This study aims to evaluate the outcomes of single intramedullary K-wire fixation in nonthumb, metacarpal shaft fractures with immediate postoperative hand mobilization without any immobilization.  This is a retrospective case series conducted from January 2019 to December 2022. We included patients with closed, simple transverse, or short oblique metacarpal shaft fracture treated with single, 1.4 mm, intramedullary K-wire fixation. Gentle postoperative range of motion exercise was encouraged in every patient without any hand, finger, or wrist motion restriction material. Clinical outcomes were evaluated with total active flexion; grip strength; disability of arm, shoulder, and hand (DASH) score; and the American Society for Surgery of the Hand Total Active Flexion (ASSH TAF) score.  This study included 34 patients, 25 males and 9 females with a mean age of 33.14 years (ranging 18–59). A total of 43 metacarpal shafts were treated. The mean DASH score at two and 6 weeks postoperative was 41.5 (ranging 19.16–60.34) and 9.58 (ranging 0.83–23.27). The mean final DASH score at last follow-up was 3.48 (ranging 0–8.33). Mean TAF at 2 weeks postoperative, 6 weeks postoperative, and at final follow-up was 203.8 (ranging 185–240), 238.2 (ranging 220–270), and 259.25 (ranging 240–270) degrees, respectively. The mean grip strength of the injured hand was 66.14 and 86.1% of the uninjured hand at 6 weeks and 3 months postoperative. There was no nonunion, malrotation, or infection. In conclusion, single intramedullary K-wire fixation gives excellent outcomes in the treatment of single or multiple, simple, metacarpal shaft fractures without the need of postoperative immobilization. Fracture of the metacarpal and phalanges are the most common fractures of the upper extremity , 2. The Antegrade intramedullary (IM) Kirschner wire (K-wire) fixation is a well-known, minimally invasive, treatment option in metacarpal fracture , 5, 10. There is still no consensus on the postoperative management of metacarpal fracture. The rehabilitation protocol varies and depends on the preference and experience of each institute. Several authors have lessened the immobilization material and promoted early mobilization after K-wire fixation , 23–28. Therefore, our objective was to evaluate outcomes along with the complications of immediate postoperative hand mobilization in metacarpal shaft fracture patients who underwent single buried antegrade IM K-wire fixation without postoperative immobilization. We hypothesized that immediate postoperative hand mobilization can be initiated after single IM K-wire fixation in metacarpal shaft fracture and the fracture unite without serious complications.We conducted a retrospective case series of patients diagnosed with metacarpal shaft fracture to evaluate the outcome of single, buried, antegrade IM K-wire fixation within 2 weeks of the initial injury. The study was conducted between January 2019 and December 2022. The inclusion criteria were as follows: (1) closed simple transverse or short oblique metacarpal shaft fractures and (2) unacceptable deformities , 18 thatSurgery was performed under wrist block which allows for intraoperative active hand motion to ensure proper fracture reduction and fixation. The patient was placed in the supine position with the pronated and extended arm on a hand table. The tourniquet was inflated to control the bleeding. The procedure was performed under fluoroscopic guidance.A 1 cm skin incision was made, starting from the base of the affected metacarpal and continued proximally. Subcutaneous vessels and tendons were retracted and protected using a Senn retractor while exposing the metacarpal base. The dorsal cutaneous branch of ulnar nerve (DCBUN) should be identified and protected when operating on the fourth or fifth metacarpal. The 2.0 mm K-wire was inserted into a universal T-handle chuck. This K-wire was used as an awl to create an entry hole at the cancellous bone of the metacarpal base, just distal to the carpometacarpal joint. This entry hole should always be in line with the central axis of the operated metacarpal.Typically, a 1.4 mm K-wire is chosen. Before bending, the pointy tip of the K-wire is cut to prevent any potential injury to the surgeon. The blunt end of the K-wire will be introduced into the medullary canal to avoid metacarpal head perforation. Once the metacarpal fracture is reduced, the K-wire is placed in position with the assistance of an image intensifier. The distal tip of the K-wire is positioned at the subchondral bone of the distal metacarpal metaphysis. The length of the implant is determined by measuring the length from the tip of the K-wire to the entry point. At the entry point, the K-wire is bent up about 45 degrees and its proximal end is twisted into a loop. Each patient's soft tissue thickness will determine the position of the loop. This loop should be located in the subcutaneous tissue when the implant was properly introduced into the metacarpal .The implant was introduced into the medullary canal through the previously made entry point. Any malrotation must be corrected before passing the K-wire through the fracture site. The K-wire was pushed to engage firmly into the subchondral bone. Intraoperative active hand motion under the image intensifier was always performed to evaluate any displacement from early hand motion. The implant was buried under the skin and the skin was closed using a nonabsorbable suture.Immediate mobilization of the wrist and all fingers was initiated after surgery without any motion restriction instrument. During the first two weeks, with no specific rehabilitation protocol from the surgeon or hand therapist, only gentle flexion and extension of all metacarpophalangeal and interphalangeal joints were allowed. The patients were permitted to resume performing light daily activities, that is, the patients can use their hand in their daily activities only as pain allows. The patients were encouraged to do gentle progressive hand and finger motions to eventually achieve full range of motion (ROM). Forceful hand clenching or pinching was strictly prohibited.The suture material was removed 2 weeks after the operation. Carefree contact with water in daily activities was allowed after the removal of the suture material. All patients were advised that no pain or discomfort should be felt in the injured site during the activities. If any discomfort is felt, the patient should discontinue that activity and rest the operated hand.Finger malrotation was assessed clinically by observing finger scissoring at full finger flexion and extension . We obseo TAF), good (120–80o TAF), or poor (<80o TAF).Functional outcomes were assessed at 2 weeks, 6 weeks, and 3 months postoperative by using the American Society for Surgery of the Hand (ASSH) TAF score , disabilt-test, Wilcoxon signed rank test, and Fisher's exact test according to data characteristics. The level of statistical significance was set at p  <  0.05.All statistical analyses were performed using STATA 16 . Data distribution was evaluated with the Shapiro–Wilk test. Continuous data with normal distribution were demonstrated using mean ± standard deviation (SD) and median with interquartile range (IQR) for non-normal distribution. Categorical data were presented with count and percentage. Inferential statistics were determined using paired This study included 34 patients, 25 males and 9 females with a mean age of 33.52 years (ranging 18–59). There were 9 patients with multiple metacarpal fracture. A total of 43 metacarpal shafts were treated. Demographic data of the patients are shown in The mean overall clinical follow-up period was 97.08 days (ranging 40–154). Healing was obtained in all patients. A sign of radiographic bone union was observed at a mean of 47.3 days (range 37–56). No infection was observed.Among 34 patients, three patients satisfied with the treatment result and declined further appointment after K-wire extraction. Four patients were lost after implant removal. All 7 patients with short follow-up showed sign of bone union and achieved excellent ASSH TAF score .There are 27 patients, and 32 metacarpals that were eligible for longer follow-up. The mean DASH score at 2 and 6 weeks postoperative was 41.5 (ranging 19.16–66.6) and 9.58 (ranging 0.83–23.27). The mean final DASH score at last follow-up was 2.66 (ranging 0–8.33). Mean TAF at 2 weeks postoperative, 6 weeks postoperative, and at final follow-up was 203.8 (ranging 185–240), 238.2 (ranging 220–270), and 259.25 (ranging 240–270) percent, respectively. Mean %TAF at 2 weeks postoperative, 6 weeks postoperative, and at final follow-up was 77.02 (ranging 68.51–88.88), 89.99 (ranging 83.01–100), and 97.97 (ranging 90.56–100) percent, respectively. Hand grip strength was assessed by digital hand grip strength dynamometer compared to the uninjured hand. The mean grip strength of the injured hand compared to the uninjured hand at 6 weeks postoperative and the last appointment were 66.96 (51.83–90.27) and 86.1 (72.79–97.87) percent, respectively .Scissoring of finger during full fingers' flexion had never been observed throughout the treatment course. There was no infection, stiffness, tendon irritation, or metacarpal head perforation observed. Five patients with fifth metacarpal fracture reported minimal postoperative paresthesia on the dorso-ulnar side of the injured hand. All patients' symptoms resolved within 3 months after surgery. One patient with fifth metacarpal shaft fracture had a dorsal callus bump at the fracture site.IM K-wire fixation in metacarpal fracture is a well-known treatment. There are many described techniques and postoperative protocols for this procedure. Abulsoud et al. were the first to report a study that describes and investigates the outcomes of fixation of metacarpal shaft fractures using a single, 1.8–2.0 mm, buried K-wire . In our According to Abulsoud et al., their study population was limited only to patients with single metacarpal shaft fractures . VariousFrom our findings and previous reported literature, immediate gentle postoperative ROM of the metacarpophalangeal joint (MPJ) and interphalangeal joint (IPJ) does not affect fracture healing or patient's hand function . ImmediaWe prefer to curl the proximal end of the K-wire into a loop and bury them under the skin to prevent tendon and soft-tissue irritation or a possibility of pin tract infection. In previously reported techniques of buried IM K-wire, the proximal end of the K-wire was frankly cut. With such a straight cut, there have been reports of tendon rupture, skin irritation, pain, and stiffness , 22, 36.In our protocol, an adequate intensity of postoperative mobilization must be tailored according to the patient's tolerability. The patient is advised to limit the affected hand activity if there is any pain at the fracture site. As a result, the patient's rehabilitation intensity increased inversely to pain. Subsequently, the patients will gradually regain their normal ROM along with the fracture healing.There were no cases of malrotation after gentle finger flexion and extension in simple metacarpal shaft fracture with single IM K-wire fixation. According to previous studies, fracture rotational malalignment was greatly influenced by the integrity of the deep transverse metacarpal ligament (DTML) , 38–42. Severe rotation deformity can be seen only if the DTML is transected, attenuated, or ruptured , 41, 43.Complications of single IM K-wire fixation in metacarpal shaft fractures include stiffness, nonunion, and metacarpal head perforation . Though To the best of our knowledge, we are the first to report outcomes of metacarpal shaft fracture fixation using single buried K-wire, with immediate postoperative hand motion without any immobilization. However, there are some drawbacks to this study and the surgical protocol. Firstly, the follow-up time was relatively short. We attempted to follow our patient for up to one year, but every patient declined further appointments when the function of their operated hand was comparable to their contralateral hand. Secondly, there is also a flaw in exposing surgeons, personnel, and the patient to radiation during the operation. After bone union, the patient requires a second operation to remove the implant. Lastly, this is a retrospective case series without comparison with other techniques. Therefore, a larger prospective comparative study with a longer follow-up period is needed to further investigate these outcomes.


This study aimed to establish an effective ultrasound diagnostic nomogram for benign or malignant lymph nodes in HIV-infected patients.The nomogram is based on a retrospective study of 131 HIV-infected patients who underwent ultrasound assess at the Shanghai Public Health Clinical Center from December 2017 to July 2022. The nomogram’s predictive accuracy and discriminative ability were determined by concordance index (C-index) and calibration curve analysis. A nomogram combining the lymph node US characteristics were generated based on the multivariate logistic regression results.Predictors contained in the ultrasound diagnostic nomogram included age (OR 1.044 95%CI: 1.014–1.074 P = 0.004), number of enlarged lymph node regions (OR 5.445 95%CI: 1.139–26.029 P = 0.034), and color Doppler flow imaging (CDFI) grades (OR 9.614 95%CI: 1.889–48.930 P = 0.006). The model displayed good discrimination with a C (ROC) of 0.775 and good calibration.The proposed nomogram may result in more-accurate diagnostic predictions for benign or malignant lymph nodes in patients with HIV infection. Whether benign or malignant tumors, lymphadenopathy is often the first manifestation of disease progression –6. LymphAs one of the most common methods available, ultrasound has the characteristics of good discrimination of lymph nodes (LNs), non-radioactive, economical, and non-invasive . The regOur study aimed to identify risk factors that may predict benign and malignant LNs in HIV-infected patients and build a nomogram by combining clinical and ultrasound features, which improves the accuracy of lymph node diagnosis by ultrasound in an objective manner.Between December 2017 and July 2022 at the Shanghai Public Health Clinical Center, Fudan University, Shanghai, China, a total of 324 HIV-infected patients underwent ultrasound examination in our hospital and were diagnosed with lymphadenopathy. Inclusion criteria were as follows: (1) HIV-infected patients, (2) one or more regions with lymphadenectasis and the diagnosis were not yet clear, (3) pathological results of needle biopsy were available, and (4) ultrasonic and pathological images were available. The exclusion criteria were: (1) inadequate or indeterminate pathological results, (2) patients were under 18 years old, and (3) incomplete follow-up data. Of all 324 patients, 166 were not diagnosed by pathology, 2 were younger than 18 years old, and 25 had incomplete follow-up data. 131 HIV-infected patients who performed ultrasound tests and had pathological results of needle biopsy were included. This study was approved by the Research Ethics Committee of Shanghai Public Health Clinical Center, Fudan University, Shanghai, China.We used Mindray Resona 7 Color Doppler Ultrasound machine. All patients were examined in the supine position, and the examined sites were fully exposed. The frequency was set to 5.0–12.0 MHz, and the mode was color Doppler mode. The US imaging characteristics of each patient were retrospectively reviewed by two independent sonographers with more than ten years of experience in lymph node imaging; neither observer was aware of the clinical nor the pathological outcome. If the sonographers had different opinions, they met to determine their final decisions by consensus. Transverse and longitudinal images of the LNs to be diagnosed were obtained from each patient. We set the filter conditions to a minimum, with a blood flow velocity color scale range of 5.4 cm/s and a maximum scanning depth of 16 cm.Lymph node vascularity was classified from grade 0 to 3 and evaluated by color Doppler flow imaging (CDFI) following the Adler criterion  . The aspNumerical variables were shown by mean ± SD, and categorized variables were summarized by absolute frequencies. Continuous variables were compared by the Student’s t-test, and categorized variables were compared by the χ2 test (or Fisher’s exact test as required) across two groups . Univariate and multivariate logistic regression models were performed to estimate the capability of different variables in predicting the malignancy of LNs. Variables with P-value < 0.05 in the univariate analysis or variables identified by clinical experience were further included in the multivariate analysis. Results were considered significant at P < 0.05. Descriptive statistics and analyses were obtained using SPSS 26.0 .Nomograms are statistical models that are ideally suited for individualized risk assessment. To provide the sonographers with a quantitative tool to diagnose the individual probability of malignant LNs, we built the diagnostic nomogram using the independent predictors selected by the multivariate logistic regression model to generate a combined indicator for estimating the likelihood of malignant LNs.The nomogram was performed using the total points as a factor. Calibration curves were plotted to assess the calibration of the diagnostic nomogram, which was evaluated by plotting the predicted versus the actual probability for quintiles of the predicted probability of malignancy within a lymph node.There were 131 patients total, including 80 (61.1%) benign and 51 (38.9%) malignant LNs. Among the patients, 112 were men, and 19 were women, with a mean age (± standard deviation) of patients of 43.82 ± 14.70 years (range: 18–76 years). Features of LNs in grayscale ultrasound were summarized in Table Table ), longer duration of HIV infection (OR 1.011 95%CI: 1.002–1.020 P = 0.020), single enlarged lymph node region (OR 5.500 95%CI: 1.412–21.422 P = 0.014), and grade 2–3 of CDFI type (OR 11.136 95%CI: 2.508–49.455 P = 0.002) were associated with malignant lymph nodes. In multivariate analysis, the older age (OR 1.044 95%CI: 1.014–1.074 P = 0.004), single enlarged lymph node region (OR 5.445 95%CI: 1.139–26.029 P = 0.034), and grade 2–3 of CDFI type (OR 9.614 95%CI: 1.889–48.930 P = 0.006) were independent risk factors for the malignant lymph node. A model incorporating the above independent predictors was developed and presented as the nomogram , the life expectancy of HIV-infected patients is significantly prolonged than before, and malignancy has become the leading cause of death among HIV-infected patients , 21. IncAs the reservoir of HIV, LNs can be infected regionally within 3–6 days and systemically within 6–25 days after the initiation of the AIDS epidemic , 23. NevCDFI technology can increase the specificity of ultrasound by providing real-time vascular assessment , 26. In The subcapsular sinus of the lymph node is the first site in the tumor-draining lymph node contacted by tumor-draining material carried in afferent lymph . Then thThere are still some limitations of our study. First, this is a retrospective study, and selection bias may occur. Second, the performance of our nomogram depends on the accuracy of the operator-reported imaging features, which may be subjectively biased. Finally, we need to perform external validation in a larger sample to obtain more objective conclusions. The model could also be improved by incorporating more valuable techniques, such as elastography and lymphangiography, which we intend to investigate in the future. However, in the current situation, it is necessary to obtain anatomopathological samples in all cases to avoid a missed diagnosis. This study greatly improves the ultrasonic diagnosis of benign and malignant lymph nodes in HIV-infected patients.This study identified the age, the number of enlarged lymph node regions, and CDFI grades as meaningful ultrasound diagnostic features and established the nomogram to give a more-accurate diagnostic prediction for benign or malignant lymph nodes in patients with HIV infection. It helps distinguish these diseases, and this easy-to-use scoring system can be conveniently applied to facilitate diagnosing HIV-infected patients with lymphadenopathy.


Larix gmelinii forests, we collected soils from light, moderate, and heavy fire disturbance forests and a natural forest as a control forest in Greater Khingan Larix gmelinii forests, in the northeast of China. The community structure and diversity of arbuscular mycorrhizal fungi was sequenced using Illumina MiSeq technology and we analyzed the correlation with the soil physicochemical characteristics. The results showed that the contents of microbial biomass content (MBC), moisture content (MC), total nitrogen (TN), and available phosphors (AP) increased significantly (p < 0.05) with increasing fire intensity (from Light to heavy fire), but available potassium (AK) decreased significantly (p < 0.05). These changes were not significant. A total of 14,554 valid sequences from all sequences were classified into 66 ASVs that belonged into one phylum, one order, four families, and four genera. The genera included Glomus, Ambispora, Paraglomus, and Acaulospora, and Glomus was the dominant genus (the genera with the five most relative abundances) in the control and heavy-fire forests. Non-metric multidimensional scaling (NMDS) analysis showed that forest fires significantly affected the community structure of arbuscular mycorrhizal fungi (p < 0.01). Redundancy analysis (RDA) showed that MBC, SOC, and AP contents significantly affected the composition structure and diversity of arbuscular mycorrhizal fungi communities. This study indicated that forest fires affected the composition and diversity of soil arbuscular mycorrhizal fungi communities through changing the soil physicochemical parameters  in cold-temperate Larix gmelinii forests. The study of soil physicochemical properties and arbuscular mycorrhizal fungi diversity in cold-temperate Larix gmelinii forests in the Greater Khingan Mountains after forest fires provides a reference basis for the revegetation and reconstruction of fire sites.Forest fires are an important disturbance factor in forest ecosystems, and obviously change the soil environment. Arbuscular mycorrhizal fungi, as a medium and bridge between vegetation and soil, play a crucial role in mediating plant nutrient uptake and regulating the productivity, stability, and succession of vegetation–soil systems. To investigate the effects of forest fires on the community structure and diversity of arbuscular mycorrhizal fungi in cold-temperate  Forest fires are one of the most important environmental factors affecting biodiversity and play a crucial role in maintaining the structure and function of forest ecosystems . The impArbuscular mycorrhizal fungi are abundantly distributed in the soil  and can Larix gmelinii forests are still lacking. Therefore, we set forests with different forest fire intensities in Huzhong National Nature Reserve of China to investigate the changes in soil physicochemical properties and arbuscular mycorrhizal fungi community structure diversity under different fire intensities.Forest fires can alter the structure and diversity of arbuscular mycorrhizal fungi communities, but the response of arbuscular mycorrhizal fungi communities was not consistent in different geographical and climatic conditions, as well as forest types and soil conditions. Lots of studies on forest fires affecting soil arbuscular mycorrhizal fungi have been carried out in temperate and tropical forests, such as in Brazil , AustralLarix gmelinii forests are the climax zonal vegetation. Forest fires caused by lightning strikes are frequent in this reserve and cause forest succession and soil environment changes. At present, studies on post-fire forestry in the Greater Khingan Mountains have mainly focused on forest carbon storage [Huzhong National Nature Reserve is located in the Greater Khingan Mountains of China, and is a typical representative of the cold-temperate forest ecosystem, where  storage , the res storage , effects storage , and soi storage . HoweverLarix gmelinii forest ecosystem in China. It is a continental monsoon climate with an average temperature of −2 to −5.5 °C and a record low of −52.3 °C [This study was located at Huzhong National Nature Reserve , Moderate- (M), and Heavy (H)-fire forests were selected in the Huzhong Reserve in 2010, and adjacent forests with the same stand type without disturbances from fire were selected as control forests. Each forest set up three replicates and an area of 20 m × 20 m for each forest. The basic information of each forests is shown in Ten soil samples were collected from 0 to 10 cm in each forest after removing humus and then mixed together into one soil sample (approximate 500 g per plot). The mixed soil samples passed through 2 mm sieve to remove plant roots and stones, and then placed in ziplock bags, backstroked in an ice box, and immediately brought to the laboratory. The soil samples were divided into two sub-parts in the laboratory, one stored at −80 °C for DNA extraction and another for determination of soil physicochemical properties.Soil microbial biomass carbon (MBC) was determined using the carbon and nitrogen analyzer method , soil pH® Soil DNA Kit DNA . Using diluted genomic DNA as a template, specific primers with Barcode AML1F (5′-ATCAACTTTCGATGGTAGGATAGA-3′) and AML2R (5′-GAACCCAAACACTTTGGGTTTCC-3′) were selected as the first pair of primers; AMV4.5NF (5′-AAGCTCGTAGTTGAATTTCG-3′) and AMDGR (5′-CCCAACTATCCCTATTAATCAT-3′) were used as the second pair of primers to amplify the fungal 18S rRNA region. In the second round of PCR, the barcode primers (AMV4.5NF/AMDGR) were used to distinguish the different PCRs. The PCR reaction volumes were: 10 × Buffer 2.5 μL, template DNA (20 ng·L−1) 2.0 μL, dNTPs (2.5 mmol·L−1) 1.5 μL, specific dNTPs (2.5 mmol·L−1) 1.5 μL, Tag enzyme 1.0 μL. The PCR reaction conditions were as follows: 95 °C for 3 min; 95 °C for 30 s, 55 °C for 30 s, 72 °C for 45 s, 30 cycles; 72 °C for 10 min. The amplified PCR products were quantified using Picogreen fluorometer, homogenized and mixed, and then sequenced by IlluminaMiseq on PE300 platform with 3 replicates for each sample. The raw data were uploaded to NCBI SRA database (sequence number: PRJNA945502).Total DNA was extracted from fresh soil samples by weighing 0.25 g and following the procedure of E.Z.N.A.The raw sequences were identified using Qiime2  softwaret-test was used to analyze alpha diversity, NMDS ranking analysis based on Bray–Curtis distance was used to analyze beta diversity, and RDA analysis and Pearson’s method were used for environmental factor association analysis. All statistics were conducted with the “microeco” package R language and the figures were also finished using the “ggplot2” R language.Microbial community alpha diversity was characterized using Chao1 index (Chao1 richness estimator), Shannon–Wiener index (Shannon–Wiener index), and Simpson index (Simpson index) . Data wep < 0.05) compared with the control forest (p < 0.05).The soil physicochemical properties  changed significantly in different fire intensities (l forest . Soil MBp < 0.05), but increased the Simpson index (p < 0.05) in the three fire intensities. Moreover, the Chao1, Shannon, and Simpson indices of soil arbuscular mycorrhizal fungi did not change within the different fire intensities (p > 0.05) (As shown in  > 0.05) .p < 0.01) in different forest fire intensities. From the The result of NMDS analysis is shown in As shown in As seen in Glomus (91.6~94.76%), followed by Ambispora (13.37~13.86%). The relative abundance of Glomus decreased in the fire group compared with the control group, while the relative abundance of Glomus increased after decreases between fire groups. In addition, Glomus was not detected in M (Moderate fire), and Acaulospora was detected only in CK (Control—blank). This shows that the community structure of L (Light fire) and M (Moderate fire) was more affected after the forest fire.From p < 0.05), the Shannon diversity index was significantly negatively correlated with MBC (p < 0.01), and the Simpson diversity index was significantly positively correlated with MBC (p < 0.01) and negatively correlated with AK (p < 0.01), indicating that AP, MBC, and AK are important soil environmental factors affecting arbuscular mycorrhizal fungi alpha diversity.The results of Pearson correlation analysis  showed tRedundancy analysis (RDA) was performed on the soil physicochemical properties for the community composition of arbuscular mycorrhizal fungi at the ASV. The first and second axes explained 91.91% and 2.16%, respectively, with a total explanation of 94.07% . The arbp < 0.05) effects on soil arbuscular mycorrhizal fungi community composition, indicating that AP, SOC, and MBC were important factors affecting soil arbuscular mycorrhizal fungi communities.As shown in Ambispora was significantly negatively correlated with soil MC content (p < 0.05) and significantly negatively correlated with soil TN content and soil AP content (p < 0.01), indicating that MC, TN, and AP were important factors affecting the genus composition of soil arbuscular mycorrhizal fungi; this might be because the MBC values of the two soils of heavy and Light fire were closer, providing a more similar soil nutrient environment for the development and reproduction of arbuscular mycorrhizal fungi, resulting in no significant difference in the diversity of arbuscular mycorrhizal fungi.It has been shown that fire has a large effect on the alpha diversity of arbuscular mycorrhizal fungi . In the p > 0.05), which may be due to the wide ecological niche of clumping mycorrhizal fungi, which can rapidly adapt to environmental changes under different fire levels [Many studies have shown ,55 that e levels ,57.Paraglomus were all present in soils burned at different intensities of fire, while Glomus, Ambispora, Acaulospora, and Archaeospora were not evenly distributed (Glomus was due to its strong environmental adaptability and unique reproductive characteristics, with a high spore production rate that allowed it to colonize different environments and be highly adaptable [Glomus propagules surviving on the soil surface [Glomus [Glomus by scorching [Glomus with the nutrients needed for growth and accelerate its growth [It has been shown that fire can change the fungal species composition and promote the dominance of specific species or enhance the spectrum . In the tributed , which idaptable . Their l surface , decreas [Glomus ; the heacorching , while is growth .Ambispora was negatively correlated with MC, TN, and AP, probably because Ambispora is more sensitive to changes in soil physicochemical properties [It has been shown that physicochemical properties such as SOC, C/N, and MBC have significant effects on fungal community composition ,63,64, aoperties , which ioperties ,65,66.Glomus was the dominant taxon in the arbuscular mycorrhizal fungi of the fire sites, and MC, TN, and AP were important soil physicochemical factors affecting the community structure composition of arbuscular mycorrhizal fungi.Different intensities of fire affected both the community structure and diversity of soil arbuscular mycorrhizal fungi. Fire significantly reduced the alpha diversity of arbuscular mycorrhizal fungi in cold-temperate forest soils, and MBC, SOC, and AP were the main influencing factors in soil physicochemical properties. Fire altered the beta diversity of arbuscular mycorrhizal fungi in cold-temperate forest soils,


Hepatitis D virus (HDV) infection causes the most severe form of chronic viral hepatitis. However, it is still unclear to what extent the underlying cirrhosis may contribute to disease progression. The aim of this study was to compare the long-term outcome of HDV infection with HBV monoinfection in a single-center cohort of both non-cirrhotic and cirrhotic patients.We retrospectively studied 175 patients with chronic hepatitis D (CHD) who were followed for at least 6 months (median of 6.3 (0.6–23.6) years). In addition, we selected 175 patients with HBV monoinfection (CHB) who were matched for gender, age, region of origin, HBeAg status, and bilirubin. Liver-related clinical end points were defined as hepatic decompensation , liver transplantation, HCC, or liver-related death.p < 0.01) in CHD patients. In a multivariate Cox regression, HDV infection was independently associated with the development of end points . However, in cirrhotic patients there were no significant differences between HBV and HDV in the development of end points. Besides, CHB patients with cirrhosis developed more frequently HCC (35.5%) than CHD patients with cirrhosis (18.5%).Clinical complications developed earlier (4.6 vs. 6.2 years) and more frequently (35.4% vs. 12.6%, Our results confirmed that HDV leads to a faster progression to cirrhosis compared to HBV. However, once cirrhosis is present, not HDV but the underlying cirrhosis is the dominate intrinsic risk factor for the development of liver-related end points and for the progression to HCC.The online version contains supplementary material available at 10.1007/s12072-023-10575-0. The hepatitis delta virus (HDV) is a defective RNA virus, which requires the hepatitis B surface antigen (HBsAg) of the hepatitis B virus for assembly virions . Thus, HThe aim of this study was to evaluate the clinical long-term outcome of CHD patients compared to matched patients monoinfected with HBV in the so far largest European, single-center cohort. In particular, it should be investigated whether the differences in the long-term clinical course of patients with CHB and CHD are confirmed in both non-cirrhotic and cirrhotic patients and whether the treatment strategies have an impact on the long-term course. One further focus should be on the development of HCC and how HDV differs from HBV.All anti-HDV-positive patients followed at Hannover Medical School as part of routine clinical follow-up were screened. Baseline was defined as the first visit at the institution. Patients were included in the study if they had detectable HBsAg and either anti-HDV IgM antibodies or HDV RNA for at least 6 months. Only patients with an available follow-up of at least 6 months with a minimum of two visits were included. Patients were excluded if they had undergone liver transplantation or suffered from HCC before the first observation. They were also excluded if they had liver disease caused by non-alcoholic steatohepatitis, alcohol, or metabolic disease. Virological parameters for hepatitis B and delta were measured as previously described. One hundred and thirty-six patients were part of a previous study . AccordiLiver-related end points were defined as hepatic decompensation , liver transplantation, HCC, or liver-related death. Cirrhosis was diagnosed based on liver histology (F5 and F6 according to the ISHAK score). If data were not available, cirrhosis was assessed based on transient elastography (≥ 14.0 kPa)  or sonogp values < 0.05 were considered as statistically significant. Continuous variables were analyzed by t tests. For nonparametric parameters Mann–Whitney U tests were used. A Chi-square test was calculated for the comparison of discrete variables. In case of an expected cell count ≤ 5, Fisher’s exact test was used instead. Parameters that were associated with the clinical long-term outcome in univariate time-depending COX-regression models (p < 0.05) were additionally compared in multivariate analysis. Multivariate COX-regression analyses were performed by using the likelihood ratio test for backward selection. Hazard ratios (HRs), including their 95% confidence intervals (CIs), were calculated for the COX-regression models. Using Kaplan–Meier analysis, we estimated the cumulative event-free survival and compared different risk groups by log-rank tests. To ensure a comprehensible matching of the groups, a propensity score was estimated from a multivariate logistic regression model, containing the above-mentioned matching variables. The subsequent individually matched propensity score was used for nearest neighbor matching of both the groups with caliper match tolerance of 0.2. Due to the low number of patients with CHB in the group of cirrhotic patients, we performed an inverse probability of treatment weighting (IPTW) in this group with subsequent calculation of the outcome using generalized estimating equations and logistic regression model.Statistical analyses and the creation of figures were performed by using SPSS software . All parameters were described as mean ± SD. p < 0.01) (Table A cohort of 350 patients was studied with a mean follow-up of 6.9 years (0.60–23.6 years). Of these patients, 175 were infected with HDV with a mean age of 38.5 years (16.0–67.9), 68.6% were male, and most of the patients were born in Eastern Europe (36.6%). The incidence of cirrhosis at the first visit was higher in patients with HDV infection (45.1%) than in HBV monoinfection (17.7%) (p < 0.01). Decompensation occurred in 68 patients  (p < 0.01). Liver transplantation and/or death was observed in 22.3% of the patients with HDV infection and in 5.7% with HBV infection (p < 0.01). Fourteen patients died  and 37 underwent liver transplantation . HCC (n = 32) was detected in 11.4% of patients with CHD and in 6.9% of patients with CHB (p = 0.1). End points developed earlier in patients infected with HDV . The poor clinical outcome of CHD patients compared to CHB patients was confirmed in Kaplan–Meier analysis (log rank: p < 0.01) , and all of them were anti-HDV positive. At least one liver-related clinical end point was observed in 84 patients; in 35.4% of the CHD patients after a mean follow-up of 4.6 years (0.50–16.2) and in 12.6% of the CHB patients after 6.18 years (0.50–17.7) (p < 0.01) and the Kaplan–Meier analysis  (supplement figure).For better comprehensibility of the matching, we also performed a propensity score matching (CHB: 129 vs. CHD: 129), which confirmed the worse course of hepatitis delta in both the Chi-square analysis analysis  in Chi-square analysis. Besides, CHB patients with cirrhosis developed more often HCC (35.5%) than CHD patients with cirrhosis (18.5%) (p = 0.04). Kaplan–Meier analysis confirmed that there were no significant differences between HBV and HDV in the development of end points (log rank p = 0.6) , LTX, and/or death (log rank p = 0.3). Even the development of HCC was not associated with HDV in the time-depending model (log rank p = 0.3).Interestingly, there were no differences in the development of liver-related clinical end points, decompensation, and liver transplantation and/or death considering patients with cirrhosis only (.6) Fig. , decompep = 0.95; OR: 1.03; 95% CI 0.41–2.56).In this group, we also wanted to provide an exact comparison of the two groups. Due to the unequal distribution of the number of patients (CHB: 31 vs. CHD: 108), the inverse probability of treatment weighting was performed. The subsequent logistic regression showed no differences in the development of end points between CHB and CHD (p < 0.01). In contrast, patients with HDV infection and IFN treatment had a favorable clinical long-term follow-up (p < 0.01), equal to patients with HBV monoinfection   Fig. b. Of notn = 350)  and ALT levels , or HBV DNA , was not associated with the clinical long-term outcome in cirrhotic or non-cirrhotic patients. In univariate COX-regression model, therapy with IFN was linked to a more benign clinical outcome , whereas treatment with NA was associated with the development of end points . Multivariate backward COX-regression model analysis revealed that HDV infection among others was an independent factor associated with the development of end points . It is important to know that age was also independently associated with the clinical long-term outcome .We next investigated which factors correlated with clinical end points in uni- and multivariate Cox-regression analysis for all patients (0) Table a. The anp = 0.61; CI 0.69–1.89), whereas therapy with IFN and therapy with NA was univariably associated with the clinical outcome ,  . In contrast, one systematic review reported an increased risk for the development of HCC in patients with HDV infection with a pooled odds ratio of 1.28 compared to patients with HBV monoinfection, but this association could be confirmed in studies published in Asia only [One further focus was the evaluation of the progression to HCC. CHB patients with cirrhosis developed more frequently HCC (35.5%) than CHD patients with cirrhosis 18.5%)  was based on the gold standard of histopathology in 60 patients only. However, patients who were suspected of having cirrhosis by fibroscan (n = 17) or sonography (n = 33) were re-examined. Importantly, they showed one or more signs of portal hypertension (see methods). Thus, the diagnosis of cirrhosis was confirmed by extensive clinical evaluation. Besides, although we had a large database of 350 patients, the numbers of patients in special subgroups were limited. Moreover, based on the retrospective character of the study, few parameters were missing, especially quantitative HBV DNA and HDV RNA. This disabled us from studying quantitative HDV RNA levels given that this information was available only for a limited number of cases and the quantitative HDV RNA assays have changed over the entire observation period, making it impossible to compare the available HDV RNA values. Additionally, serum samples for retesting of virological parameters with improved assays were not always available, and storage conditions and time might have influenced test results. Moreover, although patients were matched by gender, age, region of origin, HBeAg status and bilirubin, patients with HBV monoinfection had less progressive disease indicated by albumin, INR, and platelet count and had less frequent cirrhosis. However, there were no differences in this parameter, once cirrhosis was present. That is why we additionally performed propensity score matching and inverse probability of treatment weighting. Furthermore, as previously discussed, the benign clinical long-term outcome associated with IFN therapy in CHD patients may be biased by the administration to patients with compensated liver disease only [Several limitations of this study need to be considered. First of all, the diagnosis of cirrhosis Supplementary file2 (EPS 1179 KB)Supplementary file3 (TIF 47 KB)Supplementary file4 (DOCX 13 KB)Below is the link to the electronic supplementary material.


In recent years, improved access to effective antiretroviral therapy has meant that people living with human immune virus are living longer than before. The burden of non-communicable diseases particularly, hypertension parallels with the increase in age. Although hypertension screening is thought to be an effective indicator of overall health status and paves the way for early interventions in peoples living with human immune virus, the exact prevalence of hypertension in this population remained unknown. We aimed to report the prevalence of hypertension and examine the factors associated with hypertension among people living with human immune virus in East Africa.In this systematic review and meta-analysis, we searched PubMed, Science Direct, Scopus, Cochrane library, and Google Scholar databases for studies published until January 1, 2023. The search period was from January 10/2023, to February 10/ 2023. Random-effect models were used to calculate the pooled prevalence of hypertension. Subgroup analyses were conducted to explore potential heterogeneity. The Funnel plot and Egger’s test were used to assess publication bias.A total of 15 studies with 10,916 individuals were included in the present meta-analysis. The pooled prevalence of hypertension among people living with human immune virus was19.75% ),). The prevalence of hypertension was not differed between studies conducted 2014- 2019 and, studies conducted 2020–2022. The prevalence of hypertension was lowest in Ethiopia (16.13%) and highest in Tanzania (26.76%). Alcohol consumption (Adjusted Odds Ratio (AOR): 3.39, 95% CI: 2.35–4.43), diabetes , longer duration of HIV , male sex , obesity , and older age , were the factors associated with the presence of hypertension in people living with human immune virus.Our study shows that one in five peoples living with human immune virus have hypertension causing symptoms and impairment, therefore requiring treatment. Designing effective health screening and hypertension management intervention programs helps to prevent the occurrence of hypertension and promotes peoples’ overall quality of life.The online version contains supplementary material available at 10.1186/s12879-023-08679-x. According to the World Health Organization report in 2018, approximately 38 million people living with HIV (PLHIV) in the world, the largest number being in Africa, with 25.7 million . As a reHypertension is estimated to affect 1.13 billion people worldwide, of which two-thirds live in low and middle-income countries . CurrentFactors contributing to hypertension in PLHIV are multifactorial, inconclusive, and include traditional contributors  –21, and Evidences shows that East Africa is the second most affected region by HIV next to Southern Africa in the world . Given tWe prepared and presented this study according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)  Additio.We identified potentially eligible studies by systematically searching databases of PubMed, Science Direct, Scopus, Cochrane library and Google Scholar that are published until January 1, 2023. The keywords used during the search were:  OR (magnitude)) OR (epidemiology)) AND (hypertension)) OR (high blood pressure)) OR (increased blood pressure)) OR (elevated blood pressure)) AND (Human immunodeficiency)) OR (HIV patients)) OR (HIV/AIDS)) OR (peoples living with HIV)) AND (determinants)) OR (factors)) OR (associated factors)) OR (predictors)) AND (adults)) AND (Ethiopia). Search terms were based on PICO principles to retrieve relevant articles through the databases mentioned above. The search period was from January 10/2023, to February 10/ 2023.Two members of the study team (KDT and NAG) independently screened all titles and abstracts after the initial de-duplication. The inclusion criteria were (1) cross-sectional study, case control study, cohort study, (2) use of a validated measurement tool to assess hypertension, (3) published in English as full length article, (4) inclusion of point prevalence data for the outcome. We excluded researches with fewer than 100 participants (due to high selection bias), conference papers or abstracts, articles without full texts, and studies with data that could not be obtained from the corresponding authors.The primary outcome of interest was the prevalence of hypertension among PLHIV. Hypertension was measured as a persistent elevated blood pressure, Systolic blood pressure (SBP) ≥ 140 mmHg and/or Diastolic blood pressure (DBP) ≥ 90 mmHg, or reported uses of antihypertensive medication .We extracted data from all included studies into a customized Microsoft Excel spreadsheet.Two of the research team (KDT and NAG) independently extracted information regarding author names, study time, publication year, study location, median or mean age, prevalence of hypertension, odds ratio for factors of hypertension among PLHIV. Any disagreements between coders were resolved by the third author (GAA). We assessed all included articles for retraction or any erratum. using the appropriate JBI critical appraisal tool for prevalence studies . When re I2 index were used to evaluate heterogeneity between studies. The I2 index refers to the truly observed variation ratio [I2 respectively indicate low, medium, and high heterogeneity [A random-effects model for meta-analysis was used because of assumed heterogeneity between the studies.on ratio , and 25%ogeneity . We asseogeneity . The resOf the 2136 articles identified by applying search strategies, 1989 articles were excluded after a review of the title or abstract, leaving 147 articles assessed for full-text review, of which 15 met the eligibility criteria for this systematic review and meta-analysis.  used a cross-sectional study design [n = 12, 80%). As a screening criteria for hypertension, all studies used a cutoff point of blood pressure “SBP ≥ 140 mmHg, DBP ≥ 90 mmHg. Sander LD et al. reported the lowest prevalence of hypertension, 7.98%, whereas the highest prevalence of hypertension was reported by Memiah P et al., 2021. Therefore, fifteen studies were evaluated, and the minimum quality score given is 5 out of quality scale which is 62.50%, indicating that they are low risk and included in the analysis   and those conducted between (2020 and 2022), and by geographical regions. The prevalence of hypertension was not different in studies conducted between (2014 and 2019), and those conducted between (2020 and 2022). Regarding study location, the prevalence of hypertension was highest in Tanzania and lowest in Ethiopia (26.76% VS 16.13%). .We performed a subjective evaluation of publication bias through visual inspection of funnel plot and objective evaluation by running eggers test. The funnel plot shows a symmetrical distribution of studies from the line of effect Fig. . Eggers p > 0.050)  was associated with the pooled estimates (0) Table .Table 2MTo assess the impact of an individual study on the pooled estimates, sensitivity analysis was conducted by excluding one study alternatively. Sensitivity analyses yielded no individual study influenced the pooled prevalence of hypertension among peoples living with HIV. When one study omitted from the analysis alternatively, the pooled prevalence of hypertension ranged from 18.28% to 20.50% Fig. .Fig. 6ReTable 2 = 82.4%, p = 0.003).The result is shown in Fig. 2 = 66.8%, p = 0.049).Three studies , 32, 35 2 = 87.8%, p = 0.000).Five studies examined the associations of longer duration of HIV with hypertension in patients living with HIV. 4 –33, 37 r2 = 0.00%, p = 0.440).Three studies examined the associations of being male with hypertension among PLHIV. Two reported increased risk of hypertension with being male, and 1 revealed no associations. Male living with HIV were 1.62 times more likely to develop hypertension .Eight studies , 35, 39 2 = 97.8%, p = 0.000).Eight studies assessed the association of older age with hypertension among PLHIV. All studies revealed that older age was associated with hypertension in peoples living with HIV. Older PLHIV were 2.25 times more likely to have hypertension than younger ages , 21.2% . However2 = 96.0%) is somewhat explained by subgroup analysis by study location with no heterogeneity among studies conducted in Kenya (I2 = 24.8%) and Burundi (I2 = 0.00%). Subgroup analysis by publication year showed no significant difference between studies published between (2014 and 2019) and those published between (2020 and 2022). In areas where the prevalence of hypertension is higher among PLHIV, integrating hypertension services into routine HIV care by capitalizing the preexisting health system and following multidisciplinary approaches will be key [The subgroup analysis reported on basis of study location and publication year. Based on Country, Tanzania reported the highest prevalence of hypertension while the lowest was observed in Ethiopia (26.76% versus 16.13%). The highest heterogeneity observed in the overall meta-analysis . The eight-item questions assessing inclusion criteria, study setting and participant, exposure measurement, objectives, confounder, statically analysis, outcome measurement, and dealing confounder were used.


Streptococcus intermedius (S. intermedius) infection.To describe the clinical significance of prompt, adequate, and targeted intravenous antibiotic (IV antibiotic) therapy in the successful management of spinal epidural abscess (SEA) associated with S. intermedius infection, and gadolinium-enhanced magnetic resonance imaging (MRI) demonstrated widespread epidural abscesses in the cervical, thoracic, and lumbar spine canal. Empirical IV antibiotic therapy with vancomycin was promptly initiated, with meropenem and moxifloxacin added subsequently based on blood culture results. After 5 days of IV antibiotic treatment, the patient’s blood culture became negative. 6 weeks later, a follow-up MRI showed a decrease in the size of the abscess. The patient’s muscle strength was mostly restored after 2 months of IV antibiotic treatment.SEA is a rare, but catastrophic infection that may result in a high risk of permanent neurological disability. A 52-year-old Chinese female patient was presented to the emergency department due to 2 years of low back pain and 3 days of decreased muscle strength in the extremities. The blood culture confirmed the presence of Streptococcus intermedius infection, surgery may be non-essential, and the judicious antibiotic selection and adequate treatment duration are pivotal for successful conservative management. Furthermore, for patients who are not amenable to surgery, a comprehensive evaluation of their condition and meticulous implementation of a precise pharmacological regimen holds noteworthy clinical significance.Repeat examinations or gadolinium-enhanced MRI should be considered when initial MRI findings are not diagnostic of SEA. For extensive SEA caused by  Streptococcus intermedius (S. intermedius) with abscess formation has long been recognized. Streptococcus intermedius, also referred to as Streptococcus milleri group (SMG) (Streptococcus anginosus group (SAG). Within this group, S. intermedius has the capability to secrete a novel cytotoxin known as intermedilysin, which specifically targets human cells. Intermedilysin exhibits potent hemolytic activity exclusively toward human erythrocytes and no hemolysis on equine or goat blood agar, commonly employed in clinical microbiology. However, because this cytotoxin exhibits beta-hemolytic activity on human blood agar medium, S. intermedius is classified as a beta-hemolytic Gram-positive coccus. The first reported case of a brain abscess caused by S. intermedius dates back to 1975  is a rare yet potentially devastating pyogenic injury that occurs between the spinal dura mater, posterior longitudinal ligament, and vertebral periosteum . Rapid aup (SMG) , is a me to 1975 . Subsequa et al.  conducte9/L , 85% neutrophils , increased C-reactive protein (CRP) levels of 25.03 mg/dL , and increased procalcitonin (PCT) levels of 1.96 ng/mL . Although a fluoroscopic-guided lumbar puncture was unsuccessful, an intravenous gadolinium-enhanced magnetic resonance imaging (MRI) revealed an abnormal signal shadow ventral to the spinal canal in the cervicothoracic segment and dorsal to T12-S1 level  was initiated, followed by the addition of meropenem (1 g ivd q8h) and moxifloxacin (0.4 g ivd qd) after receiving the blood culture results. Vancomycin dosing was adjusted dynamically based on blood concentration. The patient’s clinical presentation of paraplegia and decreased muscle strength did not improve during the first 2 days of initial antibiotic treatment. The spine surgeons, in conjunction with the ICU physicians, discussed the following results: The patient’s rapidly progressing and worsening neurologic symptoms within 72 h require urgent decompression surgery. However, the cervicothoracic segment abscess located ventrally may not drain adequately. Abscesses of the lumbar segment located dorsally have relatively poor outcomes after decompression surgery, and there is no additional benefit to early surgical treatment. Patients may not tolerate the shock of prolonged decompressive surgery of multiple spinal segments. Therefore, the patient was informed that progressive neurological deterioration may not be completely resolved even after delayed surgical treatment. After obtaining informed consent, we decided to continue treatment with antibiotics for at least 6 weeks. After 5 days of IV antibiotic treatment, blood culture results were negative. Following 1 month of antibiotic treatment, the patient’s maximum temperature decreased to below 38.0°C, and her WBC count was 10 × 109/L with a CRP level of 6.19 mg/dL. After 43 days of IV antibiotic treatment, the patient exhibited grade 4 muscle strength bilaterally in the biceps, triceps, finger flexors, and finger extensors. At this time, a repeat MRI showed a significant reduction in the holospinal abscess, particularly in the upper lumbar segment  6 years prior, which was performed under combined spinal-epidural anesthesia, with no reported history of diabetes or other diseases. Upon physical examination, the patient exhibited hypoesthesia below the inguinal plane, as well as biceps, triceps, finger flexors, and finger extensors muscle strength of grade 3 on both sides, muscle strength of grade 1 in both lower limbs, no fecal incontinence, and intact rectal tone. Upon admission, the patient presented with shortness of breath and confusion. A large amount of light red, thick sputum was aspirated by sputum suction. After face mask oxygenation, oxygen saturation was around 80%. Therefore, the patient was transferred to the intensive care unit (ICU) due to type II respiratory failure and a maximum temperature of 40.8°C. Blood tests showed elevated infection markers, including a total white blood cell (WBC) count of 26.6 × 10S1 level . Blood c segment . After 4 segment , and theStaphylococcus aureus (MRSA), and the exotoxins, extracellular enzymes, and cell surface substances of MRSA can cause ischemic changes and tissue destruction in the spinal cord through various mechanisms, ultimately leading to spinal cord injury (staphylococci (7.5%), and Streptococcus anginosus group   .S. intermedius. S. intermedius is one of the three species found in the SAG, also known as the Streptococcus milleri group (SMG)  years, and four of the seven patients were women. In contrast to the case report mentioned in S. intermedius and Streptococcus griseus. The patient was treated with IV antibiotics and recovered completely. Although Gangone et al.  . SAG is up (SMG) , 14. Altup (SMG) . We repoe et al. . found nSEA is typically characterized by the triad of fever, back pain, and neurological dysfunction. However, only 0.8% of patients exhibit this classic presentation on admission, making the diagnosis of SEA challenging . The prePasteurella multocida  for the publication of any potentially identifiable images or data included in this article.DL conducted the data analysis and wrote the first draft. WL critically revised the article. WH and WZ rearranged the figures and enriched the interpretation of the figures. QL was directly responsible for the manuscript. All authors made significant contributions to the article’s conception and design.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Since the introduction of higher valency pneumococcal conjugate vaccines in 2009, recent estimates on the economic burden of pediatric pneumococcal disease (PD) in Germany have been lacking. This study estimates healthcare resource utilization (HCRU) and medical cost associated with PDs in children < 16 years old in Germany from 2014–2019.A nationally representative sample from the Institute for Applied Health Research (InGef) German claims database was used, covering approximately 5% of the total German population. Episodes of pneumococcal pneumonia (PP), all-cause pneumonia (ACP), invasive pneumococcal disease (IPD), and acute otitis media (AOM) in children aged < 16 years were identified using ICD-10-GM codes. HCRU was estimated from annual rates of outpatient visits, outpatient antibiotic prescriptions and inpatient admissions, divided by person-years (PY) at-risk. Average direct medical costs per episode were estimated as the total cost of all HCRU, divided by the total number of episodes. The Mann–Kendall test was used to assess monotonic time trends from 2014–2019. During 2014–2019, 916,805 children aged < 16 years were followed up for a total of 3,608,716 PY. The average costs per episode for out-versus inpatient care associated with PP and ACP were €67 (95% CI 58–76) versus €2,606 , and €63 (95% CI 62–63) versus €620 (95% CI 598–641), respectively. For IPD, the average medical cost per episode for out-versus inpatients were €30 (95% CI 19–42) versus €6,051 , respectively. There were no significant trends in HCRU or costs for IPD or pneumonia over the study period, except for a significant reduction in ACP outpatient visits. A significant decrease in rate of outpatient visits and antibiotic prescribing for recurrent AOM was observed, in addition to an increase in rates of hospital admissions for simple AOM. This was paralleled by a significant increase in inpatient costs per episode for treating AOM overall, and simple AOM, over the study period. The HCRU and cost per episode of pneumonia and IPD did not vary significantly from 2014–2019, but increased for AOM. The economic burden of pneumonia, IPD, and AOM remains substantial in Germany.The online version contains supplementary material available at 10.1186/s41479-023-00105-9. Streptococcus pneumoniae (S. pneumoniae), range from respiratory tract infections, such as non-invasive pneumococcal pneumonia and acute otitis media (AOM), to invasive pneumococcal disease (IPD) including bacteremic pneumonia, bacteremia without focus, and meningitis ).The database includes demographic information, diagnoses and mortality data, hospitalizations, ambulatory services and procedures, and drug prescription and dispensation data. All diagnoses are recorded using the German modification of the 10st of the respective quarter or the first day of insurance) or the date they started contributing data to the InGef research database. For those children born before 2014, study entry date was assigned as the latter of January 1st, 2014 and the date they started contributing data to the InGef research database within the study period.This retrospective cohort study included children < 16 years of age within the InGef database, between January 1, 2014 and December 31, 2019. Children who were born during the study period were included in the study from their estimated date of birth (the 1st) or the date they started contributing data to the InGef research database, until the first of the following censoring criteria: end of observation in the InGef research database , end of study year (December 31st) or end of study period .Six yearly cohorts were established (2014 to 2019). During each study year, each individual was followed-up from the start of the calendar year  sex  and underlying medical conditions. Underlying medical conditions linked to higher risk of PD were described according to the 2017/2018 German standing committee on vaccination (STIKO) recommendations for at-risk/high-risk individuals and data availability within the InGef database [Episodes of PD were identified using ICD-10-GM codes from the outpatient and inpatient data  of the InGef database. Multiple definitions were used for each manifestation of PD . Exact code definitions are outlined in the S. pneumoniae (pneumococcus) was known to have a causative role as identified from pneumococcal-specific ICD-10-GM codes. ACP was defined as pneumonia cases caused by all unknown and known pathogens  and all-cause (ACP) pneumonia—both of which excluded any IPD. PP was defined as cases where gens see .IPD included cases of meningitis, bacteremia without focus, and bacteremic pneumonia where pneumococcus was known to have a causative role as identified from pneumococcal-specific ICD-10-GM codes.Three definitions of AOM were used, AOM overall, simple, and recurrent. AOM included acute suppurative otitis media, suppurative and unspecified otitis media caused by all known and unknown pathogens through ICD-10 GM codes for AOM. Episodes were further classified as simple or recurrent, in alignment with the widely accepted definition of recurrent AOM first coined by Goycoolea in 1991 . An episFor outpatients, only diagnosis by calendar quarter is available in the database therefore at least one prescription of an antibiotic prescription or diagnostic test in the same quarter was required to accompany an outpatient diagnosis of PD. The date of first antibiotic prescription or diagnostic test was assigned as the date of diagnosis. Date of hospital admission was used to assign date of diagnosis within the inpatient data.Since the analyses were conducted by calendar year, episodes were assigned to each study year. Episodes that crossed calendar years were assigned to the year in which the episode began. For pneumonia and IPD, multiple records were considered as independent episodes if separated by ≥ 90 days . Each pnHCRU and costs were estimated for each PD manifestation. Specifically, HCRU included number of outpatient visits, number of outpatient antibiotic prescriptions, number of inpatient admissions and mean length of stay. The costs associated with each episode included costs associated with each outpatient visit, outpatient pharmacy cost of antibiotic prescriptions during each episode and costs associated with each inpatient hospital admission. Antibiotic prescriptions were captured in the outpatient setting but since indication is not recorded, all antibiotics prescribed during an episode of PD were assumed to be related. Inpatient costs were calculated using the German diagnosis-related groups reimbursement system for hospital admissions, capturing admissions with PD as the main discharge diagnosis .Rate of outpatient visits, prescriptions and rate of hospital admissions were calculated per 1,000 person-years (PY), as total amount of each resource used, divided by total amount of time with PD. 95% confidence intervals (CI) were calculated using the Clopper-Pearson method assuming a Poisson distribution . Annual The final study population included 916,805 children aged < 16 years. These individuals contributed a total of 3,608,716 PY at-risk and were followed up for a median of 4.25 (interquartile range 2.0–6.0) years.At annual cohort entry, the mean age of individuals was 6 years  Table . The majFrom 2014 through 2019 the majority of ACP (90%) patients and PP patients (67%) were seen in the outpatient setting  and €683 (95% CI 661–704), respectively. Costs per episode were much greater for inpatient versus outpatient care. Outpatient costs per episode were similar for PP and ACP: €67 (95% CI 58–76) and €63 (95% CI 62–63), for PP and ACP, respectively. This was also the case for inpatient cost per episode: €2,606  and €398 (95% CI 381–416), respectively. Pharmacy costs per episode were similar for both pneumonia definitions: PP €26 (95% CI 22–30), and ACP €21 (95% CI 21–21). The total inpatient and outpatient cost for PP and ACP, respectively, were €633,473 and €46,215,974, throughout the study period.p = 0.024  in 2014 to 5,293  in 2019, The majority of IPD patients were seen in the inpatient setting (88%) Table . The meaFrom 2014 to 2019, the average direct medical cost per IPD episode was €6,081 . Total outpatient versus inpatient costs per episode were €30 (95% CI 19–42) versus €6,051 . Pharmacy costs per episode were €17 (95% CI 9–24). The total inpatient and outpatient cost for IPD was €711,523, throughout the study period.The Mann–Kendall tests indicated no significant trends in HCRU , costs or prescribing rates for IPD over the study period Tables , 5, 6.The majority of AOM patients were seen in outpatients, regardless of whether it was simple or recurrent AOM (99%) Table . The outThe average direct medical cost per episode of AOM for outpatients and inpatients during 2014 to 2019 was €59 (CI 58–59); similar results were reported for simple and recurrent AOM. The average outpatient costs per episode were the same for all definitions of AOM: €46 (95% CI 46–46) for AOM overall and simple AOM, and €45 (95% CI 45–46) for recurrent AOM. The average pharmacy outpatient cost was similar for the three definitions, €15 (95% CI 15–15). The total inpatient and outpatient cost for AOM throughout the study period, was €19,220,490.p = 0.027), and from €10 (95% CI 8–11) to €17 (95% CI 15–19) (p = 0.051), respectively. No significant trends were found for outpatient costs.The Mann–Kendall tests indicated significant decreases in rates of outpatient visits for recurrent AOM and an increase in rates of hospital admissions for simple AOM Table . Similarp = 0.008).For AOM overall, simple AOM and recurrent AOM, the rates of antibiotic prescriptions  went from 27,097  to 26,536 , from 27,247  to 26,761 , and from 26,372  to 24,987 , respectively, from 2014 to 2019  per AOM episode have been reported to range from €332.00 in the Netherlands to €752.49 in the UK . Beyond The data from this study demonstrate that the economic burden of PD in children in Germany remains substantial following introduction of PCV13. The cost-effectiveness of PCVs in children is evident from the literature –44. HoweThere were several limitations to this study. Firstly, although the InGef database is representative of the German population in terms of age, sex, morbidity, mortality and drug usage , it is uIn addition, there are further limitations that relate to the structure of the InGef database. In the outpatient data, only yearly quarters in which diagnoses were made were available, not exact diagnosis dates which may have underestimated the number of PD episodes in the outpatient data. Antibiotic prescriptions during the quarters with the diagnoses were assumed to be related to the PD diagnosis to assign exact diagnosis dates thus potentially overestimating the number of antibiotic prescriptions being prescribed as a result of a PD episode.The purpose of the present study was to estimate the direct medical costs associated with PD in Germany. Wider societal, vaccination or other indirect costs were therefore not assessed in the present study, but are nevertheless important when considering the broader economic burden of PD. Thus, the burden of PD reported in the present study is likely an underestimate.In Germany, between 2014 and 2019, HCRU and average cost per episode did not vary significantly for pneumonia and IPD, but for AOM, the annual cost per episode increased. The economic burden of pneumonia, IPD and AOM remains substantial in children aged < 16 years in Germany.Additional file 1: Supplementary Table 1. Definitions of pneumonia cases. Supplementary Table 2. Definitions of IPD cases. Supplementary Table 3. Definitions of AOM cases.


Microbial interactions are fundamental for Earth’s ecosystem functioning and biogeochemical cycling. Nevertheless, they are challenging to identify and remain barely known. Omics-based censuses are helpful in predicting microbial interactions through the statistical inference of single (static) association networks. Yet, microbial interactions are dynamic and we have limited knowledge of how they change over time. Here, we investigate the dynamics of microbial associations in a 10-year marine time series in the Mediterranean Sea using an approach inferring a time-resolved  network from a single static network.A single static network including microbial eukaryotes and bacteria was built using metabarcoding data derived from 120 monthly samples. For the decade, we aimed to identify persistent, seasonal, and temporary microbial associations by determining a temporal network that captures the interactome of each individual sample. We found that the temporal network appears to follow an annual cycle, collapsing, and reassembling when transiting between colder and warmer waters. We observed higher association repeatability in colder than in warmer months. Only 16 associations could be validated using observations reported in literature, underlining our knowledge gap in marine microbial ecological interactions.Our results indicate that marine microbial associations follow recurrent temporal dynamics in temperate zones, which need to be accounted for to better understand the functioning of the ocean microbiome. The constructed marine temporal network may serve as a resource for testing season-specific microbial interaction hypotheses. The applied approach can be transferred to microbiome studies in other ecosystems.Video AbstractThe online version contains supplementary material available at 10.1186/s40168-023-01523-z. The t species  and ≈ 1030 cells , 6. In pal cells , 8.abiotic factors in structuring microbial communities. For example, the temperature is one of the main factors exerting selection in the ocean microbiome over spatiotemporal scales [Biotic factors can also strongly affect microbial communities [Microbial communities are highly dynamic, and their composition is determined through a combination of ecological processes: selection, dispersal, drift, and speciation . Selectil scales –15. Biotmunities . Howevermunities , 17.The vast microbial diversity and the fact that most microorganisms are still uncultured , 19 makeAs microorganisms are highly interconnected , associaPrevious studies have used temporal microbial abundance data to infer static networks summarizing all potential associations in space and time. This static abstraction assumes that the network topology does not change (static) and edges represent persistent associations assumed as interactions ; that isDespite the contribution of static networks to our understanding of microbial interactions in the ocean, it is necessary to incorporate the temporal dimension. Using a time-resolved, i.e., temporal network instead of a single static network would allow investigation of the dynamic nature of microbial associations and how they change over time, whether the change is deterministic or stochastic, and how environmental selection influences network architecture. Addressing these questions is fundamental for a better understanding of the dynamic interactions that underpin ecosystem function in the ocean. Here, we investigated marine microbial associations through time by determining a temporal network from a single static network.◦ 40′ N, 2◦ 48′ E) without major natural disturbances and little anthropogenic pressure, except for the construction of a nearby harbor between 2010 and 2012 [The BBMO is a coastal oligotrophic site in the North-Western Mediterranean Sea  monthly from January 2004 to December 2013 to determine microbial community composition and also measured ten environmental variables, which were previously described [◦C) and salinity (obtained in situ with a SAIV-AS-SD204 Conductivity-Temperature-Depth probe), day-length (hours of light), turbidity (Secchi depth in meters), total chlorophyll-a concentration , and fianalyzer ).–20 µm) and picoplankton (0.2–3 µm) using a 3-µm and 0.2-µm pore-size polycarbonate and Sterivex filters, respectively. Then, the DNA was extracted from the filters using a phenol–chloroform protocol [Sampling of microbial communities, DNA extraction, rRNA-gene amplification, sequencing, and bioinformatic analyses are explained in detail in . In shorprotocol , which hprotocol , and theprotocol  and 806Rprotocol . Amplicoprotocol , v1.10.1rrarefy function from the Vegan R-package [imputeTS R-package, v2.8 [Microbial sequence abundance tables were obtained for each size fraction for both microbial eukaryotes and prokaryotes. Before merging the tables, we subsampled each table to the lowest sequencing depth of 4907 reads with the -package , v2.4–2,-package ). We excge, v2.8 . These ige, v2.8 .Sequence taxonomy was inferred using the naïve Bayesian classifier method  together.5. This operation removed two eukaryotic ASVs and 41 bacterial ASVs from the nanoplankton, and 30 bacterial ASVs from the picoplankton  was calculated based on the p values using the p.adjust function from the stats R-package [p and q values, as suggested in other studies [First, we constructed a preliminary network using the tool eLSA , 47, as -package . We used studies . Further studies . The preSecond, for environmentally driven edge detection, we applied EnDED , combiniThird, we determined the Jaccard index, ous work , which rmentclass2pt{minime be an association between microorganisms A and B, with association duration d = , i.e., the association starts at time point t1 and ends at t2. Then, considering month m, the association e is present in the monthly subnetwork Nm, if: We determined the temporal network comprising 120 sample-specific (monthly) subnetworks through the three conditions indicated below and visualized in Fig. e is an association in the single static networkA and B are present within month mThe microorganisms m is within the duration of association, i.e., t1 ≤ m ≤ t2.With the second condition, we assumed that an association was present in a month if both microorganisms were present, i.e., the microbial abundances were non-zero for that month. However, we cannot assume that microbial co-occurrence is a sufficient condition for a microbial interaction because different mechanisms influence species and interactions, and the environmental filtering of species and interactions can differ . Using oedge density, average path length, transitivity, and assortativity based on node degree. In addition, we also computed the average strength of positive associations between microorganisms using the mean, and assortativity based on the nominal classification of nodes into bacteria and eukaryotes. Assortativity (bacteria vs. eukaryotes) is positive if bacteria tend to connect with bacteria and eukaryotes tend to connect with eukaryotes. It is negative if bacteria tend to connect to eukaryotes and vice versa. We also quantified associations by calculating their prevalence as the fraction of monthly subnetworks in which the association was present for all 10 years (recurrence) and monthly. We visualized highly prevalent associations with the circlize R-package [p values [corr.test in the psych R-package [We computed global network metrics to characterize the single static network and each monthly subnetwork using the igraph R-package . Some me-package . We testp values , with th-package . We used-package , v.0.9.2-package  for netwm is within the duration of association, i.e., t1 ≤ m ≤ t2) to infer the sample-specific subnetworks. Other methods may perform better on compositional data such as ours [We have used eLSA to estimate the duration of an association, which we used as the third condition . Theref as ours ) for com as ours . FlashWe as ours , we appl as ours , selectiSynechococcus and Prochlorococcus RNAs with the option e value 1.0e − 5. We found 2812 sequences comprising 95 different ecotypes , with 93.84–100% identity. A total of 11 BBMO ASVs obtained 63 hits with 100% identity, and within these 63 reference sequences, there were 34 different ecotypes. Most matching sequences were found for Synechococcus ASV_1. While Synechococcus ASV_5 had only two 100% hits, they did not 100% match ASV_1  and a dinoflagellate (Alveolate).From 10 years of monthly samples from the Blanes Bay Microbial Observatory (BBMO) in the Mediterranean Sea , we compWe used 1709 ASVs to infer a preliminary association network with the tool eLSA , 47. NexNext, we developed an approach to determine a temporal network. Building upon the single static network, we determined 120 sample-specific (monthly) subnetworks (see the “Methods” section for details). These monthly subnetworks represent the 120 months of the time series and together comprise the temporal network. Each monthly subnetwork contains a subset of the nodes and a subset of the edges of the single static network. We used the ASV abundances indicating the presence (ASV abundance > 0) or absence (ASV abundance = 0) as well as the estimated start and duration of associations inferred with the network construction tool eLSA , 47 for Since each monthly subnetwork was derived from the single static network, they were smaller, containing between 141 August 2005) and 571 (January 2012) nodes, median ≈354 , and total chlorophyll-a concentration to affect network topologies as indicated by correlation analyses , lost, and gained (new) in consecutive months. We found the highest loss of edges in April, pointing to a network collapse. The overall number of edges (preserved and gained) was lowest during April–September and increased towards the end of each year Fig. B. The nuWe defined summer and winter as in  and compA single static network can comprise permanent, seasonal, and temporary associations. By comparing monthly subnetworks, we identified edges that remain (preserved), appear (gained), or disappear (lost) over time Fig. B. IntuitPelagibacter ubique (SAR11 Clades Ia & II), Rhodobacteraceae, Amylibacter, Puniceispirillales (SAR116), Ascidiaceihabitans, Planktomarina, Parvibaculales (OCS116), and Kiloniella. Between April and May, we noticed a large increase in the fraction of associations including Cyanobacteria or Bacteroidetes as association partners. While Cyanobacteria associations were a small fraction during November–April, they had a dominant role from May to October along with Bacteroidetes and Alphaproteobacteria associations  was negatively associated (association score − 0.5) with other Synechococcus (bn_ASV_1 and bn_ASV_25), which, in turn, were positively associated (association score 0.8). While bn_ASV_5 appeared mainly in colder months, the other two appeared mainly in warmer months  association partners were other Cyanobacteria, which contrasted with Synechococcus and Cyanobium  connected to one Deltaproteobacteria (SAR324) ASV during the first 8 years, but the association disappeared in the last 2 years. In particular, the inferred association duration was 101 months, starting in March 2004 and ending in July 2012. After summer 2012, the Deltaproteobacteria ASV was not detected except a few reads in November and December 2012 and 2013. This Cyanobacteria example may also illustrate the dynamics of associations within other main taxonomic groups.Within the temporal network, the fraction of Cyanobacteria associations was highest in April–October Fig. A, which Thalassiosira and a Flavobacteriia starts mainly around October and often ends around March . In totPrevious work identified yearly recurrence of microbial community composition at the BBMO , 28, 64,The effects of environmental variables on network metrics are unclear , yet, ouOur preliminary network (significant associations derived with eLSA) contained 18% negative edges compared to 0.9% in the single static network (after filtering). Thus, our filtering strategy removed proportionally more negative edges. Associations may represent positive or negative interactions, but they can also indicate high niche overlap (positive association) or divergent niches (negative association) between microorganisms . We hypoWe found more highly prevalent associations within specific months than when considering all 10 years of data. Furthermore, our results indicate a potentially low number of core interactions and a vast number of non-core ones. Usually, core microorganisms are defined based on sequence abundances, as those microorganisms  appearing in all samples or habitats being under investigation . Shade aProchlorococcus and Synechococcus) with two potential eukaryote grazers (a ciliate and a dinoflagellate), 39 to other bacteria, and three between Cyanobacteria, which were all positive. Similarly, all cyanobacterial ASVs in our study connected primarily to other bacterial ASVs and featured mainly positive associations. Furthermore, Cyanobacteria displayed primarily positive associations with other microorganisms in a global ocean network [Several network-based analyses have been used to particularly study Cyanobacteria associations. For example, in the southern Californian coast, Chow et al.  identifi network . This suSynechococcus ASVs [Identifying different potential association partners for closely related Cyanobacteria may indicate adaptations to different niches. A recent study found distinct seasonal patterns for closely related bacterial taxa indicating niche partitioning at the BBMO, including cus ASVs . Our appcus ASVs .Our approach allowed us to disentangle in time the associations captured by a single static network using monthly samples for ten years. Future studies should determine whether higher sampling frequency  can capture other associations not present in our networks. Thus, our results should be considered taking into account the used (monthly) sampling frequency. In addition, certain network metrics may depend on the tool used to infer the single static network, e.g., edge density, and, therefore, should be interpreted with care. An additional consideration is that we disregarded local network patterns by using global network metrics. Future work could use the local-topological metric based on graphlets . CountinIncorporating the temporal dimension in microbial association analysis unveiled multiple patterns that often remain hidden when using single static networks. Investigating a coastal marine microbial ecosystem over 10 years revealed a 1-year-periodicity in the network topology. The temporal network architecture was not stochastic, but displayed a modest amount of recurrence over time, especially in winter. Future efforts to understand the ocean microbiome should consider the dynamics of microbial interactions as these are likely fundamental for ecosystem functioning.Additional file 1: Supplementary Figure 1. Correlation analysis. Using the temporal network, we correlated six global network metrics with environmental factors including the nutrients PO43−, NH4+, NO2−, NO3−, and SiO2. The global network metrics were: Edge density, Average positive association (Avg. pos. ass.) score, Transitivity, Average path length (Avg. path length), Assortativity (degree), and Assortativity (bacteria vs. eukaryote). Each dot is a sample-specific subnetwork and its color indicates the month it represents. Also, the linear regression line with a 0.95 confidence interval is shown in grey.Additional file 2: Supplementary Figure 2. Correlation analysis through linear regression. Using the temporal network, we correlated six global network metrics with environmental factors including the nutrients PO43−, NH4+, NO2−, NO3−, and SiO2. The global network metrics were: Edge density, Average positive association (Avg. pos. ass.) score, Transitivity, Average path length (Avg. path length), Assortativity (degree), and Assortativity (bacteria vs. eukaryote). The number, circle's size, and color in the square correspond to the Spearman correlation scores, no circle indicates non-significance.Additional file 3: Supplementary Figure 3. Global (sub)network metrics. Number of nodes, number of edges, and six selected global network metrics for each sample-specific subnetwork of the temporal network determined with FlashWeave.Additional file 4: Supplementary Figure 4. Number of preserved, gained, and lost edges in summer and winter. A) Indicates how we determined summer, shown with red dots (temperature above 17 ºC and day length above 14 hours) and winter, shown with blue dots (temperature below 17 ºC and day length below 11 hours); grey dots indicate months that are neither summer nor winter. B) Accumulation curve of ASVs per year for winter (blue) and summer (red). C) and D) number of preserved, gained, and lost edges for winter and summer, respectively. The colors of flows indicate the prevalence of an edge with 10 (light blue) being present in each year, and 1 (dark blue) appearing in only one year. An edge appears in a year if it appears in at least one monthly subnetwork in the corresponding season. In winter, most edges appear in all years , i.e., most edges are preserved in the consecutive months (we see a flow from the blue preserved box to the next blue box). In summer, compared to winter, fewer edges are present in a month , and more edges are (re)gained and lost throughout the years .Additional file 5: Supplementary Figure 5. Association prevalence increases slightly when microorganisms are taxonomically more related. We grouped the associations according to the taxonomic classification of association partners (columns) and size fractions (rows). For example “Class” groups associations between bacteria and eukaryotes, respectively, which were assigned to the same class. The grey column groups associations between bacteria and eukaryotes. The boxplot shows the association prevalence over a decade, i.e., in how many monthly subnetworks an association appears (given as a fraction from 0 to 100% = 120 networks).Additional file 6: Supplementary Figure 6. Association prevalence per month. Big bar plots: distribution of associations' prevalence for each month. For example, the bar at 100 for January indicates the number of edges that have been present in all Januarys of the ten-year time series. Small bar plots: number of nodes forming the associations with a 100% prevalence. For example, only bacteria were responsible for the edges during May, with an association prevalence of 100%. Bacteria are indicated with B or b, eukaryote with E or e. ASVs from the nano size-fraction have a capital letter , and ASVs from the pico size-fraction have a small letter .Additional file 7: Supplementary Figure 7. Association partners of Cyanobacteria. The number of Cyanobacteria associations in the temporal network (stacked bars) and the cyanobacterial sequence abundance in each month (black dashed line). Within the box, figures are split by ASVs (rows) and size fractions: picoplankton (left column) and nanoplankton (right column). The unboxed plots on the right are ASVs detected only in the nanoplankton. The height of the bar indicates the number of edges in each month for each cyanobacterial ASV. The color indicates the taxonomy of the association partner. From bottom to top, first appear bacteria, and then eukaryotes, both sorted alphabetically. The subtitle shows the number of association partners followed by an identifier (first 3 letters) for bacteria and eukaryotes.Additional file 8: Supplementary Figure 8. Microbial association partners that have been reported in the literature. Found associations in the temporal network (one association per panel and a black dot on the bottom shows presence in the monthly subnetwork) and the sequence abundance in each month (solid and dashed lines). The color and line type indicate the taxonomy of the association partners.Additional file 9: Supplementary Table 1. Number of nodes, removed isolated nodes, and number and fraction of edges in the preliminary network (A), and network obtained after removing environmentally-driven edges (B) and edges with association partners appearing more often alone than with the partner (C), which is the single static network. For comparison, we also give the minimum and maximum number of nodes and edges for the temporal network (D). We did not determine the union and intersection for the temporal network. If an ASV appeared in the nano and pico size fraction, it is counted twice. Therefore, for A-C) we also determined the number of microorganisms not considering size fraction (union) and being present in both size fractions .Additional file 10: Supplementary Table 2. Top 100 most prevalent/recurring associations.Additional file 11: Supplementary Table 3. Number of environmental factors leading to the removal of edges.Additional file 12: Supplementary Table 4. Number of environmentally-driven edges for each environmental factor and fraction considering the total number of edges (29820) in the network. In addition, we present the number of positive and negative edges and the fraction considering the number of edges removed through an environmental factor.Additional file 13: Supplementary Table 5. 100% Matching sequences from Cyanorak database for selected cyanobacterial ASVs.Additional file 14: Supplementary Table 6. Interactions found in the BBMO temporal network that have been reported in the literature. The table shows the number of associations found in the network. For example, the association between the ASVs classified as Dia. Thalassiosira and ASVs classified as F. unknown Flavobacteriia has been found 6 times in the network.