The value of West Nile virus RNA detection by real-time RT-PCR in urine samples from patients with neuroinvasive forms.

INTRODUCTION: Routine laboratory screening is based on the detection of WNV specific IgM and IgG in blood and cerebrospinal fluid. Confirmation is then classically applied by real time RT-PCR (rRT-PCR) in Cerebrospinal fluid (CSF), which often gives negative results due to too short virorachia and late sampling. rRT-PCR was applied-for the first time for routine diagnosis purpose-on urine samples. METHODS: During 2018 outbreak in Tunisia, 107 patients presented WNV neurologic symptoms and were positive for WNV serology. Of them, 95 patients were sampled for urine and 35 were sampled for CSF. Qualitative rRT-PCR was performed on both type of samples. RESULTS: WNV RNA was detected in 50.5% of urine samples (48/95) and in 2.8% of CSF samples (1/35). WNV RNA was detectable from day 1 to day 41 from symptom onset, however, positive urine rate was 53.1% during the first 10 days from symptom onset. The proportions of urine-positive and urine-negative samples, based on day of collection, showed no statistical difference (p > 0.005). Cycle threshold (Ct) values ranged from 12 to 39, with no correlation with the day of collection. The lowest Ct value was detected for urine sampled on day 5 after symptom onset. A statistically significant difference was found between age groups of confirmed and non confirmed cases (p < 0.001). DISCUSSION/CONCLUSION: Our study reported the use of rRT-PCR on urine samples as a confirmatory diagnostic tool for WNV "probable cases" during an outbreak. Our findings underlined the reliability and the rapidity of this confirmatory tool, even late, and showed its superiority on CSF investigation.

Role of hepatitis C virus core antigen assay in hepatitis C care in developing country.

BACKGROUND: The World Health Organization (WHO) aims to achieve global hepatitis C elimination by 2030, defined as diagnosis of 90% of infected individuals and treating 80% of them. Current guidelines for the screening and diagnosis of hepatitis C infection denote using a relatively cheap screen with anti-hepatitis C virus (HCV) antibody immunoassay, followed by the much costlier molecular test for HCV RNA levels using polymerase chain reaction (PCR) assay to confirm active HCV infection. Simplification of the HCV evaluation algorithm to reduce the number of required tests could considerably expand the provision of HCV treatment especially in a developing country. This study investigates the performance of hepatitis C Core Antigen (HCV Ag) test by comparing HCV Ag results versus the results obtained with HCV ribonucleic acid (RNA) PCR which is considered the gold standard for the diagnosis of HCV infection. RESULTS: Among the 109 anti-HCV positive sera, 96 were positive for both HCV Ag (> 3 fmol/L) and HCV RNA (> 15 IU/mL); 8 were negative for both tests, while the remaining 5 were positive for HCV RNA only. Considering the HCV RNA as gold standard; the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of HCV Ag test were found to be 95.05%, 100%, 100%, and 61.54%, respectively. HCV genotype was performed for 59 patients. The most common HCV genotype was genotype 1 (72.9%). Genotype 2 (15.3%) and genotype 3 (11.9%) were detected in the others samples. A high level of correlation was seen between HCV RNA and HCV Ag (r = 0.958, p < 0.001). The correlation for the samples that were genotyped 1 was significant (r = 0.966, p < 0.001). CONCLUSION: In our study, it was found that there was strong correlation between HCV RNA levels and HCV Ag levels. So, it can be used for a one-step HCV antigen test to diagnose active HCV infection.

Genetic characterization of West Nile Virus strains during neuroinvasives infection outbreak in Tunisia, 2018.

West Nile Virus (WNV) is an arbovirus transmitted by mosquito bite involving birds as reservoirs, humans and equines as accidental hosts. Eight distinct lineages (WNV-1 to WNV-8) have been identified: WNV-1 and WNV-2 infect humans and animals, and WNV-3 to WNV-8 have been identified only in vectors. WNV has been implicated in neuroinvasives infections, especially meningitis and encephalitis. Tunisia experienced three epidemics in 1997, 2003 and 2012. Serological studies on humans, equines and birds as well as the detection of the virus in the vector favour a fairly frequent circulation in the country. A new epidemic has been observed in Tunisia between August and November 2018. The obtained sequences of the VWN from Tunisia 2018 grouped in a distinct monophyletic group within the Mediterranean subtype in Cluster 1, with a maximum of 2% nucleotide divergence. These sequences were clearly distinct from the Tunisia 1997, which grouped with sequences mainly from USA in Cluster 2. This work reports the genetic characterization of the Tunisia 2018 strain in comparison with the previously identified strains in Tunisia and worldwide. The epidemic virus Tunisia 2018 was genetically close to the Mediterranean basin and Eastern Europe sequences but distinct from the Tunisia 1997 closely related to the American sequences.

Evaluation of antibody persistence after a four-dose primary hepatitis B vaccination and anamnestic immune response in children under 6 years.

Introduction. Tunisia is an intermediate hepatitis B virus (HBV) endemic country. The vaccination against hepatitis B was introduced in 1995 including four doses with a first dose administrated at birth. Decreasing the level of antibodies against hepatitis B surface antigen (anti-HBs) over time can be alarming. This study was conducted to explore the anti-HBV immune response among children under 6 years old, vaccinated according to the national vaccination schedule, by evaluating the immunological response to primary vaccination and by exploring the anamnestic immune response to a booster dose.Methods. We conducted a cross-sectional prospective study from June 2016 to June 2017 (n=180), based on voluntary participation. Children were recruited from the public pediatric ward sectors in Sahloul University Hospital of Sousse in Central Tunisia. An anti-HB titre was determined based on electro-chemiluminescence micro-particle immunoassay (ECLIA), using Elecsys Anti-HBs II kit, Roche.Results. Mean age at the time of enrollment in the study was 33±14.8 months. The seroprotection rate was 77.2 %. The anti-HB titre differed significantly between the different age groups (P=0.002). The predicting variable for having no seroprotective antibody level was older age. Children with anti-HB levels <10 IU l- 1 were offered an additional dose of HBV vaccine. Anamnestic response 1 month after the challenge dose was observed in 100 % of subjects. The probability of developing a high antibody response, following the booster dose increased in conjunction with an increased pre-booster antibody level.Conclusion. The response to a booster dose suggests the persistence of immune memory in almost all vaccinated individuals. Although a booster dose increases substantially anti-HB titre, the clinical relevance of such an increase remains unknown.

Distribution of rotavirus VP7 and VP4 genotypes circulating in Tunisia from 2009 to 2014: Emergence of the genotype G12.

Group A rotavirus (RVA) represents the most important aetiological agent of diarrhoea in children worldwide. From January 2009 to December 2014, a multi-centre study realized through 11 Tunisian cities was undertaken among children aged <5 years consulting or hospitalized for acute gastroenteritis. A total of 1127 faecal samples were collected. All samples were screened by ELISA for the presence of RVA antigen. RVA-positive samples were further analyzed by PAGE and used for G/P-genotyping by semi-nested multiplex RT-PCR. Globally, 270 specimens (24 %) were RVA-positive, with peaks observed annually between November and March. Nine different electropherotypes could be visualized by PAGE, six with a long profile (173 cases) and two with a short one (seven cases). Mixed profiles were detected in two cases. Among the 267 VP7 genotyped strains, the predominant G- genotype was G1 (39.6 %) followed by G3 (22.2 %), G4 (13 %), G9 (11.5 %), G2 (5.2 %) and G12 (5.2 %). Among the 260 VP4 genotyped strains, P[8] genotype was the predominant (74.5 %) followed by P[6] (10.4 %) and P[4] (5.5 %). A total of 257 strains (95.2 %) could be successfully G- and P-genotyped. G1P[8] was the most prevalent combination (34.4 %), followed by G3P[8] (16.3 %), G9P[8] (10.3 %), G4P[8] (8.9 %), G2P[4] (4 %), G12P[6] (2.6 %) and G12P[8] (1.9 %). Uncommon G/Pgenotype combinations, mixed infections and untypeable strains were also detected. This is the first report, in Tunisia, of multiple detection of an emerging human RVA strain, G12 genotype. This study highlighted the need for maintaining active surveillance of emerging strains in Northern Africa.

Validation of Recombinant Salivary Protein PpSP32 as a Suitable Marker of Human Exposure to Phlebotomus papatasi, the Vector of Leishmania major in Tunisia.

BACKGROUND: During a blood meal, female sand flies, vectors of Leishmania parasites, inject saliva into the host skin. Sand fly saliva is composed of a large variety of components that exert different pharmacological activities facilitating the acquisition of blood by the insect. Importantly, proteins present in saliva are able to elicit the production of specific anti-saliva antibodies, which can be used as markers for exposure to vector bites. Serological tests using total sand fly salivary gland extracts are challenging due to the difficulty of obtaining reproducible salivary gland preparations. Previously, we demonstrated that PpSP32 is the immunodominant salivary antigen in humans exposed to Phlebotomus papatasi bites and established that humans exposed to P. perniciosus bites do not recognize it. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we have validated, in a large cohort of 522 individuals, the use of the Phlebotomus papatasi recombinant salivary protein PpSP32 (rPpSP32) as an alternative method for testing exposure to the bite of this sand fly. We also demonstrated that screening for total anti-rPpSP32 IgG antibodies is sufficient, being comparable in efficacy to the screening for IgG2, IgG4 and IgE antibodies against rPpSP32. Additionally, sera obtained from dogs immunized with saliva of P. perniciosus, a sympatric and widely distributed sand fly in Tunisia, did not recognize rPpSP32 demonstrating its suitability as a marker of exposure to P. papatasi saliva. CONCLUSIONS/SIGNIFICANCE: Our data indicate that rPpSP32 constitutes a useful epidemiological tool to monitor the spatial distribution of P. papatasi in a particular region, to direct control measures against zoonotic cutaneous leishmaniasis, to assess the efficiency of vector control interventions and perhaps to assess the risk of contracting the disease.

Sequence and phylogenetic analyses of human rotavirus strains: comparison of VP7 and VP8(∗) antigenic epitopes between Tunisian and vaccine strains before national rotavirus vaccine introduction.

Group A rotaviruses (RVA) are the leading cause of severe gastroenteritis in infants and young children worldwide. Due to their epidemiological complexity, it is important to compare the genetic characteristics of vaccine strains with the RVA strains circulating before the introduction of the vaccine in the Tunisian immunization program. In the present study, the nucleotide sequences of VP7 and VP8∗ (n=31), the main targets for neutralizing antibodies, were determined. Comparison of antigenic epitopes of 11 G1P[8], 12 G2P[4], 4 G3P[8], 2 G4P[8], 1 G6P[9] and 1 G12P[8] RVA strains circulating in Tunisia from 2006 to 2011 with the RVA strains present in licensed vaccines showed that multiple amino acid differences existed in or near putative neutralizing domains of VP7 and VP8∗. The Tunisian G3 RVA strains were found to possess a potential extra N-linked glycosylation site. The Tunisian G4 RVA were closely related to the G4 vaccine strain in RotaTeq, belonging to the same lineage, but the alignment of their VP7 amino acids revealed an insertion of an asparagine residue at position 76 which is close to a glycosylation site (aa 69-71). Despite several differences detected between Tunisian and vaccine strains, which may affect binding of neutralizing antibodies, both vaccines are known to protect against the vast majority of the circulating genotypes, providing an indication of the high vaccine efficiency that can be expected in a future rotavirus immunization program.