Assessment of Immunization to Hepatitis B Vaccine among Children under Five Years in Rural Areas of Taiz, Yemen.

Background. Hepatitis B virus (HBV) infection poses a major health problem worldwide. approximately 1 million deaths annually due to cirrhosis and hepatocellular carcinoma. Objectives. This study was conducted to determine the coverage rate of HBV vaccine and assess the vaccine protective response among children under five years old in rural areas of Yemen. Methods. A cross-sectional study was conducted from January to December 2015 in four districts of countryside Yemen. The target population was children aged from 6 to 59 months. 227 children were enrolled in the study. Questionnaire was used to collect of data. Serum samples were tested for anti-HBs antibodies by enzyme linked immunosorbent assay (ELISA). Anti-HBs level ≥ 10 IU/L was considered a protective response to the vaccine. Results. The coverage rate of HBV vaccine among children was 87.3%. A total of 143 (72.2%) children responded to the vaccine with anti-HBs level ≥ 10 IU/L, while 55 (27.8%) of the children had nonprotective anti-HBs levels of <10 IU/L (P = 0.003). Conclusion. This study revealed a good coverage rate of HBV vaccine in rural areas but the protective rate against HBV infection was moderate. A considerable proportion of vaccinated children should be considered for either revaccination or booster doses.

The First Confirmed Elimination of an Onchocerciasis Focus in Africa: Abu Hamed, Sudan.

Mass treatment with ivermectin for onchocerciasis was stopped in 2012 in Abu Hamed, an isolated focus on the River Nile in northern Sudan. A 3-year posttreatment surveillance (PTS) ensued, at the end of which an evaluation was conducted in 2015 following the current World Health Organization guidelines for verification of onchocerciasis elimination. Vector black flies were collected from sentinel breeding sites and finger-prick bloodspots were collected from children ≤ 10 years of age resident in 35 communities within the focus. Polymerase chain reaction (PCR) screening of 19,191 flies from four sites for the O-150 parasite-specific marker found no flies carrying Onchocerca volvulus larvae (0%, 95% upper confidence limit [UCL] = 0.16), and serological testing of 5,266 children identified only one Ov16 seropositive child (0.019%, 95% UCL = 0.074); whose skin snips were negative when tested by O-150 PCR assay. These results indicate that for the first time in Africa, onchocerciasis elimination has been verified after a successful PTS in Abu Hamed.

Seasonal Variation in Biting Rates of Simulium damnosum sensu lato, Vector of Onchocerca volvulus, in Two Sudanese Foci.

BACKGROUND: The abundance of onchocerciasis vectors affects the epidemiology of disease in Sudan, therefore, studies of vector dynamics are crucial for onchocerciasis control/elimination programs. This study aims to compare the relative abundance, monthly biting-rates (MBR) and hourly-based distribution of onchocerciasis vectors in Abu-Hamed and Galabat foci. These seasonally-based factors can be used to structure vector control efforts to reduce fly-biting rates as a component of onchocerciasis elimination programs. METHODS: A cross-sectional study was conducted in four endemic villages in Abu-Hamed and Galabat foci during two non-consecutive years (2007-2008 and 2009-2010). Both adults and aquatic stages of the potential onchocerciasis vector Simulium damnosum sensu lato were collected following standard procedures during wet and dry seasons. Adult flies were collected using human landing capture for 5 days/month. The data was recorded on handheld data collection sheets to calculate the relative abundance, MBR, and hourly-based distribution associated with climatic factors. The data analysis was carried out using ANOVA and Spearman rank correlation tests. RESULTS: Data on vector surveillance revealed higher relative abundance of S. damnosum s.l. in Abu- Hamed (39,934 flies) than Galabat (8,202 flies). In Abu-Hamed, vector populations increased in January-April then declined in June-July until they disappeared in August-October. Highest black fly density and MBR were found in March 2007 (N = 9,444, MBR = 58,552.8 bites/person/month), and March 2010 (N = 2,603, MBR = 16,138.6 bites/person/month) while none of flies were collected in August-October (MBR = 0 bites/person/month). In Galabat, vectors increased in September-December, then decreased in February-June. The highest vector density and MBR were recorded in September 2007 (N = 1,138, MBR = 6,828 bites/person/month) and September 2010 (N = 1,163, MBR = 6,978 bites/person/month), whereas, none appeared in collection from April to June. There was a significant difference in mean monthly density of S. damnosum s.l. across the two foci in 2007-2008 (df = 3, F = 3.91, P = 0.011). Minimum temperature showed significant correlation with adult flies counts in four areas sampled; the adult counts were increased in Nady village (rs = 0.799) and were decreased in Kalasecal (rs = -0.676), Gumaiza (rs = -0.585), and Hilat Khateir (rs = -0.496). Maximum temperature showed positive correlation with black fly counts only in Galabat focus. Precipitation was significantly correlated with adult flies counts in Nady village, Abu-Hamed, but no significance was found in the rest of the sampled villages in both foci. Hourly-based distribution of black flies showed a unimodal pattern in Abu-Hamed with one peak (10:00-18:00), while a bimodal pattern with two peaks (07:00-10:00) and (14:00-18:00) was exhibited in Galabat. CONCLUSION: Transmission of onchocerciasis in both foci showed marked differences in seasonality, which may be attributed to ecology, microclimate and proximity of breeding sites to collection sites. The seasonal shifts between the two foci might be related to variations in climate zones. This information on black fly vector seasonality, ecology, distribution and biting activity has obvious implications in monitoring transmission levels to guide the national and regional onchocerciasis elimination programs in Sudan.

The impact of Merowe Dam on Simulium hamedense vector of onchocerciasis in Abu Hamed focus - Northern Sudan.

BACKGROUND: Abu Hamed, the northernmost onchocerciasis focus in the world, is located along the River Nile banks in the Nubian Desert. Hydroelectric dams can alter activity of black flies and may provide breeding sites for black fly. Merowe Dam, the largest hydropower project in Africa, was built west of Abu Hamed focus in 2009. The impact of the Dam on onchocerciasis and its black fly vectors in Abu Hamed focus was measured in this study. FINDINGS: Entomological surveys for aquatic stages and adult Simulium hamedense were conducted before and after the inception of Merowe Dam in 2007/2008 and 2010/2011. There was no black fly breeding or adult activity in the previously known breeding sites upstream of the Merowe Dam with the western most breeding site found in AlSarsaf village near the center of the focus. No adult or aquatic stages of black flies were found downstream of the Dam. CONCLUSIONS: The artificial lake of the Dam flooded all the breeding sites in the western region of the focus and no aquatic stages and/or adult black fly activity were established in the study area upstream of the Dam. The Dam seems to have positive impact on onchocerciasis and its black fly vectors in Abu Hamed focus. These outcomes of the Merowe Dam might have contributed to the recently declared interruption of onchocerciasis transmission in Abu Hamed focus. Continuous entomological surveys are needed to monitor presence of black fly vectors and its impact on the disease.

Chemotherapy in the treatment, control, and elimination of human onchocerciasis.

Onchocerciasis treatment is one of the most positive stories in tropical medicine although major challenges remain to reaching the ultimate goal of disease elimination. Such challenges are to be expected when the therapeutic goal is to kill and safely remove a large multistage, efficient, metazoan infectious agent such as Onchocerca volvulus that has an exceptionally complicated relationship with its host. Successful control of onchocerciasis has often been hampered by host reactions following chemotherapy, that can sometimes cause significant tissue pathology. Presence of other filariae, particularly Loa loa, in endemic onchocerciasis-treatment areas also poses severe problems due to adverse reactions caused by drug-induced death of the coincident microfilariae of this usually clinically benign species. Although ivermectin has been very successful, there is a need to enhance the progress toward elimination of onchocerciasis; new drugs and their efficient use are keys to this. The permanent absence of Onchocerca microfilaridermia, defined as the lack of resurgence of skin microfilarial loads after treatment, is the ultimate characteristic of a useful new chemotherapeutic agent. Several drugs are under investigation to achieve this, including the reassessment of currently available and previously tested agents, such as the antibiotic, doxycycline, which targets the adult parasites through its anti-Wolbachia endosymbiont activity. Flubendazole, a benzimidazole derivative approved for treatment of human gastrointestinal nematodes, is also being considered for repurposing as a macrofilaricide to aid in the achievement of eradication. The managerial challenges existing at the population level also need to be addressed; these include drug-distribution fatigue, the need to include noncompliant people, civil unrest in endemic areas, political cross-border issues, restrictions of age and pregnancy, and complications due to integration with other treatment programs. It is likely that a panel of chemotherapeutic options, new and old, supported by strong and effective distribution systems will be the best way to address challenges of treatment and elimination of this infection. Future research should also address management of treatment and control, and consider how new treatment paradigms can be incorporated to meet time lines set for global elimination by 2025.

Interruption of Onchocerca volvulus transmission in the Abu Hamed focus, Sudan.

Abu Hamed, Sudan, the northernmost location of onchocerciasis in the world, began community-directed treatment with ivermectin (CDTI) in 1998, with annual treatments enhanced to semiannual in 2007. We assessed the status of the parasite transmission in 2011 entomologically, parasitologically, and serologically. O-150 pool screening showed no parasite DNA in 17,537 black flies collected in 2011 (95% confidence interval upper limit [95% CI UL] = 0.023). Skin microfilariae, nodules, and signs of skin disease were absent in 536 individuals in seven local communities. Similarly, no evidence of Onchocerca volvulus Ov16 antibodies was found in 6,756 school children ≤ 10 years (95% CI UL = 0.03%). Because this assessment of the focus meets the 2001 World Health Organization (WHO) criteria for interrupted transmission, treatment was halted in 2012, and a post-treatment surveillance period was initiated in anticipation of declaration of disease elimination in this area. We provide the first evidence in East Africa that long-term CDTI alone can interrupt transmission of onchocerciasis.

Biolistic transformation of Brugia malayi.

Biolistics has become a versatile tool for direct gene transfer to various cell and tissue types. Following its successful use on the parasitic nematode Ascaris suum, we developed and evaluated biolistics in the transfection of the model filarial parasite Brugia malayi. Biolistics was proven to be an efficient strategy for transfection of all life stages of the parasite and paved the way for studies on elements essential for promoter function and gene regulation of filarial parasites. Here we present a biolistics protocol for the transfection of B. malayi based on the Biolistics PDS 1000/He system and gold microcarriers.

Polymerase chain reaction pool screening used to compare prevalence of infective black flies in two onchocerciasis foci in northern Sudan.

Onchocerciasis remains an important debilitating disease in many areas of Africa, including Sudan. The status of infection transmission in 2007 was assessed in the vectors of two disease foci in Sudan: Abu Hamed in northern Sudan, which has received at least 10 years of annual treatment and Galabat focus in eastern Sudan, where only minor, largely undocumented treatment activity has occurred. Assessment of more than 30,000 black flies for Onchocerca volvulus infectious stage L3 larvae by using an O-150 polymerase chain reaction protocol showed that black fly infectivity rates were 0.84 (95% confidence interval = 0.0497-1.88) per 10,000 flies for Abu Hamed and 6.9 (95% confidence interval = 1.1-16.4) infective flies per 10,000 for Galabat. These results provide entomologic evidence for suppressed Onchocerca volvulus transmission in the Abu Hamed focus and a moderate transmission rate of the parasite in the Galabat focus.

Use of interactive live digital imaging to enhance histology learning in introductory level anatomy and physiology classes.

Histology is one of the main subjects in introductory college-level Human Anatomy and Physiology classes. Institutions are moving toward the replacement of traditional microscope-based histology learning with virtual microscopy learning amid concerns of losing the valuable learning experience of traditional microscopy. This study used live digital imaging (LDI) of microscopic slides on a SMART board to enhance Histology laboratory teaching. The interactive LDI system consists of a digital camera-equipped microscope that projects live images on a wall-mounted SMART board via a computer. This set-up allows real-time illustration of microscopic slides with highlighted key structural components, as well as the ability to provide the students with relevant study and review material. The impact of interactive LDI on student learning of Histology was then measured based on performance in subsequent laboratory tests before and after its implementation. Student grades increased from a mean of 76% (70.3-82.0, 95% CI) before to 92% (88.8-95.3, 95% CI) after integration of LDI indicating highly significant (P < 0.001) enhancement in students' Histology laboratory performance. In addition, student ratings of the impact of the interactive LDI on their Histology learning were strongly positive, suggesting that a majority of students who valued this learning approach also improved learning and understanding of the material as a result. The interactive LDI technique is an innovative, highly efficient and affordable tool to enhance student Histology learning, which is likely to expand knowledge and student perception of the subject and in turn enrich future science careers.

Sequences necessary for trans-splicing in transiently transfected Brugia malayi.

Many genes in parasitic nematodes are both cis- and trans-spliced. Previous studies have demonstrated that a 7nt element encoded in the first intron of the Brugia malayi 70kDa heat shock protein (BmHSP70) gene was necessary to permit trans-splicing of transgenic mRNAs in embryos transfected with constructs encoding portions of the BmHSP70 gene. Here we demonstrate that this element (the B. malayi HSP70 trans-splicing motif, or BmHSP70 TSM) is necessary and sufficient to direct trans-splicing of transgenic mRNAs derived from two genes naturally containing this motif. Mutations introduced into any position of the BmHSP70 TSM abrogated its ability to direct trans-splicing. Transgenic mRNAs derived from embryos transfected with constructs containing promoters and associated downstream domains from two normally trans-spliced genes that lack a BmHSP70 TSM homologue (the B. malayi 12kDa small subunit ribosomal protein (BmRPS12) gene and the B. malayi RNA-binding protein (BmRBP1) gene), were not trans-spliced. Transfer of the BmHSP70 TSM into the first intron of the BmRPS12 gene rendered it competent for trans-splicing. Insertion of the BmHSP70 TSM into the single intron of the BmRBP1 gene did not render it trans-splicing competent. However, tagged constructs of the full-length BmRBP1 gene were trans-splicing competent. An analysis of the first exons and introns of over 200 trans-spliced B. malayi genes found homologues for the BmHSP70 TSM in roughly 25%. Thus, while the BmHSP70 TSM is necessary and sufficient to direct trans-splicing in some genomic contexts, independent trans-splicing signals are employed by other genes.

Identification of elements essential for transcription in Brugia malayi promoters.

Little is known concerning promoter structure in the filarial parasites. Recently, transient transfection methods have been developed for the human filarial parasite Brugia malayi. These methods have been employed to localize the promoter for the 70kDa heat shock protein (BmHSP70) to a region extending 394nt upstream from the initiating codon of the BmHSP70 open reading frame. Replacement mutagenesis was used to define the elements necessary for BmHSP70 promoter activity in detail. Four domains, ranging in size from six to 22 nucleotides, were found to be necessary for full promoter activity. The two most distal domains encoded a binding site for the heat shock transcription factor and a putative binding site for the GAGA transcription factor, motifs that are found in many other HSP70 promoters. However, none of the essential domains contained sequences typical of cis elements that are usually found in the core domain of a eukaryotic promoter. The largest essential domain was located at positions -53 to -32, and included the splice leader addition site. These data suggest that the regulatory domains of the BmHSP70 promoter were similar to those found in other eukaryotes, but that the core promoter domain exhibited features that appeared to be distinct from those found in most other well-characterized eukaryotic promoters. An analysis of two additional promoters of B.malayi highly transcribed genes suggests that they also lack features commonly found in most eukaryotic core promoters, suggesting that the unique features of the BmHSP70 core promoter are not confined to this gene.

Wolbachia endosymbiont levels in severe and mild strains of Onchocerca volvulus.

Epidemiological, clinical and genetic data have all suggested that the filarial parasite Onchocerca volvulus, the causative agent of onchocerciasis (or river blindness) exists as two strains in West Africa. The severe strain induces severe ocular disease in a large proportion of the infected population, while the mild strain induces little ocular disease. Although DNA probes based upon a non-coding repeat sequence family can distinguish the two strains, the underlying basis for this difference in pathogenicity is not understood. Recently, several studies have implicated products produced by the Wolbachia endosymbiotic bacterium of O. volvulus in the pathogenesis of onchocerciasis. This suggested the hypothesis that differences in the Wolbachia endosymbiont population might be responsible for the pathogenic differences noted in the two strains. To test this hypothesis, quantitative PCR assays were used to measure the amount of Wolbachia DNA per nuclear genome in a collection of well characterized samples of mild and severe strain O. volvulus. The median ratio of Wolbachia DNA to nuclear DNA was significantly greater in severe strain parasites than in mild strain parasites. These data support the hypothesis that the pathogenic differences seen in severe and mild strain O. volvulus may be a function of their relative Wolbachia burden and provide additional support to the hypothesis that Wolbachia products may play a central role in the pathogenesis of ocular onchocerciasis.

Genetic heterogeneity in Loa loa parasites from southern Cameroon: A preliminary study.

Ivermectin (or Mectizan trade mark ) is widely used by onchocerciasis and lymphatic filariasis control programs worldwide. Generally, Mectizan trade mark is both safe and well tolerated. An exception to this general pattern is in some areas co-endemic for Onchocerca volvulus and Loa loa, where a number of severe adverse reactions to Mectizan trade mark have been noted in L. loa infected individuals. The vast majority of these severe adverse events have occurred in Southern Cameroon. This suggested the hypothesis that the parasites endemic to Southern Cameroon might form a distinct population that exhibited a phenotype of eliciting severe adverse reactions in Loa-infected individuals upon Mectizan trade mark exposure. To test this hypothesis, the DNA sequences of three potentially polymorphic loci were compared among L. loa parasites from Southern Cameroon and other endemic foci in Sub-Saharan Africa. Analysis of these data suggested that parasites from Southern Cameroon were at least as genetically diverse as those from other foci. Furthermore, no polymorphisms were noted that were unique to and shared among the parasite isolates from Southern Cameroon. Although a limited number of parasite isolates were tested, these results do not appear to support the hypothesis that L. loa parasites from Southern Cameroon represent a unique, genetically isolated population.

Angiogenic activity of an Onchocerca volvulus Ancylostoma secreted protein homologue.

Angiogenesis is an important step in the development of ocular onchocercaisis. In previous studies, it has been demonstrated that Onchocerca volvulus homologues of the Ancylostoma secreted protein family have pronounced angiogenic activity. The overall goal of the current study was to determine if this angiogenic effect is exerted through a direct or indirect mechanism. These studies focused on one member of this family, OvASP-2, as this protein is expressed in microfilaria, the stage of the parasite that causes ocular onchocercaisis. Clones encoding truncated and full length open reading frames were expressed as fusion proteins with Escherichia coli maltose binding protein (MBP), and angiogenic activity was compared in vitro and in vivo with MBP alone. Truncated constructs expressing only the first 105 amino acids of OvASP-2 were as active as the full length protein in inducing new blood vessel formation. The full length fusion protein did not stimulate proliferation or production of vascular endothelial growth factor in vascular endothelial cells in vitro, indicating that OvASP-2 does not directly stimulate angiogenesis. Sequence analysis demonstrated that the gene encoding OvASP-2 contained five introns. Sequence comparisons of the genomic loci from West African blinding and non-blinding strains of O. volvulus revealed that some polymorphism existed among the various isolates tested. However, none of these polymorphisms could be used to differentiate the parasite strains, suggesting that qualitative variation in OvASP-2 could not explain the difference in ocular pathogenic potential of the two parasite strains.

Identification of mosquito avian-derived blood meals by polymerase chain reaction-heteroduplex analysis.

A polymerase chain reaction (PCR) heteroduplex assay (HDA) was developed to identify avian derived mosquito blood meals to the species level. The assay used primers amplifying a fragment of the cytochrome B gene from vertebrate but not invertebrate species. In Culex tarsalis fed on quail, PCR products derived from the quail cytochrome B gene were detected seven days post-engorgement. In an analysis of wild-caught mosquitoes, 85% of blood-fed mosquitoes produced detectable PCR products. Heteroduplex patterns obtained from bird-derived PCR products were found to permit the unambiguous identification of all species examined. No intraspecific variation in HDA patterns was found. The PCR-HDA was used to characterize blood meals in wild caught Cx. tarsalis. Of the 67 blood meals analyzed, 60% were derived from avian sources. Of the avian blood meals, 65% were derived from a single host, the common grackle.

Brugia malayi: transient transfection by microinjection and particle bombardment.

To develop a method for the introduction of DNA into filarial parasites, several methods that have proven successful in other organisms were evaluated for their ability to transform Brugia malayi. Luciferase activity was detectable in embryos bombarded with gold particles coated with a construct consisting of a luciferase reporter gene under the control of the 5S rRNA intergenic spacer (SL promoter). Similar results were seen in adult parasites and infective larvae bombarded with this construct, or in adult female parasites microinjected with the plasmid. In similar experiments employing the SL promoter driving a green fluorescent protein (GFP) reporter, expression of the reporter was detectable in the intrauterine embryos of the microinjected adult parasites, and in the sub-cuticular tissues of biolistically transfected adult female parasites. A similar pattern of GFP expression to that seen in the SL promoter construct transfected parasites was noted in parasites transfected with constructs consisting of the upstream domain derived from an aspartyl aminoacyl tRNA synthetase gene of B. malayi. The ability to transfect B. malayi embryos may provide a foundation for studies of the regulation of gene expression and function in these organisms.