Development and Application of a gp60-Based Typing Assay for Cryptosporidium viatorum.

The apicomplexan intestinal parasites of the genus Cryptosporidium take a major toll on human and animal health and are frequent causes of waterborne outbreaks. Several species and genotypes can infect humans, including Cryptosporidium viatorum, which, to date, has only been found in humans. Molecular characterization of Cryptosporidium spp., critical to epidemiological analyses, is commonly based on gp60 gene analysis, which appears to require bespoke species- or group-specific PCR primers due to extensive genetic diversity across the genus. In this study, we amplified, sequenced, and characterized the gp60 gene of C. viatorum for the first time. Moreover, we developed and validated a gp60 typing assay for this species and applied it to 27 isolates originating from Asia, Africa, and Central America. A single subtype family, XVa, was identified containing multiple alleles.

Cryptosporidium parvum infections in a cohort of veterinary students in Sweden.

In March 2013, a veterinary student tested positive for Cryptosporidium; four classmates reported similar gastrointestinal symptoms. We aimed to identify source(s) and risk factors for Cryptosporidium infection in university persons symptomatic between 21 January and 14 April 2013. Sixty-four (79%) students from a cohort of 81 fourth-year veterinary students completed questionnaires, identifying 13 cases; four were Cryptosporidium parvum GP60 subtype IIaA16G1R1b, two were IIdA24G1, seven did not submit stool samples. Thirteen cases attended the university's field clinic before symptom onset (13/37 attendees, 35%); 11 visited at least one of four farms where students recalled seeing calves with diarrhoea. C. parvum subtype IIaA16G1R1b was identified in calves at one of the farms. Entering pens of calves with diarrhoea [relative risk (RR) 7·6, 95% confidence interval (CI) 1·7-33·5] and eating in clinic cars (RR 9·1, 95% CI 1·3-65·8) were associated with being a case. Washing hands at least twice per farm visit (0 cases, P = 0·03) was protective. This outbreak investigation was notable for rapid and effective collaboration between public health, veterinary and environmental sectors, leading to swift identification of a microbiological and epidemiological link between cases, infected calves and their farms. We recommend frequent hand-washing using proper technique and dissuasion from eating in clinic cars to minimize possible exposure to contaminated surfaces.

Molecular epidemiology and clinical manifestations of human cryptosporidiosis in Sweden.

This study describes the epidemiology and symptoms in 271 cryptosporidiosis patients in Stockholm County, Sweden. Species/genotypes were determined by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) of the Cryptosporidium oocyst wall protein (COWP) and 18S rRNA genes. Species were C. parvum (n=111), C. hominis (n=65), C. meleagridis (n=11), C. felis (n=2), Cryptosporidium chipmunk genotype 1 (n=2), and a recently described species, C. viatorum (n=2). Analysis of the Gp60 gene revealed five C. hominis allele families (Ia, Ib, Id, Ie, If), and four C. parvum allele families (IIa, IIc, IId, IIe). Most C. parvum cases (51%) were infected in Sweden, as opposed to C. hominis cases (26%). Clinical manifestations differed slightly by species. Diarrhoea lasted longer in C. parvum cases compared to C. hominis and C. meleagridis cases. At follow-up 25-36 months after disease onset, 15% of the patients still reported intermittent diarrhoea. In four outbreaks and 13 family clusters, a single subtype was identified, indicating a common infection source, which emphasizes the value of genotyping for epidemiological investigations.

Occurrence of pathogens in wild rodents caught on Swedish pig and chicken farms.

A total of 207 wild rodents were caught on nine pig farms, five chicken farms and five non-farm locations in Sweden and surveyed for a selection of bacteria, parasites and viruses. Lawsonia intracellularia and pathogenic Yersinia enterocolitica were only detected in rodents on pig farms (9% and 8% prevalence, respectively) which indicate that these agents are more likely to be transmitted to rodents from pigs or the environment on infected farms. Brachyspira hyodysenteriae (1%), Brachyspira intermedia (2%), Campylobacter jejuni (4%), Campylobacter upsaliensis (2%), leptospires (7%) and encephalomyocarditis virus (9%) were also detected from rodents not in contact with farm animals. Giardia and Cryptosporidium spp. were common, although no zoonotic types were verified, and Salmonella enterica was isolated from 1/11 mice on one farm but not detected by PCR from any of the rodents. Trichinella spp. and Toxoplasma gondii were not detected.

First reported foodborne outbreak associated with microsporidia, Sweden, October 2009.

Microsporidia are spore-forming intracellular parasites that infrequently cause disease in immunocompetent persons. This study describes the first report of a foodborne microsporidiosis outbreak which affected persons visiting a hotel in Sweden. Enterocytozoon bieneusi was identified in stool samples from 7/11 case-patients, all six sequenced samples were genotype C. To confirm that this was not a chance finding, 19 stool samples submitted by healthy persons from a comparable group who did not visit the hotel on that day were tested; all were negative for microsporidia. A retrospective cohort study identified 135 case-patients (attack rate 30%). The median incubation period was 9 days. Consumption of cheese sandwiches [relative risk (RR) 4·1, 95% confidence interval (CI) 1·4-12·2] and salad (RR 2·1, 95% CI 1·1-4) were associated with illness. Both items contained pre-washed, ready-to-eat cucumber slices. Microsporidia may be an under-reported cause of gastrointestinal outbreaks; we recommend that microsporidia be explored as potential causative agents in food- and waterborne outbreaks, especially when no other organisms are identified.

Two geographically separated food-borne outbreaks in Sweden linked by an unusual Cryptosporidium parvum subtype, October 2010.

The number of sporadic cases of Cryptosporidium identified in the Stockholm county area increased above the expected limit during October 2010. Additionally, two food-borne outbreaks of cryptosporidiosis occurred in two other Swedish cities: Umeå (4 October) and Örebro (9 October). The outbreak investigations did not reveal any responsible food item, however fresh herbs were suspected. Thirty stool samples, originating from all three events, tested positive for Cryptosporidium oocysts. Polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) revealed that 27 individuals were infected with C. parvum, two with C. hominis, and one with C. felis. Using sequence analysis of the GP60 glycoprotein gene, a polymorphic marker with high intra-species diversity, we identified the same C. parvum subtype IIdA24G1 in samples from both the Umeå outbreak and the Stockholm area cases, thus indicating a possible outbreak in the Stockholm area and establishing a link between these two events. C. parvum IIdA24G1 has not previously been described in connection with a food-borne outbreak. For the outbreak in Örebro, another subtype was identified: C. parvum IIdA20G1e. These findings demonstrate that subtyping C. parvum isolates using GP60 gene amplification can be used to link cases in an outbreak investigation and we recommend its use in future similar events.

Microsporidia in duodenal biopsies from 72 HIV-infected patients with abdominal complaints.

In order to evaluate the capacity of routine histological examination to detect microsporidiosis, a retrospective study was performed on 72 duodenal biopsies from 72 HIV-infected patients with upper abdominal symptoms of unknown cause. Two light microscopic cytological staining techniques, modified trichrome stain and the fluorochrome Calcofluor, were used. Two cases of microsporidiosis were detected among the 20 patients with prolonged diarrhoea of unknown origin in whom no etiological agent had been demonstrated by stool examination, mycobacterial and cytomegalovirus culture of biopsies, and histological routine staining of duodenal biopsies. The calculated confidence interval of 3-30% corresponds to the prevalence of intestinal microsporidiosis in HIV patients with prolonged diarrhoea in various parts of the world. The findings motivate attempts to identify microsporidia using special cytological staining methods. Improved methods of species identification are needed to aid in the choice of chemotherapy.

Susceptibility of Plasmodium falciparum to chloroquine and mefloquine in Somalia.

A field study was conducted in the Malable area, in Somalia, to assess the susceptibility of Plasmodium falciparum to chloroquine and mefloquine. The in vivo response of P. falciparum to standard therapeutic regimen of chloroquine was studied in 16 children (1-12 years) using the standard WHO in vivo field test. All subjects were parasite free by day 3 and no recrudescence occurred during a 7-d follow-up. In the 24 h micro in vitro tests for chloroquine, 29 of 39 tests performed were successful. Of the 29 isolates, 3 showed distinct resistance and 2 were borderline. The drug concentration yielding 99% (EC99) inhibition was 1.64 x 10(-6) M, indicating low grade resistance. For mefloquine, 11 of 20 tests gave interpretable results and were sensitive, although with some heterogeneity. The EC50 and EC99 of 0.24 x 10(-6) M and 1.31 x 10(-6) M respectively indicate sensitivity.

Standard and reduced doses of sulfadoxine-pyrimethamine for treatment of Plasmodium falciparum in Tanzania, with determination of drug concentrations and susceptibility in vitro.

88 asymptomatic Tanzanian schoolchildren with Plasmodium falciparum parasitaemia were given all, half or quarter of the recommended standard therapeutic dose of sulfadoxine-pyrimethamine (Fansidar). All children cleared the parasites by day 3 and all remained negative during 28-42 days of follow-up. All 32 successful in vitro micro-tests showed full sensitivity. High performance liquid chromatographic methods were applied for drug determinations. Using 100 microliter capillary blood dried on filter paper for sulfadoxine determination the inter-individual variation during follow-up of the standard dose group was 2-4 fold and the median half life was 8.9 d. Sulfadoxine concentrations in the half and quarter dose groups were roughly proportional to those in the standard dose group. The median whole blood to plasma concentration ratio for sulfadoxine was 0.72 and the correlation coefficient 0.95. There was only a weak correlation (r=0.46) between plasma concentrations of sulfadoxine and pyrimethamine. The uniform efficacy of sulfadoxine-pyrimethamine in vivo, even when reduced doses were used, makes this combination a good alternative for treatment of P. falciparum in Tanzania.

Intestinal parasites in HIV-2 associated AIDS cases with chronic diarrhoea in Guinea-Bissau.

Previous studies from African countries where HIV-1 infection is prevalent have shown that infections with Cryptosporidium parvum, Isospora belli and microsporidia are frequently associated with chronic diarrhoea in AIDS patients. The information about the occurrence of these parasites in HIV-2 associated AIDS cases with chronic diarrhoea is limited. We have performed a study of stool parasites in patients from Guinea-Bissau, the country with the highest prevalence of HIV-2 in the world. Stool specimens from 52 adult patients with chronic diarrhoea of which 37 were HIV-positive and fulfilling the clinical criteria of AIDS (five HIV-1, 28 HIV-2 and four dually infected with HIV-1 and HIV-2) were screened for parasitic infections. Twenty five percent of the HIV-2 positive patients were infected with C. parvum, 11% with I. belli and 11% with microsporidia, all three parasites were seen only in HIV-positive patients. The three patients with microsporidiosis, all HIV-2 infected, are to our knowledge the first cases reported from Guinea-Bissau. Other stool parasites such as Blastocystis hominis, hookworm and Strongyloides stercoralis were observed both among HIV-positive and HIV-negative patients.

A malariometric survey in a rural community in the Muheza district, Tanzania: age profiles in the development of humoral immune responses.

A malariometric survey was carried out in a rural community situated in a malaria holoendemic endemic area of Tanzania. A random sample (n = 228) of different age groups was taken to elucidate the association between anti-Pf155/RESA and anti-Pf332 antibody responses and classical malaria indices. Parasitaemia, fever, splenomegaly, haematocrit and antimalarial consumption were assessed. Antibody responses against Pf155/RESA and Pf332 peptides were determined by ELISA. The age profiles of parasite density, splenomegaly, fever, haematocrit values and prevalence of antibody responses indicated intensive malaria exposure and the highest impact of malaria in small children. Forty-five percent of the study population had detectable chloroquine and desethyl-chloroquine blood levels, and the highest frequency and concentrations were recorded in the 12-23 months old. There was no significant association between the presence of drug and parasite density in the different age groups, although in the < 15 years old there was lower parasite prevalence among the children positive for drug in their blood (P < 0.05). High prevalence of antibody responses to all antigens was observed already at an early age, but the mean anti-Pf155/RESA and anti-Pf332 antibody levels increased significantly only in the adult group (P < 0.01). Significantly lower mean parasite densities were observed in high responders to Pf155/RESA and Pf332 peptides for the > or = 10 years old. For the 1-9 years, a similar difference was only observed in the high responders to Pf332. For the whole material, anti-Pf155/RESA and anti-Pf332 antibody levels correlated positively with age. When the effect of age was allowed for in analysing the relationship between parasite density and antibody level against the different antigens, a significant negative correlation was found only with regard to Pf332 in the > = 10 years age group. These results suggest that anti-Pf332 antibodies appear to be a better indicator for antiparasitic immunity, but both antigens are important for immune protection.

Application in clinical epidemiology of new advances in immunology and molecular biology of malaria.

The first purpose was to study the sensitivity of a spot hybridization assay for P. falciparum with a DNA probe. This assay was compared with light microscopy for the detection of low-grade parasitemia. The second purpose was to study in clinically immune individuals the seroreactivity, against a newly identified P. falciparum antigen, deposited in the erythrocyte membrane during merozoite invasion (Perlmann et al., 1984). This antigen is considered to be a potential component in a future vaccine against the blood stage of the parasite. In a holoendemic village in Yekepa area, Northern Liberia, 28 adult men with a high degree of protective immunity against malaria, were shown to have repeatedly low-grade parasitemias of varying density. The spot hybridization assay with the DNA probe was highly sensitive in detecting parasitic infection. The sensitivity was comparable to that of the examination of a blood film for about 15 min by an experienced microscopist. The seroreactivity against Pf 155 antigen varied between a high positive titer to negativity in different subjects, but the reactivity was constant over a period of 15 months for each subject despite numerous new infections and comparable protective immunity against malaria infection.

Microscopic diagnosis of sodium acetate-acetic acid-formalin-fixed stool samples for helminths and intestinal protozoa: a comparison among European reference laboratories.

The present study aimed to compare the diagnostic performance of different European reference laboratories in diagnosing helminths and intestinal protozoa, using an ether-concentration method applied to sodium acetate-acetic acid-formalin (SAF)-preserved faecal samples. In total, 102 stool specimens were analysed during a cross-sectional parasitological survey in urban farming communities in Côte d'Ivoire. Five SAF-preserved faecal samples were prepared from each specimen and forwarded to the participating reference laboratories, processed and examined under a microscope adhering to a standard operating procedure (SOP). Schistosoma mansoni (cumulative prevalence: 51.0%) and hookworm (cumulative prevalence: 39.2%) were the predominant helminths. There was excellent agreement (kappa > 0.8; p < 0.001) among the reference laboratories for the diagnosis of S. mansoni, hookworm, Trichuris trichiura and Ascaris lumbricoides. Moderate agreement (kappa = 0.54) was found for Hymenolepis nana, and lesser agreement was observed for other, less prevalent helminths. The predominant intestinal protozoa were Entamoeba coli (median prevalence: 67.6%), Blastocystis hominis (median prevalence: 55.9%) and Entamoeba histolytica/Entamoeba dispar (median prevalence: 47.1%). Substantial agreement among reference laboratories was found for E. coli (kappa = 0.69), but only fair or moderate agreement was found for other Entamoeba species, Giardia intestinalis and Chilomastix mesnili. There was only poor agreement for B. hominis, Isospora belli and Trichomonas intestinalis. In conclusion, although common helminths were reliably diagnosed by European reference laboratories, there was only moderate agreement between centres for pathogenic intestinal protozoa. Continued external quality assessment and the establishment of a formal network of reference laboratories is necessary to further enhance both accuracy and uniformity in parasite diagnosis.

Molecular typing of Cryptosporidium parvum associated with a diarrhoea outbreak identifies two sources of exposure.

An outbreak of cryptosporidiosis associated with exposure to outdoor swimming-pool water affected an estimated 800-1000 individuals. PCR products were obtained from faecal specimens from 30 individuals who tested positive for Cryptosporidium oocysts. RFLP and sequencing analyses showed that all individuals were infected with Cryptosporidium parvum. Among the infected individuals, five had just swum in an adjacent indoor pool during the same period, and had no identified contact with individuals linked to the outdoor pool. With the use of subgenotyping based on analysis of three mini- and microsatellite loci, MS1, TP14, and GP15, we could identify two sources of exposure. One subtype was associated with the outdoor pool and another with the indoor pool. These data demonstrate that the use of mini- and microsatellite loci as markers for molecular fingerprinting of C. parvum isolates are valuable in the epidemiological investigation of outbreaks.

Evaluation of Toluidine Blue O staining for the diagnosis of Pneumocystis jiroveci in expectorated sputum sample and bronchoalveolar lavage from HIV-infected patients in a tertiary care referral center in Ethiopia.

BACKGROUND: Pneumocystis pneumonia (PCP) is becoming increasingly recognized in sub-Saharan Africa. The currently recommended diagnostic methods using induced sputum (IS) and bronchoalveolar lavage (BAL) are neither technically feasible nor affordable for a wider clinical use in developing countries. Therefore, there is a need for a simple and affordable diagnostic test. METHODS: The yield of Toluidine Blue O (TBO) stain, immunofluorescence (IF), and polymerase chain reaction (PCR) for the diagnosis of Pneumocystis jiroveci were compared in 78 expectorated sputum and 118 BAL samples of 131 HIV-infected patients presenting with atypical chest X-ray and sputum smear-negative for acid-fast bacilli. RESULTS: A total of 56 (42.7%) patients tested positive for P. jiroveci by PCR, 39 (29.4%) by IF, and 28 (21.4%) by TBO stain. The sensitivity of TBO as compared to IF and PCR was 71.4% and 34.5% in expectorated sputum and 68% and 41.5% in BAL samples, respectively, with a specificity approaching 100% in both. The sputum PCR showed high concordance rate with BAL PCR. The sensitivity and specificity of sputum PCR as compared to BALPCR was 78.9% and 89%, respectively. In both TBO and IF positive BAL samples, majority were from patients who could not produce sputum (p<0.001). The density of P. jiroveci clusters in BAL samples did not correlate with prior co-trimoxazole use, immunologic status of the patient or overall mortality. CONCLUSION: Compared to IF, TBO staining has an acceptable sensitivity and very high specificity both in expectorated sputum and BAL samples. Expectorated sputum is, therefore, the most practical specimen and TBO staining an inexpensive diagnostic method to be recommended for high-HIV, resource-constrained settings. Bronchoscopy for the diagnosis of PCP is often not required for patients who can produce sputum. For patients who cannot produce sputum, however, the cost and efficacy of TBO in IS sample needs to be investigated in resource-poor countries.