Risk factors for leptospirosis and brucellosis in people living with human immunodeficiency virus who attended a referral hospital in southeastern Brazil

Abstract INTRODUCTION: Leptospirosis and brucellosis cause immunosuppression that worsens the clinical condition of people living with HIV/AIDS (PLWHA). We investigated the serological profile and risk factors of PLWHA. METHODS: Serum samples (n=238) were researched for Brucella spp. antibodies using Rose Bengal and tube agglutination tests and Leptospira spp. antibodies using the microscopic agglutination test. RESULTS: All samples were negative for Brucella spp. For leptospirosis, four samples (1.69%) were positive, and Andamana was the prevalent serovar. CONCLUSIONS: Low or no detection of these zoonoses does not reduce their importance in PLWHA. Vigilant, educational, and preventive measures should be adopted.

contaminated food, particularly milk and milk derivatives produced with unboiled or unpasteurized milk 3 . Brucella may occur either as smooth (S) or rough (R) species. These two types are based on the aspect of colonies on agar plates, which is in accordance with the cell surface and lipopolysaccharide (LPS) structure 4 . There are 10 recognized Brucella species. Among them, the S-LPS species are Brucella melitensis (B. melitensis), Brucella suis (B. suis), and Brucella abortus (B. abortus), whereas the R-LPS species are Brucella ovis (B. ovis) and Brucella canis (B. canis). All these species are clinically and epidemiologically important to animal and human health. Although underdiagnosed, > 500,000 new human cases occur annually, mainly in developing countries 2,5 .
On the contrary, leptospirosis is worldwide distributed, with the majority of cases and diseases occurring in tropical and subtropical regions, and in developing countries 6 . Several mammalian species are infected by Leptospira, but only a few act as efficient reservoirs capable of establishing long-term kidney colonization and shedding bacteria in the urine 7 . It occurs mainly in rats, the universal carriers of leptospirosis, and production animals, such as cattle and sheep. Close contact with animals increases the risk of human infection. In an urban scenario, dogs are the main source of infection for humans and are also considered sentinels and carriers for the disease 6 .
Considering the importance of brucellosis and leptospirosis as anthropozoonoses, particularly in immunosuppressed patients, the purpose of this study was to determine Brucella spp. and Leptospira spp. antibodies and related risk factors in people living with HIV/AIDS (PLWHA) in a specialized infectious disease outpatient clinic in a referral hospital in southeastern Brazil. This was a cross-sectional study and patients treated at the Domingos Alves Meira Specialized Infectious Diseases Outpatient Service (SAEI-DAM) of the Clinical Hospital (HC) of Botucatu Medical School, São Paulo State University (FMB-UNESP) were sampled. Botucatu is located in the mid-west region of São Paulo State (22º53'09" S; 48º26'42" W) with an estimated population of 146,497 8 .
The SAEI-DAM registered patients were accompanied by a multidisciplinary team. The medical record system of the HC-FMB-UNESP was used to access patient data. Among them, 300 PLWHA in several towns in the study area were identified, but only 238 patients met the study requirements: 129 (54.2%) men and 109 (45.8%) women, aged 18-76 years. No pregnant women were identified during the study period. Each patient was included in the study after obtaining an informed consent form.
Blood samples were collected using a vacutainer without anticoagulant by cephalic vein puncture to detect specific antibodies against each disease. Blood samples were centrifuged at 1,600 × g for 10 min, and the serum samples were stored at -4°C. In addition, an epidemiological questionnaire ("social and demographic characteristics", "water, garbage, and sewer variables", and "hostrelated characteristics") was applied to the PLWHA to determine the risk factors related to the studied disease.
The present study was approved by the Research Ethics Committee of the FMB-UNESP (protocol #821261).
B. abortus and B. suis antibodies were researched using the Rose Bengal test (RBT), a serum agglutination test in buffered acidantigen stained with Rose Bengal, and the slow tube agglutination test with 2-mercaptoethanol (SAT-2ME) and without 2ME (SAT) 5 .
Descriptive statistics were used to determine the absolute and relative frequencies of positive samples for one or both zoonoses, and analytical statistics were used to determine any associations with epidemiological variables. Therefore, the results of serological tests were analyzed in association with the epidemiological variables by univariate analysis using the Chi-square test (χ 2 ) and/or Fisher's exact test. Subsequently, all variables that presented p-value ≤ 0.05, in the univariate analysis, were included in the multivariate analysis and the logistic regression model 10 . All analyses were performed using Epi Info TM software, v.7.2.0.1, with a significance level (?) of 5%.
All samples were negative for antibodies against B. abortus and B. suis.
Brucellosis and leptospirosis are very important to veterinary science and public health because of their severity and lethality in humans 2 . Brucellosis is not a mandatory notifiable disease in Brazil for humans and may be underdiagnosed. In addition, no organized public health network exists in Brazil to identify human cases 5 . Globally, the prevalence of brucellosis in PLWHA ranges from 5.98% to 73.33% in Iran 2,3,12 to 66.67% in Spain 13 . This range may be related to regional cultural habits (namely, raw milk ingestion), exposure to infected animals, and/or positive family history of brucellosis 3 , and reinforces the importance of periodic serological surveys to improve disease monitoring and surveillance, especially in PLWHA. Even with negative results for the detection of B. abortus and B. suis antibodies in PLWHA in this study, its prevalence in PLWHA from developing countries may be five times higher, according to the World Health Organization (WHO) 4 .
Both humoral and cellular immune responses are required for brucellosis because the elimination of bacteria occurs in the intracellular environment. This fact increases the susceptibility of HIV/AIDS patients to Brucella infection 12 . Brucellosis is rare in PLWHA, although the eradication of intracellular bacteria is largely dependent on cellular immunity. In this way, it is hypothesized that HIV infection does not increase the incidence of brucellosis because most cases occur in asymptomatic patients with preserved immunity, and the epidemiology, clinical presentation, diagnosis, response to the therapy, and outcomes are similar to those observed in HIV-negative patients. A cross-sectional study carried out in basic health units from Alagoas State, Brazil, reported 4.4% Brucella spp. antibodies in patients with brucellosis; however, no notification of the disease was identified in the Notifiable Diseases Information System 5 .
The close contact between humans and animals is evident and may indicate a related risk factor 6 . The role of rodents in the transmission of many diseases, including leptospirosis, is widely   Although certain risk factors may be considered as indicators of the dissemination or, even, the severity of the disease in PLWHA, namely, tap water or artesian well water as "water source", "if it floods when it rains" (Table 2), and even "if the animal stays at home or in the street" (Table 3), the low prevalence and sampled population limit the adequate characterization of the possible and eligible risk factors. The association between each variable and the serology results suggests a possible risk for PLWHA that experienced floods after rain. Despite this limitation, a higher seroprevalence was observed in males from urban areas, which could be related to the occupational risk. In Pernambuco State, Brazil, the authors also reported a higher occurrence of infection in male patients 14 . In non-PLWHA, leptospirosis has a high impact as an occupational disease. This fact was observed in São Paulo State, Brazil, among blood donors (1.3% reagents) from the Donor Center of the Clinical Hospital, FMB-UNESP 9 . This finding reinforces the relevance of continuous epidemiological surveillance and health education actions to control the disease in both animals and humans.
The observed range of the serological results seems reasonable, considering the different geographic regions and variations in   environmental conditions, including rainfall, temperature and humidity, serovars, quality of the antigens, and interpretation of the results. Based on the serological results, the present study confirms that Leptospira spp. were circulating in the PLWHA population from São Paulo State, probably maintained by the animal population, even with low prevalence.

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The low or no detection of the studied zoonoses does not reduce their importance in causing disease in PLWHA. Therefore, vigilant, educational, and preventive measures should be developed and maintained for the early identification of factors that predispose to the occurrence of these zoonoses.

FINANCIAL SUPPORT
This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Coordination for the Improvement of Higher Education Personnel), Brazil.

AUTHORS' CONTRIBUTION
HL: conception and design of the study, data analysis, discussion and review; FBG: conception and design of the study, data analysis, discussion and review, final approval of the version to be submitted; ANB: conception and design of the study, data analysis, discussion and review; RCS: conception and design of the study, data analysis, statistical analysis, discussion and review, and in the final writing; KBM: drafting the article, participated in the review, discussion and analysis of the data and in the final writing; BDM: drafting the article, participated in the review, discussion and analysis of the data and in the final writing; CV: drafting the article participated in the review, discussion and analysis of the data and in the final writing.