PLOS NEGLECTED TROPICAL DISEASES RESEARCH ARTICLE Clinical profile and mortality in patients with T. cruzi/HIV co-infection from the multicenter data base of the “Network for healthcare and study of Trypanosoma cruzi/HIV co-infection and other immunosuppression conditions” Maria Aparecida Shikanai-Yasuda ID1,2,3*, Mauro Felippe Felix Mediano ID4, Christina Terra Gallafrio Novaes5, Andréa Silvestre de Sousa ID4, Ana Marli Christovam Sartori ID5, Rodrigo Carvalho Santana ID6, Dalmo Correia7, Cleudson Nery de Castro ID8, Marilia Maria a1111111111 dos Santos Severo9, Alejandro Marcel Hasslocher-Moreno ID4, Marisa a1111111111 Liliana Fernandez ID10,11, Fernando Salvador ID12, Maria Jesús Pinazo ID13, Valdes a1111111111 Roberto Bolella6, Pedro Carvalho Furtado ID7, Marcelo Corti10,14, Ana Yecê Neves Pinto15, a1111111111 Alberto Fica16, Israel Molina ID12, Joaquim Gascon ID13, Pedro Albajar Viñas ID3,17, a1111111111 Juan Cortez-Escalante ID18, Alberto Novaes Ramos, Jr ID19, Eros Antonio de Almeida20 1 Departament of Infectious and Parasitic, Faculdade de Medicina, University of São Paulo, São Paulo, Brazil, 2 Laboratory of Immunology, Hospital das Clı́nicas, Faculdade de Medicina, University of São Paulo, São Paulo, Brazil, 3 WHO Technical Group IVb on prevention, control and management of non congenital infections of the Global Network for Chagas Disease Elimination, WHO, Geneva, Switzerland, 4 Evandro OPEN ACCESS Chagas National Institute of Infectious Diseases, Oswaldo Cruz Foundation, Health Ministry, Rio de Janeiro, Citation: Shikanai-Yasuda MA, Mediano MFF, Brazil, 5 Division of Infectious Diseases, Hospital das Clı́nicas, Faculdade de Medicina, University of São Novaes CTG, Sousa ASd, Sartori AMC, Santana Paulo, São Paulo, Brazil, 6 Division of Infectious Diseases, Department of Internal Medicine, Ribeirão Preto Medical School University of São Paulo, Ribeirão Preto, Brazil, 7 Discipline of Infectious and Parasitic RC, et al. (2021) Clinical profile and mortality in Diseases, Department of Internal Medicine, Federal University of Triângulo Mineiro, Uberaba, Brazil, patients with T. cruzi/HIV co-infection from the 8 Centre for Tropical Medicine, School of Medicine, University of Brasilia, Brası́lia, Brazil, 9 Federal multicenter data base of the “Network for University of Health Sciences of Porto Alegre, Porto Alegre, Brazil, 10 Hospital de Infecciosas, Buenos Aires, healthcare and study of Trypanosoma cruzi/HIV co- Argentina, 11 National Institute of Parasitology, Departament of Clinics, Pathology and Treatment, Health infection and other immunosuppression Ministry, Buenos Aires, Argentina, 12 Department of Infectious Diseases, Vall d’Hebron University Hospital, conditions”. PLoS Negl Trop Dis 15(9): e0009809. PROSICS, Universitat Autònoma de Barcelona, Barcelona, Spain, 13 ISGlobal, Universitat de Barcelona, https://doi.org/10.1371/journal.pntd.0009809 Hospital Clı́nic, Barcelona, Spain, 14 Departamento de Medicina, Asignatura Enfermedades Infecciosas, Facultad de Medicina, Universidad Buenos Aires, Buenos Aires, Argentina, 15 Evandro Chagas Institute, Editor: Claudia Patricia Herrera, Tulane University, Health Surveillance Secretary, Health Ministry, Belém, Brazil, 16 Facultad de Medicina, Universidad Austral UNITED STATES de Chile, Valdivia, Chile, 17 Department of Control of Neglected Tropical Diseases, World Health Organization, WHO, Geneva, Switzerland, 18 Pan American Health Organization (PAHO), World Health Received: March 25, 2021 Organization (WHO), Brası́lia, Brazil, 19 Department of Community Health, School of Medicine, Federal Accepted: September 10, 2021 University of Ceará, Fortaleza, Brazil, 20 Department of Internal Medicine, Faculty of Medical Sciences, State University of Campinas, Campinas, Brazil Published: September 30, 2021 * masyasuda@yahoo.com.br, masyasuda@usp.br Copyright: © 2021 Shikanai-Yasuda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and Abstract reproduction in any medium, provided the original author and source are credited. Objective Data Availability Statement: Data set is available at: https://osf.io/j5m7p/. Chagas disease (CD) globalization facilitated the co-infection with Human Immunodefi- Funding: The author(s) received no specific ciency Virus (HIV) in endemic and non-endemic areas. Considering the underestimation of funding for this work. Trypanosoma cruzi (T. cruzi)-HIV co-infection and the risk of life-threatening Chagas Dis- Competing interests: The authors have declared ease Reactivation (CDR), this study aimed to analyze the major co-infection clinical charac- that no competing interests exist. teristics and its mortality rates. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 1 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection Methods This is a cross-sectional retrospective multicenter study of patients with CD confirmed by two serological or one parasitological tests, and HIV infection confirmed by immunoblot. CDR was diagnosed by direct microscopy with detection of trypomastigote forms in the blood or other biological fluids and/or amastigote forms in inflammatory lesions. Results Out of 241 patients with co-infection, 86.7% were from Brazil, 47.5% had <200 CD4+ T cells/ μL and median viral load was 17,000 copies/μL. Sixty CDR cases were observed. Death was more frequent in patients with reactivation and was mainly caused by CDR. Other causes of death unrelated to CDR were the manifestation of opportunistic infections in those with Acquired Immunodeficiency Syndrome. The time between the co-infection diagnosis to death was shorter in patients with CDR. Lower CD4+ cells count at co-infection diagnosis was independently associated with reactivation. Similarly, lower CD4+ cells numbers at co- infection diagnosis and male sex were associated with higher lethality in CDR. Additionally, CD4+ cells were lower in meningoencephalitis than in myocarditis and milder forms. Conclusion This study showed major features on T. cruzi-HIV co-infection and highlighted the prognos- tic role of CD4+ cells for reactivation and mortality. Since lethality was high in meningoen- cephalitis and all untreated patients died shortly after the diagnosis, early diagnosis, immediate antiparasitic treatment, patient follow-up and epidemiological surveillance are essentials in T. cruzi/HIV co-infection and CDR managements. Author summary Chagas disease (CD) is a chronic infectious disease caused by the parasite T. cruzi that accounts for a major burden due to its life-lasting morbidity in endemic and non-endemic areas. Globalization facilitated the meeting of CD and Human Immunodeficiency Virus (HIV) infection (T. cruzi/HIV co-infection). Patients with this co-infection are at risk of life-threatening CDR. This study aimed to analyze the clinical characteristics and mortal- ity rates of patients with T. cruzi/HIV co-infection. This retrospective multicenter study included 241 patients with co-infection, of those 60 with CDR. Death was more frequent in patients with reactivation and was mainly caused by CDR. Our data showed that co-infection diagnosis is close to the reactivation diagnosis and characterized by poor immune response associated to CDR, higher lethality and more severe forms of CDR (meningoencephalitis). This study emphasizes the prog- nostic role of CD4+ cells on reactivation and mortality and the need of epidemiological surveillance for co-infection and CDR. Introduction Chagas disease (CD) is a chronic infectious disease caused by T. cruzi that accounts for a major burden due to its life-long duration. It currently affects 6–7 million people not only in PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 2 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection Latin America but also in different continents, mostly due to the population movements [1,2]. T. cruzi infected people is estimated in Europe in 68,000–120,000 [3], and in the United States (US) >300,000 [4]. The control of vectorial transmission (through triatomine bugs) has improved in most Latin American territories, but the challenge now persists with oral and con- genital transmissions and with blood transfusions and organ transplantation wherever its full prevention coverage has not been reached [1,2]. The risk of CDR is increased in chronically immunosuppressed CD patients suffering from HIV infection/AIDS, neoplasia, autoimmune diseases, immunosuppression treatment and organ transplants [5,6]. The first case of T. cruzi-HIV co-infection was reported in 1990 [7], but CDR has been already cited in 1988, in a patient with AIDS [8]. Considering the worldwide estimated 38 mil- lion people living with HIV in 2019, a sustained risk of T. cruzi-HIV co-infection is still a real- ity in endemic and non-endemic areas for CD with infected immigrants [1,9]. In Brazil, the country with the highest estimated T. cruzi infected individuals (1.3–3.2 million in 2020), CDR was considered an AIDS defining condition since 2004 [10]. In WHO regions of America, an endemic region for CD, the same was defined in 2007 [11]. Co-infection cases have been reported predominantly in Brazil and Argentina, but also in Spain, USA, Chile, Colombia, Jamaica, Germany, Switzerland, Bolivia, Venezuela [5,7,12–23]. The prevalence of co-infection is estimated at 1.3%-2.3% in São Paulo State [5,24] and 5.0% in Rio Grande do Sul State [25] in Brazil and 4.2% in Argentina, varying from 1.3 to 10.5% in endemic areas [5,21,24–26] and in a restricted group of HIV infected immigrants from CD endemic areas [27], with higher rates in drug users in Argentina [21,26]. Estimates of T. cruzi- HIV co-infection cases range from 4,570–15,360, based on patients living with HIV in Brazil and Argentina [1,5,9,24–26] and in approximately 21,740 in Latin America [5]. Study on mortality from T. cruzi-HIV co-infection in Brazil using the mortality database from AIDS (103,000 deaths) and CD (about 54,000 deaths), with both diagnoses recorded in death certificates, showed that AIDS was the basic cause of death in 77.9% and CD in 17.6% [28]. Chagas Disease reactivation rates ranged from 10 to 15% among HIV co-infected people in retrospective case series [5,21] and was estimated to be about 10% in patients followed in a prospective study [6,15], needing to be better established in larger studies. The most affected sites in CDR are the CNS (74.2%) and the myocardium (16.7%). Other affected sites in 9.2% of cases are the duodenum, skin, cervix, peritoneum, pericardium, stom- ach and eye tissue. Mortality rate was 73.4% in previous cases of reactivations [5]. In these cases, CD4+ levels were generally lower than 200/μL [5,10,15,21] with an average of 98/μL in reactivation and 562/μL without reactivation [5]. This clinical profile of CDR does not differ from reactivation secondary to other forms of immunosuppression, especially chemotherapy for malignant neoplasms and those related to organ transplants. The affected sites of CDR from these other forms of immunosuppression are the central nervous system and the heart, as occurs in T. cruzi/HIV co-infection in AIDS situations, with the same clinical outcomes [6,29]. Oligosymptomatic cases and/or panniculitis/erythema nodosum could occur fre- quently in solid organ transplants. It is important to note that other infections are more frequent in AIDS than T. cruzi/HIV co-infection, requiring a differential diagnosis [5]. Brain- stem involvement on toxoplasmosis is more frequent than in T. cruzi/HIV co-infection, although CDR lesions can also occur in the brainstem, delaying the diagnosis and the specific treatment for T. cruzi [30]. An imbalance in the host-parasite relationship towards a Th2 response in case of co-infec- tion, particularly in patients with patent parasitemia [31,32], has been postulated as one of the factors associated with bidirectional influences on the interaction between HIV and T. cruzi in placenta [33] and astrocytes cells [34], leading to increased parasite multiplication. Elevated PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 3 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection parasitemia, presented in reactivation, recommends immediate investigation for CDR diagno- sis by direct microscopy in blood, other fluids or stained tissue [5,13,15,16,21], even in the absence of signs and symptoms. In fact, prospective studies reported early diagnosis of oligo- symptomatic cases with better prognosis [15,35,36]. It is noteworthy, however, that factors associated with mortality by CDR and other causes in co-infection have not been shown. Despite its relevance, data about clinical and epidemiological patterns in patients co- infected with T. cruzi and HIV are limited to a few single-center studies. In this context, a “Network for Healthcare and Study of Trypanosoma cruzi-HIV Co-infection and other immu- nosuppression conditions” was created in Brazil aiming to provide comprehensive care and study of all the cases [10]. The present international multicenter study was proposed to evalu- ate the main clinical characteristics and mortality causes of T. cruzi-HIV co-infected patients, aiming at establishing strategies for a high-quality healthcare and surveillance. Methods Ethics statement Data from medical records were anonymously analyzed and the study was approved by Insti- tutional Review Board of research centers (protocol number 095/1995 at the coordinating center, Ethics and Research Committee of Hospital das Clı́nicas, Faculdade de Medicina, Uni- versity of São Paulo, Brazil). Formal written consent was obtained from patients except for ret- rospective study participants who dropped out of follow-up. Study design This is a cross-sectional retrospective multicenter study involving 241 patients with T. cruzi— HIV co-infection from 13 health centers: 9 from Brazil, 2 from Spain, one from Argentina, and one from Chile, that were notified to the Network between 2007 and 2017 (S1 Table). Clinical- epidemiological data were collected between 1986 and 2017 using a specific case report form (S1 Text). Study population and dataset All included cases were diagnosed with T. cruzi and HIV infections, most of them were born in CD endemic regions. CD was diagnosed through serology or parasitological tests. Two of the following high pro- file anti-T. cruzi serological tests were initially applied: ELISA with non-purified epimastigotes or recombinant epi/trypomastigote antigens, indirect hemagglutination and indirect immuno- fluorescence with epimastigotes antigenic preparation. CD diagnosis was confirmed by two positive serological tests. In case of discordant result, a third confirmatory serology was applied: indirect immunofluorescence with epimastigote antigens or immunoblot with trypo- mastigotes antigens or chemiluminescence with chimeric recombinant antigens. Indirect para- sitological enrichment methods (blood culture or xenodiagnoses) were also employed for CD diagnosis and monitoring [2,15,37]. For HIV infection, a positive result by ELISA with HIV1/ HIV2 antigens was confirmed by Immunoblot with HIV1/HIV2 antigens). The CDR was diag- nosed through direct positive microscopy of parasites in the blood or other biological fluids employing methods for concentration (Blood—microhematocrit, Strout, buffy coat and cen- trifuged fluid samples) and/or amastigotes forms in stained tissular inflammatory lesions [2,15]. The report form filled by the physicians included sociodemographic, epidemiological and clinical data related to T. cruzi and HIV infections (S1 Text) by time of each infection diagnosis PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 4 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection (CD and HIV), CDR diagnoses, clinical presentation of CD, CD4+ T lymphocytes, HIV load, Polymerase Chain Reaction (PCR) for T. cruzi [22,38], CDR treatment and previous CD treat- ment. Data were submitted and validated by the researcher from each center. Duplicate cases were excluded from the analysis. Statistical analysis Descriptive statistics comprised median (interquartile range 25%-75%) for continuous vari- ables and frequencies (percentages) for categorical variables. Comparisons between groups according to reactivation status were performed using the Mann-Whitney test for continuous variables and Fisher exact test for categorical variables. Comparisons for CD4+ counts mea- sured before and after reactivation were performed using the Wilcoxon matched-pair test. Comparisons between groups presenting CDR according to their status at notification of the case to the T. cruzi/HIV co-infection Network were performed using Kruskal-Wallis test fol- lowed by Dun’s test for multiple comparisons. Additional analyses were performed dividing the sample into two groups according to the time of co-infection diagnosis (before and after 1997, when ART therapy was introduced in Brazil) using the Mann-Whitney test for continu- ous variables and Fisher exact test for categorical variables. Correlations between continuous variables were obtained by the Pearson coefficient. The associations between clinical and demographical variables with reactivation in all patients included in the study and between clinical and demographical variables with death among those CDR patients were studied using logistic regression models. Variables that pre- sented a p-value<0.20 in the univariate model were included in the multivariate model. The backwards method was used to remove variables that presented a p-value>0.05 in the multi- variate analysis, until the final model was obtained. Results The S1 Table shows the distribution of the 241 patients according to the centers (170 non-reac- tivated T. cruzi/HIV co-infection, 60 with CDR in AIDS and 11 unknown reactivation occur- rences). Most (86.7%) were from Brazilian centers. Table 1 depicts the characteristics of T. cruzi/HIV patients included in the study. Fifty-five percent of patients were male with a median age of 41.0 years (IQR 25%-75% 34–52) for the entire sample, 40.0 years for males (IQR 25%-75% 33–51), and 45.0 years (IQR 25%-75% 36.5– 53) for females (age comparison for male vs female, p = 0.05). The CD cardiac and indetermi- nate forms were the most prevalent, with the median CD4+ at co-infection diagnosis of 217 cells/μL and median viral load at co-infection diagnosis of 17,000 viral copies/μL. Almost half of patients (48.1%) were monitored by indirect parasitological enrichment methods. The outcome was death in 34.4% of cases. The interval between co-infection and reactivation diagnoses was � 7 days in 50.0% and simultaneous in 39.7% of cases. There was a moderate Spearman correlation between CD4+ count at co-infection and reactivation diagnoses (r = 0.48). There were no differences for the percentage of reactivation (p = 0.17) or death (p = 0.77) comparing the co-infection diagnosis before (n = 66) or after 1997 (n = 171). However, a statis- tically significant difference for the time between co-infection and reactivation (before 1997 median time of 11.8 months vs after 1997 median time of 0.07 months; p = 0.01) was found. No differences for the time between co-infection and death (p = 0.10) and for the time between reactivation and death (p = 0.72) were observed. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 5 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection Table 1. Characteristics of T. cruzi/HIV co-infected cases included in the study (n = 241). Variable Median (IQR 25%-75%) or % (n) Age at co-infection diagnosis (years; n = 238) 41 (34–52) Sex (%; n = 241) Male 55.2 (133) Female 44.8 (108) CD parasitology and molecular biology results (%) Indirect parasitological methods (n = 116) Positive 65.5 (76) Negative 34.5 (40) PCR (n = 17) Positive 64.7 (11) Negative 35.3 (6) CD and HIV diagnoses (n = 232) Simultaneous 16.4 (38) HIV first 55.2 (128) CD first 28.5 (66) Clinical CD presentation at co-infection diagnosis (%, n = 202) Indeterminate clinical form (%, n = 202) 47.0 (95) Cardiac disease (%, n = 202)� 47.0 (95) Digestive disease (%, n = 202)� 16.8 (34) + �� CD4 count at co-infection cells/μL; n = 219) 217 (76–410) CD4+ <200 at co-infection (%, n = 219) 47.5 (104) Viral load at co-infection (viral copies/μL; n = 180) 17,000 (572.5–198,000) CDR (%; n = 230) 26.1 (60) CDR parasitological/histopathological results (%) Direct T. cruzi detection on blood (n = 53) 62.3 (33) Direct T. cruzi detection on other fluids (n = 56) 73.2 (41) Histopathological (n = 38) 18.4 (7) Positive CDR parasitological/histopathological results (%, n = 58) One positive test 63.8 (37) Two positive tests 32.8 (19) Three positive tests 3.4 (2) Clinical presentation at reactivation (%; n = 60) Meningoencephalitis 58.3 (35) Myocarditis 16.7 (10) Meningoencephalitis and myocarditis 13.3 (8) “Others” 11.7 (7) Time between co-infection diagnosis and reactivation (months; n = 58) 0.32 (0–12.8) Time between co-infection diagnosis and reactivation (n = 58) Simultaneous 39.7 (23) � 7 days 50.0 (29) > 7 days 50.0 (29) CD4+ count at reactivation��� (cells/μL; n = 51) 67 (19–202) CD4+ <200 at reactivation (%, n = 51) 74.5 (38) VL at reactivation (viral copies/μL; n = 33) 199,000 (50,262–500,000) Antiparasitic treatment of reactivation (%; n = 60) Yes 81.7 (49) No 18.3 (11) (Continued ) PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 6 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection Table 1. (Continued) Variable Median (IQR 25%-75%) or % (n) Status at notification (%; n = 241) Death 34.4 (83) Active follow-up 43.6 (105) Lost to follow-up 22.0 (53) Time between co-infection diagnosis and death (months; n = 79) 8.7 (0.9–42.45) Time between reactivation and death (months; n = 38) 1.1 (0.3–2.9) Cause of death (n = 83) CDR 28.9 (24) Chronic CD 9.6 (8) Other opportunistic AIDS infection 27.7 (23) Other 18.1 (15) Not available 15.7 (13) CD: Chagas disease; CDR: Chagas disease reactivation; HIV: human immunodeficiency virus; IQR: interquartile interval; Indirect parasitological methods = blood culture and/or xenodiagnoses; VL: viral load, PCR: polymerase chain reaction; VL: viral load; “others”: milder oligosymptomatic febrile diseases, myelitis, erythema nodosum and a puerperal patient with T. cruzi infected newborn; � Mixed clinical presentation (10.8%; n = 22); Median CD4+ levels similar between men and women at co-infection�� (247.5 vs 188.0; p = 0.19) and at reactivation��� (75.5 vs 67.0; p = 0.31), respectively. https://doi.org/10.1371/journal.pntd.0009809.t001 Table 2 depicts the sociodemographic and clinical characteristics according to the presence of CDR. Lower CD4+ cells/μL and higher viral load/μL were found among CDR patients in comparison to non-reactivation group at the co-infection diagnosis. At the notification time, active follow-up is more frequent in non-reactivation group than in CDR. Death was more frequent in the reactivation than in the non-reactivation group (Table 2). Similarly, significant differences were shown concerning the main cause of death, the median time between co-infection diagnosis and death and median time between co-infec- tion diagnosis and death by different causes in the reactivation. Table 3 depicts the characteristics of CDR patients according to the status at notification (n = 60). Among these patients, meningoencephalitis is the most observed presentation (Table 3, n = 35, 58.3%) and seven more benign cases were classified as “others” (two mild oli- gosymptomatic febrile cases; one oligosymptomatic patient with gastritis, one patient with ery- thema nodosum, a puerperal patient with T. cruzi infected newborn, and one febrile case concomitantly to a Hodgkin lymphoma chemotherapy, a patient with myelitis limited to T3-T6). Most of the 24 CDR deaths (66.7%) occurred among those with short interval between co- infection and reactivation diagnoses (� 5 days), while the remaining occurred with an interval > 2 months. No differences of anti-parasitic treatment were shown between the group that died or was followed. Of note, out of 18 untreated and treated patients who died by CDR with known information between the time from reactivation and death, 5.6% died in the first day after the diagnosis, 11.1% < 7 days, 33.6% <15 days, and 72.2% up to 30 days after the diagnosis of reactivation. Under antiparasitic treatment, most patients with other milder forms and few severe cases with encephalitis survived. All untreated patients whose death cause was meningoencephalitis died up to the day after the reactivation diagnosis. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 7 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection Table 2. Characteristics of patients included in the study according to Chagas disease reactivation (n = 230). Variables Median (IQR 25%-75%) or % (n) p-value� Chagas Disease Reactivation Yes (n = 60) No (n = 170) Age (years; n = 58 and n = 169) 41 (34–50) 42 (34–53) 0.79 Sex (%; n = 60 and n = 170) Male 60.0 (36) 53.5 (91) 0.45 Female 40.0 (24) 46.5 (79) Clinical CD presentation (%, n = 192) Indeterminate form (%, n = 48 and n = 144) 47.9 (23) 49.3 (71) 0.99 Cardiac disease (%, n = 48 and n = 144)�� 47.9 (23) 43.8 (63) 0.62 Digestive disease (%, n = 48 and n = 144)�� 14.6 (7) 18.1 (26) 0.66 CD4+ count at co-infection (cells/μL; n = 51 and n = 163) 73 (26–241) 260 (121–443) <0.001 + CD4 <200 at co-infection (%, n = 51 and n = 163) 68.6 (35) 40.5 (66) <0.001 VL at co-infection (viral copies/μL; n = 37 and n = 138) 137,756 (21,400– 7,856 (104– <0.001 456,000) 84,500) Status at notification (%; n = 60 and n = 170) Death 66.7 (40) 20.6 (35) <0.001 Active follow-up 18.3 (11) 55.3 (94) Lost to follow-up 15.0 (9) 24.1 (41) Time between co-infection diagnosis and death (months; 4.3 (0.6–13.0) 19.3 (2.2–62.9) 0.007 n = 36 and n = 35) Cause of death (n = 40 and n = 35) CDR 60.0 (24) 0 (0.0) <0.001ƚ Chronic CD 0.0 (0) 20.0 (7) HIV opportunistic infection 17.5 (7) 42.9 (15) Other 12.5 (5) 25.7 (9) Ignored 10.0 (4) 11.1 (4) Time between co-infection diagnosis and death by cause of death (months) CDR 0.7 (0.3–8.7)��� --- --- Chronic CD --- 5.8 (1.9– --- 76.8)���� HIV opportunistic infection 9.9 (5.4–15.2)��� 5.8 (0.9– 0.57 47.9)���� Other causes 13.1 (4.8–81.2)��� 63.1 (24.3– 0.26 88.4)���� CD: Chagas disease; CDR: Chagas disease reactivation; HIV: human immunodeficiency virus; IQR: interquartile interval; VL: viral load. � Mann Whitney test for continuous and Fisher exact test for categorical variables. �� Mixed clinical presentation (n = 21, 16 non reactivated and 5 reactivated group). ɫ Reactivation vs non-reactivation for CDR (60.0% vs 0.0%; p<0.001) and HIV opportunistic infection (17.5% vs 42.9%; p = 0.02); Differences among median death times for several causes: ��� p = 0.03 -comparison among causes of death only in the reactivation group and ���� p = 0.09 –the same, in the non-reactivation group. https://doi.org/10.1371/journal.pntd.0009809.t002 Patients who died had lower CD4+ cells/μL counts and higher frequency of CD4+ count <200/μL at the co-infection diagnosis and at the reactivation time. There were no differences for the comparison of CD4+ cells/μL at co-infection and reactivation for the 51 patients that presented the information of CD4+ cells/μL at the reactivation time (p = 0.36). In CDR with PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 8 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection Table 3. Characteristics of patients that presented Chagas disease reactivation according to status at notification (n = 60). Variable Median (IQR 25%-75%) or % (n) p-value� Status at notification Death (n = 40) Being Followed Lost to Follow-Up (n = 11) (n = 9) Age (years; n = 38 and n = 11 and n = 9) 41 (33–51) 40 (34–45) 46 (41–48) 0.56 Sex (%; n = 60) Male 70.0 (28) 36.4 (4) 44.4 (4) 0.09 Female 30.0 (12) 63.6 (7) 55.5 (5) Clinical CD presentation (%; n = 48) Chronic Indeterminate form (%; n = 48) 37.9 (11) 70.0 (7) 55.6 (5) 0.20 Cardiac disease�� (%; n = 48) 58.6 (17) 30.0 (3) 33.3 (3) 0.20 Digestive disease�� (%; n = 48) 13.8 (4) 20.0 (2) 11.1 (1) 0.86 CD4+ count at co-infection (cells/μL; n = 31 and n = 11 and n = 9) 37 (19–87) 266 (84–464) 228 (56–298) 0.003 CD4+ <200 at co-infection (%, n = 31 and n = 11 and n = 9) 87.1 (27) 36.4 (4) 44.4 (4) 0.001 VL at co-infection (viral copies/μL; n = 19 and n = 11 and n = 7) 188,788 (59,900– 50,262 (1,100– 195,692 (2,000– 0.54 294,286) 1,400,000) 1,400,000) Reactivation clinical presentation (%; n = 60) Meningoencephalitis1 (n = 35) 67.5 (27) 36.4 (4) 44.5 (4) 0.008 Myocarditis2 (n = 10) 12.5 (5) 9.1 (1) 44.5 (4) Meningoencephalitis and Myocarditis3 (n = 8) 15.0 (6) 9.1 (1) 11.1 (1) 4 “Others” (n = 7) 5.0 (2) 45.4 (5) 0.0 (0) Time between co-infection diagnosis and reactivation Time (months; n = 38, 0.05 (0–7.9) 45.1 (0–99.2) 0.6 (0.2–4.6) 0.07 n = 11 and n = 9) CD4+ at reactivation (cells/μL; n = 31, n = 11 and n = 9) 38 (10–76) 171 (29–340) 240 (55–501) 0.007 CD4+ <200 at reactivation (%, n = 31, n = 11 and n = 9) 90.3 (28) 54.6 (6) 44.4 (4) 0.004 VL at reactivation (viral copies/μL; n = 17, n = 9 and n = 7) 244,037 (59,900– 68,901 (26,050– 87,574 (2,000– 0.64 456,000) 355,962) 1,400,000) Antiparasitic treatment (%, n = 60) Yes 85.0 (34) 81.8 (9) 66.7 (6) 0.46 No 15.0 (6) 18.2 (2) 33.3 (3) Time between co-infection diagnosis and death (months; n = 36) 4.3 (0.6–13.0) --- --- --- Time between reactivation and death (months; n = 38) 1.1 (0.3–2.9) --- --- CD: Chagas disease; CDR: Chagas disease reactivation; HIV: human immunodeficiency virus; IQR: interquartile interval; VL: viral load; “Others”: milder oligosymptomatic febrile diseases, myelitis, erythema nodosum and a puerperal patient with T. cruzi infected newborn; � Kruskal-Wallis test for differences in continuous variables and Fisher exact test for differences in categorical variables; �� Mixed clinical presentations (n = 5, death = 3, being followed = 2, lost to follow-up = 0). 1vs 4(p = 0.002). https://doi.org/10.1371/journal.pntd.0009809.t003 central nervous system (CNS) involvement, a higher percentage of deaths (Table 3), and lower level of CD4+ cells/μL at the reactivation time were registered over myocarditis and “others” presentations (Table 4). Table 5 shows the univariate and multivariate logistic regression analyses to assess the asso- ciations between patients’ characteristics and reactivation or death (only among CDR patients). The CD4+ count at co-infection was the only variable independently associated with reactivation whereas male sex and CD4 count at co-infection were independently associated with death in CDR patients (Table 5). Discussion This article addresses an unprecedented case series of 241 patients with T. cruzi HIV co-infec- tion, 102 previously published as single case report or case series [15,17,19,21,22,35,36,39–41]. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 9 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection Table 4. Distribution of patients according to category of CD4+ cells/μL and site of Chagas disease reactivation. CD4 + cells/μL Meningoencephalitis1 Myocarditis2 “Others”3 Meningoencephalitis + Myocarditis4 Total �100 22 3 2 5 32 101–200 3 1 1 1 6 201–350 2 3 3 0 8 >350 2 2 1 0 5 Number 29 9 7 6 51 � Median 29.0 240.0 241.0 62.0 p = 0.004 25–75% IQR 10.0–73.0 91.0–327.0 43.0–240.0 24.0–100.0 Min-Max 4–501 30–839 12–537 4–102 “Others”: milder oligosymptomatic febrile diseases, limited myelitis, erythema nodosum and a puerperal patient with T. cruzi infected newborn. � 1vs 2 (p = 0.0009); 1vs 3 (p = 0.008); 2 vs 4 (p = 0.02) by Kruskal-Wallis test, followed by Dunn test. IQR—interquartile interval. https://doi.org/10.1371/journal.pntd.0009809.t004 The observed rate of CDR in the present study (26.1%) is greater than the previous reported values, that can be explained by the lack of uniformity in the notification of cases, from previ- ous studies and some health centers. Data from reported retrospective studies are variable [5,21], and although CDR is a rare event, the data of patients from large prospective studies could lead to a more reliable rate of CDR [6,15]. Concerning CDR diagnosis, parasites search in the blood by microscope concentration methods is easily performed but the result could be negative in myocarditis or encephalitis, and the search for parasites in other fluids (pericardial and cerebrospinal) or even invasive pro- cedure with biopsy could increase the CDR diagnosis rate [6,15]. In this work, monitoring for CDR was made in 48.1% of patients with co-infection by para- sitological enrichment methods, and more rarely, by PCR. Quantitative PCR represents a safety test to monitor parasitemia in immunocompromised hosts when able to differentiate between higher parasitemia in CDR and lower parasitemia in non-reactivation episodes of the co-infection [22,38,42,43]. Therefore, its implementation is essential for CDR detection as rap- idly as possible in order to adapt immunosuppressive treatment and to start the anti-parasitic treatment. Our data demonstrated new features on morbidity and mortality of T. cruzi/HIV co-infec- tion besides interferences of CDR in the natural history of T. cruzi/HIV co-infection. Among these evidences generated by our study are: a shorter time between co-infection diagnosis and death in reactivation than in non-reactivation patients by multivariate analysis (p = 0.006); death status at notification registered more frequently in the reactivation group while “being followed” status in the non-reactivation group (p<0.001); different causes of death between reactivation (predominance of CDR) and non-reactivation group (mainly opportunistic AIDS infections) (p<0.001) with different evolution and outcomes; higher percentage of deaths in meningoencephalitis and lower in “others” non-severe cases of reactivation (p = 0.008); and CD4 counts differences among several sites of reactivation (p<0.004), with lower CD4+ counts in meningoencephalitis or “meningoencepahlitis + myocarditis” compared to myocarditis, as well as in meningoencephalitis compared to “others” (p = 0.0009, p = 0.008, and p = 0.02, respectively). We emphasized that the statistical difference of time lapsed from co-infection diagnosis to CDR or death showed a clear gap about opportunity of timely diagnose both T. cruzi and HIV infections in patients under follow-up for early detection of CDR. In addition, this association emphasizes the need of careful management of both infections by controlling adherence to ART and monitoring T. cruzi parasitemia soon after co-infection diagnosis. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 10 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection Table 5. Logistic regression model for association between clinical and demographical variables with reactivation outcome (n = 230) and death among those patients that presented CDR reactivation (n = 51). Variables Reactivation (n = 230) Univariate Multivariate OR (95% CI) p-value OR (95% CI) p-value Age (n = 227) 0.99 (0.97–1.02) 0.78 --- --- Male (n = 230) 1.30 (0.72–2.37) 0.39 --- --- CD chronic indeterminate clinical form (n = 192) 0.95 (0.49–1.82) 0.87 --- --- Cardiac clinical form (n = 192) 1.18 (0.61–2.28) 0.62 --- --- Digestive clinical form (n = 192) 0.77 (0.31–1.92) 0.58 --- --- CD4+ at co-infection (n = 214) 0.99 (0.99–0.99) <0.001 0.99 (0.99–0.99)� 0.008 CD4+ <200 at co-infection (n = 214) 3.21 (1.65–6.28) 0.001 --- --- VL at co-infection (n = 175) 1.01 (1.00–1.01) 0.01 --- --- Variables Death (n = 51) Univariate Multivariate OR (95% CI) p-value OR (95% CI) p-value Age (n = 49) 1.02 (0.96–1.08) 0.48 --- --- Male (n = 51) 4.08 (1.01–16.60) 0.04 8.98 (1.11–72.29) 0.04 CD chronic indeterminate clinical form (n = 39) 0.26 (0.06–1.23) 0.09 --- --- Cardiac disease (n = 39) 3.31 (0.71–15.44) 0.13 --- --- Digestive disease (n = 39) 0.64 (0.10–4.17) 0.64 --- --- Reactivation Presentation “Others” �� 1.0 (Reference) 1.0 (Reference) Meningoencephalitis 16.9 (2.4–118.3) 0.004 --- --- Myocarditis 12.5 (0.84–186.3) 0.07 --- Meningoencephalitis and Myocarditis 15.0 (1.03–218.3) 0.04 --- --- Parasitological treatment 1.35 (0.22–7.33) 0.80 --- --- Co-infection diagnosis to reactivation time (months) (n = 49) 0.97 (0.95–0.99) 0.01 --- --- CD4+ at co-infection (n = 42) 0.99 (0.98–0.99) 0.004 0.99 (0.98–0.99) 0.006 CD4+ <200 at co-infection (n = 42) 11.81 (2.35–59.46) 0.003 --- --- CD4+ at reactivation (n = 42) 0.99 (0.98–0.99) 0.004 --- --- CD4+ <200 at reactivation (n = 42) 7.78 (1.45–41.78) 0.02 --- --- VL at co-infection (n = 30) 0.99 (0.99–1.00) 0.31 --- --- VL at reactivation (n = 26) 1.00 (0.99–1.00) 0.43 --- --- � 95% CI (0.99465–0.99918); �� “others”: milder oligosymptomatic febrile diseases, myelitis, erythema nodosum and a puerperal patient with T. cruzi infected newborn https://doi.org/10.1371/journal.pntd.0009809.t005 In our study, HIV infection was the first diagnosed infection in 55.2% of all co-infection cases, and simultaneous diagnosis of HIV and CD only occurred in 16.4% of cases. Remark- ably, about 50% of co-infection cases were diagnosed only 7 days before reactivation diagnosis, emphasizing the need for active search for CD when HIV was first diagnosed and vice-versa upon clinical and epidemiological suspicion and serological testing or even in the absence of signs and symptoms. In fact, the greater interval between co-infection and reactivation diagno- sis before 1997 is parallel to the initial effort from the “Network for Healthcare and Study of Trypanosoma cruzi/HIV co-infection and other immunosuppression conditions” for an early diagnosis of both infections. Significant difference for the time between co-infection to reacti- vation (higher before 1997, due to active search from physician aware of CD in some centers) with no difference between co-infection diagnosis to death (p = 0.10) and reactivation time to PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 11 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection death (p = 0.72) suggest that co-infection diagnosis was early detected in the first period but not enough to modify death, probably due to the lack of timely CDR diagnosis and antipara- sitic treatment. After 1997, creation of Brazilian reference centers for HIV/AIDS increased the % of first HIV diagnosis in the co-infection diagnosis. However, CD continues to be a neglected disease and has not been the main focus for HIV specialists. Consequently, co-infec- tion has become a late diagnosis in these centers, close to reactivation diagnosis. Although it is expected that ART introduction in 1997 decreased the percentage of CDR, these data are only observed in a few centers that described CDR cases before 1997. In other centers inside and outside Brazil, most of the cases occurred after 1997. Unfortunately, about 60% of CDR cases in Brazilian centers and 90% outside Brazil after 1997 were not on ART, both by absence of HIV diagnosis or abandonment of therapy. Therefore, active search of CD has not been a priority for physicians. Thus, the spread of the need for early diagnosis of co-infection remains a challenge to restore the immune response by ART and control T. cruzi parasitemia. In agreement with most published papers, our data recognize the meningoencephalitis as the most common reactivation site, followed by myocarditis, myocarditis and meningoen- cephalitis and “others” sites [5,15,35,36]. In accordance to its severity and high lethality, we also found that meningoencephalitis was associated with lower levels of CD4+ (median = 29 cells/μL) in comparison to those patients classified as “others” forms (median = 241 cells/μL), concomitantly to more benign clinical forms and lower lethality. The description and evolution of the group classified as “others” brought new information and additional cases to those previously described [15,35,36] as oligosymptomatic and mild disease with higher CD4 counts/μL (Table 4) (Table 3). In fact, we added to previously pub- lished oligosymptomatic cases [15,35,36], three more cases presenting with febrile disease con- comitantly to a Hodgkin lymphoma, gastritis and T3-T6 myelitis. Other four reported cases had oligosymptomatic febrile disease, erythema nodosum and one is a puerperal oligosympto- matic patient with T. cruzi infected newborn [15,35,36]. We confirmed here a better outcome in this group, with 71.4% (5 out 7 patients) classified as “others” surviving after antiparasitic treatment in contrast to 12.9% (4 out 31 patients) with meningoencephalitis (Table 3). In these cases, the diagnosis needs to be actively searched since a better response to antipar- asitic treatment has been shown in severe cases [5,15,21,23]. Multivariate logistic regression analysis showed an increased risk of death in male patients with reactivation. This original data can be partially explained by the greater risk of death in males with Chagas heart disease, as documented before in non-HIV infected patients [44]. In patients with CDR who died, cardiac disease occurred in 58.6%. In addition, a Brazilian study of naïve patients for active antiretroviral therapy (ART) reported higher frequency of CD4+ counts <200 cells/μL in men than women [45] but our work did not show differences between men and women for median CD4+ levels at co-infection and reactivation diagnoses. Moreover, data analyses from mortality in South African patients with CD4+ basal and follow-up levels under control showed a 20% higher risk of death for males than females at 24 and 36 months after starting ART [46]. Better immune response to treatment was shown in women with higher increases of CD4+ counts, representing a challenge for future studies. We emphasize the role of higher CD4+ counts at the time of co-infection diagnosis as a pro- tective factor for both reactivation and mortality, confirmed by multivariate logistic regression analyses. Although high mortality and CD4+ counts lower than 200 or 300 cells/μL have been described in case report or case series of CDR, there was only one previous comparative analy- sis in the literature [21], that showed a significant association between lower CD4+cells/ μL at co-infection diagnosis and CDR in the univariate analysis [21]. However, these authors recog- nized the small number of the patients to perform a multivariate analysis. In this setting, our PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 12 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection results are partially explained by the short interval between co-infection and reactivation diag- noses in 50% of cases. In our sample, detectable viral loads (median = 199,000 viral copies/μL) and CD4+ counts (median = 67 cells/μL) at reactivation diagnosis suggest the lack of effective antiretroviral therapy. In addition, an association of lower CD4+ counts at the time of co-infec- tion diagnosis and mortality in the reactivation group was confirmed for the first time to our knowledge using a multivariate analysis. Previous report did not find significant statistical dif- ference concerning this variable [21]. We also observed that about 70% of deaths were reported within 5 days between co-infection diagnosis and death and that a shorter period of time was observed between co-infection diagnosis and death in the reactivation than in the non-reacti- vation group (p = 0.007). Remarkably, the presence of detectable viral loads (median = 17000 copies/μL) and CD4+ cells/μL (median = 217) at co-infection diagnosis in our entire sample of T. cruzi/HIV infected patients emphasizes the need of early diagnosis of co-infection and introduction of effective antiretroviral therapy. In fact, CD4+ cells play a central role in HIV/AIDS evolution since CD4+ count depletion has been linked to cellular immunity impairment and susceptibility to opportunistic infections [47]. Severe lymphopenia, unresponsiveness to in vivo and in vitro tests, and marked decrease of CD4+ cells were reported in HIV infected patients in association with opportunistic infec- tion [47]. This immunodeficiency is attributed to the impairment of CD4+-mediated viral kill- ing [48], and is reversed by antiretroviral protease inhibitor [49]. On the other hand, stimulation of CD4+ by T. cruzi antigens induces macrophage activa- tion, interferon γ secretion and increases its ability to kill the parasites [50,51]. In addition, the role of CD4+ cells (Th1 response) in protecting against increased T. cruzi parasitemia has been demonstrated by higher parasitemia in CD4+ depleted mice [52,53]. T cells have also been considered as unresponsive to secrete IFNγ and IL2 cytokines under T. cruzi antigens stimulation in a murine model of infection with parasite and leukemia virus, associated with CDR and higher parasitemia [54]. In parallel, a Th2 response was also reported in association with parasitemia in T. cruzi/HIV infected patients [31]. Although antiparasitic treatment did not cause mortality differences between treated and non-treated CDR patients, this lack of efficacy can be related to the lack of opportunity to timely receive it. Thus, patients diagnosed too late and presenting severe central nervous sys- tem or myocardium involvement do not have opportunity to have the protective effect of anti- parasitic treatment. The present study showed, for the first time, a more comprehensive data about outcome in 49 treated patients with CDR. In fact, a proportion of 72.2% of the total deaths due to CDR occurred by the 30th day after reactivation diagnosis, without the possibility to complete the recommended 60 days of antiparasitic treatment. Among 11 survivor cases, six had meningo- encephalitis and five a milder form of reactivation (“others”). In contrast, 11 patients did not receive antiparasitic treatment, the majority with meningoencephalitis. Due to the retrospec- tive nature, the reasons for this absence of treatment were not known and can be attributed to losses of follow-up, lack of drugs accessibility or impossibility of oral drug intake. In all patients who died of meningoencephalitis without antiparasitic treatment, death occurred within 36 hours after the reactivation diagnosis, so early antiparasitic treatment is mandatory in all cases. This work has limitations mainly due to the variability of the data retrospectively included by the health centers over a 31 years period, at times that differ in terms of access to full treat- ment for HIV infection (ART not available for the first cases). Differential processes for co- infection cases registration and follow-up routines, low accessibility to indirect parasitological and/or molecular methods may contribute to some issues observed in this study. In addition, although it is the largest known sample, considering the limited size of CDR group and interval PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 13 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection between co-infection and reactivation diagnoses, associations of CD4+ with reactivation and death need to be confirmed in future studies. Despite these limitations, this study highlights, in 241 T. cruzi/HIV co-infected patients with 60 reactivations, significant interference of CDR in the natural history of CD and HIV infections. In summary, these novel associations of CD4+ cells/μL with both reactivation and death as well as with the meningoencephalitis in CDR emphasize the role of these cells as risk factors for CDR and reinforce the need of a preserved immune response provided by ART in co-infection. Additionally, despite the high lethality, we reinforce the role of early antiparasitic treatment in meningoencephalitis survivors since non- treated severe cases died soon after diagnosis. This multicenter study emphasizes the crucial need for early diagnosis and immediate treat- ment of both T. cruzi and HIV infections, for monitoring HIV evolution under ART, control- ling T. cruzi parasitemia, and applying more efficient and effective tools and protocols. In addition, data gathering for a comprehensive evidence-based patient care, health professionals training and case surveillance are critical to break the underdiagnosis and epidemiological silence. Supporting information S1 Table. Distribution of 241 patients with T. cruzi/HIV co-infection (CO) according to their status of Chagas disease reactivation and to the reference centers. (PDF) S1 Text. Case report forms for T. cruzi/HIV co-infection patients. A. Portuguese B. English. (PDF) Acknowledgments The authors thank José Fernando de Castro Figueiredo (in memoriam) for collecting patients data, to the Brazilian Society of Tropical Medicine (SBMT), Vera Lúcia Carvalho da Silva and Noemia Barbosa Carvalho for collaborating with the “Network for Healthcare and Study of Trypanosoma cruzi/HIV Co-infection and other immunosuppression conditions”, Gilberto Marcelo Sperandio da Silva for the translation of the case report form to English, and Érica Tatto for supporting and writing the instructions for filling out the case report forms. PCF was an undergraduate student at Federal University of Triângulo Mineiro, Uberaba, MG, Brazil. Author Contributions Conceptualization: Maria Aparecida Shikanai-Yasuda, Alberto Novaes Ramos, Jr, Eros Anto- nio de Almeida. Data curation: Mauro Felippe Felix Mediano, Christina Terra Gallafrio Novaes, Juan Cortez- Escalante. Formal analysis: Maria Aparecida Shikanai-Yasuda, Mauro Felippe Felix Mediano. Investigation: Maria Aparecida Shikanai-Yasuda, Christina Terra Gallafrio Novaes, Andréa Silvestre de Sousa, Ana Marli Christovam Sartori, Rodrigo Carvalho Santana, Dalmo Cor- reia, Cleudson Nery de Castro, Marilia Maria dos Santos Severo, Alejandro Marcel Hasslo- cher-Moreno, Marisa Liliana Fernandez, Fernando Salvador, Maria Jesús Pinazo, Valdes Roberto Bolella, Pedro Carvalho Furtado, Marcelo Corti, Ana Yecê Neves Pinto, Alberto Fica, Israel Molina, Joaquim Gascon, Eros Antonio de Almeida. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 14 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection Methodology: Maria Aparecida Shikanai-Yasuda, Mauro Felippe Felix Mediano, Andréa Sil- vestre de Sousa. Project administration: Maria Aparecida Shikanai-Yasuda, Eros Antonio de Almeida. Software: Mauro Felippe Felix Mediano. Supervision: Maria Aparecida Shikanai-Yasuda, Mauro Felippe Felix Mediano, Andréa Silves- tre de Sousa. Validation: Maria Aparecida Shikanai-Yasuda, Mauro Felippe Felix Mediano, Christina Terra Gallafrio Novaes, Andréa Silvestre de Sousa, Ana Marli Christovam Sartori, Rodrigo Car- valho Santana, Dalmo Correia, Cleudson Nery de Castro, Marilia Maria dos Santos Severo, Alejandro Marcel Hasslocher-Moreno, Marisa Liliana Fernandez, Maria Jesús Pinazo, Val- des Roberto Bolella, Pedro Carvalho Furtado, Marcelo Corti, Ana Yecê Neves Pinto, Alberto Fica, Israel Molina, Joaquim Gascon, Pedro Albajar Viñas, Juan Cortez-Escalante, Alberto Novaes Ramos, Jr, Eros Antonio de Almeida. Visualization: Maria Aparecida Shikanai-Yasuda, Mauro Felippe Felix Mediano, Christina Terra Gallafrio Novaes, Pedro Albajar Viñas, Juan Cortez-Escalante. Writing – original draft: Maria Aparecida Shikanai-Yasuda. Writing – review & editing: Maria Aparecida Shikanai-Yasuda, Mauro Felippe Felix Med- iano, Christina Terra Gallafrio Novaes, Andréa Silvestre de Sousa, Ana Marli Christovam Sartori, Rodrigo Carvalho Santana, Dalmo Correia, Cleudson Nery de Castro, Marilia Maria dos Santos Severo, Alejandro Marcel Hasslocher-Moreno, Marisa Liliana Fernandez, Fernando Salvador, Maria Jesús Pinazo, Valdes Roberto Bolella, Pedro Carvalho Furtado, Marcelo Corti, Ana Yecê Neves Pinto, Alberto Fica, Israel Molina, Joaquim Gascon, Pedro Albajar Viñas, Juan Cortez-Escalante, Alberto Novaes Ramos, Jr, Eros Antonio de Almeida. References 1. PAHO: Chagas disease. Pan American Health Organization. Acessible 17 june 2021: https://www. paho.org/en/topics/chagas-disease (https://www.who.int/health-topics/chagas-disease#tab=tab_1 2. Dias JC, Ramos AN Jr, Gontijo ED, Luquetti A, Shikanai-Yasuda MA, Coura JR, et al. 2nd Brazilian Consensus on Chagas Disease, 2015. Rev Soc Bras Med Trop. 2016; 49 (Suppl 1):3–60. https://doi. org/10.1590/0037-8682-0505-2016. PMID: 27982292 3. Monge-Maillo B, López-Vélez R. Challenges in the management of Chagas disease in Latin-American migrants in Europe. Clin Microbiol Infect. 2017; 23(5):290–295. https://doi.org/10.1016/j.cmi.2017.04. 013 PMID: 28428122 4. Yoshioka K, Manne-Goehler J, Maguire JH, Reich MR. Access to Chagas disease treatment in the United States after the regulatory approval of benznidazole. PLoS Negl Trop Dis. 2020; 14(6): e0008398 https://doi.org/10.1371/journal.pntd.0008398 PMID: 32569280 5. Almeida EA, Ramos AN Júnior, Correia D, Shikanai-Yasuda MA. Co-infection Trypanosoma cruzi/HIV: systematic review (1980–2010). Rev Soc Bras Med Trop. 2011; 44(6):762–770. https://doi.org/10. 1590/s0037-86822011000600021 PMID: 22231251 6. Shikanai-Yasuda MA, Almeida EA, López MC, Delgado MJP. Chagas Disease: A Parasitic Infection in an Immunosuppressed Host. In: Pinazo Delgado MJ, Gascón J (eds) Chagas Disease. Chap. 13. p. 213–234. Springer, Nature, Switzerland, 2020. ISBN 978-3-030-44053-4 ISBN 978-3-030-44054-1 (eBook) https://doi.org/10.1007/978-3-030-44054-1 7. Del Castillo M, Mendoza G, Ovieda J, Perez Bianco RP, Anselmo AE, Silva M. AIDS and Chagas’ dis- ease with central nervous system tumor-like lesion. Am J Med 1990; 88:693–694. https://doi.org/10. 1016/0002-9343(90)90544-n PMID: 2111972 8. Spina-França A, Livramento JA, Machado LR, Yassuda N. Anticorpos a Trypanosoma cruzi no lı́quido cefalorraqueano. Arq Neuro-Psiquiat 1988, 46:374–378. https://doi.org/10.1590/s0004- 282x1988000400006 PMID: 3149889 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 15 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection 9. WHO: World Health Data Platform/GHO/Indicators https://www.who.int/data/gho/data/indicators/ indicator-details/GHO/estimated-number-of-people-(all-ages)-living-with-hiv. Access on 12 October 2020. 10. Ramos NA Jr, Correia D, Almeida EA, Shikanai-Yasuda MA. History, Current Issues and Future of the Brazilian Network for Attending and Studying Trypanosoma cruzi/HIV Coinfection. Journal of Inf. Develop. Countries 2010; 4: 682–688. https://doi.org/10.3855/jidc.117 11. Who case definitions of HIV for surveillance and revised clinical staging and immunological classifica- tion of HIV-related disease in adults and children. WHO Region of the Americas. WHO Press, World Health Organization, 2007. Geneva, Switzerland, 2007. ISBN 978 92 4 159562 9 (NLM classification: WC 503.1). https://www.who.int/hiv/pub/guidelines/HIVstaging150307.pdf 12. Gluckstein D, Ciferri F, Ruskin J. Chagas’ disease: another cause of cerebral mass in the Acquired Immunodeficiency Syndrome. Am J Med 1992; 92:429–432. https://doi.org/10.1016/0002-9343(92) 90275-g PMID: 1558089 13. Rocha A, Meneses ACO, Silva AM, Ferreira MS, Nishioka SA, Burgarelli MKN. et al. Pathology of patients with Chagas’ disease and acquired immunodeficiency syndrome. Amer. J. Trop. Med. Hyg. 1994; 50:261–268, 1994. https://doi.org/10.4269/ajtmh.1994.50.261 PMID: 8147485 14. Ferreira MS, Nishioka SA, Silvestre MT, Borges AS, Nunes-Araujo FR, Rocha A. Reactivation of Cha- gas’ disease in patients with AIDS: report of three new cases and review of the literature. Clin Infect Dis 1997; 25:1397–1400. https://doi.org/10.1086/516130 PMID: 9431385 15. Sartori AM, Ibrahim KY, Nunes Westphalen EV, Braz LM, Oliveira OC Jr, Gakiya E, et al. Manifestations of Chagas disease (American trypanosomiasis) in patients with HIV/AIDS. Ann Trop Med Parasitol. 2007; 101(1):31–50. https://doi.org/10.1179/136485907X154629 PMID: 17244408 16. Cordova E, Boschi A, Ambrosioni J, Cudos C, Corti M. Reactivation of Chagas Disease with Central Nervous System Involvement in HIV-Infected Patients in Argentina, 1992–2007. Int. J. Inf. Dis. 2008; 12: 587–92. https://doi.org/10.1016/j.ijid.2007.12.007. 17. Pinazo MJ, Espinosa G, Cortes-Lletget C, Posada E de J, Aldasoro E, Oliveira I, et al. Immunosuppres- sion and Chagas disease: a management challenge. PLoS Negl Trop Dis. 2013; 7(1):e1965. https://doi. org/10.1371/journal.pntd.0001965 PMID: 23349998 18. Hernández C, Cucunubá Z, Parra E, Toro G, Zambrano P, Ramı́rez JD. Chagas disease (Trypanosoma cruzi) and HIV co-infection in Colombia. Int J Infect Dis. 2014; 26:146–148. https://doi.org/10.1016/j. ijid.2014.04.002 PMID: 25080354 19. Salvador F, Sánchez-Montalvá A, Valerio L, Serre N, Roure S, Treviño B et al. Immunosuppression and Chagas disease; experience from a non-endemic country. Clin Microbiol Infect. 2015; 21(9):854–860. https://doi.org/10.1016/j.cmi.2015.05.033 PMID: 26055418 20. Castro-Sesquen YE, Gilman RH, Mejia C, Clark DE, Choi J, Reimer-McAtee MJ et al. Chagas/HIV Working Group in Bolivia and Peru. Use of a Chagas Urine Nanoparticle Test (Chunap) to Correlate with Parasitemia Levels in T. cruzi/HIV Co-infected Patients. PLoS Negl Trop Dis. 2016; 10(2): e0004407. https://doi.org/10.1371/journal.pntd.0004407 PMID: 26919324 21. Benchetrit A, Andreani G, Avila MM, Rossi D, De Rissio AM, Weissenbacher M, et al. High HIV-Trypa- nosoma cruzi Coinfection Levels in Vulnerable Populations in Buenos Aires, Argentina. AIDS Res Hum Retroviruses. 2017; 33(4):330–331. https://doi.org/10.1089/AID.2016.0068 PMID: 27875909 22. Fica A, Salinas M, Jercic MI, Dabanch J, Soto A, Quintanilla S et al. Chagas disease affecting the cen- tral nervous system in a patient with AIDS demonstrated by quantitative molecular methods. Rev Chil. Infectol. 2017; 34(1):69–76. https://doi.org/10.4067/S0716-10182017000100011. 23. Guidetto B, Tatta M, Latini V, Gonzales M, Riarte A, Tavella S et al. HIV and Chagas disease coinfec- tion, a tractable disease? Open Forum Infect Dis. 2019; 6(7):ofz307. https://doi.org/10.1093/ofid/ofz307 PMID: 31363767 24. Braga PE, Cardoso MRA, Segurado AC. Diferenças de gênero ao acolhimento de pessoas vivendo com HIV em serviço universitário de referência de São Paulo, Brasil. Cad. Saúde Pública, Rio de Janeiro, 2007; 23(11):2653–2662. https://doi.org/10.1590/s0102-311x2007001100013 25. Stauffert D, Silveira MF, Mesenburg MA, Manta AB, Dutra AD, Bicca GL et al. Prevalence of Trypano- soma cruzi/HIV coinfection in southern Brazil. Braz J Infect Dis. 2017; 21(2):180–184. https://doi.org/ 10.1016/j.bjid.2016.10.006 PMID: 27914221 26. Dolcini G, Ambrosioni J, Andreani G, Pando MA, Martı́nez Peralta L, Benetucci J. Prevalence of human immunodeficiency virus (HIV)-Trypanosoma cruzi co-infection and injectable-drugs abuse in a Buenos Aires health center. Rev. Arg. Microb. 2008; 40(3):164–166. https://pubmed.ncbi.nlm.nih.gov/ 19024504/ 27. Salvador F, Molina I, Sulleiro E, Burgos J, Curran A, Van den Eynde E, et al. Tropical diseases screen- ing in immigrant patients with HIV infection in a European country. Am J Trop Med Hyg 2013; 88:1196– 1202. https://doi.org/10.4269/ajtmh.12-0714 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 16 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection 28. Martins-Melo FR, Ramos Júnior AN, Alencar CH, Heukelbach J. Mortality Related to Chagas Disease and HIV/AIDS Coinfection in Brazil. J Trop Med. 2012, Article ID 534649. https://doi.org/10.1155/2012/ 534649 PMID: 22969814 29. Lattes R & Lasala MB. Chagas disease in immunosupressed host. Clin. Microbiol. Infect. 2014; 20(4):300–9. https://doi.org/10.1111/1469-0691.12585 PMID: 24602129 30. Lazo JE, Meneses ACO, Rocha A, Frenkel JK, Marquez JO, Chapadeiro E et al. Meningoencefalites toxoplásmica e chagásica em pacientes com infecção pelo vı́rus da imunodeficiência humana: diagnós- tico diferencial anatomopatológico e tomográfico. Rev Soc Bras Med Trop 1998; 31:163–171. https:// doi.org/10.1590/s0037-86821998000200001 31. Rodrigues DB, Correia D, Marra MD, Giraldo LE, Lages-Silva E, Silva-Vergara ML et al. Cytokine serum levels in patients infected by human immunodeficiency virus with and without Trypanosoma cruzi coinfection. Rev Soc Bras Med Trop. 2005; 38(6):483–487. https://doi.org/10.1590/s0037- 86822005000600007 PMID: 16410923 32. Tozetto-Mendoza TR, Vasconcelos DM, Ibrahim KY, Sartori AMC, Bezerra RC, Freitas VLT et al. Role of T. cruzi exposure in the pattern of T cell cytokines among chronically infected HIV and Chagas dis- ease patients. Clinics (Sao Paulo) 2017; 72(11):652–660. https://doi.org/10.6061/clinics/2017(11)02 PMID: 29236910 33. Dolcini GL, Solana ME, Andreani G, Celentano AM, Parodi LM, Donato AM, et al. Trypanosoma cruzi (Chagas’ disease agent) reduces HIV-1 replication in human placenta. Retrovirology. 2008; 5:53. https://doi.org/10.1186/1742-4690-5-53 PMID: 18593480 34. Urquiza JM, Burgos JM, Ojeda DS, Pascuale CA, Leguizamón MS, Quarleri JF. Astrocyte Apoptosis and HIV Replication Are Modulated in Host Cells Coinfected with Trypanosoma cruzi. Front Cell Infect Microbiol. 2017; 7:345. eCollection 2017. https://doi.org/10.3389/fcimb.2017.00345 PMID: 28824880 35. Sartori AM, Caiaffa-Filho HH, Bezerra RC, Guilherme CS, Lopes MH, Shikanai-Yasuda MA. Exacerba- tion of HIV viral load simultaneous with asymptomatic reactivation of chronic Chagas disease. Am J Trop Med Hyg. 2002; 67(5):521–523. https://doi.org/10.4269/ajtmh.2002.67.521 PMID: 12479555 36. Sartori AM, Sotto MN, Braz LM, Oliveira Júnior OC, Patzina RA, Barone AA, et al. Reactivation of Cha- gas disease manifested by skin lesions in a patient with AIDS. Trans R Soc Trop Med Hyg. 1999; 93 (6):631–632. https://doi.org/10.1016/s0035-9203(99)90077-9 PMID: 10717752 37. Portela-Lindoso AA, Shikanai-Yasuda MA. Chronic Chagas’ disease: from xenodiagnosis and hemocul- ture to polymerase chain reaction. Rev Saúde Publica 2003 Feb; 37(1):107–15. https://doi.org/10.1590/ s0034-89102003000100016 PMID: 12488927 38. Freitas VLT, da Silva SC, Sartori AM, Bezerra RC, Westphalen EV, Molina TD et al. Real time PCR in HIV/Trypanosoma cruzi coinfection with and without Chagas disease reactivation: association with HIV viral load and CD4 level. PLoS Negl Trop Dis 2011; 5(8): e1277. https://doi.org/10.1371/journal.pntd. 0001277 PMID: 21912712 39. Corti M. AIDS and Chagas’ disease. AIDS Patient Care STDS. 2000; 14(11):581–588. https://doi.org/ 10.1089/10872910050193752 PMID: 11155899 40. Almeida EA, Lima JN, Lages-Siva E, Guariento ME, Aoki FH, Morales AET et al. Chagas´ disease and HIV co-infection in patients without effective antiretroviral therapy: Prevalence, clinical presentation and natural history. Trans. Roy. Soc. Trop. Med. Hyg. 2010, 104 (7):447–452. https://doi.org/10.1016/j. trstmh.2010.02.004 PMID: 20303560 41. Galhardo MCG, Martins IA, Hasslocher- Moreno A, Xavier SS, Coelho JMC, Vasconcelos ACV et al. Reactivation of Trypanosoma cruzi infection in a patient with acquired immunodeficiency syndrome. Rev. Soc. Bras. Med. Trop. 1999; 32(3):291–294. https://doi.org/10.1590/s0037-86821999000300011 PMID: 10380569 42. Cura CI, Lattes R, Nagel C. Early molecular diagnosis of acute Chagas disease after transplantation with organs from Trypanosoma cruzi-infected donors. Am J Transpl. 2013; 13: 3253–3261. https://doi. org/10.1111/ajt.12487 PMID: 24266974 43. Besuschio SA, Picado A, Muñoz-Calderón A, Wehrendt DP, Fernández M, Benatar A, et al. Trypano- soma cruzi loop-mediated isothermal amplification (Trypanosoma cruzi Loopamp) kit for detection of congenital, acute and Chagas disease reactivation. PLoS Negl Trop Dis 2020; 14(8): e0008402. https://doi.org/10.1371/journal.pntd.0008402 PMID: 32797041 44. Rassi A Jr, Rassi A, Little WC, Xavier SS, Rassi SG, Rassi AG, et al. Development and validation of a risk score for predicting death in Chagas’ heart disease. N Engl J Med. 2006; 355(8):799–808. https:// doi.org/10.1056/NEJMoa053241 PMID: 16928995 45. Souza PRB, Szwarcwald CL; Castilho EA. Delay in introducing antiretroviral therapy in patients infected by HIV in Brazil, 2003–2006. Clinics São Paulo 2007; 62 (5). https://doi.org/10.1590/S1807- 59322007000500008 PMID: 17952318 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 17 / 18 PLOS NEGLECTED TROPICAL DISEASES Clinical profile and mortality in patients with T. cruzi/HIV co-infection 46. Maskew M; Brennan AT; Westreich D; McNamara L, MacPhail AP; Fox MP. Gender Differences in Mor- tality and CD4 Count Response Among Virally Suppressed HIV-Positive Patients. Journal of Women’s Health. 2013; 22(2), 113–120. https://doi.org/10.1089/jwh.2012.3585 PMID: 23350862 47. Okoye AA, Picker LJ. CD4(+) T-cell depletion in HIV infection: mechanisms of immunological failure. Immunol Rev. 2013; 254(1):54–64. https://doi.org/10.1111/imr.12066 PMID: 23772614 48. Klatzmann D, Barré-Sinoussi F, Nugeyre MT, Danquet C, Vilmer E, Griscelli C, et al. Selective Tropism of Lymphadenopathy Associated Virus (LAV) for Helper-Inducer T Lymphocytes. Science 1984; 225(4657):59–63. https://doi.org/10.1126/science.6328660 PMID: 6328660 49. Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, Markowitz M. Rapid turnover of plasma viri- ons and CD4 lymphocytes in HIV-1 infection. Nature 1995; 373(6510):123–126. https://doi.org/10. 1038/373123a0 PMID: 7816094 50. Wirth JJ, Kierszenbaum F, Sonnenfeld G, Zlotnik A. 1985. Enhancing effects of gamma interferon on phagocytic cell association and killing of Trypanosoma cruzi. Infect. Immun. 1985, 49:61–66. Google Acadêmico https://doi.org/10.1128/iai.49.1.61-66.1985 PMID: 3924832 51. Reed S. In vivo administration of recombinant interferon gamma induces macrophage activation, and prevents acute disease, immune suppression, and death in experimental Trypanosoma cruzi infections. J. Immunol. 1988; 140:4342–4347. Google Acadêmico PMID: 3131431 52. Rottenberg ME, Bakhiet M, Olsson T, Kristensson K, Mak T, Wigzell H, et al. Differential susceptibilities of mice genomically deleted of CD4 and CD8 to infections with Trypanosoma cruzi or Trypanosoma bru- cei. Infect Immun. 1993; 61(12):5129–5133. 1993 https://doi.org/10.1128/iai.61.12.5129-5133.1993 PMID: 8225589 53. Tarleton RL, Sun J, Zhang L, Postan M. Depletion of T-cell subpopulations results in exacerbation of myocarditis and parasitism in experimental Chagas’ disease. Infect Immun. 1994; 62(5): 1820–1829. https://doi.org/10.1128/iai.62.5.1820-1829.1994 PMID: 8168945 54. Silva JS, Barral-Netto M, Reed SG. Aggravation of both Trypanosoma cruzi and murine leukemia virus by concomitant infections. Am J Trop Med Hyg. 1993; 49(5):589–597. https://doi.org/10.4269/ajtmh. 1993.49.589 PMID: 8250098 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0009809 September 30, 2021 18 / 18