Prevalence of Plasmodium spp. in malaria asymptomatic African migrants assessed by nucleic acid sequence based amplification

Malaria is one of the most important infectious diseases in the world. The World Health Organization estimates that 300–500 million cases of malaria infections, with 1–3 million deaths globally occurring each year, of which 95% is caused by Plasmodium falciparum [1]. Although most cases are found in the tropical regions of Africa, Asia, Central, and South America, there is a significant increase in the number of imported cases in Europe, in particular due to the higher mobility of people in today's society [2]. Approximately 7,000 cases of imported malaria are recorded in Europe annually [3]. Due to the misleading symptoms related to initial malaria (i.e. fever and other flu-like symptoms), prompt diagnosis and treatment are of great importance. The Centers for Disease Control and Prevention recommends that malaria should be considered in the differential diagnosis of febrile patients who have traveled to a region where malaria is endemic and in any patients who experience fevers of unknown origin regardless of their travel history [2].

The current standard for diagnosis is the microscopic examination of Giemsa-stained thick and thin blood smears [4‐8]. This procedure is not expensive and in principle easy to perform, but becomes time-consuming in particular in cases of mixed infection or low parasitaemia and even with experienced microscopists examining the blood slides, misdiagnosis may occur [4, 8]. To aid malaria diagnosis, other techniques have been developed, such as immunochromatographic assays based on Plasmodium antigen detection, but some of these tests perform poorly in cases of low parasitemia [7, 9, 10] or, in contrast, lead to a false positive diagnosis of malaria due to the persistence of parasite antigen after adequate treatment [7, 11]. Molecular detection for Plasmodium with PCR has resulted in increased sensitivity and species discrimination compared to either microscopic or immunochromatographic diagnosis of malaria [12‐18]. PCR based assays are sensitive and can be converted to a quantitative format, if SYBR green or molecular probes (e.g. a Taqman probe or a molecular beacon) are used in real time format [19, 20]. Alternatively, Nucleic Acid Sequence Based Amplification (NASBA) technology can be applied, which has as advantage above real-time PCR assays that it is fast to perform; i.e. 60 minutes for NASBA compared to up to four hours for real-time PCR [21, 22]. NASBA assays, using molecular beacons as detection probes, has been developed for different Plasmodium species and has shown to be very sensitive with a detection limit of 20 parasites/ml blood [22, 23].

In the present study, NASBA using primers based on pan species Plasmodium-specific small sub-unit ribosomal RNA gene sequences [24] was used to evaluate the prevalence of Plasmodium species in a population of migrants. Furthermore, a gametocyte specific NASBA based on the detection of the mRNA encoding for the P. falciparum gametocyte specific Pfs25 gene [25] was employed to detect mature sexual transmission stages in the same study population.

In many countries, the increase in imported malaria cases from different countries is due to the increasing number of people traveling to and from endemic areas, without taking appropriate prophylaxis or immigration from low income countries [27, 28]. In the case of returning non-immune travelers, malaria infection is often very obvious with severe disease and sometimes fatal outcome. In contrast, people from disease endemic countries may have acquired semi-immunity, resulting in less serious to none manifestations of disease, as was also shown in Spain [28], and recently also in Italy [29]. However, a proportion of this population may develop disease over time due to weaning immunity and/or contribute to possible transmission. In general, people originating from malaria endemic countries may have sub-microscopic levels of parasitaemia, but may feel not ill at all or present typical features of malaria. This is also reflected in the present study. Although many study participants could not recall having had malaria in the past, when being interviewed, there was no difference between the levels of infection between these participants and those who could recall an episode of malaria. In a substantial proportion of the examined population low levels of Plasmodium parasites were found with the applied NASBA technology. Furthermore, clinical examination revealed a statistically significant association between splenomegaly and 18S NASBA positivity. Malarial splenomegaly is believed to be a consequence of an immunological dysfunction due to recurrent episodes of malaria [30, 31]. It is generally considered that prolonged exposure to malarial parasites (5–10 years) is needed to develop splenomegaly, this response is based on a overproduction of polyclonal IgM antibodies, leading to accumulation of high molecular-weight immunocomplexes and complement consumption. Long-term purification of immunocomplexes induces progressive hyperplasia of the spleen [32, 33]. Patients usually present hypersplenism, and may have anaemia, leukopaenia, thrombopaenia and even haemolytic crises, depending on the severity [34]. A significant inverse association was found between increased transaminase levels and 18S NASBA positivity; i.e. people with increased transaminase levels had a lower probability to be positive at 18S NASBA. However, the high prevalence of patients with viral hepatitis infections observed in this study could also be the consequence of other diseases.

In Italy, malaria was eradicated after a campaign launched in 1947 just at the end of World War II. The campaign was carried out by indoor treatment with DDT; houses, stables, shelters and all rural structures were treated into the mid-1950s and also later in some endemic regions, such as Sardinia. Due to the persistence of sporadic malaria cases in some areas [35, 36], the WHO declared Italy free of malaria only in 1970. In order to circumvent the potential reintroduction of malaria, a strict surveillance system was set up, which is still in place and governed by the Ministry of Health and the Istituto Superiore di Sanità (ISS, Rome, Italy), following WHO guidelines [37].

Over the last two decades, with the exception of one autochthonous case registered in 1997 in a rural area of the province of Grosseto (Tuscany), which was followed by four more cases (i.e. a cryptic case by P. falciparum in 2003 [38], two cases due to organ transplantation in 2004 [39] and a transfusional case in 2005, all cases of malaria have been imported [40‐42]). Until to 1999 the number of imported cases of malaria arose constantly, while from 2002 to 2006 a decrease between 1% and 7% per year has been registered [37] in both immigrant (-9%) and among Italian citizens(-28%). Most of the cases registered from 2002–2006 were among people from Africa (88.4%), and, in particular, from Nigeria (26%), Ghana (17%), Senegal (17%), Costa d'Ivoire (12%), and Burkina Faso (6.7%). Furthermore, additional cases were reported in 2000 [43] and in 2002 [44] in Chinese immigrants who traveled to Italy via Africa. Plasmodium falciparum was confirmed to be the most prevalent species imported in southern Europe [27, 28, 37]; in Italy, this species is responsible for 89% of the infection, P. vivax for 7.7%, P. ovale and P. malariae, 6% and 1.5%, respectively [29, 37]. In addition, mixed Plasmodium infections have also been reported in Italy, with 58% of all these cases reported from a single town, Verona [29].

The distribution and density of the three potential vectors of malaria in Italy (Anopheles labranchiae, Anopheles superpictus and Anopheles sacharovi) are also under strict surveillance by Istituto Superiore di Sanità (ISS). Anopheles labranchiae is present in several areas of Central and Southern Italy and in some regions (Tuscany, Calabria, Puglia, Sardinia and Sicily) high density have been registered [41]. The density of An. superpictus is lower and breeding sites are restricted to Calabria coasts and Sicily while although An. sacharovi has not registered since over 30 years, its presence in low density cannot be excluded in Apulia and northern Sardinia regions [37].

The combination of the presence of potential vectors and gametocyte carriers may pose a risk for re-introduction of malaria in the country. However, it should be noted that certain climatological and ecological prerequisites should probably be also met before the disease is really reintroduced [37].

This study showed that a substantial proportion of people originating from malaria endemic countries and not showing clinical signs of malaria harbor Plasmodium parasites in their blood (i.e. 32% of positivity to Plasmodium spp., 21% of population harboring P. falciparum gametocytes). If transmission prerequisites (e.g. vector, susceptible population and climatological conditions) prevail they could potential be a reservoir. Therefore, health authorities should pay special attention to the health of this potential risk group and aim to improve their health conditions.