Highly efficient and sensitive membrane-based concentration process allows quantification, surveillance, and sequencing of viruses in large volumes of wastewater.

Wastewater-based epidemiology is experiencing exponential development. Despite undeniable advantages compared to patient-centered approaches (cost, anonymity, survey of large populations without bias, detection of asymptomatic infected peoples…), major technical limitations persist. Among them is the low sensitivity of the current methods used for quantifying and sequencing viral genomes from wastewater. In situations of low viral circulation, during initial stages of viral emergences, or in areas experiencing heavy rains, the extremely low concentrations of viruses in wastewater may fall below the limit of detection of the current methods. The availability during crisis and the cost of the commercial kits, as well as the requirement of expensive materials such as high-speed centrifuge, can also present major blocks to the development of wastewater-based epidemiological survey, specifically in low-income countries. Thereby, highly sensitive, low cost and standardized methods are still needed, to increase the predictability of the viral emergences, to survey low-circulating viruses and to make the results from different labs comparable. Here, we outline and characterize new protocols for concentrating and quantifying SARS-CoV-2 from large volumes (500 mL-1 L) of untreated wastewater. In addition, we report that the methods are applicable for monitoring and sequencing. Our nucleic acid extraction technique (the routine C: 5 mL method) does not require sophisticated equipment such as automatons and is not reliant on commercial kits, making it readily available to a broader range of laboratories for routine epidemiological survey. Furthermore, we demonstrate the efficiency, the repeatability, and the high sensitivity of a new membrane-based concentration method (MBC: 500 mL method) for enveloped (SARS-CoV-2) and non-enveloped (F-specific RNA phages of genogroup II / FRNAPH GGII) viruses. We show that the MBC method allows the quantification and the monitoring of viruses in wastewater with a significantly improved sensitivity compared to the routine C method. In contexts of low viral circulation, we report quantifications of SARS-CoV-2 in wastewater at concentrations as low as 40 genome copies per liter. In highly diluted samples collected in wastewater treatment plants of French Guiana, we confirmed the accuracy of the MBC method compared to the estimations done with the routine C method. Finally, we demonstrate that both the routine C method processing 5 mL and the MBC method processing 500 mL of untreated wastewater are both compatible with SARS-CoV-2 sequencing. We show that the quality of the sequence is correlated with the concentration of the extracted viral genome. Of note, the quality of the sequences obtained with some MBC processed wastewater was improved by dilutions or enzyme substitutions suggesting the presence of specific enzyme inhibitors in some wastewater. To the best of our knowledge, our MBC method is one of the first efficient, sensitive, and repeatable method characterized for SARS-CoV-2 quantification and sequencing from large volumes of wastewater.

Metal enriched quasi-ultrafine particles from stainless steel gas metal arc welding induced genetic and epigenetic alterations in BEAS-2B cells.

Recent evidence has supported welding fume (WF)-derived ultrafine particles (UFP) could be the driving force of their adverse health effects. However, UFP have not yet been extensively studied and are currently not included in present air quality standards/guidelines. Here, attention was focused on the underlying genetic and epigenetic mechanisms by which the quasi-UFP (Q-UFP, i.e., ≤ 0.25 µm) of the WF emitted by gas metal arc welding-stainless steel (GMAW-SS) exert their toxicity in human bronchial epithelial BEAS-2B cells. The Q-UFP under study showed a monomodal size distribution in number centered on 104.4 ± 52.3 nm and a zeta potential of -13.8 ± 0.3 mV. They were enriched in Fe > Cr > Mn > Si, and displayed a relatively high intrinsic oxidative potential. Dose-dependent activation of nuclear factor erythroid 2-related factor 2 and nuclear factor-kappa B signaling pathway, glutathione alteration, and DNA, protein and lipid oxidative damage were reported in BEAS-2B cells acutely (1.5 and 9 µg/cm2, 24 h) or repeatedly (0.25 and 1.5 µg/cm2, 3 × 24 h) exposed to Q-UFP (p < 0.05). Alterations of the Histone H3 acetylation were reported for any exposure (p < 0.05). Differentially regulated miRNA and mRNA indicated the activation of some critical cell signaling pathways related to oxidative stress, inflammation, and cell cycle deregulation towards apoptosis. Taken together, these results highlighted the urgent need to better evaluate the respective toxicity of the different metals and to include the Q-UFP fraction of WF in current air quality standards/guidelines relevant to the occupational settings.

Hydroxychloroquine lung pharmacokinetics in critically ill patients with COVID-19.

Different dosage regimens of hydroxychloroquine (HCQ) have been used to manage COVID-19 (coronavirus disease 2019) patients, with no information on lung exposure in this population. The aim of our study was to evaluate HCQ concentrations in the lung epithelial lining fluid (ELF) in patients infected with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the virus that causes COVID-19. This was a retrospective, observational, multicentre, pharmacokinetic study of HCQ in critically ill COVID-19 patients. No additional interventions or additional samples compared with standard care of these patients were conducted in our teaching hospital. We included all intubated COVID-19 patients treated with crushed HCQ tablets, regardless of the dosage administered by nasogastric tube. Blood and bronchoalveolar lavage samples (n = 28) were collected from 22 COVID-19 patients and total HCQ concentrations in ELF were estimated. Median (interquartile range) HCQ plasma concentrations were 0.09 (0.06-0.14) mg/L and 0.07 (0.05-0.08) mg/L for 400 mg × 1/day and 200 mg × 3/day, respectively. Median HCQ ELF concentrations were 3.74 (1.10-7.26) mg/L and 1.81 (1.20-7.25) for 400 mg × 1/day and 200 mg × 3/day, respectively. The median ratio of ELF/plasma concentrations was 40.0 (7.3-162.7) and 21.2 (18.4-109.5) for 400 mg × 1/day and 200 mg × 3/day, respectively. ELF exposure is likely to be underestimated from HCQ concentrations in plasma. In clinical practice, low plasma concentrations should not induce an increase in drug dosage because lung exposure may already be high.

Association between Zika virus and fetopathy: a prospective cohort study in French Guiana.

OBJECTIVE: To establish the incidence of fetal central nervous system (CNS) anomalies (including microcephaly), signs of congenital infection and fetal loss in pregnant women infected with Zika virus (ZIKV) and non-infected pregnant women in western French Guiana. METHODS: This prospective cohort study was conducted between 1 January and 15 July 2016. We evaluated and compared clinical and fetal ultrasound examinations of 301 pregnant women with biological confirmation of ZIKV infection and 399 pregnant women who were negative for ZIKV infection. RESULTS: Overall, the total number of fetuses with CNS involvement was higher in the infected than in the control group (9.0% vs 4.3%; relative risk, 2.11 (95% CI, 1.18-4.13)). Anomalies of the corpus callosum and presence of cerebral hyperechogenicities were significantly more common in the infected group. There was an increased risk of microcephaly in the infected compared with the control group (1.7% vs 0.3%; relative risk, 6.63 (95% CI, 0.78-57.83)), although this was not statistically significant. When the mother was infected during the first or second trimester, there was a greater risk of severe CNS involvement, more signs of infection and intrauterine fetal death than with infection in the third trimester. The rate of vertical transmission in the exposed group was 10.9%. CONCLUSION: ZIKV infection during pregnancy is associated with a significant risk of fetal CNS involvement and intrauterine fetal death, particularly when infection occurs during the first or second trimesters. Microcephaly was not present in every case of congenital ZIKV syndrome that we observed. Until more is known about this disease, it is paramount to evaluate suspected cases by detailed neurosonography on a monthly basis, paying particular attention to the corpus callosum and the presence of hyperechogenic foci. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

[Zika virus outbreak in Latin America: what are the challenges for French Guiana in April 2016?]. / Épidémie de virus Zika en Amérique latine : quels enjeux pour la Guyane française en avril 2016 ?

Started in 2015 in Brazil, an outbreak linked to a little known arbovirus, Zika virus spread throughout Latin America. This virus, considered until recently as responsible of only mild symptoms, made mention of previously unsuspected complications, with severe neurological manifestations in adults and malformations of the central nervous system, including microcephaly, in newborns of mother infected during the pregnancy. While the continent is more accustomed to the succession of arbovirus epidemics, suspected complications and the many unknowns keys of the latter arriving raise many public health issues. French Guiana, a French territory located in the north-east of the continent, combines both European level of resources and climate and issues specific to the Amazon region and Latin America. We discuss here the issues for 2016 Zika virus epidemic in our region, many of them are generalizable to neighboring countries.

Highly heterogeneous temperature sensitivity of 2009 pandemic influenza A(H1N1) viral isolates, northern France.

We assayed the temperature sensitivity of 2009 pandemic influenza A(H1N1) viral isolates (n=23) and seasonal influenza A(H1N1) viruses (n=18) isolated in northern France in 2007/08 and 2008/09. All isolates replicated with a similar efficiency at 34 °C and 37 °C, and with a lower efficiency at 40 °C. The pandemic viral isolates showed a stronger heterogeneity in their ability to grow at the highest temperature, as compared with the seasonal isolates. No statistically significant difference in temperature sensitivity was observed between the pandemic viral isolates from severe and mild cases of influenza. Our data point to the impact of temperature sensitivity on the genetic evolution and diversification of the pandemic influenza A(H1N1) virus since its introduction into the human population in April 2009, and call for close surveillance of this phenotypic marker related to host and tissue tropism.

S-OtrH3N2 viruses: use of sequence data for description of the molecular characteristics of the viruses and their relatedness to previously circulating H3N2 human viruses.

Emergence of influenza viruses from the animal reservoir is a permanent challenge. The rapid description and immediate sharing of information on these viruses is invaluable for influenza surveillance networks and for pandemic preparedness. With the help of data generated from the World Health Organization Collaborating Centre for Reference and Research on Influenza at the United States Centers for Disease Control and Prevention, we provide here information on the swine­origin triple reassortant influenza A(H3N2) viruses detected in human cases in the north-east of the United States.

Automatic sample production by depositing solutions on filters for the organization of proficiency tests.

This article describes a device intended to produce replicas on filters by liquid deposition of anion or metal solutions. Schematically, the filters are housed in cassettes labelled automatically by means of a code. An automatic arm takes each cassette, reads the code, and deposits the amount of element required. Weighing before and after deposition allows the amount deposited to be accurately checked and determined. This automated system allows the production of replicas with high deposition regularity, replica dispersion for the most part being <1%. The samples produced can be used during proficiency tests where the assigned value is determined either by the participants or by the organizer.

Census and analysis of persistent false-negative results in serological diagnosis of human immunodeficiency virus type 1 group O infections.

Human immunodeficiency viruses (HIV) have a high level of genetic diversity. The outlier variants of HIV type 1 (HIV-1) group O are distantly related to HIV-1 group M. Their divergence has an impact on serological diagnosis, with a risk of false-negative results. In this study, we report 20 failure cases, involving patients with primary or chronic infection, in France and Cameroon between 2001 and 2008. Our results indicate that some assays detected group O infection much less efficiently than others. Two major reasons for these false-negative results were identified: the presence or absence of a group O-specific antigen (and the designed sequence) for the detection of antibodies and the greater envelope variability of group O than of group M strains. This study highlights the complexity of screening for these divergent variants and the need to evaluate test performance with a large panel of strains, due to the extensive diversity of group O variants.

First report of Litomosa spp. (Nematoda: Filarioidea) from Malagasy bats; review of the genus and relationships between species.

The presence of the filarial genus Litomosa in Malagasy bats is demonstrated by the finding of L. goodmani n. sp. from Miniopterus gleni and Litomosa sp. (male unknown) from M. manavi, both in the Special Reserve of Ankarana. These materials are compared to the 22 Litomosa species, including two Indian species originally placed in the genus Litomosoides, L. fotedari (Gupta and Trivedi, 1989) n. comb. and L. tewarii (Gupta and Trivedi, 1989) n. comb., and the new taxon L. seurati n. sp. (= L. beaucournui Bain, 1966 pro parte), type-host Rhinolophus ferrum-equinum, Algeria, distinguished by the narrow area rugosa and the female caudal extremity with two conspicuous points, instead of several small ones. The Malagasy material belongs to a group of species close to the type, L. filaria, which have a male area rugosa composed of cuticular bosses and microfilariae folded within the sheath, and which are parasitic in Vespertilionidae, Hipposideridae and Rhinolophidae from Africa and Europe. The two Malagasy species resemble L. seurati n. sp., L. beshkovi Jancev, 1971, L. chiropterum Ortlepp, 1932, L. adami Petit, 1980 and L. ottavianii Lagrange et Bettini, 1948, with the enlarged third segment of the buccal capsule. L. goodmani n. sp. is distinct with its small size and female caudal extremity with a single point, which is suppressed in old mature worms; the females of Litomosa sp. have two conical points. Relationships among Litomosa species appear to be dependent upon both the chiropteran host groups and the geographical region.

[Epidemics of acute respiratory infections in Madagascar in 2002: from alert to confirmation]. / Epidémies d'infections respiratoires aiguës à Madagascar en 2002: de l'alerte à la confirmation.

UNLABELLED: An epidemiological investigation (Ministry of Health/Institut Pasteur de Madagascar (IPM)) was conducted in July 2002, in two districts of a same province (Fianarantsoa: Fianarantsoa II and Ikongo) considering the high frequency of deaths linked with acute respiratory infection (ARI). Morbidity and mortality data was collected in the Centre de Santé de Base (CSB) which gave the alert (village of Sahafata, district Fianarantsoa II). Analysis of monthly activity reports (MAR) allowed calculation of incidence rates of ARI/pneumonia in Fianarantsoa province. Virological data was based on the analysis of nasopharyngeal samples collected during the investigations. Clinical symptoms and homogeneity of laboratory results are consistent with an origin of these epidemics being related to the circulation of an influenza virus A subtype H3N2. Attack rates were very high. CFR was significantly higher in individuals of less than 1 year and more than 65 years. This data was confirmed by posterior investigations of teams from MoH/WHO. Surprisingly, this large epidemic was due to a known influenza virus that previously circulated in countries of northern hemisphere (the year before) and even in Antananarivo weeks before. Different hypothesis could be proposed to explain such phenomenon: great restriction of exchanges between different geographical zones, nutritional status.... CONCLUSION: The epidemic episodes of acute respiratory infections in Madagascar in July 2002 were due to an influenza virus A subtype H3N2 without any genotypic or phenotypic features. Various factors, could explain the importance of the epidemic and particular high lethality found in some age groups. This epidemic illustrates the relative incapacity for a developing country, to face and manage a flu epidemic caused by a classical influenza virus.

[Influenza epidemiologic and virologic surveillance in Antananarivo from 1995 to 2002]. / Surveillance épidémiologique et virologique de la grippe à Antananarivo de 1995 à 2002.

The "Institut Pasteur de Madagascar" virology laboratory is the National WHO Centre for Influenza surveillance in Madagascar. On this surveillance collaborate the Ministry of Health with 9 sentinel centres. In the present article, the authors relate the results of influenza surveillance in Antananarivo between 1995 and 2002. Among 6341 patients with nasal and/or pharyngeal swabs, influenza virus were isolated from 427 patients (6.7%): 307 (68.4%) influenza virus A (H3N2), 124 (27.1%) influenza virus B, 8 (4.0%) influenza virus A (H1N1). The virus had been continually spreading all year long. The weak and the strong points of the influenza sentinel surveillance are also discussed in order to ameliorate the collection processes of influenzal and respiratory morbidity data.

[Genetic variability of Poliovirus: typing of strains isolated in Madagascar by restriction fragment length polymorphism (RFLP) assay]. / Variabilité génétique des Poliovirus: étude du polymorphisme de la longueur des fragments de restriction enzymatique de souches isolées à Madagascar.

The differentiation of the vaccine or wild origin of Poliovirus at the laboratory is an important step towards the process of the poliomyelitis eradication. We report herein the results obtained from Poliovirus types 3 and 2, isolated in Madagascar in 1997 and 2002 from healthy children and cases of acute flaccid paralysis, respectively. The technique used is based on the amplification of genome (RT-PCR), followed by Restriction Fragment Length Polymorphism assay (RFLP), performed in 3 different regions of the genome. In the capsid region (VP3-VP1 and VP1-2A), RFLP analysis allowed us to differentiate without ambiguity the wild or vaccine origin of the Poliovirus type 3, and to identify Vaccine-Derived Poliovirus (VDPV) type 2. In the noncapsid region, including the RNA polymerase and 3' non coding region (3Dpol-3' NTR), the VDPV were found to be recombinant with other Enteroviruses. These results confirm that RFLP assay is a reliable tool for intratypic differentiation and to study the genetic drift and recombination of Poliovirus.

[Persistence of an endemic circulation of the West Nile virus in Madagascar]. / Persistance d'une circulation endémique du virus West Nile a Madagascar.

The wide geographic distribution of the West Nile virus and the increase in virulence observed since 1994 in the Mediterranean basin, central Europe and North America, with several outbreaks of lethal encephalitis, demonstrate the importance of regular surveillance of the epidemiological data regarding this virus in the world. The Institut Pasteur de Madagascar has shown between 1975 and 1990 that this arbovirus was most abundant in Madagascar, where it had an endemic circulation. There has been no further study since that time. In order to evaluate the level of circulation, the seroprevalence of anti-West Nile antibodies in children that are 15 or less was measured on two different collections of sera. These collections came from population studies realised respectively in the region of Ambositra in the Highlands in 1996 and in the city of Mahajanga on the north west coast in 1999. The seroprevalence were 2.1% and 10.6% respectively, these results indicate that the circulation of this climatic dependent virus is still significant.

[Hemoparasites of bats in Madagascar]. / Hémoparasites des chauves-souris à Madagascar.

This study aims to evaluate the prevalence and density of haemoparasites in wild malagasy bats. Among the 440 bats, belonging to 14 species sampled in 5 localities in different bio-climatic zones of the island, 93 (21%) showed at least 1 haemoparasite with, by order of frequency, Haemoproteidae (15.7% of 440 bats), microfilariae (7.0%) and Trypanosoma (0.7%). Among these 93 bats, 92 (99%) belonged to the family Vespertilionidae. Four bat species, all endemic to the Madagascar region (Madagascar and Comoros), were found to harbour parasites: Miniopterus manavi with Haemoproteidae (38% of 129 individuals), microfilariae (23%) and Trypanosoma (2%); Myotis goudoti with Haemoproteidae (24% of 68 individuals) and microfilariae (1%); Miniopterus gleni with Haemoproteidae (23% of 13 individuals); and Triaenops furculus with Haemoproteidae (4% of 28 individuals). The sex of bats was not linked to parasite prevalence. Within Miniopterus manavi, those individuals with greater weight also had a higher prevalence of microfilariae; and within the individuals harbouring microfilariae the greatest weights corresponded to the highest density of microfilariae. Ten bat species (with 202 individuals examined) were negative for any haemoparasite. This study is the first to provide evidence of haemoparasites in Malagasy bats; it provides interesting insights, especially concerning the parasite distribution per bat species and families, the pathogenicity of this type of parasitism and the parasite transmission by arthropod vectors.

[New method of molecular typing in human Enteroviruses: characterization of Madagascar "untypable" strains]. / Nouvelle méthode de typage moléculaire des Entérovirus humains: caractérisation des souches malgaches "non sérotypables".

Enteroviruses, members of the family Picornaviridae, are responsible for a wide variety of diseases and represent a major public health hazard. Typing of non polio enterovirus (NPEV) infection is traditionally based on a serum neutralization assay. However, this method is time-consuming, labor-intensive, expensive, and may fail to identify antigenic variation. A new molecular typing involving partial sequencing of the genome has been recently developed. In this study, 46 NPEV strains were analyzed, including 37 antigenicaly "untypeable" viruses. Partial sequencing of the C-end of the viral capsid protein VP1 and pairwise identity with the prototype strains allow us to assign a serotype for all "untypeable" viruses. The results show a large number and wide variety of Coxsackieviruses A which belong to the HEV-C species and also Echoviruses and Coxsackieviruses B of the HEV-B species. This method may be useful to identify all NPEV serotypes in Madagascar and to assess the possible impact of circulating NPEV populations, as we enter the final stage of poliomyelitis eradication.

[African Swine Fever introduction into Madagascar, history and lessons from an emergence]. / Introduction de la Peste Porcine Africaine à Madagascar, histoire et leçons d'une émergence.

African Swine Fever (ASF) was diagnosed for the first time in Madagascar in 1998. ASF has apparently been introduced from the African continent to the southern part of the island with a subsequent spread to other regions except for areas in the north and in the west. The epidemic has had severe economic consequences for the home market of pork meat production. This article reviews the course of the epidemic with particular emphasis on the vectors involved in the transmission of the virus, such as the soft tick, Ornithodoros moubata porcinus. Presence of this vector and of the bushpig, Potamochoerus larvatus, as a potential wild reservoir, are some of the major obstacles in control of ASF in Madagascar. A veterinary disease surveillance system has to be urgently warranted.