Tick-borne encephalitis in Bulgaria, 2009 to 2012.

For the last 60 years, only a few cases of tick-borne encephalitis (TBE) have been detected in Bulgaria. Considering the remarkable increase in TBE morbidity in Europe over the past two decades, we conducted a study of TBE among patients with acute viral meningitis who were hospitalised in Bulgaria during 2009 to 2012. A total of 86 patients with viral meningitis of unknown aetiology during this period were tested. Acute TBE was confirmed in three of these patients. The last TBE case was detected in October 2012; the other two were diagnosed in 2009. To the best of our knowledge, these three patients are the first confirmed TBE cases reported in Bulgaria. The risk of TBE is underestimated in Bulgaria due to the low awareness of medical doctors.

Serological evidence of arboviral infection and self-reported febrile illness among U.S. troops deployed to Al Asad, Iraq.

Understanding the epidemiology of current health threats to deployed U.S. troops is important for medical assessment and planning. As part of a 2004 study among U.S. military personnel deployed to Al Asad Air Base, in the western Anbar Province of Iraq, over 500 subjects were enrolled, provided a blood specimen, and completed a questionnaire regarding history of febrile illness during this deployment (average approximately 4 months in country). This mid-deployment serum was compared to pre-deployment samples (collected approximately 3 months prior to deployment) and evaluated for seroconversion to a select panel of regional arboviral pathogens. At least one episode of febrile illness was reported in 84/504 (17%) of the troops surveyed. Seroconversion was documented in nine (2%) of deployed forces tested, with no association to febrile illness. Self-reported febrile illness was uncommon although often debilitating, and the risk of illness due to arbovirus infections was relatively low.

Prevalence of hantavirus antibody in patients with chronic renal disease in Egypt.

In Egypt, the etiology of chronic renal failure (CRF) is not well defined. A hospital-based case-control study was initiated in February 1998, to determine whether hantavirus infection is involved in chronic renal disease (CRD) in Egypt. The study enrolled 350 study patients with a history of CRF and 695 matched controls with CRD due to renal calculus or renal cancer, but with normal renal functions. Sera from cases and controls were tested for anti-hantavirus IgG using ELISA with a cell-lysate antigen from Hantaan virus prototype strain 76-118. A demographic questionnaire was completed for each study participant. Five of the 350 cases (1.4%), and seven of the 695 controls (1.0%) were antibody-positive to hantavirus, with a titer > or =1:400. The difference in antibody prevalence between the study cases and the control cases was not statistically significant (P = 0.48). All antibody-positive study cases and controls had been exposed to rodents. Data indicated that in Egypt, hantavirus seroprevalence in CRD patients is low, and hantavirus infections do not appear to be a significant cause of CRF.

Inactivation of microbes using ultrasound: a review.

Alternative methods for pasteurization and sterilization are gaining importance, due to increased consumer demand for new methods of food processing that have a reduced impact on nutritional content and overall food quality. Ultrasound processing or sonication is one of the alternative technologies that has shown promise in the food industry. Sonication alone is not very effective in killing bacteria in food; however, the use of ultrasound coupled with pressure and/or heat is promising. Thermosonic (heat plus sonication), manosonic (pressure plus sonication), and manothermosonic (heat and pressure plus sonication) treatments are likely the best methods to inactivate microbes, as they are more energy-efficient and effective in killing microorganisms. Ultrasonic processing is still in its infancy and requires a great deal of future research in order to develop the technology on an industrial scale, and to more fully elucidate the effect of ultrasound on the properties of foods.

Seroprevalence of West Nile, Rift Valley, and sandfly arboviruses in Hashimiah, Jordan.

We conducted a serosurvey among patients of a health center in Hashimiah, a Jordanian town of 30,000 inhabitants located near a wastewater treatment plant and its effluent channel. Serum samples from 261 patients >/=5 years of age were assessed for immunoglobulin G (IgG) and IgM antibodies against West Nile, sandfly Sicilian, sandfly Naples, and Rift Valley viruses; the seroprevalence of IgG antibodies was 8%, 47%, 30%, and 0%, respectively. Female participants were more likely to have been infected than male. Persons living within 2 km of the treatment plant were more likely to have been infected with West Nile (p=0.016) and sandfly Sicilian (p=0.010) viruses. Raising domestic animals within the house was a risk factor for sandfly Sicilian (p=0.003) but not for sandfly Naples virus (p=0.148). All serum samples were negative for IgM antibodies against the tested viruses. Our study is the first documentation of West Nile and sandfly viruses in Jordan and calls attention to the possible health hazards of living close to wastewater treatment plants and their effluent channels.

Seroprevalence of Helicobacter pylori among Egyptian newborns and their mothers: a preliminary report.

Helicobacter pylori is one of the most common human bacterial infections in the world and children in the developing countries acquire H. pylori infection early in life. We prospectively evaluated the prevalence of serum antibodies to H. pylori in a cohort of pregnant women and their offspring. Mothers' sera were collected during the third trimester of pregnancy and sera from their offspring were collected when they were 7-9 months and 18 months of age. Pylori-Stat, a commercially available ELISA kit, was used to detect antibodies to H. pylori in the serum of the subjects tested. Sera from 169 mothers were available for testing and 88% of these samples were positive for anti-H. pylori IgG. Of the 169 children tested, 13% of the infants 7-9 months of age and 25% of the children 18 months of age had serologic evidence of H. pylori infection. These data indicate that infection with H. pylori is common in Egypt and acquisition of infection occurs at a very young age.

Detection of Fasciola-specific excretory/ secretory (E/S) protein fraction band (49.5 kDa) and its utilization in diagnosis of early fascioliasis using different diagnostic techniques.

The objective of the present work is to evaluate Fasciola E/S antigens for diagnosis of early fascioliasis utilizing different diagnostic techniques. Using enzyme-linked immunoelectro-transfer blot (EITB), Fasciola-specific E/ S protein fraction band (49.5 kDa) was determined and electroeluted. The mono-specific antibodies against this specific fraction band were prepared by immunizing pathogen-free rabbit. Assessment of the prepared mono-specific antibodies in diagnosis of human fascioliasis was performed through the detection of E/S copro-antigens by enzyme-linked immunosorbent assay (ELISA) in stool eluates obtained from patients with confirmed fascioliasis, other parasites as well as from other healthy individuals. Serum samples were collected and tested to detect serum antibodies against Fasciola E/S antigen using EITB and counter immunoelectrophoresis (CIEP). Analysis of Fasciola adult worm E/S products by SDS/PAGE revealed a number of bands, the molecular weight (MW) of which ranged from 14-200 kDa; with three major bands (27.5, 32.5 and 55 kDa). Fasciola EIS 49.5 kDa protein fraction proved to be specific to F. gigantica. Cross reaction with S. mansoni was observed at higher MW (110-120 kDa). The sensitivity, specificity and diagnostic accuracy of EITB were 45.2%, 100% and 70.7%, respectively, while those of CIEP were 38.7%, 100% and 67.2%, respectively. ELISA technique using mono-specific anti-49.5 kDa to detect copro-antigens proved to be practical and reliable. It showed higher sensitivity (91.4%) and higher diagnostic accuracy (91.8%), while the specificity was 92.3%. In addition, ELISA had higher negative predictive value (88.9%) and fair positive predictive value (94.1%).

Evaluation of an indirect immunofluorescence assay for the detection of Leishmania promastigotes and amastigotes in sand flies and lesion fluid aspirates.

Rabbit monospecific antibody Leishmania major surface glycoprotein (gp63) was used in an indirect immunofluorescence assay (IFA) to identify promastigotes and amastigotes from sandflies and lesion fluid aspirates, respectively. Anti-gp63 fluoresced the entire periphery and flagella of promastigotes of different Leishmania species at dilutions to 1:50. Promastigotes were distinctively demonstrated in whole infected sandfly homogenate for 17 days post-infection. Amastigotes from lesion aspirates of infected BALB/c mice and from a human cutaneous leishmaniasis case were also fluoresced by the antibody. This technique could prove to be especially useful in arthropod vector surveillance efforts in which detection of the pathogen is as important as speciation of the sand fly vector.

Amebiasis in schistosomiasis endemic and non-endemic areas in Egypt.

Stool and blood specimens were collected from each of 404 and 576 individuals at Sindbis village (Qualiubia Governorate) in the Nile Delta where schistosomiasis is endemic and El-Rashda village (New Valley Governorate) in the Western Desert of Egypt where there is no schistosomiasis; respectively. Based on the microscopical examination of stool specimens, the prevalence of infection with Entamoeba (E. histolytica and/or E. dispar which are morphologically indistinguishable) was higher at Sindbis than at El Rashda village (29.3% and 20%, respectively). At Sindbis, the prevalence of Entamoeba (both species) was 35.2% (50/142) in S. mansoni infected individuals versus 26.3% (69/262) in S. mansoni negative individuals. Serum antibodies develop only against E. histolytica but not against E. dispar infection. When serological results were considered, the prevalence of E. histolytica was 4.7% in Sindbis and 3.4% at El Rashda based on those who were positive microscopically and serologically in the two villages, respectively. In other words, only 16-17% of those who were positive microscopically can be considered infected with E. histolytica as determined serologically. However, the prevalence of E. histolytica (present or past) based on those who were positive serologically whether positive or negative microscopically was 13.4% and 12.7% at the two villages, respectively. At Sindbis, the prevalence of E. histolytica infection was lower in S. mansoni negative (8.5%) than in S. mansoni positive (16.0%) individuals. These epidemiologic data suggest that: (1) S. mansoni infection may suppress the immune response of the host and therefore, the prevalence of E. histolytica based on serological testing is probably underestimated in the S. mansoni infected people and it may be higher than in the S. mansoni negative people. (2) Serological examinations can be used in determining the true prevalence of E. histolytica (present or past infections) until a routine test for detecting E. histolytica specific antigen in stool becomes available to differentiate E. histolytica from E. dispar infections.

Identification of a misleading trypanosomatid parasite from Gerbillus pyramidum and G. andersoni in a Leishmania major endemic area in north Sinai.

During an epidemiologic investigation of cutaneous leishmaniasis at a focus in north Sinai, Egypt, between June 1989 and December 1991, 897 desert rodents were trapped and examined to identify reservoir hosts for Leishmania major. Mixed forms of epimastigotes and promastigotes were isolated in Tanabe's medium from 4 Gerbillus pyramidum and 1 Gerbillus andersoni. The 2 forms were later grown and separated as distinct cultures in Schneider's medium. The isoenzyme profile of the gerbils' promastigotes was identical to Leishmania tropica but differed from those of L. major and the gerbils' epimastigotes. The protein pattern by sodium dodecyl sulfate poly acrylamide gel electrophoresis gave no conclusive results. The Hae III restriction endonuclease analysis of kinetoplast DNA of both morphological forms confirmed their similarity and distinguished them from L. tropica and L. major. The gerbils' promastigotes were 30% broader, with a smaller nucleus than those of L. tropica. Following several subcultures, epimastigotes were found to transform to promastigotes. These observations suggest that the 2 forms belong to the genus Trypanosoma. Further studies are in progress to classify this putative Trypanosoma species whose promastigote stages display isoenzyme patterns identical to L. tropica, and which can be misidentified microscopically as Leishmania promastigotes.

Leishmania major: amastigote formation in cell-free media.

The life cycle of Leishmania consists of two distinct developmental stages: the amastigote, which is the ovoid non-flagellated form found in the vertebrate host, and the promastigote, which is an elongated flagellated form found in the gut of an infected sandfly. Following its injection into the vertebrate host by the sandfly vector, the promastigote transforms into an amastigote after entering the host macrophage. The environmental cues inducing this transformation are not fully understood. Attempts to axenically develop and cultivate amastigotes from different Leishmania species have indicated that species and sometimes even strains of the same species vary in their requirements for this process (Pan et al., 1993). The majority of the available published data on transformation pertains to New World Leishmania and provides evidence that elevation in the incubation temperature and/or acidic pH can in some cases induce amastigote formation from promastigotes.

Leishmania tropica in Egypt: an undesirable import.

Cutaneous as well as visceral leishmaniasis has been previously reported in Egypt. The former clinical manifestation is attributed to Leishmania major, the latter to L. infantum. In this study, L. tropica was isolated from an Egyptian labourer returning from Saudi Arabia. Amastigotes were detected by both Giemsa staining and indirect immunofluorescence using rabbit anti-gp63. Promastigotes from Schneider's medium were typed isoenzymatically as L. tropica. In view of the emerging threat of visceralization of L. tropica, the potential risk for its transmission in Egypt is discussed.

Evaluation of GiardEIA kit for giardiasis diagnosis.

Giardia lamblia is recognized as one of the most common agents for diarrhea world wide. To date, microscopical examination of stool samples is the gold standard for giardiasis diagnosis. However, intermittence of the Giardia cycle and some medications may cause temporary disappearance of cysts from stools, thus giving false negative results. In the present study, we evaluated a commercially available enzyme immunoassay kit (GiardEIA) for the detection of Giardia copro-antigens and compared the results with those of the merthiolate-iodine-formaldehyde concentration (MIFC) microscopical examination technique. Sixty-nine fecal samples from children 2-12 years old were emulsified and allowed to react with a Giardia specific antibody, then with an enzyme conjugated antibody and the reaction was developed colorimetrically. Seventy-four percent of the parasitologically positive Giardia cases were also positive by GiardEIA while 26% of the microscopically negative cases were positive by the assay. GiardEIA gave negative results with 82% and 100% of stools with helminthic and protozoan (other than Giardia) infections, respectively. Similarly, no cross-reactivity was found with any of the bacterial agents including Shigella flexneri, pathogenic E. coli, Klebsiella spp. and Salmonella typhi. GiardEIA is a simple assay that can diagnose 24 samples in less than an hour without the need for any special equipment and can be useful in epidemiological surveys and in giardiasis outbreaks.

Viscerotropic Leishmania tropical: transformation to amastigotes in cell-free media.

Promastigotes of a viscerotropic Leishmania tropica strain, originally isolated from a U.S. veteran returning from the Gulf War, was studied for extracellular transformation to amastigotes. Promastigotes were grown in three different media namely, Schneider's Drosophila, Dulbecco's modified Eagle Medium (D-MEM) and RPMI-1640. For each medium the effect of low pH (5.8), elevated temperature (34 degrees C) and the effect of both conditions combined were compared. Cells were counted and examined for morphological changes daily. Transformation to morphologically typical amastigotes occurred in D-MEM and Schneider's media at each of the three different experimental conditions with maximum transformation occurring on day eight in D-MEM maintained at pH 5.8 and 34 degrees C. Complete transformation was not achieved in RPMI-1640 under any of the experimental conditions described. Transformed amastigotes could successfully infect murine macrophages indicating biological competence. Amastigotes could not be propagated under any of the conditions described herein.

Biochemical characterisation of human isolates of Blastocystis hominis.

SDS-PAGE and iso-enzyme analysis of 11 human isolates of Blastocystis hominis revealed at least two variants with different polypeptide patterns and two zymodemes, respectively. This is the first iso-enzyme and the second protein analysis to indicate strain differences in B. hominis.

Giardia lamblia: phospholipid analysis of human isolates.

Thin layer chromatograms for phospholipids obtained from 11 human Giardia lamblia isolates and their culture media have shown that phosphatidylcholine and sphingomyelin are the predominant phospholipid classes in all samples. A decrease in the relative percentage of the different classes, especially of phosphatidylcholine, was noticed in the medium after Giardia growth. Fatty acid analysis of the parasite phosphatidylcholine demonstrated that while oleate and palmitate were the major fatty acids in most isolates, arachidonate predominated in two of those studied. Some isolates contained small amounts of myristate, which was not present in the phosphatidylcholine of the culture medium. Moreover, stearate and linoleate predominated in phosphatidylcholine obtained from both media types. The saturated/unsaturated fatty acid ratio also varied for the different isolates. These results appear to suggest heterogeneity in the metabolic activity and utilization of lipid molecules between Giardia isolates.

Cutaneous leishmaniasis in the peace keeping force in East Sinai.

During the period from October 1982 to July 1985 cutaneous leishmaniasis was diagnosed in 113 Multinational Force and Observers (MFO) soldiers from various nations, East Sinai. Leishmania was isolated from twelve of these patients and maintained in vitro using Tanabe's medium. Intrasplenic inoculation of promastigotes from isolate cultures into hamsters resulted in dissemination patterns typical of cutaneous leishmaniasis. The excreted factor in culture when serotyped against known marker strains indicated subserotype A1B2. They also exhibited similar banding patterns to L. major L-137 for GPI, G 6-PD, MDH, NH, MPI, PGM and variant B for 6-PGDH. This leishmaniasis focus appears epidemiologically similar to that of nearby Mid Western Negev where transmission involves Phlebotomus papatasi, Psammomys, and Meriones.

Effect of immature Schistosoma mansoni worms on hamsters' plasma enzymes.

1. The pattern and activity of isocitrate, lactate and malate dehydrogenases and malic enzyme were studied in plasma of normal hamsters and hamsters at the 26th day of infection with S. mansoni. 2. Although the electrophoretic patterns of these enzymes were similar in normal and infected hamsters, their activities were higher in the latter than the former group of animals. The elevation in the enzymic activity indicates that there is tissue damage caused by the larvae at this stage.