¹Biomedical Research Laboratory. Faculty of Medicine and Nutrition, Juárez University of Durango State, Durango, Mexico;

²Institute for Scientific Research “Dr. Roberto Rivera Damm”, Juárez University of Durango State, Durango, Mexico;

³Clínica de Medicina Familiar. Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado. Predio Canoas S/N. 34079 Durango, Mexico

Corresponding Author: Dr. Cosme Alvarado-Esquivel, Faculty of Medicine and Nutrition. Av. Universidad S/N. 34000 Durango, Dgo. Mexico. Tel/Fax: +52-618-8130527. E-mail: alvaradocosme@yahoo.com.

Short title: Leptospira and liver diseases

	ABSTRACT
	INTRODUCTION
	MATERIALS AND METHODS
	RESULTS
	DISCUSSION
	CONCLUSIONS
	CONFLICT OF INTEREST
	ACKNOWLEDGEMENTS
	REFERENCES

	ABSTRACT

The seroepidemiology of Leptospira infection in patients suffering from liver disease has been poorly studied. Information about risk factors associated with infection in liver disease patients may help in the optimal planning of preventive measures. We sought to determine the association of Leptospira IgG seroprevalence and patients with liver diseases, and to determine the characteristics of the patients with Leptospira exposure. We performed a case-control study of 75 patients suffering from liver diseases and 150 age- and gender-matched control subjects. Diagnoses of liver disease included liver cirrhosis, steatosis, chronic hepatitis, acute hepatitis, and amoebic liver abscess. Sera of participants were analyzed for the presence of anti- Leptospira IgG antibodies using a commercially available enzyme immunoassay. Anti-Leptospira IgG antibodies were found in 17 (22.7%) of 75 patients and in 15 (10.0%) of 150 control subjects (OR = 2.32; 95% CI: 1.09-4.94; P=0.03). This is the first age- and gender-matched case control study about Leptospira seroprevalence in patients with liver diseases. Results indicate that Leptospira infection is associated with chronic and acute liver diseases. Results warrants for additional studies on the role of Leptospira exposure in chronic liver disease.

	INTRODUCTION

   Leptospira is a bacterium responsible of infections in humans and animals all around the world (1, 2). Infection with Leptospira causes a disease known as leptospirosis (2). This disease is considered an emerging infectious disease (3), and represents a public health problem particularly in urban centers of developing countries (1). Leptospirosis is a zoonosis (4). Leptospira is transmitted to humans by contact with Leptospira-infected animals or with environment contaminated with Leptospira (5). Skin abrasions represent the main route of Leptospira infection (6). Most infections with Leptospira are subclinical or may result in a very mild illness without any complication (5). Clinical manifestations of leptospirosis depend upon the predominant organs involved, and the case fatality ratio of leptospirosis could be 40% or higher (5). In mild cases, leptospirosis is presented as a flu-like illness (1). Fever, myalgia, and headache are common clinical manifestations of leptospirosis (6). In severe cases of the disease, patients have liver involvement with jaundice, splenomegaly, and nephritis (Weil’s disease) (5). In addition, infection with Leptospira may lead to disease in eyes (7), lungs (8), heart (6), and brain (9).

   Liver is commonly involved in leptospirosis (6, 10). Infection with Leptospira in humans has been linked to acute hepatitis (11), enlargement of and lesions in liver (12), liver damage (13), and hepatic failure (14). Morphological abnormalities of liver including severe edema with pronounced dissociation of the liver cell bands, cholestasis, cloudy swelling of hepatocytes and necrosis have been reported in leptospirosis (15, 16). The epidemiology of leptospirosis in patients with liver disease is largely unknown. To the best of our knowledge, the magnitude of Leptospira exposure in patients with liver diseases in Mexico has not been studied. Therefore, this study aimed to determine the association of Leptospira IgG seropositivity and patients suffering from liver diseases attended in a public hospital in Durango City, Mexico. Seroprevalence association with the socio-demographic, clinical, and behavioral characteristics of the patients with liver disease was also investigated.

	MATERIALS AND METHODS

   Selection and description of participants

   An age- and gender- matched case-control study was undertaken using residual serum samples from Toxoplasma gondii serosurveys in patients with liver disease (17) and general population (18) in Durango City, Mexico. Patients with liver disease (cases) and subjects without liver disease (controls) were compared for the presence of anti-Leptospira IgG antibodies. We used a 1:2 ratio of cases and controls. Thus, the group of cases included 75 patients with liver disease, and the group of controls included 150 subjects without liver disease. Patients with liver disease were attended in the Gastroenterology Department of a public hospital (Mexican Social Security Institute) in Durango City, Mexico. Of the 75 patients, 47 were males and 28 females. They were 58.65 ± 14.41 (range: 22-85) years old. Diagnoses of liver disease included liver cirrhosis in 67 patients, steatosis in four, chronic hepatitis in two, acute hepatitis in one, and amoebic liver abscess in one. Liver cirrhosis was caused by alcohol consumption in 35 patients, and by hepatitis C virus in four patients. The etiology of liver cirrhosis was unknown in 28 patients. The control group consisted of 94 males and 56 females matched with cases by age and gender. Control subjects were randomly selected from the general population of Durango City. The mean age in controls was 58.51 ± 14.47 (range: 22-88) years old and comparable with that in patients (P=0.94).

   Socio-demographic, clinical, and behavioral data of patients with liver diseases

   Data of the patients with liver disease was obtained from previously summited questionnaires (17). Socio-demographic data included age, gender, birthplace, residence, educational level, occupation and socioeconomic status. Clinical data included diagnosis and duration of liver disease, response to treatment, presence of concomitant diseases, history of blood transfusion, organ transplantation, surgery, lymph node enlargement and headache. In addition, information about the presence of impairments in memory, vision, reflexes and hearing was obtained. Liver cirrhosis was diagnosed by clinical manifestations and liver biopsy. Behavioral data included contact with animals, traveling, type of meat consumed, degree of meat cooking, consumption of unwashed raw vegetables or fruits, unpasteurized milk, untreated water, frequency of eating in restaurants or fast food outlets, soil contact, and type of flooring at home.

   Technical information

   Serum samples of patients with liver diseases and controls were tested for anti-Leptospira IgG antibodies using a commercially available enzyme-linked immunosorbent assay kit, “Leptospira IgG ELISA test” (Diagnostic Automation Inc., Calabasas, CA). This test uses purified Leptospira Patoc 1 antigen. An absorbance reading of ≥ 0.5 optical density (OD) units was used as a cut-off for seropositivity. Weakly reactive sera were those with absorbance readings between 0.5 and 1.0 OD units. Whereas, strongly reactive sera (or with high antibody levels) were those with absorbance readings > 1.0 OD units. According to the manufacturer’s package insert, this assay has a sensitivity of 100% and a specificity of 100%. All tests were performed following the manufacturer´s instructions. Positive and negative controls included in the kit were used in each run.

   Statistical analysis

   Statistical analysis was performed using the software Microsoft Excel 2013, Epi Info version 7 and SPSS version 15.0. Comparison of age between cases and controls was made with the student´s t test. We calculated odds ratios (OR) and 95% confidence intervals (CI) for the association of Leptospira seropositivity and liver diseases. The association between patients’ characteristics and Leptospira seropositivity was assessed by bivariate analysis. Comparison of the frequencies between groups was made with the Pearson’s chi-squared test and with the two-tailed Fisher exact test (for small values), and a P value <0.05 was considered statistically significant.

   Ethical aspects

   This project was approved by the Ethics Committee of the Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado in Durango City. We examined only stored sera and data of participants from previous serosurveys (17, 18). In those surveys, the purpose and procedures of the studies were explained to all participants. Written informed consent was obtained from all participants.

	RESULTS

   Anti-Leptospira IgG antibodies were found in 17 (22.7%) of 75 patients and in 15 (10.0%) of 150 control subjects. The difference in Leptospira exposure between cases and controls was statistically significant (OR = 2.32; 95% CI: 1.09-4.94; P=0.03). The frequency of high levels (≥1.0 OD units) of anti-Leptospira IgG antibodies in patients with liver disease (2/75: 2.7%) was similar to the one (4/150: 2.7%) in control subjects (P=1.00). Stratification by gender showed that the frequency of Leptospira exposure in male cases (10/47: 21.3%) was similar to that (10/94: 10.6%) in male controls (P=0.12). Whereas, the frequency of Leptospira exposure in female cases (7/28: 25.0%) was similar to that (5/56: 8.9%) in female controls (P=0.09). None of the 28 female cases and 56 female controls had high levels (≥1.0 OD units) of anti-Leptospira IgG antibodies. The frequency of high levels of anti-Leptospira IgG antibodies in male cases (2/47: 4.3%) was similar to that (4/94: 4.3%) in male controls (P=1.00). All but one seropositive controls were healthy. The ill seropositive control suffered from rheumatism.

   None of the socio-demographic characteristics of patients including age, gender, birthplace, residence, educational level, occupation and socioeconomic status was associated with Leptospira exposure (P>0.05). Concerning clinical characteristics of patients, the frequency of Leptospira exposure was similar in patients regardless the diagnosis and duration of liver disease, response to treatment, presence of concomitant diseases, history of blood transfusion, organ transplantation, surgery, lymph node enlargement, headache, and impairments in memory, vision, reflexes and hearing (Table 1). Seropositivity to Leptospira was found in 16 (23.9%) of 67 cirrhotic patients, and in one (33.3%) of 3 hepatitis patients. No seropositive cases for Leptospira were found in four patients with steatosis or in one patient with amoebic liver abscess. The only one patient with acute hepatitis had low (0.86 OD units) antibody levels against Leptospira.

   Seroprevalence of Leptospira exposure in patients with alcohol-related liver disease (9/35: 25.7%) was similar to that (8/40: 20.0%) in patients with other types of liver diseases (P=0.55). With respect to the behavioral characteristics, the frequency of Leptospira seropositivity was lower in patients with consumption of pork (13/70: 18.6%) than in those without this practice (4/5: 80.0%) (P=0.008). Similarly, the frequency of Leptospira seropositivity was higher in patients with consumption of quail meat (2/2: 100%) than in those without this practice (15/71: 21.1%) (borderline association: P=0.05). Other behavioral characteristics including contact with animals, traveling, consumption of meat other than pork or quail meat, degree of meat cooking, consumption of unwashed raw vegetables or fruits, unpasteurized milk, untreated water, frequency of eating in restaurants or fast food outlets, soil contact, and type of flooring at home did not show an association with Leptospira seropositivity.

	DISCUSSION

   Although it is well known that Leptospira is a pathogen that causes liver damage, the epidemiology of Leptospira infection in patients suffering from liver diseases is largely unknown. We are not aware of any report on the association of Leptospira infection with patients with liver cirrhosis or other chronic liver diseases. Therefore, we decided to perform this age- and gender matched case-control study to determine such association in patients with liver disease attended in a public hospital in Durango City, Mexico. We found that seroprevalence of Leptospira exposure was significantly higher in patients suffering from liver diseases than in control subjects without liver disease. Results thus indicate that Leptospira exposure is associated with liver disease in Durango City. Seroprevalence (22.7%) found in patients with liver diseases is also higher than the 4.4% seroprevalence of Leptospira exposure in waste pickers in the same Durango City reported recently (19), and the 15.6% seroprevalence reported in general population of rural Durango (20) using the same enzyme immunoassay. Acute infection with Leptospira has been linked with severe liver disease i.e., Weil’s disease (5, 13, 14), and Leptospira infection may progress into a chronic, largely asymptomatic infection (21-23). However, very little is known about the clinical picture of chronic infection with Leptospira in humans. In the present study, the great majority of patients suffered from a chronic liver disease. Therefore, the association of Leptospira exposure found in these patients suggests that Leptospira is involved in chronic liver disease. Further studies to determine the clinical features of liver disease in chronic infections with Leptospira are needed. Apart from the potential causative role of Leptospira infection in liver disease, the association of Leptospira exposure in patients with liver diseases may mean that: 1) these patients have behavioral factors that might facilitate infection with Leptospira, and 2) other unknown factors are contributing for infection in these patients. It is not clear whether Leptospira infection occurred before or during the development of liver disease. Longitudinal studies to evaluate the pathogenic role of Leptospira in chronic liver disease patients should be conducted.

   We attempted to determine sociodemographic, clinical and behavioral characteristics associated with Leptospira exposure in the liver disease patients studied. Bivariate analysis showed that Leptospira exposure was negatively associated with consumption of pork. This result means that consumption of pork did not play any contributing role in Leptospira infection in patients. On the other hand, a borderline (P=0.05) association between Leptospira seropositivity and consumption of quail meat was found. A search for this association in the medical literature found no results. We are not aware of any report about the presence of Leptospira infection in quails. However, infection with Leptospira in quails might be possible since this infection has been reported in birds (24).

   Alcohol-related toxicity and alcoholic hepatitis can occasionally produce clinical features similar to leptospirosis (25). It is unclear whether Leptospira infection may modify the severity of alcohol-related liver disease or whether these patients may have a more severe Leptospira infection. In the present study, patients with alcohol-related liver disease had similar seroprevalence of Leptospira exposure to those without this diagnosis.

   This study has some limitations. The sample size of liver disease patients was small and only few types of liver diseases among patients were present. In addition, very few subjects were positive to Leptospira and therefore statistical power was limited. Further research with larger sample sizes of patients with specific diagnoses of liver diseases to determine their association with Leptospira infection is needed.

	CONCLUSIONS

   This is the first age- and gender-matched case control study about Leptospira seroprevalence and liver diseases. Results indicate that Leptospira exposure is associated with chronic and acute liver diseases. Results warrants for further studies on the role of Leptospira exposure in chronic liver disease.

	CONFLICT OF INTEREST

   We declare that no conflict of interest exists.

	ACKNOWLEDGEMENTS

   This study was financially supported by Juárez University of Durango State. Durango, Mexico.

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