Unique human immune signature of Ebola virus disease in Guinea.

Despite the magnitude of the Ebola virus disease (EVD) outbreak in West Africa, there is still a fundamental lack of knowledge about the pathophysiology of EVD. In particular, very little is known about human immune responses to Ebola virus. Here we evaluate the physiology of the human T cell immune response in EVD patients at the time of admission to the Ebola Treatment Center in Guinea, and longitudinally until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we identify an immune signature that is unique in EVD fatalities. Fatal EVD was characterized by a high percentage of CD4(+) and CD8(+) T cells expressing the inhibitory molecules CTLA-4 and PD-1, which correlated with elevated inflammatory markers and high virus load. Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation, despite comparable overall T cell activation. Concomitant with virus clearance, survivors mounted a robust Ebola-virus-specific T cell response. Our findings suggest that dysregulation of the T cell response is a key component of EVD pathophysiology.

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa.

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.

Analysis of Diagnostic Findings From the European Mobile Laboratory in Guéckédou, Guinea, March 2014 Through March 2015.

BACKGROUND: A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. METHODS: The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcription-polymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. RESULTS: The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged <15 years had the highest proportion of Ebola virus-malaria parasite coinfections. The case-fatality ratio was high in patients aged <5 years (80%) and those aged >74 years (90%) and low in patients aged 10-19 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of <5 and ≥45 years, and, among children aged 5-14 years, malaria parasite coinfection were independent determinants of a poor EVD outcome. CONCLUSIONS: Virus load, age, and malaria parasite coinfection play a role in the outcome of EVD.

Mobile diagnostics in outbreak response, not only for Ebola: a blueprint for a modular and robust field laboratory.

We established a modular, rapidly deployable laboratory system that provides diagnostic support in resource-limited, remote areas. Developed as a quick response asset to unusual outbreaks of infectious diseases worldwide, several of these laboratories have been used as part of the World Health Organization response to the Ebola virus outbreaks by teams of the 'European Mobile Lab' project in West Africa since March 2014. Within three days from deployment, the first European mobile laboratory became operational at the Ebola Treatment Unit (ETU) in Guéckédou, southern Guinea. Deployment in close proximity to the ETU decreased the turnaround time to an average of 4 h instead of several days in many cases. Between March 2014 and May 2015, more than 5,800 samples were tested in this field laboratory. Further EMLab units were deployed to Nigeria, Liberia and Sierra Leone in the following months of the Ebola outbreak. The technical concept of the EMLab units served as a blueprint for other mobile Ebola laboratories which have been set up in Mali, Côte d'Ivoire, Sierra Leone and other countries in West Africa. Here, we describe design, capabilities and utility of this deployable laboratory system for use in response to disease outbreaks, epidemiological surveillance and patient management.

Plasmodium knowlesi infection in humans, Cambodia, 2007-2010.

Two cases of Plasmodium knowlesi infection in humans were identified in Cambodia by 3 molecular detection assays and sequencing. This finding confirms the widespread distribution of P. knowlesi malaria in humans in Southeast Asia. Further wide-scale studies are required to assess the public health relevance of this zoonotic malaria parasite.

Efficiency of fine-needle aspiration compared with other sampling techniques for laboratory diagnosis of Buruli ulcer disease.

In accordance with recent WHO recommendations, this study evaluates the sensitivities of PCR and microscopy for fine-needle aspiration (FNA) versus techniques involving swabs and punch biopsy specimens and suggests that FNA can replace punch biopsies for nonulcerative lesions and may serve as an alternative for ulcerative lesions in cases where scarred edges prevent the collection of swabs.

Comparative study of the sensitivity of different diagnostic methods for the laboratory diagnosis of Buruli ulcer disease.

BACKGROUND: Several diagnostic laboratory methods are available for case confirmation of Buruli ulcer disease. This study assessed the sensitivity of various diagnostic tests in relation to clinical presentation of the disease, type of diagnostic specimen, and treatment history. METHODS: Swab samples, 3-mm punch biopsy tissue specimens, and surgically excised tissue specimens from 384 individuals with suspected Buruli ulcer disease were obtained at 9 different study sites in Ghana and were evaluated with dry reagent-based polymerase chain reaction (PCR), microscopic examination, culture, and histopathological analysis. The study subjects presented with nonulcerative and ulcerative lesions and were divided into 3 treatment groups: (1) previously untreated patients scheduled for antimycobacterial treatment, (2) patients treated with surgery alone, and (3) patients treated with surgery in combination with previous antimycobacterial treatment. RESULTS: Of 384 suspected cases of Buruli ulcer disease, 268 were confirmed by at least 1 positive test result. The overall sensitivity of PCR (85%) was significantly higher than that of microscopic examination (57%) and culture (51%). After data were stratified by treatment group, type of lesion, and diagnostic specimen type, analysis revealed that PCR of 3-mm punch biopsy tissue specimens (obtained from previously untreated nonulcerative lesions) and of swab samples (obtained from previously untreated ulcers) had the highest diagnostic sensitivity (94% and 90%, respectively). Although duration of the disease did not significantly influence the sensitivity of any test, previous antimycobacterial treatment was significantly associated with decreased sensitivity of PCR and culture. CONCLUSIONS: Across all subgroups, PCR had the highest sensitivity. PCR assessment of 3-mm punch biopsy tissue specimens proved to be the best diagnostic tool for nonulcerative lesions, and PCR assessment of swab samples was the best diagnostic tool for ulcerative lesions. For monitoring of antimycobacterial treatment success within controlled trials, however, only culture is appropriate.

Comparison of different methods for delayed post-mortem diagnosis of falciparum malaria.

BACKGROUND: Between 10,000 and 12,000 cases of imported malaria are notified in the European Union each year. Despite an excellent health care system, fatalities do occur. In case of advanced autolysis, the post-mortem diagnostic is impaired. Quicker diagnosis could be achieved by using rapid diagnostic malaria tests. METHODS: In order to evaluate different methods for the post-mortem diagnosis of Plasmodium falciparum malaria in non-immunes, a study was performed on the basis of forensic autopsies of corpses examined at variable intervals after death in five cases of fatal malaria (with an interval of four hours to five days), and in 20 cases of deaths unrelated to malaria. Detection of parasite DNA by PCR and an immunochromatographic test (ICT) based upon the detection of P. falciparum histidine-rich protein 2 (PfHRP2) were compared with the results of microscopic examination of smears from cadaveric blood, histopathological findings, and autopsy results. RESULTS: In all cases of fatal malaria, post-mortem findings were unsuspicious for the final diagnosis, and autoptic investigations, including histopathology, were only performed because of additional information by police officers and neighbours. Macroscopic findings during autopsy were unspecific. Histopathology confirmed sequestration of erythrocytes and pigment in macrophages in most organs in four patients (not evaluable in one patient due to autolysis). Microscopy of cadaveric blood smears revealed remnants of intraerythrocytic parasites, and was compromised or impossible due to autolysis in two cases. PCR and ICT performed with cadaveric blood were positive in all malaria patients and negative in all controls. CONCLUSION: In non-immune fatalities with unclear anamnesis, ICT can be recommended as a sensitive and specific tool for post-mortem malaria diagnosis, which is easier and faster than microscopy, and also applicable when microscopic examination is impossible due to autolysis. PCR is more expensive and time-consuming, but may be used as confirmatory test. In highly endemic areas where asymptomatic parasitaemia is common, confirmation of the diagnosis of malaria as the cause of death has to rely on histopathological findings.

Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection.

BACKGROUND: In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study. METHODOLOGY: Stool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months. PRINCIPAL FINDINGS: Out of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic "gold standard", the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100). CONCLUSIONS: The use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments.

Acute undifferentiated febrile illness in rural Cambodia: a 3-year prospective observational study.

In the past decade, malaria control has been successfully implemented in Cambodia, leading to a substantial decrease in reported cases. Wide-spread use of malaria rapid diagnostic tests (RDTs) has revealed a large burden of malaria-negative fever cases, for which no clinical management guidelines exist at peripheral level health facilities. As a first step towards developing such guidelines, a 3-year cross-sectional prospective observational study was designed to investigate the causes of acute malaria-negative febrile illness in Cambodia. From January 2008 to December 2010, 1193 febrile patients and 282 non-febrile individuals were recruited from three health centers in eastern and western Cambodia. Malaria RDTs and routine clinical examination were performed on site by health center staff. Venous samples and nasopharyngeal throat swabs were collected and analysed by molecular diagnostic tests. Blood cultures and blood smears were also taken from all febrile individuals. Molecular testing was applied for malaria parasites, Leptospira, Rickettsia, O. tsutsugamushi, Dengue- and Influenza virus. At least one pathogen was identified in 73.3% (874/1193) of febrile patient samples. Most frequent pathogens detected were P. vivax (33.4%), P. falciparum (26.5%), pathogenic Leptospira (9.4%), Influenza viruses (8.9%), Dengue viruses (6.3%), O. tsutsugamushi (3.9%), Rickettsia (0.2%), and P. knowlesi (0.1%). In the control group, a potential pathogen was identified in 40.4%, most commonly malaria parasites and Leptospira. Clinic-based diagnosis of malaria RDT-negative cases was poorly predictive for pathogen and appropriate treatment. Additional investigations are needed to understand their impact on clinical disease and epidemiology, and the possible role of therapies such as doxycycline, since many of these pathogens were seen in non-febrile subjects.

Laboratory confirmation of Buruli ulcer disease in Togo, 2007-2010.

BACKGROUND: Since the early 1990s more than 1,800 patients with lesions suspicious for Buruli ulcer disease (BUD) have been reported from Togo. However, less than five percent of these were laboratory confirmed. Since 2007, the Togolese National Buruli Ulcer Control Program has been supported by the German Leprosy and Tuberculosis Relief Association (DAHW). Collaboration with the Department for Infectious Diseases and Tropical Medicine (DITM), University Hospital, Munich, Germany, allowed IS2404 PCR analysis of diagnostic samples from patients with suspected BUD during a study period of three years. METHODOLOGY/PRINCIPAL FINDINGS: The DAHW integrated active BUD case finding in the existing network of TB/Leprosy Controllers and organized regular training and outreach activities to identify BUD cases at community level. Clinically suspected cases were referred to health facilities for diagnosis and treatment. Microscopy was carried out locally, external quality assurance (EQA) at DITM. Diagnostic samples from 202 patients with suspected BUD were shipped to DITM, 109 BUD patients (54%) were confirmed by PCR, 43 (29.9%) by microscopy. All patients originated from Maritime Region. EQA for microscopy resulted in 62% concordant results. CONCLUSIONS/SIGNIFICANCE: This study presents a retrospective analysis of the first cohort of clinically suspected BUD cases from Togo subjected to systematic laboratory analysis over a period of three years and confirms the prevalence of BUD in Maritime Region. Intensified training in the field of case finding and sample collection increased the PCR case confirmation rate from initially less than 50% to 70%. With a PCR case confirmation rate of 54% for the entire study period the WHO standards (case confirmation rate ≥50%) have been met. EQA for microscopy suggests the need for intensified supervision and training. In January 2011 the National Hygiene Institute, Lomé, has assumed the role of a National Reference Laboratory for PCR confirmation and microscopy.

A genotypic approach for detection, identification, and characterization of drug resistance in Mycobacterium ulcerans in clinical samples and isolates from Ghana.

Standardized antimycobacterial therapy is considered the treatment of choice for Buruli ulcer disease. To assess the prevalence of drug resistance among clinical Mycobacterium ulcerans isolates in Ghana, we conducted a sequence-based approach to detect mutations associated with drug resistance. We subjected clinical samples to direct DNA sequencing of rpoB and rpsL genes and compared culture and whole-genome extracts regarding the efficiency of sequence analysis; 99.1% (rpoB) and 100% (rpsL) of the patients harbored M. ulcerans wild type. In one isolate (0.9%), a point mutation of the rpoB gene at codon Ser522 leading to an amino acid change was detected. Culture extracts yielded a significantly higher sequencing efficiency than whole-genome extracts. Our data suggest a low level of drug resistance in Ghana. However, mutations associated with drug resistance do occur and require monitoring. Improved techniques are necessary to enhance the efficiency of sequence analysis of whole-genome extracts.

Cutaneous leishmaniasis (Leishmania tropica) in a German tourist after travel to Greece.

We report on a German tourist returning from vacations in Southern Greece with cutaneous leishmaniasis (CL) presenting as multiple erythematosquamous lesions caused by Leishmania tropica (zymodeme MON-57). In spite of its endemicity, only few data are available on the incidence and current distribution of CL in Greece, which may allow for an assessment of the risk for travelers.

Relapsing cutaneous leishmaniasis in a patient with ankylosing spondylitis treated with infliximab.

A 31-year-old man with ankylosing spondylitis, receiving treatment with infliximab, presented with a large progressive cutaneous ulcer at the right knee. Biopsies showed Leishmania amastigotes, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis showed Leishmania infantum as the causative agent. After treatment with miltefosine, the ulcer resolved completely, and infliximab was reinstituted because of progression of spondylitis. After 1 year, there was a recurrent ulcer at the same site being positive for Leishmania DNA by PCR. Local treatment with sodium stibogluconate resulted in complete regression. Cutaneous leishmaniasis should be added to the list of opportunistic infections associated with anti-tumor necrosis factor (TNF) treatment. Despite recurrences, antileish-manial treatment may be effective in cases without alternatives to anti-TNF therapy.

Outcome of patients with buruli ulcer after surgical treatment with or without antimycobacterial treatment in Ghana.

This study assesses the frequency of recurrences and treatment outcome after surgery of buruli ulcer disease (BUD) with or without concomitant antimycobacterial treatment. Of 129 laboratory-confirmed BUD patients who underwent surgery in two treatment centers in Ghana, 79 (61%) were retrieved for follow-up 4-29 months after the initial treatment. Among 7 (9%) recurrent cases no significant association was found between recurrences and clinical or treatment specific factors including antimycobacterial treatment. In 21 (27%) patients, a reduced range of motion (ROM) of one or more joints was detected. Lesions other than nodules, joint involvement, and skin grafting were identified as independent risk factors. Functional limitations hampering daily activities were perceived by 22% of the patients. Compared with other studies the recurrence rate was relatively low, functional limitations were, however, frequent. This emphasizes the need for improvement of pre- and post-treatment wound care as well as rehabilitation programs.

Validity of malaria diagnosis in nonimmune travelers in endemic areas.

BACKGROUND: Malaria has to be considered in all febrile travelers during or after a stay in endemic areas. However, malaria diagnosis in endemic countries may be inaccurate due to limited capacity and lack of resources of local health services. To assess the validity of malaria diagnosis in travelers in endemic areas, we investigated the retrospective confirmation of malaria by detection of specific antibodies. METHODS: Sera of 105 nonimmune travelers who presented between 2003 and 2005 with a history of diagnosis and treatment of malaria during a stay in malaria-endemic countries within the previous 6 months were analyzed for antibodies against Plasmodium falciparum and Plasmodium vivax blood forms by an indirect immunofluorescence test. About 241 follow-up sera from 176 nonimmune patients with microscopically confirmed malaria served as a control group. RESULTS: Antibodies against plasmodia were detectable within 180 days after reported date of diagnosis and treatment in 16 of 105 travelers (15.2%) only. In the control group, 71.6% of analyzed sera (151 of 211) showed positive results within this interval. Within 8 to 60 days after diagnosis of malaria, the seropositivity rates were 17.9% for travelers (n = 56) and 92.4% for controls (n = 92). CONCLUSIONS: Although the sensitivity of malaria serology for retrospective confirmation of malaria is limited, the results of this analysis strongly suggest that the majority of travelers with a recent history of malaria diagnosed and treated in endemic countries did not have malaria and that diagnosis of malaria during travel in endemic areas is frequently incorrect.