Contribution of real-time PCR to Plasmodium species identification and to clinical decisions: a nationwide study in a non-endemic setting.

Treatment choice for patients with malaria in Israeli hospitals is based on microscopy and rapid diagnostic tests (RDTs). Here, we demonstrate the cumulative value of real-time polymerase chain reaction (PCR) in optimizing the treatment of malaria. Between January 2009 and December 2015, 451 samples from 357 patients were tested in our laboratory using a real-time PCR assay. Hospital laboratory results (without real-time PCR) were compared to those obtained in our laboratory. A total of 307 patients had a malaria-positive laboratory finding in the hospital. Out of those, 288 were confirmed positive and 19 negative using real-time PCR. Two negative hospital results were found to be positive by real-time PCR. More specifically, of 153 cases positive for Plasmodium falciparum by real-time PCR, only 138 (90%) had been correctly identified at the hospitals. Similarly, 66 (67%) of 99 cases positive for P. vivax, 2 (11%) of 18 cases positive for P. ovale, and 3 (30%) of 10 cases positive for P. malariae had been correctly identified. Of 10 cases of mixed infection, only one had been identified as such at the hospital. Thus, real-time PCR was required for correct identification in 81 (28%) out of 290 positive cases. In 52 (18%) of those, there was an erroneous categorization of relapsing versus non-relapsing parasites. In a nationwide study, we found that the use of real-time PCR is definitely beneficial and may change the decision regarding the choice of treatment.

Yersinia enterocolitica Infection Simulating Lymphoproliferative Disease, after Liver Transplant.

We describe a 14-year-old girl, who was 13 y after liver transplantation for biliary atresia with an unremarkable postoperative course. She presented with fever of up to 40°C, extreme fatigue, malaise, anorexia, and occasional vomiting. On physical examination the only finding was splenomegaly. Lab results showed hyperglobulinemia and an elevated sedimentation rate. Liver function tests were normal except for mild elevation of γGTP. Abdominal U/S and CT demonstrated an enlarged spleen with retroperitoneal and mesenteric lymph nodes enlargement. An exhaustive evaluation for infectious causes, autoimmune conditions, and malignancy was negative. A full recovery after 5 months prompted testing for self-limited infectious etiologies. Yersinia enterocolitica infection was diagnosed.

Internal reporting system to improve a pharmacy's medication distribution process.

PURPOSE: The current pharmacy occurrence-reporting system in an institution was reviewed, and an internal procedure that would provide data to improve the medication-use process was developed. SUMMARY: In a rural, 353-bed, tertiary care academic center, the effectiveness of a departmental occurrence-reporting system was determined over a nine-month period to increase occurrence reporting within the pharmacy and allow administrators to identify specific areas for improvement within the medication distribution process. These events were identified according to the number and type of near misses documented by pharmacy staff. The pharmacy staff was asked to complete a survey about the department's current reporting process and what the staff desired in a new occurrence-reporting system. The staff was also surveyed on which steps of the pharmacy's medication distribution process could contribute to the most errors. Initially, a paper-based error-reporting form was developed for all steps of the pharmacy distribution process except pharmacist order entry. Once the paper-based error-reporting form was introduced, the pharmacist order-entry phase of the project was begun. During the evaluation period, 203 pharmacy-dispensing errors were reported to the hospital's error-reporting system. In contrast, 1385 total pharmacy events were documented using the pharmacy's internal occurrence-reporting system. At least 204 of those reported events involved high-alert medications according to the institution's high-alert medications policy. CONCLUSION: A pharmacy internal occurrence-reporting system increased staff reporting and identified areas for improvement within the medication distribution process that may not have been recorded by a hospital-based reporting system.

Is herpes simplex virus associated with pulp/periapical inflammation?

OBJECTIVES: This study focuses on the detection of herpes simplex virus (HSV) DNA in dental pulp and inflamed periapical tissue. STUDY DESIGN: Dental pulp tissue (vital and necrotic) and periapical tissue samples were collected under strictly sterile conditions and examined for the presence of HSV DNA. Saliva samples were also examined for the presence of the viral DNA. The polymerase chain reaction assay was used to detect viral DNA. Blood samples were collected, and the enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies against HSV was carried out. RESULTS: According to the ELISA test, 19 of the 23 blood samples were IgG-positive and IgM-negative to HSV, whereas 4 were IgG-negative and IgM-negative. HSV DNA was not detected in the tissue and the saliva samples tested. CONCLUSION: HSV is not present and therefore is probably not involved in the pathology of tooth neural tissue.

Immunologic response to a single dose of tetanus toxoid in older people.

OBJECTIVES: Several studies have demonstrated that a large percentage of older people are inadequately immunized against tetanus. The aim of this study was to assess the immunity against tetanus in a group of individuals aged 69 and older and to examine the immune response to a single dose of tetanus toxoid. DESIGN: A convenience sample of 115 residents of a large retirement home, aged 69 and older, was studied. After a blood sample for anti-tetanus antibody titer, a single dose of tetanus toxoid vaccine was administered. Repeat titers were obtained 6 weeks after the vaccination and analyzed by ELISA assay. Antibody levels equal to or greater than 0.1 IU/mL were considered protective. RESULTS: Sixty-seven of 115 (58.3%) individuals had adequate antibody titers. Those individuals who reported having been vaccinated with tetanus toxoid in the past were more likely to be immunized adequately compared with those who reported having never been vaccinated (66.7% vs 39.3%, P = .02). After vaccination, 34 of 46 (73.9%) individuals with inadequate antibody titers became seropositive. Those who remained seronegative had mean prevaccination antibody titers significantly lower than those who seroconverted. Sixteen of 17 (94.1%) persons who reported having been vaccinated in the past and were found to be seronegative developed adequate antibody titers following vaccination, compared with only nine of 16 (56.2%) who reported never having been vaccinated (P = .04). There was no association between seroconversion rate and age, sex, underlying diseases, and army service. CONCLUSIONS: Most individuals will develop an adequate anti-tetanus antibody titer following administration of a single dose of tetanus vaccine. A history of past immunization is a good predictor of becoming adequately immunized. It is important that physicians follow the current recommendations for adult immunization and initiate campaigns to ensure that the older population is protected against tetanus.

Pertussis infection in fully vaccinated children in day-care centers, Israel.

We tested 46 fully vaccinated children in two day-care centers in Israel who were exposed to a fatal case of pertussis infection. Only two of five children who tested positive for Bordetella pertussis met the World Health Organization's case definition for pertussis. Vaccinated children may be asymptomatic reservoirs for infection.

[Diphtheria in a highly immunized population].

Although diphtheria vaccination is routine world-wide, outbreaks of the disease continue to occur in supposedly vaccinated populations. The incidence of diphtheria in Israel is very low, with only 3 cases reported in the past 24 years (all in unvaccinated children). In 2 of the 3 an asymptomatic carrier was identified among the patients' close contacts, presumable the source of the infection. We describe a recent case of diphtheria. It is important for physicians to be aware of the possibility of diphtheria occurring despite the high rate of vaccination in our population.

Oxidative interaction of unpaired hemoglobin chains with lipids and proteins: a key for modified serum lipoproteins in thalassemia.

We searched for a biochemical explanation to the modification of lipoproteins like low-density lipoproteins (LDL) observed in patients with the severe hemolytic anemia beta-thalassemia. Because a large fraction of the LDL surface is composed of phospholipids, we first explored the possible involvement of phospholipids in the oxidative interaction of LDL with hemoglobin (Hb), using brain extract phospholipid liposomes as a model. The relative binding affinity and oxidative interaction of three hemoglobin variants (intact Hb A and isolated beta- and alpha-chains) with LDL and liposome were compared. Studies carried out at low pH/ionic strength and under physiological conditions revealed that association of hemoglobin variants with the phospholipid liposomes is driven by electrostatic forces but their binding is not a prerequisite for oxidative interaction. Unlike phospholipid liposomes, LDL underwent only a negligible association with the Hb variants under all pH/ionic strength conditions. Nevertheless, LDL induced oxidation of Hb variants, mostly alpha-chains. The dissimilar behavior of the liposomes and LDL indicated that LDL protein apo B rather than phospholipids is the actual LDL surface component which interacts with the hemoglobin variants. This agrees with the finding that apo B protein underwent oxidative crosslinking by the hemoglobin variants among which alpha-chains were most active. We concluded from these results that the ability of hemoglobin to undergo autooxidation is the key to its oxidative reactivity toward LDL. The results of the present study indicate that the modified LDL particles observed in beta-thalassemia may reflect lipoprotein oxidation by alpha-chains in circulation.

Denaturing interaction between sickle hemoglobin and phosphatidylserine liposomes.

It is hypothesized that abnormal interaction between sickle hemoglobin (HbS) and erythrocyte membrane lipid might promote deposition of denatured hemoglobin (hemichrome) on the membrane. We compared the interaction of HbS and normal HbA with large unilamellar phosphatidylserine (PS) liposomes under low salt/pH conditions. Admixture of oxyHb and dioleoyl-PS resulted in loss of absorbance at 412 nm, the apparent first order rate constant for which was .25 +/- 0.02 hour-1 for HbA and .85 +/- 0.18 hour-1 for HbS. This was ascribable largely to formation of metHb and hemichromes and was accompanied by some actual transfer of heme from hemoglobin to lipid phase. By comparison, admixture of oxyHb with liposomes made from bovine brain PS having unsaturated acyl chains promoted even faster absorbance loss if the starting liposomal material contained detectable peroxidation by-product. In such cases, actual heme destruction developed with accompanying liberation of free iron and promotion of lipidperoxidation. Fluorescence quenching experiments indicate that hemoglobin/lipid interaction is characterized by very rapid initial electrostatic interaction, followed by development of irreversible changes. Similar changes still occur under conditions of physiologic salt/pH, but they develop much more slowly. The 3.4-fold faster oxidation of HbS versus HbA on lipid observed here represents an additional augmentation of the disparity in oxidation rates for hemoglobins in solution (1.7-fold faster for HbS than for HbA) observed previously. The accelerated promotion of Hb denaturation resulting from lipid contact may help explain deposits of hemichrome on sickle red blood cell membranes, particularly because these cells are in double jeopardy by virtue of having both the mutant HbS and abnormal amounts of peroxidized membrane lipid.

Is hemin responsible for the susceptibility of Plasmodia to oxidant stress?

Based on the unusually high and stage-dependent susceptibility of Plasmodia to oxidant stress it has been proposed that during parasite development, increasing levels of redox-active forms of iron are gradually released. The purpose of this study was to examine this proposal by using an assay monitoring the levels of available forms of iron for redox reactions. Ascorbate-driven and iron-mediated degradation of adventitious DNA served as the basis for this functional assay. Incubation of DNA with lysate from infected RBC caused massive degradation, which was dose, time- and parasite-stage dependent. In contrast, lysate from non-infected RBC did not induce DNA degradation. Likewise, lysate only from infected RBC enhanced the aerobic oxidation of ascorbate. These effects on both reaction, DNA degradation and ascorbate oxidation, could be reconstructed with hemin, instead of lysate. Also, chelators exerted similar effects on both reactions. The results suggest that increased levels of redox-active forms of iron are liberated during parasite development. We propose that hemin or hemin-like structures are the appropriate candidates which could catalyze oxidative stress and deregulate the delicate redox balance of the host-parasite system.

The effects of ascorbate-induced free radicals on Plasmodium falciparum.

Ascorbic acid has been shown to cause stage-dependent effects on the in vitro development of Plasmodium falciparum. While vitamin C marginally enhanced the development of young parasites, it proved highly destructive to the advanced forms. The present study evaluates the mechanisms by which vitamin C affects the parasite. The treatment of parasitized erythrocytes with ascorbate resulted in the conversion of added salicylate to dihydroxybenzoate products, indicating the involvement of hydroxyl radicals. There was a stage specific sensitivity, increasing conversion with progressing parasite development. This specificity could not be attributed to the altered uptake of salicylate by the parasitized erythrocyte, since salicylate uptake was similar in either parasitized or non-parasitized erythrocytes. In distinction, increased uptake of ascorbate by parasitized erythrocytes could account for an elevated oxidant stress. The treatment with ascorbate also caused the oxidation of hemoglobin to methemoglobin and the peroxidation of membrane lipids. Added catalase markedly inhibited the ascorbate-induced effects on parasite development. "Free" plasmodia were also vulnerable to treatment with ascorbate like the parasites within their host cells. These results are in accord with a free radical mechanism of damage to the infected erythrocytes. During the growth of P. falciparum the infected erythrocytes release increasing levels of iron-containing structures that are redox-active and can catalyze the formation of highly reactive oxygen derived species. The findings also indicate the multiplicity of the mode of action of ascorbate on the host-parasite system.

Correlation between destruction of malarial parasites by polymorphonuclear leucocytes and oxidative stress.

The role of reactive oxygen species (ROS) generated by polymorphonuclear leucocytes (PMNs) in the host response against malaria was investigated. Non-activated human PMNs were added to cultures of P. falciparum in microtitre cells. Parasite viability was evaluated by the incorporation of radioactive hypoxanthine. Using PMN/RBC = 1/150 (starting parasitemia was 1%) the incorporation on the second day in culture was only 61% of the control cultures. An effect could be observed already after two hours of incubation (30% reduction at a 1/50 PMN/RBC ratio). A direct contact between the effector and target cells was obligatory for the expression of the damage. Parasites within G6PD-deficient erythrocytes were more sensitive to the PMNs than normal parasitized erythrocytes. This difference could be attributed to the production of reactive oxygen intermediates in the experimental system, since G6PD-deficient erythrocytes are generally more sensitive to oxidant stress. Salicylic acid was used as a scavenger and reporter molecule for hydroxyl radical fluxes. It is converted to the corresponding dihydroxybenzoic acid derivatives, which could be detected by HPLC. Uninfected NRBC or parasitized erythrocytes containing young ring forms could trigger the PMNs to produce much less ROS than the mature forms of the parasites. Other factors associated with PMNs may inactivate the parasites, such as phagocytosis, lysosomal enzymes or degradation toxic products of the PMNs. However our results indicate that increased oxidative stress induced by PMNs interfere with the growth of P. falciparum and could play a role in human evolution of abnormal erythrocytes.

The effect of free radicals induced by paraquat and copper on the in vitro development of Plasmodium falciparum.

The role of transition metals in paraquat toxicity was studied in cultures of Plasmodium falciparum. We showed that addition of copper led to an enhancement of the plasmodium killing, whereas addition of chelating agents, such as desferrioxamine and diethylenetriamine pentaacetic acid markedly reduced the toxic effects. Parsitized G6PD deficient erythrocytes were more sensitive than parasitized normal erythrocytes to copper and to the combination of copper and paraquat.

The survival of Plasmodium Under oxidant stress.

Oxidant stress is associated with the generation of reactive oxygen-derived species, which are considered as the ultimate agents responsible for the damage of a variety of cellular components. Transition metals such as iron ions serve as catalytic centers for the repeated conversion of superoxide radicals or ascorbate to the highly reactive and deleterious hydroxyl radicals and, indeed, increasing amounts of redox-active iron become available during plasmodial development within the parasitized erythrocytes. Thus, the survival of an intracellular parasite depends on the delicate balance of oxidant stress and defense mechanisms. This balance is continuously changing and the parasite must cope with increasing oxidant stress and the decline of protective capacity.

Induction of oxidant stress by iron available in advanced forms of Plasmodium falciparum.

Oxidative stress has been incriminated as a deleterious factor in the development of malaria parasites. Various chemical reductones which can undergo cyclic oxidation and reduction, such as ascorbate have been shown to cause oxidative stress to red blood cells. This, naturally-occurring and redox-active compound, can induce the formation of active oxygen derived species, such as superoxide radicals (.O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH.). The formation of the hydroxyl radical, the ultimate deleterious species, is mediated by the redox-active and available transition metals iron and copper in the Haber-Weiss reaction. During the development of the parasite, hemoglobin is progressively digested and a concurrent release of high levels of iron-containing breakdown products takes place within the red blood cell. Indications for the progressive increase in redox-active iron during the growth of P. falciparum have been recently found in our lab: a) adventitious ascorbate proved highly detrimental to the parasite when added to the mature forms. In contrast, if the parasitized erythrocytes were in the early phase following invasion, and only low levels of iron-containing structures had been liberated, then the observed effect was a small promotion of parasite development. b) erythrocytes containing mature parasites were more potent than erythrocytes containing ring forms as a source for redox-active iron in the ascorbate-driven metal-mediated degradation of DNA. The addition of extracts from parasitized erythrocytes and ascorbate to DNA caused a dose and time dependent DNA degradation. Non-infected erythrocytes had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Deleterious synergistic effects of ascorbate and copper on the development of Plasmodium falciparum: an in vitro study in normal and in G6PD-deficient erythrocytes.

The effects of ascorbate and copper on the development of Plasmodium falciparum were studied in two modes: pretreatment of uninfected erythrocytes followed by infection by P. falciparum and treatment of parasitized erythrocytes. Pretreatment of G6PD(+) cells with ascorbate caused a slight enhancement in parasite development, while in G6PD(-) cells a suppressive effect on the plasmodia was demonstrated. Copper alone interfered with parasite growth in both cell types. The combination of copper and ascorbate arrested parasite maturation, an effect which was more pronounced in G6PD(-) cells. Synergism between copper and ascorbate was better demonstrated following the treatment of infected erythrocytes: while ascorbate alone supported parasite development and copper alone had only a marginal suppressive effect, the combination of copper and ascorbate yielded a marked inhibition of parasite growth. Ascorbate proved destructive to the parasites in the presence of adventitious copper, or on the second day of the parasite life cycle. In these cases it acted as a pro-oxidant, while in other systems, in particular in the presence of a chelator, ascorbate acted as an antioxidant and promoted parasite growth. The understanding of the role of transition metals and free radicals in parasite development and injury could shed light on novel approaches to fight malaria.

Comparison of methods for rotavirus detection in water and results of a survey of Jerusalem wastewater.

Methods for the detection of viable rotaviruses and rotavirus antigen in water were developed and compared. The methods included laboratory-developed enzyme-linked immunosorbent assays (ELISAs) with chromogenic and luminescent substrates, commercial Rotazyme and Enzygnost ELISAs, and an indirect immunofluorescent assay. Of the methods tested, the immunofluorescent assay and the Enzygnost ELISA were the most sensitive for the simian rotavirus SA-11. All of the methods were positive for human rotavirus from clinical specimens. Seeded SA-11 rotavirus was concentrated from water by absorption to and elution from Zeta Plus filters followed by organic flocculation. Interference with the assays by components of the wastewater concentrates was minimal for the ELISAs, although the undiluted organic flocs were cytotoxic for the immunofluorescent assay. A survey of Jerusalem wastewater was carried out over the course of 1 year, and samples were assayed for rotaviruses and enteroviruses. Although enteroviruses were found in almost all of the samples, all samples were negative for rotaviruses. The concentration of rotaviruses in the wastewater was thus below the detection limit of the method used.

Early acquisition of cytomegalovirus antibodies in communal settlements (kibbutzim) in Israel.

The kibbutzim (communal settlements) in Israel are unique societies. Although socioeconomic status is above average and hygienic conditions are usually excellent, infants in these communities living together in nurseries acquire infection with cytomegalovirus and Epstein-Barr virus at an early age. We have studied the occurrence of specific immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to cytomegalovirus and Epstein-Barr virus in a kibbutz in northern Israel where many of the infants and young children, living in adjacent nurseries, have suffered for weeks or months from respiratory tract illnesses. Evidence of recent or past infection with cytomegalovirus was found in all children younger than 24 months of age, while most of them did not have antibodies to Epstein-Barr virus. The latter were found in 45% of children aged 3-12 years. Similar evidence suggesting earlier acquisition of cytomegalovirus infection and immunity than of Epstein-Barr virus infection was also found in several other kibbutzim. The evidence presented suggests a different mode of transmission for cytomegalovirus and Epstein-Barr virus in the younger age groups.

Efflux of L-glutamate by synaptic plasma membrane vesicles isolated from rat brain.

Synaptic plasma membrane vesicles isolated from rat brain were loaded with L-glutamate either passively, by using a freeze-thaw technique, or by active transport. Subsequently the ion dependency of glutamate efflux from these vesicles was studied. With each of the types of loading similar results were obtained. Efflux requires the simultaneous presence of internal sodium ions and external potassium ions. The process is also stimulated by chloride ions, but either internal or external chloride ions cause stimulation. Addition of unlabeled L-glutamate stimulates efflux about 2-fold. It is concluded that efflux of L-glutamate is in many aspects symmetrical with its influx [Kanner, B. I., & Sharon, I. (1978) Biochemistry 17, 3949--3954]. It appears that in order for L-glutamate to interact with the transporter, sodium has to be present on the same side as L-glutamate whereas potassium has to be simultaneously present on the opposite site. The simplest way to account for these and the previous data is to postulate that the L-glutamate transporter catalyzes sodium and L-glutamate cotransport, while it simultaneously catalyzes antiport of potassium.