Characterising co-infections with Plasmodium spp., Mansonella perstans or Loa loa in asymptomatic children, adults and elderly people living on Bioko Island using nucleic acids extracted from malaria rapid diagnostic tests.

BACKGROUND: Regular and comprehensive epidemiological surveys of the filarial nematodes Mansonella perstans and Loa loa in children, adolescents and adults living across Bioko Island, Equatorial Guinea are lacking. We aimed to demonstrate that blood retained on malaria rapid diagnostic tests, commonly deployed for malaria surveys, could be used as a source of nucleic acids for molecular based detection of M. perstans and L. loa. We wanted to determine the positivity rate and distribution of filarial nematodes across different age groups and geographical areas as well as to understand level of co-infections with malaria in an asymptomatic population. METHODOLOGY: M. perstans, L. loa and Plasmodium spp. parasites were monitored by qPCR in a cross-sectional study using DNA extracted from a subset malaria rapid diagnostic tests (mRDTs) collected during the annual malaria indicator survey conducted on Bioko Island in 2018. PRINCIPAL FINDINGS: We identified DNA specific for the two filarial nematodes investigated among 8.2% (263) of the 3214 RDTs screened. Positivity rates of M. perstans and L. loa were 6.6% and 1.5%, respectively. M. perstans infection were more prominent in male (10.5%) compared to female (3.9%) survey participants. M. perstans parasite density and positivity rate was higher among older people and the population living in rural areas. The socio-economic status of participants strongly influenced the infection rate with people belonging to the lowest socio-economic quintile more than 3 and 5 times more likely to be L. loa and M. perstans infected, respectively. No increased risk of being co-infected with Plasmodium spp. parasites was observed among the different age groups. CONCLUSIONS/SIGNIFICANCE: We found otherwise asymptomatic individuals were infected with M. perstans and L. loa. Our study demonstrates that employing mRDTs probed with blood for malaria testing represents a promising, future tool to preserve and ship NAs at room temperature to laboratories for molecular, high-throughput diagnosis and genotyping of blood-dwelling nematode filarial infections. Using this approach, asymptomatic populations can be reached and surveyed for infectious diseases beyond malaria.

Laboratory Parameters after Treatment for Loa loa and Mansonella perstans: The Experience of a Single Referral Center for Tropical Diseases in a Non-Endemic Area.

Infections due to Loa loa and Mansonella perstans are common yet elusive neglected filariases. Parasitological cure after treatment is very difficult to assess, as adult parasites are not accessible. Therefore, outside transmission areas, patients require a long follow-up period to ascertain the therapeutic outcome, which is impractical for non-sedentary populations such as migrants. We studied the change over time of microfilaremia, eosinophil counts, and antifilarial antibodies tested with a commercial ELISA test (Bordier Affinity Products, Crissier, Switzerland), in a retrospective cohort of patients with confirmed L. loa and M. perstans infections, to evaluate the role of serology in clinical practice. After treatment, all 22 eligible patients diagnosed in our center between 2015 and 2017 reached amicrofilaremia, with microfilarial counts decreasing sharply within 2 months. Paralleling eosinophil counts, antibodies decreased in all patients, 36% of whom reached sero-reversion or near-sero-reversion in < 20 months. These findings suggest that positive serology is not just residual from a past infection, and may be used for diagnosis even when microfilaremia is negative or cannot be performed. Interestingly, antibodies and eosinophil counts increased following some, but not all, re-treatment courses. If the rise in these parameters reflects death of macrofilariae, caution is required in interpreting high eosinophil counts and antibody titers shortly after treatment, as these may reflect no need for further treatment. To optimize patients' management, it is now pivotal to ascertain the interval between treatment and macrofilarial death and therefore whether re-treatments are required for complete clearance of parasites.

Immune response in Mansonella ozzardi infection modulated by IL-6/IL-10 axis in Amazon region of Brazil.

Mansonellosis is an endemic disease in the South and Central America. In Brazil, one of the etiological agents is Mansonella ozzardi. This filarial infection is yet poorly understood, with a controversial morbity, presenting since a oligosymptoms, malaria-like signs or without complaint in humans. The knowledge of the human immune response to microfilariae infection is limited mainly by different evolutionary cycles of the parasite in the host. In addition, the prevalence of this filarial parasite infection is high in several regions of Amazonas State. A cross-sectional study was conducted in an endemic area for microfilariae of M. ozzardi (MF) infection in the Amazonas State, Brazil. Proinflammatory and regulatory cytokines (IL-2, IL-4, IL-6, IL-10, TNF, IFN-gamma, and IL-17A) were measured in cryopreserved serum using the Cytometric Bead Array techniques (CBA) in 54 patients diagnosed with M. ozzardi infection and 55 individuals without the infection were included in the study (Controls). The IL-4, IL-6 and IL-10 level increased in infected patients with MF infection, while IL-17A increased in control only. When we compared controls to patients with high or low parasite load, the increased level of IL-6 and IL-10 were maintained. IL-6 contributes to the proinflammatory activity and IL-10 modulates Th1, Th2 and Th17 immune response. Furthermore, IL-4 was detected as a marker in the MF infection and MF patients with low parasite load, indicating the action of the Th2 cell response. The complex network of cytokines acting during M. ozzardi infection depends on a fine balance to determine a host protective effect or filarial persistence. Therefore, these results suggest that the immune response in MF infection is modulated by IL-6/IL-10 axis.

Successful long-term maintenance of Mansonella perstans in an in vitro culture system.

BACKGROUND: Approximately 114 million people are infected with Mansonella perstans in large proportions of Africa. In contrast to other filariae that infect humans, M. perstans-infected individuals show no distinct pathology or specific clinical picture, indicating a well-tuned adaptation to the host. In addition, since M. perstans adult worms reside in serous cavities which are difficult to access, research has been hindered and there is a paucity of knowledge about the biology of M. perstans, especially the development of the different life stages as well as M. perstans-driven immune responses. Thus in this study, an in vitro culture system was developed which allows an in-depth analysis of M. perstans. RESULTS: Culicoides species were caught in Ediki (Kumba), Southwest Region within Cameroon following a blood meal on a microfilaremic donor that had 1500 microfilariae/ml of peripheral blood and kept in captivity for 12 days at 23 °C. In a pilot experiment, 15 infective larvae were obtained from the midges and co-cultured with a confluent monolayer of monkey kidney epithelial cells (LLC-MK2) in DMEM medium supplemented with 10% FBS for up to 77 days. The resulting survival rates of 33% revealed that the cell-conditioned medium was suitable for long-term maintenance of M. perstans worms. To confirm these preliminary observations, 249 infective larvae were cultured for 50 days and their development was monitored daily and microscopically graded for motility. In total, 170 (68.3%) filariae survived and 124 (49.8%) larvae moulted between days 21-30 to become L5 stage larvae which were motile and showed continuous vigorous movement. CONCLUSION: We have established an in vitro culture system for the generation and long-term maintenance of viable M. perstans worms. This technique will be an important tool to study parasite biology and development, the role in host immunity, and might be helpful to discover novel treatment strategies against this filariae.

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.

New records of Mansonella ozzardi: a parasite that is spreading from the state of Amazonas to previously uninfected areas of the state of Acre in the Purus River region

Mansonella ozzardi infections are common in the riverside communities along the Solimões, Negro and Purus Rivers in the state of Amazonas (AM). However, little is known about the presence of this parasite in communities located in regions bordering AM and the state of Acre. The prevalence rate of M. ozzardi infections was determined in blood samples from volunteers according to the Knott method. A total of 355 volunteers from six riverine communities were enrolled in the study and 65 (18.3%) were found to be infected with M. ozzardi. As expected, most of the infections (25%) occurred in individuals involved in agriculture, cattle rearing and fishing and an age/sex group analysis revealed that the prevalence increased beginning in the 40-50-years-of-age group and reached 33% in both sexes in individuals over 50 years of age. Based on the described symptomatology, articular pain and headache were found to be significantly higher among infected individuals (56 and 65% prevalence, respectively, p < 0.05). Sera from volunteers were subjected to ELISA using a cocktail of recombinant proteins from Onchocerca volvulus to evaluate the specificity of the test in an endemic M. ozzardi region. No cross-reactions between M. ozzardi-infected individuals and recombinant O. volvulus proteins were detected, thus providing information on the secure use of this particular cocktail in areas where these parasites are sympatric.

New records of Mansonella ozzardi: a parasite that is spreading from the state of Amazonas to previously uninfected areas of the state of Acre in the Purus River region.

Mansonella ozzardi infections are common in the riverside communities along the Solimões, Negro and Purus Rivers in the state of Amazonas (AM). However, little is known about the presence of this parasite in communities located in regions bordering AM and the state of Acre. The prevalence rate of M. ozzardi infections was determined in blood samples from volunteers according to the Knott method. A total of 355 volunteers from six riverine communities were enrolled in the study and 65 (18.3%) were found to be infected with M. ozzardi. As expected, most of the infections (25%) occurred in individuals involved in agriculture, cattle rearing and fishing and an age/sex group analysis revealed that the prevalence increased beginning in the 40-50-years-of-age group and reached 33% in both sexes in individuals over 50 years of age. Based on the described symptomatology, articular pain and headache were found to be significantly higher among infected individuals (56 and 65% prevalence, respectively, p < 0.05). Sera from volunteers were subjected to ELISA using a cocktail of recombinant proteins from Onchocerca volvulus to evaluate the specificity of the test in an endemic M. ozzardi region. No cross-reactions between M. ozzardi-infected individuals and recombinant O. volvulus proteins were detected, thus providing information on the secure use of this particular cocktail in areas where these parasites are sympatric.

Genetic characterization of atypical Mansonella (Mansonella) ozzardi microfilariae in human blood samples from northeastern Peru.

DNA sequence comparisons are useful for characterizing proposed new parasite species or strains. Microfilariae with an atypical arrangement of nuclei behind the cephalic space have been recently described in human blood samples from the Amazon region of Peru. Three blood specimens containing atypical microfilariae were genetically characterized using three DNA markers (5S ribosomal DNA, 12S ribosomal DNA, and cytochrome oxidase I). All atypical microfilariae were clustered into the Mansonella group and indistinguishable from M. ozzardi based on these DNA markers.

Expanded numbers of circulating myeloid dendritic cells in patent human filarial infection reflect lower CCR1 expression.

APC dysfunction has been postulated to mediate some of the parasite-specific T cell unresponsiveness seen in patent filarial infection. We have shown that live microfilariae of Brugia malayi induce caspase-dependent apoptosis in human monocyte-derived dendritic cells (DCs) in vitro. This study addresses whether apoptosis observed in vitro extends to patent filarial infections in humans and is reflected in the number of circulating myeloid DCs (mDCs; CD11c(-)CD123(lo)) in peripheral blood of infected microfilaremic individuals. Utilizing flow cytometry to identify DC subpopulations (mDCs and plasmacytoid DCs [pDCs]) based on expression of CD11c and CD123, we found a significant increase in numbers of circulating mDCs (CD11c(+)CD123(lo)) in filaria-infected individuals compared with uninfected controls from the same filaria-endemic region of Mali. Total numbers of pDCs, monocytes, and lymphocytes did not differ between the two groups. To investigate potential causes of differences in mDC numbers between the two groups, we assessed chemokine receptor expression on mDCs. Our data indicate that filaria-infected individuals had a lower percentage of circulating CCR1(+) mDCs and a higher percentage of circulating CCR5(+) mDCs and pDCs. Finally, live microfilariae of B. malayi were able to downregulate cell-surface expression of CCR1 on monocyte-derived DCs and diminish their calcium flux in response to stimulation by a CCR1 ligand. These findings suggest that microfilaria are capable of altering mDC migration through downregulation of expression of some chemokine receptors and their signaling functions. These observations have major implications for regulation of immune responses to these long-lived parasites.

[Occurrence of Mansonella ozzardi (Nematoda, Onchocercidae) in riverine communities of the Purus river, Boca do Acre municipality, Amazonas State, Brazil]. / Ocorrência da Mansonella ozzardi (Nematoda, Onchocercidae) em comunidades ribeirinhas do rio Purus, Município de Boca do Acre, Amazonas, Brasil.

The study aimed to estimate the prevalence of Mansonella ozzardi and calculate the parasitic infection rate in simuliid blackflies. The research was conducted in communities on the Purus River, Boca do Acre municipality, Amazonas State, Brazil. Prevalence was measured using the thick smear method. Captured blackflies were dissected to verify the parasitic infection rate. M. ozzardi prevalence was 27.30% (77/282). The study showed higher prevalence in men (31.97%) than women (22.22%), farmers (48.99%), and individuals in the 38-47 (60.00%), 48-57 (66.66%), and 58-67-year age brackets (75.00%). Microfilaremia was higher in individuals 58 to 67 years of age (average= 58.41 mf/40 microl), men (41.44 mf/40 microl), and farmers (49.94 mf/40 microl). Only the simuliid Cerqueirellum amazonicum was found infected with a parasitic infection rate of 0.98%.

Ocorrência da Mansonella ozzardi (Nematoda, Onchocercidae) em comunidades ribeirinhas do rio Purus, Município de Boca do Acre, Amazonas, Brasil / Occurrence of Mansonella ozzardi (Nematoda, Onchocercidae) in riverine communities of the Purus river, Boca do Acre municipality, Amazonas State, Brazil

Este estudo teve o objetivo de estimar as prevalências de Mansonella ozzardi e calcular taxa de infecção parasitária nos simulídeos. O trabalho foi realizado em comunidades ribeirinhas do rio Purus, Boca do Acre, Amazonas, Brasil. As prevalências foram obtidas por meio do método de gota espessa de sangue obtido por porção digital. Os simulídeos coletados foram dissecados para estimar a taxa de infecção parasitária. A prevalência de M. ozzardi foi de 27,3 por cento (282/77). Foram observadas maiores prevalências nos homens (31,97 por cento) que nas mulheres (22,22 por cento), nos agricultores (48 por cento) e nos indivíduos entre: 38-47 (60 por cento), 48-57 (66,66 por cento) e 58-67 (75 por cento). A microfilaremia foi maior nos indivíduos entre 58-67 anos (média = 58,41mf/40µL), sexo masculino (41,44mf/40µL) e nos agricultores (49,94mf/40µL). Somente o simulídeo Cerqueirellum amazonicum foi encontrado infectado com taxa de infecção parasitária de 0,98 por cento.

The study aimed to estimate the prevalence of Mansonella ozzardi and calculate the parasitic infection rate in simuliid blackflies. The research was conducted in communities on the Purus River, Boca do Acre municipality, Amazonas State, Brazil. Prevalence was measured using the thick smear method. Captured blackflies were dissected to verify the parasitic infection rate. M. ozzardi prevalence was 27.30 percent (77/282). The study showed higher prevalence in men (31.97 percent) than women (22.22 percent), farmers (48.99 percent), and individuals in the 38-47 (60.00 percent), 48-57 (66.66 percent), and 58-67-year age brackets (75.00 percent). Microfilaremia was higher in individuals 58 to 67 years of age (average= 58.41mf/40µl), men (41.44mf/40µl), and farmers (49.94mf/40µl). Only the simuliid Cerqueirellum amazonicum was found infected with a parasitic infection rate of 0.98 percent.

Analysis of the 24-h microfilarial periodicity of Mansonella perstans.

The Mansonella perstans microfilarial (mf) periodicity in peripheral blood was analysed in a group of 32 healthy individuals from an endemic community in Uganda. The majority of individuals had maximum mf intensity during the first and minimum during the last 12 h of the day. Mean mf ratios (i.e. time-specific counts in relation to mean count for the individual) indicated a weak but significant diurnal periodicity with peak intensity around 0800 h. Trigonometric analysis of the ratios, assuming a harmonic wave pattern of periodicity, gave a periodicity index of 11.1 and a peak time of 0645 h. The higher mf intensities in the early morning appeared to coincide with the preferred biting hour of the vectors. The observed weak pattern of mf periodicity indicates that the sampling time of blood specimens for diagnosis will have only minor effect on diagnostic sensitivity.

Mansonella perstans causing symptomatic hypereosinophilia in a missionary family.

Mansonella perstans is rarely pathogenic. The rare reports of symptomatic cases, however, include severe complications. Three cases of symptomatic hypereosinophilia with multi-organ involvement are described in a missionary family returning from tropical Africa. Pathogenicity may be related to the induction of hypereosinophilia rather than direct host-parasite interactions.

Abnormal depolarizing patterns in three patients with filarial infection.

Several authors have described a particular potential of automated depolarization analysis in detecting malaria infection as part of the routine full blood count (FBC) performed by the Cell-Dyn 4000 analyzer. In these cases, abnormal depolarizing patterns are due to the presence of leukocyte-associated malaria hemozoin, a pigment which depolarizes the laser light. In this report we describe samples from three individual patients who did not have malaria infection but showed abnormal depolarizing events. Further investigation determined that these samples were from patients infected by the nematode Mansonella perstans. The observed depolarizing pattern consisted of a normal depolarizing eosinophil population and in addition an abnormal depolarizing population that showed a close "linear" relationship between "granularity" (90 degrees depolarization) and "lobularity" (90 degrees polarization). This atypical population was smaller than normal leukocytes and thus clearly different from the patterns associated with malaria infection. Abnormal depolarization patterns of M. perstans clearly do not reflect leukocyte-associated malaria hemozoin. It is possible however that the erythrocyte-lysing agent used to facilitate leukocyte analysis by the instrument may have caused microfilaria fragmentation and thus the distinctive "straight-line" features of the abnormal scatter plots

Loa loa and Mansonella perstans infections in Ijebu north, western Nigeria: a parasitological study.

The prevalence and intensity of Loa loa and Mansonella perstans were studied in three villages of the Ijebu north area in Ogun State, western Nigeria. Blood samples were collected by finger-pricking from 373 (181 males, 192 females) subjects with an age range of 4 to 55 years. The blood samples were examined microscopically for the presence of microfilariae (mf). L. loa and M. perstans were present in the blood samples in 39 (10.5%) and 12 (3.2%) of the subjects, respectively. Neither of the infections were found to be sex-dependent. The geometric mean of the L. loa intensity was 1.8 mf per 50 microl of blood, while that of M. perstans was 1.5 mf per 50 microl. The prevalence of mixed infections of both L. loa and M. perstans was 1.0%.

Mansonella ozzardi infection in Bolivia: prevalence and clinical associations in the Chaco region.

A cross-sectional survey carried out in the Chaco region of Bolivia showed that 26% (77 of 296) and 0.7% (2 of 298) of the rural population of the Camiri and Villa Montes areas, respectively, harbored Mansonella ozzardi microfilariae (mf). No significant differences were observed between sexes. The lowest prevalence (9%) was in the 0-14-year-old age group, with no children <11 months of age infected. The prevalence increased sharply in the 25-34-year-old age group (32%), and continued increasing in the older age classes. Microfilaremia, ranging from 1 to 305 mf/20 microl of blood, was lowest in 0-14-year-old children (geometric mean concentration = 1.1 mf/20 microl), and increased with age (>100 mf/20 microl in people >44 years old). An expected increasing sensitivity with the blood volume examined was observed. No significant association between clinical symptoms (fever, skin rash, pruritus, headache, lymphedema, elephantiasis, and articular pain) and microfilaremia was observed.