James Lee Crainey

Outstanding insecurities concerning the use of an Ov16-based ELISA in the Amazonia onchocerciasis focus

In a recent issue of Memórias do Instituto Oswaldo Cruz, published in Rio de Janeiro in February 2014 (109: 87-92), Adami et al. have published a survey reporting Mansonella parasite prevalence in the Amazon Region. This report makes a useful contribution to the existing knowledge of filarial parasite distribution within the Amazon area, parasite prevalence rates in relation to age and occupation and provides observations on the possible clinical impact of Mansonella ozzardi. Their publication also provides an account of what appears to be a novel ELISA that has recently been used in the Simuliidae and Onchocerciasis Laboratory of the Oswaldo Cruz Institute, Rio de Janeiro, Brazil. We are concerned that the publication of this ELISA may have created an excessively positive impression of the effectiveness of the onchocerciasis recrudescence serological surveillance tools that are presently available for use in the Amazonia onchocerciasis focus. In this letter we have, thus, sought to highlight some of the limitations of this ELISA and suggest how continuing insecurities concerning the detection of antibodies to Onchocerca volvulus within the Amazonia onchocerciasis focus might be minimised.

Multidrug resistant Pseudomonas aeruginosa survey in a stream receiving effluents from ineffective wastewater hospital plants

BackgroundMulti-drug resistant forms of Pseudomonas aeruginosa (MDRPA) are a major source of nosocomial infections and when discharged into streams and rivers from hospital wastewater treatment plants (HWWTP) they are known to be able to persist for extended periods. In the city of Manaus (Western Brazilian Amazon), the effluent of three HWWTPs feed into the urban Mindu stream which crosses the city from its rainforest source before draining into the Rio Negro. The stream is routinely used by Manaus residents for bathing and cleaning (of clothes as well as domestic utensils) and, during periods of flooding, can contaminate wells used for drinking water.Results16S rRNA metagenomic sequence analysis of 293 cloned PCR fragments, detected an abundance of Pseudomonas aeruginosa (P. aeruginosa) at the stream’s Rio Negro drainage site, but failed to detect it at the stream’s source. An array of antimicrobial resistance profiles and resistance to all 14 tested antimicrobials was detected among P. aeruginosa cultures prepared from wastewater samples taken from water entering and being discharged from a Manaus HWWTP. Just one P. aeruginosa antimicrobial resistance profile, however, was detected from cultures made from Mindu stream isolates. Comparisons made between P. aeruginosa isolates’ genomic DNA restriction enzyme digest fingerprints, failed to determine if any of the P. aeruginosa found in the Mindu stream were of HWWTP origin, but suggested that Mindu stream P. aeruginosa are from diverse origins. Culturing experiments also showed that P. aeruginosa biofilm formation and the extent of biofilm formation produced were both significantly higher in multi drug resistant forms of P. aeruginosa.ConclusionsOur results show that a diverse range of MDRPA are being discharged in an urban stream from a HWWTP in Manaus and that P. aeruginosa strains with ampicillin and amikacin can persist well within it.

Molecular Verification of New World Mansonella perstans Parasitemias

We obtained ribosomal and mitochondrial DNA sequences from residents of Amazonas state, Brazil, with Mansonella parasitemias. Phylogenetic analysis of these sequences confirm that M. ozzardi and M. perstans parasites occur in sympatry and reveal the close relationship between M. perstans in Africa and Brazil, providing insights into the parasite’s New World origins.

A novel polyclonal antibody-based sandwich ELISA for detection of Plasmodium vivax developed from two lactate dehydrogenase protein segments

BackgroundImmunoassays for Plasmodium detection are, presently, most frequently based on monoclonal antibodies (MAbs); Polyclonal antibodies (PAbs), which are cheaper to develop and manufacture, are much less frequently used. In the present study we describe a sandwich ELISA assay which is capable of detecting P. vivax Lactate Dehydrogenase (LDH) in clinical blood samples, without cross reacting with those infected with P. falciparum.MethodsTwo recombinant proteins were produced from different regions of the P. vivax LDH gene. Two sandwich ELISA assay were then designed: One which uses mouse anti-LDH 1-43aa PAbs as primary antibodies (“Test 1”) and another which uses anti-LDH 35-305aa PAbs (“Test 2”) as the primary antibodies. Rabbit anti-LDH 1-43aa PAbs were used as capture antibodies in both ELISA assays. Blood samples taken from P. vivax and P. falciparum infected patients (confirmed by light microscopy) were analysed using both tests.Results“Test 2” performed better at detecting microscopy-positive blood samples when compared to “Test 1”, identifying 131 of 154 positive samples (85%); 85 positives (55%) were identified using “test 1”. “Test 1” produced one false positive sample (from the 20 malaria-free control) blood samples; “test 2” produced none. Kappa coefficient analysis of the results produced a value of 0.267 when microscope-positive blood smears were compared with “test 1”, but 0.734 when microscope-positive blood smears were compared with the results from “test 2”. Positive predictive value (PPV) and negative predictive value (NPV) were observed to be 98% and 22% respectively, for “Test 1”, and 99% and 45%, for “test 2”. No cross reactivity was detected with P. falciparum positive blood samples (n = 15) with either test assay.ConclusionBoth tests detected P. vivax infected blood and showed no evidence of cross-reacting with P. falciparum. Further studies will need to be conducted to establish the full potential of this technique for malaria diagnostics. As well as representing a promising new cost-effective novel technique for P. vivax diagnosis and research, the method for developing this assay also highlights the potential for PAb-based strategies for diagnostics in general.

New molecular identifiers for Simulium limbatum and Simulium incrustatum s.l. and the detection of genetic substructure with potential implications for onchocerciasis epidemiology in the Amazonia focus of Brazil.

The Amazonia onchocerciasis focus of southern Venezuela and northern Brazil is the larger of the two remaining Latin American onchocerciasis foci where disease transmission still occurs and is often regarded as the most challenging of all the Latin American foci to eliminate onchocerciasis. The site is home to a population of over 20,000 semi-nomadic, hunter-gatherer Yanomami people and is made-up of a mosaic of rainforest and savannah ecologies, which are influenced by the areas undulating terrain and rich geological diversity. At least six blackfly vectors have been implicated in onchocerciasis transmission in this focus; however, because of the difficulty in their routine identification the relative importance of each has been obscured. Simulium limbatum and Simulium incrustatum s.l. have both been recorded as vectors in the Amazonia focus, but they are difficult to discriminate morphologically and thus the ecological range of these species, and indeed the presence of S. limbatum in the Amazonia focus at all, have remained controversial. In the work described here, we report 15 S. incrustatum s.l. CO1 sequences and 27 S. limbatum sequences obtained from field-caught adult female blackflies collected from forest and savannah localities, inside and just outside the Amazonia focus. Phylogenetic analysis with the sequences generated in this study, showed that both the S. limbatum and the S. incrustatum s.l. CO1 sequences obtained (even from specimens living in sympatry) all fell into discrete species-specific bootstrap-supported monophyletic groups and thus confirmed the utility of the CO1 gene for identifying both these species inside the Amazonia focus. As the S. limbatum-exclusive cluster included CO1 sequences obtained from forest-caught and morphologically identified specimens these results provide the clearest evidence yet of the presence of S. limbatum inside the Amazonia focus. The question, however, of whether S. limbatum is actually a vector in the focus still remains unanswered as the data presented here also suggest that S. limbatum found in the savannahs adjacent to, but outside the Amazonia focus (and which represent the only S. limbatum population to be unambiguously incriminated as a host of Onchocerca volvulus), are genetically distinct from those living inside the focus. These findings highlight the need for a clearer picture of the vector taxonomy inside the Amazonia onchocerciasis focus.

The mitogenome of Onchocerca volvulus from the Brazilian Amazonia focus

We report here the first complete mitochondria genome of Onchocerca volvulus from a focus outside of Africa. An O. volvulus mitogenome from the Brazilian Amazonia focus was obtained using a combination of high-throughput and Sanger sequencing technologies. Comparisons made between this mitochondrial genome and publicly available mitochondrial sequences identified 46 variant nucleotide positions and suggested that our Brazilian mitogenome is more closely related to Cameroon-origin mitochondria than West African-origin mitochondria. As well as providing insights into the origins of Latin American onchocerciasis, the Brazilian Amazonia focus mitogenome may also have value as an epidemiological resource.

Mansonella ozzardi mitogenome and pseudogene characterisation provides new perspectives on filarial parasite systematics and CO-1 barcoding

Despite the broad distribution of M. ozzardi in Latin America and the Caribbean, there is still very little DNA sequence data available to study this neglected parasite’s epidemiology. Mitochondrial DNA (mtDNA) sequences, especially the cytochrome oxidase (CO1) gene’s barcoding region, have been targeted successfully for filarial diagnostics and for epidemiological, ecological and evolutionary studies. MtDNA-based studies can, however, be compromised by unrecognised mitochondrial pseudogenes, such as Numts. Here, we have used shot-gun Illumina-HiSeq sequencing to recover the first complete Mansonella genus mitogenome and to identify several mitochondrial-origin pseudogenes. Mitogenome phylogenetic analysis placed M. ozzardi in the Onchocercidae “ONC5” clade and suggested that Mansonella parasites are more closely related to Wuchereria and Brugia genera parasites than they are to Loa genus parasites. DNA sequence alignments, BLAST searches and conceptual translations have been used to compliment phylogenetic analysis showing that M. ozzardi from the Amazon and Caribbean regions are near-identical and that previously reported Peruvian M. ozzardi CO1 reference sequences are probably of pseudogene origin. In addition to adding a much-needed resource to the Mansonella genus’s molecular tool-kit and providing evidence that some M. ozzardi CO1 sequence deposits are pseudogenes, our results suggest that all Neotropical M. ozzardi parasites are closely related.

Mansonellosis: current perspectives

Mansonellosis is a filarial disease caused by three species of filarial (nematode) parasites (Mansonella perstans, Mansonella streptocerca, and Mansonella ozzardi) that use humans as their main definitive hosts. These parasites are transmitted from person to person by bloodsucking females from two families of flies (Diptera). Biting midges (Ceratopogonidae) transmit all three species of Mansonella, but blackflies (Simuliidae) are also known to play a role in the transmission of M. ozzardi in parts of Latin America. M. perstans and M. streptocerca are endemic in western, eastern, and central Africa, and M. perstans is also present in the neotropical region from equatorial Brazil to the Caribbean coast. M. ozzardi has a patchy distribution in Latin America and the Caribbean. Mansonellosis infections are thought to have little pathogenicity and to be almost always asymptomatic, but occasionally causing itching, joint pains, enlarged lymph glands, and vague abdominal symptoms. In Brazil, M. ozzardi infections are also associated with corneal lesions. Diagnosis is usually performed by detecting microfilariae in peripheral blood or skin without any periodicity. There is no standard treatment at present for mansonellosis. The combination therapy of diethylcarbamazine plus mebendazole for M. perstans microfilaremia is presently one of the most widely used, but the use of ivermectin has also been proven to be very effective against microfilariae. Recently, doxycycline has shown excellent efficacy and safety when used as an antimicrobial against endosymbiotic Wolbachia bacteria harbored by some strains of M. perstans and M. ozzardi. Diethylcarbamazine and ivermectin have been used effectively to treat M. streptocerca infection. There are at present no estimates of the disease burden caused by mansonellosis, and thus its importance to many global health professionals and policy makers is presently limited to how it can interfere with diagnostic tools used in modern filarial disease control and elimination programs aimed at other species of filariae.

Geographical distribution and species identification of human filariasis and onchocerciasis in Bioko Island, Equatorial Guinea

Human filariae are vector-borne parasites and the causative agents of various diseases, including human onchocerciasis and lymphatic filariasis. Onchocerciasis causes a spectrum of cutaneous and ophthalmologic manifestations (including blindness) and has long been a major public health problem in Bioko Island (Equatorial Guinea). Bioko Island has been included in the WHOs Onchocerciasis Control Program since 1987. In Bioko Island, the specificity and sensitivity of clinical Onchocerca volvulus diagnosis is key. The objective of this work was to update onchocerciasis elimination progress in Bioko Island, after 18 years of mass ivermectin intervention, and the general filariasis situation through a rapid and accurate molecular method. A cross-sectional study was conducted in Bioko Island from mid-January to mid-February 2014. A total of 543 subjects were included in the study. Whole blood and one skin snip (from lumbar regions) were analysed with a real time PCR assay. Two other skin biopsies were analysed by an expert microscopist. All positive samples were confirmed by sequencing. Traditional microscopic examination of the skin biopsies failed to detect any microfilariae. However, 11 (2.03%) infections were detected using PCR assay, including one O. volvulus, two Mansonella streptocerca, seven Mansonella perstans and one Loa loa infections. PCR assays in blood detected 52 filariae-positive individuals (9.6%) which harboured M. perstans or L. loa. The low prevalence of O. volvulus confirms the success of the Onchocerciasis Control Programme and suggests that Mass Drug Administration in Bioko Island can be interrupted in the near future. The very high prevalence of M. perstans found in skin snips assays raises doubts about the reliability of microscope-based diagnosis of O. volvulus infections.

The genomic architecture of novel Simulium damnosum Wolbachia prophage sequence elements and implications for onchocerciasis epidemiology

Research interest in Wolbachia is growing as new discoveries and technical advancements reveal the public health importance of both naturally occurring and artificial infections. Improved understanding of the Wolbachia bacteriophages (WOs) WOcauB2 and WOcauB3 [belonging to a sub-group of four WOs encoding serine recombinases group 1 (sr1WOs)], has enhanced the prospect of novel tools for the genetic manipulation of Wolbachia. The basic biology of sr1WOs, including host range and mode of genomic integration is, however, still poorly understood. Very few sr1WOs have been described, with two such elements putatively resulting from integrations at the same Wolbachia genome loci, about 2 kb downstream from the FtsZ cell-division gene. Here, we characterize the DNA sequence flanking the FtsZ gene of wDam, a genetically distinct line of Wolbachia isolated from the West African onchocerciasis vector Simulium squamosum E. Using Roche 454 shot-gun and Sanger sequencing, we have resolved >32 kb of WO prophage sequence into three contigs representing three distinct prophage elements. Spanning ≥36 distinct WO open reading frame gene sequences, these prophage elements correspond roughly to three different WO modules: a serine recombinase and replication module (sr1RRM), a head and base-plate module and a tail module. The sr1RRM module contains replication genes and a Holliday junction recombinase and is unique to the sr1 group WOs. In the extreme terminal of the tail module there is a SpvB protein homolog—believed to have insecticidal properties and proposed to have a role in how Wolbachia parasitize their insect hosts. We propose that these wDam prophage modules all derive from a single WO genome, which we have named here sr1WOdamA1. The best-match database sequence for all of our sr1WOdamA1-predicted gene sequences was annotated as of Wolbachia or Wolbachia phage sourced from an arthropod. Clear evidence of exchange between sr1WOdamA1 and other Wolbachia WO phage sequences was also detected. These findings provide insights into how Wolbachia could affect a medically important vector of onchocerciasis, with potential implications for future control methods, as well as supporting the hypothesis that Wolbachia phages do not follow the standard model of phage evolution.