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Background: Accurate malaria diagnosis constitutes a challenging task, necessitating the need for the implementation of targeted and effective diagnostic tools. The purpose of the current study was to evaluate the effectiveness of two different molecular methodologies in terms of sensitivity for the detection of External Quality Assessment (EQA) Plasmodium samples. Methods: A total of 104 lyophilized blood samples from 14 different UK-NEQAS (National External Quality Assessment Site) (2016–2021) and eight WHO-NEQAS distributions (2017–2020) were analyzed. An in-house multiplex PCR protocol, followed by single target real-time PCR protocols for all five Plasmodium species, was implemented. Results: The multiplex PCR had a success rate of 10/16 and 20/28 for P. vivax and P. falciparum species, respectively. On the other hand, the respective results for real-time PCR had a success rate of 13/16 (P. vivax), 28/28 (P. falciparum), 5/8 (P. malariae), 8/10 (P. ovale), and 10/14 (P. knowlesi). Plasmodium falciparum samples displayed the highest sensitivity of detection, 0.02 parasites/μL. Plasmodium vivax samples displayed a 0.1 parasites/μL cutoff value, greater than the respective value for whole blood samples, while P. ovale species displayed a respective cutoff value of 0.05 parasites/μL. Due to the limited number of tested samples, data obtained for P. malariae and P. knowlesi species samples were inconclusive. Conclusions: Real-time PCR comprises a credible molecular methodology in terms of sensitivity assessment and detection of low parasitemia levels of Plasmodium sp. in EQA lyophilized blood samples.

Leishmania infantum is considered the predominant Leishmania species responsible for visceral leishmaniasis (VL) in Greece but limited molecular-typing-based studies have been performed so far. We retrospectively analyzed data and serum samples collected from 3661 individuals suspected for VL in a sixteen-year period, from 2005 to 2020, to study the seasonality and demographic characteristics of VL cases and to define the L. infantum genotypes circulating in the country. Serum samples were tested with immunofluorescence assay and/or molecular assay. qPCR Leishmania-positive samples were subjected to genotypic analysis based on polymorphisms in 12 microsatellite regions of the internal transcribed spacers (ITSs) 1 and 2. We diagnosed 219 definite (6%, sample with a positive molecular assay and/or antibody titer ≥ 1:400) and 230 probable (6.3%, sample with antibody titer between 1:100 and 1:200) VL cases. Data analysis revealed that amongst VL-definite cases, the age group (≥65) constitutes the most affected factor, since 36.9% of the VL cases belonged to this age group. Amongst the VL definite cases, the most frequently reported symptoms were fever (83%), splenomegaly (49%), and hepatomegaly (40%), but this was not the case for immunocompromised patients that developed non-typical symptoms of leishmaniasis. Although no statistically significant differences in the overall seasonality of VL cases were observed, February and June showed a significantly higher proportion of VL cases compared to August and December. Genotyping of ITS1 and ITS2 regions revealed that all VL cases belong to ITS type A of L. infantum. Our study provides epidemiological information on VL and demonstrates for the first time, providing genotypic data, the circulation of ITS type A L. infantum in Greece.

Greece has been malaria-free since 1974, after an intense malaria control program. However, as Greece hosts migrant populations from P. vivax malaria-endemic countries, there is a risk of introducing the disease to specific vulnerable and receptive areas of the country. Knowledge of the genetic diversity of P. vivax populations is essential for understanding the dynamics of malaria disease transmission in a given region. We used nine highly polymorphic markers to genotype 124 P. vivax-infected archived DNA samples from human blood specimens referred to the NMRL from all over Greece throughout 2015–2019. The genotypic variability of the samples studied was noted, as they comprised several unique haplotypes, indicative of the importation of a large number of different P. vivax strains in the country. However, only a few events of local transmission were recorded. Genotyping revealed and confirmed the same clusters as those identified through epidemiological investigation. In only one introduction event was the index case found. No sustained/ongoing malaria transmissions in/between the studied regions or during consecutive years or additional foci of local transmission were observed. Genotyping is an important component in assisting malaria surveillance, as it provides information concerning the patterns of introduction and the effectiveness of implemented malaria control and elimination measures.

Human cases of West Nile virus (WNV) infections have been recorded in Greece since 2010, with seasonal outbreaks (summer-autumn) on an almost annual basis, caused mainly by the WNV lineage 2 strain (Nea Santa-Greece-2010). National Public Health Organization (NPHO) in Greece is annually implementing enhanced surveillance of human WNV infection, in order to promptly identify human cases of WNV infection and monitor distribution in terms of time and place. Entomological surveillance activities were carried out on a national basis in 2019 and 2020, under NPHO coordination and the collaboration of several private subcontractors, along with the Unit of Medical Entomology, Laboratory for Surveillance of Infectious Diseases (LSID). The aim was to monitor mosquito species composition, abundance, and WNV circulation in mosquito pools of Culex pipiens s.l. species. Adult mosquito traps were placed in selected sites; collected samples were morphologically characterized and pooled by date of collection, location, and species types. Female Culex pipiens s.l. pools were tested for WNV and WNV infection rates (MIR and MLE) were estimated. Highest mean number of female Culex pipiens s.l. species was recorded in Central Macedonia both for 2019 and 2020. Six hundred and fifty-nine mosquito pools (147 in 2019 and 512 in 2020) of female Culex pipiens s.l. were examined for WNV presence. The highest MLE was detected in Western Macedonia in 2019 and in Thessaly in 2020. Here, we present data on the mosquito species composition in the studied areas and WNV detection in mosquitoes from areas in Greece where the specific national mosquito surveillance program was implemented, for two years, 2019 and 2020.

Greece, a malaria-free country since 1974, has experienced re-emergence of Plasmodium vivax autochthonous malaria cases in some agriculture areas over the last three years. In early 2012, an integrated control programme (MALWEST Project) was launched in order to prevent re-establishment of the disease. In the context of this project, the rapid diagnostic tests (RDT) of SD Bioline Malaria Ag Pf/Pan that detects hrp-2 and pan-LDH antigens were used. The aim of this study was to assess the field application of the RDT for the P. vivax diagnosis in comparison to light microscopy and polymerase chain reaction (PCR). A total of 955 samples were tested with all three diagnostic tools. Agreement of RDT against microscopy and PCR for the diagnosis of P. vivax was satisfactory (K value: 0.849 and 0.976, respectively). The sensitivity, specificity and positive predictive value of RDT against PCR was 95.6% (95% C.I.: 84.8-99.3), 100% (95% C.I.: 99.6-100.0) and 100% (95% CI: 91.7-100.0) respectively, while the sensitivity, specificity and positive predictive value of RDT against microscopic examination was 97.4% (95% C.I.: 86.1-99.6), 99.4% (95% C.I.: 98.6-99.8) and 86.1% (95% CI: 72.1-94.7), respectively. Our results indicate that RDT performed satisfactory in a non-endemic country and therefore is recommended for malaria diagnosis, especially in areas where health professionals lack experience on light microscopy.