Deep Sequencing Reveals Occult Mansonellosis Coinfections in Residents From the Brazilian Amazon Village of São Gabriel da Cachoeira.

Mansonella ozzardi and Mansonella perstans infections both cause mansonellosis but are usually treated differently. Using a real-time polymerase chain reaction assay and deep sequencing, we reveal the presence of mansonellosis coinfections that were undetectable by standard diagnostic methods. Our results confirm mansonellosis coinfections and have important implications for the disease's treatment and diagnosis.

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.

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

BACKGROUND: Multi-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. RESULTS: 16S 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. CONCLUSIONS: Our 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.

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

BACKGROUND: Immunoassays 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. METHODS: Two 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. CONCLUSION: Both 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.

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.

Filarial disease in the Brazilian Amazon and emerging opportunities for treatment and control.

Following the successful eradication of Wuchereria bancrofti, there are now just three species of conventional microfilaremic human filarial parasites endemic to the Brazilian Amazon region: Mansonella ozzardi, Mansonella perstans and Onchocerca volvulus. The zoonotic filarial parasite Dirofilaria immitis is also found in the Amazon region as are several sylvatic filarial parasites, some of which have been recorded causing zoonoses and some of which have never been recorded outside the region. Onchocerca volvulus is only found in the Amazonia onchocerciasis focus in the Brazilian state of Roraima where it affects the people of the Yanomami tribe living around the densely forested Venezuela border region. Mansonella ozzardi is by far the most common filarial parasite in Brazil and has a broad but patchy distribution throughout the western Amazon region. Recorded in the Brazilian states of Acre, Roraima, Matto Grosso, and within almost every municipality of Amazonas state, it is believed that pollution of the urban stream and river systems prevents the development of the simuliid vectors of M. ozzardi and explains the parasite's reduced distribution within urban areas and an absence of recent reports from the state capital Manaus. Decades of WHO-led periodic ivermectin treatment of Yanomami tribe's people have resulted in the partial suppression of O. volvulus transmission in this focus and has also probably affected the transmission of M. ozzardi in the region. Mansonella perstans, O. volvulus and very probably M. ozzardi infections can all be treated and most likely cured with a 4-6-week treatment course of doxycycline. The Brazilian Ministry of Health does not, however, presently recommend any treatment for mansonellosis infections and thus parasitic infections outside the Amazonia focus are typically left untreated. While the long treatment courses required for doxycycline-based mansonellosis therapies preclude their use in control programmes, new fast-acting filarial drug treatments are likely to soon become available for the treatment of both onchocerciasis and mansonellosis in the Amazon region. Filarial disease management in the Brazilian Amazon is thus likely to become dramatically more viable at a time when the public health importance of these diseases is increasingly being recognized.

The search for better treatment strategies for mansonellosis: an expert perspective.

INTRODUCTION: Four species of the Mansonella genus infect millions of people across sub-Saharan Africa and Central and South America. Most infections are asymptomatic, but mansonellosis can be associated with nonspecific clinical manifestations such as fever, headache, arthralgia, and ocular lesions (M. ozzardi); pruritus, arthralgia, abdominal pain, angioedema, skin rash, and fatigue (M. perstans and perhaps Mansonella sp. 'DEUX'); and pruritic dermatitis and chronic lymphadenitis (M. perstans). AREAS COVERED: We searched the PubMed and SciELO databases for publications on mansonelliasis in English, Spanish, Portuguese, or French that appeared until 1 May 2023. Literature data show that anthelmintics - single-dose ivermectin for M. ozzardi, repeated doses of mebendazole alone or in combination with diethylcarbamazine (DEC) for M. perstans, and DEC alone for M. streptocerca - are effective against microfilariae. Antibiotics that target Wolbachia endosymbionts, such as doxycycline, are likely to kill adult worms of most, if not all, Mansonella species, but the currently recommended 6-week regimen is relatively impractical. New anthelmintics and shorter antibiotic regimens (e.g. with rifampin) have shown promise in experimental filarial infections and may proceed to clinical trials. EXPERT OPINION: We recommend that human infections with Mansonella species be treated, regardless of any apparent clinical manifestations. We argue that mansonellosis, despite being widely considered a benign infection, may represent a direct or indirect cause of significant morbidity that remains poorly characterized at present.

Dirofilaria immitis is endemic in rural areas of the Brazilian Amazonas state capital, Manaus.

The canine filarial parasite Dirofilaria immitis has not been reported in Brazil´s Amazonas state capital, Manaus, for over a century. Here, we report one imported and 27 autochthonous D. immitis infections from a microfilarial survey of 766 domestic dog blood samples collected between 2017 and 2021 in Manaus. An Overall prevalence estimate of 15.44% (23/149) was calculated from our two rural collection sites; a prevalence of 1.22% (4/328) was estimated at our periurban collection site, and an overall prevalence of 0.35% (1/289) was calculated from our two urban clinic collections. Our data suggest that in the urban areas of Manaus, where the parasites are very likely vectored by the same species of mosquito that historically vectored Wuchereria bancrofti (Culex quinquefasciatus), prevalence levels are very low and possibly maintained by an influx from rural areas where sylvatic reservoirs and/or more favorable vector transmission dynamics maintain high prevalences.

Molecular detection of Mansonella mariae incriminates Simulium oyapockense as a potentially important bridge vector for Amazon-region zoonoses.

OBJECTIVE: To assess the emergent zoonotic disease risk posed by the voracious human-biting blackfly species Simulium oyapockense in the peripheral regions of an expanding urban centre situated deep in the Brazilian Amazon rainforest. METHODS: We performed nine human landing catches at three periurban sites surrounding the Brazilian Amazon town of São Gabriel da Cachoeira. Using the detection of non-human primate filarial parasites as an indicator of the zoonotic disease threat posed by a biting insect, we screened 3328 S. oyapockense blackflies for the presence of zoonotic filarial DNA with an ITS-1 PCR assay and Sanger sequencing. RESULTS: Between 98 and 100% of the biting insects captured during our nine collections were identified as S. oyapockense; at our three collection sites and during our three seasonally-distinct collections this species was captured at rates between 28 and 294 blackflies per hour. PCR screening of the march-collected S. oyapockense detected infectious-stage (L3) Mansonella mariae parasites (which are only known to infect non-human primates) in >0.15% of the tested head samples. CONCLUSIONS: Our results show that residents of the periurban regions of São Gabriel da Cachoeira are routinely exposed to the bites of S. oyapockense blackflies which have previously fed on non-human primates.

Correction: SARS-CoV-2 in the Amazon region: A harbinger of doom for Amerindians.

[This corrects the article DOI: 10.1371/journal.pntd.0008686.].

SARS-CoV-2 in the Amazon region: A harbinger of doom for Amerindians.

As the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic continues to expand, healthcare resources globally have been spread thin. Now, the disease is rapidly spreading across South America, with deadly consequences in areas with already weakened public health systems. The Amazon region is particularly susceptible to the widespread devastation from Coronavirus disease 2019 (COVID-19) because of its immunologically fragile native Amerindian inhabitants and epidemiologic vulnerabilities. Herein, we discuss the current situation and potential impact of COVID-19 in the Amazon region and how further spread of the epidemic wave could prove devastating for many Amerindian people living in the Amazon rainforest.

Blackflies in the ointment: O. volvulus vector biting can be significantly reduced by the skin-application of mineral oil during human landing catches.

BACKGROUND: Standard human landing catches (sHLCs) have historically been a key component of Onchocerca volvulus transmission monitoring, but expose health-workers to potentially hazardous vector bites. Novel human-bait-free trapping methods have been developed, but do not always work where they are needed and may not generate O. volvulus surveillance data that is directly comparable with historic data. METHODOLOGY: Simuliid sHLCs and mineral-oil protected HLCs (mopHLCs) were performed in a rural village of Amazonas state, Brazil. A four-hour direct comparisons of sHLCs and mopHLCs was carried-out using six vector collectors, each of whom used one leg for a sHLC and one for a mopHLC. Two-person collection teams then exclusively performed either mopHLCs or sHLCs for a further set of 12 four-hour collections. Following the completion of all collections, simuliid-bite mark estimates were made from legs used exclusively in sHLCs and legs used exclusively in mopHLCs. PRINCIPAL FINDINGS: All of the 1669 captured simuliids were identified as the O. volvulus vector Simulium oyapockense. Overall, mopHLC simuliids captured per hour (S/H) rates were lower than those obtained with sHLC trapping (15.5 S/H versus 20 S/H). Direct comparisons of simuliid capture rates found that vector-collectors captured simuliids significantly more efficiently ([Formula: see text]: 20.5 S/H) with mopHLC trapping than with sHLC trapping ([Formula: see text]: 16.4 S/H): P-value = 0.002. MopHLCs performed in isolation were, however, observed to capture vectors less efficiently ([Formula: see text]: 13.4 S/H) than sHLCs performed under similar conditions ([Formula: see text]: 19.98 S/H). All six vector collectors had significantly higher simuliid capture per counted bite mark (SC/CBM) rates using mopHLCs than they were observe to have using sHLCs ([Formula: see text]: 21 SC/CBM versus [Formula: see text]: 1 SC/CBM; p-value = 0.03125). CONCLUSIONS: Vector collectors captured significantly more simuliids per counted bite mark with mopHLCs than with sHLCs. Further investigations into the utility of mopHLCs for onchocerciasis xenomonitoring and beyond are merited.

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.

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 WHO's 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.

A field trial of a PCR-based Mansonella ozzardi diagnosis assay detects high-levels of submicroscopic M. ozzardi infections in both venous blood samples and FTA card dried blood spots.

BACKGROUND: Mansonella ozzardi is a poorly understood human filarial parasite with a broad distribution throughout Latin America. Most of what is known about its parasitism has come from epidemiological studies that have estimated parasite incidence using light microscopy. Light microscopy can, however, miss lighter, submicroscopic, infections. In this study we have compared M. ozzardi incidence estimates made using light microscopy, with estimates made using PCR. METHODS: 214 DNA extracts made from Large Volume Venous Blood Samples (LVVBS) were taken from volunteers from two study sites in the Rio Solimões region: Codajás [n = 109] and Tefé [n = 105] and were subsequently assayed for M. ozzardi parasitism using a diagnostic PCR (Mo-dPCR). Peripheral finger-prick blood samples were taken from the same individuals and used for microscopic examination. Finger-prick blood, taken from individuals from Tefé, was also used for the creation of FTAcard dried blood spots (DBS) that were subsequently subjected to Mo-dPCR. RESULTS: Overall M. ozzardi incidence estimates made with LVVBS PCRs were 1.8 times higher than those made using microscopy (44.9% [96/214] compared with 24.3% [52/214]) and 1.5 times higher than the PCR estimates made from FTAcard DBS (48/105 versus 31/105). PCR-based detection of FTAcard DBS proved 1.3 times more sensitive at diagnosing infections from peripheral blood samples than light microscopy did: detecting 24/105 compared with 31/105. PCR of LVVBS reported the fewest number of false negatives, detecting: 44 of 52 (84.6%) individuals diagnosed by microscopy; 27 of 31 (87.1%) of those diagnosed positive from DBSs and 17 out of 18 (94.4%) of those diagnosed as positive by both alternative methodologies. CONCLUSIONS: In this study, Mo-dPCR of LVVBS was by far the most sensitive method of detecting M. ozzardi infections and detected submicroscopic infections. Mo-dPCR FTAcard DBS also provided a more sensitive test for M. ozzardi diagnosis than light microscopy based diagnosis did and thus in settings where only finger-prick assays can be carried-out, it may be a more reliable method of detection. Most existing M. ozzardi incidence estimates, which are often based on light microscope diagnosis, are likely to dramatically underestimate true M. ozzardi parasitism incidence levels.

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.