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Rapid and Non-destructive Detection and Identification of Two Strains of <i>Wolbachia</i> in <i>Aedes aegypti</i> by Near-Infrared Spectroscopy
by Maggy T. Sikulu-Lord, Marta F. Maia, Masabho P. Milali, Michael Henry, Gustav Mkandawile, Elise A. Kho, Robert A. Wirtz, Leon E. Hugo, Floyd E. Dowell, Gregor J. DevineThe release of Wolbachia infected mosquitoes is likely to form a key component of disease control strategies in the near future. We investigated the potential of using near-infrared spectroscopy (NIRS) to simultaneously detect and identify two strains of Wolbachia pipientis (wMelPop and wMel) in male and female laboratory-reared Aedes aegypti mosquitoes. Our aim is to find faster, cheaper alternatives for monitoring those releases than the molecular diagnostic techniques that are currently in use. Our findings indicate that NIRS can differentiate females and males infected with wMelPop from uninfected wild type samples with an accuracy of 96% (N = 299) and 87.5% (N = 377), respectively. Similarly, females and males infected with wMel were differentiated from uninfected wild type samples with accuracies of 92% (N = 352) and 89% (N = 444). NIRS could differentiate wMelPop and wMel transinfected females with an accuracy of 96.6% (N = 442) and males with an accuracy of 84.5% (N = 443). This non-destructive technique is faster than the standard polymerase chain reaction diagnostic techniques. After the purchase of a NIRS spectrometer, the technique requires little sample processing and does not consume any reagents.
by Peter J. Hotez
Optimization of a Membrane Feeding Assay for <i>Plasmodium vivax</i> Infection in <i>Anopheles albimanus</i>
by Andrés F. Vallejo, Kelly Rubiano, Andres Amado, Amy R. Krystosik, Sócrates Herrera, Myriam Arévalo-HerreraIntroduction
Individuals exposed to malaria infections for a long time develop immune responses capable of blocking Plasmodium transmission to mosquito vectors, potentially limiting parasite spreading in nature. Development of a malaria TB vaccine requires a better understanding of the mechanisms and main effectors responsible for transmission blocking (TB) responses. The lack of an in vitro culture system for Plasmodium vivax has been an important drawback for development of a standardized method to assess TB responses to this parasite. This study evaluated host, vector, and parasite factors that may influence Anopheles mosquito infection in order to develop an efficient and reliable assay to assess the TB immunity.Methods/Principal Findings
A total of 94 P. vivax infected patients were enrolled as parasite donors or subjects of direct mosquito feeding in two malaria endemic regions of Colombia (Tierralta, and Buenaventura). Parasite infectiousness was assessed by membrane feeding assay or direct feeding assay using laboratory reared Anopheles mosquitoes. Infection was measured by qPCR and by microscopically examining mosquito midguts at day 7 for the presence of oocysts.Best infectivity was attained in four day old mosquitoes fed at a density of 100 mosquitos/cage. Membrane feeding assays produced statistically significant better infections than direct feeding assays in parasite donors; cytokine profiles showed increased IFN-γ, TNF and IL-1 levels in non-infectious individuals. Mosquito infections and parasite maturation were more reliably assessed by PCR compared to microscopy.Conclusions
We evaluated mosquito, parasite and host factors that may affect the outcome of parasite transmission as measured by artificial membrane feeding assays. Results have led us to conclude that: 1) optimal mosquito infectivity occurs with mosquitoes four days after emergence at a cage density of 100; 2) mosquito infectivity is best quantified by PCR as it may be underestimated by microscopy; 3) host cellular immune response did not appear to significantly affect mosquito infectivity; and 4) no statistically significant difference was observed in transmission between mosquitoes directly feeding on humans and artificial membrane feeding assays.
Performance Testing of PCR Assay in Blood Samples for the Diagnosis of Toxoplasmic Encephalitis in AIDS Patients from the French Departments of America and Genetic Diversity of <i>Toxoplasma gondii</i>: A Prospective and Multicentric Study
by Daniel Ajzenberg, Isabelle Lamaury, Magalie Demar, Cyrille Vautrin, André Cabié, Stéphane Simon, Muriel Nicolas, Nicole Desbois-Nogard, Rachida Boukhari, Homayoun Riahi, Marie-Laure Dardé, Patrice Massip, Michel Dupon, Pierre-Marie Preux, Anaïs Labrunie, Marie-Paule BoncoeurBackground
Toxoplasmic encephalitis in patients with AIDS is a life-threatening disease mostly due to reactivation of Toxoplasma gondii cysts in the brain. The main objective of this study was to evaluate the performance of real-time PCR assay in peripheral blood samples for the diagnosis of toxoplasmic encephalitis in AIDS patients in the French West Indies and Guiana.Methodology/Principal Findings
Adult patients with HIV and suspicion of toxoplasmic encephalitis with start of specific antitoxoplasmic therapy were included in this study during 40 months. The real-time PCR assay targeting the 529 bp repeat region of T. gondii was performed in two different centers for all blood samples. A Neighbor-Joining tree was reconstructed from microsatellite data to examine the relationships between strains from human cases of toxoplasmosis in South America and the Caribbean. A total of 44 cases were validated by a committee of experts, including 36 cases with toxoplasmic encephalitis. The specificity of the PCR assay in blood samples was 100% but the sensitivity was only 25% with moderate agreement between the two centers. Altered level of consciousness and being born in the French West Indies and Guiana were the only two variables that were associated with significantly decreased risk of false negative results with the PCR assay.Conclusion/Significance
Our results showed that PCR sensitivity in blood samples increased with severity of toxoplasmic encephalitis in AIDS patients. Geographic origin of patients was likely to influence PCR sensitivity but there was little evidence that it was caused by differences in T. gondii strains.Trial Registration
Concomitant Immunity Induced by Persistent <i>Leishmania major</i> Does Not Preclude Secondary Re-Infection: Implications for Genetic Exchange, Diversity and Vaccination
by Michael A. Mandell, Stephen M. BeverleyBackground
Many microbes have evolved the ability to co-exist for long periods of time within other species in the absence of overt pathology. Evolutionary biologists have proposed benefits to the microbe from ‘asymptomatic persistent infections’, most commonly invoking increased likelihood of transmission by longer-lived hosts. Typically asymptomatic persistent infections arise from strong containment by the immune system, accompanied by protective immunity; such ‘vaccination’ from overt disease in the presence of a non-sterilizing immune response is termed premunition or concomitant immunity. Here we consider another potential benefit of persistence and concomitant immunity to the parasite: the ‘exclusion’ of competing super-infecting strains, which would favor transmission of the original infecting organism.Methodology / Principle Findings
To investigate this in the protozoan parasite Leishmania major, a superb model for the study of asymptomatic persistence, we used isogenic lines of comparable virulence bearing independent selectable markers. One was then used to infect genetically resistant mice, yielding infections which healed and progressed to asymptomatic persistent infection; these mice were then super-infected with the second marked line. As anticipated, super-infection yielded minimal pathology, showing that protective immunity against disease pathology had been established. The relative abundance of the primary and super-infecting secondary parasites was then assessed by plating on selective media. The data show clearly that super-infecting parasites were able to colonize the immune host effectively, achieving numbers comparable to and sometimes greater than that of the primary parasite.Conclusions / Significance
We conclude that induction of protective immunity does not guarantee the Leishmania parasite exclusive occupation of the infected host. This finding has important consequences to the maintenance and generation of parasite diversity in the natural Leishmania infectious cycle alternating between mammalian and sand fly hosts.
Antibody-Mediated Neutralization of the Exotoxin Mycolactone, the Main Virulence Factor Produced by <i>Mycobacterium ulcerans</i>
by Jean-Pierre Dangy, Nicole Scherr, Philipp Gersbach, Melanie N. Hug, Raphael Bieri, Claudio Bomio, Jun Li, Sylwia Huber, Karl-Heinz Altmann, Gerd PluschkeBackground
Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, causes extensive tissue destruction by inducing apoptosis of host cells. In this study, we aimed at the production of antibodies that could neutralize the cytotoxic activities of mycolactone.Methodology/Principal Findings
Using the B cell hybridoma technology, we generated a series of monoclonal antibodies with specificity for mycolactone from spleen cells of mice immunized with the protein conjugate of a truncated synthetic mycolactone derivative. L929 fibroblasts were used as a model system to investigate whether these antibodies can inhibit the biological effects of mycolactone. By measuring the metabolic activity of the fibroblasts, we found that anti-mycolactone mAbs can completely neutralize the cytotoxic activity of mycolactone.Conclusions/Significance
The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target.
<i>Clonorchis sinensis</i> Co-infection Could Affect the Disease State and Treatment Response of HBV Patients
by Wenfang Li, Huimin Dong, Yan Huang, Tingjin Chen, Xiangzhan Kong, Hengchang Sun, Xinbing Yu, Jin XuBackground
Clonorchis sinensis (C. sinensis) is considered to be an important parasitic zoonosis because it infects approximately 35 million people, while approximately 15 million were distributed in China. Hepatitis B virus (HBV) infection is a major public health issue. Two types of pathogens have the potential to cause human liver disease and eventually hepatocellular carcinoma. Concurrent infection with HBV and C. sinensis is often observed in some areas where C. sinensis is endemic. However, whether C. sinensis could impact HBV infection or vice versa remains unknown.Principal Findings
Co-infection with C. sinensis and HBV develops predominantly in males. Co-infected C. sinensis and HBV patients presented weaker liver function and higher HBV DNA titers. Combination treatment with antiviral and anti-C. sinensis drugs in co-infected patients could contribute to a reduction in viral load and help with liver function recovery. Excretory-secretory products (ESPs) may, in some ways, increase HBV viral replication in vitro. A mixture of ESP and HBV positive sera could induce peripheral blood mononuclear cells (PBMCs) to produce higher level of Th2 cytokines including IL-4, IL-6 and IL-10 compared to HBV alone, it seems that due to presence of ESP, the cytokine production shift towards Th2. C. sinensis/HBV co-infected patients showed higher serum IL-6 and IL-10 levels and lower serum IFN-γ levels.Conclusions/Significance
Patients with concomitant C. sinensis and HBV infection presented weaker liver function and higher HBV DNA copies. In co-infected patients, the efficacy of anti-viral treatment was better in patients who were prescribed with entecavir and praziquantel than entecavir alone. One possible reason for the weaker response to antiviral therapies in co-infected patients was the shift in cytokine production from Th1 to Th2 that may inhibit viral clearance. C. sinensis/HBV co-infection could exacerbate the imbalance of Th1/Th2 cytokine.
by Katarina Resman Rus, Luka Fajs, Miša Korva, Tatjana Avšič-ŽupancHemorrhagic fever with renal syndrome (HFRS) and Crimean-Congo hemorrhagic fever (CCHF) are common representatives of viral hemorrhagic fevers still often neglected in some parts of the world. Infection with Dobrava or Puumala virus (HFRS) and Crimean-Congo hemorrhagic fever virus (CCHFV) can result in a mild, nonspecific febrile illness or as a severe disease with hemorrhaging and high fatality rate. An important factor in optimizing survival rate in patients with VHF is instant recognition of the severe form of the disease for which significant biomarkers need to be elucidated. To determine the prognostic value of High Mobility Group Box 1 (HMGB1) as a biomarker for disease severity, we tested acute serum samples of patients with HFRS or CCHF. Our results showed that HMGB1 levels are increased in patients with CCHFV, DOBV or PUUV infection. Above that, concentration of HMGB1 is higher in patients with severe disease progression when compared to the mild clinical course of the disease. Our results indicate that HMGB1 could be a useful prognostic biomarker for disease severity in PUUV and CCHFV infection, where the difference between the mild and severe patients group was highly significant. Even in patients with severe DOBV infection concentrations of HMGB1 were 2.8–times higher than in the mild group, but the difference was not statistically significant. Our results indicated HMGB1 as a potential biomarker for severe hemorrhagic fevers.
by Daniel S. Mason, Michael Marks, Oliver Sokana, Anthony W. Solomon, David C. Mabey, Lucia Romani, John Kaldor, Andrew C. Steer, Daniel EngelmanBackground
Scabies and impetigo are common, important and treatable skin conditions. Reports from several Pacific island countries show extremely high prevalence of these two conditions, but for many countries, including the Solomon Islands, there is a paucity of epidemiological data.Methodology
Ten rural villages in the Western Province of the Solomon Islands were included in the study, chosen so that data collection could be integrated with an existing project investigating clinical and serological markers of yaws. All residents were eligible to participate, and 1908 people were enrolled. Participants were interviewed and examined by a paediatric registrar, who recorded relevant demographic information, and made a clinical diagnosis of scabies and/or impetigo, severity and distribution.Principal Findings
The total unweighted prevalence of scabies was 19.2% (95% confidence interval [CI] 17.5–21.0), and age and gender weighted prevalence 19.2% (95%CI 16.7–21.9). The adult prevalence of scabies was 10.4% (95%CI 8.2–13.2), and the highest prevalence was found in infants < 1 year of age (34.1%, adjusted odds ratio [AOR] compared with adults: 3.6, 95%CI 2.2–6.0) and children aged 1–4 years (25.7%, AOR 2.6, 95%CI 1.7–3.9). Scabies affected two or more body regions in 80.9% of participants, and 4.4% of scabies cases were classified as severe. The total unweighted prevalence of active impetigo was 32.7% (95%CI 30.6–34.8), and age and gender weighted prevalence 26.7% (95%CI 24.2–29.5). The highest prevalence was found in children aged 1–4 years (42.6%, AOR compared with adults: 4.1, 95%CI 2.9–5.8). Scabies infestation was associated with active impetigo infection (AOR 2.0, 95%CI 1.6–2.6); with 41.1% of active impetigo cases also having scabies.Conclusions and Significance
Scabies and impetigo are very common in the rural Western Province of the Solomon Islands. Scabies infestation is strongly associated with impetigo. Community control strategies for scabies may reduce the burden of both conditions and their downstream complications.
Evolution of the Transmission-Blocking Vaccine Candidates Pvs28 and Pvs25 in <i>Plasmodium vivax</i>: Geographic Differentiation and Evidence of Positive Selection
by Ricardo A. Chaurio, M. Andreína Pacheco, Omar E. Cornejo, Ester Durrego, Craig E. Stanley Jr., Andreína I. Castillo, Sócrates Herrera, Ananias A. EscalanteTransmission-blocking (TB) vaccines are considered an important tool for malaria control and elimination. Among all the antigens characterized as TB vaccines against Plasmodium vivax, the ookinete surface proteins Pvs28 and Pvs25 are leading candidates. These proteins likely originated by a gene duplication event that took place before the radiation of the known Plasmodium species to primates. We report an evolutionary genetic analysis of a worldwide sample of pvs28 and pvs25 alleles. Our results show that both genes display low levels of genetic polymorphism when compared to the merozoite surface antigens AMA-1 and MSP-1; however, both ookinete antigens can be as polymorphic as other merozoite antigens such as MSP-8 and MSP-10. We found that parasite populations in Asia and the Americas are geographically differentiated with comparable levels of genetic diversity and specific amino acid replacements found only in the Americas. Furthermore, the observed variation was mainly accumulated in the EGF2- and EGF3-like domains for P. vivax in both proteins. This pattern was shared by other closely related non-human primate parasites such as Plasmodium cynomolgi, suggesting that it could be functionally important. In addition, examination with a suite of evolutionary genetic analyses indicated that the observed patterns are consistent with positive natural selection acting on Pvs28 and Pvs25 polymorphisms. The geographic pattern of genetic differentiation and the evidence for positive selection strongly suggest that the functional consequences of the observed polymorphism should be evaluated during development of TBVs that include Pvs25 and Pvs28.
The Schistosomiasis Clinical Trials Landscape: A Systematic Review of Antischistosomal Treatment Efficacy Studies and a Case for Sharing Individual Participant-Level Data (IPD)
by Amélie M. Julé, Michel Vaillant, Trudie A. Lang, Philippe J. Guérin, Piero L. OlliaroBackground
Schistosomiasis control mainly relies on preventive chemotherapy with praziquantel (PZQ) distributed through mass drug administration. With a target of 260 million treatments yearly, reliably assessing and monitoring efficacy is all-important. Recommendations for treatment and control of schistosomiasis are supported by systematic reviews and meta-analyses of aggregated data, which however also point to limitations due to heterogeneity in trial design, analyses and reporting. Some such limitations could be corrected through access to individual participant-level data (IPD), which facilitates standardised analyses.Methodology
A systematic literature review was conducted to identify antischistosomal drug efficacy studies performed since 2000; including electronic searches of the Cochrane Infectious Diseases Group specialised register and the Cochrane Library, PubMed, CENTRAL and Embase; complemented with a manual search for articles listed in past reviews. Antischistosomal treatment studies with assessment of outcome within 60 days post-treatment were eligible. Meta-data, i.e. study-level characteristics (Schistosoma species, number of patients, drug administered, country, etc.) and efficacy parameters were extracted from published documents to evaluate the scope of an individual-level data sharing platform.Principal findings
Out of 914 documents screened, 90 studies from 26 countries were included, enrolling 20,517 participants infected with Schistosoma spp. and treated with different PZQ regimens or other drugs. Methodologies varied in terms of diagnostic approaches (number of samples and test repeats), time of outcome assessment, and outcome measure (cure rate or egg reduction rate, as an arithmetic or geometric mean), making direct comparison of published data difficult.Conclusions
This review describes the landscape of schistosomiasis clinical research. The volume of data and the methodological and reporting heterogeneity identified all indicate that there is scope for an individual participant-level database, to allow for standardised analyses.
Accuracy of Mobile Phone and Handheld Light Microscopy for the Diagnosis of Schistosomiasis and Intestinal Protozoa Infections in Côte d’Ivoire
by Jean T. Coulibaly, Mamadou Ouattara, Michael V. D’Ambrosio, Daniel A. Fletcher, Jennifer Keiser, Jürg Utzinger, Eliézer K. N’Goran, Jason R. Andrews, Isaac I. BogochBackground
Handheld light microscopy using compact optics and mobile phones may improve the quality of health care in resource-constrained settings by enabling access to prompt and accurate diagnosis.Methodology
Laboratory technicians were trained to operate two handheld diagnostic devices (Newton Nm1 microscope and a clip-on version of the mobile phone-based CellScope). The accuracy of these devices was compared to conventional light microscopy for the diagnosis of Schistosoma haematobium, S. mansoni, and intestinal protozoa infection in a community-based survey in rural Côte d’Ivoire. One slide of 10 ml filtered urine and a single Kato-Katz thick smear from 226 individuals were subjected to the Newton Nm1 microscope and CellScope for detection of Schistosoma eggs and compared to conventional microscopy. Additionally, 121 sodium acetate-acetic acid-formalin (SAF)-fixed stool samples were examined by the Newton Nm1 microscope and compared to conventional microscopy for the diagnosis of intestinal protozoa.Principal Findings
The prevalence of S. haematobium, S. mansoni, Giardia intestinalis, and Entamoeba histolytica/E. dispar, as determined by conventional microscopy, was 39.8%, 5.3%, 20.7%, and 4.9%, respectively. The Newton Nm1 microscope had diagnostic sensitivities for S. mansoni and S. haematobium infection of 91.7% (95% confidence interval (CI) 59.8–99.6%) and 81.1% (95% CI 71.2–88.3%), respectively, and specificities of 99.5% (95% CI 97.0–100%) and 97.1% (95% CI 92.2–99.1%), respectively. The CellScope demonstrated sensitivities for S. mansoni and S. haematobium of 50.0% (95% CI 25.4–74.6%) and 35.6% (95% CI 25.9–46.4%), respectively, and specificities of 99.5% (95% CI 97.0–100%) and 100% (95% CI 86.7–100%), respectively. For G. intestinalis and E. histolytica/E. dispar, the Newton Nm1 microscope had sensitivity of 84.0% (95% CI 63.1–94.7%) and 83.3% (95% CI 36.5–99.1%), respectively, and 100% specificity.Conclusions/Significance
Handheld diagnostic devices can be employed in community-based surveys in resource-constrained settings after minimal training of laboratory technicians to diagnose intestinal parasites.
by Myrna C. Bonaldo, Ieda P. Ribeiro, Noemia S. Lima, Alexandre A. C. dos Santos, Lidiane S. R. Menezes, Stephanie O. D. da Cruz, Iasmim S. de Mello, Nathália D. Furtado, Elaine E. de Moura, Luana Damasceno, Kely A. B. da Silva, Marcia G. de Castro, Alexandra L. Gerber, Luiz G. P. de Almeida, Ricardo Lourenço-de-Oliveira, Ana Tereza R. Vasconcelos, Patrícia BrasilBackground
Zika virus (ZIKV) is an emergent threat provoking a worldwide explosive outbreak. Since January 2015, 41 countries reported autochthonous cases. In Brazil, an increase in Guillain-Barré syndrome and microcephaly cases was linked to ZIKV infections. A recent report describing low experimental transmission efficiency of its main putative vector, Ae. aegypti, in conjunction with apparent sexual transmission notifications, prompted the investigation of other potential sources of viral dissemination. Urine and saliva have been previously established as useful tools in ZIKV diagnosis. Here, we described the presence and isolation of infectious ZIKV particles from saliva and urine of acute phase patients in the Rio de Janeiro state, Brazil.Methodology/Principal Findings
Nine urine and five saliva samples from nine patients from Rio de Janeiro presenting rash and other typical Zika acute phase symptoms were inoculated in Vero cell culture and submitted to specific ZIKV RNA detection and quantification through, respectively, NAT-Zika, RT-PCR and RT-qPCR. Two ZIKV isolates were achieved, one from urine and one from saliva specimens. ZIKV nucleic acid was identified by all methods in four patients. Whenever both urine and saliva samples were available from the same patient, urine viral loads were higher, corroborating the general sense that it is a better source for ZIKV molecular diagnostic. In spite of this, from the two isolated strains, each from one patient, only one derived from urine, suggesting that other factors, like the acidic nature of this fluid, might interfere with virion infectivity. The complete genome of both ZIKV isolates was obtained. Phylogenetic analysis revealed similarity with strains previously isolated during the South America outbreak.Conclusions/Significance
The detection of infectious ZIKV particles in urine and saliva of patients during the acute phase may represent a critical factor in the spread of virus. The epidemiological relevance of this finding, regarding the contribution of alternative non-vectorial ZIKV transmission routes, needs further investigation.
Relative Contribution of Dengue IgG Antibodies Acquired during Gestation or Breastfeeding in Mediating Dengue Disease Enhancement and Protection in Type I Interferon Receptor-Deficient Mice
by Pei Xuan Lee, Li Ching Ong, Eshele Anak Libau, Sylvie AlonsoDengue virus (DENV) causes a spectrum of diseases ranging from self-limiting dengue fever to severe conditions such as haemorrhagic fever and dengue shock syndrome. Antibody-dependent enhancement (ADE) is thought to explain the occurrence of severe dengue whereby pre-existing binding but non-neutralising antibodies enhance DENV infection. The ADE phenomenon is supported by epidemiological findings that infants that born to dengue immune mothers are at greater risk to develop severe dengue upon primary infection. The role of maternally acquired dengue-specific antibodies in disease enhancement was recently recapitulated in a mouse model where mice born to DENV1-immune mothers experienced enhanced disease severity upon DENV2 infection. Here, this study investigates the relative contribution of maternal dengue-specific antibodies acquired during gestation and breastfeeding in dengue disease. Using a surrogate breastfeeding mother experimental approach, we showed that majority of the maternal dengue-specific antibodies were acquired during breastfeeding and conferred an extended enhancement window. On the other hand, in the context of homologous infection, breastfeeding conferred protection. Furthermore, measurement of dengue-specific antibody titres over time in mice born to dengue immune mothers revealed a biphasic pattern of antibody decay as reported in humans. Our work provides evidence of the potential contribution of breast milk-acquired dengue-specific IgG antibodies in enhancement and protection against dengue. Should such contribution be established in humans as well, it may have important implications for the development of guidelines to dengue-immune breastfeeding mothers.
Development and Application of a Loop-Mediated Isothermal Amplification (LAMP) Approach for the Rapid Detection of <i>Dirofilaria repens</i> from Biological Samples
by Donato Antonio Raele, Nicola Pugliese, Domenico Galante, Laura Maria Latorre, Maria Assunta CafieroDirofilariasis by Dirofilaria repens is an important mosquito vector borne parasitosis, and the dog represents the natural host and reservoir of the parasite. This filarial nematode can also induce disease in humans, and in the last decades an increasing number of cases have been being reported. The present study describes the first loop mediated isothermal amplification (LAMP) assay to detect D. repens DNA in blood and mosquitoes. Two versions of the technique have been developed and described: in the first, the amplification is followed point by point through a real time PCR instrument (ReT-LAMP); in the second, the amplification is visualized by checking UV fluorescence of the reaction mixture after addition of propidium iodide (PI-LAMP). The two variants use the same set of 4 primers targeting the D. repens cytochrome oxidase subunit I (COI) gene. To assess the specificity of the method, reactions were carried out by using DNA from the major zoonotic parasites of the family of Onchocercidae, and no amplification was observed. The lower limit of detection of the ReT-LAMP assay was 0.15 fg/μl (corresponding to about 50 copy of COI gene per μl). Results suggest that the described assay is specific, and its sensitivity is higher than the conventional PCR based on the same gene. It is also provide a rapid and cost-effective molecular detection of D. repens, mainly when PI-LAMP is applied, and it should be performed in areas where this emerging parasitosis is endemic.
A Knowledge, Attitudes and Practices Survey Conducted Three Years after Halting Ivermectin Mass Treatment for Onchocerciasis in Guatemala
by Frank O. Richards Jr., Robert E. Klein, Oscar de León, Renata Mendizábal-Cabrera, Alba Lucía Morales, Vitaliano Cama, Carol G. Crovella, Carlos E. Díaz Espinoza, Zoraida Morales, Mauricio Sauerbrey, Nidia RizzoBackground
Mass drug administration (MDA) with ivermectin for onchocerciasis was provided in Guatemala’s Central Endemic Zone (CEZ) over a 24 year period (1988–2011). Elimination of Onchocerca volvulus transmission was declared in 2015 after a three year post MDA surveillance period (2012–2014) showed no evidence of recrudescence. The purpose of the present study was to evaluate the knowledge, attitudes and practices (KAP) towards onchocerciasis and ivermectin among residents in the post endemic CEZ. A major interest in this study was to determine what community residents thought about the end of the ivermectin MDA program.Methodology/Principal Findings
A total of 148 interviews were conducted in November 2014 in four formerly hyperendemic communities using a standard questionnaire on smart phones. The majority (69%) of respondents knew that the MDA program had ended because the disease was no longer present in their communities, but a slight majority (53%) was personally unsure that onchocerciasis had really been eliminated. Sixty-three percent wanted to continue to receive ivermectin because of this uncertainty, or because ivermectin is effective against intestinal worms. Eighty-nine percent of respondents said that they would seek medical attention immediately if a family member had symptoms of onchocerciasis (especially the presence of a nodule), which is a finding very important for ongoing surveillance.Conclusions/Significance
Many respondents wanted to continue receive ivermectin and more than half did not believe onchocerciasis had been eliminated. The ministry of health outreach services should be prepared to address ongoing concerns about onchocerciasis in the post endemic CEZ.
by Bronwyn A. Clayton, Deborah Middleton, Rachel Arkinstall, Leah Frazer, Lin-Fa Wang, Glenn A. MarshPerson-to-person transmission is a key feature of human Nipah virus outbreaks in Bangladesh. In contrast, in an outbreak of Nipah virus in Malaysia, people acquired infections from pigs. It is not known whether this important epidemiological difference is driven primarily by differences between NiV Bangladesh (NiV-BD) and Malaysia (NiV-MY) at a virus level, or by environmental or host factors. In a time course study, ferrets were oronasally exposed to equivalent doses of NiV-BD or NiV-MY. More rapid onset of productive infection and higher levels of virus replication in respiratory tract tissues were seen for NiV-BD compared to NiV-MY, corroborating our previous report of increased oral shedding of NiV-BD in ferrets and suggesting a contributory mechanism for increased NiV-BD transmission between people compared to NiV-MY. However, we recognize that transmission occurs within a social and environmental framework that may have an important and differentiating role in NiV transmission rates. With this in mind, ferret-to-ferret transmission of NiV-BD and NiV-MY was assessed under differing viral exposure conditions. Transmission was not identified for either virus when naïve ferrets were cohoused with experimentally-infected animals. In contrast, all naïve ferrets developed acute infection following assisted and direct exposure to oronasal fluid from animals that were shedding either NiV-BD or NiV-MY. Our findings for ferrets indicate that, although NiV-BD may be shed at higher levels than NiV-MY, transmission risk may be equivalently low under exposure conditions provided by cohabitation alone. In contrast, active transfer of infected bodily fluids consistently results in transmission, regardless of the virus strain. These observations suggest that the risk of NiV transmission is underpinned by social and environmental factors, and will have practical implications for managing transmission risk during outbreaks of human disease.
Evaluation of Six Commercially Available Rapid Immunochromatographic Tests for the Diagnosis of Rabies in Brain Material
by Elisa Eggerbauer, Paola de Benedictis, Bernd Hoffmann, Thomas C. Mettenleiter, Kore Schlottau, Ernest C. Ngoepe, Claude T. Sabeta, Conrad M. Freuling, Thomas MüllerRabies is a neglected zoonotic disease that causes an estimated 60,000 human deaths annually. The main burden lies on developing countries in Asia and Africa, where surveillance and disease detection is hampered by absence of adequate laboratory facilities and/or the difficulties of submitting samples from remote areas to laboratories. Under these conditions, easy-to-use tests such as immunochromatographic assays, i.e. lateral flow devices (LFD), may increase surveillance and improve control efforts. Several LFDs for rabies diagnosis are available but, except for one, there are no data regarding their performance. Therefore, we compared six commercially available LFDs for diagnostic and analytical sensitivity, as well as their specificity and their diagnostic agreement with standard rabies diagnostic techniques using different sample sets, including experimentally infected animals and several sets of field samples. Using field samples the sensitivities ranged between 0% up to 100% depending on the LFD and the samples, while for experimentally infected animals the maximum sensitivity was 32%. Positive results in LFD could be further validated using RT-qPCR and sequencing. In summary, in our study none of the tests investigated proved to be satisfactory, although the results somewhat contradict previous studies, indicating batch to batch variation. The high number of false negative results reiterates the necessity to perform a proper test validation before being marketed and used in the field. In this respect, marketing authorization and batch release control could secure a sufficient quality for these alternative tests, which could then fulfil their potential.
An mRNA Vaccine Encoding Rabies Virus Glycoprotein Induces Protection against Lethal Infection in Mice and Correlates of Protection in Adult and Newborn Pigs
by Margit Schnee, Annette B. Vogel, Daniel Voss, Benjamin Petsch, Patrick Baumhof, Thomas Kramps, Lothar StitzRabies is a zoonotic infectious disease of the central nervous system (CNS). In unvaccinated or untreated subjects, rabies virus infection causes severe neurological symptoms and is invariably fatal. Despite the long-standing existence of effective vaccines, vaccine availability remains insufficient, with high numbers of fatal infections mostly in developing countries. Nucleic acid based vaccines have proven convincingly as a new technology for the fast development of vaccines against newly emerging pathogens, diseases where no vaccine exists or for replacing already existing vaccines. We used an optimized non-replicating rabies virus glycoprotein (RABV-G) encoding messenger RNA (mRNA) to induce potent neutralizing antibodies (VN titers) in mice and domestic pigs. Functional antibody titers were followed in mice for up to one year and titers remained stable for the entire observation period in all dose groups. T cell analysis revealed the induction of both, specific CD4+ as well as CD8+ T cells by RABV-G mRNA, with the induced CD4+ T cells being higher than those induced by a licensed vaccine. Notably, RABV-G mRNA vaccinated mice were protected against lethal intracerebral challenge infection. Inhibition of viral replication by vaccination was verified by qRT-PCR. Furthermore, we demonstrate that CD4+ T cells are crucial for the generation of neutralizing antibodies. In domestic pigs we were able to induce VN titers that correlate with protection in adult and newborn pigs. This study demonstrates the feasibility of a non-replicating mRNA rabies vaccine in small and large animals and highlights the promises of mRNA vaccines for the prevention of infectious diseases.
by Suleiman Hussein Suleiman, EL Sammani Wadaella, Ahmed Hassan FahalSurgical intervention is an integral component in the diagnosis and management of mycetoma. Surgical treatment is indicated for small, localised lesions and massive lesions to reduce the mycetoma load and to enable better response to medical therapy. It is also a life-saving procedure in patients with massive disease and sepsis. Surgical options for mycetoma treatment range from a wide local surgical excision to repetitive debridement excisions to amputation of the affected part. Adequate anaesthesia, a bloodless field, wide local excision with adequate safety margins in a suitable surgical facility, and expert surgeons are mandatory to achieve the best surgical outcome. Surgical intervention in mycetoma is associated with considerable morbidity, deformities, and disabilities, particularly in advanced disease. These complications can be reduced by educating patients to seek medical advice earlier when the lesion is small, localised, and amenable to surgery. There is no evidence for mycetoma hospital cross infection. This communication is based on the authors’ experience in managing over 7,200 mycetoma patients treated at the Mycetoma Research Centre, University of Khartoum, Sudan.