The American Journal of Tropical Medicine and Hygiene - Volume 75, Issue 2, August 2006

In May 2003, we studied amodiaquine (AQ) efficacy in children < 5 years of age with uncomplicated falciparum malaria in Magoda and Mpapayu (with insecticide treated nets [ITNs]) and Mgome (without ITNs) in Muheza, Tanzania. The trial involved 28 days of follow-up, and data were adjusted by polymerase chain reaction (PCR) genotyping of msp1 and msp2 genes. Additionally, Pfcrt codon 72–76 polymorphisms were studied by PCR and sequence-specific oligonucleotide probe (SSOP) ELISA. In 54 cases with complete follow-up, a significant difference in late treatment failure (LTF) rates was seen (60.7% in ITN versus 88.5% in non-ITN villages, P = 0.02) before PCR correction. However, after PCR correction, 23 cases (60.5%) were confirmed as reinfections, giving a true LTF rate of 21.4% (6/28) and 34.6% (9/26) in the above settings, respectively. Frequency of Pfcrt CVIET haplotype mutation pretreatment was high (97.0%); the remaining samples were CVMNK. We conclude that AQ alone is no longer effective in the study area.

A controlled randomized trial of antihelminthic treatment was undertaken in 1996–1997 in a rural area of Madagascar where populations were simultaneously infected with Ascaris lumbricoides and Plasmodium falciparum. Levamisole was administered bimonthly to 164 subjects, randomized on a family basis, whereas 186 were controls. While levamisole proved to be highly effective in reducing Ascaris egg loads in the treated group (P < 10−3 at all bimonthly visits), subjects more than 5 years of age, treated with levamisole had a significant increase in their P. falciparum densities compared with controls (P = 0.02), whereas there was no effect of anti-helminthic treatment on children 6 months to 4 years of age. The demonstration of a clear negative interaction between Ascaris infection and malaria parasite density has important implications. Single community therapy programs to deliver treatments against several parasitic infections could avoid an increase of malaria attacks after mass treatment of ascariasis.

Neutralizing antibodies to glycosylphosphatidylinositols (GPIs), which are Plasmodium falciparum surface protein anchor molecules implicated in malaria pathogenesis, are thought to protect against symptomatic malaria. Index cases of severe malaria in Malian children 3 months to 14 years of age were matched by age and residence to uncomplicated malaria and healthy controls. Serum antibodies to GPI (IgM and IgG) were measured at the time of severe malaria and after the malaria transmission season. The mean optical density values for IgM and IgG antibodies were higher in children with severe or uncomplicated malaria compared with healthy controls. Similarly, higher percentages of children with IgM and IgG antibodies to GPI were observed in the severe malaria group compared with matched healthy controls. IgG antibody levels to GPI were highest among children with cerebral malaria and children who died. The IgG antibody levels to GPI peaked during periods of malaria transmission and decreased after malaria transmission ended. A direct correlation between age and parasitemia and IgG antibodies to GPI was observed. In summary, higher levels of IgM and IgG antibodies to GPI in young children were associated with disease severity and were short-lived.

The World Health Organization recommends that pregnant women in malaria-endemic areas receive ≥ 2 doses of intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp/SP) in the second and third trimesters of pregnancy to prevent maternal anemia, placental parasitemia, and low birth weight (LBW). In 2001, a program evaluation in Koupéla District, Burkina Faso demonstrated that despite widespread use of chloroquine chemoprophylaxis, the burden of malaria during pregnancy remained high. In 2003, the Burkina Faso Ministry of Health piloted a program of IPTp/SP (three doses) and accelerated distribution of insecticide-treated nets (ITN) to pregnant women in Koupéla District. In 2004, a follow-up program evaluation was conducted. Coverage with ≥ 1 doses of IPTp/SP was high among women attending antenatal clinics (ANCs) (96.2%) and delivery units (DUs) (93.5%); ITN ownership was moderately high (ANC = 53.9%, DU = 61.6%). In multivariate analysis, ≥ 1 dose of IPTp/SP was associated with a significant reduction in the prevalence of peripheral parasitemia at ANCs (risk ratio [RR] = 0.49, P = 0.008), ≥ 2 doses of IPTp/SP were associated with a reduction in the prevalence of placental parasitemia (RR = 0.56, P = 0.02), and three doses of IPTp/SP were associated with a reduced risk of LBW (RR = 0.51, P = 0.04). The proportions of women at ANCs with peripheral parasitemia and anemia were significantly lower in 2004 than in 2001 (RR = 0.53, P = 0.001 and RR = 0.78, P = 0.003, respectively). The proportions of women at DUs with peripheral and placental parasitemia were also significantly lower in 2004 than in 2001 (RR = 0.66, P < 0.0001 and RR = 0.71, P = 0.0002, respectively). These data suggest that a package of IPTp/SP and ITNs is effective in reducing the burden of malaria during pregnancy in Burkina Faso.

Antigenic variation in Plasmodium falciparum erythrocyte membrane protein 1, caused by a switch in transcription of the encoding var gene, is an important feature of malaria. In this study, we quantified the relative abundance of var gene transcripts present in P. falciparum parasite clones using real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional RT-PCR combined with cloning and sequencing, with the aim of directly comparing the results obtained. When there was sufficient abundance of RNA for the real-time RT-PCR assay to be operating within the region of good reproducibility, RT-PCR and real-time RT-PCR tended to identify the same dominant transcript, although some transcript-specific issues were identified. When there were differences in the estimated relative amounts of minor transcripts, the RT-PCR assay tended to produce higher estimates than real-time RT-PCR. These results provide valuable information comparing RT-PCR and real-time RT-PCR analysis of samples with small quantities of RNA as might be expected in the analysis of field or clinical samples.

Knowledge of the baseline malaria transmission in a given environment is important to guide malaria control interventions. However, in Uganda, recent information on malaria transmission intensity is lacking. Therefore, a 1-year entomological study was conducted in seven ecologically different sites throughout the country to assess spatial and temporal patterns in malaria transmission intensity. Anopheles gambiae sensu stricto was the main vector in five of the seven study sites, and An. funestus was the most important vector in the two other sites. In a peri-urban village, An. arabiensis contributed substantially to malaria transmission. Clear differences in annual entomological inoculation rates (AEIR) were observed between the study sites, ranging from 4 infective bites per person per year in the southwestern part of the country to >1,500 infective bites per person per year in a swampy area near the Nile River. Between villages with parasite prevalences of ≥ 80% in children between 1 and 9 years old, a 4-fold difference in AEIR was observed. Based on the observed behavior of the vectors, insecticide-treated bed nets will be highly effective in controlling malaria. However, in the high transmission areas, additional measures will be needed to reduce the malaria burden to acceptable levels.

This study investigated the impact of single, double, and triple insecticide sprayings on indoor infestation of Triatoma dimidiata in Jutiapa, Guatemala. Up to three successive insecticide sprayings were applied in villages, where the indoor infestation index was > 5% before each spraying round or located adjacent to the persistently infested villages. Among 64 villages with single spraying, the mean indoor infestation index reduced from 20.8% to 1.4% after 12 months, but rose to 8.1% after 33 months. In 40 double-sprayed villages, it decreased from 41.9% to 11.9% by the first spraying and to 4.8% by the second spraying. For 12 villages with triple spraying, it reduced from 40.6% to 13.2%, 10.9%, and 4.1% through each spraying round. Geographic analysis showed that originally highly infested villages were spatially clustered and were likely to remain infested after the sprayings. Indoor infestation of T. dimidiata can be controlled with less than three rounds of spraying.

We used polymerase chain reaction (PCR)-based DNA profiling to determine the person from whom Anopheles funestus and An. gambiae collected in natural human habitations obtained their blood meals. Less than 20% of human hosts contributed to > 50% of all blood meals, and 42% were not bitten at all, including people in the age group bitten most often. As expected, bites were unevenly distributed by age (young adults > older adults > children). Use of untreated bed nets by adults, but not children, seemed to redirect bites to children. Multiple blood meals in a single gonotrophic cycle occurred frequently enough to be epidemiologically important (14% for An. funestus and 11% for An. gambiae). Mosquitoes that did not bite a person who slept in the collection house can affect estimation of entomological risk. Mosquito–human interactions did not differ across ecologically and epidemiologically distinct highland and lowland sites.

Anopheles darlingi is the most important malaria vector in the Amazon basin of South America, and is capable of transmitting both Plasmodium falciparum and P. vivax. To understand the genetic structure of this vector in the Amazonian region of Peru, a simple polymerase chain reaction (PCR)–based test to identify this species of mosquito was used. A random amplified polymorphic DNA–PCR was used to study genetic variation at the micro-geographic level in nine geographically separate populations of An. darlingi collected in areas with different degrees of deforestation surrounding the city of Iquitos. Within-population genetic diversity in nine populations, as quantified by the expected heterozygosity (HE), ranged from 0.27 to 0.32. Average genetic distance (FST) among these populations was 0.017. These results show that the nine studied populations are highly homogeneous, suggesting that strategies can be developed to combat this malaria vector as a single epidemiologic unit.

Anopheles gambiae females in a relatively isolated hut and all larvae from larval habitats within 100 m of the hut were collected in August 2001 in western Kenya. Among 42 aquatic habitats, 16 had A. gambiae larvae. Two hundred fifty larvae and 58 adults were genotyped using nine microsatellite markers to infer sibling relationship between the larvae and maternity between the females and larvae. The pairwise genetic relatedness of A. gambiae larvae per habitat ranged from −0.4112 to 0.9375, indicating that full siblings, half siblings, and genetically unrelated individuals presented at those habitats with multiple larvae. From a likelihood analysis, it was estimated that 56.6% of females had larvae in multiple habitats. These results substantiate that one A. gambiae female uses multiple breeding sites for oviposition, and thus, average genetic relatedness for breeding sites with high larval populations tends to be low.

A previously developed method to identify members of the Culex pipiens complex exploiting polymorphisms in a nuclear intron (acetylcholinesterase [ACE] based-assay) cannot differentiate the two forms of Cx. pipiens: form pipiens and form molestus. Notably, the two forms seem to differ extensively in behavior and physiology and likely have very different epidemiologic importance. Because they are morphologically indistinguishable, molecular methods are critical for the evaluation of their relative importance. Although the two forms of Cx. pipiens have been distinguished using a panel of microsatellite loci, such a protocol is laborious and expensive. We developed a rapid assay based on polymorphisms in the flanking region of a microsatellite locus. Used in conjunction with the ACE-assay, this new assay allows the identification of pure and hybrid populations of the two Cx. pipiens forms as well as those including Cx. quinquefasciatus. We discuss the usefulness of the method as well as limitations to its application.

Recently, two techniques, polymerase chain reaction (PCR) amplification and sequencing of cytochrome b gene (cyt b gene sequencing) and polymorphism-specific PCR (PS-PCR) were recommended for Leishmania species identification. Before this study, however, the accuracy of these methods had not been tested against the multilocus enzyme electrophoresis, the current gold standard technique on this task. Therefore, a trial was done for the first time to compare the results obtained by these techniques, using 17 Argentinean Leishmania stocks in independent assays. For all the stocks examined, the same results at species level were obtained by the three techniques. Among them, 14 were assigned to L. (Viannia) braziliensis, and three to L. (V.) guyanensis. The two techniques, cyt b gene sequencing and PS-PCR, were able to distinguish between all the proven species responsible for leishmaniases in Argentina. Thus, both techniques were validated and could be used independently for the species designation of Leishmania parasites in the country.

Seventeen Leishmania stocks isolated from cutaneous lesions of Pakistani patients were studied by multilocus enzyme electrophoresis and by polymerase chain reaction amplification and sequencing of the cytochrome b (Cyt b) gene. Eleven stocks that expressed nine zymodemes were assigned to L. (Leishmania) major. All of them were isolated from patients in the lowlands of Larkana district and Sibi city in Sindh and Balochistan provinces, respectively. The remaining six, distributed in two zymodemes (five and one), isolated from the highland of Quetta city, Balochistan, were identified as L. (L.) tropica. The same result at species level was obtained by the Cyt b sequencing for all the stocks examined. No clear-cut association between the clinical features (wet or dry type lesions) and the Leishmania species involved was found. Leishmania (L.) major was highly polymorphic compared with L. (L.) tropica. This difference may be explained by the fact that humans may act as a sole reservoir of L. (L.) tropica in anthroponotic cycles; however, many wild mammals can be reservoirs of L. (L.) major in zoonotic cycles.

Mucosal leishmaniasis (ML) is often clinically silent until reaching a highly advanced state. In this prospective study, 6 of 220 patients with early cutaneous leishmaniasis were diagnosed with mucosal involvement by otorhinolaryngological examination (a rate similar to the reported rate of late ML). Detection of early ML may represent an important strategy in preventing severe mucosal destruction in human leishmaniasis.

A patient with an ulcerated cutaneous leishmaniasis of the pinna had suppurative otochondritis after a first unsuccessful course of treatment with meglumine antimoniate. Although the Leishmania ulceration healed after a second course of meglumine antimoniate, and despite three oral dicloxacillin or pristinamycin courses, the otochondritis extended and an abscess developed. Pus from the abscess revealed a pure culture of Pseudomonas aeruginosa. Five days of oral ciprofloxacin plus rifampin led to a marked improvement. The P. aeruginosa isolate was sensitive to ciprofloxacin but fully resistant to rifampin. Healing with minimal mutilation was obtained at the end of a six-week course of multiple antibiotic therapy. Pseudomonas aeruginosa otochondritis was a co-factor of cartilage mutilation in this patient. Thus, infection with P. aeruginosa should be promptly treated when present in tender cutaneous or mucosal leishmaniasis lesions near cartilaginous areas.

Previous studies have shown that variation in the distribution of vectors associated to the transmission of Leishmania species may be related to climatic changes. However, the potential implications of these ecological changes in human health need to be further defined in various endemic populations where leishmaniasis carries a substantial burden of disease such as in Northeastern Colombia. Herein, we report the impact of El Niño Southern Oscillation climatic fluctuations during 1985–2002 in the occurrence of cases of leishmaniasis in two northeastern provinces of Colombia. During this period, we identified that during El Niño, cases of leishmaniasis increased, whereas during La Niña phases, leishmaniasis cases decreased. This preliminary data show how climatic changes influence the occurrence of leishmaniasis in northeastern Colombia and contributes to the growing body of evidence that shows that the incidence of vector-borne diseases is associated with annual changes in weather conditions.

The potential relationship between Schistosoma mansoni and anemia was examined using data obtained by the Schistosomiasis Control Initiative (SCI) before (baseline) and 1 year after (follow-up) a chemotherapeutic treatment program in Uganda. Changes in hemoglobin (Hb) levels in 2,788 children in relation to their schistosomiasis and/or hookworm infection intensity category and baseline anemia status were analyzed. At baseline, significant predictors of childhood anemia were intensities of S. mansoni and hookworm infection. At follow-up, moderate or heavy hookworm as well as heavy S. mansoni infections were important. Children heavily infected with S. mansoni or hookworm had significantly lower Hb counts at baseline compared with those not infected. Among anemic children at the baseline survey, a significant increase in Hb counts of 0.834 g/dL after treatment was found. Our results suggest that anemia is associated with schistosomiasis and hookworm in Ugandan children and that such anemia shows a significant improvement after chemotherapy.

Some have claimed that triclabendazole, a safe and efficacious drug for the treatment of fascioliasis, also exhibits antischistosomal properties, but results are conflicting. We assessed the effect of triclabendazole and its two main metabolites against two different strains of Schistosoma mansoni harbored in mice. Low worm burden reductions (18.6–35.9%) were observed in mice infected with an Egyptian strain of S. mansoni and treated with a single dose of 120 mg/kg 3 days before infection or single/double doses of 120–200 mg/kg 7 weeks after infection. Triclabendazole failed to significantly reduce hepatic and intestinal tissue egg loads, and eggs of all developmental stages were observed. Administration of 400 mg/kg of either triclabendazole, triclabendazole sulphone, or triclabendazole sulfphoxide to mice infected with a Liberian strain of S. mansoni resulted in worm burden reductions < 10%. In comparison, high worm burden reductions (82–100%) were observed in S. mansoni–infected mice treated with single oral doses of 400, 500, or 500 mg/kg twice a day praziquantel, regardless of the S. mansoni strain. We conclude that triclabendazole and its main metabolites display weak and inconsistent schistosomicidal activities.

The eastern Qinghai-Tibet plateau of China is a highly endemic region of echinococcosis where Echinococcus granulosus sensu stricto (sheep strain), Echinococcus multilocularis, and Echinococcus shiquicus are distributed sympatrically. We developed a polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) method for the identification of the three species in this region. The PCR-RFLP showed the dual infection of animals with different Echinococcus spp. The first case was a domestic dog concurrently infected with adults of E. granulosus and E. multilocularis. The second case was a plateau pika (Ochotona curzoniae) harboring metacestodes of E. multilocularis and E. shiquicus in the liver. The high susceptibility of some mammalian hosts to the parasites and the high prevalence of the three co-endemic species probably increase the chance of mixed infections in the eastern Tibetan plateau.