Use of multiplex real-time PCR for detection of common diarrhea causing protozoan parasites in Egypt

Mahmoud M. El Sibaei1, Magda Y. Abdel Hamid1 , Khalifa E. Khalifa1, Ranyia A. S. Tawfik1, John T. Nazeer1, Heidrun von Thien2, Egbert Tannich2 Parasitology Departmet, Faculty of Medicine Ain Shams University1, Department of Molecular Parasitology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany2

Diarrhoea is an important cause of morbidity and mortality, worldwide. Giardia intestinalis, Cryptosporidium spp and Entamoeba histolytica are the most common diarrheacausing parasitic protozoa. Diagnosis of these parasites is usually performed by microscopy. However, microscopy lacks sensitivity and specificity. Replacing microscopy with more sensitive and specific nucleic acid based methods is hampered by the higher costs, in particular in developing countries.

Multiplexing the detection of more than one parasite in a single test by real time PCR has been found to be very effective and would decrease the cost of the test. In the present study multiplex real-time PCR was used for the simultaneous detection of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp among Egyptian patients complaining of diarrhea and in a representative control group

Material and Methods

1. Faecal specimens:
Fresh stool samples were collected from 396 patients complaining of diarrhea and 202 apparently healthy individuals. The samples were collected over a period of one year from October 2010 to October 2011 in Cairo and the Egyptian governorates Fayoum and Benha, respectively. The patients and the healthy controls aged between 6 months and 60 years.

2. Microscopy:
After collection, stool samples were divided into 2 portions: 1. The first portion was preserved frozen at -20 C for further processing by real-time PCR 2. The second portion was examined by: Direct saline and/or iodine mounts Formol-ethyl acetate concentration technique. Modified Ziehl Neelsen on direct fresh smears as well as on formol-ethyl acetate concentrates to detect Cryptospordium oocysts.

3. Real-time Multiplex PCR:

DNA was extracted from all samples using standard protocols. The primers were designed to amplify : - A 172 bp fragment from SSU rRNA of both Entamoeba histolytica and Entamoeba dispar . - A 62 bp fragment from SSU rRNA of Giardia Intestinalis. - A 138 bp fragment from the genomic DNA of Cryptosporidium parvum Taqman probes were designed to detect the amplification products from the 4 parasites specifically

3. Real-time Multiplex PCR:

Amplification reactions were performed in single tubes, in final volumes of 25 L containing Qiagen HotstarTaq master mix, the primers and probes of E. histolytica/dispar, Giardia intestinalis and Cryptosporidium parvum specific primers. As an internal control to detect possible PCR inhibition of amplification by stool contents, a specific primer and probe set targeting Phocin herpes virus 1( PhHV-1) were included with each run

3. Real-time Multiplex PCR:

Amplification consisted of 3 minutes at 95C followed by 40 cycles of 30 seconds at 95C, 30 seconds at 55C, and 30 seconds at 72C. Fluorescence was measured during the annealing step of each cycle. Amplification, detection, and data analysis were performed with the Rotor gene 6000 real-time detection system.

Results

Results of microscopy and multiplex real-time PCR for the detection of enteric protozoa among 396 patients with diarrhea and 202 control subjects
Microscopy Diarrheal cases Organism E. histolytica/dispar Giardia intestinalis Cryptosporidium spp No 32 110 4 11 0 0 239 % 8.0 27.8 1.0 2.8 0 0 60.4 Control No 0 1 0 0 0 0 201 % 0 0.25 0 0 0 0 99.75 Multiplex real-time PCR Diarrheal cases No 5a 141 8 2a 3 1a 236 % 1.3 35.6 2.0 0.5 0.75 0.25 59.6 Control No 1b 4 3 0 0 0 194 % 0.5 2.0 1.5 0 0 0 96.0

Combined Giardia and E. histolytica/dispar

Combined Giardia and Cryptosporidium spp Combined Three parasites Negative for the three parasites

Sensitivity, specificity, positive and negative predictive values of microscopy compared to multiplex real-time PCR for detection of Entamoeba histolytica/dispar, Giardia intestinalis and Cryptosporidium spp in the stool of 396 diarrheal patients
Multiplex real-time PCR Microscopy

E. histolytica/dispar 100 % 91 % 18.6 % 100 %

Giardia intestinalis 57.8 % 85.5 % 70.2 % 77.5 %

Cryptosporidium spp 33.3 % 100 % 100 % 98 %

Sensitivity Specificity PPV NPV

The results indicate that among diarrheal patients in Egypt Giardia intestinalis is the most common protozoan parasite, with prevalence rates of 30.5% and 37.1%, depending of the method used (microscopy vs. multiplex real-time PCR). Cryptosporidium spp was detected in 1% of the diarrheal patients by microscopy and in 3 % by real-time PCR. While Entamoeba histolytica/ dispar was detected in 10.8% by microscopy. Less than one fifth of them (2%) were found true positive for Entamoeba dispar by real time PCR. Entamoeba histolytica DNA was not detected in any of the diarrheal patients

In comparison with multiplex real-time PCR, microscopy exhibited many false positive and negative cases with the three parasites giving sensitivities and specificities of 100% and 91% for Entamoeba histolytica/dispar, 57.8% and 85.5% for Giardia intestinalis, and 33.3% and 100% for Cryptosporidium spp. For Entamoeba histolytica/dispar the many false positive findings confirm the low positive predicative value of microscopy (18.6 %).

This may be due to misdiagnosis of other Entamoeba species such as Entamoeba coli, E. hartmanni, or the morphologically identical Entamoeba moshkovskii. These findings confirm the limitation of microscopy for the differentiation of the various Entamoeba spp and calling into question prevalence rates of Entamoeba histolytica previously recorded on the basis of microscopy.

Conclusion

The present study has shown that the implementation of multiplex real-time PCR for the simultaneous detection target DNA in a closed tube system would be beneficial for the rapid and accurate diagnosis of common diarrhea causing protozoa. For developing countries, although the reagents costs are still high compared to microscopy, multiplex real time PCR not only simplifies the detection of several enteric pathogens, but also reduces the cost of unnecessary treatment following misdiagnosis. Pooling of samples to a reference laboratory would reduce the running cost of the test.