In the second part of the study, the first part's experimental results were verified by subjecting at least one-quarter udder of eight buffaloes to ultrasonography seven times during mid-July to mid-August for obtaining ultrasonic values at different somatic cell levels. The echo textural characteristics [mean numerical pixel values (NPVs) and pixel heterogeneity ] were evaluated using 16 ultrasonographic images of each buffalo with Image ProPlus software. The effects of SCC, days in milk (DIM), scanning order (SO), season, as well as the scanning plane and udder quarter (SP + UQ) on both the PSD and NPVs of the mammary gland were significant (p < 0.05). The correlation coefficient between pre-milking sagittal PSD and somatic cell score (SCS) was the highest  with fitted linear model: y = 0.19445x . In addition, SCC and ultrasonic of udder quarter were followed for 1 month, confirming that pre-milking sagittal PSD of mammary gland value could explain the SCC variation in milk. The current study demonstrated that the ultrasonographic examination of the udder could be one of the complementary tools for diagnosing subclinical mastitis in buffaloes.The aim of the present study was to determine whether the echotextural features of the mammary gland parenchyma in buffaloes during lactation at different somatic cell levels could be used to diagnose mastitis. This study was divided into two parts. In the first experiment, experimental buffaloes ( Buffalo is the second-largest milk-producing livestock, with an average of 118.162 million tons of buffalo milk per year over the last decade, according to the FAO statistical database . AlthougEarly and accurate diagnosis of the disease is an essential part of treatment. Nowadays, many different methods for diagnosing mastitis are being practiced based on SCC and plate-culture techniques . SCC is Ultrasonography is a non-invasive technique that can examine udder for hematoma, abscess, inflammation, milk stones, tissue proliferation, congenital malformation, mucosal lesions, and foreign bodies . In receUltrasonography facilitates researchers in examining the same animals repeatedly and readily, providing grayscale and two-dimensional images of tissues in real time to evaluate their structure, echogenicity, and homogeneity. Therefore, the objective of this study was to develop a rapid method to detect mastitis by establishing the relationship between somatic cells and mammary echotextural characteristics in buffaloes.n = 65, test-day records = 94) was tested in the first part and the validation group  with at least one-quarter udder was tested in the second part to verify the results of the first part. All lactating buffaloes were housed in the free-stall pens and fed a total mixed rations (TMR) diet. DIM of all the buffaloes enrolled in this study were between 5 and 349 days with the parity between 1 and 6 and milked twice a day, morning and evening, in a parallel milking parlor (6 × 2). The milking machine parameters were set to an operational vacuum of 50 kPa, 64 ± 3 pulsation cycles/min at a ratio of 60:40, which were maintained every 3 months. Data for individual buffalo data were obtained from herd management software .The present investigation was carried out in the buffalo farm of Jinniu Animal Husbandry Co. Ltd., China, from November 2020 to September 2021. The experimental animals were divided into two groups: experimental group .It was challenging to interpret the sensitivity difference between 5- and 7.5-MHz probes in examining the mammary parenchyma . PreviouEach buffalo was scanned 16 times, and a total of 1,710 ultrasound images were captured and saved in bitmap (BMP) format. Subsequently, images were transferred using universal serial bus (USB) for computer analysis with USB software Image-Pro Plus 6.0  to analyze, process, and edit grayscale images by pixel value statistics and calculate area to get the intensity values. Areas with lactiferous ducts, gland cisterns, and visible blood vessel formations were not considered for the grayscale analysis . Grayscan = 54) was examined based on SCC tests  of quarter milk samples. Mastitis can transfect to other quarter udder regions if not treated. Collecting milk samples and mammary glands ultrasound examination were carried out seven times at an interval of 6 and 1 day, respectively and followed up for 30 days to obtain mammary parenchyma echotexture images at different somatic cell levels.To verify the echotextural variable's accuracy in detecting mastitis, at least one of the four udders of eight buffaloes of parity –5 with dThe data from 94 test-day records from 65 buffaloes were included in calculating the fixed effects using SAS's general linear model (GLM) procedure implemented in SAS version 9.4  . The maiijklmn is the NPVs or PSD; μ is the population mean of the model; DIM is fixed effect of lactation period ; parity is fixed effect of the number of lactations ; season is fixed effect season of measurement ; SCC is fixed effect of somatic cell counts ; SO is fixed effect of SO ; UQ + SP is fixed effect of scanning plane for each quarter ; SO × (UQ + SP) is the fixed effect of the interaction between SO and UQ + SP; and eijklmn is the random residual.where Y2  was used, using only data from the 94 test-day records from 65 buffaloes. Pearson correlations analysis was performed using the PROC CORR in SAS   . DIM, paMean values of mammary gland echotexture at different somatic cell levels  were obtained after 1-month follow-up and compared by ANOVA using SAS  . The datp < 0.0001; DIM, p = 0.0101; SO, p = 0.0003; Season, p < 0.0001; UQ + SP, p < 0.0001; NPVs: SCC, p = 0.0478; DIM, p < 0.0001; SO, p = 0.0406; Season, p < 0.0001; UQ + SP, p < 0.0001). Whereas, parity and SO × (UQ + SP) on PSD were not found to have an effect on any of the measurements , p = 0.2901). Parity and SO × (UQ + SP) were found to have an effect on NPVs , p = 0.0283).p < 0.05). NPV value in spring was significantly lower than that in other seasons . PSD were greater after than before milking, but NPVs were the opposite . PSD and NPVs were highest between 5 and 90 days of lactation .Mean PSD was the lowest in the SCC <200,000 cells/ml and increases with SCC increase , but non-significant with NPVs (p > 0.05), respectively , compared with pre-milking coronal PSD , post-milking coronal PSD , and post-milking sagittal PSD . The high value of adjusted R2 (0.82 to 0.84) indicated that the linear model of PSD fitting explained sufficient variability in SCC in milk. Pre-milking sagittal PSD had explained higher variation  and fitted better than other models  between different levels of SCC. The pre-milking sagittal PSD value reached the lowest value at SCC <200,000 cells/ml and significantly lower than other levels .All the data were reported as the average of the same SCC level in seven ultrasonic testing and SCC levels varied in 24 out of 32 mammary glands. The PSD value for most quarter udder increases with somatic cells . A one-wThe quality of feed, milking equipment, health conditions, and management systems of buffalo are all-inferior to those of cows, which significantly affects the prevention, diagnosis, and treatment of mastitis in buffalo, especially at the farm level. California mastitis test for diagnosing mastitis has the advantage of being quick and easy to perform on the farm. However, it results in subjective and indirect determination, which instigates finding more accurate techniques . The effUdder fibrosis and atrophy due to chronic mastitis had hyperechoic cordial bands representing the fibrous tissues, replacing the glandular tissues in buffaloes . The ultCurrently, SCC, either from the whole udder or quarter, has been the best indicator of the mammary glands' inflammatory status , 35. A pThe present study found that examining the structures of the mammary gland parenchyma with vertical positioning showed better results than the horizontal direction, contradictory to findings in goats . HoweverOur study showed that echotextural characteristics of the mammary parenchyma were influenced by many factors, including DIM, seasons, and measurement methods. Therefore, these factors should be considered while interpreting the ultrasonic-related effects. In our study, PSD values of the mammary gland were highest at 5–90 days of lactation and in summer. Coincidentally, Riekerink et al.  found thInterestingly, the results of our study showed that, before milking, the NPVs values were significantly higher than after milking, which was different from the results in cows . In agrep < 0.05). The correlation coefficient between pre-milking sagittal PSD and SCC was the highest . The pre-milking sagittal PSD of mammary gland value could explain the SCC variation in milk well. Computer-assisted pixel analysis of mammary gland ultrasonograms examination of the udder can aid in diagnosing subclinical mastitis in buffaloes. However, further research is needed to validate the current study's findings and statistical methods with large sample size and data.Despite the availability of different traditional and novel diagnostic tools for early mastitis diagnosis, there is no gold for testing as all tools have limitations; hence, a multifaceted approach is advised to improve udder health. Ultrasound is an economical, rapid tool for detecting mastitis on dairy farms. The PSD and NPVs of mammary were significantly associated with SCS, DIM, SO, season, as well as the scanning plane and udder quarter (The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Ethical Committee of the Hubei Research Center of Experimental Animals. Written informed consent was obtained from the owners for the participation of their animals in this study.LY and XZ: conceptualization. XZ, ZA, KN, and WW: methodology. PN and SG: software and data curation. XZ and MA: investigation. XZ: writing—original draft preparation. MA: writing—review and editing. LY: supervision, project, and funding acquisition. All authors have read and agreed to the published version of the manuscript.China Agriculture Research System of MOF and MAR supported this research work.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.


Background: The lactobacillus-rich microbiome forms a defense system against infections. Babies are born sterile and acquire their microbiome from exposure to the mothers' vaginal and rectal microbiota. Bacterial vaginosis (BV), which is characterized by a deficit of the Lactobacilli genera, may predispose women and their babies to an increased frequency of illness.Objective: To determine the effect of BV on HIV-infected women's post-delivery health as well as the morbidity and mortality of the exposed infant at birth, 6 months, and at 12 months of life.Study Design: A retrospective cohort study was conducted using previously collected data to investigate whether there was an association between BV-HIV-1 infected mothers and subsequent infant morbidity and mortality over a 12-month period.Methods: Data for this analysis were extracted from the original data set. Women were categorized into two groups according to whether they had a positive or negative laboratory-based diagnosis of BV using the Nugent method. The two groups were compared for socio-demographic characteristics, prior to the pregnancy experience in their current pregnancy outcome and at post-delivery morbidity, and for the duration of hospital stay. BV-exposed and unexposed infants were compared in terms of morbidity and mortality at birth, and in the periods between birth and 6 months, and between 6 and 12 months, respectively, based on prospectively collected data of the mother's past and present illness, and clinical examination at scheduled and unscheduled visits during the follow-up period of the original study. The generalized estimating equation (GEE) was used to analyze the longitudinally collected data. We used the Kaplan-Meier (KM) method to generate the cumulative hazard curve and compared the mortality in the first year of life between the two groups.Results: In total, 365 patients were included in the study. Exposure to BV was associated with an adverse maternal condition  and maternal hospital admission  but was not linked to any neonatal morbidity at birth. There was a higher frequency of gastro-intestinal morbidity among BV-exposed infants. At 6 months, infants of BV-exposed mothers had higher odds of bloody stool , dehydration , vomiting , and mouth ulcers . At 12 months, exposure to BV was associated with dehydration  and vomiting . KM survival analysis showed non-significant higher trends of deaths among BV-exposed infants (P = 0.65).Conclusion: This study demonstrates differences in maternal and infant morbidity outcomes associated with exposure to BV. Further research is required to determine whether treatment for maternal BV mitigates maternal and infant morbidity. Lactobacillus bifidus and an overgrowth of anaerobic polybacteria that include Clostridiales, Fusobacterium nucleatum, Gardnerella vaginalis, Mycoplasma hominis, and Bacteroides urealyticus, among others. BV may co-exist with other anerobic bacteria, such as Streptococcus, Staphylococcus, and Enterobacteriaceae, typically found in the lower genital tract , also referred to as vaginal dysbiosis, is characterized by altered vaginal biota, which typically has a deficit of al tract .BV has been shown to be a risk factor for pre-term births . A pre-tin-utero stress. In the same study, independent of meconium staining and chorioamnionitis, BV-exposed term babies were at an increased risk of respiratory distress requiring ventilatory support, admission to a neonatal intensive care unit (NICU), and neonatal sepsis  and Trichomonas . BV incrral load .African women carry a disproportionate burden of BV. In the meta-analysis published by Torrone et al., the prevalence of BV among HIV-negative women in sub-Saharan Africa was >40% higher in the high-risk groups, compared to women recruited at community level or at health clinics, but the difference was not significant (49.5 vs. 35.2%) . An equaL. genera that form an important defense mechanism in the infant gut and other mucous membranes, we hypothesized that it may be correlated with an increase in infectious disease morbidity beyond the newborn period.At birth, infants are sterile, and current birthing practices are designed to optimize newborn exposure to maternal biota for which there is already immunologic protection through transplacental immunoglobulin transfer and through breastfeeding, thereby reducing the risk of infection, which is one of the leading causes of newborn deaths , 22. TheHIV-exposed infants have increased vulnerability. In the first instance, BV increases the risk of maternal HIV acquisition , 17. FurThere is emerging evidence that child survival is closely linked to a mother's vital status; the younger a child is when a mother dies, the more likely it is that the child will die . Nguyen This analysis is based on a retrospective cohort study design. The study methods that were used for the original study have previously been described . BrieflyThe original study recruited 425 women, 401 of whom had live births. This study is restricted to 365 pairs who had complete matching information in terms of the BV status of the mother and requisite neonatal characteristics and included 180 women with BV and their exposed infants and 185 without BV. A total of 36 mother baby pairs were excluded because of missing information on the BV status as depicted by the flowchart shown in Pregnant women attending Nairobi City Council clinics were offered HIV testing as an integrated pregnancy assessment and those who tested positive were offered participation in the study. At 32 weeks of pregnancy, after obtaining informed consent, women were subjected to a standard interview and physical examination that included a pelvic examination using a speculum to facilitate collection of vaginal and cervical secretions for microscopy and gram staining for BV, in addition to screening for sexually transmitted diseases and genital shedding of HIV. Cervical and vaginal samples were collected separately using sterile Dacron swabs. To determine the viral load and CD4—8 cell counts, 15 ml of blood was collected in purple-top vacutainers at enrollment.Women were encouraged to deliver at Kenyatta National Hospital (KNH) and nurse midwives of the study team provided 24-h care to facilitate this process. At delivery, a standard form was used to collect delivery data and a cord blood sample.The mother-baby pairs were followed up with at 6, 10, and 14 weeks, and then monthly until the child was 1 year old, and thereafter every 3 months until 24 months of age or death of the infant. Blood samples were collected at pre-determined time points during the follow-up for HIV testing.At each visit, interviews were conducted, and information was gathered using a standard tool on any symptoms of illness since the previous encounter and during the current visit, and of any illness or hospital visits/admissions in the period since the last clinic visit. This was done regardless of whether it was a scheduled or unscheduled visit due to illness. If the participants had sought medical care elsewhere, the take-home medical records or discharge summaries were examined, and information was abstracted to ensure completeness of documentation for the morbidities. The mother-baby pairs underwent a physical examination, their clinical characteristics were documented using a standard tool and the relevant study samples were collected by the team of study doctors, which included an obstetrics registrar and three consultants, two of whom are pediatricians, and one who is a medicine-pediatrics specialist.To capture all morbidity events, the participants had unrestricted access to care in the study clinic that operated for 4 days a week and they received careful instructions on how to navigate the hospital for other services outside clinic hours. The study participants received a standard transport reimbursement for every visit. The women were encouraged to attend the clinic from where they were enrolled for the child's follow-up, and to make sure the child received all the scheduled immunizations.p = 0.02). The 2-year cumulative mortality of the children was 24.4 and 20.0% in the breastfeeding and formula arms, respectively (p = 0.30)  , 24. HIVFor purposes of this study, the following data were retrieved from the main dataset.We abstracted data on demographic characteristics , past reproductive health , baseline viral load, whether they had BV or not, and delivery-related complications , as well as the history and duration of hospitalization.We abstracted information on their status at delivery including, anthropometric measurements , Apgar score at 1 and 5 min after birth, and estimated gestational age using the Dubowitz scores. Newborns with an estimated gestation of >37 completed weeks were classified as premature. Any breathing issues were classified as respiratory distress. Information was abstracted on whether the newborn had jaundice, conjunctivitis, lymphadenopathy, a skin rash, respiratory distress, or any other abnormalities after birth.Data was abstracted on presence of common childhood morbidities, including but not limited to pneumonia, ear infection, blood in stool, lymphadenopathy, encephalopathy, sepsis, conjunctivitis, dehydration, wheezing, hematologic conditions, cold, otitis, fever, cough, diarrhea, thrush, vomiting, difficulty in feeding, heat rash, fungal rush, eczema/dermatitis, scabies, and mouth ulcer.Women were categorized as having BV or not using the Nugent criterion  as descrThe study protocol was approved by the ethics review boards of the University of Washington and the University of Nairobi.Women who had BV and their exposed infants were compared to those who did not have BV. Mean values were used for characteristics with normal distribution, medians for the skewed data, and proportions for the categorical data. Effects of BV were measured and reported as a relative risk (RR) or odds ratio (OR).To estimate the risk of BV of mothers during birth on (1) whether they needed hospital admission after facility delivery and (2) had adverse maternal condition, we estimated the RR.P-value to check for significance.We assessed the viral load data for normality using the histogram and found that it violated the bell shape required for normal distribution; therefore, we calculated the median viral load of 39,482 copies per ml. We then assessed the women with a viral load count more than the median, and less than the median, using chi-square statistics to check for statistical significance. We performed a log transformation of the viral load data to determine whether there was an association of the BV with HIV and used t-statistics to calculate the t-test was performed, whereas curves that deviated from the assumption were considered non-normal data, and the non-parametric and Mann-Whitney U tests were performed to determine any associations. All tests were two-tailed. For the normal data, we reported data as the mean and standard deviation, whereas for the non-normal data, we reported data as the median and inter-quantile range.We compared BV-exposed and unexposed infants. For continuous data, we performed normality checks through standardized normal probability plots. Curves showing the bell shape of the normal distribution were considered normal data, and a p-values using the Fisher's exact test. For the infant data obtained at 6 and 12 months after birth, we used the Generalized Estimating Equation (GEE) because the measures were repeated, and we had to control for the correlation. We employed the independence correlation structure, according to Hardin and Hilbe  censored subjects had the same risk as those remaining in the analysis; thus, censoring was non-informative; and (2) timing of exposure was known exactly; thus, infants exposed to BV were known before birth by checking their mothers' BV status. All cases of deaths from birth were included in the analysis.P < 0.05 were considered statistically significant.All statistical analyses were performed using R version 3.6.3  . Analysip = 0.18). The majority of the women were married, 81% of the BV-exposed group and 73.4% of the unexposed group, and these differences were not significant (p = 0.18) (The mean age was 23.5 years (range 17–39) for the BV-exposed infants and 24.1 years (15–38) for the unexposed infants. Among BV-exposed women 27.2% had not completed the 8 years of primary education, 19.6% had completed 8 years, and 53.3% had >8 years of education compared to 35.9, 17.2, and 46.9%, respectively among the unexposed women, however these differences were not significant ( = 0.18) .p = 0.74). Furthermore, 28.3% of the BV-exposed women reported having had an STD compared to 30.7% of the unexposed women, showing a non-significant difference (p = 0.71)  .p = 0.25). The mean log viral load was 10.64 and 10.37 copies per ml for women afflicted with BV and those who were not, respectively, but results were not significant (p = 0.19) as shown in A total of 79 (53%) BV-exposed women and 76 (46%) unexposed women had a high viral load, higher than the calculated median (39482); the association of a higher viral load with BV was not statistically significant  of the 165 BV-exposed women and seven (4%) of the 177 unexposed women. The risk for an adverse maternal condition was significantly associated with BV RR = 2.45 [  = 0.04] .P = 0.02] as shown in p = 0.3). BV-exposed women had a longer duration of admission compared to unexposed women, a higher maximum admission of 21 days against 16 for the unexposed. The mean hospital admission duration was 1.3 ± 3.2 days among women with BV and 0.7 ± 2.7 days among women with BV (p = 0.91).Among 331 women who delivered in hospital, 48 (15%) required extended hospital admission, 19.3% were BV exposed, and 9.7% were unexposed to BV, RR = 1.99  and vomiting OR 1.64 , p = 0.03], signs of dehydration , and mouth ulcers . Babies exposed to BV were less likely to have lymphadenopathy  and fever  ,  = 0.04] .p = 0.03] and vomiting  decreased. Babies exposed to BV were less likely to have lymphadenopathy , difficulty in feeding [OR 0.78 ], and hepatomegaly . The other characteristics assessed were not significant at the 0.05 level , 05 level .BV-exposed and unexposed infants had similar incidence of conditions affecting the respiratory and other systems at 6 and 12 months.p = 0.39]. We compared the survival between infants whose mothers were exposed to BV and those whose mothers were not, using the KM (p = 0.65).In the BV-exposed pregnancies, there were two still births and two intrapartum deaths, in contrast to zero still births and three intrapartum deaths. Over the first year, there were 14 deaths in the first year, (9 [5%]) of 180 in the exposed group, and (5 [2.8%]) in the 185 unexposed group, showing a risk difference of 2.2%. The relative risk of death in the exposed group was RR = 1.85 [(95% CI 0.63–5.41), g the KM . The graIn this study, we examined the association of maternal BV with a mothers' condition and infants' morbidities. Our analysis showed an increase in the frequency of maternal hospital admissions among women with BV compared to non-exposed subjects. In our literature review, we did not find any publication that directly reports on maternal perinatal adverse outcome associated with BV, and reports have universally focused on fetal/neonatal outcome. The closest article was a systematic review on the use of probiotics to improve maternal microbiota, which demonstrated a reduction in gestational diabetes and better blood sugar control. Prolonged hospital admission has a cost implication and increases the risk of nosocomial infections .Exposure to BV did not show any association with any neonatal morbidities, as previously reported . HoweverThe most novel observation from this study was the link between BV and morbidities among infants at 6 and 12 months after birth. The children exposed to BV had significantly more symptoms of gastrointestinal diseases. Additionally, there was increased mortality among infants exposed to BV, although, the latter did not achieve statistical significance. These observations support our hypothesis that babies exposed to BV have Lactobacillus-deficient biota characterizing BV, making them more vulnerable to gastrointestinal infections . It is pFouhy et al. in the 2012 comprehensive review of the composition of early intestinal microbiota observe that the gut is sterile at birth and is progressively colonized by bacteria achieving an adult like microbiome by the age of 2 . The iniStudies contrasting babies born vaginally with those born by caesarian section show that the gut is first colonized with the mother's vaginal and fecal microbes, which quickly helps to establish the dominant number of lactobacillus within a few hours of delivery. In contrast, Cesarean babies are first colonized with maternal skin microbes with a predominance of staphylococci. It is reasonable to assume that BV-exposed infants were colonized with bacteria typical of vaginal dysbiosis .A number of studies have looked at the long-term impact of being born vaginally vs. Cesarean, therefore acquiring different gut biota, and found worse outcomes in allergy and metabolic conditions, such as asthma and obesity among those born by Cesarean . We haveUnexposed subjects had higher odds of manifesting lymphadenopathy, hepatomegaly, difficulty feeding, and fever, and may reflect reverse causality. The BV-exposed children may have had greater access to antimicrobial therapy, which would have treated a variety of illness beyond what they were reported to have, while the unexposed children went on to have a full expression of their illness.p = 0.65); however, the graph showed a trend toward a higher mortality rate in the BV-exposed group. Therefore, this means that the risk of mortality still exists among infants whose mothers are exposed to BV. Our study was not powered to fully evaluate this. Future studies regarding BV and mortality are needed to confirm our results and to elucidate the underlying associations.There has not been any published literature linking child mortality directly to BV. However, there is an indirect link through the related adverse pregnancy outcome, including pre-term birth, prematurity, and pregnancy complications, which are factors directly related to neonatal and infant mortality . Our KapWe believe the results of our study are credible. The diagnosis of BV was made using the Nugent method, the gold standard, and therefore, there is clarity on the infants' exposure. The technologist who examined the slides was part of the team that developed the Nugent method for diagnosing BV. We used the Nugent method to diagnose BV, which has the advantage of having a standardized scoring system, and therefore, reduces variability in reporting. The alternative method would have been to culture the bacteria, but at the time, there was limited microbiology culture capacity, and diagnosis of BV was not the primary endpoint of the study. Since then, the development of the field of genomics has expanded on opportunities to describe the microbiota, and in the process, has expanded the spectrum of bacteria identified to be part of the human microbiome , 13. StuThe infants were seen frequently for both scheduled and unscheduled visits as needed, and a standard tool was used to collect the data, and therefore, we are confident regarding the documentation about the presence or absence of a specific morbidity. The clinics were run by very qualified staff; thus, the data collected was of high quality, there were few cases with missing data, and therefore, the results are generalizable. For example, of the 401 mother and infant pairs in the original study, the prevalence of BV as reported by Nduati et al.  was 47%.In this study, the syndromic treatment of women with abnormal vaginal discharge, which covers for BV, and prompts the treatment of children's illness, means that effects of BV are attenuated toward null. As observed earlier, participants received transport reimbursement and the research clinic provided a safe space when HIV was still new and very stigmatized. Both BV-exposed and unexposed babies had a median number of eight visits during the first year of life. With this, we were not able to establish the correct frequency of hospital visits due to genuine morbidities.This paper reported many findings that are consistent with those reported in the literature and has added further knowledge in the area of BV. While previous studies have focused only on the effects of BV on infants, our study explored the effects of BV on both the mother and baby in the context of HIV. This has an implication for infants who are more vulnerable to several infections due to a compromised immune status. Our study has shown independence in terms of association of BV with HIV and recommends for BV to be researched alone. Future research is recommended to gain deeper insights into the predictors of BV.https://osf.io/yxbhg/files/.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: The studies involving human participants were reviewed and approved by University of Nairobi and Washington Institution Review Board. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Rosacea is a chronic inflammatory skin disease characterized by facial redness and bothersome symptoms. It can exert significant psychological effects and impair the quality of life of patients. To investigate the prevalence and risk predictors of anxiety and depression in rosacea patients, we conducted a cross-sectional study in an outpatient setting. Consecutive patients completed a questionnaire, which included questions on sociodemographic information and severity of signs and symptoms; they also completed the Patient Health Questionnaire and the Generalized Anxiety Disorder scale. Disease burden was assessed using Dermatology Life Quality Index (DLQI), Willing-to-Pay, and Time trade-off. Multivariate analysis was conducted to determine the risk factors for anxiety and depression. A total of 774 patients completed the survey. The prevalence of anxiety was 53.9% (95% CI: 50.4–57.4%) and that of depression was 58.1% (95% CI: 54.7–61.6%). The factors associated with anxiety were age, gender, the need to make appearances at work, severity of self-reported symptoms, the number of rosacea signs and adaptive behaviors, and disease burden. Depression was associated with younger age, more severe self-reported symptoms, more adaptive behaviors, and higher disease burden. After adjusting for demographics, the risk of anxiety or depression increased in young patients who had severe self-reported symptoms, high DLQI scores, and many adaptive behaviors. Taken together, there is a high prevalence of anxiety and depression among Chinese rosacea patients. Younger rosacea patients who have more severe self-reported symptoms and higher disease burden are prone to anxiety and depression. Rosacea is a chronic inflammatory cutaneous disease with an estimated worldwide prevalence of 5.5% . In ChinRosacea patients indeed often experience various symptoms of emotional stress, including depression, anxiety, social phobia, low self-esteem, and frustration –11. ThisRosacea not only has severe physical and mental health effects, but also imposes a heavy burden on the patients. For the assessment of skin diseases, clinicians commonly use measures/tools, such as the Dermatology Life Quality Index (DLQI) , 17, WilIn this study, we aimed to investigate the prevalence of anxiety and depression among rosacea patients and to identify the possible demographic/clinical characteristics associated with anxiety and depression in rosacea patients. Identification of the predictors for anxiety/depression in rosacea patients may be valuable to clinicians in daily practice.This was a cross-sectional, single center study of 774 rosacea patients conducted at the dermatology outpatient department of Xiangya Hospital of Central South University from May 2018 to August 2019. The inclusion criteria included: (1) patient who visited the hospital for the first time and was independently diagnosed with rosacea by two board-certified dermatologists according to the diagnostic criteria proposed by the National Rosacea Society Expert Committee ; (2) proThe survey consisted of two parts: a self-administered questionnaire and a face-to-face interview. The self-administered questionnaire included questions on patients' demographic information, patients' self-reported signs and symptoms, symptoms of anxiety/depression, and disease burden-related measures , whereas the face-to-face interview included questions on clinical information. The severity of rosacea symptoms was assessed by patients themselves and clinicians using a Visual Analog Scale (VAS) –a horizoWe evaluated anxiety and depression separately using a seven-item Generalized Anxiety Disorder scale (GAD-7) and a nine-item Patient Health Questionnaire (PHQ-9) , 25. TheQuality of life: We utilized the DLQI to assess the effect of rosacea on the quality of life of the patients. The DLQI scores range from 0 to 30, with grade 1 (0~1) indicating no e?ect on the patient's quality of life, grade 2 (2~5) indicating a mild effect, grade 3 (6~10) indicating a moderate effect, grade 4 (11~20) indicating a severe effect, and grade 5 (21~30) indicating very severe impact on the patient's life.Economic burden: We used WTP, which has been widely used in previous studies, to measure the economic burden of rosacea on the patients , 21, 27.Psychological burden: To further assess the psychological burden of rosacea on the patients, we used TTO as previously described . We askeAs described previously , a definCategorical variables were described as absolute and relative frequencies, whereas continuous variables were expressed as means, standard deviations, and medians. The chi-square test was used to compare the prevalence of anxiety and depression among patients with different sociodemographic and clinical characteristics. The chi-square test and Spearman's correlation analysis were used to assess the relationship between anxiety and depression and disease burden. Variables that were identified as significant using univariate analysis were further investigated using multivariate analysis. Multivariate analysis was performed by using multiple logistic regression  to identify variables that were independently associated with anxiety and depression among rosacea patients. Relative risk estimates were calculated as odds ratios (ORs) with 95% confidence intervals (CIs). A two-tailed 5% significance level was used in this study. All analyses were performed using SPSS for Windows .A total of 774 patients were enrolled in this study; 417  suffered from anxiety, whereas 450  had depression. Among the patients who had anxiety, 20.0% (95% CI: 17.2–22.9%) reported moderate to severe symptoms. For the patients who had depression, 25.8% (95% CI: 22.8–28.9%) reported moderate to severe symptoms.n = 601) of the patients have had rosacea for less than a year. Furthermore, 29.1% of the patients (n = 225) had more than six adaptive behaviors .The demographic and clinical information of the participants are summarized in Factors associated with anxiety were younger age, female gender, and higher requirement for appearances in the workplace. Factors linked to depression were younger age and an unmarried status. Moreover, we found that patients who had more rosacea signs displayed a significant association with anxiety, but no association with depression. It is widely accepted that patients have identified many factors that can easily induce or exacerbate rosacea, thus making them consciously avoid these triggers in daily life, named “adaptive behaviors.” Patients who had anxiety or depression tended to have more adaptive behaviors to avoid recurrence and exacerbation of rosacea.Additionally, we found that the patients' self-reported signs and symptoms, rather than the clinically evaluated ones, were significantly associated with both anxiety and depression in a logistic regression model before and after adjusting for demographics. As shown in n = 572) of the patients (DLQI score≥6). The mean ± SD total DLQI score of patients who had anxiety and depression was 14.034 ± 7.511 and 13.336 ± 7.497, respectively, and 8.048 ± 6.348 and 8.407 ± 6.791 for the patients who did not have anxiety and those who did not have depression, respectively. We used WTP and TTO to further evaluate the economic and psychological burden of rosacea on the patients, respectively. Overall, we revealed that the relationship of anxiety/depression with disease burden correlated in a significant way  of the patients enrolled in this study.Based on the insights from previous studies, some patients who have rosacea can be defined as a “high-burden (HB)” population who are characterized by an adverse quality of life, adaptive behaviors, and other economic and psychosocial factors . Accordir = 0.163, P = 0.000) or depression .As mentioned previously, we have determined that patients with anxiety or depression had a significantly positive result in three domains that are indicative of HB . Further, we also found that there is a positive correlation between the final domain of adaptive behaviors and anxiety  , 29. BasConsidering the high incidence of anxiety and depression among rosacea patients, especially in HB patients, it is necessary to identify patients who have anxiety or depression to avoid serious consequences such as suicide. Our results highlighted that younger age, more severe self-reported symptoms, higher DLQI score, and having more adaptive behaviors are predictive factors for the presence and severity of anxiety and depression.Since utilizing GAD-7 and PHQ-9 scales that have numerous questions is not practical in a clinical setting due to the restricted duration of clinical visits, asking questions using the VAS would be of simple and value. Therefore, to screen for anxiety and depression, we recommend the use of the following questions in clinical practice:Score the effect of rosacea on your sleep using the VAS.Score the effect of rosacea on your daily life using the VAS.Describe the severity of your symptoms  using the VASHow many lifestyle changes have you made due to rosacea?These questions will help physicians to recognize patients who have psychological burdens and o?er them psychological counseling earlier.This study has some limitations. Firstly, as this was a single-center survey, the sample population is not representative of the whole population of China. Secondly, the choice of scale for the assessment of anxiety and depression may have the potential to skew the detection of the presence and severity of anxiety and depression in rosacea patients. Lastly, we overlooked the prevalence of anxiety and depression in rosacea patients after proper treatments, and thus not being able to provide comprehensive data to fully demonstrate that rosacea can cause anxiety and depression. However, these limitations are unlikely to confound the comparison of the groups evaluated in this study. We will continue to explore these issues more thoroughly and comprehensively in further studies.In conclusion, the proportion of rosacea patients who have anxiety or depression is large. However, the psychological distress experienced by these patients is usually neglected by clinicians. Our study revealed that younger age, more severe self-reported symptoms, and higher disease burden may indicate the possibility of anxiety or depression, especially when clinical signs and self-reported symptoms are inconsistent or if patients have apparently impaired quality of life. Psychological intervention should be taken into consideration when prescribing atreatment regimen.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by The protocol for this study was established, according to the ethical guidelines of the Helsinki Declaration and approved by the ethical committee of the Xiangya Hospital, Central South University, China (IRB number 201404361). The patients/participants provided their written informed consent to participate in this study.MC and ZD were responsible for writing and revising the article, as well as the experiment design. YH were responsible for the guidance on experiment design and statistical methods. JL were responsible for the participant enrolment and the management and quality control of the research process. All authors gave their scientific contribution and have approved the final 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.


Post-traumatic stress disorder (PTSD) is one of the most commonly reported mental health consequences, followed by disasters and traumatic events, either natural or man-made. At present, there are no unified results for the prevalence rate of PTSD in patients suffering from acute trauma and related influencing factors. Therefore, the purpose of this study is to systematically evaluate the existing literatures, thus obtaining a comprehensive estimation of the combined prevalence rate of PTSD and related factors in trauma patients, so as to provide evidence support for clinical disease prediction models and intervention strategies.Published articles will be retrieved from PubMed, Embase, Cochrane Library, Web of Science, China Biology Medicine Database, China National Knowledge Infrastructure, China Science and Technology Journal Database, and Wanfang Database. Research reports will be searched in March 2021. STATA 14.0 software will be applied for data analysis. Mantel–Haenszel fixed effect model or DerSimonian–Laird random effect model will be selected to estimate the pooled prevalence of PTSD in patients with acute trauma and associated factors.We will disseminate the findings of this systematic review and meta-analysis via publications in peer-reviewed journals.The results of this analysis can be used to establish a risk prediction model of PTSD in patients experiencing acute trauma, so as to provide intervention strategies.DOI 10.17605/OSF.IO/Z275U. Severe trauma can induce systemic reaction, with local manifestations of swelling, tenderness and pain, deformity and dysfunction in the case of fracture and dislocation, and fatal asphyxia, disturbance of consciousness and shock in severe cases. As a common post-traumatic complication, PTSD has serious impacts on the recovery of patients’ social function. Related studies have revealed that, by improving clinicians’ understanding of PTSD, early screening and formulating effective, scientific and correct intervention measures, and clarifying relevant pathogeneses of PTSD, it is of great significance to diagnose and treat PTSD, so as to recover patients’ social function.In recent years, with the rapid development of society, transportation, industry, sports, and agriculture have led to an increasing incidence of injuries.,11 At present, no effective treatment for PTSD is found. About 50% of patients are delayed to the chronic course of disease that in patients with PTSD is more than 10 years, which seriously affects the patients’ life quality. It is very important to prevent the occurrence of PTSD. The risk factors of PTSD include demography, disease severity, psychosocial factors, and so on. However, the current research results are not consistent.In recent years, more and more studies on PTSD patients with acute trauma abroad have proved that patients with acute trauma are the victims of PTSD.In order to better understand the impacts of PTSD on patients with acute trauma, and to guide clinical practices and future researches, we conducted a systematic review and meta-analysis. The specific purpose of this systematic review and meta-analysis is to quantitatively summarize the prevalence of PTSD in patients with acute trauma. On the other hand, we try to investigate the potential predictors and related factors of these diseases.22.1This protocol has been registered on Open Science Framework (grant number: DOI 10.17605/OSF.IO/Z275U). This report will be based on the preferred reporting items for systematic review and meta-analysis protocols.2.2A systematic review of published research that reports the prevalence and associated factors of PTSD in acute trauma patients will be considered. A search of research reports will be made by the following databases: PubMed, Embase, Cochrane Library, Web of Science, China Biology Medicine Database, China National Knowledge Infrastructure, China Science and Technology Journal Database, and Wanfang Database. Research reports in March 2021 will be included. These search terms are summarized in Table 2.32.3.1All relevant research reports that will be available on the search until March 2021 will be included based on the following inclusion criteria:1.The subjects were patients with acute trauma, aged over 18 years;2.The contents of the study are the risk factors or predictive factors of PTSD;3.Using one or more PTSD symptom assessment tools or PTSD diagnostic criteria;4.Case-control study, cohort study and cross-sectional study;5.The results of the study involve the specific values of odds ratio and 95% confidence interval of risk factors.2.3.21.Experimental, qualitative, and psychometric studies;2.Repeatedly published literatures;3.The full text cannot be obtained normally or the extracted data are affected.2.4The process of the selection is exhibited in Figure 2.5We have 2 goals in this systematic review and meta-analysis: to determine the common prevalence of PTSD in patients with acute trauma and to estimate the related factors of PTSD in patients with acute trauma. The combined prevalence of PTSD will be calculated using STATA 14.0. The estimated value of the combined effects of the factors related to PTSD will be calculated. The odds ratio will be prepared from the searched research reports using 2 tables.2.6 Newcastle–Ottawa Scale score ≥6 means that the literature quality is better.The quality of the research reports included will be assessed using the Newcastle–Ottawa Scale for quality assessment.2.7 If there is significant statistical heterogeneity, we will apply the DerSimonian–Laird random effect model.The data analysis will be conducted through STATA 14.0, and we will use OR and 95%CI to represent. Mantel–Haenszel fixed effect model or DerSimonian–Laird random effect model will be adopted to estimate the pooled prevalence of PTSD in patients with acute trauma and associated factors. If there are no findings of statistical heterogeneity, the Mantel–Haenszel fixed effect model is adopted for data synthesis.2.8χ2 test and I2 test. When P < .1 and/or I2 > 50%, the random effect model is adopted for the combined analysis. Otherwise, the fixed effect model is used for the combined analysis.The prevalence of the reported researches will be checked for heterogeneity by performing a heterogeneity 2.92.9.1According to trauma type, gender, mean age, study type, study quality, and time between trauma occurrences, we make a subgroup analysis2.9.2We conducted meta-regression analyses to explore source of heterogeneity on a range of study characteristics . We first conducted univariate meta-regression, followed by multivariable analysis including factors that reached statistical significance in the univariate models.2.9.3To determine the stability of the outcome measures, each outcome measure was analyzed by performing sensitivity analysis.2.9.4We will evaluate the possibility of publication bias using funnel plots and Egger test of bias will be taking as a complement.2.10We will try our best to ensure the integrity of the data. If the included data is not complete, we will take every effort to contact the corresponding author of the article, including sending emails or making a phone call. If the corresponding author cannot be contacted, we will remove the experiment with incomplete data. After data integrity is assured, intention analysis therapy and sensitivity analysis will be carried out.2.11The content of this article does not involve moral approval or ethical review and will be presented in print or at relevant conferences.3–20 Among them, the symptoms of separation mainly include insufficient consciousness, slow response, and split amnesia. The repeated re-experience of injury events refers to the refeeling of traumatic events and related people and objects. The improvement of alertness is mainly manifested by anxiety, sleep disorders, irritability, and increased vigilance. There is a growing recognition that PTSD has adverse effects on health outcomes, including readmission rates, return to work rates, activities of daily living, feelings of rehabilitation, and opioid application.–24 The need for adequate screening and early intervention for patients with PTSD symptoms is growing.The manifestations of PTSD are various, including the separation of symptoms, repeated repetition of injury events, deliberate avoidance and so on.The purpose of this study was to determine the combined prevalence of PTSD in patients with acute trauma and to estimate the combined effects of related factors as well. The study will try to fill this gap by providing new evidence-based results to attract the attention of decision makers. The results of this study are expected to determine problems for future research.Conceptualization: Lang Wang.Data curation: Furong Tang, Jianghong Tan, Xi Guo.Formal analysis: Furong Tang.Funding acquisition: Lang Wang.Project administration: Lang Wang.Resources: Xi Guo.Software: Jianghong Tan, Xi Guo.Supervision: Xi Guo, Jinlian Huang, Jinhua Yi.Validation: Jinlian Huang, Jinhua Yi.Visualization: Jianghong Tan, Jinlian Huang.Writing – original draft: Lang Wang, Furong Tang, Jianghong Tan.Writing – review & editing: Lang Wang, Furong Tang, Jianghong Tan.


Chlamydia psittaci infection in humans, also known as psittacosis, is usually believed to be an uncommon disease which mainly presents as community-acquired pneumonia (CAP). It is usually sporadic, but outbreaks of infection may occasionally occur. In outbreaks, diagnosis and investigations were usually hampered by the non-specificity of laboratory testing methods to identify C. psittaci. In this study, we use metagenomic next-generation sequencing (mNGS) in the diagnosis of a family outbreak of psittacosis under COVID-19. Three members of an extended family of 6 persons developed psittacosis with pneumonia and hepatic involvement with common symptoms of fever and weakness. Two newly purchased pet parrots, which had died successively, were probably the primary source of infection. Imagings show lung consolidations and infiltrates, which are difficult to be differentiated from CAP caused by other common pathogens. mNGS rapidly identified the infecting agent as C. psittaci within 48 h. The results of this work suggest that there are not characteristic clinical manifestations and imagings of psittacosis pneumonia which can differentiate from CAP caused by other pathogens. The use of mNGS can improve accuracy and reduce the delay in the diagnosis of psittacosis especially during the outbreak, which can shorten the course of the disease control. Family outbreak under COVID-19 may be related to the familial aggregation due to the epidemic. To our knowledge, this is the first reported family outbreak of psittacosis in China, and the first reported psittacosis outbreak identified by the method of mNGS in the world. Chlamydia psittaci. Traditionally, psittacines (parrot-type birds) have been considered as the host of the causative bacterium. In addition, many other birds, including wild birds and commercially raised poultry, have also been involved [Psittacosis is a zoonotic infectious disease caused by the bacterium involved . Pathogeinvolved . PneumonPsittacosis is usually sporadic , 4, but Chlamydia psittaci, and molecular detection. Laboratory diagnosis requires meeting any one of the following three criteria: (1) IgM antibody against C. psittaci titre detected by MIF of 1:16 or higher; (2) a fourfold or greater increase in antibody titre between serum samples collected 2 weeks apart, using a micro-immunofluorescence (MIF) or complement fixation test (CFT) and (3) isolation of Chlamydia psittaci from respiratory secretions [Psittacosis can be diagnosed by serology, isolation of cretions . Polymercretions . BecauseMetagenomic next-generation sequencing (mNGS) is a new tool, which can precisely and rapidly identify potential pathogens, regardless of whether they are bacterial, viral, fungal, or parasitic . Recent We herein describe the clinical features of a family outbreak of psittacosis pneumonia under COVID-19 which was diagnosed by mNGS after exposure to sick pet parrots, and demonstrate that mNGS is an effective diagnostic method. The family outbreak under COVID-19 may be correlated to the familial aggregation due to the epidemic. To our knowledge, this is the first report of family outbreak of psittacosis identified by mNGS in China.We conducted a retrospective case review of 3 members of an extended family of 6 persons admitted successively within 1 week to the Second Affiliated Hospital of Chongqing Medical University, a tertiary teaching hospital in Chongqing, China, with psittacosis pneumonia in December 2020. For each case, data on symptoms, results of laboratory tests, dynamic and comprehensive computed tomography imagings, and clinical course of the disease were extracted from electronic medical records. Additional data on treatments, outcomes, and relevant follow-up data were also collected.This study was approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University and conducted in accordance with the Declaration of Helsinki.Sample processing and DNA extraction: Clinical samples  were collected by following the standards of aseptic processing procedures and treated with enzymes for liquefaction within 24–48 h from collection at 4°C. To selectively enrich the microbial nucleic acid, the samples were processed using the Vision Medicals’ Patho-NET technology to remove host gDNA before DNA extraction. Up to 600 uL samples were transferred to new sterile tubes. Cell wall of microbes was broken by vortex with glass beads following described conditions. DNA was extracted from 300 uL-treated sputum or BALF using the TIANamp Micro DNA Kit  following the manufacturer’s operational manual. The extracted DNA specimens were used for the construction of DNA libraries.mNGS was conducted using the following operational steps ,20Samplg for 10 min at 4°C. Plasma samples were transferred to new sterile tubes. DNA was extracted from 300 uL of plasma using the TIANamp Micro DNA Kit  following the manufacturer’s operational manual. The extracted DNA specimens were used for the construction of DNA libraries.(2) Library construction: DNA libraries were constructed through transposase mediated methods . The quality of the DNA libraries was assessed using a Qsep1 bio-fragment analyzer  to measure the adapters and the sizes of fragments before sequencing. The size of qualified library was 300∼500 bp without adapters and PCR dimers, and concentration of the library was greater than 0.5 ng/uL. Finally, qualified DNA libraries were pooled together and sequenced on Nextseq 550 Dx sequencing platform .(3) Data Analysis: High-quality sequencing data were generated by removing low-quality, and short (length < 40 bp) reads, followed by computational substractions of human host sequences mapped to the human reference genome (hg38 and YH sequences) using Burrows–Wheeler Alignment. The remaining data by the removal of low-complexity reads were classified by simultaneously aligning to four Microbial Genome Databases, consisting of viruses, bacteria, fungi, and parasites. The classification reference databases were downloaded and optimized from public database, such as NCBI, EBI or Genbank. In the end, the multi-parameters of Species in Microbial Genome Databases were calculated and exported and professionals with microbiology and clinical background will interpret the result.While for clinical sample (blood): Volume of 3–4 mL of blood sample was collected from patients, placed in cell-free blood collecting tubes and stored at room temperature before plasma separation and centrifuged at 1600C. psittaci pneumonia successively in Chongqing, China in December, 2020. Among them, two were women, one was man. Their median age was 56 (range 46–72) years  years . All patAll patients had similar symptoms: recurrent fever higher than 38.5°C and weakness. Two patients had chill, cough and poor appetite, and one patient had myalgia at the onset of their illness. The median time from the onset of the illness to admission to our department was 4.7 (range 3–7) days .The first patient admitted to our hospital on 22 December 2020 was the 72-year-old female with the chief complaints of weakness for 4 days and fever for 8 h. Chest CT examination performed in our hospital on 22 December 2020 showed signs of consolidations with bronchograms which were compatible with pneumonia on the left lower lobe and the right upper lobe.The second patient admitted to our hospital on 23 December 2020 was a 40-year-old female who was the elder daughter-in-law of the first patient, with the chief complaints of fever for 7 days and cough for 2 days. Chest CT examinations performed in another hospital on 21 December 2020 showed signs of infiltrates, reticular shadows and consolidations with bronchograms on the right lower lobe and the right upper lobe which were compatible with pneumonia.The third patient admitted to our hospital on 28 December 2020 was the 46-year-old male who was the younger son of the first patient, with the chief complaints of weakness for 3 days and fever for 1 d. Chest CT examinations performed in our hospital on 28 December 2020 showed signs of consolidations with bronchograms on the right lower lobe which were compatible with pneumonia.The signs on physical examination of the patients were rare and non-specific, only weakened respiratory sounds, and little moist rales on auscultation on the affected side were found in the first patient (the eldest patient).9/L, percentage of neutrophils of 76.3%, C-reactive protein (CRP) level of 133 mg/L, and procalcitonin (PCT) level of 0.45 ng/mL. All the three patients had elevated lactate dehydrogenase levels, two patients had elevated alanine aminotransferase and aspartate aminotransferase levels, and one patient had elevated creatine kinase levels and hypokalaemia .After admission to our hospital, two younger patients (patient 2 and patient3) underwent bronchoscopy, and bronchoalveolar lavage fluid (BALF) was collected for mNGS. The blood and sputum samples, while not BALF of the elderly patient, were simultaneously sent for mNGS due to the age and relatively poor condition. The typical bronchoscopy findings were hyperaemic tracheal and bronchial mucosa with oedema, with little thin white secretions in the segmental bronchi .Figure C. psittaci sequences that covered 0.03% of the total C. psittaci genome were detected by mNGS in the blood sample of the first patient, and 379 specific C. psittaci sequences that covered 2.95% of the total C. psittaci genome were detected by mNGS in the sputum sample of the first patient. Two specific C. psittaci sequences that covered 0.11% of the total C. psittaci genome were detected by mNGS in the BALF sample of the second patient. A total of 779 specific C. psittaci sequences that covered 2.34% of the total C. psittaci genome were detected by mNGS in the BALF sample of the third patient  to control the symptoms, but it didn’t work. Then clinician in that hospital added a cephalosporin (cefdinir). The first and third patients didn’t go to hospital, and took some antipyretics (paracetamol) by themselves. However, the clinical condition didn’t improve but gradually deteriorated.C. psittaci infection was confirmed, the antibiotic was changed to minocycline [.Patients were given empirical antibiotic therapy with β-lactam/β-lactamase inhibitor combinations and quinolones on admission according to the CAP management guideline . The mNGocycline . Minocycocycline .After minocycline therapy was initiated, patients’ fever generally disappeared within 3 days, and their cough and general conditions, including weakness and poor appetite, gradually improved. The percentage of neutrophils reduced to normal. Procalcitonin and C-reactive protein dropped sharply to near normal . ConsoliChlamydia psittaci [Psittacosis is a common disease in birds caused by the infection of psittaci , 24. Howpsittaci , 4. Therpsittaci . There wpsittaci , 44–47. C. psittaci can be divided into various serology, namely IF, CFT, ELISA/EIA, and IPA tests, culture, and molecular detections. Culture has high specificity and is often used to confirm the diagnosis, but is generally used as a supplement to other tests and requires a high-level biosafety laboratory. Hence culture cannot be routinely performed in most diagnostic laboratories and hospitals. Serological tests were used in most reports [C. psittaci is not available in most hospitals in China, including many tertiary hospitals. PCR is a highly specific targeted test, but it is usually performed only when clinicians suspect the infection of C. psittaci, which is difficult to diagnose clinically.In this paper, we describe the first reported family outbreak of human psittacosis in China, to our knowledge. As to the reasons for relatively fewer reports of human psittacosis in China, especially outbreaks, except a lower rate of pathogen carriers among Asian parrots than among European parrots , the lim reports , 41, 49. reports . Molecul reports , 38, 41.In recent years, mNGS, another molecular detection method came into our view. The advantage of mNGS is that it has a wide detection range and does not need to specify the suspected causative microorganism priorly, and can be used in the diagnosis of encephalitis, meningitis, and lower respiratory tract infections , 52. In Legionella pneumophila, Chlamydia pneumoniae, Mycoplasma pneumoniae via serology, and other pathogen through blood culture, sputum culture on admission which were negative, and giving empiric treatment with antibiotics for CAP which didn’t work, they were given mNGS of blood and bronchoalveolar lavage fluid to detect the pathogen, especially after a family cluster trend was observed. The results of mNGS returned to us within 48 h and identified C. psittaci in all the three patients. After we got these results, we retrospected the contact history of birds and poultry, a definite exposure history of two sick pet parrots, which had died 1 week before the admission, was traced. The use of mNGS can improve accuracy and reduce the delay in the diagnosis of psittacosis especially during the outbreak, which permits a timely study of the spread of the disease and shortens the course of the disease control.In our study, it was lucky for these three patients in this family outbreak. After screening other common pathogens for CAP including COVID-19, H1N1 flu virus, parainfluenza virus, With the use of mNGS, there is a growing number of human psittacosis identified in clinic practice in China, especially among fever of unknown origin and atypical pneumonia caused by rare pathogens. Due to the non-specific symptoms and imaging manifestations, psittacosis is easily underdiagnosed and misdiagnosed. This may be another reason for the low report rate in China.The social isolation and restrictions on recreational measures due to COVID-19 may lead to increased demand for pets. Among them, pet birds especially parrots, will increase the risk of psittacosis. At the same time, the epidemic of COVID-19 decreases unnecessary travel which leads to relatively increase of familial aggregation and the possibility of family outbreak of psittacosis.Our three cases of psittacosis pneumonia, diagnosed with the help of mNGS, have similar clinical features and characteristics with previously reported cases , 53. PreC. psittaci has higher pathogenicity and faster reproduction rate than other Chlamydiales species, so C. psittaci causes more severe inflammatory reactions. Inflammatory lesions representing, as consolidation with bronchograms and ground-glass opacity close to the pleura on CT, were mainly in the lower lobe of the lung in all the three patients, and also in the upper lobes in two patients, which were more extensive on radiography than would be expected from clinical signs. In the meantime, the manifestations under bronchoscope, including congestion and oedema of bronchial mucosa, and the amount of secretions in the airway, were lighter than would be expected depending on the range of consolidations, which are different from CAP with similar CT imagings caused by bacteria. The characteristic appearances of psittacosis under bronchoscope have not been reported previously.The laboratory data of patients in our paper showed normal or slightly elevated leucocytes, neutrophils, and PCT, along with high CRP levels, which are consistent with those usually showed in psittacosis. Knittler and Sachs  reportedC. psittaci belongs to the family Chlamydiaceae in the order Chlamydiales [C. psittaci infections, with a minimum inhibitory concentration of 0.03–0.06 mg/L and a minimum bactericidal concentration of 0.06–0.25 mg/L in vitro [As mydiales , tetracymydiales . Tetracymydiales . Minocycin vitro . BecauseC. psittaci infection. As a result, many psittacosis index patients are discovered late and often in hospital. mNGS can quickly and objectively detect more and more rare pathogenic microorganisms in clinical specimens, without the need for specific amplification, and has an advantage in the diagnosis of rare pathogenic bacteria in difficult cases. The use of mNGS can improve the accuracy and reduce the delay in the diagnosis of psittacosis especially during the outbreak, which permits a timely study of the spread of the disease and shortens the course of the disease control. It may be a better testing method for C. psittaci than culture, PCR and various serological methods, particularly in outbreaks of psittacosis. However, the cost of mNGS is relatively high in clinic, so it can not completely replace the current conventional identification methods. And limitations to the use of mNGS technology exist, despite its widespread use. There is no authoritative guide to the interpretation of the mNGS report. Detection of a broad spectrum of pathogens by mNGS has blunted the diagnosis of pathogenicity resulting in the inability to distinguish between background, colonization and microbial infection, and pollution [In outbreaks of psittacosis, there is often a considerable delay between the onset of symptoms in patients and the laboratory diagnostic test, especially when the history is not taken carefully and the bird contact is unknown, standard diagnosis in patients with pneumonia usually does not include tests for ollution . Hence, The main limitation of this study is that it included only three cases of psittacosis pneumonia. This relatively small sample size is insufficient to investigate all the relevant features of psittacosis pneumonia. The study was a retrospective study and we did not use CFT or MIF to confirm the diagnosis. Given the poor response of CAP to empirical antibiotics, mNGS can shorten the time to diagnosis and enable the initiation of targeted antibiotic therapy earlier.


Using the amplitude of low-frequency fluctuation (ALFF) and functional connectivity (FC) algorithm to study the alteration of brain function in hemodialysis patients with end-stage renal disease (ESRD).t-test. Then, we set the altered ALFF brain areas as seed regions of interest (ROIs), and FC analysis was used to investigate the functional integration patterns between the seed ROI and the voxels within the whole brain.We recruited 20 patients with ESRD on regular hemodialysis and 17 healthy controls (HCs). All of the participants underwent resting-state fMRI (rs-fMRI), neuropsychological tests, and blood biochemical examination. The individual ALFF values between the two groups were tested by an independent sample The ALFF values of the right precuneus and angular gyrus (RAG) in the ESRD group were lower than those in the HC subjects, but the right precentral gyrus showed higher ALFF values in patients. Hemoglobin (Hb) was negatively correlated with the ALFF values of the right precentral gyrus, and the ALFF values of the right precuneus were negatively correlated with line-tracing test (LTT) scores in patients with ESRD. Patients with ESRD show decreased connectivity between the RAG and the left precuneus, right superior frontal gyrus (RSFG), and the connectivity within the RAG was weak. In addition, FC in the RAG-right cuneus, right precuneus-left supramarginal gyrus was enhanced in the patient group.Our research suggested that, in hemodialysis patients with ESRD, the brain areas with abnormal spontaneous brain activity and FC are mainly located in the default mode network (DMN) regions. Hb and the LTT results were correlated with abnormal spontaneous brain activity. These findings provide additional evidence to understand the possible underlying neuropathological mechanisms in patients with ESRD. Cognitive impairment is common in patients with end-stage renal disease (ESRD), especially in those who have received hemodialysis –3. TheseMagnetic resonance imaging is a non-invasive technique, and plays a significant role in revealing the neuropathological mechanism of cognitive dysfunction. Recently, researchers have used many MRI methods to study the neuropathological changes in patients with ESRD, including structural, functional, and perfusion MRI –16. By uTo our knowledge, previous ALFF studies found that patients with ESRD have abnormal spontaneous brain activity –24. For We hypothesize that hemodialysis patients with ESRD may have abnormal spontaneous brain activity and functional integration impairment at the same time. The purpose of our research was to study the functional connectivity patterns between the brain regions with altered ALFF value and the voxels within the whole brain in hemodialysis patients with ESRD, and to analyze the correlation between the results and clinical biomarkers.2). The patients were required to have received treatment with conventional hemodialysis for over three months. The etiology and hemodialysis duration were collected from electronic medical records of the patients.This prospective study was approved by the Ethics Committee of Guangdong Second Provincial General Hospital. Written informed consent was obtained from all the subjects before they were included in this research. We enrolled 20 patients with ESRD from the Department of Organ Transplantation in our hospital and 17 healthy volunteers from the community. All of the subjects were younger than 50 years in order to avoid possible confounding factors. All of the patients were diagnosed as stage 5 of chronic kidney disease, the glomerular filtration rate was almost completely reduced (<15 ml/min/1.73 m+), phosphate (P), and calcium (Ca2+). Before MRI examination, all of the subjects were asked to complete a questionnaire, including age, sex, years of education, and a series of neuropsychological tests.We excluded patients with drug abuse, diabetes, mental illness, alcoholism, major neurological diseases , left-handedness, head rotation >1.0°, or translation >1.0 mm during MR scanning. All of the patients completed blood biochemical tests within 24 h before MR examination, including serum creatinine, uric acid, urea levels, Hb, Kalium , Self-rating Anxiety Scale (SAS), digit symbol test (DST), line tracing test (LTT), number connection test type A and B (NCT-A/B), serial dotting test (SDT), and Montreal Cognitive Assessment (MoCA). The SAS and SDS were used to exclude patients with anxiety and depression. The sensitivity of the MoCA scale in screening mild cognitive impairment is higher than Mini-mental State Examination (MMSE) . NCT-A/B2, matrix = 64 × 64, and total volume = 240. There were 33 axial slices with 3.5 mm thickness and 0.7 intersection gap in total. The duration of each rs-fMRI scan was 8 min. All of the subjects were asked several questions intended to check the extent of their cooperation after scanning.Magnetic resonance imaging data were obtained using a 3.0 Tesla MR scanner  in the Department of Medical Imaging, Guangdong Second Provincial General Hospital. All of the subjects were instructed to keep their eyes closed and awake, and to not think of anything systematically during MR scanning. In order to detect clinically silent lesions, conventional imaging sequences (such as T1-weighted images and T2-FLAIR images) were performed for every subject. The rs-fMRI data were collected using the echo-planar image (EPI) sequence, which is sensitive to blood oxygenation level-dependent (BOLD) contrast. The parameters of EPI sequence were as follows: repetition time (TR) = 2,000 ms, echo time (TE) = 30 ms, flip angle = 90°, field-of-view (FOV) = 230 × 230 mmhttp://restfmri.net/forum/DPARSF]. Because the signal is unstable because of an incomplete T1 relaxation at the beginning of acquisition, and the subject needs to adapt to the scanning state, the first 10 time points of each set of fMRI data need to be removed. There were only 230 volumes which were left for analysis. Next, the slice timing, head motion realignment, and spatial normalization to the standard Montreal Neurological Institute (MNI) template with a resampled voxel size of 3 × 3 × 3 mm3 were conducted. The maximum translation of head movement in any of the x, y, or z direction of all subjects was <1.0 mm, or the maximum rotation about three axes during scanning were <1.0°. In our study, one healthy control (HC) and three patients were eliminated because of an excessive head motion. And then, the normalized data were smoothed with a 4-mm full-width at half maximum. Finally, nuisance covariates were regressed out from the fMRI data, including global mean signals, head motion parameters, cerebrospinal fluid signals, and white matter signals.Image preprocessing was conducted using a MATLAB toolbox known as Data Processing Assistant for Resting-State [DPARSF ; http://Amplitude of low-frequency fluctuation analysis was performed by using a similar toolkit as previously described . After pSeed-based FC analysis was used in our study. The regions showed that ALFF differences between patients with ESRD and controls were selected as regions of interest (ROIs) for FC analysis. The fMRI time series of all voxels within the region with abnormal ALFF were averaged to obtain the reference time series of each ROI. Next, a bandpass filter (0.01–0.1 Hz) was performed for each time series. The Pearson's correlation coefficients of the time course between each seed and each voxel of the whole brain were calculated. Fisher's r to z transformation was used to improve the normality of the correlation coefficients.t-tests were used to analyze the differences in age, education level, MoCA, SDS, SAS, DST, NCT-A/B, LTT, and SDT between patients and HCs. The difference in sex was calculated by the chi-squared test.The statistical analysis of clinical and demographic data were carried out by the software SPSS version 22.0 . Independent two-sample t-test was used to assess the ALFF differences between patients and HCs . A partial correlation coefficient was used to analyze the association between ALFF values and neuropsychological test scores, and chemical results in the patient group. Age, sex, and duration of education were set as control variables. P-value < 0.05 was considered to be significantly correlated .Taking age, duration of education, and sex as confounding covariates, voxel-by-voxel two-sample t-test was applied to analyze the Z score FC maps within the ESRD group and the healthy group . Then, compared the Z score FC maps between patients with ESRD and HCs by using two-sample t-tests, with the threshold of significance differences set at p < 0.05 using FDR correction in the REST software (http://www.restfmri.net). To study the association between FC intensity of regions and neuropsychological test scores, and chemical results in patients with ESRD, we also used the Partial correlation analysis as aforementioned .A one-sample P > 0.05). The score in the MoCA of the ESRD group was lower than that of the HC group (p < 0.05).The results of demographic, neuropsychological, and blood biochemical tests are shown in r = −0.487, p = 0.040, r = −0.511, p = 0.036, Relative to controls, the ALFF values of the right precuneus and angular gyrus (RAG) were significantly decreased in patients with ESRD. But the right precentral gyrus exhibited increased ALFF values in patients with ESRD compared with controls ; Table 2Z scores of abnormal FC regions and any clinical data in patients.The three seed ROIs, in which increased/decreased ALFF values were found in hemodialysis patients with ESRD, were chosen for FC analysis. Relative to the control group, the patient group showed decreased connectivity between the RAG and the left precuneus, right superior frontal gyrus (RSFG), but increased connectivity between the RAG and right cuneus; the connectivity within the RAG was weak ; Table 3The results of ALFF and FC analysis indicated that some important areas of DMN in hemodialysis patients with ESRD have altered spontaneous brain activity and functional integration impairment. The DMN brain areas are responsible for brain functions such as memory, attention, motion, and language and emotion management . The finIn our research, relative to the control group, the ALFF values of the right precuneus and RAG were decreased, but increased in the right precentral gyrus in the patient group. As a vital component of the DMN, the precuneus contributes to self-processing, the extract of episodic memory, and visuospatial imagination . The AG In many neuropsychological tests, a lower score on MoCA was found in the patient group relative to HC. This may indicate that mild cognitive impairment had developed in patients in ESRD who were undergoing hemodialysis. Unexpectedly, after being corrected by multiple comparisons, MoCA scores exhibited no correlation with the ALFF values in the aforementioned brain regions. However, the correlation coefficient between the ALFF values of the right precuneus and the LTT results was negative in patients with ESRD. This might indicate that the altered spontaneous brain activity of the precuneus might affect the psychomotor speed and visuospatial ability of hemodialysis patients. Our results were similar to those of previous studies; Gu et al. also found that the MoCA scores were significantly lower in the ESRD group, and the ALFF values of the bilateral anterior cingulate gyri were positively correlated with the MoCA scores . Luo et Z scores of abnormal FC in the brain regions. These inconsistent results may be attributed to the small sample size and different methodology.Importantly, we found that the ALFF values of the right precentral gyrus were negatively correlated with Hb. Some previous studies have shown that anemia is associated with cognitive impairment in patients with ESRD , 24, 36.Another important finding was that the ESRD groups showed decreased connectivity between the RAG with the left precuneus and RSFG, and the connectivity within the RAG was weak. The decreased connectivity may be because of the interruption of neuronal connection in the brain networks, and the dysfunction of functional integration in the selected areas of the DMN that might be related to cognitive impairment –43. ThesWe acknowledge that some limitations existed in our study. First, the sample size was small, which may have an adverse effect on the statistical analysis and reduced the persuasiveness of our research results. Second, we used a cross-sectional study; however, it is necessary to explore the changes in brain activity in patients with ESRD before and after renal transplantation by a longitudinal study. Third, we did not collect the results of blood biochemical tests for HCs, and the health status obtained by inquiry showed a lack of objective evidence. At last, the influence of blood pressure and the etiology of nephropathy on brain function in hemodialysis patients with ESRD was not assessed in our study.Using resting-state ALFF and FC analysis, our study showed that the hemodialysis patients with ESRD had serious abnormalities of spontaneous brain activity and functional integration in the DMN regions, particularly in the AG and precuneus, which may be associated with cognitive impairment. These findings offer additional evidences to realize the possible underlying neuropathological mechanisms in hemodialysis patients with ESRD.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Ethics Committee of Guangdong Second Provincial General Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.HS, GJ, and XQ designed and carried out the experiment. The clinical data were collected by SM. The results of neuropsychological tests and MRI data were obtained and sorted out by ML, YY, and KH. HS, SF, and KH processed and analyzed the complete data. The manuscript was written by HS and GJ. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China , the Science and Technology Planning Project of Guangdong (202002030234), and the National Natural Science Foundation of Guangdong Province (2018A0303130129).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.


However, none of them showed a significant difference between the SCLC and NC groups, and only the IgM autoantibody against JAK2 showed a higher expression in LUSC than in NC (p = 0.046). Through logistic regression analysis, with the five IgM autoantibodies and carcinoembryonic antigen (CEA), one diagnostic model was constructed for LUAD. The model yielded an AUC of 0.827 . The diagnostic efficiency was superior to that of either CEA (AUC = 0.692) or IgM autoantibodies alone (AUC = 0.698). Notably, the accuracy of this model in early-stage LUAD reached 83.02%. In conclusion, we discovered and identified five novel IgM indicators and developed a multi-analyte model combining IgM autoantibodies and CEA, which could be a valuable and powerful auxiliary diagnostic tool and might improve the accuracy of early detection of LUAD.Immunoglobulin M (IgM) autoantibodies, as the early appearing antibodies in humoral immunity when stimulated by antigens, might be excellent biomarkers for the early detection of lung cancer (LC). We aimed to develop a multi-analyte integrative model combining IgM autoantibodies and a traditional tumor biomarker that could be a valuable and powerful auxiliary diagnostic tool and might improve the accuracy of early detection of lung adenocarcinoma (LUAD). A customized protein array based on cancer driver genes was constructed and applied in the discovery cohort consisting of 68 LUAD patients and 68 normal controls (NCs); 31 differentially expressed IgM autoantibodies were identified. The top 5 candidate IgM autoantibodies [based on the area under the receiver operating characteristic curve (AUC) ranking], namely, TSHR, ERBB2, survivin, PIK3CA, and JAK2, were validated in the validation cohort using enzyme-linked immunosorbent assay (ELISA), which included 147 LUAD samples, 72 lung squamous cell carcinoma (LUSC) samples, 44 small cell lung carcinoma (SCLC) samples, and 147 NCs. These indicators presented diagnostic capacity for LUAD, with AUCs of 0.599, 0.613, 0.579, 0.601, and 0.633, respectively ( LUAD, a% of cancCurrently, low-dose spiral CT (LDCT) and pathological tissue biopsy are used to screen and detect LC patients clinically, but the former has high false-positive rates and the latter is traumatic, which causes some excessive diagnosis, unnecessary tests, invasive procedures, and, rarely, radiation-induced cancers . In receTumors are the products of the malignant transformation of normal cells, which are characterized by continuous proliferation and metastasis in the body. The prominent feature of tumor cells in immunology is the appearance of certain tumor-associated antigens that are not visible or have low expressions in normal cells of the same type . Due to In the present study, we aimed to screen valuable IgM autoantibody indicators for LUAD by protein array and verify them in another sample cohort with enzyme-linked immunosorbent assay (ELISA). Ultimately, the five candidate IgM autoantibodies and CEA were integrated to construct a diagnostic model to improve the diagnostic efficiency for LUAD. The diagnostic model might be able to improve the treatment status of LC patients and increase their survival rate.All serum samples included in this study were obtained from the Specimen Biobank in Henan Key Medical Laboratory of Tumor Molecular Biomarkers collected from a provincial hospital in Zhengzhou, Henan Province, China, between 2016 and 2019. Two independent sample cohorts  were used in this research. The discovery cohort consisted of 68 LUAD patients (LUADs) and 68 normal controls (NCs) matched by gender and age. In addition, 147 LUADs, 147 matched NCs, 72 LUSC patients, and 44 SCLC patients were included in the validation cohort. The blood samples of all LC patients were drawn upon their first diagnosis without any other cancers, antitumor treatment, and autoimmune diseases. All NCs were individuals who had a health checkup without history of cancer, pulmonary diseases, and autoimmune diseases. The sera were extracted and stored according to standard protocols .The human protein array assay was commissioned to BC Biotechnology Co., Ltd.  based on the conception of our laboratory. The protein chip contained 154 recombinant proteins or protein fragments, 11 of which , with potential diagnostic value, were reported in our previous research , 21; theIn order to eliminate the deviation caused by the inconsistency of the background value between different samples, the ratio of the foreground value to the background value of each protein (F median/B median) was calculated, namely, the signal-to-noise ratio (SNR). Statistical analyses of the samples were based on the SNR value. The tests for the test samples were repeated 30 times at different time points, different chips, and different positions to evaluate the stability of the operation.p < 0.05]. In view of the optimal choice in terms of the number of indicators when constructing the model, we set the top 5 based on AUC rankings  among the 31 differential autoantibodies as the candidate IgM indicators  of each autoantibody in LUADs and NCs was applied based on the SNR value of each samples. Thirty-one IgM autoantibodies were screened and showed higher distinguishing values in LUADs .Given the insufficiency of a single IgM autoantibody for LUAD detection, we selected and matched the populations (83 LUADs and 83 NCs) with CEA results from the validation cohort to construct a diagnostic model that combines candidate IgM autoantibodies and CEA. Binary logistic regression was adopted to combine the IgM autoantibodies with CEA. It was found that the AUC of CEA alone was only 0.692 and that of the five IgM autoantibodies (combined) was 0.698. However, the AUC of the model that included both five IgM autoantibodies and CEA reached 0.827 (vs. advanced stage (0.803) and without lymph node metastasis (0.565) vs. with lymph node metastasis (0.900) vs. without distant metastasis (0.557) vs. with distant metastasis (0.905), all p < 0.05]  (p > 0.05)  and CEA for LUADs is exhibited in . 83 NCs Table 3.In recent studies, the EarlyCDT-Lung test has achieved remarkable results in distinguishing LC patients from high-risk subjects. The panel, which includes seven IgG autoantibodies, demonstrated sensitivity and specificity values for newly diagnosed LC of about 40% and 90%, respectively –24. RegrA fundamental target in cancer research is to understand the mechanisms of cell transformation. Cancer driver genes are involved in tumorigenesis and development across cancer types . Hence, ERBB2 is a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases, and its IgG autoantibody has been extensively assessed as a potential diagnostic biomarker for gastric cancer  and breaIn phase II of the present study, we found that single-autoantibody indicators always showed an insufficient diagnostic ability, as in previous studies –37. MoreInterestingly, the diagnostic performance of the model with five IgM autoantibodies and CEA (AUC = 0.827) was a little higher than that of the model with three IgG autoantibodies and CEA (AUC = 0.781). Additionally, for early-stage LUAD, the model with five IgM autoantibodies and CEA showed great diagnostic value, but the model with three IgG autoantibodies and CEA had no diagnostic value. This would justify IgM autoantibodies as having the advantage of potentially identifying malignant lesions at an earlier stage than IgG autoantibodies, as reported in the study of Pedchenko et al. , where hOur study displayed several novel features. Firstly, we adopted a new modality of a customized protein array based on cancer driver genes to improve the possibility of screening potential IgM autoantibodies, which yielded satisfactory results. Secondly, ELISA was applied to validate the candidate IgM autoantibodies in an independent validation cohort with the aim of identifying robust LUAD biomarkers. Thirdly, the model constructed with the combination of IgM autoantibodies and CEA improved the diagnostic value of the existing clinical auxiliary diagnostic method, which implied that multi-analyte tests could ameliorate the problem of the insufficient diagnostic value of biomarkers.There were certain limitations in this retrospective study. In future work, we will longitudinally explore the levels of these IgM indicators using pre-diagnostic longitudinal sera from LUADs. Moreover, we will verify the performance of the diagnostic model in a larger sample cohort to evaluate its capability in the early stage of LUAD.In conclusion, we performed a meaningful approach of a protein array assay to identify novel IgM autoantibodies for the detection of LUAD. The diagnostic model that combined five IgM autoantibodies and CEA may be a more accurate method for the early detection of LUAD and possess certain predictive power for the severity of tumor.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Medical Ethics Committee of Zhengzhou University. The patients/participants provided written informed consent to participate in this study. Written informed consent was obtained from individual(s) for the publication of any potentially identifiable images or data included in this article.LD was responsible for study conception and design. LD and JZ provided administrative support. TW, LP, FL, XiZ, and XW contributed to the provision of study materials or patients. XuZ, JL, YW, ML, and DJ collected and organized data. XuZ analyzed and interpreted the data and wrote the manuscript. All authors contributed to the article and approved the final manuscript.This work was supported by the Leading Talents of Science and Technology Innovation in Henan Province (grant no. 20420051008), the National Natural Science Foundation of China (grant no. 8167291), the Major Project of Science and Technology in Henan Province (grant no. 16110311400), the Key Project of Discipline Construction of Zhengzhou University (grant no. XKZDQY202009), and the Project of Basic Research Fund of Henan Institute of Medical and Pharmacological Sciences (grant no. 2020BP0202).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.