Detection of Intestinal Protozoa in HIV Patients: A Comparative Study of Microscopy and Multiplex Real-Time PCR

Introduction: Intestinal protozoa are traditionally diagnosed through microscopic examination, which is labor-intensive and often requires repeated sampling. Recent molecular approaches, particularly multiplex Real-Time PCR (RT-PCR), provide a faster alternative for parasite detection. This study aimed to compare the sensitivity and specificity of direct smear and multiplex RT-PCR methods against trichrome staining for intestinal protozoa detection. Methods: A cross-sectional study was conducted using 51 stool samples collected from HIV-naïve patients, HIV patients on therapy, and healthy volunteers at General Hospital Dr. Saiful Anwar, Malang, Indonesia. The samples consisted of 41 HIV-positive individuals (17 HIV-naïve and 24 on therapy) and 10 healthy volunteers. Stool specimens were examined using direct smear, trichrome staining, and multiplex RT-PCR. Results: Microscopic examination identified Entamoeba histolytica, Blastocystis hominis, coccidian parasites, and non-pathogenic amoebae. In contrast, multiplex RT-PCR detected only B. hominis in both HIV and non-HIV samples. Compared with trichrome staining, direct smear demonstrated 36.6% sensitivity and 80.0% specificity, while multiplex RT-PCR showed higher sensitivity (63.4%) but lower specificity (40.0%). Differences among methods may be influenced by limited sample size, population characteristics, clinical status, and technical or biological factors affecting microscopy and molecular examinations. Conclusion: Combining molecular diagnostics with conventional microscopy may improve the diagnostic accuracy of intestinal protozoa infections and support more effective treatment strategies and better clinical outcomes in HIV patients.