HIV-1-Subtypisierung mittels Multi-Region Hybridisation Assay in Bevölkerungsgruppen mit unterschiedlichem sexuellen Risikoverhalten in Mbeya Region, Tansania

Seit der Entdeckung der zwei Typen des Human Immunodeficiency Virus (HIV) in den Jahren 1983 und 1986 gibt es zahlreiche kontroverse Diskussionen über deren Ursprung. Erst die Erforschung des zweiten Primate Immunodeficiency Virus (PIV), des Simian Immuno-deficiency Virus (SIV), ergab neue Erkenntnisse, die die Abstammung des HIV aus SIV weitgehend belegen konnten, obwohl noch heute diesbezügliche Zweifel bestehen. Aufgrund der großen genetischen Variabilität dieser Viren, wurden sie in verschiedene Stämme, Gruppen, Subtypen und Subsubtypen eingeteilt. Ihre Klassifikation und Nomenklatur wurde aufgrund der – vor allem in den 1990er-Jahren gemachten – neuen wissenschaftlichen Erkenntnisse über das HIV-Genom häufig geändert, bis im April 2000 ein Overview of subtypes in the primate immunodeficiency viruses publiziert wurde. Er dient seither als Grundlage für die Nomenklatur von HIV und SIV, wie sie auch in dieser Dissertation Verwendung findet. Die Forschung mit HIV-Subtypen machte es möglich, die epidemiologischen Ausbreitungs-wege von HIV zu eruieren und neue Erkenntnisse zur HIV-Infektion, zu ihren Übertragungs-wegen, zur Prävention und schließlich zu geeigneten Public-Health-Maßnahmen zu erlangen. Die bis noch vor wenigen Jahren diskutierte Frage, ob HIV-Superinfektionen und -Mehrfach-infektionen überhaupt möglich sind und wie die Rekombinanten entstehen, konnte ebenfalls durch die Subtypenforschung beantwortet werden. Diesbezügliche Studien werden seit einigen Jahren auch in Mbeya Region im Südwesten Tansanias durchgeführt. Die Daten dieser Dissertation basieren zum Teil auf jenen der fünfjährigen HIV Superinfection Study (HISIS), die im August 2000 in Mbeya Region begonnen wurde. HISIS befasst sich vor allem mit der Erforschung von HIV-Superinfektionen und deren Mehrfachinfektionen und Intersubtyp-Rekombinationen. Hierzu bedarf es eines Studienortes, in dem unterschiedliche HIV-Subtypen prävalent sind. Dies ist in Mbeya Region mit den Subtypen A, C und D der Fall. Die zweite Voraussetzung ist eine Studienpopulation, die eine hohe Prävalenz dieser Subtypen aufweist. Hierfür wurde eine offene Kohorte von über 700 weiblichen Prostituierten gebildet, aus der ich von September 2000 bis Mai 2001 bei 626 Studienteilnehmerinnen Daten für die vorliegende Arbeit gewinnen konnte. Um diese Populationsgruppe (so genannte High-Risk-Group, HRG) mit einem sehr hohen Risiko hinsichtlich des sexuellen Verhaltens studieren zu können, habe ich für meine Dissertation eine Vergleichspopulation untersucht, die sich in dieser Variable (sexuelles Risikoverhalten) von der HRG unterscheidet (so genannte Non-High-Risk-Group, NRG). Dafür konnte ich Blutproben und soziologische Daten von 757 Blutspendern (Oktober 2000 bis August 2001) und von 351 antenatal clinic attendees (Februar bis August 2001) gewinnen. Um diese HIV-Subtypisierung mit hoher Qualität und relativ geringen Kosten durchführen zu können, wurde von M. Hölscher, dem Mitbetreuer dieser Dissertation, der Multi-Region Hybridisation Assay (MHA) entwickelt. Dieses molekularbiologische Verfahren konnte ich in der vorliegenden Studie zum ersten Mal bei einem größeren Stichprobenumfang einsetzen: 341 Proben aus Mbeya Region. Diese Dissertation konnte nachweisen, dass der MHA eine ausreichende Sensitivität besitzt, um ihn zur HIV-1-Subtypen-Bestimmung bei transversalen Studien einzusetzen, wobei die Sensitivität von der Anzahl der pro Blutprobe gewonnen mononukleären Zellen abhängig ist. In meiner Arbeit hat sich bestätigt, dass in Mbeya Region HIV-1-Mehrfachinfektionen – mit einer Prävalenz von 14 % innerhalb der Studienpopulation – als auch Einfachinfektionen mit rekombinanten Subtypen – mit einer Prävalenz von 32 % innerhalb der Studienpopulation –vorkommen. Dies bedeutet, dass es sich nur bei etwas mehr als die Hälfte aller in Mbeya Region zirkulierenden HI-Viren um reine Subtypen handelt. Die Prävalenz der HIV-1-Subtypen war wie folgt: Subtyp C: 63 %, Subtyp A: 24 %, Subtyp D: 13 %. Konsekutiv sind dadurch C-Rekombinanten (AC und CD) häufiger präsent als die anderen. Auffällig ist die Tatsache, dass unter den Rekombinanten der Subtyp C seltener und die Subtypen A und D häufiger vorkommen als unter den reinen Subtypen. Ich konnte mit dieser Arbeit beweisen, dass mit einem erhöhten Risiko hinsichtlich des sexuellen Verhaltens die Gefahr von Mehrfachinfektionen steigt (p = 0,0196). Aufgrund der Datenlage darf vermutet werden, dass für die Entstehung von Rekombinationen HIV-1-Mehrfachinfektionen Voraussetzung sind. Ein Einfluss des Geschlechts auf die Prävalenz von Mehrfachinfektionen und Rekombinanten konnte – unter Berücksichtigung des geringen Stichprobenumfangs bei den weiblichen und männlichen Blutspendern – in dieser Arbeit nicht festgestellt werden. Ein Einfluss des Alters auf die Prävalenz von Mehrfachinfektionen und Rekombinanten zeigte sich nur in der Risikogruppe der Prostituierten und nicht in den Populationsgruppen antenatal clinic attendees und Blutspender. Die Dissertation basiert auf den Daten meiner Querschnittsstudie, die ich vom August 2000 bis zum August 2001 in Mbeya Region durchgeführt habe. Sie soll den Einfluss der Variablen sexuelles Risikoverhalten auf die Verteilung der HIV-1-Subtypen und deren Rekombinationen und Mehrfachinfektionen eruieren. Obwohl einige signifikante Assoziationen festzustellen waren, kann keine Aussage über deren kausaler Zusammenhang erfolgen, da es sich hierbei ausschließlich um eine transversale Studie handelt. Zur Eruierung der kausalen Inferenz sind longitudinale Kohortenstudien geeignet, die mit Verlaufsdaten von Individuen arbeiten. Des Weiteren können diese Studien mittels Interventionen (Aufklärung, Kondombenutzung, medizinische Betreuung und Versorgung u.a.) eine Verminderung des Risikos hinsichtlich des sexuellen Verhaltens herbeiführen, was die Inzidenz für Neu- und Superinfektionen erfolgreich senken würde. Eine entsprechende Studie wurde in Mbeya Region von der Abteilung für Infektions- und Tropenmedizin der Ludwig-Maximilians-Universität München in Kooperation mit der Muhimbili University of Dar es Salaam an über 700 Prostituierten von August 2000 bis Juni 2005 durchgeführt

RLEP LAMP for the laboratory confirmation of leprosy: towards a point-of-care test

BACKGROUND Nucleic acid-based amplification tests (NAAT), above all (q)PCR, have been applied for the detection of Mycobacterium leprae in leprosy cases and household contacts with subclinical infection. However, their application in the field poses a range of technical challenges. Loop-mediated isothermal amplification (LAMP), as a promising point-of-care NAAT does not require sophisticated laboratory equipment, is easy to perform, and is applicable for decentralized diagnosis at the primary health care level. Among a range of gene targets, the M. leprae specific repetitive element RLEP is regarded as highly sensitive and specific for diagnostic applications.~ METHODS: Our group developed and validated a dry-reagent-based (DRB) RLEP LAMP, provided product specifications for customization of a ready-to-use kit (intended for commercial production) and compared it against the in-house prototype. The assays were optimized for application on a Genie® III portable fluorometer. For technical validation, 40 \textquotedblmust not detect RLEP\textquotedbl samples derived from RLEP qPCR negative exposed and non-exposed individuals, as well as from patients with other conditions and a set of closely related mycobacterial cultures, were tested together with 25 \textquotedblmust detect RLEP\textquotedbl samples derived from qPCR confirmed leprosy patients. For clinical validation, 150 RLEP qPCR tested samples were analyzed, consisting of the following categories: high-positive samples of multibacillary (MB) leprosy patients (> 10.000~bacilli/extract), medium-positive samples of MB leprosy patients (1.001-10.000~bacilli/extract), low-positive samples of MB leprosy patients (1-1.000 bacilli/extract), endemic controls and healthy non-exposed controls; each n = 30.~ RESULTS: Technical validation: both LAMP formats had a limit of detection of 1.000 RLEP copies, i.e. 43-27 bacilli, a sensitivity of 92% (in-house protocol)/100% (ready-to-use protocol) and a specificity of 100%. Reagents were stable for at least 1~year at 22~°C. Clinical validation: Both formats showed a negativity rate of 100% and a positivity rate of 100% for high-positive samples and 93-100% for medium positive samples, together with a positive predictive value of 100% and semi-quantitative results. The positivity rate for low-positive samples was 77% (in-house protocol)/43% (ready-to-use protocol) and differed significantly between both formats.~ CONCLUSIONS: The ready-to-use RLEP DRB LAMP assay constitutes an ASSURED test ready for field-based evaluation trials aiming for routine diagnosis of leprosy at the primary health care level

Trends in the incidence of AIDS-defining and non-AIDS-defining cancers in people living with AIDS: a population-based study from Sao Paulo, Brazil

People living with AIDS are at increased risk of developing certain cancers. Since the introduction of the highly active antiretroviral therapy (HAART), the incidence of AIDS-defining cancers (ADCs) has decreased in high-income countries. The objective of this study was to analyse trends in ADCs and non-AIDS-defining cancers (NADCs) in HIV-positive people with a diagnosis of AIDS, in comparison to the general population, in Sao Paulo, Brazil. A probabilistic record linkage between the 'Population-based Cancer Registry of Sao Paulo' and the AIDS notification database (SINAN) was conducted. Cancer trends were assessed by annual per cent change (APC). In people with AIDS, 2074 cancers were diagnosed. Among men with AIDS, the most frequent cancer was Kaposi's sarcoma (469;31.1%), followed by non-Hodgkin lymphoma (NHL;304;20.1%). A decline was seen for ADCs (APC = -14.1%). All NADCs have increased (APC = -7.4%/year) significantly since the mid-2000s driven by the significant upward trends of anal (APC = -24.6%/year) and lung cancers (APC = -15.9%/year). In contrast, in men from the general population, decreasing trends were observed for these cancers. For women with AIDS, the most frequent cancer was cervical (114;20.2%), followed by NHL (96;17.0%). Significant declining trends were seen for both ADCs (APC = -15.6%/ year) and all NADCs (APC = -15.8%/ year), a comparable pattern to that found for the general female population. Trends in cancers among people with AIDS in Sao Paulo showed similar patterns to those found in developed countries. Although ADCs have significantly decreased, probably due to the introduction of HAART, NADCs in men have shown an opposite upward trend

Treatment Outcome of Patients with Buruli Ulcer Disease in Togo

Background Following introduction of antimycobacterial treatment of Buruli ulcer disease (BUD),several clinical studies evaluated treatment outcomes of BUD patients, in particular healing times, secondary lesions and functional limitations. Whereas recurrences were rarely observed, paradoxical reactions and functional limitations frequently occurred. Although systematic BUD control in Togo was established as early as 2007, treatment outcome has not been reviewed to date. Therefore, a pilot project on post-treatment follow-up of BUD patients in Togo aimed to evaluate treatment outcomes and to provide recommendations for optimization of treatment success. Methodology/Principal Findings Out of 199 laboratory confirmed BUD patients, 129 could be enrolled in the study. The lesions of 109 patients (84.5%) were completely healed without any complications, 5 patients (3.9%) had secondary lesions and 15 patients (11.6%) had functional limitations. Edema, category III ulcers >15cm, healing times >180 days and a limitation of movement at time of discharge constituted the main risk factors significantly associated with BUD related functional limitations (P180 days and limitation of movement at discharge constituted the main risk factors for functional limitations in Togolese BUD patients. Standardized treatment plans, patient assessment and follow-up, as well as improved management of medical records are recommended to allow for intensified monitoring of disease progression and healing process, to facilitate implementation of therapeutic measures and to optimize treatment success

Determination of clusters and factors associated with dengue dispersion during the first epidemic related to Dengue virus serotype 4 in Vitoria, Brazil

Dengue occurrence is partially influenced by the immune status of the population. Consequently, the introduction of a new Dengue virus serotype can trigger explosive epidemics in susceptible populations. The determination of clusters in this scenario can help to identify hotspots and understand the disease dispersion regardless of the influence of the population herd immunity. The present study evaluated the pattern and factors associated with dengue dispersion during the first epidemic related to Dengue virus serotype 4 in Vitoria, Espirito Santo state, Brazil. Data on 18,861 dengue cases reported in Vitoria from September 2012 to June 2013 were included in the study. The analysis of spatial variation in temporal trend was performed to detect clusters that were compared by their respective relative risk, house index, population density, and income in an ecological study. Overall, 11 clusters were detected. The time trend increase of dengue incidence in the overall study population was 636%. The five clusters that showed a lower time trend increase than the overall population presented a higher incidence in the beginning of the epidemic and, compared to the six clusters with higher time trend increase, they presented higher relative risk for their inhabitants to acquire dengue infection (P-value = 0.02) and a lower income (P-value < 0.01). House index and population density did not differ between the clusters. Early increase of dengue incidence and higher relative risk for acquiring dengue infection were favored in low-income areas. Preventive actions and improvement of infrastructure in low-income areas should be prioritized in order to diminish the magnitude of dengue dispersion after the introduction of a new serotype

Implementation of a National Reference Laboratory for Buruli Ulcer Disease in Togo

Background: In a previous study PCR analysis of clinical samples from suspected cases of Buruli ulcer disease (BUD) from Togo and external quality assurance (EQA) for local microscopy were conducted at an external reference laboratory in Germany. The relatively poor performance of local microscopy as well as effort and time associated with shipment of PCR samples necessitated the implementation of stringent EQA measures and availability of local laboratory capacity. This study describes the approach to implementation of a national BUD reference laboratory in Togo. Methodology: Large scale outreach activities accompanied by regular training programs for health care professionals were conducted in the regions "Maritime'' and "Central,'' standard operating procedures defined all processes in participating laboratories (regional, national and external reference laboratories) as well as the interaction between laboratories and partners in the field. Microscopy was conducted at regional level and slides were subjected to EQA at national and external reference laboratories. For PCR analysis, sample pairs were collected and subjected to a dry-reagent-based IS2404-PCR (DRB-PCR) at national level and standard IS2404 PCR followed by IS2404 qPCR analysis of negative samples at the external reference laboratory. Principal Findings: The inter-laboratory concordance rates for microscopy ranged from 89% to 94%; overall, microscopy confirmed 50% of all suspected BUD cases. The inter-laboratory concordance rate for PCR was 96% with an overall PCR case confirmation rate of 78%. Compared to a previous study, the rate of BUD patients with non-ulcerative lesions increased from 37% to 50%, the mean duration of disease before clinical diagnosis decreased significantly from 182.6 to 82.1 days among patients with ulcerative lesions, and the percentage of category III lesions decreased from 30.3% to 19.2%. Conclusions: High inter-laboratory concordance rates as well as case confirmation rates of 50% (microscopy), 71% (PCR at national level), and 78% (including qPCR confirmation at external reference laboratory) suggest high standards of BUD diagnostics. The increase of non-ulcerative lesions, as well as the decrease in diagnostic delay and category III lesions, prove the effect of comprehensive EQA and training measures involving also procedures outside the laboratory

Evidence for significant influence of host immunity on changes in differential blood count during malaria

Background: Malaria has been shown to change blood counts. Recently, a few studies have investigated the alteration of the peripheral blood monocyte-to-lymphocyte count ratio (MLCR) and the neutrophil-to-lymphocyte count ratio (NLCR) during infection with Plasmodium falciparum. Based on these findings this study investigates the predictive values of blood count alterations during malaria across different sub-populations. Methods: Cases and controls admitted to the Department of Infectious Diseases and Tropical Medicine from January 2000 through December 2010 were included in this comparative analysis. Blood count values and other variables at admission controlled for age, gender and immune status were statistically investigated. Results: The study population comprised 210 malaria patients, infected with P. falciparum (68%), Plasmodium vivax (21%), Plasmodium ovale (7%) and Plasmodium malariae (4%), and 210 controls. A positive correlation of parasite density with NLCR and neutrophil counts, and a negative correlation of parasite density with thrombocyte, leucocyte and lymphocyte counts were found. An interaction with semi-immunity was observed; ratios were significantly different in semi-immune compared to non-immune patients (P <0.001). The MLCR discriminated best between malaria cases and controls (AUC = 0.691; AUC = 0.741 in non-immune travellers), whereas the NLCR better predicted severe malaria, especially in semi-immune patients (AUC = 0.788). Conclusion: Malaria causes typical but non-specific alterations of the differential blood count. The predictive value of the ratios was fair but limited. However, these changes were less pronounced in patients with semi-immunity. The ratios might constitute easily applicable surrogate biomarkers for immunity

Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease-Towards a Point-of-Care Test

Background As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP),a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents. Methodology/Principal Findings Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR),IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%;100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%;cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays. Conclusions/Significance Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level

Laboratory Confirmation of Buruli Ulcer Disease in Togo, 2007–2010

Buruli ulcer disease (BUD) is an emerging disease particularly affecting children under the age of 15 years. Due to scarring and contractures BUD may lead to severe functional disability. Introduction of antimycobacterial treatment necessitated the laboratory confirmation of BUD, and WHO recommends confirmation of at least 50% of patients with suspected BUD by polymerase chain reaction (PCR). In Togo, cases have been reported since the early 1990s. However, less than five percent were laboratory confirmed. Since 2007, the German Leprosy and Tuberculosis Relief Organization (DAHW) has supported the Togolese National Buruli Ulcer Control Program in the area of training, treatment and laboratory confirmation of BUD. In close collaboration of DAHW and the Department for Infectious Diseases and Tropical Medicine, University Hospital, Munich (DITM), diagnostic samples from Togolese patients with suspected BUD were subjected to PCR. Out of 202 suspected BUD cases 109 BUD patients (54%) were PCR confirmed over a period of three years. Whereas the PCR case confirmation rate initially was below 50%, intensified training measures for health staff in the field of clinical diagnosis and collection of diagnostic samples ultimately resulted in 69% PCR confirmed cases. Our findings confirm the prevalence of BUD in Maritime Region

O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis

Background: Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. Methods: A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. Results: Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. Conclusions: The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis