Severe Sepsis With Septic Shock as a Consequence of a Severe Hospital-Acquired Pneumonia Resulting From Legionella Pneumophila in Children: A Case Series and Literature Review

Legionella pneumophila is responsible for hospital or community-acquired pneumonia in adults. Immunocompromised patients with Legionella pneumophila infection are associated with rapidly severe clinical course and high mortality rates. Legionella pneumophila infection is rare in children, especially combined with extrapulmonary manifestations. In this report, we describe 2 children of severe hospital-acquired pneumonia and septic shock resulting from Legionella pneumophila. Standardized metagenomics next-generation sequencing allowed early diagnosis. Appropriate antibiotic therapy and timely extracorporeal life support were effective in achieving complete recovery. This is the rst report of children with septic shock related to Legionella pneumophila infections diagnosed by metagenomics next-generation sequencing and recovered successfully. There were 2 cases about septic shock resulting from Legionella pneumophila. One was a six-month girl with congenital biliary atresia who underwent liver transplantation. She was suspected for rejecting the liver and admitted to hospital. The other one was a ve-year-old boy with Burkitt lymphoma who was in the end of early chemotherapy. They both presented with fever, cough or shortness of breath during hospitalization. And they were transferred to PICU because of worsening dyspnea and decreased blood pressure. Patients were diagnosed by severe sepsis with septic shock likely resulting from hospital-acquired pneumonia. Metagenomics next-generation sequencing indicated L. pneumophila in blood and sputum. Aggressive intravenous uids resuscitation and vasopressors were initiated on arrival to PICU, and they were placed on mechanical ventilation and continuous renal replacement therapy. Intravenous antibiotic therapy followed by azithromycin. Finally, the patients recovered without any long-term sequelae. at


Abstract Background
Legionella pneumophila is responsible for hospital or community-acquired pneumonia in adults. Immunocompromised patients with Legionella pneumophila infection are associated with rapidly severe clinical course and high mortality rates. Legionella pneumophila infection is rare in children, especially combined with extrapulmonary manifestations. In this report, we describe 2 children of severe hospital-acquired pneumonia and septic shock resulting from Legionella pneumophila. Standardized metagenomics next-generation sequencing allowed early diagnosis. Appropriate antibiotic therapy and timely extracorporeal life support were effective in achieving complete recovery. This is the rst report of children with septic shock related to Legionella pneumophila infections diagnosed by metagenomics next-generation sequencing and recovered successfully.

Case presentation
There were 2 cases about septic shock resulting from Legionella pneumophila. One was a six-month girl with congenital biliary atresia who underwent liver transplantation. She was suspected for rejecting the liver and admitted to hospital. The other one was a ve-year-old boy with Burkitt lymphoma who was in the end of early chemotherapy. They both presented with fever, cough or shortness of breath during hospitalization. And they were transferred to PICU because of worsening dyspnea and decreased blood pressure. Patients were diagnosed by severe sepsis with septic shock likely resulting from hospital-acquired pneumonia. Metagenomics next-generation sequencing indicated L. pneumophila in blood and sputum.
Aggressive intravenous uids resuscitation and vasopressors were initiated on arrival to PICU, and they were placed on mechanical ventilation and continuous renal replacement therapy. Intravenous antibiotic therapy followed by azithromycin. Finally, the patients recovered without any long-term sequelae.

Conclusions
Though sepsis or sepsis shock caused by Legionella pneumophila is rare in children, it can occur at high-risk population. Metagenomics nextgeneration sequencing is useful for conforming hard-to-culture pathogens and severely ill patients. The report remind pediatric physicians that we should be aware that Legionella pneumophila can cause severe sepsis or sepsis shock, especially in immunocompromised children. It is signi cant to select appropriate samples and pathogen detection methods in the early stage of disease.

Background
Sepsis is the leading causes of childhood mortality worldwide and identi ed a highest mortality of 22.8% in developing countries [1] . Although studies demonstrate the impact of staphylococcal and streptococcal infections as leading pathogens for sepsis, only approximatively 50% children admitted to pediatric intensive care unit (PICU) with a clinical diagnosis of sepsis have a microbiologically con rmed infection [2] . Because the majority of pediatric sepsis befall very early, it is very important to identify the pathogen as soon as possible [3] . Legionella pneumophila (L. pneumophila) is an important pathogen of severe pneumonia in immunocompromised patients. It also can occur extrapulmonary manifestations. L. pneumophila in patients is di cult to identi ed because of nonspeci c clinical symptoms. Although a variety of methods have been developed to detect L. pneumophila, it is still di cult to isolate it from blood as the gold standard [4] . As far as we know, reports about L. pneumophila with sepsis or septic shock in children are extremely rare. We describe 2 children who had severe sepsis with septic shock as a consequence of a severe hospitalacquired pneumonia (HAP) resulting from L. pneumophila. We veri ed L. pneumophila infection through metagenomics next-generation sequencing (mNGS) of blood and sputum. This is the rst presentation of children with septic shock related to L. pneumophila infection diagnosed by mNGS in the English-language. It is important to remind pediatricians that it does occur in children, particularly in immunocompromised patients. And it is useful that we can veri ed pathogeny by mNGS for critically ill patients in the early course of illness.

Case Presentation Case1
A six-month girl with congenital biliary atresia underwent liver transplantation. Engraftment on day 28 posttransplant was complicated by an increased alanine aminotransferase (ALT) level. She had no fever, cough and other symptoms while at home. She was suspected for rejecting the liver and admitted to hospital. On admission, her immunosuppression included tacrolimus (1.2 mg every 12 h)and methylprednisolone (12 mg every day).Her abnormal laboratory results included an ALT level of 283 U/L (normal: 7-40 U/L) and an aspartate aminotransferase (AST) level of 112.7 U/L (normal: 13-35 U/L). Empiric intravenous therapy with high-dose methylprednisolone( 80 mg intravenously every day) was initiated, along with oral tacrolimus continuously (1.2 mg every 12 h).Her ALT and AST were decreased gradually during hospitalization.
On the 25th hospital day, she presented with fever, cough with whitish sputum, and shortness of breath. Coarse crackles were heard over both lung elds. Laboratory data revealed a white blood cell(WBC) count of 25.45 × 10 9 /L with neutrophil predominance (79%)(normal: 4-10 × 10 9 /L), Creactive protein(CRP) 51.26 mg/dl (normal: 0-3.5 U/L)and procalcitonin(PCT) 0.22 ng/mL(normal: 0-0.1 U/L). Computed tomography scan of the chest showed patchy in ltrations were seen in the upper lobe of right lung and lower lobe of left lung (Fig. 1, A). After the culture of blood and sputum, empiric antibiotics with meropenem (80 mg every 8 h), micafunginand (30 mg every day) and vancomycin (160 mg every 12 h) were administered for 6 days. Pathogenic serological and antibody-based assays, blood culture and respiratory culture were negative.
On the 31th admission, the patient needed mechanic ventilation because of worsening dyspnea. She was transferred to PICU. Her vital signs on arrival to PICU were as follows: blood pressure of 80/33 mmHg (mean arterial pressure of 48.7 mmHg), a respiratory rate of 50 breaths/minute, a heart rate of 240 beats/minute, a temperature of 38.3℃, and an oxygen saturation by pulse oximetry of 85% while breathing ambient air. Physical examination revealed with rales on auscultation, low skin temperature of limbs and capillary re ll time 3 seconds. After a critical care medicine evaluation was requested, the patient was diagnosed by severe sepsis with septic shock likely resulting from HAP. Aggressive intravenous uids resuscitation and vasopressors (norepinephrine, vasopressin) were initiated on arrival to PICU, and the patient was subsequently intubated and placed on mechanical ventilation because of worsening tachypnea and increased FiO 2 requirements. Meanwhile, she was treated with continuous renal replacement therapy (CRRT) to eliminate in ammatory factors for 2 days. Empiric antibiotics with meropenem (370 mg every 8 h), caspofungin (30 mg every day) and vancomycin (140 mg every 8 h) were continued to be administered, along with oral tacrolimus and methylprednisolone.
We sent samples of blood and sputum to BGI Group (Beijing, China) for mNGS. Though Serum antibody, blood culture and respiratory culture were negative, mNGS indicated L. pneumophila in blood and sputum. For blood, there was 1044 raw reads and 516 raw reads for sputum of L.
pneumophila. Intravenous antibiotic therapy followed by azithromycin (80 mg every day) for 10 days. On day three of PICU admission, vasopressors were titrated off. Computed tomography scan of the chest disclosed decreased in ltrations over the both lung (Fig. 1, B). The patient was extubated on the 5th day of PICU. Finally, after a 2-week stay in the PICU, her abnormal laboratory tests have returned to normal, and the patient recovered without any long-term sequelae.

Case2
A ve-year-old boy with Burkitt lymphoma was in the end of early chemotherapy. He was presented with multiple ulcers in oral mucosa for 10 days.
Abnormal laboratory tests showed a WBC count of 0.16 × 10 9 /L with percentage of neutrophil 0.06%, hemoglobin 108 g/L (normal: 115-150 U/L) and a platelet count of 56 × 10 9 /L(normal: 125-350 U/L). He was admitted to the hospital because of the diagnosis of myelosuppression ( °) and ulcerative stomatitis. On admission, the patient was in the state of tracheotomy. The WBC and percentage of neutrophils decreased signi cantly, empiric antibiotics with meropenem (500 mg every 8 h), sulfamethoxazole (400 mg Q6H) and trimethoprim (80 mg Q6H) and vancomycin (330 mg every 8 h) were administered for 6 days.
On the 3th hospital day, the patient occurred fever, cough with yellow sputum at the tracheotomy. Widespread patchy in ltrations were seen in various segments of both lungs on computed tomography of the thorax (Fig. 2, C). On the 5th hospital day, the respiratory symptoms of the patient gradually worsening. He was transferred to PICU. His physical examinations at PICU admission included: blood pressure of 87/53 mmHg (mean arterial pressure of 61.0 mmHg), a respiratory rate of 40 breaths/minute, a heart rate of 160 beats/minute, a temperature of 36.3℃, multiple ulcers in oral mucosa, rales on auscultation, low skin temperature of limbs and capillary re ll time 5 seconds. Laboratory ndings showed a WBC count of 0.14 × 10 9 /L with 0.21% neutrophil, hemoglobin 99 g/L and a platelet count of 39 × 10 9 /L, CRP 281.36 mg/dl, PCT49.34 ng/mL and interleukin-6 (IL-6) 19259.97 pg/mL (normal: 1.7-16.6 pg/mL). Arterial blood gas (ABG)showed a pH of 7.408 (normal:7.35-7.45), a pCO 2 level of 56.7 mmHg (normal: 35-45 mmHg), and a paO 2 level of 33.10 mmHg (normal: 80-100 mmHg), Lactate 2.52 mmol/L (normal: 0-2 mmol/L). The patient was diagnosed by severe sepsis with septic shock likely resulting from HAP. Aggressive intravenous uids resuscitation and vasopressors (norepinephrine, vasopressin) were initiated on arrival to PICU. And he was complicated by respiratory distress so that he required mechanic ventilation. He was treated with CRRT to eliminate IL6 for 6 days. On the 6th hospital day, the patient suddenly developed ventricular brillation with cyanosis and weak heart sound. An oxygen saturation by pulse oximetry of 88% while mechanic ventilation fall to 60%. Blood pressure fall to 60/40 mmHg.He was treated with cardiopulmonary resuscitation and intermittent intravenous injections of 1:10000 adrenaline. He recovered after 15 minutes. Serum antibody, blood culture and respiratory culture were negative.
We also sent samples of blood and sputum to BGI Group (Beijing, China) for mNGS. mNGS indicated Legionella pneumophila in blood and sputum.
For blood, there was 2280 raw reads and 3372326 raw reads for sputum of L. pneumophila. He received Azithromycin (245 mg every day) therapy for 10 days. On the 12th hospital day, vasopressors were titrated off. On the 15th day of admission, chest radiography disclosed decreased in ltrations over the both lung (Fig. 2, D). And the patient was extubated. On the 28th hospital day, he was transferred to general ward because of stable vital signs and normal laboratory examination. He had no discomfort follow up 2 months.

Discussion And Conclusions
In this report, we describe unusual cases of severe HAP and septic shock resulting from L. pneumophila. We identi ed L. pneumophila through mNGS of blood and sputum early in the course of illness. As we know, this is the rst presentation of children with HAP and septic shock related to L. pneumophila infection in the English-language. Although authors have pointed out the importance of mNGS, especially in critically ill patients [5] , it is also the rst report that patients with HAP and septic shock involved L. pneumophila were diagnosed by mNGS in children.
HAP is a frequent complication of hospitalization. It has an assignable mortality rate of more than 8% [6] . The spectrum of causative organisms in HAP is more likely to involve gram-negative bacilli such as Pseudomonas aeruginosa or Staphylococcus aureus and Streptococcus pneumoniae in adults, not Legionella [7] . L. pneumophila can be isolated in HAP of immunocompromised adults, but Legionella pneumophila is rarely isolated in children [8] . Furthermore, culturing L. pneumophila requires a speci c medium, which makes it di cult to obtain a positive result [9,4] . Therefore, it is di cult to identify bacteria when patients have sepsis and septic shock because of HAP [2] . Sepsis and septic shock are the main causes of childhood mortality all over the world. If doctors can conform pathogeny of sepsis, the mortality will decrease. As a result, pediatric doctors do not tend to systematically pursue diagnostic testing for L. pneumophila. Pediatric data from studies suggest that severe diseases of L. pneumophila occur in children with malignant neoplasms, organ transplantation, underlying pulmonary disease and immunosuppression with corticosteroids [10][11][12] .
L. pneumophila belongs to Legionella which found in the environment, such as freshwater environments, moist soil and so on [13] . Human infection most commonly occurs as a result of contaminated manmade water sources, whirlpool spa humidi ers and evaporative condensers [13] . Generally, human infection is incidental and usually asymptomatic or mild and unrecognized. But individuals at higher risk for developing serious Legionnaires' disease are olders, severe combined immune de ciency, cancer or organ transplantation [14] . Since the identi cation of L. pneumophila in 1977,research on adults indicates that the incidence rate is up to 1.4 case noti cations per 100,000 individuals [15] . Over decades, L. pneumophila infection has been reported increasingly in immunocompromised patients, but our understanding of its epidemiology depends on case reports [16] . A patient with L. pneumophila infection shows pneumonic as well as a variety of extrapulmonary manifestations, such as headache, encephalopathy, relative bradycardia, abdominal pain, hepatic involvement, electrolyte imbalance and other multisystemic ndings [17] . Severe extrapulmonary ndings of L. pneumophila infection are rare, especially in children [18] .
In this report, our patients presented with HAP and septic shock resulting from L. pneumophila. Review the literature, extrapulmonary ndings involved sepsis and septic shock are few in adults [19][20][21][22] . The clinical data of patients with sepsis and septic shock related to L. pneumophila are depicted in Table 1. There are four patients in total who are adults who are mostly olders with chronic disease or bad habits. They had sepsis and septic shock along with respiratory symptoms or pneumonia. The primary antibiotics for L. pneumophila infection are quinolones or beta-lactam therapy. In this paper, our immunocompromised patients had liver transplantation or lymphadenoma. They had higher risk for developing severe Legionnaires' disease. We chose azithromycin as the main antibiotic treatment. In addition, we take advantage of CRRT to remove in ammatory mediators, as well as improving circulation and clinical manifestations. The advantages of CRRT for sepsis and sepsis shock have been reported in the literature [23] .
For L. pneumophila infection, the time to detection is still crucial for the nal outcome of the disease, especially for at-high-risk populations. In addition to clinical manifestations, laboratory tests are also essential for diagnosis; therefore, speci c detection methods include serological and antibody-based detection, culture, urine antigen detection and nucleic acid ampli cation detection [4] . These methods have been developed to evaluate L. pneumophila using sputum or respiratory secretions, blood or tissue, urine or serum samples [4] . However, culture on a prescribed growth medium is still the standard reference method for the diagnosis and identi cation of L. pneumophila, it is very di cult to isolate bacteria from samples [4] . mNGS is a high-throughput pathogen identi cation method, which is superior to the existing microbial diagnosis methods in the identi cation of culturable pathogens [24] . From Table 1, there were many ways to detect pathogeny; the positive rate of blood culture was very low. In this report, polymerase chain reaction (PCR) and serologic testing were performed as well as culture of blood and sputum at PICU admission. And there was a negative consequence. We sent samples of blood and sputum for mNGS just as described in the literature [5] . Finally, we identi ed the presence of L. pneumophila with the help of mNGS. Under azithromycin treatment, the patients recovered at last. Taken together, sepsis or sepsis shock caused by L. pneumophila is rare, especially in children. The detection time of pathogen is very important at high-risk population. mNGS is useful for conforming hard-to-culture pathogens, and it has short turnaround time. Therefore, it is useful for severely ill patients especially. It is sensitive but expensive, so doctors must assess the value of mNGS for identifying pathogens. In a few words, pediatric physicians should be aware that L. pneumophila can cause sepsis or sepsis shock, especially in immunocompromised children. It is signi cant to select appropriate samples and pathogen detection methods in the early stage of disease.

Consent for publication
We obtained the written consent for publication from the guardian of the patient.

Availability of data and materials
The data used in this report are available from the corresponding author on reasonable request.