Comparison of most probable number-PCR and most probable number-foci detection method for quantifying infectious Cryptosporidium parvum oocysts in environmental samples

Microbial contamination of public water supplies is of significant concern, as numerous outbreaks, including Cryptosporidium, have been reported worldwide. Detection and enumeration of Cryptosporidium parvum oocysts in water supplies is important for the prevention of future cryptosporidiosis outbreaks. In addition to not identifying the oocyst species, the U.S. EPA Method 1622 does not provide information on oocyst viability or infectivity. As such, current detection strategies have been coupled with in vitro culture methods to assess oocyst infectivity. In this study, a most probable number (MPN) method was coupled with PCR (MPN-PCR) to quantify the number of infectious oocysts recovered from seeded raw water concentrates. The frequency of positive MPN-PCR results decreased as the oocyst numbers decreased. Similar results were observed when MPN was coupled to the foci detection method (MPN-FDM), which was done for comparison. For both methods, infectious oocysts were not detected below 10(3) seeded oocysts and the MPN-PCR and MPN-FDM estimates for each seed dose were generally within one-log unit of directly enumerated foci of infection. MPN-PCR estimates were 0.25, 0.54, 0 and 0.66 log(10) units higher than MPN-FDM estimates for the positive control, 10(5), 10(4) and 10(3) seed doses, respectively. The results show the MPN-PCR was the better method for the detection of infectious C. parvum oocysts in environmental water samples.     

Influence of temperature on Cryptosporidium parvum oocyst infectivity in river water samples as detected by tissue culture assay

Cryptosporidium parvum oocysts were stored in 1-ml aliquots of filtered river water at -20, 4, 10, and 21-23 C in the dark. Oocysts were also added to filter-sterilized river water samples and stored at 21-23 C. The infectivity of oocysts stored under different conditions was assayed at weekly intervals through infection of human adenocarcinoma ileocecal (HCT-8) cell monolayers. Wells containing between 10 and 100 foci of infection were enumerated by immunofluorescent microscopy, and the number of infective oocysts was calculated. No infectious oocysts were detected after 1 wk at -20 C. The number of infective oocysts stored at 4 C decreased 5-fold, and the number of those stored at 10 C decreased 2.5-fold after 14 wk. The infectivity of oocysts stored in potassium dichromate (positive control) at 4 C decreased 2-fold over 14 wk. The number of infective oocysts in filter-sterilized and non-filter-sterilized river water stored at 21-23 C decreased by 3.3 and 2.6 log units, respectively, over 12 wk, and no foci of infection were detected at 14 wk. The results show that as temperature increased from 4 to 23 C, the duration of oocyst infectivity decreased.     

Homologous interference of lymphocytic choriomeningitis virus: detection and measurement of interference focus-forming units.

Lymphocytic choriomeninigitis (LCM) virus defective interfering (DI) particles form foci of protected cells in a monolayer under an agarose-containing overlay medium. Foci originate from one cell dually infected with at least 1 interference focus-forming unit and infectious virus. As a result, an interfering factor is produced and released which interacts with neighboring cells, thereby protecting them against cytopathic lysis by challenge virus. The property of individual LCM virus DI particles to induce countable foci has been made the basis of quantitative assay that is comparable in every respect to the plaque assay of infectious virus and is much more sensitive and probably more accurate than other procedures used to measure LCM virus DI particles. LCM virus was passaged, undiluted, 10 times in cell cultures. When yields were analyzed as to concentrations of PFU and interference focus-forming units, both entities were found to fluctuate with the pattern expected from theoretical considerations.     

Colorimetric focus-forming assay with automated focus counting by image analysis for quantification of infectious hepatitis C virions

Hepatitis C virus (HCV) infection is the leading cause of liver transplantation in Western countries. Studies of HCV infection using cell culture-produced HCV (HCVcc) in vitro systems require quantification of infectious HCV virions, which has conventionally been performed by immunofluorescence-based focus-forming assay with manual foci counting; however, this is a laborious and time-consuming procedure with potentially biased results. In the present study, we established and optimized a method for convenient and objective quantification of HCV virions by colorimetric focus-forming assay with automated focus counting by image analysis. In testing different enzymes and chromogenic substrates, we obtained superior foci development using alkaline phosphatase-conjugated secondary antibody with BCIP/NBT chromogenic substrate. We additionally found that type I collagen coating minimized cell detachment during vigorous washing of the assay plate. After the colorimetric focus-forming assay, the foci number was determined using an ELISpot reader and image analysis software. The foci number and the calculated viral titer determined by this method strongly correlated with those determined by immunofluorescence-based focus-forming assay and manual foci counting. These results indicate that colorimetric focus-forming assay with automated focus counting by image analysis is applicable as a more-efficient and objective method for quantification of infectious HCV virions.     

Quantitation of Cryptosporidium parvum infection in cell culture using a colorimetric in situ hybridization assay

A quantitative colorimetric in situ hybridization assay was developed for detecting Cryptosporidium parvum infection in cell cultures using a digoxigenin-labeled probe targeting 18S rRNA. Intra-cellular developmental stages of C. parvum such as trophozoites and meronts were clearly discerned by light microscopy as localized areas of dark purple/black precipitate against a colorless background. Infections developed focally and the term infectious focus was applied to each cluster of developmental stages. There were no significant differences in the number of infectious foci following 24 h or 48 h incubation. However, 24 h and 48 h dose response curves were significantly different when infectivity was measured as the number of developmental stages per monolayer, with an average of 5.3-fold more stages following 48 h incubation. When infectivity was expressed as the number of infectious foci per inoculum oocyst converted to a percentage, it was demonstrated that the rate of infection decreased with increasing oocyst age. Oocysts of the Iowa isolate that were 7-10 days old demonstrated 7.8+/-2.4% infectivity (mean +/- standard deviation) compared to 4.2+/-0.8% for 21-28 day-old oocysts and 1.4+/-1.3% for 42-70 day-old oocysts. The assay also detected infection with other genotype 2 oocysts and a genoptye 1 isolate. This assay provides a direct quantitative approach for measuring C. parvum infectivity in cell culture.     

Replication of Radiation-Induced Murine Leukemia Virus in Normal and Transformed Mouse Cells

Autonomous radiation-induced leukemia virus (RadLV) replication could be detected in mouse 3T3 cells by the development of interference with murine sarcoma virus (MSV), the appearance of covert helper activity for defective MSV, and by the induction of cytopathic effect type foci in MSV-transformed, leukemia virus-negative (S+L-) cells. A chronic infection of either 3T3 or S+L- cells with RadLV could be established. Both RadLV infectivity and helper activity were demonstrated in the same peak at a buoyant density of 1.16 g/cm(3). Additionally a soluble inhibitor of MSV focus formation was found which could be separated from infectious RadLV. Examination of cell clones derived from chronically infected 3T3 cells showed that essentially every cell was infected and produced both infectious RadLV and low levels of inhibitor. Quantitative comparisons of autonomously replicating RadLV in normal 3T3 and S+L- cells suggested that RadLV may consist of several populations of virus of varying replicative potential. Apparently 99% of RadLV can be assayed only as helper units in normal cells or as replicative units in S+L- cells. To explain the atypical results, a model for RadLV deficiency is proposed.     

Visualization and Detection of Infectious Coxsackievirus Replication Using a Combined Cell Culture-Molecular Beacon Assay

Rapid detection of infectious viruses is of central importance for public health risk assessment. By directly visualizing newly synthesized viral RNA with molecular beacons (MBs), we have developed a generalized method for the rapid and sensitive detection of infectious viruses from cell culture. An MB, CVB1, specifically targeting the 5′ noncoding region of the enterovirus genome was designed and synthesized. Introduction of MB CVB1 into permeabilized cells highly infected with coxsackievirus B6 resulted in brightly fluorescent cells that can be easily visualized with a fluorescence microscope. In contrast, no detectable signal was observed with noninfected cells or with nonspecific MBs. The number of fluorescent cells also increased in a dose-responsive manner, enabling the direct quantification of infectious viral dosages by direct counting of fluorescent foci. As little as 1 PFU of infectious coxsackievirus B6 was detected within 6 h postinfection. When combined with nuclease-resistant MBs, this method could be useful not only for the real-time detection of infectious viruses but is also useful to study the life cycle of viral processing in vivo.     

Visualization and detection of infectious coxsackievirus replication using a combined cell culture-molecular beacon assay

Rapid detection of infectious viruses is of central importance for public health risk assessment. By directly visualizing newly synthesized viral RNA with molecular beacons (MBs), we have developed a generalized method for the rapid and sensitive detection of infectious viruses from cell culture. An MB, CVB1, specifically targeting the 5' noncoding region of the enterovirus genome was designed and synthesized. Introduction of MB CVB1 into permeabilized cells highly infected with coxsackievirus B6 resulted in brightly fluorescent cells that can be easily visualized with a fluorescence microscope. In contrast, no detectable signal was observed with noninfected cells or with nonspecific MBs. The number of fluorescent cells also increased in a dose-responsive manner, enabling the direct quantification of infectious viral dosages by direct counting of fluorescent foci. As little as 1 PFU of infectious coxsackievirus B6 was detected within 6 h postinfection. When combined with nuclease-resistant MBs, this method could be useful not only for the real-time detection of infectious viruses but is also useful to study the life cycle of viral processing in vivo.     

Improving the rate of infectivity of Cryptosporidium parvum oocysts in cell culture using centrifugation.

Centrifugation was evaluated as a method to improve infectivity assays of Cryptosporidium parvum in cell culture using the focus detection method, an immunofluorescence-based method for detecting infectious C. parvum oocysts in vitro. Human ileocecal adenocarcinoma (HCT-8) cells were grown for 48 hr on 13-mm cover slips in 24-well microtiter plates and infected with bleach-treated C. parvum oocysts. Plates were centrifuged at 228 g for 10 min and incubated at 37 C for 5, 12, 18, 24, and 48 hr. Foci of infection were stained by immunofluorescence and enumerated using epifluorescent microscopy. Results were compared to noncentrifuged controls. Foci in centrifuged samples could be enumerated after 18 hr. According to most probable number (MPN) analysis, the number of infectious oocysts estimated at 48 hr (13,326 infectious oocysts) was reached by 18 hr in centrifuged samples. After 48 hr, there was no significant difference (P < 0.05) between centrifuged and noncentrifuged samples enumerated by number of foci or the MPN of infectious oocysts. Centrifugation may expedite detection during C. parvum infectivity assays. Furthermore, multiwell plate formats are more cost effective than traditional chamber slides.     

Adenovirus Type 12-Rat Embryo Transformation System

Adenovirus type 12 (Huie) inoculated into cultures of primary whole rat embryo produced foci of morphologically altered cells. The number and identification of these transformed areas was dependent upon the calcium concentration of the medium; more foci appeared in 0.1 mm than in 1.8 mm calcium. Cell lines derived from these inoculated cultures did not yield infectious virus, and also were similar to cell lines derived from adenovirus type 12-induced tumors with respect to morphology, presence of virus-specific tumor antigen, and oncogenicity. Dose-response curves revealed that transformation of rat embryo cells by adenovirus type 12 followed one-hit kinetics, and that approximately 7 x 10(5) infectious virus particles were required for one transformation event. Our results indicate that the transformation system described for adenovirus type 12 is reproducible, and that previous difficulties experienced in developing such a system may well be explained by the higher calcium concentration of the tissue culture media used.     

Hold everything! Holding policies for protecting plasma supplies

In spite of advances in testing technologies for detecting infections such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV), occasionally blood or plasma is collected that is potentially infectious, but is not detected as such by existing screening tests. We consider the effect of a holding policy for further reducing the number of potentially infectious units that are released for fractionation. The policy dictates a holding period during which all donated units are stored. If a donor tests positive for the infection in question at a subsequent donation, then all of that donor's units currently in storage are discarded. Otherwise, donated units are released at the end of the holding period. In the case of a single disease, we determine optimal holding periods as well as policies that are as effective as the best screening tests currently available.     

Radiologic diagnosis of bone metastases recurrence in patients with prostate cancer treated strontium-89

Aiming to evaluate efficiency of 89SrCl (Metastron) in patients with metastatic lesion of the skeleton in prostate cancer we have performed a follow-up scintigraphy of the skeleton with 99mTc-methylendiphosphonate (MDP) and MRI with quantitative study of metastatic foci. 12 patients with prostate cancer (on the average 11 +/- 6 bone metastases were examined using scintigraphy of the skeleton with 99mTc-MDP and MRI study in T1, T2 and proton density modes. Investigations were performed before injected as a single dose of 150 MBq (4 mCi). At all the stages there was made a quantitative study of foci of pathological uptake of 99mTc-MDP compromising numbers of foci, focus parameters, intensity of 99mTc-MDP accumulation in the pathological part relatively the contralateral region as well as quantification of MRI signals from metastatic areas in signal intensity units. In 3 month 4 patients with extensive metastatic skeletal lesion (> 12) showed a considerable decrease of number of foci of pathological 99mTc-MDP uptake (on average to 6 +/- 3). In the remained metastatic foci there was noted a decrease of dimensions and 99mTc-MDP uptake intensity at an average by 29.8 +/- 15%, improvement in T1 intensity by 113 +/- 55.6 units. In 2 patients who initially presented a "superscan" pattern on 99mTc-MDP bone scintigraphy the 89SrCl treatment converted this of low intensity had demonstrated their complete regression. Results of radiologic follow-up of bone metastases in prostate cancer using MRI and bone scintigraphy with 99mTc-MDP argue that systemic radiotherapy with 89SrCl induces significant regress of metastatic process that involves all volume of the metastases.     

Clinico-epidemiologic characteristics of infectious hepatitis in a populated area where hepatitis had not been reported for a long time

The analysis of the clinico-epidemiological characteristic of infectious hepatitis in a populated area in the Kashkadarya region of the Uzbek SSR, where the cases of infectious hepatitis were not formerly registered for a long time, is presented. The epidemic process was found to take and intensive course with the formation of a large number of foci with 2 or more patients, an in the age groups of 20-29, 40 years and older a comparatively high morbidity rate, exceeding that among the total population 2-3 times, was registered. A low proportion of patients under 14 years (21.1%) was probably due to the planned preventive gamma globulin immunization carried out in this area.     

Novel probiotic Bifidobacterium longum subsp. infantis CECT 7210 strain active against rotavirus infections

Rotavirus is the leading cause of severe acute gastroenteritis among children worldwide. It is well known that breast-feeding and vaccination afford infants protection. Since breast-feeding has drastically decreased in developed countries, efforts have been focused on the potential use of probiotics as preventive agents. In this study, a novel Bifidobacterium longum subsp. infantis strain was isolated from infant feces and selected, based on its capacity to inhibit in vitro rotavirus Wa replication (up to 36.05% infectious foci reduction) and also to protect cells from virus infection (up to 48.50% infectious foci reduction) in both MA-104 and HT-29 cell lines. Furthermore, studies using a BALB/c mouse model have proved that this strain provides preliminary in vivo protection against rotavirus infection. The strain has been deposited in the Spanish Type Culture Collection under the accession number CECT 7210. This novel strain has the main properties required of a probiotic, such as resistance to gastrointestinal juices, biliary salts, NaCl, and low pH, as well as adhesion to intestinal mucus and sensitivity to antibiotics. The food safety status has been confirmed by the absence of undesirable metabolite production and in acute ingestion studies of mice. Overall, these results demonstrate that Bifidobacterium longum subsp. infantis CECT 7210 can be considered a probiotic able to inhibit rotavirus infection.     

Specificity of Agrobacterium-mediated delivery of maize streak virus DNA to members of the Gramineae

Parameters affecting the efficiency of agroinfection of maize streak virus (MSV) in maize have been determined. Monomeric units, cloned at a number of sites in the MSV genome were not infectious but multimeric units containing partial duplications were equally as infectious as complete tandem dimeric clones. Inoculation of tandem dimeric units conjugated into different strains of Agrobacterium showed that both A. tumefaciens and A. rhizogenes were able to transfer DNA to maize and this ability was Ti (or Ri) plasmid-specific. Nopaline strains of A. tumefaciens and both agropine and mannopine A. rhizogenes strains efficiently transferred MSV DNA to maize. A number of strains were capable of MSV DNA transfer to other members of the Gramineae, providing information which may be essential for Agrobacterium-mediated transformation of monocotyledonous plants.     

Technetium-99m-labeled ceftizoxime loaded long-circulating and pH-sensitive liposomes used to identify osteomyelitis

Osteomyelitis is an infectious disease located in the bone or bone marrow. Long-circulating and pH-sensitive liposomes containing a technetium-99m-labeled antibiotic, ceftizoxime, (SpHL-(99m)Tc-CF) were developed to identify osteomyelitis foci. Biodistribution studies and scintigraphic images of bone infection or non infection-bearing rats that had been treated with these liposomes were performed. A high accumulation in infectious foci and high values in the target-non target ratio could be observed. These results indicate the potential of SpHL-(99m)Tc-CF as a potential agent for the diagnosis of bone infections.     

Quantification of Shiga toxin 2‐encoding bacteriophages,by real‐time PCR and correlation with phage infectivity

Aims: To evaluate a qPCR‐based protocol for the enumeration of Shiga toxin (Stx) 2 phages and to compare the results of qPCR with the number of infective Stx phage particles. Methods and Results: An approach based on qPCR was applied to count Stx phages in five phage lysates of known titre. The number of viral particles from each phage lysate was determined by electron microscopy using latex spheres. The infectivity of the Stx phages was evaluated onto three bacterial host strains, by double agar layer assay and plaque blot hybridization. The number of phage particles detected by electron microscopy correlates with the number calculated by qPCR in all the phages assayed. The number of infectious phages was from 1 to 3 log₁₀ units below the numbers obtained by qPCR and electron microscopy. Conclusions: The approach allows accurate quantification of Stx phages with a high recovery. The number of infectious phages is always below the number of phage particles detected by qPCR. Significance and Impact of the Study: The qPCR method is a good approach to enumerate Stx phages. However, these results should be carefully considered when related to the number of infectious phages for each lysate that could be applied in real samples, because values of infectious particles are always below the number of Stx phages detected by qPCR.     

Certain data concerning the analysis of the foci of infectious hepatitis in cases of subsequent occurrence of the disease in families

Materials of epidemiological analysis of familial infectious hepatitis nidality in 104 families are presented. The data obtained pointed to the preponderant affection of children constituting 97.1% of primary and 87.7% of subsequent cases in familial foci. Infection was brought to the families by schoolchildren in 57.7% of the cases, and by organized preschoolchildren--in 31.7% of the cases. The greatest affection of schoolchildren aged from 7 to 14 years in the foci with subsequent occurrence of the disease (77.7%) was explained by their marked age activity. This led to the conclusion that mass prophylaxis with alpha-globulin of these particular groups of children was of primary importance. The differences of the specific ratio of adults who contracted the disease in familial foci and in the region as a whole pointed to the possibility of infection of adults outside the family and on the prevalence in them of serum hepatitis.     

Quantitative Characteristics of the Transformation of Hamster Cells by PARA (Defective Simian Virus 40)-Adenovirus 7

An in vitro method for the quantitative measurement of transformation in hamster embryo fibroblasts by the PARA [defective simian virus 40 (SV40)]-adenovirus 7 hybrid has been developed. Transformation by PARA particles followed one-hit kinetics with a ratio of 1 focus-forming unit per 250 plaque-forming units. The method of viral adsorption had a direct effect upon the total number of foci which developed but not on the quantitative aspects of this assay. A fluorescent-focus assay was developed which provided a direct correlation of the observed morphological transformation and the presence of the PARA genome. This fluorescent-focus assay utilized detection of the SV40 tumor antigen, which was present in all foci transformed by PARA. Single foci induced by PARA were isolated and grown into cell lines. Two types of foci were observed and isolated; the first contained cells having a cuboidal or SV40-type morphology, and the second consisted of epithelial or adenovirus-type transformed cells. Both types contained the SV40 tumor and SV40 surface antigens as determined by the indirect fluorescence technique; however, only the epithelial cells contained the adenovirus 7 tumor antigen. All five cell lines which were injected into weanling Syrian hamsters were found to be oncogenic. These cell lines induced antibodies to both SV40 and adenovirus 7 tumor antigens in tumor-bearing animals.     

Qualitative and quantitative analysis of phosphorylated ATM foci induced by low-dose ionizing radiation

We examined the formation of phosphorylated ataxia telangiectasia mutated (ATM) foci in exponentially growing normal human diploid cells exposed to low doses of X rays. Phosphorylated ATM foci were detected immediately after irradiation, and the number of foci decreased as the time after irradiation increased. The kinetics of phosphorylated ATM foci was comparable to that of phosphorylated histone H2AX. We found that there were fewer spontaneous phosphorylated ATM foci than that phosphorylated histone H2AX foci. Notably, significant numbers of phosphorylated histone H2AX foci, but not phosphorylated ATM foci, were detected in the S-phase cells. The induction of foci showed a linear dose-response relationship with doses ranging for 10 mGy to 1 Gy, and the average number of phosphorylated ATM foci per gray was approximately 50. The average size of the foci was comparable for the cells irradiated with 20 mGy and 1 Gy, and there was no significant difference in the kinetics of disappearance of foci, indicating that DNA double-strand breaks are similarly recognized by DNA damage checkpoints and are repaired irrespective of the dose.