Preliminary antigenic characterisation of an adult worm vomit preparation of Fasciola hepatica by infected human sera

Fascioliasis is an emerging/re-emerging vector-borne disease with the widest known distribution. Approximately 17 million people are infected around the world, being the Andean region the most affected area. There is an important necessity to develop sensitive and specific diagnostic tools to treat patients early and to avoid complications. In this paper we evaluated the immune response of infected humans against two antigenic preparations: the total soluble extract (FhTSE) and the adult worm vomit (FhAWV) in order to identify antigenic fractions specific for Fasciola hepatica. Both preparations were processed by SDS-PAGE and Western blot with human sera with fascioliasis (F), other parasitosis and healthy individuals. In the immunoblot of FhTSE, sera F recognised 16 bands with MW between eight and 110 kDa, from which those of 8, 9, 10, 38, 45 and 57 kDa were specific. In the preparation FhAWV, sera F recognised nine bands with MW from eight to 85 kDa, from which those of 8, 12, 15 and 24 kDa were specific. Some bands of cross-reaction were evident with sera from patients with other parasitoses, more frequent with the FhTSE. Bands within the MW mentioned, particularly that of eight kDa, have been shown to be specific by others, and deserve additional characterisation for their potential use in immunodiagnosis.

T CD4+ cells count among patients co-infected with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1): high prevalence of tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM)

INTRODUCTION: HIV positive patients co-infected with HTLV-1 may have an increase in their T CD4+ cell counts, thus rendering this parameter useless as an AIDS-defining event. OBJECTIVE: To study the effects induced by the co-infection of HIV-1 and HTLV-1 upon CD4+ cells. MATERIAL AND METHODS: Since 1997, our group has been following a cohort of HTLV-1-infected patients, in order to study the interaction of HTLV-1 with HIV and/or with hepatitis C virus (HCV), as well as HTLV-1-only infected asymptomatic carriers and those with tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). One hundred and fifty HTLV-1-infected subjects have been referred to our clinic at the Institute of Infectious Diseases "Emílio Ribas", São Paulo. Twenty-seven of them were also infected with HIV-1 and HTLV-1-infection using two ELISAs and confirmed and typed by Western Blot (WB) or polymerase chain reaction (PCR). All subjects were evaluated by two neurologists, blinded to the patient's HTLV status, and the TSP/HAM diagnostic was based on the World Health Organization (WHO) classification. AIDS-defining events were in accordance with the Centers for Disease Control (CDC) classification of 1988. The first T CD4+ cells count available before starting anti-retroviral therapy are shown compared to the HIV-1-infected subjects at the moment of AIDS defining event. RESULTS: A total of 27 HIV-1/HTLV-1 co-infected subjects were identified in this cohort; 15 already had AIDS and 12 remained free of AIDS. The median of T CD4+ cell counts was 189 (98-688) cells/mm³ and 89 (53-196) cells/mm³ for co-infected subjects who had an AIDS-defining event, and HIV-only infected individuals, respectively (p = 0.036). Eight of 27 co-infected subjects (30%) were diagnosed as having a TSP/HAM simile diagnosis, and three of them had opportunistic infections but high T CD4+ cell counts at the time of their AIDS- defining event. DISCUSSION: Our results indicate that higher T CD4+ cells count among HIV-1/HTLV-1-coinfected subjects was found in 12% of the patients who presented an AIDS-defining event. These subjects also showed a TSP/HAM simile picture when it was the first manifestation of disease; this incidence is 20 times higher than that for HTLV-1-only infected subjects in endemic areas.

Expression of circulating leucocytes before, during and after myiasis by Dermatobia hominis in experimentally infected rats

Expression of circulating white blood cells was investigated in rats (Rattus norvegicus) experimentally infected with larvae of Dermatobia hominis, the human bot fly. Leucocytes were counted prior to infection (control group) as well as at 6, 10, 15, 20 and 28 days post-infection (dpi) and at 7, 15, 30 and 60 days post-larval emergence (dple). Total leucocyte numbers did not differ markedly among the groups. Significant differences were registered when values from control and animals harboring each larval stage of D. hominis were compared; with crescent rank: L1-, L2-, control and L3-infected groups. Leucocyte numbers were significantly higher in the control, 15, 20 or 28 dpi groups than in the 6 dpi animals. Higher counts were observed in control, L2- or L3-infected rats than L1-infected animals. Neutrophils, eosinophils and both large and small lymphocytes were also counted and analyzed. Basophils and monocytes were insufficient in number to permit statistical studies. These results stimulate the continuity of the studies about the host-parasite relationship in the dermatobiosis.

Prevalence of intestinal microsporidiosis in Human Immunodeficiency Virus-infected patients with diarrhea in major United States cities

To determine the prevalence of intestinal microsporidiosis in HIV-infected patients, we performed a prospective study of HIV-infected patients with diarrheal illnesses in three US hospitals and examined an observational database of HIV-infected patients in 10 US cities. Among 737 specimens from the three hospitals, results were positive for 11 (prevalence 1.5%); seven (64%) acquired HIV through male-to-male sexual contact, two (18%) through male-to-male sexual contact and injection drug use, and one (9%) through heterosexual contact; one (9%) had an undetermined mode of transmission. Median CD4 count within six months of diagnosis of microsporidiosis was 33 cells/µL (range 3 to 319 cells/µL). For the national observational database (n = 24,098), the overall prevalence of microsporidiosis was 0.16%. Prevalence of microsporidiosis among HIV-infected patients with diarrheal disease is low, and microsporidiosis is most often diagnosed in patients with very low CD4+ cell counts. Testing for microsporidia appears to be indicated, especially for patients with very low CD4+ cell counts.

A comparison of the immune parameters of dogs infected with visceral leishmaniasis using Western blot and neutralization techniques

The Western blot technique was used to demonstrate the presence of antibodies in the blood of dogs that presented canine visceral leishmaniasis. This technique was used against some specific molecules present in the lysate of the promastigote form of Leshmania chagasi.Through the association of the results of the Western blot technique with the morphological alterations seen as a result of the serum neutralization technique performed in McCoy cells (which mimetizes the macrophage) it was possible to observe the role of some molecules of great relevance in determining the disease in symptomatic dogs as well as that of some other molecules associated with asymptomatic infected dogs that may become transmitters as well as differentiating them as asymptomatic resistant dogs. In the sera analyses carried out during the immunobloting a variation of 9 to 27 immunoreacting bands was observed, which were then compared using Dice's similarity coefficient. In the dendrogram constructed on the basis of the coefficient, 50% similarity was observed among the total number of reagent bands with the promastigote lysate, thus creating five groups. The main difference observed related to the clinical condition of the dogs: symptomatic and asymptomatic dogs were found in separate groups. The asymptomatic group of dogs was distributed in two different places in the dendrogram because they presented two different behavior patterns regarding the cellular morphology in the serum neutralization reaction: the presence or absence of cellular lysis. According to this analysis it is possible to evaluate the immune status and associate it with specific markers observed in the reaction found in the Western blot strips.

Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

Quantitative toxoplasma gondii oocyst detection by a modified Kato Katz test using Kinyoun staining (KKK) in ME49 strain experimentally infected cats

We detected Toxoplasma gondii oocysts in feces of experimentally infected cats, using a Kato Katz approach with subsequent Kinyoun staining. Animals serologically negative to T. gondii were infected orally with 5x10² mice brain cysts of ME49 strain. Feces were collected daily from the 3rd to the 30th day after challenge. Oocysts were detected by qualitative sugar flotation and the quantitative modified Kato Katz stained by Kinyoun (KKK). In the experimentally infected cats, oocysts were detected from the 7th to 15th day through sugar flotation technique, but oocysts were found in KKK from the 6th to 16th day, being sensitive for a larger period, with permanent documentation. The peak of oocysts excretion occurred between the 8th to 11th days after challenge, before any serological positive result. KKK could be used in the screening and quantification of oocysts excretion in feces of suspected animals, with reduced handling of infective material, decreasing the possibility of environmental and operator contamination.

Clinical and histological characteristics of HIV and hepatitis C virus-co-infected patients in Brazil: a case series study

Hepatitis C virus (HCV) is an important factor contributing to morbidity and mortality in patients co-infected with HIV and HCV. In addition, liver biopsy is an important tool in the clinical management of these patients. Although liver biopsy is controversial, it is recommended for all patients. Data regarding the clinical and histological characteristics of these patients are scarce not only in Brazil but in Latin America as a whole. With the goal of better understanding these characteristics and the benefit of liver biopsy indications in this disease setting, data collected from 234 patients followed from 1996 to 2004 at Casa da AIDS, São Paulo, were analyzed. The following variables were extracted from the patients' medical files at the time of liver biopsy: sex, age, hepatitis C infection risk factors, hepatitis C infection duration, ALT levels, CD4+ T cell counts, history of alcohol abuse, history of antiretroviral therapy, HCV genotype, and liver histological alterations. CONCLUSIONS: 1 - Hepatitis C virus 1 and 3 were the most frequently identified genotypes and were diagnosed in 72% and 25.5% of cases respectively; 2 - Structural liver alterations were found to be mild or absent in 48.2% (113/234) of the analyzed patients; 3 - Fifty-three patients (23%) had normal ALT levels and 4 - Significant liver architectural changes (F2-F3) were evident in 22.5% of the patients with normal ALT levels.

Etiological agents of diarrhea in patients infected by the human immunodeficiency virus-1: a review

Despite the importance of understanding the epidemiology of agents responsible for infectious diarrhea in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) population, the number of articles about this subject is relatively few. The current article summarizes published data on bacterial, fungal, viral and parasitic enteropathogens in the HIV/AIDS seropositive subjects in different countries, regions and localities. In general, there is a great difference in the frequencies of etiological agents due to factors which include immune status, geographical location, climate and socioeconomic conditions. It is important to stress that a great prevalence of infection by emergent agents has been reported in the more advanced stages of AIDS. Therefore, to establish specific treatment depends directly on knowledge of these agents and risk factors associated to their distribution. Moreover, the colonization by potential pathogenic agents verified in these individuals is high thus implicating that they act as carriers. Finally, public health measures of control and prevention must take into consideration the regional previously identified enteropathogens, especially in areas where HIV prevalence is high.

Muscular strength decrease in Rattus norvegicus experimentally infected by Toxocara canis

The muscular strength of experimental infected Rattus norvegicus with 3rd. stage Toxocara canis larvae was investigated. Fifty Wistar rats, divided in three groups (G1 - 20 rats infected by 300 eggs of T. canis; G2 - 20 rats infected by 2,000 eggs of T. canis and G3 - 10 rats without infection) had been used. Ten and 30 days after infection the muscular strength in the fore-feet of the rats was checked; at the same time, the body weight was determined. No significative differences in the body weight were noted among the infected and control rats in both occasions. Otherwise, an impairment on the muscular strength was observed in rats infected with T. canis 30 days after inoculation.

Cytokine and nitric oxide production by mouse macrophages infected with brazilian flaviviruses

The Flaviviridae family, Flavivirus genus includes viruses that are transmitted to vertebrates by infected mosquitoes or ticks. The genus Flavivirus includes a variety of viruses that cause diseases such as acute febrile illness, encephalitis, and hemorrhagic fever. Flaviviruses primarily infect blood monocytes and tissue macrophages, which have been shown to be permissive, supporting viral replication and serving as virus reservoirs. On the other hand, these cells may have an important antiviral activity related to modulation by cytokine production and by the capacity of these cells to synthesize reactive free radicals such as nitric oxide (NO) which can have a microbicidal effect. The present study was performed in order to determine the production of cytokines interleukin-1beta (IL-1β), tumor necrosis factor -alpha (TNF-α), transforming growth factor- beta (TGF-β) and interferon -alpha (IFN-α) and NO by macrophages infected with one of four Brazilian flaviviruses, Bussuquara virus (BUSV), Yellow Fever virus (YFV), Rocio virus (ROCV) and Encephalitis Saint Louis virus (SLEV), and to verify the possible antiviral effect of NO during macrophage infection with ROCV. Moreover, we asked if the different viruses were able to regulate bacterial lipopolysaccharide (LPS) induced cytokine production. Our results showed that YFV and SLEV reduced the production of IL-1β and TGF-β by LPS-stimulated macrophages, while ROCV only diminished LPS-stimulated TGF-β synthesis. On the other hand, BUSV more likely favored an enhancement of the LPS-induced production of IL-1β by macrophages. Additionally, while most of the viruses stimulated the production of IFN-α, none of them altered the production of TNF-α by murine macrophages. Interestingly, all viruses induced synthesis of NO that was not correlated with antiviral activity for ROCV.

HTLV- I infection is not associated with a higher risk of death in peruvian HIV-infected patients

Limited and contradictory information exists regarding the prognosis of HIV/HTLV-I co-infection. Our goal was to estimate the effect of HTLV-I infection on mortality in HIV-infected patients at a HIV reference center in Peru. We studied a retrospective cohort of HIV-infected patients, who were exposed or unexposed to HTLV-I. Exposed patients were Western Blot (WB) positive for both retroviruses. Unexposed patients were WB positive for HIV, and had least one negative EIA for HTLV-I. These were selected among patients who entered our Program immediately before and after each exposed patient, between January 1990 and June 2004. Survival time was considered between the diagnosis of exposure to HTLV-I and death or censoring. Confounding variables were age, gender, baseline HIV clinical stage, baseline CD4+ T cell count, and antiretroviral therapy. We studied 50 exposed, and 100 unexposed patients. Exposed patients had a shorter survival compared to unexposed patients [median survival: 47 months (95% CI: 17-77) vs. 85 months (95% CI: 70-100), unadjusted p = 0.06]. Exposed patients had a higher rate of mortality compared to unexposed patients (HIV/HTLV-I (24/50 [48%]) vs. HIV only (37/100 [37%]), univariable p = 0.2]. HTLV-I exposure was not associated to a higher risk of death in the adjusted analysis: HR: 1.2 (0.4-3.5). AIDS clinical stage and lack of antiretroviral therapy were associated to a higher risk of dying. In conclusions, HTLV-I infection was not associated with a higher risk of death in Peruvian HIV-infected patients. Advanced HIV infection and lack of antiretroviral therapy may explain the excess of mortality in this population.

Distribution of hepatitis B virus genotypes and viral load levels in Brazilian chronically infected patients in São Paulo city

The objective of the present study was to evaluate the serum viral load in chronically infected Hepatitis B virus (HBV) patients and to investigate the distribution of HBV genotypes in São Paulo city. Quantitative HBV-DNA assays and HBV genotyping have gained importance for predicting HBV disease progression, have been employed for assessing infectivity, for treatment monitoring and for detecting the emergence of drug resistance. Twenty-nine Brazilian patients with suspected chronic hepatitis B were studied, using real time PCR for viral load determination and direct DNA sequencing for the genotyping. The serology revealed chronic HBV infection in 22 samples. The HBV-DNA was positive in 68% samples (15/22). The phylogenetic analysis disclosed that eleven patients were infected with HBV genotype A, two with genotype F and two with genotype D. Thus, the genotype A was the most prevalent in our study.

Molecular characterization of Cryptosporidium spp. from HIV infected patients from an urban area of Brazil

Cryptosporidium spp. are important cause of enteric disease in humans, but may also infect animals. This study describes the relative frequency of several Cryptosporidium species found in human specimens from HIV infected patients in the São Paulo municipality obtained from January to July 2007. Sequence analysis of the products of nested-PCR based on small subunit rRNA and Cryptosporidium oocyst wall protein coding genes revealed 17 (63.0%) isolates of C. hominis, four (14.8%) C. parvum, five (18.5%) C. felis and one (3.7%) C. canis. These findings suggest that, in urban environments of Brazil, the cat adapted C. felis may play a potential role in the zoonotic transmission of cryptosporidiosis whereas the anthroponotic transmission of cryptosporidiosis caused by C. hominis seems to predominate.