The Use of Quinacrine in Nitroimidazole-resistant Giardia Duodenalis: An Old Drug for an Emerging Problem.

Background: There is little evidence regarding the management of refractory giardiasis after treatment with nitroimidazoles. This study estimates the proportion of persistent giardiasis in 3 hospitals in Barcelona, describes associated risk factors and genotype, and evaluates the efficacy rate of quinacrine in those with persistent giardiasis. Methods: A clinical, prospective, observational study was conducted in patients with giardiasis treated with nitroimidazoles. Those with persistent giardiasis were provided quinacrine. Molecular characterization of Giardia isolates was performed by polymerase chain reaction amplification of a fragment of tpi and bg genes. Results: Seventy-seven patients were recruited and treated with nitroimidazoles, and in 14 of 71 (20%) of patients followed up, Giardia persisted. Refractory giardiasis was associated with malaise (P = .007) and anorexia (P = .02), with previous giardiasis (P = .03), and with previous antibiotic (P = .02) or antiparasitic(P = .04) use. Quinacrine had an effectiveness rate of 100% in refractory giardiasis (n = 13; 95% confidence interval = 75-100). Molecular characterization showed that 17 (25%) Giardia isolates belonged to assemblage A, and 31 (43%) belonged to assemblage B. In refractory giardiasis, assemblage A and B were found responsible in 4 and 6 cases, respectively. Conclusions: Almost 20% of patients presented persistent giardiasis, belonging to both assemblages A and B, after nitroimidazole. Short course of quinacrine was effective in treating refractory cases. Further controlled studies should evaluate its efficacy and safety.

Identification of free-living amoebae and amoeba-associated bacteria from reservoirs and water treatment plants by molecular techniques.

The occurrence of free-living amoebae (FLA) was investigated in 83 water samples from reservoirs and water treatment plants, with culture positive in 64 of them (77.1%). Polymerase chain reaction (PCR) of partial 18S rRNA gene and ITS region was performed in order to identify amoeba isolates, and the presence of Legionella pneumophila , Mycobacterium spp., Pseudomonas spp., and Microcystis aeruginosa was investigated in 43 isolates of amoebae by multiplex PCR. Of the isolated amoebae, 31 were Acanthamoeba spp., 21 were Hartmannella vermiformis, 13 were Naegleria spp., and one was Vanella spp. T2, T4, and T5 genotypes of Acanthamoeba have been identified, and T4 isolates were grouped into five subgenotypes and graphically represented with a Weblog application. Inside amoebae, L. pneumophila was detected in 13.9% (6/43) of the isolates, and Pseudomonas spp. and Mycobacterium spp. were detected in 32.6% (14/43) and 41.9% (18/43), respectively. No statistical correlation was demonstrated between FLA isolation and seasonality, but the presence of intracellular bacteria was associated with warm water temperatures, and also the intracellular presence of Mycobacterium spp. and Pseudomonas spp. were associated. These results highlight the importance of amoebae in natural waters as reservoirs of potential pathogens and its possible role in the spread of bacterial genera with interest in public and environmental health.

Two cases of zoonotic cryptosporidiosis in Spain by the unusual species Cryptosporidium ubiquitum and Cryptosporidium felis.

INTRODUCTION: Two cases of infection by zoonotic transmission of unusual species of Cryptosporidium were detected in 2010-2011 in Spain (León and Zaragoza). MATERIALS AND METHODS: Cryptosporidium spp. was detected by microscopic examination of modified Ziehl-Neelsen stained fecal smears. PCR-RFLP of the SSUrDNA gene and sequencing of the amplified fragment confirmed the species. RESULTS: C. ubiquitum and C. felis were identified in samples from an immunocompetent child and from a HIV-positive adult, respectively. CONCLUSIONS: This is the first report of human infection by C. ubiquitum (cervine) and autochthonous C. felis, identified in Spain.

Intestinal myiasis due to Eristalis tenax: report of a new case in Spain.

We describe a new case of accidental intestinal myiasis by Eristalis tenax in Spain. Only about 20 cases have been reported worldwide, two of them occurring in Spain. A 51-year-old patient with nonspecific abdominal pain and occasional diarrhoea expelled larvae in her stool. Macroscopic analysis of these larvae revealed morphology compatible with that of Eristalis tenax. The larva analysis showed its autofluorescence as parasitological feature described for the first time.

[Prevalence of Giardia duodenalis assemblage B in humans in Zaragoza and León, Spain]. / Prevalencia de Giardia duodenalis genotipo B en humanos en Zaragoza y León, España.

OBJECTIVES: To study Giardia duodenalis assemblages circulating in the health areas of two hospitals in Zaragoza and León (Spain). METHODS: A total of 211 stool samples with Giardia were genotyped by PCR of the tpi gene. RESULTS: Assemblage B was the most prevalent, both in Zaragoza (84,7%) and León (95,1%). The remaining isolates were identified as AII+B. CONCLUSIONS: We detected the spread of G. duodenalis assemblage B in Zaragoza and in León, with an increase in its prevalence in Zaragoza compared to previous studies.

Genetic characterization of Cryptosporidium species from humans in Spain.

Several species of Cryptosporidium have been associated with infection. Cryptosporidium parvum and Cryptosporidium hominis are the main agents of cryptosporidiosis in humans. Stool samples from 108 Cryptosporidium-infected patients were submitted to PCR-RFLP analysis for a 553-bp fragment of Cryptosporidium oocyst wall protein (COWP) gene and an 826-864 bp fragment of the small-subunit ribosomal RNA (SSU-rRNA) gene. Ninety-two patients were immunocompetent children and 16 were HIV-infected adults. C. hominis was detected in 69 patients (59 immunocompetent and 10 HIV-infected); C. parvum, in 34 patients (28 immunocompetent and 6 HIV-infected); and C. meleagridis and C. felis in one patient each (both immunocompetent children). Three samples yielded negative results. C. parvum was significantly more frequent in children from rural areas than in those of urban residence (p=0.010). As far as we know, this is the first surveillance study about the molecular characterization of Cryptosporidium in humans performed in Spain. The finding of zoonotic species infecting humans calls for further research on this subject.

Molecular characterization of Cryptosporidium isolates from pigs in Zaragoza (northeastern Spain).

A total of 142 stool specimens from pigs on 24 farms from the province of Zaragoza (northeastern Spain) were screened for Cryptosporidium spp. Samples were first analysed by routine techniques (formalin-ethyl acetate sedimentation method and modified Ziehl-Neelsen stain) selecting those microscopically positive for genetic characterization. Cryptosporidium species and genotypes were determined by a nested PCR-RFLP technique at the 18S ribosomal DNA locus and sequencing of the PCR-positive secondary products. Cryptosporidium oocysts were microscopically identified in the faeces of 32 pigs (22.5%) from 15 farms (62.5%). Infected animals included 23 weaned piglets (30.7%), 5 fattening pigs (11.9%) and 4 sows (16%). Diarrhoea was not detected in any of the infected pigs. The molecular characterization was successfully performed in 26 samples from 14 farms. Cryptosporidium suis was found in 10 specimens from 7 farms (nine weaned piglets and one sow) and the Cryptosporidium pig genotype II in 16 samples from 10 farms (13 weaned piglets and 3 fattening pigs). Both C. suis and the pig genotype II were concurrently detected on three farms.

[Cryptosporidium hominis diarrhea outbreak and transmission linked to diaper infant use]. / Brote de diarrea y transmisión de Cryptosporidium hominis asociados al uso de pañal en niños.

BACKGROUND AND OBJECTIVE: On the basis of several cases of cryptosporidiosis detected in a child day-care center, we stablished the extent of the outbreak and investigated causes of parasite transmission. PATIENTS AND METHOD: A retrospective cohort study was designed on all children attending day-care center and care givers to determine their infection status and identify risk factors associated to the outbreak. RESULTS: 24 cases of cryptosporidiosis were detected, with an attack rate of 0.46 (24/52); 12 of them were parasitologycally confirmed. All care givers were negative for Cryptosporidium and none of them reported symptoms of acute gastroenteritis. Transmission pattern was compatible with person to person modes. Among the factors investigated, two were associated with the risk of disease: diaper wear (relative risk = 2.06; p = 0.059); and diarrhea in relatives (relative risk = 2.05; p = 0.01). In all confirmed cases, Cryptosporidium hominis (previously known as C. parvum, genotype 1), was identified. CONCLUSIONS: Cryptosporidiosis should be considered as a possible cause of outbreaks of gastroenteritis at day-care centers. Increasing care on diaper changing practices, specially over children with diarrhea, may be the key factor to prevent transmission of Cryptosporidium.

Multilocus typing and population structure of Cryptosporidium from children in Zaragoza, Spain.

A multilocus typing approach with eight variable-number tandem-repeat (VNTR) loci and the GP60 gene was used to analyze the inter- and intra-species variation of 44 Cryptosporidium isolates from pediatric patients in Zaragoza city (NE, Spain). Restriction and sequence analyses of the SSU rRNA gene revealed that Cryptosporidium transmission is mostly anthroponotic in this area, with the predominance of Cryptosporidium hominis (n: 41) over Cryptosporidium parvum (n: 3). GP60 subtyping showed limited genetic diversity and four subtypes were identified, including IbA10G2 (n: 35), IaA24R3 (n: 6), IIaA15G1R1 (n: 1) and IIaA15G2R1 (n: 2). Five out of eight VNTR loci showed a discriminatory power higher than the GP60 gene, although each locus had a predominant allele exhibited by more than 50% of isolates. All but four alleles were associated to either C. hominis or C. parvum and linked alleles at different loci were found. Multilocus typing substantially increased the discriminatory power (Hunter-Gaston index: 0.807, 95% CI, 0.683-0.926) and revealed that genetic diversity is much higher than that reported by GP60 sequencing, since 17 multilocus subtypes (MLTs) were identified. Nearly half of the specimens were allocated to a single major MLT. However, no more than three specimens were allocated to each of the remaining MLTs. Both phylogenetic and population analyses revealed a population clustering of C. hominis according to the GP60 subtype, which indicates the robustness of this marker to differentiate genetic subpopulations. Subpopulations had an overall clonal genetic structure, although traces of genetic flow between them were also observed.

Intestinal parasites in HIV-seropositive patients in the Continental Region of Equatorial Guinea: its relation with socio-demographic, health and immune systems factors.

BACKGROUND: Prevalence of intestinal parasitic infections in HIV-positive people and its association with clinical and socioeconomic factors has been investigated on the mainland of Equatorial Guinea, in order to define the precise measures for improvement of their quality of life. METHODS: In August 2010, 273 HIV-positive and 60 HIV-negative were recruited. A sample of faeces, T-CD4+ cell counts, and clinical, socioeconomic and demographic data were collected from each patient. Stool samples were analysed by microscopy and immunochromatography. Data were analysed by Pearson's χ2 test and the risk of each factor was measured by odds ratio bivariate analysis. RESULTS: Two hundred and ten (76.9%) HIV-positive participants were infected by intestinal parasites and 16 parasite species were identified; 246 (48.9%) were pathogenic helminths, 159 (22.9%) pathogenic protozoa and 142 (28.2%) opportunistic protozoa. Trichuris trichiura, Entamoeba histolytica/dispar/moshkovskii, Ascaris lumbricoides and Giardia duodenalis were the most prevalent parasites; 52 (86.7%) of HIV-negative participants were parasitized. HIV was related to co-infection by Entamoeba spp., E. nana and Blastocystis hominis. CONCLUSIONS: The high rates of parasitic infections found highlights the urgent need of environmental sanitation, health education and water distribution actions, as well as early diagnosis and treatment of intestinal parasites.

Prevalence of intestinal parasites in HIV-positive patients on the island of Bioko, Equatorial Guinea: its relation to sanitary conditions and socioeconomic factors.

The prevalence of intestinal parasitic diseases and their associated factors has been investigated in HIV populations from the Island of Bioko, Equatorial Guinea. The feces of 310 participants from the island of Bioko (260 HIV-positive and 50 HIV-negative) were analyzed by microscopic observation. Immunochromatography was also used to diagnose Giardia, Entamoeba histolytica and Cryptosporidium spp. In addition, patients were asked for sociodemographic, economic and academic status, and CD4+ T cell counts were recorded. For HIV-positive patients, the prevalence of infection by intestinal parasites was 81.5% (212/260), 83.8% (218/260) by pathogenic helminths and 55.4% (168/260) by pathogenic protozoa (E. histolytica/dispar and Giardia duodenalis). Gender association was found between the infection by Ascaris and Schistosoma, a higher proportion being found in women; and between Entamoeba and the place of residence, a higher proportion being observed in the urban belt. Strongyloides stercoralis and Chilomastix mesnili appeared only in the people of this group, all the cases of Chilomastix being in females. For HIV-negative participants, the prevalence of infection by intestinal parasites was 74.0% (37/50), 90.0% (45/50) by pathogenic helminths and 66.0% (43/50) by pathogenic protozoa. Gender, educational level and low hygiene were associated with intestinal parasitic infection. When comparing the two groups (HIV-positive and HIV-negative), statistical association between HIV co-infection and infection by Giardia and Entamoeba was found. Diarrhea was also associated with intestinal parasitic infection in the HIV-positive group. Not only do our findings reflect high rates of intestinal parasitic infections in HIV-positive people, but also in the HIV-negative group, suggesting a closer relationship between sanitary status and living conditions than with immune status, and thus they highlight the need to carry out health education policies in the population. In addition, Schistosoma intercalatum/guineensis has been detected for the first time as an autochthonous parasite on the island.

Evaluation of in vitro efficacy of combined riboflavin and ultraviolet a for Acanthamoeba isolates.

PURPOSE: To evaluate in vitro the amoebicidal effects of riboflavin and ultraviolet A (UVA) collagen cross-linking. DESIGN: Experimental study, laboratory investigation. METHODS: Two different strains of Acanthamoeba species were tested identically. Four treatment groups were considered: group 1 consisted of 0.1% riboflavin and 30-minute UVA irradiation; group 2 consisted of 0.1% riboflavin and 60-minute UVA irradiation; group 3 consisted of no riboflavin and no UVA exposure; group 4 consisted of 0.1% riboflavin and no UVA exposure. The application of UVA was performed under the parameters used for in vivo corneal collagen cross-linking. RESULTS: In all cases, cysts and trophozoites were detected 24 hours after treatment at a radial distance from the center of the seeding point more than 5 mm, indicating that the amoebae were viable. All treated and untreated groups of amoebae from the 2 strains exhibited growth (radii of 14 to 15 mm in groups 1, 3, and 4; radius of 12 mm in group 2). The final morphologic features of the 2 strains of trophozoites that received treatment were similar to those of the initial seeding group and the untreated control group. CONCLUSIONS: The results obtained in our study show that a single dose (30 or 60 minutes) of cross-linking cannot achieve eradication in the 2 different Acanthamoeba strains examined. However, in vitro results do not always indicate in vivo efficacy, so future studies should test the validity of this treatment for Acanthamoeba keratitis.

Identification and molecular characterization of Cryptosporidium and Giardia in children and cattle populations from the province of Álava, North of Spain.

The prevalence of and factors associated with the protozoan enteropathogens Cryptosporidium and Giardia have been investigated in selected children and cattle populations from the province of Álava (Northern Spain). The presence of these organisms was detected in fecal samples using commercially available coproantigen-ELISA (CpAg-ELISA) and immunochromatographic (ICT) assays. A total of 327 caregivers of children participants were asked to answer questions on risk factors potentially associated to the prevalence of Cryptosporidium and Giardia, including water-use practices, water sports and contact with domestic or pet animals. Molecular analyses were conducted using a nested-PCR technique to amplify the small-subunit (SSU) rRNA gene of Cryptosporidium and the triosephosphate isomerase (tpi) gene of Giardia. Cryptosporidium oocysts and Giardia cysts were found in 3 and 16 samples using the CpAg-ELISA, and in 5 and 9 samples using the ICT test, respectively. Cryptosporidium and Giardia were also found in 7 and 17 samples by CpAg-ELISA, and 4 and 14 samples by ICT, respectively, of 227 cattle fecal samples. The overall Cryptosporidium and Giardia infection prevalences, based on a Bayesian approach accounting for the imperfect sensitivities and specificities of both diagnostic tests, were estimated to 1.0% (95% BCI: 0.2%-2.8%) and 3.1% (1.5%-5.3%) in children and 3.0% (0.5%-9.2%) and 1.4% (0.0%-6.4%) in cattle, respectively. In humans, a single Cryptosporidium isolate was characterized as C. hominis. Of seven Giardia isolates, four were identified as assemblage B, two as assemblage A-II and one was a mixed assemblage B+A-II infection. No Cryptosporidium or Giardia isolates could be obtained from cattle samples. Although limited, these results seem to suggest that cattle are unlikely to be an important reservoir of zoonotic Cryptosporidium and/or Giardia in the province of Álava.

Vitamin B12 and folic acid in children with intestinal parasitic infection.

OBJECTIVE: To determine prospectively plasma levels of vitamin B12 and folic acid in children with intestinal parasitic infection before and three months after antiparasitic treatment. METHODS: 3036 stool samples were collected from 1959 children and 939 cello-tape anal swabs were taken from 688 children for intestinal parasite investigation. Of these, 155 children were identified as having a parasitic infection; however, only 86 were followed up during this study: 26 children with Giardia lamblia infection were treated with tinidazole and metronidazole, pyrantel pamoate was used in the treatment of 40 children with Enterobius vermicularis, and 20 patients infected with Cryptosporidium parvum received only symptomatic treatment. Vitamin B12 and folic acid levels were measured by radioimmunoassay, before and three months after the completion of treatment. RESULTS: Vitamin B12 serum concentrations did not show any significant differences among the three groups. There was a significant increase in vitamin B12 serum concentrations after three months of anti-parasitic treatment (630.57+/-200.97 vs. 667.97+/-181.55 pg/dL, p = 0.002, n = 86). Paired analysis in each group showed only significant increases for vitamin B12 in the Giardia lamblia group and in the Enterobius vermicularis group. No statistically significant differences were found for folic acid serum concentrations before and three months after treatment. CONCLUSIONS: Patients with symptomatic infection by Giardia lamblia and Enterobius vermicularis have lower vitamin B12 levels than asymptomatic patients. This could reflect a more affected intestinal mucous. These results could present the opportunity to treat these parasitic infections and to use vitamin B12 supplementation in symptomatic children with Giardia lamblia and Enterobius vermicularis infection.

Serum mineral levels in children with intestinal parasitic infection.

Parasitic infections are highly prevalent in the general population. A relation between a parasitic infection and absorption of minerals is not an easy task. Serum levels of copper, zinc and magnesium were prospectively measured in 64 children with intestinal parasitic infection. Thirty-nine children with Enterobius vermicularis were treated with pyrantel pamoate and 25 children with Giardia lamblia with tinidazole and metronidazole. Three months after treatment, significant differences in serum copper, zinc and magnesium were seen in patients with E. vermicularis infection, and in serum magnesium levels in patients with G. lamblia. Although the pathogenic mechanism is not clear, these findings could reflect a deficiency related to malabsorption due to mucous affection. Early detection and treatment of intestinal parasitosis could avoid these serum mineral deficiencies.