After 24-day hospitalization, the patient recovered well and discharged from the hospital. Throughout the whole course, the fetus was stable and there was no sign of threatened premature delivery.

Discussion And Conclusions
Psittacosis is a zoonotic disease, and animal-to-human transmission might occur through direct contact with golden bird, parrot, pigeon, turkey and other birds, or inhalation of aerosols from their excrement dust [8,9]. Recently, it has been reported that peacock, duck and chicken can also carry pathogens and result in transmission [10,11]. C. psittaci is rarely documented as the pathogen in hospitalized CAP. The reported incidence by the organism, ranging from 0-2.1%, might be underestimated, because the detection of psittacosis is rarely incorporated in the routine microbiological diagnostics in CAP, and serological method is also rarely available [12]. Reviewing the history of travel, occupation, contact, and cluster, revealed that most of these patients had family clusters or pet bird contact history. This is con rmed in our case, and the patient had a parrot contact history before the onset of the disease.
Diagnosis of psittacosis is often based on the clinical examination, epidemiological history, and laboratory tests, including direct (culture or PCR) or indirect (serology) methods. It is a challenge to identify C. psittaci as the pathogen due to the lack of effective diagnostic methods and its elusiveness. Given the complexity and slow-growth nature, the culture of C. psittaci (biosafety level 3) is almost unavailable, and the clinical criteria for a diagnosis of psittacosis is a positive PCR result or a four-fold rise of antibody titer in complement xation test (CFT) con rmed by micro-immuno uorescence [3]. However, to date, with the wide application of NGS [13], the detection rate of C. psittaci may be increased [6]. However, there is no uniform standard for the interpretation of NGS results, and to date, it can only be used as a clinical clue and needs further con rmation.
Psittacosis during pregnancy is very rare and can lead to numbers of maternal and fetal mortality [14]. Most mothers suffered multiple organ failure, including severe hypoxemia, hepatic dysfunction, renal dysfunction and disseminated intravascular coagulation(DIC). Moreover, more than half of the fetuses died and, if alive, they would be born prematurely [15]. In our case, although the mother had multiple organ failure (severe respiratory failure, acute kidney injury, cardiogenic shock, liver function injury and coagulation dysfunction), the baby had been relatively healthy, and there was no sign of threatened premature delivery throughout the course of the disease. Therefore, early recognition and proper management may improve the prognosis of mother and fetus.
Tetracycline, especially doxycycline, is the rst-line medicine, and may show signi cant improvement after 3 day administration [3]. Macrolides can be used as an alternative antibiotic agent, but are less effective in severe pregnant cases [16]. In the present case, we chose another tetracycline agent. The woman in our case had the regimen of the combination of azithromycin and tigecycline, which was remarkably effective. The patient recovered well and discharged home. No recurrence of symptoms and signs was reported in a two-month of outpatient follow-up.
In summary, psittacosis may not be underestimated due to limited diagnostical methods. C. psittaci infection in pregnant women is very rare, but critical. Early diagnosis and correct treatment may save the mother and fetus. The introduction of NGS and other effective assays might increase the detection rate. The new tetracycline agent, tigecycline, may also be an effective alternative and should be further evaluated in psittacosis. A: X-Ray on the 9th October; B: X-Ray on 13th October, which revealed signi cant improvement