Chemotherapeutic effects on larval stages of Schistosoma mansoni during infection and reinfection of mice

The sensitivity of the larval stages of Schistosoma mansoni to chemotherapy with praziquantel and oxamniquine was tested in mice during primary and secondary infections and after different intervals from cercarial exposure. Worm recovery by perfusion of the porto-mesenteric system, followed by counting and a morphometric study of the parasite, allowed the conclusion that the relative resistance of the larval stages of S. mansoni to schistosomicide drugs, demonstrated in primary infections, also persists when the host is already infected. This indicates that a therapeutic failure may result when an infected host is treated some time after being re-infected, because of the presence of migrating, drug-resistant, immature forms of the parasite. Key-words : Schistosoma mansoni. Larval stages. Praziquantel. Oxamniquine. Resumo A susceptibilidade dos estágios larvais do Schistosoma mansoni aos esquistossomicidas praziquantel e oxamniquine foi testada em camundongos durante infecção primária ou secundária, e após diferentes intervalos de tempo após a exposição cercariana. A avaliação foi feita pela contagem dos vermes após recuperação destes por perfusão do sistema porto-mesentérico e pelo estudo morfométrico dos mesmos. O estudo revelou que a relativa resistência das formas larvais aos esquistossomicidas, já demonstrada em infecção primária, persiste no caso de hospedeiros já infectados. Este fato indica que uma falha terapêutica pode resultar quando o tratamento é feito em hospedeiros re-infectados recentemente, em virtude dos mesmos apresentarem formas migrantes e imaturas do parasita, as quais são particularmente resistentes aos esquistossomicidas. Palavras-chaves: Schistosoma mansoni. Estágio Larval. Praziquantel. Oxamniquine. 1. Laboratório de Patologia Experimental do Centro de Pesquisas Gonçalo Moniz da Fundação Oswaldo Cruz, Salvador, BA, Brazil. 2. Laboratório de Imunopatologia do Centro de Pesquisas Aggeu Magalhães da Fundação Oswaldo Cruz, Recife, PE, Brasil. Financial support: PRONEX Address to: Dr. Zilton Andrade. Rua Waldemar Falcão 121 Brotas, 40295-001 Salvador, BA, Brasil. e-mail:zilton@cpqgm.fiocruz.br Recebido para publicação em 27/7/2002 Aceito em 26/2/2003 Oxamniquine and praziquantel are the two drugs used in Brazil for the treatment of schistosomiasis. Large scale treatment programs using either drug have been carried out in several Brazilian endemic areas 11 . Treatment efficacy is considered satisfactory, but failures have been registered with variable frequency. Such therapeutic failures are often attributed to the development of drug resistance by the worms and to early post treatment re-infection 6 . However, in areas of high transmission, another possibility may occur. Treated individuals, even though apparently cured, may re-establish the infection in consequence of the existence of migrating larval forms of Schistosoma mansoni, which are usually resistant to chemotherapy, as experimentally demonstrated . Sabath et al16 have shown that schistosomula are far less susceptible than adult worms to six schistosomicides at curative doses, including praziquantel and oxamniquine. However, these studies were performed in mice with experimental primary infections treated at different points of time after cercarial exposure. Since the effect of chemotherapy is influenced by the immune state of the host3 4 15, the response of the larval stages of S. mansoni to schistosomicides should also be tested during reinfection. The present investigation studied the effects of oxamniquine or praziquantel on different larval stages of S. mansoni in mice with primary infection compared to mice submitted to re-infection.

Oxamniquine and praziquantel are the two drugs used in Brazil for the treatment of schistosomiasis. Large scale treatment programs using either drug have been carried out in several Brazilian endemic areas 10 11 13 . Treatment efficacy is considered satisfactory, but failures have been registered with variable frequency. Such therapeutic failures are often attributed to the development of drug resistance by the worms and to early post treatment re-infection 2 6 7 . However, in areas of high transmission, another possibility may occur. Treated individuals, even though apparently cured, may re-establish the infection in consequence of the existence of migrating larval forms of Schistosoma mansoni, which are usually resistant to chemotherapy, as experimentally demonstrated 16 21 .
Sabath et al 16 have shown that schistosomula are far less susceptible than adult worms to six schistosomicides at curative doses, including praziquantel and oxamniquine. However, these studies were performed in mice with experimental primary infections treated at different points of time after cercarial exposure. Since the effect of chemotherapy is influenced by the immune state of the host 3 4 15 , the response of the larval stages of S. mansoni to schistosomicides should also be tested during reinfection.
The present investigation studied the effects of oxamniquine or praziquantel on different larval stages of S. mansoni in mice with primary infection compared to mice submitted to re-infection.

MATERIAL AND METHODS
Two main experiments were performed. The first experiment involved 185 outbred Swiss mice of both sexes, weighing 18-22g, that were infected transcutaneously with 100 S. mansoni cercariae of the Feira de Santana strain 1 . This strain has been maintained through successive passages in laboratoryraised Bimphalaria glabrata. The infected animals were randomly separated into three groups: Group 1: consisted of 25 mice that were not subjected to drug treatment, and served as untreated controls. Animals were sacrificed 40 days after cercarial exposure, when the worms were recovered from the portalmesenteric venous system by perfusion, according to Duvall and DeWitt 8 .
Group 2: included 80 mice treated with oxamniquine. The drug was suspended in distilled water and administered by gavage in a single dose of 100mg/kg/ bw. at 5, 10, 20 or 30 days following infection, 20 different animals being treated at each time point. Thirty days after treatment (35-60 days after infection), the animals were anesthetized and sacrificed by severing the abdominal aorta. Worms were recovered by perfusion of the portal system. Group 3: also with 80 mice, received praziquantel, administered by gavage in a dose of 400 mg/kg/w, otherwise following the same steps as for the previous Group. Another approach was made with 153 Swiss mice, which were at first submitted to transcutaneous infection with 30 S. mansoni cercariae. Thirty to fortyfive days afterwards, the animals were exposed to reinfection with 50 cercariae. Only mice passing viable eggs in the stools were used. Similarly to the first experiment, re-infected animals were separated into three groups, as follows: a) untreated re-infected controls (n = 13); b) treated with oxamniquine (100mg/kg, n = 64); and c) treated with praziquantel (400mg/kg, n = 64). Chemotherapies were administered by gavage one time only, 10, 20 or 30 days following re-infection, for sub-groups of animals, according to the intervals after re-infection. Twenty days after treatment, re-infected animals were anesthetized and sacrificed for recovery of worms. Untreated controls were sacrificed 30 days after re-infection.
An additional group was also included in this experiment, consisting of an extra non-treated control group with primary infection for comparing the effect of the cumulative infections (n = 12, 30 cercariae).
Drug efficacy was determined through the percentage of worm load reduction, by using the formula by Sabah et al 15 (R*=100 -(t x 100/c), where t = median of worms recovered from the treatment group and c = median of worms recovered from the untreated group, and comparing treated and non-treated animals in their respective groups and sub-groups. Median numbers of worms were compared by means of the Mann Whitney test.
In an attempt to separate the young and old worms from infections made at different times, a morphometric analysis was performed. Male and female worms were recovered from mice with one single infection, and from those infected and re-infected, including treated and non-treated controls. The worms were fixed in neutral 10% formalin, stained with carmine chloride, dehydrated in alcohol, cleared in creosote and mounted on glass slides with a 1:1 mixture of Canadian balsam and creosote for microscopic examination. Considering sexual dimorphism, morphometric evaluation was made separately for male and female worms. A Leica Quantimet 500C (Leica Cambridge, U.K.) system, with a Sigma-Scan Measurement semi automatic morphometric device (Jandel Scientific, SF, USA) was used. The variables considered were: corporeal length; number of testicular masses; and the presence of eggs in uterus.

RESULTS
The first experiment revealed that the sensitivity of S. mansoni larvae to both oxamniquine and praziquantel varied according to their developmental stage. Oxamniquine yielded poor results during the first 20 days of infection, the percentage reduction of worm-load was 28-34% in treated as compared to non-treated mice (Table 1). However, 30 days after exposure, the percentage reduction was 86 to 100%, which was statistically significant when compared to pre-treatment. The p value varied from 0.001 to < 0.0001. For the other periods the effect of chemotherapy was the same. Regarding praziquantel, efficacy was 48 to 52% for the animals treated until 10 days after cercarial exposure, dropping to 19% on the 30 th day. For the two first experimental points, praziquantel did not improve the efficacy in further reducing worm load (p = 0.9440). However, between the 10 th to 30 th days, reduction in worm load was significant. Compared to oxamniquine, the efficacy of praziquantel did not essentially differ on the 10 th day period (p = 0.07), although the latter seemed more effective on the 20 th day of infection (p <0.0001), while better effects occurred with the former by the 30 th day, p <0.0001. (Table 1).
During the second experiment the percentage reduction of worm load was greater for both treatment groups when compared to the re-infected controls. (Figures 1b, c, d, e, f and g; and Table 2). Oxamniquine was seen to be very effective by the 10 th day following re-infection, presenting a reduction of 64%. These figures decreased subsequently by the 20 th day post infection (p=0.0409), while still varying with time, a few mice were totally cured among those treated later on. Thus, no variation in the chemotherapy action was demonstrated between 20 and 30 days following cercarial exposure (p = 0.1031). In fact, praziquantel  reduction of worm-load for the animals re-infected and treated reached 75%. Praziquantel was more effective than oxamniquine in reducing worm load during re-infection, although not at a statistically significant level. A 76% reduction occurred when treatment was given at the 10 th , 20 th (74%) and 30 th day following re-infection (76%). All animals treated after re-infection showed statistically significant differences in worm-load reduction when compared with the re-infected but untreated control group. The mean male worm length for the control group of single infection was 7,958 x 10 -3 mm, with a confidence interval between 7,644.5 and 8,272.2 x10 -3 . The figures for females were: 10,457.4 x 10 -3 with confidence interval of 9,536.1 to 11,378.7 x 10 -3 . To evaluate worm length from a second infection, the values obtained were distributed in quartiles. In quartile 25 it was noted that the length of male and female worms was 6,300 x 10 -3 and 7,356 x 10 -3 , respectively. It was considered that worms of both sex with body length equal or inferior to these figures represented worms from the second infection. Figure  2 is based on such data.

DISCUSSION
The present study shows that the effect of the schistosomicide drugs, praziquantel and oxamniquine, upon migrating schistosomula in mice differs according to the drug used and the time of infection. Furthermore, several authors have reported that the larval stages of S. mansoni are less susceptible to chemotherapy than young and adult worms 3 16 21 . These differences were now seen to be accentuated when the treatment was made after several different periods following re-infection.
It has been demonstrated that the drug-induced killing of adult worms is mediated by the host immune system 3 4 15 . The schistosomicides administered cause focal areas of damage to the worm tegument 12 18 , exposing target antigens to host antibodies, which ultimately provoke the worm death 3 9 17 . Unfortunately, the role played by the immune system cannot be enhanced by means of artificial antigenic stimulation 19 .
There are few and contradictory experimental data concerning the participation of the immune response during the drug killing of immature schistosomes. Studies made with immunofluorescence labeling after recovering of lung-stage larvae, soon after treatment with praziquantel, revealed mild tegumental lesions with exposure of parasite antigens 9 . However, transfer of the immune serum from 6 to 7-week-old infections to intact mice carrying 25-day-old schistosome infections did not enhance praziquantel chemotherapy of these infections 3 .
Some reduction in worm load in re-infected animals is to be expected since infected mice are not only immunologically protected from a new cercarial Some studies have demonstrated that the migrating schistosomula of a re-infection can be diverted into portal collateral vessels toward peripheral tissues, where they may die as a result of being out of their natural habitat 14 20 . However, although these factors are important, they alone do not explain the present results. A significant worm load reduction occurred after treatment in all treated and re-infected groups. In addition, there were also marked differences in relation to the period after re-infection when the treatment was made. In this regard, the 64% reduction in worm load observed with oxamniquine treatment starting on day 10 after re-infection, can be confronted with that of 28% in controls with single infections. This latter result also contrasts with the general observation, derived from the treatment of single infections, which revealed that the more immature the schistosomula, the more resistant they are to schistosomicides. In animals exposed to a second infection, praziquantel exhibited greater homogeneity of positive results, with a percentage of worm reduction in the order of 75%, in comparison to the 32 to 64% obtained with oxamniquine ( Table 1). The number of testicular masses evaluated in the different groups, did not show significant differences in regard to re-infection or treatment, although oxamniquine revealed a greater reduction in egg production.
In spite of the observation that schistosomicide drugs were effective against migrating S. mansoni larvae during re-infection, the effect was always partial.
Therefore, these data support the possibility that a therapeutic failure may be due to the presence of migrating forms of S. mansoni, which escaped being killed by drugs within the organism of a treated individual.