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.

Eosinophil-associated processes underlie differences in clinical presentation of loiasis between temporary residents and those indigenous to Loa-endemic areas.

BACKGROUND: Loa loa has emerged as an important public health problem due to the occurrence of immune-mediated severe posttreatment reactions following ivermectin distribution. Also thought to be immune-mediated are the dramatic differences seen in clinical presentation between infected temporary residents (TR) and individuals native to endemic regions (END). METHODS: All patients diagnosed with loiasis at the National Institutes of Health between 1976 and 2012 were included. Patients enrolled in the study underwent a baseline clinical and laboratory evaluation and had serum collected and stored. Stored pretreatment serum was used to measure filaria-specific antibody responses, eosinophil-related cytokines, and eosinophil granule proteins. RESULTS: Loa loa infection in TR was characterized by the presence of Calabar swelling (in 82% of subjects), markedly elevated eosinophil counts, and increased filaria-specific immunoglobulin G (IgG) levels; these findings were thought to reflect an unmodulated immune response. In contrast, END showed strong evidence for immune tolerance to the parasite, with high levels of circulating microfilariae, few clinical symptoms, and diminished filaria-specific IgG. The striking elevation in eosinophil counts among the TR group was accompanied by increased eosinophil granule protein levels (associated with eosinophil activation and degranulation) as well as elevated levels of eosinophil-associated cytokines. CONCLUSIONS: These data support the hypothesis that differing eosinophil-associated responses to the parasite may be responsible for the marked differences in clinical presentations between TR and END populations with loiasis.

[Long-lasting souvenir from Cameroon - Persistent, massive hypereosinophilia in amicrofilaremic infection with Loa loa]. / Langlebiges Souvenir aus Kamerun ­ persistierende, massive Hypereosinophilie bei amikrofilarämischer Infektion mit Loa loa.

INTRODUCTION: Diagnosis of a polysymptomatic, rare parasitosis requires collaboration of internal specialists, tropical disease specialists, parasitologists and dermatologists. HISTORY: The course of disease is shown in a 66-year-old woman who regularly travels to Cameroon and presented with remarkable hypereosinophilia and pruritus with urticarial swellings. FINDINGS AND DIAGNOSIS: Using interdisciplinary diagnostics based on travel history, symptoms and laboratory results an occult amicrofilaraemic Loa loa infection with immunological hyperreaction to the parasite antigen, reactive hypereosinophilia and high antibody titers was diagnosed. THERAPY AND COURSE: Anthelmintic therapy was inducted with ivermectin and diethylcarbamazine. Treatment with ivermectin alone resulted in a prompt regression of symptoms and decrease of eosinophil levels and antibody titers. CONCLUSIONS: Parasitic diseases like L. loa infections are extremely rare in Europe but should be considered as differential diagnosis at an early stage when patients present with appropriate travel history and clinical findings. There is a lack of standardized therapy and follow-up recommendations. A precise recording of all new diagnoses with therapy progress/response should be established in an international registry.

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.

New promising method to assess microfilarial Loa loa load on the peripheral blood.

Loiasis is a vector-borne parasitic disease caused by the filarial Loa loa (L. loa). Definitive diagnosis can be done by identifying and counting microfilariae in the peripheral blood by microscopy and with L.loa-specific PCR. An additional diagnostic method is the detection of L.loa-specific antibodies. Accurate methods are needed to automate quantification of microfilaria (mf) in peripheral blood. Indeed, the treatment procedure depends on the microfilarial L. loa load in blood. We report the first documented use of flow cytometry as a new method to count microfilaraemia in peripheral blood from a patient with L. loa infection. The diagnosis of loiasis was strongly suspected based on clinical presentation and rapidly confirmed by identifying typical features of L. loa in the peripheral blood. This diagnosis was achieved by flow cytometry using a specific fluorescence pattern for microfilaraemia count. The current report highlights the potential of flow cytometry to assess microfilarial L. loa load from a patient with loiasis infection.

Mouse models of Loa loa.

Elimination of the helminth disease, river blindness, remains challenging due to ivermectin treatment-associated adverse reactions in loiasis co-infected patients. Here, we address a deficit in preclinical research tools for filarial translational research by developing Loa loa mouse infection models. We demonstrate that adult Loa loa worms in subcutaneous tissues, circulating microfilariae (mf) and presence of filarial biomarkers in sera occur following experimental infections of lymphopenic mice deficient in interleukin (IL)-2/7 gamma-chain signaling. A microfilaraemic infection model is also achievable, utilizing immune-competent or -deficient mice infused with purified Loa mf. Ivermectin but not benzimidazole treatments induce rapid decline (>90%) in parasitaemias in microfilaraemic mice. We identify up-regulation of inflammatory markers associated with allergic type-2 immune responses and eosinophilia post-ivermectin treatment. Thus, we provide validation of murine research models to identify loiasis biomarkers, to counter-screen candidate river blindness cures and to interrogate the inflammatory etiology of loiasis ivermectin-associated adverse reactions.

Ivermectin treatment of Loa loa hyper-microfilaraemic baboons (Papio anubis): Assessment of microfilarial load reduction, haematological and biochemical parameters and histopathological changes following treatment.

BACKGROUND: Individuals with high intensity of Loa loa are at risk of developing serious adverse events (SAEs) post treatment with ivermectin. These SAEs have remained unclear and a programmatic impediment to the advancement of community directed treatment with ivermectin. The pathogenesis of these SAEs following ivermectin has never been investigated experimentally. The Loa/baboon (Papio anubis) model can be used to investigate the pathogenesis of Loa-associated encephalopathy following ivermectin treatment in humans. METHODS: 12 baboons with microfilarial loads > 8,000mf/mL of blood were randomised into four groups: Group 1 (control group receiving no drug), Group 2 receiving ivermectin (IVM) alone, Group 3 receiving ivermectin plus aspirin (IVM + ASA), and Group 4 receiving ivermectin plus prednisone (IVM + PSE). Blood samples collected before treatment and at Day 5, 7 or 10 post treatment, were analysed for parasitological, hematological and biochemical parameters using standard techniques. Clinical monitoring of animals for side effects took place every 6 hours post treatment until autopsy. At autopsy free fluids and a large number of standard organs were collected, examined and tissues fixed in 10% buffered formalin and processed for standard haematoxylin-eosin staining and specific immunocytochemical staining. RESULTS: Mf counts dropped significantly (p<0.05) in all animals following ivermectin treatment with reductions as high as (89.9%) recorded; while no significant drop was observed in the control animals. Apart from haemoglobin (Hb) levels which recorded a significant (p = 0.028) drop post treatment, all other haematological and biochemical parameters did not show any significant changes (p>0.05). All animals became withdrawn 48 hours after IVM administration. All treated animals recorded clinical manifestations including rashes, itching, diarrhoea, conjunctival haemorrhages, lymph node enlargement, pinkish ears, swollen face and restlessness; one animal died 5 hours after IVM administration. Macroscopic changes in post-mortem tissues observed comprised haemorrhages in the brain, lungs, heart, which seen in all groups given ivermectin but not in the untreated animals. Microscopically, the major cellular changes seen, which were present in all the ivermectin treated animals included microfilariae in varying degrees of degeneration in small vessels. These were frequently associated with fibrin deposition, endothelial changes including damage to the integrity of the blood vessel and the presence of extravascular erythrocytes (haemorrhages). There was an increased presence of eosinophils and other chronic inflammatory types in certain tissues and organs, often in large numbers and associated with microfilarial destruction. Highly vascularized organs like the brain, heart, lungs and kidneys were observed to have more microfilariae in tissue sections. The number of mf seen in the brain and kidneys of animals administered IVM alone tripled that of control animals. Co-administration of IVM + PSE caused a greater increase in mf in the brain and kidneys while the reverse was noticed with the co-administration of IVM + ASA. CONCLUSIONS: The treatment of Loa hyper-microfilaraemic individuals with ivermectin produces a clinical spectrum that parallels that seen in Loa hyper-microfilaraemic humans treated with ivermectin. The utilization of this experimental model can contribute to the improved management of the adverse responses in humans.

Parasitological, Hematological and Biochemical Characteristics of a Model of Hyper-microfilariaemic Loiasis (Loa loa) in the Baboon (Papio anubis).

BACKGROUND: Loiasis, a filarial infection caused by Loa loa usually thought to cause relatively minor morbidity, can cause serious and often fatal reactions in patients carrying very high levels of circulating Loa loa microfilariae (mf) following administration of microfilaricidal drugs. An experimental model of this condition would greatly aid the definition of the optimal management of this important clinical presentation. METHODOLOGY/PRINCIPLE FINDINGS: Fifteen baboons (Papio anubis) were infected with 600 infective larvae (L3) isolated from Chrysops vector flies. Animals were observed for any clinical changes; blood samples were collected every 1-2 months for 22 months, and analysed for parasitological, hematological and biochemical profiles using standard techniques. All animals became patent but remained clinically normal throughout the study. The parasitological pre-patent period was between 4-8 months, with a majority (60%) of animals becoming patent by 5 months post infection (MPI); all animals were patent by 8 MPI. Microfilarial loads increased steadily in all animals and reached a peak at 18 MPI. By 10 MPI >70% of animals had mf >8,000 mf/mL, and at 18 MPI >70% of animals had mf >30,000 mf/mL with 50% of these animals with mf >50,000 mf/mL. Absolute eosinophil, creatinine, Ca2+ and K+ levels were generally above normal values (NV). Positive associations were seen between microfilariaemia and eosinophilia, Hb, Ca2+, and gamma-GT values, whilst significant negative associations were seen between microfilariaemia and potassium, glucose and mononuclear leukocyte levels. CONCLUSIONS: Infection of splenectomised baboons with L. loa can induce levels of circulating microfilariae, and corresponding haematological profiles, which parallel those seen in those humans in danger of the severe post-microfilariacide clinical responses. Utilization of this experimental model could contribute to the improved management of the loiasis related adverse responses in humans.

Clinical Features of Imported Loiasis: A Case Series from the Hospital for Tropical Diseases, London.

We retrospectively analyzed the background, clinical features, and treatment response of 50 cases of imported loiasis who presented between 2000 and 2014 to the Hospital for Tropical Diseases (HTD), London, United Kingdom. Of them, 29 were migrants from, and 21 were visitors to, countries where the disease is endemic. Clinical features differed between these groups. Migrants experienced fewer Calabar swellings (odds ratio [OR] = 0.12), more eye worm (OR = 3.4), more microfilaremia (OR = 3.5), lower filarial antibody levels, and lower eosinophil counts (P < 0.05 for all tests). Among 46 patients who were started on treatment at HTD, 33 (72%) received diethylcarbamazine (DEC) monotherapy as first-line treatment, and among 26 patients who were followed up after treatment, seven (27%) needed a second course of treatment. There were 46 courses of treatment with DEC, and 20 (43%) of them had reactions. All patients with microfilaremia > 3,000 microfilariae/mL and all those with an elevated C-reactive protein (CRP) (≥ 5 mg/L) before treatment had reactions (P = 0.10 and P = 0.01, respectively). These data suggest that monotherapy with DEC may not be the optimal treatment for patients with loiasis, particularly for those with a high microfilarial load.

Loa loa: experimental infection in two species of African primates.

Four species of primates, baboon (Papio anubis), patas monkeys (Erythrocebus patas), green monkey (Cercopithecus aethiops) and chimpanzee (Pan troglodytes) were inoculated with third-stage larvae of a human strain of Loa loa from Cameroon, West African. The baboon and patas monkeys developed patent infections after 135 to 148 days; the green monkeys and chimpanzee did not. In each animal which became patent, microfilaremia rose rapidly to high levels. In the baboon, but not in the patas monkeys, there was a suppression of microfilaremia during the 4th month of patency. After splenectomy, microfilariae reappeared in the peripheral blood in large numbers. In both baboon and patas monkeys, the microfilariae of Loa loa maintain the diurnal periodicity so characteristic of their behavior in man.

Lymphadenitis caused by Loa loa.

The World Health Organization's Collaborating Centre for the Histopathology of Filarial Diseases of Man contains specimens of lymph node from 13 patients infected by the filarial nematode Loa loa. Ten of these nodes have distinctive microscopic features characterized by distended sinuses that contain histiocytes and eosinophils and by atrophy of lymphoid follicles. Less striking features include fibrosis of capsule and trabeculae, dilated lymphatic vessels of capsule and medulla, and inflammatory cell infiltrates. We believe that these changes, although in themselves nonspecific, are characteristic of lymphadenitis caused by Loa loa. These 10 lymph nodes were removed from the inguinal region--one from each of 10 native Zairians . At the time of herniorrhaphy the nodes in eight patients were found to be enlarged and were removed for diagnosis. Seven of the 10 patients were infected with Dipetalonema perstans as well as Loa loa, and one of these seven had three filarial infections--L. loa, D. perstans and D. streptocerca . Lymph nodes from other patients infected by other filariae that were available for study at the AFIP did not have these histopathologic features.

Loiasis: "Calabar" swellings and involvement of deep organs.

The authors report clinical and histopathologic changes in six patients with symptomatic loiasis. One patient had cutaneous swellings, three patients presented with hydrocele, one patient developed bowel obstruction, and one had generalized fatal loiasis. The first five patients had localized lesions provoked by adult worms; all were surgically removed. The sixth patient died of disseminated loiasis that included a severe loal encephalitis. The authors discuss the mechanism of "Calabar" swellings, the reaction to adult Loa loa worms and loal encephalitis.

Survival of Loa loa following transplantation from drills (Mandrillus leucophaeus) into jirds (Meriones unguiculatus): parasitology and pathology.

Two drills infected with Loa loa maintained a microfilaraemia for four and a half years ranging from less than 1 mf/100 microliters to 1150 mf/100 microliters. No significant tissue reactions to the adult worms were seen at autopsy. Adult worms were transplanted into the peritoneal cavities of naive jirds when a persistent microfilaraemia first developed by 17 days. Retransplantation of adult worms into naive jirds produced a microfilaraemia and microfilariae in the peritoneal cavities of three out of five animals. These three animals were all negative for circulating parasites by eight and a half months. The tissue reactions to the worms in the jirds are described, including a granulomatous response surrounding adults and a myositis involving microfilariae.

Loasis as a possible trigger of African endomyocardial fibrosis: a case report from Nigeria.

A Nigerian boy, previously in good health, presented with a two-day history of fever with chills; followed on the third day by periorbital swelling, urticarial rash and itching; and on the sixth day by dyspneoa, abdominal swelling and leg swelling. There was clinical, radiological and electrocardiographic evidence of dominant right-sided heart failure. Loa-loa was isolated from the blood and eosinophilia was marked but both were cured by diethylcarbamazine therapy. Heart failure, however, persisted and ended fatally 25 1/2 months later. Endomyocardial fibrosis, more severe on the right sided chambers, but affecting both ventricles was diagnosed. Evidence is presented from the literature to indicate loasis as the trigger of endomyocardial damage in this patient.

Ocular findings after ivermectin treatment of patients with high Loa loa microfilaremia.

Hemorrhages in the palpebral conjunctiva (HPCs) have been recorded in patients living in an area endemic for loiasis who developed serious reactions after ivermectin treatment. A study was designed to evaluate the frequency of these lesions, and to identify risk factors associated with their appearance. The conjunctivae of 1,682 patients who complained of reactions were systematically examined. HPCs were found in 41 patients. The initial mean Loa loa microfilaremia in the individuals with HPCs was 14,900 microfilariae (mf) per mL, as compared with 14.5 mf/mL in the other patients. Mansonella perstans microfilaremia and male gender were also associated with HPCs. Post-treatment fundus examinations were performed on 37 patients, and a close relationship was found between the occurrence of HPCs and the presence of retinal lesions. The vascular pathological processes leading to the ocular lesions may be similar to those which occur at the cerebral level in patients harboring high L. loa microfilaremia who develop neurologic troubles after ivermectin treatment. Retinal lesions may represent a special feature of the Loa-related encephalopathies useful for differential diagnosis, and the HPCs may be useful as an alarm sign to identify those individuals who might develop serious reactions after ivermectin treatment.

Nephropathy in Cameroon: evidence for filarial derived immune-complex pathogenesis in some cases.

This study was carried out in two parts. Part 1 consisted of an epidemiological survey of 1011 subjects aged 3-65 yrs from 2 adjoining villages hyper-endemic for Onchocerciasis and 890 subjects in a control area, relatively free from this infection but otherwise with a similar parasitological profile. There was a significantly higher prevalence of proteinuria in subjects from the onchocercal zone than in controls (observed difference greater than 5 1/2 times its standard error). Part 2 comprised detailed investigations, including renal biopsy, of 63 consecutive patients admitted into hospital with severe proteinuria and/or renal failure from a caption area extending into the onchocercal zone. There were a variety of causative factors, but in 9 cases filarial antigen was demonstrable in the immune-complex deposits in the kidney. A plenum of renal histopathological changes were seen in patients with onchocerciasis. The significance of these findings is discussed.

A case of loiasis.

The prevalence of Loa loa infections in non-endemic areas such as Korea is very low, even though it is quite common in the endemic regions of West and Central Africa. We describe a patient who presented with temporary localized edema (classical Calabar swellings) after travelling to Cameroon and in whom the diagnosis of loiasis was made by ELISA. This is the second reported case of loiasis in Korea. As international travel is becoming more frequent, Loa loa infection should be considered in the differential diagnosis for patients with eosinophilia and Calabar swellings in Korea.

Loa loa presenting in a ThinPrep Pap test: case report and review of parasites in cervicovaginal cytology specimens.

Parasites other than Trichomonas vaginalis may occasionally present in Pap tests obtained during gynecologic examination. We present a case of Loa loa found on a Pap test from an apparently healthy 19-yr-old woman who had immigrated to the US at the age of 15 from Cameroon. We discuss the cytologic features from this case and then briefly review Loa loa and the presence of parasites in Pap tests and other cervicovaginal specimens.

Innovative tools for assessing risks for severe adverse events in areas of overlapping Loa loa and other filarial distributions: the application of micro-stratification mapping.

BACKGROUND: The wide distribution of Loa loa infection (loiasis) throughout the Democratic Republic of Congo (DRC) is a major obstacle to the plans to eliminate onchocerciasis and lymphatic filariasis (LF) because the standard drug regime is dependent on ivermectin, which cannot be used in co-endemic areas due to the risk of severe adverse events (SAEs). A better understanding of the micro-epidemiology, overlapping low and high risk areas, and how they relate to SAEs is critical to ensure safe and effective treatment. FINDINGS: Based on published data from the Bas Congo Province in DRC, this study used geographical information systems (GIS) to re-map and analyse onchocerciasis and loiasis prevalence (<20%, 20 to 40%, >40%) at 144 sites in relation to health district areas reporting SAEs. The new maps highlighted the contrasting patterns of the high prevalence sites, and significant geographical overlap between low onchocerciasis and high loiasis sites. Statistical analyses found that sites with medium to high loiasis prevalence were 10 to 16 times more likely to be in a SAE area than those with low prevalence of loiasis. Sites where both onchocerciasis and loiasis prevalence was >20% were also associated with SAE areas. CONCLUSIONS: Collaborative efforts between the national onchocerciasis and LF programmes are critical as plans to scale interventions are moving forward and thus, alternative strategies needed in loiasis co-endemic areas which may include the new L. loa test and treat strategy using the Cellscope, or interventions such as integrated vector management, or anti Wolbachia therapy using doxycycline.