ABSTRACT
Objective To understand the prevalence of Cryptosporidium infection in diarrhea infants under 2 years old in Wuhan City,so as to provide the epidemiological evidence for the prevention and treatment of cryptosporidiosis. Methods The fecal samples from infants under 2 years old with diarrhea were collected in Hubei General Hospital and Central South Hospital in Wuhan City,Hubei Province from August 2014 to July 2015. The fecal samples were stored in 2.5%potassium dichromate at 4℃after filtered. The DNA was extracted from the fecal pellets with the phenol-chloroform method. The Cryptosporidium species were detected by a nested PCR assay targeting the SSU rRNA gene of the parasite. All the positive PCR products were sequenced on ABI 3100 automated sequencer,and the amplified sequences were compared to homologous sequences in the NCBI database by using the Basic Local Alignment Search Tool(BLAST). Phylogenetic analyses were performed by using the software MEGA (version 4.0)based on the Neighbour-Joining method. Results The human stool specimens(n=298)were screened for the presence of Cryptosporidium by nested PCR. The infection rate of Cryptosporidium was 3.02%(9/298). The infection rate of Cryp-tosporidium was 5.93%(7/118)in the infants between 1-2 years old,and the infection rate was 1.11%(2/180)in the infants un-der 1 year old,and there was a significant difference between the two groups(χ2 =4.13,P<0.05). The nine samples which were positive by nested PCR were successfully sequenced and compared with the reference sequences in GenBank. The results revealed the nine positive specimens were all infected with C. parvum,and two of them were co-infected with C. hominis. Neigh-bor-joining trees were constructed from the aligned partial SSU rRNA sequences of these nine isolates,and in the SSU rRNA lo-cus,the nine isolates were grouped with C. parvum. Conclusion There exists Cryptosporidium infection in the infants under 2 years old with diarrhea in Wuhan City,and the main species of Cryptosporidium is C. parvum.
ABSTRACT
Background: Cryptosporidiosis is one of the most difficult protozoan infections to treat, with only two drugs i.e. nitazoxanide and paromomycin known for treatment with variable response in different patients. Human cryptosporidiosis is accounted mainly by C. hominis and C. parvum. These two species or their subtypes are known to differ in clinical manifestations, and may differ in their response to drugs. So, we planned the study to see the effect of nitazoxanide and paromomycin on different isolates of Cryptosporidium in vitro. Methods: MDCK cell lines were used for in vitro growth of parasite and cytotoxicity of drugs to MDCK cells was determined by MTT assay after 3, 12 and 24 hours of drug exposure. Efficacy of non-toxic drug concentrations (<25% cytotoxic) on 12 Cryptosporidium isolates (7 C. hominis and 5 C. parvum) was determined at three different life cycle stages (in vitro growth, invasion and oocyst) by quantitative RT-PCR. Unpaired t-test was used to calculate the difference response of Cryptosporidium isolates to nitazoxanide and paromomycin. Results and conclusions: Cytotoxicity of nitazoxanide and paromomycin increased in dose and time dependent manner. After 24 hours of drug exposure, >25% cytotoxic effect was seen with nitazoxanide and paromomycin at concentrations of more than, 25μg/ml and 6mg/ml, respectively. Nitazoxanide was more effective than paromomycin in decreasing in vitro growth, invasion inhibition and reducing oocyst viability of Cryptosporidium isolates. Drugs effect was higher on growth inhibition followed by invasion inhibition and least in decreasing oocyst viability. Different isolates had variable response to drugs; cumulatively C. parvum isolates were more susceptible at particular drug concentrations than C. hominis isolates.
ABSTRACT
Objective: Genetical characterization of human Cryptosporidium isolates to determine species diversity. Patients and Methods: A cross-sectional study in Valparaiso, Chile, was performed. A total of 458 patients participated in the study: 259 immunodeficient (HIV, cancer, renal transplant hyper-IgM syndrome, HIV and unintended pregnancy) and 178 immunocompetent individuals provided stool samples and 21 patients bile samples. Results: We obtained 29 (6.3%) positive samples. 25 (9.7%) derived from immunodeficient patients: 18 (7.3%) from HIV patients and 7 from patients with other immunodeficiencies. The remaining 4 (2.2%) samples originated from immunocompetent individuals. Cryptosporidium genotyping was performed by nested polymerase chain reaction (PCR) and restriction fragments length polymorphism and/or PCR followed by sequencing of the SSU rRNA from oocysts in stool samples. 4 species were identified: C. parvum, C. hominis, C. muris, and C. meleagridis. In immunodeficient patients, 16 C. parvum, 8 C. hominis, and 1 C. muris strain were identified. In immunocompetent participants, 3 C. hominis and 1 C. meleagridis isolate were found. Conclusion: The results indicate that zoonotic and anthroponotic transmission occurs and that C. parvum is the predominant species in our study population. Cryptosporidium species of zoonotic transmission accounted for 62% of the human infections detected in this study.
Objetivo: Caracterizar genéticamente Cryptosporidium spp para determinar la diversidad de especies en seres humanos. Pacientes y Métodos: estudio transversal realizado en Valparaíso, Chile, Un total de 458 pacientes participaron del estudio; 259 inmunodeficientes (pacientes con infección por VIH, oncológicos, con trasplante renal, síndrome de hiper IgM y una mujer embarazada sin infección por VIH) y 178 inmunocompetentes proporcionaron muestras fecales y 21 muestras de bilis. Resultados: Se obtuvieron 29 (6,3%) muestras positivas; 25 (9,7%) de inmunodeficientes: 18 (7,3%) de pacientes con infección por VIH y 7 con otras inmunodeficiencias; los restantes 4 (2,2%) fueron de personas inmunocompetentes. La genotipificación de Cryptosporidium se efectuó mediante reacción de polimerasa en cadena (RPC) anidada y el polimorfismo de la longitud de los fragmentos de restricción y/o RPC - secuenciación de la SSU ARNr, a partir de ooquistes en la muestra fecal. Se identificaron 4 especies: C. parvum, C. hominis, C. muris y C. meleagridis. En pacientes inmunodeficientes, se caracterizaron 16 C. parvum, 8 C. hominis y un C. muris; en inmunocompetentes: 3 C. hominis y un C. meleagridis. Conclusión: Los resultados indican que se produce transmisión zoonótica y antroponótica y que C. parvum es la especie predominante en este estudio. Las especies de Cryptosporidium de transmisión zoonótica representan el 62% en los seres humanos participantes de este estudio.
Subject(s)
Adolescent , Adult , Aged , Child , Female , Humans , Male , Middle Aged , Pregnancy , Young Adult , Cryptosporidiosis/parasitology , Cryptosporidium/classification , Immunocompetence , Immunocompromised Host , Chile , Cross-Sectional Studies , Cryptosporidium/genetics , Cryptosporidium/isolation & purification , Genotype , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , /geneticsABSTRACT
Cardiobacterium hominis, a fastidious gram-negative bacillus rarely causing endocarditis, was isolated from blood culture of a 41-year-old man with subacute bacterial endocarditis. The patient had undergone an operation of valve replacement 4 years before. Growth of the organism was only detected in 1 of 6 blood cultures after 7 days incubation. The isolate showed typical characteristics of C. hominis, i.e., tear-drop like cells and a weak positive indole reaction. The isolate was susceptible to many antimicrobial agents, but penicillin G and streptomycin therapy failed to cure the disease.