Length and caliber of the rectosigmoid colon among patients with Chagas disease and controls from areas at different altitudes

Introduction: In this study, we investigated radiological changes in the sigmoid colon in chagasic patients by comparing their colon lengths and caliber with those of non-chagasic living in the same region and non-chagasic living at high altitudes. Methods: A total of 317 individuals were evaluated using clinical, serological and radiological methods and divided into three groups: 1) one hundred and nine non-chagasic individuals from Uberaba, Brazil; 2) sixty-one non-chagasic from Puno, Peru; 3) one hundred forty-seven chagasics examined in Uberaba, being 62 without megacolon (3A), 72 with megacolon (3B) and 13 with doubtful diagnosis of megacolon (3C). Results: In group 2, the sigmoid colon had a signifi cantly larger caliber (p=0.001) and the rectosigmoid colon was longer (p<0.001) than group 1. In subgroup 3A, the sigmoid colon (p<0.001) and rectum (p<0.001) had a signifi cantly larger caliber and the rectosigmoid was longer (p<0.001) than that of the non-chagasic individuals. In subgroup 3B, the rectosigmoid was longer in all patients, and the caliber of the sigmoid was signifi cantly larger than that of subjects in subgroups 3A and 3C (p<0.001). Conclusions: Morphometric analysis confi rms that Chagas disease may increase the caliber and length of the rectosigmoid. Our results suggest that altitude, ethnicity and diet may have infl uenced the size and length of the rectosigmoid of andean patients.

Human Chagas disease (HCd), which affects between 2 and 3 million Brazilians, is an important societal problem 1 , with 75-90 million Latin Americans at risk of becoming infected 2 .
The association between megacolon and Trypanosoma cruzi was reported by Chagas and Villela 3 .The etiopathogenesis of megacolon in HCd is a lesion caused by T. cruzi in the intramural nerve plexus of the autonomic nervous system in the large intestine.Decreased numbers of neurons (hypoganglionosis) is necessary and suffi cient for development of megacolon 4,5 .Intramural denervation leads to dyskinesia of the affected segment and typically culminates in dilatation and elongation of the terminal colon, which is responsible for deposition.Moreover, secretion, absorption, and motility are altered, leading to chagasic megacolon 5 .
Altitude is thought to infl uence colon caliber and length.A study by Frisancho 6 showed that dolichomegacolon of those living in the Andes mountains has several specifi c clinical, radiological, and anatomopathological characteristics as well as complications that distinguish it from other types of megacolon, particularly chagasic megacolon 7,8 .
In the Andes, megacolon is common among inhabitants of the South American regions located 3,000m above sea level; this condition is thought to be unrelated to HCd.Increased colon caliber and length are attributed to racial factors, cocaine use, and vitamin B defi ciency among patients in Sucre, Bolivia 9 .These morphological changes were also seen in patients in Peruvian hospitals located at altitudes >3,000m above sea level in the Andes 10 .Several possible causes of megacolon in Andean patients have been proposed, including anatomical elements, longer sigmoid colon, and aggravating factors including dietary habits, low atmospheric pressure, and racial factors.
Radiological analysis of colonic segments is difficult because of diversity in measurement methods 11,12 , and no standards exist to describe normal or abnormal colopathy.Therefore, to examine variations, it is necessary to compare normal colons to those of patients with HCd.We conducted a

METHODS
radiological investigation to analyze the rectosigmoid colon and compare differences between controls and patients with HCd.The rectosigmoid colon was chosen for the analysis because it is easily accessible and generally affected by HCd.Therefore, the aim of this study was to examine the radiological alterations within the sigmoid colon in patients with HCd and compare the measurements with those in control individuals in Brazil and control individuals living at high altitudes.
The study was conducted in region with altitude of 762 feet above sea level in Brazil, and at the mountainous region of Peru at an altitude of 3,850 feet above sea level.

Patient selection
The Brazilian individuals were selected from among patients with chronic HCd who were seeking outpatient services.The patients underwent a clinical examination in which all data were collected in a standard form prepared for this study.They were then subjected to a series of complementary examinations such as repeated serology for HCd, blood cultures for T. cruzi, and tests to characterize the clinical form of HCd.
Control individuals (Group 1), were from several clinical departments from Clinics Hospital, and did not have complaints related to HCd.Subjects from high-altitude regions (Group 2) were selected from those living within Puno, Peru.These individuals were chosen from employees and patients at the Puno's Hospital; their family members were also selected.Individuals in both groups with diarrhea, constipation for >3 days, or evidence of infl ammatory or tumor-like intestinal lesions and those who were <20 or >60 years of age were excluded from the study.
A clinical evaluation was previously conducted in patients and controls to rule out secondary causes of megacolon dysfunction, such as congenital or toxic megacolon, intestinal parasitic disease, cancer, or metabolic causes (diabetes mellitus and hypothyroidism).

Serological exams for human Chagas disease
Blood samples were collected from all patients, and serological reactions such as indirect hemagglutination (IHA), indirect immunofl uorescence reaction (IFR), and enzyme-linked immunosorbent assay (ELISA) were conducted to diagnose HCd.Only samples that were positive for at least 2 of the 3 performed tests were considered seropositive.
Blood samples of the patients from Puno were acquired via fi nger prick, collected on Whatman ® qualitative fi lter paper, Grade 1 (Sigma-Aldrich, USA), and tested using the same serological tests.

Radiological exams
Colopathy was diagnosed by measuring the caliber and length of the rectosigmoid colon.
Thoracic teleradiography was used to evaluate the heart.Conventional radiography of the abdomen with the patient in the supine position was used to evaluate the distribution of intestinal gas before administration of opaque enema.These tests were performed using medium-sized devices at 500mA and 125kVp without an image intensifi er.
In Group 1, the colon test was preceded by intestinal preparation with administration of a laxative the day before and an aqueous enema 4h before the test.In all other groups, contrast colonoscopy (opaque enema) was conducted without intestinal preparation, according to the simplifi ed technique reported by Ximenes et al. 13 .In all patients, 300mL of barium sulfate was diluted in 900mL of water for a total volume of 1,200mL.Radiography was performed for each patient in the supine, prone, and side positions for a total of 3 radiographs each.The technique reported by Ximenes et al. 13 was also used in Puno.
To diagnose megacolon, we determined whether the colon was enlarged by dilatation or elongation.We identifi ed different sigmoid parameters and classifi ed them as I, II, III, or undetermined based on the Farrar guidelines to determine the morphology of the sigmoid colon in control individuals in Uberaba 11 .
To measure the length of the rectosigmoid colon, a curvimeter was used and passed through the central axis of the colon using a backward motion from the anus up to the left iliac crest, which was assumed to be the beginning of the sigmoid colon.The caliber and length of the sigmoid colon and rectum were measured using a ruler based on the results of the side radiograph of the rectum.These measurements were always conducted at the level of the third sacral vertebra or where the sigmoid colon had a larger diameter.The caliber of the rectum was measured below the S3 level to the sigmoid colon.All measurements were conducted twice at different time points by the same investigator so that reproducibility of this method could be ensured.

Statistical analysis
Quantitative variables are expressed as medians.Those measurements were compared among groups (Groups 1, 2, 3A, 3B, and 3C) using the Kruskal-Wallis, non-parametric analysis of variance (ANOVA) test, and Dunn's multiple comparison test or the Mann-Whitney U test wherever appropriate.The possible effects of age or height on the measured rectosigmoid parameters were evaluated using Spearman's correlation, while the effect of gender in each group was evaluated using the Mann-Whitney U test.To compare categorical variables among groups, the χ 2 test with Yates's correction was used as needed or Fisher's exact test was employed.Probabilities < 5% (p < 0.05) were considered statistically signifi cant.

Ethical considerations
This study was approved by the Institucional ethics committee of our Federal University.The study participants were informed of the tests to be performed and provided written consent.
A total of 317 patients with HCd and controls were divided into 3 groups and examined from 2000 to 2005.Analysis of subject age showed that individuals with Chagas tended to be older than controls (p < 0.005).There was no age difference between the HCd subgroups.
In control individuals (Group 1), the caliber of the sigmoid colon was 2-6cm with a median of 3.1cm.There was a signifi cant gender-related difference (p = 0.029) in caliber (median, 3.3cm in males vs. 3cm in females).The caliber of the rectum was 2.5-8.5 cm with a median of 4.5cm.Individual gender, age, and height did not affect caliber.The length of the rectosigmoid colon was 32-75cm.The median length was 57cm and was signifi cantly infl uenced by patient gender, age, and height (Table 1).
Based on the tests performed on 109 control individuals (Group 1) and using a 99% percentile for comparison with other groups, we considered the following as the upper normal limits: sigmoid caliber, 4.5cm; rectum caliber, 7.4cm; and rectosigmoid length, 74cm.
In control individuals (Group 2), the caliber of the sigmoid colon was 3.2-8.0cmwith a median of 5cm.The caliber of the rectum was 2.5-8cm with a median of 4cm.In 15 (24.6%) individuals, the rectum had a higher caliber than that of the sigmoid colon.The length of the rectosigmoid colon was 45-84cm with a median of 65cm (Table 1).None of these measurements were infl uenced by gender, age, or height.
We observed that the caliber of the sigmoid colon was signifi cantly greater in subjects from Puno than in those from Uberaba (5.0cm and 3.1cm, respectively; p=0.001).Moreover, their rectosigmoid colons were larger (65cm in Puno vs. 57cm in Uberaba; p<0.001) (Table 1 and Figure 1).
The measurements of only 3 (2.7%)patients in this group exceeded the limits established for Group 1, whereas 41 (66.1%) individuals in Group 2 had measurements that exceeded the normal limits (Table 1).
Calibers of the sigmoid colon (3-4cm) and rectum (5-45cm) and the length of the sigmoid colon (65cm) of the patients with HCd but without megacolon (subgroup 3A) were signifi cantly higher than those of the control individuals from Group 1, who had measurements of 3.1, 4.5, and 57cm, respectively (Table 1 and Figure 2).
All 72 patients diagnosed with megacolon (subgroup 3B) had increased rectosigmoid colon length.The calibers of the rectums were measured in 54 patients with megacolon and were 3.0-13.5cm(median, 6.1cm).The calibers of the rectums were 3.5-10cm (median, 6.3cm).No patients had a caliber <3.5cm, and the caliber did not exceed 7.4cm in 39 (72.2%) patients (Table 2).All patients from subgroup 3B showed a large increase in sigmoid colon length, making this measurement impossible.When there were large alterations, the calibers of both the rectum and sigmoid colon increased.
All patients with an unconfi rmed diagnosis of megacolon (subgroup 3C) had a rectosigmoid length that exceeded the normal limit (74cm).None was <80cm, and the median was 88.5cm.We observed that 41.6% of the calibers of the sigmoid colon and 25% of the calibers of the rectum exceeded the maximum normal limits established in this study (Table 2).The calibers of the sigmoid colon and rectum were signifi cantly higher in subgroup 3B than in subgroup 3A (p < 0.001) (Table 2).
When the length of the sigmoid colon increased signifi cantly (subgroup 3B), the calibers of the rectum and sigmoid colon increased and were higher than the normal limits in 11 (20.3%)patients.The lengths of the rectosigmoid colon were higher in subgroup 3B patients (with megacolon) than in subgroup 3C patients.Additionally, the sigmoid calibers were signifi cantly higher in subgroup 3B patients; however, there was no difference in rectum calibers (Table 2).Of the 13 patients in subgroup 3C, only 1 (7.7%) had a simultaneous increase in sigmoid and rectum calibers, while this simultaneous increase was observed in 12 (22.2%)patients in subgroup 3B.In subgroup 3C, there were 5 (41.6%) patients with calibers higher than the normal limits, while this was observed in only 3 (7.4%)patients of subgroup 3B.A caliber increase in either the sigmoid colon or the rectum was observed in 7 (58.3%)patients of subgroup 3C and in 39 patients of subgroup 3B.

DISCUSSION
The chronic phase of HCd includes a period of subpatent parasitemia after the fi rst 60 days of acute infection 14 .The factors infl uencing the clinical variability of the phases of HCd have not been elucidated, but genetic variability of both the host and the parasite may be important 15 .
The most frequently observed alterations of the digestive form of HCd include esophagopathy and colopathy, which cause swallowing diffi culties and constipation, respectively 16,17 .Colopathy manifests as prolonged constipation, which may have serious complications such as fecaloma and volvulus.
This study aimed to contribute to the diagnosis of colopathy, particularly small dilatations, by proposing the standard for measuring rectosigmoid dimensions.Thus, we established normal limits for sigmoid and rectum calibers and rectosigmoid lengths.
Compared to our results, studies have reported different median sigmoid and rectum values.Rezende et al. 18 found that the diameter of a normal sigmoid colon should be <6cm.Hernandez et al. 19 studied approximately 60 individuals and identifi ed a mean sigmoid caliber of 4.38, rectum caliber of 6.2cm, and rectosigmoid length of 51.2cm; all of these values are lower than those found in this study.However, the maximum values in control individuals are similar.This similarity suggests that the differences in limits established by earlier previous studies may be due to the measurement methodology.
Occurrence of megacolon in Puno and other regions within the Andes represents a special situation that requires further study, despite studies that have already been conducted by researchers from Bolivia, Argentina, Chile, and Peru.These studies describe changes in the colon in those living in the Andes and the existence of another type of endemic nonchagasic megacolon.They draw attention to the frequency of volvulus 20,21 and changes in intestinal transit 7 in certain countries of the Andes, which motivated us to include this population in our study.
The unique significant difference in rectosigmoid measurements between patients from Uberaba and those from Puno was that the patients from Puno had rectums with smaller calibers.
The initial hypothesis that dolichomegacolon in the Andes is due to altitude appears to have no basis because there are no reports of megacolon occurrence in other populations living at the same or even higher altitudes such as in the Alps or the Himalayas 10 .However, we unexpectedly observed a higher frequency of intestinal movement in patients from Puno, with a mean interval of 0.73 days in contrast to the interval of 2 days observed in controls from Uberaba.This difference may be related to the smaller caliber of the rectum observed in Puno despite the higher sigmoid caliber.However, other factors such as diet, cocaine use, and race must be considered and investigated further.
Patients with HCd have greater calibers of the sigmoid colon and rectum and grater rectosigmoid lengths than those of control individuals.Sigmoid colon caliber was signifi cantly greater among individuals with HCd and evidence of megacolon than among individuals without megacolon and those with unconfi rmed diagnosis.A similar study by Castro 12 of 291 patients showed similar fi ndings regarding sigmoid measurements; however, the authors observed that the mean diameters of the rectums in patients with HCd did not differ signifi cantly from those of control individuals.
We used morphometry to confi rm the presence of megacolon in both cases classifi ed as unconfi rmed and others previously considered normal.In these cases, calibers of the sigmoid colon and rectum and rectosigmoid colon lengths were signifi cantly higher in control individuals (8%, 3.2%, and 16.1%, respectively), that were higher than normal.In 16 (25.8%)patients, morphometry showed incipient organ enlargement.Such fi ndings show that colopathy occurs more frequently than what can be evaluated using conventional radiology.
In addition to being relevant for diagnosis, the use of morphometry revealed the importance of rectosigmoid elongation in pathophysiology of the megacolon.It showed that even in cases of advanced megacolon, sigmoid and rectum calibers may remain normal, which confi rms the results of earlier studies showing that elongation may precede dilatation 22 .In contrast to the esophagus, elongation of the megacolon occurs earlier and more frequently, the fecal content is retained, and an intestinal transit delay occurs 22 .
Among the 3 radiological measurements obtained, only the rectosigmoid length had increased in all radiographs with a megacolon diagnosis.Even among the 13 patients for whom diagnosis was unconfi rmed after radiography, morphometry enabled detection of alterations, thus allowing the diagnosis of megacolon.The 10 patients with a colon >74cm (subgroup 3A) were considered to have incipient megacolon.However, without using morphometry, it is extremely diffi cult to identify such patients.
The clinical type of megacolon could be determined in 50 of the 62 patients with HCd who were diagnosed as not having megacolon and in whom measurements showed incipient megacolon.Of these, 14 were undetermined, 9 were cardiac, 10 were mixed, and 17 were digestive, with 3 (13%) showing abnormal values of the colon on morphometry.Esophagopathy was observed in 27 subjects, while an increased rectosigmoid caliber or length was seen in 10 (37%).
In summary, our results suggest that factors such as diet (corn, potatoes, and starchy foods), cocaine use, ethnicity, and altitude may have infl uenced rectosigmoid calibers and lengths of Andean patients.Morphometry revealed that HCd may increase rectosigmoid caliber and length.Moreover, measurement results are dependent on the observer.

FIGURE 1 -
FIGURE 1 -Different morphologies of the rectosigmoid colon: increased sigmoid caliber and length (A), increased rectum caliber only (B), increased rectum caliber with normal sigmoid caliber (B) and (D), and elongated sigmoid (C).

FIGURE 2 -
FIGURE 2 -Normal (A) and altered morphometry (B).Normal rectosigmoid colon of a patient from Uberaba (C) and a patient from Puno (D).
Lopes GP et al -Length and caliber of the colon in Chagas disease A total of 317 individuals were examined and divided into 3 groups as follows: Group 1: one hundred nine individuals with negative serological results for HCd from Triângulo Mineiro and Alto Paranaíba, State of Minas Gerais, Brazil (35 men with a median age of 45 years and 74 women with a median age of 42 years); Group 2: sixty-one individuals with negative serological results for HCd from the region of Puno, Peru (40 men with a median age of 37 years and 21 women with a median age of 36 years); and Group 3: one hundred forty-seven patients with HCd from Triângulo Mineiro, Brazil (median age, 58 years; 62 without megacolon [subgroup 3A], 72 with megacolon [subgroup 3B], and 13 with an unconfi rmed diagnosis of megacolon [subgroup 3C]).

TABLE 2 -
Comparisons between chagasics without diagnosis of megacolon (subgroup 3A) and those diagnosed with megacolon (subgroup 3B) and between chagasics with megacolon (subgroup 3B) and those with doubtful diagnosis of megacolon (subgroup 3C).