Detection of Plasmodium falciparum male and female gametocytes and determination of parasite sex ratio in human endemic populations by novel, cheap and robust RTqPCR assays.

BACKGROUND: The presence of Plasmodium falciparum gametocytes in peripheral blood is essential for human to mosquito parasite transmission. The detection of submicroscopic infections with gametocytes and the estimation of the gametocyte sex ratio are crucial to assess the human host potential ability to infect mosquitoes and transmit malaria parasites. AIM AND OBJECTIVES: The aim of this work was to develop sensitive and cheap Real Time qPCR assays for large-scale epidemiological surveys, based on detection and amplification of gametocyte sex specific transcripts selected from the literature: the female-specific pfs25 and pf glycerol kinase (pfGK) and the male-specific pfs230p and pf13 transcripts. METHODS: RTqPCR assays were used to test the gametocyte- and sex-specific expression of the target genes using asexual stages of the gametocyteless parasite clone F12 and FACS purified male and female gametocytes of the PfDynGFP/P47mCherry line. Assays were performed on 50 blood samples collected during an epidemiological survey in the Soumousso village, Burkina Faso, West-Africa, and amplification of the human housekeeping gene 18S rRNA was employed to normalize RNA sample variability. RESULTS: SYBR Green assays were developed that showed higher sensitivity compared to Taqman assays at a reduced cost. RTqPCR results confirmed that expression of pfs25 and pfs230p are female and male-specific, respectively, and introduced two novel markers, the female-specific pfGK and the male-specific pf13. A formula was derived to calculate the ratio of male to female gametocytes based on the ratio of male to female transcript copy number. Use of these assays in the field samples showed, as expected, a higher sensitivity of RTqPCR compared to microscopy. Importantly, similar values of gametocyte sex-ratio were obtained in the field samples based on the four different target combinations. CONCLUSION: Novel, sensitive, cheap and robust molecular assays were developed for the detection and quantification of female and male P. falciparum gametocytes. In particular, the RTqPCR assays based on the female-specific pfs25 and the newly described male gametocyte-specific pf13 transcripts, including normalization by the human 18S, reliably assess presence and abundance of female and male gametocytes and enable to determine their sex-ratio in human subjects in endemic areas.

Imported human Schistosoma japonicum: A report on two cases in Filipino migrants present in Italy and a systematic review of literature.

BACKGROUND: Schistosoma japonicum is endemic in the Philippines, China, and Indonesia, and is the third-most common schistosoma species. The infection can be asymptomatic for years but, if left untreated, can lead to irreversible complications. METHOD: We report the results of a systematic review of the literature on imported S. japonicum infection and describe two previously unpublished cases diagnosed in Filipino migrants in Italy. RESULTS: Twenty-five imported cases of S. japonicum schistosomiasis were retrieved. All patients but one were migrants. Most subjects acquired the infection in Philippines (n = 18, 72%). Median age at diagnosis was 46 years. Median period of residence in non-endemic countries before diagnosis was 14.5 years. Cases of prevalent hepatosplenic involvement were 10 (40%), those with prevalent intestinal involvement were 10 (40%), whereas five (20%) had overlapping manifestations. Ten patients suffered from cirrhosis; two underwent liver transplantation. Three patients presented with acute abdomen due to intestinal complications, leading to explorative laparotomy. In all cases, but one, the diagnosis was based on a histological examination of biopsy specimen, revealing S. japonicum ova. Seventeen patients were treated with praziquantel, and in three of them, possible treatment failures occurred. CONCLUSIONS: S. japonicum infection is uncommonly reported in non-endemic areas, but is probably underestimated because of a low threshold awareness of clinicians and unavailability of specific diagnostic tools. Viable S. japonicum adults may persist for decades, indicating that migrants or travellers previously exposed in areas with high-risk areas can harbour viable worms and deserve treatment.