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1.  Towards Improving Point-of-Care Diagnosis of Non-malaria Febrile Illness: A Metabolomics Approach 
PLoS Neglected Tropical Diseases  2016;10(3):e0004480.
Non-malaria febrile illnesses such as bacterial bloodstream infections (BSI) are a leading cause of disease and mortality in the tropics. However, there are no reliable, simple diagnostic tests for identifying BSI or other severe non-malaria febrile illnesses. We hypothesized that different infectious agents responsible for severe febrile illness would impact on the host metabololome in different ways, and investigated the potential of plasma metabolites for diagnosis of non-malaria febrile illness.
We conducted a comprehensive mass-spectrometry based metabolomics analysis of the plasma of 61 children with severe febrile illness from a malaria-endemic rural African setting. Metabolite features characteristic for non-malaria febrile illness, BSI, severe anemia and poor clinical outcome were identified by receiver operating curve analysis.
Principal Findings
The plasma metabolome profile of malaria and non-malaria patients revealed fundamental differences in host response, including a differential activation of the hypothalamic-pituitary-adrenal axis. A simple corticosteroid signature was a good classifier of severe malaria and non-malaria febrile patients (AUC 0.82, 95% CI: 0.70–0.93). Patients with BSI were characterized by upregulated plasma bile metabolites; a signature of two bile metabolites was estimated to have a sensitivity of 98.1% (95% CI: 80.2–100) and a specificity of 82.9% (95% CI: 54.7–99.9) to detect BSI in children younger than 5 years. This BSI signature demonstrates that host metabolites can have a superior diagnostic sensitivity compared to pathogen-detecting tests to identify infections characterized by low pathogen load such as BSI.
This study demonstrates the potential use of plasma metabolites to identify causality in children with severe febrile illness in malaria-endemic settings.
Author Summary
In the tropics, malaria is commonly attributed to be the cause of most childhood fevers, while in fact this condition is more commonly caused by other pathogens that are clinically indistinguishable from malaria. These so-called non-malaria febrile illnesses include bacterial bloodstream infections, which are associated with a higher mortality than malaria. Most health care facilities in the tropics have malaria diagnostic tests available, but tests for non-malarial febrile illnesses are extremely limited. There is the critical need for new tests that can address the question ‘if a febrile patient is not suffering from malaria, then what is it and what treatment will be effective?’ Using metabolomics, we have comprehensively screened the biochemical profile of patients with severe febrile illness for biological markers of non-malaria febrile illness. The results show that severe malaria and non-malaria febrile illness trigger a distinct metabolic response in the host. We demonstrate that this pathophysiological difference can be exploited for differential diagnosis of severe febrile illness and identification of patients with bacterial bloodstream infections.
PMCID: PMC4778767  PMID: 26943791
2.  Diagnosis of Bacterial Bloodstream Infections: A 16S Metagenomics Approach 
PLoS Neglected Tropical Diseases  2016;10(2):e0004470.
Bacterial bloodstream infection (bBSI) is one of the leading causes of death in critically ill patients and accurate diagnosis is therefore crucial. We here report a 16S metagenomics approach for diagnosing and understanding bBSI.
Methodology/Principal Findings
The proof-of-concept was delivered in 75 children (median age 15 months) with severe febrile illness in Burkina Faso. Standard blood culture and malaria testing were conducted at the time of hospital admission. 16S metagenomics testing was done retrospectively and in duplicate on the blood of all patients. Total DNA was extracted from the blood and the V3–V4 regions of the bacterial 16S rRNA genes were amplified by PCR and deep sequenced on an Illumina MiSeq sequencer. Paired reads were curated, taxonomically labeled, and filtered. Blood culture diagnosed bBSI in 12 patients, but this number increased to 22 patients when combining blood culture and 16S metagenomics results. In addition to superior sensitivity compared to standard blood culture, 16S metagenomics revealed important novel insights into the nature of bBSI. Patients with acute malaria or recovering from malaria had a 7-fold higher risk of presenting polymicrobial bloodstream infections compared to patients with no recent malaria diagnosis (p-value = 0.046). Malaria is known to affect epithelial gut function and may thus facilitate bacterial translocation from the intestinal lumen to the blood. Importantly, patients with such polymicrobial blood infections showed a 9-fold higher risk factor for not surviving their febrile illness (p-value = 0.030).
Our data demonstrate that 16S metagenomics is a powerful approach for the diagnosis and understanding of bBSI. This proof-of-concept study also showed that appropriate control samples are crucial to detect background signals due to environmental contamination.
Author Summary
Bacterial bloodstream infection (bBSI) is one of the biggest causes of mortality in critically ill patients and standard diagnosis is still done by blood culture methods. Parallel deep sequencing of the 16S ribosomal RNA genes (16S metagenomics) is a new and rapidly evolving research field for profiling bacterial communities. We designed a 16S metagenomics approach for the identification of bacteria in the blood of patients with a bBSI, and evaluated its performance in 75 children with severe febrile illness in Burkina Faso. In addition to superior sensitivity compared to standard blood culture, 16S metagenomics revealed important novel insights into the nature of bBSI. Patients with acute malaria or recently recovered from acute malaria are at increased risk of presenting polymicrobial bloodstream infection, which was in itself a significant risk for non-survival. This proof-of-concept study shows that 16S metagenomics is a powerful approach to diagnose and understand bBSI but also that appropriate control samples are crucial for correct data interpretation.
PMCID: PMC4771206  PMID: 26927306
3.  Patient satisfaction with a teleradiology service in general practice 
BMC Family Practice  2016;17:17.
Accessibility to secondary health services is not always easy for patients who live at a great distance of hospital. In these circumstances, transferring diagnostic tools and treatment options to primary care could prove beneficial for patients. To do so, the quality of medical care and the costs and benefits of the approach need to be assessed. However, the patient perspective is equally important, offering important insights.
In a cross-sectional study we investigate the satisfaction of patients toward a new teleradiology facility offered a general practice on Ameland, an island in the Netherlands.
A questionnaire was created based on the Dutch version of the Patient Satisfaction Questionnaire III and completed by all patients after receiving an x-ray in primary care between June 1, 2007 and June 1, 2009. Those who received more than one x-ray in that period were included only once. The technical and interpersonal skills of doctors were rated out the sum score of the questionnaire namely 25 and 30, respectively. Analysis of variance (ANOVA) was used to analyze the differences between the means of the satisfaction subscales and the patient characteristics.
The response proportion was after reminder 65 % (381/587 patients). Satisfaction with the technical skills of the doctor providing the teleradiology service was 22.4 ± 3.7, while satisfaction with the interpersonal skills of the doctor during the diagnostic phase was 26.8 ± 3.8. Island residents, the elderly, and those with no history of trauma were more satisfied with the technical and interpersonal aspects of the consultation than non-residents, younger patients, and those with a history of trauma.
Patients in the island community of Ameland experienced high levels of satisfaction with the teleradiology service offered in primary care.
Electronic supplementary material
The online version of this article (doi:10.1186/s12875-016-0418-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4748486  PMID: 26864118
Teleradiology; Patient satisfaction; General practice; Family practice
4.  Performance of Microscopy for the Diagnosis of Malaria and Human African Trypanosomiasis by Diagnostic Laboratories in the Democratic Republic of the Congo: Results of a Nation-Wide External Quality Assessment 
PLoS ONE  2016;11(1):e0146450.
The present External Quality Assessment (EQA) assessed microscopy of blood parasites among diagnostic laboratories in the Democratic Republic of the Congo. The EQA addressed 445 participants in 10/11 provinces (October 2013–April 2014). Participants were sent a panel of five slides and asked to return a routinely stained slide which was assessed for quality of preparation and staining. Response rate was 89.9% (400/445). For slide 1 (no parasites), 30.6% participants reported malaria, mostly Plasmodium falciparum. Only 11.0% participants reported slide 2 (Plasmodium malariae) correctly, 71.0% reported “malaria” or “Plasmodium falciparum” (considered acceptable). Slide 3 contained Plasmodium falciparum (109/μl) and Trypanosoma brucei brucei trypomastigotes: they were each reported by 32.5% and 16.5% participants respectively, 6.0% reported both. Slide 4 (Trypanosoma) was recognised by 44.9% participants. Slide 5 (Plasmodium ovale) was correctly reported by 6.2% participants, another 68.8% replied “malaria” or “Plasmodium falciparum” (considered acceptable). Only 13.6% of routine slides returned were correctly prepared and stained. The proportion of correct/acceptable scores for at least 4/5 slides was higher among EQA-experienced participants compared to first time participants (40.9% versus 22.4%, p = 0.001) and higher among those being trained < 2 years ago compared to those who were not (42.9% versus 26.3%, p = 0.01). Among diagnostic laboratories in Democratic Republic of the Congo, performance of blood parasite microscopy including non-falciparum species and Trypanosoma was poor. Recent training and previous EQA participation were associated with a better performance.
PMCID: PMC4720473  PMID: 26788725
6.  Diagnostic performance of a novel loop-mediated isothermal amplification (LAMP) assay targeting the apicoplast genome for malaria diagnosis in a field setting in sub-Saharan Africa 
Malaria Journal  2015;14:396.
New diagnostic tools to detect reliably and rapidly asymptomatic and low-density malaria infections are needed as their treatment could interrupt transmission. Isothermal amplification techniques are being explored for field diagnosis of malaria. In this study, a novel molecular tool (loop-mediated isothermal amplification—LAMP) targeting the apicoplast genome of Plasmodium falciparum was evaluated for the detection of asymptomatic malaria-infected individuals in a rural setting in The Gambia.
A blood was collected from 341 subjects (median age 9 years, range 1–68 years) screened for malaria. On site, a rapid diagnostic test (RDT, SD Bioline Malaria Antigen P.f) was performed, thick blood films (TBF) slides for microscopy were prepared and dry blood spots (DBS) were collected on Whatman® 903 Specimen collection paper. The TBF and DBS were transported to the field laboratory where microscopy and LAMP testing were performed. The latter was done on DNA extracted from the DBS using a crude (methanol/heating) extraction method. A laboratory-based PCR amplification was done on all the samples using DNA extracted with the Qiagen kit and its results were taken as reference for all the other tests.
Plasmodium falciparum malaria prevalence was 37 % (127/341) as detected by LAMP, 30 % (104/341) by microscopy and 37 % (126/341) by RDT. Compared to the reference PCR method, sensitivity was 92 % for LAMP, 78 % for microscopy, and 76 % for RDT; specificity was 97 % for LAMP, 99 % for microscopy, and 88 % for RDT. Area under the receiver operating characteristic (ROC) curve in comparison with the reference standard was 0.94 for LAMP, 0.88 for microscopy and 0.81 for RDT. Turn-around time for the entire LAMP assay was approximately 3 h and 30 min for an average of 27 ± 9.5 samples collected per day, compared to a minimum of 10 samples an hour per operator by RDT and over 8 h by microscopy.
The LAMP assay could produce reliable results the same day of the screening. It could detect a higher proportion of low density malaria infections than the other methods tested and may be used for large campaigns of systematic screening and treatment.
PMCID: PMC4599330  PMID: 26450599
Malaria diagnosis; LAMP; RDT; Microscopy; PCR; Field-based; Resource-limited settings
7.  Diagnosis of neglected tropical diseases among patients with persistent digestive disorders (diarrhoea and/or abdominal pain ≥14 days): Pierrea multi-country, prospective, non-experimental case–control study 
BMC Infectious Diseases  2015;15:338.
Diarrhoea still accounts for considerable mortality and morbidity worldwide. The highest burden is concentrated in tropical areas where populations lack access to clean water, adequate sanitation and hygiene. In contrast to acute diarrhoea (<14 days), the spectrum of pathogens that may give rise to persistent diarrhoea (≥14 days) and persistent abdominal pain is poorly understood. It is conceivable that pathogens causing neglected tropical diseases play a major role, but few studies investigated this issue. Clinical management and diagnostic work-up of persistent digestive disorders in the tropics therefore remain inadequate. Hence, important aspects regarding the pathogenesis, epidemiology, clinical symptomatology and treatment options for patients presenting with persistent diarrhoea and persistent abdominal pain should be investigated in multi-centric clinical studies.
This multi-country, prospective, non-experimental case–control study will assess persistent diarrhoea (≥14 days; in individuals aged ≥1 year) and persistent abdominal pain (≥14 days; in children/adolescents aged 1–18 years) in up to 2000 symptomatic patients and 2000 matched controls. Subjects from Côte d’Ivoire, Indonesia, Mali and Nepal will be clinically examined and interviewed using a detailed case report form. Additionally, each participant will provide a stool sample that will be examined using a suite of diagnostic methods (i.e., microscopic techniques, rapid diagnostic tests, stool culture and polymerase chain reaction) for the presence of bacterial and parasitic pathogens. Treatment will be offered to all infected participants and the clinical treatment response will be recorded. Data obtained will be utilised to develop patient-centred clinical algorithms that will be validated in primary health care centres in the four study countries in subsequent studies.
Our research will deepen the understanding of the importance of persistent diarrhoea and related digestive disorders in the tropics. A diversity of intestinal pathogens will be assessed for potential associations with persistent diarrhoea and persistent abdominal pain. Different diagnostic methods will be compared, clinical symptoms investigated and diagnosis-treatment algorithms developed for validation in selected primary health care centres. The findings from this study will improve differential diagnosis and evidence-based clinical management of digestive syndromes in the tropics.
Trial registration; identifier: NCT02105714.
PMCID: PMC4539676  PMID: 26282537
Bacteria; Diagnosis-treatment algorithm; Helminths; Intestinal protozoa; Neglected tropical diseases; Persistent diarrhoea; Côte d’Ivoire; Indonesia; Mali; Nepal
8.  Fracture diagnostics, unnecessary travel and treatment: a comparative study before and after the introduction of teleradiology in a remote general practice 
BMC Family Practice  2015;16:53.
Teleradiology entails attainment of x-rays in one location, transfer over some distance and assessment at another location for diagnosis or consultation. This study documents fracture diagnostics, unnecessary trips to the hospital, treatment and number of x-rays for the years 2006 and 2009, before and after the introduction of teleradiology in a general practice on the island of Ameland in the north of the Netherlands.
In a retrospective, descriptive, observational before and after study of the introduction of x-ray facilities in an island-based general practice, we compared the number of accurately diagnosed fractures, unnecessary trips, treatments and number of x-rays taken in 2006 when only a hospital x-ray facility was available 5 hours away with those in 2009 after an x-ray facility became available at a local general practice. All patients visiting a general practice on the island of Ameland in 2006 and 2009 with trauma and clinical suspicion of a fracture, dislocation or sprain were included in the study. The initial clinical diagnoses, including those based on the outcomes of x-rays, were compared for the two years and also whether the patients were treated at home or in hospital.
A total of 316 and 490 patients with trauma visited a general practice in 2006 and 2009, respectively. Of these patients, 66 and 116 were found to have fractures or dislocations in the two years, respectively. In 2006, 83 x-rays were ordered; in 2009, this was 284. In 2006, 9 fractures were missed; in 2009, this was only 2. In 2006, 15 patients with fractures or dislocations were treated at the general practice; in 2009, this had increased to 77.
Since the introduction of teleradiology the number of missed fractures in patients visiting the general practice with trauma and the number of the unnecessary trips to a hospital are reduced. In addition more patients with fractures and dislocations can be treated in the general practice as opposed to the hospital.
PMCID: PMC4424890  PMID: 25943473
Teleradiology; Family practice; General practice; Diagnosis; Treatment; Trauma fractures
9.  Antibiotic prescription behaviours in Lao People's Democratic Republic: a knowledge, attitude and practice survey 
To assess the antibiotic prescribing practices of doctors working in the Lao People's Democratic Republic and their knowledge of local antibiotic resistance patterns.
Doctors attending morning meetings in 25 public hospitals in four provinces were asked to complete a knowledge, attitude and practice survey. The questionnaire contained 43 multiple choice questions that the doctor answered at the time of the meeting.
The response rate was 83.4% (386/463). Two hundred and seventy doctors (59.8%) declared that they had insufficient information about antibiotics. Only 14.0% (54/386) recognized the possibility of cephalosporin cross-resistance in methicillin-resistant Staphylococcus aureus. Most participants had no information about local antibiotic resistance for Salmonella Typhi (211/385, 54.8%) and hospital-acquired pneumonia (253/384, 65.9%). Unnecessary antibiotic prescriptions were considered as harmless by 115 participants and 148 considered locally-available generic antibiotics to be of poor quality. Nearly three-quarters (280/386) of participants agreed that it was difficult to select the correct antibiotics. Most participants (373/386) welcomed educational programmes on antibiotic prescribing and 65.0% (249/383) preferred local over international antibiotic guidelines.
Doctors in the Lao People's Democratic Republic seem to favour antibiotic prescribing interventions. Health authorities should consider a capacity building programme that incorporates antibiotic prescribing and hospital infection control.
PMCID: PMC4431558  PMID: 26229186
10.  Toxocariasis Diagnosed in International Travelers at the Institute of Tropical Medicine, Antwerp, Belgium, from 2000 to 2013 
PLoS Neglected Tropical Diseases  2015;9(3):e0003559.
Although infection with Toxocara canis or T. catis (commonly referred as toxocariasis) appears to be highly prevalent in (sub)tropical countries, information on its frequency and presentation in returning travelers and migrants is scarce. In this study, we reviewed all cases of asymptomatic and symptomatic toxocariasis diagnosed during post-travel consultations at the reference travel clinic of the Institute of Tropical Medicine, Antwerp, Belgium. Toxocariasis was considered as highly probable if serum Toxocara-antibodies were detected in combination with symptoms of visceral larva migrans if present, elevated eosinophil count in blood or other relevant fluid and reasonable exclusion of alternative diagnosis, or definitive in case of documented seroconversion. From 2000 to 2013, 190 travelers showed Toxocara-antibodies, of a total of 3436 for whom the test was requested (5.5%). Toxocariasis was diagnosed in 28 cases (23 symptomatic and 5 asymptomatic) including 21 highly probable and 7 definitive. All but one patients were adults. Africa and Asia were the place of acquisition for 10 and 9 cases, respectively. Twelve patients (43%) were short-term travelers (< 1 month). Symptoms, when present, developed during travel or within 8 weeks maximum after return, and included abdominal complaints (11/23 symptomatic patients, 48%), respiratory symptoms and skin abnormalities (10 each, 43%) and fever (9, 39%), often in combination. Two patients were diagnosed with transverse myelitis. At presentation, the median blood eosinophil count was 1720/μL [range: 510–14160] in the 21 symptomatic cases without neurological complication and 2080/μL [range: 1100–2970] in the 5 asymptomatic individuals. All patients recovered either spontaneously or with an anti-helminthic treatment (mostly a 5-day course of albendazole), except both neurological cases who kept sequelae despite repeated treatments and prolonged corticotherapy. Toxocariasis has to be considered in travelers returning from a (sub)tropical stay with varying clinical manifestations or eosinophilia. Prognosis appears favorable with adequate treatment except in case of neurological involvement.
Author Summary
Toxocariasis is a zoonosis of worldwide distribution caused by dog (Toxocara canis) or cat (T. catis) roundworm that can be fully asymptomatic or may cause significant disease such as a the systemic syndrome called visceral larva migrans as well as neurological or eye manifestations. Toxocariasis prevails in tropical areas, but information about this disease in travelers and migrants is scarce. In this study, we describe in detail a case series of 28 international travelers, mostly adults, diagnosed with toxocariasis from 2000 to 2013 at the reference travel clinic of the Institute of Tropical Medicine of Antwerp, Belgium. We found this infection in all types of travelers returning from any part of the world. Clinical symptoms, when present, varied widely and an increase of the blood eosinophil count was almost always present. Morbidity was substantial and 2 patients had severe neurological complications. Diagnosis was difficult in travelers because the illness often resembled other tropical infections. Recovery was, however, complete, either spontaneously or with anti-parasitic drugs, except in both cases with neurological involvement. Toxocariasis is one of the numerous parasitic infections to consider in travelers returning from the tropics with any type of symptoms or with an increased blood eosinophil count.
PMCID: PMC4351981  PMID: 25746418
11.  Multi Locus Variable-Number Tandem Repeat (MLVA) Typing Tools Improved the Surveillance of Salmonella Enteritidis: A 6 Years Retrospective Study 
PLoS ONE  2015;10(2):e0117950.
Surveillance of Salmonella enterica subsp. enterica serovar Enteritidis is generally considered to benefit from molecular techniques like multiple-locus variable-number of tandem repeats analysis (MLVA), which allow early detection and confinement of outbreaks. Here, a surveillance study, including phage typing, antimicrobial susceptibility testing and MLVA on 1,535 S. Enteritidis isolates collected between 2007 and 2012, was used to evaluate the added value of MLVA for public health surveillance in Belgium. Phage types PT4, PT8, PT21, PT1, PT6, PT14b, PT28 and PT13 dominate the Belgian S. Enteritidis population. The isolates of S. Enteritidis were most frequently susceptible to all antibiotics tested. 172 different MLVA profiles were detected, of which 9 frequent profiles included 67.2% of the S. Enteritidis population. During a serial passage experiment on selected isolates to investigate the in vitro stability of the 5 MLVA loci, no variations over time were observed indicating that the MLVA profiles were stable. The MLVA profile of isolates originating from different outbreaks in the Democratic Republic of the Congo (DRC) between 2010 and 2011 were distinct from any of the MLVA profiles found in Belgian isolates throughout the six year observational period and demonstrates that MLVA improves public health surveillance of S. Enteritidis. However, MLVA should be complemented with other subtyping methods when investigating outbreaks is caused by the most common MLVA profile.
PMCID: PMC4334661  PMID: 25693200
12.  SMS photograph-based external quality assessment of reading and interpretation of malaria rapid diagnostic tests in the Democratic Republic of the Congo 
Malaria Journal  2015;14:26.
The present External Quality Assessment (EQA) assessed reading and interpretation of malaria rapid diagnostic tests (RDTs) in the Democratic Republic of the Congo (DRC).
The EQA consisted of (i) 10 high-resolution printed photographs displaying cassettes with real-life results and multiple choice questions (MCQ) addressing individual health workers (HW), and (ii) a questionnaire on RDT use addressing the laboratory of health facilities (HF). Answers were transmitted through short message services (SMS).
The EQA comprised 2344 HW and 1028 HF covering 10/11 provinces in DRC. Overall, median HW score (sum of correct answers on 10 MCQ photographs for each HW) was 9.0 (interquartile range 7.5 – 10); MCQ scores (the % of correct answers for a particular photograph) ranged from 54.8% to 91.6%. Most common errors were (i) reading or interpreting faint or weak line intensities as negative (3.3%, 7.2%, 24.3% and 29.1% for 4 MCQ photographs), (ii) failure to distinguish the correct Plasmodium species (3.4% to 7.0%), (iii) missing invalid test results (8.4% and 23.6%) and (iv) missing negative test results (10.0% and 12.4%). HW who were trained less than 12 months ago had best MCQ scores for 7/10 photographs as well as a significantly higher proportion of 10/10 scores, but absolute differences in MCQ scores were small. HW who had participated in a previous EQA performed significantly better for 4/10 photographs compared to those who had not. Except for two photographs, MCQ scores were comparable for all levels of the HF hierarchy and non-laboratory staff (HW from health posts) had similar performance as to laboratory staff. Main findings of the questionnaire were (i) use of other RDT products than recommended by the national malaria control programme (nearly 20% of participating HF), (ii) lack of training for a third (33.6%) of HF, (iii) high proportions (two-thirds, 66.5%) of HF reporting stock-outs.
The present EQA revealed common errors in RDT reading and interpretation by HW in DRC. Performances of non-laboratory and laboratory staff were similar and dedicated training was shown to improve HW competence although to a moderate extent. Problems in supply, distribution and training of RDTs were detected.
PMCID: PMC4318196  PMID: 25626915
Malaria RDT; External quality assessment; Short message service; DRC
13.  Harmonization of malaria rapid diagnostic tests: best practices in labelling including instructions for use 
Malaria Journal  2014;13:505.
Rapid diagnostic tests (RDTs) largely account for the scale-up of malaria diagnosis in endemic settings. However, diversity in labelling including the instructions for use (IFU) limits their interchangeability and user-friendliness. Uniform, easy to follow and consistent labelling, aligned with international standards and appropriate for the level of the end user’s education and training, is crucial but a consolidated resource of information regarding best practices for IFU and labelling of RDT devices, packaging and accessories is not available.
The Roll Back Malaria Partnership (RBM) commissioned the compilation of international standards and regulatory documents and published literature containing specifications and/or recommendations for RDT design, packaging and labelling of in vitro diagnostics (IVD) (which includes RDTs), complemented with a questionnaire based survey of RDT manufacturers and implementers. A summary of desirable RDT labelling characteristics was compiled, which was reviewed and discussed during a RBM Stakeholder consultation meeting and subsequently amended and refined by a dedicated task force consisting of country programme implementers, experts in RDT implementation, IVD regulatory experts and manufacturers.
This process led to the development of consensus documents with a list of suggested terms and abbreviations as well as specifications for labelling of box, device packaging, cassettes, buffer bottle and accessories (lancets, alcohol swabs, transfer devices, desiccants). Emphasis was placed on durability (permanent printing or water-resistant labels), legibility (font size, letter type), comprehension (use of symbols) and ease of reference (e.g. place of labelling on the box or cassette packaging allowing quick oversight). A generic IFU template was developed, comprising background information, a template for procedure and reading/interpretation, a selection of appropriate references and a symbol key of internationally recognized symbols together with suggestions about appropriate lay-out, style and readability.
The present document together with its additional files compiled proposes best practices in labelling and IFU for malaria RDTs. It is expected that compliance with these best practices will increase harmonization among the different malaria RDT products available on the market and improve their user-friendliness.
Electronic supplementary material
The online version of this article (doi:10.1186/1475-2875-13-505) contains supplementary material, which is available to authorized users.
PMCID: PMC4302121  PMID: 25519980
Malaria RDT; Harmonization; Best practices; Labeling; Instructions for use
14.  Intermediate Susceptibility to Ciprofloxacin among Salmonella enterica Serovar Typhi Isolates in Lima, Peru 
Journal of Clinical Microbiology  2014;52(3):968-970.
Thirty-three Salmonella enterica serovar Typhi blood isolates from Lima, Peru (2008 to 2012), were fully susceptible to trimethoprim-sulfamethoxazole, chloramphenicol, ceftriaxone, and tetracycline; 8/33 (24.2%) showed intermediate susceptibility to ciprofloxacin carrying mutations in the quinolone resistance-determining region of the gyrA gene (Ser83-Phe and Asp87-Asn) and in the gyrB gene (Ser464-Phe).
PMCID: PMC3957797  PMID: 24371234
15.  Rotavirus Surveillance in Kisangani, the Democratic Republic of the Congo, Reveals a High Number of Unusual Genotypes and Gene Segments of Animal Origin in Non-Vaccinated Symptomatic Children 
PLoS ONE  2014;9(6):e100953.
Group A rotavirus (RVA) infections form a major public health problem, especially in low-income countries like the Democratic Republic of the Congo (COD). However, limited data on RVA diversity is available from sub-Saharan Africa in general and the COD in particular. Therefore, the first aim of this study was to determine the genetic diversity of 99 RVAs detected during 2007–2010 in Kisangani, COD. The predominant G-type was G1 (39%) and the most predominant P-type was P[6] (53%). A total of eight different G/P-combinations were found: G1P[8] (28%), G8P[6] (26%), G2P[4] (14%), G12P[6] (13%), G1P[6] (11%), G9P[8] (4%), G4P[6] (2%) and G8P[4] (1%). The second aim of this study was to gain insight into the diversity of P[6] RVA strains in the COD. Therefore, we selected five P[6] RVA strains in combination with the G1, G4, G8 (2x) or G12 genotype for complete genome analysis. Complete genome analysis showed that the genetic background of the G1P[6] and G12P[6] strains was entirely composed of genotype 1 (Wa-like), while the segments of the two G8P[6] strains were identified as genotype 2 (DS-1-like). Interestingly, all four strains possessed a NSP4 gene of animal origin. The analyzed G4P[6] RVA strain was found to possess the unusual G4-P[6]-I1-R1-C1-M1-A1-N1-T7-E1-H1 constellation. Although the majority of its genes (if not all), were presumably of porcine origin, this strain was able to cause gastro-enteritis in humans. The high prevalence of unusual RVA strains in the COD highlights the need for continued surveillance of RVA diversity in the COD. These results also underline the importance of complete genetic characterization of RVA strains and indicate that reassortments and interspecies transmission among human and animal RVAs strains occur regularly. Based on these data, RVA vaccines will be challenged with a wide variety of different RVA strain types in the COD.
PMCID: PMC4072759  PMID: 24968018
16.  Bacterial Sepsis in Patients with Visceral Leishmaniasis in Northwest Ethiopia 
BioMed Research International  2014;2014:361058.
Background and Objectives. Visceral leishmaniasis (VL) is one of the neglected diseases affecting the poorest segment of world populations. Sepsis is one of the predictors for death of patients with VL. This study aimed to assess the prevalence and factors associated with bacterial sepsis, causative agents, and their antimicrobial susceptibility patterns among patients with VL. Methods. A cross-sectional study was conducted among parasitologically confirmed VL patients suspected of sepsis admitted to the University of Gondar Hospital, Northwest Ethiopia, from February 2012 to May 2012. Blood cultures and other clinical samples were collected and cultured following the standard procedures. Results. Among 83 sepsis suspected VL patients 16 (19.3%) had culture confirmed bacterial sepsis. The most frequently isolated organism was Staphylococcus aureus (68.8%; 11/16), including two methicillin-resistant isolates (MRSA). Patients with focal bacterial infection were more likely to have bacterial sepsis (P < 0.001). Conclusions. The prevalence of culture confirmed bacterial sepsis was high, predominantly due to S. aureus. Concurrent focal bacterial infection was associated with bacterial sepsis, suggesting that focal infections could serve as sources for bacterial sepsis among VL patients. Careful clinical evaluation for focal infections and prompt initiation of empiric antibiotic treatment appears warranted in VL patients.
PMCID: PMC4033396  PMID: 24895569
17.  Invasive Salmonella enterica Serotype Typhimurium Infections, Democratic Republic of the Congo, 2007–2011 
Emerging Infectious Diseases  2014;20(4):701-704.
Infection with Salmonella enterica serotype Typhimurium sequence type (ST) 313 is associated with high rates of drug resistance, bloodstream infections, and death. To determine whether ST313 is dominant in the Democratic Republic of the Congo, we studied 180 isolates collected during 2007–2011; 96% belonged to CRISPOL type CT28, which is associated with ST313.
PMCID: PMC3966400  PMID: 24655438
Salmonella Typhimurium; ST313; CRISPOL; MLST; Central Africa; bacteremia; bacteria; Democratic Republic of the Congo
18.  Gambiense Human African Trypanosomiasis and Immunological Memory: Effect on Phenotypic Lymphocyte Profiles and Humoral Immunity 
PLoS Pathogens  2014;10(3):e1003947.
In mice, experimental infection with Trypanosoma brucei causes decreased bone marrow B-cell development, abolished splenic B-cell maturation and loss of antibody mediated protection including vaccine induced memory responses. Nothing is known about this phenomenon in human African trypanosomiasis (HAT), but if occurring, it would imply the need of revaccination of HAT patients after therapy and abolish hope for a HAT vaccine. The effect of gambiense HAT on peripheral blood memory T- and B-cells and on innate and vaccine induced antibody levels was examined. The percentage of memory B- and T-cells was quantified in peripheral blood, prospectively collected in DR Congo from 117 Trypanosoma brucei gambiense infected HAT patients before and six months after treatment and 117 controls at the same time points. Antibodies against carbohydrate antigens on red blood cells and against measles were quantified. Before treatment, significantly higher percentages of memory B-cells, mainly T-independent memory B-cells, were observed in HAT patients compared to controls (CD20+CD27+IgM+, 13.0% versus 2.0%, p<0.001). The percentage of memory T-cells, mainly early effector/memory T-cells, was higher in HAT (CD3+CD45RO+CD27+, 19.4% versus 16.7%, p = 0.003). After treatment, the percentage of memory T-cells normalized, the percentage of memory B-cells did not. The median anti-red blood cell carbohydrate IgM level was one titer lower in HAT patients than in controls (p<0.004), and partially normalized after treatment. Anti-measles antibody concentrations were lower in HAT patients than in controls (medians of 1500 versus 2250 mIU/ml, p = 0.02), and remained so after treatment, but were above the cut-off level assumed to provide protection in 94.8% of HAT patients, before and after treatment (versus 98.3% of controls, p = 0.3). Although functionality of the B-cells was not verified, the results suggest that immunity was conserved in T.b. gambiense infected HAT patients and that B-cell dysfunction might not be that severe as in mouse models.
Author Summary
African trypanosomes are parasites that cause sleeping sickness in humans. In mice models, trypanosomiasis causes loss of the spleen memory B-cell precursors, of the host memory response and of protection against certain pathogens, built up by vaccination. The phenomenon has never been studied in human sleeping sickness, but if occurring, revaccination after treatment would be required. We show that gambiense human sleeping sickness is associated with a relevant increase in memory T- and B- cells in peripheral blood, in particular T-independent memory B-cells. As measles vaccination is included in standard vaccination programs, we measured measles antibody concentrations, which, although slightly lower in sleeping sickness patients than in controls, exceeded in 95% of patients the minimum level considered protective. Anti-red blood cell IgM titres, measured to assess the T-cell independent antibody response, were one titre lower in patients than in controls, but normalized after treatment. Overall, our results in gambiense HAT patients do not suggest trypanosomiasis associated massive memory cell destruction, or loss of antibody levels, although the antibody's protective capacity remains to be confirmed.
PMCID: PMC3946376  PMID: 24603894
19.  Frequency of Severe Malaria and Invasive Bacterial Infections among Children Admitted to a Rural Hospital in Burkina Faso 
PLoS ONE  2014;9(2):e89103.
Although severe malaria is an important cause of mortality among children in Burkina Faso, data on community-acquired invasive bacterial infections (IBI, bacteremia and meningitis) are lacking, as well as data on the involved pathogens and their antibiotic resistance rates.
The present study was conducted in a rural hospital and health center in Burkina Faso, in a seasonal malaria transmission area. Hospitalized children (<15 years) presenting with T≥38.0°C and/or signs of severe illness were enrolled upon admission. Malaria diagnosis and blood culture were performed for all participants, lumbar puncture when clinically indicated. We assessed the frequency of severe malaria (microscopically confirmed, according to World Health Organization definitions) and IBI, and the species distribution and antibiotic resistance of the bacterial pathogens causing IBI.
From July 2012 to July 2013, a total of 711 patients were included. Severe malaria was diagnosed in 292 (41.1%) children, including 8 (2.7%) with IBI co-infection. IBI was demonstrated in 67 (9.7%) children (bacteremia, n = 63; meningitis, n = 6), 8 (11.8%) were co-infected with malaria. Non-Typhoid Salmonella spp. (NTS) was the predominant isolate from blood culture (32.8%), followed by Salmonella Typhi (18.8%), Streptococcus pneumoniae (18.8%) and Escherichia coli (12.5%). High antibiotic resistance rates to first line antibiotics were observed, particularly among Gram-negative pathogens. In addition, decreased ciprofloxacin susceptibility and extended-spectrum beta lactamase (ESBL) production was reported for one NTS isolate each. ESBL production was observed in 3/8 E. coli isolates. In-hospital mortality was 8.2% and case-fatality rates for IBI (23.4%) were significantly higher compared to severe malaria (6.8%, p<0.001).
Although severe malaria was the main cause of illness, IBI were not uncommon and had higher case-fatality rates. The high frequency, antibiotic resistance rates and mortality rates of community acquired IBI require improvement in hygiene, better diagnostic methods and revision of current treatment guidelines.
PMCID: PMC3925230  PMID: 24551225
20.  Accuracy of PfHRP2 versus Pf-pLDH antigen detection by malaria rapid diagnostic tests in hospitalized children in a seasonal hyperendemic malaria transmission area in Burkina Faso 
Malaria Journal  2014;13:20.
In most sub-Saharan African countries malaria rapid diagnostic tests (RDTs) are now used for the diagnosis of malaria. Most RDTs used detect Plasmodium falciparum histidine-rich protein-2 (PfHRP2), though P. falciparum-specific parasite lactate dehydrogenase (Pf-pLDH)-detecting RDTs may have advantages over PfHRP2-detecting RDTs. Only few data are available on the use of RDTs in severe illness and the present study compared Pf-pLDH to PfHRP2-detection.
Hospitalized children aged one month to 14 years presenting with fever or severe illness were included over one year. Venous blood samples were drawn for malaria diagnosis (microscopy and RDT), culture and complete blood count. Leftovers were stored at −80 °C and used for additional RDT analysis and PCR. An RDT targeting both PfHRP2 and Pf-pLDH was performed on all samples for direct comparison of diagnostic accuracy with microscopy as reference method. PCR was performed to explore false-positive RDT results.
In 376 of 694 (54.2%) included children, malaria was microscopically confirmed. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value were 100.0, 70.9, 69.4 and 100.0%, respectively for PfHRP2-detection and 98.7, 94.0, 91.6 and 99.1%, respectively for Pf-pLDH-detection. Specificity and PPV were significantly lower for PfHRP2-detection (p <0.001). For both detection antigens, specificity was lowest for children one to five years and in the rainy season. PPV for both antigens was highest in the rainy season, because of higher malaria prevalence. False positive PfHRP2 results were associated with prior anti-malarial treatment and positive PCR results (98/114 (86.0%) samples tested).
Among children presenting with severe febrile illness in a seasonal hyperendemic malaria transmission area, the present study observed similar sensitivity but lower specificity and PPV of PfHRP2 compared to Pf-pLDH-detection. Further studies should assess the diagnostic accuracy and safety of an appropriate Pf-pLDH-detecting RDT in field settings and if satisfying, replacement of PfHRP2 by Pf-pLDH-detecting RDTs should be considered.
PMCID: PMC3896846  PMID: 24418119
21.  A Systematic Review and Meta-Analysis of the Performance of Two Point of Care Typhoid Fever Tests, Tubex TF and Typhidot, in Endemic Countries 
PLoS ONE  2013;8(12):e81263.
In the absence of well-equipped laboratory infrastructure in many developing countries the accurate diagnosis of typhoid fever is challenging. Rapid diagnostic tests (RDT) with good performance indicators would be helpful to improve clinical management of suspected cases. We performed a systematic literature review and meta- analysis to determine the performance of TUBEX TF and Typhidot for the diagnosis of typhoid fever using PRISMA guidelines.
Titles and abstracts were reviewed for relevance. Articles were screened for language, reference method and completeness. Studies were categorized according to control groups used. Meta-analysis was performed only for categories where enough data was available to combine sensitivity and specificity estimates. Sub-analysis was performed for the Typhidot test to determine the influence of indeterminate results on test performance.
A total of seven studies per test were included. The sensitivity of TUBEX TF ranged between 56% and 95%, Specificity between 72% and 95%. Meta-analysis showed an average sensitivity of 69% (95%CI: 45–85) and an average specificity of 88% (CI95%:83–91). A formal meta-analysis for Typhidot was not possible due to limited data available. Across the extracted studies, sensitivity and specificity estimates ranged from 56% to 84% and 31% to 97% respectively.
The observed performance does not support the use of either rapid diagnostic test exclusively as the basis for diagnosis and treatment. There is a need to develop an RDT for typhoid fever that has a performance level comparable to malaria RDTs.
PMCID: PMC3864786  PMID: 24358109
22.  External Quality Assessment of Reading and Interpretation of Malaria Rapid Diagnostic Tests among 1849 End-Users in the Democratic Republic of the Congo through Short Message Service (SMS) 
PLoS ONE  2013;8(8):e71442.
Although malaria rapid diagnostic tests (RDT) are simple to perform, they remain subject to errors, mainly related to the post-analytical phase. We organized the first large scale SMS based external quality assessment (EQA) on correct reading and interpretation of photographs of a three-band malaria RDT among laboratory health workers in the Democratic Republic of the Congo (DR Congo).
Methods and Findings
High resolution EQA photographs of 10 RDT results together with a questionnaire were distributed to health facilities in 9 out of 11 provinces in DR Congo. Each laboratory health worker answered the EQA by Short Message Service (SMS). Filled-in questionnaires from each health facility were sent back to Kinshasa. A total of 1849 laboratory health workers in 1014 health facilities participated. Most frequent errors in RDT reading were i) failure to recognize invalid (13.2–32.5% ) or negative test results (9.8–12.8%), (ii) overlooking faint test lines (4.1–31.2%) and (iii) incorrect identification of the malaria species (12.1–17.4%). No uniform strategy for diagnosis of malaria at the health facility was present. Stock outs of RDTs occurred frequently. Half of the health facilities had not received an RDT training. Only two thirds used the RDT recommended by the National Malaria Control Program. Performance of RDT reading was positively associated with training and the technical level of health facility. Facilities with RDT positivity rates >50% and located in Eastern DR Congo performed worse.
Our study confirmed that errors in reading and interpretation of malaria RDTs are widespread and highlighted the problem of stock outs of RDTs. Adequate training of end-users in the application of malaria RDTs associated with regular EQAs is recommended.
PMCID: PMC3742745  PMID: 23967211
23.  Irregular Migration as a Potential Source of Malaria Reintroduction in Sri Lanka and Use of Malaria Rapid Diagnostic Tests at Point-of-Entry Screening 
Case Reports in Medicine  2013;2013:465906.
Background. We describe an irregular migrant who returned to Sri Lanka after a failed people smuggling operation from West Africa. Results. On-arrival screening by Anti-Malaria Campaign (AMC) officers using a rapid diagnostic test (RDT) (CareStart Malaria HRP2/PLDH) indicated a negative result. On day 3 after arrival, he presented with fever and chills but was managed as dengue (which is hyperendemic in Sri Lanka). Only on day 7, diagnosis of Plasmodium falciparum malaria was made by microcopy and CareStart RDT. The initially negative RDT was ascribed to a low parasite density. Irregular migration may be an unrecognized source of malaria reintroduction. Despite some limitations in detection, RDTs form an important point-of-entry assessment. As a consequence of this case, the AMC is now focused on repeat testing and close monitoring of all irregular migrants from malaria-endemic zones. Conclusion. The present case study highlights the effective collaboration and coordination between inter-governmental agencies such as IOM and the Ministry of Health towards the goals of malaria elimination in Sri Lanka.
PMCID: PMC3703740  PMID: 23861687
24.  False Positivity of Non-Targeted Infections in Malaria Rapid Diagnostic Tests: The Case of Human African Trypanosomiasis 
In endemic settings, diagnosis of malaria increasingly relies on the use of rapid diagnostic tests (RDTs). False positivity of such RDTs is poorly documented, although it is especially relevant in those infections that resemble malaria, such as human African trypanosomiasis (HAT). We therefore examined specificity of malaria RDT products among patients infected with Trypanosoma brucei gambiense.
Methodology/Principal Findings
Blood samples of 117 HAT patients and 117 matched non-HAT controls were prospectively collected in the Democratic Republic of the Congo. Reference malaria diagnosis was based on real-time PCR. Ten commonly used malaria RDT products were assessed including three two-band and seven three-band products, targeting HRP-2, Pf-pLDH and/or pan-pLDH antigens. Rheumatoid factor was determined in PCR negative subjects. Specificity of the 10 malaria RDT products varied between 79.5 and 100% in HAT-negative controls and between 11.3 and 98.8% in HAT patients. For seven RDT products, specificity was significantly lower in HAT patients compared to controls. False positive reactions in HAT were mainly observed for pan-pLDH test lines (specificities between 13.8 and 97.5%), but also occurred frequently for the HRP-2 test line (specificities between 67.9 and 98.8%). The Pf-pLDH test line was not affected by false-positive lines in HAT patients (specificities between 97.5 and 100%). False positivity was not associated to rheumatoid factor, detected in 7.6% of controls and 1.2% of HAT patients.
Specificity of some malaria RDT products in HAT was surprisingly low, and constitutes a risk for misdiagnosis of a fatal but treatable infection. Our results show the importance to assess RDT specificity in non-targeted infections when evaluating diagnostic tests.
Author Summary
Rapid diagnostic tests (RDT) for malaria are currently rolled-out as the backbone of parasite-based diagnosis, and their diagnostic accuracy is sufficiently high to substitute microscopy. One decade ago, attention has been given to occurrence of limited false positivity in a number of malaria RDTs, but false positivity of RDTs has remained poorly documented since then. In the last years, the number of available RDT products has dramatically increased and test performance has improved. False positivity may therefore not be perceived as a problem anymore. In this manuscript, we demonstrate that specificities of malaria rapid diagnostic tests detecting parasite antigens are seriously affected by human African trypanosomiasis (sleeping sickness), with values down to 11%. Malaria constitutes the main differential diagnosis of human African trypanosomiasis, and the false-positive results for malaria RDTs increase the risk of misdiagnosis or delayed diagnosis of human African trypanosomiasis which is a fatal but treatable infection.
PMCID: PMC3636101  PMID: 23638201
25.  Bloodstream Infection among Adults in Phnom Penh, Cambodia: Key Pathogens and Resistance Patterns 
PLoS ONE  2013;8(3):e59775.
Bloodstream infections (BSI) cause important morbidity and mortality worldwide. In Cambodia, no surveillance data on BSI are available so far.
From all adults presenting with SIRS at Sihanouk Hospital Centre of HOPE (July 2007–December 2010), 20 ml blood was cultured. Isolates were identified using standard microbiological techniques; antibiotic susceptibilities were assessed using disk diffusion and MicroScan®, with additional E-test, D-test and double disk test where applicable, according to CLSI guidelines.
A total of 5714 samples from 4833 adult patients yielded 501 clinically significant organisms (8.8%) of which 445 available for further analysis. The patients’ median age was 45 years (range 15–99 y), 52.7% were women. HIV-infection and diabetes were present in 15.6% and 8.8% of patients respectively. The overall mortality was 22.5%. Key pathogens included Escherichia coli (n = 132; 29.7%), Salmonella spp. (n = 64; 14.4%), Burkholderia pseudomallei (n = 56; 12.6%) and Staphylococcus aureus (n = 53; 11.9%). Methicillin resistance was seen in 10/46 (21.7%) S. aureus; 4 of them were co-resistant to erythromycin, clindamycin, moxifloxacin and sulphamethoxazole-trimethoprim (SMX-TMP). We noted combined resistance to amoxicillin, SMX-TMP and ciprofloxacin in 81 E. coli isolates (62.3%); 62 isolates (47.7%) were confirmed as producers of extended spectrum beta-lactamase. Salmonella isolates displayed high rates of multidrug resistance (71.2%) with high rates of decreased ciprofloxacin susceptibility (90.0%) in Salmonella Typhi while carbapenem resistance was observed in 5.0% of 20 Acinetobacter sp. isolates.
BSI in Cambodian adults is mainly caused by difficult-to-treat pathogens. These data urge for microbiological capacity building, nationwide surveillance and solid interventions to contain antibiotic resistance.
PMCID: PMC3612098  PMID: 23555777

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