Seasonal profile and level of CD 4 + lymphocytes in the occurrence of cryptosporidiosis and cystoisosporidiosis in HIV / AIDS patients in the Triângulo Mineiro region , Brazil

Patients with AIDS are particularly susceptible to infection with intestinal coccidia. In this study the prevalence of infections with Cryptosporidium sp and Cystoisospora belli were evaluated among HIV/AIDS patients in the Triângulo Mineiro region, Brazil. Between July 1993 and June 2003 faecal samples from 359 patients were collected and stained by a modified Ziehl-Neelsen method, resulting in 19.7% of positivity for coccidian (8.6% with Cryptosporidium sp, 10.3% with Cystoisospora belli and 0.8% with both coccidian). Patients with diarrhoea and T CD4+ lymphocyte levels < 200 cells/mm3 presented higher frequency of these protozoans, demonstrating the opportunistic profile of these infections and its relationship with the immunological status of the individual. It was not possible to determine the influence of HAART, since only 8.5% of the patients positive for coccidian received this therapy regularly. Parasitism by Cryptosporidium sp was more frequent between December and February and thus was characterised by a seasonal pattern of infection, which was not observed with Cystoisospora belli. Key-words: Cryptosporidium sp. Cystoisospora belli. Isospora belli. HIV/AIDS.

A high prevalence of gastrointestinal infections has been observed in AIDS patients since the first cases were described, especially those caused by opportunistic parasites.The rapid dissemination of human immunodefiency virus (HIV) has increased considerably the incidence of these entomopathogens, especially Cryptosporidium sp and Cystoisospora belli, which are responsible for potentially severe diarrhoeic profiles with morbidity directly related to the degree of immunodepression 20 24 .The clinical profile may be presented as a self-limiting infection, acute or chronic diarrhoeic syndromes, with debilitating diarrhoea frequently accompanied by weight loss, dehydration, abdominal pain and poor absorption syndrome 1 7 11 .The chronic nature of these infections contributes to the increase in morbidity and mortality in these patients.Cryptosporidiosis may be associated with gastrointestinal syndrome and respiratory infections and recurrences are common.Infection rates vary from 0.6 -85% for Cryptosporidium 13 and 0.2 -20% for Cystoisospora belli 18 .
In Brazil, the prevalence of cryptosporidiosis in HIV/AIDS patients varies between 6.4-9.1% while for cystoisosporidiosis the rate is 4.4-18.0% 1 2 8 19 21 .Cystoisoporidiosis is estimated to be the second most important gastrointestinal protozoan disease in AIDS patients, exceeded only by cryptosporidiosis 13 .
In this study the frequency and seasonal profile of cryptosporidiosis and cystosporidiosis were evaluated among HIV/AIDS patients attended at the Hospital Escola da Universidade Federal do Triângulo Mineiro (UFTM).Possible associations based on examination of clinical and laboratory data of the patients were also investigated.

MATERIAL AND METHODS
Data were examined from previous studies on Cryptosporidium sp and Cystoisospora belli in faecal samples (diarrhoeic or not) from HIV/AIDS patients attended in the outpatients clinic or infirmary of the UFTM Department of Infectious and Parasitic Diseases between July 1993 -June 2003.This institution provides the regional reference centre for treatment of HIV/AIDS patients in Uberaba and the surrounding region.The examinations were performed in the parasitology laboratory, where the samples were submitted to sedimentation by the formalin-ether (Ritchie's) method.Faecal smears were made form the sediment, stained by a modified Ziehl-Neelsen method and examined under the 100X objective of the light microscope to look for Cryptosporidium sp and Cystoisospora belli.Samples were considered to be negative when no oocysts were detected after observation of at least 100 microscopic fields.
Clinical data, quantification of T CD4 + lymphocytes, confirmatory examination of HIV infection and use of antiretroviral therapy (HAART) were obtained from the participants.

RESULTS
In all 638 faecal samples were examined from 359 patients (1.7 samples/patient).The ages of members of the study group varied from 2 -72 with a mean of 33.3 (± 10.42).Males comprised 241 (67.2%) of the participants and females 118 (32.8%).
Eleven of the 14 (78.6%)patients who presented coccidian in the faeces had diarrhoea and a T CD4+ lymphocyte count equal to or less than 200 cells/mm 3 (Table 2).The mean T CD4 + lymphocyte count in patients with Cryptosporidium sp was 82.3 ± 65.8 cells/mm 3 and in those infected with Cystoisospora belli 98.0 ± 151.2 cells/mm 3 .Only 8.5% (6/71) of the patients who presented coccidian in the faeces were receiving HAART.
The frequency distribution of coccidian per year of observation showed a higher occurrence of patients infected with Cryptosporidium sp during 1997 (23%) and with Cystoisospora belli in 2000 (18.8%) (Figure1).When seasonal distribution of the two coccidian in HIV/AIDS patients over a 10-year period was evaluated, seasonal distribution of Cryptosporidium sp was found to peak between December and February.A slight rise in Cystoisospora belli infections was seen from June -August (Figure 2).

DISCUSSION
Factors such as immunological status of the patient, use or not of highly active antiretroviral therapy (HAART), level of basic sanitation and climatic diversities of the different regions, as well as methodological differences between the various studies, have all been used to explain differences in prevalence values of these coccidioses in the literature.
When occurrence of diarrhoeic syndrome was evaluated as an indicator of the presence of these coccidian within the study population, a higher positivity was observed in patients with diarrhoea (21.4%), than in non-diarrhoeic individuals (15.8%).Similar results were reported by Rigo & Franco (2000), who found higher rates of both cryptosporidiosis (18.3%) and cystoisosporidiosis (6.6%) in HIV patients with diarrhoea than in non-diarrhoeic individuals (1.6%).According to these authors, this data illustrates the risk of overestimating the frequency of Cryptosporidium sp and Cystoisospora belli infection rates when only patients with diarrhoea are included in the study population.
In immunocompromised patients a T CD4+ lymphocyte level above 200 cells/mm 3 is considered to be a limiting factor for opportunistic infections.Despite T CD4+ lymphocyte levels being quantified in only 17.8% of patients, the opportunistic character of intestinal coccidial infections could be perceived in 78.6% of the patients which presented coccidian in the faeces showing T CD4+ lymphocyte counts ≤ 200 cells/mm 3 .This fact was closely related to the cases of cryptosporidiosis, corroborating results already reported in the literature 3 .This association was also detected in cases of cystoisosporiadosis, although the high standard deviation in the T CD4 + lymphocyte counts observed in patients with Cystoisospora belli, demonstrated that this coccidiann suffers less from the host immune response than Cryptosporidium.
According to the literature, the numbers of opportunistic infections in HIV/AIDS patients 13 has diminished considerably since the introduction of HAART in 1996, including cases of cryptosporidiosis.However, in this study it was impossible to determine the influence of this therapy on infections with Cryptosporidium sp and Cystoisospora belli, since only 8.5% of patients which presented coccidian regularly received HAART.
In the Triângulo Mineiro region, as well as other parts of the world 16 17 23 a seasonal pattern was noted for Cryptosporidium sp, with a higher occurrence during the hottest months of the year (January-March).Although frequency of cystoisosporidiosis cases was somewhat higher in the months of June -August, it was not possible to show seasonal behaviour of infection by this coccidian in the studied area, and this remains to be established elsewhere 4 .
The survey of Cryptosporidium sp and Cystoisospora belli in HIV/AIDS patients attended the UFTM, from June 1993 -June 2003, showed that these parasites occur at a similar frequency in the studied population and that are closely linked to the presence of diarrhoeic syndrome (particularly in cryptosporidiosis), with a drop in T CD4 + lymphocyte counts.However, the influence of T CD4+ lymphocytes in infection with Cystoisospora belli needs to be better evaluated due to the reduced number of samples screened in the present study and to the possible recurrence of cases after treatment.These factors may be related to the immune response of the patient or even the presence of extraintestinal cysts, which are less susceptible to treatment 10 15 .

Figure 1 -Figure 2 -
Figure 1 -Annual distribution of Cryptosporidium spp and Cystoisospora belli in HIV/AIDS patients attended in at UFTM, Brazil, June 1993 -June 2003.