Evaluation of staining techniques, antigen detection and nested PCR for the diagnosis of cryptosporidiosis in HIV seropositive and seronegative patients

Abstract

The study was designed to determine the efficacy of modified Ziehl–Neelsen (ZN), safranine methylene blue (SM) staining, antigen detection ELISA and a nested PCR assay (specific for Cryptosporidium parvum) for detection of Cryptosporidium in HIV seropositive and seronegative patients with diarrhoea. Cryptosporidium was detected in 10 (4.9%), 9 (4.4%), 39 (18.9%) and 27 (13.1%) of 206 HIV seropositive and 7 (4.6%), 6 (3.9%), 21 (13.7%) and 17 (11.1%) of 153 HIV seronegative patients by ZN staining, SM staining, antigen detection ELISA and PCR, respectively. None of the 50 apparently healthy control subjects was found to be infected with Cryptosporidium by any of the techniques. Based on the criteria of ‘true positive’ samples positive by at least any two techniques out of ZN staining, antigen detection and PCR, sensitivity of ZN and SM staining techniques was 37% and 33.3% in HIV seropositive and 41.2% and 35.3% in seronegative patients, respectively. Sensitivity of antigen detection ELISA was 92.6% and 94.1% in HIV seropositive and seronegative patients, respectively, while sensitivity of PCR was 100% each in HIV seropositive and seronegative patients. Specificity of all three techniques, i.e. ZN, SM staining and PCR was 100% in both HIV seropositive and seronegative patients while specificity of antigen detection was 92.2% and 96.3% in HIV seropositive and seronegative patients, respectively. The staining techniques were found less sensitive as compared to antigen detection and PCR for detection of Cryptosporidium in HIV seropositive patients with CD4 count >200 cells/μl.