PHARE: a bioinformatics pipeline for compositional profiling of multiclonal Plasmodium falciparum infections from long-read Nanopore sequencing data.

BACKGROUND: The emergence of drug-resistant clones of Plasmodium falciparum is a major public health concern, and the ability to detect and track the spread of these clones is crucial for effective malaria control and treatment. However, in endemic settings, malaria infected people often carry multiple P. falciparum clones simultaneously making it likely to miss drug-resistant clones using traditional molecular typing methods. OBJECTIVES: Our goal was to develop a bioinformatics pipeline for compositional profiling in multiclonal P. falciparum samples, sequenced using the Oxford Nanopore Technologies MinION platform. METHODS: We developed the 'Finding P. falciparum haplotypes with resistance mutations in polyclonal infections' (PHARE) pipeline using existing bioinformatics tools and custom scripts written in python. PHARE was validated on three control datasets containing P. falciparum DNA of four laboratory strains at varying mixing ratios. Additionally, the pipeline was tested on clinical samples from children admitted to a paediatric hospital in the Central African Republic. RESULTS: The PHARE pipeline achieved high recall and accuracy rates in all control datasets. The pipeline can be used on any gene and was tested with amplicons of the P. falciparum drug resistance marker genes pfdhps, pfdhfr and pfK13. CONCLUSIONS: The PHARE pipeline helps to provide a more complete picture of drug resistance in the circulating P. falciparum population and can help to guide treatment recommendations. PHARE is freely available under the GNU Lesser General Public License v.3.0 on GitHub: https://github.com/Fippu/PHARE.

Development and evaluation of PlasmoPod: A cartridge-based nucleic acid amplification test for rapid malaria diagnosis and surveillance.

Malaria surveillance is hampered by the widespread use of diagnostic tests with low sensitivity. Adequate molecular malaria diagnostics are often only available in centralized laboratories. PlasmoPod is a novel cartridge-based nucleic acid amplification test for rapid, sensitive, and quantitative detection of malaria parasites. PlasmoPod is based on reverse-transcription quantitative polymerase chain reaction (RT-qPCR) of the highly abundant Plasmodium spp. 18S ribosomal RNA/DNA biomarker and is run on a portable qPCR instrument which allows diagnosis in less than 30 minutes. Our analytical performance evaluation indicates that a limit-of-detection as low as 0.02 parasites/µL can be achieved and no cross-reactivity with other pathogens common in malaria endemic regions was observed. In a cohort of 102 asymptomatic individuals from Bioko Island with low malaria parasite densities, PlasmoPod accurately detected 83 cases, resulting in an overall detection rate of 81.4%. Notably, there was a strong correlation between the Cq values obtained from the reference RT-qPCR assay and those obtained from PlasmoPod. In an independent cohort, using dried blood spots from malaria symptomatic children living in the Central African Republic, we demonstrated that PlasmoPod outperforms malaria rapid diagnostic tests based on the PfHRP2 and panLDH antigens as well as thick blood smear microscopy. Our data suggest that this 30-minute sample-to-result RT-qPCR procedure is likely to achieve a diagnostic performance comparable to a standard laboratory-based RT-qPCR setup. We believe that the PlasmoPod rapid NAAT could enable widespread accessibility of high-quality and cost-effective molecular malaria surveillance data through decentralization of testing and surveillance activities, especially in elimination settings.

Molecular characterization of measles viruses in Central African Republic, 2012-2016.

Despite being preventable through vaccination, measles is still one of the most important causes of morbidity and mortality in young children in Africa. In 2015, several African countries, including the Central African Republic (CAR), began implementing national measles elimination programs. However, measles remains a public health problem in Africa, particularly in the CAR. A retrospective study was conducted at the Institut Pasteur de Bangui, using blood samples (n = 255) and oral swabs (n = 7) collected between January 2012 and December 2016 from measles IgM-positive cases, to attempt genotyping of circulating measles virus strains. Overall, 50 samples were positive by real-time polymerase chain reaction, and 40 sequences of acceptable quality were obtained. The phylogenetic analysis showed that 38 strains belonged to genotype B3 suggesting that this genotype was endemic in the CAR during the study period. No genotype B2 sequences were detected, suggesting that this genotype is no longer present in the CAR.

Rubella epidemiology in the Central African Republic, 2015-2016 and molecular characterization of virus strains from 2008-2016.

OBJECTIVES: Rubella cases in the Central African Republic (CAF) are currently identified during measles surveillance. This study aimed to investigate rubella epidemiology between 2015 and 2016 and to provide baseline genotype data for monitoring future rubella control efforts. METHODS: 831 measles IgM negative or equivocal sera from 2015/2016 were tested for rubella IgM antibodies and 350 rubella IgM positive sera collected between 2008 and 2016 were selected for PCR and sequencing. RESULTS: 411 of the 831 sera (49.5%) were rubella IgM positive and most cases (n=391, 95.1%) occurred between January and April. Most patients were between 5 and 9 years old (50.2%) and more than half of the rubella cases (56.7%) originated from the capital Bangui. Genotype information was obtained for 37 of the 350 selected rubella IgM-positive specimens, with the majority of the patients originating from Bangui (n=24, 64.9%) and sequences covering all years except 2009. Phylogenetic analysis identified genotypes 1E (n=12), 1G (n=5) and 2B (n=20), with 2B being detected from 2014 onwards. CONCLUSIONS: Our study confirmed the important role of rubella as a rash and fever disease in CAF and provided comprehensive data on rubella epidemiology and the first information on rubella genotypes in the country.

Epidemiologic profile of measles in Central African Republic: A nine year survey, 2007-2015.

INTRODUCTION: Measles remains a major public health problem in many developing countries in which vaccination coverage is poor, as is the case in the Central African Republic (CAR). At the beginning of the 2000s, a surveillance system was established in the country, and samples from suspected cases are regularly tested in the laboratory for serological confirmation. Since 2007, when case-by-case monitoring with standardized laboratory databases and monitoring, was set up, no assessment have been performed. Therfore, 9 years later it seemed appropriate to make a first assessment. The aim of the study reported here was to describe the epidemiology of measles in the CAR on the basis of surveillance and laboratory data. METHOD: A descriptive retrospective study was conducted, based on the databases of the measles surveillance programme and of the Institut Pasteur laboratory in Bangui during the period 2007-2015. RESULTS: During this study period, the surveillance programme notified 3767 cases. Of these, 2795 (75%) were sent for laboratory confirmation, and 24.6% (687/2795) were confirmed serologically. Of the 1797 cases of measles declared during this period by the surveillance programme, 1110 (61.8%) were confirmed clinically or by epidemiological linkage. The majority of confirmed cases (83.7%; 575/687) occurred in children under 10 years, over half of whom (44.2%; 304/687) were aged 1-4 years. Epidemics occurred regularly between 2011 and 2015, with > 10% of laboratory-confirmed cases. The rate of laboratory investigation was < 80% between 2011 and 2013 but nearly 100% in the other years. CONCLUSION: Measles remains a common, endemic illness in the CAR. Improved detection will require better measles surveillance, increased vaccination coverage, revision of the investigation forms to include the WHO case definition and training of the health personnel involved in case-finding in the field.

Epidemiology of primary rubella infection in the Central African Republic: data from measles surveillance, 2007-2014.

BACKGROUND: Although rubella is generally considered a benign childhood disease, infection of a pregnant woman can cause foetal congenital rubella syndrome, which results in embryo-foetal disease and malformations. The syndrome is still a public health problem in developing countries where the vaccine has not yet been introduced, such as the Central African Republic (CAR). The aim of the study reported here was to define the epidemiology of primary rubella infection, in order to determine its effect on morbidity rates in the country. METHODS: Data derived from epidemiological surveillance of measles and rubella were analysed retrospectively between 1 January 2007 and 31 December 2014. The database includes cases of suspected measles, according to the WHO clinical case definition. In this algorithm, samples that are negative or doubtful by ELISA for measles (presence of immunoglobulin M) are tested in another ELISA for detection of rubella-specific IgM. Descriptive analyses were conducted for socio-demographic characteristics, including age, sex and health region, for patients tested for rubella. RESULTS: Of the sera tested for rubella, 30.2 % (425/1409) were positive, 62.3 % (878/1409) were negative, and 7.5 % (106/1409) were doubtful. Among the 425 positive cases, 213 (50.1 %) were female and 212 (40.9 %) were male with a sex ratio of 1.03. The mean age was 8 years (range, 6-37 years). The highest prevalence (47.3 %; 116/425) was seen in 2007 and the lowest (8.9 %; 11/425) in 2012. Primary infections were always more frequent during the first 3 months of the year, with a peak at the same time, between January and February which is the hottest period of the year in the CAR. In both sexes, rubella IgM was rarely found before the age of 1 year (0.5 %; 2/425). The highest rate (43.5 %; 185/425) was observed at ages 5-9 years; however, at least 8 % (18/213) of girls aged 15 or more had primary infections. CONCLUSIONS: Sentinel sites for surveillance of congenital rubella syndrome are urgently needed, and introduction of vaccination against rubella in the Expanded Programme of Immunization should be considered, to ensure immunization of girls of reproductive age.

Measles outbreak in Northern Central African Republic 3 years after the last national immunization campaign.

BACKGROUND: Despite huge efforts to promote widespread vaccination, measles remains an important cause of morbidity and mortality worldwide, especially in African children. In March 2011, an abnormally high number of cases were reported from the Ouham Prefecture, Central African Republic to the national measles case-based surveillance system. In response, reactive vaccination activities were implemented. The aims of this study were to investigate this outbreak and describe the response. METHODS: Measles cases were defined according to WHO recommendations. In the first weeks of the outbreak, blood samples were collected and sent to the Institut Pasteur in Bangui for laboratory confirmation by detection of IgM antibodies against measles virus. In addition, a portion of viral RNA was amplified from 5 IgM positive patient samples and the amplicons were sequenced for phylogenetic analysis. RESULTS: Between March and September 2011, 723 clinical cases originated from the Ouham Prefecture, including 2 deaths, were reported. Amongst 59 blood samples collected, 49 were positive for the detection of IgM. A high number of self-declared vaccinated subjects (31%) were found amongst the cases. Most of the cases were under 5 years. The causative virus was found to belong to genotype B3.1. In response, 2 sub-national supplementary immunization activities were quickly conducted and limited this outbreak to mainly 2 sub-prefectures. CONCLUSIONS: This outbreak was the largest epidemic of measles in CAR since 2002. Its occurrence, 3 years after the last national immunization campaign, highlights the necessity to pursue efforts and improve and extend immunization programs in order to reach measles elimination goal in Africa.