93 research outputs found

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

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    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

    Plasmodium knowlesi infection in a returning German traveller from Thailand: a case report on an emerging malaria pathogen in a popular low-risk travel destination

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    Background: Thailand is a major destination for German travellers with more than 760,000 arrivals in 2015. At the same time, malaria is a concern in travel recommendations with regard to this destination. The World Malaria Report of 2016 mentions only P. falciparum and P. vivax as prevalent species for Thailand, however, P. knowlesi infections have been occasionally reported in Thailand. In German travellers, only five cases of P. knowlesi infections have been reported to date. Case presentation: A 45-year-old German male tourist travelled to Thailand from 25 December 2016 to 13 January 2017. On 14 January he developed fever with no other symptoms, and presented on 17 January at the Division for Tropical Medicine and Infectious Diseases in Munich, Germany. Malaria was diagnosed, primarily based on a single parasite in the thin smear microscopy, while commercial rapid diagnostic testing remained negative. Only the result of a differential PCR assay revealed P. knowlesi infection. Conclusions: P. knowlesi has to be considered in travellers returning from Thailand. Cases may present with an extremely low parasitaemia. This is in contrast to the assumption that P. knowlesi was likely to cause high parasitaemia due to its short replication cycle

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

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    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

    Treatment Outcome of Patients with Buruli Ulcer Disease in Togo

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    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

    Severe Plasmodium knowlesi infection with multi-organ failure imported to Germany from Thailand/Myanmar

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    During the last two decades human infections with Plasmodium knowlesi are increasingly diagnosed in South East Asia and have also been reported in travellers. A severe case of imported P. knowlesi infection in a 73-year old German is presented, who had been travelling through Myanmar and Thailand for three weeks. Microscopy showed a parasitaemia of 3% and different parasite stages including band-forms resembling Plasmodium malariae. Due to the clinical picture of severe malaria and the microscopical aspect (combination of parasites resembling P. malariae and Plasmodium falciparum), P. knowlesi was suspected. The patient was treated with intravenous quinine; he was put on mechanical ventilation and catecholamines due to cardiorespiratory failure. Parasitaemia was cleared rapidly but renal function deteriorated resulting in intermittent haemodialysis. The patient was hospitalized for six weeks but he recovered completely without any physical sequelae. Plasmodium knowlesi mono-infection was confirmed by molecular methods later on. Plasmodium knowlesi infection has to be taken into account in feverish travellers returning from Thailand/Myanmar. Moreover this species can cause life-threatening or even lethal complications. Accordingly severe P. knowlesi infection should be treated like severe P. falciparum infections

    Infection with Mansonella perstans Nematodes in Buruli Ulcer Patients, Ghana.

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    During August 2010-December 2012, we conducted a study of patients in Ghana who had Buruli ulcer, caused by Mycobacterium ulcerans, and found that 23% were co-infected with Mansonella perstans nematodes; 13% of controls also had M. perstans infection. M. perstans co-infection should be considered in the diagnosis and treatment of Buruli ulcer

    Detection of Gastrointestinal Pathogens from Stool Samples on Hemoccult Cards by Multiplex PCR

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    Purpose. Up to 30% of international travelers are affected by travelers' diarrhea (TD). Reliable data on the etiology of TD is lacking. Sufficient laboratory capacity at travel destinations is often unavailable and transporting conventional stool samples to the home country is inconvenient. We evaluated the use of Hemoccult cards for stool sampling combined with a multiplex PCR for the detection of model viral, bacterial, and protozoal TD pathogens. Methods. Following the creation of serial dilutions for each model pathogen, last positive dilution steps (LPDs) and thereof calculated last positive sample concentrations (LPCs) were compared between conventional stool samples and card samples. Furthermore, card samples were tested after a prolonged time interval simulating storage during a travel duration of up to 6 weeks. Results. The LPDs/LPCs were comparable to testing of conventional stool samples. After storage onHemoccult cards, the recovery rate was 97.6% for C. jejuni, 100% for E. histolytica, 97.6% for norovirus GI, and 100% for GII. Detection of expected pathogens was possible at weekly intervals up to 42 days. Conclusion. Stool samples on Hemoccult cards stored at room temperature can be used in combination with a multiplex PCR as a reliable tool for testing of TD pathogens

    Active case finding: comparison of the acceptability, feasibility and effectiveness of targeted versus blanket provider-initiated-testing and counseling of HIV among children and adolescents in Cameroon

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    Background: Children and adolescents still lag behind adults in accessing antiretroviral therapy (ART), which is largely due to their limited access to HIV testing services. This study compares the acceptability, feasibility and effectiveness of targeted versus blanket provider-initiated testing and counseling (PITC) among children and adolescents in Cameroon. Methods: During a 6-month period in three hospitals in Cameroon, we invited HIV-positive parents to have their biological children (6 weeks-19 years) tested for HIV (targeted PITC). During that same period and in the same hospitals, we also systematically offered HIV testing to all children evaluated at the outpatient department (blanket PITC). Children of consenting parents were tested for HIV, and positive cases were enrolled on ART. We compared the acceptability, feasibility and effectiveness of targeted and blanket PITC using Chi-square test at 5% significant level. Results: We enrolled 1240 and 2459 eligible parents in the targeted PITC (tPITC) and blanket PITC (bPITC) group, and 99.7% and 98.8% of these parents accepted the offer to have their children tested for HIV, respectively. Out of the 1990 and 2729 children enrolled in the tPITC and bPITC group, 56.7% and 90.3% were tested for HIV (p < 0.0001), respectively. The HIV positivity rate was 3.5% (CI: 2.4-4.5) and 1.6% (CI: 1.1-2.1) in the tPITC and bPITC (p = 0.0008), respectively. This finding suggests that the case detection was two times higher in tPITC compared to bPITC, or alternatively, 29 and 63 children have to be tested to identify one HIV case with the implementation of tPITC and bPITC, respectively. The majority (84.8%) of HIV-positive children in the tPITC group were diagnosed earlier at WHO stage 1, and cases were mostly diagnosed at WHO stage 3 (39.1%) (p < 0.0001) in the bPITC group. Among the children who tested HIV-positive, 85.0% and 52.5% from the tPITC and bPITC group respectively, were enrolled on ART (p = 0.0018). Conclusions: The tPITC and bPITC strategies demonstrated notable high HIV testing acceptance. tPITC was superior to bPITC in terms of case detection, case detection earliness and linkage to care. These findings indicate that tPITC is effective in case detection and linkage of children and adolescents to ART
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