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CURRENT DEVELOPMENT IN THE DIAGNOSIS OF PARASITIC INFECTION INCLUDING PCR METHODS

DIAGNOSTIC METHODS IN PARASITOLOGY

equipments.CONVENTIONAL METHODS • Need trained staffs.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. Cultivation methods : slow growth . Low sensitivity 3. 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.

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

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.es/Genetica/tema29/PCR.unex. bacterias or parasites .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 .swf .Disease causing viruses.

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

isolation of parasite in tissue culture or mouse requires 4-6 weeks before resukt is known 2.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. Sampling of fetal blood increases risk of abortion 3. Serological detection in mother and fetus is only 50-80% sensitive .

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 congenital infection Major breakthrough in the use amniotic fluid as sample.

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 .Simple body fluid such as CSF and peripheral blood can be used.Toxoplasma gondii • PCR diagnosis of toxoplasmosis in the immunocompromised . no need for brain biopsies .

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

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

The red arrow shows the diagnostic band for Cryptosporidium parvum zoonotic genotype (size: 435 bp). Lane S: Molecular base pair standard (100-bp ladder). Lane 1: C. parvum positive fecal specimen.A: Agarose gel (2%) analysis of a PCR diagnostic test for detection of Cryptosporidium parvum DNA. Black arrows show the size of standard bands. PCR was performed using standard ABI protocol. .

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 .

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% .Placental Malaria • Placental infection with Plasmodim falciparum contribute to maternal morbidity.

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. expensive and difficult .Visceral leishmaniasis (kala-azar) • Routine diagnosis is by direct microscopy or culture • Microscopic detection of amastigote in Geimsa stained spleen. And inconvenient to patient (c) Isolation of parasites by culture is time consuming.

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 .

CI.1 B. two of the remaining nine patients were PCR positive. 99%. however.1 and S.8 to 100%). respectively) tool for the diagnosis of VL. CI.Evaluation of PCR for Diagnosis of Indian Kala-Azar and Assessment of Cure R. Kumar. making 58 (96. all patients became PCR negative. 94 to 100%). In the majority of patients.1 R. Singh. At the 3-month follow-up. At the 6-month follow-up. 96. 73 to 93%) became negative immediately after treatment and continued to be negative at 3 and 6 months of follow-up. confidence interval [CI]. The pretreatment PCR result was positive for 100 of 101 patients (sensitivity. 100%. just after the end of treatment.* • 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. CI. One patient who was PCR negative immediately after the end of treatment relapsed 11 months later. .1 P. 51 (85%. and at 3 and 6 months of follow-up. Maurya. Sundar1. Salotra. None of the 150 negative controls tested were PCR positive (specificity. Of 60 patients who were treated at our center. it can identify a successful disease outcome.2 M. its translation into the field setting remains a major challenge. The PCR assay was performed with DNA isolated from the peripheral blood of parasitologically confirmed cases of VL before the initiation of treatment. Rai. K. This limited prospective study with VL patients suggests that the PCR assay is a highly sensitive and specific (99% and 100%.7%. 87 to 100%) patients PCR negative.

PCR LABORATORY • Sample Handling • DNA preparations Laboratory Mixing Site Thermocycler Amplification Detection Documentation R&D .