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4.  Diagnosis of Persistent Fever in the Tropics: Set of Standard Operating Procedures Used in the NIDIAG Febrile Syndrome Study 
PLoS Neglected Tropical Diseases  2016;10(11):e0004749.
In resource-limited settings, the scarcity of skilled personnel and adequate laboratory facilities makes the differential diagnosis of fevers complex [1–5]. Febrile illnesses are diagnosed clinically in most rural centers, and both Rapid Diagnostic Tests (RDTs) and clinical algorithms can be valuable aids to health workers and facilitate therapeutic decisions [6,7]. The persistent fever syndrome targeted by NIDIAG is defined as presence of fever for at least one week. The NIDIAG clinical research consortium focused on potentially severe and treatable infections and therefore targeted the following conditions as differential diagnosis of persistent fever: visceral leishmaniasis (VL), human African trypanosomiasis (HAT), enteric (typhoid and paratyphoid) fever, brucellosis, melioidosis, leptospirosis, malaria, tuberculosis, amoebic liver abscess, relapsing fever, HIV/AIDS, rickettsiosis, and other infectious diseases (e.g., pneumonia). From January 2013 to October 2014, a prospective clinical phase III diagnostic accuracy study was conducted in one site in Cambodia, two sites in Nepal, two sites in Democratic Republic of the Congo (DRC), and one site in Sudan (clinicaltrials.gov no. NCT01766830). The study objectives were to (1) determine the prevalence of the target diseases in patients presenting with persistent fever, (2) assess the predictive value of clinical and first-line laboratory features, and (3) assess the diagnostic accuracy of several RDTs for the diagnosis of the different target conditions.
doi:10.1371/journal.pntd.0004749
PMCID: PMC5094701  PMID: 27812090
6.  Molecular Surveillance Identifies Multiple Transmissions of Typhoid in West Africa 
PLoS Neglected Tropical Diseases  2016;10(9):e0004781.
Background
The burden of typhoid in sub-Saharan African (SSA) countries has been difficult to estimate, in part, due to suboptimal laboratory diagnostics. However, surveillance blood cultures at two sites in Nigeria have identified typhoid associated with Salmonella enterica serovar Typhi (S. Typhi) as an important cause of bacteremia in children.
Methods
A total of 128 S. Typhi isolates from these studies in Nigeria were whole-genome sequenced, and the resulting data was used to place these Nigerian isolates into a worldwide context based on their phylogeny and carriage of molecular determinants of antibiotic resistance.
Results
Several distinct S. Typhi genotypes were identified in Nigeria that were related to other clusters of S. Typhi isolates from north, west and central regions of Africa. The rapidly expanding S. Typhi clade 4.3.1 (H58) previously associated with multiple antimicrobial resistances in Asia and in east, central and southern Africa, was not detected in this study. However, antimicrobial resistance was common amongst the Nigerian isolates and was associated with several plasmids, including the IncHI1 plasmid commonly associated with S. Typhi.
Conclusions
These data indicate that typhoid in Nigeria was established through multiple independent introductions into the country, with evidence of regional spread. MDR typhoid appears to be evolving independently of the haplotype H58 found in other typhoid endemic countries. This study highlights an urgent need for routine surveillance to monitor the epidemiology of typhoid and evolution of antimicrobial resistance within the bacterial population as a means to facilitate public health interventions to reduce the substantial morbidity and mortality of typhoid.
Author Summary
Typhoid fever, a serious bloodstream infection caused by the bacterium Salmonella Typhi, is a major cause of disease and death around the world. There have been limited data on the epidemiology of typhoid in many countries in sub-Saharan African, including Nigeria. Recent evidence, however, showed that typhoid was an important cause of bacteraemia in children residing in two regions of Nigeria. Here, we analyzed the whole genome sequences of 128 S. Typhi isolates from two studies in order to elucidate the population structure and characterize the genetic components of antimicrobial resistance. We found that the multiple S. Typhi genotypes identified were closely related to other S. Typhi from neighboring regions of Africa and that multidrug resistance (MDR) was common among these isolates, and in many cases was associated with the IncHI1 plasmid known to cause MDR typhoid. These results provide evidence that typhoid was established in Nigeria as a result of several independent introductions into the country and that there has been extensive exchange of S. Typhi in and around the region of West Africa. This study emphasizes the importance of surveillance to improve our understanding of the epidemiology of typhoid, which is needed to underpin public health measures to reduce the spread of disease and facilitate patient management.
doi:10.1371/journal.pntd.0004781
PMCID: PMC5033494  PMID: 27657909
7.  Organizing the Donation of Convalescent Plasma for a Therapeutic Clinical Trial on Ebola Virus Disease: The Experience in Guinea 
Although convalescent plasma (CP) transfusion was prioritized among potential Ebola treatments by the World Health Organization, there were concerns on the feasibility of its implementation. We report on the successful organization of donor mobilization and plasma collection as part of the Ebola-Tx clinical trial from November 2014 to July 2015 in Conakry, Guinea. Project implementation registers, tools and reports, mission reports, and minutes of research team meetings were used to reconstruct the sequence of events on how donor mobilization was organized, plasmapheresis was set up, and how effective this approach was in collecting CP. An initial needs assessment of the Guinean National Blood Transfusion Center resulted in targeted training of staff on site, resulting in autonomy and independent production of CP within 3 months. The Conakry Ebola Survivors Association played a direct role in donor mobilization and organization of CP donations. A total of 98 Ebola survivors were screened for plasma donation, of which 84 were found eligible for plasmapheresis. Of these, 26 (30.9%) were excluded. The remaining 58 donors made a total of 90 donations, corresponding to 50.9 L of CP. This sufficed to treat the 99 eligible patients enrolled in the trial. Within a poor resource emergency context, transfusion capacity could be rapidly improved through the strengthening of local capacities and gradual transfer of skills coupled with active involvement of Ebola survivors. However, large-scale plasma collection or multisite studies may require further adaptations of both strategy and logistics. The Ebola-Tx trial was funded by the European Union and others.
doi:10.4269/ajtmh.15-0890
PMCID: PMC5014273  PMID: 27430546
8.  Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events 
Wong, Vanessa K | Baker, Stephen | Pickard, Derek J | Parkhill, Julian | Page, Andrew J | Feasey, Nicholas A | Kingsley, Robert A | Thomson, Nicholas R | Keane, Jacqueline A | Weill, François-Xavier | Edwards, David J | Hawkey, Jane | Harris, Simon R | Mather, Alison E | Cain, Amy K | Hadfield, James | Hart, Peter J | Thieu, Nga Tran Vu | Klemm, Elizabeth J | Glinos, Dafni A | Breiman, Robert F | Watson, Conall H | Kariuki, Samuel | Gordon, Melita A | Heyderman, Robert S | Okoro, Chinyere | Jacobs, Jan | Lunguya, Octavie | Edmunds, W John | Msefula, Chisomo | Chabalgoity, Jose A | Kama, Mike | Jenkins, Kylie | Dutta, Shanta | Marks, Florian | Campos, Josefina | Thompson, Corinne | Obaro, Stephen | MacLennan, Calman A | Dolecek, Christiane | Keddy, Karen H | Smith, Anthony M | Parry, Christopher M | Karkey, Abhilasha | Mulholland, E Kim | Campbell, James I | Dongol, Sabina | Basnyat, Buddha | Dufour, Muriel | Bandaranayake, Don | Naseri, Take Toleafoa | Singh, Shalini Pravin | Hatta, Mochammad | Newton, Paul | Onsare, Robert S | Isaia, Lupeoletalalei | Dance, David | Davong, Viengmon | Thwaites, Guy | Wijedoru, Lalith | Crump, John A | De Pinna, Elizabeth | Nair, Satheesh | Nilles, Eric J | Thanh, Duy Pham | Turner, Paul | Soeng, Sona | Valcanis, Mary | Powling, Joan | Dimovski, Karolina | Hogg, Geoff | Farrar, Jeremy | Holt, Kathryn E | Dougan, Gordon
Nature genetics  2015;47(6):632-639.
The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species.
doi:10.1038/ng.3281
PMCID: PMC4921243  PMID: 25961941
9.  Microbiological, clinical and molecular findings of non-typhoidal Salmonella bloodstream infections associated with malaria, Oriental Province, Democratic Republic of the Congo 
BMC Infectious Diseases  2016;16:271.
Background
In sub-Saharan Africa, non-typhoidal Salmonella (NTS) can cause bloodstream infections, referred to as invasive non-typhoidal Salmonella disease (iNTS disease); it can occur in outbreaks and is often preceded by malaria. Data from Central Africa is limited.
Methods
Clinical, microbiological and molecular findings of NTS recovered in a blood culture surveillance project (2009–2014) were analyzed.
Results
In March-July 2012 there was an epidemic increase in malaria infections in the Oriental Province of the Democratic Republic of the Congo (DRC). In one referral hospital, overall hospital admissions in June 2012 were 2.6 times higher as compared to the same period in the years before and after (336 versus an average of 128 respectively); numbers of malaria cases and blood transfusions were nearly three- and five-fold higher respectively (317 versus 112 and 250 versus 55). Case fatality rates (in-hospital deaths versus all admissions) peaked at 14.6 %. Salmonella Typhimurium and Salmonella Enteritidis together accounted for 88.9 % of pathogens isolated from blood cultures collected during an outreach visit to the affected districts in June 2012. Children infected with Salmonella Enteritidis (33 patient files available) tended to be co-infected with Plasmodium falciparum more often than children infected with Salmonella Typhimurium (40 patients files available) (81.8 % versus 62.5 %). Through the microbiological surveillance project (May 2009–May 2014) 113 unique NTS isolates were collected (28.5 % (113/396) of pathogens); most (95.3 %) were recovered from children < 15 years. Salmonella Typhimurium (n = 54) and Salmonella Enteritidis (n = 56) accounted for 47.8 % and of 49.6 % NTS isolates respectively. Multilocus variable-number tandem-repeat analysis (MLVA) revealed more heterogeneity for Salmonella Typhimurium than for Salmonella Enteritidis. Most (82/96, 85.4 %) NTS isolates that were available for antibiotic susceptibility testing were multidrug resistant. All isolates were susceptible to ceftriaxone and azithromycin.
Conclusion
During the peak of an epidemic increase in malaria in the DRC in 2012, a high proportion of multidrug resistant Salmonella Typhimurium and Salmonella Enteritidis were isolated from blood cultures. Overall, the two serovars showed subtle differences in clinical presentation and genetic diversity.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-016-1604-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s12879-016-1604-1
PMCID: PMC4902913  PMID: 27286886
Bloodstream infections; Salmonella; Democratic Republic of the Congo; Antibiotic; Molecular typing; Symptoms; Children; Malaria
10.  Severe anaemia associated with Plasmodium falciparum infection in children: consequences for additional blood sampling for research 
Malaria Journal  2016;15:304.
Background
Plasmodium falciparum infection may cause severe anaemia, particularly in children. When planning a diagnostic study on children suspected of severe malaria in sub-Saharan Africa, it was questioned how much blood could be safely sampled; intended blood volumes (blood cultures and EDTA blood) were 6 mL (children aged <6 years) and 10 mL (6–12 years). A previous review [Bull World Health Organ. 89: 46–53. 2011] recommended not to exceed 3.8 % of total blood volume (TBV). In a simulation exercise using data of children previously enrolled in a study about severe malaria and bacteraemia in Burkina Faso, the impact of this 3.8 % safety guideline was evaluated.
Methods
For a total of 666 children aged >2 months to <12 years, data of age, weight and haemoglobin value (Hb) were available. For each child, the estimated TBV (TBVe) (mL) was calculated by multiplying the body weight (kg) by the factor 80 (ml/kg). Next, TBVe was corrected for the degree of anaemia to obtain the functional TBV (TBVf). The correction factor consisted of the rate ‘Hb of the child divided by the reference Hb’; both the lowest (‘best case’) and highest (‘worst case’) reference Hb values were used. Next, the exact volume that a 3.8 % proportion of this TBVf would present was calculated and this volume was compared to the blood volumes that were intended to be sampled.
Results
When applied to the Burkina Faso cohort, the simulation exercise pointed out that in 5.3 % (best case) and 11.4 % (worst case) of children the blood volume intended to be sampled would exceed the volume as defined by the 3.8 % safety guideline. Highest proportions would be in the age groups 2–6 months (19.0 %; worst scenario) and 6 months–2 years (15.7 %; worst case scenario). A positive rapid diagnostic test for P. falciparum was associated with an increased risk of violating the safety guideline in the worst case scenario (p = 0.016).
Conclusions
Blood sampling in children for research in P. falciparum endemic settings may easily violate the proposed safety guideline when applied to TBVf. Ethical committees and researchers should be wary of this and take appropriate precautions.
doi:10.1186/s12936-016-1356-9
PMCID: PMC4890332  PMID: 27251128
Child; Blood specimen collection; Blood volume; Practice guidelines as topic
11.  Phylogenomic Analysis Reveals an Asian Origin for African Burkholderia pseudomallei and Further Supports Melioidosis Endemicity in Africa 
mSphere  2016;1(2):e00089-15.
Sporadic melioidosis cases have been reported in the African mainland and Indian Ocean islands, but until recently, these regions were not considered areas where B. pseudomallei is endemic. Given the high mortality rate of melioidosis, it is crucial that this disease be recognized and suspected in all regions of endemicity. Previous work has shown that B. pseudomallei originated in Australia, with subsequent introduction into Asia; however, the precise origin of B. pseudomallei in other tropical regions remains poorly understood. Using whole-genome sequencing, we characterized B. pseudomallei isolates from Madagascar and Burkina Faso. Next, we compared these strains to a global collection of B. pseudomallei isolates to identify their evolutionary origins. We found that African B. pseudomallei strains likely originated from Asia and were closely related to South American strains, reflecting a relatively recent shared evolutionary history. We also identified substantial genetic diversity among African strains, suggesting long-term B. pseudomallei endemicity in this region.
ABSTRACT
Burkholderia pseudomallei, an environmental bacterium that causes the deadly disease melioidosis, is endemic in northern Australia and Southeast Asia. An increasing number of melioidosis cases are being reported in other tropical regions, including Africa and the Indian Ocean islands. B. pseudomallei first emerged in Australia, with subsequent rare dissemination event(s) to Southeast Asia; however, its dispersal to other regions is not yet well understood. We used large-scale comparative genomics to investigate the origins of three B. pseudomallei isolates from Madagascar and two from Burkina Faso. Phylogenomic reconstruction demonstrates that these African B. pseudomallei isolates group into a single novel clade that resides within the more ancestral Asian clade. Intriguingly, South American strains reside within the African clade, suggesting more recent dissemination from West Africa to the Americas. Anthropogenic factors likely assisted in B. pseudomallei dissemination to Africa, possibly during migration of the Austronesian peoples from Indonesian Borneo to Madagascar ~2,000 years ago, with subsequent genetic diversity driven by mutation and recombination. Our study provides new insights into global patterns of B. pseudomallei dissemination and adds to the growing body of evidence of melioidosis endemicity in Africa. Our findings have important implications for melioidosis diagnosis and management in Africa.
IMPORTANCE Sporadic melioidosis cases have been reported in the African mainland and Indian Ocean islands, but until recently, these regions were not considered areas where B. pseudomallei is endemic. Given the high mortality rate of melioidosis, it is crucial that this disease be recognized and suspected in all regions of endemicity. Previous work has shown that B. pseudomallei originated in Australia, with subsequent introduction into Asia; however, the precise origin of B. pseudomallei in other tropical regions remains poorly understood. Using whole-genome sequencing, we characterized B. pseudomallei isolates from Madagascar and Burkina Faso. Next, we compared these strains to a global collection of B. pseudomallei isolates to identify their evolutionary origins. We found that African B. pseudomallei strains likely originated from Asia and were closely related to South American strains, reflecting a relatively recent shared evolutionary history. We also identified substantial genetic diversity among African strains, suggesting long-term B. pseudomallei endemicity in this region.
doi:10.1128/mSphere.00089-15
PMCID: PMC4863585  PMID: 27303718
Burkholderia; epidemiology; infectious disease; melioidosis; phylogeography; population genetics
12.  Towards Improving Point-of-Care Diagnosis of Non-malaria Febrile Illness: A Metabolomics Approach 
PLoS Neglected Tropical Diseases  2016;10(3):e0004480.
Introduction
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.
Methodology
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.
Conclusions
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.
doi:10.1371/journal.pntd.0004480
PMCID: PMC4778767  PMID: 26943791
13.  Diagnosis of Bacterial Bloodstream Infections: A 16S Metagenomics Approach 
PLoS Neglected Tropical Diseases  2016;10(2):e0004470.
Background
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).
Conclusions/Significance
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.
doi:10.1371/journal.pntd.0004470
PMCID: PMC4771206  PMID: 26927306
14.  Patient satisfaction with a teleradiology service in general practice 
BMC Family Practice  2016;17:17.
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/s12875-016-0418-y
PMCID: PMC4748486  PMID: 26864118
Teleradiology; Patient satisfaction; General practice; Family practice
15.  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.
doi:10.1371/journal.pone.0146450
PMCID: PMC4720473  PMID: 26788725
17.  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.
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/s12936-015-0926-6
PMCID: PMC4599330  PMID: 26450599
Malaria diagnosis; LAMP; RDT; Microscopy; PCR; Field-based; Resource-limited settings
18.  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.
Background
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.
Methods/Design
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.
Discussion
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
ClinicalTrials.gov; identifier: NCT02105714.
doi:10.1186/s12879-015-1074-x
PMCID: PMC4539676  PMID: 26282537
Bacteria; Diagnosis-treatment algorithm; Helminths; Intestinal protozoa; Neglected tropical diseases; Persistent diarrhoea; Côte d’Ivoire; Indonesia; Mali; Nepal
19.  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.
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/s12875-015-0268-z
PMCID: PMC4424890  PMID: 25943473
Teleradiology; Family practice; General practice; Diagnosis; Treatment; Trauma fractures
20.  Antibiotic prescription behaviours in Lao People's Democratic Republic: a knowledge, attitude and practice survey 
Abstract
Objective
To assess the antibiotic prescribing practices of doctors working in the Lao People's Democratic Republic and their knowledge of local antibiotic resistance patterns.
Methods
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.
Findings
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.
Conclusion
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.
doi:10.2471/BLT.14.142844
PMCID: PMC4431558  PMID: 26229186
21.  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.
doi:10.1371/journal.pntd.0003559
PMCID: PMC4351981  PMID: 25746418
22.  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.
doi:10.1371/journal.pone.0117950
PMCID: PMC4334661  PMID: 25693200
23.  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.
Background
The present External Quality Assessment (EQA) assessed reading and interpretation of malaria rapid diagnostic tests (RDTs) in the Democratic Republic of the Congo (DRC).
Methods
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).
Results
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.
Conclusions
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.
doi:10.1186/s12936-014-0535-9
PMCID: PMC4318196  PMID: 25626915
Malaria RDT; External quality assessment; Short message service; DRC
24.  Harmonization of malaria rapid diagnostic tests: best practices in labelling including instructions for use 
Malaria Journal  2014;13:505.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1475-2875-13-505
PMCID: PMC4302121  PMID: 25519980
Malaria RDT; Harmonization; Best practices; Labeling; Instructions for use
25.  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).
doi:10.1128/JCM.02663-13
PMCID: PMC3957797  PMID: 24371234

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