Polymicrobial and Monomicrobial Necrotizing Fasciitis: Clinical, Laboratory, Radiology, Pathological Hallmark and Differences, a Retrospective Analysis

Background: Necrotizing fasciitis(NF) is a life-threatening infection with high morbidity and mortality rates which should be diagnosed and treated with surgical and antibiotic therapy. Many studies have addressed NF and its subtypes, but few have reviewed the clinical, radiological and pathological differences between the poly-microbial and the mono-microbial groups. Objective: The objective of our study is to describe a relatively large cohort of patients with NF and study and compare the clinical, radiological and pathological differences between the poly-microbial(Pm) and the mono-microbial(Mm) groups. Methods: The charts of hospitalized patients with NF diagnosis from 2002-2019 at the Rabin Medical Center were reviewed. The primary outcome was all-cause mortality at 90 days, secondary outcomes included duration of hospitalization, intensive care unit(ICU) admission, LRINEC score and the need for vasopressor use. Results: 81 patients with NF were included in the study, 54(66.6%) had Mm growth and 27(33.3%) had Pm growth. There were no signicant differences between the two groups in the 90 days mortality, and moreover in hospital mortality was also insignicantly different. In a multivariate analysis, we found that 90 days mortality was more prevalent in the Mm group compared to Pm group. In addition, we found that in hospital mortality, ICU admission and vasopressors use were more frequent among the Mm-group compared to the Pm-group. Conclusions: our study is the rst to compare the differences between the two most prevalent entities of NF. The results demonstrate better prognosis for Pm-NF, with minimal ICU stay, lower mortality, and lower use of vasopressors.


Background
Necrotizing fasciitis (NF) is an infrequent fatal soft tissue infection rst described by Jones [1] in 1871.
The annual incidence of Necrotizing fasciitis in the US [2] is 0.04 per 1000 person years, and varies between different populations [3]. The mortality rate due to NF is high, ranging from 30%-70% [4,5], thus making early diagnosis crucial for the reduction of morbidity and mortality.
NF is divided into several subtypes shown below, based upon the microbiological pro les of the infection, with the rst two being the most frequent.
Type I NF, is a poly-microbial infection involving aerobic and anaerobic organisms, its incidence is still debatable. Some studies have found type I to be the most prevalent, accounting for almost 80% of all necrotizing soft tissue infections (NSTI) [5][6][7], while others found it to be responsible for 30-55% of all ]. Predisposing factors for type I include diabetic ulcers, hemorrhoids, and rectal ssures. Gas in the tissue is often a sign correlated with type 1 NF [10].
Type II NF contributes 10%-40% of NSTI [8,9] and is usually caused by a Gram positive mono-microbial infection, the most common being Group A Streptococcus. [3,11], followed by Staphylococcus aureus [10,11]. Type II NF may occur in all age groups and among individuals with no comorbidities [12].
Other types of NF are rather scarce and their de nition is still debated.
Type III NF is characterized by some as a clostridial infection caused by the clostridia [13], while other de ne Type III as mono-microbial infection primarily caused by Vibrio Spp [14].
Type IV NF caused by fungal infection and occurring mostly after traumatic wounds and burns, with Aspergillus and zygomycetes being the most frequent causative organisms [14].
Mono-microbial NF caused by Gram negative bacteria has been reported more frequently in recent years [15][16][17], and it is an emerging cause of NF. Several studies have found that mono-microbial Gramnegative bacteria account for ca. 50% of the mono-microbial NF cases, with the predominant causative organisms being Klebsiella pneumonia [18], and Escherichia coli. [16,17,19]. A higher 30 day mortality rate was attributed to Gram negative mono-microbial NF as opposed to Gram positive mortality rate (42.1% vs 30.8% respectively) [17].
While many studies have addressed necrotizing fasciitis and its subtypes, only a few have reviewed the clinical, radiological and pathological differences between the types. The aim of our research is to study a relatively large cohort of patients with NF and compare the clinical, radiological and pathological differences between the poly-microbial and mono-microbial groups.

Methods
The charts of all hospitalized patients with NF diagnosis (screening for ICD-10 NF related codes including m72.6) [20] between 2002 to 2019 at the Rabin Medical Center were reviewed.
The diagnosis of NF was con rmed for all patients with intraoperative clinical ndings such as presence of necrotic fascia, purulent discharge and/or histopathologic veri cation supporting the diagnosis.
Aerobic and anaerobic cultures were obtained for all patients from both the blood and the soft infected tissues prior to the antibiotic treatment.
All blood and tissue cultures were processed at the microbiology laboratory at Rabin Medical Center. Bacterial identi cation and susceptibility testing were done by routine methods. Until 2012 all isolations were performed by manual biochemical testing, followed by VITEK-II in inconclusive cases. As of 2012 all isolations were veri ed by either MALDI-TOF or VITEK-II. Susceptibility tests were performed using disk diffusion and E-test methods and interpreted according to CLSI criteria.
Positive culture was de ned as the isolation of a bacterial pathogen from at least one blood culture and/or infected soft tissue (obtained intraoperative or by skin biopsy) and evidence of necrotic fascia during an operation or pathology features consistent with the diagnosis (extensive tissue destruction, The patients were assigned into two groups according to the NF classi cation, Group I was consistent with Type 1 NF and included patients with Polymicrobial infection (Pm group) and group 2 included type II NF based on the mono-microbial (Mm group) growth. Although type 3 NF is considered to be caused by mono-microbial organisms, our study found that all the causative mono-microbial organisms to belonged to type II NF, hence group 2 is equal in our study to Type II NF.
Clinical and laboratory data collected for all patients at presentation included fever, blood pressure, heart rate, white blood cells count, platelet count, C-reactive protein, creatinine, serum glucose levels, lactate, creatinine kinase (CK), albumin, and sodium.
Imaging , clinical examination, number of surgeries, duration of hospitalization, intensive care unit (ICU), amputation, use of vasopressors, death date data and Charlson comorbidity index were also collected [22].
The modi ed Chralson comorbidity index (CCI) predicts 10 years survival in patients. This index is in practice to assess patient comorbidity risk and consists of 9 different comorbidities.
All patients had their medical record checked for surgeries, related anatomically to the infection site, which were performed in the last 30 days In order to assess sepsis severity, quick SOFA (qSOFA) score was calculated upon admission to the hospital. qSOFA score was rst introduced in 2016, and was validated internally and externally for predicting mortality in patients diagnosed with sepsis. qSOFA Score ≥2 suggests high risk of poor outcome (in a scale of 0-3) [23,24].
In addition, immunosuppression status was collected for all patients. Positive immunosuppression status was determined when patients were receiving more the 20mg prednisone and or cytotoxic drug/chemotherapy. LRINEC score is in use to distinguish patients with severe cellulitis or abscess vs necrotizing fasciitis.
LRINEC score includes the following laboratory data: C-reactive protein, WBC, Hb, Na, creatinine and glucose, a score equal or above is the agreed cut off to rule-in NF. We used the LRINEC score, for laboratory risk assessment [25]. In order to assess the severity of the NF, we calculated in our study the LRINEC score at admission to the Emergency Department (ED) for all patients.
The study protocol was approved by the local ethics committee.
The primary outcome was all-cause mortality at 90 days. Secondary outcomes included length of hospitalization, intensive care unit (ICU) admission, LRINEC score and the need for vasopressor use, and amputation by 90 days. We compared for these outcomes the poly-microbial group with the monomicrobial one.

Statistics
Data are expressed as means ± standard deviations (SD), median ± interquartile range (IQR), or number and percentage. We compared patient characteristics between poly vs. mono bacterial groups using ttest, chi-square and non-parametric tests. To estimate the association of the study outcomes with type of bacterial growth, we conducted a forward stepwise logistic regression of the dependent variable. Each set of covariates (demographic, medical history, laboratory, etc.) was entered as separate block to the model. The nal model was selected based on model goodness of t using the c-statistic and plausible clinical explanation. For the primary outcome of 90 days mortality, we used Cox regression, using the same stepwise method described above. Data analysis was performed using SPSS version 25.0.

Results
Eighty-one patients with NF were included in the study, 54 (66.6%) had mono-microbial growth and 27 (33.3%) had poly-microbial growth. All patients in our cohort had a bacterial growth, from blood infection or wound infection, or both. Gram-positive bacteria were the most common group of pathogens in the mono-microbial group (31/54, 57.4%), with Streptococcus pyogenes being the most common pathogen isolated (18/54, 33.3%).
Among the Gram-negative bacteria causing Mm NF, E.coli was the most common one, isolated in 7 cases (7/22, 13%). In the Pm group, Gram-negatives predominated (38/54 (70%)), with E.coli being the most common pathogen (17 (31.5%) isolations). Table 1 depicts all pathogen isolations, it should be noted that some patients had simultaneously several bacterial isolations; hence, the number of isolations depicted in Table 1 is larger than the number of patients.  Table 2 lists the epidemiological characteristic of our cohort. We use the qSOFA to assess sepsis severity and found no signi cant differences between the Mm and Pm groups (1.67 ± 0.9 vs. 1.56 ± 0.9 mean + SD, p = 0.64, respectively). Clinical ndings like redness and swelling were signi cantly more common in the Mm group (p = 0.04, p = 0.01), whereas confusion was more common in the Pm group (p = 0.04) ( Table 3). Laboratory values were similar for the Mm and Pm groups. With CK at normal range in both groups (Table 4).    The results regarding the clinical outcomes are shown in Table 6. In the primary outcome (90 days mortality) there were no signi cant difference between the two groups. However, the length of hospitalization period was higher among the PM group patients than among the Mm group patients (32.   In a multivariate analysis for clinical outcomes between the Mm and PM groups, adjusted for age and sex, we found that 90 days mortality was more prevalent in the Mm groups compared to the Pm group (Table 7). In addition, we found that in hospital mortality, ICU admission and vasopressors use were more frequent among the Mm groups compared to the Pm group. In Fig. 1 of in-hospital mortality strati ed by microbial type, we found that more patients in the Pm group survived longer at day 60 compared to the Mm group (p = 0.25),however this nding did not differ signi cantly.

Discussion
This is the rst study comparing the presentation, treatment and prognosis of patients with Mm vs. Pm NF. We aimed to nd and describe the differences between the types of NF; so that the study will shed new information and will show ways to achieve a faster diagnosis of this clinical devastating infection.
Our study found clinical and radiological differences at presentation in those groups. Redness and swelling in the site of infection were signi cantly more common in the Mm group whereas non-speci c ndings (confusion) were more common in the Pm group. In line with previous studies, overweight patients were at risk for necrotizing soft tissue infections in general and more likely to have polymicrobial than mono-microbial infections [26].
The clinical differences which we found between the groups are in accordance with the current literature [26][27][28], where Mm NF, especially strep group A pathogens, are often located in the extremities and in more exposable places, while Pm NF is more complex and causes deeper infections ( involving the perineum, sacrum) where redness and swelling cannot be seen. Pain was common in both groups and it is typical for NF to see pain that is non-proportional to ndings in the physical examination.
Several radiological ndings in our study were overlapping in both groups. Among those non-speci c ndings we noticed uid collection, edema, and liquefaction. Our ndings correlate with the current radiological pattern for NF which usually includes with uid collections along the deep fascial sheaths, a non-enhancement of the muscular fascia and extension of edema into the intramuscular septa, vascular thrombosis, subcutaneous gas, uid collections along the deep fascial sheaths, low attenuation areas in the deeper fascial planes suggestive of fat in ltrate and liquefaction necrosis, with facial involvement and lack of fascial enhancement being the most indicative signs for NF [20].
In our study, the CT ndings were consistent with the literature [30][31]; free air cases in the tissue were more common in the Pm group, re ecting the anaerobic component causing this nding. [32] When checking NF outcomes, we found that there were lower ICU hospitalization, lower use of vasopressors, and lower in hospital mortality rates among the Pm group compared to the Mm group. Several studies have found that the Mm NF due to gram negative bacteria is associated with higher mortality rates [33][34]. Group B Streptococcus NF infections were also previously described as associated with higher mortality rates [25]. Our Mm group included six cases of NF caused by group B Streptococcus. Still, historically group A Streptococcus accounts for most of the group, and the higher mortality rate found in our study among the Mm group could be attributed to an epidemiological shift of invasive GAS strains as was described in Sweden. [26,35] Our analysis found a higher rate of surgical interventions performed on the Pm group compared to the Mm group. This nding has not been described previously in the literature. While it could explain the higher mortality rate among the Mm group, we think that the distinct radiographic features among the Pm group patients lead to prompt surgical intervention and to subsequent interventions following early intervention.
Although not statistically signi cant, our analysis found overall mortality to be lower among the Pm group compared to the Mm group. A possible explanation of the lower mortality could be due to the fact that the poly-microbial group underwent signi cantly more surgeries than the mono-microbial group, and this nding could account for the mortality differences between the groups.
Limitation: our study has several limitations. First, NF is a rare disease; hence, the number of cases in this cohort is small, however -most of the published studies dealing with NF are of the same size. Secondly, the retrospective nature of this study limits us to the available medical records, hence, decision-making data on the reasons why some patients underwent more surgeries than others did, and on the nature of the medical treatments including ventilation support, hemodynamic support and antibiotic treatment, are not noted. Our study is based on the medical charts, lled by medical staff, it is possible that there were some mistakes; however, it is unlikely that such errors would have occurred more often in a particular group.
In conclusion, our study is the rst to compare the differences between the two most prevalent entities of NF. The results demonstrate better prognosis for Pm NF, with minimal ICU stay, lower mortality, and lower use of vasopressors. As discussed above, those ndings could be due to the radiological characteristics which make Pm NF unique and thus its diagnosis is not delayed. But it is also possible that the presentation of the majority of the Pm group with skin and soft tissue infections helped the physician reach a quicker diagnosis. Either way, those results require further investigation to assess their true impact. We believe that identifying the unique features of each entity can be helped by prompt and accurate diagnosis, and by elucidating the best treatment for each entity; thus additional studies are required in order to achieve these objectives.

Figure 1
Kaplan-Meire of overall mortality strati ed by microbial type (p=0.23)