Incidence and risk factors of SARS-CoV-2 infection among workers in a public health laboratory in Tunisia.

The aim of this study was to measure the extent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among workers at the Institut Pasteur de Tunis (IPT), a public health laboratory involved in the management of the COVID-19 pandemic in Tunisia, and to identify risk factors for infection in this occupational setting. A cross-sectional survey was conducted on IPT workers not vaccinated against coronavirus disease 2019 (COVID-19). Participants completed a questionnaire that included a history of reverse transcription-polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 infection. Immunoglobulin G antibodies against the receptor-binding domain of the spike antigen (anti-S-RBD IgG) and the nucleocapsid protein (anti-N IgG) of the SARS-CoV-2 virus were detected by enzyme-linked immunoassay (ELISA). A multivariate analysis was used to identify factors significantly associated with SARS-CoV-2 infection. A total of 428 workers were enrolled in the study. The prevalence of anti-S-RBD and/or anti-N IgG antibodies was 32.9% [28.7-37.4]. The cumulative incidence of SARS-CoV-2 infection (positive serology and/or previous positive RT-PCR test) was 40.0% [35.5-44.9], while the proportion with asymptomatic infection was 32.9%. One-third of the participants with RT-PCR-confirmed infection tested seronegative more than 90 days postinfection. Participants aged over 40 and laborers were more susceptible to infection (adjusted OR [AOR] = 1.65 [1.08-2.51] and AOR = 2.67 [1.45-4.89], respectively), while tobacco smokers had a lower risk of infection (AOR = 0.54 [0.29-0.97]). The SARS-CoV-2 infection rate among IPT workers was not significantly different from that detected concurrently in the general population. Hence, the professional activities conducted in this public health laboratory did not generate additional risk to that incurred outside the institute in day-to-day activities.

First Report of Two Jaculus Rodents as Potential Reservoir Hosts of Leishmania Parasites in Tunisia.

This study shows, for the first time, natural Leishmania infection among Jaculus spp. in an endemic region of Tataouine, South Tunisia. To better characterize the transmission cycles in this complex focus of mixed transmission, Leishmania detection and species identification were performed by direct examination, internal transcribed spacer-1 (ITS1)-PCR-restriction fragment length polymorphism (RFLP), and sequencing of Jaculus (J.) jaculus (Linnaeus, 1758) and J. hirtipes (Lichtenstein, 1823) rodent species, which are frequently encountered in this area. Leishmania parasites were observed in 19 (41.3%) smears, while DNA parasites were detected in 28 (60.9%) Jaculus spp. spleens; among them, 12 (54.5%) were from 22 J. jaculus individuals and 16 (66.7%) were from 24 J. hirtipes individuals. Leishmania parasites were confirmed as Leishmania (L.) killicki (syn. L. tropica) in two J. hirtipes individuals (4.3%) and L. major (n = 24; 52.2%) in 10 J. jaculus and 14 J. hirtipes individuals. This finding represents the first evidence of natural infection with Leishmania parasites in rodents belonging to the Jaculus genus, providing the rationale to consider them as potential reservoir hosts of Old World Leishmania parasites in Tunisia and North Africa.

Evaluation of the Taxonomic Status of Lesser Egyptian Jerboa, Jaculus jaculus: First Description of New Phylogroups in Tunisia.

The taxonomy of the Lesser Egyptian jerboa, Jaculus (J.) jaculus (Dipodinae subfamily), was recently reevaluated, and the taxonomic status was defined by the presence of two cryptic species, J. jaculus (Linnaeus 1758) and J. hirtipes (Lichtenstein, 1823), with a higher genetic divergence in the sympatric North African populations than in other studied parapatric populations. Using phylogenetic analysis of the cytochrome b (Cytb) gene from 46 specimens, we confirmed the new status in Tunisia; rodents were collected from two different biotopes belonging to the same locality at the ecological level (mountainous vs. Saharan) in the south of the country. The study of the eye lens weight of these specimens allowed the definition of a cutoff value (58.5 g), categorizing juveniles from adults. Moreover, this study confirmed the phylotaxonomic status of J. jaculus in Tunisia, as recently illustrated, into two distinct species, J. jaculus and J. hirtipes, and recorded for the first time the presence of two phylogroups among each of these rodent species. The lack of clear micro-geographical structure and biotope specificity between the two rodent species and their phylogroups was also highlighted.

A prospective cohort study of Cutaneous Leishmaniasis due to Leishmania major: Dynamics of the Leishmanin skin test and its predictive value for protection against infection and disease.

BACKGROUND: Leishmanin Skin Test (LST) is considered as a useful indicator of past infection by Leishmania parasites. However, the temporal dynamics of a positive LST under different epidemiologic scenarios and whether it relates to the protection against the recurrence of an overt disease are not fully documented. METHODOLOGY/PRINCIPAL FINDINGS: We report here on a population based prospective study conducted on 2686 individuals living in two foci located in Central Tunisia, to assess over a one-year epidemiologic season, the incidence of Leishmania (L.) major infection and disease and changes in LST reactivity. The two foci were both endemic for Cutaneous Leishmaniasis (CL) due to L. major, but contrasted in their history for this disease (ie: an old focus versus a recent focus). We found that most infections occurred in the new focus (290/1000; 95% CI: 265-315 person-years) with an incidence rate of CL lesions 2.4 times higher than in the old focus. Likewise, the rates of LST reactivity reversion and loss, in the new focus, were 99/1000[38-116] person-years and 14/1000[8-21] person-years, respectively. Loss of LST reactivity was not noticed in the old focus. Interestingly, the incidence rates of symptomatic infection did not differ significantly according to the LST status at enrolment (negative versus positive) between the combined foci and the new one. CONCLUSIONS/SIGNIFICANCE: Our findings confirm LST as a good tool for assessing L. major cryptic infection. However, the instability of the LST positivity in new foci should be considered as an important confounder of the outcome of this infection when developing a research protocol for vaccine trial.

Evaluation of the influenza-like illness surveillance system in Tunisia, 2012-2015.

BACKGROUND: This study was initiated to evaluate, for the first time, the performance and quality of the influenza-like illness (ILI) surveillance system in Tunisia. METHODS: The evaluation covered the period of 2012-2015 and used different data sources to measure indicators related to data quality and completeness, representativeness, timeliness, simplicity, acceptability, flexibility, stability and utility. RESULTS: During the evaluation period, 485.221 ILI cases were reported among 6.386.621 outpatients at 268 ILI sentinel sites. To conserve resources, cases were only enrolled and tested for influenza during times when the number of patients meeting the ILI case definition exceeded 7% (10% after 2014) of the total number of outpatients for the week. When this benchmark was met, five to 10 patients were enrolled and sampled by nasopharyngeal swabs the following week. In total, The National Influenza Center (NIC) received 2476 samples, of which 683 (27.6%) were positive for influenza. The greatest strength of the system was its representativeness and flexibility. The timeliness of the data and the acceptability of the surveillance system performed moderately well; however, the utility of the data and the stability and simplicity of the surveillance system need improvement. Overall, the performance of the Tunisian influenza surveillance system was evaluated as performing moderately well for situational awareness in the country and for collecting representative influenza virologic samples. CONCLUSIONS: The influenza surveillance system in Tunisia provided pertinent evidence for public health interventions related to influenza situational awareness. To better monitor influenza, we propose that ILI surveillance should be limited to sites that are currently performing well and the quality of data collected should be closely monitored and improved.

Presence of Sergentomyia (Parrotomyia) lewisi (Diptera: Psychodidae) in Tunisia.

In Tunisia, 17 phlebotomine sand fly species are reported, belonging to genera Phlebotomus and Sergentomyia. However, subsequent to faunal studies and outbreaks of leishmaniasis in different regions of the country, unrecognized sand fly species could exist. Indeed, we report in this study the presence of Sergentomyia (Parrotomyia) lewisiParrot 1948. A brief collection of phlebotomine sand flies was undertaken in August 2016 in Khbina locality, in Sidi Bouzid governorate situated in Central Tunisia, which constitutes an old focus of Zoonotic Cutaneous Leishmaniasis. Sand flies were collected, using CDC light traps that were placed overnight, in different biotopes (inside habitations, outdoors, and within animal shelters). Specimens were collected and morphologically identified. Measurements were taken with an ocular micrometer. Two female specimens of the species Se. (Parrotomyia) lewisi were collected. One specimen is here described and measured. A comparison of its taxonomic characters to the holotype from Sudan and a specimen from Algeria is also presented. This species was until now only reported from Sudan, Ethiopia, Algeria, and Morocco. It is here described for the first time in Tunisia, which raises the Tunisian sand fly fauna to seven subgenera and 18 species.

First detection of Leishmania DNA in Psammomys obesus and Psammomys vexillaris: Their potential involvement in the epidemiology of leishmaniasis in Tunisia.

Leishmaniasis, a public health problem in Tunisia, are diseases caused by different Leishmania species. Cutaneous leishmaniasis is present from the North to the South under different forms, due to Leishmania (L.) major, L. infantum or L. tropica. Whereas, Psammomys (P.) obesus is the confirmed reservoir host of L. major, those of L. tropica and dermotropic L. infantum wait to be identified. Importantly, P. vexillaris species have been recently highlighted; however, no studies have been carried out to explore its potential role in leishmaniasis epidemiology. Seventy two rodents were collected from Central and South-West of Tunisia between 2007 and 2010. Using several methods, 43 animals were identified as P. obesus and 29 as P. vexillaris. Leishmania kinetoplast DNA was detected in liver samples by real-time PCR in 18 P. obesus and in 8 P. vexillaris. Then, the direct sequencing of the amplified internal transcribed spacer 1, allowed the identification of L. infantum DNA in five P. obesus and in three P. vexillaris, as well as L. tropica DNA in three other P. vexillaris. Whereas, PCR fluorescent fragment length analysis of the 7 spliced leaders, allowed identifying L. major among infected P. obesus and P. vexillaris, and interestingly co-infection (L. major/L. infantum) among two P. obesus. We report here for the first time, the infection of P. obesus, from Central Tunisia, by L. infantum. Suggesting that P. obesus the known reservoir host of L. major, may also serve as reservoir host for L. infantum and thus play a role in the spread of sporadic cutaneous or visceral leishmaniasis in this region. Of equal importance, this work establish for the first time, the natural infection of P. vexillaris by different Leishmania species, suggesting its potential epidemiological role as reservoir host.

Natural infection of Ctenodactylus gundi by Leishmania major in Tunisia.

Incriminating new rodent species, as reservoir hosts of Leishmania parasites is crucial for understanding the transmission cycle of cutaneous leishmaniasis in Tunisia. Ctenodactylus (C.) gundi was previously described as extremely abundant in all Tunisian Leishmania (L.) tropica foci in south Tunisia besides its presence in L. major endemic area. The aim of this study was to detect Leishmania species parasites among C. gundi in two endemic regions in Tunisia: Sidi Bouzid and Tataouine. Total DNA was isolated from the spleens and the livers of 92C. gundi. Leishmaniasis clinical manifestations were detected among 11 rodents (12%). Leishmania parasites were detected in 30 (32.6%) rodents using direct exam method. Leishmania DNA was detected in 40 (43.5%) C. gundi by combining results among spleens and livers using ITS1-PCR. Positive samples were confirmed to be L. major except for only one specimen which was L. tropica. These results demonstrated, for the first time, the high natural infection rate of C. gundi with L. major parasites in Tunisia. Hence, C. gundi should be considered as potential reservoir host of Leishmania parasites causing cutaneous leishmaniasis in Tunisia.

Blood Meal Analysis of Phlebotomine Sandflies (Diptera: Psychodidae: Phlebotominae) for Leishmania spp. Identification and Vertebrate Blood Origin, Central Tunisia, 2015-2016.

During the time periods of June 2015 and from July to August 2016, sandflies were collected among seven collection sites of the three leishmaniasis endemic villages of Sidi Bouzid, Tunisia. A total of 690 sandflies were captured and identified (380 males and 310 females). Four species belonging to genus Phlebotomus (Ph.) and two species belonging to genus Sergentomyia were identified. Leishmania DNA was detected in four out of 310 females (one Ph. sergenti and three Ph. papatasi). The overall sensitivity of the Prepronociceptin gene detection reached 76%. The concurrent presence of Ph. papatasi and Ph. sergenti vectors, the analysis of blood-meals, together with the detection of L. major in Ph. papatasi, confirms the ultimate conditions for the transmission of the disease in center Tunisia. These results expand the known epidemiological area of distrubtion of leishmaniasis and its vectors in this part of Tunisia, highlighting the need for ongoing entomological and parasitological surveillance.

Insight into the global evolution of Rodentia associated Morbilli-related paramyxoviruses.

One portion of the family Paramyxoviridae is a group of Unclassified Morbilli-Related Viruses (UMRV) recently recognized in wild small mammals. At a global level, the evolutionary history of these viruses is not properly understood and the relationships between UMRV and their hosts still remain largely unstudied. The present study revealed, for the first time, that Rodentia associated UMRV emerged from a common ancestor in southern Africa more than 4000 years ago. Sequenced UMRV originating from different regions in the world, clustered into four well-supported viral lineages, which suggest that strain diversification occurred during host dispersal and associated exchanges, with purifying selection pressure as the principal evolutionary force. In addition, multi-introductions on different continents and islands of Rodentia associated UMRV and spillover between rodent species, most probably Rattus rattus, were detected and indicate that these animals are implicated in the vectoring and in the worldwide emergence of this virus group. The natural history and the evolution dynamics of these zoonotic viruses, originating from and hosted by wild animals, are most likely shaped by commensalism related to human activities.

Taxonomic status and origin of the Egyptian weasel (Mustela subpalmata) inferred from mitochondrial DNA.

The Egyptian weasel (Mustela subpalmata) is a small mustelid with a distribution restricted to the lower Nile Valley and the Nile Delta. Traditionally considered a subspecies of the least weasel (M. nivalis), it is currently recognized as a separate species based on morphology. Here we present the first genetic assessment of the taxonomic status of the Egyptian weasel by comparing mitochondrial DNA (Cytochrome b gene and control region) sequences to those of least weasels from the western Palearctic, with a focus on the Mediterranean region. Our results provide no evidence to support the view that the Egyptian weasel is genetically distinct from the least weasel, as we found that, for both Cytochrome b and control region, haplotypes were shared between the two taxa. Specifically, the Cytochrome b and control region haplotypes detected in the Egyptian weasel were also present in M. nivalis from Turkey and Malta, two populations genetically analysed here for the first time. Our results suggest that the Egyptian weasel is distinct from the least weasel populations currently living in the Maghreb, which were inferred to be the result of an earlier colonization of North Africa, but the genetic data alone do not allow us to determine whether the Egyptian weasel is native or introduced. Nevertheless, the observed genetic patterns, together with the weasel fossil record in Israel and the unique commensal lifestyle of the Egyptian weasel, are consistent with the hypothesis that the Egyptian population is a relict of past range expansion from the Levant into Egypt. We suggest that the large size and characteristic sexual dimorphism of the Egyptian weasel are likely to represent ecotypic variation, but genomic studies are required to clarify the extent of its functional genetic divergence.

Ecological Niche Modeling for the Prediction of the Geographic Distribution of Cutaneous Leishmaniasis in Tunisia.

Cutaneous leishmaniasis is a very complex disease involving multiple factors that limit its emergence and spatial distribution. Prediction of cutaneous leishmaniasis epidemics in Tunisia remains difficult because most of the epidemiological tools used so far are descriptive in nature and mainly focus on a time dimension. The purpose of this work is to predict the potential geographic distribution of Phlebotomus papatasi and zoonotic cutaneous leishmaniasis caused by Leishmania major in Tunisia using Grinnellian ecological niche modeling. We attempted to assess the importance of environmental factors influencing the potential distribution of P. papatasi and cutaneous leishmaniasis caused by L. major. Vectors were trapped in central Tunisia during the transmission season using CDC light traps (John W. Hock Co., Gainesville, FL). A global positioning system was used to record the geographical coordinates of vector occurrence points and households tested positive for cutaneous leishmaniasis caused by L. major. Nine environmental layers were used as predictor variables to model the P. papatasi geographical distribution and five variables were used to model the L. major potential distribution. Ecological niche modeling was used to relate known species' occurrence points to values of environmental factors for these same points to predict the presence of the species in unsampled regions based on the value of the predictor variables. Rainfall and temperature contributed the most as predictors for sand flies and human case distributions. Ecological niche modeling anticipated the current distribution of P. papatasi with the highest suitability for species occurrence in the central and southeastern part of Tunisian. Furthermore, our study demonstrated that governorates of Gafsa, Sidi Bouzid, and Kairouan are at highest epidemic risk.

First report of naturally infected Sergentomyia minuta with Leishmania major in Tunisia.

BACKGROUND: Many sand fly species are implicated in the transmission cycle of Leishmania parasites around the world. Incriminating new sand flies species, as vectors of Leishmania is crucial to understanding the parasite-vector transmission cycle in different areas in Tunisia and surrounding countries. FINDINGS: Seventy-four unfed females belonging to the genera Sergentomyia and Phlebotomus were collected in South Tunisia between June and November 2014, using sticky papers. PCR-RFLP (Restriction Fragment Length Polymorphism) analysis of the internal transcribed spacer 1 (ITS1) was used for Leishmania parasites detection and identification. Leishmania (L.) major (Yakimoff & Shokkor, 1914) was identified within two Sergentomyia (S.) minuta (Rondani, 1843) and one Phlebotomus papatasi (Scopoli, 1786). CONCLUSION: This is the first report of L. major identified from S. minuta in Tunisia. This novel finding enhances the understanding of the transmission cycle of L. major parasites of cutaneous leishmaniasis in an endemic area in South Tunisia.

Spatio-temporal Genetic Structuring of Leishmania major in Tunisia by Microsatellite Analysis.

In Tunisia, cases of zoonotic cutaneous leishmaniasis caused by Leishmania major are increasing and spreading from the south-west to new areas in the center. To improve the current knowledge on L. major evolution and population dynamics, we performed multi-locus microsatellite typing of human isolates from Tunisian governorates where the disease is endemic (Gafsa, Kairouan and Sidi Bouzid governorates) and collected during two periods: 1991-1992 and 2008-2012. Analysis (F-statistics and Bayesian model-based approach) of the genotyping results of isolates collected in Sidi Bouzid in 1991-1992 and 2008-2012 shows that, over two decades, in the same area, Leishmania parasites evolved by generating genetically differentiated populations. The genetic patterns of 2008-2012 isolates from the three governorates indicate that L. major populations did not spread gradually from the south to the center of Tunisia, according to a geographical gradient, suggesting that human activities might be the source of the disease expansion. The genotype analysis also suggests previous (Bayesian model-based approach) and current (F-statistics) flows of genotypes between governorates and districts. Human activities as well as reservoir dynamics and the effects of environmental changes could explain how the disease progresses. This study provides new insights into the evolution and spread of L. major in Tunisia that might improve our understanding of the parasite flow between geographically and temporally distinct populations.

RAPD-PCR reveals genetic polymorphism among Leishmania major strains from Tunisian patients.

BACKGROUND: Zoonotic cutaneous leishmaniasis caused by Leishmania (L.) major is endemoepidemic in the Center and South of Tunisia. The clinical course of the disease varies widely among different patients and geographic regions. Although genetic diversity in L. major parasites has been suggested as a potential factor influencing their pathogenic variability, little information on genetic polymorphism among L. major strains is available in the literature. This work aimed to estimate the genetic variability within different isolates of L. major. METHODS: Our sample comprised 39 isolates (confirmed as L. major by restriction fragment length polymorphism typing) from patients experiencing the same clinical manifestations but living in different regions of Tunisia where L. major is endemic. Random amplified polymorphic DNA (RAPD) PCR marker polymorphism was estimated by calculating Nei and Li's genetic distances and by an analysis of molecular variance (AMOVA). RESULTS: Analysis of the genetic diversity among the isolates revealed a high level of polymorphism (43 %) among them. AMOVA indicated that the highest variability (99 %) existed within the study regions. CONCLUSIONS: Our results revealed a heterogeneous genetic profile for L. major with similar clinical manifestations occurring within the different geographical regions. Additional L. major isolates from patients, insect vectors, and reservoir hosts from different endemic foci should be collected for further analysis.

Genotype profile of Leishmania major strains isolated from tunisian rodent reservoir hosts revealed by multilocus microsatellite typing.

Zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania (L.) major parasites represents a major health problem with a large spectrum of clinical manifestations. Psammomys (P.) obesus and Meriones (M.) shawi represent the most important host reservoirs of these parasites in Tunisia. We already reported that infection prevalence is different between these two rodent species. We aimed in this work to evaluate the importance of genetic diversity in L. major parasites isolated from different proven and suspected reservoirs for ZCL. Using the multilocus microsatellites typing (MLMT), we analyzed the genetic diversity among strains isolated from (i) P. obesus (n = 31), (ii) M. shawi (n = 8) and (iii) Mustela nivalis (n = 1), captured in Sidi Bouzid, an endemic region for ZCL located in the Center of Tunisia. Studied strains present a new homogeneous genotype profile so far as all tested markers and showed no polymorphism regardless of the parasite host-reservoir origin. This lack of genetic diversity among these L. major isolates is the first genetic information on strains isolated from Leishmania reservoirs hosts in Tunisia. This result indicates that rodent hosts are unlikely to exert a selective pressure on parasites and stresses on the similarity of geographic and ecological features in this study area. Overall, these results increase our knowledge among rodent reservoir hosts and L. major parasites interaction.

Leishmania major infection among Psammomys obesus and Meriones shawi: reservoirs of zoonotic cutaneous leishmaniasis in Sidi Bouzid(central Tunisia).

A study was undertaken between November 2008 and March 2010, in the focus of cutaneous leishmaniasis of Central Tunisia, to evaluate the role of Psammomys obesus (n=472) and Meriones shawi (n=167) as reservoir hosts for Leishmania major infection. Prevalence of L. major infection was 7% versus 5% for culture (p=not signifiant [NS]), 19% versus 16% for direct examination of smears (p=NS), and 20% versus 33% (p=NS) for Indirect Fluorescent Antibody Test among P. obesus and M. shawi, respectively. The peak of this infection was in winter and autumn and increased steadily with age for the both species of rodents. The clinical examination showed that depilation, hyper-pigmentation, ignition, and severe edema of the higher edge of the ears were the most frequent signs observed in the study sample (all signs combined: 47% for P. obesus versus 43% for M. shawi; p=NS). However, the lesions were bilateral and seem to be more destructive among M. shawi compared with P. obesus. Asymptomatic infection was ~40% for both rodents. This study demonstrated that M. shawi plays an important role in the transmission and the emergence of Leishmania major cutaneous leishmaniasis in Tunisia.

First report of natural infection of least weasel (Mustela nivalis Linnaeus, 1776) with Leishmania major in Tunisia.

Zoonotic cutaneous leishmaniasis, caused by Leishmania major (L. major), is endemic in Tunisia. Several rodents have been identified as reservoir hosts of parasites. This study reports, for the first time, the natural infection with L. major zymodeme MON-25 in a specimen of least weasel: Mustela nivalis Linnaeus, 1776 (M. nivalis) collected in Sidi Bouzid. This finding justifies further research on larger samples of this animal to verify its role as a potential reservoir host for cutaneous leishmaniasis in Tunisia.