Giuseppe Dettori

Development of a Real-Time PCR Assay for Detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for Routine Clinical Diagnosis

ABSTRACT A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasites—Plasmodium falciparum, P. ovale, and P. vivax—was devised and evaluated using 122 whole-blood samples from patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms and fever. The assay was compared to conventional microscopy and to an established nested-PCR assay. The specificity of the new assay was confirmed by sequencing the PCR products from all the positive samples and by the lack of cross-reactivity with Toxoplasma gondii and Leishmania infantum DNA. Real-time PCR assay showed a detection limit (analytical sensitivity) of 0.7, 4, and 1.5 parasites/μl for P. falciparum, P. vivax, and P. ovale, respectively. Real-time PCR, like nested PCR, brought to light errors in the species identification by microscopic examination and revealed the presence of mixed infections (P. falciparum plus P. ovale). Real-time PCR can yield results within 2 h, does not require post-PCR processing, reduces sample handling, and minimizes the risks of contamination. The assay can therefore be easily implemented in routine diagnostic malaria tests. Future studies are warranted to investigate the clinical value of this technique.

Detection and Isolation of Mycobacterium avium Subspecies paratuberculosis from Intestinal Mucosal Biopsies of Patients with and without Crohn's Disease in Sardinia

OBJECTIVES:Sardinia is an island community of 1.6 million people. There are also about 3.5 million sheep and one hundred thousand cattle in which Johnes disease and Mycobacterium avium subspecies paratuberculosis infection are endemic. The present study was designed to determine what proportion of people in Sardinia attending for ileocolonoscopy with or without Crohns disease were infected with this pathogen.METHODS:Mycobacterium avium subspecies paratuberculosis was detected by IS900 PCR on DNA extracts of fresh intestinal mucosal biopsies as well as by isolation in culture using supplemented MGIT media followed by PCR with amplicon sequencing.RESULTS:Twenty five patients (83.3%) with Crohns disease and 3 control patients (10.3%) were IS900 PCR positive (p = 0.000001; Odds ratio 43.3). Mycobacterium avium subspecies paratuberculosis grew in cultures from 19 Crohns patients (63.3%) and from 3 control patients (10.3%) (p = 0.00001; Odds ratio 14.9). All patients positive by culture had previously been positive by PCR. Mycobacterium avium subspecies paratuberculosis first appeared in the liquid cultures in a Ziehl Neelsen (ZN) staining negative form and partially reverted through a rhodamine-auramine positive staining form to the classical ZN positive form. This resulted in a stable mixed culture of all 3 forms illustrating the phenotypic versatility of these complex chronic enteric pathogens.CONCLUSIONS:Mycobacterium avium subspecies paratuberculosis was detected in the majority of Sardinian Crohns disease patients. The finding of the organism colonizing a proportion of people without Crohns disease is consistent with what occurs in other conditions caused by a primary bacterial pathogen in susceptible hosts.

Mycobacterium avium Subspecies paratuberculosis Infection in Cases of Irritable Bowel Syndrome and Comparison with Crohn's Disease and Johne's Disease: Common Neural and Immune Pathogenicities

ABSTRACT Mycobacterium avium subsp. paratuberculosis causes Johnes disease, a systemic infection and chronic inflammation of the intestine that affects many species, including primates. Infection is widespread in livestock, and human populations are exposed. Johnes disease is associated with immune dysregulation, with involvement of the enteric nervous system overlapping with features of irritable bowel syndrome in humans. The present study was designed to look for an association between Mycobacterium avium subsp. paratuberculosis infection and irritable bowel syndrome. Mucosal biopsy specimens from the ileum and the ascending and descending colon were obtained from patients with irritable bowel syndrome attending the University of Sassari, Sassari, Sardinia, Italy. Crohns disease and healthy control groups were also included. Mycobacterium avium subsp. paratuberculosis was detected by IS900 PCR with amplicon sequencing. Data on the potential risk factors for human exposure to these pathogens and on isolates from Sardinian dairy sheep were also obtained. Mycobacterium avium subsp. paratuberculosis was detected in 15 of 20 (75%) patients with irritable bowel syndrome, 3 of 20 (15%) healthy controls, and 20 of 23 (87%) people with Crohns disease (P = 0.0003 for irritable bowel syndrome patients versus healthy controls and P = 0.0000 for Crohns disease patients versus healthy controls). One subject in each group had a conserved single-nucleotide polymorphism at position 247 of IS900 that was also found in isolates from seven of eight dairy sheep. There was a significant association (P = 0.0018) between Mycobacterium avium subsp. paratuberculosis infection and the consumption of hand-made cheese. Mycobacterium avium subsp. paratuberculosis is a candidate pathogen in the causation of a proportion of cases of irritable bowel syndrome as well as in Crohns disease.

Epidemiological trend of human leptospirosis in Italy between 1994 and 1996

In the three-year period 1994–1996, 222 reports on human cases of leptospirosis were received by the Italian Ministry of Health. The average annual number of reports was 29.2% lower than in the preceding eight years. In all cases but two the infections were thought to have been acquired in Italy. As in previous years, the majority of cases was observed in the northern regions of the country (83.8%), mostly in males (88.9%). Cases occurred in all age groups, but were more common in the working-age population (15–64 years). There was no common-source outbreaks. The typical leptospiral seasonal course, with a peak in August, was observed. During 1994, leptospirosis was the reported cause of death in 19 patients. Mortality was higher among males than females. The overall fatality rate was 22.6%. During the study period, a total of 126 cases of leptospirosis were confirmed by the National Centre for Leptospirosis or one of the 12 Regional Leptospira Laboratories. Of the 103 patients for whom information on place of residence, contact with animals, occupational and recreational activities was available, 98 (95.1%) were people who live in rural areas or devote themselves to occupational or recreational activities at risk. The likely source of infection and the mode of exposure were known for 55 patients. Forty-five patients (81.8%) were likely infected by contaminating water (43 cases) or soil (2 cases), ten (18.2%) by direct contact with animals or animal urine. Both running (51.2%) and stagnant water (27.9%) have been reported as a source of infection. Rodents were implicated in 50.0% of the 10 cases involving animals. In comparison with the preceding eight-year period, the risk of contracting leptospirosis was found to have increased for recreational activities (from 34.7 to 38.2%) and decreased for occupational activities (from 45.8 to 32.7%). A large number of infections, however, was ascribed to accidental events (25.5%). As in the previous period, besides fever, the involvement of the liver was the most frequent clinical manifestation (70.8%). Influenza-like symptoms were the only signs of illness in 15.1% of cases. Infections by 9 different serogroups were detected. The most frequent antibodies were those against serovars icterohaemorrhagiae, poi, copenhageni and bratislava. The presence of co-agglutinins against serovars belonging to different serogroups prevented the identification of the presumptive infecting serogroup in 19.8% of subjects.

Genetic Polymorphisms Influence Plasmodium ovale PCR Detection Accuracy

ABSTRACT Detection of Plasmodium ovale by use of a nested PCR assay with a novel Plasmodium ovale primer set was superior to detection of Plasmodium ovale by real-time PCR assays. Nested PCR was also better at detecting P. malariae. The detection of P. ovale in many patients first admitted >2 months following their return to Italy indicated that P. ovale relapses are common.

Hepatitis C virus core antigen: Analytical performances, correlation with viremia and potential applications of a quantitative, automated immunoassay

BACKGROUND Testing for hepatitis C virus core antigen (HCV Ag) may represent a complementary tool to anti-HCV and HCV-RNA in the diagnosis and monitoring of HCV infection. OBJECTIVE To evaluate the performance characteristics of the automated Abbott ARCHITECT HCV Ag assay. STUDY DESIGN Five sites analyzed over 3000 routine serum samples from populations at different risk, comparing HCV Ag results with anti-HCV screening and supplemental assay results and with HCV-RNA. RESULTS The HCV Ag assay showed a specificity of 100%, a good precision (CV<10%) and excellent dilution linearity (r>0.999). The sensitivity (3 fmol/L) corresponds to 700-1100 IU/mL of HCV-RNA. A non-linear correlation with HCV-RNA was found: r=0.713 vs. Siemens bDNA (523 specimens), r=0.736 vs. Roche Cobas TaqMan (356 specimens) and r=0.870 vs. Abbott Real-Time PCR (273 specimens). HCV Ag quantitation was equally effective on different HCV genoypes (239 for genotype 1/1a/1b/1c, 108 for genotype 2/2a/2c, 86 for genotype 3/3a, 50 for genotype 4/4a/4c/4d). Testing of subjects at high risk for HCV and with potential or actual impairment of the immune system identified 2 cases negative for anti-HCV and positive for HCV Ag on 361 hemodialyzed (0.6%) and 7 cases on 97 (7.2%) among transplant recipients. HCV Ag positivity anticipated anti-HCV seroconversion in all three cases of acute hepatitis C. CONCLUSIONS HCV Ag may be used as reflex testing on anti-HCV positive individuals to confirm or exclude an active infection, and on subjects with acute hepatitis or belonging to high risk groups.

Broadly Reactive Nested Reverse Transcription-PCR Using an Internal RNA Standard Control for Detection of Noroviruses in Stool Samples

ABSTRACT We developed a nested reverse transcription-PCR (nRT-PCR) for the detection of noroviruses in stools, using random primers for RT, the JV12/JV13 primer pair in the first round of nPCR, and a set of nine inner primers for the second, comprising the reverse sequences of primers SR46, SR48, SR50, and SR52, and five novel oligonucleotide sequences (113-1, 113-2, 115-1, 115-2, and 115-3). The specificity of the nRT-PCR was confirmed by testing 61 stools containing enteric viruses other than noroviruses. In comparative assays on either stools or RNA dilutions from two genogroup I and three genogroup II (GII) norovirus-positive samples, nRT-PCR was always at least as sensitive as RT-PCR and Southern hybridization. With some of the samples tested, the increase in sensitivity was 10-fold or higher. For GII viruses, the detectable range of nRT-PCR was estimated to be 8.4 × 104 to 2 RNA viral particles. When used on 85 stools from pediatric patients with acute gastroenteritis negative for viruses by electron microscopy and cell culture, the nRT-PCR detected norovirus in 19 samples (22.3%), while it failed to detect one reference RT-PCR-positive sample containing a Desert Shield strain. Sixteen of the 19 nRT-PCR-positive samples gave concordant results with reference RT-PCR and Southern hybridization, and all with sequence analysis. Partial sequencing of the polymerase region revealed that from January to April 2000 all GII strains except two (Rotterdam- and Leeds-like viruses) formed a tight cluster related to Hawaii virus. The nRT-PCR described could prove suitable for large epidemiological studies and for specialized clinical laboratories performing routine molecular testing.

Evaluation of a real-time polymerase chain reaction assay for the laboratory diagnosis of giardiasis.

A real-time polymerase chain reaction (PCR) assay was evaluated in comparison with the combination of conventional methods (microscopic examination and antigen detection assay) during the period 2006 to 2008 on 771 fecal samples belonging to 386 patients to assess its usefulness for an accurate laboratory diagnosis of giardiasis. The real-time PCR assay detected Giardia intestinalis DNA in 195 samples (106 patients), including 26 samples (21 patients) negative by the conventional assays. Among the 21 patients, in 8 cases, giardiasis was previously diagnosed also by conventional methods in additional samples of the same patients, whereas in 13, it would have been undiagnosed if real-time PCR assay was not used. The real-time PCR assay demonstrated a detection limit of 2 cysts per reaction and 100% specificity and sensitivity compared to conventional methods. A genotype analysis targeting the beta-giardin gene allowed to identify 53 samples (23 patients) containing genotype A and 59 samples (45 patients) containing genotype B.

Infective colitis associated with human intestinal spirochetosis

Aim:  Our study reports the detection and identification of intestinal spirochetosis in patients with colonic diseases in a tertiary‐care hospital over a 12‐year period, and includes a description of all cases we diagnosed.

Evaluation of a real-time polymerase chain reaction assay for the detection of Dientamoeba fragilis ☆

The diagnostic value of a real-time polymerase chain reaction (PCR) assay targeting the 5.8S rDNA of Dientamoeba fragilis was investigated as compared with conventional parasitologic methods including cultivation testing 959 fecal samples from 491 patients attending a tertiary-care hospital and suspected of having an intestinal parasitosis. The real-time PCR assay revealed 117 additional D. fragilis-positive samples as compared with conventional methods, showing 100% sensitivity and specificity in our experience. On the whole, D. fragilis infection was detected in 186 samples from 105 patients (21.4%, third in frequency among the diagnosed intestinal parasitoses). The evaluated real-time PCR assay represents an effective tool to obtain both an accurate diagnosis and a reliable epidemiologic picture of dientamoebiasis.