Social determinants of health (SDH) include factors such as income, education, and race, that could significantly affect the human immunodeficiency virus and acquired immunodeficiency syndrome (HIV/AIDS). Studies on the effects of SDH on HIV/AIDS are limited, and do not yet provide a systematic understanding of how the various SDH act on important indicators of HIV/AIDS progression. We aimed to evaluate the effects of SDH on AIDS morbidity and mortality.A retrospective cohort of 28.3 million individuals was evaluated over a 9-year period (from 2007 to 2015). Multivariable Poisson regression, with a hierarchical approach, was used to estimate the effects of SDH—at the individual and familial level—on AIDS incidence, mortality, and case-fatality rates.A total of 28,318,532 individuals, representing the low-income Brazilian population, were assessed, who had a mean age of 36.18 (SD: 16.96) years, 52.69%  were female, 57.52%  were pardos, 34.13%  were white/Asian, 7.77%  were black, and 0.58%  were indigenous. Specific socioeconomic, household, and geographic factors were significantly associated with AIDS-related outcomes. Less wealth was strongly associated with a higher AIDS incidence  and mortality . Lower educational attainment was also greatly associated with higher AIDS incidence , mortality  and case-fatality rates . Being black was associated with a higher AIDS incidence , mortality  and case-fatality rates . Overall, also considering the other SDH, individuals experiencing greater levels of socioeconomic deprivation were, by far, more likely to acquire AIDS, and to die from it.In the population studied, SDH related to poverty and social vulnerability are strongly associated with a higher burden of HIV/AIDS, most notably less wealth, illiteracy, and being black. In the absence of relevant social protection policies, the current worldwide increase in poverty and inequalities—due to the consequences of the COVID-19 pandemic, and the effects of war in the Ukraine—could reverse progress made in the fight against HIV/AIDS in low- and middle-income countries (LMIC).10.13039/100000060National Institute of Allergy and Infectious Diseases (NAIDS), 10.13039/100000002National Institutes of Health (NIH), US Grant Number: 1R01AI152938. Various definitions of social determinants of health (SDH) express the idea that the living and working conditions of population groups are related to and influence their health, and that socioeconomic inequalities may lead to health inequalities. To investigate studies that evaluated possible associations between SDH and outcomes related to HIV infection/AIDS, we used the Medline and Embase electronic databases through keywords: 'social determinants of health' AND ('human immunodeficiency virus' OR 'acquired immune deficiency syndrome'), with the addition of filters for study type AND , and publication type ('article' OR 'article in press'). The search was performed without any language restrictions, until December 20, 2022. Additional searches were then made from the references found in the identified articles.We found 145 searches and identified 15 studies that, to a certain extent, observed that geographic, socioeconomic, and individual level factors were associated with HIV/AIDS outcomes. However, the results were limited to specific populations, used only cross-sectional or aggregate data, had a relatively small sample size, and findings were often inconsistent.While different HIV/AIDS outcomes and SDH were assessed in these studies, no study has been conducted to evaluate the effects of a wide range of SDH on several AIDS-related outcomes  in a low- and middle-income country (LMIC), such as Brazil, using large cohort data and robust analytical methods.Our findings show the relevant role of SDH associated with socioeconomic vulnerability in all of the outcomes assessed. Our study provided a unique opportunity to use the largest single, longitudinal dataset of socioeconomic and health data existing in any LMIC, to assess the effects of SDH on AIDS-related outcomes  in a LMIC. To the best of our knowledge, this is the first nationwide, longitudinal study, with an large individual sample to show the effect of SDH on AIDS-related outcomes, with strong and consistent findings. They were all influenced by SDH, with an emphasis on geographic (region and area of residence), socioeconomic , and living condition (home infrastructure) factors.Our findings show that the most economically disadvantaged groups of the population—the poor, black people, the illiterate, and those with inadequate housing—are at greater risk of becoming sick, and dying from AIDS. Understanding the influence of SDH on the health/disease process of AIDS is urgent, to tackle the disease, especially in countries characterized by large economic and health disparities, such as Brazil. Strategies aimed at health education, prevention of infection, timely diagnosis, early initiation of antiretroviral therapy (ART), and adherence to treatment, are crucial to tackle the disease, and are already applied worldwide. However, direct investments in the health sector should be coupled with those to improve social inequalities, focusing on socioeconomic determinants.The human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) are responsible for a decades-long epidemic, resulting in more than 40 million deaths.,,11,,,,,9,The worst AIDS-related outcomes are still tied to social inequalities, which impede progress towards ending the HIV/AIDS epidemic.Despite Brazil’s pioneering epidemic responseOur study aims to evaluate the effects of SDH on AIDS-related outcomes, namely incidence, mortality, and case-fatality rates, using a large cohort of individuals over a long study period, in Brazil.This retrospective cohort study was based on de-identified, linked, registry data of 28.3 million Brazilians, from 2007 to 2015. The population studied was identified by selecting individuals from the 100 Million Brazilians Cohort,The process of selecting individuals into the cohort is shown in This study adheres to Brazilian research regulations, and its protocolCadÚnico), used to identify people eligible for social protection programs in Brazil; data on all AIDS diagnoses recorded in the Brazilian Ministry of Health Information System for Notifiable Diseases (SINAN); and data on mortality recorded in the Mortality Information System (SIM), from 2007 to 2015.The study cohort comprises linked data from individuals listed in the Unified Registry of Brazilians  was defined as years from the date of admission to the cohort until the time of AIDS diagnosis (incidence), AIDS-related death , death from other causes, or end of the study timeframe. For case-fatality rates, follow-up commenced from the date of AIDS diagnosis.The analysis used a hierarchical approachBolsa Família—if qualified as a beneficiary); Block 3: household conditions ; or Block 4: behavioral factors . Age, sex, year of admission into the cohort, and municipal level data on AIDS endemicity and mortality were included as adjustment variables in all analyses (The outcome variables available were i) new AIDS cases, defined by adapted CDC, Rio de Janeiro/Caracas, and death criteria; ii) AIDS-related death as the underlying cause, considering the International statistical classification of diseases and related health problems, 10th revision (ICD-10) codes related to AIDS (B20–B24)  originally had five response categories . However, the yellow/Asian category represents a very small proportion of the Brazilian population. In our cohort, only 0.47% of individuals self-identified as yellow/Asian, and this segment also represents a small number of cases and deaths from AIDS , the majority were women , young adults (24–34.9 years old) , live in the Northeast  and Southeast  regions, and urban areas . With regards to socioeconomic status (Block 2), the majority had elementary school education , were pardo , classified as level 5 of wealth  , and were Bolsa Família Program beneficiaries . They lived in houses with favorable conditions (Block 3), with the majority living in houses constructed with bricks , with electricity , a low domiciliary density (up to 2 residents per room) , a public water supply network , public garbage collection , and a sanitary sewage system   and Southeast . Higher proportions of AIDS cases were observed in men than in the general population , were young adults (24–34.9 years old) , resident in urban areas , had an elementary school education , were black , and had the lowest level of wealth . Similarly, AIDS-related deaths were higher among men  and young adults (24–34.9 years old) , in the Southeast , resident in urban areas , had an elementary school education , were black , and had the lowest level of wealth . In both cases, few differences in concentration were observed for household conditions (Block 3)  ; the AIDS mortality rate was 6.90 per 100,000 py ; and the AIDS case-fatality rate was 6.90 per 100 py  . There wThe hierarchical approach allowed the selection of the SDH that produce significant effects for AIDS outcomes. For the AIDS incidence and mortality outcomes, the same set of variables was maintained in the final models: all variables from blocks 1  and 2 (socioeconomic status) of analysis were maintained in the final models; for block 3 (household conditions), the variables water supply and waste disposal were excluded in the intra-block analyses (models 1) and the variable sewage system was excluded in the final inter-block analyses (models 3), all other variables were maintained in the final models .Table 2EFor the AIDS case-fatality rates outcome, all variables from blocks 2 (socioeconomic status) and 4  of the analysis were maintained in all hierarchical selection steps. The variable area of residence  was excluded in the final inter-block analyses (model 4). For block 3 (household conditions), the intra-block analyses (model 1) suggested the exclusion of the variables household material, water supply and waste disposal, and in the final model (model 4) the variable electrical power was also excluded, while the other variables (household density and sewage system) were maintained in the final model (model 4) .Table 3EFrom these selection steps, we obtained the final models with the adjusted effect of SDH on AIDS incidence, mortality , and casThe region of residence was associated with incidence and mortality; individuals living in the South  or Central-West  of Brazil were most likely to become AIDS cases. People living in the Southeast  and South  of Brazil experienced a higher risk of AIDS-related mortality . The areWith regards to socioeconomic indicators, illiterate people had a 46% higher risk of becoming an AIDS case  and a 176% higher risk of an AIDS-related death , when compared to those who had completed higher education. Similarly, the risk was higher for those who had only completed elementary school, both for incidence  and mortality  . For peoBlack people had a 53% higher risk of becoming an AIDS case , and a 69% higher risk of an AIDS-related death  than those who self-identified as white or of Asian heritage . Among PWealth was closely related to AIDS outcomes, with the risk strongly increasing as the wealth level decreases. Individuals with lower wealth levels were 55% more likely to become an AIDS case , and 99% more likely to die  . Among PBolsa Família receipt also showed associations with all outcomes under study. Individuals who received Bolsa Família for less than two years had a higher risk of becoming ill  and dying from AIDS , when compared to those who did not meet the criteria for receiving the benefit. A protective effect was observed for long-term receipt, with those receiving the benefit for between 5 and 10 years, or more than 10 years, having a lower risk of becoming ill  and dying from AIDS  (The duration of ctively) . Among Pctively) .All sensitivity analyses produced similar estimates, demonstrating the robustness of the results. Different model specifications—in terms of alternative sets of individual-level and aggregate-level variables—did not significantly change the SDH effect estimates, and analyses performed solely with a small set of municipalities with a high quality of vital information, produced similar results. In the analyses for potential changes over time of the association between SDH and AIDS-related outcomes, the effects of all SDH remained similar throughout the study period. In some SDH, such as education, race/skin color, wealth levels, and length of time receiving Bolsa Família, there was a slight effect on the increase , different sexual risk behaviors, and access to healthcare. Previous studies have shown that individuals with less education tend to be exposed to more risky sexual behaviors,In our study, people who self-identified as black had higher AIDS-related incidence, mortality, and case-fatality rates. Similarly, in high-income countries, incidence and mortalityWith regards to other SDH, household-related conditions  had significant relationships with AIDS-related incidence, mortality, and case-fatality rates, with higher risks among those living in conditions of lower socioeconomic status.Analysis of the Bolsa Família conditional cash transfer (CCT) program, showed expected effects. For individuals who received a CCT for a short period of time (less than 2 years), a positive association was observed with AIDS outcomes, which may be related to the characteristics of greater social vulnerability of individuals eligible for the program. On the other hand, for those who received the CCT for a longer period of time (5 years or more), a protective effect was observed, revealing a longer-term positive effect of the program for AIDS outcomes. This may be due to the poverty-relief effect of the program’s cash allowances, and conditionalities to receive these benefits, such as the need to monitor their health at primary health care services, and school attendance for children and adolescents. This brings families closer to the services that make the diagnosis, and health education actions, which enable advancement in the level of schooling. Systematic reviews have already demonstrated that the CCT may reduce risky sexual behaviors (such as sex work), and consequent HIV infection.In the analyses of factors associated with AIDS case-fatality rates, the effects of several SDH decreased when we included behavioral and other proximal variables in the model . This was expected, due to their mediation effectsOur results reveal that the entire chain of AIDS-related events is influenced by SDH, whether through a higher risk of infection , or a greater risk of acquiring an advanced disease, and dying from it . It is understood, therefore, that SDH, especially wealth levels, education, and race/skin color, are strongly associated with the dynamics of HIV/AIDS infection and survival.This study has a number of limitations. The first is that the study cohort focuses on the poorest half of the Brazilian population. While this could limit the representativeness of the study population, it may also be considered a strength of our analyses, considering that SDH mainly exert their effects on individuals with a lower socioeconomic status. This large cohort includes a high number of extremely poor individuals, who are usually not considered, or underrepresented, in traditional, observational, or randomized studies.Another limitation is heterogeneity in Brazil’s 5570 municipalities’ ability to deliver effective and timely AIDS diagnoses, as well as adequate access to health services and follow-up of PLWHA. Thus, living in a municipality with higher or lower endemicity, health surveillance capacity, health infrastructure, and socio-environmental conditions may contribute to different levels of detection and management of HIV/AIDS outcomes. In order to correct for this possible bias, first we assessed the dependency of outcomes at the municipal, state, or region of residence level. Second, we assessed the adjustment for county variables representing health infrastructure and socio-environmental conditions. And third, we evaluated estimates restricted to counties with adequate quality of vital information. We detected no change in the estimates for the different models evaluated as a sensitivity analysis (The analysis of case-fatality rates also has specific limitations, since AIDS may have a relatively long survival period.Our results show—for the first time in such a comprehensive way and with a large number of individuals—that SDH play a major role in the burden of HIV/AIDS in a highly unequal MIC, such as Brazil, and wealth, education, and race have a particularly strong effect on AIDS incidence, mortality, and case-fatality rates.These findings have important implications for HIV/AIDS prevention and control programs in other LMICs. This study lends evidence to assertions that investments to reduce social inequalities, with a focus on the most important SDH that affect HIV/AIDS, should be promoted and coupled with current biomedical and behavioral interventions. Investments in public health policies are required, to expand access, and ensure equity of care, allowing those with greater social vulnerabilities to be prioritized. HIV/AIDS policies also need to broaden their view on social vulnerabilities and health prevention actions, and the discrimination that the poor, black people, and the illiterate suffer to access health services, should be effectively addressed.Without the implementation of significant interventions to improve SDH, especially social protection programs, there is a risk that the current increase in poverty rates and social vulnerabilities—partly due to the COVID-19 pandemic, the effects of the war in the Ukraine, and the global inflation crisis—could reverse progress made in the fight against HIV/AIDS over the last decades, and hamper achievement of the HIV/AIDS-related Sustainable Development Goals.DR, ID, LES, and JM developed the study concept. MLB, MYI, and CASTS collected the data. IL, DR, AFS, and JP designed the study and investigation. IL, AFS, LM, and NSG have accessed and verified the data. IL, AFS, LM, and NSG analyzed the data, and wrote the first draft. DR, ID, LES, JM, and LM supervised and validated the study process. RA managed the project, resources, and software. All authors had access to all the data reported in the study. All authors contributed to data interpretation and validation, reviewed, and edited the manuscript.https://cidacs.bahia.fiocruz.br/en/platform/cohort-of-100-millionbrazilians. The linkage protocols are explained in the referenced publications, and the codes are available at: https://gitHub.com/gcgbarbosa/cidacs-rl. Individual-level data will not be available for sharing, due to confidentiality and ethical issues.The protocol for creation of the 100 Million Brazilians Cohort, and the 100 Million Brazilians Cohort profile is available in the publications referenced in the article. Further material is available at: The authors declare no competing interests.


IgG4-related disease (IgG4-RD) is a newly discovered systemic disease that can affect any organ or tissue in the body. IgG4-related kidney disease (IgG4-RKD) is relatively rare but essential to IgG4-RD. However, there are few reports of IgG4-RD mimicking malignant ureteral tumors leading to hydronephrosis. We report here a rare case of IgG4-RD involving the ureter.An 87-year-old man presented to our nephrology department with anorexia, nausea, and acute kidney injury in November 2020. Urinary computed tomography (CT) examination revealed a right lower ureter mass with right renal and ureter hydronephrosis. The serum level of IgG4 was 1890 mg/dL, and the concurrently renal biopsy revealed extensive infiltration of IgG4-positive plasma cells in renal interstitium, which was diagnosed as IgG4-associated tubule-interstitial nephritis(IgG4-TIN). The renal function improved significantly after double-J tube implantation of the right ureter and moderate-dose hormone therapy. The serum IgG4 decreased to the normal range, and the right lower ureter mass almost disappeared after one year of low-dose hormone maintenance therapy.IgG4-RD can present as a mass in the renal pelvis and (or) ureter, leading to hydronephrosis. Therefore, early recognition of this disease is significant. Most patients respond well to hormonal therapy to avoid surgical treatment due to misdiagnosis as malignant tumors, causing secondary harm to patients. IgG4-RD is a newly discovered systemic disease in the past 20 years, which can affect any organ or tissue in the body . IgG4-RKAn 87-year-old male presented with a history of hypertension for over 50 years. He had type 2 diabetes for more than ten years, coronary heart disease for more than ten years, and "right lobectomy" for "right lung cancer" 16 years ago. The patient had a history of chronic renal insufficiency with basal serum creatinine around 187 μmol/L. In November 2020, he came to our outpatient department due to anorexia and nausea for half a month. His biochemical test results suggested that serum creatinine(Scr) was 428 μmol/L, urea nitrogen(BUN) was 20.9 mmol/L, the complement C3 was 0.53 g/L, C4 was standard, the IgG was 25.65 g/L, the blood eosinophil was 0.73 × 109/L, the total IgE was 453.3 IU/ mL, and the 24-h urinary protein quantification was 2.38 g. Urinary CT examination revealed a mass in the right lower ureter with the right renal and ureter hydronephrosis. Ultrasonography showed generalized superficial lymph node enlargement throughout the body. Due to the patient's previous history of lung cancer resection, the right ureteral mass was suspected to be a ureteral malignant tumor. We performed a urine examination three times to find abnormal cells, which were all negative. Cystoscopy and right ureteroscopy were performed by the urology department, which showed local bulging of the right ureteral orifice, ranging from about 2.5 × 1.0 cm, and stenosis of the right ureteral orifice, and an F6 double-J tube was indwelled in the lower segment of the right ureter. A biopsy was taken on a right ureteral orifice bulge, and a histopathological examination revealed the extruded and deformed fibrous tissue  and European League Against Rheumatism (EULAR) classification criteria for IgG4-related disease , this caIn 2001, Hamano et al. found that patients with Autoimmune pancreatitis (AIP) had elevated serum IgG4 concentrations and massive IgG4-positive plasma cell infiltration in the diseased tissues . In 2007Combined with this patient, the authors of this paper searched for reports worldwide in the past 20 years on IgG4-RD involving the renal pelvis and(or) ureter Table  1, 4, 88–35. We According to the reports of Ramasamy V et al.  and Lei In conclusion, IgG4-RD can present as a mass in the renal pelvis and (or) ureter, leading to hydronephrosis and resembling a ureteral tumor. Although IgG4-RD may have many clinical manifestations with multiple organ involvement, but there are few reports on such participation of the ureter. Therefore, early recognition of this disease is significant. Most patients respond well to hormonal therapy to avoid surgical treatment due to misdiagnosis as malignant tumors, causing secondary harm to patients.


The aim of the study was to evaluate the differences in clinical profile, laboratory parameters, and ophthalmological signs, and symptoms between patients with high IgG4 Graves orbitopathy and patients with normal IgG4 Graves orbitopathy.This was a prospective observational study. We recruited adult patients with Graves Orbitopathy(GO) referred to our clinic for further diagnostics and treatment. Eventually, 60 patients with GO were enrolled in the study. All patients underwent ophthalmological assessment, magnetic resonance imaging (MRI) of the orbits, and laboratory tests, including IgG4 serum concentration measurement. High IgG4 GO was diagnosed if the IgG4 concentration exceeded 135 mg/dl. We used both the clinical activity score (CAS) and magnetic resonance imaging (MRI) to assess the activity of GO. Eventually, active GO was defined according to MRI results.Among 60 GO patients, 15 (25%) patients had elevated IgG4 levels. Patients in the high IgG4 group had a higher prevalence of active GO by MRI than patients with normal IgG4 . They also had a higher eosinophile count in peripheral blood, a lower bilirubin level, a more frequent lower eyelid retraction, and a lower prevalence of glaucoma. There were no statistically significant differences between the groups in CAS. Patients with active GO, had higher median IgG4 level . The receiver operating characteristic (ROC) analysis for IgG4 as a marker of active GO revealed the following results: AUC 0.848 for the cut-off value of 54.2 mg/dl, sensitivity 79.5%, specificity 87.5%, positive predictive value 94.6%, negative predictive value 59.1%.We demonstrated that IgG4 is a marker of GO activity. Certain differences in the clinical profile of patients with high IgG4 GO, and normal IgG4 GO were observed. More data is needed to establish whether patients with high IgG4 GO are GO patients with particularly active disease or actually represent a distinct clinical entity related to IgG4-Related Disease. Human immunoglobulin G4 (IgG4) usually constitutes less than five percent of the total amount of IgG in the serum. This makes it the least abundant IgG subclass but certainly not the least important . It has 3,6. Typical manifestations are pancreatitis, chronic sclerosing sialadenitis, dacryoadenitis, thyroiditis, mediastinal or retroperitoneal fibrosis, periaortitis, cholangitis, inflammatory pseudotumor, and Mikulicz disease , especially in a subgroup of patients with Graves orbitopathy (GO) . Graves So far, the role of IgG4 in GD and orbitopathy has been assessed to a limited extent. According to a recent review, the average prevalence of elevated IgG4 levels in GD patients is around 10% (5.4% in patients without GO and 17.6% in patients with orbitopathy). While some studies linked elevated IgG4 levels with the occurrence of GO and its activity, better response to thiamazole, or increased thyroid antibody levels, other studies failed to find such associations , 15. TheIn our study, we aimed to evaluate whether IgG4 can be used as a marker of GO activity in clinical practice and assess the differences in clinical profile, laboratory parameters, and ophthalmological signs and symptoms between patients with GO and high IgG4 serum concentration and patients with GO and normal IgG4 serum concentration.This was a prospective observational study. It was conducted at the Department of Endocrinology, Metabolism, and Internal Medicine of the Poznan University of Medical Sciences . We recruited all adult patients (>= 18 years old) diagnosed with Graves orbitopathy, who were referred to our department for diagnostics and treatment of the disease. Exclusion criteria were: 1) systemic immunosuppressive or immunomodulatory treatment at the time of admission or in the previous 6 months; 2) Chronic kidney disease stages 4 and 5; 3) Liver failure; 4) Active neoplastic disease or suspicion of malignant disease; 5) Active acute or chronic infections; 6) Any immunodeficiency disorder 7) pregnancy.Eventually, 60 patients with GO were enrolled in the study, of whom 25 underwent previous thyroidectomy, 28 were treated with radioiodine in the past (5 underwent both thyroidectomy and radioiodine therapy), and 6 were still on antithyroid medication. All patients were screened for human immunodeficiency virus, hepatitis B, and hepatitis C. Patients who were carriers of those viruses were excluded from the study.A detailed history of treatment and comorbidities, including smoking status, was obtained from each patient.An extensive ophthalmologic examination was performed in each patient by an experienced ophthalmologist who was blinded to IgG4 results throughout the study. Hertl’s exophthalmometer was used for precise exophthalmos measurements. Patients were evaluated according to the Clinical Activity Score (CAS), based on the following symptoms 1) Periorbital or retroorbital pain or pressure; 2) Pain with lateral, downward, or upward movement, and following signs 1) Swelling of the eyelids; 2) Redness of the eyelids; 3) Conjunctival injection; 4) Chemosis; 5) Inflammation of the caruncle or plica. Intraocular pressure (IOP) values were obtained in each patient with Goldmann applanation tonometry. IOP values over 21 mm Hg were considered diagnostic for glaucoma. However, it is worth noting that some patients have been diagnosed with glaucoma before entering the study and at the time of admission had IOP <21 on IOP lowering medication. Patient’s best corrected visual acuity was measured and defined according to the WHO definitions of vision impairment: mild – visual acuity worse than 6/12 to 6/18; moderate – visual acuity between 6/18 and 6/60; severe – visual acuity between 6/60 and 3/60, blindness – visual acuity worse than 3/60. Extraocular muscle involvement was defined as restriction in eyeball motility resulting in double vision in either primary or extreme gaze positions. Optic nerve involvement was defined as pallor or choking of the optic disc with preserved or diminished visual acuity; Corneal involvement was defined as the presence of stippling, ulceration, clouding, or perforation of the cornea.Fasting (overnight) venous blood samples were taken from each patient. Detailed laboratory analysis was conducted, including thyroid-related hormones , renal function (creatinine), liver function , electrolytes , metabolic profile  and inflammatory process markers (C-reactive protein) and complete blood count with differential (CBC-D).The CBC-D was measured by the flow cytometry based hematology analyzer Sysmex-XN 1000 (Sysmex Europe GmbH) TRAb levels were evaluated by the radioimmunological method with a commercially available radioimmunoassay kit (Brahms GmbH). The rest of the parameters mentioned above were assessed with the use of the COBAS 8000 analyzer (Roche).Finally, IgG4 serum concentration was measured by an immune–enzymatic assay using commercially available ELISA kits . Based on IgG4 levels, we divided patients into two groups – GO with high IgG4 and GO with normal IgG4 serum concentration. We applied the widely recognized cut-off value diagnostic for IgG4-RD (135 mg/dl), which was also used by the majority of previous studies on the role of IgG4 in GD.Each patient underwent thyroid ultrasound. The volume of each thyroid lobe was calculated with the use of the following formula: 0.52 x length x width x depth. The cumulative volume of the thyroid gland was obtained after adding together volumes of the right and left lobe. For differential diagnosis and to assess the activity of GO, we performed a magnetic resonance imaging (MRI) of the orbits in each patient. MRI was performed by Magnetom Skyra 3T scanner. Patients with signs of active inflammatory process in dedicated sequence of the MRI of the orbits were placed in the active GO group, while patients without signs of active inflammation were placed in the inactive GO group. Active disease was diagnosed if the MRI revealed swollen extraocular muscles with increased signal intensity in the STIR(short tau inversion recovery) or T2-weighted sequences. If the imaging test did not show any abnormalities or just swelling of the extraocular muscles without increased signal intensity then inactive GO was diagnosed.Every patient signed a written informed consent to participate in the study. The project was conducted in accordance with the declaration of Helsinki . The stuThe statistical analyses were performed using PQ Stat v.1.8.4. The Shapiro-Wilk test was used to check for normality. We assessed the equality of variances with Levene’s test. We used the unpaired student’s T-test to compare continuous, normally distributed data with homogenous variances. The Mann-Whitney U test was used to compare nonparametric data. The comparison of qualitative variables was carried out with the use of Fisher’s exact test. Pearson’s test was used for the correlation analysis of parametric data, while the Spearman test was used for the correlation analysis of nonparametric data. Data are presented as mean ± Standard Error (SE) or median . The P level <0.05 was considered significant. All tests were two-tailed. The ROC (receiver operating characteristic) curves were built. AUC was calculated with the DeLong method. The P level <0.05 was considered significant. All tests were two-tailed.A total number of 60 patients were enrolled in the study. We divided patients into the high IgG4 group (IgG4 serum concentration >135 mg/dl) and normal IgG4 group (IgG4 serum concentration <135 mg/dl). 15 (25%) patients were placed in the high IgG4 group and 45 (75%) in the normal IgG4 group. Patients did not differ significantly in age, gender, or smoking status. There were also no statistically significant differences between those two groups in the number of patients treated with levothyroxine or antithyroid medications. The groups also did not differ in the number of patients who previously were treated with thyroidectomy radioiodine or received intravenous glucocorticoid pulse therapy in the past (>6 months ago). The thyroid volume was comparable among those two groups . There was no statistically significant difference in IgG4 serum concentrations between patients previously treated with methylprednisolone pulse therapy compared to patients who have never been treated  vs. 60.3  mg/dl, P=0.275). A detailed summary of patient’s ultrasound results, history and demographic data are presented in Patients in the high IgG4 group had a higher prevalence of active GO than patients in the normal IgG4 group . Patients in the elevated IgG4 group had also a higher eosinophile count (240 (190–320) vs 130 (70–250) cells/μl, P=0.041) and a lower total bilirubin level . Other biochemical parameters, including complete blood count, TRAb, TPOAb, TgAb, TSH, fT3, fT4, ALT, AST, creatinine, electrolytes, and lipid profile parameters, did not differ significantly. A full comparison of biochemical parameters is presented in We performed a detailed ophthalmologic examination to check for differences in ocular signs and symptoms between the high IgG4 and normal IgG4 groups. Patients in the high IgG4 group had more frequently lower eyelid retraction . They suffered less often from glaucoma . We also observed that patients with elevated IgG4 levels tended to manifest conjunctival injection more frequently (60% vs. 28.89%), but the result failed to achieve statistical significance (P=0.060). There were no statistically significant differences between the groups in CAS (and its components), intraocular pressure (IOP), visual acuity, proptosis, soft tissue involvement, extraocular muscle involvement, optic nerve involvement, GO signs . Findings from the extensive ophthalmological examination are summarized in We divided patients into groups based on the activity of the disease. Patients with active disease had a higher IgG4 level  vs 43.45  mg/dl, P<0.001) – We performed a receiver operating characteristic (ROC) analysis to evaluate the potential of IgG as a diagnostic marker of GO activity Figure 2Sixty patients with GO were enrolled in our study. 15 (25%) of our patients had IgG4 serum concentration elevated over 135 mg/dl, which is more prevalent than the reported average of IgG4 elevation in GO 17.6%) . Some au.6% . SomIn our study, 15 (100%) of the patients with high IgG4 levels had active GO compared to only 29 of 45 (64.44%) in the normal IgG4 group. Other studies have also reported a higher prevalence of active GO in elevated IgG4 patients , 19. SomHowever, our study is the only one where GO activity was assessed with the combined use of MRI with CAS. Other studies used only CAS to divide patients into active and inactive GO groups. Even though CAS is undoubtedly a great diagnostic tool, it has to be interpreted cautiously. Some papers report that its sensitivity can be as low as 55% and the PPV is 65% . Other rInterestingly, in our study 8 out of 15 patients in the high IgG4 group had a CAS score of <3, but all of them had signs of active disease on MRI of the orbits. To a certain extent this could be explained by a small number of cases with severe or sight threating threating GO.However, this also shows that we cannot rely solely on CAS in diagnosing active GO. There is a significant group of patients who present with troublesome symptoms , which could still be treated with immunosuppressive/immunomodulatory drugs. If we relied solely on the CAS score, they might be falsely pronounced “inactive” and thus do not receive proper treatment. Based on this observation, we hypothesize that IgG4 might be a very sensitive marker of the inflammatory process, which is also elevated in patients, who do not show typical signs of active orbitopathy like inflammation of the conjunctiva, plica, eyelids, or chemosis.,but have active disease in the extraocular muscles and periorbital soft tissue. IgG4 serum concentration measurement would help to avoid false negative (“inactive”) diagnoses in patients with active disease and low CAS scores, especially in situations when access to MRI is limited, the patient has contraindications to MRI, or the results of imaging studies are ambiguous.Various serum markers in GO have been studied recently, including Th2-derived chemokines  , 27. OurSome researchers described that patients with high IgG4 GD had high TRAb levels, which positively correlated with IgG4 serum concentration , 22. OthWe did not find any associations between CAS and IgG4 levels, which is in concordance with results reported in other papers , 22. HowPatients with GD and elevated IgG4 levels were found to have lower echogenicity of the thyroid on ultrasound examination , 29. UnfA recent study by Li et al. reported that IgG4 levels decreased after intravenous methylprednisolone treatment and that patients with high IgG4 levels had better treatment outcomes. Our study was not longitudinal in design, and thus we could not check for post-treatment results. However, patients enrolled in our study, who were previously treated with methylprednisolone pulse therapy (>6 months ago) did not differ in IgG4 levels from treatment naïve patients.Previous studies did not find any differences in ocular signs or symptoms other than CAS between patients with elevated IgG4 and normal IgG4 levels , 22. HowAn interesting novel finding from our study is that patients in the elevated IgG4 group had lower bilirubin levels than GO patients with normal IgG4 values. Previous studies did not check for bilirubin levels. Historically, only high bilirubin serum concentrations were considered disturbing findings. However, more recently, low serum bilirubin levels have been reported as a risk factor for adverse outcomes like stroke or coronary artery disease, diabetes, cerebral deep white matter lesions, and many others –37. BiliOur study has several limitations. Firstly, it was a single-center study. Secondly, most of the patients referred to our hospital had a history of the longstanding disease, and most of them had active GO, while patients with inactive GO were underrepresented. In addition, the majority of our patients were already after thyroidectomy or radioiodine treatment, and we had no histopathological data either from thyroid specimens or from orbital biopsies. We also did not assess total IgG serum concentration, however previous studies have shown that total IgG does not offer an adequate estimation of IgG4 levels.This study shows that patients with active GO have higher IgG4 serum concentrations than patients with inactive GO. Moreover, patients with high IgG4 GO have more frequently active GO, higher eosinophil count in peripheral blood, and lower bilirubin levels. We demonstrate that IgG4 serum concentrations can be used as a useful diagnostic tool to detect patients with active GO. Apart from disease activity, frequent lower eyelid retraction, and a decreased prevalence of glaucoma, we did not find many differences in clinical features between high IgG4 GO, and normal IgG4 GO. Thus, the debate if patients with high IgG4 GO are GO patients with particularly active disease or actually represent a distinct clinical entity related to IgG4-RD will probably continue until more studies, especially with histopathological examinations of thyroid specimens or orbital biopsies, emerge.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Bioethics Committee at the Poznan University of Medical Sciences. The patients/participants provided their written informed consent to participate in this study.MO, ES-P, and MR contributed to the conception and design of the study. MO, AO-K, and PA organized the database. MO performed the statistical analysis. MO wrote the first draft of the manuscript. AO-K and PA contributed to the first draft of the introduction section. All authors contributed to manuscript revision, read, and approved the submitted version.


Human immunodeficiency virus (HIV) related depression has seriously affected the quality of life and treatment outcomes of patients living with HIV (PLWH), which has become a hot topic in recent years. This study aims to discover the main keywords, predict frontier topics, and give meaningful suggestions for researchers by bibliometric analysis.Publications between 1999 and 2022 on depression in HIV/AIDS were searched in the Web of Science core collection. Microsoft Excel 2010 and VOSviewer were utilized to key contributors . VOSviewer and CiteSpace were used to analyze the knowledge evolution, collaborative maps, hot topics, and keywords trends in this field.In total, 8,190 publications were included in the final analysis. From 1999 to 2021, the number of published articles roughly presents a steadily increasing trend. The United States, South Africa, and the United Kingdom were three key contributing countries/regions to this field. University Calif San Francisco (United States), University Calif Los Angeles (United States), and Johns Hopkins University (United States) were three key contributing institutions. Safren, Steven A. was the most productive and highest cited author. AIDS Care was the top prolific journal. Antiretroviral therapy and adherence, men has sex with men, mental health, substance abuse, stigma, and Sub-Saharan Africa were the central topics regarding the depression-related research in HIV/AIDS.This bibliometric analysis reported the publication trend, major contributing countries/regions, institutions, authors, journals and mapped the knowledge network of depression-related research on HIV/AIDS. In this field, topics such as “adherence,” “mental health,” “substance abuse,” “stigma,” “men who have sex with men” and “South Africa” have attracted considerable attention. HIV/AIDS ranks as the top 10 causes of disease-related life loss in adolescents  and adults  in 2019, a significant increase compared to 1999  infection and its’ caused acquired  ~20–40% . Prior s ~20–40% . Further ~20–40% . Depress ~20–40% .Many scholars are devoted to studying the association between HIV and depression. As early as 2001, Ciesla et al. have evaluate the relationship between HIV infection and depression through a meta-analysis . Leah H Bibliometrics is the cross-disciplinary science of quantitative analysis of the keyword clusters and hotspot bursts in a given field by mathematical, bibliographical, and statistical methods . It typiIdentify key contributors  and how they collaborate.Discover main topics and knowledge evolution based on keywords and co-cited references.Overview this field, predict frontier topics, and give meaningful suggestions for researchers.2.2.1.On September 06, 2022, we conducted an advanced search on the Web of Science Core Collection (WoSCC). Search formulas were “TS = (depress* OR depression OR depressed OR depressive disorder OR mood disorder)” AND “TS = (HIV OR HIV AND AIDS OR human-immunodeficiency-virus OR acquired immune deficiency syndrome),” to identify publications primarily focused on HIV in the field of depression. The date ranged from 1999/01/01 to 2022/09/06, language type was set as English, and the document type was limited to articles and reviews. First, two authors (Xiaoyu Du and Qian Zhang) independently searched the WoSCC and then screened the title, abstract, or full manuscript to identify whether these publications met this research topic. Second, the other two senior scholars (Jing Liu and Jia Chen) read and evaluated the discrepancies between the former authors, and discussed with them until a consensus was reached. All publications were exported as the format of “Full Record and Cited References,” and then used for bibliometric parameters extraction.2.2.Firstly, we used VOSviewer  to extract the following bibliometric parameters, mainly including the following elements, e.g., title, keywords, authors, institutions, countries/regions, published journals, publication year, total citations (TC), citations per publication (CPP) and cited references. Then we used Microsoft Excel 2010  to identify the publication trend, the top contributors . And we mapped the collaboration network between authors, institutions, and countries by VOSviewer, to demonstrate their scientific influence in this field. CiteSpace (Version 6.1. R1) were used to map the co-cited references network and identify references with highly citation burst. Co-occurrence of author keywords and keywords with citation burst were used to visualize the knowledge evolution, hot topics and potential research frontiers in this filed. In the map of VOSviewer and CiteSpace, the node size positively related to the number of publications, and the line width positively associated with their link strength.3.3.1.3.2.3.3.3.4.3.5.3.6.n = 683 publications) was the top prolific journal, followed by AIDS Behavior (n = 615 publications) and JAIDS-J Acquired immune deficiency syndromes (n = 224 publications). However, in terms of CPP, AIDS (n = 153 publications) has recorded the highest CPP (n = 46.4 CPP), followed by JAIDS-J Acquired immune deficiency syndromes  and AIDS Patient Care and STDS . Co-cited journals were journals frequently cited in a series of selected publications. Of which, AIDS Behavior , AIDS Care , and AIDS  ranked the first three.3.7.Citation is a quotation from one article to another article. The number of citations to the literature has been used as an indicator of the value, interest and utility of different articles in terms of the advancement of knowledge . Table 23.8.Co-citation is defined as two publications being cited by a third at the same time . The ana3.9.n = 2,703), “depression” , and “mental health” (n = 641). Further, to decipher potential research frontiers in this field, we used the overlay map of keywords occurrence by VOSviewer and keyword citation burst by CiteSpace. Frequently used keywords can be detected during a particular period. Keyword citation burst is an increase in the frequency and number of keyword occurrences within a certain period of time (n = 49.9). In the early stage, “stress,” “quality of life,” “cocaine” received lots of attention. And then keywords like “social support,” “adherence,” “risk behavior” got much attention. In recent years (2010-now), keywords have received more attention , which suggests that these keywords are likely to become frontier hot topics in the future.VOSviewer and CiteSpace were used to conduct keyword co-occurrence analysis to present major hotspots and frontiers in this field. To count the number of keyword occurrences more accurately, we used a thesaurus  to merge of time . Figure 4.4.1.In this study, 8,190 publications were included in the final analysis. We observed from 4.2.Lancet, a very authoritative journal. The top most cited articles, all collaborations between the University Queensland, University Washington, and Harvard University, were published in Lancet. They analyzed the global burden of disease and mortality, which suggested that HIV/AIDS and depression were the top 10 leading diseases for global disease burden in recent decades . We reasoned that this may be attributed to the top prolific author, Safren, SA . He has established a close collaboration with Mayer, KH. and Mimiaga, MJ, and Pence, BW, all of them ranked the top 10 prolific authors in 4.4.Analysis of keywords can help to quickly understand the knowledge network of a field. As shown in In this study, we visualized the knowledge networks and research paradigms of depression-related research among HIV/AIDS. The results showed 83 keywords appeared more than 40 times in 4.4.1.n = 346),” “antiretroviral therapy (ART) (n = 322),” “medication adherence (n = 101),” “highly active antiretroviral therapy (HARRT) (n = 94),” and “cognition (n = 81).” Since the inception of ART, the lifespan of HIV patients has been greatly improved. Adherence to ART plays a significant role in the effectiveness of treatment  in PLWH in Khayelitsha, South Africa ,” “sexual risk behavior (n = 45),” “sexual orientation (n = 61),” “condom use (n = 47)” and “childhood sexual abuse (CSA) (n = 31).” MSM remain the high-risk group for HIV, accounting for 69% of new HIV-positive diagnoses in the United States in 2020 ,” “social support (n = 266),” “discrimination (n = 63),” “China (n = 132),” and “COVID-19 (n = 61).” Stigma and discrimination are one of the biggest issues facing PLWH ,” “alcohol use (n = 149),” “drug use (n = 70),” and “methamphetamine (n = 55).” Approximately 14% of the global burden of disease is caused by neuropsychiatric diseases, of which, depression, alcohol use, and substance use are the top three reasons ,” “quality of life (n = 258),” “stress (n = 141),” and “suicide (n = 68).”There has been substantial evidence that the depression and anxiety experienced by HIV-infected patients are related to neurobiological changes caused by the ongoing attack on the central nervous system by the virus ,” “women (n = 283),” “pregnancy (n = 88),” “adolescents (n = 56),” “uganda (n = 94),” and “Kenya (n = 60).” Under this cluster, they focus on adolescents and women living with HIV. Globally, there are 1.6 million adolescents living with HIV, 70% of whom are living in Eastern and Southern Africa  were not included in this study. Second, the indicators for the quality of an article, an author, or an institution only included the number of publications, TC, and CPP. Other metrics are widely used in other studies, such as H-index, Impact Factor, CiteScore, etc. Third, due to the included criteria of language and publication types, only articles or reviews in English were included in this analysis. However,“English” remains the most common language used in published academic literatures worldwide, future research should integrate multiple non-English language  publications to provide a more comprehensive analysis of this field.5.This bibliometric analysis provides an overview of depression-related research on HIV/AIDS from 1999 to 2022. This study reported the publication trend, major contributing countries/regions, authors, institutions, journals and mapped the knowledge network of depression-related research on HIV/AIDS. In this field, topics such as “adherence,” “mental health,” “substance abuse,” “stigma,” “men who have sex with men” and “south africa” have attracted considerable attention. Further work should emphasize the following initiatives: (1) strengthen the co-operation between authors, institutions, and countries. (2) exploring different interventions to improve adherence. (3) developing more organizations to help PLWH reduce stigma and provide social support, especially in MSM (4) increasing investments to low-resource settings, especially in Sub-Saharan Africa.The original contributions presented in the study are included in the article/XD and QZ conceived of the study, participated in its design, and drafted the manuscript. JL and JH involved in study design, obtained data and contributed to interpretation, and helped to draft the manuscript. JC provided the theoretical frameworks and performed much of the editing of the manuscript and provided the fund support. All authors contributed to the article and approved the submitted version.This study was supported by the Hunan Science and Technology Innovation Platform and Talent Plan (grant: 2017TP1004).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.


Concrete is now a prevalent type of synthetic rock, and its production and usage have major environmental implications. Yet, assessments of ordinary concrete have rarely considered that concrete itself is potential habitat for a globally important microbial guild, the endolithic microbes, which live inside rocks and other mineralized substrates. We sought evidence that many common concrete structures harbor endolithic microbial communities and that these communities vary widely depending on the conditions imposed by the concrete. In Summer 2022, we obtained samples from various concrete structures found throughout Lubbock, Texas, USA and subjected the internal (non-surface) portions of each sample to controlled microbial life detection tests including culture tests, DNA quantifications, DNA amplification tests, and ATP assays. The great preponderance of positive life detection results from our concrete samples suggests that most modern concrete hosts cryptic endolith communities composed of bacteria, sometimes co-occurring with fungi and/or archaea. Moreover, many of these microbes are viable, culturable, and identifiable via genetic analysis. Endolith signatures varied widely across concrete samples; some samples only yielded trace evidence of possibly dormant microbes while other samples contained much more microbial biomass and diversity, on par with some low-biomass soils. Pre-cast masonry units and fragments of poured concrete found underwater generally had the most endolith signatures, suggesting that concrete forms and environmental positioning affect endolithy. Endolith biosignatures were generally greater in less dense and less alkaline concrete samples. So, concrete endolith communities may be as ubiquitous and diverse as the concrete structures they inhabit. We propose further research of concrete endoliths to help clarify the role of modern concrete in our rapidly urbanizing biosphere. Being a cheap, workable, and open-ended composite material, poured and pre-cast concrete is used for everything from setting fence posts to building massive infrastructures such as highways and dams. It has become the most abundant human-made material on Earth, with production rates far exceeding that of any other building material including steel and wood Concrete has a long and intensive history of scientific study and entrepreneurial innovation, mostly aimed at improving its material properties and structural utility 1.2.Imagining the insides of concrete as microbial habitat first requires understanding that concrete is synthetic rock, analogous to naturally occurring conglomerate rocks Coombes et al. 1.3.Our primary objective here was to survey common forms concrete gathered from various settings within a city environment for endolithic life, then use the survey results to test some basic hypotheses about concrete endoliths. Concrete is as variable as it is ubiquitous, so instead of conducting narrow analyses of particular types of concrete in select settings, we performed a broad microbiological investigation on a wide variety of real-world concrete structures. We designed this survey to determine if and how frequently the concrete present in our built environments is naturally inhabited by endolithic bacteria, fungi, and archaea. Although there is compelling evidence that the internal portions of concrete structures or ‘endo-concrete’ environments can host at least some types of microbes in certain capacities, there is still relatively little evidence of concrete endoliths, and there are several reasons to think that most concrete is inhospitable to microbes. So, we further tested the ‘concrete endolith hypothesis’ by noting if and when we obtained positive life-detection results from our concrete samples. We reasoned that, if concrete is suitable habitat for endolithic microbes, then we should be able to cultivate microbes and/or successfully detect biosignature molecules from at least some endo-concrete samples.Assuming that we would obtain positive life-detection results during our survey, we also tested the ‘endolith-concrete interaction hypothesis.’ This stated that endolithic life detection results inside concrete will vary by the physicochemical characteristics of the respective concrete substrates and/or by the general environments and forms in which the concrete existed because concrete conditions affect resident microbes. So, we tested for significant differences in life detection results (culture test frequencies and biosignature molecule quantities) among different categories of concrete and for significant relationships between biological variables and concrete physicochemical variables.To supplement our survey, we explored using scanning electron microscopy (SEM) for life detection. While unable to incorporate SEM micrographs into our main survey, we deemed it worthwhile to search for visual evidence of endoliths in a subset of our concrete samples. Such evidence would help corroborate the main life detection tests and help determine the feasibility of SEM in visualizing rock-embedded microbes without significant sample manipulation.Lastly, we hoped to verify fungi among the viable microbes that exist deep within ordinary concrete. To our knowledge, there is no evidence of incidental endolithic fungi in ordinary concrete, but like many other microbiomes, endo-concrete microbiomes may include both prokaryotic and eukaryotic constituents, with important implications for how these communities may function. Our survey positioned us to cultivate fungi along with other endo-concrete microbes, so we will attempt to verify that some of the microbes we culture are indeed fungi using DNA sequencing.2.2.1.We collected concrete samples throughout Lubbock, Texas, USA  between June–August 2022. Lubbock is in the Southern Great Plains region and has a semi-arid climate with a mean annual precipitation of 485 mm, a mean annual low temperature of 8.3 °C, a mean annual high temperature of 23.3 °C, and a mean annual temperature of 15 °C We did not collect samples without permission or samples whose basic origins and histories we could not ascertain through personal communication with vested parties. We restricted our samples to ‘ordinary’ or ‘common’ concrete made with Portland cement (the most common concrete binder). We did not collect concrete made with highly specialized concrete mixes or samples of pure mortar, nor did we collect samples of asphalt, brick, or other similar materials. The bulk samples were typically between 10–25 kg and had an effective thickness of > 60 mm, ensuring that we could extract an adequate number of sub-samples of sufficient size. It was not feasible to collect samples that were actively in use, so we collected leftover and recently discarded concrete (often from demolition sites).2.2.From each large bulk sample of poured concrete, we extracted several sub-samples as cylindrical cores  using a drill press and a diamond coring bit. We avoided coring from the edges of the bulk samples and trimmed 5 mm off the top of each core and at least 5 mm from its bottom with a tile saw, leaving a 50 mm core that contained none of the original surface portions. From each pre-cast CMU sample, we cut out several cuboids from the face shells with a tile saw and trimmed off 5 mm from the tops and bottoms. When we trimmed off 5 mm from the surfaces of our samples, we removed material that was likely inhabited by endoliths, particularly endoliths that colonized the concrete after it solidified, However, for this study, we wanted to analyze the endolithic zone of the concrete samples without the confounding effects of the epilithic surface portions. Additionally, we extracted one cylindrical core from the corner of each CMU for compressive strength analysis (discussed below). As an initial sterilization step, we wiped all sub-samples designated for biological analysis with 95% ethanol immediately after trimming and then stored these temporarily in clean plastic bags.One sub-sample (core or cuboid) from each bulk sample was pulverized to make it suitable for biological and chemical analysis. We pulverized each sub-sample with a stainless-steel mortar and pestle custom-made to crush large, rocky samples to various particle sizes while minimizing contamination . We sterAfter the sub-samples were pulverized, we sieved each sample in a laminar flow hood. We passed the sample through a sterilized, stainless steel, 1-mm sieve and reserved both the fine (< 1 mm) and coarse (1–10 mm) fractions in separate sterile containers. We stored the fine fractions at −20 °C and the coarse fractions at 4 °C until subsequent analyses. We left other sub-samples and portions of each bulk sample intact for different analyses.2.3.We used a large tile saw to expose a complete cross-section of each bulk sample. We sprayed a 1% phenolphthalein pH indicator solution onto the freshly exposed face and noted the color change inward from the surfaces of the sample as a measure of carbonation depth. Although sometimes prone to underestimates, the carbonation depths shown after phenolphthalein application reveal the spatial extent to which acidifying carbonation reactions have gradually penetrated the otherwise highly alkaline concrete structure from the outside in, bringing some of the outermost material to a pH of 8.6 or below −3). We averaged the densities of sub-samples from the same bulk sample to calculate the sample's mean bulk density (g cm−3). We measured the compressive strength of one intact core  from each bulk sample using an FX Test Pilot 250/300 Forney Press. We divided the pressure required to fail the concrete specimen by the top surface area of the core to calculate compressive strength . This procedure closely matched standard concrete strength testing procedures  except we used relatively small cores that we drilled out of pre-existing structures and cut to match our other sub-samples instead of using large, molded, test cylinders with greater height-to-diameter ratios. We obtained strength measurements below the normal range for concrete materials  of each cylindrical and cuboidal sub-sample to determine the sub-sample bulk density  with 20 mL of deionized water in a 50-mL centrifuge tube, vortexed the tube on medium speed for five minutes, incubated it at room temperature for 24 hours, then measured the pH of the solution with a calibrated digital pH meter after filtering it through coarse filter paper 2.4.To test the concrete endolith hypothesis, we performed a three-media culture test on each concrete sample. These tests, also known as growth media sterility tests, do not detect dead, inviable, or unculturable microbes. In a laminar flow hood, we poured several coarse fragments (1–5 mm) from each pulverized concrete sample onto three, 100 × 15 mm Petri dishes containing 25 mL of solidified malt extract agar (MEA), potato dextrose agar (PDA), or quarter-strength tryptic soy agar (TSA). We used coarsely pulverized concrete to inoculate the plates because finely pulverized concrete coated the growth media and interfered with colony formation. We shook the plates so that the small fragments rolled across the surface of the solid media, then dumped out any fragments that did not adhere to the agar. We sealed all plates with laboratory film to prevent desiccation, left them agar-side down for 24 hours at 25 °C, then dark-incubated them agar-side up for an additional six days. After the seven-day incubation, we photographed each plate and noted the presence or absence of visible microbial on the surface of the media. We also noted the level of visible microbial growth as minimal growth , or abundant growth . We selected these media types because they have general purpose, non-selective nutrient profiles that accommodate the growth of a wide variety of microbes. We used the MEA and PDA plates to cultivate any yeasts and molds, and we used the TSA plates to cultivate bacteria, although we noted any type of microbial growth on any of the plates as a positive life detection result, as long as there were no signs of contamination.We used several controls during the culture tests to assess and manage contamination. We made all media plates in-house and in multiple batches, so before using plates from a batch, we incubated a subset of the batch while the plates were still sealed. We never opened these ‘batch controls’ since their initial preparation, so any growth in these plates indicated that the batch was possibly contaminated during preparation and thus unusable for testing. During inoculations, we prepared ‘workspace control plates’ by opening, manipulating, and resealing extra sets of plates in the same manner as the plates inoculated with concrete, except we left these plates empty. Growth on these plates suggested contamination during the inoculation steps, and we discarded all associated plates. We also inoculated plates with fragments of concrete samples that were autoclaved at 121 °C for 30 minutes three times over five days. These ‘sterilized concrete’ plates served as negative controls that displayed the physicochemical effects of concrete on the growth media without the influence of viable microbes. We took growth on these plates to mean that these and their associated plates were likely contaminated and unusable in the final test results. During final inspections, we discarded and replaced plates that looked compromised , had obvious signs of contamination , and had growths emanating only from the edges of the plates (in case these resulted from contamination during plate handling).2.5.To test the concrete endolith hypothesis using biosignature molecules, we extracted double-stranded DNA (dsDNA) from our concrete samples. As in other studies that attempted to extract genomic DNA from troublesome substrates e.g., , we atteBased on the results of the digestion experiment, we followed (with minor modifications) a non-kit-based protocol that involves an extended pre-digestion/pre-treatment lysis step that uses both EDTA and acetic acid. This DNA extraction protocol was posted by Kiledal and Maresca To each tube containing either concrete, sterilized glass, or live soil, we added a pre-treatment/lysis solution containing 5 mL of 0.5 M EDTA, 150 µL of 20 mg/mL nuclease-free water Proteinase K solution, 138 µL of 20% sodium dodecyl sulfate (SDS), and 200 µL glacial acetic acid. We incubated these tubes at 55 °C for 24 hours on a shaker unit set for gentle agitation (85 rpm). After incubation, we added 5 mL of nuclease-free water to each tube to increase the volume of supernatant. After vortexing at maximum speed for 10 minutes and centrifuging at 5,000 RPM for three minutes, we transferred the supernatants (~15 mL) into sterile tubes for DNA solubilization and binding. To each supernatant volume, we added 5 µL of 1-mg/mL yeast RNA solution and 125 µL of silica suspension (a silicon dioxide powder and water solution prepared by isolating medium-sized particles after large particles settled out after one hour and fine particles were pipetted out after 12 hours). We also added 30 mL of Qiagen Buffer QG amended with 25 mM sodium chloride solution and Triton X-100 surfactant. After mixing the solutions and allowing them to bind for 24 hrs at room temperature with gentle agitation, we centrifuged the tubes for five minutes at 5,000 RPM to pellet the silica-bound DNA. We decanted the supernatants and washed the silica pellets in 10 mL 80% ethanol to precipitate the DNA. After centrifuging for 30 minutes at 5,000 RPM at 4 °C, we decanted the supernatants and resuspended the pellets in 1 mL of 80% ethanol. We centrifuged the suspensions at 13,000 RPM for three minutes and pipetted off the supernatants. Then, we air-dried the pellets in a flow hood and resuspended them in 40 µL 10 mM Tris (pre-warmed to 60 °C) for five minutes and with gentle rotation. We centrifuged those suspensions for three minutes at 13,000 RPM and transferred the DNA-containing supernatants to sterile microtubes. We repeated this elution two more times to increase DNA recovery.−1 extract), then multiplied that by the extract volume (50 µL) to find the total recovered DNA per sample (ng). We divided that value by the mass of the original concrete sample (10 g) to obtain the sample DNA concentration .We concentrated our 120-µL DNA extracts by vacuum centrifuging (Savant Speed Vac Concentrator) for 4 hours at ~40 °C to dehydrate the volumes below 50 µL. We rehydrated the extracts with nuclease-free water back up to 50 µL to equalize the volumes so that we could accurately compare the yields between each of our DNA samples. We quantified the DNA using a Qubit® 3.0 fluorometer (Thermo Fisher Scientific) in ‘High Sensitivity dsDNA’ mode and following the manufacturer's instructions. We recorded the DNA concentration . We selected universal primers to maximize the chances of amplification . Like our culture tests, we noted which DNA extracts from which concrete samples had any successful amplification of microbial DNA and which extracts showed successful amplification of bacterial, fungal, or archaeal DNA specifically. We calculated the overall frequency of successful amplifications and the frequency of successful amplifications of each barcode type for all samples and among samples of each concrete category.2.7.We quantified ATP content of each sample using a rapid commercial and medical hygiene monitoring system. In a laminar flow hood, we inserted an UltraSnap™ Surface ATP Test Device (Hygiena) into each sample container. We tilted and rotated each container for 30 seconds so that its contents  touched the pre-wetted swab end of each device. We also swabbed the inner side and bottom walls of the containers for 30 seconds. After sampling, we immediately re-inserted the swab into the test device where it was inundated with a liquid-stable reagent containing luciferin and luciferase, which bind to ATP and luminesce. Immediately after swab inundation, we inserted the test device into a luminometer . We recorded ATP contents Relative Light Units (RLUs), which is directly proportional to the amount of ATP collected by the test device.2.8.n = 5), concrete sample group (n = 25), and positive control group (n = 4) using Kruskal-Wallis tests (α = 0.05).We evaluated the concrete endolith hypothesis using results from the culture tests, DNA quantifications, amplification tests, and ATP assays. We determined if the frequency of positive results of a given test was greater than zero for concrete samples (or sub-groups of samples). We also compared DNA concentrations and ATP contents between our negative control group  and the DNA amplification tests produced categorical results for each sample (amplification failed vs. amplification successful) and for each of the three test primers. To determine if endolithic communities in concrete are different among the general types of concrete found in urban landscapes, we noted if the frequencies of any growth on culture plates, abundant growth on culture plates, and successful amplification of microbial barcodes were unequal among the concrete categories. We also determined if these test results showed any patterns when the samples were sorted by continuous and non-repeating physicochemical parameters of the concrete  and then displayed as ordinal heatmap tables or categorical table matrices. We tested this hypothesis statistically by comparing DNA concentrations and ATP contents among the five types of concrete samples using Kruskal-Wallis tests (α = 0.05). To assess the relationships between concrete endolith community variables and concrete physicochemistry, we linearly regressed microbial DNA concentrations and ATP contents against concrete density, strength, carbonation proportion, and pH (α = 0.05). We performed all statistical tests using IBM SPSS Version 28.0.0.0.2.9.2 for structures resembling intact cells or other forms of biological debris and saved the notable micrographs.To explore microscopy-based detection of concrete endoliths, we examined one small, randomly selected fragment from the freshly pulverized portions of three concrete samples  using a Hitachi S-4300SE/N SEM . In a laminar flow hood and using sterilized tools and equipment, we affixed each fragment to a 26-mm diameter, aluminum specimen holder using a double-sided carbon tape, and transported the mounted specimen to the microscope in a sealed, sterile container. To understand if concrete endoliths could be imaged in-situ and near to their natural state, we imaged the fragments uncoated, unstained, and without drying using the low-vacuum environmental scanning electron microscopy (ESEM) mode. As we traced the electron beam in a loose grid pattern across the surface of each fragment, we visually scanned the areas between 12–1200 µm2.10.To confirm fungal taxa within our concrete samples, we inoculated MEA plates with fragments from three concrete samples  before a 7-day incubation. We excised tissue from one fungal-like colony from each plate onto fresh plates, then, from the resulting pure cultures, excised and froze small pieces of tissue. We extracted genomic DNA from the tissues using the DNeasy Plant Mini Kit (Qiagen) following the protocol of Kaur et al. 3.3.1.−3, but most of the poured chunks, slabs, and structures had similar densities around 2.3 g cm−3. As a category, CMUs had distinctly lower densities (x = 1.6 g cm−3) and were noticeably more porous than any other type of concrete. We tested compressive strength using a non-standard method, so the absolute strength readings were generally low, between 2–12 MPa . However, relative to each other, the readings were generally as expected in that the samples from the soundest structures  had the highest compressive strengths (8–12 MPa) while the samples that were not designed for high standalone strength on a per gram basis (namely the CMU samples) had the lowest compressive strengths (2–4 MPa). In terms of carbonation, our samples ranged from having extremely shallow carbonation depths  to complete carbonation . Our more standardized expressions of carbonation depths, carbonation proportions, ranged from 0 to 1. Most sub-samples from the submerged, belowground, ground-level, and aboveground poured samples were <30% carbonated. In contrast, pre-cast CMU samples had a mean carbonation proportion of 0.73; most CMUs showed no highly alkaline regions in cross-section. Most concrete samples were highly alkaline (median pH = 11.8) when the samples were prepared as aqueous solutions. Similar to carbonation measurements, there was considerable variation in pH within concrete categories and no clear pattern except that samples of poured concrete had a noticeably higher mean pH (x = 11.7) than that of pre-cast CMU samples (x = 9.6).The physicochemical properties of all 25 concrete samples are summarized in 3.2.The frequencies of positive culture test results are summarized in Moreover, culture test results showed some patterns when sorted by some physicochemical variables . Abundan3.3.We observed that 24-hour soaking in 0.5M EDTA, ZymoBIOMICS™ Lysis Solution, and 5% acetic acid induced micro-cracking and some mineral dissolution in small concrete fragments . Submersx = 0.1 ng µL−1), concrete samples (x = 5.7 ng µL−1), and live soil positive control samples . The ranking of mean DNA concentrations among these general treatment groups matched our prediction that endo-concrete substrates host low-biomass microbial communities, but other observations confounded these results. The post-hoc pairwise comparison of concrete samples and negative controls revealed only a marginally significant difference (p = 0.051), and some concrete extracts measured lower than some extracts from sterilized control samples, indicating that some concrete samples had less DNA than what would be expected from background contamination. Still, several concrete samples yielded 2-172 times more DNA than any negative control sample.Observing that EDTA and acetic acid can partially digest concrete samples, we followed a direct DNA extraction protocol that involves pre-treating the concrete in a solution containing both EDTA and acetic acid  for an extended period of time i.e., . We isolR2 values show that any one of these physicochemical variables only explained about 30–45% of the variation in endogenous DNA concentrations. The relationship between DNA and compressive strength was not significant (p = 0.09). We compared DNA concentrations among the five types of sampled concrete and found no significant differences (p = 0.389). However, we noticed that the highest DNA yields (between 100–300 ng dsDNA g−1 concrete) were all from CMU samples while no samples from any other concrete type exceeded 40 ng dsDNA g−1 concrete , followed by archaeal DNA (5 samples), then fungal DNA (4 samples). For many samples, bacterial DNA was the only type of DNA to amplify, but all of the samples that contained archaeal and/or fungal DNA also contained bacterial DNA. All of the negative control extracts failed to amplify while all of the positive control extracts amplified all three types of target DNA. As predicted by the endolith-concrete interaction hypothesis, we noted differences in amplification patterns among the various categories of concrete  and high compressive strengths (>8.8 MPa).Amplification results seemed to vary only loosely with concrete physicochemistry . Amplifi3.5.x = 288 RLUs). However, as with DNA concentrations, there was considerable variation within the concrete sample group. We did not detect ATP for 13 of the 25 concrete samples, while seven samples had ATP readings between 1–2 RLUs, and three samples had ATP readings between 7–42 RLUs. That is, most concrete samples were as sterile as the sterilized controls, but several had more than would be expected from sterile substances, and the two most ATP-rich concrete samples  overlapped with the two least fertile soil samples . However, we did not statistically test for significant differences between negative control, concrete, and positive control samples because there was no variation in the negative control group, and the values in the concrete group were steeply skewed.We detected ATP biosignatures in 48% of concrete samples, a positive overall life detection result. On average, we detected 3.6 RLUs of ATP in concrete samples, which is between the sterilized glass negative control samples  and the live soil positive control samples .We used ATP as another molecular biosignature with moderate success. Bioluminescence-based ATP quantification systems, such as the one used here, are popular because they offer rapid detection of a wide range of biological contaminants Finally, we found SEM to be a useful tool for detecting concrete endoliths. Microscopy was not part of our primary survey, but we acknowledged that the concrete endolith hypothesis could benefit from micrographs, which are incredibly valuable as visual microbial biosignatures, especially when used in conjunction with other life detection tools 4.2.or be effectively devoid of life. Our stratified sampling of concrete types ensured that our final set of samples captured some of the incredible diversity of concrete within our study area. As predicted, we observed a wide range of endolithic life detection results among those concrete structures.Our endolith-concrete interaction hypothesis considered that modern cities hold an unaccountable variety of concrete structures. Depending on its unique features, a concrete structure may support rich microbial communities The five concrete categories showed noteworthy dissimilarities in the culture tests , DNA quaLower concrete densities seem to favor microbes  and 3a. none, minimal, and abundant) are somewhat arbitrary, and the amplification result categories (failed vs. successful amplification) are purely qualitative. Yet, when sorted by sample pH, instances of abundant culture growth , and all had unique sizes and shapes, so we relied on a drill press corer and tile saws to extract usable sub-samples out of our bulk samples. These tools are powerful enough to manipulate solid concrete but are not intended to carry out microbiologically sensitive tasks. Attempting to clean these power tools was difficult and time consuming, but these tools allowed us to (1) definitively isolate the internal portions of each sub-sample and cut off any surface portions, (2) control the final dimensions of the sub-samples, and (3) know precisely from what part of the bulk samples we obtained the sub-samples. Moreover, it was easy to apply sterilizing ethanol and flaming to the large, solid sub-samples . Before analysis, most of our sub-samples required pulverization, which exposes the internal portions of a solid sample, making the sample vulnerable to environmental contamination as well as direct contamination from the pulverizing instrument Contamination was not a major concern during our physicochemical measurements, but we carefully considered false readings during life detection procedures. Culture tests are highly sensitive life-detection tools, and in any given test, a huge diversity of microbes can be reliably detected using a small set of non-selective media types We encountered some problems with contamination during DNA testing; our negative control samples yielded very low but detectable amounts of DNA. We assume that DNA was from contaminant microbes, possibly from the silicon dioxide used during the extractions. That powder had high chemical purity, but it was the only material that we did not decontaminate ourselves nor could we presume it to be pre-sterilized or self-sterilizing. Fourteen of 25 concrete samples overlapped with the negative controls in terms of DNA yield. This could mean that many of our concrete samples had undetectably low amounts of endogenous DNA and their final DNA readings reflect background contaminants. Alternatively, and despite some precautions, the carbonate- and calcium-rich concrete may have inhibited the DNA extraction, artificially reducing DNA yields to negligible levels and producing false negatives We could not amplify DNA from our negative control extracts while the positive control extracts showed amplification by all test primers. Thus, we did not encounter any obvious signs of contamination, but PCR can amplify even trace amounts of contaminant DNA along with endogenous DNA, a typical problem for low-biomass environmental samples Unlike DNA extraction and quantification, our ATP procedure was simple and rapid, so the risk of contamination was relatively low. In fact, we suspect that we obtained more false negatives in these results . We believe that our assay method could be improved in several ways to be more standardized and sensitive. For example, instead of timed swabbings of dry concrete fragments, analyzing concrete solutions  may offer more standardization (if solutions are consistently prepared) and sensitivity (if swabs optimized for fluids and/or low-biomass samples are used).5.This study gathered compelling evidence that the insides of ordinary concrete are often inhabited by various microbes, thereby revealing a novel microbial guild and a new type of urban microbiome. We corroborated the few studies that previously demonstrated microbial life within ordinary concrete There are many options for future work in this area, but the most important questions can be answered by refining life detection methods, exploring new methods, conducting more in-depth microbiome analyses, and analyzing other types of concrete samples. Methodological refinements  will minimize false results, and new methods  will elucidate other aspects of concrete endolith communities . Microscopy may be especially suited to differentiating the different types of endoliths recognized by Golubic et al. Such studies could also pinpoint incidental microbes with physiologies that either degrade concrete (e.g., silica solubilization The ultimate goal is to reevaluate concrete's position in the biosphere. Some have articulated concrete's role in large-scale phenomena such as environmental modification, planetary urbanization, and the global proliferation of built environments