Sporozoite vaccine induces genetically restricted T cell elimination of malaria from hepatocytes.

The target of the CD8+ T cell-dependent immunity that protects mice immunized with irradiation-attenuated malaria sporozoites has not been established. Immune BALB/c mice were shown to develop malaria-specific, CD8+ T cell-dependent inflammatory infiltrates in their livers after challenge with Plasmodium berghei sporozoites. Spleen cells from immune BALB/c and C57BL/6 mice eliminated hepatocytes infected with the liver stage of P. berghei in vitro. The activity against infected hepatocytes is not inhibited by antibodies to interferon-gamma and is not present in culture supernatants. It is genetically restricted, an indication that malaria antigens on the hepatocyte surface are recognized by immune T effector cells. Subunit vaccine development will require identification of the antigens recognized by these T cells and a method of immunization that induces such immunity.

Immunodot assay of Plasmodium falciparum sporozoites in mosquitoes using a direct binding membrane system.

We describe a membrane based immunodot assay for the detection of Plasmodium falciparum sporozoites mixed with mosquitoes. A crude sodium dodecyl sulfate extract of mosquitoes and sporozoites is passed through a bi-layered membrane system, the top layer being a polyvinyldiene difluoride hydrophilic pre-filter which screens out debris but allows the passage of antigen. Sporozoite, as well as mosquito, proteins are bound to the hydrophobic membrane below. This membrane was probed with a monoclonal antibody to the repeat region of the P. falciparum circumsporozoite protein, a peroxidase labeled second antibody and a tetramethyl-benzidine substrate. The method detects as few as 10 sporozoites/mosquito or 100 sporozoites in a pool of 10.

Polymerase chain reaction amplification from Plasmodium falciparum on dried blood spots.

We report a simple method for the polymerase chain reaction (PCR) amplification of whole blood samples collected on filter paper. The blood spot was used directly in the PCR after treatment with methanol. We evaluated this assay using clinical samples collected from subjects in a Plasmodium falciparum vaccine trial and from samples collected during a hospital-based study in Thailand. Specimens prepared from heparinized blood samples were successfully amplified following pretreatment with heparinase. Sensitivity was 100% when compared with thick blood film results in the vaccine trial (range = 4-60 parasites/microliters, median = 8/microliters) and 94.6% (range = 3-133,988 parasites/microliters, median = 616/microliters) in the hospital study.

Analysis of the immunoglobulins responsible for the circumoval precipitation reaction.

Two morphologically distinct types of circumoval precipitates (COP) have been observed in human Schistosoma japonicum infections. An elongated segmented COP occurs in chronic human infections. An unsegmented "reaction of recent infection" (RRI) occurs in serum from humans with recently acquired infections and is morphologically similar to the reaction observed in the sera of mice infected with S. japonicum. Sera from infected mice and humans were separated by G-200 chromatography to determine whether the unsegmented "RRI" was due to IgM antibody and the segmented COP reaction due to IgG. There was an elevation of the 19S fraction of sera of mice with 10 and 16 week infections. In addition, the murine 7S fraction was elevated in the 16 week infections. The COP activity was confined to the 7S fraction in the murine sera. Sera from Philippine patients which produced reactions of recent infection (acute sera), segmented COP reactions (chronic sera), and mixed reactions (believed to be from a transition stage between acute and chronic schistosomiasis) were tested. All human sera had elevation of both the 19S and 7S fractions of the acute serum. However, COP-reactive antibodies were confined to the 7S fraction of sera from the transition stage and acute stage infections. The results suggest that although IgM antibodies do in certain cases participate in the COP and produce reactions of recent infection, antibody class is not responsible for the different morphology of this reaction.

Efficacy of purified schistosoma japonicum egg antigens for ELISA serodiagnosis of human Schistosomiasis japonica: specificity and sensitivity.

At present, there is no consensus that purified schistosome egg antigens offer any advantage in the diagnosis of schistosomiasis by enzyme linked immunosorbent assay (ELISA). Previously, we demonstrated by multiple techniques that the major serologic antigens in Schistosoma japonicum soluble egg antigen (SEA) are glycoproteins, and that the glycoproteins with highest specificity and sensitivity are hydrophobes. We therefore tested these materials for their specificity, sensitivity and cost effectiveness in the ELISA. In this study we used five SEA fractions that varied in their purity and antigenicity. The order of immunologic specific activity in the ELISA, measured by titration of a standard sera pool, was: hydrophobic glycoproteins (highest), crude SEA glycoproteins, hydrophilic glycoproteins, crude SEA, and SEA proteins (lowest). Complexity (purity) of these materials were (in rank order), hydrophilic glycoproteins (purest), hydrophobic glycoproteins, crude glycoproteins, SEA proteins, and crude SEA (most complex). Epidemiologic sensitivity in the ELISA was tested on limited but well characterized populations. At high antigen coating concentration (0.5 microgram/well), the only antigen fraction with poor sensitivity was SEA proteins. There was little difference in epidemiologic sensitivity between the purer fractions with highest immunologic sensitivity (hydrophobic glycoproteins and crude SEA glycoproteins) and the crude SEA which possesses intermediate immunologic sensitivity. Differences in epidemiologic sensitivity were most pronounced when wells were coated at an antigen concentration (0.1 microgram/well) where crude SEA began to fail. Specificity for all preparations, assessed by reactivity with sera from patients with other trematode infections and with cestode and nematode infections, was excellent. The clinical sensitivity of the ELISA employing crude S. japonicum SEA is so high, and the specificity so good, that the increased immunologic sensitivity of partially purified antigens had little effect on epidemiologic sensitivity. This is not true for the S. mansoni ELISA where crude antigens had inferior sensitivity and specificity.

Acridine orange detection of Plasmodium falciparum malaria: relationship between sensitivity and optical configuration.

Blood samples collected from five volunteers participating in a P. falciparum infectivity trial were examined to determine the efficacy of the acridine orange technique. Several lens configurations were tested for efficiency in the diagnosis of malaria using this system. There was no significant difference in the sensitivity for detecting positive specimens or number of parasites among three lens configurations: a 50x long working distance objective (0.34 mm) with either a 10x ocular (total magnification 500x) or a 12.5x ocular (625x) and a 750x configuration using a 50x objective with a shorter working distance (0.24 mm). All three lens configurations were significantly better than the 1,000x configuration using a commonly available 100x oil immersion objective. The results achieved using this lens still exceeded the sensitivity of the thick blood film.

Acridine orange diagnosis of Plasmodium falciparum: evaluation after experimental infection.

The value and role of the acridine orange/microhematocrit tube method (quantitative buffy coat [QBC] analysis) in the diagnosis of malaria remains controversial. To establish the true sensitivity of this test in comparison with the thick blood film, we studied 49 subjects who were experimentally infected with Plasmodium falciparum in 10 malaria vaccine and infectivity trials. Diagnosis was made by the acridine orange staining method 1-3 days earlier than by the thick blood film in 23 subjects (47%) and at the same time as the thick blood film in 20. On the other hand, diagnosis was made by thick blood film earlier than by the acridine orange staining method in six individuals. There were no false positive results using acridine orange among 584 specimens studied. Diagnosis was made using acridine orange at a parasitemia of less than 11 parasites/microliters of blood in 65% of cases. Where available, the acridine orange assay is clearly preferable in terms of speed and accuracy to the thick blood film for diagnosis with parasitemias of less than 150/microliters of blood, and perhaps as important, for ruling out infection with P. falciparum in a symptomatic patient.

Amodiaquine less effective than chloroquine in the treatment of falciparum malaria in the Philippines.

Amodiaquine was compared to chloroquine in two groups of Filipino patients with uncomplicated falciparum malaria. Every patient received 25 mg/kg of base orally given over three days. In a hospital study, all eight patients receiving chloroquine cleared their parasitemia by day 6, but six of eight patients receiving amodiaquine failed to clear parasitemia and in four patients there was no response at all (RIII resistance); this difference was significant (P less than 0.01). In a village based study, there was initial clearing of parasitemia in each patient. However, recrudescent infection occurred in all five patients receiving amodiaquine (RI resistance). Five of six falciparum infections were sensitive to chloroquine, while parasitemia reappeared in one patient. In this village, resistance to amodiaquine was significantly more common than resistance to chloroquine (P less than 0.05). To our knowledge, this is the first report of amodiaquine being substantially worse than chloroquine in the treatment of Plasmodium falciparum infection.

Plasmodium falciparum-infected Anopheles stephensi inconsistently transmit malaria to humans.

Malaria was transmitted to only 5 of 10 volunteers bitten by 1-2 Anopheles stephensi carrying sporozoites of the 3D7 clone of the NF54 strain of Plasmodium falciparum in their salivary glands. Parasites were detectable by culture in blood taken 7-10 days following exposure and by thick blood film 14-16.5 days after exposure. Infectivity did not correlate with the numbers of sporozoites in the salivary glands.

QBC and Giemsa-stained thick blood films: diagnostic performance of laboratory technologists.

This study compared the proficiency of military medical technicians in the diagnosis of malaria using Giemsa-stained thick blood films (GSF) and the QBC system. Fourteen technicians, with no previous experience of the QBC and limited experience in interpreting GSF, were given training in the 2 techniques and then tested on their ability to make a malaria diagnosis when provided with previously prepared GSF and QBC specimens (9 positive and 10 negative). The students achieved a sensitivity of 75% and 84%, respectively, with the QBC and GSF; specificity was 84% and 76%. There was no difference in the concordance of the 2 techniques with the actual diagnosis (80% vs. 81%).

Prevalence of renal involvement in Schistosoma japonicum infection.

244 outpatients and 100 hospitalized patients with confirmed Schistosoma japonicum infection were prospectively surveyed for the presence of nephropathy. There was no association between schistosomiasis and renal disease in the outpatient group. Three hospitalized patients had evidence of significant nephropathy, but this number was not significantly higher than in a control group of 100 hospitalized age and sex-matched control patients without schistosomiasis. One schistosomiasis patient with severe nephrotic syndrome underwent percutaneous renal biopsy. Neither S. japonicum antigen nor antibody was found in the biopsy specimen. 64 of the 100 hospitalized patients had portal hypertension; in 28 patients there was hepatic decompensation. Only one of these hepatosplenic patients had evidence of renal disease. Thus renal involvement was uncommon in patients presenting various manifestations of chronic S. japonicum infection, including those with severe hepatosplenic disease. These results contrast markedly with S. mansoni infection, in which nephropathy associated with advanced liver disease is a distinct, well-recognized clinical entity.

Reversal of drug-resistant falciparum malaria by calcium antagonists: potential for host cell toxicity.

Agents capable of reversing multidrug resistance (mdr) in falciparum malaria were investigated for potentiation of chloroquine accumulation and toxicity in a cell culture system. Verapamil, its analog RO11-2933, and desipramine caused a dose-dependent increase in the accumulation of chloroquine (CQ) within human and mouse hepatocytes but not human lung cells. Only those cells in which drug accumulation was enhanced by reversing agents reacted positively for P-glycoprotein (PgP)--the putative mediator of the enhanced drug efflux characteristic of mdr. Clinically achievable concentrations of verapamil (0.4 microM) and desipramine (1 microM) increased CQ accumulation within primary mouse hepatocytes by more than 50%. A well-differentiated normal human cell line (Hep-G2) was killed in media containing a combination of supraphysiological concentrations of CQ and verapamil but survived the same concentrations of either drug alone. Reversing agents may block PgP-mediated drug export from normal tissues as well as from MDR cells. Iatrogenic toxicity resulting from this accumulation of potentially toxic drugs such as CQ within normal cells could complicate the reversal of mdr in vivo.

Chloroquine and quinine: a randomized, double-blind comparison of efficacy and side effects in the treatment of Plasmodium falciparum malaria in the Philippines.

Chloroquine (25 mg/kg over 3 d) was compared to quinine (10 mg/kg 3 times daily for 5 d) in 20 adult Filipino males with uncomplicated Plasmodium falciparum malaria in a double-blind, randomized trial. Asexual parasitaemia was cleared in all patients, with no statistically significant difference (P = 0.13) in the rate of clearance between the chloroquine-treated patients (76.1 +/- 29.3 h) and those receiving quinine (60.3 +/- 12.5 h). The duration of fever was also comparable (chloroquine 46.3 +/- 24.7 h; quinine 43.2 +/- 20.0 h; P = 0.76) and 40% of patients in each treatment group experienced mild side effects. Chloroquine, however, is cheaper and easier to administer. In vitro results were strikingly different. P. falciparum parasites from 4 quinine-treated patients were all sensitive to this compound in vitro, whereas 4 of the 5 isolates from the chloroquine group were resistant. Further comparisons of these two antimalarials are indicated, especially in cerebral malaria, and drug use policies should be based on clinical and parasitological response to treatment.

Cell washing versus immediate reinfusion of intraoperatively shed blood during abdominal aortic aneurysm repair.

Significant hematologic changes are known to occur following intraoperative autotransfusion of shed blood, but the clinical importance of cell washing prior to reinfusion has not been substantiated. To evaluate these changes and their relationship to the use of blood bank products and postoperative morbidity, 26 patients undergoing elective abdominal aortic aneurysm repair were prospectively randomized to reinfusion with washed shed blood or to the use of a collection system in which filtered, but unwashed, whole blood was reinfused intraoperatively. Each patient was evaluated with respect to standard metabolic and hematologic laboratory parameters preoperatively, immediately postoperatively, and 12 to 18 hours postoperatively. Patient demographic data were similar for both groups. Perioperative survival was 100% for both groups. Total blood loss and blood volume autotransfused were significantly greater in the unwashed cell group compared with the washed cell group (p = 0.00014 and p = 0.00011, respectively). Hemoglobin, fibrinogen, prothrombin time, and partial thromboplastin time levels were not significantly different between the two groups at any time perioperatively; fibrin split product and d-dimer levels were significantly higher in the unwashed cell group postoperatively (p = 0.016 and p < 0.001, respectively). Serum free hemoglobin levels were significantly higher in the immediate postoperative period in the unwashed cell group compared with the washed cell group (p = 0.0013); by 12 to 18 hours postoperatively, this difference was not significant. Haptoglobin levels were significantly lower in the unwashed cell group at both postoperative times (123 +/- 86 mg/dL versus 41 +/- 50 mg/dL, p = 0.0086; 102 +/- 66 mg/dL versus 24 +/- 36 mg/dL, p = 0.0001); however, there was no perioperative renal failure in either group. Furthermore, homologous blood product use was not significantly different between the two groups, with an average of 1.5 +/- 2.5 units of packed red blood cells given to patients in the unwashed cell group versus 0.8 +/- 1.7 units in the washed cell group (p = 0.419). Overall complications were higher and critical care and total hospital stays were longer in the unwashed cell group but did not result from autotransfusion of unwashed blood. We conclude that the intraoperative reinfusion of unwashed shed blood is safe and effective, causing transient hematologic abnormalities that normalize in the early postoperative period, and is not associated with increased mortality, or hematologic, cardiopulmonary, or renal complications.

Exposure to lead in stained glass work. An environmental evaluation.

An environmental evaluation was conducted to determine lead exposure in a group of crafts people who produce stained glass and Tiffany glass. The environmental evaluation consisted of air sampling for potential lead emissions from solder and of work area dusts. In addition, the completion of a questionnaire, observation of work practices and noting of other details relevant to hazardous exposures were carried out. Lead concentrations in air were found to be well below the ACGIH TLV-TWA of 150 micrograms/m3. High lead concentrations were found in the work area dust samples. Exposure to high concentrations of lead could occur by ingestion as a result of neglect of basic hygiene precautions.

Rapid diagnosis of Brugia malayi and Wuchereria bancrofti filariasis by an acridine orange/microhematocrit tube technique.

A microhematocrit tube technique for diagnosis of human filariasis has been previously described. A system incorporating heparin, EDTA, and acridine orange into a microhematocrit tube (Quantitative Blood Count, QBC) has been commercially developed for the quantitation of blood counts and has been used for the diagnosis of malaria. We evaluated this test for its usefulness in the diagnosis of filariasis. Upon centrifugation, the parasites were concentrated in the area of the buffy coat and could be observed through the wall of the tube. The parasites were concentrated further by a plastic float that expands the buffy coat and confines the parasites to the periphery of the tube. Acridine orange stains the DNA of the parasite, and morphologic characteristics can be examined by fluorescence microscopy. The terminal and subterminal nuclei and long cephalic space of Brugia malayi, as well as the short cephalic space and caudal nuclei of Wuchereria bancrofti, were easily recognized and differentiated from each other. Microfilariae were detected in samples diluted to a level of approximately 50/ml.

Relevance of carcinogenicity bioassays in mice in assessing potential health risks associated with exposure to methylene chloride.

Considerable research has been conducted to identify possible mechanisms of the carcinogenicity of methylene chloride in rodents, and to ascertain whether the observed increased incidences of liver and lung tumours in mice exposed to this substance, are relevant in assessing the potential hazards and risks to human health. On the basis of a study that purported to show qualitative differences between murine and human tissues, in the subcellular localization of the Theta-class glutathione S-transferase enzyme responsible for converting methylene chloride to a putative highly unstable, but reactive genotoxic metabolite, it was suggested that the mouse is an inappropriate model for human health risk assessment. However, other studies conducted in vitro with intact cells do not support the hypothesis that a putatively reactive metabolite of methylene chloride must be generated only within the nucleus in order to be able to interact with genomic DNA. Moreover, investigations employing semi-quantitative methods of mRNA hybridization are not convincing in identifying the subcellular localization of active Theta class glutathione S-transferase, and do not support the hypothesis of the differential subcellular localization of this enzyme within the nucleus of mouse, but not human cells. There is therefore, insufficient evidence to support the view that qualitative differences between humans and mice in the subcellular distribution of Theta-class glutathione S-transferase, renders carcinogenicity studies conducted with mice irrelevant in human hazard identification and risk assessment.

Comparison of washed and unwashed specimens in the Plasmodium falciparum in vitro microculture drug assay.

The dose response of Plasmodium falciparum isolates in the standard in vitro assay for drug resistance was compared using blood specimens which were centrifuged and washed before cultivation. Washing of the cultures increased the success of cultivation by greater than 100%. Eight cultures which grew using both methods gave similar results in the determination of resistance or sensitivity. The ED50 as determined by probit analysis, was approximately 50% higher in parasites which had been washed before cultivation.

In vitro drug response of Plasmodium falciparum in the Philippines: increased resistance to amodiaquine.

A long term study was carried out at San Lazaro Hospital, Manila, Philippines, monitoring the in vitro response of Plasmodium falciparum to chloroquine, amodiaquine, mefloquine, and quinine. The in vitro effective dose giving 50% inhibition of schizogony was: 0.68 X 10(-6) M/liter blood for chloroquine; 0.18 X 10(-6) for amodiaquine; 0.2 X 10(-6) for mefloquine; and 1.12 X 10(-6) for quinine. The percent of isolates determined to be resistant in vitro was 85.2% for chloroquine, and 1.2% for both mefloquine and quinine. These figures were relatively unchanged over the course of 3 years studied. The in vitro resistance rate to amodiaquine increased from 5.1% in 1982 to 22.2% in 1984.

HLA antigens and malaria at San Lazaro Hospital Manila, Philippines.

Human leucocyte antigens (HLA) were used as genetic markers in an attempt to determine possible host genetic susceptibility or resistance to malarial infections. HLA-A and B typing on lymphocytes from 68 confirmed P. falciparum and 77 P. vivax cases was compared with that found in 66 control subjects with no known history of malaria. A significant deviation was observed in the distribution of HLA-B27. This phenotype was absent in the P. falciparum group although found present in the P. vivax group (10%) and the control group (11%). Also, the combination of A9(w24) and B5 was significantly higher among the P. falciparum group than that found in the P. vivax and control groups. These findings require confirmation but do suggest the possibility of genetic susceptibility and that extensive genetic studies might be worth investigating.