In vitro maintenance of Mansonella perstans microfilariae and its relevance for drug screening.

BACKGROUND: Mansonellosis arises from infections with threadlike filarial nematodes in millions of individuals, especially in sub-Saharan Africa. Since infections present no overt clinical symptoms but attenuate immune responses that might lead to increased susceptibility and worsened disease course of concomitant infections, it is truly a neglected tropical disease. Nevertheless, only few studies focus on identifying suitable safe drugs for its control and little is known about the requirements for in vitro maintenance of the Mansonella perstans transmission stage. This study, therefore, evaluated the survival of M. perstans microfilariae (mf) using in vitro conditions that have been shown to promote survival of Loa loa, a closely related filarial nematode. Furthermore, the in vitro microfilaricidal effect of 15 agents was assessed on this helminth. METHODS: The ability of two basic culture media; Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute (RPMI-1640) supplemented with 10% fetal bovine serum (FBS) and a monkey kidney epithelial cell line (LLC-MK2) to support the survival of M. perstans microfilariae was investigated. Subsequently, 6 anti-helminthics, 5 anti-malarials, 1 anti-microbacterial, 2 trypanocidals and 1 anti-cancer agent were tested in vitro against mf. The suitability of the culture media as well as the effect of the anti-infective agents on mf survival was assessed by scoring their motility. RESULTS: FBS supplement and additional LLC-MK2 cells significantly improved the survival of mf in DMEM and RPMI-1640 culture. In detail, RPMI-1640 supplemented with 10% FBS and LLC-MK2 cells sustained the maintenance of mf for at least 20 days (100.00 ±â€¯0.00% survival). In co-cultures with LLC-MK2 cells without serum, M. perstans mf were maintained in DMEM and RPMI-1640 medium with a motility above 99% by day 5. Mefloquine displayed the highest microfilaricidal effect in vitro followed by artesunate. CONCLUSION: Both RPMI and DMEM in the presence of LLC-MK2 cells are suitable for the maintenance of M. perstans mf in vitro. In absence of the feeder cells, the addition of 10% FBS to RPMI-1640 medium improved the parasite survival rate and motility. The microfilaricidal activity of mefloquine and artesunate on M. perstans mf was documented for the first time in this study and can therefore be considered as reference for further screening of agents against this parasite stage.

Update on the biology and ecology of Culicoides species in the South-West region of Cameroon with implications on the transmission of Mansonella perstans.

BACKGROUND: Culicoides (Diptera; Ceratoponidae) are tiny, stout, blood-sucking flies with a near worldwide distribution. When present, they are often considered a biting nuisance but in addition, they are involved in the transmission of pathogens to humans, domestic and wild animals. Data on Culicoides species in the South-West region of Cameroon dates back to the 1950s. Over the decades, ecological transformation due to agriculture and deforestation may have affected the population dynamics of Culicoides and therefore our study provides an update of their bio-ecology in the region. Furthermore, the role of various Culicoides species in the transmission of parasitic filariae of the genus Mansonella remains inconclusive in this region. This study was designed to address these unknown issues and expand on current scientific knowledge. RESULTS: Eight species of Culicoides (C. bedfordi, C. inornatipennis, C. fulvithorax, C. grahamii, C. imicola, C. milnei, C. neavei and C. kumbaensis) were collected using light traps and human baits. Culicoides grahamii was the most abundant species, followed closely by C. milnei. Three species (C. milnei, C. grahamii and C. inornatipennis) were common in all observed larval development sites. Only four species (C. inornatipennis, C. fulvithorax, C. grahamii and C. milnei) were collected on humans. Anthropophilic species were more abundant (P < 0.001) in the evening (4-7 pm) when compared to the morning collections (6-9 am). After overnight fly collections using a drop trap with a human microfilaremic donor, C. milnei emerged as the potential host for transmitting Mansonella perstans. Substantial heterogeneity was observed between the trap visiting cycles of the various species (P < 0.001). The biting cycle of the main vector, C. milnei, showed two peaks (10-11 pm and 4-5 am), the highest being 10-11 pm. CONCLUSIONS: The Culicoides fauna of the South-West region of Cameroon has not changed significantly since the 1950s. Culicoides milnei was demonstrated to be the major vector of M. perstans in this part of Cameroon. It is essentially a nocturnal species which peaks in abundance between 10 and 11 pm.

Inflammatory and regulatory CCL and CXCL chemokine and cytokine cellular responses in patients with patent Mansonella perstans filariasis.

Mansonella perstans (Mp) filariasis is present in large populations in sub-Saharan Africa, and to what extent patent Mp infection modulates the expression of immunity in patients, notably their cellular cytokine and chemokine response profile, remains not well known. We studied the spontaneous and inducible cellular production of chemokines (C-X-C motif) ligand 9 (CXCL9) [monokine induced by interferon (IFN)-γ (MIG)], CXCL-10 [inducible protein (IP)-10], chemokine (C-C motif) ligand 24 (CCL24) (eotaxin-2), CCL22 [macrophage-derived chemokine (MDC)], CCL13 [monocyte chemotactic protein-4 (MCP-4)], CCL18 [pulmonary and activation-regulated chemokine (PARC)], CCL17 [thymus- and activation-regulated chemokine (TARC)] and interleukin (IL)-27 in mansonelliasis patients (Mp-PAT) and mansonelliasis-free controls (CTRL). Freshly isolated peripheral mononuclear blood cells (PBMC) were stimulated with helminth, protozoan and bacterial antigens and mitogen [phytohaemagglutinin (PHA)]. PBMC from Mp-PAT produced spontaneously (without antigen stimulation) significantly higher levels of eotaxin-2, IL-27, IL-8, MCP-4 and MDC than cells from CTRL, while IFN-γ-IP-10 was lower in Mp-PAT. Helminth antigens activated IL-27 and MCP-4 only in CTRL, while Ascaris antigen, Onchocerca antigen, Schistosoma antigen, Entamoeba antigen, Streptococcus antigen, Mycobacteria antigen and PHA stimulated MIG release in CTRL and Mp-PAT. Notably, Entamoeba antigen and PHA strongly depressed (P < 0·0001) eotaxin-2 (CCL24) production in both study groups. Multiple regression analyses disclosed in Mp-PAT and CTRL dissimilar cellular chemokine and cytokine production levels being higher in Mp-PAT for CCL24, IL-27, IL-8, MCP-4, MDC and PARC (for all P < 0·0001), at baseline (P < 0·0001), in response to Entamoeba histolytica strain HM1 antigen (EhAg) (P < 0·0001), Onchocerca volvulus adult worm-derived antigen (OvAg) (P = 0·005), PHA (P < 0·0001) and purified protein derivative (PPD) (P < 0·0001) stimulation. In Mp-PAT with hookworm co-infection, the cellular chemokine production of CXCL10 (IP-10) was diminished. In summary, the chemokine and cytokine responses in Mp-PAT were in general not depressed, PBMC from Mp-PAT produced spontaneously and selectively inducible inflammatory and regulatory chemokines and cytokines at higher levels than CTRL and such diverse and distinctive reactivity supports that patent M. perstans infection will not polarize innate and adaptive cellular immune responsiveness in patients.

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.

Mansonella ozzardi: a neglected New World filarial nematode.

Mansonella ozzardi (Nematoda: Onchocercidae) is an understudied filarial nematode, originally described by Patrick Manson in 1897, that can be transmitted by two families of dipteran vectors, biting midges (most of them members of the genus Culicoides) and black flies (genus Simulium). With a patchy geographic distribution from southern Mexico to northwestern Argentina, human infection with M. ozzardi is highly prevalent in some of the Caribbean islands, along riverine communities in the Amazon Basin, and on both sides of the border between Bolivia and Argentina. There is no clinical entity unequivocally associated with M. ozzardi infection, although fever, arthralgia, headache, cold lower extremities, and itchy cutaneous rashes are occasionally mentioned in case report series. More recently, ocular manifestations (especially keratitis) have been associated with mansonelliasis, opening an important area of investigation. Here, we briefly review the biology, epidemiology, pathogenesis, and clinical aspects of M. ozzardi infection and point to some existing knowledge gaps, aiming to stimulate a research agenda to help filling them.

Ecological Drivers of Mansonella perstans Infection in Uganda and Patterns of Co-endemicity with Lymphatic Filariasis and Malaria.

BACKGROUND: Mansonella perstans is a widespread, but relatively unknown human filarial parasite transmitted by Culicoides biting midges. Although it is found in many parts of sub-Saharan Africa, only few studies have been carried out to deepen the understanding of its ecology, epidemiology, and health consequences. Hence, knowledge about ecological drivers of the vector and parasite distribution, integral to develop spatially explicit models for disease prevention, control, and elimination strategies, is limited. METHODOLOGY: We analyzed data from a comprehensive nationwide survey of M. perstans infection conducted in 76 schools across Uganda in 2000-2003, to identify environmental drivers. A suite of Bayesian geostatistical regression models was fitted, and the best fitting model based on the deviance information criterion was utilized to predict M. perstans infection risk for all of Uganda. Additionally, we investigated co-infection rates and co-distribution with Wuchereria bancrofti and Plasmodium spp. infections observed at the same survey by mapping geographically overlapping areas. PRINCIPAL FINDINGS: Several bioclimatic factors were significantly associated with M. perstans infection levels. A spatial Bayesian regression model showed the best fit, with diurnal temperature range, normalized difference vegetation index, and cattle densities identified as significant covariates. This model was employed to predict M. perstans infection risk at non-sampled locations. The level of co-infection with W. bancrofti was low (0.3%), due to limited geographic overlap. However, where the two infections did overlap geographically, a positive association was found. CONCLUSIONS/SIGNIFICANCE: This study presents the first geostatistical risk map for M. perstans in Uganda. We confirmed a widespread distribution of M. perstans, and identified important potential drivers of risk. The results provide new insight about the ecologic preferences of this otherwise poorly known filarial parasite and its Culicoides vector species in Uganda, which might be relevant for other settings in sub-Saharan Africa.

CD4+CD25hiFOXP3+ cells in cord blood of neonates born from filaria infected mother are negatively associated with CD4+Tbet+ and CD4+RORγt+ T cells.

BACKGROUND: Children who have been exposed in utero to maternal filarial infection are immunologically less responsive to filarial antigens, have less pathology, and are more susceptible to acquire infection than offspring of uninfected mothers. Moreover children from filaria infected mothers have been shown to be less responsive to vaccination as a consequence of an impairment of their immune response. However, it is not well known how in utero exposure to parasite antigens affects cellular immune responses. METHODOLOGY: Here, 30 pregnant women were examined for the presence of microfilaria of Loa loa and Mansonella perstans in peripheral blood. At delivery, cord blood mononuclear cells (CBMC) were obtained and the CD4+T cells were phenotyped by expression of the transcription factors Tbet, RORγt, and FOXP3. RESULTS: No significant difference was observed between newborns from infected versus uninfected mothers in the frequencies of total CD4+T cells and CD4+T cells subsets including CD4+Tbet+, CD4+RORγt+ T and CD4+CD25hiFOXP3+ T cells. However, there was a negative association between CD4+CD25hiFOXP3+T cells and CD4+Tbet+ as well as CD4+RORγt+ T cells in the infected group only (B = -0.242, P = 0.002; B = -0.178, P = 0.013 respectively). CONCLUSION: Our results suggest that filarial infection during pregnancy leads to an expansion of functionally active regulatory T cells that keep TH1 and TH17 in check.

Endemic mansonellosis in Emohua local government area, Nigeria: human parasitaemia and Culicoides biting patterns.

BACKGROUND & OBJECTIVES: The study was aimed at elucidating the prevalence and intensity of Mansonella perstans microfilaraemia in the Emohua Local Government Area, Nigeria, and ascertaining the abundance, circadian, and the annual biting patterns of the Culicoides vector. METHODS: Thick smear of 50 µl finger-prick blood stained with Giemsa was examined microscopically in a cross-sectional study. Vector landing collection on human bait was employed in a longitudinal study of the vector biting patterns, carried out between July 2005 and August 2006. RESULTS: Of 1486 individuals examined, 11.2% of both males and females were positive for M. perstans microfilaraemia. Microfilaraemia appeared early in life. The overall geometric mean intensity among those with positive microfilaraemia was 117 mf/ml (121 mf/ml for males and 113 mf/ml for females). The differences in geometric mean intensity between different age groups were statistically significant (one-way analysis of variance; p <0.05), being highest in the oldest age group (266 mf/ml). A total of 1183 female Culicoides sp were caught from September 2005 to August 2006. The abundance of Culicoides sp was seasonal. The circadian biting activity had a broad peak between 0700 and 1200 hrs. The monthly biting rates ranged from zero bite per person per month in January 2006 to 1151 bites per person per month in June 2006. The annual biting rate was 7382 bites per person per year. CONCLUSION: Majority of those with positive microfilaraemia were poor socioeconomically, underscoring the need for health education and application of effective control measures against Culicoides biting midges in Emohua.

[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.

Passage of ingested Mansonella ozzardi (Spirurida: Onchocercidae) microfilariae through the midgut of Aedes aegypti (Diptera: Culicidae).

When virus and microfilariae are ingested concurrently by a mosquito, microfilariae (mf) may penetrate the mosquito midgut and introduce virus directly into the mosquito hemocoel, allowing mosquitoes to become infectious much sooner than normal and enhancing transmission of viruses by mosquitoes. Mansonella ozzardi (Manson) is a benign filarial nematode parasite of humans in Latin America and is transmitted by black flies (Diptera: Simuliidae) and biting midges (Diptera: Ceratopogonidae). Because M. ozzardi and dengue are sympatric, we wanted to know whether M. ozzardi mf had the ability to penetrate the midgut of Aedes aegypti (L.) (Diptera: Culicidae) and thus play a potential role in the enhancement of dengue transmission. To test this, the F1 progeny from locally collected Ae. aegypti were fed on M. ozzardi-infected human males in an endemic village in northern Trinidad. Mosquitoes were dissected at various times after feeding and examined for mf in the midguts and thoraces. Microfilariae penetrated the midguts of 43% of 63 mosquitoes that ingested mf. Overall, 11% of mf penetrated the midgut by 17 h after being ingested. The intensity of midgut penetration was positively correlated to the numbers of mf ingested. Because midgut penetration is a key requirement for mf enhancement to occur, the potential exists that M. ozzardi could be involved in the enhancement of dengue virus transmission.

[Mansonella perstans filariasis]. / La filariosi da Mansonella perstans.

Mansonella perstans filariasis is widely present in Africa and equatorial America and its pathogenicity has been recently reconsidered. Although M. perstans infection has been considered a minor filariasis, remaining asymptomatic in most of infected subjects, more recent studies have shown that M. perstans is capable of inducing a variety of clinical features, including angioedemas, swellings like the "Calabar swellings" of loiasis, pruritus, fever, headache, pain in bursae and/or joint synovia, or in serous cavities. It is likely that some of the pathological changes observed are induced by the immune response to the infection. Eosinophilia is present in many cases of infection. Moreover M. perstans filariasis is difficult to be treated. Effective treatment is lacking and there is no consensus on optimal therapeutic approach. The most commonly used drug is diethylcarbamazine (DEC) that is however often ineffective. Although other drugs have been tried (e.g. praziquantel, ivermectin), none has proven to be reliably and rapidly effective. Mebendazole seemed more active than DEC in eliminating the infection, with a comparable rate of overall responses. Thiabendazole evidenced a small, but significant activity against the infection. Combination treatments (DEC plus mebendazole) resulted in a significantly higher activity compared with the single drugs.

Acanthocheilonema viteae in Mastomys coucha: chemotherapeutic and chemoprophylactic role of vitamin A in experimental filarial infection.

The role of vitamin A was evaluated for its chemotherapeutic and chemoprophylactic action against Acanthocheilonema viteae infection in Mastomys coucha. Vitamin A was administered for 10 days, five days before infection and five days post infection. On day 0 experimental animals as well as controls were infected with L3, the infective stage. Establishment of the worms revealed significantly less percentage of worm recovery over untreated controls. Cell-mediated response was found to be the cause of this reduction in worm recovery, whereas humoral response was not significant as IgG, IgA and IgM titres were low.

[Mansonella perstans filariasis after stay in Cameroon. A 19-year-old patient born in Cameroon, in Switzerland for the last 10 years]. / Mansonella perstans-Filariose nach Aufenthalt in Kamerun. 19-jahrige Patientin, geboren in Kamerun, seit zehn Jahren in der Schweiz.

After a spending three weeks in Cameroon, a 19-year-old woman presented in the emergency room with sudden lancinating pain in the lower abdomen, predominantly on the right side. A blood smear tested for presumed malaria showed subperiodic microfilariae of the species Mansonella perstans. The patient was treated with mebendazole (Vermox).

Mansonella ozzardi: the course of patency in experimentally-infected patas monkeys.

Twenty-five of 30 patas monkeys (Erythrocebus patas) inoculated with varying numbers (35 to 135) of third-stage larvae of Mansonella ozzardi developed patent infections in an average of 163 days. There was no correlation between the size of the inoculum and the length of the prepatent period. Ten of the monkeys were monitored thereafter by regular blood examination for extended periods of time in order to characterize the onset, course and duration of patency. Typically, with the onset of patency, microfilaremias increased steadily, peaking at about 20 weeks and then decreased slowly stabilizing at low levels for up to 48 weeks. Thereafter microfilariae disappeared from the blood and occasionally reappeared in scanty numbers. Laparotomies and followup studies indicated that the spleen was involved in the suppression of peripheral microfilaremia as had been observed earlier in patas monkeys infected with Loa loa. In ten monkeys splenectomized after the initial "wave" of microfilaremia, it was observed that (a) 30% of the animals remained amicrofilaremic, (b) another 30% reestablished patent infections but microfilaremias were lower than presplenectomy levels, and (c) in the remaining 40%, levels of microfilaremia equaled or exceeded pre-splenectomy levels. Patent infections persisted for up to 212 weeks. One monkey splenectomized prior to inoculation with 87 larvae developed a patent infection with microfilaremia which persisted for 156 weeks. Three monkeys with low and high levels of microfilaremia bled at four hour intervals over a 28 hour period showed no evidence of periodicity in the microfilaria.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of Culicoides barbosai as a vector of Mansonella ozzardi in Haiti.

Studies in the southern peninsula of Haiti showed that the biting midge, Culicoides barbosai, was capable of supporting the development of Mansonella ozzardi to the infective stage. The known vector, Culicoides furens, also was encountered. Both species showed distinct biting site preferences, i.e., 98% of the midges that engorged on the arms and head were C. barbosai whereas C. furens was collected mostly from the lower legs. Nine days after engorgement, 19 infective larvae were recovered from 13 C. barbosai versus six larvae from four C. furens. It was judged that C. barbosai may be as importantly involved in the natural transmission cycle as C. furens in this community.

[Absence of response of Mansonella ozzardi microfilaria in the diethylcarbamazine provocation test]. / Défaut de réponse des microfilaires Mansonella ozzardi au test de provocation par le diéthyl-carbamazine.

One hundred and twelve inhabitants of an area endemic for M. ozzardi received 20 cmm capillary blood samples by finger prick before and 30 minutes after the ingestion of 100 mg of diethylcarbamazine citrate. No significant variations in microfilaremia were detected. Thus, a provocative dose of DEC would not appear to be helpful in sampling for mansonellosis in Haiti.

Crypto-periodicity in Mansonella ozzardi.

Studying nycthemeral microfilarial density in eight carriers of Mansonella ozzardi, Nathan et al. concluded that there is an absence of periodicity in M. ozzardi in Trinidad. Re-examination of the results obtained shows evidence of crypto-periodicity. Two of the eight patients showed highly significant periodicity but the biorhythms appear to be almost out of phase, the respective peaks being at 18.00 hours and 02.00 hours. The six other patients showed no obvious periodicity; the calculated acrophases (peak hours), instead of being randomly spaced, regrouped with the preceding ones: late afternoon in two subjects and in the second half of the night for the other four. It is concluded that the apparent non-periodicity is due to the co-existence of two periodic forms, but these are markedly out of phase.