[Show abstract][Hide abstract] ABSTRACT: Microscopy, being relatively easy to perform at low cost, is the universal diagnostic method for detection of most globally important parasitic infections. As quality control is hard to maintain, misdiagnosis is common, which affects both estimates of parasite burdens and patient care. Novel techniques for high-resolution imaging and image transfer over data networks may offer solutions to these problems through provision of education, quality assurance and diagnostics. Imaging can be done directly on image sensor chips, a technique possible to exploit commercially for the development of inexpensive "mini-microscopes". Images can be transferred for analysis both visually and by computer vision both at point-of-care and at remote locations.
Here we describe imaging of helminth eggs using mini-microscopes constructed from webcams and mobile phone cameras. The results show that an inexpensive webcam, stripped off its optics to allow direct application of the test sample on the exposed surface of the sensor, yields images of Schistosoma haematobium eggs, which can be identified visually. Using a highly specific image pattern recognition algorithm, 4 out of 5 eggs observed visually could be identified.
As proof of concept we show that an inexpensive imaging device, such as a webcam, may be easily modified into a microscope, for the detection of helminth eggs based on on-chip imaging. Furthermore, algorithms for helminth egg detection by machine vision can be generated for automated diagnostics. The results can be exploited for constructing simple imaging devices for low-cost diagnostics of urogenital schistosomiasis and other neglected tropical infectious diseases.
[Show abstract][Hide abstract] ABSTRACT: Some protozoans are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent. Acanthamoeba is an exception since its cyst wall contains cellulose. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent beta-1,4-linked hexose backbones of these molecules. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. The identification of Acanthamoeba spp., which is based primarily on morphological and biochemical features, is labor-intensive and requires cloning and axenization. We describe a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of Trichoderma reesei cellulase to protozoan cyst wall cellulose. A recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from T. reesei cellulases was coupled to the fluorescent dyes Alexa Fluor 350 and Alexa Fluor 568 or was labeled with biotin using EZ-Link sulfo-NHS-biotin. No staining reaction was observed with chitin-containing preparations of fungi. Thus, the recombinant CBDs can be used as a marker to distinguish between cellulose and chitin. This allows rapid identification of Acanthamoeba cyst wall cellulose in paraffin or frozen sections of infected tissues.
Applied and Environmental Microbiology 10/2009; 75(21):6827-30. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Web-based virtual microscopy has enabled new applications within pathology. Here, we introduce and evaluate a network of academic servers, designed to maximize image accessibility to users from all regions of Europe. Whole-slide imaging was utilized to digitize the entire slide set (n = 154) for the slide seminars of the 21st European Congress of Pathology. The virtual slides were mirrored to five academic servers across Europe using a novel propagation method. Functionality was implemented that automatically selects the fastest server connection in order to optimize the slide-viewing speed ( http://www.webmicroscope.net/ECP2007). Results show that during 6 months of monitoring the uptime of the network was 100%. The average viewing speed with the network was 3.1 Mbit/s, as compared to 1.9 Mbit/s using single servers. A good viewing speed (>2Mbit/s) was observed in 32 of 37 countries (86%), compared to 25 of 37 (68%) using single servers. Our study shows that implementing a virtual microscopy network spanning a large geographical area is technically feasible. By utilizing existing academic networks and cost-minimizing image compression, it is also economically feasible.
Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin 04/2009; 454(4):421-9. · 2.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Free-living amebae (FLA) are known to occur worldwide in water-related biotopes, but only limited information is available on these organisms in developing countries and so far no information on their presence is available from Nicaragua. The aims of this study were to evaluate the prevalence of potentially pathogenic Acanthamoeba spp. and Naegleria spp. in different water sources to which the population of Le6n municipality is exposed. Since pathogenic amebae are thermotolerant, we were especially interested in the occurrence of FLA in geothermal areas. Water samples were collected from Le6n area in Nicaragua: 88 samples were from rivers and springs, 111 from wells, 74 from water taps and 21 from water tanks in urban and suburban Le6n and from three nearby geothermal areas of San Jacinto, Posoltega and Tipitapa. Amebae were identified using morphological and physiological criteria, immunohistochemical staining procedures and molecular methods. Indirect immunofluorescent test was performed on cysts and trophozoites fixed on microscopical slides and incubated for 30 min at room temperature in separate experiments with the following antibodies: rabbit-anti N fowleri/N lovanensis (Nf-Pab), mouse monoclonal antibody anti N. fowleri (Nf-5D12u), rabbit antibodies against Acanthamoeba spp. And fluorescent in situ hybridization (FISH) was performed using 18S rRNA-targeted fluorescent oligonucleotide probes. Probes: GSP for the detection of Acanthamoeba and NAEG1088 for the detection of Naegleria. Free-living amebae were recovered from approximately 43 % of the samples. Acanthamoeba spp was found in 21% of samples from León municipality and in 2% of samples from geothermal areas. Amoeboflagellates were found in 10 % of samples from Le6n and in 19% in geothermal areas. Fifty three percent of tested wells in the geothermal area contained thermotolerant amoeboflagellates. Naegleria spp. was identified in 24 out of 39 (61.5 %) of isolated amoeboflagellates. Twelve of them were assigned to N. lovanenesis while none of the isolates could be identified as N fowleri. However, the common presence of thermotolerant Naegleria in water, specially N. lovanensis, which is an indicator species for N. fowleri, suggests that also this pathogenic amoeba may pose a risk to public health in the area. On the other side, direct pathogenicity, free-living amebae are receiving increasing attention as reservoirs and potential vehicles for the transmission of bacteria in the environment. Thus the information provided in this study may serve as base-line for future studies on the role of free-living amebae e.g. in waterborne-disease outbreaks in the region. Among such potentially important enteropathgens are Vibrio cholerae, E. coli 0157, and Helicobacterpylori.
Revista de biologia tropical 07/2008; 56(2):439-46. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of exposed surface carbohydrates in chemoreception in Schistosoma mansoni females was investigated in vitro. Sexually mature females were treated with peanut (PNA), soybean (SBA) or wheat germ (WGA) agglutinin, and their migration in response to excretory-secretory (ES) products of males was measured in a bioassay system. Random distribution of untreated and lectin-treated females was similar (p=>0.05). PNA-treated females were unresponsive for 1.0 h, after which they exhibited slight repulsion to ES products. SBA-treated females did not respond to ES products for 3.0 h. WGA-treated females were unresponsive for 1.5h, after which they exhibited slight attraction to male ES products. Attempts were made to restore chemoattraction by using specific sugar inhibitors of lectin-binding; chemoattraction was best restored in n-acetyl-d-galactosamine (GalNAc)-treated females. These data suggest that GalNAc-containing receptors, possibly GP50, the major SBA-binding schistosome glycan, mediate chemoreception in schistosome females. These findings may explain the observed differences in morbidity between individuals with different blood groups. Individuals with blood group B or O may afford partial protection because of their natural anti-A or by mounting a more efficient antibody response directed against GalNAc-containing structures shared by blood group A red blood cells.
[Show abstract][Hide abstract] ABSTRACT: The basis for correctly assessing the burden of parasitic infections and the effects of interventions relies on a somewhat shaky foundation as long as we do not know how reliable the reported laboratory findings are. Thus virtual microscopy, successfully introduced as a histopathology tool, has been adapted for medical parasitology.
Specimens containing parasites in tissues, stools, and blood have been digitized and made accessible as a "webmicroscope for parasitology" (WMP) on the Internet (http://www.webmicroscope.net/parasitology).These digitized specimens can be viewed ("navigated" both in the x-axis and the y-axis) at the desired magnification by an unrestricted number of individuals simultaneously. For virtual microscopy of specimens containing stool parasites, it was necessary to develop the technique further in order to enable navigation in the z plane (i.e., "focusing"). Specimens were therefore scanned and photographed in two or more focal planes. The resulting digitized specimens consist of stacks of laterally "stiched" individual images covering the entire area of the sample photographed at high magnification. The digitized image information (approximately 10 GB uncompressed data per specimen) is accessible at data transfer speeds from 2 to 10 Mb/s via a network of five image servers located in different parts of Europe. Image streaming and rapid data transfer to an ordinary personal computer makes web-based virtual microscopy similar to conventional microscopy.
The potential of this novel technique in the field of medical parasitology to share identical parasitological specimens means that we can provide a "gold standard", which can overcome several problems encountered in quality control of diagnostic parasitology. Thus, the WMP may have an impact on the reliability of data, which constitute the basis for our understanding of the vast problem of neglected tropical diseases. The WMP can be used also in the absence of a fast Internet communication. An ordinary PC, or even a laptop, may function as a local image server, e.g., in health centers in tropical endemic areas.
[Show abstract][Hide abstract] ABSTRACT: Intravascular Schistosoma mansoni worms seem to take up immunoglobulins from blood by surface Fc-receptors, but the process whereby bound immunoglobulins are processed by the parasite is poorly understood. We here present morphological data suggesting that two distinct main processes are involved: Host immunoglobulins were seen at two distinct locations in the parasite: in the frontal part of the enteric tube, the oesophagus, and as a fine granular staining at the surface and in the subtegumental region. The latter staining pattern corresponds to host immunoglobulin localization in discrete organelle-like aggregates tentatively identified as 'discoid or elongate bodies' at the ultrastructural level using immunogold staining. Immunoglobulin uptake by intravascular worms was also demonstrated in vivo after passive administration of 125I-labelled rabbit and mouse immunoglobulins. Radiolabelled immunoglobulins were taken up by the worms and shown to localize as fine strands running perpendicular to the parasite surface. Our results suggest that intravascular schistosomes take up host immunoglobulins both as part of their enteric digestion and by a surface Fc-receptor-mediated mechanism, involving transport and processing within organelles, 'elongate bodies'. Immunoglobulins taken up by intravascular schistosomes form a distinct organelle-like granules, which seem to be processed within the excretory system of the parasite.
[Show abstract][Hide abstract] ABSTRACT: The presence of anti-tissue antibodies in sera from patients with genitourinary carcinomas and non-malignant genitourinary diseases was studied by indirect immunofluorescence. An increased incidence of various tissue antibodies was observed in carcinomas of the urinary bladder, prostate and kidney when compared to controls. There was a trend towards association between anti-nuclear antibodies and metastatic undifferentiated tumors. A low degree of malignancy was associated with antibodies against connective tissue “reticulin”. A striking similarity between tissue antibody “patterns” in carcinoma patients and patients with chronic renal inflammation was seen. This points to inflammation as being an important factor in tissue antibody production.
International Journal of Cancer 07/2006; 19(3):332 - 336. · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Based on stool microscopy, an E. histolytica/E. dispar prevalence of 18.6% was found in León, Nicaragua about 10 years ago. Since then, new non-microscopic methods have been developed to discriminate between pathogenic E. histolytica and nonpathogenic E. dispar. The main objectives of the present study were to evaluate the true prevalence of E. histolytica among individuals with diarrhea and to assess the diagnostic procedures carried out at the health center level.
A descriptive study was carried out on patients with diarrhea. Parasite detection was performed by conventional microscopy on native preparations or concentrated and stained specimens, Triage Parasite Panel and by PCR for both E. histolytica and E. dispar.
In 134 individuals with diarrhea, the prevalence of intestinal parasites was 69% as detected by direct stool examination. E. histolytica/E. dispar was found in eight (6%) of the samples, but the health centers reported 24%. In the Triage Parasite Panel only one case of E. histolytica/E. dispar was found. Analysis by PCR showed E. dispar in ten (7.5%) and E. histolytica in two cases (1.5%). The detection of intestinal coccidia and Dientamoeba fragilis required additional staining methods.
PCR results showed that E. histolytica is a rare finding in patients with diarrhea. At the health centers, E. histolytica, E. histolytica/E. dispar were clearly overdiagnosed, with the consequence of overtreatment.
Archives of Medical Research 06/2006; 37(4):529-34. · 2.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the absence of egg excretion, laboratory diagnosis of recently acquired schistosomiasis is dependent on serology. 42 of 83 Swedish adventure tourists to sub-Saharan Africa had serum anti-schistosome antibodies indicating recent infection. There is little doubt regarding the specificity and sensitivity of serology for the demonstration of infection, but there is a need for alternative serological methods which could be more widely used than the standard immunofluorescence assay (IFA) for antibodies against gut-derived antigens (anti-GAA). We present results suggesting that 40/42 anti-GAA positive cases also react with keyhole limpet haemocyanin (KLH), a readily available commercial antigen. High anti-GAA titres were seen for more than 2 y despite treatment with praziquantel. Thus we are faced with several questions. How likely is it that positive serology means treatment failure? What is the risk involved in chronic infection? What is the prospect for monitoring treatment outcome by serology? We conclude that there is a need for better information on the risk of becoming infected, for improved methods for testing and for monitoring the therapeutic effects in adventure tourists.
[Show abstract][Hide abstract] ABSTRACT: Our preliminary observation, that sera from schistosomiasis patients react with carcinomas, raised the possibility of antigenic cross-reactivity. We here extend this observation to show that mice experimentally infected with Schistosoma mansoni react with human urothelial and transitional bladder carcinomas and also with a gastric carcinoma cell line, AGS. To identify cross-reacting epitopes, we looked for the expression of carcinoma markers in schistosome worms and eggs using monoclonal antibodies against tumour antigens MUC1, Tn and TF (also known as the oncofetal Thomsen-Friedenreich antigen or T antigen). Immunohistochemical staining showed that the TF-epitope is present in adult intravascular S. mansoni worms and eggs deposited in tissues of infected animals. The localization of TF-immuno-reactive material in schistosomes was seen at the parasite surface between male and female worms and around trapped eggs in the liver. This localization is consistent with the antigen being secreted. Mice experimentally infected with S. mansoni, developed circulating antibodies against the TF-epitope (identified as Gal(beta1-3) GalNAc-O-R) as seen in ELISA using TF-expressing asialoglycophorin (AGP) as antigen. The observed anti-TF response in S. mansoni-infected mice reflects the complexity of host-parasite interactions in this infection.
[Show abstract][Hide abstract] ABSTRACT: Giardia lamblia is a protozoan parasite infecting the upper mammalian small intestine. Infection relies upon the ability of the parasite to attach to the intestine via a unique cytoskeletal organelle, the ventral disk. We determined the composition and structure of the disk throughout the life cycle of the parasite and identified a new disk protein, SALP-1. SALP-1 is an immunodominant protein related to striated fiber-assemblin (SFA). The disk is disassembled during encystation and stored as four fragments in the immobile cyst. Serial Analysis of Gene Expression (SAGE) showed that the mRNA levels of the disk proteins decreased in encystation but two-dimensional protein gels showed that the protein levels were more constant. The parasite emerges without a functional disk but the four disk fragments are quickly reassembled into two new disks on the dividing, early excysting form. Thus, disk proteins are stored within the cyst, ready to be used in the rapid steps of excystation.
Molecular and Biochemical Parasitology 07/2005; 141(2):199-207. · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Secretory IgA (sIgA) antibodies are important in the host defence against the intestinal protozoan parasite Giardia intestinalis. However, few antigens have been identified. In this study 100 milk and saliva samples from lactating women, living in an endemic region (León, Nicaragua), were screened for the presence of antibodies against G. intestinalis. Most milk and saliva samples contained anti-Giardia antibodies (59% and 52%, respectively), with a mean sIgA content 50 times higher in milk than in saliva. The positive samples reacted with trophozoite membrane, flagella and cytoplasmic antigens. Western blot analysis showed that milk and saliva anti-Giardia sIgA recognized up to 16 different Giardia proteins in the molecular weight region 20-165 kDa. Two-dimensional Western blotting showed that the major immunoreactive proteins were the same as the immunoreactive proteins identified by serum from acute giardiasis patients in a non-endemic country. The major difference was a stronger reactivity against the variant surface proteins (VSPs) in the milk samples. Milk sIgAs also recognized recombinant Giardia proteins such as alpha-1 giardin, ornithine carbamoyl transferase, VSP-4EX, arginine deaminase and alpha-enolase. These antigens will be important targets in the development of new immunodiagnostic tools and vaccines.
[Show abstract][Hide abstract] ABSTRACT: The ability of salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars of Salmonella enterica and five different isolates of axenic Acanthamoeba spp. In gentamicin protection assays, Salmonella enterica serovar Dublin was internalized more efficiently than Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium in all of the amoeba isolates tested. The bacteria appeared to be most efficiently internalized by Acanthamoeba rhysodes. Variations in bacterial growth conditions affected internalization efficiency, but this effect was not altered by inactivation of hilA, a key regulator in the expression of the invasion-associated Salmonella pathogenicity island 1. Microscopy of infected A. rhysodes revealed that S. enterica resided within vacuoles. Prolonged incubation resulted in a loss of intracellular bacteria associated with morphological changes and loss of amoebae. In part, these alterations were associated with hilA and the Salmonella virulence plasmid. The data show that Acanthamoeba spp. can differentiate between different serovars of salmonellae and that internalization is associated with cytotoxic effects mediated by defined Salmonella virulence loci.
Applied and Environmental Microbiology 07/2004; 70(6):3706-14. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: KLH (Keyhole limpet hemocyanin) is highly immunogenic, and crossreactive epitopes occur widely in nature. In schistosomiasis, infected hosts generate antibodies reactive with KLH. This is of diagnostic importance but we lack detailed information on the immunogen-carrying molecules and their distribution in the worm. We used anti-KLH antibodies to localize cross-reacting epitopes in the various developmental stages of the parasite in experimental Schistosoma mansoni infection. The staining results show KLH crossreactivity in the life stages of the parasite. By immunoblotting we show that KLH-crossreactive antigenic epitopes in the parasite eggs are carbohydrates, also recognized by antibodies against soluble schistosome egg antigens. The localizations in the larval stages and in adult worms suggest that crossreacting antigenic epitopes are secretory products.
Journal of Histochemistry and Cytochemistry 11/2003; 51(10):1367-73. · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The protective effect of anti-Giardia antibodies in mother's milk on the acquisition of Giardia infection in their children during the first 2 y of life was analysed as part of a prospective study on infant diarrhoea in a group of 307 mothers and children in Leòn, Nicaragua. Among 24 children acquiring infection within the first 6 months, 23 were born to mothers lacking antibodies. These children also developed more severe diarrhoea. A significant difference between children born to mothers with and without antibodies with respect to the age at which the first Giardia infection was acquired was demonstrated by survival analysis and log rank test (p = 0.036). In conclusion, children born to non-immune mothers are at significantly higher risk of acquiring Giardia infection and developing giardiasis with more severe symptoms compared with children of immune mothers.
[Show abstract][Hide abstract] ABSTRACT: Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent beta-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.
Applied and Environmental Microbiology 06/2002; 68(5):2503-8. · 3.95 Impact Factor