Virological failure after 1 year of first-line ART is not associated with HIV minority drug resistance in rural Cameroon .

OBJECTIVES: The aim of this study was to describe clinical and virological outcomes in therapy-naive HIV-1-positive patients treated in a routine ART programme in rural Cameroon. METHODS: In a prospective cohort, 300 consecutive patients starting first-line ART were enrolled and followed for 12 months. Among 238 patients with available viral load data at Month 12, logistic regression was used to analyse risk factors for virological failure (≥1000 HIV RNA copies/mL) including clinical, immunological and virological parameters, as well as data on drug adherence. Population sequencing was performed to detect the presence of drug-resistance mutations in patients with virological failure at Month 12; minority drug-resistance mutations at baseline were analysed using next-generation sequencing in these patients and matched controls. RESULTS: At Month 12, 38/238 (16%) patients experienced virological failure (≥1000 HIV RNA copies/mL). Patients with virological failure were younger, had lower CD4 cell counts and were more often WHO stage 3 or 4 at baseline. Sixty-three percent of patients with virological failure developed at least one drug-resistance mutation. The M184V (n = 18) and K103N (n = 10) mutations were most common. At baseline, 6/30 patients (20%) experiencing virological failure and 6/35 (17%) matched controls had evidence of minority drug-resistance mutations using next-generation sequencing (P = 0.77). Lower CD4 count at baseline (OR per 100 cells/mm(3) lower 1.41, 95% CI 1.02-1.96, P = 0.04) and poorer adherence (OR per 1% lower 1.05, 95% CI 1.02-1.08, P < 0.001) were associated with a higher risk of virological failure. Unavailability of ART at the treatment centre was the single most common cause for incomplete adherence. CONCLUSIONS: Virological failure after 1 year of ART was not associated with minority drug resistance at baseline but with incomplete adherence. Strategies to assure adherence and uninterrupted drug supplies are pivotal factors for therapy success.

Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections.

We present a rigorously validated and highly sensitive confirmatory real-time RT-PCR assay (1A assay) that can be used in combination with the previously reported upE assay. Two additional RT-PCR assays for sequencing are described, targeting the RdRp gene (RdRpSeq assay) and N gene (NSeq assay), where an insertion/deletion polymorphism might exist among different hCoV-EMC strains. Finally, a simplified and biologically safe protocol for detection of antibody response by immunofluorescence microscopy was developed using convalescent patient serum.

Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction.

We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.5­6.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation.

Recombination dynamics of human parechoviruses: investigation of type-specific differences in frequency and epidemiological correlates.

Human parechoviruses (HPeVs) are highly prevalent RNA viruses classified in the family Picornaviridae. Several antigenically distinct types circulate in human populations worldwide, whilst recombination additionally contributes to the genetic heterogeneity of the virus. To investigate factors influencing the likelihood of recombination and to compare its dynamics among types, 154 variants collected from four widely geographically separated referral centres (UK, The Netherlands, Thailand and Brazil) were typed by VP3/VP1 amplification/sequencing with recombination groups assigned by analysis of 3Dpol sequences. HPeV1B and HPeV3 were the most frequently detected types in each referral region, but with marked geographical differences in the frequencies of different recombinant forms (RFs) of types 1B, 5 and 6. HPeV1B showed more frequent recombination than HPeV3, in terms both of evolutionary divergence and of temporal/geographical indicators of population separation. HPeV1 variants showing between 10 and 20% divergence in VP3/VP1 almost invariably fell into different recombination groups, compared with only one-third of similarly divergent HPeV3 variants. Substitution rates calculated by beast in the VP3/VP1 region of HPeV1 and HPeV3 allowed half-lives of the RFs of 4 and 20 years, respectively, to be calculated, estimates fitting closely with their observed lifespans based on population sampling. The variability in recombination dynamics between HPeV1B and HPeV3 offers an intriguing link with their markedly different seasonal patterns of transmission, age distributions of infection and clinical outcomes. Future investigation of the epidemiological and biological opportunities and constraints on intertypic recombination will provide more information about its influence on the longer term evolution and pathogenicity of parechoviruses.

Type 1 wild poliovirus and putative enterovirus 109 in an outbreak of acute flaccid paralysis in Congo, October-November 2010.

An outbreak of flaccid paralysis syndrome in adults is ongoing in Congo. Molecular analysis of faecal, throat and cerebrospinal samples identified wildtype 1 poliovirus and an additional enterovirus C strain related to enterovirus 109 as the cause. As of 22 November, the cumulative number of cases was 409, of which 169 (41.3%) were fatal. This is one of the largest wild type 1 poliovirus outbreaks ever described associated with an unusually high case fatality rate.

GloPID-R report on chikungunya, o'nyong-nyong and Mayaro virus, part 5: Entomological aspects.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) chikungunya (CHIKV), o'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group has been established to investigate natural history, epidemiology and clinical aspects of infection by these viruses. Here, we present a report dedicated to entomological aspects of CHIKV, ONNV and MAYV. Recent global expansion of chikungunya virus has been possible because CHIKV established a transmission cycle in urban settings using anthropophilic vectors such as Aedes albopictus and Aedes aegypti. MAYV and ONNV have a more limited geographic distribution, being confined to Africa (ONNV) and central-southern America (MAYV). ONNV is probably maintained through an enzootic cycle that has not been characterized yet, with Anopheles species as main vectors and humans as amplification hosts during epidemics. MAYV is transmitted by Haemagogus species in an enzootic cycle using non-human primates as the main amplification and maintenance hosts, and humans becoming sporadically infected when venturing in or nearby forest habitats. Here, we focused on the transmission cycle and natural vectors that sustain circulation of these viruses in their respective locations. The knowledge of the natural ecology of transmission and the capacity of different vectors to transmit these viruses is crucial to understand CHIKV emergence, and to assess the risk that MAYV and ONNV will expand on wide scale using anthropophilic mosquito species not normally considered primary vectors. Finally, the experts identified knowledge gaps and provided adapted recommendations, in order to address future entomological investigations in the right direction.

Detection of influenza A(H1N1)v virus by real-time RT-PCR.

Influenza A(H1N1)v virus was first identified in April 2009. A novel real-time RT-PCR for influenza A(H1N1)v virus was set up ad hoc and validated following industry-standard criteria. The lower limit of detection of the assay was 384 copies of viral RNA per ml of viral transport medium (95% confidence interval: 273-876 RNA copies/ml). Specificity was 100% as assessed on a panel of reference samples including seasonal human influenza A virus H1N1 and H3N2, highly pathogenic avian influenza A virus H5N1 and porcine influenza A virus H1N1, H1N2 and H3N2 samples. The real-time RT-PCR assay for the influenza A matrix gene recommended in 2007 by the World Health Organization was modified to work under the same reaction conditions as the influenza A(H1N1)v virus-specific test. Both assays were equally sensitive. Clinical applicability of both assays was demonstrated by screening of almost 2,000 suspected influenza (H1N1)v specimens, which included samples from the first cases of pandemic H1N1 influenza imported to Germany. Measuring influenza A(H1N1)v virus concentrations in 144 laboratory-confirmed samples yielded a median of 4.6 log RNA copies/ml. The new methodology proved its principle and might assist public health laboratories in the upcoming influenza pandemic.

GloPID-R report on chikungunya, o'nyong-nyong and Mayaro virus, part 2: Epidemiological distribution of o'nyong-nyong virus.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) chikungunya (CHIKV), o'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group has been established to identify gaps of knowledge about the natural history, epidemiology and medical management of infection by these viruses, and to provide adapted recommendations for future investigations. Here, we present a report dedicated to ONNV epidemiological distribution. Two large-scale ONNV outbreaks have been identified in Africa in the last 60 years, interspersed with sporadic serosurveys and case reports of returning travelers. The assessment of the real scale of ONNV circulation in Africa remains a difficult task and surveillance studies are necessary to fill this gap. The identification of ONNV etiology is made complicated by the absence of multiplex tools in co-circulation areas and that of reference standards, as well as the high cross-reactivity with related pathogens observed in serological tests, in particular with CHIKV. This is a specific obstacle for seroprevalence studies, that necessitate an improvement of serological tools to provide robust results. The scarcity of existent genetic data currently limits molecular epidemiology studies. ONNV epidemiology would also benefit from reinforced entomological and environmental surveillance. Finally, the natural history of the disease deserves to be further investigated, with a specific attention paid to long-term complications. Considering our incomplete knowledge on ONNV distribution, GloPID-R CHIKV, ONNV and MAYV experts recommend that a major effort should be done to fill existing gaps.

GloPID-R report on Chikungunya, O'nyong-nyong and Mayaro virus, part I: Biological diagnostics.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) Chikungunya (CHIKV), O'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group is investigating the natural history, epidemiology and medical management of infection by these viruses, to identify knowledge gaps and to propose recommendations for direct future investigations and rectification measures. Here, we present the first report dedicated to diagnostic aspects of CHIKV, ONNV and MAYV. Regarding diagnosis of the disease at the acute phase, molecular assays previously described for the three viruses require further evaluation, standardized protocols and the availability of international standards representing the genetic diversity of the viruses. Detection of specific IgM would benefit from further investigations to clarify the extent of cross-reactivity among the three viruses, the sensitivity of the assays, and the possible interfering role of cryoglobulinaemia. Implementation of reference panels and external quality assessments for both molecular and serological assays is necessary. Regarding sero-epidemiological studies, there is no reported high-throughput assay that can distinguish among these different viruses in areas of potential co-circulation. New specific tools and/or improved standardized protocols are needed to enable large-scale epidemiological studies of public health relevance to be performed. Considering the high risk of future CHIKV, MAYV and ONNV outbreaks, the Working Group recommends that a major investigation should be initiated to fill the existing diagnostic gaps.

Evidence for multiple sylvatic transmission cycles during the 2016-2017 yellow fever virus outbreak, Brazil.

OBJECTIVES: Since December 2016, Brazil has experienced an unusually large outbreak of yellow fever (YF). Whether urban transmission may contribute to the extent of the outbreak is unclear. The objective of this study was to characterize YF virus (YFV) genomes and to identify spatial patterns to determine the distribution and origin of YF cases in Minas Gerais, Espírito Santo and Rio de Janeiro, the most affected Brazilian states during the current YFV outbreak. METHODS: We characterized near-complete YFV genomes from 14 human cases and two nonhuman primates (NHP), sampled from February to April 2017, retrieved epidemiologic data of cases and used a geographic information system to investigate the geospatial spread of YFV. RESULTS: All YFV strains were closely related. On the basis of signature mutations, we identified two cocirculating YFV clusters. One was restricted to the hinterland of Espírito Santo state, and another formed a coastal cluster encompassing several hundred kilometers. Both clusters comprised strains from humans living in rural areas and NHP. Another NHP lineage clustered in a basal relationship. No signs of adaptation of YFV strains to human hosts were detected. CONCLUSIONS: Our data suggest sylvatic transmission during the current outbreak. Additionally, cocirculation of two distinct YFV clades occurring in humans and NHP suggests the existence of multiple sylvatic transmission cycles. Increased detection of YFV might be facilitated by raised awareness for arbovirus-mediated disease after Zika and chikungunya virus outbreaks. Further surveillance is required, as reemergence of YFV from NHPs might continue and facilitate the appearance of urban transmission cycles.

The European Virus Archive goes global: A growing resource for research.

The European Virus Archive (EVA) was created in 2008 with funding from the FP7-EU Infrastructure Programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry. Within three years, it developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. In 2014, the H2020 Research and Innovation Framework Programme (INFRAS projects) provided support for the transformation of the EVA from a European to a global organization (EVAg). The EVAg now operates as a non-profit consortium, with 26 partners and 20 associated partners from 21 EU and non-EU countries. In this paper, we outline the structure, management and goals of the EVAg, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. Organisations or individuals who would like to be considered as contributors are invited to contact the EVAg coordinator, Jean-Louis Romette, at jean-louis.romette@univmed.fr.

Retrospective diagnosis of two rabies cases in humans by high throughput sequencing.

BACKGROUND: Rabies is prevalent in 150 countries and is definitely found in Russian Federation. The average registered incidence of rabies infection among animals in Russia is 3000 cases per year. At least 500,000 cases of animal bites and scratches are registered in the Russia every year, but only 2-4 cases of rabies infection in humans are reported per year. This relatively low incidence of rabies infection among humans is the result of a well-organized program of rabies surveillance and control as well as the readily available vaccination and immunoglobulin therapies. However, physician awareness of rabies infection in patients with acute encephalopathy is low, and some cases of rabies remain undiagnosed. OBJECTIVES: A retrospective study of autopsy materials from patients who died of encephalitis of unknown etiology in the Astrakhan region of Russia in 2003. STUDY DESIGN: A broad-range polymerase chain reaction (PCR) analysis followed by high throughput sequencing were used for the diagnosis. RESULTS: Two cases of rabies were detected and subsequently confirmed using a fluorescent antibody test, an enzyme-linked immunosorbent assay (ELISA) and a mouse inoculation test. Two strains of rabies virus were isolated and characterized using virological methods. The entire genome of each strain was sequenced.

Similar virus spectra and seasonality in paediatric patients with acute respiratory disease, Ghana and Germany.

Epidemiological differences between tropical and temperate regions regarding viruses causing acute respiratory infection are poorly understood. This is in part because methodological differences limit the comparability of data from these two regions. Using identical molecular detection methods, we tested 1174 Ghanaian and 539 German children with acute respiratory infections sampled over 12 months for the 15 most common respiratory viruses by PCR. A total 43.2% of the Ghanaian and 56.6% of the German children tested positive for at least one respiratory virus. The pneumoviruses respiratory syncytial virus and human metapneumovirus were most frequently detected, in 13.1% and 25.1% within the Ghanaian and German children, respectively. At both study sites, pneumoviruses were more often observed at younger ages (p <0.001). In the Ghanaian rainy season, enveloped viruses were detected twice as often as non-enveloped viruses (prevalence rate ratio (PR) 2.0, 95% CI 1.7-2.4). In contrast, non-enveloped viruses were more frequent during the Ghanaian dry season (PR 0.6, 95% CI 0.4-0.8). In Germany, enveloped viruses were also more frequently detected during the relatively colder winter season (PR 1.6, 95% CI 1.2-2.1) and non-enveloped viruses during summer (PR 0.7, 95% CI 0.5-0.9). Despite a distance of about 5000 km and a difference of 44° latitude separating Germany and Ghana, virus spectra, age associations and seasonal fluctuation showed similarities between sites. Neither respiratory viruses overall, nor environmentally stable (non-enveloped) viruses in particular were more frequent in tropical Ghana. The standardization of our sampling and laboratory testing revealed similarities in acute respiratory infection virus patterns in tropical and temperate climates.

Human-associated Staphylococcus aureus strains within great ape populations in Central Africa (Gabon).

The risk of serious infections caused by Staphylococcus aureus is well-known. However, most studies regarding the distribution of (clinically relevant) S. aureus among humans and animals took place in the western hemisphere and only limited data are available from (Central) Africa. In this context, recent studies focused on S. aureus strains in humans and primates, but the question of whether humans and monkeys share related S. aureus strains or may interchange strains remained largely unsolved. In this study we aimed to evaluate the distribution and spread of human-like S. aureus strains among great apes living in captivity. Therefore, a primate facility at the International Centre for Medical Research of Franceville (Gabon) was screened. We detected among the primates a common human S. aureus strain, belonging to the spa-type t148. It was isolated from three different individuals of the western lowland gorilla (Gorilla gorilla gorilla), of which one individual showed a large necrotizing wound. This animal died, most probably of a staphylococcal sepsis. Additionally, we discovered the t148 type among chimpanzees (Pan troglodytes) that were settled in the immediate neighbourhood of the infected gorillas. A detailed analysis by pulsed field gel electrophoresis showed that the gorilla and chimpanzee isolates represented two closely related strains. To our knowledge, this is the first report of a human-associated S. aureus strain causing disease in great apes. The simultaneous detection in gorillas and chimpanzees indicated an interspecies transmission of this S. aureus strain. Our results recommend that protection of wild animals must not only be based on habitat conservation, but also on the assessment of the risk of contact with human pathogens.