Identification and characterisation of Mansonella perstans in the Volta Region of Ghana.

Mansonella spp. have been reported to have a wide global distribution. Despite the distribution and co-occurrence with other filarial parasites like Wuchereria bancrofti, Onchocerca volvulus and Loa loa, it is given little attention. There are few surveillance programmes for assessing the distribution of mansonellosis, due to the associated mild to no symptoms experienced by infected people. However, addressing this infection is critical to the onchocerciasis control program as current rapid diagnostic tools targeting O. volvulus have the tendency to cross react with Mansonella species. In this study we identified and characterised M. perstans from five sites in two districts in the Volta Region of Ghana and compared them to samples from other regions. Night blood smears and filter blood blots were obtained from individuals as part of a study on lymphatic filariasis. The Giemsa-stained smears were screened by microscopy for the presence of filarial parasites. Genomic DNA was extracted from blood blots from 39 individuals that were positive for M. perstans and Nested PCR targeting the internal spacer 1 (ITS-1) was conducted. Of these, 30 were sequenced and 24 sequences were kept for further analysis. Phylogenetic analysis of 194 nucleotide positions showed no differences in the samples collected. The similarities suggests that there could be one species in this area. However, more robust studies with larger sample sizes are required to draw such conclusions. We also observed a clustering of the samples from Ghana with reference sequences from Africa and Brazil, suggesting they could be related. This study draws further attention to a neglected infection, presents the first characterisation of M. perstans in Ghana and calls for more population-based studies across different geographical zones to ascertain species variations and disease distribution.

Molecular Surveillance Detects High Prevalence of the Neglected Parasite Mansonella ozzardi in the Colombian Amazon.

BACKGROUND: Mansonellosis is an undermapped insect-transmitted disease caused by filarial nematodes that are estimated to infect hundreds of millions of people. Despite their prevalence, there are many outstanding questions regarding the general biology and health impacts of the responsible parasites. Historical reports suggest that the Colombian Amazon is endemic for mansonellosis and may serve as an ideal location to pursue these questions. METHODS: We deployed molecular and classical approaches to survey Mansonella prevalence among adults belonging to indigenous communities along the Amazon River and its tributaries near Leticia, Colombia. RESULTS: Loop-mediated isothermal amplification (LAMP) assays on whole-blood samples detected a much higher prevalence of Mansonella ozzardi infection (approximately 40%) compared to blood smear microscopy or LAMP performed using plasma, likely reflecting greater sensitivity and the ability to detect low microfilaremias and occult infections. Mansonella infection rates increased with age and were higher among men. Genomic analysis confirmed the presence of M. ozzardi that clusters closely with strains sequenced in neighboring countries. We successfully cryopreserved M. ozzardi microfilariae, advancing the prospects of rearing infective larvae in controlled settings. CONCLUSION: These data suggest an underestimation of true mansonellosis prevalence, and we expect that these methods will help facilitate the study of mansonellosis in endemic and laboratory settings.

Inaccurate recording of Mansonella perstans in free-ranging primates outside its endemic area in Brazil?

In the study undertaken by Souza et al. [Primates 64(1):153-159, 2022; https://doi.org/10.1007/s10329-022-01038-5 ], published in the most recent volume of this journal, the blood samples of two Alouatta guariba clamitans (Primates, Atelidae) from two municipalities in the state of Rio Grande do Sul, southern Brazil were reported to be positive for Mansonella perstans. This is the first reported finding of M. perstans in A. guariba clamitans, as well as the first time that M. perstans has been recorded in Brazil outside the Amazon region. We would like to express our concern about this finding, specifically with respect to the geographical distribution of M. perstans in Brazil, as, up until this study, this filaria had only been found in the upper Rio Negro region in São Gabriel da Cachoeira, Amazonas, Brazil. Moreover, species identification was performed using partial sequences of three gene fragments, namely internal transcribed spacer 2, 12S, and 18S, yet neither the phylogenetic trees nor the BLAST alignments of these sequences provided supporting evidence that they belong to M. perstans.

Whole genome analysis of two sympatric human Mansonella: Mansonella perstans and Mansonella sp "DEUX".

Introduction: Mansonella species are filarial parasites that infect humans worldwide. Although these infections are common, knowledge of the pathology and diversity of the causative species is limited. Furthermore, the lack of sequencing data for Mansonella species, shows that their research is neglected. Apart from Mansonella perstans, a potential new species called Mansonella sp "DEUX" has been identified in Gabon, which is prevalent at high frequencies. We aimed to further determine if Mansonella sp "DEUX" is a genotype of M. perstans, or if these are two sympatric species. Methods: We screened individuals in the area of Fougamou, Gabon for Mansonella mono-infections and generated de novo assemblies from the respective samples. For evolutionary analysis, a phylogenetic tree was reconstructed, and the differences and divergence times are presented. In addition, mitogenomes were generated and phylogenies based on 12S rDNA and cox1 were created. Results: We successfully generated whole genomes for M. perstans and Mansonella sp "DEUX". Phylogenetic analysis based on annotated protein sequences, support the hypothesis of two distinct species. The inferred evolutionary analysis suggested, that M. perstans and Mansonella sp "DEUX" separated around 778,000 years ago. Analysis based on mitochondrial marker genes support our hypothesis of two sympatric human Mansonella species. Discussion: The results presented indicate that Mansonella sp "DEUX" is a new Mansonella species. These findings reflect the neglect of this research topic. And the availability of whole genome data will allow further investigations of these species.

Emergence of Mansonella sp. in free-ranging primates in southern Brazil.

Mansonellosis is a neglected and emerging tropical disease. Among all zoonotic filarial diseases, it is probably the most prevalent and least studied, with approximately 114 million people infected. The parasites of Mansonella spp. are among the most common blood parasitemias and are widely found in Africa and Latin America. Through molecular analysis of blood samples from free-ranging primates Sapajus nigritus (n 33) and Alouatta guariba clamitans (n 5) in the southern states of Brazil (Santa Catarina and Rio Grande do Sul), we identified samples positive for Mansonella perstans in two specimens of A. guariba clamitans. A fragment of 578 bp from the ITS intergenic region (5.8S-ITS2-28S) was targeted for an initial PCR screening. Subsequently, positive samples were subjected to other PCR assays targeting a fragment of the 12S and the 18S genes. This is the first record of molecular detection of the agent in this host in the Pampa Biome. With a wide distribution across Brazil and Argentina, these primates may represent a potential wild reservoir for the zoonotic agent of mansonellosis. Entomological and transmission studies are essential to avoid the urbanization of mansonellosis and to understand the cycles of agents in different environmental scenarios.

Targeting a highly repetitive genomic sequence for sensitive and specific molecular detection of the filarial parasite Mansonella perstans from human blood and mosquitoes.

BACKGROUND: Mansonella perstans is among the most neglected of the neglected tropical diseases and is believed to cause more human infections than any other filarial pathogen in Africa. Based largely upon assumptions of limited infection-associated morbidity, this pathogen remains understudied, and many basic questions pertaining to its pathogenicity, distribution, prevalence, and vector-host relationships remain unanswered. However, in recent years, mounting evidence of the potential for increased Mansonella infection-associated disease has sparked a renewal in research interest. This, in turn, has produced a need for improved diagnostics, capable of providing more accurate pictures of infection prevalence, pathogen distribution, and vector-host interactions. METHODOLOGY/PRINCIPAL FINDINGS: Utilizing a previously described pipeline for the discovery of optimal molecular diagnostic targets, we identified a repetitive DNA sequence, and developed a corresponding assay, which allows for the sensitive and species-specific identification of M. perstans in human blood samples. Testing also demonstrated the ability to utilize this assay for the detection of M. perstans in field-collected mosquito samples. When testing both sample types, our repeat-targeting index assay outperformed a ribosomal sequence-targeting reference assay, facilitating the identification of additional M. perstans-positive samples falsely characterized as "negative" using the less sensitive detection method. CONCLUSIONS/SIGNIFICANCE: Through the development of an assay based upon the systematic identification of an optimal DNA target sequence, our novel diagnostic assay will provide programmatic efforts with a sensitive and specific testing platform that is capable of accurately mapping M. perstans infection and determining prevalence. Furthermore, with the added ability to identify the presence of M. perstans in mosquito samples, this assay will help to define our knowledge of the relationships that exist between this pathogen and the various geographically relevant mosquito species, which have been surmised to represent potential secondary vectors under certain conditions. Detection of M. perstans in mosquitoes will also demonstrate proof-of-concept for the mosquito-based monitoring of filarial pathogens not vectored primarily by mosquitoes, an approach expanding opportunities for integrated surveillance.

Comparison of three PCR-based methods to detect Loa loa and Mansonella perstans in long-term frozen storage dried blood spots.

OBJECTIVES: Loa loa and Mansonella perstans are two very common filarial species in Africa. Although microscopy is the traditional diagnostic method for human filariasis, several polymerase chain reaction (PCR) methods have emerged as an alternative approach for identifying filarial parasites. The aim of this study is to compare three molecular methods and decide which is the most suitable for diagnosing human loiasis and mansonellosis in non-endemic regions using dried blood spot (DBS) as a medium for sample collection and storage. METHODS: A total of 100 DBS samples, with their corresponding thin and thick blood smears, were selected for this study. Microscopy was used as the reference method to diagnose and calculate the microfilaraemia. Filarial DNA was extracted using the saponin/Chelex method and the DNA isolated was assayed by Filaria-real time-PCR, filaria-nested PCR, and cytochrome oxidase I PCR. All PCR products were subsequently purified and sequenced. The statistical values for each molecular test were calculated and compared. RESULTS: Overall, 64 samples were identified as negative by all tests and a further 36 samples were positive by at least one of the methods used. The sensitivity and specificity were similar for the different molecular methods, all of which demonstrated good agreement with microscopy. CONCLUSIONS: Based on this study, and from a practical point of view (single and short amplification round), the optimal technique for diagnosing filarial infection in non-endemic regions is filaria-real time-PCR, which presents high sensitivity and specificity and is also able to detect a wide range of human filariae.

Molecular Epidemiology of Mansonella Species in Gabon.

Mansonella perstans, a filarial nematode, infects large populations in Africa and Latin America. Recently, a potential new species, Mansonella sp "DEUX," was reported. Carriage of endosymbiotic Wolbachia opens treatment options for Mansonella infections. Within a cross-sectional study, we assessed the prevalence of filarial infections in 834 Gabonese individuals and the presence of the endosymbiont Wolbachia. Almost half of the participants (400/834 [48%]) were infected with filarial nematodes, with Mansonella sp "DEUX" being the most frequent (295/400 [74%]), followed by Loa loa (273/400 [68%]) and Mansonella perstans (82/400 [21%]). Being adult/elderly, male, and living in rural areas was associated with a higher risk of infection. Wolbachia carriage was confirmed in M. perstans and Mansonella sp "DEUX." In silico analysis revealed that Mansonella sp "DEUX" is not detected with currently published M. perstans-specific assays. Mansonella infections are highly prevalent in Gabon and might have been underreported, likely also beyond Gabon.

Detection of DNA of filariae closely related to Mansonella perstans in faecal samples from wild non-human primates from Cameroon and Gabon.

BACKGROUND: The Onchocercidae is a family of filarial nematodes with several species of medical or veterinary importance. Microfilariae are found in the blood and/or the dermis and are usually diagnosed in humans by microscopy examination of a blood sample or skin biopsy. The main objectives of this study were to evaluate whether filariae DNA can be detected in faecal samples of wild non-human primates (NHPs), whether the detected parasites were closely related to those infecting humans and whether filarial DNA detection in faeces is associated with co-infections with nematodes (Oesophagostumum sp. and Necator sp.) known to cause blood loss while feeding on the host intestinal mucosa. METHODS: A total of 315 faecal samples from 6 species of NHPs from Cameroon and Gabon were analysed. PCRs targeted DNA fragments of cox1 and 12S rDNA genes, to detect the presence of filariae, and the internal transcribed spacer 2 (ITS2), to detect the presence of Oesophagostomum sp. and Necator sp. infections. RESULTS: Among the 315 samples analysed, 121 produced sequences with > 90% homology with Onchocercidae reference sequences. However, 63% of the 12S rDNA and 78% of the cox1 gene sequences were exploitable for phylogenetic analyses and the amplification of the 12S rDNA gene showed less discriminating power than the amplification of the cox1 fragment. Phylogenetic analyses showed that the cox1 sequences obtained from five chimpanzee DNA faecal samples from Gabon and two from Cameroon cluster together with Mansonella perstans with high bootstrap support. Most of the remaining sequences clustered together within the genus Mansonella, but the species could not be resolved. Among the NHP species investigated, a significant association between filarial DNA detection and Oesophagostomum sp. and Necator sp. infection was observed only in gorillas. CONCLUSIONS: To our knowledge, this is the first study reporting DNA from Mansonella spp. in faecal samples. Our results raise questions about the diversity and abundance of these parasites in wildlife, their role as sylvatic reservoirs and their potential for zoonotic transmission. Future studies should focus on detecting variants circulating in both human and NHPs, and improve the molecular information to resolve or support taxonomy classification based on morphological descriptions.

High Prevalence of Mansonella ozzardi Infection in the Amazon Region, Ecuador.

We reviewed Giemsa-stained thick blood smears, obtained through the national malaria surveillance program in the Amazon region of Ecuador, by light microscopy for Mansonella spp. microfilariae. Of 2,756 slides examined, 566 (20.5%) were positive. Nested PCR confirmed that the microfilariae were those of M. ozzardi nematodes, indicating that this parasite is endemic to this region.

In Silico Identification of Novel Biomarkers and Development of New Rapid Diagnostic Tests for the Filarial Parasites Mansonella perstans and Mansonella ozzardi.

Mansonelliasis is a widespread yet neglected tropical infection of humans in Africa and South America caused by the filarial nematodes, Mansonella perstans, M. ozzardi, M. rodhaini and M. streptocerca. Clinical symptoms are non-distinct and diagnosis mainly relies on the detection of microfilariae in skin or blood. Species-specific DNA repeat sequences have been used as highly sensitive biomarkers for filarial nematodes. We have developed a bioinformatic pipeline to mine Illumina reads obtained from sequencing M. perstans and M. ozzardi genomic DNA for new repeat biomarker candidates which were used to develop loop-mediated isothermal amplification (LAMP) diagnostic tests. The M. perstans assay based on the Mp419 repeat has a limit of detection of 0.1 pg, equivalent of 1/1000th of a microfilaria, while the M. ozzardi assay based on the Mo2 repeat can detect as little as 0.01 pg. Both LAMP tests possess remarkable species-specificity as they did not amplify non-target DNAs from closely related filarial species, human or vectors. We show that both assays perform successfully on infected human samples. Additionally, we demonstrate the suitability of Mp419 to detect M. perstans infection in Culicoides midges. These new tools are field deployable and suitable for the surveillance of these understudied filarial infections.

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.

Sensitivity of diagnostic methods for Mansonella ozzardi microfilariae detection in the Brazilian Amazon Region

BACKGROUND The human filarial worm Mansonella ozzardi is highly endemic in the large tributaries of the Amazon River. This infection is still highly neglected and can be falsely negative when microfilariae levels are low. OBJECTIVES This study investigated the frequency of individuals with M. ozzardi in riverine communities in Coari municipality, Brazilian Amazon. METHODS Different diagnostic methods including polymerase chain reaction (PCR), blood polycarbonate membrane filtration (PCMF), Knott's method (Knott), digital thick blood smears (DTBS) and venous thick blood smears (VTBS) were used to compare sensitivity and specificity among the methods. Data were analysed using PCMF and Bayesian latent class models (BLCM) as the gold standard. We used BLCM to calculate the prevalence of mansonelliasis based on the results of five diagnostic methods. FINDINGS The prevalence of mansonelliasis was 35.4% by PCMF and 30.1% by BLCM. PCR and Knott methods both possessed high sensitivity. Sensitivity relative to PCMF was 98.5% [95% confidence interval (CI): 92.0 - 99.7] for PCR and 83.5% (95% CI: 72.9 - 90.5) for Knott. Sensitivity derived by BLCM was 100% (95% CI 93.7 - 100) for PCMF, 100% (95% CI: 93.7 - 100) for PCR and 98.3% (95% CI: 90.6 - 99.9) for Knott. The odds ratio of being diagnosed as microfilaremic increased with age but did not differ between genders. Microfilariae loads were higher in subjects aged 30 - 45 and 45 - 60 years. MAIN CONCLUSIONS PCMF and PCR were the best methods to assess the prevalence of mansonelliasis in our samples. As such, using these methods could lead to higher prevalence of mansonelliasis in this region than the most commonly used method (i.e., thick blood smears).

Sensitivity of diagnostic methods for Mansonella ozzardi microfilariae detection in the Brazilian Amazon Region.

BACKGROUND: The human filarial worm Mansonella ozzardi is highly endemic in the large tributaries of the Amazon River. This infection is still highly neglected and can be falsely negative when microfilariae levels are low. OBJECTIVES: This study investigated the frequency of individuals with M. ozzardi in riverine communities in Coari municipality, Brazilian Amazon. METHODS: Different diagnostic methods including polymerase chain reaction (PCR), blood polycarbonate membrane filtration (PCMF), Knott's method (Knott), digital thick blood smears (DTBS) and venous thick blood smears (VTBS) were used to compare sensitivity and specificity among the methods. Data were analysed using PCMF and Bayesian latent class models (BLCM) as the gold standard. We used BLCM to calculate the prevalence of mansonelliasis based on the results of five diagnostic methods. FINDINGS: The prevalence of mansonelliasis was 35.4% by PCMF and 30.1% by BLCM. PCR and Knott methods both possessed high sensitivity. Sensitivity relative to PCMF was 98.5% [95% confidence interval (CI): 92.0 - 99.7] for PCR and 83.5% (95% CI: 72.9 - 90.5) for Knott. Sensitivity derived by BLCM was 100% (95% CI 93.7 - 100) for PCMF, 100% (95% CI: 93.7 - 100) for PCR and 98.3% (95% CI: 90.6 - 99.9) for Knott. The odds ratio of being diagnosed as microfilaremic increased with age but did not differ between genders. Microfilariae loads were higher in subjects aged 30 - 45 and 45 - 60 years. MAIN CONCLUSIONS: PCMF and PCR were the best methods to assess the prevalence of mansonelliasis in our samples. As such, using these methods could lead to higher prevalence of mansonelliasis in this region than the most commonly used method (i.e., thick blood smears).

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.

Atypical Mansonella ozzardi Microfilariae from an Endemic Area of Brazilian Amazonia.

Mansonellosis is endemic in several regions of Africa, the Caribbean, and Latin America. Mansonella ozzardi and Mansonella perstans have been reported in Latin America, including the Amazon region. A morphological and molecular microfilariae study was performed in Pauini (Brazil). Blood samples were collected from 40 individuals, and were analyzed by Giemsa-stained blood film and by two different nested polymerase chain reactions which detect internal transcribed spacer-1 and the major sperm protein gene. By microscopy, 14 of 40 were positive: 11 as M. ozzardi and three as M. perstans-like infections. Both molecular methods detected 19 positive cases as M. ozzardi, including those 14 individuals detected by microscopy, without detectable genetic differences among any of the 19 positive samples. Molecular techniques showed an improvement of mansonellosis diagnosis and may become an effective tool to evaluate the present status of M. ozzardi and M. perstans in Latin America.

Molecular Epidemiology of Blood-Borne Human Parasites in a Loa loa-, Mansonella perstans-, and Plasmodium falciparum-Endemic Region of Cameroon.

The study of the interactions among parasites within their hosts is crucial to the understanding of epidemiology of disease and for the design of effective control strategies. We have conducted an assessment of infections with Loa loa, Mansonella perstans, Wuchereria bancrofti, and Plasmodium falciparum in eastern Cameroon using a highly sensitive and specific quantitative polymerase chain reaction assay using archived dried whole blood spots. The resident population (N = 1,085) was parasitized with M. perstans (76%), L. loa (39%), and P. falciparum (33%), but not with W. bancrofti Compared with single infections (40.1%), coinfection was more common (48.8%): 21.0% had L. loa-M. perstans (Ll(+)/Mp(+)/Pf(-)), 2.7% had L. loa-P. falciparum (Ll(+)/Pf(+)/Mp(-)), 15.1% had M. perstans-P. falciparum (Mp(+)/Pf(+)/Ll(-)), and 10.0% had L. loa-M. perstans-P. falciparum (Ll(+)/Mp(+)/Pf(+)). Interestingly, those with all three infections (Ll(+)/Mp(+)/Pf(+)) had significantly higher L. loa microfilaria (mf) counts than either single Ll(+) (P = 0.004) or double Ll(+)/Mp(+) (P = 0.024) infected individuals. Of those infected with L. loa, the mean estimated counts of L. loa mf varied based on location and were positively correlated with estimated intensities of M. perstans mf. Finally, at a community level, heavy L. loa infections were concentrated in a few individuals whereby they were likely the major reservoir for infection.

Mansonella, including a Potential New Species, as Common Parasites in Children in Gabon.

BACKGROUND: Like other tropical African countries, Gabon is afflicted by many parasitic diseases, including filariases such as loiasis and mansonellosis. This study aimed to assess the prevalence of these two filarial diseases in febrile and afebrile children using quantitative real-time PCR and standard PCR assays coupled with sequencing. METHODOLOGY/PRINCIPAL FINDINGS: DNA from blood specimens of 1,418 Gabonese children (1,258 febrile and 160 afebrile) were analyzed. Overall, filarial DNA was detected in 95 (6.7%) children, including 67 positive for M. perstans (4.7%), which was the most common. M. perstans was detected in 61/1,258 febrile children (4.8%) and 6/160 afebrile children (3.8%, P = 0.6). Its prevalence increased statistically with age: 3.5%, 7.7% and 10.6% in children aged ≤ 5, 6-10 and 11-15 years, respectively. M. perstans prevalence was significantly higher in Koulamoutou and Lastourville (12% and 10.5%, respectively) than in Franceville and Fougamou (2.6% and 2.4%, respectively). Loa loa was detected in seven febrile children including one co-infection with M. perstans. Finally, 21 filarial DNA positive were negative for M. perstans and Loa loa, but ITS sequencing could be performed for 12 and allowed the identification of a potential new species of Mansonella provisionally called "DEUX". Mansonella sp. "DEUX" was detected only in febrile children. CONCLUSIONS/SIGNIFICANCE: Further study should be performed to characterize Mansonella sp. "DEUX" and evaluate the clinical significance of mansonellosis in humans.

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.