CURRENT DEVELOPMENT IN THE DIAGNOSIS OF PARASITIC INFECTION INCLUDING PCR METHODS

DIAGNOSTIC METHODS IN PARASITOLOGY

CONVENTIONAL METHODS
Need trained staffs, equipments,slow throughput BUT gold standard Rapid molecular tests being developed

MOLECULAR DIAGNOSTIC
Methods to study primary structure (Sequence of DNA Every organism contains some unique, species specific DNA sequences Molecular diagnostic make the species specific DNA visible Needed if traditional methods provide poor results 1. Microscopy gives false positive/ negative results 2. Low sensitivity 3. Cultivation methods : slow growth

MOLECULAR METHODS
Advantages High sensitivity & specificity Detect pathogens , not immune response Quick results High gransport tolerations Easy to perform Cost effective

Disadvantages Expensive : PCR PCR can fail : contamination and false positive Do not distinguish dead and living parasites

MOLECULAR DIAGNOSIS METHODS

1. MOLECULAR HYBRIDISATION (a) RNA-RNA hybrids (b)RNA-DNA hybrids (c)In situ Hybridization(analyze prepared cells / histologic sections) 2. POLYMERASE CHAIN REACTION TECHNIQUES (a) Conventional PCR (b) Nested PCR (c) Multiplex PCR (d) RT-PCR (e) Real time PCR 3. COMBINED TECHNIQUES (a) Serological and molecular techniques (b) Immunocapture PCR (IC PCR)

POLYMERASE CHAIN REACTION

A technique used to amplify the number of copies of a specific region of DNA fragment This technique can be used to identify - Disease causing viruses, bacterias or parasites - A criminal suspect (RFLP or DNA finger printing) Uses the basic elements of DNA replication processes (DNA is unwound and each strand of the parent molecule is used as a template to produce a complementary daughter strand Minute quantities of DNA required Rapid

http://genuex.unex.es/Genetica/tema29/PCR.swf

 By heating and cooling - Target DNA is denatured to provide single stranded template DNA - Temperature lower , primers will anneal to flanking regions - Bases are added to 3 end of annealed primers by DNA polymerase The cycles are repeated, amount of DNA increase exponentially

Toxoplasma gondii
Toxoplasmosis : ingestion of oocysts Congenital infection during pregnancy risks of fetus becoming infected during benign acute infection requires screening on mother and fetus preferably in utero Major drawbacks of conventional diagnosis
1. isolation of parasite in tissue culture or mouse requires 4-6

weeks before resukt is known 2. Sampling of fetal blood increases risk of abortion 3. Serological detection in mother and fetus is only 50-80% sensitive

Toxoplasma gondii
PCR diagnosis of congenital infection Major breakthrough in the use amniotic fluid as sample,obviating the need for fetal blood Reduced risk of fetal loss
Infection in the immunocompromised (AIDS patients,transplantation patients) Diagnosis is mainly by imaging scans which often cannot distinguish other brain diseases Serology is ambiguous and not useful Direct detection of parasite in the blood or CSF by conventional means is time consuming and not sensitive

Toxoplasma gondii
PCR diagnosis of toxoplasmosis in the immunocompromised - Simple body fluid such as CSF and peripheral blood can be used, no need for brain biopsies - PCR reported to be very efficient 100% sensitive Sensitivity and specificity depends not only on target sequence but also primer design Sample preparation and DNA isolation methods not standardized among laboratories in the world

Toxoplasma gondii
Five targets are routinely used in PCR 1. 35-fold repititive gene B1 2. Single copy P30 gene, coding for the major surface protein antigen P30, nested PCR is favored 3. 110-fold repititive ssu rRNA 4. Single copy tubulin gene 5. Single copy tubulin gene

Cryptosporidium parvum
Usefullness of PCR approach in detecting oocyst in stool - conventional lab diagnosis (poor sensitivity, not suitable for early detection) - PCR (sensitive, early detection critical for treatment especially in immunocompromised patients)

A: Agarose gel (2%) analysis of a PCR diagnostic test for detection of Cryptosporidium parvum DNA. PCR was performed using standard ABI protocol. Lane S: Molecular base pair standard (100-bp ladder). Black arrows show the size of standard bands.

Lane 1: C. parvum positive fecal specimen. The red arrow shows the diagnostic band for Cryptosporidium parvum zoonotic genotype (size: 435 bp).

NESTED PCR ASSAY for detection Cryptosporidium parvum Oocysts

These sophisticated new "nested PCR" systems are significantly more sensitive than other currently available PCR technologies. A schematic of nested PCR is shown in the figure

Placental Malaria
Placental infection with Plasmodim falciparum contribute to maternal morbidity, low birth weight and pre term delivery Post partum microscopy of placental blood films has been used as a more accurate indicator
Sensitivity detecting Plasmodium falciparum in peripheral blood Microscopy Immunoassay PCR

42%

80%

99.9%

Visceral leishmaniasis (kala-azar)

Routine diagnosis is by direct microscopy or culture Microscopic detection of amastigote in Geimsa stained spleen, bone marrow/lymph node aspirates is simple and cheap Disadvantages :
(a) Spleen aspiration is danger under field conditions (b) Bone marrow and lymph node aspirates are of limited sensitivity. And inconvenient to patient (c) Isolation of parasites by culture is time consuming, expensive and difficult

Visceral leishmaniasis (kala-azar)

PCR > sensitive than microscopy Blood may be used for initial PCR screening If PCR on blood is negative, PCR on lymph node / bone marrow should be performed PCR is cheaper test compared to microscopy detection

Evaluation of PCR for Diagnosis of Indian Kala-Azar and Assessment of Cure R. Maurya,1 R. K. Singh,1 B. Kumar,1 P. Salotra,2 M. Rai,1 and S. Sundar1,* ABSTRACT

This study was done to evaluate PCR with Ld1 primers for the diagnosis of Indian visceral leishmaniasis (VL) and to assess its role in prediction of the disease outcome. The PCR assay was performed with DNA isolated from the peripheral blood of parasitologically confirmed cases of VL before the initiation of treatment, just after the end of treatment, and at 3 and 6 months of follow-up. The pretreatment PCR result was positive for 100 of 101 patients (sensitivity, 99%; confidence interval [CI], 94 to 100%). None of the 150 negative controls tested were PCR positive (specificity, 100%; CI, 96.8 to 100%). Of 60 patients who were treated at our center, 51 (85%; CI, 73 to 93%) became negative immediately after treatment and continued to be negative at 3 and 6 months of follow-up. At the 3-month follow-up, two of the remaining nine patients were PCR positive, making 58 (96.7%; CI, 87 to 100%) patients PCR negative. At the 6-month follow-up, all patients became PCR negative. One patient who was PCR negative immediately after the end of treatment relapsed 11 months later. This limited prospective study with VL patients suggests that the PCR assay is a highly sensitive and specific (99% and 100%, respectively) tool for the diagnosis of VL. In the majority of patients, it can identify a successful disease outcome; however, its translation into the field setting remains a major challenge.

PCR LABORATORY
Sample Handling DNA preparations

Laboratory Mixing Site

Thermocycler Amplification

Detection Documentation

R&D