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Dipetalogaster maximus (Hemiptera, Triatominae) for xenodiagnosis of patients with serologically detectable Trypanosoma cruzi infection
524 TRANSACTIONS OFTHEROYALSOCIETYOFTROPICALMEDICINEANDHI’GIENE,VOL.~~,NO.~,~~~~
Dipetalogaster
(Hemiptera, Triatominae) for xenodiagnosis of cruzi infection serologically detectable Trypanosoma
maximus
patients with
C. C. CUBA*, N. J. ALVARENGA**,A. C. BARRETO*,P. D. MARSDEN*,V. MACEDO* AND M. P. GAMA*** * Faculty of Health Sciences, University of Brasilia, ** Institute of Biology, University of Brasilia ** * Institute of Exact Sciences, University of Brasilia
Summary In patients serologically positive for Trypanosoma cruzi infection the three bug species/instar combinations used in xenodiagnosis showed third-instar Dipetalogaster maximus to be more efficient in detecting circulating trypanosomes than the first instar of the same species which, in turn, is more sensitive than third-instar Triatoma infestans. The sensitivity of the pool technique of faecal examination compared with individual dissection was investigated. Four pool examinations (the product of 20 bugs) were equivalent to 10 individual bug dissections. Because of the ease of providing large numbers of bugs for mass xenodiagnosis, firstinstar D. maximus has replaced third-instar T. infestans in our routine work. The value of thirdinstar D. maximus as a xenodiagnostic agent needs further investigation. Introduction The necessity for new investigations to establish a standard technique for xenodiagnosis as the method for detecting subpatent parasitaemia in patients chronically infected with Trypanosoma cruzi has recently received attention in the literature (CERISOLA et al., 1974; MILES et al., 1975; SALGADO, 1969; MINTER et al., 1977, 1978). Preliminary studies using Dipetalogaster maximus for xenodiagnosis of Brazilian patients known to have been positive to xenodiagnosis previously, have established that this species is more successful in detecting subpatent parasitaemia than is Triatoma infestans (BARRETO et al., 1978; CUBA et al., 1978). In this study we used these two species of bug in simultaneous xenodiagnosis and report the results of the following specific investigations : (i) to confirm that the degree of susceptibility of the bugs used in the test was in the decreasing sequence: D. maximus third-instar > D. maximus first-instar > T. infestans third-instar. Further, to verify that these relative susceptibilities occurred also in patients showing only positive serology, not a previously positive xenodiagnosis. (ii) to compare the “pool” technique of examining bugs with that of individual dissection. In our routine laboratory such a “pool” technique is essential because of the factor of technician time. It has been used in other xenodiagnostic laboratories (CERISOLA et al., 1974). Material and Methods The results of xenodiagnosis of about 200 patients, carried out as part of an epidemiological study in the Municipality of Mambai, GoiBs, Brazil, are
Brazil
analysed in this paper. All these patients had three serological tests positive for T. cruzi, namely the indirect fluorescent antibody test (IFAT), the indirect haemagglutination test (IHAT) and the complement fixation test (CFT). These tests were carried out on paired sera at two reference laboratories (Drs. M. Camargo, Sgo Paulo, and J. Cerisola, Buenos Aires, Argentina). The number of patients for whom various species/instar combinations were available for test differs. but the smallest groun (i.e. that in which all combinations were usei si&&aneously) was 159 (Table IV). Specimens of D. maximus and T. infestans were bred in the insectaries of the Tropical Medicine Unit of the University of Brasilia. Unfed bugs of the appropriate species and instars were used for xenodiagnosis in the following manner: for each patient, 10 first-instar and 10 third-instar D. maximus and 40 third-instar T. infestans were applied at the same time. The 60 triatomines were applied in six boxes, each holding 10 bugs, to the forearms and legs of the patients and left for 30 min. After the test the boxes were kept at 26°C for 30 to 40 days. Boxes of bugs were examined as follows: one box of third-instar T. infestans and the two boxes containing the first and third-instar D. maximus; the bugs were examined individually. Initially, this was done by searching for Trypanosoma cruzi in an expressed faecal drop and if this proved negative the contents of the terminal intestinal tract were examined by dissection. In the remaining three boxes, containing 30 third-instar Triatoma infestans, the dissected contents of the intestinal tract were harvested in the form of two pools of faeces, each from half of the live bugs in the box. Dead bugs were not examined in either procedure and mortalities were not noted, thus reproducing the routine method of examination in our laboratory. The results obtained were analysed statistically using the Student t test and x’) estimations, employing a level of significance of 5%. Results Tables I and II show results obtained from the individual examination of bugs in terms of the This study was supported by funds of the Conselho National de Desenvolvimento Cientifico e Tecnolbgico (CNPq) SIP,O8-067 Brazil.
525
C. C. CUBA et d.
Table I-Results of xenodiagnosis in 190 patients D. maximus (all individually dissected)
using 10 third-instar
T. injestans and 10 first-instar
10 x T. injestans III No. of bugs used per patient, with species and instar
Patients
Patients
+ ve
Total (7:)
- ve
Patients
-t-ve
14
28
42 (22.1)
Patients
- ve
22
126
148 (77.8)
36 (18.9)
154 (81.0)
190 (lOO*O)
10 : D. maximus I Total ( ‘i(,) Note: x: 7 80.20
Table instar
P < 0.05
II-Results of xenodiagnosis in 175 patients D. maximus (individually dissected)
using
10 third-instar
T. injestans and 10 third-
10 ,/ T. injestans III No. of bugs used per patient, with species and instar
Patients
+ ve
Patients
- ve
Total (‘;b)
Patients
ve
31
53
84 (48.0)
Patients
- ve
2
89
91 (52.0)
10 ’ D. maximus III 33 (18.8)
Total ( ‘:;,) Note: -y;? =-: 34.39
Table instar
175 (100.0)
142 (81.1)
J? < 0.05
III-Results showing number combinations (166 patients)
of individual
bugs infected
in xenodiagnosis
with three species/
Results Species and instar of bug
Number examined
No. Positive
D. maximus III
1413
274
19.3
D. maximus I
1255
58
4.6*
T. injestans III
1458
46
3.1”
0i”’
In this table T. injestans examined by the pool method are not recorded. (*) Difference statistically significant, P < 0.05 Table
IV-Comparative
results
of xenodiagnosis
in 159 patients,
Pool procedures Bug species and instar”
Individual dissection : (‘,) positive
T. injestans III
19.5**
D. maximus I
23.89t
D. maximus III
49.05
(*) Batches of 10 insects (**) Difference not statistically (t) Difference not statistically
according
to technique
using 30 third-instar
used
T. injestans:
‘;,, Positive ‘Jo positive ‘:; Positive in 4 pools in 2 pools in 6 pools (Product of 10 bugs) (Product of 20 bugs) (Product of 30 bugs)
significant, P < 0.05 significant, P < 0.05
12.58
21*38**
27.67
526 Table V-The infected with
D. maximus
positive
FOR XENODIAGNOSIS OF T. cruzi INFECTION
rate in xenodiagnosis
for each species and instar
of bugs used in 90 patients
T. cruzi
Results Manner of examinations
No. of triatomines used, with species and instar 10 D. maximus
III
Individually
30 T. injestans
III
01 PosiZve
No. of patients i- ve 80
88.8
Pooled faeces
43
47.7
10 D. maximus I
Individually
dissected
36
40*
10 T. injestans
III
Individually
dissected
32
35+5*
(*) Difference
statistically
significant,
dissected
I? < 0.05
number of patients with positive xenodiagnosis when tested with the three species/instar combinations. When 10 first-instar D. maximus were used in the test, significantly more positive isolates of Trypanosoma cruzi were made from the same patients than when 10 third-instar Triatoma injestans were employed (Table I). When 10 third-instar D. maximus were used, a significantly higher proportion of however, isolates was made than with either first-instar D. maximus or third-instar T. injestans (Tables I and II). Table III shows the number of individual bugs infected during the test on 166 patients. Once again the ranking is third-instar D. maximus > firstinstar D. maximus > third-instar T. injestans. Table IV compares the pool method with individual dissection of the three species/instar combinations in 159 patients. Six sets of pooled results were available from the 30 third-instar T. injestans. These are expressed cumulatively in pairs in the table. Thus, the first pool pair had a result inferior to the individual dissection technique. Using the same species and instar the pool products of 20 bugs (four pools) equals individual dissection of 10 bugs. The result of six pools significantly surpasses individual dissection. In Table V the results of the four species/instar combinations examined for xenodiagnosis in this investigation are expressed in order of sensitivity according to the number of positive diagnoses they achieved in the 90 patients shown to have a positive xenodiagnosis. Discussion That D. maximus, a species found only in Baja California, Peninsula of Mexico, should exhibit such a sensitivity to infection with Brazilian strains of Trypanosoma cruzi (BARRETO et al., 1978) is interesting in relation to the usual finding that local vectors are the best agents to use in xenodiagnosis (ZELEDON, 1974). However D. maximus sucks more blood than any other triatomine. Mean figures published elsewhere (CUBA et al., 1978) are: firstinstar D. maximus-93.8 mg; third-instar D. Triatoma maximus-469.4 mg; and third-instar infestans--53 * 1 mg.
DIAS (1940) was the first to compare the susceptibility of different species of triatomines to infection with Trypanosoma cruzi in dogs. He showed that the two important Brazilian vectors P. megistus and Triatoma injestans were more susceptible to infection cruzi tha with a Brazilian strain of Trypanosoma the Venezuelan species Rhodnius prolixus. A Venezuelan strain of T. cruzi infected a larger proportion of R. prolixus than of the Brazilian triatomines. However, these studies involved small numbers of bugs and were poorly controlled. More recently, RYCKMAN (1965) in California and LITTLE et al. (1966) in Mexico fed various species of triatomines from different geographical regions on animals infected with local strains of T. cruzi and conclude that the local species of triatomine was more susceptible to the native strain of T. cruzi than were exotic species. It appears that a complex of factors, particularly genetic, are responsible for the relationship between parasite and triatomine (MILES
et al., 1975; MAUDLIN,
1976; MINTER
et al.,
1978). The results presented here confirm our previous observations (CUBA et aZ., 1978). There is a strong correlation between the species and instar used in the test and the number of positive results found, both in terms of number of patients positive and number of bugs infected in the procedure. This has been shown with other species/instar combinations (PIFANO et al., et al., 1978).
1973;
MILES
et al.,
1975;
MINTER
When the same test conditions are observed, first-instar D. maximus are equal in value to thirdinstar Triatoma injestans as a xenodiagnostic agent. Because of the ease of producing large numbers of bugs from a battery of gravid females, first-instar D. maximus have replaced third-instar T. injestans in our laboratory for mass xenodiagnosis. Such bugs should be used at least seven days after hatching to obtain maximum feeding (Barreto et al., in preparation). The comparison of the pool technique with that of individual examination of third-instar T. injestans shows that the former is less sensitive if the same number of bugs are used but by using a large number of bugs a result similar or superior to individual dissection can be obtained (Table IV). The amount of technician time necessary to
c. c. CUBA
examine six pools (the product of 30 bugs) is equivalent to that used to examine 10 bugs individually. CERISOLA et al. (1974) have shown an increase in sensitivity when the number of bugs examined by the pool technique is increased. Table V demonstrates that the most effective agent in these investigations was third-instar D. maximus and the value of this species and instar will be explored in further studies. The evidence so far suggests that for maximal results, such as those necessary for evaluating specific chemotherapy, the use of this species and instar might be advantageous. References Barreto, A. C., Marsden, P. D., Cuba, C. C. & Alvarenga, N. J. (1978). Estudo preliminar sobre maximus (Uhler, o emprego de Dipetalogaster 1894) (Triatominae) na tecnica do xenodiagnostico em forma cr6nica de Doenca de Chaaas. Revista do Instituto de Medic&a Paula, 20, 183-189.
Tropical
de Stio
Cerisola, J. A., Rohwedder, R., Segura, E. L., Del Prado, C. E., Alvarez, M. & De Martini, C. J. W. (1974). El Xenodiagndstico. Buenos Aires, Argentina: Ministerio de Bienestar Social, 128 pp. Cuba, C. C., Alvarenga, N. J., Barreto, A. C., Marsden, I’. D. & Chiarini, C. (1978). Nuevos estudios comparatives entre Dipetalogaster maximus y Triatoma infestans en xenodiagnostico de la infection chagasica cronica humana. Revista do Instituto de Medicina Paulo, 20, 145-151.
Tropical
de Sao
cruzi”.
Bras&Medico,
54, 859-
861. Little, J. W., Tay, J. & Biagi, F. (1966). A study on the susceptibility of triatomid bugs to some Mexican strains of Trypanosoma cruzi. Journal of Medical
Entomolonv,
3, 252-255.
Maudlin,
I. (1976).%heritance of susceptibility to T. cruzi infection in Rhodnius prolixus. hlature. 262, 214-215. Miles, M. A., Patterson, J. W., Marsden, P. D. & Minter, D. M. (1975). A comparison of Rhodnius
527
prolixus, Triatoma infestans and Panstrongylus megistus in the xenodiagnosis of a chronic Trypanosoma (Schizotrypanum) cruzi infection in a Rhesus monkey (Macaca mulata). Transactions of the Royal Society of Tropical Medicine and Hygiene, 69, 377-382.
Minter, D. M., Minter-Goedbloed, E. & Ferro Vela, C. (1977). Quantitative studies with firstinstar triatomines in the xenodiagnosis of Trypanosoma (Schizotrypanum) cruzi in experi-_ - -. mentally and naturally infected hosts. Transactions of the Roval Societv of Tropical Hygiene, 71, 330-541. d d *
Medicine
and
Minter, D. M., Minter-Goedbloed, E. & Marshall, T. F. de C. (1978). Comparative xenodiagnosis with three triatomine species of different hosts with natural and experimental chronic infections cruzi. Transwith Trypanosoma (Schizotrypanum) actions of the Royal Societv of Tropical and Hygiene, 72; 84-91. - . -
Medicine
Pifano, F., Morrell, J. R. & De Ortiz, M. D. (1973). Estudio comparative entre el Rhodnius prolixus Stal 1859 y el Triatoma pallidipennis (Stal. 1872) (Pinto, 1927) en la prueba xenodiagnostica real&da en -cases cronicos de enfermedad de Venezolanos de Medicina Chaeas. Archives Tropical
y Parasitologia
Medica,
5, 85-94.
Ryckman,
R. E. (1965). Epizootiology of Trypanosoma cruzi in south western North America. V: Host-parasite specificity between Trypanosoma cruzi and Triatominae (Kinetoplastida : Trypanosomidae) (Hemiptera: Triatominae).
Journal
Dias, E. (1940). Xenodiagnosticos seriados em caes venezuelanas de infectados corn amostras “Schizotrypanum
et d.
of Medical
Entomology,
2, 96-99.
Salgado, A. A. (1969). Consideraciones sobre metodologia y sensibilidad de1 xenodiagnostico. Boletin Chileno de Parasitologia, 24, 9-13. Zeledon, R. (1974). Epidemiology, modes of transmission and reservoir hosts of Chagas’ disease. In : Trypanosomiasis and Leishmaniasis with special reference to Chagas’ Disease. Ciba Foundation Symposium, No. 20 (new series), Amsterdam: Elsevier, pp. 51-77.
1
Accepted
for publication
19th December,
1978.
Dipetalogaster
(Hemiptera, Triatominae) for xenodiagnosis of cruzi infection serologically detectable Trypanosoma
maximus
patients with
C. C. CUBA*, N. J. ALVARENGA**,A. C. BARRETO*,P. D. MARSDEN*,V. MACEDO* AND M. P. GAMA*** * Faculty of Health Sciences, University of Brasilia, ** Institute of Biology, University of Brasilia ** * Institute of Exact Sciences, University of Brasilia
Summary In patients serologically positive for Trypanosoma cruzi infection the three bug species/instar combinations used in xenodiagnosis showed third-instar Dipetalogaster maximus to be more efficient in detecting circulating trypanosomes than the first instar of the same species which, in turn, is more sensitive than third-instar Triatoma infestans. The sensitivity of the pool technique of faecal examination compared with individual dissection was investigated. Four pool examinations (the product of 20 bugs) were equivalent to 10 individual bug dissections. Because of the ease of providing large numbers of bugs for mass xenodiagnosis, firstinstar D. maximus has replaced third-instar T. infestans in our routine work. The value of thirdinstar D. maximus as a xenodiagnostic agent needs further investigation. Introduction The necessity for new investigations to establish a standard technique for xenodiagnosis as the method for detecting subpatent parasitaemia in patients chronically infected with Trypanosoma cruzi has recently received attention in the literature (CERISOLA et al., 1974; MILES et al., 1975; SALGADO, 1969; MINTER et al., 1977, 1978). Preliminary studies using Dipetalogaster maximus for xenodiagnosis of Brazilian patients known to have been positive to xenodiagnosis previously, have established that this species is more successful in detecting subpatent parasitaemia than is Triatoma infestans (BARRETO et al., 1978; CUBA et al., 1978). In this study we used these two species of bug in simultaneous xenodiagnosis and report the results of the following specific investigations : (i) to confirm that the degree of susceptibility of the bugs used in the test was in the decreasing sequence: D. maximus third-instar > D. maximus first-instar > T. infestans third-instar. Further, to verify that these relative susceptibilities occurred also in patients showing only positive serology, not a previously positive xenodiagnosis. (ii) to compare the “pool” technique of examining bugs with that of individual dissection. In our routine laboratory such a “pool” technique is essential because of the factor of technician time. It has been used in other xenodiagnostic laboratories (CERISOLA et al., 1974). Material and Methods The results of xenodiagnosis of about 200 patients, carried out as part of an epidemiological study in the Municipality of Mambai, GoiBs, Brazil, are
Brazil
analysed in this paper. All these patients had three serological tests positive for T. cruzi, namely the indirect fluorescent antibody test (IFAT), the indirect haemagglutination test (IHAT) and the complement fixation test (CFT). These tests were carried out on paired sera at two reference laboratories (Drs. M. Camargo, Sgo Paulo, and J. Cerisola, Buenos Aires, Argentina). The number of patients for whom various species/instar combinations were available for test differs. but the smallest groun (i.e. that in which all combinations were usei si&&aneously) was 159 (Table IV). Specimens of D. maximus and T. infestans were bred in the insectaries of the Tropical Medicine Unit of the University of Brasilia. Unfed bugs of the appropriate species and instars were used for xenodiagnosis in the following manner: for each patient, 10 first-instar and 10 third-instar D. maximus and 40 third-instar T. infestans were applied at the same time. The 60 triatomines were applied in six boxes, each holding 10 bugs, to the forearms and legs of the patients and left for 30 min. After the test the boxes were kept at 26°C for 30 to 40 days. Boxes of bugs were examined as follows: one box of third-instar T. infestans and the two boxes containing the first and third-instar D. maximus; the bugs were examined individually. Initially, this was done by searching for Trypanosoma cruzi in an expressed faecal drop and if this proved negative the contents of the terminal intestinal tract were examined by dissection. In the remaining three boxes, containing 30 third-instar Triatoma infestans, the dissected contents of the intestinal tract were harvested in the form of two pools of faeces, each from half of the live bugs in the box. Dead bugs were not examined in either procedure and mortalities were not noted, thus reproducing the routine method of examination in our laboratory. The results obtained were analysed statistically using the Student t test and x’) estimations, employing a level of significance of 5%. Results Tables I and II show results obtained from the individual examination of bugs in terms of the This study was supported by funds of the Conselho National de Desenvolvimento Cientifico e Tecnolbgico (CNPq) SIP,O8-067 Brazil.
525
C. C. CUBA et d.
Table I-Results of xenodiagnosis in 190 patients D. maximus (all individually dissected)
using 10 third-instar
T. injestans and 10 first-instar
10 x T. injestans III No. of bugs used per patient, with species and instar
Patients
Patients
+ ve
Total (7:)
- ve
Patients
-t-ve
14
28
42 (22.1)
Patients
- ve
22
126
148 (77.8)
36 (18.9)
154 (81.0)
190 (lOO*O)
10 : D. maximus I Total ( ‘i(,) Note: x: 7 80.20
Table instar
P < 0.05
II-Results of xenodiagnosis in 175 patients D. maximus (individually dissected)
using
10 third-instar
T. injestans and 10 third-
10 ,/ T. injestans III No. of bugs used per patient, with species and instar
Patients
+ ve
Patients
- ve
Total (‘;b)
Patients
ve
31
53
84 (48.0)
Patients
- ve
2
89
91 (52.0)
10 ’ D. maximus III 33 (18.8)
Total ( ‘:;,) Note: -y;? =-: 34.39
Table instar
175 (100.0)
142 (81.1)
J? < 0.05
III-Results showing number combinations (166 patients)
of individual
bugs infected
in xenodiagnosis
with three species/
Results Species and instar of bug
Number examined
No. Positive
D. maximus III
1413
274
19.3
D. maximus I
1255
58
4.6*
T. injestans III
1458
46
3.1”
0i”’
In this table T. injestans examined by the pool method are not recorded. (*) Difference statistically significant, P < 0.05 Table
IV-Comparative
results
of xenodiagnosis
in 159 patients,
Pool procedures Bug species and instar”
Individual dissection : (‘,) positive
T. injestans III
19.5**
D. maximus I
23.89t
D. maximus III
49.05
(*) Batches of 10 insects (**) Difference not statistically (t) Difference not statistically
according
to technique
using 30 third-instar
used
T. injestans:
‘;,, Positive ‘Jo positive ‘:; Positive in 4 pools in 2 pools in 6 pools (Product of 10 bugs) (Product of 20 bugs) (Product of 30 bugs)
significant, P < 0.05 significant, P < 0.05
12.58
21*38**
27.67
526 Table V-The infected with
D. maximus
positive
FOR XENODIAGNOSIS OF T. cruzi INFECTION
rate in xenodiagnosis
for each species and instar
of bugs used in 90 patients
T. cruzi
Results Manner of examinations
No. of triatomines used, with species and instar 10 D. maximus
III
Individually
30 T. injestans
III
01 PosiZve
No. of patients i- ve 80
88.8
Pooled faeces
43
47.7
10 D. maximus I
Individually
dissected
36
40*
10 T. injestans
III
Individually
dissected
32
35+5*
(*) Difference
statistically
significant,
dissected
I? < 0.05
number of patients with positive xenodiagnosis when tested with the three species/instar combinations. When 10 first-instar D. maximus were used in the test, significantly more positive isolates of Trypanosoma cruzi were made from the same patients than when 10 third-instar Triatoma injestans were employed (Table I). When 10 third-instar D. maximus were used, a significantly higher proportion of however, isolates was made than with either first-instar D. maximus or third-instar T. injestans (Tables I and II). Table III shows the number of individual bugs infected during the test on 166 patients. Once again the ranking is third-instar D. maximus > firstinstar D. maximus > third-instar T. injestans. Table IV compares the pool method with individual dissection of the three species/instar combinations in 159 patients. Six sets of pooled results were available from the 30 third-instar T. injestans. These are expressed cumulatively in pairs in the table. Thus, the first pool pair had a result inferior to the individual dissection technique. Using the same species and instar the pool products of 20 bugs (four pools) equals individual dissection of 10 bugs. The result of six pools significantly surpasses individual dissection. In Table V the results of the four species/instar combinations examined for xenodiagnosis in this investigation are expressed in order of sensitivity according to the number of positive diagnoses they achieved in the 90 patients shown to have a positive xenodiagnosis. Discussion That D. maximus, a species found only in Baja California, Peninsula of Mexico, should exhibit such a sensitivity to infection with Brazilian strains of Trypanosoma cruzi (BARRETO et al., 1978) is interesting in relation to the usual finding that local vectors are the best agents to use in xenodiagnosis (ZELEDON, 1974). However D. maximus sucks more blood than any other triatomine. Mean figures published elsewhere (CUBA et al., 1978) are: firstinstar D. maximus-93.8 mg; third-instar D. Triatoma maximus-469.4 mg; and third-instar infestans--53 * 1 mg.
DIAS (1940) was the first to compare the susceptibility of different species of triatomines to infection with Trypanosoma cruzi in dogs. He showed that the two important Brazilian vectors P. megistus and Triatoma injestans were more susceptible to infection cruzi tha with a Brazilian strain of Trypanosoma the Venezuelan species Rhodnius prolixus. A Venezuelan strain of T. cruzi infected a larger proportion of R. prolixus than of the Brazilian triatomines. However, these studies involved small numbers of bugs and were poorly controlled. More recently, RYCKMAN (1965) in California and LITTLE et al. (1966) in Mexico fed various species of triatomines from different geographical regions on animals infected with local strains of T. cruzi and conclude that the local species of triatomine was more susceptible to the native strain of T. cruzi than were exotic species. It appears that a complex of factors, particularly genetic, are responsible for the relationship between parasite and triatomine (MILES
et al., 1975; MAUDLIN,
1976; MINTER
et al.,
1978). The results presented here confirm our previous observations (CUBA et aZ., 1978). There is a strong correlation between the species and instar used in the test and the number of positive results found, both in terms of number of patients positive and number of bugs infected in the procedure. This has been shown with other species/instar combinations (PIFANO et al., et al., 1978).
1973;
MILES
et al.,
1975;
MINTER
When the same test conditions are observed, first-instar D. maximus are equal in value to thirdinstar Triatoma injestans as a xenodiagnostic agent. Because of the ease of producing large numbers of bugs from a battery of gravid females, first-instar D. maximus have replaced third-instar T. injestans in our laboratory for mass xenodiagnosis. Such bugs should be used at least seven days after hatching to obtain maximum feeding (Barreto et al., in preparation). The comparison of the pool technique with that of individual examination of third-instar T. injestans shows that the former is less sensitive if the same number of bugs are used but by using a large number of bugs a result similar or superior to individual dissection can be obtained (Table IV). The amount of technician time necessary to
c. c. CUBA
examine six pools (the product of 30 bugs) is equivalent to that used to examine 10 bugs individually. CERISOLA et al. (1974) have shown an increase in sensitivity when the number of bugs examined by the pool technique is increased. Table V demonstrates that the most effective agent in these investigations was third-instar D. maximus and the value of this species and instar will be explored in further studies. The evidence so far suggests that for maximal results, such as those necessary for evaluating specific chemotherapy, the use of this species and instar might be advantageous. References Barreto, A. C., Marsden, P. D., Cuba, C. C. & Alvarenga, N. J. (1978). Estudo preliminar sobre maximus (Uhler, o emprego de Dipetalogaster 1894) (Triatominae) na tecnica do xenodiagnostico em forma cr6nica de Doenca de Chaaas. Revista do Instituto de Medic&a Paula, 20, 183-189.
Tropical
de Stio
Cerisola, J. A., Rohwedder, R., Segura, E. L., Del Prado, C. E., Alvarez, M. & De Martini, C. J. W. (1974). El Xenodiagndstico. Buenos Aires, Argentina: Ministerio de Bienestar Social, 128 pp. Cuba, C. C., Alvarenga, N. J., Barreto, A. C., Marsden, I’. D. & Chiarini, C. (1978). Nuevos estudios comparatives entre Dipetalogaster maximus y Triatoma infestans en xenodiagnostico de la infection chagasica cronica humana. Revista do Instituto de Medicina Paulo, 20, 145-151.
Tropical
de Sao
cruzi”.
Bras&Medico,
54, 859-
861. Little, J. W., Tay, J. & Biagi, F. (1966). A study on the susceptibility of triatomid bugs to some Mexican strains of Trypanosoma cruzi. Journal of Medical
Entomolonv,
3, 252-255.
Maudlin,
I. (1976).%heritance of susceptibility to T. cruzi infection in Rhodnius prolixus. hlature. 262, 214-215. Miles, M. A., Patterson, J. W., Marsden, P. D. & Minter, D. M. (1975). A comparison of Rhodnius
527
prolixus, Triatoma infestans and Panstrongylus megistus in the xenodiagnosis of a chronic Trypanosoma (Schizotrypanum) cruzi infection in a Rhesus monkey (Macaca mulata). Transactions of the Royal Society of Tropical Medicine and Hygiene, 69, 377-382.
Minter, D. M., Minter-Goedbloed, E. & Ferro Vela, C. (1977). Quantitative studies with firstinstar triatomines in the xenodiagnosis of Trypanosoma (Schizotrypanum) cruzi in experi-_ - -. mentally and naturally infected hosts. Transactions of the Roval Societv of Tropical Hygiene, 71, 330-541. d d *
Medicine
and
Minter, D. M., Minter-Goedbloed, E. & Marshall, T. F. de C. (1978). Comparative xenodiagnosis with three triatomine species of different hosts with natural and experimental chronic infections cruzi. Transwith Trypanosoma (Schizotrypanum) actions of the Royal Societv of Tropical and Hygiene, 72; 84-91. - . -
Medicine
Pifano, F., Morrell, J. R. & De Ortiz, M. D. (1973). Estudio comparative entre el Rhodnius prolixus Stal 1859 y el Triatoma pallidipennis (Stal. 1872) (Pinto, 1927) en la prueba xenodiagnostica real&da en -cases cronicos de enfermedad de Venezolanos de Medicina Chaeas. Archives Tropical
y Parasitologia
Medica,
5, 85-94.
Ryckman,
R. E. (1965). Epizootiology of Trypanosoma cruzi in south western North America. V: Host-parasite specificity between Trypanosoma cruzi and Triatominae (Kinetoplastida : Trypanosomidae) (Hemiptera: Triatominae).
Journal
Dias, E. (1940). Xenodiagnosticos seriados em caes venezuelanas de infectados corn amostras “Schizotrypanum
et d.
of Medical
Entomology,
2, 96-99.
Salgado, A. A. (1969). Consideraciones sobre metodologia y sensibilidad de1 xenodiagnostico. Boletin Chileno de Parasitologia, 24, 9-13. Zeledon, R. (1974). Epidemiology, modes of transmission and reservoir hosts of Chagas’ disease. In : Trypanosomiasis and Leishmaniasis with special reference to Chagas’ Disease. Ciba Foundation Symposium, No. 20 (new series), Amsterdam: Elsevier, pp. 51-77.
1
Accepted
for publication
19th December,
1978.