Medical Parasitology

.- =ay be used, however, for the detection and cultivation of - :::::estinal amebas and flagellates (Entamoeba histolytica, =_=:amoeba fragilis, Trichomonas hominis, etc.) and the and tis sue flagellates (Leishmania donovani, -" ;>aDo soma cruzi, etc.).

e~f9

MEDICAL

PARASITOLOGY

The field of parasitology in its broadest sense is concerned with those organisms, either animal or plant, living on or in other organisms which serve as their hosts. Although it is true that bacteria, rickettsiae, fungi, spirochetes, and filtrable viruses are also parasites, parasitology in its restricted sense includes only animal parasites which, by their residence in or attachment to the body of the host, serve as agents or vectors of disease. Some parasites may cause little or no damage to the host. These should not be ignored, however, since careful differentiation of pathogenic from nonpathogenic types is necessary if diagnostic confusion and mistakes are to be avoided. Importance of Morphologic Identification: The recognition and differentiation of the animal parasites of man demands primarily a knowledge of their morphology. Except in a comparatively few instances the diagnostic cultural, biochemical, and serologic procedures so commonly utilized by the bacteriologist are not available to the parasitologist. Inasmuch as the diagnosis of a parasitic infection rests upon the demonstration of the parasite - and since this demonstration depends upon morphologic recognition - it is essential that those responsible for the handling of clinical specimens be well-trained microscopists. Proficiency in this phase of laboratory work is gained only by extensive practical experience with properly collected clinical specimens. The diagnosis and differentiation of the diseases caused by proto.zoa, whether intestinal or systemic, present particular difficulties. This is especially true with respect to the differentiation of the intestinal amebas. These difficulties may be overcome only if the microscopist entrusted with the examinations has a thorough knowledge of the morphology and the habitat of the various organisms parasitizing man and animals. Cultivation Technics: The use of cultivation procedures as a means of diagnosis in parasitology is limited. Effective methods are available *Associate Professor of Parasitology, University Rico School of Medicine, San Juan, Puerto Rico.

of Puerto

- _::, .opc Technics: -erologic tests are often of practical value in demonstra·-:l:e presence of a particular protozoan or helminthic -:-a.site. The complement fixation test for amebiasis; the - -.: ~ement fixation. agglutination, and precipitin tests for aniasis; the complement fixation test for Trypanosoma , infections; and the dye test for toxoplasmosis have - ••ed of distinct diagnostic benefit. Serologic tests as well ; ..;:~adermal tests may help in the diagnosis of certain he1- -:hic infections, such as trichinosis, echinococcosis, and .' tosomiasis, where it is difficult and sometimes impos- e to demonstrate the causative organisms. ~:

patho10gic Technics: Certain parasites produce characteristic tissue changes cl::make possible a histopathologic diagnosis. Biopsy of " rectum and/ or the urinary bladder and liver is often the re 'able method of diagnosis in schistosomiasis. Muscle ;sy is indicated for trichinosis.

:::-_"emiology: T::e solution of problems associated with the epidemi- - -' prevention, and control of parasitic diseases of man ::':::'res a knowledge of the geographic distribution, life ;:-::-:es, and habits not only of parasites but of their vectors --:. ::os s as well. The elimination of parasitic infections -=. a community thus depends upon the combined efforts of -~ '-' oratory worker, the field investigator, and the ~:-'an.

Or: the following pages information pertinent to the more --~ant parasites of man is presented, as well as a con~=:ion of the diseases produced by them and a description -" :aboratory methods and procedures employed for their -~on in clinical specimens. For more extensive informathis subject the following references should be consulted: :aust, E. C., Animal Agents and Vectors of Human :>:'sease. Lea and Febiger, 1955. _ :aust, E. C., and Russell, P. F., Craig and Faust's ..inical Parasitology, 6th ed. Lea and Febiger, 1957. ·ckie, T.T., Hunter, G.W., and Worth, C.B., A ual of Tropical Medicine, 2nd ed. Saunders, 1954. ?essoa, S. B., Parasitologia Medica, 5th ed. Livraria :::Gt, Guanabara, Koogan. Rio de Janeiro, 1958.

~

Sporozoites (Through coelomic

_____ Oocyst

reach

salivary

Saliva from ,~ mosquito injected into hu an host

cavity

Pre- erythrocytic _ exo-erythrocytic ••

_________

in the liver

glands)

t

MeroLites~

(Penetrates stomach wal1 of mosquito and encysts)

ENDOGENOUS PHASE IN MAN

EXOGENOUS PHASE IN MOSQUITO

t

Sexual Cycle (Sporogony)

Ookinete

1

(Enters red cell)

/ Segmenter

i

Asexual Cycle (Schizogony)

CLINICAL MALARIA

Schizont '~

j

Signet Ring Mature /Trophozoite Trophozoite

Human

~Microgamete Zygote (fertilization) ~-----

(r:f)

(in stomach) Macrogamete

A<

blood _Microgametocyte enters mosquito __ Macrogametocyte

(" ) ~.

Length of Sexual Cycle (in mosquito)

Species

P. vivax (tertian) P. malariae (Quartan (malignant P. falciparum

tertian)

(r:f)

(differentiation) (" )

Incubation

7 - 10 days 18 - 21 days 7 - 10 days

(Prepatent) Period (in man) Pre-erythrocytic Cycle 8 - 15 days 23 days (averaf!e) 8 - 15 days

Plasmodium vivax (benign tertian, vivax malaria). World-wide. Prevalent in temperate zones. Commonest type.

Enlarged pale cell; SchUffner's dots (eosinophilic) in mature forms. Multiple infections common.

All forms peripheral blood.

Plasmodium malariae (quartan malaria). Comparatively rare. Temperate zone and subtropics.

Not enlarged; calor normal. Multiple infections rare.

All forms peripheral blood.

Not enlarged; color normal. Maurer's dots red, larger and fewer than SchUffner's. Multiple infections very common.

Only rings and gametocytes in peripheral blood.

Plasmodium falciparum {malignant tertian. estivo-autumnal malaria}. Predominates throughout tropical regions.

of the different

(0 .'.

plasmodia

Cycle

Has a predl:'eCUQIl for reucu;.ocytes but does Dot attack mature erythrocytes. This limits the parasitemia to 8.000 to 20,000 per cu. mm . of blood.

in

Attacks primarily older erythrocytes which already are about to be removed by the normal process of blood destruction. Parasitemias less than 10,000 per cu. mm. of blood.

in

Ring Trophozoite characteristics

Length of Asexual (in man)

48 hours 72 hours 36 - 48 hours

P .L-as;.~ed Erythrocyte

*The invasive

and cycle

parallel

the marked

Macrogametocyte variations

Microgametocyte

in the severity

Invades all erythrocytes regardless of age. Parasitemias may reach 500,000 per cu. mm. P. falciparum also causes red cells to agglutinate, forming thrombi and emboli. and causing them to adhere to the capillary walls, with resulting capillary obstruction and severe ischemia.

of the disease.

Found world-wide, incidence depending on the sanitation level. Entamoeba histolytica and Dientamoeba fragilis, 9-50% of population. Giardia lamblia, 15"/0. Balantidium coli, comparatively rare; mainly an infection of swine. Parasite

Entamoeba histolytica Giardia lamblia Balantidium coli Dientamoeba fragilis

Enters

Man

Infective Form

Life CyC'1ein Man

Becomes Mouth trophoz cite-

Cyst

by

in contamination intestine

?

May invade mucosa and/or other organs
Exit

Cysts andlor trophozoites in feces

Reservoir

Host

Man, dog*, cat*. rat*. pig*, monkey*. Man. Man, pig, monkey.

Trophozoites in feces

? - Probably

Non-pathogenic protozoa are commonly found in the feces of man. They are of diagnostic interest must be distinguished from. the pathogenic forms. The commoner organisms: Amoebae - Entamoeba can a, Iodamoeba biitschlii; Flagellates - Chilomastix mesnili, Trichomonas hominis.

man.

only because they coli, Endolimax

Trichomonas vaginalis (Trichomonas genitalisJ, known only in the trophozoite stage, inhabits the human vagina and urethra of male and female. It is thought to produce a vaginitis, with severe itching and mucopurulent discharge. Some workers believe the streptococci usually associated with the parasite cause the infection. T. vaginalis is worldwide in distribution, occurring in 10-40"/0 of women examined. It is transferred probably by coitus. A flagellate identical with T. vaginalis is found in the vagina of monkeys.

Glardiasis. G. lamblia Balantidiasis. Bal. coli Dientamoeba diarrhea. D. fralZilis Amebiasis. E. histolytica

Diarrhea.

abdominal

pain, dis comfo rt.

Limited to intestine where parasites sembling amebic dysentery. Not proved to invade tissues. amebiasis.

localize .. with pathology and symptoms

Diarrhea,

abdominal pain, discomfort

re-

as in mild

Exam.u:e feces by smear, cancentratlOD. Examine feces by smear .. concentration, culture. Examine feces by smear, culture.

F lask- shaped ulcers formed in mucosa of large bowel which appear as pinpoint Examine feces by smear, concentradots on surface, with little inflammation but extensive undermining below surface. Localize in cecum and appendix. anemia. There is no tion, culture. Blood: Leukocytosis, eosinophilia in uncomplicated protozoan infections. Symptoms: Dysentery, bloody Abscess and ulcer gas distention; appetite material (as for diarrhea, or constipation; abdominal pain and discomfort, feces ). disturbance, weight loss, headache, nervous manifestations, localized tenderness. Complications: 'Liver abscess (single or multiple) with liver enlargement and conPain, swelling, leukocytosis, Lung abscess primary or gestion. anernia, fever. secondary to liver abscess. Peritonitis (bacterial) with usual manifestations. manifestations depending upUlcerations and abscesses of other organs or tissues, on site infected. 2 mos. -years. ) (lncubation Period: Acute~ 8- 10 days; chronic, Prevention: Protect food and water supply. Find and treat carriers. Toxoplasma gondii is a crescent-shaped protozoon found intracellularly in tissues and free in body fluids~ which causes toxoplasmosis. Method of transmission to adults is unknown. The fetus in utero is infected across the placenta. May be symptomatic or latent. Acute form involves all the viscera and is usually quickly fatal. Subacute or chronic toxoplasmosis shows predominantly C.N.S. manifestations and eye signs. Diagnostic laboratory methods: Isolation of the organism from C.S.F. or autopsy material. The complement fixation test and the Sabin-Feldman dye test are the most reliable serologic procedures. C. S. F. protein in subacute and chronic forms is high. usually over 500 mg./! 00 m!.

Parasite and Distribution

Fly Host

Leishmania donovani. Phlebotomus Asia, Africa, Brazil, (sandfly) Middle East. Mediterranean basin. Leishmania tropica. Phlebotomus Asia. S. and Cent. Am. , (sandfly) Middle East. Europe. Leishmania Phlebotomus brasiliensis. (sandfly) Central and S. America.

Enters

Man

injected with bite of fly.

Fly bites; metacyclic trypanosome injected with saliva.

Trypanosoma cruzi. Central and S. America, United States.

Metacyclic trypanosome in feces contaminates skin.

Disease and Etioloe:v Kala-azar (visceral le ishmaniasis). L. donovani. Oriental sore (cutaneous leishmaniasis ). L. trooica. American leishmaniasis (espundia, uta. forest yaws; mucocutaneous leishmaniasis) . L. brasiliensis. Sleeping sicknesS. West African T. gambiense; East African T. rhodesiense. Chagas' disease. T. cruzi.

Exit

Becomes leishmania in endothelial cells of skin. Becomes leishmania in endothelial cells of skin and (secondary) mucous membranes of nasopharynx .. Trypanosome in lymph and blood; later in spinal fluid.

Becomes leishmania in tissue cells and trypanosome in blood stream.

FLAGELLATE

Sucked

Becomes

into

leptomonas

fly

in the

with

intestine

blood

of the fly. Becomes crithidia and metacyclic trypanosome in the intestine and salivary !!lands of fly. Becomes crithidia and metacyclic trypanosome in the intestine of the bue.

of host.

Sucked into bug with blood of host.

----D.FECTIONS

OF

hyperplasia of cells of the reticuloIrregular fever spleen and liver. sweating, diarrhea, edema, 2-9 months.) (Incubation Period:

Itching Endothelial cells and lymphoid tissue of skin parasitized. red papule ~ scaling ----.. crusted ulcer ~enlargernent of Several (Incubation Period: May be multiple. ulcer ----..healing. davs to months. Iproducing Initial lesions similar to oriental sore, but this enlarges, Destructive and deforming secona weeping "saucer" ulceration. Have fever, pain, dary lesions occur at mucocutaneous junctions. lnoculable and auto-inoculable. malaise, and anemia. Unknown.) (Incubation Period:

Local lesion at fly bite followed by fever. adenitis, rash, transitory May fulminate (T. rhodesiense) or go on to meningoedemas. with mental and physical wasting encephalitis and meningomyelitis, (Incubation Period: leading to coma and death (T. gambiense). 1-2 weeks.) rhodesiense, T. !!ambiense. 1-3weeks;T. with tissue cell degenAcute (children) and chronic (adult) disease. eration leading to fibrosis (heart. brain, live-r), enlarged liver and (Incubation Period: May end in cardiac failure and death. spleen. 1-2 weeks.)

_.

Reservoir Host

Man, dog, cat, monkey. rodents. Man, dog, cat, monkey, rodents.

Man, domestic animals. Man,

wild game animals. Man, dog, cat, fox. armadillo. opossum (Dide lphis), rodents.

iAC

Clinical Features causing Chronic febrile disease, endothelial system. particularly (spiking twice a day) with chills, cachexia, leukopenia. anemia.

Cycle in Fly

Man, dog, cat, monkey, rodents.

leptomonas

BLOOD

I

Becomes leishmania in cells of reticuloendothelial system.

Plug of

Trypanosoma "Glossina palpalis gambiense. Central and W. Africa. (tsetse flv) Trypanosoma Glossina rhodesiense. morsitans Central and E. Africa. (tsetse fly) Panstrongylus megistus (kissing bug) and other reduviid bugs

Life Cycle in Man

Lab. Diagnosis Blood: Thick and thin smears. culture. Napier and Chopra Sternal marrow serum tests. and splenic aspirate: Stained smears. Stained Ulcer curettings: culture. smears,

Ulcer curettings: culture. smears,

Stained

Blood: Thick and thin smears. concentration, culture. Spinal fluid: Smears of sediment. Lymph node fluid: Smears, culture. Blood: Thick and thin smears, concentration, culture. See p. 322. Xenodiagnosis:

Parasite and Distribution Enterobius vermicularis. World-wide. Trichuris trichiura. World-wide. Trichinella spiralis. Pork - eating countries.

Man Ingests Egg

After Liberation

in Duodenum

Adult Goes To

Exit

Reservoir

Larvae become adult in ileum or lower bowel.

Periana region to lay eggs.

-'"ggs on perIanal skin; in feces.

Man

Larvae attach to villi and mature.

Cecum to lay eggs.

Eggs in feces.

Man

Feeding of uncooked garbage to hogs perpet-

Hog, rat, flesh-eating mammals.

Larvae in raw pork (ingested also by rats). Egg

Larvae penetrate mucosa, mature, (Cycle ends in females produce larvae; enter blood man.) stream; travel over body; encapsulate in striated muscles (diaphragm, intercostals. eye, etc. ). Ascaris Duodenum and lumbricoides. right heart jejunum to lay .J. World-wide. eggs .. (StrongyNecator Larvae Filariform alveoli of lungs Ioicles penetrates americanus, * larvae pene- .enter wall to lay eggs blood~ Ancylostoma trate skin. up trathea to esophagus which hatch to duodenale. *§ Egg (rare). stream ,rhabditiform Strongyloides swall}wed ~ intestine larvae. ) stercoralis. t

Host

uates the

organism. Eggs in feces.

Man

Man

Rhabditiformt larvae in feces.

Man,

dog

*Hookworm Soil Cycle: Rhabditiform larva-+ filariform larva~enter man under favorable conditions. tStrongyloides larvae may become filariform larvae in the bowel or perianal surface and reinfect directly (hyperinfection). Usually pass to soil: Rhabditiform larva---+adult or filariform (infective) larva ~ man (under (Soil Cycle) favorable conditions). adult ---+- egg_ rhabditiform larva §Found in tropical and subtropical climates where sanitation is poor and favorable conditions exist for the parasite.

t

t

Disease and Eliology pinEnterobiasis. worm infection. Enterobius vermicularis Trichuriasis. Trichuris trichiura Trichinosis. Trichinella spiralis

C!.icical Features Symptomless to slight s)'IDptoms oi nervousness, itching and irritation. Eosinophilia. Secondary occur. Depend on worm load. resulting in cachexia. Worms: --diarrhea,

Symptomless

insomnia. perianal bacterial infection may

to protracted,

debilitating

diarrhea,

Lab. D"agnosis Cellulose tape swab: p. 321. Feces: Direct smear, concentration. Feces: Direct, standardized smear to count eggs (see p. 318) . Biopsy material: Digestion and examination. Feces: Direct smear (rarely), concentration, intradermal test. Feces: Direct smear, concentration. Sputum: Direct smear. Feces: Direct standardized smear to count eggs (see p. 318).

Irritate and inflame intestinal mucosa, with nausea, vomiting, pain. Larvae: Muscu1ar pains, edema of eyes, face, hands. Irregular, persistent fever; breathing, swallowing, speech difficulties, rash, eosinophilia. Encysted larvae: Cachexia, toxic edema, dehydration, collapse. Ascariasis. Larvae: Pneumonitis with cough, hemorrhages. lung consolidation. Ascaris Adults: Symptomless to serious intllstinal complications (obstruction, lumbricoides perioration). Nausea, pain, vomitin~, systemic toxemia, eosinophilia. Hookworm infection. Larval ground itch with edema, erythema, vesicular or papular eruption. Necator americanus, Lung: Reaction similar to but milder than Strongyloides or Ascaris types. Ancylostoma Intestine: Heavy infections cause hypochromic microcytic anemia and duodenale eosinophilia. Strongyloidiasis. Skin: Petechial hemorrhages, congestion, edema. Intense pruritus, no Feces: Direct smear, Strongyloides inflammation. Lung: Larvae cause hemorrhage, cellular iniiltration, concentration. Intestine: Catarrhal inflammation of mucosa; stercoralis bronchial pneumonitis. Abdominal pain, tenderness, nervous m ani watery, mucoid diarrhea. festations, eosinophilia. Hyperinf'ection may cause dea th or chronic invalidism (see next page). Prevention: Trichinella - Proper meat handling. The U. S. GOZJer'7'.J11ent does not ~nspect jor' Tr'~ch~nella. Hookworm, Ascaris, Strongyloides - Protect food and water supplies. Prevent soil pollution by proper disposal oi feces. Eliminate infection reservoirs. Enterobius - Personal cleanliness, prevent dispersion of eggs, clean surroundings, mass treatment of family.

in U. S. ; Echinococcus Parasite and Distribution

l!ranulosus,

Entry of Egg In Inte s tine

Hymenolepis nana (dwarf tapeworm). World-wide. Taenia saginata (beef tapeworm). World-wide.

Ingestion (man).

Taenia solium (pork tapeworm~ World-wide. Echinococcus granulosus (hydatid worm). Sheep-raising countries. Diphyllobothrium Iatum (fish tapeworm). North Europe, Great Lakes region of North America.

Ingestion (hogs, man). ngestion (sheep, accidental ingestion in man). Water flea ingests freeswimming larva (coracidium) hatched from el!l!.

Disease and Etiology

3-160/0in endemic areas; Taenia solium,

Intermediate

of Larva

onc hosphere (1st

Maturation of Worm

Exit

Reservoir

(Entire cycle in one Eggs host.) Larva leaves in villi, attaches to lower feces to intestine, becomes adult. Into blood stream, Man ingests larva liberGravover body to encyst ated in intestine from raw id in muscles or organs beef; larva attaches to seg(cysticercus). intestine, becomes adult. ments per As for T. As for T. saginata. saginata. anus (Cycle ends in man.)

Hosts

Man; rats and mice; gerbille in Africa.

Definitive: Man. Intermediate: Cattle. buffalo, giraffes, llamas, goats. Man.

stage larva).

Larva encysts in hemal cavity, becomes procercoid larva.

Into blood stream, over body to muscles and organs (forms hydatid cys t). (Cycle ends in man.) Fresh-water fish eats water flea; larva escapes in intestine, enters muscles, becomes 3rd stage larva sparl!anum\.

Clinical

Sheep dog ingests from sheep carcass. Worm heads attach to intestinal wall; become adult.

Eggs Definitive: Dogs, in cats, wolves, ja.okals. feces Intermediate: Man, sheep, hogs, cattle.

Man ingests raw fish with Eggs sparganum; larva liberin ated in intestine, attaches feces to intestinal wall, becomes adult.

Features

Hymenolepiasis. Hymenolepis nana. Taeniasis saginata. Taenia saJ:(inata. Taeniasis solium. Taenia solium.

Symptomless to severe systemic toxeroia, depending on worm load. with or without diarrhea, and pain. nervous manifestations, Eosinophilia, May produce convulsions, insomnia, dizziness. Abdominal and hunger pains, chronic indigestion, weight loss, persistent diarrhea or alternating with constipation; nervous manifestations. Eosinophilia. (Incubation Period: 10-12 weeks.) Symptoms may vary with Cysticercosis: Intestinal: Same as T. saginata. Foreign body response with inflamnumber of larvae and site in tissues. mation followed by fibrosis and necrosis of parasite, later calcification. 10-12 weeks.) (Incubation Period: If in brain, epilepsy and convulsions.

Hydatid disease. Echinococcus granulosus.

Symptoms depend on location and type of cyst: Unilocular cysts usually cause inflammatory and fibrous tissue response, eosinophilia; pressure Hydatid fluid, escaping, can produce allergic reaction. symptoms if large. Alveolar cysts produce metastases wlth secondary cyst formation; a Osseous cysts cause rapid erosion of bone. Spontaneous malignant growth. cyst rupture mav cause violent reaction reScultinl! in death. Pain, weight loss, diarrhea. Symptomless to marked systemic toxemia. Severe macrocytic anemia like pernicious anemia found in a small number of cases; 700/0 from Finland where there is a tendency to this type of 10-12 weeks.) (Incubation Period: anemia; eosinophilia.

Diphyllobothriasis (fish tapeworm disease). Diphyllobothrium latum.

in the U. S.).

Hos t

Into duodenal villi, becoming cysti$:ercoid larva.

Hatches Ingestion (cattle).

Entry

hiJ:(hwhere pork is eaten (rare

Definitive

Host

Definitive: Man, fish-eating mammals. Intermediate: Diaptomus, various "pike" fish.

Lab. Diagnosis Feces: Direct smear, concentration. Direct smear Feces: not reliable. Recovery of gravid segments. Direct smear Feces: Recovery not reliable. Reof gravid segments. covery of larva by excision' and identification. Cyst contents, urlne, sputum: Direct smear. Blood: Complement fixation and precipitin. Intradermal test. X-rays. Feces: Direct smear, concentration.

Parasite and Distribution Schistosoma haematobium. Asia Minor, Tropical and N. Africa.

Schistosoma mansoni. P. Rico, Brazil, Venezuela, N. and Cent. Africa. Schistosoma japonicum. Japan, E. Asia, P.!. Fasciolopsis buski. E. and S. Asia. Fasciola hepatica. World-wide, sheepraising countries. Clonorchis sinensis. S. Asia. Paragonimus westermani. America~ Asia, N. and Cent. Africa.

Etiology esical schistosomiasis Schistosoma haematobium. Intestinal schistosomiasis. Schistosoma mansoni, Schistosoma japonicum.

Definitive Host Habitat Vesical and pelvic venous plexuses draining bladder.

Cercaria penetrates skin.

Exit Egg in urine or feces.

Intermediate Host Cvcle Outside Man Egg hatches to miracidium in water. .Invades appropriate snail. (Clonorchis larva ingested by snail.)

Branches of inferior mesenteric veins draining rectum and sigmoid.

Branches of superior mesenteric veins draining intestine.

Man, monkeys. Mouse, hamster (experimental).

in

In snail, becomes sporocyst, redia (except Schistosoma), cercaria.

Man, horses, cows dogs, cats, mice, water- buffalo.

~

Cercaria swarms from snail. Ingested metacercaria on water plant. Ingested metacercaria in raw fish. Ingested metacercaria in crayfish, crab.

+

Intestinal wall, abdominal cavity, diaphragm and liver capsule to distal bile caoillaries. Bile ducts.

Encysted in lungs, cavity, liver.

pleural

Egg in bile to

Egg in sputum or feces.

Encysts on water plants (Fasciola and Fasciolopsis), or Invades fish (Clonorchis), crayfish, or crab (Paragonimus), or directly attacks man (Schistosoma) .

)!un penetrallon: yractlcauy symp omless; 1 cnmg, erytnema, petectuae. Migration: Acute clinical episode with visceral and cutaneous hemorrhages; urticaria, asthma, transient pneumonic infiltrations of lungs. Deposition of eggs: Foreign body reaction; inflammation with fibrosis~ calcification or cyst formation with ulceration which may produce polyps or malignant changes. Bacterial infeclion often follows. Eosinophilia. S. haematobium eggs in bladder wall cause pain~ urinary frequency~ hematuria. S. mansoni and S. japonicum eggs in intestines cause abdominal pain, diarrhea with blood, mucus, and pus (schistosomal dysentery). If eggs go to secondary sites, they cause urethritis, cirrhosis, splenomegaly, infection of other organs or tissues. (Incubation Period: 1-3 months.) Fasciolopsiasis. Localized inflammation of jejunum or duodenum, followed frequently by ulceraFasciolopsis tion at sites of worm attachment. Diarrhea with foul- smelling stools; abdominal buski. pain. In severe infections eosinophilia, ascites, anorexia, nausea, vomiting, toxemia, prostration. (Incubation Period: 3-4 months.) Fascioliasis Worm migrations cause tissue necrosis and fibrosis. Vomiting, coughing, hepatica. Fasci- generalized abdominal pain. Urticaria, diarrhea, jaundice, irregular fever, ola hepatica. edema, anemia, and eosinophilia. (Incubation Period: 3-4 months.) Clonorchiasis. Parallel degree of infection. Vast numbers of parasites cause diarrhea, jaundice. Clonorchis cachexia, eosinop~lia. Proliferation and desquamation of biliary epithelium. sinensis. dilatation and thickening of the wall occur. Severe symptoms of liver d)rsfunction, recurring icterus with hepatomel!aly follow. (Incubation Period: 3 months.) Paragonimiasis. Lung: Parasites are embedded in subpleural cysts (eggs act as foreign bodies) Paragonimus with inflammation and fibrous capsule formation. Chronic cough with fever, brown sputum, hemoptysis, severe chest pain, bronchopneumonia or pleural fluid. westermani. May enter any organ to produce symptoms, e.g., abdominal pain, diarrhea, eosinophilia. Similar lesions in other tissues. (Incubation Period: 3 months.l

Sheep, cattle. other herbivores. Man, dogs, cats.

Man~ wildcats, foxes~ wolves, dogs, rats, pigs, weasels.

Urine: Direct smear, sedimentation for S. haematobium. Feces: Direct smear, concentration for S. mansoni or japonicurn. Rectal biopsy. Blood: Precipitin (circumoval or CHR) test (see p. 321). Feces: Direct concentration.

smear,

Feces: Direct smear, concentration. Duodenal drainage: Direct smear.

Sputum: Direct smear. Feces: Direct smear~ concentration.

Parasite and Distribution Wuchereria bancrofti. Tropics and subtropics. Loa loa. Tropical Africa. Onchocerca volvulus. Tropiclll Africa and Central A~erica. Dracunculus medinensis. Afrioa, India, Middle East, East Indies. Disease and Etiology Bancroft's filariasis. W. bancrofti.

Insect Host Culex and other mosquitoes. Chrysops I (de.er fly). Simulium (buffalo I gnat). Cyclops (water flea).

Enters MaI\ Filariform larva .injected with

Matures to Adult In lymph vessels and nodes becomes adult filaria. In subcutaneous tissues becomes adult filaria.

insect bite. Cyclops containing larva ingested by host.

Digested out of Cyclops in intestines; migrates intb tissues.

Exit Microfilaria Goes to MicroBlood stream (nocturnal or no filaria periodicity). is taken Blood stream (di- up by urnal periodicity). fly when (Remains in biting. subcutaneous tissues.) (Adult female worm dischar ges larva through skin "blister" into water. )

Fly Cycle Becomes infective larva in fly thoracic muscles. Migrates to proboscis.

Reservoir

Man

lnf ecti ve larva develops in Cyclops.

Filaria in afferent lymphatics or lymph nodes (of lower extremity, vulva, scrotum, epididymis, mammary gland) cause inflammation followed by intensely fibrotic re· action involving whole area in a maSs of scar tissue. Pain, fever, chills, toxemia. 'Blood: Thick eosinophilia. Chronic stage varies from microscopic lesion to lymph varicosity to and thin smears, marked elephantiasis. Non·pitting edema on pressure. (Inc. Per. : Months to years.) concentration Loaiasis. Fugitive swellings of skin due to migration of worm and edema. Common over nose. (see p. 322). Loa loa. Eosinophilia. May have albuminuria. (Inc. Per.: Months to years.) Nodule aspirate; Onchocerciasis. Small cutaneous fibrous skin nodules with filariae in center. Microfilariae may ioskin section. O. volvulus. vade eye, cause inflammation and blindness. Eosinophilia. IInc. Per. : Mos. to vrs.) Dracontiasis. Asymptomatic until reddish papular lesion appears on skin. Becomes a blister which Smear or ulcer washing. X-ray D. medinensis. bursts, liberating larvae. Itching, rash, asthma-like symptoms develop. Eosino· of worm in tissue. Iphilia. (Inc. Per.: 8-12 months.) Preventlon: f>rotectlOn agamst fly blte (Dracunculus: agamst Cyclops mgestlOn). EradlcatlOn of arthropods. Chemoprophylaxis and/or chemotherap of i abitants in e demic a.reas. __ ~ _

LABORA TOR Y EXAMINA FOR PARASITES

TION

FECES (See also p. 267.) I"••.lmen Collection: ollect specimen in a clean, dry container and examine loon as possible, preferably while still warm. Specimens hollowing administration of oil, bismuth, or barium are until factory. Repeated examinations are advisable; at least 1111'( • la six should be examined on consecutive days since the Ilml' of appearance of parasites in the feces is variable. If 1I\\I1'UH or blood streaks are present these must be examined 111"(' trophozoites or cysts may be' distributed irregularly IIIl ollghout the specimen. 'rvation and Shipment of Specimens to Be Examined for Trophic and Encysted Protozoa (Method of Brooke and Goldman): 1\ Iteagent: Modified Schaudinn's solution is prepared by ,nlxing 5 ml. of glacial acetic acid, 1. 5 ml. glycerol, and 03.5 ml. Schaudinn's solution (two parts saturated aqueous on re uric chloride and one part ~5"!o ethyl alcoho'1). Heat the above solution to 75° C. ;. while stirring, slowly cleI 5 Gm. polyvinyl alcohol (PVA) powder (Dupont's l~lvanol®, Grades 71-24 or 71-30). This final solution Hhould be clear and free of lumps after cooling. It is LIB d at room temperature, and lasts for several months. 11. I>reparation of Specimens: 1. ne specimen should be sent without above fixative to be used for detection of protozoan cysts and helminthic ova. This specimen can be used for temporar.y or permanent smears or for concentratioJ:! procedures (see p. 318). 2. Second specimen is pr'epared by thoroughly mixing with three or more parts of fixative in a small vial. To prepare slides a small amount of this fecal mixture (recent or months old) is spread thinly over about onethird of the slide. After drying for three or more hours at 37° C. or overnight at room temperature, the smear is stained by the iron hematoxylin method (see p. 319). This procedure is particularly suitable for the preservation and staining of the trophic forms of the intestinal protozoa. To obtain satisfactory stained fecal smears containing cystic s·tages the excess clear PVA solution is decanted from the vial and a small amount of the remaining fixed fecal material is placed on a piece of facial tissue or toilet paper. The excess PVA solution is allowed to absorb for five to ten minutes, leaving a moist fecal residue. Gently scrape up

1"'('

a small amount of the residue with the sharp edge of a broken applicator stick and smear with gentle brushing strokes on a slide. Drop smear immediately into 700/0 alcohol to which iodine has been added to produce a port wine color, and stain by the iron hematoxylin method (see p, 319), To insure satisfactory preparations of both cystic and trophic forms, the preparation of the two types of smears is recommended. Standardized Direct Fecal Smear Examination: Follow directions given by Beaver strictly (J. Parasito!., 35:125-135, 1949; 36:451-456, 1950). Place a glass slide over a calibrated photometer (a Weston Master is just as good) and set the reading at 20 f. c. Add a drop of saline and with the tip of an applicator comminute feces progressively until the needle moves down to the preset reading. A 2 mg. preparation is preferable. Cover with a coverglass and examine all the microscopic fields, counting the various eggs and looking for cysts and trophozoites of protozoa. A drop of iodine solution (Lugol's, Craig's, or D'Antoni's) may help identify some of the protozoa. This method thus replaces the ordinary direct saline smear, permitting the quantitation of worm loads as well as examination for all signs of parasites. Concentration: Parasites are often so few in number that they cannot be detected WIthout employing a concentration procedure. A. Formalin-Ether Sedimentation Technic (Ritchie): 1. Emulsify portion of stool (a mass 2 to 3 cm. in diameter) in 30 to 50 ml. saline. (For specimens that have previously been preserved in formalin, start with 2, below. Tap water may be used instead of saline in this case if desired. ) 2. Strain about 10 ml. of the emulsion through two layers of wet gauze into a 15 ml. centrifuge tube with a conical tip. The remainder of the emulsion may be used for other concentration technics, direct smears, or for preservation. 3. Centl'ifuge at moderate speed for a few minutes and then decant supernatant. 4. Resuspend sediment in' fresh saline, centrifuge, decant as before. Repeat as necessary, if supernatant is particularly cloudy. 5. Add 10 ml. of 100/0formalin to the sediment, mix thoroughly, and allow to stand for five minutes. 6. Add 3 ml. of ether, stopper the tube, and shake vigorously. 7. Centrifuge at low speed for about two minutes. Four layers should result: (1) at the bottom, small amount of sediment containing most of the parasites and eggs;

(2) a layer of formalin; (3) a plug of fecal debris on top of the formalin; and (4) at the top, a layer of ether. 8. Free the plug of debris from the sides of tube by ringing with an applicator stick, and decant the top three layers. 9. Mix the remaining sediment with the small amount of fluid that drains back from the sides of the tube and prepare iodine or unstained mounts in the usual manner for microscopic examination. B. Zinc Sulfate Centrifugal Flotation Technic (Faust, D'Antoni, and Sawitz): 1. Emulsify 1 ml. of feces in 10 ml. of tap water. 2. Filter through two layers of gauze. 3. Centrifuge mixture for one minute at 2600 r. p. m. and pour off supernatant fluid. 4. Add fresh saline, mix well, and centrifuge again. Repeat this process three or four times. 5. For the final emulsification, substitute 33"10 zinc sulfate solution (sp. gr. 1.80) for the saline.' 6. Centrifuge the suspension for one minute at top speed. 7. Eggs and cysts rise to surface (trophozoites destroyed). 8. Disturbing the supernatant as little as possible, transfer several loopsful of the surface film to a glass slide. Add a drop of iodine, mix, cover with coverglass, and examine microscopically. He'denhain's Iron Hematoxylin Staining Method For Intestinal Protozoa: There are numerous modifications of the iron-hema-oxylin stain, anyone of which will give good results if properly handled. The process of decolorization is the most n.a. step. This can be mastered only by experience. A. Staining Solutions: 1. Schaudinn's fixative - Two parts saturated mercuric chloride in water, one part 950/0 ethyl alcohol. Before using add 4 ml. glacial acetic acid to 100 ml. of the stock solution. 2. Hematoxylin stain (stock solution) - Dissolve 10 Gm. hematoxylin powder in 100 ml. absolute ethyl alcohol. Let stand several weeks to ripen before use. For use, 5 ml. of the ripened hematoxylin is added to 95 ml. distilled water. 3. Mordant (iron alum) - Dissolve 2 Gm. ferric ammonium sulfate in 100 ml. distilled water. B Procedure: 1. ~l1akethin fecal smear on glass sI ide with toothpick or applicator. 2, Before drying occurs, immerse slide in Schaudinn's fluid which has been heated to 45° C. Fix for five to 15 minutes at this temperature or for 30 minutes at room temperature.

3. Staining700/0 iodine alcohol 70"/0 alcohol 500/0 alc-ohol Tap water (running) Distilled water 20/0aqueous alum (mordant) Tap water (running) Distilled water O. 50/0hematoxylin Differentiate in cold 20/0 aqueous iron alum (a critical step) Tap water (running) 500/0 alcohol 70"/.alcohol' 950/0 alcohol 1000/0 alcohol Carbol xylol Xylol

5 minutes 2 minutes 2 minutes 2 minutes Rinse 10 minutes at 30·C. 5 minutes Rinse 10 minutes at 3D·C.

Examine centrifugate directly under the microscope. e smears, stain with Giemsa's stain, and examine. Cul-e some of the sediment, as for blood. Inoculate guinea ?:gs or mice if necessary.

Wet preparation may be examined directly for TrichomoCultures can be made by experienced tech=i :ans.

z= vaginalis.

15 to 20 minutes 2 minutes 2 minutes 2 minutes 2 minutes 5 minutes 5 minutes

Mount in xylol-balsam, xylol-Damar with a coverglass of No. 1 thickness.

or Permount®

Cultivation of Intestinal Protozoa: Numerous types of special media have been developed for the cultivation of the intestinal amebas and flagellates as well as for the ciliate Balantidium coli. Among these are Boeck-Drbohlav's Locke-egg-serum medium, Nelson's egg yolk-alcoholic extract medium, and Balamuth's egg yolk infusion-liver extract medium. Consult standard texts for details of preparation.

Collect urine in clean, dry container, avoiding fecal contamination. Study centrifuged sediment. Trophozoites of Trichomonas vaginalis, eggs of Schistosoma haematobium, and intact scolices or hooklets of Echinococcus granulosus may appear in the urine.

Examine for 'parasites directly under' the microscope, or study sediment after centrifuging (if sputum is sufficiently fluid). Larvae of Strongyloides stercoralis, scolices of Echinococcus granulosus, or eggs of Paragonimus westermani may be present in the sputum.

GRAHAM CELLULOSE TAPE TECHNIC FOR DIAGNOSIS OF ENTEROBIASIS The adhesive side of a small strip or loop of cellulose :..a;>e e. g., Scotch Tape®j is pressed over the anal and peria.::.a: surfaces with the help of a tongue depressor. Then place

:a;;e with adhesive side down in a drop of toluene on a micro5:: .:c slide. Examine for eggs or worms which have ade~eC.to the tap e.

IMMUNOLOGIC TESTS (See p. 342.) tradermal tests for Trichinella spiralis (Bachmann and Echinococcus granulosus (Casoni test) are specific, =c some diagnostic laboratories are prepared to make them. -":gen for the former is made from extracts of larvae =--;;ested from muscles of laboratory-infected rats, whlle the ~-er antigen consists of fluid obtained from hydatid cysts ,. :.n man or sheep .. r::e circumoval and CHR (cercarien-hiillen-reaktion) tests =e soecific in the diagnosis and follow-up of schistosomiasis. _- ,,~living eggs or cercariae of the species under suspiare incubated overnight in blood serum from the patient. -",:>e ~ precipitate will form around them. The circumo~ :e . is species-specific and becomes negative several after successful therapy. Intradermal tests with s: '-e extracts of cercariae or adult schistosomes are helpful • :n screening cas es since they are genus- specific but not s-= ·es-specific. . _-':'~ough reliable complement fixation, precipitin, and - -- _:ination tests have been developed for the diagnosis of : :-a.:.of the parasitic infections of man, they have had only ~;eC. application. Comparatively few laboratories are e' ._?;>ed to perform these tests. Satisfactory antigens are • S'

often difficult to prepare, tainable.

and control sera are often not ob-

Small pieces of deltoid, biceps, or gastrocnemius muscle are removed from the vicinity of their tendinous attachment under local anesthesia. (1) Examine small pieces of muscle compressed between two glass slides microscopically for encysting or encysted larvae. (2) Digest muscle in artificial gastric juice (pepsin and hydrochloric acid) and examine sediment for motile larvae.

Laboratory-bred reduviid bugs (obtainable from Gorgas Memorial Laboratory, Panama City, Panama) are fed on patients suspected of having Chagas' disease. Two weeks later, they are examined for the presence of the parasite.

Combined Thin and Thick Films: A. Making Films: Cleanse finger or ear lobe thoroughly with acetone and allow to dry. Prick skin deeply to cause a few drops of blood to flow freely. On one end of a meticulously clean slide make a thin smear as for a blood count. For the preparation of the thick film deposit a large drop of blood at the other end of the slide and spread it out evenly with the corner of another slide to a diameter of about 20 mm. The film should not be too thick since it may crack and peal when dry. Dry slide in a flat position so that distribution of blood will be even. Protect from dust and insects; avoid excessive heat. Allow to dry in air for at least eight to 12 hours or for two hours in incubator at 37° C. B. Staining Films: Fix thin-filmed end of slide in methyl alcohol for two to three minutes. THE TIUCK FILM MUST NOT BE FIXED. Immerse slide for 30 minutes in a mixture of one drop of concentrated Giemsa's stain to each ml. of neutral distilled water. Wash off and dry in air. Examine with oil-immersion objective. (For further details, see references.) Hematoxylin stain is preferred for microfilariae since their sheaths (if present) stain more distinctly.

C

centration of Microfilariae (Knott Technic): Thoroughly shake 2 ml. blood with 10 ml. of a 2% soluof formaldehyde. Centrifuge for five minutes at 2000 ~ ::>. m. Decant supernatant and examine sediment by pre;>'L:.:ng dried thick films stained with Giemsa's stain or hema. xylin. :e..-.;m Tests for Leishmania donovani: The Napier aldehyde test and the Chopra antimony test a..-e based on an increase in the euglobulin fraction of the ::U)(x! serum. Sometimes, but not always, they aid in diag:::osis. (See references for further details. ) ::::tt3"es for Blood Flagellates: .:... Leishmania donovani: Culture sedimented cells from citrated blood, bone marrow, liver or spleen pulp (obtained by puncture) in N. N. N. (Novy, MacNeal, Nicolle) medium at 20° to 24° C. for one to four weeks. :So Leishmania tropica and L. brasiliensis: Culture scrapings or aspirated material from lesions as for L. donovani. (Not found in blood. ) C Trypanosoma gambiense and T. rhodesiense: Culture centrifuged citrated blood, lymph node aspiration fluid, or spinal fluid in special media developed for these organisms. D. Trypanosoma cruzi: Culture blood as for Leishmania donovani.

Trophozoite

ll,u

Iodamoeba

Cyst biitschlii

lO.u

Trophozoite 9p Dientamoeba

(no cyst) fragilis

...~; Trophozoite Giardia

14JJ

lamblia

Trophozoite 18 J.l ~ Chilomastix ~esnili

Trophozoite (no cyst) 12p Trichomonas hominis

Diphyllobothrium latum

,

Hymenolepis

nana

{~1:

!4Scolex ~~

~.?

1 mm.

~:; Scolex

;.1;.~)

o. :3

mm.

Gravid Proglottid 6 X 12 mm. Gravid Proglottid 6 X 20 mm.

Adult fluke

60 X 30

mm. C:cmorchis

sinensis

Egg 31X

\\.

161'

~

)

~

~~

'a'';'~

~~~

Leptomonas 15-251' - .::?3D0soma

Crithidia 15-251'

"\

i~

cruzi

15-2"::7

~

~lk ~~ "~~ ~;. Trypanosoma gambiense

Diphyllobothrium latum

,

Hymenolepis

nana

{~1:

!4Scolex ~~

~.?

1 mm.

~:; Scolex

;.1;.~)

o. :3

mm.

Gravid Proglottid 6 X 12 mm. Gravid Proglottid 6 X 20 mm.

Adult fluke

60 X 30

mm. C:cmorchis

sinensis

Egg 31X

\\.

161'

~

)

~

~~

'a'';'~

~~~

Leptomonas 15-251' - .::?3D0soma

Crithidia 15-251'

"\

i~

cruzi

15-2"::7

~

~lk ~~ "~~ ~;. Trypanosoma gambiense