Fresh Blood Examination Research Articles

Trypanosoma cruzi experimental congenital transmission associated with TcV and TcI subpatent maternal parasitemia

The congenital transmission of Chagas disease is associated with an increase in parasitemia during pregnancy, maternal and fetal immunity, and populations of Trypanosoma cruzi. In this study, the biological behavior of TcI and TcV (isolated from a human congenital case) strains and their potential for experimental congenital transmission were evaluated in female BALB/C mice. Parasitemia was estimated by fresh blood examination, semiquantitative microhematocrit, and hemoculture, while congenital transmission was evaluated by culture in the liver infusion tryptose medium and by polymerase chain reaction (PCR) of the pups' tissues on postnatal day 7 and of the pups' blood sample at 30 days after birth. Infection was detected in 100 % of the females. Both strains showed subpatent parasitemia, which was higher for TcV infection. The presence of amastigote nest was detected only in an animal infected with TcI. The inflammatory process was more frequent (p = 0.001) in the tissues of the animals infected with TcV (58.6 %) than TcI (31.1 %). The fertility rates of females mated after 35 days postinfection were similar (90 % for TcV, 88.9 % for TcI; p = 0.938). Parasitemia did not change during pregnancy. The average number of pups/female was greater (p = 0.03) in mice with TcV infection (8.30) than in those with TcI infection (4.78). Congenital transmission was detected exclusively by PCR in 50.9 % of the pups, 46.6 % for TcV and 58.1 % for TcI. The PCR positivity for TcI was higher in the blood than in the tissue (p = 0.003). These results demonstrate the T. cruzi experimental congenital infection associated with subpatent maternal parasitemia of TcI and TcV.

In vivo susceptibility to benznidazole of Trypanosoma cruzi strains from the western Brazilian Amazon

To assess the susceptibility of Trypanosoma cruzi strains from Amazon to benznidazole. We studied 23 strains of T. cruzi obtained from humans in the acute phase of Chagas disease, triatomines and marsupials in the state of Amazonas and from chronic patients and triatomines in the state of Paraná, Brazil. The strains were classified as TcI (6), TcII (4) and TcIV (13). For each strain, 20 Swiss mice were inoculated: 10 were treated orally with benznidazole 100 mg/kg/day (TBZ group) for 20 consecutive days and 10 comprised the untreated control group (NT). Fresh blood examination, haemoculture (HC), PCR, and ELISA were used to monitor the cure. The overall cure rate was 60.5% (109/180 mice) and varied widely among strains. The strains were classified as resistant, partially resistant or susceptible to benznidazole, irrespective of discrete typing units (DTUs), geographical origin or host. However, the TcI strains from Amazonas were significantly (P = 0.028) more sensitive to benznidazole than the TcI strains from Paraná. The number of parasitological, molecular and serological parameters that were significantly reduced by benznidazole treatment also varied among the DTUs; the TBZ group of mice inoculated with TcIV strains showed more reductions (8/9) than those with TcI and TcII strains. Benznidazole resistance was observed among natural populations of the parasite in the Amazon, even in those never exposed to the drug.

Biological behavior of different Trypanosoma cruzi isolates circulating in an endemic area for Chagas disease in the Gran Chaco region of Argentina

The biological behavior of the different Trypanosoma cruzi strains is still unclear and the importance of exploring the relevance of these differences in natural isolates is of great significance. Herein we describe the biological behavior of four T. cruzi isolates circulating sympatrically in a restricted geographic area in Argentina endemic for Chagas Disease. These isolates were characterized as belonging to the Discrete Typing Units (DTUs) TcI, TcIII, TcV and TcVI as shown by Multilocus Enzyme Electrophoresis and Multilocus Sequence Typing. In order to study the natural behavior of the different isolates and to preserve their natural properties, we developed a vector transmission model that allows their maintenance in the laboratory. The model consisted of serial passages of these parasites between insect vectors and mice. Vector-derived parasite forms were then inoculated in C57BL/6J mice and number of parasite in peripheral blood, serological response and histological damage in acute and chronic phases of the infection were measured. Parasites from DTUs TcI, TcIII and TcVI were detected by direct fresh blood examination, while TcV parasites could only be detected by Polimerase Chain Reaction. No significant difference in the anti-T. cruzi antibody response was found during the chronic phase of infection, except for mice infected with TcV parasites where no antibodies could be detected. Histological sections showed that TcI isolate produced more damage in skeletal muscle while TcVI induced more inflammation in the heart. This work shows differential biological behavior among different parasite isolates obtained from the same cycle of transmission, permitting the opportunity to formulate future hypotheses of clinical and epidemiological importance.

Real-time PCR strategy for parasite quantification in blood and tissue samples of experimental Trypanosoma cruzi infection

The lack of an accurate diagnosis has been a serious obstacle to the advancement of the anti-Trypanosoma cruzi chemotherapy and long-term infection can result in different health risks to human. PCRs are alternative methods, more sensitive than conventional parasitological techniques, which due to their low sensitivities are considered unsuitable for these purposes. The aim of this study was to investigate a sensitive diagnostic strategy to quantify blood and cardiac tissues parasites based on real-time PCR tools during acute and chronic phases of murine Chagas disease, as well as to monitor the evolution of infection in those mice under specific treatment. In parallel, fresh blood examination, immunological analysis and quantification of cardiac inflammation were also performed to confront and improve real-time PCR data. Similar profiles of parasitemia curves were observed in both quantification techniques during the acute phase of the infection. In contrast, parasites could be quantified only by real-time PCR at 60 and 120 days of infection. In cardiac tissue, real-time PCR detected T. cruzi DNA in 100% of infected mice, and using this tool a significant Pearson correlation between parasite load in peripheral blood and in cardiac tissue during acute and chronic phases was observed. Levels of serum CCL2, CCL5 and nitric oxide were coincident with parasite load but focal and diffuse mononuclear infiltrates was observed, even with significant (p<0.05) reduction of parasitism after 60 days of infection. Later, this methodology was used to monitor the evolution of infection in animals treated with itraconazole (Itz). Itz-treatment induced a reduction of parasite load in both blood and cardiac muscle at the treatment period, but after the end of chemotherapy an increase of parasitism was detected. Interestingly, inflammatory mediators levels and heart inflammation intensity had similar evolution to the parasite load, in the group of animals treated. Taken together, our data show that real-time PCR strategy used was suitable for studies of murine T. cruzi infection and may prove useful in investigations involving experimental chemotherapy of the disease and the benefits of treatment in relation to parasitism and inflammatory response.

Biological behaviour in mice of Trypanosoma cruzi isolates from Amazonas and Paraná, Brazil

The biological behaviour of 23 Trypanosoma cruzi isolates in Swiss mice was compared. Nineteen isolates were obtained from patients in the acute phase of Chagas disease (13), sylvatic reservoir hosts (Didelphis marsupialis) (3), and triatomine bugs (Rhodnius robustus) (3) from four regions of the State of Amazonas (AM). Four isolates were obtained from chronic chagasic patients in the State of Paraná (PR): three autochthones, and one allochthone from the State of Minas Gerais. Only one isolate was unable to infect the mice. The AM and PR isolates showed the largest number of significant differences from each other. The former had lower mean values in the pre-patent (5.4days) and patent (4.6days) periods (PP), with the parasitaemia (Pmax) reaching a peak of 9.9×104 blood trypomastigotes (BT)/mL of blood by the 7th day following inoculation. The AM isolates also had higher positivity to fresh-blood examination (FBE) (84.1%) compared to haemoculture (HC) (58.7%) and polymerase chain reaction (PCR) (33.3%), in addition to higher mortality (2.9%). The PR isolates had higher values for PP (18.5days) and Pmax (99.9×104BT/mL) as well as higher positivity to FBE (87.2%), HC (100%), and PCR (83.3%). The correlations between the biological behaviour of the T. cruzi isolates and the clinical and epidemiological characteristics of Chagas disease are discussed.

Different forms of administration of biotherapy 7dH in mice experimentally infected by Trypanosoma cruzi produce different effects

To evaluate the effects of different forms of administration of the blood trypomastigotes biotherapy 7dH in mice experimentally infected with Trypanosoma cruzi. Male swiss mice were inoculated with 1400 blood trypomastigotes of the Y strain of T. cruzi and allocated into 5 treatment groups: IC (distilled water); TCBZ (benznidazole); TBA(7dH) (biotherapy 7dH 20 days after infection); TBB(7dH)7 (biotherapy 7dH seven days before infection); TBB(7dH)30 (biotherapy 7dH 30 days before infection). Parasitological parameters assessed included pre-patent and patent periods, parasitemia peak, total parasitemia, mortality and survival rates. Cure index was obtained by fresh blood examination, hemoculture and polymerase chain reaction (PCR). The TBB(7dH)7 group showed a reduction in parasitemia peak, parasitemia area under the curve and total parasitemia. TBB(7dH)30 showed a tendency to increased pre-patent and survival periods, peak parasitemia was increased without increased total parasitemia. TBA(7dH) did not present significant alterations in the parasitological parameters analyzed. Biotherapy 7dH given before infection (7 or 30 days) produces different effects suggesting modulation of the host's immune system. The effects range from reduced parasitemia to its effective increase. The use of biotherapy to treat T. cruzi infection including dose, potency and schedule deserves further investigation.

Trypanosoma cruzi TcIII / Z3 genotype as agent of an outbreak of Chagas disease in the Brazilian Western Amazonia

Chagas' disease is an emerging and neglected disease in the Brazilian Amazon region, where T. cruzi I predominates among the acute cases of the disease; and T. cruzi III/Z3, a population cluster from sylvatic areas of the Amazon basin, is rarely associated with human infections. On 23rd April 2007, the Foundation for Health Surveillance of the State of Amazonas, Brazil reported an outbreak of acute Chagas disease in the municipality of Coari on the Solimões River banks. Fresh blood examination confirmed the infection in 25 patients. Parasite culture in LIT medium was successful for 18 isolates. Molecular characterization was performed by PCR of the non-transcribed spacer of the mini-exon and by sequencing of the mitochondrial cytochrome c oxidase subunit II (COII) gene. The T. cruzi isolates were all from genotype Z3, and sequencing revealed that all isolates had equal COII sequences compatible with TcIII type, suggesting a single source of infection. To our knowledge, this is the first outbreak of acute cases caused uniquely by the genotype TcIII/Z3. Wild vectors harbouring TcIII stocks contribute to transmission when the triatomine species reaches human food chain or when humans invade the forest environment, where sylvatic cycle constitutes a reservoir of parasites that might be associated with specific epidemiological and clinical traits of the emergent Chagas disease in the Amazon.

A Hemogram of Clarias batrachus Parasitized by Two Species of Hemoflagellates (Trypanosoma batrachi Qadri, 1962 and Trypanoplasma haematalis n.sp.) as an Indicator of Infectivity

The blood of a freshwater catfish, Clarias batrachus Linn. was scanned for hemoflagellate infectivity by examination of fresh blood (hanging drop preparations and hematocrit) and stained smears. Trypanosoma batrachiQadri, 1962 occurring at a prevalence of 29.5% (intensity: 4 to 20 forms/20 min scan) is reported as a new locality record (Bareilly, Uttar Pradesh, India). A new species of monomorphic biflagellate was also found in 8.1% of the infected fish concurrently with Trypanosoma. Based on its morpho-cytostatistical features, flagellar length, and AF:PF ratio, the new species is named Trypanoplasma haematalis n.sp. A few selected hematological parameters (Hb%, ESR, PCV, and MCH) of infected fish were estimated to ascertain the validity as an indicator of flagellate infection under natural and experimentally-induced conditions, by inoculation of 3 ml of infected blood in laboratory-reared, healthy uninfected fish. In either case, the blood parameters recorded significant changes up to a maximum of 55.79% (Hb%, Group B3), 151.04% (ESR, Group B3), 65.45% (PCV, Group B3), and 106.26% (MCH, Group B3) suggesting that hemograms of fishes may be utilized as indicators of some infestation, which offers scope to corroborate it with flagellate infectivity.

Accidental infection by Trypanosoma cruzi follow-up by the polymerase chain reaction: case report

We report a case of accidental infection by Trypanosoma cruzi in a 42-year-old female patient who presented an inoculation chagoma. Laboratory confirmation was based on examination of fresh blood, Giemsa-stained blood smear, immunoenzyme test (EIA-IgG), indirect immunofluorescence (IIF-IgM, IgG) and polymerase chain reaction (PCR). Only the PCR gave a positive result, and the EIA test was inconclusive. Two treatments with benznidazole were necessary. PCR was the only technique that continued to give positive results for approximately two months (65 days, or 2.2 months) following the second treatment and negative results from 96 days (3.2 months) to 850 days (28.3 months). We concluded that the presence of an inoculation chagoma and use of PCR were important and decisive for diagnosis and follow-up of the case.

Trypanosoma cruzi: blood parasitism kinetics and their correlation with heart parasitism intensity during long-term infection of Beagle dogs

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.

Usefulness of the polymerase chain reaction for monitoring cure of mice infected with different Trypanosoma cruzi clonal genotypes following treatment with benznidazole

The capacity of the polymerase chain reaction (PCR) to detect the DNA of Trypanosoma cruzi was evaluated in 90 blood samples from BALB/c mice infected with T. cruzi cloned stocks of genotypes 19 and 20 (T. cruzi I) and 39 and 32 (T. cruzi II), and treated with benznidazole. The results from the fresh blood examination, hemoculture, and ELISA allowed to group the treated animals into: cured (TC), dissociated (DIS) and non-cured (NC). The PCR detected T. cruzi DNA in 50.9%, 58.3% and 100.0% of the samples from TC, DIS and NC mice, respectively. These DNA possibly derives from live T. cruzi or from recently lysed parasites, suggests that these animals are in fact not cured. The difference between the PCR results and results obtained using other techniques was statistically significant and independent of the parasite genotype. The PCR described has therefore potential to be used in cure control of treated patients.

Persistence of PCR-positive tissue in benznidazole-treated mice with negative blood parasitological and serological tests in dual infections with Trypanosoma cruzi stocks from different genotypes

To assess different methodologies to better define an early post-therapeutic cure criterion after benznidazole treatment in BALB/c mice following mixed infection with dual Trypanosoma cruzi genotypes. According to the classical cure criteria, animals were classified as treated not cured (TNC = 76.4%), treated cured (TC = 12.5%) and dissociated (DIS = 11.1%) using parasitological [fresh blood examination (FBE), blood culture (BC) and blood PCR] and serological methods [conventional serology (CS-ELISA) and non-conventional serology (NCS-FC-ALTA)]. Tissues were also evaluated by PCR. FBE was able to detect patent parasitaemia in only 18.1% of TNC and therapeutic failure was detected in 79.1% and 97.2% of TNC by BC and blood PCR, respectively. CS-ELISA should not be used before 3 months after treatment since it may lead to false-negative results. At 3 months after treatment with benznidazole, NCS-FC-ALTA was more efficient for categorizing the groups of treated mice. In the TNC group, although a decreased frequency of PCR-positive tissue was observed in several host tissues, increased positivity was also observed, despite the T. cruzi genotype combination. All TC animals presented at least two positive tissue-PCR results. Our results confirm that NSC-FC-ALTA and blood PCR are the most suitable methods to early detect therapeutic failure in acute murine T. cruzi infection. Additionally, our data show that BC positivity is highly dependent upon the T. cruzi genotype combination. Moreover, our findings demonstrated that PCR tests performed on tissues from animals considered cured after benznidazole treatment still detected T. cruzi DNA, most probably indicating residual infection.

Cost-effectiveness of Algorithms for Confirmation Test of Human African Trypanosomiasis

The control of Trypanosoma brucei gambiense human African trypanosomiasis (HAT) is compromised by low sensitivity of the routinely used parasitologic confirmation tests. More sensitive alternatives, such as mini-anion exchange centrifugation technique (mAECT) or capillary tube centrifugation (CTC), are more expensive. We used formal decision analysis to assess the cost-effectiveness of alternative HAT confirmation algorithms in terms of cost per life saved. The effectiveness of the standard method, a combination of lymph node puncture (LNP), fresh blood examination (FBE), and thick blood film (TBF), was 36.8%; the LNP-FBE-CTC-mAECT sequence reached almost 80%. The cost per person examined ranged from euro1.56 for LNP-FBE-TBF to euro2.99 for LNP-TBF-CTC-mAECT-CATT (card agglutination test for trypanosomiasis) titration. LNP-TBF-CTC-mAECT was the most cost-effective in terms of cost per life saved. HAT confirmation algorithms that incorporate concentration techniques are more effective and efficient than the algorithms that are currently and routinely used by several T.b. gambiense control programs.

Validité, coût et faisabilité de la mAECT et CTC comme tests de confirmation dans la détection de la Trypanosomiase Humaine Africaine

ResumeObjectifs Evaluer la validité, le coût et la faisabilité de deux tests parasitologiques pour la confirmation de la maladie du sommeil; la mini Anion Exchange Centrifugation Technique (mAECT) et la Capillary Tube Centrifugation (CTC).Méthodes Au cours d'une campagne de dépistage de la maladie du sommeil en 2004 nous avons examiné 6502 personnes à Kwamouth en République Démocratique du Congo (RDC). Les personnes testées positives au Card Agglutination Test for Trypanosomiasis sur sang total (CATT) ont reçu une ponction ganglionnaire, un examen de sang frais, une goutte épaisse colorée, un examen de mAECT, un examen de CTC et une titration de CATT. La sensibilité et spécificité des tests de confirmation ont été calculées en utilisant la combinaison de tous les tests parasitologiques comme référence standard. Le coût de chaque méthode a été calculé et leur faisabilité a étéévaluée par des interviews structurées avec les techniciens.Résultats La sensibilité des méthodes parasitologiques classiques était de 44,8% (IC 95%: 36,8–53,0), pour la CTC 56,5% (IC 95%: 48,3–64,5) pour la mAECT 75,3 % (IC 95%: 67,7–81,9). Le coût par test était de 2,82€ pour la mAECT et 0,76€ pour la CTC. Le temps pour l'obtention du résultat était de 29,78 min pour la mAECT et 18,25 min pour la CTC. Ces deux tests ont été jugés faisables sous les conditions de terrain.Conclusion L'utilisation individuelle ou en combinaison de la CTC et de la mAECT comme tests de confirmation sur les personnes au CATT sur sang total apporterait une amélioration considérable de la détection des cas de trypanosomiase humaine africaine. Les deux tests se sont avérés faisables dans des conditions opérationnelles lorsque la disponibilité d'un courant de 220 V pouvait être garantie. Vue que le test mAECT est plus sensible mais considérablement plus coûteux, les analyses de coût‐efficacité de la faisabilité devraient guider au choix du meilleur algorithme.

Trypanosomatidae de importancia en salud pública en animales silvestres y sinantrópicos en un area rural del municipio Tovar del estado Mérida, Venezuela.

Chagas disease and leishmaniasis are important public health problems due to their high frequency and broad distribution in Latin America. Understanding of the roles of reservoir animals is crucial for a global assessment of the epidemiology of these diseases. To identify parasites classed as Trypanosomatidae as they occurred in sylvatic animals, and to establish rates of coinfection. Sylvatic animals were systematically captured in the rural area of El Carrizal, Merida State, Venezuela, betweenJuly, 1998 and February, 2000. The captures were made in Tomahawk type homemade traps, placed 15 nights per month throughout the study period. Blood was extracted from each captured and anesthetized animal by means of cardiac puncture. The search for trypanosomatids was undertaken by fresh blood examination, Giemsa stained blood smears and by means of blood-agar culture. Occasional xenodiagnoses were made to check diagnostic accuracy. The isolates obtained in culture media were identified by restriction fragment analysis and hybridization with specific probes. Three species of sylvatic animals (n = 215) were captured: Rattus spp. (135), Sigmodon hispidus (73) and Didelphis marsupialis (7). From them, three species of Trypanosomatidae were identified: Leishmania (Viannia) guyanensis, Trypanosoma cruzi and Trypanosoma lewisi. Trypanosoma. cruzi was identified in D. marsupialis (4/7), S. hispidus (1/73) and Rattus spp. (1/ 135), whereas L. (V.) guyanensis and T. lewisi were identified only in Rattus spp., 1/135 and 12/ 135, respectively. The coexistence of these genetically related hemoflagellates in sylvatic hosts was important for understanding the immunological interactions that may be established in reservoir animals, and the possible implications that this may have for the susceptible host. Finally, the identification of L. (V.) guyanensis in Rattus spp and T. cruzi in S. hispidus constituted the first reports of this relationship in Venezuela.

Infectivity for mice of Trypanosoma cruzi I and II strains isolated from different hosts

In this paper, the infectivity for mice of Trypanosoma cruzi I and II strains isolated from sylvatic animals, triatomines, and humans is determined using fresh blood examination, hemoculture, culture of macerated organs, and polymerase chain reaction (PCR). Six strains were considered to have low infectivity (9.1-18.2%), five medium (27.3-45.4%), and one high (100.0%). Infectivity of T. cruzi strains isolated from sylvatic animals was significantly higher than that of strains isolated from humans and triatomines (p=0.0141). No significant difference was observed between the infectivity of T. cruzi I and II strains. The parasite was detected by fresh blood examination in one strain, by hemoculture and culture of macerated organs in four strains, and by PCR in all strains. We conclude that the infectivity is related to the host from which the strains were isolated, but the infectivity is not related to the genetic group of the parasite. We also conclude that the majority of the strains studied have low and medium infectivity for mice, and that PCR is an important tool to detect T. cruzi in strains with this biological characteristic.

Evaluation of the rabbit as a model for Chagas' disease. I. Parasitological studies.

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, 72 animals have been inoculated by intraperitoneal and conjunctival route with bloodstream forms, vector-derived metacyclic trypomastigotes and tissue culture trypomastigotes of Trypanosoma cruzi strains Y, CL and Ernane. In 95.6% of the animals trypomastigotes had been detected at the early stages of infection by fresh blood examination. The course of parasitemia at the acute phase was strongly influenced by the parasite strain and route of inoculation. At the chronic phase parasites had been recovered by xenodiagnosis and/or hemoculture in 40% of the examined animals. The xenodiagnosis studies have shown selective interactions between the T. cruzi strains and the four species of vectors used, inducing significant variability in the results. The data herein present are consistent with the parasitological requirements established for a suitable model for chronic Chagas' disease.

A nitroimidazole-thiadiazole derivative with curative action in experimental Trypanosoma cruzi infections.

A nitroimidazole-thiadiazole derivative [(2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4- thiadiazole)] displays a marked curative effect in mice experimentally infected with Trypanosoma cruzi. When doses of 50 or 100 mg kg−1 were used for 20 consecutive days, parasitological cures (assessed by fresh blood examination, haemoculture, sub-inoculation, re-inoculation and indirect immunofluorescent test) were obtained in 100% of the animals. High curative levels were also observed with a single dose (500 mg kg−1) of this drug. Such cure rates were not observed when similar treatment schedules were used with standard and- T. cruzi drugs such as 5-nitrofuran and 2-nitroimidazoIe derivatives, including those already being used clinically.

The effect of temperature, age and density of gametocytes, and changes in gas composition on exflagellation of Leucocytozoon simondi.

The rate of initiation of exflagellation of microgametocytes of Leucocytozoon simondi was studied in relation to temperature, age and density of gametocytes, and changes in gas composition. Observations were made in vitro through examination of fresh blood, and thin blood films were prepared at appropriate intervals. An inverse relationship between temperature and the time required for initiation of ex-flagellation was demonstrated. There was a decrease in the time required for initiation of exflagellation as the temperature increased from 15 to 26C. Between 26 and 40C exflagellation usually occurred in 1–1½ min. Exflagellation at 40C, which approximates the body temperature of the host, indicates that a drop in temperature per se is not necessary for the initiation of exflagellation. Gametocytes appear to be capable of exflagellation for about 5 days postmaturity. Differences in density of parasitemia do not affect the time for initiation of exflagellation. The presence of O2 and a decrease in CO2 are important stimuli for exflagellation. The effect of the above factors on the initiation of exflagellation is discussed in relation to the uptake of infected blood by the simuliid vector of the parasite, and compared with the situation in related Haemosporina

Relapsing fever in Abyssinia

1. 1. In a series of cases of relapsing fever in Abyssinia hepatitis and dyspnoea were the outstanding clinical signs. 2. 2. Differential leucocyte counts revealed an increase of monocytes and a diminution of polymorphonuclear leucocytes within a few days of the arsenic injection. The differential count appeared to have no prognostic significance. 3. 3. Intravenous injections in fairly large doses acted more rapidly in removing spirochaetes from the peripheral blood than either intramuscular or smaller intravenous injections. 4. 4. There is a progressive diminution in the number of spirochaetes in the peripheral blood after injection, as revealed by 4-hourly blood slides. 5. 5. Motility tests were carried out. The spirochaetes in specimens of blood taken 6 hours after injection were still motile at a time when thick films revealed no parasites. 6. 6. The shape of the spirochaete in a fresh blood preparation differed from that in a stained slide. 7. 7. The intermediate host was proved to be the louse, Pediculus corporis. Larger numbers of spirochaetes were found in the gut contents of the lice than in the blood films of the patients. 8. 8. Other cases of hepatitis occurred during the time of this epidemic. The possibility of their connection with relapsing fever is discussed. 9. 9. The pyrexial course tended to be of longer duration than that of other types of relapsing fever. 10. 10. A satisfactory diagnostic technique for the large out-patient clinic consisted of examination of fresh blood and simultaneous staining of multiple thick films on the same slide. 11. 11. It is important to administer a large initial dose of arsenic intravenously; small injections had little beneficial effect even when repeated, and were sometimes followed by relapses. No relapses occurred after the larger doses.