The application of metagenomic next-generation sequencing in diagnosing Chlamydia psittaci pneumonia: a report of five cases

Background Chlamydia psittaci pneumonia is a zoonotic infectious disease caused by Chlamydia psittaci. Diagnostic tools, including culture, serologic test and PCR-based methods, are available but prone to false negative results. Case presentation This report included five cases of Chlamydia psittaci pneumonia. Symptoms and signs common to all 5 cases included fever, coughing, generalized muscle ache, and most notably, inflammatory infiltration of the lungs upon chest CT and X-ray. Metagenomic next-generation sequencing (mNGS) revealed the presence of Chlamydia psittaci in biopsy lung tissue in 3 cases and bronchoalveolar lavage fluid in the remaining 2 cases. Three patients responded to doxycycline plus moxifloxacin; two patients responded to moxifloxacin alone. Conclusions mNGS could be used to diagnose Chlamydia psittaci pneumonia.


Background
Chlamydia psittaci is an obligatory intra-cellular Gramnegative bacterium that typically infects birds, but could occasionally cause psittacosis in humans when contaminated aerosols from infected birds are inhaled. Chlamydia psittaci pneumonia in humans is underestimated due to low awareness of the disease and atypical clinical presentation in majority of the cases [1][2][3]. The low sensitivity and complex procedure of Chlamydia psittaci culture causes it hardly routinely performed in most diagnostic laboratories. Other laboratory testing included serological assay and polymerase chain reaction (PCR) based methods, but both have questionable sensitivity and specificity [2]. Untargeted metagenomic nextgeneration sequencing (mNGS) has been increasingly used in the diagnosis of infectious diseases, particularly when conventional diagnostic approaches have limitations [4]. Here we report 5 cases of Chlamydia psittaci pneumonia, in which the diagnosis was established with mNGS. All together, mNGS was conducted in a total of 120 pneumonia cases in the index period. Demographical and basic clinical features of the 5 cases are summarized in Table 1. generalized muscle ache and malaise for 10 days. On the 5th day after the onset of symptoms, she visited a local hospital. A chest computed tomography (CT) scan showed inflammatory infiltration in the lower lobe of the left lung. She was treated with moxifloxacin (0.4 g, I.V., qd) plus meropenem (1 g, I.V., q12h) for 3 days, but fever continued (highest body temperature 39.4°C). A repeat CT scan suggested progression of pulmonary infection. Moxifloxacin was replaced with linezolid (600 mg, I.V., q12h), with continuing meropenem treatment. On the second day after linezolid/meropenem, fever subsided. However, other symptoms and signs continued.

Case #2
A 45-year-old man was transferred to us with fever, productive coughing, generalized muscle ache and malaise for 7 days. On the 4th day of the onset, he visited a local hospital. A chest CT scan showed consolidation in the right upper lobe (Fig. 2a). Test results included: WBC count 8.22*10 9 /L, neutrophil count 7.65*10 9 /L, 93.1% neutrophils, 4.0% lymphocytes (count 0.33*10 9 /L), CRP 226 mg/L, ESR 42 mm/h, and PCT 1.712 ng/ml. A de novo diagnosis of diabetes was also established based on repeated testing of fasting blood glucose and hemoglobin A1c. Treatment with ceftazidime (2 g, I.V., q8h) and levofloxacin (500 mg, I.V., qd) was initiated for 3 days, but symptoms did not dissipate. Upon transferring, the body temperatures was 39.5°C, with crackles in both lungs. The patient received ertapenan (1 g, I.V., qd) and oseltamivir (75 mg, P.O., q12h), and fever subsided in 3 days. One days later, however, he developed high fever again (40°C), with signs of respiratory failure (PaO 2 40 mmHg). On the 4th day of arrival to our hospital, a chest CT scan showed bilateral diffuse infiltration (Fig. 2b).

Case #4
A 66-year-old woman was transferred to us with fever, rigor, dry cough and dizziness for 6 days. At the beginning of the illness, she received levofloxacin and cefotaxime in a local hospital but symptoms persisted. A chest CT scan upon transferring showed consolidation in the right upper lobe (Fig. 4a, b). Laboratory test showed 5.47*10 9 /L WBC count, 69.8% neutrophils (3.82*10 9 /L), 19.4% lymphocytes (1.06*10 9 /L), 124 mg/L CPR, 17 mm/ h ESR, and 0.13 ng/ml PCT. Treatment with moxifloxacin (0.4 g, I.V., qd) was initiated, and body temperature returned to normal within 2 days. A percutaneous lung biopsy was conducted; the sample testing with mNGS (IngeniGen-Hangzhou) detected Chlamydia psittaci (Table 1). Later, we learned that she had close contact with a large poultry farm on a daily basis. Moxifloxacin treatment lasted for 10 days. CT scan 8 days after moxifloxacin treatment initiation (Fig. 4c) and 16 days after discontinuation showed gradual infiltrate absorption (Fig. 4d).

Case #5
A 61-year-old woman presented with fever, rigor, weakness, productive coughing and dizziness for 3 days. A chest CT scan showed patchy infiltration and consolidation of both lungs (Fig. 5a, b, c). Laboratory test showed 6.88*10 9 /L WBC count, 68.4% neutrophils (4.70*10 9 /L), 22.7% lymphocytes (1.56*10 9 /L), 96.7 mg/L CRP, 37 mm/ h ESR, and normal PCT. She disclosed close contact with a pet parrot. She was treated with moxifloxacin and her body temperature returned to normal on the next day. Analysis of lung biopsy tissue with mNGS (Ingeni-Gen-Hangzhou) reported Chlamydia psittaci (Table 1). A CT scan 8 days later showed partial absorption of the pulmonary infiltrate (Fig. 5d, e, f).

Discussion and conclusions
Chlamydia psittaci can be classified into 10 genotypes, with varying preference for host species [5]. Genotype A and E could infect humans. After entry via contaminated aerosols, Chlamydia psittaci spreads to the reticuloendothelial system. The lungs are the most common sites of Chlamydia psittaci infection. Chlamydia psittaci pneumonia is estimated to account for approximately 1% of community-acquired pneumonia cases [3,6]. Symptoms mimics that of influenza, and typically include fever, fatigue, headache, myalgia, and coughing [7,8]. In 4 out of the 5 cases in this report, patients had contacts with birds (parrot and pigeon) or poultry, suggesting the need to investigate such exposure upon suspected cases. Two out of the 5 patients had diabetes; and the other 3 were otherwise healthy, suggesting that Chlamydia psittaci could infect human subjects regardless of underlying diseases.
Chlamydia psittaci infection tends to be overlooked due to relatively low awareness by physicians. Laboratory testing for Chlamydia psittaci includes culture, serological assay, and PCR. Culture is time consuming, and most formidably, requires P3 facility [6]. Serological tests are only appropriate for retrospective diagnosis because sera from the both acute and convalescent phase of the illness are required [9]. PCR-based testing is the most specific and fastest method but only sensitive in the acute phases of the infection [2]. The current case series indicated that mNGS could be used to diagnose Chlamydia psittaci infection. If using a set of universally accepted standards, mNGS could even provide semiquantitative information (based on sequence reads) about the load of Chlamydia psittaci, such information could be critically important in determining whether a specific microbe is the causative pathogen(s) in polymicrobial samples. The limitations of mNGS analysis, for infection with a Chlamydia psittaci or any other agent, include host background sequences. In future studies,  targeted sequencing and host depletion methods could be used to minimize the human host background, workflow quality control procedures could be optimized to reduce false positives. It is also likely that Chlamydia psittaci is merely present in the sample and not the culprit of infection. The fact that many other pathogens, including Candida albicans, have been identified in the 5 cases illustrate the complexity and a need to integrate the mNGS results into the overall clinical scenario. Lack of verification with serologic tests and/or culture is a significant limitation in the current study. These findings therefore must be interpreted with caution.
In the current series, mNGS was conducted only after initial empirical antibiotic treatment failed to control the infection in 4 out of the 5 cases. Considering the feasibility and cost of mNGS, we believed the timing of mNGS is appropriate, and recommend mNGS testing only if patients do not respond to treatments against other more common causes.
The specific reads for Chlamydia psittaci ranged from 2 to 225 in the 5 cases. Comparison across cases is not possible since the assay was conducted by several different companies. In case #1 and #5, the detected Chlamydia psittaci reads were 6 and 2, respectively. We speculate that the relatively low specific reads of Chlamydia psittaci in Case #1 may reflect the therapeutic effects of moxifloxacin, albeit not adequate. In Case #5, the low specific reads of Chlamydia psittaci may reflect the loss of biological activity of pathogens and degradation of nucleic acid during the process of sample collection and transportation or the treatments that the patient received prior to sample collection.
Recommended treatment for Chlamydia psittaci pneumonia included tetracycline, macrolide and quinolones [10]. Treatment must continue for at least 10-14 days to prevent relapse. In case #1, moxifloxacin was used as an initial treatment for 3 days, but no significant improvement was obtained. In our opinion, the lack of response to moxifloxacin in this case could be due to several reasons, including: 1) possible superinfection by other agents that could be readily controlled by doxycycline; 2) relative insensitivity of the Chlamydia psittaci isolate in this specific case to moxifloxacin. Indeed, recommended first-line treatment of Chlamydia psittaci pneumonia is doxycycline and not quinolones [11].
In conclusion, the current series suggested that mNGS could be used in diagnosing Chlamydia psittaci infection. This preliminary finding should be examined with diagnostic trial in the future.