Ronald H. Guderian

Albendazole treatment of children with ascariasis enhances the vibriocidal antibody response to the live attenuated oral cholera vaccine CVD 103-HgR.

Because concurrent infections with geohelminth parasites might impair the immune response to oral vaccines, we studied the vibriocidal antibody response to the oral cholera vaccine CVD 103-HgR in children infected with Ascaris lumbricoides and investigated the effect of albendazole pretreatment on the postvaccination response. Children with ascariasis were randomized to receive either 2 sequential doses of 400 mg of albendazole or placebo. After the second dose, CVD 103-HgR was given, and serum vibriocidal antibody levels were measured before and 10 days after vaccination. Postvaccination rates of seroconversion were greater in the treatment group that received albendazole (P=.06). Significantly greater rates of seroconversion and geometric mean titer were observed in the albendazole group in subjects with non-O ABO blood groups. A significant association was observed between vibriocidal seroconversion rates and treatment group, suggesting that A. lumbricoides infections impair the immune response to oral cholera vaccine, particularly in subjects of non-O blood groups.

Evidence for hybridization by multilocus enzyme electrophoresis and random amplified polymorphic DNA between Leishmania braziliensis and Leishmania panamensis/guyanensis in Ecuador.

ABSTRACT. The taxonomic attribution of four Leishmania stocks isolated from humans in Ecuador has been explored by both multilocus enzyme electrophoresis and random amplified polymorphic DNA. For three loci, MLEE results showed patterns suggesting a heterozygous state for a diploid organism, while the corresponding homozygous states are characteristic of the Leishmania panamensis/guyanensis complex and Leishmania braziliensis. RAPD profiles exhibited for several primers a combination of the Leishmania panmensis/guyanensis complex and L. braziliensis characters. These data hence suggest that the four stocks are the results of hybridization between L. panamensis/guyanensis and L. braziliensis. MLEE data show that the results cannot be attributed to either mixture of stocks, or an F1 in the framework of a simple Mendelian inheritance.

Onchocerca volvulus Glycolytic Enzyme Fructose-1,6-Bisphosphate Aldolase as a Target for a Protective Immune Response in Humans

ABSTRACT To identify potential vaccine candidates for the prevention of infection with the filarial nematode Onchocerca volvulus, we screened an O. volvulus L3 stage cDNA library with sera from putatively immune (PI) subjects, and a prominent immunogenic clone of 1,184 nucleotides was identified. It contained an open reading frame of 363 amino acids encoding the glycolytic enzyme fructose 1,6 bisphosphate aldolase (Ov-fba-1). Immunolocalization experiments demonstrated that the protein was most abundantly expressed in metabolically active tissues, including body wall muscle and the reproductive tract of adult female worms. Immunoelectron microscopy of L3 demonstrated binding in the region where the cuticle separates during molting, in the channels connecting the esophagus to the cuticle, and in the basal lamina surrounding the esophagus and the body cavity. Among subjects from areas where this organism is endemic specific humoral and cellular immune responses to recombinant protein were observed in both PI and infected subjects, whereas responses were not observed among subjects who had not been exposed to O. volvulus. Despite the absence of differential responsiveness in parasite-exposed human populations, when the recombinant was tested for protective efficacy in a mouse chamber model, a reduction in survival of larvae by ca. 50% was seen. This observation provides support for the further study of this parasite enzyme as a vaccine candidate in larger animal models.

Early Human Infection with Onchocerca volvulus Is Associated with an Enhanced Parasite-Specific Cellular Immune Response

The immune response after early exposure to or infection with Onchocerca volvulus was investigated in an autochthonous focus caused by the migration of infected persons to a previously unaffected area in Ecuador. Peripheral blood mononuclear cell (PBMC) proliferative and cytokine responses (interferon [IFN]-gamma and interleukin [IL]-5) to filarial antigens were measured in 14 subjects with serologic evidence of exposure and in 7 subjects with evidence of dermal microfilarial DNA and were compared with responses in 43 subjects with chronic O. volvulus infections. PBMC proliferative and cytokine responses (IFN-gamma and IL-5) to parasite antigens were elevated in the early exposure/infection group, compared with those in the chronic infection group. Addition of an IL-10-neutralizing antibody to filaria antigen-stimulated cultures resulted in significantly elevated proliferative responses in the chronic infection group. The findings suggest that early exposure and early parasite patency are associated with a vigorous cellular response, but, as infections become chronic, the cellular response becomes down-regulated, partly through an IL-10-dependent mechanism.

Treatment of human pulmonary paragonimiasis with triclabendazole: clinical tolerance and drug efficacy

An open clinical trial to determine the efficacy and tolerability of postprandial doses of triclabendazole against Paragonimus mexicanus in 62 patients with pulmonary paragonimiasis from the Ecuadorian Amazon region was performed. Praziquantel was used as therapeutic control. Patients were allocated at random to the following 4 therapeutic regimens: triclabendazole, 5 mg/kg once daily for 3 d (16 patients), 10 mg/kg twice on one day (15 patients), and 10 mg/kg in a single dose (16 patients), and praziquantel, 25 mg/kg thrice daily for 3 d (15 patients). Clinical tolerance, based on the frequency and severity of adverse reactions, was superior in all 3 triclabendazole regimens to that of praziquantel. No alteration was observed in hepato-renal functions or haematological values. The clinical symptoms resolved at a comparable rate in all 4 treatment groups. A more rapid parasitological response to treatment, as determined by the reduction in the average number of parasite eggs found in sputum, was seen in patients treated with triclabendazole than with praziquantel. By day 90, 60 patients had no egg detected in their sputum; 2 patients, treated with a single dose of 10 mg/kg, had a few and were re-treated with triclabendazole (5 mg daily for 3 d). On day 365, none of the patients had eggs in their sputum. Triclabendazole can be recommended as an alternative drug of choice for the treatment of pulmonary paragonimiasis; it is as effective as praziquantel in clearing infections and better tolerated.

Interruption of infection transmission in the onchocerciasis focus of Ecuador leading to the cessation of ivermectin distribution.

Introduction: A clinically significant endemic focus of onchocerciasis existing in Esmeraldas Province, coastal Ecuador has been under an ivermectin mass drug administration program since 1991. The main transmitting vector in this area is the voracious blackfly, Simulium exiguum. This paper describes the assessments made that support the decision to cease mass treatment. Methodology and Principle Findings: Thirty-five rounds of ivermectin treatment occurred between 1991–2009 with 29 of these carrying >85% coverage. Following the guidelines set by WHO for ceasing ivermectin distribution the impact on parasite transmission was measured in the two vector species by an O-150 PCR technique standard for assessing for the presence of Onchocerca volvulus. Up to seven collection sites in three major river systems were tested on four occasions between 1995 and 2008. The infectivity rates of 65.0 (CI 39–101) and 72.7 (CI 42–116) in 1995 dropped to zero at all seven collection sites by 2008. Assessment for the presence of antibodies against O. volvulus was made in 2001, 2006, 2007 and 2008 using standard ELISA assays for detecting anti-Ov16 antibodies. None of total of 1810 children aged 1–15 years (between 82 and 98% of children present in the surveyed villages) tested in the above years were found to be carrying antibodies to this antigen. These findings were the basis for the cessation of mass drug treatment with ivermectin in 2009. Significance: This fulfillment of the criteria for cessation of mass distribution of ivermectin in the only known endemic zone of onchocerciasis in Ecuador moves the country into the surveillance phase of official verification for national elimination of transmission of infection. These findings indicate that ivermectin given twice a year with greater than 85% of the community can move a program to the final stages of verification of transmission interruption.

Impact of long-term treatment of onchocerciasis with ivermectin in Ecuador: potential for elimination of infection

BackgroundOnchocerciasis is a leading cause of blindness worldwide, hence elimination of the infection is an important health priority. Community-based treatment programs with ivermectin form the basis of control programs for the disease in Latin America. The long-term administration of ivermectin could eliminate Onchocerca volvulus infection from endemic areas in Latin America.MethodsA strategy of annual to twice-annual treatments with ivermectin has been used for onchocerciasis in endemic communities in Ecuador for up to 14 years. The impact of ivermectin treatment on ocular morbidity, and O. volvulus infection and transmission was monitored in seven sentinel communities.ResultsOver the period 1990–2003, high rates of treatment coverage of the eligible population were maintained in endemic communities (mean 85.2% per treatment round). Ivermectin reduced the prevalence of anterior segment disease of the eye to 0% in sentinel communities and had a major impact on the prevalence and transmission of infection, with possible elimination of infection in some foci.ConclusionThe distribution of ivermectin in endemic communities in Ecuador might have eliminated ocular morbidity and significant progress has been made towards elimination of the infection. A strategy of more frequent treatments with ivermectin may be required in communities where the infection persists to achieve the objective of elimination of the infection from Ecuador. The elimination of the infection from an endemic country in Latin America would be a major public health achievement and could stimulate the implementation of elimination strategies in other endemic countries.

A new TNFA promoter allele identified in South American Blacks

Numerous DNA sequence polymorphisms have been identified near the TNFA and TNFB genes (Wilson et al. 1992; Udalova et al. 1993; Takashiba et al. 1993; D’Alfonso and Richiardi 1994; Hamann et al. 1995). Recently, one of these mutations (TNF2, G to A at nucleotide –308) has been associated with pathology in malaria and leishmania (McGuire et al. 1994; Cabrera et al. 1995). Potentially, mutations of this nature may alter TNFA expression and influence clinical manifestations associated with infectious diseases. Human genomic DNA was prepared from whole blood (Zimmerman et al. 1993) collected from an Ecuadoran study population (N = 85) comprised of Chachi Amerindians (CA; N = 37) and South American Blacks (SAB; N = 48). Polymerase chain reaction (PCR) conditions were identical to those described previously (Zimmerman et al. 1993). The upstream, (+)-strand primer for the TNFA promoter (TNFAp) amplicons was TNFA1, 59TTCCTGCATCCTGTCTGGAAG-39; downstream, (–)strand primers include TNFA2 59-CAGCGGAAAACTTCCTTGGTG-39 and TNFA3, 59-CGGATCATGCTTTCAGTGCTC-39. The temperature cycling conditions for both amplification strategies presented below were 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s for 40 cycles. Following PCR amplification of a 327 base pair (bp) TNFAp amplicon (nucleotides –396 to –69; primers TNFA1 to TNFA2) from each study subject, heteroduplex analysis identified a new TNFAp allele in four individuals by unique electrophoretic mobility (unpublished data) when compared with previously identified TNFAp alleles. Three of the four individuals were not related and were studied further to characterize the DNA sequence polymorphism. For DNA sequence analysis, PCR of a 599 bp TNFAp amplicon was performed. This larger product (nucleotides –396 to +203; primers TNFA1 to TNFA3) included the proximal promoter, the transcription start site, the 59 untranslated region and the initiating methionine codon. Following PCR amplification, amplicons were cloned (pCRII; Invitrogen, La Jolla, CA) and sequenced (ABI 373 a automated DNA sequencer; Applied Biosystems Division of Perkin Elmer, Foster City, CA). Figure 1 shows a comparison of the new TNFAp allele, TNFAp-4, with those reported previously (Wilson et al. 1992; Takashiba et al. 1993; D’Alfonso and Richiardi 1994; Hamann et al. 1995). The TNFAp-4 sequence is the consensus of nine individual plasmid clones from the three unrelated study subjects, sequenced in both directions. TNFAp-4 differs from TNFAp-1 (Nedwin et al. 1985) by a single G to A transition at position –244 (100% concordance between subjects). This mutation introduces a Dde I restriction endonuclease cleavage site (C/TNAG; nucleotides –247 to –243) which is diagnostic for TNFAp-4. Upon Dde I treatment, TNFAp amplicons from each of the previously identified study subjects produced the expected 151 and 174 bp fragments following agarose gel electrophoresis and ethidium bromide staining (unpublished data). Alleles TNFAp-2 (G to A, –308) and TNFAp-3 (G to A, –238) also differ by single point mutations when compared with TNFAp-1. Results from this study cannot verify the finding that nucleotide –376 (G to A; Hamann et al. 1995) exhibits DNA sequence polymorphism. Interestingly, the mutations characterizing both TNFAp-3 and TNFAp-4 occur within a putative Y box (D’Alfonso and Richiardi 1994), a DNA sequence motif involved in regulating expression of major histocompatibility complex class II genes. This represents the fourth TNFAp allelic sequence observed in multiple individuals. Allelic frequencies of the four TNFAp alleles within this Ecuadoran study population are shown in Table 1. Results (Table 1) illustrate that TNFAp-4 is present only in SAB (f = 0.042). Of the other The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U42625

Chagas disease in Ecuador: evidence for disease transmission in an Indigenous population in the Amazon region.

Two well-defined synthetic peptides TcD and PEP2 were used in a sero-epidemiological study for the detection of Trypanosoma cruzi infections in an indigenous group in the Amazon region of Ecuador. Of the 18 communities studied along the Rio Napo, province of Napo, 15 (83.3%) were found to be positive for T. cruzi infection. Of the 1,011 individuals examined 61 (6.03%) resulted positive. A prevalence of infection of 4.8% was found in children aged 1-5 years. The prevalence of infection increased with age, with adults 50 years or older showing a maximum prevalence of 18.8%. Autochthonous transmission of T. cruzi is present among this isolated indigenous population.

Immunoglobulin kappa chain allotypes (KM) in onchocerciasis.

GM and KM allotypes, powerful tools for genetic characterization of human populations, have been shown to play an important role in genetic predisposition to some infectious diseases. Two diverse racial groups--Afro-Ecuadorians and Amerindians--living in a single restricted geographical area of Ecuador, appear to have different risk factors for acquisition and clinical expression of onchocerciasis, a disease caused by the filarial parasite Onchocerca volvulus. In this study, GM and KM allotypes were determined in 25 Afro-Ecuadorians and 24 Amerindians infected with Onchocerca volvulus (INF) and in putative immune individuals (PI). In Afro-Ecuadorians, the frequency of the homozygous KM 3 phenotype was significantly decreased in INF as compared with the PI group (20 vs. 68%; P= 0.0012), while the frequency of the heterozygous KM 1,3 phenotype was increased in INF as compared with the PI subjects (48 vs. 9%; P= 0.0044). These results suggest that in Afro-Ecuadorians KM 3 is associated with a lower relative risk (resistance), whereas KM 1,3 is associated with an increased risk (susceptibility) of onchocerciasis.