Background
Cryptosporidium parvum, the protozoan parasite, causes a significant enteric disease in immunocompromised hosts such as HIV patients. Severe chronic infections may develop in immunocompromised hosts with lymphocyte or gammaglobulin deficiencies which suggest that both cell mediated and humoral immune responses are involved in resolution of infections and development of protection [
1]. Serologic surveys in immunocompetent individuals or HIV/AIDS patients show varying levels of anti
Cryptosporidium antibodies. Detectable IgG levels against
Cryptosporidium were reported in 86% in Australia [
2], 26% in Britain [
3], 64% in Peru [
4] and 64% in Venezuela [
4] in healthy individuals. A previous study [
5] reported
Cryptosporidium specific positive IgG response in 5 (100%) HIV patients and 12 (100%) immunocompetent patients. In another study [
6] IgG antibodies were observed in 13 out of 15 (86.7%) immunocompetent and all the 26 (100%) AIDS patients studied and IgM response was observed in 14 out of 15 (93.3%) immunocompetent and 4 out of 26 (15.4%) HIV positive patients. A positive IgG and IgM response was reported in all 16 (100%) immunocompetent and 24 (100%) AIDS patients studied [
7]. In a similar study [
8] positive IgG, IgM and IgA response was observed in all the 4 (100%) immunocompetent and 4 (100%) HIV positive patients, studied. The role of antibody responses in protection is still not clear. In the study conducted in Alabama [
5], antibodies to
Cryptosporidium were detected in sera from 5 AIDS subjects with persistent cryptosporidiosis. The fact that some AIDS patients with persistent symptomatic infection with
Cryptosporidium have a high antibody titre to
C. parvum supports the theory that specific serum antibody alone is not sufficient to control the infection [
9]. Studies comparing the IgG, IgM and IgA response in HIV seropositive and seronegative patients to
C. parvum are scarce and reported in very limited numbers of subjects. In India the number of HIV infected individuals is growing at an alarming rate with 2.47 million people infected with HIV till the end of year 2006 [
10]. Although,
Cryptosporidium has been reported in 4.6%-12% HIV patients from different geographical areas in India [
11‐
17], reports regarding IgG, IgM or IgA response to
C. parvum in HIV seropositive or seronegative subjects are totally lacking. The present study was aimed to compare serum IgG, IgM and IgA responses to crude soluble antigen of
C. parvum in HIV seropositive and seronegative patients co-infected with
Cryptosporidium and to correlate the responses with symptomatology.
Discussion
In the present study, Cryptosporidium specific serum antibody (IgG, IgM and IgA) response was studied in doubling serum dilutions (1:10-1:40) to assess and compare the response in different study groups. We found that 1:20 dilution had the maximum sensitivity and specificity and was subsequently taken for interpretation of results.
In the present study, IgG antibody response was found positive in all 11 (100%) HIV seropositive
Cryptosporidium positive (Gp I) and 10 (100%) HIV seronegative
Cryptosporidium positive (Gp III) including 4 (100%) post-transplant (Gp IIIa) patients while in the
Cryptosporidium negative groups (Gp II, IV and V), 3 (12%) patients with other parasitic infections showed positive response. In the present study, no difference was observed between number of patients showing positive response in HIV seropositive
Cryptosporidium positive (Gp I) and HIV seronegative
Cryptosporidium positive (Gp III) patients (p > 0.05). However, positive response was observed in significantly higher number of
Cryptosporidium infected individuals both HIV seropositive (Gp I) and seronegative (Gp III) when compared to
Cryptosporidium un-infected HIV seropositive (Gp II), patients with other parasitic infections (Gp V) and HIV seronegative healthy (Gp IV) individuals. Our findings are similar to previous study whereby serum IgG positivity was reported to be 100% in AIDS patients with cryptosporidiosis, 87.7% in immunocompetent patients with cryptosporidiosis, 5% in presumably un-infected healthy individuals and 50% in patients with other parasitic individuals [
6]. Other studies have also reported positive antibody response in 100% cryptosporidiosis patients with or without HIV [
7,
8]. Based on assessment of mean OD values, previous studies [
24‐
26] found mean IgG levels higher in sera from HIV infected patients with chronic cryptosporidiosis as compared to healthy controls or HIV infected patients without cryptosporidiosis. In healthy individuals, IgG seropositivity has been found to vary in number of individuals in different parts of the world. IgG antibody response was found positive in 86% in Australia [
2], 26% in Britain [
3] and 64% in Peru [
4] and 64% in Venezuela [
4] in healthy individuals. Although, studies regarding IgG antibody response in transplant patients are not available, in the present study, no difference in number of patients with IgG positive response was observed between the two sub-groups in HIV seronegative
Cryptosporidium positive (post-transplant and presumably immunocompetent) patients (p > 0.05).
In the present study, IgM antibody response was found positive in 2 (18.2%) HIV seropositive
Cryptosporidium positive (Gp I) and 2 (20%) HIV seronegative
Cryptosporidium positive (Gp III) including 1 (100%) post-transplant (Gp IIIa) patient while in the
Cryptosporidium negative groups (Gp II, IV and V), 2 (8%) patients with other parasitic infections showed positive response. No significant difference was observed between number of patients showing positive response in any of the group. Based on assessment of mean OD values, previous studies from France [
24‐
26] found IgM levels higher in sera from HIV infected patients with chronic cryptosporidiosis as compared to healthy controls or HIV infected patients without cryptosporidiosis. Another study found IgM levels higher in sera from children with chronic cryptosporidiosis as compared to healthy controls [
27]. IgM serpositivity rate of 15.5% and 19.8% has been reported in healthy children from Peru and Venezuela, respectively [
4]. Another study reported serum IgM positivity to be 15.4% in AIDS patients with cryptosporidiosis, 93.3% in immunocompetent patients with cryptosporidiosis, 13% in presumably un-infected healthy individuals and 22.6% in patients with other parasitic individuals [
6]. In contrast, some studies have reported IgM positivity rate of 100% in both HIV positive and negative patients with cryptosporidiosis [
7,
8].
In the present study, IgA antibody response was found positive in 7 (63.6%) HIV seropositive
Cryptosporidium positive (Gp I) and 5 (50%) HIV seronegative
Cryptosporidium positive (Gp III) including 1(25%) post-transplant (Gp IIIa) patient while 4 (20%) HIV seropositive
Cryptosporidium negative, 2 (10%) HIV seronegative
Cryptosporidium negative healthy and 2 (8%) patients with other parasitic diseases were found positive for IgA response. A positive response was observed in significantly higher number of
Cryptosporidium infected individuals when compared to
Cryptosporidium un-infected irrespective of HIV status. In agreement with our findings, a previous study has reported IgA positive response in 100% both in HIV positive and negative patients with cryptosporidiosis [
8]. Based on assessment of mean OD values, previous studies found IgA levels higher in sera from HIV infected patients with chronic cryptosporidiosis as compared to healthy controls or HIV infected patients without cryptosporidiosis [
24‐
26] and in sera from children with chronic cryptosporidiosis as compared to healthy controls [
27].
The difference in seropositivity in HIV infected as well as healthy subjects in reports from different geographical areas may be attributed to the duration of illness and type of antigen used to study the antibody response.
No reports are available regarding prevalence of anti-
Cryptosporidium antibodies in India for comparison. However, varying rates of
Cryptosporidium positivity in both HIV (4.6-12%) and non-HIV (.06-13%) subjects are reported from different geographical locations in India [
20].
Sensitivity and specificity of IgG ELISA by using
Cryptosporidium parvum crude soluble antigen (CCA) was 100% and 95.4%, respectively, in the present study, which is similar to an earlier study [
28] which reported the sensitivity and specificity by using two antigens (recombinant form of 27kDa and a partially purified fraction from 17 kDa oocysts antigen) as 90 and 92% and 90 and 94%, respectively. In the present study, sensitivity and specificity of IgM and IgA ELISA was found 19% and 97% and 57.1% and 87.7%, respectively. However, there are no previous reports regarding sensitivity and specificity of IgM or IgA ELISA in cryptosporidiosis.
On comparison of antibody response in HIV seropositive patients with and without diarrhoea, the number of IgG, IgM and IgA seropositive patients with history of diarrhoea was not significantly different from that of patients without history of diarrhoea in both HIV seropositive
Cryptosporidium positive and HIV seropositive
Cryptosporidium negative groups. Earlier studies show controversial role of IgG antibodies in cryptosporidiosis. Study done in B-cells depleted BALB/c mouse model indicates that the specific IgG antibody response does not play the major role in the resolution of infection with
Cryptosporidium [
29]. The fact that some AIDS patients with persistent symptomatic infection with
Cryptosporidium have a high antibody titre to
C. parvum also supports the theory that specific serum antibody alone is not sufficient to control the infection [
9]. In contrast, Experimental study in healthy volunteers reported that seropositivity was higher in persons who were given infection dose >20 fold higher than persons who were seronegative for
Cryptosporidium [
30]. However, the authors suggest that serum antibody may simply be a marker of an effective secretory and/or cellular response to infection. In another study, Frost et al, (2005) reported that in HIV positive individuals, a strong IgG response to the 27-kDa antigen group was associated with a reduced risk of diarrhea [
31]. There are no reports available regarding role of IgM antibodies in protection. Previous study shows that HIV positive patients with chronic cryptosporidiosis had higher levels of serum IgA to soluble
Cryptosporidium antigen compared with HIV positive persons who cleared the infection but secretory IgA antibodies were higher in HIV positive persons who cleared the infection as compared to HIV positive persons with chronic cryptosporidiosis. This suggests that secretory antibody but not serum antibody may be playing role in protection. It has been suggested that secretory IgA is responsible for the recovery from an effective immune response at the mucosal surface [
32]. However, the mechanism of diarrhoea in cryptosporidiosis is not well-understood and is suggested to be due to disrupted mucosal architecture and intestinal dysfunction resulting from the infection and the host response to the infection besides other factors [
33,
34] suggesting that antibody responses may not be the only factors playing significant role in protection from symptomatic cryptosporidiosis, and other innate and cellular immune responses may also be contributing in the protection.
The number of IgG, IgM and IgA positive patients with CD4 counts <200 cells/μl was not significantly different from that of patients with CD4 counts >200 cells/μl in both HIV seropositive
Cryptosporidium positive and HIV seropositive
Cryptosporidium negative groups which shows that production of antibodies may not be affected by CD4 counts in HIV patients. In agreement with our findings, a previous study reported no significant difference in peak antibody response to
Cryptosporidium antigens with respect to HIV status, CD4 cell count or history of diarrhea [
35].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KK participated in study design, performed analysis and interpretation of data and drafted the manuscript. SK contributed to analysis and interpretation of data and writing of manuscript. AW contributed to analysis and interpretation of data and writing of manuscript. NM conceived the study, participated in its design, contributed to analysis and interpretation of data and corrected draft copies of manuscript.