The American Journal of Tropical Medicine and Hygiene - Volume 41, Issue 5, November 1989

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Freeze-fracturing has been used to study the formation of the triple layer pellicular complex of budding sporozoites of Plasmodium falciparum in the early occyst. Sporozoites are formed from sporoblasts within the oocyst. The outer membrane of the sporozoites is derived from the single plasma membrane of the sporoblast while the inner 2 membranes are formed anew at the base of the differentiating sporozoites. A dense collar of intramembranous particles located on the P face of the outer membrane encircles the base of each budding sporozoite. The fact that this collar of intramembranous particles is located in the same region where the inner membranes of the sporozoites first make their appearance strongly suggests that the 2 are related, and that the collar may be related to either membrane synthesis or to membrane organization and assembly.

We conducted a cross-sectional study to determine the serological response to malaria in an HIV-1 infected population and in a control population in a region of high malaria transmission. The study group consisted of 66 hospitalized patients with clinical acquired immunodeficiency syndrome (AIDS) and 70 trauma patients without clinical AIDS (controls). Mean optical densities of antibody produced against RESA-4, RESA-8, RESA-11, (PNAN)5 and (NAAG)5 synthetic peptides of Plasmodium falciparum were compared between HIV-1 seropositive and HIV-1 seronegative patients using non-parametric statistics. HIV-1 seropositive patients with clinical AIDS had significantly less antibody to the synthetic P. falciparum ring stage peptide, RESA-8 (P = 0.001), than a comparable group of seronegative patients. Antibody levels were also low for the other ring stage peptides, RESA-4 (P = 0.024) and RESA-11 (P = 0.024). Although not statistically significant, antibody levels among the HIV-1 seropositive trauma patients were higher than among the HIV-1 seronegative trauma patients. During HIV-1 infection, a polyclonal B cell activation may occur as noted in the HIV-1 seropositive trauma patients, but with increased immunosuppression in advanced clinical AIDS, B cell stimulation appears to be diminished. This results in decreased production of malaria antibody.

Gerbils were maintained on a low-fiber (5%) or a high-fiber (20%) diet in which the major fiber source was cellulose. Animals in the low-fiber diet group were significantly more likely to become infected when inoculated with 100 Giardia lamblia cysts than were animals in the high-fiber group. No differences were detected in gastrointestinal transit, gastric, and small intestinal luminal pH, or in duodenal mucus blanket acidic glycoprotein between animals in the high- and the low-fiber diet groups at the time of cyst inoculation. The fiber content of the diet after cyst inoculation determined the infection rate. These data suggest that the dietary fiber effect occurred during trophozoite colonization of the small intestine. When infected animals on the low-fiber diet were placed on the high-fiber diet for 24 hr, trophozoite clearing occurred in the lower small intestine. In the jejunum, the number of trophozoites attached to the mucosal surface decreased, while the number associated with luminal mucus increased. We conclude that the fiber-induced mucus secretion and the bulk movement of the insoluble fiber reduced the attachment of trophozoites to the intestinal mucosa, which decreased the probability of trophozoites establishing and sustaining colonization of the mucosa.

We performed serological and pathological studies on 495 patients with Chagas' disease from different areas of Bolivia. Eighty-nine Trypanosoma cruzi strains, isolated by xenodiagnosis, were characterized by 12 isoenzyme loci and were related to the presence of cardiac changes and enteric disease with megacolon. There was a high heterogeneity of human zymodemes, presenting evidence of 2 predominant zymodemes genetically dissimilar from each other and ubiquitous in Bolivia. The frequencies of these predominant zymodemes among strains from patients were compared to strains from triatomine bugs previously studied. We observed mixtures of different zymodemes within the same patient, a phenomenon seen previously in Bolivian patients. There was no apparent difference of pathogenicity between the 2 more frequent zymodemes isolated from humans.

To identify Trypanosoma cruzi target antigens in overt Chagas' heart disease, a parasite λgt11 cDNA library was screened with the serum of a patient with a severe chagasic heart involvement (JL). Using a phage dot array immunoassay, 5 highly antigenic clones, JL1, JL5, JL7, JL8, and JL9, were probed with sera from clinically characterized T. cruzi infected subjects. The correlation of cloned T. cruzi antigen recognition with the clinical status of the subjects led to the identification of a recombinant antigen, JL5, that reacted predominantly with sera from patients with Chagas' heart disease. The antigenic determinant of the JL5 recombinant was a small 35 amino acid peptide. The nucleotide and the deduced amino acid sequence, together with other experimental data, allowed identification as the C-terminal portion of a T. cruzi P ribosomal protein. The C-terminal undecapeptide in JL5, EDDDMGFGLFD, was highly homologous to the same region of the human P protein SD(D/E)DMGFGLFD. The latter sequence has been identified as the P protein epitope in systemic lupus erythematosus (SLE). Positive SLE sera reacted with the JL5 recombinant phage, suggesting that the T. cruzi P protein might induce antibodies with a similar specificity to that of P antibodies in SLE.

To search for the sequential compromise of the spinal cord, nerves, and skeletal muscle in mice chronically infected with Trypanosoma cruzi, animals were subjected to electromyographic investigation, end-plate recordings, and histological studies at 7, 15, 37, 60, 90, 120, 180, 270, and 360 days postinfection. Electromyographic studies showed signs of motor unit remodeling as early as 15 days postinfection, when diminished duration and amplitude of motor unit potentials pointing to a primary muscle involvement were found. Thereafter, certain features of denervation, reinnervation, and primary muscle involvement were often found to coexist. Low miniature end-plate potentials with normal frequency and acetylcholine quantum content were found in end-plate recordings made at the phrenic-diaphragm in vitro. Double end-plate potentials were observed in most of the tested muscle fibers from day 90 postinfection. All these features suggest post-synaptic damage of the end-plate and the presence of reinnervation after day 90 postinfection. Histological studies disclosed inflammatory infiltrates consisting of lymphocytes and macrophages, with vasculitis as the main lesion in the hamstring muscles; intracellular parasites were seen in 25% of the cases. Neuropathic features, as expressed by type fiber grouping and grouped muscle fiber atrophy, were found. On nerve examination epineural, perineural, and endoneural vasculitis were seen. Digestion chambers and myelin ovoids (axonal degeneration) were observed. In teased fiber preparations, segmental internodal and paranodal demyelination and remyelination were found. The lumbar inflammatory spinal cord failed to show grey or white matter infiltrates. However, spinal roots and dorsal root ganglia were densely affected by inflammatory cells.

A histopathological and immunofluorescence (IMF) study of the choroid plexus was performed in 8 cases of hepatosplenic schistosomiasis mansoni and in 20 cases which had resulted in death with no evidence of liver or brain involvement by schistosomiasis or other disease process, and in which renal disease and arterial hypertension were also excluded (control group). IgA, IgG, IgM, C3, and C1q were investigated. Positive IMF in the choroid plexus was found in 75% of the schistosomiasis group. IgA and IgG were the immunoglobulins (Ig) most frequently found. C3 was also commonplace. Histologic examination of the choroid plexus showed changes in 87.5% of the schistosomiasis group. The most frequently found change was characterized by focal, linear, occasionally nodular, subepithelial deposition of a homogeneous, acidophilic, and PAS positive substance, apparently in relation to the epithelial basement membrane, with thickening of this structure. In the control group, the IMF in the choroid plexus was negative in all cases, and only 2 cases (10%) presented histopathological changes of the choroid plexus with a pattern similar to that of the schistosomiasis group. The demonstration of the deposition of Ig and fractions of the complement system, and of histological changes in the choroid plexus in a liver disease which is known to exhibit circulating immune complexes and glomerulopathy with deposition of Ig and fractions of the complement system suggests an etiopathogenetic relationship between both findings.

Sera from humans with chronic Schistosoma mansoni infections (CHS) were chromatographed with CNBr-activated Sepharose 4 B conjugated with NP-40 extracts obtained from live 3 hr schistosomula. Both unbound (CHSUB) and bound (CHSB) fractions which contained IgG and IgM isotypes were characterized by ELISA, immunofluorescence, and complement-mediated in vitro killing assays. ELISA data showed that the CHSB fraction recognized schistosomula NP-40 extracts, whereas the CHSUB fraction did not. However, both CHSUB and CHSB fractions recognized 8 M urea adult worm extracts and 8 M urea egg extracts. By indirect immunofluorescence assay, the CHSB fraction recognized epitopes on the surface of live schistosomula 3 hr-29 days of age, whereas the CHSUB fraction showed surface fluorescence only on 24- and 29-day-old worms. The CHSB fraction mediated 95% killing of schistosomula in a complement dependent in vitro assay, the CHSUB fraction and the unfractionated CHS did not exhibit killing ability. The CHSUB fraction was able to titrate out the killing ability of the CHSB fraction in in vitro cytotoxic assays when mixed with the CHSB fraction at increasing concentrations. In passive immunization experiments, the CHSB fraction provided ∼30% passive protection in mice when injected 1 day or 6 days after challenge and 20% protection when injected at 15 days, but failed to provide protection when administered ≥24 days after challenge. Unfractionated CHS failed to mediate passive protection.

We investigated the presence of the circulating anodic antigen (CAA) in the urine of schistosomiasis patients. This genus specific antigen was hitherto demonstrated only in the serum of schistosomiasis patients. The urine of 80 patients with Schistosoma mansoni infections, 33 patients with S. haematobium infections, and 2 patients with mixed S. haematobium and S. mansoni infections were screened by a quantitative enzyme-linked immunosorbent assay (ELISA). CAA was demonstrated in 81% of those with intestinal schistosomiasis and in 97% of those with urinary schistosomiasis. CAA titers were < 1:0.2–1:51.2. Results were compared with circulating cathodic antigen (CCA) titers in urine obtained in an indirect hemagglutination assay (IHA). CCA was generally not detectable in the urine of patients with S. haematobium infection, but was demonstrated in the urine of 85% of the patients with S. mansoni infection. Both CAA titers and CCA titers correlated positively with the number of S. mansoni eggs excreted in the feces, but CAA titers did not show a significant correlation with the number of S. haematobium eggs in urine. Both antigen titers showed a moderate correlation with the serum CAA level in schistosomiasis mansoni. The discovery of CAA in the urine of the majority of schistosomiasis patients tested suggests the use of urine samples for non-invasive immunodiagnosis of the disease.

Trimeresurus popeorum, a dark green pit viper, is commonly found in Southeast Asia. This study describes the clinical picture and blood studies of 51 patients bitten by this snake. Affected limbs were swollen; and hemorrhagic blebs in fingers and toes were found in 12 patients. Lymphangitis was observed in 4 instances. Six individuals exhibited hypofibrinogenemia of 0–84 mg/dl, and 2 cases developed thrombocytopenia and bleeding. The presence of venom in the blood of these patients was demonstrated. Positive fibrin degradation products of 40–320 µg/ml were observed in 6 cases with hypofibrinogenemia, and in 8 other cases. Nineteen patients had short euglobulin lysis times of 51.8 ± 24.7 min. Hyperfibrinogenemia of 626.7 ± 288.9 mg/dl was found in 18 cases. Apart from bleeding, there were no systemic symptoms. Hypofibrinogenemia became normal in 3–12 days. The clinical course in all patients was uneventful, and none received antivenin.

Five murine monoclonal antibodies (Mabs) reactive against the prM glycoproteins of DEN-3 and -4 were used to passively protect mice in vivo against lethal challenge with homologous and heterologous dengue virus serotypes. Four of the 5 prM-reactive monoclonals cross-protected mice against heterologous challenge, whereas 1 protected against challenge with only the homologous serotype. Although in vitro binding to virions was readily demonstrated, only 2 of the prM Mabs had detectable neutralizing activity. The neutralizing activity could not be enhanced by anti-mouse immunoglobulin or complement. However, 4 of the 5 prM Mabs fixed complement. This is the first report of prM-specific Mabs that are protective in mice.

Ribavirin was evaluated as a potential therapeutic for Crimean-Congo hemorrhagic fever (CCHF). Viral yields for strains of CCHF virus from Europe, Asia, and Africa in African green monkey kidney (Vero) cells were markedly reduced by this drug. Some CCHF viral strains appeared more sensitive than others, but in general, ribavirin doses as low as 5 µg/ml caused a transient reduction of viral yields. A further reduction in viral yields was induced by a dose of 25 µg/ml, and evidence of viral replication was not demonstrated in cells treated with 50 or 250 µg/ml. In contrast, a dose of ribavirin at least 9 times greater was required to induce a comparable inhibitory effect on the yields of Rift Valley fever virus, for which the drug has been shown to inhibit replication in monkeys and rodents.

The newly recognized pathogenic virus-like infectious agent (VLIA), originally reported in patients with AIDS but also known to be pathogenic in previously healthy non-AIDS patients and in non-human primates, was cultured in cell-free conditions using a modified SP-4 medium and classified as a member of the order Mycoplasmatales, class Mollicutes. The infectious microorganism is tentatively referred to as Mycoplasma incognitus. M. incognitus has the unique biochemical properties of utilizing glucose both aerobically and anaerobically, as well as having the ability to metabolize arginine. Among all known human mycoplasmas, these specific biochemical characteristics were found previously only in a rarely isolated species, M. fermentans. In comparison with M. fermentans, M. incognitus appears to be even more fastidious in cultivation requirements and fails to grow in all tested mycoplasma media other than modified SP-4 medium. In addition, M. incognitus grows much more slowly, has a smaller spherical particle size and occasional filamentous morphology, and forms only irregular and very small colonies with diffuse edges on agar plates. Antigenic analysis using polyclonal and monoclonal antibodies and DNA analysis of sequence homology and restriction enzyme mappings in M. incognitus, M. orale, M. hyorhinis, M. hominis, M. pneumoniae, M. fermentans, M. arginini, M. genitalium, M. salivarium, Ureaplasma urealyticum, and Acholeplasma laidlawii revealed that M. incognitus is distinct from other mycoplasmas, but is most closely related to M. fermentans.

Monoclonal antibodies (Mabs) were developed against antigens from a pure culture of Mycoplasma incognitus grown in modified SP-4 medium. All the Mabs obtained were shown to react only with M. incognitus, and not with other species of human mycoplasma. The Mabs identified M. incognitus immunohistologically in thymus, liver, spleen, lymph node, or brain from 22 patients with AIDS, as well as in 2 placentas delivered by patients with AIDS. Using an 35S-labeled DNA probe specific for M. incognitus and in situ hybridization technique, we also identified M. incognitus-specific genetic material in these tissues. Furthermore, ultrastructural studies of the specific areas of tissues which were highly positive for M. incognitus antigens revealed characteristic structures of mycoplasma organisms. These mycoplasma-like particles could be identified intracellularly and extracellularly. Histopathology of the tissues infected by M. incognitus varied from no pathological changes to fulminant necrosis with or without an associated inflammatory reaction. M. incognitus, a novel pathogenic mycoplasma, was cytopathic and cytocidal.