SCACM Medical Parasitology: Neglected Tropical Diseases

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LYNNE S. GARCIA, MS, CLS, FAAM

[email protected]

SCACM

Medical Parasitology: Neglected Tropical Diseases

SPONSOR: MEDICAL CHEMICAL CORPORATION

www.med-chem.com

Consultant for: Medical Chemical, Meridian Biosciences, Savyon, AAB, CAP, Genetic Signatures, BioMérieux,

TechLab, Nanosphere 1

“TROPICAL” DISEASES ?THE MALARIA CAPERS, THE THORN IN THE STARFISH

NEW GUINEA TAPEWORMS AND JEWISH GRANDMOTHERS

“Tropical diseases are as American as the heart attack; yellow fever lived happily for centuries in Philadelphia; malaria liked it fine in Washington, not to mention in the Carolinas. The Ebola virus stopped over in Baltimore and Taenia solium settled in Brooklyn.” Most tropical diseases are now considered cosmopolitan.

Dr. Peter Hotez

Baylor College of

Medicine

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TROPICAL PARASITOLOGY ?

IN THE PAST to NOW …….

Malaria: California, North Carolina, Holland, marshlands of London

Hookworms: U.S. southerners, California miners, Alpine Swiss tunnel workers

Filariasis/elephantiasis: Charleston, North Carolina

American Trypanosomiasis: Texas to Detroit and Canada; triatomid bugs - other states; >300,000 cases suspected; serologic evidence of exposure

Leishmaniasis: American integumentary: South U.S. to North Argentina 3

WHAT CAN WE EXPECT IN THE

21ST CENTURY ?

Cataclysmic hot and cold storms; hot summers; more drought, floods, disease

World population 9 billion by 2025

Political turmoil and terrorism

People moving to U.S. and other industrialized nations

CO2 level double by 2100

10-20% of coastal land inundated

Wonderful world for insects and diseases they carry!

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MAJOR NEGLECTED TROPICAL

DISEASES (Ranked by Prevalence)

NTD Global Prevalence Population at risk

Ascariasis 807 million 4.2 billion

Trichuriasis 604 million 3.2 billion

Hookworm 576 million 3.2 billion

Schistosomiasis 207 million 779 million

Lymphatic Filariasis 120 million 1.3 billion

Trachoma 84 million 590 million

Onchocerciasis 37 million 90 million

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MAJOR NEGLECTED TROPICAL

DISEASES (Ranked by Prevalence)

NTD Global

Prevalence

Population at

risk

Leishmaniasis 12 million 350 million

Chagas’ disease 8-9 million 25 million

Leprosy 0.4 million Not Known

African Trypanosomiasis

0.3 million 60 million

Buruli ulcer (M. ulcerans) 0.05 million Not Known

Dracunculiasis 0.1 million Not Known6

Disability Adjusted Life

Years

DALY = YLL + YLD

Disability Adjusted Life Years

Time is used as the common metric for

mortality and health states

YLL Years of life lost due to mortality

YLD Equivalent years of healthy life

lost due to disability 7

KEY POINTS NEGLECTED TROPICAL DISEASES

Among most common infections of the poorest people

Nonemerging, ancient conditions

Chronic and disabling, profound stigma in society

High morbidity, low mortality

Disability-adjusted life years (DALYs) NTD = to those of HIV/AIDS, malaria, and tuberculosis

Coendemicity with HIV/AIDS and malaria

Ranking of the “gang of four” for death, DALYs– HIV/AIDS

– NTDs

– Malaria

– Tuberculosis 8

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Entamoeba

histolytica

Clinical Symptoms

▪ Intestinal: diarrhea, dysentery

▪ Extraintestinal: right upper quadrant pain, fever

Clinical specimens

▪ Intestinal: stool, sigmoidoscopy

▪ Extraintestinal: liver aspirate, biopsy, serology

Therapy

▪ Intestinal: Iodoquinol, Diloxanide furoate (cysts)

▪ Symptomatic: Metronidazole (trophozoites)9

ENTAMOEBA HISTOLYTICA

ENTAMOEBA DISPAR

Note: Ingested RBCs

Entamoeba histolytica (pathogen)

Entamoeba dispar (non-pathogen)

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REPORTING

Entamoeba histolytica Complex

If cysts or no ingested RBCs (trophs) are seen or immunoassay is not available:

▪ Entamoeba histolytica/E. dispar/E. moshkovskii, E. bangladeshi, Entamoeba histolytica COMPLEX

NOTE: Entamoeba moshkovskii (nonpathogen)

looks like Entamoeba histolytica/E. dispar; the

name is currently not added to the overall report.

It is more rare and controversy per pathogenicity

(Australia indicates some symptomatic patients)11

Blastocystis spp. (Stramenopile)

Pathogenic

• Central body form, large size range, quantitate

• Multiple nuclei around central body area

• 9/17 subtypes human, HALF PATHOGENIC, rare dissemination (IC)

• Associated with Irritable Bowel Syndrome (4 ST), urticaria; troph

adhesion to epithelium, release of cysteine proteases

• Mucosal sloughing, ↑intestinal permeability, pro-inflammatory cytokine response; subtype variations in pathogenicity

• Common: 1 (Giardia/Dientamoeba 2, 3)

• Prevalence 2016 Lebanon: Blastocystis, 63%; D. fragilis, 60.6%; Giardia,

28.5%; Cryptosporidium, 10.4% 12

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Giardia lamblia

(duodenalis, intestinalis)

Pathogen

• Teardrop shape, spoon

• Two nuclei, stain pale

• Curved median bodies

• Linear axonemes

• Pathogen, 19,733 in 2005

• Water, food borne

• Typical motility, but caught in

mucus

•Fecal immunoassays may

require 2 stools for POS13

Dientamoeba fragilis: Pathogen

• Very pleomorphic, 1 or 2 nuclei

• Nuclei fragmented chromatin or solid

• Pathogenic, transmitted via helminth eggs

• Cyst: animal reservoir, permanent stain

• As common or more common than Giardia

Cyst

~1%

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Pentatrichomonas hominis (NP)

Trichomonas vaginalis (P)

• No cyst forms

• Note different position of undulating membrane

• Nonpathogen (GI tract) and pathogen

(urinary/genital tract) (possible urine

contamination with stool – sexual abuse!!)

NP

P

P. hominis

T. vaginalis

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Cryptosporidium spp. – Clinical

10,500 Cases Reported in 2010

C. hominis (human), C. parvum (animals/humans) C. ubiquitum (zoonotic)

Immunocompetent – GI tract

▪ Self-limiting, profuse watery diarrhea

▪ Cramping pain, nausea, anorexia

Immunocompromised - Disseminated

▪ Severe diarrhea (3-6 liters/day), weeks

▪ HIV patients, CD4 cell count marker

▪ 180-200 cells/mm3 or higher, good

▪ Transplants, water outbreaks 16

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CRYPTOSPORIDIUM SPP.

C. hominis, C. parvum

Modified acid-fast:

stool specimen;

note

sporozoites, 4-6 µm

FA combo reagent

for

Cryptosporidium

and Giardia

Cyclospora 8-10

µm; Crypto 4-6

µm; Artifact ~1

µm; Mod acid-fast

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Cyclospora cayetanensis (Lab confirmed) 1,110 Cases (1997-2008)*

*Does not include year of big outbreaks, 1996 – U.S.

Immunocompetent – GI tract

▪ Malaise, fever, watery diarrhea

▪ Fatigue, anorexia, vomiting, weight loss

Immunocompromised – May disseminate

▪ Relapses for many weeks – in sputum

▪ Up to 12 weeks, biliary disease – AIDS

▪ TMP-SMX effective

BioFire FilmArray (Crypto, Cyclo, E. histo)

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North American Cyclospora

cayetanensis Outbreaks

1990 Tap water 1995, 1996 Fresh Guatemalan raspberries 1996 Basil-Pesto pasta salad, fresh basil 1997 US, Canada raspberries, mesclun 1997 Import voluntarily suspended 1998 US, Canada, fruit salad, raspberries 1999 US, chicken pasta, tomato basil salad 2000 Imported raspberries 2001 Canada, Thai basil 2004 Guatemalan snow peas 2005 US, fresh basil 2009 Cruise ship – multiple countries 2013 US, primarily cilantro, salad mix 2015 US, 29 states, 476 cases, Aug 17, ‘15

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MICROSPORIDIA

Pathogen (now Fungi)

Group of obligate intracellular, sporesfungi: 10 cases up to 1985

Phylum Microspora, 160 genera, 1,300 spp

Genera (8), 14 species = human pathogens

Possibilities include person-to-person and animal-to-person – Insects??? (water & foodborne; widespread antibodies)

Questions remain (reservoir hosts, congenital infections): as of 2015 20

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Microsporidia Diagnosis

Order: Stool & Urine

Modified trichrome stains (chromotrope)

10X amount of chromotrope 2R, dye in routine Wheatley’s trichrome (O&P)

Tissue Gram stains recommended

PAS, silver stains acceptable, H&E NO

Calcofluor, but non specific (stool)

Fecal immunoassays under development; available in Europe 21

Microsporidia – Polar Tubule

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MICROSPORIDIA

Intestinal Tissue Urine: Calcofluor White

Spores in NA aspirate

Spores, muscle Corneal button Corneal stroma

Eye Infections Cytospin

Gram stain

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Microsporidia Genera – Clinical(Eye infections mimic herpes virus stromal

keratitis)

Enterocytozoon bieneusi - IMPORTANT• Enteritis, cholangitis, cholecystitis,

pneumonia, bronchitis, sinusitis, rhinitis

Encephalitozoon intestinalis - IMPORTANT• Enteritis, cholangitis, cholecystitis, nephritis, urinary

tract infection, sinusitis, rhinitis, bronchitis, keratoconjunctivitis, disseminated

Pleistophora, Trachipleistophora, Vittaforma, Anncaliia, Microsporidium, Brachiola, Tubulinosema

• Hepatitis, peritonitis, encephalitis, urinary tract, intestinal, keratoconjunctivitis, sinusitis, rhinitis, disseminated infection 24

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Naegleria fowleriPrimary Amebic Meningoencephalitis

(PAM) – Neti pot sinus irrigation

28-year-old male developed PAM after a history of

irrigating sinuses daily with tap water and Neti pot

Admitted with severe headache, vomiting, fever, neck and back pain; CSF = bacterial meningitis; antibiotics

Wet mount of CSF = amebae; patient expired

51-year-old female PAM after 3 days of altered mental status, nausea, vomiting, high fever

Died 4 days later; Neti pot use; faucets PCR +

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PATHOGENIC FREE-LIVING

AMEBAE: Acanthamoeba

Environment Soil, air, fresh water, salt water, sewage

Washing the face in pond water, sand/dust

in eye, inhalation, traumatic injection, entry

through existing wounds or lesions

Disseminated InfectionsSkin, brain, bones

Rhinosinusitis, keratitis, otitis, vasculitis, endophthalmitis reported in HIV infected persons

Skin lesions present in absence of CNS involvement

ImmunocompromisedAIDS, lung, kidney, or liver transplants

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PATHOGENIC FREE-LIVING AMEBAE

AGAR PLATE CULTURE

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NEGLECTED TROPICAL DISEASES

Arctic Canada, Alaska

Trichinosis (Trichinella spiralis nativa)

– Inuit, 150,000 indigenous people (rely on hunting, fishing); Europe – horse meat

– Absence of fruits, vegetables (sea mammals, polar bears)

– 60% polar bears infected, walrus often the cause

Toxoplasmosis: 60% pos (seal, caribou)

Echinococcosis

– Cystic: (E. granulosus) (wolves, sled dogs), 12,000 Inuit

– Alveolar: (E. multilocularis) now moving into Canada, Dakotas, Montana, Wyoming

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ECHINOCOCCUS

MULTILOCULARIS

(Alveolar Hydatid Disease)

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ECHINOCOCCOSIS

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MAJOR NTDS – U.S., Canada

(Ranked by Prevalence) * No data: Dientamoeba

Blastocystis

NTD Estimated No. of Cases

Giardiasis* 424,120 - 2,120,600

Toxocariasis 323,000 - 500,000

Chagas’ Disease 150,000

Strongyloidiasis 68,000

Cysticercosis 2,800 – 41,400

Cystic Echinococcosis 12,000

Congenital Toxoplasmosis 400 – 4,000 per year

Trichinellosis 1,500

Alveolar Echinococcosis <10032

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NEGLECTED TROPICAL DISEASES

Mexico and the Caribbean

Onchocerciasis: (Onchocerca volvulus) southern states

of Chiapas, Oaxaca

– Use of ivermectin widely used, no new cases of blindness

Hookworm, other soil transmitted helminths

Cutaneous leishmaniasis cases on the increase

Chagas’ disease: >1,500 new cases (1990-2005)

Amebiasis (Entamoeba histolytica)

Caribbean: Lymphatic filariasis, schistosomiasis,

hookworm33

NEGLECTED TROPICAL DISEASES – U.S.

Soil Transmitted Helminths (1)

Toxocariasis

Toxocariasis (ingestion of Toxocara eggs – dog ascarid parasite)

– Visceral Larva Migrans

– Ocular Larva Migrans (retinoblastoma)

– Covert Toxocariasis (asthma); link in Europe

1970’s: 4.6-7.3% children (African-Americans –30%), various areas within U.S.

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NEGLECTED TROPICAL DISEASES – U.S.

Soil Transmitted Helminths (2)

Toxocariasis

1990’s: Connecticut rural, urban areas – 10%

serology pos; poor Hispanic children (up to 50%

in Bridgeport), 27.5% playgrounds in Wallingford

contaminated with Toxocara eggs (2005)

New York City: 5% children tested for lead were

positive for previous T. canis infection (1987)

Possibility of 500,000 inner city American

children have/had toxocariasis (major East Coast

and Southern cities)35

TOXOCARIASIS

VLM, OLM, NLM

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NEGLECTED TROPICAL DISEASES – U.S.Soil Transmitted Helminths (3)

Strongyloidiasis

Strongyloidiasis (enteritis and diarrhea; fatal disseminated disease in immunocompromised)

Chronic >40 yrs, steroids, transplants

Endemic rural Appalachian region of U.S. (6.8 mil); estimated 68,000 cases (Strongyloides stercoralis)

Eastern Kentucky and Tennessee: 1-4% infection

Worldwide: 30 million in Asia, Africa, Americas

Sputum Stool

Colon

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Strongyloides stercoralisCulture good sensitivity (serology fair) – results vary

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DIROFILARIA SPP.

IN U.S.

Dog heartworm, mosquitoes

Human subcutaneous nodules, lung parenchyma “coin lesions” –x ray

Ocular, inflammation, pain, blurring

No microfilariae in blood, serologies poor

Surgical/autopsy worm ID

Often misdiagnosed, harmful interventions

Emerging zoonosis in US, many dogs positive

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RARE INFECTIONS in U.S.

Baylisascaris procyonis – Raccoon

Serology: CDC

Raccoon ascarid, zoonotic disease; eggs/juveniles

Human: egg/dirt ingestion; not reportable disease

–Young children, VLM, NLM

–Lethargy, coma, blindness, death

–Larval growth (2 mm); very vigorous

Diagnosis: (larvae in tissues); raccoon latrines, many extremely resistant eggs; not killed 44ºC

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LIVER AND LUNG TREMATODES

(Flukes)

>50 million people infected, >1.1 billion exposed

Aquaculture: 48.2% (2012), water/snail exchange

Life cycles tend to be complex with one or more intermediate hosts as well as definitive hosts (require freshwater snail in life cycle)

Humans serve as the definitive host - immigrants

Ingestion of metacercariae encysted on plant material or within fish, crabs, crayfish, etc.

Most well known infections: Clonorchis sinensis, Opisthorchis, Fasciola, and Paragonimus spp.

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CLONORCHIS SINENSIS (Chinese Liver

Fluke)

Pathogenic: Yes, worm burden, cholangiocarcinoma

Acquired: Ingestion of infective metacercariae encysted in raw or poorly cooked freshwater fish (aquaculture)

Body site: Bile ducts and liver

Symptoms: None to acute pancreatitis, biliary tract obstruction

Clinical specimen: Stool

Epidemiology: China, Japan, Korea, Malaysia, Singapore, Taiwan, Vietnam, human to human; animal to human (dogs, cats, fish-eating mammals)

Control: Improved hygiene, fecal waste disposal, adequate cooking of freshwater fish 43

BLOOD TREMATODES

(Flukes)

200 – 300 million people in 77 countries

Rounded with separate sexes, blood vessels, non-operculated eggs, no encysted metacercariae

Life cycles tend to be complex, requiring freshwater snail in life cycle

Humans serve as the definitive host

Skin penetration by cercariae released from the freshwater snail.

Most well known infections: Schistosoma mansoni, S. haematobium, S. japonicum 44

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SCHISTOSOMA SPP.

(Blood Flukes)

Potential diagnostic problem: premature hatching

Concentration: use saline, not water (stool, urine)

Note expansion of miracidium larva once

released from shell.

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BABESIA SPP.

Babesia divergens: Europe

– Rare, but 42% mortality

Babesia microti: US, parts of Europe, Japan

– Northeast, south to New Jersey; 5% mortality

– Most cases mild; more severe in immunosuppressed

WA1, CA1, MO1 (Babesia divergens-like), states

– Very serious in splenectomized patients

– Related to canine pathogen, B. gibsoni

WA1, CA5: Now Babesia duncani

– More pathogenic than B. microti

Ixodes

scapularis

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Other blood parasites: Babesia spp.

Maltese Cross varies

Artifacts Plasmodium falciparum

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LEISHMANIASIS – KEY FACTS~350 Million in 88 countries at risk

Cutaneous: macrophages of skin

Old World: L. tropica, L. major, L. aethiopica

New World: L. mexicana, L. braziliensis

Mucocutaneous: macrophages of skin and buccal cavity or nasopharynx

L. braziliensis

Visceral: macrophages of spleen, liver, bone marrow (reticuloendothelial system)

L. donovani, L. infantum

American integumentary: cut/mucocut presentations on American continent

South U.S. to North Argentina

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CUTANEOUS LEISHMANIASIS

: CLINICAL PRESENTATION

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INTEGUMENTARY LEISHMANIASIS

L. mexicana Complex

Common throughout Mexico, Belize, Guatemala and southern United States (Arizona, Texas)

Forest rodents (wood rats) are important hosts

Prolonged exposure = “chicleros” live in forest collecting chewing gum latex (30% infected in first year), timber cutters, road builders, farm workers

Two culture-positive and four PCR-pos rodents Leishmania-positive. Isolates extend geographic and ecologic range of enzootic leishmaniasis in the United States and represent a new host record. Now in Tucson, Arizona. South US to North Argentina.

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MUCOCUTANEOUS/INTEGUMENTARY

LEISHMANIASIS

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CUT/MUCOCUT LEISHMANIASIS:

DIAGNOSIS

Organism isolation by smear or culture; histology

Lesion sampling: dental broach, slit scrape method, aspiration of lesion edge (multiple samples required), biopsy

Tissue sampling better than other smearing techniques; culture positive in 80% of cases

LD bodies seen in about 30% of patients; many lymphocytes/plasma cells in wet ulcerative lesions; dry lesions = granulomas / fewer cells

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AMERICAN TRYPANOSOMIASIS

(No longer South American)

Chagas’ Disease – Trypanosoma cruzi, MD suspicion

Transmission –feces of triatomid bugs (kissing bugs,

reduviid bugs); U.S. bugs infected; POTENTIAL; possible

anaphylaxis; Chagas Detect Plus Rapid (InBios) POC

Bug’s feces contact site of bug bite or mucus membranes;

transfusions, congenital infections

Mexico, Central, South, and North America (Texas, LA,

Georgia, California); rural areas; dogs, cats, opossums,

rodents, armadillos important reservoir hosts

Latin America: #1 impact on health, social systems; dogs

sentinel animals for organism reintroduction 53

CONFIRMED BLOOD DONATIONS:

CHAGAS’ DISEASE

Spread of Triatomid Bugs

>300,000 infected with T. cruzi in US; 300 new

congenital infections in US/yr; Canada also high

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AMERICAN TRYPANOSOMIASIS–

CLINICAL DISEASE

Initial infection often asymptomatic –years later chronic

form of disease; acute disease, children

Unilateral periorbital edema (Romaña’s sign)

Lesion, fever, lymphadenopathy, myocarditis,

hepatosplenomegaly, meningoencephalitis

Chronic disease = cardiomyopathy, congestive heart

failure or arrhythmias; dilation of esophagus or colon;

“MEGASYNDROME”

xenodiagnosis

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TOXOPLASMA

GONDII

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INFECTIONS IN HIV+ INDIVIDUALS

Toxoplasmosis

Toxoplasmosis; enhance HIV-1 replication

15.8% US positive for IgG antibodies; at risk for reactivation of latent infection

–Oocysts in cat feces, rare/raw meats,

in utero, transfusions, transplantation

–Most infections asymptomatic

–Severe CNS problems in compromised

Diagnosis problems: serologies

Tissue isolation hard to interpret57

SUMMARY

“Tropical Parasitology” may be somewhat misleading, since these diseases do not seem to be confined to particular areas of the world.

The world is not as compartmentalized as we may think.

Potential changes in the 21st century will support the spread of these diseases in many areas of the world.

The Neglected Tropical Diseases (NTDs) and their impact on global health will become more widely recognized and more important.

The expansion of mass drug delivery systems and vaccine development will continue to be supported by the need to control and eliminate these infections.

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10 LEADING CAUSES OF GLOBAL DALYs*DALYs (the number of healthy life years lost from

disability or premature death) lost annually.

Lower respiratory infections 91.4 million

HIV/AIDS 84.5 million

Unipolar depression 67.3 million

Diarrheal diseases 62 million

Ischemic heart disease 58.6 million

NEGLECTED TROPICAL DISEASES 56.6 million

Cerebrovascular diseases 49.2 million

MALARIA 46.5 million

Road traffic accidents 38.7 million

Tuberculosis 34.7 million59

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