Tropheryma whipplei, Helicobacter pylori, and Intestinal Protozoal Co-Infections in Italian and Immigrant Populations: A Cross-Sectional Study.

Tropheryma whipplei (TW), Helicobacter pylori (HP), and intestinal protozoa (IP) are widespread pathogens with similar routes of transmission and epidemiological risk factors. Epidemiological data on co-infection between TW, HP, and IP are scarce. We aim to more deeply investigate the co-infection rate for these pathogens, evaluating the risk factors and symptoms. Methods: This is a cross-sectional study conducted at the IRCCS Sacro Cuore Don Calabria Hospital in Northern Italy, a referral center for tropical and Whipple's disease (WD). Stored stool samples from 143 subjects previously tested for TW DNA by real-time PCR were explored for HP and IP DNA detection. The virulence factor cagA was investigated in HP-positive patients. Results: A history of migration was reported significantly more in TW-positive than in negative subjects (34.1% vs. 9.1%, p = 0.001) and in HP-infected than in those non-infected (59.1% vs. 9.1%, p < 0.001). The HP infection rate differed significantly between TW-infected and uninfected groups (31.8% vs. 8.1%, p = 0.001), while no difference was observed for IP infection. Significantly higher TW intestinal colonization was found in HP-infected patients than in non-infected (63.6% vs. 24.8%, p < 0.001). In addition, the proportion of Blastocysts positive finding was also significantly higher in HP-infected than in non-infected (40.9% vs. 17.4%, p = 0.018). Conclusions: The present study is the first to report a high TW and HP co-infection rate. To reduce the risk of morbidity from a chronic infection of either pathogen, clinicians may consider TW-HP molecular screening on the same stool sample for patients with suspected HP disease or WD, particularly in case of travel history.

Preliminary comparison between an in-house real-time PCR vs microscopy for the diagnosis of Loa loa and Mansonella perstans.

Infections with the filarial nematodes Loa loa and Mansonella perstans are among the most neglected filarial infections. L. loa is endemic in 11 countries of Central and West Africa and loiasis is estimated to affect about 20 million people. M. perstans infection is widespread in more than 30 countries of sub-Saharan Africa. Due to the difficulty in diagnosing loiasis and M. perstans mansonellosis on a clinical basis, the diagnosis of infection with L. loa and M. perstans relies on laboratory techniques. Definitive diagnosis is based on the detection, identification, and quantification of circulating microfilariae (mf) by microscopy of concentrated blood. However, this is impractical for screening purposes as it requires expert laboratory personnel, considerable blood manipulation, and is time consuming, especially for the final issue of negative result reports, which are very common in the population visited outside endemic areas. The aim of the current work is the preliminary evaluation of the performance of the in-house real-time PCR described by Ta and colleagues compared to the routine microscopic approach for the screening of filarial infections in the clinical setting outside endemic areas, using samples from patients accessing the dedicated outpatient clinics for migrants and travelers of a reference centre for tropical diseases in Northern Italy.

Droplet Digital PCR for the Detection of Plasmodium falciparum DNA in Whole Blood and Serum: A Comparative Analysis with Other Molecular Methods.

BACKGROUND: The estimation of Plasmodium falciparum parasitaemia can vary according to the method used. Recently, droplet digital PCR (ddPCR) has been proposed as a promising approach in the molecular quantitation of Plasmodium, but its ability to predict the actual parasitaemia on clinical samples has not been largely investigated. Moreover, the possibility of applying the ddPCR-sensitive method to serum samples has never been explored. METHODS: We used, for the first time, ddPCR on both blood and serum to detect the DNA of P. falciparum in 52 paired samples from 26 patients. ddPCR was compared with loop-mediated isothermal amplification (LAMP) and rtPCR. The correlation between the ddPCR results, microscopy, and clinical parameters was examined. RESULTS: ddPCR and microscopy were found to be strongly correlated (ρ(26) = 0.83111, p < 0.0001) in blood. Samples deviating from the correlation were partially explained by clinical parameters. In serum samples, ddPCR revealed the best performance in detecting P. falciparum DNA, with 77% positive samples among malaria subjects. CONCLUSION: Absolute quantitation by ddPCR can be a flexible technique for Plasmodium detection, with potential application in the diagnosis of malaria. In particular, ddPCR is a powerful approach for Plasmodium DNA analysis on serum when blood samples are unavailable.

Assessment of real-time PCR for Helicobacter pylori DNA detection in stool with co-infection of intestinal parasites: a comparative study of DNA extraction methods.

BACKGROUND: Many studies reported high prevalence of H. pylori infection among patients co-infected with intestinal parasites. Molecular approach for the DNA detection of those microbes in stool have been proposed. However there are a few reports that evaluated the effect of bead-beating in relation to the H. pylori outcome. Therefore, we developed and evaluated two TaqMan-based real-time PCR (rt-PCR) qualitative assays for the detection of ureC (glmM) and cagA of Helicobacter pylori on DNA extracted by three procedures. RESULTS: The two PCRs were analysed on 100 stool samples from patients who were screened for intestinal parasites. Three DNA extraction procedures were used: 1) automation with bead beating, 2) automation without bead beating and 3) hand column. The specificity of the new assays was confirmed by sequencing the PCR products and by the lack of cross-reactivity with other bacteria or pathogens DNA. Rt-PCR assays showed a detection limit of 10^4 bacteria/200 mg stool. The ureC_PCR with bead beating process was compared to conventional stool antigen test (SAT), with 94.12 and 93.75% of respectively sensitivity and specificity. However, the discordant samples were confirmed by DNA sequencing suggesting a potential higher sensitivity and specificity of PCR. CONCLUSIONS: Our findings showed that the automation with bead-beating -suggested procedure for intestinal parasitic infections- can reach highly sensitive results in H. pylori detection on stool compared also with SAT. Thus, this work can provide new insights into the practice of a clinical microbiology laboratory in order to optimize detection of gastro-intestinal infections. Further studies are needed to better define the clinical value of this technique.

Improved detection of DNA Schistosoma haematobium from eggs extracted by bead beating in urine.

Diagnosis of Schistosoma haematobium relies primarily on microscopical analysis of urine. The method is time consuming and requires some expertise. Genus-specific real-time PCRs have been developed, but we still observed low sensitivity. In the present study, in order to achieve a more sensitive DNA detection of eggs of S. haematobium in urine samples, we wanted to develop a novel protocol of DNA extraction using mechanic disruption of eggs by bead beating as supplementary step. We tested Schistosoma spp. internal transcribed spacer 2 real-time PCR after both methods with and without bead beating. First, we preliminary assessed the DNA detection after bead beating using dilution of 2, 10, 50, and 90 eggs/10 mL, and the Ct value analysis showed significant improved DNA detection per each point of egg concentration using the novel supplementary step. Twenty microscopy positive and five microscopy negative urine samples were used to validate the procedure. All urines came from imported cases and admitted at center for tropical medicine, and were examined by microscopy. PCR results after novel method with bead beating showed 100% to be positive for S. haematobium, compared with 85% positive by our standard extraction procedure. Results confirmed mechanic disruption of eggs by bead beating before DNA extraction to be highly effective method for the detection of S. haematobium DNA in urine.

A diagnostic study comparing conventional and real-time PCR for Strongyloides stercoralis on urine and on faecal samples.

Strongyloides stercoralis is a soil-transmitted helminth with a wide distribution in tropical and subtropical areas. The diagnosis of S. stercoralisinfection can be challenging, due to the low sensitivity of microscopic examination of stool samples and coproculture. In the last decade, different in-house molecular biology techniques for S. stercoralis have been implemented. They demonstrated good accuracy, although sensitivity does not seem sufficiently high yet. Recently, a novel PCR technique has been evaluated for the detection of S. stercoralis DNA in urine. Aim of this work was to compare the sensitivity of the real-time PCR (qPCR) on feces routinely used at the Centre for Tropical Disease (CTD) of Negrar, Verona, Italy, with that of the novel based PCR on urine. As secondary objective, we evaluated a Urine Conditioning Buffer ® (Zymoresearch) with the aim of improving nucleic acid stability in urine during sample storage/transport at ambient temperatures. Patients attending the CTD and resulting positive at routine screening with serology for S. stercoralis were invited, previous written consent, to supply stool and urine samples for molecular biology. A convenience sample of 30 patients was included. The sensitivity of qPCR on feces resulted 63%, and that of based PCR on urine was 17%. In all the samples treated with the Urine Conditioning Buffer ® there was no detectable DNA. In conclusion, the sensitivity of the novel technique resulted low, and needs further implementation before being considered as a valid alternative to the validated method.