The American Journal of Tropical Medicine and Hygiene - Volume 44, Issue 6, June 1991

This issue completes the first volume of the Journal produced through the efforts of the new editorial staff in Atlanta. Start-up has not been without problems. As a result, issues have been late, but we hope that from the next issue forward delivery will be back on schedule. I would like to take this opportunity to provide the membership of the Society with a status report on the Journal.

Since last fall, when we began assuming our responsibilities, we have received, accessioned, initiated and in many cases completed the review process for approximately 350 manuscripts, 182 of these during the first five months of 1991. Initial peer reviews are indicating that almost 60% of the articles received are acceptable for publication if modified to meet changes suggested by referees. Approximately 50% of the papers received are eventually revised and returned for inclusion in the Journal.

Eight previously untyped Bunyamwera serogroup bunyaviruses that had been isolated from mosquitoes collected in California and Oregon between 1969 and 1985, were identified by cross-neutralization tests. Four viruses from Anopheles freeborni and a virus from Aedes sierrensis collected in Butte County in the Central Valley of California in 1970–71 were shown to belong to the Northway serotype. The existence of a Northway serotype virus in California had been inferred from previous serologic surveys of deer and horses, but these are the first Northway serotype viruses to be identified from the contiguous United States or from An. freeborni. This is also the first isolation of any virus from Ae. sierrensis. Two viruses from Culiseta inornata collected in Umatilla County, Oregon in 1969 may represent a new serotype in the Bunyamwera serocomplex of the Bunyamwera serogroup. The name “Stanfield” is proposed for this serotype, which is closely related to the Cache Valley and Northway bunyavirus serotypes. A virus from Ae. taeniorhynchus collected in San Diego County, California in 1985 was confirmed to belong to the Main Drain serotype. Previously, Main Drain serotype viruses in California have been associated principally with Culicoides variipennis.

Small mammals were trapped during a 21-month period at 27 farm sites in 15 localities within and beyond the known endemic area for Argentine hemorrhagic fever (AHF). Prevalence of Junin virus (JV) was assessed by antigen-capture enzyme immunoassay (ELISA) on samples of body fluids and/or organs from 3, 282 captured rodents. Infection in rodent populations was variable (0–3.7%) among localities but, in all cases, was lower than previously reported rates. Overall prevalence was 1.4% in the AHF epidemic area, 0.6% in the historic (currently low incidence of AHF) area, and 0.4% in two localities beyond the previously defined endemic area. These low values underestimate the actual prevalence of JV, as ELISA validation by virus isolation indicated a sensitivity of 30% and a specificity of 99%. Of 37 positive rodents, 28 (76%) were of two species: Calomys musculinus (23 animals) and C. laucha (5 animals). Antigen also was found in three Akodon azarae, four Bolomys obscurus, one Mus musculus, and one Oxymycterus rufus, and JV was isolated from two Oligoryzomys flavescens. Three of these rodent species (B. obscurus, O. flavescens, and O. rufus) have heretofore not been implicated in JV maintenance in the field. Evidence suggests that the AHF endemic area may continue to expand northward.

Five anopheline species, Anopheles deaneorum, An. albitarsis, An. triannulatus, An. oswaldoi, and An. mediopunctatus were compared to An. darlingi for susceptibility to infection by P. falciparum in Costa Marques, Rondonia, Brazil. Laboratory reared F1 An. darlingi and anopheline test species were allowed to feed at the same time on falciparum malaria patients who had gametocytes in their blood, and who had not yet been treated with quinine. Mosquitoes were dissected and examined for occysts on day 9, and for sporozoites on days 16–20 after feeding. Anopheles mediopunctatus had higher mean numbers of oocysts and oocyst positive rates than An. darlingi. The oocyst positive rate and the mean number of oocysts in An. deaneorum and An. darlingi were similar. Anopheles triannulatus and An. oswaldoi had fewer oocysts than An. darlingi. The salivary gland sporozoite infection rate was similar for An. mediopunctatus and An. deaneorum and much lower for An. triannulatus and An. oswaldoi when compared to An. darlingi. Anopheles albitarsis developed oocysts, but sporozoites did not invade the salivary glands. In relative levels of susceptibility to P. falciparum, An. darlingi was equal to An. mediopunctatus which was greater than An. deaneorum, which was greater than An. triannulatus, which was greater than An. oswaldoi.

Anopheles darlingi fed on eight falciparum malaria patients with gametocytes before and after treatment with quinine sulfate or quinine sulfate plus tetracycline became infected. Quinine and quinine plus tetracycline had no apparent sporontocidal or gametocytocidal effect on late stage immature and mature gametocytes. Plasmodium falciparum gametocytes are persistent and infected mosquitoes for up to 21 days after patients were treated with quinine plus tetracycline. Sporogonic development was similar for groups of mosquitoes fed before and after patients were treated with these schizontocides. The percentages of infected mosquitoes that developed salivary gland infections were also similar for groups of mosquitoes fed before and after treatment. Twenty-four hours after treatment with 45 mg of primaquine phosphate, falciparum malaria patients were not infective to An. darlingi.

Different non-radioactive probe labeling and detection systems were used with pAnaI, a species-specific oligonuleotide probe that distinguishes male Anopheles gambiae and An. arabiensis mosquitoes. Comparisons have been made between the performance of each technique with respect to sensitivity and specificity against DNA dot-blots and mosquito squashes. Their relative costs, economy, and ease of use were analyzed in an attempt to develop an appropriate non-radioactive system for use in the field.

Enzyme-labeled probes that were detected directly by label activity proved more suitable than probes requiring reporter molecules for detection. Binding of reporter molecules to mosquito squashes caused the appearance of false positives and, in addition, their binding to nylon filters caused high background coloration.

Chemiluminescent detection provided an attractive alternative to colorimetric detection. Both systems analyzed were rapid, simple, and economic. However, less severe treatment of filters was required for reprobing with chemiluminescence. The greatest sensitivity achieved was with chemiluminescent detection in which the limit of detection was 0.15 ng of target DNA. This study suggests that a synthetic DNA probe coupled to a chemiluminescent detection system should provide a sufficiently simple, sensitive, and reliable technique for insect vector identification in the field.

Monoclonal antibodies (MAbs) directed against different epitope regions on three sexual stage-specific gamete surface proteins of Plasmodium falciparum, Pfs 25, Pfs 230, and Pfs 48/45, were used to study the genetic diversity of these epitopes among fresh isolates of P. falciparum from Malaysia, using immunofluorescence microscopy (IFA). Among 45 Malaysian isolates, one epitope of Pfs 25, designated region I, showed evidence of variable reactivity with MAbs among different isolates; the Pfs 25 epitope, region II, was universally recognized by MAbs in all isolates. Two apparently distinct epitope regions of Pfs 230 were defined by MAbs, one of which was universally recognized by MAbs among the 45 isolates; the other was conserved in all but three isolates. The epitope regions of gamete-surface protein Pfs 48/45, designated regions I, IIa, IIb, IIc, III, and IV, were examined for reactivity by IFA in 33 isolates. Epitope regions I, IIb, III, and IV were conserved in all isolates; regions IIa and IIc existed in variant forms.

The role of circulating peripheral blood momonuclear cells (PBMC) in mediating protective immunity was examined during an immunization trial in Saimiri monkeys. Three engineered constructs representing different but overlapping regions of the circumsporozoite (CS) protein of Plasmodium vivax were used to immunize the Saimiri monkeys. Monkeys were randomly placed into three immunization groups: rPvCS2, rPvCS3, and LCV3 (representing three different but overlapping portions of the P. vivax CS protein) and two control groups: an alum adjuvant control group and an unimmunized control group. Collections of PBMC were made throughout the study at weeks 0, 2, 8, challenge (week 16), and two weeks after challenge. Proliferative responses to all immunogens and pokeweed mitogen were measured in all monkeys. Fourteen of 18 monkeys immunized with either rPvCS2 or rPvCS3 responded on the day of challenge to the appropriate immunogen with a stimulation index > 2. Immunization with LCV3, which represents the repeat region only, elicited a specific response in only one monkey. However, monkeys in both control groups also responded to rPvCS2 and rPvCS3, regardless of immunization, suggesting the presence of epitopes in rPvCS2 and rPvCS3 capable of associating with differing MHC antigens. Furthermore, the frequency of these cells in the periphery was increased by immunization, as demonstrated by a greater number of responding monkeys in the rPvCS2 and rPvCS3 immunized groups.

Between 1987 and 1990, susceptibility of Plasmodium falciparum to chloroquine and to Fansidar® was measured in vivo in 151 volunteers using the standard 7-day test. All volunteers lived in Arso PIR, Irian Jaya. A 25 mg/kg dose of chloroquine base was administered over a three-day period to 92 volunteers positive for P. falciparum rings (> 10 rings/200 white blood cells). Fifty volunteers (54%) showed results consistent with resistance. Twenty-nine were classified RII, and 21 RIII. In November 1989, a single curative dose of Fansidar® was administered to 59 volunteers divided among three groups with 18 months, four years, and life-long exposure to endemic malaria. The proportion of volunteers in each group still positive for P. falciparum on day 7 of followup was 54%, 0%, and 14%, respectively. Thus, immune status profoundly effected clinical response to Fansidar®. Standard in vitro microtests were also performed on parasites from 11 volunteers against chloroquine, amodiaquine, quinine, pyrimethamine/sulfadoxine, and mefloquine. Nine of ten isolates showed in vitro growth consistent with resistance to chloroquine. Tests with other drugs showed few isolates with results considered indicative of susceptibility. Arso PIR has a severe drug resistance problem.

Ninety-four leishmanial isolates from the Brazilian Amazon Region (Amapá, Amazonas, Pará, and Rondônia) were identified and classified using specific monoclonal antibodies and an indirect radioimmunoassay (serodeme analysis); eighty-two were also characterized by enzyme electrophoresis (zymodeme analysis), the results of which were subjected to a numerical phenetic analysis. Six isolates from humans (3), Didelphis marsupialis (1), Lutzomyia olmeca nociva (1), and Lu. reducta (1) showed reactivity patterns and isoenzyme profiles similar to those obtained with the Leishmania amazonensis reference strains, and were identified as this species. Eighty-six stocks were classified as members of the L. braziliensis complex; of these, 61 were L. guyanensis or variants, which presented three serodeme subtypes, but whose isoenzyme profiles were all similar to the reference strain. A total of 15 isolates were distinguished as L. braziliensis or variants and were classified into five serodeme subtypes. The isolate from Psychodopugus davisi appeared, from the numerical analysis, to be a distinct parasite species. Ten isolates showed reactivity patterns and isoenzyme profiles similar to those obtained with the L. naiffi reference strain. A parasite isolated from Ps. claustrei appeared to be different from all reference strains by both techniques, and was classified as probably being a new species. The importance of these results with respect to the taxonomic status of the New World Leishmania, and their implications for both clinical and epidemiologic data are discussed.

Characterization of Leishmania colombiensis sp.n. is presented, which on the basis of biological and molecular criteria, appears to be a new member of the L. braziliensis complex. A total of nine isolates of the new parasite were made in Colombia and Panama between 1980 and 1986: two from human cases of cutaneous leishmaniasis, six from phlebotomine sand flies, and one from a sloth. Although most closely related to L. lainsoni, L. colombiensis sp.n. is clearly distinguishable from other members of the genus by its reactivity with monoclonal antibodies, isoenzyme electrophoresis, and restriction endonuclease fragment patterns of kinetoplast DNA (k-DNA).

With the aim of identifying and differentiating Trypanosoma cruzi from Trypanosoma rangeli, culture epimastigotes from 30 Honduran trypanosomatid isolates were analyzed by susceptibility to complement lysis, reactivity to lectins, reactivity to monoclonal antibodies specific for T. cruzi, and isoenzymatic electrophoretic patterns. Using these four methodologies, 27 of the 30 trypanosomatid isolates, as well as 5 clones, were identified as T. cruzi, whereas the remaining three isolates were classified as T. rangeli. None of the isolates presented mixed trypanosome species. Results indicate that both trypanosomatid species circulate in Honduras and that any of the four methods employed may be used to reliably differentiate T. cruzi from T. rangeli.

Five cases of encephalitis following treatment with diethylcarbamazine (DEC) were observed in Congolese patients with Loa loa filariasis. Two cases had a fatal outcome and one resulted in severe sequelae. The notable fact was that this complication occurred in three patients hospitalized before treatment began, with whom particularly strict therapeutic precautions were taken, i.e., initial dose less than 10 mg of DEC, very gradual dose increases, and associated anti-allergic treatment. This type of drug-induced complication may not be that uncommon in highly endemic regions. It occurs primarily, but not exclusively, in subjects presenting with a high microfilarial load. The relationship between the occurrence of encephalitis and the decrease in microfilaremia is evident. The pathophysiological mechanisms are discussed in the light of these observations and the few other comments on this subject published in the literature.

Conventional methods for diagnosis of Wuchereria bancrofti infection are insensitive and often impractical because of the need for night blood collections. A sensitive and specific antigen detection assay has been developed for W. bancrofti, which is based on a monoclonal antibody (AD12) that binds to a repeated epitope on a 200 kDa adult worm excretion product present in sera from infected humans. The only formal evaluation of this assay to date was performed with sera from India. In the present study, we have evaluated the performance of the AD12 antigen assay in two laboratories with sera collected in endemic and non-endemic areas in Egypt. Antigen was detected in 57 of 59 (97%) sera from microfilaremic subjects, and in 22 of 139 asymptomatic and amicrofilaremic subjects who reside in a highly endemic area. Antigen titers were significantly correlated with microfilaria counts (r = 0.41, P < 0.01). Filarial antigen was not detected in most sera from amicrofilaremic subjects with clinical filariasis. Comparative antigen test results obtained from laboratories in Cairo and St. Louis agreed in 170 of 173 sera tested. Filarial antigen was not detected in sera from Egyptians with no history of residence in filariaendemic areas. Specifically, nonendemic sera from patients with other parasitic infections (schistosomiasis, fascioliasis, ascariasis, etc.) were uniformly negative in the assay. We conclude that the AD12 filarial antigen assay is sensitive and specific for W. bancrofti infection in Egypt.

Wistar rats inoculated intraperitoneally with 10 viable metacestodes of Taenia crassiceps without adjuvant once on day 0 showed strong resistance to challenge with 200 eggs of T. taeniaeformis on day 30. When rats were killed one month after challenge, there were 80.4% and 46.1% reductions in the number of cystic and total metacestodes of T. taeniaeformis in the liver, respectively. When five rats were killed 16 months after challenge, they showed almost complete immunity against the challenge, with 99.4% and 91.1% reductions in the number of cystic and total metacestodes, respectively. There were only a few degenerated, pin-point metacestodes of T. taeniaeformis in the liver of all five rats; one harbored one cystic metacestode as well. However, there were no such reductions in rats injected initially with cyst fluid antigens of T. crassiceps with Freund's complete adjuvant. An additional experiemnt was carried out using 500 eggs of T. taeniaeformis in order to confirm the vaccine effect against higher egg dose. There were 96.6%, 87.9%, 83.9%, and 79.3% reductions in the number of cystic metacestodes in rats initially inoculated with 10 viable, 10 formalized, and 10 frozen metacestodes, and injected with sodium deoxycholate-solubilized metacestode antigens, respectively. It is strongly suggested that rats singly dosed with 10 viable or non-viable, intact metacestodes of T. crassiceps without adjuvant became highly resistant to challenge infection with eggs of T. taeniaeformis, which resulted in almost no cystic metacestode establishment.

We have examined the Mycobacterium leprae phenolic glycolipid-I (PG-I) antigen levels in the sera of 45 multibacillary leprosy patients commencing chemotherapy. The PG-I antigen levels correlated with the bacterial and morphological indices, but not with the serum IgM anti-PG-I antibody levels. Antigen levels were significantly higher in patients with diffuse skin infiltration, but did not vary significantly with other parameters reflecting the duration and extent of untreated disease. The PG-I antigen levels in 27 patients examined serially decreased consistently over the first year of multidrug therapy.