Background: We performed a meta-analysis to evaluate the outcomes of minimally invasive surgery and open surgery in the simultaneous resection of colorectal cancer and synchronous colorectal liver metastases.Methods: A systematic literature search up to April 2021 was done and 13 studies included 1,181 subjects with colorectal cancer and synchronous colorectal liver metastases at the start of the study; 425 of them were using minimally invasive surgery and 756 were open surgery. They were reporting relationships between the outcomes of minimally invasive surgery and open surgery in the simultaneous resection of colorectal cancer and synchronous colorectal liver metastases. We calculated the odds ratio (OR) or the mean difference (MD) with 95% CIs to assess the outcomes of minimally invasive surgery and open surgery in the simultaneous resection of colorectal cancer and synchronous colorectal liver metastases using the dichotomous or continuous method with a random or fixed-effect model.Results: Minimally invasive surgery in subjects with colorectal cancer and synchronous colorectal liver metastases was significantly related to longer operation time , less blood loss , less blood transfusion needs , shorter length of hospital stay , lower overall complications , higher overall survival , and higher disease-free survival  compared to open surgery.Conclusions: Minimally invasive surgery in subjects with colorectal cancer and synchronous colorectal liver metastases may have less blood loss, less blood transfusion needs, shorter length of hospital stay, lower overall complications, higher overall survival, and higher disease-free survival with longer operation time compared with the open surgery. Furthers studies are required to validate these findings. Colorectal cancer is one of the main reasons for cancer-related mortality in the world. The frequency and death of colorectal cancer are the third and the second, respectively, of all types of cancer in the world . AccordiThe present study followed the meta-analysis of studies in the epidemiology statement , which wIncluded studies were that with statistical measures of relationship  between the outcomes of minimally invasive surgery and open surgery in the simultaneous resection of colorectal cancer and synchronous colorectal liver metastases.Only human studies in any language were considered. Inclusion was not restricted by study size or type. Publications excluded were review articles and commentary and studies that did not supply a degree of relationship. The articles were included in the meta-analysis when the next inclusion criteria were met:The study was a randomized control trial or retrospective study.The target population is subjects with colorectal cancer and synchronous colorectal liver metastases.The intervention program was the minimally invasive surgery and open surgery.The study included comparisons between the outcomes of minimally invasive surgery and open surgery in the simultaneous resection of colorectal cancer and synchronous colorectal liver metastases.The exclusion criteria were as follows:Studies that did not compare minimally invasive surgery to open surgery.Studies with diseases other than colorectal cancer and synchronous colorectal liver metastases.Studies did not focus on the effect on comparative results.A protocol of search strategies was prepared according to the PICOS principle , and we Data were abbreviated based on the following; study associated and subject associated features onto a homogeneous form, the primary author last name, study period, publication year, country, the studies region, and design of the study; type of the population, the total number and subjects number, demographic data and clinical and treatment features; the evaluation period associated to measurement, quantitative method and qualitative method of assessment, source of information, and assessment of the outcomes; and statistical analysis MD or relative risk, with 95% CI of relationship among the outcomes of minimally invasive surgery and open surgery in the simultaneous resection of colorectal cancer and synchronous colorectal liver metastases . If a stThe main result concentrated on measuring minimally invasive surgery and open surgery in the simultaneous resection of colorectal cancer and synchronous colorectal liver metastases. An assessment of the outcomes of minimally invasive surgery and open surgery in the simultaneous resection of colorectal cancer and synchronous colorectal liver metastases was extracted forming a summary.Sensitivity analyses were limited only to studies reporting the relationship between the outcomes of minimally invasive surgery and open surgery in the simultaneous resection of colorectal cancer and synchronous colorectal liver metastases. For subcategory and sensitivity analysis, we compared the effect of minimally invasive surgery to open surgery.I2 index; the I2 index is from 0 to 100%. Values of about 0, 25, 50, and 75% indicate no, low, moderate, and high heterogeneity, respectively , and qualitatively, by visual examination of funnel plots of the logarithm of ORs or MDs vs. their SE .The dichotomous or continuous method with random-effect or fixed-effect models was used to calculate the OR or MD and 95% CI. We used the Chi-square test to perform biological heterogeneity analyses between different studies. We calculated the ectively . When I2their SE . All p-vA total of 2,534 unique studies were identified, of which 13 studies (between 2011 and 2021) fulfilled the inclusion criteria and were included in the study –31.The 13 studies included 1,181 subjects with colorectal cancer and synchronous colorectal liver metastases at the start of the study; 425 of them were using minimally invasive surgery and 756 were open surgery. All studies evaluated the outcomes of minimally invasive surgery and open surgery in the simultaneous resection of colorectal cancer and synchronous colorectal liver metastases.Study size ranged from 14 to 444 subjects with colorectal cancer and synchronous colorectal liver metastases at the start of the study. The details of the 13 studies are shown in p = 0. 01) with high heterogeneity (I2 = 84%), less blood loss  with high heterogeneity (I2 = 90%), less blood transfusion needs  with no heterogeneity (I2 = 0%), shorter length of hospital stay  with low heterogeneity (I2 = 41%), lower overall complications  with no heterogeneity (I2 = 0%), higher overall survival  with no heterogeneity (I2 = 0%), and higher disease-free survival  with no heterogeneity (I2 = 0%) compared to open surgery as shown in Minimally invasive surgery in subjects with colorectal cancer and synchronous colorectal liver metastases was significantly related to longer operation time . Though, most of the comprised studies were evaluated to be of a low methodological quality. All studies did not have selective reporting bias, and no articles had incomplete result data and selective reporting.Based on the visual examination of the funnel plot and on quantitative measurement by the Egger regression test, there was no indication of publication bias  and the type of associated colorectal surgery since no enough data in the 13 selected papers described these parameters in more details. It is very relevant to know if the type of liver surgery performed was local resections or major hepatectomies (three or more liver segments). It seems essential to describe the type of liver and colorectal resections and whether or not there are significant differences with respect to major or minor hepatectomies in both branches. Hence, this suggests the further description of such parameters in the upcoming studies with more details.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.JG: administrative support, provision of study materials or subjects, and manuscript writing. FG: administrative support, collection and assembly of data, and manuscript writing. QX: provision of study materials or subjects, data analysis, and interpretation. XZ: collection and assembly of data, data analysis and interpretation, and manuscript writing. ZL: conception and design, and final approval of the manuscript. All authors have read and approved the manuscript.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.


Introduction: The most frequent causes of tinnitus associated with hearing loss are noise-induced hearing loss and presbycusis. The mechanism of tinnitus is not yet clear, although several hypotheses have been suggested. Therefore, we aimed to analyze characteristics of chronic tinnitus between noise-induced hearing loss and presbycusis. Materials and Methods: This paper is a retrospective chart review and outpatient clinic-based study of 248 patients with chronic tinnitus from 2015 to 2020 with noise-induced or presbycusis. Pure tone audiometry (PTA), auditory brainstem response (ABR), distortion product otoacoustic emissions (DPOAE), transient evoked otoacoustic emissions (TEOAE), and tinnitograms were conducted. Results: PTA showed that hearing thresholds at all frequencies were higher in patients with noise-induced hearing loss than the presbycusis group. ABR tests showed that patients with presbycusis had longer wave I and III latencies (p < 0.05 each) than patients with noise-induced hearing loss. TEOAE tests showed lower values in patients with noise-induced hearing loss than presbycusis at 1.5, 2, 3, and 4 kHz (p < 0.05 each). DPOAE tests showed that response rates in both ears at 1.5, 2, and 3 kHz were significantly higher in patients with presbycusis than noise-induced hearing loss (p < 0.05 each). Discussion: This study showed that hearing thresholds were higher, the loudness of tinnitus was smaller, and the degree of damage to outer hair cells was lower in patients with presbycusis than with noise-induced hearing loss. Moreover, wave I and III latencies were more prolonged in patients with presbycusis despite their having lower hearing thresholds. These phenomena may reflect the effects of aging or degeneration of the central nervous system with age. Further studies are needed to evaluate the etiologies of tinnitus. Tinnitus is one of the most common otologic diseases with a condition in which an individual recognizes sounds in the absence of external sound stimulation . There aHowever, in some situations, a single hypothesis cannot accurately explain the cause of tinnitus. Moreover, although tinnitus affects large numbers of people and reduces their quality of life, evidence-based, multidisciplinary clinical practice guidelines are not yet clear . DespiteThis study consisted of a retrospective chart review of patients with chronic tinnitus who visited Kyunghee Medical Center from 2015 to 2020 with noise-induced or presbycusis. The chronic tinnitus was defined as duration >3 months. Patients with noise-induced hearing loss were defined as those with a history of exposure to noise, such as workers in the mining and machinery industries. The hearing threshold was ≥25 dB, as calculated using the six-division method ((500 Hz + 2 × 1 kHz + 2 × 2 kHz + 4 kHz)/6) on PTA ,10. PatiAll patients with tinnitus were evaluated by tests of pure tone audiometry (PTA), auditory brainstem response (ABR), distortion product otoacoustic emissions (DPOAE), and transient evoked otoacoustic emissions (TEOAE). Tinnitograms were performed to get the pitch and loudness of tinnitus. In all frequencies, we selected threshold and added another 10 dBHL and presented pure tone or noise, according to the patients’ description on the characteristics of their tinnitus. We requested the patient to answer when realizing that the sound presented was similar to their tinnitus, and in the frequency indicated by the patient as similar to their tinnitus, the stimulus was presented noise, with an initial intensity of 10 dBHL below the patient’s threshold. We increased the intensity in steps of 2 dBHL, and the patients realized that the intensity was similar to the presented tinnitus ,12. The t-tests, and response rates on tests of DPOAE were compared using the MEDCALC program [p-values < 0.05 considered statistically significant.Sound-to-noise ratios (SNRs) on TEOAE tests were compared in the two groups using  program . All othp > 0.05 each). Concomitant symptoms, such as vertigo, autophonia, and ear fullness, were more frequent in patients with presbycusis, but the differences were not statistically significant (p > 0.05 each)  .p < 0.05) (p > 0.05). Loudness was significantly greater in patients with noise-induced than with presbycusis (p < 0.05)  . The mea < 0.05) .p < 0.05 each). Wave V latency also tended to be longer in patients with presbycusis, but the difference was not statistically significant (p > 0.05). In addition, I-III IPL and III-V IPL were significantly longer in patients with presbycusis than with noise-induced hearing loss (p < 0.05 each) (ABR tests showed that wave I and wave III latencies were significantly longer in patients with presbycusis than with noise-induced hearing loss (05 each) .p < 0.05 each) (p < 0.05 each)  . On DPOA05 each) .Many studies have evaluated the mechanisms, causes, and treatments of tinnitus. However, the evaluation of tinnitus through audiological examination is still limited in its ability to fully characterize the tinnitus, and there are many areas that should be studied in the future . The basThe PTA tests in our study found that hearing thresholds at all frequencies were lower in patients with presbycusis than with noise-induced hearing loss. In general, patients with presbycusis showed a downward pattern toward the high-frequency region, whereas patients with noise-induced hearing loss showed a downward pattern in both directions around 4 kHz. In addition, the loudness of tinnitus was significantly lower in patients with presbycusis than with noise-induced hearing loss. Patients with noise-induced hearing loss in the study reported more severe hearing loss and more severe subjective tinnitus than patients with presbycusis. It showed the same results as previous studies .Tinnitus pitch did not differ significantly in the two groups of patients. Moreover, the frequency of the pitch did not match the worst hearing threshold in either group. Similar results have been observed in previous studies, and one study found that both the pitch and loudness of tinnitus in the two groups of patients were lower than in this study. In that study, tinnitus pitch and loudness were 3 kHz and 45 dB, respectively, in patients with presbycusis and 4 kHz and 40 dB, respectively, in patients with noise-induced hearing loss . AdditioABR tests showed that wave I, III, and V latencies were slightly longer in patients with presbycusis and noise-induced hearing loss than in normal subjects. Moreover, wave I and wave III latencies were significantly longer in patients with presbycusis than with noise-induced hearing loss. Since the results of ABR tests are dependent on the lesion site, these findings seem to be related to differences in the pathogenesis of hearing loss and tinnitus in the two groups. Although these findings are consistent with previous results, no consensus has been reached on the magnitudes of these differences . ABR hasOAE tests can evaluate the degree of damage to the outer hair cells, with the DPOAE tests being better able to select higher frequencies than the TEOAE tests . In thisIn conclusion, this study showed that hearing thresholds were higher, the loudness of tinnitus was smaller, and the degree of damage to outer hair cells was lower in patients with presbycusis than with noise-induced hearing loss. Moreover, wave I and III latencies were more prolonged in patients with presbycusis, despite their having lower hearing thresholds. These phenomena may reflect the effects of aging or degeneration of the central nervous system with age. Further studies are needed to evaluate the etiologies of tinnitus.


Fusobacterium nucleatum, Streptococcus sanguinis, F. Hwasookii, Anaerococcus mediterraneensis and further species enriched in primary CRCs. The primary CRC of one patient was enriched for F. alocis, S. anginosus, Parvimonas micra and Gemella sp. 948. Enrichment of Escherichia coli strains IAI1, SE11, K‐12 and M8 was observed in metastases together with coliphages enterobacteria phage φ80 and Escherichia phage VT2φ_272. Virome analysis showed that phages were the most preponderant viral species (46%), the main families being Myoviridae, Siphoviridae and Podoviridae. Primary CRCs were enriched for bacteriophages, showing five phages  together with their pathogenic hosts in contrast to normal tissues. The most frequently detected, and Blast‐confirmed, viruses included human endogenous retrovirus K113, human herpesviruses 7 and 6B, Megavirus chilensis, cytomegalovirus (CMV) and Epstein–Barr virus (EBV), with one patient showing EBV enrichment in primary tumour and metastases. EBV was PCR‐validated in 80 pairs of CRC primary tumour and their corresponding normal tissues; in 21 of these pairs (26.3%), it was detectable in primary tumours only. The number of viral species was increased and bacterial species decreased in CRCs compared with normal tissues, and we could discriminate primary CRCs from metastases and normal tissues by applying the Hutcheson t‐test on the Shannon indices based on viral and bacterial species. Taken together, our results descriptively support hypotheses on microorganisms as potential (co)risk factors of CRC and extend putative suggestions on critical microbiome species in CRC metastasis.The paucity of microbiome studies at intestinal tissues has contributed to a yet limited understanding of potential viral and bacterial cofactors of colorectal cancer (CRC) carcinogenesis or progression. We analysed whole‐genome sequences of CRC primary tumours, their corresponding metastases and matched normal tissue for sequences of viral, phage and bacterial species. Bacteriome analysis showed  This work analysed the presence of sequences of bacteria and viruses in primary colorectal cancer (CRC) tissues, compared with corresponding normal colorectal tissues and metastases. We observed a higher viral species diversity and lower bacterial species diversity in primary CRCs than in matched normal tissues. Some microbial species were enriched in primary CRCs and some metastases, particularly phages.  AcMNPVautographa californica nucleopolyhedrovirusCDCrohn’s diseaseCMVcytomegalovirusCRCcolorectal cancerEBVEpstein–Barr virusEMCVencephalomyocarditis virusHERV‐K113human endogenous retrovirus K113HHV‐6Bhuman herpesvirus 6BHHV‐7human herpesvirus 7HPVhuman papillomavirusHPyV7human polyomavirus 7IBDinflammatory bowel diseaseJCVJC virusSV40simian virus 40UCulcerative colitisVHDVirus‐Host DatabaseWGSwhole‐genome sequencing1Fusobacterium nucleatum, Bacteroides fragilis, Streptococcus bovis and Enterococcus faecalis are the most frequently cited [Colorectal cancer (CRC) is the third most prevalent form of cancer worldwide, with a global incidence of over 1.4 million cases annually , 3. Abouly cited .Polyomaviridae family [Moreover, viruses could play a yet underestimated role in the carcinogenic process. In the past decades, several studies have pointed out an increased prevalence of viral infections in patients suffering from CRC or associated inflammatory conditions, such as inflammatory bowel disease (IBD), which include Crohn’s disease (CD) and ulcerative colitis (UC) . IBD is e family , 20, 21.Caudovirales  has been observed in patients affected by these diseases [Recent microbiome work has expanded beyond human‐infecting viruses and has highlighted, for example, the importance of bacteria‐infecting viruses (phages) in CRC and IBD. For instance, an increased prevalence of phage species belonging to the order of diseases , 24, 25.diseases , 31, 32.diseases . This asdiseases . Furtherdiseases , 35, faediseases . The fewdiseases , 38.The paucity of microbiome studies based on intestine tissues has contributed to the poor understanding of the conditions that foster CRC. Specifically, there is certainly a lack of research based on metastases. About one‐quarter of CRC cases progress towards metastasis , 40; thuIn this study, we (re‐)analysed whole‐genome sequences (WGS), which we generally described in previous work , to asse22.1All of the samples were completely anonymized and handled exclusively in strictly anonymized conditions. Tissues had been generally obtained from the biobank of the Medical Faculty Mannheim (Dept. of Surgery), University of Heidelberg, Germany, and the biobanking and tissue sampling approved by the Ethical Committee of this institution. The primary colorectal tumours, matched healthy colorectal tissues, and corresponding metastases re‐analysed here were described previously , the ana2.2Ensembl database. Viral reference sequences (n = 10 384) were retrieved from the National Center for Biotechnology Information (NCBI) database using Entrez utilities (https://www.ncbi.nlm.nih.gov/books/NBK25497/) and were concatenated into a single sequence  using mingle v. 2.3 [bwa v. 0.7.17 [Genomic sequences from the human assembly issue 38 built 12 (GChr38) were obtained from the e v. 2.3 . Human a. 0.7.17 .trimmomatic v. 0.38 [bwa‐mem v. 0.7.17 [sambamba v. 0.6.7 [Mingle to determine the corresponding viral species. Reads mapping to phage φX174, which was used as quality control in the libraries’ preparation, were discarded.Sequencing adapters and reads with quality below 33 phred‐score units were removed with  v. 0.38 , 45, 46.. 0.7.17 . De‐duplv. 0.6.7 , and onlsamtools v. 1.9‐18 and qualimap v. 2.2.1 [blast+ v. 2.9.0 [clustal omega v. 1.2.4 [blastx v. 2.9.0 [delly v. 0.8.3 [The quality of the alignment and sample coverage was assessed with v. 2.2.1 , 50. blav. 2.9.0 , 52 was v. 1.2.4 . To furtv. 2.9.0 . Sequencv. 0.8.3 .m of each dNTP, 1 μm of forward primer (5´‐CCGCTCCTACCTGCAATATCA‐3´), 1 μm of reverse primer (5´‐CAATAACGGCAGCAAGCTTG −3´), 1 mm MgCl2, 1.25 units of enzyme, 100 ng template DNA and water to 25 μL. The amplification conditions were as follows: 5 min at 95 °C followed by 30 cycles of 1 min at 95 °C, 1 min at 58 °C, 1 min at 72 °C and final extension for 7 min at 72 °C.In a series of 80 colorectal primary tumours and corresponding normal colorectal tissues, EBV DNA was detected as a validation by end‐point PCR targeting EBNA1 as previously described . BrieflyTo address the question of cell types primarily affected within these tissues, the presence of EBV was detected by in situ hybridization with an EBER‐specific peptide nucleic acid probe, in conjunction with a PNA detection kit (Dako) following the manufacturer’s protocol as described . Images 2.3spades v. 3.13.0 [Virus‐Host Database (VHD) [PhageAI database [Reads not mapping to either the human chromosomes or the composite virus chromosomes were assembled with . 3.13.0 . Sequencse (VHD) . Classifdatabase .2.4julia v. 1.3.1  and bash v. 5.0.3 languages. Plotting, statistical analysis and hierarchical clustering were performed with r v. 3.6.1 [r package Qsutils [r package iNEXT [t‐test. The Hutcheson t‐test [r with the package ecoltest .Data processing was performed with v. 3.6.1 . The Shav. 3.6.1  and its  Qsutils . Species Qsutils , 65 was ge iNEXT . Statistn t‐test  was used33.1Myoviridae (n = 154), Siphoviridae (n = 79) and Podoviridae (n = 29) as the most represented , including the aforementioned HERV‐K113 (an endogenous retrovirus) and encephalomyocarditis virus (EMCV). There were 73, 404 and 24 viral species present either in normal tissues, primary colorectal tumours, or metastases only, respectively, and an additional three (27.3%) were common to both primary colorectal tumours and metastases: EBV, Qinghai Himalayan marmot astrovirus and Tipula oleracea nudivirus. EBV was present in the lung metastasis together with AcMNPV, Synechococcus phage S‐SM2 and HERV‐K113.Twelve cases previously described  with matidae n = 54, Siphoidae n = 54, SiphoSiphoviridae (n = 11), Myoviridae (n = 10) and Podoviridae (n = 7) as the most represented  and 15 (53.6%) were observed in primary colon tumours. Stratification of the phagial species by tissue type showed that one species (Enterobacteria phage λ) was common to all tissues types, one (Shigella phage SfII) was common to normal colorectal tissues and liver metastases. Enterobacteria phage VT2φ_272 and enterobacteria phage φ80, Shigella phage SfIV and enterobacteria phage mEp460 were confirmed specifically in one metastasis each. An additional 16 species were present only in primary carcinomas, and six were observed only in normal colorectal tissues. Overall, 87.5%, 70.6% and 100.0% of phages in in normal tissues, primary colorectal tumours and liver metastases were temperate, respectively. Filtering confirmed HERV‐K113, AcMNPV, phage λ (but not Synechococcus phage S‐SM2) as the most preponderant viral species across all tissue entities. HHV‐7 was present collectively in five patients, and specifically in the normal colorectal tissue and primary colorectal carcinomas of two patients as well as in the primary colorectal carcinoma and liver metastasis of another patient. HHV‐6B was confirmed collectively in four patients, and specifically in the normal colon tissue and primary colorectal carcinoma of one patient as well as the normal colon tissue and liver metastasis of another patient. CMV was present specifically in the primary colorectal carcinoma tissues of two patients. Torque teno (TT) viruses 16 and 24 were also confirmed in primary colorectal carcinomas. We also confirmed SV40 and HPyV7 in one case each . EMCV was specifically present in liver metastasis tissue. Of the aforementioned giant viruses, only megavirus chiliensis was confirmed in two patients. The phage crAssphage was confirmed in the normal colon tissues and the primary colorectal tumours of three patients. EBV was confirmed after BLAST filtering in the primary colorectal tumour and the lung metastasis of the same patient. Himalayan marmot astrovirus and Tipula oleracea nudivirus were not confirmed in the lung metastasis after Blast filtering.To avoid the possibility of false‐positive detection, we further filtered the data with the highly sensitive Blast alignment . To gain additional information on putative cell types infected with EBV within colorectal carcinoma tissues, the presence of EBV was further investigated in a subset of 5 samples using EBER staining (ISH)  and either liver or lung metastases  as well as primary colorectal tumours from either liver or lung metastases .Next, we sought to determine whether we could differentiate primary colorectal tumour tissues, corresponding normal colorectal tissues, and metastases based on the viral content. We used rarefaction analysis to calculate the estimated number of species normalized for the sample size, and the Shannon index to have a comparative value. Overall, there were an estimated 124 (95% CI: 60–296), 232 (95% CI: 99–674) and 57 (95% CI: 26–157) normalized viral species, in the normal, primary colorectal tumour and metastatic tissues, respectively  and Klebsiella pneumoniae concomitantly in primary colon tumour and liver metastasis. There were no bacterial species observed in the lung metastatic tissue. We found Fusobacterium nucleatum enriched only in the primary colorectal tumour sections of three patients in contrast to corresponding normal colorectal tissues, whereas Fusobacterium hwasookii was present in the primary colorectal tumour sections of two patients. Porphyromonas gingivalis was present in the normal colorectal tissue and primary colon carcinoma sections of one patient. Anaerococcus mediterraneensis and Prevotella denticola were observed in the primary colorectal tumours of two patients, as opposed to corresponding normal tissues. Among the 122 species observed only in primary colorectal tumours, the families Bacillaceae and Clostridiaceae represented the most represented, with 20 species each (16.4%), followed by the Streptococcaceae with 17 species (13.9%). Bacteroides fragilis was prevalent in normal colorectal tissues and also present in primary colorectal tumour sections. Streptococcus anginosus was observed in the normal colorectal tissue of one patient, and in the primary colorectal tumours as opposed to their matched normal tissues of two patients. Furthermore, among the species observed only in primary colorectal tumours, S. sanguinis, Filifactor alocis, Gemella sp. oral taxon 928 and Parvimonas micra were present in high frequency in one patient and were represented by a number of sequence assemblies in the range of 468–147, compared to a mean of 5.7, indicating their highly specific representation.We identified 518 bacterial species, corresponding to 143 bacterial families in our sample set. Overall, there were 391, 217 and 10 bacterial species in normal colorectal tissues, primary colorectal tumours and liver metastases, respectively, corresponding to a ratio of phages over bacteria of 0.02, 0.08 and 0.60. There were 122 species present only in primary colon tumour tissues, four strains of t‐test allowed discriminating normal tissues from both primary colorectal tumours  and metastases ; similarly, primary colorectal tumours could be discriminated from metastases .The normalized species count, obtained by rarefaction analysis, was 2370 (95% CI: 1701–3383), 1011 (95% CI: 693–1543) and 40 (95% CI: 14–209) for the normal, primary colorectal carcinoma and metastatic tissues . Our results indicated a certain level of parallelism between the presence of the host and that of the associated phage Table . Half of4The present work is one of the very few, if not the first so far, that carried out microbiome analysis on primary colorectal carcinoma and corresponding metastases in comparison to matched normal tissues. Although, certainly, our analysis from whole genomes so far is largely bioinformatic, our study suggests that some microorganisms, at least their sequences, are more prevalent in some primary colorectal tumours or metastases, this pertaining to viruses and bacteria, and—potentially most interesting since these were not yet systematically investigated in such a setting—(bacterio‐)phages.E. coli as a host, for example enterobacteria phage φ80 and Escherichia phage VT2φ 272 which, after Blast filtering, we even found specifically in metastatic tissues. We observed over half of the most frequent phages especially in primary tumours, two of which (Bacillus prophage PfEFR‐5 and Streptococcus phage φARI0462) showing their specific host  in primary tumour tissues only, not in corresponding normal tissues, which suggests that this might not have been due to contamination. This is hypothesized since the primary colorectal cancers and corresponding normal colorectal tissues were both acquired simultaneously, being exposed to the same microbiologic milieu of the same intestinal lumen within the same patient under the same settings, and therefore, they would have been exposed to the same contaminants. Interestingly, some of the hosts of the phages specifically observed in primary colorectal tumour tissues are known pathogens. For instance, Bacillus prophage PfEFR‐5 infects Bacillus cereus, a pathogen associated with food poisoning [B. cereus and S. pneumoniae in tumour samples only, in contrast to the corresponding normal colorectal tissue of the very same patient, suggested a sort of ‘linkage disequilibrium’ between these tissue types.In both primary colorectal cancers and metastases, we observed several species of phages, for example phage λ or crAssphage, crAssphage having been reported as the most common virus in the colon , 69, 70.oisoning . The obsF. nucleatum, S. anginosus and F. alocis, S. sanguinis, P. micra and Gemella oral taxon 928 confirm the results of other research groups [F. nucleatum, S. anginosus and S. sanguinis at high prevalence in cancer tissues  [Fusobacterium spp. are included in the passenger/driver model of CRC and have been reported to promote the formation of metastases [F. nucleatum and P. micra are commonly enriched in CRC sections [Fusobacterium hwasookii and Porphyromonas gingivalis in colorectal cancer tissues, together with high prevalence of Fusobacteria and Bacteroidetes [F. alocis and P. micra are prevalent at sites of periodontitis (a chronic inflammation of the gum that results in tooth loss) and in oral squamous cell carcinomas [S. sanguinis has been reported in cases of occult colon carcinomas [Prevotella denticola was enriched in colorectal cancer tissues [Klebsiella pneumoniae, although commonly associated with pneumonia, has been reported as a risk factor for colorectal cancer [S. anginosus is an opportunistic bacterium commonly observed in the oral and intestinal flora, but it is also enriched in esophageal and gastric cancers [Gemella is a genus of commensal bacteria of the mouth and gut whose main members (G. morbillorum and G. haemolysans) are usually isolated in abscesses and endocarditis [Anaerococcus mediterraneensis has been recently isolated in a case of vaginosis [The bacterial species we observed enriched, or exclusively, in primary colorectal carcinomas have been reported to show oncogenic or pro‐inflammatory potential. Our tumour‐specific findings of h groups , 79, 80.han 105) . Fusobactastases , 82. F. sections , 84, 85,roidetes . F. alocrcinomas , 88. S. rcinomas , 90, 91.l cancer , 94. S.  cancers , 96, and cancers . Gemellacarditis , 99, 100aginosis , suggestE. coli was commonly observed in our samples, we also observed an enrichment of some E. coli strains specifically in metastatic tissue. E. coli is both a commensal of the human gut but also an opportunistic species [E. coli, unbalancing the gut microbiome. On the other hand, phages can piggyback pathogenic species invading inner tissues. Therefore, our detection of coliphages might reflect these scenarios, and further data are needed to discriminate between these possibilities. Interestingly, it has been shown that this bacterium can use α‐haemolysin to invade the colon epithelium in a process known as ‘focal leak’ [E. coli (C25 and HBTEC‐1) have been reported to transcytose colonic cells [Although  species . Thus, lal leak’ . Specifiic cells . The poric cells . It has ic cells . The intic cells , 108, whic cells . In turnic cells . An impaic cells .The gut and the liver share a special relationship due to their connection via the portal vein, the biliary tract and systemic circulation. Hence, the presence of pathogenic bacteria in the gut might be reflected in the liver especially if there are leakages in the mucosal barrier in the intestine. Such a hypothesis is backed by extensive literature, demonstrating a linkage between intestinal dysbiosis and liver disease due to an increased permeability of the intestinal barrier. If certain bacteria or phages (viruses), or their disbalance, can be pathogenic for the colorectal tissue, they might affect the liver, too, in a similar or also slightly different way as compared to the intestinal tissue. Specifically, if particular pathogens, or their interaction, are able to induce inflammation in the gut, they might also induce inflammatory processes in the liver. However, with the exception of some specific systemically infecting viruses such as HCV, EBV, and others which are able to induce pro‐oncogenic pathways in cells including hepatocytes , 114, stNevertheless, the implications for the presence of bacteria and phages in the liver are still poorly understood. It is feasible to assume that these microorganisms might induce a chronic inflammation that can foster liver disease, albeit more experimental data are required to determine whether microorganisms might also induce metastasis. The fact that the ratio of phages to bacteria was much higher in metastases than in colorectal tissues might reflect the fact that phages are more likely to cross the gut epithelial barrier. Recent metagenomic analysis suggested that the induction of temperate phages might be responsible for propagating intestinal dysbiosis, resulting in an increased amount of phages in relation to bacteria , 116. ThEnterobacteriaceae family  in the inflamed gut [E. coli, in addition to B. fragilis, E. faecalis and F. nucleatum, all of which we found represented in our tissues analysed, have been suggested to be able to act as pathogens, questioning their in part uncritical use as probiotics [Enterobacteriaceae family, including strains of E. coli and K. pneumoniae, have been shown to release a genotoxin (colibactin) able to induce genetic damage, fostering the insurgence of cancer [Furthermore, recent metagenomic studies have reported the increased prevalence of members of the amed gut . Our obsobiotics , 121. Sef cancer , 123.per se, favouring the expansion of bacteria that promote inflammation or even carcinogenesis [The oncogenic potential of these bacteria, together with, at least in part, an enrichment in primary colorectal carcinomas we observed herein, suggest a putatively complex scenario of several infectious components which, in their interaction, might lead to a netto support of carcinogenesis and/or CRC progression. Towards this end, phages such as the ones we identified here could further support the growth of pathobionts, which can cause damage to intestinal cells, local inflammation and more. Thus, phages are increasingly acknowledged as a putative risk factor for CRC, whose importance has been underestimated in the past . As to fogenesis . Again, ogenesis , areas oogenesis . Finallyogenesis . Phages,ogenesis , 126. Thogenesis , 130. Teogenesis  are the Taken together, there are several mechanisms by which phages, and phage‐host interactions, impact the microbiome and pro‐inflammatory, or even pro‐carcinogenic/‐metastatic, conditions in the intestine. Thus, our findings can encourage mechanistic studies on particular bacteriophages as to their putative function in CRC carcinogenesis or progression in the future, especially since, despite the intense research ongoing to describe the microbial communities in the human intestinal tract of CRC and IBD , 38, 131Similar to the bacteria, many of the viral species we observed enriched in primary colorectal tumours and liver metastases, besides phages, have been reported to possess oncogenic and/or pro‐inflammatory potential. This is certainly true for EBV which we found in whole‐genome sequencing, including even in one metastasis after Blast filtering, and also by PCR in an independent series in which about a quarter of the analysed primary tumour/normal colorectal tissue sample pairs showed a specific positivity in the carcinomas. Regarding putative pro‐carcinogenic mechanisms, EBV has been reported to act several fold , 133: itP‐value = 0.115) [We observed other viruses with oncogenic potential. CMV infects preferentially neoplastic epithelium, where it can reach a prevalence of over 40% compared to < 6% in the surrounding normal tissues , and tum= 0.115) .HERV‐K113 is an endogenous retrovirus with a prevalence of up to 30% , and, unWe also observed EMCV, a zoonotic pathogen that has a seroprevalence ranging from two per cent to over 30% , 177. EMc‐MYC to identify breast cancer patients at a higher risk of mortality [Taking advantage of the observed presence of microorganisms within our samples, we tried to apply statistical methods to distinguish between normal colorectal, primary colorectal carcinoma and metastasis tissue types. Previous studies have applied the Shannon index for diagnostic purposes, for instance applying the genetic variability of ortality , 183. InThe much lower Shannon indices observed in metastatic sections was unsurprising since these tissues are expected to be rather sterile. Thus, the presence of some microorganisms in liver metastases still appears counter‐intuitive. Only HERV‐K113, an endogenous retrovirus, displayed a profile characterized by high sequence coverage associated with integration in the host’s chromosomes Fig. , thus, tCertainly, our study has limitations, including methodological ones. First, it was initially designed for whole‐genome screening. Consequently, no viral enrichment, or general enrichment for microbial genomes, had been carried out, nor included, in the processing of the samples. Thus, the low microbial biomass could have introduced a bias in the results, generating lower than expected findings in sequence copies, or in individual sequence coverage, of microbial sequences as compared to sequences from human chromosomes . Lower s5In conclusion, this is the first work carrying out metagenomic analysis on CRC metastases as compared to corresponding primary CRC tumours and normal colorectal tissues and that attempted to apply microbial richness calculations to differentiate these tissues. We showed that the microbial landscape might be used to differentiate primary colorectal tumours from nonmalignant tissue and metastases, in particular by using the Shannon index. Moreover, we highlighted particular species, especially including the previously not extensively considered species of (bacterio‐)phages, for future functional studies as to how they could contribute to colorectal carcinogenesis or even progression and metastasis, for example by creating permissive or nonpermissive microbial environments. Extending this analysis to broader sets of available samples, for example in multicentre approaches, could foster a better understanding of infectious agents as a potentially complex interplay of cofactors for CRC carcinogenesis and progression, and assess whether the use of microbial analysis could support precision medicine to pinpoint patients at increased risk for CRC or CRC metastasis.The authors declare no conflict of interest.LM mainly performed and coordinated the analyses within this paper. LM, TR, and JJML performed all aspects of the bioinformatics analysis, MLA acquired the samples and consents, coordinated the aspects associated with their collection, and led wet laboratory sample analysis and PCR. SD and HJD performed and interpreted EBER stainings. HA initiated the idea of virome, phage and bacteriome analysis in association with CRC carcinogenesis and metastasis, and coordinated the research and funding. All authors participated in the discussion and interpretation of data, the writing and/or revision, and approval of the manuscript.Fig. S1. Visualization of viral integration of human endogenous retrovirus K113 (HERV‐K113).Table S1. Read counts stratified by tissue type.Click here for additional data file.


Childhood hearing loss is a global epidemic most prevalent in low- and middle-income countries where hearing healthcare services are often inaccessible. Referrals for primary care services to central hospitals add to growing lists and delays the time-sensitive treatment of childhood hearing loss.To compare a centralised tertiary model of hearing healthcare with a decentralised model through district hearing screening for children in the Western Cape province, South Africa.A central paediatric tertiary hospital in Cape Town and a district hospital in the South Peninsula region.A pragmatic quasi-experimental study design was used with a 7-month control period at a tertiary hospital (June 2019 to December 2019). Decentralising was measured by attendance rates, travelling distance, number of referrals to the tertiary hospital and hearing outcomes. There were 315 children in the tertiary group and 158 in the district group. Data were collected from patient records and an electronic database at the tertiary hospital.p < 0.001). Travel distance to the district hospital was significantly shorter (p < 0.001). Number of referrals to the tertiary hospital decreased significantly during the intervention period (p < 0.001). Most children in both the tertiary and district groups  passed initial hearing screening bilaterally.Attendance rate at the district hospital was significantly higher (Hearing screening should be conducted at the appropriate level of care to increase access, reduce patient travelling distances and associated costs and reduce the burden on tertiary-level hospitals. Referrals for primary care services such as hearing screening at central tertiary-level hospitals add to growing waiting lists for specialised care such as diagnostic hearing assessments and hearing aid fittings. Direct referrals to a central tertiary hospital often imply that parents and caregivers must travel further to access hearing healthcare infrastructure, which may in turn lead to poor follow-up rates, late diagnoses and late access to hearing technology. Childhood hearing loss impedes speech, language and academic development,15 and early auditory stimulation is crucial to minimise the adverse effects of hearing loss in children.16In LMICs such as South Africa, healthcare facilities are typically tiered into three main levels of care: primary such as point-of-entry clinics, secondary that includes district and regional hospitals and tertiary which encompasses specialised services.17 Innovative service delivery models, with an emphasis on decentralisation, are required to develop sustainable services in these settings.18 Decentralisation is the transfer of responsibility for planning, management and financing from central to peripheral levels of government and has been a key health sector reform in a wide range of LMICs over the past decade.19 Despite being implemented as a strategy across many health systems, the impact of decentralisation on health equity is still unclear.20 However, it has been suggested that in order to minimise such inequity, government, health sectors and communities must address socio-economic and financial barriers and implement complementary mechanisms alongside decentralisation.20Access to sustainable hearing healthcare services in LMICs is an important public health priority.21 is disproportionate to the lack of hearing healthcare services available and current efforts to reach underserved communities are inadequate.5 If hearing healthcare services are not available at primary-level healthcare clinics, many communities in LMICs do not have access to these services at all22 and tertiary-level services are being overburdened with screening services that should be conducted at a lower level of care. Therefore, approaches that incorporate the delivery of community-based hearing care in order to decentralise hearing healthcare services is a priority.24The growing burden of hearing loss in LMICsThis study aimed to compare a centralised tertiary model of hearing healthcare to a decentralised model through district hearing screening for children in the Western Cape province, South Africa. The effects of a decentralised model of hearing healthcare were measured in terms of attendance rates for initial hearing screening, patient travelling distance, number of referrals to a tertiary-level hospital and hearing outcomes.A pragmatic quasi-experimental study design was implemented, with a 7-month control group receiving standard hearing service provision at a tertiary hospital (from June 2018 to December 2018), compared with a 7-month intervention group where hearing screening was offered at a district hospital (from June 2019 to December 2019).25 and is situated in the Southern Peninsula of the Western Cape province, South Africa. The metropole incorporates eight health subdistricts with eight district-level hospitals of which only three have audiology services. Victoria Hospital is a district hospital with 159 beds in the South Peninsula health district of the metropolitan region and currently has no audiology services. No audiological services are available at any of the primary healthcare clinics or maternity and obstetric units (MOU) in this area, which result in referrals for initial hearing screening of older children based on risk factors or concerns for hearing loss. All patients aged 0–13 years who are from the district hospital catchment area and who need audiology services are referred directly to Red Cross War Memorial Children’s Hospital, which is a central tertiary-level hospital in Cape Town.The Cape Town metropole has a population of 4 067 774The Western Cape has three tertiary academic hospitals. Red Cross War Memorial Children’s Hospital is one of two dedicated paediatric tertiary-level academic hospitals in sub-Saharan Africa and serves as a central referral hospital for paediatric patients across the entire Western Cape who require specialised healthcare services. The Department of Audiology at this tertiary facility assesses and provides hearing rehabilitation for approximately 300 children per month. Referrals are received from district hospitals, primary level clinics and MOUs. Both the district and tertiary hospitals in this study are situated in a LMIC and serve mostly children from the public healthcare sector who do not have access to private medical insurance.Consecutive sampling was used to select participants for both the tertiary and district groups.All patients who were referred to the tertiary hospital via email for initial hearing screening from the district hospital catchment area during the control period (June 2018 to December 2018), and who attended their hearing screening appointment at the tertiary hospital, were included in the tertiary group, regardless of the reason for referral. These patients were retrospectively selected from the audiology departmental electronic database at the tertiary hospital to form the tertiary group of 315 paediatric patients.All consecutive referrals for initial hearing screening from facilities that fell within the district hospital catchment area were sent via email to the tertiary hospital during the intervention period (from June 2019 to December 2019). These referrals were selected for the decentralised hearing screening project at the district hospital. Only referrals who met the specified inclusion criteria for the district hearing screening project were included in the district group. The primary method of hearing screening for the district group utilised otoacoustic emissions (OAEs), which assesses cochlear function, therefore, referrals for initial screening of high-risk patients who presented with risk factors for retro-cochlear pathology or auditory neuropathy spectrum disorder  were excluded and booked at the tertiary hospital. Patients with known middle ear pathology such as otitis media or otorrhoea were also excluded from the district group, as they were likely to fail screening because of middle ear abnormality and would have been better served at the tertiary hospital with a diagnostic hearing assessment.As a result of limited time and space available at the district hospital, only 10–15 paediatric patients were booked per afternoon twice per month for the 7-month intervention period, which equated to a sample size of 190 referred patients. Parents of referred children were contacted telephonically by the tertiary hospital’s audiology clerk to arrange an appointment for a hearing screening at the district hospital during the intervention period (from June 2019 to December 2019). Children who attended their initial hearing screening appointment at the district hospital were included and formed the district group of 158 patients. The hearing screening at the district hospital was conducted by two audiologists from the tertiary hospital. Most of the hearing screening appointments coincided with routine follow-up paediatrician visits at the district hospital.An electronic patient database from the Department of Audiology at the tertiary hospital was used to retrospectively review data of the patients from the district hospital catchment area who were referred to the tertiary hospital for initial hearing screening during the control period (from June 2018 to December 2018). Data included demographic information, reason for referral, initial hearing screening results and number of children from the district hospital catchment area who were referred directly to the tertiary hospital. Only initial OAE hearing screening results were included for the tertiary group, as diagnostic testing was carried out on the same day at the tertiary hospital if a patient referred OAE screening unilaterally or bilaterally, instead of scheduling a rescreen 2 weeks later at the tertiary hospital. Diagnostic assessment results were also included for those children who referred initial OAE screening unilaterally or bilaterally in the tertiary group. The same electronic patient database was used to review the number of children from the district hospital catchment area who were referred to the tertiary hospital for initial hearing screening during the 7-month intervention period at the district hospital (from June 2019 to December 2019).A hearing screening data sheet for the 7-month intervention period at the district hospital (from June to December 2019) was used to record patient data in terms of demographics, geographical area of residence, reason for referral, OAE screening results and need for further diagnostic testing. Patients in the district group who referred the initial screening unilaterally or bilaterally underwent tympanometry to check their middle ear status and were referred to the paediatrician at the district hospital on the same day as the initial hearing screening in order to treat any middle ear pathology. These patients were rescreened at the district hospital after 2 weeks, and if another unilateral or bilateral refer result was obtained on the rescreen, they were referred for diagnostic hearing assessment at the tertiary hospital.10The Maico Eroscan® OAE test system was used for initial hearing screening during both the control and intervention periods. The system incorporates a screening function with a four-frequency (2000 hertz [Hz] – 5000 Hz) low-to-high distortion-product OAE testing protocol and conducts a fast, automatic test showing a pass or refer result. The signal-to-noise ratio is set at 6 decibels [dB], and a pass result is obtained if three frequencies pass. The reliability and validity of OAEs for use in a screening setting are well-established.t-test was utilised for parametrical numerical data. A p-value of ≤ 0.05 was considered significant.Data were entered into Microsoft Excel 2016  and descriptive analysis was performed. Data were imported into the Statistical Package for the Social Sciences (SPSS)  for inferential analysis. Pearson’s Chi-square test was utilised for categorical data, whereas Student’s The study was approved by the University of Pretoria Research Ethics Committee of the Faculty of Humanities (HUM024/0419), the University of Cape Town Human Research Ethics Committee (365/2019), Red Cross War Memorial Children’s Hospital Ethics Committee (RCC203) and the Western Cape Health Research sub-directorate (WC_201906_023). The tertiary hospital in this study has an Outreach Policy Agreement with all Western Cape Health Facilities, which was used in conjunction with a letter requesting institutional permission from the district hospital to conduct an outreach OAE-screening service there twice per month for 7 months. A letter of informed consent was issued to the caregivers of participants prior to data collection. Informed assent was obtained from children over the age of 7 years.The mean age of patients at the time of initial hearing screening was 48.4 months  and 52.3 months  in the tertiary and district groups, respectively. The tertiary and district groups were similar in terms of age, gender and language distribution .p < 0.001).An attendance rate of 83.2% (158/190) was found during the 7-month intervention period for patients attending the district hearing screening project, which was significantly higher than the attendance rate of 70.2% (315/449) for patients from the district hospital catchment area who were seen for initial hearing screening at the tertiary hospital during the control period (p < 0.001).The mean travel distance for patients in the district group commuting from home to the district hospital was 12.6 km . This distance was significantly shorter than the travel distance of 19.1 km , which patients would have had to travel from home to the tertiary hospital (p < 0.001).A total of 1729 patients were referred from facilities across the Western Cape to the tertiary hospital during the control period (from June 2018 to December 2018), of which 449 (26.0%) referrals were for initial hearing screening from the district hospital catchment area. Throughout the intervention period (from June 2019 to December 2019), during which the district screening project was being conducted, the tertiary hospital received a total of 1601 referrals from facilities across the Western Cape province, with a significant decrease to 114 (7.1%) referrals for initial hearing screening from the district hospital catchment area (n = 315), 115 referrals (36.5%) were received for reasons that were excluded from the intervention period analysis. When excluding these 115 referrals, the most common reasons for referral in the tertiary group were speech delay (35.0%) and behavioural or school-related concerns (28.5%) (n = 200). In the district group, speech delay (33.5%) and meningitis (33.5%) were the most common reasons for referral (n = 158).The reasons for referral for initial hearing screening are depicted . During n = 248/315, 78.7%) passed the initial OAE screening bilaterally. The number of patients who required diagnostic assessment in the tertiary group were 67 (21.3%). Of the 67 patients who required diagnostic assessment, 54 (80.6%) attended their appointments. Half of the patients  were diagnosed with mild conductive hearing loss.Outcomes of the initial OAE hearing screenings for the tertiary group and diagnostic assessment results for patients who referred initial OAE screening unilaterally or bilaterally from June 2018 to December 2018 are presented . For then = 127/158, 80.4%) passed OAE screening bilaterally, whilst less than 10% referred OAE screening in both ears. The follow-up attendance rate for rescreening at the district hospital 2 weeks after the initial screening was 80.8% (n = 21/26). The total number of patients in the district group that needed referral to the tertiary hospital for specialised diagnostic assessment were 15 , of which 11  attended the diagnostic hearing assessment appointment. Of these 11 patients, nearly half  presented with mild conductive hearing loss.Outcomes of the initial OAE screenings from the intervention period at the district hospital and the diagnostic assessment results for patients referred to the tertiary hospital after a unilateral or bilateral refer result on rescreening at the district hospital, are also presented . For theThis study explored the effect of decentralising hearing healthcare services from a tertiary-level hospital to a district-level hospital in the Western Cape province, South Africa. Decentralised hearing screening resulted in increased attendance rates for initial hearing screening, shorter travelling distances for patients and decreased referral rates to a tertiary-level hospital.26 Furthermore, especially in severely resource-constrained settings typical of LMICs, non-attendance delays the identification, diagnosis and timeous intervention of healthcare conditions.27 The Health Professions Council of South Africa Early Hearing Detection and Intervention Guidelines28 suggest that a 70% and higher follow-up return rate for hearing screening is considered ideal, but that the feasibility of attaining a high follow-up rate is influenced by various factors such as access to healthcare facilities and personal constraints such as poverty.28Attendance rates were significantly higher for initial hearing screening at the district hospital when compared with initial screening at the tertiary hospital. Non-attendance can result in underutilisation of healthcare provider time and can lead to longer appointment waiting time for patients.29 Patients who needed referral to the tertiary hospital for specialised diagnostic assessment had an attendance rate of 73.3%, which is in line with a previous South African community-based hearing screening study that found an attendance rate for diagnostic assessments of 75.8%.29The follow-up attendance rate for rescreening at the district hospital two weeks after the initial screening was high (80.8%). This could be attributed to the fact that the second screening was also conducted at a community level and coincided with a paediatrician visit to follow up on middle ear pathology for the majority of patients who referred OAE screening bilaterally. A high follow-up attendance rate (89.4%) for hearing screening was also found in a recent South African community-based study when the rescreening was conducted at a community-level as opposed to a public healthcare institution.12 The costs involved in attending healthcare appointments, both in terms of time taken off from work and travel costs for patients with limited resources, remain a further challenge in accessing healthcare in LMICs.30 Therefore, primary healthcare is an important strategy employed in South Africa, in order to provide more accessible patient-centred services closer to home.30 Community delivered hearing healthcare models have been identified as an important strategy to increase the accessibility and affordability of hearing healthcare in underserved communities.31Patient travelling distance was significantly shorter to the district hospital as opposed to the tertiary hospital. Access to services is one of the leading barriers to hearing healthcare in underserved communities.The inaccessibility of hearing healthcare services at a primary- or district-level, which adds severe strain on tertiary-level specialised services, may be alleviated by decentralising services. The results of this study corroborate this. The number of direct referrals for initial hearing screening from the district hospital catchment area to the tertiary hospital significantly decreased after implementation of the decentralised hearing screening project at the district hospital. The decreased number of referrals to the tertiary hospital for initial hearing screening support decreased waiting times and improved capacity to provide specialised diagnostic hearing assessments and intervention to patients requiring tertiary-level care.24 however, utilising OAEs in a screening setting is advantageous in terms of time taken to conduct and minimal training that is required.More than 80% of children who attended the initial hearing screening during the intervention period at the district hospital passed initial OAEs bilaterally. This high pass rate is a positive outcome for the premise of decentralising hearing screening services to a more appropriate level of care. The majority of patients (78.7%) in the tertiary group also passed initial OAE screening, which supports the premise that hearing outcomes are similar for initial hearing screening regardless of the level of care where hearing screening is conducted. Telehealth applications are available for hearing assessment of older children,29 A possible reason for the higher referral rate is the method of screening. Otoacoustic emissions screening is sensitive to middle ear pathology and it is more likely to fail in the presence of abnormal middle ear function.32The referral rate for diagnostic hearing assessment at the tertiary hospital for the children who attended hearing screening during the intervention period at the district hospital was 9.5%. This percentage is higher than the reported referral rate of a South African community-based hearing and vision screening study of 5.4%, which utilised smartphone-based pure tone audiometry screening.Referral for diagnostic testing in the tertiary group (21.3%) was twice as high in the district group (9.5%). The higher number of diagnostic assessments in the tertiary group were because of the fact that no opportunity for rescreening after two weeks was provided, as all patients who referred initial screening unilaterally or bilaterally or those for whom OAE screening results could not be elicited, underwent diagnostic assessment on the same day in order to minimise follow-up appointments at the tertiary hospital.31 This would in turn minimise the burden on district level staff and address the problem of preventative hearing loss in children at grassroots level.31Providing hearing screening at a district level increased access to medical treatment for all children who presented with middle ear pathology as evidenced by abnormal tympanometry results on the day of initial OAE screening. These children were assessed and treated by the paediatrician on the same day, instead of waiting for months to get an ENT appointment at the tertiary hospital. Thus, middle ear pathology was treated timeously and effectively at a more appropriate level of care, decreasing the added burden to long tertiary waiting lists. Early identification of middle ear pathology is a primary-level healthcare service, and it would be more appropriate to refer children even closer to home to their nearest community healthcare centres for treatment.26 Community healthcare workers and nurses can be trained to screen for hearing loss using mobile health technology via home-based visits to reach vulnerable communities in LMICs,33 thereby improving access to hearing healthcare services and reducing the demands on the limited number of hearing healthcare professionals in South Africa.33 In addition no sample size calculation was conducted and group size was pragmatically determined by number of patients over the specified time periods.A limitation of this study was that tertiary-level audiologists conducted the hearing screening at the district hospital during the intervention period. Future studies should assess the training needs of community healthcare workers and nurses to conduct hearing screening at district hospital facilities. The premise of task-shifting through community-based hearing screening programmes has been proposed as a way to improve access to hearing healthcare.Decentralised hearing screening programmes conducted at the appropriate level of care can increase access to hearing healthcare, reduce patient travelling distances and associated costs and reduce the burden on tertiary-level hospitals. Accessible hearing screening yields higher attendance rates, leading to more effective and timeous treatment of the adverse effects of childhood hearing loss.


Argonaute proteins are key players in small RNA-guided gene silencing processes. Ago2 is the member of the Argonaute subfamily with slicer endonuclease activity and is critical for microRNA homeostasis and indispensable for biological development. However, the impact of Ago2 dysregulation in the retina remains to be fully explored. In this study, we studied the role of Ago2 in mouse retina.Ago2 disruption mouse model. An ERG was carried out to determine the retinal function. Spectral domain optical coherence tomography, fundus photographs, and immunostaining were performed to investigate the retinal structure. A quantitative RT-PCR assay was used to determine the expression of noncoding RNAs.We explored the function of Ago2 in the mouse retina through an adeno-associated virus-mediated Both silencing and overexpression of Ago2 in mouse retina resulted in significant retinal morphological alterations and severe impairment of retinal function, mainly with a thinned outer nuclear layer, shortened inner segment/outer segment, and diminished ERG responses. Furthermore, Ago2 disruption resulted in alterations of noncoding RNAs in retina.Our finding demonstrated that Ago2 interruption led to severe retinal degeneration, suggested that Ago2 homeostasis contributed to retinal structural and functional maintenance. As types of small RNAs, microRNAs (miRNAs), short interfering RNAs (siRNA) and PIWI-interacting RNAs typically assemble with Argonaute proteins into the RNA-induced silencing complex (RISC).,It has been reported that the RISC assembly of miRNAs and siRNAs share a similar manner.Ago2 possess catalytic activity and contributes “slicer” activity to RISC.,–The retina, a key part of the central nervous system, is critical in transducing light into electrophysiological signals that ultimately form vision. The miRNA-guided gene silencing is essential for retinal development, maintenance and homeostasis.miR-183/96/182 (miR-183C),–miR-204,,miR-124a,miR-9, and miR-23a.,Tug1) was the first reported lncRNA in retina,Rncr2),Six3os and Vax2os1 were also the lncRNAs that received the early attention.Meg3, once implicated in cancers, has been reported to be linked to the retina.As the widely studied subclass of noncoding RNAs, miRNAs has been well-characterized in retina, including ,––Ago2 has been reported to be a candidate master regulator during miRNA biogenesisHerein, to elucidate the regulatory function of Ago2 in the retina, we used adeno-associated virus (AAV)-mediated interference of Ago2 in vivo. We found that Ago2 disruption led to severe retinal degeneration, including shortened inner segment/outer segment (IS/OS), thinned outer nuclear layer (ONL), and impaired ERG response. Moreover, some noncoding RNAs expressed in retina were dysregulated. Our results indicate that Ago2 is indispensable for retinal structure and functional maintenance.C57BL/6J mice were bred in the animal facility of Ophthalmology & Optometry in Wenzhou Medical University and were maintained under a 12-hour light–dark cycle and had free access to food and water. All experiments were carried out according to the ARVO Statement.Ago2-EGFP and AAV-Ago2-3Flag vectors. To generate the AAV-shAgo2-EGFP, the target sequence GCACACGCTCTGTGTCAAT was cloned into the GV478 vector containing pU6-MCS-CAG-EGFP. The control AAV-EGFP was the GV478 vector with nonsense sequence CGCTGAGTACTTCGAAATGTC. To generate AAV-Ago2-3Flag, the target Ago2 sequence (2622 bp) was amplified by primer TACCGGACTCAGATCTCGAGATGTACTCGGGAGC-CGGCCCCGTTC and GATCCCGGGCCCGCGGTACCGTAGC-AAAGTACATGGTGCGCAGTGTG, then, the PCR product was cloned into the Xho I and Kpn I sites of a GV411 vector containing pCMV-betaGlobin-MCS-3Flag-SV40 PolyA. The GV411 empty vector with no target sequence was used as control AAV-3Flag. The titers of AAV-shAgo2-EGFP and AAV-EGFP were 1.23 × 1012 and 1.30 × 1012 TU/mL, respectively. The titers of AAV-3Flag and AAV-Ago2-3Flag were 1.09 × 1012 and 1.08 × 1012 TU/mL, respectively.The silencing and overexpression of Ago2 in mouse retina has been achieved through subretinal injection of AAV-sh,Ago2-EGFP (1.23 × 1013 TU/mL)/AAV-Ago2-3Flag (1.08 × 1013 TU/mL) for the right eyes and an equal dose of the control vector AAV-EGFP/AAV-3Flag for the left eyes were injected slowly into the subretinal or intravitreal space using a blunt 5-µL Hamilton syringe held in a micromanipulator. All injections were performed bilaterally. For the subretinal injection, the presence of a retinal detachment indicated the successful injection.The subretinal injection of AAV was performed as previously described.Ago2-EGFP/AAV-EGFP and AAV-Ago2-3Flag/AAV-3Flag injected mice were dilated with 0.5% tropicamide and anesthetized using pentobarbital sodium. Fundus photography and SD-OCT were then performed. For SD-OCT measurements, images crossing through the optic nerve were obtained and collected for each eye. The thicknesses of the different retinal layers were measured using Insight software .AAV-sh2 stimulus intensity with different interstimulus intervals. Photopic ERG was measured at 0.7 to 3.3 log cd-s/m2 with different interstimulus intervals after 10 minutes of light adaptation with a background illumination of 5.0 log cd/m2.Scotopic and photopic responses in both eyes of the mouse were recorded with a Phoenix Micron IV as previously described. Briefly, mice were dark-adapted overnight and pupils were dilated using 0.5% tropicamide. Then, the mice were anesthetized intraperitoneally with pentobarbital sodium. The ground electrodes and referential needle were punctured into the tail and cheek, respectively. Scotopic ERG was recorded at −1.7 to 3.1 log cd-s/mWhole eyeballs of mice were isolated and fixed in 4% (wt/vol) paraformaldehyde in PBS for 20 minutes. Retinas were dissected and refixed with 4% (wt/vol) paraformaldehyde for an extra 15 minutes. Retinas were then dehydrated in 30% (wt/vol) sucrose and embedded in embedding medium . 12-µm-thick cryosection slides were obtained and stored at −80 °C. For immunostaining, cryosection slides were placed at room temperature, washed with PBS, blocked in blocking buffer  for 45 minutes, treated with primary antibody at 4 °C overnight, and then incubated with secondary antibody at room temperature for 1 hour. Then primary and secondary antibodies were used as follows: rabbit anti-Cone arrestin , mouse anti-Rhodopsin , rabbit anti-Recoverin , rabbit anti-Pkcα , rabbit anti–glutamine synthetase , mouse anti–glial fibrillary acidic protein , mouse anti–cellular retinaldehyde binding protein , mouse anti-GFP , goat anti-Flag , and guinea pig anti-vGlut1 . Donkey anti-rabbit IgG conjugated to Alexa Fluor 488 , donkey anti-mouse IgG conjugated to Alexa Fluor 594 , Donkey anti-rabbit IgG conjugated to Alexa Fluor 680 , and goat anti-guinea pig IgG conjugated to Alexa Fluor 568  were used as secondary antibodies. Samples were stained with 4,6-diamidino-2-phenylindole and visualized using laser-scanning confocal microscope . Images were taken at 200 to 400 µm location in reference to the optic nerve.Ago2-EGFP/AAV-EGFP and AAV-Ago2-3Flag/AAV-3Flag. Retinas were dissected and placed in TRIzol reagent. RNA was extracted using the RNeasy Mini Kit (Qiagen). For miR-183C, cDNA was synthesized using 1 µg of total RNA, TaqMan miRNA RT probe  and M-MLV reverse transcriptase, and quantified using a TaqMan miRNA probe, U6 snRNA was used as the reference gene. For other miRNAs, cDNA was synthesized and quantified using Bulge-Loop miRNA qRT-PCR Starter Kit , U6 was used as an internal control. For mRNAs and lncRNAs detection, cDNA was synthesized with M-MLV reverse transcriptase and random primers, and quantified using SYBR Green Master Mix (Roche). Gapdh was used as the reference gene. The relative expression of RNAs was calculated by using equation 2−ΔΔCT. The sequences of the sense and antisense primers are listed in To harvest retinal RNA, eyeballs were isolated from mice treated with AAV-shRetinas were isolated, collected, and lysed in lysis buffer containing 1 × phenylmethanesulfonyl fluoride. Protein was extracted and quantified using a BCA protein assay kit (Invitrogen). Proteins were separated using SDS-PAGE and then analyzed by anti-Ago2  and anti-GAPDH .U test and 2-way ANOVA were adopted.The values shown in the graphs represent averages of several independent experiments and the actual number of samples for each experiment stated in the figure legends. Data are presented as means ± SEM. The Mann–Whitney To determine the Ago2 expression pattern during retinal development, we performed Western blots of E18.5, P1, P7, P14, P30, and P60 retinas. We found that the expression of Ago2 proteins were stable from E18.5 to P14, and decreased gradually from P14 to adulthood A, B.Ago2-EGFP and its control AAV-EGFP into the subretinal space of wild-type mice at P30 (Ago2 expression (decreased to 42.70%)  D. Wester 42.70%) E. These Ago2-EGFP–treated retinas (Ago2-EGFP treated retina (We first surveyed alterations in retinal morphology and structure after Ago2 deletion , 2. Fund retinas C, left.  retinas . Nuclei d retina A. In addd retina B.To better understand these defects in retinal structure, we examined photoreceptors, bipolar neurons and synapses by immunostaining in P60 retinas C–E. ImmuNext, we evaluated retinal function in Ago2-deficient retinas at P60 by ERG. Of note, Ago2-deficient mice displayed an abolished scotopic response lacking a- and b-wave amplitudes at each stimulus intensity A. This f,–,,miR-183C,–miR-204,,miR-124miR-9 and miR-23a,,As the major player of RISC, the Ago2 protein directly binds to miRNATug1,Rncr2,Six3osVax2os expression is restricted to the ONL of the ventral retina.We then investigated the expression of retina-associated lncRNAs reported previously,Ago2-3Flag and its control AAV-3Flag into wild-type mice through subretinal injection at P30 (Ago2-3Flag group (It has been reported that Ago2 could bind and stabilize mature miRNAs, and thereby enhance miRNAs abundance and guide more mRNA silencing.n at P30 A, B. Retn at P30 C, right.n at P30 . Westernag group D, E. TheAgo2-3Flag group (Ago2-3Flag–treated retina. However, the INL seemed to be unaffected (However, no obvious abnormalities fundus was observed in AAV-ag group C, left. affected . This fiaffected B.Ago2-3Flag treated retina. The overexpression of Ago2 led to retinal degeneration similar to that of the AAV-shAgo2-EGFP–treated retina. However, the impairment after Ago2 overexpression was less severe.Furthermore, immunostaining for Cone-arrestin, Recoverin, Pkcα, and vGlut1showed that architecture defects mainly happened in the outer retina and that the inner retina remains relatively intact after Ago2 overexpression C–E. To iSimilar to the Ago2 silencing experiment, overexpression of Ago2 mainly affected the scotopic ERG response A, B, indAgo2-EGFP or AAV-Ago2-3Flag into the subretinal space of mice. As expected, silencing or overexpression of Ago2 led to a decreased retinal thickness and ERG response. And, the retinal degeneration caused by Ago2 silencing was more serious than Ago2 overexpression. This phenomenon is similar to a previous finding.We disrupted Ago2 expression in the retina by introducing AAV-shOCT and immunostaining assays showed that silencing of Ago2 resulted in severe defects in the outer retina, and the inner retina thickness remained relatively normal, indicating that retinal degeneration mainly occurred in the photoreceptors first and then extended to the inner retina.ERG results showed that a-wave amplitudes were decreased in Ago2-deficient mice, especially in the scotopic response, and an immunostaining assay also showed decreased signals of Recoverin and absent signals of Rhodopsin. These results indicated that the effect of Ago2 was mainly on the photoreceptor layer. Compared with cones, rods were more susceptible to Ago2 deletion. This phenomenon may be due to the large portion of rods in the photoreceptors. In contrast, it has been reported that subretinally injected AAV vectors displayed a preference for rods as the photoreceptor matured. So rods were disrupted first and, then, loss of the outer segments of rod could make the AAV display a preference for cones.For the 2-way injections, subretinal injection usually has a direct effect on the resident cells and tissues in the subretinal space, such as photoreceptors.Ago2-EGFP and AAV-Ago2-3Flag were introduced into the retina via intravitreal injection at P30. Retinal cross-sections showed the successful absorption of AAVs by the inner retina at P60, especially the ganglion cell (Ago2 did not alter the fundus and retinal thickness obviously (−/−/Nrl−/−. They also compared subretinal injection and intravitreal injection and found that both of these 2 methods could cause positive CNGA3 expression in cones.The direct damage to photoreceptors in our results may be due to the subretinal injection method, which can directly affect photoreceptors. To investigate the effect of Ago2 on the inner retina, AAV-shion cell A, S10A. bviously B, C. Int,,As the key regulator in miRNA homeostasis, Ago2 binds to miRNAs directly and becomes a component of the ribonucleoprotein complex that regulates miRNA function.miR-183/96 double KO model.,miR-183/96 was mainly on cones.miR-182 KOmiR-183 KOmiR-182 or miR-183 deletion led to a decreased ERG response but did not affect the retinal morphology. Target disruption of Rncr3, the dominant source of miR-124a, caused reduced cones.miR-211 results in progressive cone dystrophy, but does not affect the function of rods.miR-9 and upregulated target Bcan is responsible for disorganized MG phenotype. The increased miR-9 after Ago2 silencing also led to MG activation in our study, implying that miRNAs are essential for glia homeostasis and retinal architecture.In addition, the phenotypes caused by Ago2 disruption were similar to that reported in ,,Among these factors involved in miRNA biogenesis and function, Ago2, Dicer, and Dgcr8 have been reported to be absolute requirement for mouse development, and disruption of Ago2, Dicer, or Dgcr8 resulted in early embryonic death.,Dgcr8-null ES cells showed that Dgcr8 is essential for miRNA biogenesis and silencing of ES cell self-renewal.Dicer-null mice have been reported to die at E7.5.,,,,The embryonic–lethal phenotype caused by Ago2 may be because, compared with the other RISC components PACT and TRBP,Taken together, our findings demonstrated that the silencing or overexpression of Ago2 in mice led to a severe retinal degeneration phenotype. And, for the first time, suggested that Ago2 homeostasis is essential for retinal maintenance and function.


The current trends are promising exciting clinical applications within 5–10 years. Local inner ear therapy via a hearing prosthesis has already been realized and alternative stimulus optimization protocols are being developed. Special attention is being paid in the area of pediatric intervention since the quality of life of young patients is always a highly considered factor.The aim of this special issue, entitled ‘Advances in Pediatric and Adult Cochlear Implant and Middle Ear Prostheses’, is to report the Hearing loss is reported to be among the most prevalent disabilities in Western countries, as it has been estimated to involve up to 30% of the adult population ,2,3. AccThe restoration of hearing and the rehabilitation strategies for hearing loss are based on the data from the correct identification of the causes that affect the auditory system and determine the treatment possibilities. Modern technology offers numerous solutions, including the rehabilitation and treatment of cases that present moderate, severe or profound sensorineural hearing impairment. Currently, hearing impairment is overcome by technological solutions that are related to hearing aids (HAs), implantable hearing aids, cochlear implants (CIs) and, more rarely, auditory brainstem implants.Cochlear implants are considered among the most effective neural prostheses ever developed. Presently, CI technology is further evolving at a great pace in terms of hardware and software solutions. From the single-channel processors used in the early 1960s, the CI devices are nowadays equipped with multiple channels, allowing for the stimulation of multiple cochlear locations. The CI stimulus processing strategies have also evolved, targeting a higher performance index regarding auditory pathway activation ,5,6,7.The paragraphs below summarize the most distinct current trends and underline what is expected in the near future in the field of hearing prostheses and hearing restoration.Size and connectivity optimization: Future steps could be directed toward the optimization of the size/volume and the increased connectivity of the hearing restoration device. Intelligent connectivity is considered to be a trend in vogue since there are numerous advantages to the users when CIs and HAs can interact intelligently with other everyday life devices . Currently, CI and HA users can moderately interact with their smartphones using Bluetooth-dedicated channels. Interesting ideas within this field include the possibilities of self-fitting a device using a smartphone. The implications from such a technological step are vast and, in combination with advances in teleaudiology, can result in an affordable rural assistance model [A. ce model ,9.Local ear therapy: This aspect is mostly related to CIs but could eventually be expanded to middle-ear-implantable devices. It is likely that in the near future, CIs may undergo several structural changes from using normal electrodes to electrodes containing films or reservoirs that release agents that can perform local cochlear therapy (intra-cochlear administration of drugs). In this context, it could be possible to even further enhance auditory rehabilitation, for example, by releasing local growth factors/neurotrophins targeting specific spiral ganglion neurons. This approach could boost a CI’s neural performance by stimulating the neuronal response to electric stimulation; on the other hand, it could allow for the protection of the inner ear structures from cochlear mechanical trauma that is caused by the insertion of the CI electrodes [B. ectrodes ,5,6,7,10Globalization of the prostheses solution: In order to offer the above-mentioned hearing restoration and rehabilitation solutions on a larger global scale, a reduction in the total costs should be attained within the near future [C. r future . IndirecConcerning other restoration and rehabilitation solutions for hearing losses that are related to physical damage to the external or the middle ear, there are several interesting fields of research that could possibly yield new clinical solutions in the future. For instance, bioactive materials were proposed by several authors for improving the adaptation or resolution of cartilage or bony tissue defects ,13. In tFinally, the application of nanomedicine and nanotechnologies could offer further solutions for the treatment of hearing disorders. In fact, nanoparticles (NPs) were proposed as carriers of various drugs for local inner ear therapy. Ideally, NPs should have a series of specific features, such as the capacity to permeate the round or oval window membrane, the ability to identify specific targets or the controlled release of their therapeutic loads in a safe way ,15,16,17


Dear Editor,Sudden hearing loss in cases of SARS-CoV-2 is rare, and it is even less common as a first symptom. For the first time in the literature, we present a case of SARS-CoV-2 accompanied by bilateral sudden hearing loss in a peritoneal dialysis patient.3/µL), high C-reactive protein (CRP) levels , and high D-dimer concentration (838 ng/ml). Favipiravir, enoxaparin, and meropenem were started. Immediately after the nasopharyngeal and oropharyngeal swab results were found positive, samples were taken from four consecutive dialysates with six hours of waitings. The samples were studied after being centrifuged at 4000 rpm for 10 min. The SARS-CoV-2 PCR results were negative in all peritoneal effluent samples. The main complaint that brought the patient to the hospital was bilateral ear pain and hearing loss. Bilateral serous otitis media were observed on otoscopic examination. Moderate hearing loss of mixed type  was observed in the audiogram. Type-B appearance in both ears was seen in tympanometry. Methylprednisolone 56 mg/day was started to be administered orally. No intratympanic steroids were given. Methylprednisolone was continued by decreasing 8 mg daily. During an examination four days later, it was observed that the patient’s complaints and the appearance of serous otitis were reduced. Mild conductive hearing loss was observed on the audiogram. She did not need oxygen during her hospitalization. Hypervolemia was not observed. The 4 × 2000 mL CAPD/DPCA 17  treatment was continued. Hearing loss improved, and the CRP regressed to 35 mg/L. The patient, who had no additional complaints, was discharged on the seventh day of hospitalization.A 23-year-old female patient had been on a continuous ambulatory peritoneal dialysis program for the last 5 years. Three days prior to presenting to the emergency room, she started experiencing sudden onset bilateral ear pain and hearing loss. She was admitted to the pandemic ward after infiltrative ground-glass densities showing bilateral consolidation consistent with SARS-CoV-2 pneumonia were observed in thoracic CT images. SARS-CoV-2 PCR results were positive in the nasopharyngeal and oropharyngeal swabs. She had severe lymphopenia (0.43 × 10The coexistence of sudden hearing loss and SARS-CoV-2 is rare, but it has been reported with increasing frequency . Profoun


Within hearing care, significant disparities persist despite the highly prevalent nature of age-related hearing loss and relatively few trials include representation of racial/ethnic minorities. HEARS is an affordable, accessible hearing care intervention delivered by older adult peer mentors. The HEARS randomized controlled trial (NCT03442296) is a community-engaged RCT with an embedded human-centered design practitioner. Recruitment efforts occurred over 18 months in partnership with 13 affordable housing and social centers. The cohort (n=151) includes 43% (n=65) who self-identify as African American and 63.6% (n=96) with <$25,000 annual household income. The cohort represents the largest to-date of African American and low-income older adults with hearing loss. Recruitment efforts entailed 470.5 staff hours and $4,917.26 in supplies, equating to 1.4 hours and $14.13 per 1 individual screened and 3.1 hours and $32.56 for 1 participant randomized. Community-engaged research, partnered with human-centered design, may offer critical approaches to increasing representation within behavioral intervention trials.


Lichtheimia spp. in a 21-year-old multi-trauma patient is presented. The patient was hospitalized in the intensive care unit and underwent craniotomy due to brain injuries. Two weeks following the initial procedure, pus drained from the surgical wound was microscopically examined and cultured, yielding Lichtheimia spp. Imaging showed parietal, temporal and frontal abscesses at the right side. The patient was commenced on amphotericin B and underwent surgical debridement, while histopathological examination of the affected tissue demonstrated broad, aseptate hyphae, findings typical for mucormycetes. The patient passed away due to heavy traumatic injuries after 2 months. It is speculated that direct inoculation was the portal of entry for infection, and that high steroid use for 2 weeks following inoculation contributed to the severity of infection that developed. Isolated cerebral mucormycosis in immunocompetent hosts is an extremely rare, but severe disease. Diagnosis is established through direct microscopy, histopathology and/or cultures. PCR-based techniques are useful either to detect mucormycetes in tissues, especially when cultures are negative, or to accurately identify the fungi grown in cultures at the species level. A high suspicion index, especially in the necrotic lesions of traumas, is of the utmost importance for early diagnosis. Appropriate surgical debridement, as well as antifungal therapy, including amphotericin B, represents the treatment of choice.Isolated post-traumatic cerebral mucormycosis represents an extremely rare and severe disease. A case of isolated cerebral mucormycosis infection caused by  Mucorales, such as Mucor, Rhizopus, Rhizomucor or Lichtheimia [Aspergillus species, are commonly found in the soil and high-humidity climates and can, therefore, cause primary cutaneous infection via direct inoculation [Mucormycosis represents a severe infection caused by the fungi of the order htheimia ,2. The iculation ,3. The vMucorales are ubiquitous, and morphologically appear as broad, aseptate or sparsely septate ribbon-like hyphae. Eleven genera and about 27 species under Mucorales are associated with human infections. Rhizopus arrhizus is the most common agent causing mucormycosis across the globe, followed by Lichtheimia, Apophysomyces, Rhizomucor, Mucor and Cunninghamella species [Isolated cerebral mucormycosis represents a rare invasive fungal infection, most commonly affecting immunocompromised patients or patients with a history of intravenous drug abuse . The fun species ,5. MucorLichtheimia spp. in a 21-year-old multi-trauma patient is presented, aiming to report infection due to a rather rare species, causing life-threating disease.An extremely rare case of isolated cerebral mucormycosis infection caused by A 21-year-old female, with unremarkable medical history was brought to the Emergency Department due to a motorcycle accident. Upon presentation, she was hemodynamically unstable  and disorientated.The patient was treated according to the Advanced Trauma Life Support guidelines (ABCs). She was intubated and transferred to the intensive care unit (ICU), while an initial computer tomography (CT) scan revealed subarachnoid hemorrhage, epidural and subdural hematoma, as well as free air in the left eye socket and multiple fractures in the frontal, parietal, temporal bone and the sinus cavity. Furthermore, she had sustained fractures of the left radius and ulna and the right humerus and radius.Due to mydriasis, a second CT scan was performed the same day, revealing exacerbation of the hematomas and edema, as well as the displacement of the midline structures. At that point, she underwent urgent bilateral decompressive craniotomy and hematoma removal.The patient remained in the ICU. Neuromonitoring was regularly performed during the postoperative period, while she was commenced on vancomycin and meropenem, as well as steroids for the brain edema. She continued to be hemodynamically unstable and was supported. On the 15th postoperative day, pus was drained from the surgical wound and was microscopically examined and cultured.Lichtheimia spp. was yielded from the cultures, while liposomal amphotericin B was switched to 7 mg/kg due to the severity and the location of the infection.Direct examination with a fluorescent brightener (Blankophor P) revealed wide hyphae, aseptate with right-angle branching, typical for mucormycetes . Hence, At that point, a new CT scan revealed parietal, temporal and frontal abscesses at the right side . The patFollowing the surgical debridement, Posaconazole (300 mg per day) was added to the regimen. The patient passed away due to the heavy traumatic brain injuries 2 months after the second surgery, while on antifungal treatment. Cerebral mucormycosis, is a severe infection associated with high mortality ,6. DiagnLichtheimia spp. in a healthy female, without a history of intravenous drug use. The spread of the mold should be considered to be due to direct inoculation, making this case remarkable, while there have been only three similar cases of post-traumatic isolated cerebral mucormycosis in the literature so far [The present report presented a case of isolated cerebral mucormycosis due to e so far ,9,10. Mucorales, while Lichtheimia species can be found in farming products, such as hay and straw, as well as processed and unprocessed food products, including flour and fermented soybeans [Lichtheimia spp. (formerly Absidia) seem to account for approximately 5% of all mucormycoses, However, this mold is not uncommon in cases of mucormycosis in Europe [Lichtheimia species have been implicated in the form of occupational hypersensitivity pneumonitis termed farmer’s lung disease [Soil is believed to be the main habitat of most soybeans ,6. In gln Europe ,6,7. TheThe main predisposing factors for mucormycosis are hematological malignancies, organ transplantation, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), hematopoietic stem-cell transplant, lymphoid malignancies, neutropenia, hereditary immune deficiencies, immunosuppressive medications, diabetes mellitus, intravenous drug abuse and mechanical breakdown of the blood–brain barrier through surgery or trauma ,6,11. ThMucorales after injury [Mucorales may cause necrosis, restricting circulation in several vessels [Spreading through disrupted cutaneous barriers, following major trauma, represents a major route of infection in immunocompetent hosts ,16,17,18r injury ,15,18. Ir injury . Thus, n vessels .Lichtheimia infections had been diagnosed in the facility; hence, inoculation most probably occurred during the initial trauma and not during surgery, since the air filtration system in the operating room meets the highest standards. The infected sites in the reported patient were the right parietal, temporal and frontal lobe, as shown in the CT scan (Isolated cerebral mucormycosis mainly occurs in young adults, while headache, fever, focal weakness and altered mental status represent the main symptoms. It is also of note that in most cases, the infection is localized in the basal ganglia ,17,18. T CT scan . Regarding imaging techniques indicating diagnosis, the modality of choice is brain MRI with special sequences, including gradient echo and susceptibility weighting, to assess for microhemorrhage, indicating a potentially invasive process . HoweverFor the definite diagnosis of cerebral mucormycosis, tissue is required. The specimen should be sent for microscopy, histology and culture . The chaMucorales growth and binding to endothelial cells, thereby accelerating angioinvasion, the control of hyperglycemia and acidemia is critical [It is of note that steroid use in patients with cerebral mucormycosis has been associated with worse outcomes ,6. Furthcritical ,2,17.Regarding the treatment of such infections, liposomal amphotericin B plays a pivotal role, since it has been documented that patients with isolated cerebral mucormycosis not receiving this agent had a fatal outcome ,7. The pIsolated cerebral mucormycosis represents about 15% of cerebral mucormycosis cases, while it mainly results from an episode of fungemia . The maiIn conclusion, post-traumatic isolated mucormycosis is an extremely rare but life-threatening disease. A high suspicion index, especially in necrotic lesions in trauma patients, is pivotal for early diagnosis. Definite diagnosis through microscopy, histology and/or cultures are of paramount importance, while PCR testing may be extremely useful, especially in doubtful cases and those that antifungal treatment has already been given. Appropriate surgical debridement, as well as antifungal therapy including liposomal amphotericin B and/or isavuconazole represent the treatment of choice.