Thesis (Ph. D.)--Illinois State University, 2005. / Title from title page screen, viewed September 26, 2006. Dissertation Committee: David Williams (chair), Craig Gatto, Radheshyam Jayaswal, Wade Nichols, Laura Vogel, Brian Wilkinson. Includes bibliographical references (leaves 150-166) and abstract. Also available in print.

Bilharziasis ranks with Tuberculosis and Malaria as the three most important endemic diseases of man in Rhodesia. The prominence given to bilharziasis in the endemic diseases pattern arises from its high prevalence, particularly in the African population. It is probable that bilharziasis was endemic in the country even prior to the arrival in the conntry of the European settlers in 1890, but it was not until Orpen (1915) described the results of a small survey that local infections were proven. He reported 182 (31 percent) urinary infections in 592 African prisoners examined in the Salisbury gaol. In the ensuing 15 years there was only scanty evidence of the prevalence of the disease. However, in the decade 1931 to 1940 the Annual Public Health Reports of Southern Rhodesia indicated increasing prevalence, and this stimulated interest in the problem and led to the establishment in 1939 of a specialised laboratory to study the parasites causing the disease. The unpublished records of this laboratory indicate that in more recent years the disease has increased not only in prevalence but also in its intensity in infected individuals. Surveys show that the urinary form of the disease is more widespread than the intestinal form. Both forms are becoming more prevalent but the rapid increase of the latter, considered by most authorities to be the more severe, indicates that a greater importance must be accorded to it in the future. Introduction, p. 1.

A esquistossomose mansÃnica à considerada uma doenÃa de grande importÃncia em saÃde pÃblica, porque, embora tenha uma distribuiÃÃo focal, ainda à prevalente em extensas Ãreas do globo. Nesses focos, a doenÃa pode determinar formas clÃnicas graves, reduzindo, drasticamente, a capacidade laborativa e os anos de vida, com qualidade, dos indivÃduos portadores. Recentemente, medidas de controle, baseadas em tratamento em massa, saneamento e manejo do meio ambiente, fornecimento de Ãgua de qualidade e controle quÃmico de caramujos, tÃm propiciado um impacto significativo na morbidade desta parasitose, primariamente, em virtude de reduÃÃo na carga de parasitas na populaÃÃo e, secundariamente, pela queda da prevalÃncia. A Ãrea endÃmica da esquistossomose no CearÃ, identificada em 1977, compreendia um total de 2.972 localidades, distribuÃdas em 16 municÃpios. AÃÃes de controle foram desenvolvidas, de forma sistemÃtica e uniforme, em todas as localidades endÃmicas do CearÃ, nos Ãltimos 20 anos. Algumas localidades responderam, de forma espetacular, Ãs medidas de controle, enquanto outras nÃo apresentaram a mesma resposta. Este trabalho teve como objetivo investigar quais os fatores responsÃveis pela manutenÃÃo da transmissÃo da esquistossomose, numa Ãrea sob o impacto permanente de medidas de controle. Em particular, comparou-se a execuÃÃo de prÃticas e de fatores de risco ambientais, da densidade de caramujos e do percentual de infecÃÃo natural de caramujos, em localidades de alta e baixa transmissÃo. Observou-se, que alguns fatores de risco eram mais frequentes nas localidades de alta transmissÃo e que outros eram distribuÃdos, igualmente, nos dois grupos de localidades. Adicionalmente, as localidades de baixa transmissÃo estavam num estÃgio de urbanizaÃÃo menos avanÃado que as localidades de alta transmissÃo, de forma que o contato com a natureza e o consequente risco de exposiÃÃo aos fatores ambientais era mais frequente entre os indivÃduos que viviam nas localidades de baixa transmissÃo. No entanto, nas localidades de alta transmissÃo, o ambiente oferecia maior risco pois, tanto a densidade como as estimativas da frequÃncia da infecÃÃo natural dos caramujos eram maiores, proporcionando, assim, riscos adicionais para a transmissÃo da esquistossomose. / Schistosomiasis caused by Schistosoma mansoni is a very important disease from a public health perspective. Although, the disease is focused its distribution is worldwide. The infection can determine clinical disease which hamper the laborious capacity of individuals and reduce the quality-adjusted life years. Recently, control programmes based in treatment, sanitation, supply of clean water and chemical control of snails have had a significant impact on the morbidity of this parasitosis, mainly due to reduction of the intensity and prevalence of infection. The endemic area for schistosomiasis in Ceara, was delimited in 1977, and it includes 2,972 villages over 16 municipalities. The control programme has been implemented, systematically and uniformly, in all endemic villages of Ceara for 20 years. Some villages have displayed a dramatic decrease in the intensity and prevalence of infection, although other did not. This study aims to describe the factors involved in the maintenance of transmission of schistosomiasis in an area under pressure of control over 20 years. Specifically, we compared the prevalence of risky behaviors, environmental risk factors, density and natural infection rate of snails from villages with high transmission with those from low transmission villages. We have concluded that some risk factors were more prevalent in high transmission villages, and others had similar prevalence in both group of villages. Moreover, the low transmission villages were in a lower degree of urbanization, in order that the man-water contact were more frequent there. Despite a lower exposure to water in the high transmission area, the likelihood of infection was higher because the environment exposes individual to a higher burden of the parasite expressed by a higher density and natural infection rate of snails in the higher transmission area.

Die Schistosomiasis ist nach wie vor eine der häufigsten parasitären Erkrankungen der Welt und verursacht erhebliche gesundheitliche und wirtschaftliche Folgen, insbesondere in ärmeren, ländlichen Regionen. Durch Immunreaktionen auf die im Wirt abgelegten Eier des Parasiten können sich chronische Verlaufsformen manifestieren. Dabei kann es zu irreversiblen Schäden kommen. Um dies zu verhindern sind eine frühe und sichere Diagnose sowie eine Behandlung mit Praziquantel (PZQ) unabdingbar. Zudem spielt der zuverlässige Nachweis der Schistosomiasis eine Schlüsselrolle bei der Überwachung, Prävention und Kontrolle der Erkrankung. In epidemiologischen Studien findet am häufigsten die mikroskopische Kato-Katz (KK)-Methode zum Nachweis von Schistosoma mansoni Eiern im Stuhl Anwendung. Dieses Verfahren ist äußerst spezifisch und bietet die Möglichkeit der Quantifizierung, wodurch die Intensität der vorliegenden Infektion bestimmt werden kann. Die Sensitivität der Testmethode ist jedoch nur moderat, insbesondere bei einer niedrigen Infektionsintensität. Zudem kann eine Infektion erst nach der Präpatenzzeit nachgewiesen werden. Der ebenfalls häufig eingesetzte urinbasierte Point-of-Care Circulating Cathodic Antigen (POC-CCA)-Test weist zwar eine höhere Sensitivität aber geringere Spezifität als das KK-Verfahren auf. Als hochsensitive und sehr spezifische Methode zur Diagnose der Schistosomiasis hat sich der Nachweis von Schistosoma-spezifischer DNA mittels Real-Time PCR herausgestellt. Allerdings wird für die Durchführung dieser Technik ein gut ausgestattetes Labor benötigt, das sich in der Regel nicht in unmittelbarer Nähe zum Patienten im Feld befindet. Daher ist es besonders wichtig, über praktikable und schnelle Konservierungsmethoden zu verfügen, die bevor die Extraktion und Amplifikation der DNA stattfindet, einen einfachen Transport und eine einfache Lagerung des Probenmaterials ermöglichen. Das Ziel des ersten Teils der vorliegenden Arbeit war, die Sensitivität und Spezifität der klassischerweise verwendeten KK-Methode und des POC-CCA-Tests mit der Real-Time PCR- Methode unter Verwendung von Stuhlproben, Urinproben, Serumproben sowie auf Filterpapier getrocknete Blutproben (dried blood spots – DBSs) zu vergleichen. Zudem wurde die Anwendbarkeit der Real-Time PCR aus Serum- und Urinproben zur Therapiekontrolle überprüft. Die dazu notwendigen Studien wurden alle in der Region Mwanza in Tansania durchgeführt, welche als hochendemisch für S. mansoni gilt. Für die Untersuchungen zur stuhlbasierten Real-Time PCR wurden als Studienteilnehmer Schulkinder gewählt. Aufgrund der erforderlichen Blutabnahme wurden die anderen Teilstudien nur mit erwachsenen Probanden durchgeführt. Unter Verwendung der KK-Methode als Goldstandard erzielten die Real-Time PCR aus Stuhlproben und der POC-CCA-Test sehr hohe Sensitivitäten von 99,5% bzw. 89,7%, jedoch nur geringe Spezifitäten von 29,55% und 22,73%. Die KK-Methode weist bekanntermaßen nur eine geringe bis moderate Sensitivität auf und ist daher nicht gut als Referenz geeignet. Deshalb wurde zusätzlich eine latente Klassenanalyse angewandt, um die tatsächlich Erkrankten zu ermitteln und anhand dieser die diagnostische Güte der verwendeten Tests zu bestimmen. Hier zeigte der POC-CCA-Test die höchste Sensitivität (99,5%) sowie eine Spezifität von 63,4%. Der Real-Time PCR-Test hatte eine Sensitivität von 98,7% und die höchste Spezifität (81,2%). Die Spezifität der KK-Technik betrug 72,8%, die Sensitivität war signifikant niedriger (89,7%) als bei den anderen beiden Methoden. Diese Ergebnisse verdeutlichen, dass der POC-CCA-Schnelltest empfindlicher ist als die KK-Methode und zum Screening von S. mansoni-Infektionen eingesetzt werden kann. Die Stuhl-PCR war zwar ebenfalls hochsensitiv und zeigte unter den drei getesteten Diagnoseverfahren die höchste Spezifität, aber aufgrund der höheren Kosten und der komplizierten Anwendung sollte für epidemiologische Untersuchungen in Hochprävalenzregionen der POC-CCA-Test bevorzugt werden. Bei unklaren Diagnosen kann die Real-Time PCR-Methode als Bestätigungstest Anwendung finden. In der Teilstudie zur serum- und urinbasierten Real-Time PCR in einer endemischen Region vor und nach der Behandlung mit PZQ wurden folgende Ergebnisse erzielt: Unter Verwendung einer kombinierten Referenz aus den Ergebnissen des parasitologischen KK-Tests und / oder der serumbasierten PCR konnte zu Studienbeginn eine Prävalenz von S. mansoni von 77,1% ermittelt werden. In Bezug auf die Sensitivität zeigte der DNA-Nachweis aus Serum (96,3%) und der POC-CCA-Assay (77,8%) die höchsten Ergebnisse. Die urinbasierte Real-Time PCR zeigte die geringste Empfindlichkeit (33,3%). Durch die Behandlung mit Praziquantel wurde eine signifikante Reduktion der S. mansoni Prävalenz erreicht. Zwanzig Wochen nach Therapie konnte durch die KK-Methode keine, mit dem POC-CCA-Test 33,3% und mit der serumbasierten Real-Time PCR 58,3% Infektionen festgestellt werden. Die Analyse der mittels der serumbasierten PCR bestimmten mittleren Ct-Werte im zeitlichen Verlauf zeigte, dass dieser eine Woche nach der Behandlung signifikant abnahm (von 30,3 auf 28) und 20 Wochen später über den Basiswert (34,9) anstieg. Der Ct-Wert ist umgekehrt proportional zur DNA-Ausgangsmenge, die in die PCR eingesetzt wurde. Dies deutet darauf hin, dass kurz nach der Therapie ein DNA-Anstieg zu verzeichnen war und 20 Wochen später weniger DNA als zu Beginn der Studie nachweisbar war. Dieser DNA-Verlauf lässt verschiedene Interpretationsmöglichkeiten zu. Die Daten zeigen jedoch, dass die serumbasierte Real-Time PCR eine ausgezeichnete diagnostische Genauigkeit aufweist. Da die nachgewiesene DNA jedoch keine Rückschlüsse auf das Parasitenstadium zulässt und es sich hierbei auch um DNA aus im Gewebe verbliebenen Eiern oder Reinfektionen handeln könnte, ist diese Methode in Hochprävalenz- Regionen nicht zur Therapiekontrolle geeignet. Die Verwendung von Urin zum DNA-Nachweis erzielte keine vielversprechenden Ergebnisse. Die Sensitivität der Real-Time PCR aus DBSs war ebenfalls sehr gering (45,4%) und kann ohne weitere ausführliche Testung hinsichtlich Lagertemperatur, Lagerdauer, verschiedener Filterpapierarten und Extraktionsmethoden nicht empfohlen werden. Zusammenfassend zeigten die Ergebnisse dieser Studien, dass sowohl die stuhl- als auch die serumbasierte Real-Time PCR bei der Erkennung und Bewertung der Infektionsprävalenz, einem wichtigen Aspekt epidemiologischer Studien, deutlich empfindlicher ist als das mikroskopische KK-Verfahren. Aufgrund des hohen Kosten- und Personalaufwandes und der Notwendigkeit eines gut ausgestatteten Labors wird sich diese Methode aber nicht zum Screening in hochendemischen Ländern durchsetzen. Sie kann jedoch einen Mehrwert bei der Diagnose der Schistosomiasis bieten, vor allem bei frühen oder leichten Infektionen. Zudem kann diese hochsensitive und spezifische Methode als Bestätigungstest bei unklaren Diagnosen herangezogen werden. Im zweiten Teil dieser Arbeit wurden malakologische Untersuchungen zur Identifizierung potenzieller Übertragungsorte für die Schistosomiasis rund um die im Viktoriasee gelegene Insel Ijinga durchgeführt. Diese Analysen fanden innerhalb eines Pilotprojektes zur Eliminierung der Erkrankung auf der Insel Ijinga statt, wobei ein intensiviertes Behandlungsprotokoll, welches die gesamte Inselbevölkerung einschloss, Anwendung fand. Die Kontrolle der Praziquanteleffektivität nach mehreren Behandlungsrunden bringt eine Reihe diagnostischer Herausforderungen mit sich. Hier könnte die Beurteilung der Schistosoma-Infektion in den Zwischenwirtschnecken vor und nach der Therapie als Indikator für den Erfolg der Maßnahme dienen. Zu diesem Zweck erfolgte zunächst eine Baseline-Untersuchung, bei der Schnecken an Uferregionen gesammelt wurden, an denen die Inselbewohner häufigen Wasserkontakt hatten. Die Schnecken wurden anhand morphologischer Merkmale identifiziert und mithilfe der Real-Time PCR-Methode auf Infektionen mit S. mansoni untersucht. Insgesamt wurden 35,4% (279/788) S. mansoni- positive Zwischenwirtschnecken (Biomphalaria) detektiert. Dies verdeutlicht, dass an den meisten Wasserkontaktstellen um die Insel Ijinga ein potentielles Risiko für die Übertragung der Schistosomiasis besteht. Die mithilfe der KK-Methode ermittelte Gesamtprävalenz von S. mansoni in der humanen Bevölkerung betrug 68,9%. Nachdem die Bewohner der Insel viermal mit PZQ behandelt wurden, zeigte sich in der kontinuierlich überwachten Sentinelgruppe eine Reduktion der Prävalenz auf 28,7%. Zu diesem Zeitpunkt wurde ebenfalls die Analyse der Schnecken wiederholt und es konnten 16,8% (57/350) Schnecken mit einer S. mansoni Infektion nachgewiesen werden. Die Reduktion der Infektionshäufigkeit in den Schnecken vor und nach der viermaligen Behandlung der Bevölkerung war signifikant (χ² = 74.335, p < 0,001). Dies deutet darauf hin, dass die intermediären Wirtsschnecken zur Überwachung von Kontrollmaßnahmen verwendet werden können. / Schistosomiasis remains one of the most common parasitic infections in the world, causing significant health and economic consequences, particularly in rural areas. Immune reactions to the parasite's eggs deposited in the host can lead to chronic progression. This may result in to irreversible damage. In order to prevent this, an early and reliable diagnosis and a concomitant therapy will be indispensable. In addition, the reliable detection of schistosomiasis plays a key role in monitoring, prevention and control of the disease. In epidemiological studies, the microscopic Kato-Katz (KK) method is most frequently used to detect eggs in stool. This method is highly specific and offers the possibility of quantification, which allows to determine the intensity of the existing infection. However, especially at low infection intensities, the sensitivity of the test method is rather moderate. Furthermore, infections can only be detected after the prepatent period. The also frequently used urine-based Point-of-Care Circulating Cathodic Antigen (POC-CCA) test shows a higher sensitivity but lower specificity than the KK method. The detection of schistosome-specific DNA through Real-Time PCR has proven to be a highly sensitive and very specific method for the diagnosis of schistosomiasis. However, this assay requires a well-equipped laboratory, which is usually not located in the immediate vicinity of the patient in the field. Therefore, it is particularly important to have practical and rapid preservation methods that allow easy transport and storage of the sample material before DNA extraction and amplification takes place. The aim of the first part of this dissertation was to compare the sensitivity and specificity of the classical KK method and the POC-CCA test with the Real-Time PCR method using stool samples, urine samples, serum samples and blood samples dried on filter paper (dried blood spots – DBS). In addition, tests were conducted regarding the applicability of Real-Time PCR from serum and urine samples for therapy control. The necessary studies were all carried out in the Mwanza region of Tanzania, which is considered to be highly endemic for S. mansoni. Schoolchildren were selected as study participants for the studies on stool-based Real-Time PCR. Due to the required blood collection, the other partial studies were conducted with adults only. Using the KK method as gold standard, the Real-Time PCR from stool samples and the POC-CCA test achieved very high sensitivities of 99.5% and 89.7%, respectively, but only low specificities of 29.55% and 22.73%. The KK method is known to have low to moderate sensitivity and is therefore not well suited as a reference. For this reason, a latent class analysis was carried out to determine the true patients and the diagnostic quality of the tests used. The POC-CCA test showed the highest sensitivity (99.5%) and a specificity of 63.4%. The Real-Time PCR test had a sensitivity of 98.7% and the highest specificity (81.2%). The specificity of the KK technique was 72.8%, the sensitivity was significantly lower (89.7%) than with the other two methods. These results show that the POC-CCA rapid test is more sensitive than the KK method and can be used to screen for S. mansoni infections. Although stool PCR is also highly sensitive and shows the highest specificity of the three diagnostic methods tested, the POC-CCA test should be preferred for epidemiological investigations in high prevalence regions on account of the higher costs and complicated application. If the diagnosis is unclear, the Real-Time PCR method can be used as a confirmatory test. The following results were obtained in the partial study regarding the use of serum and urine-based Real-Time PCR in an endemic region before and after treatment with Praziquantel (PZQ): Using a combined reference of the results of the parasitological KK test and / or the serum-based PCR, a prevalence of S. mansoni of 77.1% could be determined at the beginning of the study. In terms of sensitivity, DNA detection from serum (96.3%) and the POC-CCA assay (77.8%) showed the highest results. Urine-based Real-Time PCR showed the lowest sensitivity (33.3%). Treatment with Praziquantel significantly reduced the prevalence of S. mansoni. Twenty weeks after therapy, no infections could be detected with the KK method, 33.3% with the POC-CCA test and 58.3% with the serum-based Real-Time PCR. Analysis of mean Ct-values determined by serum-based Real-Time PCR over time showed that it decreased significantly (from 30.3 to 28) one week after treatment and increased above the baseline value (34.9) 20 weeks later. The Ct-value is inversely proportional to the initial amount of DNA added to the PCR. This suggests that shortly after therapy there was an increase in DNA and 20 weeks later less DNA was detectable compared to the beginning of the study. This DNA progression offers various interpretation possibilities. However, the data show that serum-based Real-Time PCR has excellent diagnostic accuracy. Yet, since the detected DNA does not allow conclusions to be drawn about the parasite stage and this could also be DNA from eggs remaining in the tissue or reinfections, this method is not suitable for therapy control in high prevalence regions. The use of urine for DNA detection did not yield promising results. The sensitivity of the Real-Time PCR from DBSs was also very low (45.4%) and cannot be recommended without further extensive testing with regard to storage temperature, storage time, different filter paper types and extraction methods. In summary, the results of these studies showed that Real-Time PCR is significantly more sensitive than microscopy in the detection and evaluation of infection prevalence, an important aspect of epidemiological studies. However, due to the high costs and personnel involved and the need for a well-equipped laboratory, this method will not be accepted for screening in high-endemic countries. Yet, it can provide added value in the diagnosis of schistosomiasis, especially in early or light infections. In addition, this highly sensitive and specific method can be used as a confirmatory test for unclear diagnoses. The second part of this dissertation describes malacological investigations to identify potential transmission sites for schistosomiasis around Ijinga Island located on Lake Victoria. These analyses took place as part of a pilot project to eliminate the disease on this island, using an intensified treatment protocol that included the entire island population. The control of Praziquantel effectiveness after several rounds of treatment poses a number of diagnostic challenges. Here, the assessment of the schistosoma infection in the intermediate host snails before and after the therapies could serve as an indicator for the success of the measure. For this purpose, a baseline study was carried out, in which snails were collected from shore regions where the islanders had frequent contact with water. The snails were identified on the basis of morphological characteristics and examined for infections with S. mansoni using the Real-Time PCR method. A total of 35.4% (279/788) S. mansoni positive intermediate host snails (Biomphalaria) were detected. This shows that there is a potential risk of schistosomiasis transmission at most water contact points around Ijinga. The total prevalence of S. mansoni in the human population determined by the KK method was 68.9%. After treating the community with Praziquantel four times, the prevalence of S. mansoni in the continuously monitored sentinel group was reduced to 28.7%. At this time, the analysis of the snails was repeated and 16.8% (57/350) snails with a S. mansoni infection were detected. The reduction in the frequency of infection in the snails before and after the fourfold treatment of the population was significant (χ² = 74,335, p < 0.001). This suggests that the intermediate host snails can be used to monitor control measures.

Die Schistosomiasisprävalenz und Krankheitslast in der Region Mwanza ist trotz staatlicher Maßnahmen anhaltend hoch. Zur Vorbereitung einer integralen Eliminationsstudie auf der Insel Ijinga sollte erfasst werden, ob die Bevölkerung über relevante Kenntnisse und Motivation verfügt, um sich an nachhaltiger Schistosomiasiskontrolle zu beteiligen. Zudem sollten lokal priorisierte Maßnahmen, sowie hemmende und unterstützende Faktoren identifiziert werden, um geplante Maßnahmen bestmöglich an die lokalen Begebenheiten anzupassen. Von Februar bis März 2016 wurde eine quantitative Befragung von 356 Schulkindern, sowie 393 Erwachsenen durchgeführt. Anhand logistischer Regression wurden Faktoren ermittelt die mit einem relevanten Kenntnisstand assoziiert sind. Unter Schulkindern wurden des Weiteren Faktoren bestimmt, die mit einer hohen Motivation an Therapiekampagnen teilzunehmen, einhergehen. Nur ein geringer Teil der Bevölkerung (17% der Kinder und 48% der Erwachsenen) verfügte über ein Mindestmaß an relevantem Wissen, um sich an nachhaltiger Schistosomiasiskontrolle zu beteiligen. Assoziierte Faktoren waren bei den Kindern die Informationsquelle Schule, der Besuch der 6. und 7. Klassen und eine zurückliegende Diagnose. Bei Erwachsenen bestand ein Zusammenhang zu verschiedenen Informationsquellen (Schule, Gesundheitswesen, lokale Medien), zum Alter, Bildungsstand und zur Präsenz von Symptomen. Nur 40% der Kinder hatten eine hohe Motivation an Therapiekampagnen teilzunehmen. Assoziierte Faktoren waren die wahrgenommene eigene Vulnerabilität und Schwere der Erkrankung, eine zurückliegende Diagnose, sowie eine erwartete Heilung durch Praziquantel. Die aktuelle Wissensvermittlung in der 6. und 7. Schulklasse erfolgt zu spät und zu punktuell, um über einen theoretischen Kenntnisstand hinaus zur Verhaltensänderung zu führen. Eine systematische Aufnahme von Schistosomiasis in verschiedene Sektoren könnte das Bewusstsein in der Bevölkerung stärken. Zur nachhaltigen Einbindung der Bevölkerung ist ein partizipativer Ansatz, priorisierte WASH Interventionen und eine Stärkung des dezentralen Gesundheitssystems zur frühen Fallfindung und Therapie sinnvoll. / Despite ongoing national control measures, the prevalence and disease burden of schistosomiasis remains high in Mwanza region. A study was conducted on Ijinga Island to determine whether the population has enough relevant knowledge and motivation to engage in sustainable schistosomiasis control. We furthermore wanted to explore the local communities` priorities. The results of the study were used to inform integral control interventions. From February to March 2016, a quantitative survey was conducted among 356 school children and 393 adults. Logistic regression was used to determine factors associated with relevant knowledge. Among schoolchildren, we furthermore determined associated factors with a high intention to participate in mass drug administration programs of praziquantel, the backbone of the national control strategy. Only a small proportion of the population (17% of children and 48% of adults) had a minimum level of relevant knowledge to engage in sustainable schistosomiasis control. Associated factors among children were having heard about schistosomiasis at school, being enrolled in 6th or 7th grade and a previous own diagnosis of schistosomiasis. In adults, relevant knowledge was associated to several sources of information (school, healthcare, local media), age, a high level of education and the own presence of symptoms. Only 40% of the children demonstrated high intention to participate in treatment campaigns. Factors associated were previous diagnosis, perceived general risk of disease transmission by lake water, perceived own vulnerability of getting infected, perceived danger of the disease and the perceived effectiveness of medicaments to cure the disease. The current education on schistosomiasis in the 6th and 7th grades occurs too late and too punctual to change behavior beyond a theoretical level of knowledge. In general, practical knowledge on preventive measures such as taking anti-schistosomiasis drug need to be impacted in schoolchildren to increase their participation in the control program. A systematic uptake of schistosomiasis into different sectors could raise awareness among the population. Participatory approaches, locally prioritized interventions such as water, sanitation and hygiene provision and the strengthening of decentralized early diagnosis and treatment could contribute to engage the communities in sustainable schistosomiasis control.

Law Pui-ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 98-105). / Abstracts in English and Chinese. / Statement --- p.i / Acknowledgments --- p.ii / Abstract --- p.iii / Table of contents --- p.viii / List of figures --- p.xv / List of tables --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Schistosomiasis --- p.1 / Chapter 1.1.1 --- Disease burden --- p.1 / Chapter 1.1.2 --- Causative agents --- p.1 / Chapter 1.1.3 --- Transmission --- p.2 / Chapter 1.1.4 --- Pathology of the disease --- p.3 / Chapter 1.1.5 --- Control and therapy --- p.3 / Chapter 1.2 --- Schistosomiasis in China --- p.5 / Chapter 1.3 --- Schistosoma japonicum --- p.6 / Chapter 1.3.1 --- Life cycle of S. japonicum --- p.6 / Chapter 1.3.2 --- Biology of S. japonicum cercaria --- p.9 / Chapter 1.3.3 --- Transformation of cercaria to schistosomulum --- p.10 / Chapter 1.3.4 --- Cercarial stage-specific antigens --- p.12 / Chapter 1.4 --- Aim of study --- p.14 / Chapter Chapter 2 --- Materials and methodology --- p.15 / Chapter 2.1 --- Materials --- p.15 / Chapter 2.1.1 --- Schistosoma japonicum cercaria cDNA library --- p.15 / Chapter 2.1.2 --- Cercarial stage-specific clones --- p.15 / Chapter 2.1.3 --- Adult worm and cercaria RNA --- p.18 / Chapter 2.1.4 --- "Snail intermediate host, Oncomelania hupensis" --- p.18 / Chapter 2.1.5 --- Bacterial strains --- p.18 / Chapter 2.1.6 --- Chemicals --- p.19 / Chapter 2.1.7 --- Kits and reagents --- p.21 / Chapter 2.1.8 --- Nucleic acids --- p.22 / Chapter 2.1.9 --- Solutions --- p.22 / Chapter 2.1.10 --- Enzymes --- p.24 / Chapter 2.1.11 --- Primers --- p.25 / Chapter 2.1.12 --- Antibodies --- p.26 / Chapter 2.2 --- Methodology --- p.27 / Chapter 2.2.1 --- Identification of cercarial stage-specific genes --- p.27 / Chapter 2.2.1.1 --- Sequence analysis of cercarial stage-specific clones --- p.27 / Chapter 2.2.1.2 --- "Confirmation of stage-specific expression of the selected gene, 20H8 and sjCa8, by RNA dot blot" --- p.29 / Chapter 2.2.1.2.1 --- Cloning of S. japonicum glyceraldehyde-3-phosphate dehydrogenase (sjGAPDH) (GenBank accession no. U75571) --- p.29 / Chapter 2.2.1.2.1.1 --- Reverse transcription and PCR amplification of sjGAPDH --- p.29 / Chapter 2.2.1.2.1.2 --- Cloning of sjGADPH in pBluescript II KS (-) --- p.29 / Chapter 2.2.1.2.1.3 --- Sequence verification of cloned sjGAPDH --- p.30 / Chapter 2.2.1.2.2 --- Synthesis of Digoxigenin (DIG)-labeled probe --- p.31 / Chapter 2.2.1.2.2.1 --- Synthesis of DIG-labeled probe by PCR --- p.31 / Chapter 2.2.1.2.2.2 --- Estimation of concentration of DIG-labeled probe --- p.32 / Chapter 2.2.1.2.3 --- RNA dot blot --- p.33 / Chapter 2.2.1.2.3.1 --- Transferring RNA to the membrane using the BIO-RAD blotting manifold --- p.33 / Chapter 2.2.1.2.3.2 --- Hybridization of DIG-labeled probe to detect sjGAPDH --- p.33 / Chapter 2.2.1.2.3.3 --- Detection of the chemiluminescent signal --- p.33 / Chapter 2.2.1.2.3.4 --- Stripping membrane for reprobing --- p.34 / Chapter 2.2.2 --- Characterization of the cercarial stage-specific gene and gene product of 20H8 --- p.35 / Chapter 2.2.2.1 --- Cloning of full-length cDNA of 20H8 --- p.35 / Chapter 2.2.2.1.1 --- 5'RACE of 20H8 --- p.35 / Chapter 2.2.2.1.2 --- Cloning of full-length 20H8 into pBluescript II SK(-) --- p.36 / Chapter 2.2.2.2 --- Analysis of DNA sequence and deduced amino acid sequence of 20H8 --- p.37 / Chapter 2.2.2.3 --- Demonstration of the immunogenicity and antigenicity of 20H8 --- p.38 / Chapter 2.2.2.3.1 --- Expression of 20H8 in E. coli --- p.38 / Chapter 2.2.2.3.1.1 --- "Cloning of 20H8 in an E coli expression vector, pET32a+" --- p.38 / Chapter 2.2.2.3.1.2 --- Expression of recombinant 20H8 protein in E. coli --- p.39 / Chapter 2.2.2.3.2 --- Purification and concentration of recombinant 20H8 protein --- p.40 / Chapter 2.2.2.3.3 --- Production of antiserum --- p.40 / Chapter 2.2.2.3.4 --- Evaluation of immunogenicity of recombinant 20H8 protein --- p.41 / Chapter 2.2.2.3.5 --- Evaluation of antigenicity of recombinant 20H8 protein --- p.42 / Chapter 2.2.2.4 --- Immunolocalization of 20H8 in cercaria --- p.43 / Chapter 2.2.2.4.1 --- Collection of S. japonicum cercaria --- p.43 / Chapter 2.2.2.4.2 --- Immunofluorescence staining of cercaria --- p.43 / Chapter 2.2.3 --- Characterization of the cercarial stage-specific gene and gene product of sjCa8 --- p.45 / Chapter 2.2.3.1 --- Analysis of DNA sequence and deduced amino acid sequence of sjCa8 --- p.45 / Chapter 2.2.3.2 --- Demonstration of the immunogenicity and antigenicity of sjCa8 --- p.46 / Chapter 2.2.3.2.1 --- Expression of sjCa8 in E. coli --- p.46 / Chapter 2.2.3.2.2 --- Purification and concentration of recombinant sjCa8 protein --- p.46 / Chapter 2.2.3.2.3 --- Production of antiserum --- p.46 / Chapter 2.2.3.2.4 --- Evaluation of the immunogenicity of sjCa8 --- p.47 / Chapter 2.2.3.2.5 --- Evaluation of the antigenicity of sjCa8 --- p.47 / Chapter 2.2.3.3 --- Demonstration of calcium-binding property of sjCa8 --- p.48 / Chapter 2.2.3.3.1 --- Calcium-dependent electrophoretic mobility shift --- p.48 / Chapter 2.2.3.3.2 --- Ruthenium red assay --- p.48 / Chapter 2.2.3.4 --- Immunolocalization of sjCa8 in cercaria --- p.49 / Chapter Chapter 3 --- Results --- p.50 / Chapter 3.1. --- Identification of cercarial stage-specific genes --- p.50 / Chapter 3.1.1 --- Identification of cercarial stage-specific genes by microarray --- p.50 / Chapter 3.1.2 --- "Confirmation of stage-specific expression of the selected gene, 20H8 and sjCa8, by RNA dot blot" --- p.54 / Chapter 3.2. --- Characterization of the cercarial stage-specific gene and gene product of 20H8 --- p.57 / Chapter 3.2.1 --- Cloning of full-length cDNA of 20H8 --- p.57 / Chapter 3.2.2 --- Analysis of DNA sequence and deduced amino acid sequence of 20H8 --- p.59 / Chapter 3.2.3 --- Demonstration of the immunogenicity and antigenicity of 20H8 --- p.62 / Chapter 3.2.3.1 --- Expression of 20H8 in E. coli --- p.62 / Chapter 3.2.3.2 --- Purification and concentration of recombinant 20H8 protein --- p.64 / Chapter 3.2.3.3 --- Production of antiserum --- p.65 / Chapter 3.2.3.4 --- Evaluation of immunogenicity of recombinant 20H8 protein --- p.66 / Chapter 3.2.3.5 --- Evaluation of antigenicity of recombinant 20H8 protein --- p.67 / Chapter 3.2.4 --- Immunolocalization of 20H8 in cercaria --- p.68 / Chapter 3.3. --- Characterization of the cercarial stage-specific gene and gene product of sjCa8 --- p.70 / Chapter 3.3.1 --- Analysis of DNA sequence and deduced amino acid sequence of sjCa8 --- p.70 / Chapter 3.3.2 --- Demonstration of the immunogenicity and antigenicity of sjCa8 --- p.75 / Chapter 3.3.2.1 --- Expression of sjCa8 in E. coli --- p.75 / Chapter 3.3.2.2 --- Purification and concentration of recombinant sjCa8 protein --- p.76 / Chapter 3.3.2.3 --- Production of anti-sjCa8 serum --- p.77 / Chapter 3.3.2.4 --- Evaluation of immunogenicity of sjCa8 protein --- p.77 / Chapter 3.3.2.5 --- Evaluation of antigenicity of sjCa8 protein --- p.78 / Chapter 3.3.3 --- Demonstration of calcium-binding property of sjCa8 --- p.79 / Chapter 3.3.3.1 --- Electrophoretic motility shift --- p.80 / Chapter 3.3.3.2 --- Ruthenium red binding assay --- p.80 / Chapter 3.3.4 --- Immunolocalization of sjCa8 in cercaria 81 --- p.81 / Chapter Chapter 4 --- Discussion --- p.83 / Chapter 4.1 --- Identification of cercarial stage-specific genes --- p.83 / Chapter 4.1.1 --- Identification of cercarial stage-specific genes by microarray --- p.83 / Chapter 4.1.2 --- "Confirmation of stage-specific expression of the selected genes, 20H8 and sjCa8" --- p.85 / Chapter 4.2 --- Characterization of the cercarial stage-specific gene and gene product of 20H8 --- p.86 / Chapter 4.2.2 --- Analysis of DNA sequence and deduced amino acid sequence of 20H8 --- p.86 / Chapter 4.2.3 --- Demonstration of the immunogenicity and antigenicity of 20H8 --- p.88 / Chapter 4.2.4 --- Immunolocalization of 20H8 in cercaria --- p.90 / Chapter 4.3 --- Characterization of the cercarial stage-specific gene and gene product of sjCa8 --- p.91 / Chapter 4.3.1 --- Analysis of DNA sequence and deduced amino acid sequence of sjCa8 --- p.91 / Chapter 4.3.2 --- Demonstration of the immunogenicity and antigenicity of sjCa8 --- p.93 / Chapter 4.3.3 --- Demonstration of calcium-binding property of sjCa8 --- p.94 / Chapter 4.3.4 --- Immunolocalization of sjCa8 in cercaria --- p.94 / Chapter 4.4 --- Conclusions --- p.95 / References --- p.96

Urogenital schistosomiasis, due to Schistosoma haematobium, is one of the helminth infections of public health importance in sub-Saharan Africa where children carry the heaviest burden of infection. Treatment with the drug praziquantel is the only widely available tool for control. Current schistosome control programs are essentially school-based and exclude younger pre-school children. However, increasing reports of significant infection levels in this age group call for the inclusion of these young children in control programs. The hygiene hypothesis suggests that helminth infections, including schistosome infection, may protect against atopy, implying that helminth eradication may carry a detrimental burden of immune disorders. However, this conjecture is controversial. This study was designed to investigate the relationship between schistosome infection and atopy. In a comparative epidemiological approach between two schistosome endemic areas with differing infection levels, it is shown that atopy is inversely associated with current schistosome infection intensity but not with cumulative history of infection. This inverse relationship was subsequently shown to be associated with the levels of soluble CD23, the low affinity IgE receptor, but not with the polyclonal IgE stimulation. However, while praziquantel treatment was associated with an increase in schistosome-specific responses, a decline in atopic responses was observed in these communities, suggesting that treatment differentially affect anti-schistosome and atopic responses. In addition, the study has shown that praziquantel treatment of preschool age children increases, quantitatively and qualitatively, their schistosome-specific antibody responses purportedly associated with resistance to schistosome infection/reinfection.

published_or_final_version / Community Medicine / Master / Master of Philosophy

Previous studies on the influence of abiotic factors on the distribution of bilharzia intermediate host snails of the family Planorbidae are reviewed. Much of this work is basically descriptive and relatively few attempts have been made to examine the effects of these factors on snails in their natural habitats - to identify the factors precisely and to measure their critical levels. An account is given of recent studies on Eiomphalaria pfeifferi (Krauss) in two climatically different regions of southeastern Africa, at 6S5m altitude on the eastern Transvaal escarpment and 19m on the coastal peneplain of northern Zululand. These have shown the species to undergo a similar annual life-cycle of three overlapping generations in both areas. Further, in these areas where the snail has a discontinuous distribution, two abiotic factors, stream geology and water temperature, were found to be important in limiting its distribution. The longitudinal distribution of B. pfeifferi and another host snail Bulinus (Physopsis) globosus (Morelat), extended upstream in a perennial watercourse, the Gladdespruit, on the escarpment only as far as permanently lentic habitats were available. These habitats, usually detached from the channel, characterized the stream's lowest physical zone with its substratum of exposed granite, as opposed to the sandy-bottomed lotic zones upstream. The limit of the snails' occurrence lay close to the junction of these two substratum types. Granite is resistant to erosion but contains soft inclusions which erode more quickly than the hard matrix causing uneven weathering. This results in the formation of depressions in the stream bed (i.e. pools, potholes and backwaters) which provide the permanently lentic conditions necessary for the snails' survival. Temperature recordings made on this plain, upstream of the snails' limit, suggest that the water temperature here exceeded the critical lower levels required for a positive intrinsic rate of natural increase sufficiently often to permit the species to survive here, though not optimally. Above this point however, current velocities are continually in excess of the maximum tolerated (0.3 m/sec.) so that in this situation at least, current velocity is a dominant factor. A contrasting situation exists on the coastal peneplain of northern Zululand where B. pfeifferi occurs in some lentic habitats and not in others. This discontinuous distribution is shown to be related to the prolonged duration of temperatures above the level for optimal increase. The apparently suitable habitats from which B. pfeifferi was absent were found to be both very shallow and to experience these prolonged high temperatures during spring and early summer. This corresponds to the maturation period of the B. pfeifferi 1st generation as defined by Appleton (1974) and causes reduced fecundity probably through impaired gonad development. The density of its filial (2nd) generation is correspondingly reduced. A statistically significant partial-correlation (at a 1% level) was found between the fecundity of the 1st generation and increasing periods of temperatures above the species' optimal limit in the habitats involved during its maturation period (spring). It is interesting to nate here that B. (Ph.) globosus, which is known to be better adapted to high temperatures than B. pfeifferi (Shiff & Husting, 1966) occurred in all the waterbodies concerned. Both these factors therefore play important roles in determining the country-wide distribution pattern of the host snails. This distribution is closely correlated with the availability of permanently lentic habitats. In river systems, which form the main environment for these snails, such stable habitats occur almost entirely in low-gradient reaches over hard bed-rock which is resistant to erosion. Current velocities above 0.3 m/sec. render steeper reaches and those over soft, non-resistant rock and sand (unstable substrata) unsuitable and therefore constitute a most important limiting factor. Temperature however becomes a dominant factor in permanently lentic waterbodies. Biomphalaria pfeifferi is adversely affected by high temperature regimes and where such regimes occur in waterbodies which are too shallow to permit temperature gradient to develop, the species cannot survive whereas B. (Ph.) globosus can do so. This intolerance of high temperature regimes on the part of B. pfeifferi accounts for the species' adoption of a discontinuous distribution over the coastal plain of south-eastern Africa. A comparable pattern has been reported for the species over this plain northwards to approximately 16°S latitude in Mozambique and is probably due to a similar high-temperature effect. Further northwards in equatorial latitudes B. pfeifferi is restricted to the more elevated and cooler areas above an altitude of 500 - 600 m, probably because the very high temperature regimes prevailing in the lowlands there exclude the species altogether.

The present study investigates the infection rates of parasites, morbidity, and the living conditions of street children and orphans in Mwanza city, northern Tanzania. A high percentage of orphans and street children in Mwanza city is infected with one or more parasites. A significantly higher rate of infections with S. mansoni in street children as compared with orphans could be observed. The prevalence of S. mansoni determined by POC CCA test was 65.9% for orphans and 94.5% for street children. 19.2% of the orphans tested positive for S. mansoni in Kato Katz. Of the street children, 77.1% showed positive test results in Kato-Katz. Only 1.3% of the orphans stated in the questionnaire that they use the lake to wash, whereas 91.1% of the street children named the lake as at least one of their options for washing. Protozoal infections used as a marker for hygiene were at a comparable level for both groups. Microscopy showed positive results for G. intestinalis in 8.2% and for E. histolytica/dispar in 23% of orphans and 8.1% for G. intestinalis, and 23.8% for E. histolytica/dispar in street children. Through ultrasonography, we observed no signs of severe PPF and only a few mild PPF patterns. Most street children use the lake to wash and often do not have access to adequate sanitation. However, everyone in the study group indicated having access to safe drinking water. Overall, we found the general hygienic conditions for both groups to be inadequate. With the help of simple public health measures, like improve sanitation and regular mass drug administration, the overall situation would likely be considerably improved. / Die vorliegende Studie untersucht Infektionen mit verschiedenen Parasiten, die Morbidität und Lebensbedingungen von Straßenkindern und Waisen in Mwanza, einer Stadt im Norden Tansanias. Ein hoher Prozentsatz von Waisen und Straßenkindern in Mwanza leidet an Infektionen mit einem oder mehreren Parasiten. Es konnte eine signifikant höhere Rate von Infektionen mit S. mansoni bei Straßenkindern im Vergleich zu Waisen festgestellt werden. Die Prävalenz von Infektionen mit S. mansoni, ermittelt durch POC-CCA-Tests, betrug 65,9% bei Waisen und 94,5% bei Straßenkindern. 19,2% der Waisen waren im Kato-Katz- Test S. mansoni positiv. Bei den Straßenkindern zeigten 77,1% positive Testergebnisse im Kato-Katz-Test. Nur 1,3% der Waisen gaben in dem Fragebogen an, den See zum Waschen zu benutzen, während es 91,1% bei den Straßenkindern waren. Protozoeninfektionen, die als Marker für die Hygieneumstände, unter denen die Kinder leben verwendet wurden, waren bei beiden Gruppen auf vergleichbarem Niveau. Die Mikroskopie zeigte positive Ergebnisse für G. intestinalis bei 8,2% und für E. histolytica/dispar bei 23% der Waisen und bei 8,1% für G. intestinalis und 23,8% für E. histolytica/dispar bei den Straßenkindern. Durch Ultraschall konnten keine Anzeichen für schwere PPF festgestellt werden, und nur bei wenigen Kindern zeigten sich leichte PPF-Muster. Die meisten Straßenkinder benutzen den See um sich zu waschen und haben oft keinen Zugang zu angemessener Sanitärversorgung. Alle Teilnehmer der Studie gaben jedoch an, Zugang zu sicherem Trinkwasser zu haben. Insgesamt stellten wir fest, dass die allgemeinen hygienischen Bedingungen für beide Gruppen unzureichend sind. Mit Hilfe einfacher öffentlicher Gesundheitsmaßnahmen, wie bessere sanitäre Einrichtungen und regelmäßige Entwurmung, könnte die Gesamtsituation für Straßen- und Waisenkinder erheblich verbessert werden.