Co-circulation of Toscana virus and Leishmania infantum in a focus of zoonotic visceral leishmaniasis from Central Tunisia.

In the Mediterranean basin, sand flies are vectors of Leishmania parasites and phleboviruses affecting humans and animals. In this study, we aimed to investigate phlebovirus and Leishmania parasites circulating in a focus of zoonotic visceral leishmaniasis (ZVL) located in a highly irrigated area within the arid Central Tunisia, known mainly to be endemic for zoonotic cutaenous leishmaniasis (ZCL) caused Leishmania major and transmitted by Phlebotomus papatasi. Sand flies were collected using CDC light traps in the village of Saddaguia, an emergent focus of ZVL located in Central Tunisia during September-October 2014, 2015, and 2016. Pools of live female sand flies were screened for phleboviruses and Leishmania by nested PCR in the polymerase gene and kinetoplast minicircle DNA, respectively. Dead sand flies were identified morphologically to species level. Sand flies of the subgenus Larroussius mainly Phlebotomus perfiliewi, Phlebotomus perniciosus, and Phlebotomus longicuspis were predominant in this ZVL focus compared to P. papatasi. A total of 1932, 1740, and 444 sand flies were tested in 2014, 2015 and 2016, respectively. Pathogen screening performed on 4116 sand flies distributed in 148 pools revealed the presence of Leishmania infantum and Toscana virus. The minimum infection rates of sand flies with TOSV in 2014, 2015, and 2016 were 0.05%, 011%, and 0.22%, respectively. The minimum infection rates of sand flies with L. infantum in 2014, 2015, and 2016 were 0.25%, 012%, and 0.79%, respectively. No L. major was detected during the 3-years investigation in this ZVL focus. Our results showed clearly the endemic co-circulation of TOSV and L. infantum in this emergent ZVL focus. However, no co-infection of TOSV and L. infantum was detected in any of the sand fly pools investigated during the three years period. TOSV was isolated from positive pools in 2015. Phylogenetic analysis showed that the Tunisian strains of TOSV belonged to the sublineage A. Based on the present findings, our results provided strong evidence that TOSV and L. infantum are transmitted by the same predominant sand fly species of the subgenus Larroussius, and subsequently, humans and dogs could be co-infected through co-infected or successive infected bites. Our results showed clearly that the development of irrigation in arid areas contributed significantly to the establishment of stable transmission cycles of L. infantum and TOSV and subsequently to the emergence of a ZVL focus within this arid bio-geographical area characterized by the presence of multiple foci of ZCL located outside the study site. Thus, more studies are needed to better understand the impact of RNA viruses shared by vectors and reservoir hosts of L. infantum on the development of zoonotic visceral leishmaniasis.

Phleboviruses associated with sand flies in arid bio-geographical areas of Central Tunisia.

An entomological investigation was carried out in 2014 at two sites located in Central Tunisia, one irrigated and another non-irrigated situated in arid bio-geographical areas. Sand flies of the subgenus Larroussius namely Phlebotomus perfiliewi, Phlebotomus perniciosus, and Phlebotomus longicuspis are the most abundant sand fly species in the irrigated site. However, in the non-irrigated site, Phlebotomus papatasi of the Phlebotomus genus is the most abundant species. A total of 3191 sand flies were collected and pooled with up to 30 specimens per pool based on sex, trapping location and collection date, were tested for the presence of phleboviruses by nested reverse transcriptase polymerase chain reaction in the polymerase gene and sequenced. Of a total of 117 pools, 4 were positive, yielding a minimum infection rate of sand flies with phleboviruses of 0.12%. Phylogenetic analysis performed using partial nucleotide and amino acid sequence in the polymerase gene showed that these phleboviruses belonged to four different clusters corresponding to Toscana virus (TOSV), Saddaguia virus (SADV), Sandfly Fever Sicilian Virus (SFSV) and Utique virus (UTIV). This study provides more evidence that the abundance of P. perfiliewi is associated with the development of irrigation in arid bio-geographical areas of Central Tunisia which may have led to the emergence of phleboviruses. We report the first detection of TOSV from sand flies collected from Central Tunisia.

Serologic evidence of exposure to Rift Valley fever virus detected in Tunisia.

Rift Valley fever virus (RVFv) is capable of causing dramatic outbreaks amongst economically important animal species and is capable of causing severe symptoms and mortality in humans. RVFv is known to circulate widely throughout East Africa; serologic evidence of exposure has also been found in some northern African countries, including Mauritania. This study aimed to ascertain whether RVFv is circulating in regions beyond its known geographic range. Samples from febrile patients (n = 181) and nonfebrile healthy agricultural and slaughterhouse workers (n = 38) were collected during the summer of 2014 and surveyed for exposure to RVFv by both serologic tests and PCR. Of the 219 samples tested, 7.8% of nonfebrile participants showed immunoglobulin G reactivity to RVFv nucleoprotein and 8.3% of febrile patients showed immunoglobulin M reactivity, with the latter samples indicating recent exposure to the virus. Our results suggest an active circulation of RVFv and evidence of human exposure in the population of Tunisia.

Infection of sand flies collected from different bio-geographical areas of Tunisia with phleboviruses.

An entomological investigation performed in 2013 covering different bio-geographical areas varying from humid in the north to the arid in the center showed that sand flies of the subgenus Larroussius including Phlebotomus perniciosus, Phlebotomus perfiliewi, and Phlebotomus longicuspis are abundant and widely distributed in Tunisia. A total of 3992 collected and pooled with up to 30 specimens per pool based on sex, trapping location and collection data were tested for the presence of phleboviruses by nested reverse transcriptase polymerase chain reaction and sequencing. Of a total of 135 pools, 23 were positive, yielding and minimum infection rate of 0.6%. Phylogenetic analysis performed using partial amino acid sequence in the polymerase gene showed that all these phleboviruses were grouped in one cluster clearly distinct from but closely related to Massilia virus and Granada virus. This putative novel virus, tentatively called Saddaguia virus (SADV), is widely distributed in Tunisia. Together with Toscana, Punique, and Utique viruses, SADV is the fourth recognized phlebovirus to be transmitted by sand flies in Tunisia. The medical and public health interest of SADV remains to be investigated.

Enterovirus detection in stool specimen: relevance for poliovirus and enterovirus surveillance.

Detection of enterovirus genome by PCR in clinical samples is now extensively used for the diagnostic of enterovirus infections given its rapidity and high sensitivity. In contrast, its use in surveillance programs targeting specific enterovirus serotypes remains less frequent. The most sensitive protocols are those amplifying in the 5'untranslated region (5'UTR). However the possibility to use sequence analysis of the 5'UTR amplicons for serotype identification is not yet well established. In this report, stool samples from polio suspected cases and their healthy contacts were tested. The results of direct detection of enterovirus genome by PCR and serotype identification based on sequence analysis of the PCR products in the 5'UTR were compared to those of standard cell-culture-based protocols. Standard protocols detected enterovirus isolates in 7.4% of cases while 9.8% of samples were positive by PCR. Serotype identification based on sequence analysis of amplicons showed concordant results with serotypes determined on virus isolates by seroneutralisation or sequencing in the VP1 gene in 39% of cases only. These results confirm that the use of PCR amplification from stool samples improves the sensitivity of enterovirus detection but do not recommend the use of sequence analysis of the 5'UTR PCR product to determine enterovirus serotype.

Localization of saturated karst aquifer with magnetic resonance sounding and resistivity imagery.

To answer one of the main questions of hydrogeologists implementing boreholes or working on pollution questions in a karst environment--i.e., where is the ground water?--numerous tools including geophysics are used. However, the contribution of geophysics differs from one method to the other. The magnetic resonance sounding (MRS) method has the advantage of direct detection of ground water over other geophysical methods. Eight MRSs were implemented over a known karst conduit explored and mapped by speleologists to estimate the MRS ability to localize ground water. Two direct current resistivity imageries (DC-2D imagery) were also implemented to check their capability to map a known cave. We found that the MRS is a useful tool to locate ground water in karst as soon as the quantity of water is enough to be detected. The threshold quantity is a function of depth and it was estimated by forward modeling to propose a support graph to hydrogeologists. The measured MRS's signals could be used to calculate transmissivity and permeability estimators. These estimators were used to map and to draw a cross section of the case study site, which underline accurately the known karst conduit location and depth. We also found that the DC-2D imagery could underline the karst structures: It was able to detect the known cave through its associated faults. We prepared a computer simulation to check the depth of such a cave to induce resistivity anomaly which could be measured in similar conditions.