Diagnostic performance of capillary and venous blood samples in the detection of Loa loa and Mansonella perstans microfilaraemia using light microscopy.

BACKGROUND: Loa loa and Mansonella perstans-the causative agents of loiasis and mansonellosis-are vector-borne filarial parasites co-endemic in sub-Saharan Africa. Diagnosis of both infections is usually established by microscopic analysis of blood samples. It was recently established that the odds for detecting Plasmodium spp. is higher in capillary (CAP) blood than in venous (VEN) blood. In analogy to this finding this analysis evaluates potential differences in microfilaraemia of L. loa and M. perstans in samples of CAP and VEN blood. METHODS: Recruitment took place between 2015 and 2019 at the CERMEL in Lambaréné, Gabon and its surrounding villages. Persons of all ages presenting to diagnostic services of the research center around noon were invited to participate in the study. A thick smear of each 10 microliters of CAP and VEN blood was prepared and analysed by a minimum of two independent microscopists. Differences of log2-transformed CAP and VEN microfilaraemia were computed and expressed as percentages. Furthermore, odds ratios for paired data were computed to quantify the odds to detect microfilariae in CAP blood versus in VEN blood. RESULTS: A total of 713 participants were recruited among whom 52% were below 30 years of age, 27% between 30-59 years of age and 21% above 60 years of age. Male-female ratio was 0.84. Among 152 participants with microscopically-confirmed L. loa infection median (IQR) microfilaraemia was 3,650 (275-11,100) per milliliter blood in CAP blood and 2,775 (200-8,875) in VEN blood (p<0.0001), while among 102 participants with M. perstans this was 100 (0-200) and 100 (0-200), respectively (p = 0.44). Differences in linear models amount up to an average of +34.5% (95% CI: +11.0 to +63.0) higher L. loa microfilaria quantity in CAP blood versus VEN blood and for M. perstans it was on average higher by +24.8% (95% CI: +0.0 to +60.5). Concordantly, the odds for detection of microfilaraemia in CAP samples versus VEN samples was 1.24 (95% CI: 0.65-2.34) and 1.65 (95% CI: 1.0-2.68) for infections with L. loa and M. perstans, respectively. CONCLUSION: This analysis indicates that average levels of microfilaraemia of L. loa are higher in CAP blood samples than in VEN blood samples. This might have implications for treatment algorithms of onchocerciasis and loiasis, in which exact quantification of L. loa microfilaraemia is of importance. Furthermore, the odds for detection of M. perstans microfilariae was higher in CAP than in VEN blood which may pre-dispose CAP blood for detection of M. perstans infection in large epidemiological studies when sampling of large blood quantities is not feasible. No solid evidence for a higher odds of L. loa microfilariae detection in CAP blood was revealed, which might be explained by generally high levels of L. loa microfilaraemia in CAP and VEN blood above the limit of detection of 100 microfilariae/ml. Yet, it cannot be excluded that the study was underpowered to detect a moderate difference.

Mansonella perstans microfilaremic individuals are characterized by enhanced type 2 helper T and regulatory T and B cell subsets and dampened systemic innate and adaptive immune responses.

The filarial nematode Mansonella perstans is endemic throughout Africa, northern South America and the Caribbean. Interestingly, M. perstans-infected individuals present no distinct clinical picture associated with certain pathology. Due to its relatively silent nature, research on this tropical disease has been neglected, especially M. perstans-driven immune responses. A hindrance in obtaining data on M. perstans-specific responses has been the inability to obtain adult worms since their habitats in serous cavities are difficult to access. Thus, in this study, for the first time, we used Mansonella perstans worm antigen extract as stimulant to obtain filarial-specific recall and immunoglobulin responses from M. perstans microfilaremic individuals (Mp MF+) from Cameroon. Moreover, systemic immune profiles in sera and immune cell composition in peripheral blood from Mp MF+ and amicrofilaremic individuals (Mp MF-) were obtained. Our data reveal that Mp MF+ individuals showed significantly reduced cytokine (IL-4, IL-6 and IL-12p70) and chemokine levels (IL-8 and RANTES), but significantly higher MIP-1ß as well as increased M. perstans-specific IgG4 levels compared to Mp MF- individuals. In contrast, upon re-stimulation with worm antigen extract, IFN-γ, IL-13, IL-10 and IL-17A secretion was enhanced in cell cultures from Mp MF+ individuals when compared to those from cultures of healthy European individuals. Moreover, analysis of immune cell composition in peripheral blood from Mp MF+ individuals revealed increased type 2 helper T (Th2), natural killer (NK), regulatory B and T cell (Breg and Treg) subsets but decreased type 1 regulatory T (Tr1) cells. In summary, this study deciphers for the first time, M. perstans-specific immune responses using worm antigen extract and shows that patent M. perstans infections have distinct Th2, Breg and Treg subsets accompanied with reduced systemic innate and adaptive immune responses and dominant filarial-specific IgG4 levels.

Imported Infections with Mansonella perstans Nematodes, Italy.

We report 74 patients in Italy infected with Mansonella perstans nematodes, a poorly described filarial parasite. M. perstans nematodes should be included in the differential diagnosis for patients with eosinophilia from disease-endemic countries. Serologic analysis is useful for screening, and testing for microfilaremia in peripheral blood should be performed for parasite-positive patients.

The relationship between microfilaraemic and amicrofilaraemic loiasis involving co-infection with Mansonella perstans and clinical symptoms in an exposed population from Gabon.

The relationship between the frequency of loiasis objective symptoms and microfilaraemic or amicrofilaraemic infection was assessed in 1148 exposed patients also infected, or not, with Mansonella perstans. Filarial infections were detected by direct microscopy, leucoconcentration and serology, with prevalence values of 39.5% Loa loa, 5.6% M. perstans and 3.4% co-infection with both filarial species. Amicrofilaraemic or occult loiasis (OL) predominated among L. loa-infected individuals, with a prevalence of 58.2%. Hypermicrofilaraemia (>8000 microfilariae (mf)/ml) was found in 18.4% of L. loa microfilaraemic patients, with 25.7% of them harbouring more than 30,000 mf/ml. Up to 34% of patients with OL showed evidence of Calabar swelling, compared with 26.3% of microfilaraemic patients (P= 0.03). Overall 5.3% of patients presented with adult worm migration across the eye, representing 16.3% of microfilaraemic individuals and 11.4% of amicrofilaraemic patients (P= 0.13). This symptom was similarly found in patients with more than 30,000 mf/ml (22%), those with microfilaraemia between 8 and 30,000 mf/ml (15.4%) and also in individuals with low or without microfilaraemia (16.1%) (P= 0.7). Five (14.3%) hypermicrofilaraemic patients did not present any L. loa-specific objective symptoms, as well as all the patients with single M. perstans infection. The presence of adult eye worm migration as a strong predictor of high microfilaraemia density would obscure the real burden of L. loa hypermicrofilaraemia in exposed individuals. For epidemiological purposes and control strategies, the mapping of L. loa in endemic areas should also take into account the group of patients with occult loiasis.

[Ocular manifestations in mansonelliasis]. / Acometimento ocular em pacientes com mansonelose.

PURPOSE: To evaluate eye manifestations in mansonelliasis patients from Coari, AM, rural area on the middle Amazonas River. METHODS: Cross-sectional study consisted of evaluation of 543 subjects from Coari rural area on Solimões River right margin. All subjects were submitted to blood test for microfilaria and the subjects who had a positive test also were submitted to skin biopsies and ophthalmologic examination. Histopathology and PCR research for microfilaria were done on eye biopsies of suspect lesions. RESULTS: Mansonella ozzardi was found in peripheral blood of 103 (18.9%) subjects. Ninety-five mansonelliasis patients were examined. Punctate keratitis was observed in 12 of them, nummular keratitis in one subject and sclerosing keratitis in another one. Skin biopsies were negative in all subjects. Conjunctival and limbal biopsies were done in five mansonelliasis patients and corneal biopsy in another three patients. Conjunctival and limbal biopsies of two patients and corneal biopsy of one patient showed no microfilaria or alterations due to its presence on histopathology. PCR showed no evidence of microfilaria in the other biopsies. CONCLUSION: Association between Mansonella ozzardi infection and corneal lesions with no evident etiology was observed in this study, but the lack of conclusive findings on histopathology and PCR make us doubt the corneal involvement in mansonelliasis. Large studies of high mansonelliasis prevalence populations and extensive employment of microfilaria identification tests in ocular tissue are needed to evaluate the microfilaria corneal pathogenicity.

Acometimento ocular em pacientes com mansonelose / Ocular manifestations in mansonelliasis

OBJETIVOS: Avaliar as alterações oculares observadas em indivíduos com mansonelose residentes na área rural do município de Coari, AM, na região do médio Amazonas. MÉTODOS: Foi conduzido um estudo de corte transversal envolvendo 543 moradores residentes na área rural do município de Coari, na margem direita do rio Solimões. Todos os indivíduos foram submetidos a exame de gota espessa para pesquisa de microfilária e os indivíduos positivos foram submetidos a exame oftalmológico. Lesões oculares suspeitas foram biopsiadas e enviadas para exame histopatológico. RESULTADOS: Mansonella ozzardi foi encontrada no sangue periférico em 103 (18,9 por cento) indivíduos. Noventa e cinco pacientes com mansonelose foram examinados, observando ceratite puntata em 12 (11,7 por cento) destes, ceratite numular em um indivíduo (1 por cento) e esclerosante em outro (1 por cento). Biópsia de pele foi negativa em todos os indivíduos. Biópsia de conjuntiva e limbo foi realizada em cinco pacientes com mansonelose e biópsia de córnea em outros três pacientes. Duas biópsias de conjuntiva e limbo e uma biópsia de córnea foram enviadas para exame histopatológico, não sendo observadas microfilárias ou alterações sugestivas da presença destas nas peças estudadas. O restante das biópsias foi enviado para pesquisa de DNA do parasita por PCR, não se encontrando microfilária. CONCLUSÕES: Associação entre infecção por Mansonella ozzardi e lesões corneanas sem etiologia aparente foi verificada neste estudo, mas a ausência de achados específicos no exame histopatológico e a não identificação de microfilárias por PCR em lesões suspeitas põem em dúvida a o acometimento corneano na mansonelose. Estudos mais amplos de populações com alta prevalência de M. ozzardi e a realização de outros testes para identificação de microfilárias em tecido ocular são necessários para investigar a patogenicidade da microfilária ao tecido ocular.

PURPOSE: To evaluate eye manifestations in mansonelliasis patients from Coari, AM, rural area on the middle Amazonas River. METHODS: Cross-sectional study consisted of evaluation of 543 subjects from Coari rural area on Solimões River right margin. All subjects were submitted to blood test for microfilaria and the subjects who had a positive test also were submitted to skin biopsies and ophthalmologic examination. Histopathology and PCR research for microfilaria were done on eye biopsies of suspect lesions. RESULTS: Mansonella ozzardi was found in peripheral blood of 103 (18.9 percent) subjects. Ninety-five mansonelliasis patients were examined. Punctate keratitis was observed in 12 of them, nummular keratitis in one subject and sclerosing keratitis in another one. Skin biopsies were negative in all subjects. Conjunctival and limbal biopsies were done in five mansonelliasis patients and corneal biopsy in another three patients. Conjunctival and limbal biopsies of two patients and corneal biopsy of one patient showed no microfilaria or alterations due to its presence on histopathology. PCR showed no evidence of microfilaria in the other biopsies. CONCLUSION: Association between Mansonella ozzardi infection and corneal lesions with no evident etiology was observed in this study, but the lack of conclusive findings on histopathology and PCR make us doubt the corneal involvement in mansonelliasis. Large studies of high mansonelliasis prevalence populations and extensive employment of microfilaria identification tests in ocular tissue are needed to evaluate the microfilaria corneal pathogenicity.

Onchocercosis mansonella co-infection presenting as a subcutaneous nodule in a child.

A 11-year-old girl presented with a subcutaneous lesion on the forehead, thought to be an implantation dermoid cyst. Microscopic examination revealed an onchocercoma. Microfilariae were found in the blood indicating a co-infection with Mansonella perstans. This case demonstrates the diagnostic difficulties of a parasitic co-infection uncommonly encountered in Europe.

Prevalence of Mansonella perstans infections among the nomadic Fulanis of northern Nigeria.

The study was undertaken to investigate the prevalence and clinical manifestations of Mansonella perstans infection in the nomadic Fulanis of northern Nigeria. Physical examination of 755 consenting nomads for clinical manifestations of M. perstans infection was carried out between June 1996-September 2000. This was followed by parasitological examination of blood samples collected by finger prick method to identify microfilariae (mf). Data collected was stratified by locality, age and sex, and analyzed using the Chi-square test. Overall, 66 (8.7 %) of 755 nomads were infected. Infection was higher in men (9.7 %) than in women (6.5 %) (p<0.05) and occurred in all age groups with highest prevalence in persons in the 4th (14.1 %) and 5th (17.1 %) decades of life. Fifty-six (81.8 %) of the 66 nomads with mf had clinical manifestations with periodic dizziness (18.2 %), body itching (15.2 %) was the most prevalent while back pain (7.6 %) was the least. The findings show that mansonelliasis is an important health problem in nomads. This underscores the need to establish a well-articulated Filariasis Control Programme for this group whose major health problems are rooted in their occupation and amplified by several contingencies of the environment.

First report of presumed parasitic keratitis in Indians from the Brazilian Amazon.

PURPOSE: To describe corneal disorders in patients with systemic parasitic disease. METHODS: Cross-sectional study consisted of ophthalmologic examinations, peripheral blood tests and skin biopsies in 496 subjects (395 Indians and 101 non-indians) from the upper Negro River, Brazilian Amazon. RESULTS: Mansonella ozzardi was detected in the blood of 140 (28.23%) subjects. Seventy-six (55.07%) of them also presented multiple nummular opaque superficial lesions in the cornea. None of the examined patients presented onchocercal-related skin lesions or positive skin biopsies for filaria. CONCLUSION: There was a significant association between mansonelliasis and keratitis.

Estudio de un grupo de estudiantes africanos parasitados con Manzonella perstans en Cuba (área no endémica) durante 1979 a 1982 / Study of a group of African students parasited with Manzonella perstans in Cuba (non-endemic area) during 1979-1982

Se estudiaron 60 casos de Manzonella perstans por un período de 3 años mediante estudios clínicos y parasitológicos de sangre. Se observó que el 31,2% se negativizó y que la microfilaremia media disminuyó de 49,3 en el inicio del estudio a 32,9 al final en los casos que aún continuaban positivos. Por la microfilaremia persistente que traduce una relativa longevidad de los parásitos adultos, se recomienda la vigilancia de estos individuos al llegar a un área no endémica donde existan los vectores de esta enfermedad

Estudio de un grupo de estudiantes africanos parasitados con Manzonella perstans en Cuba (área no endémica) durante 1979 a 1982 / Study of a group of African students parasited with Manzonella perstans in Cuba (non-endeic area) during 1979-1982

Se estudiaron 60 casos de Manzonella perstans por un período de 3 años mediante estudios clínicos y parasitológicos de sangre. Se observó que el 31,2

se negativizó y que la microfilaremia media disminuyó de 49,3 en el inicio del estudio a 32,9 al final en los casos que aún continuaban positivos. Por la microfilaremia persistente que traduce una relativa longevidad de los parásitos adultos, se recomienda la vigilancia de estos individuos al llegar a un área no endémica donde existan los vectores de esta enfermedad

Adult Mansonella perstans in the abdominal cavity in nine Africans.

Adult Mansonella perstans infected the abdominal cavity of nine patients seen at Karawa Hospital in the Ubangi territory of Zaire. In four patients the worms were removed at laparotomy, and in the other five they were removed at autopsy. Twelve adult worms were identified in the nine patients. None of the worms caused symptoms or contributed to the patient's death. Worms were in the hernial sac in three patients, and one each was in connective tissue beside a reactive mesenteric lymph node, in peripancreatic connective tissue, in perirenal connective tissue, in hepatic portal connective tissue, on the serosal surface of the small intestine, and in connective tissue adjacent to rectum. The diameter of male worms was 45 microns to 60 microns and of female worms, 80 microns to 125 microns. One female worm was removed intact. It was 6 cm long and had a bifurcated tail characteristic of M. perstans.

Microfilariae of Mansonella ozzardi in human skin biopsies.

Microfilariae of Mansonella ozzardi were seen in specimens of skin taken by biopsy from 4 of 6 subjects previously shown to be positive for microfilariae in the blood. While most of the microfilariae were confined to small blood vessels in the papillary and superficial reticular dermis, some were in perivascular spaces and a few were noted in the dermal interstitium. There was no microscopic evidence that the minimal cutaneous disease seen could be directly attributed to the microfilariae.