Severe Community-acquired Pneumonia Caused by Type 7 Human Adenovirus in Immuno competent Adults

Severe community-acquired pneumonia (SCAP) caused by human adenovirus type 7 (HAdV-7) in immunocompetent adults has been of increasing concern recently. Clinical understanding of SCAP caused by HAdV-7 in adults remains limited, though the pathogen has been adequately identied by metagenomic next-generation sequencing (mNGS). We conducted a retrospective review of all patients with SCAP caused by HAdV-7 in immunocompetent adults admitted to Hospital, a hospital in China, between July 2017 and April 2020. Clinical manifestation, laboratory ndings, serial radiological characteristics, mNGS results, treatments and outcomes of these patients were collected and analyzed.

Severe community acquired pneumonia (SCAP) requires admission to intensive care unit(ICU), occupying 20% of the admission of community-acquired pneumonia (CAP) and the core pathogens include streptococcus pneumoniae, atypical pathogens and so on [1].The case fatality rate of CAP is as high as 21% ~ 54% [2] and human adenoviruses (HAdVs) account for about 4.8% of CAP [3]. HAdVs pneumonia typically is limited to children, immunocompromised hosts, and usually a cluster erupts [4], but with the development of metagenomic next-generation sequencing (mNGS) technology, HAdVs pneumonia has been increasingly found to be involved in sporadic cases and outbreaks of SCAP in adults. Though it is generally self-limited, once it has progressed to severe pneumonia, the fatality rate is extremely high [5,6].
HAdVs, ubiquitous in the environment, are non-enveloped, double-stranded DNA viruses, To date, 69 HAdVs genotypes have been recognized and are assigned to seven subgroups (A-G) according to genetic and biochemical characteristics. HAdV-3 and HAdV-7 in subgenus B were not only widespread, but also prone to severe pneumonia in children. HAdV-7 caused more severe clinical manifestations than HAdV-3, even leading to death [7,8].
There are few international reports on the epidemiological and clinical features of adult severe adenovirus pneumonia [9,10]. However, HAdV pneumonia in immunocompetent adults and risk factors of disease progression to SCAP have not been well studied. This paper reports 7 cases of adult severe HAdVs pneumonia con rmed by mNGS technology in Drum Tower Hospital a liated to medical college of Nanjing University in Jiangsu Province, China, and analyzes their clinical features and prognosis.

Study population
We conducted a retrospective review of 7 critically ill patients with severe HAdVs pneumonia admitted to Nanjing Drum Tower Hospital, a tertiary hospital in Nanjing, China, between July 2017 and April 2020.
The requirement for informed consent by individual patients was waived given the retrospective nature of the study. The criteria for severe CAP was de ned by modi ed American Thoracic Society criteria [11] and all patients were positive for Human adenoviruses (HAdVs) DNA fragments by mNGS in bronchoalveolar lavage uid (BALF) and blood specimens.

Mngs Technology
Sequencing were performed at BGISEQ-100 platform (Beijing Genomics Institute, Wuhan, China). MNGS was conducted using the following operational steps: Collection and management of specimens: After obtaining the informed consent of the patient and his/her family for blood and BALF mNGS, 3 mL of peripheral venous blood of the patient was extracted or 1.5 mL of BALF was collected. Blood samples were stored at room temperature for 3-5 min and centrifuged at 4,000 RPM for 10 min within 8 h. BALF samples: 1 g glass beads with a diameter of 0.5 mm were added and mixed for 30 min. After treatment, the specimens were frozen at -20℃. DNA extraction: 0.3 ml of treated blood or alveolar lavage uid was performed, and DNA extraction was performed using TIANamp Micro DNA Kit (Beijing Tiangen Biotechnology Co., LTD.). Library construction and sequencing: Agilent 2100 Bioanalyzer quality control library was used to insert 200 ~ 300 bp fragments, which were cycled to form a single-chain ring structure. The cycled library was replicated by rolling ring (RCA) to produce DNA nanospheres (DNB).The prepared DNB was loaded into the sequencing chip and BGISEQ50 was used for sequencing (BGI Shenzhen Co., LTD.). Data analysis and detection value signi cance: Low quality and < 35 bp in length sequencing data were removed using BWA software (HTTP: / / Bio-BWA).Sourceforge.Net /) alignment removes human host sequences from high-quality data. After further removing the low-complexity sequence, it was compared with the dedicated microbial large database, and the compared data were classi ed and arranged according to viruses, bacteria, fungi and parasites, etc., to report the standard reference for pathogenic bacteria [12].The database includes 6,350 species of bacteria whose sequences are known (including 174 species of Mycobacterium and 137 species of Mycoplasma/chlamydia/ricketia), 1,798 DNA viruses, 1,064 fungal species and 234 parasitic species.

Diagnostic Criteria For Scap Caused By Hadvs
Patients diagnosed with SCAP caused by HAdVs had to meet the following three criteria: (1) meet the criteria for SCAP [13]; (2) have speci c fragment DNA of HAdVs in BALF and/or blood specimen identi ed using mNGS; and (3) excluded other etiological pathogen, including bacteria, fungi and other viruses, no other causative infectious location identi ed.

Clinical Data Collection
Investigators collected clinical data through the electronic medical record system in Nanjing Drum Tower Hospital, a tertiary hospital in Nanjing, China, including demographic data (age, gender), underling diseases, clinical symptoms, such as fever, cough, sputum, dyspnea, chest pain, dyspnea, diarrhea, myalgia and sore throat), laboratory ndings, arterial blood gas analysis, the discovery of microorganisms (including HAdV type) and chest imaging data, drug therapy, respiratory support, complications, and overall prognosis were also recorded.
All the patients had type I respiratory failure and received different oxygen therapy at admission, 1 with high-ow nasal cannula (HFNC), 1 with noninvasive ventilation, other 5 with tracheal intubation and mechanic ventilation. Among them, 3 patients deteriorated rapidly and received extracorporeal membrane oxygenation (ECMO) treatment.
CT scan changes at different stages with SCAP caused by HAdVs were showed in Table 3 and Fig. 1. At early stage, with median 5 days from onset of illness, consolidation (85.71%) and patchy ground-glass opacity (GGOs) (85.71%) were the most common ndings in severe HAdV CAP. Small nodules were revealed as predominant in two patients (28.57%). Lesion involvement was often limited to unilateral lung and the most commonly involved lobe was the right lower lobe (n = 4, Figs

Discussion
In the absence of appropriate diagnostic methods, the incidence of CAP due to virus infection is far underestimated [14].In recent years, with the development of molecular diagnostic methods, more and more CAPs caused by viruses have been recognized. HAdV-caused CAP has been con rmed to account for 1-7% in immunocompetent adults = [4].In this study, all the patients con rmed HAdV-7 infection through mNGS in BALF and blood specimens and clinically characterized by SCAP.
Severe adenovirus CAP is clinically di cult to differentiate from bacterial and mycoplasma pneumonia.
Combine age, season, underlying disease and risk factors, clinical symptoms or signs, chest imaging features, laboratory tests, etc., to preliminarily infer the possible pathogen in clinical practice. Studies have shown that the immunocompetent HAdV SCAP patients, mainly for different degrees of fever, irritation dry cough, and headache, fatigue, chest tightness, poor appetite, diarrhea and other non-speci c systemic toxaemia. Most of the symptoms are not serious, a few can quickly progress to severe pneumonia [15,16]. Common laboratory ndings include leukopenia, lymphopenia, elevated transaminases and occasionally leukocytosis with neutrophilia [17]. From above all, these symptoms and laboratory ndings are common in severe viral infections and lack speci city.
Therefore, in the absence of etiological detection, the diagnosis of adenovirus pneumonia is mainly dependent on imaging features. It has been reported that GGO is the early characteristic imaging feature and consolidation is the typical CT appearance in HAdVs pneumonia [18]. The patient's imaging abnormalities in this study appeared early and advanced rapidly. In early stage from onset, CT scans showed the lesion were dominated by consolidation, GGO and other focal morphology, mostly in the lower lobes in unilateral lung. As the disease progressed, the lesions spread from unilateral to bilateral lungs, and the consolidation shadow enlarged and becomes the most prominent feature in progressive stage. After about two weeks, the patient's condition gradually improved and the lesions gradually absorbed, partly remaining brous lesions.
Currently, clinical HAdVs diagnosis mainly relies on speci c nucleic acid sequence PCR and antibody detection, but low positive rate and often di culty in differentiating clinical subtypes [19]. MNGS technology, a new type of pathogen detection technology, collects the wall of segments of nucleic acid in specimen, ampli cation detection after its sequence, then through bioinformatics analysis and the quantitative analysis of sequence number and coverage can be obtained. MNGS has the merit of high sensitivity, fast screening and has been widely used in bacteria, fungi, virus and mycoplasma detection [20,21]. HAdV-7 was identi ed in blood and BALF of these 7 patients in this study by mNGS, with high median DNA detection sequence and mean coverage above 90%, indicating that mNGS has good credibility in detecting HAdV-7. It is indicated that HAdV ran into the bloodstream after pulmonary infection to cause systemic symptom, so DNA sequence of digits and coverage in BALF were more than those in blood HAdV-7 detection, even so, the median detection sequence in blood specimen is still up to 1038, average coverage of 97.7%, suggesting even in blood high sensitivity of mNGS for HAdV-7 were obtained.
The optimal treatment for severe adenovirus infections has not been established. Cidofovir has been proven to be effective in treating adenovirus pneumonia. [9,[22][23][24]. However, it is expensive, di cult to obtain in China, and has serious side effects, mainly including renal and hematological toxicity. Ribavirin is a broad-spectrum antiviral drugs of nucleoside analogues that are less toxic and have shown e cacy in treating severe adenovirus infections in children and immunocompromised adults [4,25]. In this study, 6 patients were treated with ribavirin and 1 with cidofovir for antiretroviral therapy, with no signi cant difference in e cacy and mortality. All 7 patients were survived, but patients treated with cidofovir had complications of chronic renal failure and required long-term dialysis.
The pathogenesis of severe adenovirus pneumonia in adults is still unclear. The possible mechanism is that HAdV could bind to the cell receptors of target cells, causing the expression and release of in ammatory mediators such as interleukins and chemokines. Excessive release of pro-in ammatory cytokines (such as IL-6, IL-10, TNF-, IFN-, etc.) can cause tissue damage, leading to acute lung injury and multiple organ dysfunction [26]. All the 7 patients in in this study were healthy young healthy adults before their illness and had no underlying disease. However, the count of CD4 + T cell and mHLA-DR% dramatically decreased at admission, suggesting that both cellular immunity and humoral immunity were both impaired. Previous studies have shown that most of the acute damage for body caused by adenovirus infection may be due to immune system disorders [27,28].These ndings may partly explain the self-limiting nature of severe HAdV infection whatever antiviral therapy use, and suggest that host immune response to infection is a major factor in the pathogenesis of lung injury. But the mechanism by which adenovirus impairs cellular and humoral immunity in a healthy body is not clear and further research is needed.
Our study has several limitations. First, there were a relatively small number of cases because of the relatively low incidence of in immunocompetent adults. Second, the study was a retrospective study, no dynamic monitoring of laboratory tests and viral loading were made. Finally, the study is a single-center study and the results may be limited.
In conclusion, the number of adenovirus pneumonia in adult patients has been increasing in recent years. It is recommended to routinely conduct chest CT scan for suspected adenovirus patients to detect early pulmonary lesions, and pay attention to pathogenic screening, especially mNGS technology, for timely detection, early antiviral and respiratory function support treatment.

Con ict of interest statement
All work has been approved by all co-authors. Ying Xu and Qin Gu made substantial contributions to the conception and design; the acquisition of data was performed Ying Xu; the analysis and interpretation of data were performed by Ying Xu and Ning Liu; Ying Xu and Qin Gu wrote the draft of the article and revised it critically for intellectual content. Mingran Shao re-evaluated and added content on the CT imaging outcomes. The nal version was approved by all authors. No con icts of interest exist in the submission of this manuscript. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously and is not under consideration for publication elsewhere, in whole or in part.

Ethical approval
This study was approved by the Ethical Committee of Drum Tower Hospital a liated with the Medical School of Nanjing University.

Funding source
Financial funding support was received from the Medical Technology Development Project of Nanjing (Grant Nos. YKK 15055).

Figure 1
1A-1E A 24-year-old male patient was admitted due to "fever for 8 days, cough and wheezing for 2 days".
A chest CT scan showed lamellar ground glass opacities (GGOs) of the left lower lung on day 6 after onset (1A).On the 8th day after onset, there were large consolidation shadows in the left lung and multiple GGOs in the right lung (1B).The oxygenation index (OI) was 72 mmHg (1 mmHg = 0.133 kPa), and ECMO treatment was performed. On the 18th day after onset, the left lung consolidation had undergone absorption, and the right lower lung had developed small consolidation shadows (1C). The patient was weaned from ECMO. On the 32nd day after onset, the shadows of the bilateral lung consolidation had undergone more absorption, and remaining brous foci were observed (1D). At the follow-up 3 months after discharge, the bilateral lung lesions had mostlydissipated, leaving a few brous foci (1E). 2A-2D A 14-year-old male patient was admitted due to "fever for 5 days, chest tightness for 3 days". A chest CT scan showed consolidation of the lower lobe of the right lung with multiple GGOs on the 5th day after onset (2A). On the 8th day after onset, consolidation was more advanced in the right middle and lower lobe, and few GGOs appeared in the left lung (2B). The OI was 162 mmHg. On the 14th