The American Journal of Tropical Medicine and Hygiene - Volume 60, Issue 6, June 1999

An epizootiologic investigation testing for the presence of tick-borne relapsing fever spirochetes in rodent and tick hosts was conducted at Eagle Lake in Lassen County, California. Six of 211 O. hermsii ticks and none of 180 rodents were polymerase chain reaction (PCR)-positive for Borrelia spirochetes. Sequencing of the PCR-amplified flagellin gene fragment suggests that the spirochetes from Eagle Lake represent a previously undescribed strain of Borrelia hermsii.

Twenty-seven rickettsiae were isolated and/or detected from 100 Amblyomma variegatum ticks collected on Guadeloupe in the French West Indies. In this study, the polymerase chain reaction procedure appeared to be more sensitive in detecting rickettsiae in ticks than the shell-vial technique. Sequencing a portion of the outer membrane protein A-encoding gene showed that these rickettsiae appeared to be identical to Rickettsia africae, a member of the spotted fever group rickettsiae recently described as an agent of African tick-bite fever occurring in sub-Sahelian Africa. A high seroprevalence of antibodies to R. africae was demonstrated among mammals, particularly humans, cattle, and goats. These results and a recently reported case of an infection due to R. africae on Guadeloupe demonstrate that R. africae is present on this island. Although this disease has been underdiagnosed there, it may be frequent and may exist on other Caribbean islands where A. variegatum has propagated dramatically over recent years.

We tested single serum samples from 631 intravenous (i.v.) drug users from inner-city Baltimore, Maryland for serologic evidence of exposure to spotted fever group rickettsiae. A total of 102 (16%) individuals had titers > or = 64 to Rickettsia rickettsii by an indirect immunofluorescence assay. Confirmation that infection was caused by R. akari was obtained by cross-adsorption studies on a subset of serum samples that consistently resulted in higher titers to R. akari than to R. rickettsii. Current i.v. drug use, increased frequency of injection, and shooting gallery use were significant risk factors for presence of group-specific antibodies reactive with R. rickettsii. There was a significant inverse association with the presence of antibodies reactive to R. rickettsii and antibodies reactive to the human immunodeficiency virus. This study suggests that i.v. drug users are at an increased risk for R. akari infections. Clinicians should be aware of rickettsialpox, as well as other zoonotic diseases of the urban environment, when treating i.v. drug users for any acute febrile illness of undetermined etiology.

In June 1994, 18 people developed serologically confirmed histoplasmosis following cave exploration associated with the annual National Speleological Society Convention in Bracketville, Texas. Six others had an undiagnosed illness suspected to be histoplasmosis. Two persons were hospitalized. We conducted a survey of convention attendees and a nested case-control study of those entering caves. We also conducted a histoplasmin skin test survey of a subgroup of the society, the Texas Cavers Association, who were attending a reunion in October 1994. Among the national convention attendees, exposure to two caves was identified as responsible for 22 (92%) of the 24 cases; 12 (75%) of 16 people exploring one cave (Cave A) and 10 (77%) of 13 exploring a separate cave (Cave B) developed acute histoplasmosis. Additional risk-factors included fewer years of caving experience, longer time spent in the caves, and entering a confined crawl space in Cave A. Of 113 participants in the separate skin test survey, 68 (60%) were found to be skin test positive, indicating previous exposure to Histoplasma capsulatum. A positive skin test was significantly associated with male sex and more years of caving experience. Those less experienced in caving associations should be taught about histoplasmosis, and health care providers should pursue histories of cave exposure for patients with bronchitis or pneumonia that does not respond to initial antibiotic therapy.

To characterize the dengue epidemic that recently occurred in Malaysia, we sequenced cDNAs from nine 1993-1994 dengue virus type-3 (DEN-3) isolates in Malaysia (DEN-3 was the most common type in Malaysia during this period). Nucleic acid sequences (720 nucleotides in length) from the nine isolates, encompassing the precursor of membrane protein (preM) and membrane (M) protein genes and part of the envelope (E) protein gene were aligned with various reference DEN-3 sequences to generate a neighbor-joining phylogenetic tree. According to the constructed tree, the nine Malaysian isolates were grouped into subtype II, which comprises Thai isolates from 1962 to 1987. Five earlier DEN-3 virus Malaysian isolates from 1974 to 1981 belonged to subtype I. The present data indicate that the recent dengue epidemic in Malaysia was due to the introduction of DEN-3 viruses previously endemic to Thailand.

The global resurgence of malaria has raised concerns of the possible reintroduction of indigenous transmission in the United States. The Centers for Disease Control and Prevention's National Malaria Surveillance System, using data supplied by state and local health departments (SLHDs), is maintained to detect local malaria transmission and monitor trends in imported cases. To determine the completeness of reporting of malaria cases to SLHDs, cases identified by local surveillance systems were compared with those identified through active case detection conducted at all laboratories that receive clinical specimens from 11 metropolitan areas in Arizona, California, New Mexico, and Texas. Of the 61 malaria cases identified through either local surveillance or active case detection, 43 (70%) were identified by SLHDs (range by metropolitan area = 50-100%) and 56 (92%) through active case detection. High percentages of cases were identified by SLHDs in New Mexico (80%) and San Diego County (88%), where laboratories are required to send positive blood smears to the SLHD laboratory for confirmation. Completeness of reporting, calculated using the Lincoln-Peterson Capture-Recapture technique, was 69% for SLHD surveillance systems and 89% for laboratory-based active case detection. The high percentage of cases identified by the 11 SLHDs suggests that the National Malaria Surveillance System provides trends that accurately reflect the epidemiology of malaria in the United States. Case identification may be improved by promoting confirmatory testing in SLHD laboratories and incorporating laboratory-based reporting into local surveillance systems.

Infection with hepatitis C virus (HCV) has become the most important public health problem in Egypt. In Egypt, viral hepatitis along with infection with Schistosoma mansoni is the major cause of chronic liver disease and liver cirrhosis. Although HCV infection is highly prevalent in Egypt, very little information is available on the distribution of the different genotypes of HCV. Our aims in this study were first to determine the prevalence of viral and parasite infections in patients with chronic liver disease and then to assess the distribution of HCV genotypes in these patients. In the present study, 151 individuals (50 with chronic liver disease, 51 with chronic diseases of organs other than the liver, and 50 apparently healthy persons) were investigated. The last 2 groups served as control groups. These individuals were subjected to routine liver function tests and detection of serum antibodies to bilharziasis, hepatitis B surface antigen (HBsAg), and HCV. Furthermore, the presence of hepatitis G virus (HGV) and HCV in the serum samples were tested for by a reverse transcription polymerase chain reaction (RT-PCR). Prevalence of different genotypes of HCV in patients positive for HCV were determined by RT-PCR using type-specific primers. Results of the study revealed that 84, 74, 12, and 20% of patients with chronic liver disease were positive for Schistosoma mansoni, HCV, HBsAg, and HGV, respectively, as compared to 51, 43.1, 2, and 4% of patients with other chronic diseases and 22, 6, 0, and 0% of apparently healthy individuals. One hundred percent of patients with chronic liver disease, 72.5% of those with other diseases, and 26% of normal controls were shown to have at least one of the studied infectious agents. Two or more of the agents were highly coincident in patients with chronic liver disease. In Egypt, HCV genotype 4a is highly prevalent, where it contributed 85% of the tested samples in comparison to 10, 2.5, and 2.5% for subtypes 1b, 2a, and 3a, respectively. In conclusion, these results suggest that in Egypt, HCV along with schistosomal parasite infection is the major risk factor for chronic liver disease. In most Egyptian patients, HCV genotype 4 is highly prevalent.

Epidemiologic studies of helminthic infections have shown that susceptibility to these parasites frequently aggregates in families, suggesting the possible involvement of genetic factors. This paper presents a genetic epidemiologic analysis of Ascaris lumbricoides infection in the Jirel population of eastern Nepal. A total of 1,261 individuals belonging to a single pedigree were assessed for intensity of Ascaris infection at two time points. Following an initial assessment in which all individuals were treated with albendazole, a follow-up examination was performed one year later to evaluate reinfection patterns. Three measures of worm burden were analyzed, including eggs per gram of feces, direct worm counts, and worm biomass (weight). For all traits, variance component analysis of the familial data provided unequivocal evidence for a strong genetic component accounting for between 30% and 50% of the variation in worm burden. Shared environmental (i.e., common household) effects account for between 3% and 13% of the total phenotypic variance.

Treatment with praziquantel reduces the prevalence and intensity of Schistosoma mansoni infection. However, reversibility of periportal fibrosis of the liver, which potentially leads to fatal complications, is not unequivocally substantiated. In the Nile District of Uganda, 460 patients were parasitologically (Kato-Katz method) and ultrasonographically examined during October 1991, October 1992, and May 1994. Treatment with praziquantel at a dosage of 40 mg per kilogram of body weight was given in October 1991 and October 1992 to 460 individuals (group A). Another 192 patients were seen during the baseline study in October 1991 and missed the follow-up in October 1992 but took part in the second follow-up in May 1994. Thus, they received praziquantel only once in October 1991 (group B) and had an interval of 2.7 years until the next investigation in May 1994. Periportal thickening (PT) of the liver was assessed by ultrasound at each time point. Praziquantel therapy reduced the prevalence of S. mansoni in group A from 84% in 1991 to 31% in 1992 and 30% in 1994. The respective intensities of infection (geometric means of egg output) were 81 eggs per gram (epg) of stool in 1991, 31 epg in 1992, and 30 epg in 1994. Periportal thickening was found in 46% of patients in 1991, 32% of patients in 1992, and 35% of patients in 1994. Reversibility of PT was influenced by age (markedly lower reversibility in individuals older than 30 years) and sex (women and girls responded less favorably than did men and boys). Surprisingly, no significant difference was detected between group A and group B with respect to reversibility of PT The outcome between the 2 groups did not differ significantly. This may indicate that a single dose of praziquantel (as given to group B) may have a longer lasting effect than previously thought, that is, more than 2.5 years.

Recent evidence suggest that resistance to praziquantel (PZQ) may be developing. This would not be surprising in countries like Egypt where the drug has been used aggressively for more that 10 years. The classic phenotype of drug resistance is a significant increase in the 50% effective dose value of isolates retrieved from patients not responding to the drug. In a previous publication, we reported that such phenotypes have been isolated from humans infected with Schistosoma mansoni. Since the action of PZQ may be dependent upon the drug and host factors, most notably the immune system, we analyzed the quantitative effects of PZQ on single worms that differed in their response to PZQ when maintained in mice. Our hypothesis was that the in vitro action of the drug would correlate with it in vivo action. We confirmed this hypothesis and conclude that the in vitro action of the drug is related to its in vivo action. Knowing this relationship will assist in our ability to detect or survey for the PZQ resistant phenotype in human populations.

The new oral fixed combination artemether-lumefantrine (CGP 56697) has proved to be an effective and well-tolerated treatment of multi-drug resistant Plasmodium falciparum malaria, although cure rates using the four-dose regimen have been lower than with the currently recommended alternative of artesunate-mefloquine. Two six-dose schedules (total adult dose = 480 mg of artemether and 2,880 mg of lumefantrine) were therefore compared with the previously used four-dose regimen (320 mg of artemether and 1,920 mg of lumefantrine) in a double-blind trial involving 359 patients with uncomplicated multidrug-resistant falciparum malaria. There were no differences between the three treatment groups in parasite and fever clearance times, and reported adverse effects. The two six-dose regimens gave adjusted 28-day cure rates of 96.9% and 99.12%, respectively, compared with 83.3% for the four-dose regimen (P < 0.001). These six-dose regimens of artemether-lumefantrine provide a highly effective and very well-tolerated treatment for multidrug-resistant falciparum malaria.

We have studied the reversal of activity against Plasmodium falciparum of WR99210, a triazine antimalarial drug, and of the pro-drug PS-15 by folic acid (FA) and folinic acid (FNA). Folic acid and FNA inhibit the growth of P. falciparum in vitro at concentrations > 10(-4.5) and 10(-3.5) mol/L, respectively. The activity of pyrimethamine against Kenyan strains M24 and K39 is reduced 10-12-fold by 10(-5) mol/L of FA, and virtually eliminated by 10(-5) mol/L of FNA. Folates do not antagonise the action of WR99210 against Kenyan strains, and only partially antagonize the action of WR99210 action against the Southeast Asian strains V1/S and W282. Similarly, FA and FNA exerted weak or no antagonism of the action of PS-15. The inability of folates to antagonize the action of WR99210 can be explained in terms of high drug-enzyme affinity, but this does not account for the inability of FA and FNA to antagonize PS-15. These results suggest that action of PS-15 against P. falciparum is primarily due to a non-folate mechanism.

Malaria antibody detection is valuable in providing retrospective confirmation of an attack of malaria. Blood bank screening is another area were malaria serology is potentially useful. In the present study, we tested the presence of antibodies to Plasmodium falciparum in sera from blood bank donors of non-endemic and malaria-endemic areas of Venezuela. Sera from 1,000 blood donors were tested by an indirect immunofluorescent antibody (IFA) assay and an IgG-ELISA for the presence of malaria antibodies using a synchronized in vitro-cultured Venezuelan isolate of P. falciparum as the antigen source. A selected group of positive and negative sera (n = 100) was also tested by a dot-IgG-ELISA. Positive results (reciprocal titer > or = 40) were found in 0.8% and 3.8% of blood donors when tested by the IFA assay and in 0.8% and 2% (optical density > or = 0.2) when tested by the IgG-ELISA in Caracas (non-endemic area) and Bolivar City (endemic area), respectively. The presence of anti-malarial antibodies in some sera from non-endemic areas such as Caracas reflects the increased potential risk of post-transfusional malaria in those areas due to the mobility of the blood donors. The data obtained indicate the need to implement new blood donor policy in blood banks in developing areas. Our results also indicate that the IFA assay is the most reliable test to use in malaria serodiagnosis.

In Southeast Asia, schistosomiasis japonica is an important cause of hepatic fibrosis and gastrointestinal hemorrhage. Reliable methods to investigate portal hypertension (PHT) clinically and epidemiologically on community level are lacking. Doppler sonography is an established tool for investigating PHT in hospital settings. In Leyte, The Philippines, 137 individuals underwent color Doppler sonography, stool examination, and serology for hepatitis B and C, liver cell injury and cholestasis. A total of 85% of the study population had been infected with Schistosoma japonicum. Sonographically, periportal liver fibrosis was seen in 25% and reticular echogenicities (network pattern) in 44%. Portal blood flow was decreased or portosystemic collaterals were present in 10% (adults throughout) and correlated with periportal fibrosis, but not with network lesions. Chronic viral hepatitis was rare. Thus, hepatic lesions are frequent in adults but not in children in areas endemic for S. japonicum. Periportal liver fibrosis indicates a risk of PHT, and network pattern fibrosis apparently does not. Doppler sonography is suitable for research under tropical field conditions.

In an initial cross-sectional survey, serum, urine, and stool samples were collected from 370 participants representing about 10% of the population (n = 4,438) in Behbeet village, 50 km south of Cairo, Egypt, an area well known to be endemic solely for Schistosoma haematobium. Diagnosis was approached in two parallel ways. The first approach, which simulated actual conditions in many endemic areas in Egypt, was based on physical examination and urine and stool microscopic analysis. The second approach was based on two advanced immunodiagnostic assay systems. One system detected antibodies to species-specific microsomal antigens, the other detected circulating schistosomal antigens. Microsomal antigens from S. haematobium and S. mansoni were used to detect antibodies in the Falcon assay screening test (FAST)-ELISA and the enzyme-linked immunoelectrotransfer blot (EITB). Circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) were quantified in serum and urine samples in a sandwich ELISA using monoclonal antibodies. Parasitologically, the prevalence of S. haematobium was 7.01% in females and 25.82% in males, giving an overall prevalence of 15.8%. The combination of urine CCA and serum CAA for detecting circulating antigens and the combination of the S. haematobium adult worm microsomal antigens (HAMA) FAST-ELISA and the HAMA EITB for detecting antibodies significantly improved the sensitivity of detecting S. haematobium circulating antigens and antibodies. Also, including a medical examination as an integral part of field studies and correlating immunodiagnostic results with other clinical and investigational data allowed us to calculate an accurate estimation of S. haematobium prevalence in this area of low endemicity.

Follow-up stool examinations were carried out on two groups of the subjects who were screened negative (group 1) or positive (group 2) for Strongyloides stercoralis by the agar plate culture. This technique could detect S. stercoralis larvae in 87.5-96.4% of the subjects in group 2 and 0-5.9% of the subjects in group 1 on various days of the eight-week and four-week follow-up periods, respectively. The detection rate on each day of examination was not statistically different from that on the first day in both groups. Quantitative measurement of S. stercoralis larvae excreted in the feces of the subjects in group 2 by the standard direct smear method of Beaver and others revealed slight to marked fluctuations of the larval output in individual subjects. From the results of both stool examination methods, it could be implied that 52% of S. stercoralis-infected individuals had low-level infection.

The host immune response in onchocerciasis is believed to contribute to the clinical manifestations of infection. Mazzotti and chronic inflammatory reactions might be mediated by mechanisms involving specific IgE and reactivity of mast cells and basophils to the parasite antigens. In this report, we show that Onchocerca volvulus antigens are capable of inducing histamine release. Three types of extracts were prepared from the parasite: soluble total, surface, and cuticular collagen. Soluble extracts released histamine in all individuals with onchocerciasis at significantly higher levels (P < 0.05) than those found in endemic controls, but similar levels to those found in patients with mansonellosis. However, cuticular collagen induced significantly (P < 0.01) higher histamine release in patients with onchocerciasis than in those with mansonellosis. No reactivity against human type IV collagen was observed. Implications derived from the presence of sensitized basophils in the pathogenesis of onchocerciasis are discussed.

The occurrence of renal abnormalities was investigated in patients with onchocerciasis in comparison to individuals without onchocerciasis in Guinea. Serum creatinine levels, excretion of urinary marker proteins, and kidney size by ultrasound were determined. A high prevalence of glomerular as well as tubular dysfunctions was observed; however, no association with onchocerciasis could be detected. We also hypothesized that patients with hyperreactive onchocerciasis might be prone to develop immune-mediated glomerular disorders; however, this could not be verified. Following treatment with ivermectin, a slight but significant increase in the excretion of urinary albumin and alpha1-microglobulin was seen five days after treatment in all treated patients, whereas levels of proteinuria were significantly higher five days after treatment only in patients with high microfilarial densities. Our results indicate that ivermectin can cause glomerular and tubular disturbances in patients with onchocerciasis; however, these are minor and do not seem to be clinically relevant.

Following intravenous inoculation with horse blood-infected with the agent of human granulocytic ehrlichiosis (HGE) from a human fatality, two rhesus macaques (Macaca mulatta) exhibited pyrexia and lethargy on days 4-12 postinfection (PI). Hematology revealed neutropenia, thrombocytopenia, and anemia, with ehrlichial morulae in monocytes and neutrophils on days 4-12. Blood was polymerase chain reaction (PCR)-positive on days 4-12 and bone marrow was PCR-positive on day 11. There was a minor increase in gamma-glutamyl transpeptidase on day 12 and serum interferon-gamma levels increased by day 18. Seroconversion occurred on day 20 PI to a titer of 100 by day 22. Western blot bands characteristic of HGE included 25-, 44-, 80-, 94-, 105-, and 125-kD bands. There was generalized lymphohistiocytic infiltration in the liver, spleen, lymph nodes, and other tissues. The liver had focal hepatocyte apoptosis. There was HGE DNA (by PCR) only in the spleen. Comparable findings were not observed in a monkey that received uninfected horse blood as a control. This animal model of human disease is important for further studies of HGE diagnosis, management, and pathogenesis.

Merozoite surface antigen 1 (MSA1) is a promising candidate for vaccine development against malaria parasites. Here, we report the complete nucleotide sequence of the gene encoding the precursor to this major surface antigen of Plasmodium berghei strain ANKA using cDNA library screening and polymerase chain reaction techniques. A single open reading frame of 5,376 basepairs encoding a protein with a calculated molecular mass of 197 kD was defined. The protein contains a putative signal peptide of 19 amino acids, a membrane anchor sequence of 18 residues, and shows two epidermal growth factor-like domains rich in Cys residues at the C-terminus. There are four repeat sequences of oligopeptides in the molecule: tetrapeptide (Ser-Thr-Thr-Thr), tripeptide (Pro-Thr-Pro and Pro-Ala-Ala), and dipeptide (Ser-Gly). Furthermore, three nine-residue stretches of a motif (Ala-Ser-Asn-Pro-Gly-Ala-Ser-Ala-Ser) are located near each other. All of these repeat sequences are unexceptionally located in the variable regions when compared with other MSA1 molecules. The molecule displays 79% overall identity to the analogous antigen of P. yoelii yoelii strain YM, 70% to that of P. chabaudi chabaudi strain AS, and 38% to that of P. falciparum strain Wellcome.