Case report: fatal pneumonia caused by new sequence type Legionella pneumophilia serogroup 1

Abstract Introduction: Legionnaires’ disease is caused by Legionella bacteria, and commonly manifests as pneumonia and has a high fatality rate. Patient concerns: This case study reports on the fatal incident of a patient, initially diagnosed with pneumonia, and subsequently diagnosed with Legionnaires’ disease caused by a new sequence type (ST) of Legionella. Diagnosis: It is speculated that the patient acquired Legionnaires’ disease from a contaminated water source. Legionnaires’ disease was diagnosed using the Legionella urinary antigen assay and bacterial cultures of respiratory secretions; Legionella pneumophilia Type 1 was also identified through serological testing. Sequence-based typing of the cultured bacterium revealed it to be a previously unidentified species, and it was named ST2345 new-type. Interventions: In addition to the treatment of Legionnaires’ disease, blood samples taken on the second day of admission showed a co-infection of Candida tropicalis, which was treated with anti-fungal treatment. The patient improved after a week, however, on the seventh day of administration lower respiratory secretions showed the growth of Klebsiella pneumonia, indicative of ventilator-associated pneumonia. Outcomes: Despite active treatment, the patient passed away due to multiple organ failure. As this was a fatal case, further research is needed to determine whether the critical condition of this case was related to the virulence of the novel Legionella strain. Conclusion: A key finding of this study is that treatment for suspected Legionnaires’ disease must be administered rapidly, as infection with Legionella may give rise to secondary pathogenic infections.


Introduction
Legionnaires' disease is a disease caused by Legionella bacteria that primarily presents as pneumonia and can affect multiple organs. It commonly manifests as severe pneumonia and has a high case fatality rate. Due to its complex and diverse clinical presentation, imaging and routine laboratory tests lack the required specificity, and hence Legionnaires' disease is prone to misdiagnosis and improper treatment. [1] Here, we report a case of a new sequence type (ST) of fatal Legionnaires' disease diagnosed using the Legionella urinary antigen assay and bacterial culture of respiratory secretions. As this was a fatal case, this paper aims to investigate the cause of death of this patient with Legionnaires' disease, summarize the experience of treatment failure, and report on this new-ST Legionella.

Chief complaints
The patient was a 59-year-old male who was admitted to our hospital on July 19, 2012 due to "fever with progressive dyspnea for 6 days."

History of present illness
The patient developed a high fever 6 days prior and his body temperature reached a maximum of 39.6°C. He also presented with a cough, yellow sputum, and bloody sputum, accompanied by progressive dyspnea. His body temperature dropped after intravenous infusion of cefoxitin and dexamethasone but rose again, and he was admitted to our hospital for treatment.

History of past illness
Two weeks before the onset of the disease, the patient received root canal treatment (including drilling and rinsing) at a private dental clinic due to toothache. He had a history of cirrhosis, had been smoking for 30 years, and his smoking index was 30 packyears.

Imaging examinations
A pulmonary computed tomography (CT) scan showed diffuse ground-glass opacity of the right lung with lower lobe consolidation; right lower lobe cavities with fluid level; right pleural effusion (Fig. 1).

Microbiological identification of the causative agent
After admission, the lower respiratory tract secretion aspirated through endotracheal intubation was orange-yellow in color (Fig. 2). The secretion was positive for Legionella cultured in buffered charcoal yeast extract (BCYE) agar and Legionella-selective Glycine Vancomycin Polymyxin Cycloheximide (GVPC) agar (Fig. 3). Serological testing identified the strain as Legionella pneumophila Type I (Fig. 4). The blood samples on the second day of admission showed the growth of Candida tropicalis, and serum Legionella antibody IgM was positive on the ninth day of admission. L pneumophila Type I was cultured and isolated from the respiratory secretions of this patient, which were sent for further sequence-based typing (SBT). More specifically, PCR amplification was performed on this Legionella strain, and seven L pneumophila housekeeping gene loci were detected: flaA, pilE, asd, mip, mompS, proA, and neuA. This sequencing result was compared to the SBT database of the European Working Group for Legionella Infections (http://www.ewgli.org), and the specific housekeeping allelic profile was: 2, 10, 57, 28, 2, 1, and 6. The results indicate that this Legionella strain is a new sequence type (ST) that has not been reported internationally. The sequencing result of this strain was uploaded to the database, and the strain was named ST2345 new-type.
2.7.1. Diagnosis. After admission, the patient was diagnosed with severe community-acquired pneumonia.

Treatment.
As oxygenation could not be maintained by oxygen mask and non-invasive ventilation, mechanical ventilation was performed via endotracheal intubation. Imipenem/ cilastatin was combined with moxifloxacin intravenous infusion for anti-infective treatment. The patient developed septic shock after admission and was given symptomatic vasopressors and organ support therapy. The patient was positive for Legionella urinary antigen, which suggested the patient had contracted Legionnaires' disease, and doxycycline nasal combination therapy was administered. The patient's blood culture suggested the growth of Candida tropicalis (C tropicalis), and voriconazole antifungal treatment was added. After 1 week of treatment the patient's condition had improved slightly, the septic shock was corrected, and the vasopressor was gradually reduced and stopped. However, the patient's body temperature did not return to normal, fluctuating between 37°C and 38.5°C. Furthermore, his oxygenation was poor, and the concentration of inhaled oxygen had to be above 70% to ensure that his PO2 remained at approximately 60 mmHg; the lower respiratory tract secretions gradually turned from orange to bloody. The patient developed high fever again on the seventh day of admission and his body temperature was 40°C. Culture of the lower respiratory secretions showed the growth of Klebsiella pneumoniae, elevated procalcitonin 54.77 ng/mL, and endotoxin 201.4 pg/mL. Repeat bedside chest X-ray revealed the absorption of the primary lesions in the right lung, and exudative lesions in the left lung (Fig. 5). Secondary ventilator-associated pneumonia was considered. Despite active treatment, the patient passed away due to multiple organ failure.

Discussion
Legionnaires' disease often develops into severe pneumonia. Nearly 50% of hospitalized patients with Legionella infections need to be admitted to the ICU, and its case fatality rate is between 5% to 30%. [2] The patient in this case presented with multiple organ failure upon admission, and subsequently developed septic shock. Although anti-Legionella drugs (moxifloxacin, doxycycline) were administered, the patient continued to have a high fever, which still could not be brought down to normal levels using an ice blanket. Causes of the high fever may  [3] whereas oral doxycycline has a weaker efficacy, which may be less effective than combined intravenous medication. 2. Secondary double infections may have caused long-term fever. On the second day of admission (40 hours after admission), the patient's blood culture showed a positive result that indicated the growth of C tropicalis. In the later stages of treatment (seventh day of hospital stay), the culture of lower respiratory tract secretions indicated the growth of K pneumoniae, presence of C-reactive protein, an elevated procalcitonin index compared to before, and appearance of ventilator-

of infections by various pathogens in patients with
Legionnaires' disease, including K pneumoniae, Staphylococcus aureus, and Enterobacter. [4] In the same year, Cohen et al. found among 10 cases of fatal nosocomial Legionnaires' disease that 4 cases had positive sputum cultures; the positive bacteria were S aureus, K pneumoniae, and Citrobacter. [5] In 2002, Tan et al reported 6 cases of Legionnaires' disease with other bacteremic coinfections and inferred that patients with Legionnaires' disease are prone to bacteremia. [6] Our research group found in animal experiments of Legionnaires' disease that other non-Legionella bacteria could be cultured from the lungs and liver of mice during the acute phase of the disease, which mainly included Escherichia coli, S aureus, and Citrobacter, whereas the control group showed no bacterial growth. Hence, our findings further validate this view. [7] Currently, the mechanisms underlying how Legionnaires' disease gives rise to other secondary pathogenic infections is unknown. There is a hypothesis that the translocation of intestinal bacteria may occur during the acute phase of Legionnaires' disease. [5] Therefore, the issue of double infections in patients with Legionella infection should not be ignored, and close attention should be paid to double infections, early discovery, and early active treatment. With respect to the high fever caused by double infections, when the patient's condition deteriorates, we should apply hemofiltration in the early stages in addition to antibiotics. This is because there is an outbreak of inflammatory mediators during the acute phase of Legionella infection, which will cause elevated levels of inflammatory mediators, thus resulting in patients with persistently high fever and poor condition. [8][9][10][11] Hemofiltration can remove inflammatory mediators and might improve prognosis. The patient in this case had a root canal treatment two weeks before disease onset and had a history of dental restoration. The source of infection may have been water contaminated by Legionella in the pipelines of the dental clinic. We isolated and cultured the pipe water from the pipeline network in the dental clinic, but the results were negative. As there was a delay between the time of our isolation and culture and the patient's disease onset, we believe that the dental clinic may have disinfected the water source, which led to our negative results. This case suggests that we should trace the history of suspected environmental exposure in cases of suspected Legionnaires' disease, such as airconditioned environments, hot spring bathing, pipeline repairs, exposure to contaminated drinking water, rainwater, etc. [12,13] This case also suggests that dental clinics and similar venues should regularly disinfect their water source to prevent Legionella infections in patients.
Due to the rapid development of Legionnaires' disease, inappropriate initial treatments can significantly increase the patient's fatality rate. The cause of death in this patient was related to his delayed diagnosis and delayed treatment. As the treatment for Legionella was not administered before the patient was hospitalized, this meant he had missed the window for early  treatment and his condition progressed rapidly, manifesting as severe pneumonia with severe respiratory failure and multiple organ involvement. Thus, the subsequent treatment for Legionella, which was administered after the patient was hospitalized, was not effective. Therefore, we urge physicians to pay closer attention to the clinical presentation of such patients and to the features of suspected Legionella cases. If cases of adult community-acquired pneumonia show clinical symptoms, such as fever with bradycar-dia, acute onset headache, non-drug-induced disturbance of consciousness/lethargy, non-drug-induced diarrhea, shock, acute liver and kidney function damage, hyponatremia, hypophosphatemia, and no response to b-lactam antibiotics, then the possibility of Legionnaires' disease should be considered. [14][15][16][17][18][19][20][21] This patient was diagnosed with severe pneumonia with rapid disease progression, manifesting as severe respiratory failure with multiple organ involvement and hyponatremia. Thus, Legionella infection  should be suspected. Furthermore, we found that the patient's sputum was a distinct orange-yellow color (Fig. 2). We have also previously observed that the sputum of patients with Legionnaires' disease similarly presented this distinct color. Therefore, such orange-yellow sputum may be instructive in the clinical diagnosis of Legionella infection. This patient was suspected of Legionella infection after admission. Thus, Legionella urinary antigen assay and respiratory secretion culture were performed immediately, which confirmed the pathogenic diagnosis as a L pneumophila Type 1 infection. The L pneumophila Type I isolated from this respiratory secretion culture was further subjected to sequencebased typing (SBT). The sequencing results were recorded on the SBT database of the European Working Group for Legionella Infections (http://www.ewgli.org), and the strain was named ST2345 new-type. The SBT method is a gene sequence typing technique for Legionella that is currently widely applied in the molecular typing of Legionella. Most of the Legionella cases reported internationally are of the ST1 type. The Legionella strain cultured from this patient was named ST2345 new-type, which implies that this is the first discovery of this ST internationally. It also indicates that the Legionella contracted by this patient has unique features. As this is a fatal case, further research is needed to verify whether the critical condition of this case was related to the high virulence of this strain.