Laboratory Diagnosis & Treatment of Parasitic Diseases

Bradford McGwire, MD, PhDDivision of Infectious DiseasesCenter for Microbial Interface Biologybrad.mcgwire@osumc.edu

Learning Objectives

Describe and differentiate the principles of laboratory diagnosis for parasitic diseases

Recognize general classes of anti-parasitic agents, describe their underlying mechanisms of action, predict their clinical side effect profiles, and accurately evaluate their role in the therapy of parasitic infections

Laboratory Methods for Diagnosing Parasitic Disease

Macroscopic examination

Microscopic examination Wet mountPermanent stainsStool concentrates

Serologic examination Antibody responseAntigen detection

Nucleic acid hybridization Probes and amplification techniquesDetectionIdentification

Culture

Animal inoculation

Xenodiagnosis

Body Sites, Specimen Collection & Diagnostic Procedures for Selected Parasitic Infections – Blood, bone marrow and CNS

Infecting Organism Specimen Options Collection Methods Diagnostic Procedure

Blood

Plasmodium spp., Babesia spp., filaria, Leishmania, Toxoplasma, Trypanosoma spp.

Whole blood, anticoagulated Venipuncture

Microscopic examination (Giemsa stain) or acridine orange fluorescent stain

Thin filmThick film

Blood concentration (filaria)Serology

AntibodyAntigen

PCR

Bone Marrow

Leishmania spp., Trypanosoma cruzi

Aspirate Sterile Microscopic examination (Giemsa stain)Culture

Serum Venipuncture Serology (antibody)

PCR

Central Nervous System

Acanthamoeba spp., Naegleria spp.,trypanosomes,Toxoplasma gondii

Spinal fluid Sterile

Microscopic examination Wet mountPermanent stain

CultureSerology (antibody)PCR

Serum Venipuncture

Blood Films: “Thin” & “Thick” Preparations

This Giemsa stained slide depicts an example of properly prepared thick and thin film blood smears to be examined. Gustav Giemsa (1867 – 1948) was by trade both a chemist and a pharmacist. It was in 1902 that he developed a staining technique that was useful in the identification of malarial parasites such as Plasmodium falciparum.

“Thin” Blood Films: P. vivax & P. falciparum

These thin film Giemsa stained micrographs show ring-form Plasmodium vivax and P. falciparum trophozoites. As the parasite increases in size, the ring morphology of the early trophozoite disappears, and becomes what is referred to as a mature trophozoite, which undergoes further transformation maturing into a schizont.

CDC/ Steven Glenn, Laboratory & Consultation Division

“Thick” Blood Film: P. malariae & P. falciparum

This thick film Giemsa stained micrograph reveals a mixed P. falciparum and P. malariae parasitic infection. In the field of view note the presence of a growing Plasmodium malariae trophozoite (Lt), and a Plasmodium falciparum gametocyte (Rt), indicative of a mixed Plasmodium spp. infection.

CDC/ Steven Glenn, Laboratory & Consultation Division

“Thin & Thick” Blood Films: Babesia spp.

Babesia sp. in a thin blood smear stained with Giemsa. Note the tetrads, a dividing form characteristic for Babesia.

Babesia sp. in thick blood smears stained with Giemsa.

“Thick” Blood Film: Wuchecheria bancrofti

This is a micrograph of a Wuchereria bancrofti microfilaria in a thick blood smear using Giemsa stain technique. W. bancrofti, the most common filarial parasite in humans, is one of the causative agents for lymphatic filariasis. Lymphatic filariasis affects an estimated 120 million people in tropical areas of the world.

CDC/Dr. Mae Melvin

Culture: Acanthamoeba & Naegleria spp.

Trophozoites of Acanthamoeba spp. from culture. Notice the slender, spine-like acanthapodia.

Trophozoite of N. fowleri in culture.

Tissue & Body Fluids: Acanthamoeba & Naegleria spp.

Trophozoite of Acanthamoeba spp. in tissue, stained with hematoxylin and eosin (H&E).

Trophozoite of N. fowleri in CSF, stained with hematoxylin and eosin (H&E).

Cutaneous Ulcers

Leishmania spp., Acanthamoeba spp.

Aspirate Sterile plus smears Microscopic examination (Giemsa stain)CultureSerology (antibody)PCR

BiopsySterile, nonsterile to

histology

Serum Venipuncture

Eye

Acanthamoeba spp., Loa loa Microsporidia

Corneal scrapingsSterile saline, air-dried

smear

Microscopic examination Wet mountPermanent stain

Corneal biopsy Sterile saline Culture

Infecting Organism Specimen Options Collection Methods Diagnostic Procedure

Body Sites, Specimen Collection & Diagnostic Proceduresfor Selected Parasitic Infections

Tissue & Corneal Scraping: Leishmania spp. & Acanthamoeba spp.

Leishmania spp. amastigotes in a Giemsa-stained tissue scraping.

Trophozoites of Acanthamoeba spp. in a corneal scraping, stained with H&E.

Entamoeba histolytica

Fresh stool Preserved stool Sigmoidoscopy material

Waxed container Formalin, PVA Fresh, PVA Schaudinn smears

Microscopic examination Wet mountPermanent stains

Serum VenipunctureSerology

Antigen (stool)Antibody (serum)

CulturePCR

Giardia spp. Fresh stool Preserved stool Duodenal contents

Waxed container Formalin, PVA Entero-Test or aspirate

Microscopic examination Wet mountPermanent stains

Antigen IFAEIA

Culture

Cryptosporidium spp.

Fresh stool Preserved stool Biopsy

Waxed container Formalin, PVA Saline

Microscopic examination (acidfast)Antigen

IFAEIA

Infecting Organism Specimen Options Collection Methods Diagnostic Procedure

Body Sites, Specimen Collection & Diagnostic Proceduresfor Selected Parasitic Infections – Intestinal Tract

MicrosporidiaFresh stool Preserved stool

Duodenal contents Biopsy

Waxed container Formalin, PVA Aspirate Saline

Microscopic Giemsa stainGram stainChromotrope stain

Pinworm Anal impression smear Cellophane tape Macroscopic examinationMicroscopic examination(eggs)

Helminths Fresh stool Preserved stool Waxed container Formalin, PVA Macroscopic examination(adults)Microscopic examination(larvae and eggs)

Infecting Organism Specimen Options Collection Methods Diagnostic Procedure

Body Sites, Specimen Collection & Diagnostic Proceduresfor Selected Parasitic Infections – Intestinal Tract

“Wet” Mount & Stain: Entamoeba spp.

Cyst of E. histolytica/E. dispar in an unstained concentrated wet mount of stool. Notice the chromatoid bodies with blunt, rounded ends (arrow).

Trophozoite of E. histolytica/E. dispar stained with trichrome.

Cellulose Tape & “Wet” Mount: Enterobius vermicularis (Pinworm)

Eggs of E. vermicularis in a cellulose-tape preparation. Eggs of E. vermicularis in a wet mount.

Macroscopic Examination & “Wet” Mount: Ascaris lumbricoides

Adult female A. lumbricoides. Unfertilized egg of A. lumbricoides in an unstained wet mount of stool.

Infecting Organism Specimen Options Collection Methods Diagnostic Procedure

Liver, Spleen

E. histolytica, Leishmania spp.

Aspirates Sterile, collected in four separate aliquots (liver) Microscopic

examination Wet mountPermanent stains

Serology AntigenAntibody

Culture

Biopsy Sterile; nonsterile to histology

Serum Venipuncture

Lung

Rarely: amebae, ( E. histolytica), trematodes ( Paragonimus westermani), Larvae (Strongyloides stercoralis), or cestode hooklets

Sputum Induced, no preservative Microscopic examination

Giemsa stainGram stain

Hematoxylin and eosin

Lavage No preservative

Transbronchial aspirate Air-dried smears

Brush biopsy Air-dried smears

Open lung biopsy Fresh squash preparation; nonsterile to histology

Body Sites, Specimen Collection & Diagnostic Proceduresfor Selected Parasitic Infections

Infecting Organism Specimen Options Collection Methods Diagnostic Procedure

Muscle

Trichinella spiralis, T. cruzi Biopsy Nonsterile to histology Microscopic examination (permanent stains)

Serum Venipuncture Serology AntibodyAntigen

Skin

Onchocerca volvulus, Leishmania spp. Cutaneous larval migrans

Scrapings Aseptic, smear, or vialMicroscopic examination

Wet mountPermanent stains

Skin snip No preservative

Biopsy Nonsterile to histology

Serum Venipuncture Serology (antibody)Culture ( Leishmania spp.)

Body Sites, Specimen Collection & Diagnostic Proceduresfor Selected Parasitic Infections – tissue

Tissue Biopsy: Paragonimus spp. & Trichinella spp.

Eggs of Paragonimus spp. taken from a lung biopsy stained with hematoxylin and eosin (H&E). These eggs measured 80-90 µm by 40-45 µm. The species was not identified in this case.

Encysted larvae of Trichinella spp. in muscle tissue, stained with hematoxylin and eosin (H&E). The image magnification is 400x.

Infecting Organism Specimen Options Collection Methods Diagnostic Procedure

Urogenital System

Trichomonas vaginalis

Vaginal discharge Saline swab, culture medium Microscopic examination Wet mountPermanent stains

Antigen (IFA)CultureSerology (antibody)Nucleic acid probe

Prostatic secretions Saline swab, culture medium

Urethral discharge Saline swab, culture medium

Schistosoma haematobium Urine Single unpreserved specimen Microscopic examination

Biopsy Nonsterile to histology

Body Sites, Specimen Collection & Diagnostic Proceduresfor Selected Parasitic Infections – urogenital tract

“Wet” Mount: Trichomonas vaginalis & Schistosoma spp.

Trophozoite of T. vaginalis in a vaginal smear, stained with Giemsa.

Egg of S. haematobium in a wet mount of urine concentrates, showing the characteristic terminal spine.

Organism Gene Target Sensitivity (%) Comment

Plasmodium vivax Circumsporozoite gene 91 – 96 Dried blood-spotted filter paper samples are used.

Leishmania species kDNA minicircle sequence 87 – 100 Results are compared to culture and microscopy of biopsy specimens.

Trypanosoma cruzi kDNA minicircle sequence 100Results are compared to serology and xenodiagnosis of blood samples.

Toxoplasma gondii B1 repetitive gene P30 major surface antigen Recombinant DNA sequences

46 – 99PCR of BAL, blood, CSF, and amniotic fluid show great potential for diagnosis of toxoplasmosis.

Entamoeba histolytica P145 tandem repeat sequence SSU rRNA 96 – >90

Results are compared to microscopic diagnosis of stool samples. Test may distinguish pathogenic from nonpathogenic strains.

Examples of Techniques for Detection of Parasitic Infections Based on PCR Analysis

Chemotherapeutic Strategies That Exploit Differences Between Parasite & Host

Unique Site of Attack Drug Organism

Drug-concentrating mechanism unique to parasite Chloroquine Plasmodium spp.

Folic acid pathway (parasite unable to use exogenous folate) Pyrimethamine or Trimethoprim/Sulfamethoxazole

Plasmodium or Toxoplasma spp.

Inhibitor of trypanothione-dependent mechanisms for reducing oxidized thiol groups

Arsenicals,Difluoromethylornithine Trypanosomes

Interference with neuromediators unique to parasites Pyrantel pamoate,Diethylcarbamazine Ascaris spp.

Interacts with chloride channels, resulting inhyperpolarization of cells, paralysis, and death of parasites Ivermectin Filaria

Interaction with tubulin unique to parasites Benzimidazoles Many helminths

Inhibition of topoisomerase II Pentamidine Trypanosomes

Inhibition of pyruvate ferredoxin oxidoreductase Nitazoxanide Cryptosporidium and Giardia

Mechanisms of Action & Clinical Indications for the Major Anti-parasitic Agents – Anti-protozoal Agents

Drug Class Mechanism of Action Examples Clinical Indications

Heavy metals: arsenical and antimonials

• Inactivate sulfhydryl groups.• Disrupt glycolysis.

Melarsoprol, sodium stibogluconate, meglumine antimonate

Trypanosomiasis, Leishmaniasis

Aminoquinoline analogues

• Accumulate in parasitized cells.

• Interfere with DNA replication.

• Bind to ferriprotoporphyrin IX.

• Raise intravesicular pH.• Interfere with hemoglobin

digestion

Chloroquine, mefloquine, quinine, primaquine, halofantrine, lumafantrine

Malaria prophylaxis and therapy Radical cure (exoerythrocytic-primaquine only)

Folic acid antagonists• Inhibit dihydropteroate

synthetase and dihydrofolate reductase

Sulfonamides, pyrimethamine,trimethoprim

Toxoplasmosis, malaria, cyclosporiasis

Inhibitors of protein synthesis

• Block peptide synthesis at levelof ribosome

Clindamycin, spiramycin,paromomycin, tetracycline, doxycycline

Malaria, babesiosis, amebiasis, cryptosporidiosis, leishmaniasis, onchocerciasis

Mechanisms of Action & Clinical Indications for the Major Anti-parasitic Agents – Anti-protozoal Agents

Drug Class Mechanism of Action Examples Clinical Indications

Diamidines• Bind DNA• Interfere with uptake

and function of polyamines

PentamidinePneumocystosisLeishmaniasisTrypanosomiasis

Nitroimidazoles

• Interact with DNA • Inhibit metabolism of

glucose and interfere with mitochondrial function

Metronidazole, benznidazole,tinidazole

AmebiasisGiardiasisTrichomoniasis American trypanosomiasis (Chagas disease)

Nitrofurans

• Depletion of glutathione, trypanothione and metallothionein.

• Oxidative stress.

NifurtimoxChagas diseaseLate-stage African trypanosomiasis (T.b. gambiense)

Mechanisms of Action & Clinical Indications for the Major Anti-parasitic Agents – Anti-protozoal Agents

Drug Class Mechanism of Action Examples Clinical Indications

Sesquiterpenes

• React with heme, causing free-radical damage to parasite membranes (artemisinins).

• Inhibit methionine aminopeptidase type 2 (fumagillin).

• Inhibit RNA and DNA synthesis (fumagillin).

Artemisinin, artemether, artesunate Fumagillin

Malaria (artemisinins) Schistosomiasis Gastrointestinal and ocular microsporidiosis (fumagillin)

Hydroxynaphthoquinone (Atovaquone)+ anti-folate (Proguanil) combination

• Atovaquone Inhibition of electron transport system in mitochondria of parasites, thus blocking nucleic acid synthesis and inhibiting replication

• Proguanil Selective inhibition of plasmodial dihydrofolate reductase. Lowers effective concentration at which atovaquone causes collapse of mitochondrial membrane potential.

Malarone (Atovaquone-Proguanil)

Malaria prophylaxis and therapy(P. falciparum – active against all stages of development; P. vivax and P. ovale – active against erythrocytic stages)

Ornithine analogue • Inhibits ornithine decarboxylase.• Interferes with polyamine metabolism Difluoromethylornithine African

trypanosomiasis

Mechanisms of Action & Clinical Indications for the Major Anti-parasitic Agents – Anti-protozoal Agents

Drug Class Mechanism of Action Examples Clinical Indications

Phosphocholine analogue

• Disruption of lipid metabolism Miltefosine Leishmaniasis

Acetanilide • Unknown Diloxanide furoate Intestinal amebiasis

Sulfated naphthylamine

• Inhibits sn-glycerol-3- phosphate oxidase and glycerol-3-phosphate dehydrogenase causing decreased ATP synthesis

Suramin African trypanosomiasis

Thiazolides• Inhibit pyruvate-

ferredoxin oxidoreductase

Nitazoxanide Cryptosporidiosis,Giardiasis

Mechanisms of Action & Clinical Indications for the Major Anti-parasitic Agents – Anti-helminthitic Agents

Drug Class Mechanism of Action Examples Clinical Indications

Benzimidazoles

• Inhibit fumarate reductase.• Inhibit glucose transport.• Disrupt microtubular

function.

Mebendazole,thiabendazole,albendazole

Broad-spectrum anthelmintic: nematodes, cestodes

Tetrahydropyrimidine• Blocks neuromuscular

action• Inhibits fumarate reductase

Pyrantel pamoate Ascariasis, pinworm, hookworm

Piperazines• Cause neuromuscular

paralysis• Stimulates phagocytic cells.

Piperazine,diethylcarbamazine

Ascariasis and pinworm infections

Avermectins• Block neuromuscular action• Hyperpolarize nerve and

muscle cells• Inhibit filarial reproduction

IvermectinFilarial infections, strongyloidiasis, ascariasis, scabies

MOA – Praziquantel

Before exposure to praziquantel, the schistosome is capable of avoiding the numerous antibodies directed toward surface and internally located antigens. A, Cross-section of the dorsal surface of a normal male schistosome. Within 1 to 2 seconds after exposure to praziquantel, the muscles of the schistosome contract because of a drug-induced influx of calcium ions into the schistosome tegument. B, The change in permeability of the schistosome surface toward external ions initiates the appearance of small holes and balloon-like structures, making the parasite vulnerable to antibody-mediated adherence of host leukocytes that kill the helminth.

Mechanisms of Action & Clinical Indications for the Major Anti-parasitic Agents – Anti-helminthitic AgentsDrug Class Mechanism of Action Examples Clinical Indications

Pyrazinoisoquinoline

• Calcium agonist • Causes tetanic muscular

contractions • Causes tegumental

disruption. • Provides synergy with host

defenses.

PraziquantelBroad-spectrum anthelmintic:cestodes, trematodes

Phenol • Uncouples oxidative phosphorylation Niclosamide Intestinal tapeworm

Quinolone• Alkylates DNA• Inhibits DNA, RNA, and

protein synthesis Bithionol, oxamniquine

Paragonimiasis, schistosomiasis

Organophosphate• Anticholinesterase • Blocks neuromuscular

action Metrifonate Schistosomiasis

Sulfated naphthylamidine

• Inhibits glycerophosphate oxidase and dehydrogenase Suramin Onchocerciasis

Lab Diagnosis and Treatment of Parasitic Disease Quiz

Thank you for completing this module

• I hope that I was able to teach the subject clearly.• If you have any questions, write to me: brad.mcgwire@osumc.edu

References

Primary Reference Source: -Medical Microbiology, 7th Ed. Murray, Rosenthal & Pfaller; Chapter 76, pages 715 – 721; Chapter 77, pages 722 – 725; Chapter 78, pages 726 – 727; Chapter 79, pages 728 – 736; Chapter 80, pages 737 – 744; Chapter 81, pages 745 – 758; Chapter 82, pages 759 – 777; Chapter 83, pages 778 – 795; Chapter 84, pages 796 – 805; Chapter 85, pages 806 – 816; Chapter 86, pages 823 – 824 (focus on Scabies); Chapter 80, pages 737 – 744.

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