Antioxidant activity and apoptotic induction as mechanisms of action of Withania somnifera (Ashwagandha) against a hepatocellular carcinoma cell line.

Objective To evaluate the antioxidant and apoptotic inductive effects of Withania somnifera (Ashwagandha) leaf extract against a hepatocellular carcinoma cell line. Methods After treating HepG2cells with Ashwagandha water extract (ASH-WX; 6.25 mg/ml-100 mg/ml), cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Antioxidant activities (total antioxidant, glutathione S-transferase and glutathione reductase), Fas-ligand level, tumour necrosis factor-α (TNF-α) level and caspase-3, -8, and -9 activities were measured. Molecular modelling assessed the binding-free energies of Ashwagandha in the cyclin D1 receptor. Results The MTT assay demonstrated increased cytotoxicity following treatment of HepG2 cells with ASH-WX compared with control untreated cells and theIC50was 5% (approximately 5.0 mg/ml). Antioxidant activities, Fas-ligand levels and caspase-3, -8 and -9 activities significantly increased, while TNF-α level significantly decreased following ASH-WX treatment compared with control untreated cells. Molecular docking analysis revealed a good prediction of binding between cyclin D1 and Ashwagandha. There was significant accumulation of ASH-WX-treated HepG2cells in the G0/G1 and G2/M phases compared with the control untreated cells. Conclusion Ashwagandha could be a powerful antioxidant and a promising anticancer agent against HCC.

Learning about Barriers to Care for People Living with HIV in Egypt: A Qualitative Exploratory Study.

The purpose of this study was to identify obstacles health care workers face in providing care for people living with HIV and AIDS (PLWHA). Based on these findings, health authorities can design interventions to support health care workers in providing better medical care for PLWHA. Thirty in-depth interviews were conducted with physicians and nurses in one 300-bed tertiary care public hospital in Giza, Egypt. Thematic analysis was conducted by 2 investigators. Five main themes were identified (1) fear of infection; (2) disbelief in effectiveness of infection control measures to protect against HIV; (3) misconceptions regarding medical care for PLWHA; (4) fear of secondary stigma; and (5) moral judgments toward PLWHA and negative connotations related to HIV. Interventions targeting health care workers should be multidimensional, including knowledge and skills building as well as value and attitude change. Reducing stigma among health care workers will improve access to care for PLWHA.

Seroprevalence of rubella antibodies among adult Egyptian females aged 20-30 years. Is there a need for rubella vaccination?

INTRODUCTION: Egypt adopted a comprehensive strategy to eliminate measles and rubella by conducting a catch up campaign (in 2008) targeting children and young adults in the age group 10-19 years. This study aimed to explore the seroprevalence of rubella among females aged 20 to 30 years in order to provide the Ministry of Health with information to develop future strategies for rubella supplemental immunization activities among women of childbearing age before marriage and conception. METHODS: A total of 339 females in the age group 20 to 30 were selected. The study group comprised women who attended the central laboratory of the Ministry of Health for checking up before travelling abroad as pre-travel requirements for visa application. The collected serum samples were tested for rubella-specific IgG antibodies. RESULTS: The overall prevalence of rubella antibodies in the study group was 88.2%. Around 5.0% of females, who reported that they had been vaccinated, were susceptible to rubella. Age, history of measles, mumps and rubella (MMR) vaccination and past history of rubella infection were considered factors associated with seropositivity for rubella. CONCLUSION: The seroprevalence rate of rubella antibodies among our female study group was considered low.

Genotypic characterization of Egypt enterotoxigenic Escherichia coli isolates expressing coli surface antigen 6.

INTRODUCTION: One approach to control enterotoxigenic Escherichia coli (ETEC) infections has been to develop vaccines focused on inducing protective immunity against surface expressed antigenic factors. One such factor is coli surface antigen 6 (CS6); ETEC isolates expressing CS6 may also simultaneously co-express surface antigens CS4 or CS5. However, there is little information regarding the inter-relationships of isolates expressing the CS6 antigen alone or in combination with CS4 or CS5. METHODOLOGY: A total of 62 CS6-associated ETEC isolates were evaluated for their antimicrobial susceptibility, mechanisms of resistance, toxin genes, colonization factor expression, and XbaI-pulsed-field gel electrophoretic profiles. RESULTS: We observed 46 XbaI profiles; 31 were exclusive to ETEC expressing CS6 alone and 15 among the ETEC co-expressing CS4 or CS5. Nearly half (47%) of these isolates were resistant to ampicillin, a third (37%) of the isolates were resistant to trimethoprim-sulfamethoxazole, and 24% of the isolates were tetracycline-resistant. A blaTEM gene was detected in 24 (83%) ampicillin-resistant isolates. Trimethoprim-sulfamethoxazole-resistant isolates (n = 23) carried either sulI (n = 1, 4%), sulII (n = 8, 35%) or both genes (n = 10, 43%); 4 had no detectable sul gene. CONCLUSIONS: Our results show a lack of clonality among Egypt CS6 E. coli isolates and supports the use and the further research on vaccines targeting this cell surface antigen.

Case control study to identify risk factors for acute hepatitis C virus infection in Egypt.

BACKGROUND: Identification of risk factors of acute hepatitis C virus (HCV) infection in Egypt is crucial to develop appropriate prevention strategies. METHODS: We conducted a case-control study, June 2007-September 2008, to investigate risk factors for acute HCV infection in Egypt among 86 patients and 287 age and gender matched controls identified in two infectious disease hospitals in Cairo and Alexandria. Case-patients were defined as: any patient with symptoms of acute hepatitis; lab tested positive for HCV antibodies and negative for HBsAg, HBc IgM, HAV IgM; and 7-fold increase in the upper limit of transaminase levels. Controls were selected from patients' visitors with negative viral hepatitis markers. Subjects were interviewed about previous exposures within six months, including community-acquired and health-care associated practices. RESULTS: Case-patients were more likely than controls to have received injection with a reused syringe (OR=23.1, CI 4.7-153), to have been in prison (OR=21.5, CI 2.5-479.6), to have received IV fluids in a hospital (OR=13.8, CI 5.3-37.2), to have been an IV drug user (OR=12.1, CI 4.6-33.1), to have had minimal surgical procedures (OR=9.7, CI 4.2-22.4), to have received IV fluid as an outpatient (OR=8, CI 4-16.2), or to have been admitted to hospital (OR=7.9, CI 4.2-15) within the last 6 months. Multivariate analysis indicated that unsafe health facility practices are the main risk factors associated with transmission of HCV infection in Egypt. CONCLUSION: In Egypt, focusing acute HCV prevention measures on health-care settings would have a beneficial impact.

Pandemic (H1N1) 2009 and Hajj Pilgrims who received Predeparture Vaccination, Egypt.

In Egypt, vaccination against pandemic (H1N1) 2009 virus was required of pilgrims departing for the 2009 Hajj. A survey of 551 pilgrims as they returned to Egypt found 542 (98.1% [weighted]) reported receiving the vaccine; 6 (1.0% [weighted]) were infected with influenza virus A (H3N2) but none with pandemic (H1N1) 2009 virus.

Effects of hand hygiene campaigns on incidence of laboratory-confirmed influenza and absenteeism in schoolchildren, Cairo, Egypt.

To evaluate the effectiveness of an intensive hand hygiene campaign on reducing absenteeism caused by influenza-like illness (ILI), diarrhea, conjunctivitis, and laboratory-confirmed influenza, we conducted a randomized control trial in 60 elementary schools in Cairo, Egypt. Children in the intervention schools were required to wash hands twice each day, and health messages were provided through entertainment activities. Data were collected on student absenteeism and reasons for illness. School nurses collected nasal swabs from students with ILI, which were tested by using a qualitative diagnostic test for influenza A and B. Compared with results for the control group, in the intervention group, overall absences caused by ILI, diarrhea, conjunctivitis, and laboratory-confirmed influenza were reduced by 40%, 30%, 67%, and 50%, respectively (p<0.0001 for each illness). An intensive hand hygiene campaign was effective in reducing absenteeism caused by these illnesses.

Discovery and phylogenetic analysis of novel members of class b enterotoxigenic Escherichia coli adhesive fimbriae.

Enterotoxigenic Escherichia coli (ETEC) is recognized to be a common cause of acute watery diarrhea in children from developing countries. Colonization factors (CFAs) have been identified predominantly in ETEC isolates secreting heat-stable enterotoxin (ST) or cosecreting ST with a heat-labile toxin (LT). We hypothesized that LT-only-secreting ETEC produces unique colonization factors not previously described in ST and LTST-secreting ETEC. A set of degenerate primers based on nucleotide sequence similarities between the major structural genes of CS20 (csnA), CS18 (fotA), CS12 (cswA), and porcine antigen 987 (fasA) was developed and used to screen a collection of 266 LT-secreting ETEC isolates in which no known CFA was detected. PCR-amplified products of different molecular masses were obtained from 49 (18.4%) isolates. Nucleotide sequence analysis of the PCR amplicons followed by GenBank nucleotide BLASTn analysis revealed five novel DNA sequences; translated amino acid BLASTx analysis confirmed sequence similarity to class 1b major structural proteins encoded by csnA, fotA, and fasA. Strains expressing the novel CFAs were phylotyped and analyzed using multilocus sequence typing (MLST; Achtman scheme), and the types detected were compared to those of a collection of archived global E. coli strains. In conclusion, application of the degenerate primer sets to ETEC isolates from surveillance studies increased the total number of ETEC isolates with detectable CFAs by almost 20%. Additionally, MLST analysis suggests that for many CFAs, there may be a requirement for certain genetic backgrounds to acquire and maintain plasmids carrying genes encoding CFAs.

Acute respiratory illness and health-seeking behavior in Egyptian villages: enhancing pandemic preparedness by understanding local realities.

The emergence of influenza viruses has raised awareness worldwide about influenza pandemic risks. Pandemic preparedness emphasizes development of risk communication and surveillance systems. The objective was to explore community classification of Acute Respiratory Illness (ARI) and health-seeking behaviors. Twenty in-depth interviews and 18 focus group discussions were conducted with caretakers in Egypt. The interviews were recorded, transcribed, and analyzed using the thematic analysis. ARI terminology contained few terms, usage of them was complex, and microorganism not part of illness explanation. Severe ARI was associated with social stigma. Homecare included extensive use of medications. In mild and severe ARI, health-seeking determinants varied. Classification of ARI parallels World Health Organization case definitions for ARI, facilitating risk communication. Homecare practices are social norms that can be expanded to include messages on ARI. Risk communication strategies and surveillance systems need to consider socio-cultural understanding of ARI.

Studies on West Nile virus infection in Egypt.

We conducted a prospective cohort study to determine prevalence and incidence of West Nile virus (WNV) in Egypt. Cohorts were established in Upper (UE), Middle (ME), and Lower (LE) Egypt. Additionally, a cross-sectional serosurvey was performed in the North (NS) and South (SS) Sinai. Cohorts were bled initially and 1 year later. Sera were tested for WNV-IgG by ELISA and positive sera were confirmed by plaque reduction neutralization test (PRNT). Sentinel chicken flocks placed in the above sites were bled monthly for virus isolation and serology. Mosquitoes were collected monthly from the above sites and tested for WNV. Human seroprevalence rates were 35%, 27%, 14%, 1% and 7% in UE, ME, LE, NS and SS, respectively. Seroconversion rates were 18%, 17% and 7% in UE, ME and LE, respectively; 49% of the seroconverters reported undiagnosed febrile illness. Sentinel chickens showed seroconversion in all study sites. WNV was isolated from both sentinel chickens and mosquitoes in cohort sites. This study demonstrates that WNV was actively circulating during the study period in different areas in Egypt and causing febrile illness in a considerable proportion of individuals in the study sites.

Zoonotic transmission of avian influenza virus (H5N1), Egypt, 2006-2009.

During March 2006-March 2009, a total of 6,355 suspected cases of avian influenza (H5N1) were reported to the Ministry of Health in Egypt. Sixty-three (1%) patients had confirmed infections; 24 (38%) died. Risk factors for death included female sex, age > or = 15 years, and receiving the first dose of oseltamivir >2 days after illness onset. All but 2 case-patients reported exposure to domestic poultry probably infected with avian influenza virus (H5N1). No cases of human-to-human transmission were found. Greatest risks for infection and death were reported among women > or = 15 years of age, who accounted for 38% of infections and 83% of deaths. The lower case-fatality rate in Egypt could be caused by a less virulent virus clade. However, the lower mortality rate seems to be caused by the large number of infected children who were identified early, received prompt treatment, and had less severe clinical disease.

HIV prevalence and risk behaviors of male injection drug users in Cairo, Egypt.

OBJECTIVE: To measure HIV prevalence and related risk behaviors among male injection drug users (IDUs) in Cairo, Egypt in the context of the first survey wave of an integrated biological and behavioral surveillance system. DESIGN AND METHODS: Given the hidden nature of injection drug use, we used the peer-referral methodology of respondent-driven sampling in a cross-sectional study to recruit a sample of male IDUs in Cairo between May and August 2006. Behavioral data were collected through face-to-face interviews and serum was obtained for HIV antibody testing. Population estimates were produced using respondent-driven sampling Analysis Tool. RESULTS: The study enrolled 413 male IDUs. The population estimated HIV prevalence was 0.6% (95% confidence interval 0.1-1.8). More than half (53.0%) reported injecting drugs with used needles or syringes and nearly one-third (32.4%) shared their used needle or syringe with one or more persons in the preceding month. Overall, 70.5% had sex in the preceding year, of whom 9.4% reported sex with male partners and 13.2% reported sex with commercial sex workers in the preceding 12 months. Ever use of a condom during sex was low with all partner types and only 5.8% ever had an HIV test. CONCLUSION: This first survey wave of integrated biological and behavioral surveillance system in Egypt to track the HIV epidemic among male IDUs found relatively low prevalence of infection compared to global estimates, though the figure is many times higher than the general population. In addition, risky injection practices and unprotected sex were high with sexual networks including men who have sex with men, female sex workers, wives, and other regular and casual partners. The respondent-driven sampling method was effective in recruiting male IDUs and the results are being used to inform surveillance and prevention programs.

Antigenic and genetic diversity of highly pathogenic avian influenza A (H5N1) viruses isolated in Egypt.

Highly pathogenic avian influenza A virus (H5N1) has diverged antigenically and genetically since its initial detection in Asia in 1997. Viruses belonging to clade 2.2 in particular have been reported in numerous countries with the majority occurring in Egypt. Previous reports identified antigenic similarities between viruses belonging to clade 2.2. However, poultry and human viruses isolated in northern Egypt during 2007 and 2008 were found to be antigenically distinct from other clade 2.2 viruses from this country. Genetic analysis of the hemagglutinin revealed a high degree of nucleotide and amino acid divergence. The antigenic changes in Egyptian viruses isolated during 2007-08 necessitated that two of these strains be considered as potential H5N1 pre-pandemic vaccine candidates.

Cost-benefit analysis of a rotavirus immunization program in the Arab Republic of Egypt.

BACKGROUND: The availability of rotavirus vaccines makes the implementation of a national immunization program an important decision requiring economic considerations. METHODS: A cost-benefit analysis of a national rotavirus immunization program in Egypt, from the perspective of the Ministry of Health and Population, and a cost-effectiveness analysis, from a societal perspective, were conducted. RESULTS: For a birth cohort of 1.9 million children, a vaccination program was estimated to prevent 1,140,496 episodes of diarrhea, 438,395 outpatient visits, and 47,508 hospitalizations and to save 2873 lives, resulting in direct Ministry of Health and Population medical savings of $2,481,792 (14,369,578 Egyptian pounds [LE]). On the basis of a $9.18 (53 LE) single-dose cost, rotavirus vaccine introduction would cost the Ministry of Health and Population $34,203,445.87 (198,037,951.56 LE) in health expenditures. This equates to an incremental cost of $30.22 (174.95 LE) per infection prevented. Vaccination would prevent the loss of 94,993 disability-adjusted life-years, resulting in an incremental cost-effectiveness ratio of $363 per disability-adjusted life-year. CONCLUSIONS: The introduction of rotavirus vaccine to the national immunization program was not found to be cost saving based strictly from the Ministry of Health and Population perspective; however, the potential benefits of long-term health and economic gains from reduced mortality and morbidity, decreased direct costs of care for families, and indirect societal costs should be considered in such decisions.

Shedding of vaccine viruses with increased antigenic and genetic divergence after vaccination of newborns with monovalent type 1 oral poliovirus vaccine.

For the final stages in the eradication of poliovirus type 1 (P1), the World Health Organization advocates the selective use of monovalent type 1 oral poliovirus vaccine (mOPV1). To compare the immunogenicity of mOPV1 with that of trivalent OPV (tOPV) in infants, a study was performed in Egypt in 2005. Newborns were vaccinated with mOPV1 or tOPV immediately after birth and were challenged with mOPV1 after 1 month. Vaccination with mOPV1 at birth resulted in significantly higher seroconversion against P1 viruses and lower excretion of P1 viruses than vaccination with tOPV. Intratypic differentiation of the viruses shed by the newborns revealed the presence of remarkably high numbers of antigenically divergent (AD) P1 isolates, especially in the mOPV1 study group. The majority of these AD P1 isolates (71%) were mOPV1 challenge derived and were shed by newborns who did not seroconvert to P1 after the birth dose. Genetic characterization of the viruses revealed that amino acid 60 of the VP3 region was mutated in all AD P1 isolates. Isolates with substitution of residue 99 of the VP1 region had significantly higher numbers of nonsynonymous mutations in the VP1 region than isolates without this substitution and were preferentially shed in the mOPV1 study group. The widespread use of mOPV1 has proven to be a powerful tool for fighting poliovirus circulation in the remaining areas of endemicity. This study provides another justification for the need to achieve high vaccination coverage in order to prevent the circulation of AD strains.

Community transmission of hepatitis B virus in Egypt: results from a case-control study in Greater Cairo.

BACKGROUND: To identify current risk factors for hepatitis B virus (HBV) transmission in Greater Cairo. METHODS: A 1:1 matched case-control study was conducted in two 'fever' hospitals in Cairo. Acute hepatitis B cases were patients with acute hepatitis, positive HBs antigen, and high anti-HBc IgM titres. Control subjects were acute hepatitis A patients (positive anti-HAV IgM) or relatives of patients diagnosed with acute hepatitis C, identified at the same hospitals, with no past HBV infection (negative anti-HBc) and matched to cases on the same age and sex. Conditional logistic regression was used to identify factors associated with acute hepatitis B. RESULTS: Between April 2002 and June 2006, 233 cases and 233 controls were recruited to the study. In multivariate analysis, factors associated with an increased HBV risk in males were illiteracy [odds ratio (OR) = 6.1, 95% confidence interval (CI) = 2.8-13.1], shaving at barbers (OR = 2.1, 95% CI = 1.1-3.9) and injecting drug use (IDU) (OR = 3.4, 95% CI = 1.0-11.4). In females, factors associated with an increased HBV risk were illiteracy (OR = 2.2, 95% CI = 1.0-5.0), recent (<1 year) marriage (OR = 42.0, 95% CI = 3.8-463.9 compared with single women) and giving birth (OR = 3.7, 95% CI = 1.0-13.9). CONCLUSION: In this study, HBV transmission took place primarily in the community, whether as a result of recent marriage (presumably first sexual intercourse), shaving at barbershops or IDU, and was more common among illiterates. Health promotion campaigns should be carried out to increase awareness about community transmission of HBV. In addition to routine immunization for infants and other populations, premarital screening might be useful to identify at-risk spouses in order to propose targeted immunization.

Monovalent type 1 oral poliovirus vaccine in newborns.

BACKGROUND: In 1988, the World Health Assembly resolved to eradicate poliomyelitis. Although substantial progress toward this goal has been made, eradication remains elusive. In 2004, the World Health Organization called for the development of a potentially more immunogenic monovalent type 1 oral poliovirus vaccine. METHODS: We conducted a trial in Egypt to compare the immunogenicity of a newly licensed monovalent type 1 oral poliovirus vaccine with that of a trivalent oral poliovirus vaccine. Subjects were randomly assigned to receive one dose of monovalent type 1 oral poliovirus vaccine or trivalent oral poliovirus vaccine at birth. Thirty days after birth, a single challenge dose of monovalent type 1 oral poliovirus vaccine was administered in all subjects. Shedding of serotype 1 poliovirus was assessed through day 60. RESULTS: A total of 530 subjects were enrolled, and 421 fulfilled the study requirements. Thirty days after the study vaccines were administered, the rate of seroconversion to type 1 poliovirus was 55.4% in the monovalent-vaccine group, as compared with 32.1% in the trivalent-vaccine group (P<0.001). Among those with a high reciprocal titer of maternally derived antibodies against type 1 poliovirus (>64), 46.0% of the subjects in the monovalent-vaccine group underwent seroconversion, as compared with 21.3% in the trivalent-vaccine group (P<0.001). Seven days after administration of the challenge dose of monovalent type 1 vaccine, a significantly lower proportion of subjects in the monovalent-vaccine group than in the trivalent-vaccine group excreted type 1 poliovirus (25.9% vs. 41.5%, P=0.001). None of the serious adverse events reported were attributed to the trial interventions. CONCLUSIONS: When given at birth, monovalent type 1 oral poliovirus vaccine is superior to trivalent oral poliovirus vaccine in inducing humoral antibodies against type 1 poliovirus, overcoming high preexisting levels of maternally derived antibodies, and increasing the resistance to excretion of type 1 poliovirus after administration of a challenge dose. (Current Controlled Trials number, ISRCTN76316509.)

Survey of poliovirus antibodies during the final stage of polio eradication in Egypt.

BACKGROUND: Egypt provides ideal conditions for poliovirus (PV) transmission (high population density, high contact rates and low sanitation and hygiene in some areas). Despite excellent program performance, wild poliovirus type 1 (PV1) continue to circulate in 2004. To investigate potential causes for the persistence, we conducted a serological study. METHODS: Seroprevalence surveys were conducted in "polio-endemic" regions (Greater Cairo and Upper Egypt) and in one control region (Lower Egypt) in December 2004. Sera collected from infants aged 6-11 months were tested for antibodies to poliovirus by neutralization assay. RESULTS: A total of 973 subjects were tested. Seroprevalence to PV type 1 (PV1), PV type 2 (PV2) and PV type 3 (PV3) was 99, 99 and 91%, respectively. Significant variation in PV3 seroprevalence was found (range: 76-100%). Region, density, maternal education, socioeconomic status (SES), stunting and diarrhea were significant risk factors for lower seroprevalence in the univariate analysis. CONCLUSIONS: Our study suggested that uniformly high immunity levels (>96%) were required to interrupt PV1 transmission in the last remaining reservoirs (last PV1 was isolated in mid-January 2005 in Egypt). It further suggests substantial regional differences in OPV immunogenicity, with rural areas and low SES achieving the lowest seroprevalence to PV3.

Brucellosis as a cause of acute febrile illness in Egypt.

To develop better estimates of brucellosis incidence, we conducted population-based surveillance for acute febrile illness (AFI) in Fayoum governorate (population 2347249), Egypt during two summer periods (2002 and 2003). All hospitals and a representative sample of community healthcare providers were included. AFI patients without obvious etiology were tested for brucellosis by culture and serology. Incidence estimates were calculated adjusting for sampling methodology and study period. Of 4490 AFI patients enrolled, 321 (7%) met the brucellosis case definition. The estimated annual incidence of brucellosis per 100000 population was 64 and 70 in 2002 and 2003, respectively. The median age of brucellosis patients was 26 years and 70% were male; 53% were initially diagnosed as typhoid fever. Close contact with animals and consumption of unpasteurized milk products were associated with brucellosis. The high incidence of brucellosis in Fayoum highlights its public health importance, and the need to implement prevention strategies in humans and animals.

Retrospective serosurvey of leptospirosis among patients with acute febrile illness and hepatitis in Egypt.

The epidemiologic status of leptospirosis in Egypt has not been well defined because of difficulties in disease diagnosis. A retrospective study was conducted to detect leptospiral antibodies among undiagnosed acute febrile illness (AFI) and hepatitis cases. Approximately 16% of both AFI (141/886) and acute hepatitis (63/392) cases showed seroreactivity to Leptospira IgM by ELISA and microscopic agglutination test (MAT). Canicola, Djasiman, Grippotyphosa, Pyrogenes, Icterohemorrhagiae, and Pomona were the most commonly reactive serovars among patients with AFI. Djasiman, Grippotyphosa and Icterohemorrhagiae were the most reactive among patients with acute hepatitis. This study represents the first systematic report of Leptospira associated with patients with AFI and hepatitis in Egypt. Physicians need to have increased awareness about the importance of leptospirosis in the differential diagnosis of AFI and acute hepatitis in Egypt. In addition, laboratory capacity should be developed at fever hospitals to diagnose leptospirosis.