Sensitive poliovirus detection using nested PCR and nanopore sequencing: a prospective validation study.

Timely detection of outbreaks is needed for poliovirus eradication, but gold standard detection in the Democratic Republic of the Congo takes 30 days (median). Direct molecular detection and nanopore sequencing (DDNS) of poliovirus in stool samples is a promising fast method. Here we report prospective testing of stool samples from suspected polio cases, and their contacts, in the Democratic Republic of the Congo between 10 August 2021 and 4 February 2022. DDNS detected polioviruses in 62/2,339 (2.7%) of samples, while gold standard combination of cell culture, quantitative PCR and Sanger sequencing detected polioviruses in 51/2,339 (2.2%) of the same samples. DDNS provided case confirmation in 7 days (median) in routine surveillance conditions. DDNS enabled confirmation of three serotype 2 circulating vaccine-derived poliovirus outbreaks 23 days (mean) earlier (range 6-30 days) than the gold standard method. The mean sequence similarity between sequences obtained by the two methods was 99.98%. Our data confirm the feasibility of implementing DDNS in a national poliovirus laboratory.

Use of a rapid digital microfluidics-powered immunoassay for assessing measles and rubella infection and immunity in outbreak settings in the Democratic Republic of the Congo.

The Democratic Republic of the Congo (DRC) has a high measles incidence despite elimination efforts and has yet to introduce rubella vaccine. We evaluated the performance of a prototype rapid digital microfluidics powered (DMF) enzyme-linked immunoassay (ELISA) assessing measles and rubella infection, by testing for immunoglobulin M (IgM), and immunity from natural infection or vaccine, by testing immunoglobulin G (IgG), in outbreak settings. Field evaluations were conducted during September 2017, in Kinshasa province, DRC. Blood specimens were collected during an outbreak investigation of suspected measles cases and tested for measles and rubella IgM and IgG using the DMF-ELISA in the field. Simultaneously, a household serosurvey for measles and rubella IgG was conducted in a recently confirmed measles outbreak area. DMF-ELISA results were compared with reference ELISA results tested at DRC's National Public Health Laboratory and the US Centers for Disease Control and Prevention. Of 157 suspected measles cases, rubella IgM was detected in 54% while measles IgM was detected in 13%. Measles IgG-positive cases were higher among vaccinated persons (87%) than unvaccinated persons (72%). In the recent measles outbreak area, measles IgG seroprevalence was 93% overall, while rubella seroprevalence was lower for children (77%) than women (98%). Compared with reference ELISA, DMF-ELISA sensitivity and specificity were 82% and 78% for measles IgG; 88% and 89% for measles IgM; 85% and 85% for rubella IgG; and 81% and 83% for rubella IgM, respectively. Rubella infection was detected in more than half of persons meeting the suspected measles case definition during a presumed measles outbreak, suggesting substantial unrecognized rubella incidence, and highlighting the need for rubella vaccine introduction into the national schedule. The performance of the DMF-ELISA suggested that this technology can be used to develop rapid diagnostic tests for measles and rubella.

Measles seroprevalence after reactive vaccination campaigns during the 2015 measles outbreak in four health zones of the former Katanga Province, Democratic Republic of Congo.

BACKGROUND: Measles continues to circulate in the Democratic Republic of Congo, and the country suffered from several important outbreaks over the last 5 years. Despite a large outbreak starting in the former province of Katanga in 2010 and the resulting immunization activities, another outbreak occurred in 2015 in this same region. We conducted measles seroprevalence surveys in four health zones (HZ) in the former Katanga Province in order to assess the immunity against measles in children 6 months to 14 years after the 2015 outbreak. METHODS: We conducted multi-stage cluster surveys stratified by age group in four HZs, Kayamba, Malemba-Nkulu, Fungurume, and Manono. The age groups were 6-11 months, 12-59 months, and 5-14 years in Kayamba and Malemba-Nkulu, 6-59 months and 5-14 years in Manono and Fungurume. The serological status was measured on dried capillary blood spots collected systematically along with vaccination status (including routine Extended Program of Immunization (EPI), and supplementary immunization activities (SIAs)) and previous self-reported history of suspected measles. RESULTS: Overall seroprevalence against measles was 82.7% in Kayamba, 97.6% in Malemba-Nkulu, 83.2% in Manono, and 74.4% in Fungurume, and it increased with age in all HZs. It was 70.7 and 93.8% in children 12-59 months in Kayamba and Malemba-Nkulu, and 49.8 and 64.7% in children 6-59 months in Fungurume and Manono. The EPI coverage was low but varied across HZ. The accumulation of any type of vaccination against measles resulted in an overall vaccine coverage (VC) of at least 85% in children 12-59 months in Kayamba and Malemba-Nkulu, 86.1 and 74.8% in children 6-59 months in Fungurume and Manono. Previous measles infection in 2015-early 2016 was more frequently reported in children aged 12-59 months or 6-59 months (depending on the HZ). CONCLUSION: The measured seroprevalence was consistent with the events that occurred in these HZs over the past few years. Measles seroprevalence might prove a valuable source of information to help adjust the timing of future SIAs and prioritizing support to the EPI in this region as long as the VC does not reach a level high enough to efficiently prevent epidemic flare-ups.

Genotypes of rubella virus and the epidemiology of rubella infections in the Democratic Republic of the Congo, 2004-2013.

Rubella is a viral infection that may cause fetal death or congenital defects, known as congenital rubella syndrome (CRS), during early pregnancy. The World Health Organization (WHO) recommends that countries assess the burden of rubella and CRS, including the determination of genotypes of circulating viruses. The goal of this study was to identify the genotypes of rubella viruses in the Democratic Republic of the Congo (DRC). Serum or throat swab samples were collected through the measles surveillance system. Sera that tested negative for measles IgM antibody were tested for rubella IgM antibody. Serum collected within 4 days of rash onset and throat swabs were screened by real-time RT-PCR for rubella virus RNA. For positive samples, an amplicon of the E1 glycoprotein gene was amplified by RT-PCR and sequenced. 11733 sera were tested for rubella IgM and 2816 (24%) were positive; 145 (5%) were tested for the presence of rubella RNA by real-time RT-PCR and 10 (7%) were positive. Seventeen throat swabs were analyzed by RT-PCR and three were positive. Sequences were obtained from eight of the positive samples. Phylogenetic analysis showed that the DRC rubella viruses belonged to genotypes 1B, 1E, 1G, and 2B. This report provides the first information on the genotypes of rubella virus circulating in the DRC. These data contribute to a better understanding of rubella burden and the dynamics of rubella virus circulation in Africa. Efforts to establish rubella surveillance in the DRC are needed to support rubella elimination in Africa. J. Med. Virol. 88:1677-1684, 2016. © 2016 Wiley Periodicals, Inc.

Strengthening laboratory capacity through the surveillance of rotavirus gastroenteritis in Central Africa: the Surveillance Épidémiologique en Afrique Centrale (SURVAC) Project.

OBJECTIVES: The goal of the SURVAC pilot project was to strengthen disease surveillance and response in three countries; Cameroon (CAE), Democratic Republic of the Congo (DRC) and Central African Republic (CAR). METHODS: Seven laboratories involved in rotavirus surveillance were provided with equipment, reagents and supplies. CDC and WHO staff provided on-site classroom and bench training in biosafety, quality assurance, quality control (QC), rotavirus diagnosis using Enzyme Immunoassay (EIA) and genotyping of rotavirus strains using the Reverse Transcription Polymerase-chain reaction (RT-PCR). All laboratory data were reported through WHO/AFRO. RESULTS: Twenty-three staff members were trained on RT-PCR for rotavirus genotyping which was introduced for the first time in all three countries. In CAE, the number of samples analysed by EIA and RT-PCR increased tenfold between 2007 and 2013. In DRC, this number increased fivefold, from 2009 to 2013 whereas in CAR, it increased fourfold between 2011 and 2013. All laboratories passed WHO proficiency testing in 2014. CONCLUSION: Laboratory capacity was strengthened through equipping laboratories and strengthening a subregional laboratory workforce for surveillance of rotavirus gastroenteritis. Each of the three countries generated rotavirus surveillance and genotyping data enabling the mapping of circulating genotypes. These results will help monitor the impact of rotavirus vaccination in these countries.

Molecular surveillance of rotavirus infection in the Democratic Republic of the Congo August 2009 to June 2012.

BACKGROUND: Rotavirus is a major cause of severe diarrhea worldwide. It causes 453,000 deaths in children annually. In the Democratic Republic of the Congo, sentinel site surveillance of rotavirus gastroenteritis started in 2009 and aimed to document burden of rotavirus diarrhea and identify circulating rotavirus genotypes. METHODS: Between August 2009 to June 2012, stool samples were collected in Kinshasa and Lubumbashi, from children <5 years of age who met the WHO case definition for rotavirus gastroenteritis. Rotavirus antigen detection was performed using an enzyme immunoassay technique and rotavirus strains were characterized using a multiplex reverse transcription polymerase chain reaction assay. RESULTS: During the study period, 1614 stool samples were screened for rotavirus by enzyme immunoassay and 990 (61%) were positive. Of these, the genotype was determined in 330 (33%) samples. The most common genotypes found in the samples analyzed were G1P[8] in 2009 (28%) and 2012 (33%), G2P[4] (33%) in 2010 and G2P[6] (28%) in 2011. Uncommon strains like G8P[6] (5%), G6P[6] (5%), G12P[6] (3%), G12P[8] (3%) and G8P[8] (2%) were also detected. CONCLUSIONS: In Democratic Republic of the Congo, 61% of the diarrhea in children in <5 years of age was caused by rotavirus infection and a variety of rotavirus genotypes were detected. Implementation of rotavirus genotyping at the national level has improved the timely identification of rotavirus strains. These results will help decision makers in Democratic Republic of the Congo plan the implementation of a rotavirus vaccination program.