Performance evaluation of Baermann techniques: The quest for developing a microscopy reference standard for the diagnosis of Strongyloides stercoralis

BackgroundSoil-transmitted helminths (STH) are common in low and middle income countries where there is lack of access to clean water and sanitation. Effective diagnosis and treatment are essential for the control of STH infections. However, among STH parasites, Strongyloides stercoralis is the most neglected species, both in diagnostics and control strategies. Diagnostic methods cover different approaches, each with different sensitivities and specificities, such as serology, molecular techniques and microscopy based techniques. Of the later, the Baermann technique is the most commonly used procedure. In the literature, several ways have been described to perform the Baermann method, which illustrates the overall lack of a ‘(gold) reference standard’ method for the diagnosis of S. stercoralis infection. In this study we have evaluated the performance of three Baermann techniques in order to improve the reference standard for the microscopic diagnosis of S. stercoralis infection thereby facilitating individual case detection, mapping of the disease and proper evaluation of treatment responses.Methods/Principal findingsA community based cross sectional study was conducted at Zenzelima, Bahir Dar Zuria Ethiopia. A total of 437 stool samples were collected and analyzed by the following procedures: conventional Baermann (CB), modified Baermann (MB), and modified Baermann with charcoal pre-incubation (MBCI). The diagnostic sensitivity and Negative Predictive Value (NPV) of each technique was calculated using the combination of all the three techniques as a composite reference standard. Our result indicated that larvae of S. stercoralis were detected in 151 (34.6%) stool samples. The prevalence of S. stercoralis infection based on the three diagnostic methods was 9.6%, 8.0%, and 31.3% by CB, MB, and MBCI respectively. The sensitivity and NPV for CB, MB, and MBCI were 26.7% and 70.8%, 22.1% and 69.6%, and 87.0% and 93.2%, respectively. The MBCI showed significant difference (P- value = <0.001) in the sensitivity and NPV values when compared with CB and MB values. The agreement between CB, MB, and MBCI with the composite reference standard was 31.8%, 26.7%, 89.6%, respectively.Conclusion/SignificanceOur results suggest the superior performance of MBCI. It is relatively easy to implement, simple to perform and comparatively cheaper. The CB is by far the commonly used method in routine diagnostic although this technique significantly underestimates the true burden of the disease and thereby contributing to the exclusion of S. stercoralis from the control strategies. Therefore, MBCI is recommended as a routine microscopy-based diagnostic test for S. stercoralis infection, particularly in settings where molecular procedures are not available.     

Phylogenetic characterisation of Taenia tapeworms in spotted hyenas and reconsideration of the “Out of Africa” hypothesis of Taenia in humans

The African origin of hominins suggests that Taenia spp. in African carnivores are evolutionarily related to the human-infecting tapeworms Taenia solium, Taenia saginata and Taenia asiatica. Nevertheless, the hypothesis has not been verified through molecular phylogenetics of Taenia. This study aimed to perform phylogenetic comparisons between Taenia spp. from African hyenas and the congeneric human parasites. During 2010–2013, 233 adult specimens of Taenia spp. were collected from 11 spotted hyenas in Ethiopia. A screening based on short DNA sequences of the cytochrome c oxidase subunit 1 gene classified the samples into four mitochondrial lineages designated as I–IV. DNA profiles of nuclear genes for DNA polymerase delta (pold) and phosphoenolpyruvate carboxykinase (pepck) showed that lineages II and III can be assigned as two independent species. Common haplotypes of pold and pepck were frequently found in lineages I and IV, suggesting that they constitute a single species. Morphological observations suggested that lineage II is Taenia crocutae, but the other lineages were morphologically inconsistent with known species, suggesting the involvement of two new species. A phylogenetic tree of Taenia spp. was reconstructed by the maximum likelihood method using all protein-coding genes of their mitochondrial genomes. The tree clearly demonstrated that T. crocutae is sister to T. saginata and T. asiatica, whereas T. solium was confirmed to be sister to the brown bear tapeworm, Taenia arctos. The tree also suggested that T. solium and T. arctos are related to two species of Taenia in hyenas, corresponding to lineages I + IV and III. These results may partially support the African origin of human-infecting Taenia spp., but there remains a possibility that host switching of Taenia to hominins was not confined to Africa. Additional taxa from African carnivores are needed for further testing of the “Out of Africa” hypothesis of Taenia in humans.     

Effect of Drought and Weed Management on Maize Genotypes and the Tensiometric Soil Water Content of an Eutric Nitisol in South Western Nigeria

In the dry savannas of West and Central Africa, where low soil fertility, unpredictable rainfall, weed competition and recurrent drought are major constraints to maize production, the development of tropical maize genotypes with high and stable yields under drought and low-nitrogen condition is very important since access to these improved genotypes may be the only affordable alternative to many small scale farmers. Field trials were conducted in 2002 and 2003 at Ikenne southwestern Nigeria to investigate the effect of weed pressures and drought stress on 2 maize (Zea mays L.) hybrids (9134-14, 9803-9) and 2 open-pollinated varieties (STREVIWD, IYFDCO1). Irrigation was withdrawn 4 weeks after planting (about four weeks to mid-flowering) in the drought stress while the adjacent watered treatment had irrigation throughout the growing period. The weed pressures were the completely weeded plots (hand weeding every week) and weedy plots (weeded once, 2 weeks after planting). The experiment was a split plot in a randomized complete block design with four replicates. Drought stress reduced the stover weight and grain yield of the maize cultivars by 6% and 34% respectively. Weed-free plots had maize with higher agronomic traits than unweeded treatments. Hybrid 9803-9 was more susceptible to drought and weed stress as indicated in the stover weight and grain yield. STREVIWD an open-pollinated variety (OPV) and Hybrid 9134-14 had superior performances in terms of grain yield and shorter anthesis silking interval. Soil moisture content was higher in the unweeded plots while the uptake of moisture was highest in drought susceptible hybrid 9803-9. Irrespective of the genotypes, maize (hybrid and OPV) was more tolerant to drought in a weed-free environment than in unweeded conditions. There existed a negative but significant correlation between weed biomass and chlorophyll content (−0.29, P < 0.01), grain yield (−0.45, P < 0.05), ear plant⁻¹ (−0.27, P < 0.05) and kernel-number (−0.366 P < 0.01).     

Identification of resistance-associated proteins in closely-related maize lines varying in aflatoxin accumulation

Aspergillus flavus infection of maize and subsequent contamination with carcinogenic aflatoxins poses serious health concerns, especially in developing countries. Maize lines resistant to A. flavus infection have been identified; however, the development of commercially-useful aflatoxin-resistant maize lines has been hindered due to a lack of breeding markers. To identify maize resistance-associated proteins (RAPs) as potential markers for breeding, 52 BC1S4 lines developed from crosses between five African maize inbreds and five temperate aflatoxin-resistant lines were screened using the kernel screening assay. Five pairs of closely-related lines that had 75?C94% genetic similarity within each pair and which varied within each pair in aflatoxin accumulation were selected for proteomic investigation. Kernel embryo and endosperm protein profile differences within the pair and across pairs were compared using two-dimensional polyacrylamide gel electrophoresis. Differentially expressed (??1.5-fold) RAPs were sequenced through tandem mass spectrometry and were identified as antifungal, stress-related, storage or regulatory proteins. Sequence homology analysis highlighted several proteins in maize that confer resistance to A. flavus infection and/or aflatoxin production.     

A USA–Africa collaborative strategy for identifying, characterizing, and developing maize germplasm with resistance to aflatoxin contamination

Aflatoxin contamination of maize by Aspergillus flavus poses serious potential economic losses in the US and health hazards to humans, particularly in West Africa. The Southern Regional Research Center of the United States Department of Agriculture, Agricultural Research Service (USDA-ARS-SRRC) and the International Institute of Tropical Agriculture (IITA) initiated a collaborative breeding project to develop maize germplasm with resistance to aflatoxin accumulation. Resistant genotypes from the US and selected inbred lines from IITA were used to generate backcrosses with 75% US germplasm and F₁ crosses with 50% IITA and 50% US germplasm. A total of 65 S₄ lines were developed from the backcross populations and 144 S₄ lines were derived from the F₁ crosses. These lines were separated into groups and screened in SRRC laboratory using a kernel-screening assay. Significant differences in aflatoxin production were detected among the lines within each group. Several promising S₄ lines with aflatoxin values significantly lower than their respective US resistant recurrent parent or their elite tropical inbred parent were selected for resistance-confirmation tests. We found pairs of S₄ lines with 75–94% common genetic backgrounds differing significantly in aflatoxin accumulation. These pairs of lines are currently being used for proteome analysis to identify resistance-associated proteins and the corresponding genes underlying resistance to aflatoxin accumulation. Following confirmation tests in the laboratory, lines with consistently low aflatoxin levels will be inoculated with A. flavus in the field in Nigeria to identify lines resistant to strains specific to both US and West Africa. Maize inbred lines with desirable agronomic traits and low levels of aflatoxin in the field would be released as sources of genes for resistance to aflatoxin production.     

Molecular identification of unilocular hydatid cysts from domestic ungulates in Ethiopia: implications for human infections

To identify the etiologic agents of cystic echinococcosis in Ethiopia, unilocular hydatid cysts were collected from 11 sheep, 16 cattle and 16 camels slaughtered in abattoirs of Aweday, Jijiga, Haramaya and Addis Ababa during June 2010 to February 2011. A PCR-based DNA sequencing of the mitochondrial cytochrome oxidase c subunit 1 gene (cox1) was conducted for 40 cysts. The majority of cysts (87.5%) were identified as Echinococcus granulosus sensu stricto and the rest as Echinococcus canadensis. The fertile cysts of E. granulosus s.s. were found only from sheep, although it occurred in all the host species. The predominance of E. granulosus s.s. has important implications for public health since this species is the most typical causative agent of human cystic echinococcosis worldwide. The major cox1 haplotype of E. granulosus s.s. detected in Ethiopia was the same as that has been reported to be most common in Peru and China. However, a few cox1 haplotypes unique to Ethiopia were found in both of the two Echinococcus species. The present regional data would serve as baseline information in determining the local transmission patterns and in designing appropriate control strategies.     

Resistance to Striga hermonthica in a maize inbred line derived from Zea diploperennis

Breeding for resistance to Striga in maize (Zea mays), with paucity of donor source and known mechanisms of resistance, has been challenging. Here, post-attachment development of S. hermonthica was monitored on two maize inbreds selected for field resistance and susceptibility reactions to Striga at the International Institute of Tropical Agriculture. Haustorial invasion of the parasite into roots of these inbreds was examined histologically. Morphological differences were observed between roots of the susceptible and the resistant inbreds. The resistant maize had fewer Striga attachments, delayed parasitic development and higher mortality of attached parasites compared with the susceptible inbred. Striga on the susceptible inbred usually penetrated the xylem and showed substantial internal haustorial development. Haustorial ingress on the resistant inbred was often stopped at the endodermis. Parasites able to reach resistant host xylem vessels showed diminished haustorial development relative to those invading susceptible roots. These results suggest that the resistant inbred expresses a developmental barrier and incompatible response against Striga parasitism.     

Molecular marker-based genetic diversity assessment of Striga-resistant maize inbred lines

Striga-resistant maize inbred lines are of interest to maize breeding programs in the savannas of Africa where the parasitic weed is endemic and causes severe yield losses in tropical maize. Assessment of the genetic diversity of such inbred lines is useful for their systematic and efficient use in a breeding program. Diversity analysis of 41 Striga-resistant maize inbred lines was conducted using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers to examine the genetic relationships among these lines and to determine the level of genetic diversity that exists within and between their source populations. The two marker systems generated 262 and 101 polymorphic fragments, respectively. Genetic similarity (GS) values among all possible pairs of inbred lines varied from 0.45 to 0.95, with a mean of 0.61±0.002 for AFLPs, and from 0.21 to 0.92, with a mean of 0.48±0.003, for SSRs. The inbred lines from each source population exhibited a broad range of GS values with the two types of markers. Both AFLPs and SSRs revealed similar levels of within population genetic variation for all source populations. Cluster and principal component analysis of GS estimates with the two markers revealed clear differentiation of the Striga-resistant inbred lines into groups according to their source populations. There was clear separation between early- and late-maturing Striga-resistant inbred lines. Considering the paucity of germplasm with good levels of resistance to Striga in maize, the broad genetic diversity detected within and among source populations demonstrates the genetic potential that exists to improve maize for resistance to Striga.     

Comparisons of methods for introgressing exotic germ plasm into adapted sorghum

The incorporation of exotic germ plasm into breeding populations can broaden and diversify the genetic base of adapted genotypes. To more effectively utilize the genetic resources existing in Sorghum bicolor (L.) Moench, a rapid and efficient method of incorporating exotic genotypes into adapted populations is needed. Therefore, this study was conducted to compare the effectiveness of backcrossing to a broad-based population versus backcrossing to an inbred line for developing improved lines from adapted x exotic crosses. A wild sorghum, a cultivated landrace, and a converted sorghum line were crossed to an inbred line (CK60) and a broad-based population (KP9B). After two generations of backcrossing to the respective adapted parent, 50 F₂ lines were derived from each of the backcross generations of every mating and evaluated at three test environments. Backcrossing to an inbred line (CK60) gave fewer high-yielding segregates and generated less genetic variation than backcrossing to a population (KP9B). Also, the number of agronomically acceptable lines derived from each CK60 mating was fewer than that derived from the corresponding mating with KP9B. Overall, the use of a broad-based population as an adapted recurrent parent for introgressing exotic genotypes may provide good opportunities for developing suitable inbred lines from adapted x exotic backcrosses.Contribution no. 93-499-J from the Kansas Agricultural Experiment Station     

Comparison of trait associations in adapted × exotic matings of sorghum developed by two introgression methods

The association among six traits in the F₂ lines derived from adapted × exotic backcrosses of sorghum developed via two introgression methods was studied using principal component analysis. The first principal component defined a hybrid index in matings of the wild accession (12–26) but not in matings of the cultivated sorghum genotypes (Segeolane and SC408), no matter which adapted parent was used. This component accounted for 27–42% of the total variation in each mating. The recombination spindle was wide in all matings of CK60 and KP9B, which indicated that the relationships among traits were not strong enough to restrict recombination among the parental characters. The index scores of both CK60 and KP9B matings showed clear differentiation of the backcross generations only when the exotic parent was the undomesticated wild accession (12–26). None of the distributions of the first principal component scores in any backcross population was bimodal. The frequency of recombinant genotypes derived from a mating was determined by the level of domestication and adaptation of the exotic parent and the genetic background of the adapted parent. Backcrossing to a population (KP9B) was found to be superior to backcrossing to an inbred line (CK60) to produce lines with an improved adapted phenotype.Contribution no. 93-574-J from the Kansas Agricultural Experiment Station.