Molecular features of hookworm larvae (Necator spp.) raised by coproculture from Ugandan chimpanzees and Gabonese gorillas and humans.

Species composition of Necator hookworms was surveyed in (i) Ugandan chimpanzees living around farms and villages at Bulindi, (ii) Gabonese gorillas under habituation in Moukalaba-Doudou National Park (MDNP), and (iii) Gabonese villagers living adjacent to MDNP. Internal transcribed spacers (ITS) of rDNA and partial cytochrome c oxidase subunit 1 (Cox1) gene of mtDNA were analyzed from larvae obtained by coproculture. Three ITS types (I, II and III) and three Cox1 haplotype groups (A, B and C) were demonstrated. ITS type I and Cox1 haplotype group A, representing Necator americanus, were demonstrated in the hookworm larvae from Gabonese gorillas and humans, but not from Ugandan chimpanzees. Type II and haplotype groups B and C, presumably representing N. gorillae, were found in larvae from Ugandan chimpanzees and Gabonese gorillas and humans. These features were overall similar with those found previously in the Central African Republic. Meanwhile, type III was proven in a larva from a Gabonese gorilla as the first demonstration from a non-human primate. Cox1 haplotypes obtained from Ugandan chimpanzees formed a subgroup within group B, presumably reflecting dispersal and diversification processes of the apes.

Adult hookworms (Necator spp.) collected from researchers working with wild western lowland gorillas.

BACKGROUND: In general, studies on the diversity of strongylid nematodes in endangered host species are complicated as material obtained by non-invasive sampling methods has limited value for generic and species identification. While egg morphology barely allows assignment to family, the morphology of cultivated infective third stage larvae provides a better resolution at the generic level but cannot be used for exact species identification. Morphology-based taxonomic approaches greatly depend on the examination of adult worms that are usually not available. METHODS: Hookworm parasites in two European researchers, who participated in gorilla research in the Central African Republic, were expelled after anthelmintic treatment to the faeces, collected and morphologically examined. A male worm discharged naturally from a wild bonobo (Pan paniscus) in Congo was also examined for comparison. RESULTS: Two species of Necator were identified in researchers' faecal material: Necator americanus (Stiles, 1902) and N. gorillae Noda & Yamada, 1964; the latter species differed in having a smaller body, smaller buccal cavity and shorter spicules with spade-shaped membranes situated distally. Males of N. gorillae also possessed unusual cuticular thickenings on the dorsal side of the prebursal region of the body. These characters, shared with the male worm from the bonobo, correspond well to the description of N. gorillae described from gorillas in Congo. CONCLUSIONS: Based on the morphology of the hookworms recovered in this study and previous molecular analyses of larvae developed from both humans and western lowland gorillas (Gorilla gorilla gorilla) from this locality, we conclude that the researchers became infected with gorilla hookworms during their stay in the field. This is the first report of infection with a Necator species other than N. americanus in humans.

Schistosoma japonicum and occult blood loss in endemic villages in Leyte, the Philippines.

Schistosoma japonicum has been related to anemia, but the mechanisms mediating this relationship remain unresolved. The primary objective of this study was to assess the role of occult blood loss in mediating S. japonicum-associated anemia after adjusting for age, sex, socioeconomic status (SES), and other helminth infections. The secondary objective was to identify intensity categories of risk for occult blood loss for Trichuris and hookworm after adjustment for the presence of other helminth infections. The role of occult blood loss in mediating S. japonicum-associated anemia was studied cross-sectionally in 729 individuals 8-30 years old in Leyte, The Philippines. Three stool specimens were examined in duplicate for helminth eggs. Hemoglobin, fecal occult blood loss, and anemia were measured and related to the presence and intensity of helminths. Multivariate models were made to adjust for confounding by other helminths and SES. In multivariate models, hemoglobin significantly decreased with increasing infection intensity of S. japonicum, hookworm, and T. trichuria (P < 0.0031, P < 0.0001, and P < 0.0001, respectively). Individuals with higher intensities S. japonicum and T. trichuria were significantly more likely to be fecal occult positive (odds ratio [OR] = 3.54; P = 0.008 and OR = 2.68; P = 0.013, respectively), although this was not true for individuals with hookworm. Additionally, individuals with higher intensities of S. japonicum, hookworm, and T. trichuria were all more likely to be anemic (OR = 3.7, P = 0.0002; OR = 5.3, P = 0.0003; and OR = 1.6, P = 0.021, respectively). It is likely that occult blood loss plays a role only at heavier intensity S. japonicum infections and some other mechanism, such as anemia of inflammation, may be contributing to anemia.

A modified agar plate method for detection of Strongyloides stercoralis.

The agar plate method is a new technique with high detection rates for coprological diagnosis of human strongyloidiasis. This report details modifications of the technique and establishes a standardized procedure. We recommend that all plates should be carefully observed using a microscope because macroscopic observation can lead to false negative results. It is also advisable to pour formalin solution directly into microscopically positive dishes to collect worms by sedimentation. This procedure enables one to observe worms otherwise hidden. Sealing dishes with adhesive tape prevents larvae from crawling out of the dishes, eliminating any possibility in the reduction of detection rates, and greatly improves the safety conditions for the technician performing the procedure. We consider the agar plate method to be superior to the filter paper method in detecting Strongyloides, and we believe that it will eventually become the technique of choice.

Evaluation of the skin test for hookworm as an epidemiological tool.

The usefulness of a skin test with larval Necator americanus antigen for assessment of hookworm prevalence was evaluated in an endemic area of Costa Rica. In comparison with standard coprologic techniques employed to survey the population, the skin test detected 83% of infections, showing a fairly satisfactory sensitivity. The overall specificity of the test was 50%, i.e., random. No correlation was found between skin reactivity and hookworm burden. The sensitivity of the test increased moderately with age, but its specificity decreased significantly at the same time. False positive reactions were more numerous among persons formerly infected with hookworm who had been negative for as long as 5 years. There was an indication of cross reactivity with intestinal nematodes other than hookworm. The test was used to detect hookworm infected persons in the community for selective treatment, in comparison with mass treatment of all the people in another village. The selective administration of an anthelminthic drug to only skin test positive persons did not achieve the same drop in community hookworm prevalence as did the indiscriminate treatment of the whole population.

Influence of intestinal parasitism on lactose absorption in well-nourished African children.

Hydrogen breath tests were performed in Gabon (Central Africa) after a loading dose of lactose in 67 well-nourished African children (50 with intestinal parasites and 17 unparasitized) and in 18 unparasitized young adults. All had normal nutritional status, and none had diarrhea or digestive symptoms. Parasites that were found included Ascaris lumbricoides in 76% of the parasitized children, Trichuris trichiura in 58%, Giardia in 24%, Entamoeba histolytica in 20%, Schistosoma intercalatum in 16%, and Necator Americanus in 14%. A similar proportion of parasitized (64%) or unparasitized (62.8%) subjects were lactose malabsorbers. Giardia infection was associated with a higher, but not significantly different, proportion of lactose intolerance (10 of 12, 83.3%). The presence of infection with A. lumbricoides or T. trichiura did not increase the percentage of lactose malabsorption. These data indicate that a decrease of lactase activity in well-nourished African children is not related to the presence or the importance of Ascaris or other intestinal parasites if the nutritional status is normal.

The growth and migration of Necator americanus following infection of neonatal hamsters.

Necator americanus was studied in neonatally infected hamsters in order to determine precisely the growth and migration of the parasite in this laboratory host. Most larvae stayed at the skin infection site for at least 48 hours following administration of larvae and the movement to the lungs commenced on day 3. There was no significant growth at the skin site or during the first two days in the lungs. 98% of the larvae were recovered from the lungs by day 6 and showed signs of some growth and development. Moulting larvae were seen in the lungs on days 7 and 8, but intestinal worms, which were first detected on day 7, were all L4 larvae. These worms were significantly longer than the lung stages and henceforth grew rapidly. Over 80% of the worms were recovered from the intestine on day 9, only small numbers of larvae persisting in the lungs until day 12. Moulting worms were observed in the intestine on days 17 to 21, after which growth continued and did not slow until about the fifth week. Small quantities of eggs were occasionally detected as early as day 34 and continuous egg production commenced in the seventh week of infection reaching a peak by about the 10th week.

Hookworm (Necator americanus) infection and storage iron depletion.

The relationship between iron status and the intensity of infection with hookworm was investigated in a rural population on Karkar Island, Mandang Province, Papua New Guinea. There was a significant negative correlation between plasma ferritin level and hookworm burden, which was strongest in males. In contrast, there was no correlation between plasma ferritin and hookworm egg count, and no consistent correlation between haemoglobin level or haematocrit and either measure of hookworm intensity. The results suggest that the role of hookworm in the aetiology of anaemia may be difficult to assess without the accurate measurement of hookworm burden.

Necator americanus in neonatally infected hamsters. The time-course of infection and antibody response to the surface antigens of L4 and adult worms.

Neonatal hamsters were infected with a hamster-adapted strain of Necator americanus, and the time-course of infection was followed by worm and faecal egg counts. Parasite eggs were first recorded during the 6th week of infection, increasing rapidly thereafter to peak in weeks 7-10. Male hamsters excreted more eggs than females, but both sexes were equally susceptible to infection and harboured comparable worm burdens. Faecal egg counts declined from week 10 onwards and this was associated with a loss of worms from animals with heavy infections. Low level infections were stable over the first 114 d of infection but worm fecundity nevertheless still declined over this period. Both hamster sexes responded similarly to surface antigens on adult worms, the antibody levels rising from week 5 onwards to reach a plateau in weeks 6-7, which persisted until the experiments were terminated. The major antigens recognised on the surface of adult worms had molecular masses corresponding to 25 kDa, 32 kDa, a doublet with the heaviest polypeptide resolving at 46 kDa, and a triplet with the heaviest at 67 and 93kDa. In contrast L4 had only 2 major cuticular antigens resolving at 41 and 93kDa. The 93kDa molecule on L4 and adult worms may be antigenically related.

Hookworm infection in a rural community in south India and its association with haemoglobin levels.

Faecal samples were obtained from 1113 persons living in a rural area in South India, and the hookworm ova load (Necator americanus) was determined using Kato's thick smear method. Evidence of hookworm infection was present in 92%, 77% having a count of under 100 epg, 11% a count of 1000 to 1999 epg, and 4% having counts between 2000 and 12,000 epg. Females had significantly higher ova counts than males on the average, but age did not appear to have any effect. Haemoglobin was also estimated: 80% of adult males, 87% of adult females and 90% of children were anaemic. There was a significant negative association between ova load and haemoglobin level, and the decrease in haemoglobin for a doubling of the ova load was estimated by regression analysis to be 0.18, 0.29 and 0.16 g/dl in adult males, adult females and children, respectively. There was nearly perfect agreement in the ranking of 10 clusters by mean ova count and mean haemoglobin level or percentage with anaemia.

Population biology of hookworms in west Bengal: analysis of numbers of infective larvae recovered from damp pads applied to the soil surface at defaecation sites.

The aims of this study were to analyze the seasonal distribution of infective larvae on the soil surface, to determine whether numbers of infective larvae near faeces were related to the faecal egg count of individuals, and to relate the distribution of larvae to environmental characteristics. Larvae were recovered from damp pads, applied to the soil surface in an annulus around fresh, identified stools of individuals who were participating in a larger epidemiological study. This provided an estimate of exposure to infection at the time of defaecation. Transmission was restricted to the rainy season and large aggregations of larvae were encountered earlier rather than later in the rainy season. Frequency distributions for the number of larvae extracted from each pad showed a high degree of aggregation, with most monthly counts showing good fits to the negative binomial probability distribution. Despite variations in monthly sampling means, the degree of aggregation in the population of larvae was remarkably stable over the 18 month sampling period (k of negative binomial = 0.01 to 0.08). Estimates of the degree of aggregation of the parasites in the human population were also available, and comparisons suggest that the infective larvae were much more aggregated than the parasitic stages. There was no relationship between the mean daily egg output of individuals and the number of larvae which developed and were recovered from the soil surface near the faeces. Thus, people who contribute large numbers of eggs to the environment are not necessarily those who are the greatest source of infection for others.(ABSTRACT TRUNCATED AT 250 WORDS)

The epidemiology and population biology of Necator americanus infection in a rural community in Zimbabwe.

Baseline data from an epidemiological study of hookworm infection in a rural community in Zimbabwe are presented. The infection status of an age-stratified sample of the community was assessed using anthelmintic expulsion techniques. Necator americanus was the only helminth parasite found to be present. The age-prevalence and intensity profiles rose asymptotically to an adult prevalence of about 80% and adult mean burden of 7.7 worms per host. The overall mean burden was 4.8 worms per host. The frequency distribution of N. americanus was overdispersed and well described by the negative binomial distribution with a value for the aggregation parameter, k, of 0.346. Separate estimates of k were lower in males and older hosts. The distribution patterns were difficult to reconcile with any simple process of age-dependent acquisition of an effective immune response. A significant negative correlation was recorded between per caput fecundity and worm burden, providing evidence for a density-dependent regulation of female worm fecundity. The basic reproductive rate (R0 congruent to 2) was found to be similar to estimates from other geographical areas.

A comparison of post-mortem analysis of helminths with faecal egg counts.

Intestinal helminth infections are very common amongst residents of the Colombo area. Most common by post-mortem examination of 104 cases of sudden death were Trichuris trichiura (97.1%), Necator americanus (88.5%), Enterobius vermicularis (77.9%) and Ascaris lumbricoides (40.4%). Trematode and cestode infections were not encountered except a single case of H. diminuta infection. A comparison of the results obtained by the direct smear method for examining stools missed cases harbouring gravid females of up to 3 Ascaris lumbricoides, 29 Trichuris trichiura and 66 Necator americanus. The direct smear revealed all the helminths present except threadworms in only 30% of the cases. The correlation between worm loads calculated from ova counts and actual numbers harboured was fairly close in 19 out of 35 cases for Ascaris and 37 out of 78 cases for Necator americanus. Highly erroneous results were obtained in many instances when worm loads were calculated from the results of ova counts.

A novel method for the isolation of nematode larvae using pluronic F-68-treated cellulose strips.

A new method is described for the isolation of cultured nematode larvae. This allows effective separation of larvae from fecal contamination, exsheathed larvae from cast sheaths, and viable larvae from nonviable larvae. The method involves the use of cellulose strips and has been assessed using larvae from 2 hookworm species, Necator americanus and Ancylostoma ceylanicum. Pretreatment of the cellulose strips with 1.0% (w/v) of the nonionic surfactant, Pluronic F-68, significantly increased larval recovery of both species.

Hookworm infections of schoolchildren in southern Thailand.

A study of hookworm infections of schoolchildren was conducted in Nakhon Si Thammarat Province, southern Thailand. Of the 2,940 hookworms that were recovered from the children, almost all (99.9%), were Necator americanus, only three (0.1%) were identified as Ancylostoma duodenale, and all were female worms. An estimation of the worm burden of and the worm expulsion from the schoolchildren indicated there were 17 cases of light intensity hookworm infection. Fifteen cases (88.2%) expelled worms in numbers that corresponded with the worm burden that was estimated from the number of eggs per gram of feces. Two cases (11.8%) expelled more worms than predicted. In 16 moderate intensity cases, five (31.3%) expelled worms in a quantity that corresponding with the estimated worm burden. Eleven cases (68.7%) expelled fewer worms than predicted. All cases of heavy intensity infection expelled fewer worms than predicted.

Humans and great apes cohabiting the forest ecosystem in central african republic harbour the same hookworms.

BACKGROUND: Hookworms are important pathogens of humans. To date, Necator americanus is the sole, known species of the genus Necator infecting humans. In contrast, several Necator species have been described in African great apes and other primates. It has not yet been determined whether primate-originating Necator species are also parasitic in humans. METHODOLOGY/PRINCIPAL FINDINGS: The infective larvae of Necator spp. were developed using modified Harada-Mori filter-paper cultures from faeces of humans and great apes inhabiting Dzanga-Sangha Protected Areas, Central African Republic. The first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA and partial cytochrome c oxidase subunit 1 (cox1) gene of mtDNA obtained from the hookworm larvae were sequenced and compared. Three sequence types (I-III) were recognized in the ITS region, and 34 cox1 haplotypes represented three phylogenetic groups (A-C). The combinations determined were I-A, II-B, II-C, III-B and III-C. Combination I-A, corresponding to N. americanus, was demonstrated in humans and western lowland gorillas; II-B and II-C were observed in humans, western lowland gorillas and chimpanzees; III-B and III-C were found only in humans. Pairwise nucleotide difference in the cox1 haplotypes between the groups was more than 8%, while the difference within each group was less than 2.1%. CONCLUSIONS/SIGNIFICANCE: The distinctness of ITS sequence variants and high number of pairwise nucleotide differences among cox1 variants indicate the possible presence of several species of Necator in both humans and great apes. We conclude that Necator hookworms are shared by humans and great apes co-habiting the same tropical forest ecosystems.

Rapid detection and identification of human hookworm infections through high resolution melting (HRM) analysis.

BACKGROUND: Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. METHODS: Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. CONCLUSION: The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species.