In 1998, 152 patients presented with fever, headache, and myalgia, followed by cervical adenopathy in a rural region near the city of Nova Serrana, in Minas Gerais, the second most populated Brazilian state. Then, 134 patients developed GN 7 to 10 days after the infection.4Balter S. Benin A. Pinto S.W. et al.Epidemic nephritis in Nova Serrana, Brazil. Histologic examination of renal biopsy samples of patients with worse clinical presentation showed a postinfectious GN pattern, with varying severity, proliferative and crescentic glomerular disease.4Balter S. Benin A. Pinto S.W. et al.Epidemic nephritis in Nova Serrana, Brazil. The GN outbreak was attributed to consuming unpasteurized cheese contaminated with S zooepidemicus.4Balter S. Benin A. Pinto S.W. et al.Epidemic nephritis in Nova Serrana, Brazil. Most (90%) patients were adults, 72% were hospitalized, 98% had edema, all had hematuria, 58% had serum creatinine levels greater than 1.2 mg/dl, and 7.5% needed acute hemodialysis.5Pinto S.W. Sesso R. Vasconcelos E. et al.Follow-up of patients with epidemic poststreptococcal glomerulonephritis. Two years after the acute episode, 42% of subjects had hypertension, 12% had serum creatinine levels greater than 1.2 mg/dl, and 34% had increased microalbuminuria.5Pinto S.W. Sesso R. Vasconcelos E. et al.Follow-up of patients with epidemic poststreptococcal glomerulonephritis. Five years after the acute episode, 5 patients (3.7% of the original sample) required chronic hemodialysis.6Five-year follow-up of patients with epidemic glomerulonephritis due to Streptococcus zooepidemicus. Although the proportion of patients with hypertension and microalbuminuria had decreased, there were more patients with reduced renal function over the 5 years.6Five-year follow-up of patients with epidemic glomerulonephritis due to Streptococcus zooepidemicus. An improvement of the eGFR was observed 10 years after the outbreak, with mean levels a little more than those after 2 years of the disease onset, and associated with a decreased proportion of patients with albuminuria and increased hypertension rates.3Pinto S.W. Mastroianni-Kirsztajn G. Sesso R. Ten-year follow-up of patients with epidemic post infectious glomerulonephritis.MethodsStudy Design and Sampling

This follow-up study consisted of 2 analysis subsets as follows: (i) a cross-sectional analysis of patients initially affected during the PSGN outbreak in 1998 and recontacted for a new clinical and laboratory evaluation in 2019; and (ii) a retrospective cohort analysis of the eGFR trajectory for all patients with serum creatinine values available in the last evaluation (2019) and taking into account their creatinine values in the 4 previous evaluations (1998, 2000, 2003, and 2008). The local nephrology team saved the medical record data of the affected patients with their contact addresses with 1 of the authors of this article (SWP). Therefore, 47 subjects were found for reevaluation 20 years after the outbreak, in the current study. The serum creatinine-based eGFR available from previous surveys of these 47 patients was used to assess their evolution.

A total of 253 cases of GN were reported in the state of Minas Gerais, Brazil, from December 1997 to July 1998. Most (92%) patients resided in Nova Serrana and Quilombo da Gaia (a small village in the city of São Gonçalo do Pará, 5%), both small towns in the center-west region of the state (populations of 27,500 and 12,000, respectively). The complete description of the outbreak has already been published.4Balter S. Benin A. Pinto S.W. et al.Epidemic nephritis in Nova Serrana, Brazil. First, GN was defined by at least 3 symptoms, namely systolic blood pressure higher than 140 mmHg or diastolic blood pressure higher than 90 mmHg, edema, and at least trace hematuria or 30 mg/dl proteinuria in a previously healthy patient. Therefore, 134 patients presented with criteria for GN, and their findings in prior surveys have been previously published.3Pinto S.W. Mastroianni-Kirsztajn G. Sesso R. Ten-year follow-up of patients with epidemic post infectious glomerulonephritis., 4Balter S. Benin A. Pinto S.W. et al.Epidemic nephritis in Nova Serrana, Brazil., 5Pinto S.W. Sesso R. Vasconcelos E. et al.Follow-up of patients with epidemic poststreptococcal glomerulonephritis., 6Five-year follow-up of patients with epidemic glomerulonephritis due to Streptococcus zooepidemicus. Next, a new clinical survey was conducted from July to September 2019 (20 years after the outbreak). After confirming their addresses in the local medical files, a research team member (SWP) contacted the original GN patients. All contacted patients were invited for a home clinical reassessment by clinical examination and collection of laboratory tests. The participants signed written informed consent. The Ethics Committee of the Federal University of São Paulo approved this study.Procedures and Variables Collected

The medical team visited the participants in their homes, collected the clinical information, obtained early morning blood (drawn after fasting), and recently voided urine samples for analysis. An interim history was obtained, and a physical examination was performed.

The study variables included gender, age, comorbidities (diabetes, obesity, and systemic arterial hypertension [SAH]), laboratory values (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, hematocrit, hemoglobin, uric acid, glycated hemoglobin, glucose, and proteinuria), and the use of angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers. Blood pressure was measured with a mercury sphygmomanometer while the patient was sitting after 5 minutes of rest. The average of 3 measurements taken with 1-minute intervals was used in the analysis. Hypertension was defined as systolic blood pressure was higher than or equal to 140 mmHg, or diastolic blood pressure was higher than or equal to 90 mmHg (or use of antihypertensive medication). Diabetes was defined as glycated hemoglobin was higher than 7% or by hypoglycemic drug use. The creatinine-based chronic kidney disease-epidemiology collaboration equation, without the race correction, was used to estimate eGFR.18Levey A.S. Stevens L.A. Schmid C.H. et al.A new equation to estimate glomerular filtration rate. We observed eGFR evolution among the participants enrolled in the last visit (2019) and used their available serum creatinine results in the previous assessments since the outbreak. Proteinuria measurements (24-hour albuminuria at the 2003 visit and protein-to-creatinine ratio at the 2019 visit) and hypertension status (present or not) were obtained in the 2003 and 2019 follow-up assessments.Laboratory Analyses

Blood and urine laboratory analysis was conducted in the same laboratory using the same methodology as in all surveys performed since 1998. Blood and urine samples were appropriately stored and transported to the reference laboratory at 4°C. When examined within 1 day, urine samples were kept at 4°C; otherwise, they were frozen at −20°C. In all surveys performed since 1998, blood samples were analyzed for serum creatinine (alkaline picrate method) using a spectrophotometer. Albuminuria values were assessed by radioimmunoassay using gamma counter equipment (Gamma C12; Diagnostic Products Corporation, Los Angeles, CA) in 2003, and the values were considered abnormal if greater than 20 mg per 24 hours. Urine protein was tested in 2019 using a colorimetric assay kit (Gold Análise Diagnóstico LTDA, Minas Gerais, Brazil) and expressed as protein-to-creatinine ratio; the values were considered abnormal if greater than 150 mg/g.

Statistical Analysis

The χ2 test was used for the comparison of categorical variables. t-test or the Mann-Whitney test (whenever appropriate) were used to compare continuous variables. We evaluated the variables associated with CKD by logistic regression analysis; odds ratios and 95% CI were calculated. CKD was defined as eGFR less than 60 ml/min per 1.73 m2 and/or the presence of proteinuria equal to or greater than 150 mg/g. All clinically plausible variables with a P-value lower than 0.20 to the outcome in the univariate method were included in the multivariate logistic regression analysis. After using a manual backward elimination, a final multivariate model was proposed to adjust for all significant variables in the model. The average eGFR reduction was calculated by subtracting the eGFR in 2019 from the 2008 value, and then dividing by the number of years in the range for patients with measurements in both evaluations. We evaluated the eGFR trajectory over the 20-year follow-up in the available sample at the last survey (n = 47) by mixed-effect linear regression analysis, taking into account their measurements in the 1998, 2000, 2003, 2008, and 2019 surveys, with the eGFR being the outcome of this analysis. The random effect was the subject identification variable. The other covariates were fixed (follow-up time, gender, age at baseline, presence of hypertension, and albuminuria [higher than 20 mg/24h] in 2003). Interaction terms between covariates and time were assessed in the models.

We considered the presence of hypertension and albuminuria values from the 2003 assessment in the analysis because that was the visit with the most data available. Similarly, all variables associated with the outcome in the univariate method with a P-value lower than 0.20 were included in the multivariate linear mixed-effect model analysis. A final multivariate model was proposed after using a manual backward elimination to adjust for all significant variables in the model. Tests were 2-sided and statistical significance was set at P < 0.05. All data were analyzed using STATA/IC 16.1 software program (StataCorp, College Station, TX USA).

ResultsOf the 134 confirmed cases of PSGN seen in 1998, 12 died as follows: 3 in the acute phase of illness (the causes of death were sepsis, cerebrovascular accident, and respiratory failure, respectively), 6 died while in a chronic dialysis program before 2 years of follow-up, and 3 with normal renal function died (due to congestive heart failure, myocardial infarction, and stroke) between 2 and 5 years of follow-up. Some cases of the 122 surviving patients could not be located 20 years after the outbreak (n = 65) or did not accept to undergo another medical evaluation (n = 10), thereby leaving 47 patients for the present study (Figure 1). These participants did not differ significantly from the 87 who were not re-examined regarding gender and mean age at presentation. The mean (range) duration of follow-up of the 47 participants was 20.5 years (20.5–20.6).

Figure 1Flow chart of the study population during the follow-up.

Table 1 shows the major sample characteristics as well as clinical and laboratory renal outcomes at 20 years of follow-up. The participants’ mean (SD) age was 56.6 (15.1) years, 12 (25.5%) were 65 years or older, and 62% were women. During the outbreak, 5 (10.6%) patients were 18 years old or younger. Most study participants were nondiabetic (85%) and not obese (94%). Hypertension was present in 34 (72%) patients, 21 (44.7%) had eGFR less than 60 ml/min per 1.73 m2, and 8 of 43 (18.6%) patients with measured proteinuria had values greater than 150 mg/g. The whole group’s median (range) of proteinuria was 80 mg/g (30–1450 mg/g). The median (range) proteinuria was 215 mg/g (160–1450 mg/g) in the subgroup with proteinuria greater than 150 mg/g, and 70 mg/g (30–130 mg/g) in the subgroup with proteinuria less than 150 mg/g. Moreover, 25 (53%) patients had CKD (low eGFR and/or proteinuria). Eight patients had proteinuria greater than 150 mg/g; 4 (16%) of these had eGFR lower than 60 ml/min per 1.73 m2, and the other 4 (16%) had eGFR higher than 60 ml/min per 1.73 m2. The presence of CKD was higher among those older than 65 years than those younger than 65 years (11 of 12, 91.7%; and 14 of 35, 40%, respectively, P = 0.002). Two of the 25 (8%) CKD patients were younger than 18 years during the outbreak (12.0 and 13.6 years, respectively). There was no significant association between diabetes, obesity, and degree of proteinuria (Supplementary Results).

Table 1Characteristics of patients with post-streptococcal glomerulonephritis at 20 years of follow-up (n = 47)

ACEIs, angiotensin-converting enzyme inhibitor; ARBs, angiotensin receptor blocker; eGFR, estimated glomerular filtration rate; HDLc, high-density lipoprotein cholesterol; LDLc, low-density lipoprotein cholesterol

Table 2 shows the features of patients with and without CKD 20 years after the outbreak. Patients with CKD were older (mean age, 62.9 vs. 50.1 years, P = 0.003) and had higher median proteinuria (95 vs. 70 mg/g, P = 0.020) than those without CKD. In the logistic regression univariate analysis (Table 3), increasing age was associated with CKD and remained so after adjusting for other confounders in the multivariate model (odds ratio: 1.07; 95% CI: 1.02–1.13; P = 0.011). Diabetes mellitus was marginally associated with CKD only in the univariate analysis (odds ratio: 6.63; 95% CI: 0.73–60.21; P = 0.093).

Table 2Characteristics of patients with and without chronic kidney disease after 20 years of the post-streptococcal glomerulonephritis outbreak (n = 47)

BP, blood pressure; CKD, chronic kidney disease; HbA1C, glycated hemoglobin; HDLc, high-density lipoprotein cholesterol; LDLc, low-density lipoprotein cholesterol.

CKD = eGFR<60ml/min/1.73 m2 and/or proteinuria, protein-to-creatinine ratio >150 mg/g.

Table 3Univariate and multivariate logistic regression analysis for variables associated with chronic kidney disease after 20 years of the post-streptococcal glomerulonephritis outbreak (n = 47)

ACEIs, angiotensin-converting enzyme inhibitor; ARBs, angiotensin receptor blocker; BP, blood pressure; CI, confidence interval; HDLc, high-density lipoprotein cholesterol; LDLc, low-density lipoprotein cholesterol; OR, odds ratio; VLDLc, very low-density lipoprotein cholesterol.

Table 4 describes the observed mean eGFR values, albuminuria, and hypertension rates at each assessment visit during the follow-up. A substantial increase in mean eGFR values occurred from baseline until 2008 and a pronounced decline in 2019, with a mean eGFR loss of −3.2 ml/min per 1.73 m2 per year (95% CI: −3.7 to −2.7) in the last 11 years of follow-up (calculated from 46 patients with measurements on both occasions).

Table 4Descriptive analysis of the observed eGFR, albuminuria, and hypertension rates at each follow-up visit for post-streptococcal glomerulonephritis patients from 1998 to 2019

NA, not available.

eGFR values are mean±SD. In parenthesis is the number of patients evaluated.

Hypertension is defined as blood pressure ≥140/90 mmHg or the use of antihypertensive drugs.

Albuminuria: values >20 μg/min in the 2000 and 2003 visits, >30 mg/g creatinine in 2008. In 2019 proteinuria was assessed as protein-to-creatinine ratio >150 mg/g.

Figure 2 shows the predicted mean eGFR values during each visit using the mixed-effects regression model in the cohort of 47 patients. The median number of serum creatinine measures per patient during the follow-up was 3 (minimum of 2, maximum of 4). The predicted mean eGFR values were similar to the observed values and had the same course over time, showing a tendency to increase from 1998 to 2000 and 2008 and a subsequent marked decline in 2019.

Figure 2Estimated glomerular filtration rate at follow-up visits in post-streptococcal glomerulonephritis patients during 20-year follow-up using mixed-effects regression analysis.

Show full caption

eGFR, estimated glomerular filteration rate.

Values are mean and 95% CI.

Predicted mean eGFR and number of patients (in parenthesis) at each visit: 59 ml/min per 1.73 m2 (n = 47), 89 ml/min per 1.73 m2 (n = 36), 85 ml/min per 1.73 m2 (n = 30), 101 ml/min per 1.73 m2 (n = 46), and 65 ml/min per 1.73m2 (n = 47) in 1998, 2000, 2003, 2008, and 2019, respectively.

Univariate mixed-effects regression analysis evaluating variables associated with the trajectory of eGFR showed that older age at baseline and hypertension, but not the albuminuria level after 5 years, were associated with a significantly lower eGFR between groups during the follow-up (Table 5). Multivariate analysis confirmed that older age at baseline (coefficient: −1.05 ml/min per 1.73 m2 per year; 95% CI: −1.28 to −0.81; P 2; 95% CI: −14.67 to −0.88; P = 0.027) remained significantly associated with lower eGFR after adjusting for other confounders. Nevertheless, when the models included the interaction terms between these risk factors and follow-up time, these terms were not statistically significant (Supplementary Table S1, S2, and S3). The main effects of age and hypertension on eGFR decline were not different between groups, resulting in curves with similar eGRF trajectories (Figure 3 and 4). In univariate analysis, the mean eGFR in 2019 was not significantly different from the mean value during the outbreak; however, after adjusting for age and hypertension, it was 11/min per 1.73 m2 greater at the end of the study (P = 0.028). In a further analysis, including data of all 67 available individuals, the results on eGFR trajectories remained similar (Supplementary Table S4).

Table 5Univariate and multivariate analysis by mixed-effect linear regression for variables associated with the estimated glomerular filtration rate trajectory 20 years after the post-streptococcal glomerulonephritis outbreak (n = 47)

Figure 3Estimated glomerular filtration rate at follow-up visits by baseline age tertile in post-streptococcal glomerulonephritis patients during 20-year follow-up using mixed-effects regression analysis.

Show full caption

Values are mean and 95% CI.

Age tertile 1: 11 to 27.9 years; age tertile 2: 28 to 39.9 years; age tertile 3: 40 to 70.9 years.

Figure 4Estimated glomerular filtration rate at follow-up visits by hypertension classification in post-streptococcal glomerulonephritis patients during 20-year follow-up using mixed-effects regression analysis.

Show full caption

Values are mean and 95% CI.

The presence of hypertension was ascertained at the 2003 visit.

Discussion

This is the longest follow-up study of adult patients with PSGN. During the 20-year follow-up, patients recovered their eGFR 2 years after the acute phase and continued improving after 10 years; however, there was a marked decline in mean eGFR after 20 years. The mean annual eGFR reduction represented an accelerated drop during the last 11 years. In addition, about half of the sample studied had CKD. Older age at disease onset and the occurrence of hypertension after 5 years were associated with a lower eGFR trajectory.

Long-term follow-up studies to properly evaluate the prognosis after acute PSGN have several obstacles. One such obstacle is the difficulty of asymptomatic individuals adhering to surveillance examinations years after the acute phase of the disease.14Potter E.V. Lipschultz S.A. Abidh S. et al.Twelve to seventeen-year follow-up of patients with poststreptococcal acute glomerulonephritis in Trinidad. Another hurdle is keeping patients’ records and an organized research network group in resource-limited regions to allow their periodic monitoring. The only 2 endemic PSGN studies with an extensive follow-up time, 1 of 12 to 17 years14Potter E.V. Lipschultz S.A. Abidh S. et al.Twelve to seventeen-year follow-up of patients with poststreptococcal acute glomerulonephritis in Trinidad. and the other of 6 to 18 years,19White A.V. Hoy W.E. McCredie D.A. Childhood post-streptococcal glomerulonephritis as a risk factor for chronic renal disease in later life. were mainly conducted among children affected by group A beta-hemolytic Streptococci. Most GN patients (90%) in the acute phase of the current study outbreak were adults with a mean age of 37 years, and 65% were women.4Balter S. Benin A. Pinto S.W. et al.Epidemic nephritis in Nova Serrana, Brazil. The most common first symptoms were edema (98%) and high blood pressure (80%). Three of the 10 patients who needed acute hemodialysis (10%) died during the acute phase.4Balter S. Benin A. Pinto S.W. et al.Epidemic nephritis in Nova Serrana, Brazil.Duca et al.20Duca E. Teodorovici G. Radu C. et al.A new nephritogenic streptococcus. reported the first cases of pharyngitis caused by group C S zooepidemicus in Romania; about 90% of the patients were adults and one-third presented GN. As in the few previous reports of human disease due to S zooepidemicus,20Duca E. Teodorovici G. Radu C. et al.A new nephritogenic streptococcus., 21Barnham M. Ljunggren A. McIntyre M. Human infection with Streptococcus zooepidemicus (Lancefield group C): three case reports., 22Centers for Disease Control
Group C streptococcal infections associated with eating homemade cheese-New Mexico., 23Edwards A.T. Roulson M. Ironside M.J. A milk-borne outbreak of serious infection due to Streptococcus zooepidemicus (Lancefield Group C)., 24Kuusi M. Lahti E. Virolainen A. et al.An outbreak of Streptococcus equi subspecies zooepidemicus associated with consumption of fresh goat cheese., 25Bordes-Benitez A. Sanchez-Onoro M. Suarez-Bordon P. et al.Outbreak of Streptococcus equi subsp. zooepidemicus infections on the island of Gran Canaria associated with the consumption of inadequately pasteurized cheese. the origin of the infection reported in the Minas Gerais outbreak was associated with the consumption of unpasteurized milk,4Balter S. Benin A. Pinto S.W. et al.Epidemic nephritis in Nova Serrana, Brazil. Patients affected by PSGN due to S zooepidemicus are primarily adults and not rarely need dialysis or intensive care unit admission in the early phase of the disease.21Barnham M. Ljunggren A. McIntyre M. Human infection with Streptococcus zooepidemicus (Lancefield group C): three case reports.,23Edwards A.T. Roulson M. Ironside M.J. A milk-borne outbreak of serious infection due to Streptococcus zooepidemicus (Lancefield Group C).,25Bordes-Benitez A. Sanchez-Onoro M. Suarez-Bordon P. et al.Outbreak of Streptococcus equi subsp. zooepidemicus infections on the island of Gran Canaria associated with the consumption of inadequately pasteurized cheese.Short-term recovery after acute PSGN has been a frequent outcome,8Epidemic acute glomerulonephritis at Red Lake.,26Treatment of acute nephritis: the immediate results and the outcome ten years later in eighty-nine cases. and most studies suggest a benign long-term prognosis of PSGN in children.27Kanjanabuch T. Kittikowit W. Eiam-Ong S. An update on acute postinfectious glomerulonephritis worldwide. Nevertheless, some studies conducted nearly 4 decades ago reported a relevant progression to CKD,3Pinto S.W. Mastroianni-Kirsztajn G. Sesso R. Ten-year follow-up of patients with epidemic post infectious glomerulonephritis.,28Hoy W.E. White A.V. Dowling A. et al.Post-streptococcal glomerulonephritis is a strong risk factor for chronic kidney disease in later life. mainly among adults.1Baldwin D.S. Gluck M.C. Schacht R.G. Gallo G. The long-term course of poststreptococcal glomerulonephritis.,3Pinto S.W. Mastroianni-Kirsztajn G. Sesso R. Ten-year follow-up of patients with epidemic post infectious glomerulonephritis.,12Lien J.W. Matheus T.H. Meadows R. Acute post-streptococcal glomerulonephritis in adults: a long-term study.,29Rodriguez-Iturbe B. Garcia R. Rubio L. et al.Epidemic glomerulonephritis in Maracaibo. Evidence for progression to chronicity. In a predominantly adult population with PSGN (n = 60), persistent proteinuria, hypertension, or reduction of kidney function were found in 50% of the patients more than 7 years after the acute phase.1Baldwin D.S. Gluck M.C. Schacht R.G. Gallo G. The long-term course of poststreptococcal glomerulonephritis. Progression to chronic glomerular disease has been found in kidney biopsies conducted about 4 years after the initial event among adult patients.29Rodriguez-Iturbe B. Garcia R. Rubio L. et al.Epidemic glomerulonephritis in Maracaibo. Evidence for progression to chronicity. Nonetheless, misclassification error may partly account for patients’ variable course in long-term PSGN studies because many were based on undocumented infections. Also, nonstandardized and imprecise methods have been used to evaluate kidney function during the follow-up.8Epidemic acute glomerulonephritis at Red Lake.,26Treatment of acute nephritis: the immediate results and the outcome ten years later in eighty-nine cases.,27Kanjanabuch T. Kittikowit W. Eiam-Ong S. An update on acute postinfectious glomerulonephritis worldwide.,30Rammelkamp Jr., C.H. Stetson C.A. Krause R.M. et al. Different pathways by which Streptococci could initiate and maintain glomerular injury have been postulated since the classical theoretical concept of glomerular trapping of immune complex and complement activation as other structural similarities between streptococcal fractions and kidney structural elements being responsible by a nephritogenic role.31Rodriguez-Iturbe B. Batsford S. Pathogenesis of poststreptococcal glomerulonephritis a century after Clemens von Pirquet.