Barbara Castagna

High genetic polymorphism among Giardia duodenalis isolates from Sahrawi children

Human giardiasis, the gastrointestinal infection caused by two genetically different groups (or assemblages) of Giardia duodenalis, is very common worldwide, and its prevalence is higher in developing countries. However, few surveys in these regions have been performed to include a genetic characterization of the parasite, which is necessary to unravel the complex epidemiology of the infection. In this work, we screened 120 faecal samples collected from Sahrawi children in 2003-2005, and found 41 (34.2%) of them to be positive, using immunofluorescent microscopy, for the presence of G. duodenalis cysts. Molecular characterization of the isolates was performed by RFLP and/or sequence analysis of the triose phosphate isomerase (tpi) and the glutamate dehydrogenase (gdh) genes. The results disclosed an unexpectedly high genetic polymorphism among isolates of both assemblages A and B, and a large percentage of the sequences (50% for the tpi gene, and 90% for the gdh gene) from assemblage B isolates characterized by the presence of overlapping nucleotide peaks at specific positions in the chromatograms, which can be attributed to mixed infections or to allelic sequence heterozygosity of single cysts. Notably, this phenomenon was not observed in sequences from assemblage A isolates. These results suggest that the genetic structure is different in isolates of assemblages A and B.

Rapid identification and antimicrobial susceptibility testing of Gram‐positive cocci in blood cultures by direct inoculation into the BD Phoenix system

Rapid identification and antimicrobial susceptibility testing (AST) of the causative agent(s) of bloodstream infections are essential for the selection of appropriate antimicrobial therapy. To speed up the identification and AST of the causative agent, the fluid from blood culture bottles of a Bactec 9240 instrument (Becton Dickinson) containing Gram-positive cocci was mixed with saponin. After a 15-min incubation, the bacteria were harvested and transferred to the appropriate panel of a BD Phoenix automated microbiology system (Becton Dickinson) for identification and AST. With this approach (referred to as the direct method), we concordantly/correctly identified 56 (82%) of 68 monomicrobial cultures using the results obtained with the method currently used in our laboratory (current method) as comparator. Two (3%) isolates could not be identified and ten (15%) were misidentified. Complete agreement, concerning clinical susceptibility categories and MIC values, between the AST results determined with the direct method and the current method was found for 32 (55%) of 58 isolates. The E-test indicated that the direct method yielded a correct susceptibility profile for 13 of the remaining 26 blood culture isolates. Therefore, a concordant/correct susceptibility profile (with all antimicrobial agents tested) was obtained for 45 (77%) of 58 cultures. The overall error rate amounted to 1.9%, with the majority (1.3%) of errors being minor. Importantly, the results obtained with the direct method were available 12-24h earlier than those obtained with the current method.

Parasite prevalence in a village in Burkina Faso: the contribution of new techniques

INTRODUCTION Parasites are a major public health problem in developing countries. A coproparasitological and immunoparasitological study was conducted in Burkina Faso, in the rural village of Touguri, in November and December 2011. The coproparasitologic analysis was conducted in the pediatric population and seroprevalence surveys were conducted in the adult population to research intestinal, blood, and helminth parasites. METHODOLOGY The coproparasitologic study was performed on stool samples using two diagnostic methods - standard microscopy and the FLOTAC technique. The total of 49 stool samples analyzed were obtained from children between two months and eleven years of age. The serology study was carried out to evaluate the prevalence of P. falciparum, Echinococcus spp., Tenia solium, and A. lumbricoides using different immunological techniques such as ELISA and Western Blot techniques. The study population included 85 adult patients between 15 and 70 years of age. RESULTS Results of coproparasitological analyses showed Hymenolepis nana as the only helminth found, in 28.6% of the total number of patients. Results of serological evaluation revealed a practically null prevalence of Echinococcus, Taenia solium, and Ascaris lumbricoides, and a 77.64% prevalence of Plasmodium falciparum. CONCLUSIONS Despite the small number (especially in terms of coprological samples) of individuals examined, this study showed that the parasite prevalence in a rural area of Burkina Faso has a significant impact in the general population, particularly in children. Another finding was that FLOTAC had a higher sensitivity than the widely used ethyl ether-based concentration technique for coprological sample analysis.

Matrix metalloproteinase (MMP)-9: A realiable marker for inflammation in early human trichinellosis

Matrix Metalloproteinases (MMPs) are involved in many physiological and pathological processes. As regards parasitic infections, the role of these proteins has been particularly studied in malaria, neurocysticercosis and angiostrongyloidosis. Recently, we evaluated serum levels of MMP-9 and -2 (gelatinases) in mice experimentally infected with Trichinella spiralis or Trichinella pseudospiralis, which cause different degrees of myositis and we found their significant increase in the former and, at a lesser extent, in the latter, thus suggesting the possibility that these gelatinases, particularly MMP-9, represent a marker of inflammation. Our aim was to evaluate the levels of MMP-9 and 2 in trichinellosis patients, to assess their possible clinical significance. Serum samples from 31 Trichinella britovi-infected individuals (20 males and 11 females), living in Tuscany, Central Italy, were analysed for MMP-9 and MMP-2 serum levels. Patients acquired infection with Trichinella after consuming raw or undercooked meat of wild boar. Their median age was 49±0.33years (range from 7 to 91). Sera was collected before starting anti-inflammatory treatment, aliquoted and stored at -20°C until use. Sera from healthy subjects was considered as controls. The gelatinolytic activity of MMPs was analysed by gelatin zymography on 8% polyacrylamide-SDS gels containing 0.1% porcine gelatin, under non-reducing conditions. Clear bands corresponding to the digested areas were evaluated with an appropriate software. MMP-9 levels were additionally determined in 15 patients using a commercial ELISA kit for human MMP-9. The zymographic analysis of the gels showed the presence in serum samples of gelatinase bands at approximately 125-kDa, 92-kDa and 72-kDa, corresponding to the MMP-9/Neutrophil gelatinase-associated lipocalin (NGAL) complex and proenzyme forms of MMP-9 and MMP-2, respectively. A significant (p<0.01) increase in gelatinolytic activity in patients compared to the control group was observed for pro-MMP-9 in 25 out of 31. The mean increase in activity was 39.25%±16.67%. No significant differences were observed for pro-MMP-2 activity. The MMP-9 levels detected by ELISA showed significant correlation with zymographic data (r2=0.62, p<0.003) and were higher in more affected patients (suffering diarrhea, facial edemas and myalgia). In conclusion, MMP-9 might be considered as a marker of inflammation in T. britovi patients. On the contrary, MMP-2 did not result significantly different in patients, compared to controls.

Low Anisakis-specific IgE prevalence in dyspeptic patients in Italy – a retrospective study

Abstract The aims of this case-control study were to determine the prevalence of Anisakis-specific IgE in patients reporting chronic or acute gastrointestinal (GI) symptoms and to investigate the correlation with raw fish ingestion habits. A group of patients undergoing gastric endoscopy and a control group of healty subjects answered a self-administered questionnaire on their food habits, presence of symptoms (both allergic and not allergic), and general life style. The presence of anti-Anisakis IgE has been evaluated using a serum immunoCAP assay. Our data show a low prevalence of IgE directed against Anisakis allergens in Italy in dyspeptic patients, despite the high consumption of poorly cooked fish. These findings does not correlate with the results of studies from other Mediterranean countries, such as Spain, for example. The general prevalence of Anisakis allergens sensitization in Italy could be further investigated through screenings in the allergic population, especially on those patients who claim to have developed a fish allergy and with history of raw fish consumption. Moreover, the attention should be moved on recent allergic reactions associated with fishing ingestion. This could in fact indicate a recent encounter with the parasite. Finally, we must underline that the evaluation of Anisakis-specific IgG would have probably shown a difference in terms of exposure between the two groups; thus, it might be useful to detect also this antibody class in future population-based studies.