versão impressa ISSN 0074-0276
Mem. Inst. Oswaldo Cruz v.103 n.1 Rio de Janeiro fev. 2008 Epub 31-Jan-2008
Penélope E PalominosI; Rose GasnierI; Rubens RodriguezII; Aventino A AgostiniII; Carlos Graeff-TeixeiraI, +
ILaboratório de Parasitologia Molecular, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) and Laboratório de Biologia Parasitária, Faculdade de Biociências da PUCRS, Avenida Ipiranga 6690, 2º andar, Sala 20, 90690 900 Porto Alegre, Brasil
IIInstituto de Patologia de Passo Fundo, Universidade de Passo Fundo, Passo Fundo, RS, Brasil
Abdominal angiostrongyliasis (AA) is a zoonotic nematode infection caused by Angiostrongylus costaricensis, with widespread occurrence in the Americas. Although the human infection may be highly prevalent, morbidity is low in Southern Brazil. Confirmed diagnosis is based on finding parasitic structures in pathological examination of biopsies or surgical resections. Serology stands as an important diagnostic tool in the less severe courses of the infection. Our objective is to describe the follow up of humoral reactivity every 2-4 weeks up to one year, in six individuals with confirmed (C) and ten suspected (S) AA. Antibody (IgG) detection was performed by ELISA and resulted in gradually declining curves of reactivity in nine subjects (56%) (4C + 5S), that were consistently negative in only three of them (2C + 1S) after 221, 121 and 298 days. Three individuals (2C + 1S) presented with low persistent reacitivity, other two (1C + 1S) were serologically negative from the beginning, but also presenting a declining tendency. The study shows indications that abdominal angiostrongyliasis is usually not a persistent infection: although serological negativation may take many months, IgG reactivity is usually declining along time and serum samples pairing may add valuable information to the diagnostic workout.
Key words: abdominal angiostrongyliasis - Angiostrongylus costaricensis - eosinophilic gastroenteritis - zoonosis
Abdominal angiostrongyliasis (AA) is a nematode infection caused by Angiostrongylus costaricensis. Wild rodents are the definitive hosts and larval stages develop in terrestrial mollusks (Morera 1971). The parasitosis occurs mainly in the Americas, from Southern USA to Northern Argentina and Southern Brazil (Ubelaker & Hall 1979, Agostini et al. 1984, Demo & Pessat 1986). Imported infections have been diagnosed in USA, Europe and probably in Africa (Baird et al. 1987, Silvera et al. 1989, Vázquez et al. 1993). Accidental human infection may present as acute abdominal disease that usually affects the ileo-cecum transition with an inflammatory reaction mainly produced by secreted antigens and the presence of eggs in small capillaries. The disease may complicate with intestinal obstruction due to extensive eosinophilic infiltration and intestinal perforation, peritonitis and sepsis, secondary to arterial thrombosis and necrosis (Céspedes et al. 1967). Detection of parasite structures in examination of biopsies or surgical specimens allows the definitive diagnosis. In the absence of intra-arterial worms or eggs, histological findings such as severe eosinophilic infiltration, granulomatous reaction and eosinophilic vasculitis lead to a suspected diagnosis (Graeff-Teixeira et al.1991a). Serology is essential for the diagnostic workout in uncomplicated clinical cases, since fecal elimination of parasitic structures have never been reported. Previous evaluations of IgE, IgM, IgA or isotypes of IgG anti-crude adult worm antigens did not result a better diagnostic tool than an IgG-ELISA currently in use (Geiger et al. 2001). Our objective is to report the follow up of IgG anti-A. costaricensis production in patients from the endemic area in Southern Brazil.
PATIENTS, MATERIAL AND METHODS
Patients - A number of pathology laboratories and clinical services in the Brazilian southernmost state, Rio Grande do Sul, are included in a vigilance network looking for suspected or confirmed new cases of AA, according to previously described histopathological criteria (Graeff-Teixeira et al. 1991a). Briefly, the detection of parasite structures (intra-arterial worms or eggs) is the mainstay for a confirmed diagnosis, while the intense eosinophillic inflammatory response in the intestinal wall, the eosinophilic vasculitis (especially in arteries) and granulomatous reaction (including granulomatous arteritis) support the suspicion of A. costaricensis infection (Agostini et al. 1984). From 1999 to 2003, six patients with confirmed abdominal angiostrongyliasis (group C) and ten with suspected diagnosis (group S) had a serological follow-up for 298 ± 156 (media ± standard deviation) and 409 ± 212 days, respectively (Table). The collection of blood samples were scheduled as follows: one initial sample immediately after the diagnosis and samples every two weeks for three months and every month up to one year. Time zero was the date of the surgical procedure. Large variation in the intervals for sample collection occurred because patients and health services did the sampling at their convenience, not necessarily as scheduled by the research protocol. Serum was immediately separated, eventually stored locally at -20ºC and kept in ice for transportation, never longer than two days, before final storage at -80ºC at PUCRS laboratories.
The parasite and ELISA - A. costaricensis, Santa Rosa and Nova Itaberaba strains, are maintained in vivo at the laboratory through passages in Swiss mice or Oligoryzomis nigripes and Biomphalaria glabrata (Esteio strain). Details of antigen preparation and ELISA methodology are described elsewhere (Graeff-Teixeira et al. 1997, Geiger et al. 2001). In brief, the immunoenzymatic assay was performed using crude female worm antigens with sensitivity and specificity of 76% and 91%, respectively. Results of the ELISA are expressed as a ratio: average optical density from duplicates/cut-off value as described by Geiger et al. (2001).
Bioethics - Informed consent was obtained from all adult participants and from parents or legal guardians of minors, with the name of the appropriate institutional review board having approved the project. The investigation protocol was approved by the ethical committee (CEP-PUCRS, 15 december 1999) and performed according to Brazilian regulation (Resolução MS-CNS 196/96).
The individual IgG reactivity is shown graphically in Figs 1, 2, 3 and 4. Nine of them (4 from group C and 5 from group S) presented gradually declining curves, although only three (2 from group C and 1 from group S) had a consistently negative ELISA after 221, 121 and 298 days (214 ± 89) (Table). Two patients (NGP-C and ABN-S) were serologically negative from the beginning, but both showed a tendency to reduce the reactivity, especially ABN-S (Fig. 4). Other two patients (EC-C and EF-S) were persistently positive at very low antibody titers (Fig. 4). Some individuals (MDP-C in Fig. 1; MF-S and IW-S in Fig. 3) presented a low initial reactivity level before a sharp increase at 53 ± 10 days. IB-C (Fig. 1) showed a striking reduction at 157 days and another peak of reactivity at 220 days with gradual decline up to 506 days. In spite of clinical follow-up data not being presented because they were not available all the time from most of the patients, convalesce was usually symptom-free and all of them survived.
Sero-epidemiological studies have demonstrated accidental human infection with A. costaricensis to be very frequent, while morbidity is usually very low (Graeff-Teixeira et al. 2005). Severe cases requiring surgical treatment for perforation and/or intestinal obstruction are rare in Southern Brazil as can be illustrated by the low number of cases with definitive diagnosis in the present study.
As indicated by a preliminary evaluation, antibody response gradually decreases along time, indicating that the worms do not survive long time in humans (Geiger et al. 2005). The inappropriateness of the classic wisdom that the infection courses with high morbidity in a less adapted host, is illustrated by the situation in human AA, where cycle is not completed and adult worms do not survive a long time, but infection only rarely manifests its more extreme "virulence" or "pathogenicity". This situation fits in the proposition of alternative models to describe host-parasite coevolutionary status, like the "titer-time curve" of parasite transmission forms made available for the infection of new hosts, thereby favoring parasite species survival in nature (Garnik 1992).
Because of the difficulties for detection of L1 in stools, serology is a key diagnostic methodology to reveal the presence sub-clinical or asymptomatic courses of AA. Serology may also help the follow-up of patients leading to early detection of complications requiring surgical treatment. One patient (MDP-C, Fig. 1) had a dramatic increase in reactivity coincident with a recurrence of severe intestinal inflammatory lesions leading to a second surgical intervention. IB-C (Fig. 1) also showed a second peak which was not related to new lesions, suggesting reinfection. On the other hand, a decrease in antibody titer is apparently a good prognostic sign.
Several patterns of reactivity may be delineated from these data: (1) a predominant gradually decreasing reactivity (EB-C, Fig. 1 and OB-S, Fig. 2); persistently positive in (2) high (LG-S and MF-S, Fig. 3) and (3) low titers (DS-S, Fig. 3; EC-C and EF-S, Fig. 4); and (4) delayed peak reactivity (MDP-C, Fig. 1; MF-S and IW-S, Fig. 3).
The similarity of reactivity curves in several patterns both from confirmed and suspected abdominal angiostrongyliasis add validation to clinical and histopathological criteria as previously established (Agostini et al. 1984, Graeff-Teixeira et al. 1991a).
The persistence of antibodies for many months, either in high or low titers, was not previously detected, since all patients in the evalution by Geiger et al. (2001) were serologically negative from three months onwards. Although rare, the most intriguing situations are the persistent high titers, like the curve from LG-S (Fig. 3) or LW-S (Fig. 2), suggesting persistence of infection for a longer than expected time.
Part of the problems of sensitivity of immunological methods may be explained by the low responders. Variability and diversity of antibody response to a parasite infection should be taken in consideration when diagnostic methods are under analysis. More extensive observations with clinical, parasitological and serological follow-up are required for better evaluation of prognostic value of serological and other molecular methods in abdominal angiostrongyliasis.
To Ingrid Porto and Juliano Romanzini for technical help. Serum samples collections were made possible through cooperation from the following Institutions and individuals: Ana CA Silva, Antonio C Laitano, Caroline P Andrade, Fabiane Rigotti, Fabiane W Cidade, Maria F Pellicioli, Márcia CF Silva, Pablo B Bubols, Paula B Terraciano, Rafael L Maurer, Renata Ben, Roberta K Barbosa (FABIO/IPB PUCRS); Aventino A Agostini and Rubens Rodriguez (Instituto de Patologia de Passo Fundo); Ana M Gaiger (Lab Pat Reunidos, P.Alegre); Alex Guerini (Hospital Nonoai); Altair Collares (Hospital de Sarandi); Altair Copatti and Glaé Lameira (Hospital Santo Antônio, Três de Maio); Anencir F Silva, Irmã Romana, Rosane Perios and Nídia Perios (Hospital Cândido Godoi); Auristela Barros (SM Saúde, Planalto), Carlos Reichel (FAMED, PUCRS), Carlos Muneroli and Tânia Otto (Hospital Comunitário de Carazinho); Clóvis Bringuenti (Laboratório Vital, Nonoai); Eglê de Almeida (Hospital Cristo Redentor, Marau); Gilberto Mayer (Hospital São Vicente, Passo Fundo); Janice P Zanella (Laboratório Sta Maria, Planalto); João Mesquita (Hospital Cesar Santos, Passo Fundo); José HM Silva (Lab das Clínicas, Passo Fundo), Paulo Sommer and Ursula Bockwinkel (Hosp Porto Xavier); Paulo Valiatti (Lab Fontoura Xavier) and Laboratório Biolabor (Marau).
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Received 19 October 2007
Accepted 11 February 2008
Finantial support: PUCRS (FABIO, IPB and Hospital São Lucas), CNPq (477782/2004-3, 307872/2004-1), FAPERGS