DNA sequence analysis suggests that cytb-nd1 PCR-RFLP may not be applicable to sandfly species identification throughout the Mediterranean region.

Molecular methods are increasingly used for both species identification of sandflies and assessment of their population structure. In general, they are based on DNA sequence analysis of targets previously amplified by PCR. However, this approach requires access to DNA sequence facilities, and in some circumstances, it is time-consuming. Though DNA sequencing provides the most reliable information, other downstream PCR applications are explored to assist in species identification. Thus, it has been recently proposed that the amplification of a DNA region encompassing partially both the cytochrome-B (cytb) and the NADH dehydrogenase 1 (nd1) genes followed by RFLP analysis with the restriction enzyme Ase I allows the rapid identification of the most prevalent species of phlebotomine sandflies in the Mediterranean region. In order to confirm the suitability of this method, we collected, processed, and molecularly analyzed a total of 155 sandflies belonging to four species including Phlebotomus ariasi, P. papatasi, P. perniciosus, and Sergentomyia minuta from different regions in Spain. This data set was completed with DNA sequences available at the GenBank for species prevalent in the Mediterranean basin and the Middle East. Additionally, DNA sequences from 13 different phlebotomine species (P. ariasi, P. balcanicus, P. caucasicus, P. chabaudi, P. chadlii, P. longicuspis, P. neglectus, P. papatasi, P. perfiliewi, P. perniciosus, P. riouxi, P. sergenti, and S. minuta), from 19 countries, were added to the data set. Overall, our molecular data revealed that this PCR-RFLP method does not provide a unique and specific profile for each phlebotomine species tested. Intraspecific variability and similar RFLP patterns were frequently observed among the species tested. Our data suggest that this method may not be applicable throughout the Mediterranean region as previously proposed. Other molecular approaches like DNA barcoding or phylogenetic analyses would allow a more precise molecular species identification.

Validation of rK39 immunochromatographic test and direct agglutination test for the diagnosis of Mediterranean visceral leishmaniasis in Spain.

BACKGROUND: Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is endemic in Europe with Mediterranean countries reporting endemic status alongside a worrying northward spread. Serological diagnosis, including immunochromatographic test based on the recombinant antigen rK39 (rK39-ICT) and a direct agglutination test (DAT) based on the whole parasite antigen, have been validated in regions with high VL burden, such as eastern Africa and the Indian subcontinent. To date, no studies using a large set of patients have performed an assessment of both methods within Europe. METHODOLOGY/PRINCIPAL FINDINGS: We selected a range of clinical serum samples from patients with confirmed VL (including HIV co-infection), Chagas disease, malaria, other parasitic infections and negative samples (n = 743; years 2009-2015) to test the performance of rK39-ICT rapid test (Kalazar Detect Rapid Test; InBios International, Inc., USA) and DAT (ITM-DAT/VLG; Institute of Tropical Medicine Antwerp, Belgium). An in-house immunofluorescence antibody test (IFAT), was included for comparison. Estimated sensitivities for rK39-ICT and DAT in HIV-negative VL patients were 83.1% [75.1-91.2] and 84.2% [76.3-92.1], respectively. Sensitivity was reduced to 67.3% [52.7-82.0] for rK39 and increased to 91.3% [82.1-100.0] for DAT in HIV/VL co-infected patients. The in-house IFAT was more sensitive in HIV-negative VL patients, 84.2% [76.3-92.1] than in HIV/VL patients, 79.4% [73.3-96.2]. DAT gave 32 false positives in sera from HIV-negative VL suspects, compared to 0 and 2 for rK39 and IFAT, respectively, but correctly detected more HIV/VL patients (42/46) than rK39 (31/46) and IFAT (39/46). CONCLUSIONS/SIGNIFICANCE: Though rK39-ICT and DAT exhibited acceptable sensitivity and specificity a combination with other tests is required for highly sensitive diagnosis of VL cases in Spain. Important variation in the performance of the tests were seen in patients co-infected with HIV or with other parasitic infections. This study can help inform the choice of serological test to be used when screening or diagnosing VL in a European Mediterranean setting.

Prevalence of HIV-1 dual infection in long-term nonprogressor-elite controllers.

INTRODUCTION: Human immunodeficiency virus type 1 (HIV-1) dual infection (DI) in long-term nonprogressor-elite controller patients (LTNP-EC) has been described only in sporadic cases and then, consequences in disease progression are not clearly established. To fill-up this limited knowledge, we analyzed, for the first time, the prevalence, host genetic polymorphisms, and clinical consequences of HIV-1 DI in a group of LTNP-EC. METHODS: For DI detection, nucleotide sequences in env gene from viruses from 20 LTNP-EC were analyzed by maximum likelihood. Epidemiological and clinical parameters and host factors of patients were also studied. RESULTS: DI was detected in 4 (20%) of the 20 LTNP-EC, of which 3 maintained the elite controller status. CD4⁺ T-cell counts were not different between single and DI patients although higher CD8⁺ T-cell counts were observed in DI patients, and, consequently, the CD4⁺/CD8⁺ ratios were lower in LTNP-EC DI patients. CONCLUSIONS: Prevalence of HIV-1 DIs in LTNP-EC is similar to other groups of HIV-1 patients; in addition, DI was not associated with loss of disease control in the patients. These DI LTNP-EC patients showed, in comparison with single infected patients, higher numbers of CD8⁺ T cells and lower CD4⁺/CD8⁺ ratios.

Low-replicating viruses and strong anti-viral immune response associated with prolonged disease control in a superinfected HIV-1 LTNP elite controller.

OBJECTIVE: To study the causes for the lack of clinical progression in a superinfected HIV-1 LTNP elite controller patient. METHODOLOGY AND PRINCIPAL FINDINGS: We studied host genetic, virological and immunological factors associated with viral control in a SI long term non progressor elite controller (LTNP-EC). The individual contained both viruses and maintained undetectable viral loads for >20 years and he did not express any of the described host genetic polymorphisms associated with viral control. None of four full-length gp160 recombinants derived from the LTNP-EC replicated in heterologous peripheral blood mononuclear cells. CTL responses after SI were maintained in two samples separated by 9 years and they were higher in breadth and magnitude than responses seen in most of 250 treatment naïve patients and also 25 controller subjects. The LTNP-EC showed a neutralization response, against 4 of the 6 viruses analyzed, superior to other ECs. CONCLUSIONS: The study demonstrated that a strong and sustained cellular and humoral immune response and low replicating viruses are associated with viral control in the superinfected LTNP-EC.