[LAB DIAGNOSIS] LAB MODULE 3 UNIT 2 AND 3

Question 1

 In surveys of infected population, as well as individual cases, it is sometimes desirable to estimate the intensity of infection by counting the number of eggs in the feces.

Answer 1

EGG COUNTING PROCEDURES

Question 2

 Egg counts provide a reasonable estimate of the number of adult worms present.

Answer 2

EGG COUNTING PROCEDURES

Question 3

 Egg counts before treatment may help determine whether treatment is needed and counts after treatment assess its success based on egg reduction rate (ERR) as a consequence of reduction of worm burden.

Answer 3

EGG COUNTING PROCEDURES

Question 4

 This procedure uses a measured amount of stool which has been sieved
through a wire mesh and pressed under cellophane paper soaked in glycerine-
malachite green solution.

Answer 4

A. Kato-Katz method

Question 5

 A uniform amount of stool is examined through the use of a template with a
uniform-sized hole in the middle.

Answer 5

A. Kato-Katz method

Question 6

A hole of 6 mm on a 1.5 mm thick template will deliver (?)

Answer 6

41.7
mg of stool

Question 7

o a hole of 9 mm on a 1.0 mm thick template, ?

Answer 7

50 mg

Question 8

o a hole of 6.5 mm on a 0.5 mm thick template, ?

Answer 8

20 mg

Question 9

 All eggs seen in the whole preparation are counted.

Answer 9

A. Kato-Katz method

Question 10

 The total egg count is multiplied with a factor depending on the amount of
stool used.

Answer 10

A. Kato-Katz method

Question 11

o The total number of eggs counted is multiplied by 24 for a
(1) template; by 20 for (2) template; and by 50 for
(3) template.

Answer 11

1. 41.7 mg
2. 50 mg
3. 20 mg

Question 12

 Consistency of the stool is the main determinant for the sensitivity of this
technique, since well-formed stools yield higher egg counts than moist ones.

Answer 12

A. Kato-Katz method

Question 13

 The technique can only be done on fresh formed stools and not on liquid and
preserved samples.

Answer 13

A. Kato-Katz method

Question 14

 The only human nematodes for which it is reasonably possible to correlate egg
production with adult worm burdens are Ascaris lumbricoides, Trichuris
trichiura, and the hookworms, all of which are soil-transmitted helminths
(STH).

Answer 14

A. Kato-Katz method

Question 15

 Note: The procedure is also useful for assessing the intensity of infection with
Schistosoma.

Answer 15

A. Kato-Katz method

Question 16

 Owing to its simplicity and relatively low cost, the Kato-Katz technique is
recommended by the World Health Organization (WHO) for epidemiological
surveys and surveillance pertaining to soil-transmitted helminthiasis and
intestinal schistosomiasis control programs.

Answer 16

A. Kato-Katz method

Question 17

uses a counting chamber with two compartments,
each with a grid etched onto the upper surface.

Answer 17

B. McMaster technique

Question 18

This enables a known volume of fecal suspension (2 x 0.15 ml) to be examined
microscopically.

Answer 18

B. McMaster technique

Question 19

 A known weight, 2 gms, of feces and a known volume, 28 m l , of flotation fluid
are used to prepare the suspension with a total volume approximately 30 ml.

Answer 19

B. McMaster technique

Question 20

 When filled with a suspension of feces in flotation fluid, much of the debris will
sink while eggs float to the surface, where they can easily be seen and those
under the grid counted.

Answer 20

B. McMaster technique

Question 21

 The number of eggs per gram of feces (EPG) can be calculated by multiplying
the number of eggs under the marked areas by a simple conversion factor.

Answer 21

B. McMaster technique

Question 22

 Multiply the number of eggs counted in one compartment by 100, or by 50 if
both compartments are used, to give the number of eggs per gram (EPG) of
feces.

Answer 22

B. McMaster technique

Question 23

has been developed to easily carry out the flotation of the fecal
sample (fresh or fixed) in a centrifuge, the translation of the apical portion of
the floating suspension, and the subsequent examination under the
microscope.

Answer 23

C. FLOTAC® and Mini-FLOTAC®

Question 24

These techniques use the FLOTAC® apparatus, a cylindrical-shaped device
made of polycarbonate amorphous thermoplastic, with two 5-ml flotation
chambers, which allows up to 1 g of stool to be prepared for microscopic
analysis.

Answer 24

C. FLOTAC® and Mini-FLOTAC®

Question 25

 There are two versions of the FLOTAC apparatus:

Answer 25

o FLOTAC-100, which permits a maximum magnification of ×100
o FLOTAC-400, which permits a maximum magnification of ×400

Question 26

 However, the main limitation of (?) is the complexity of the technique
which involves centrifugation of the sample in a specific device

Answer 26

FLOTAC

Question 27

 Centrifugation can take place in either a large volume centrifuge or in
benchtop centrifuge with rotor for microtiter plate, an equipment that is often
not available in some laboratories.

Answer 27

C. FLOTAC® and Mini-FLOTAC®

Question 28

 The (?) comprises two physical components, namely the
base, the reading disc and two accessories, the key and the microscope adaptor that are useful for assembly of apparatus and examination under a microscope.

Answer 28

Mini-FLOTAC® apparatus

Question 29

 There are two 1-ml flotation chambers, which are designed for optimal
examination of fecal sample suspensions in each flotation chamber (total
volume = 2 ml).

Answer 29

C. FLOTAC® and Mini-FLOTAC®

Question 30

 (?) be used in combination with Fill-FLOTAC® .

Answer 30

Mini-FLOTAC®

Question 31

 It is composed of a graduated container and a lid.

Answer 31

Mini-FLOTAC®

Question 32

 On the top of the lid, there are two holes with screw caps: a central one (with
a large screw cap) for the collector/homogenizer pole and one (with a small
screw cap) to which a tip is attached for passage of fecal suspension.

Answer 32

Mini-FLOTAC®

Question 33

 The conical collector permits measurement of 2 g of feces (Fill-FLOTAC 2).

Answer 33

Mini-FLOTAC®

Question 34

 The filter in the lower part of the lid has 250-µm holes to ensure an optimal
filtration of the fecal suspension.

Answer 34

Mini-FLOTAC®

Question 35

 The (?) enables the first four consecutive steps of the Mini-FLOTAC
technique, i.e. sample collection and weighing, homogenization, filtration and
filling.

Answer 35

Fill-FLOTAC®

Question 36

 The technique is a dilution technique in which 4 ml (~4 gm) of feces
(determined by displacement) is suspended in 56 ml of 0.1 N sodium
hydroxide.

Answer 36

D. STOLL’S EGG COUNTING TECHNIQUE

Question 37

 The sodium hydroxide saponifies fat and frees eggs from fecal debris.

Answer 37

D. STOLL’S EGG COUNTING TECHNIQUE

Question 38

 The amount of diluted stool used for egg counting is measured by Stoll
pipettes calibrated at 0.075 mL and 0.15 mL

Answer 38

D. STOLL’S EGG COUNTING TECHNIQUE

Question 39

 The constant used to multiply the total egg count depends on the amount of
stool examined.

Answer 39

D. STOLL’S EGG COUNTING TECHNIQUE

Question 40

Materials and reagents:
 Stool displacement flask etched lines at 56 and 60 ml.
 Pipettes for 0.150 and 0.0.075 ml
 0.1 N sodium hydroxide
 Glass beads
 Glass slide and coverslip

Answer 40

D. STOLL’S EGG COUNTING TECHNIQUE

Question 41

1. In a calibrated Stoll flask, add 0.1 N sodium hydroxide to the (?).

Answer 41

56-ml mark

Question 42

2. Add fresh fecal material to the flask so that the level of fluid rises to the 60-ml mark. This amount of feces is equivalent to (?).

Answer 42

4 g of feces

Question 43

3. Add a few (?), and shake vigorously to make a uniform suspension. If the
   specimen is hard, the mixture may be placed in a refrigerator overnight before shaking
   to aid in mixing.

Answer 43

glass beads

Question 44

4. With a calibrated pipette, quickly remove [?] (or 0.075 ml) of suspension and
   transfer it to a slide.

Answer 44

0.15 ml

Question 45

5. Do not use a (?); place the slide on a mechanical stage, and count all of the
   eggs.

Answer 45

coverslip

Question 46

6. Multiply the egg count by 100 if (1) stool suspension is used (or 200 if [2] is
   used) to obtain the number of eggs per gram of stool.

Answer 46

1. 0.15 ml
2. 0.075 ml

Question 47

7. The estimate (eggs per gram) obtained will vary according to the (?) of the stool.

Question 48

The following correction factors should be used to convert the estimate to a formed-
stool basis:
mushy formed ……………………… ?
mushy ………………………………….. ?
mushy diarrheic ……………………. ?

Answer 48

1.5
2
3

Question 49

 Egg counts on liquid specimens are generally unreliable; the most accurate
counts are obtained with use of (??.

Answer 49

formed or semi-formed specimens

Question 50

 crude but useful semi-quantitiative method based on the assumption that an ideal fecal smear contains (?) of stool

Answer 50

1 to 2 mg

Question 51

 An estimated (?) stool in a standard smear is covered by 22 by 22 mm
coverslip and all eggs in the entire preparation are counted.

Answer 51

1 to 2 mg

Question 52

 The number of eggs per gram (EPG) is obtained by multiplying the total counts
by 667 if the standard smear has (?) stool (1000 mg divided by 1.5 )

Answer 52

1.5 mg

Question 53

o the factor is 500 if the smear has (?) stool, etc. Obviously,
results are subject to large variation if the total egg count per
coverslip is low.

Answer 53

2 mg

Question 54

1. Microscopic examination
2. Colonoscopy, Sigmoidoscopy, or Proctoscopy
3. Egg count test (Kato-Katz)

Answer 54

A. Trichuris trichiura

Question 55

– detection of the characteristic eggs in stools

Answer 55

Microscopic examination

Question 56

a. Direct fecal smear (DFS)
b. Kato thick smear (KTS) – not used in protozoans; ova
c. Kato-Katz

Answer 56

Trichuris trichiura Microscopic examination

Question 57

o Modified kato-thick

Answer 57

Kato-Katz

Question 58

o To count egg per gram

Answer 58

Kato-Katz

Question 59

o With sheaving of stool (debris removal – more refined stool sample)

Answer 59

Kato-Katz

Question 60

o To easily count parasite

Answer 60

Kato-Katz

Question 61

1. Place paper
2. Place sheave on top of stool sample
3. Get a slide and place a template (board or plastic that gives
   volume of stool being examined)
   a. Thickness of 1,5mm
   b. Diameter of hole: 6.5
   c.
4. Fill out the hole on the slide when template is removed

Answer 61

Kato-Katz

Question 62

o Stool concentation techniques (sedimentation and flotation) or Formalin-
ether acetate sedimentation

Answer 62

Kato-Katz

Question 63

 Adult worms

Answer 63

Colonoscopy, Sigmoidoscopy, or Proctoscopy

Question 64

 Adult worms – visible on macroscopic examination of the:

Answer 64

o intestinal mucosa.
o intestinal tract down to and including the rectum in heavy infections

Question 65

 identifying intensity of infection
o to determine the dose
o to follow-up effect of treatment

Answer 65

Egg count test (Kato-Katz)

Question 66

1. Microscopic examination
2. Duodenal aspiration

Answer 66

Capillaria philippinensis

Question 67

– recovery of the characteristic eggs in stools; larva; adult

Answer 67

Microscopic examination

Question 68

 Direct fecal smear (DFS)
 Kato thick smear (KTS)
 Kato-Katz
 Stool concentation techniques (sedimentation and flotation) or Formalin-ether acetate
sedimentation

Answer 68

Capillaria philippinensis Microscopic examination

Question 69

1. Histopathologic examination.
2. Serological test

Answer 69

Capillaria hepatica

Question 70

 Eggs - identified on the basis of their characteristic morphology on liver biopsy specimens

Answer 70

Histopathologic examination.

Question 71

 true human infection – no eggs are found in the stool

Answer 71

Capillaria hepatica

Question 72

 presence eggs in stool during (O&P) examinations – spurious infection following consumption of
egg-laden animal liver

Answer 72

Capillaria hepatica

Question 73

 unembryonated eggs – non-infectious

Answer 73

Capillaria hepatica

Question 74

o merely pass through the digestive tract with feces

Answer 74

 unembryonated eggs – non-infectious

Question 75

 IFAT

Answer 75

Serological test

Question 76

– testing of human sera for the detection of early Capillaria hepatica infection

Answer 76

IFAT

Question 77

1. Muscle biopsy
2. Beck’s Xenodiagnosis
3. Serological tests
4. Bachman intradermal test
5. Molecular methods
6. Radiological examination
7. Other laboratory findings

Answer 77

Trichinella spiralis

Question 78

• demonstration of spiral larvae

Answer 78

Muscle biopsy

Question 79

• Definitive diagnostic exam

Answer 79

Muscle biopsy

Question 80

• can only be done when encystment of the parasite has occurred (7 days after infection)

Answer 80

Muscle biopsy

Question 81

• Parasitized muscles

Answer 81

Muscle biopsy

Question 82

 diaphragm, pectoral gluteus, deltoid, biceps, & gastrocnemius

Answer 82

Parasitized muscles

Question 83

 At least 1 gram of muscle

Answer 83

Parasitized muscles

Question 84

 preferably near tendon insertion

Answer 84

Parasitized muscles

Question 85

o chosen for taking diagnostic muscle biopsies
o easily accessible

Answer 85

 diaphragm, pectoral gluteus, deltoid, biceps, & gastrocnemius

Question 86

• Muscle fibers

Answer 86

Muscle biopsy

Question 87

 digested with trypsin

Answer 87

Muscle fibers

Question 88

 mounted on a glass slide

Answer 88

Muscle fibers

Question 89

 examined under microscope

Answer 89

Muscle fibers

Question 90

o a teased preparation of muscle tissue is prepared in a drop of saline
solution

Answer 90

 examined under microscope

Question 91

o squeezed between two glass slides

Answer 91

 examined under microscope

Question 92

o muscle tissue is stained with safranin

Answer 92

 examined under microscope

Question 93

• diagnosis of trichinosis

Answer 93

Beck’s Xenodiagnosis

Question 94

• Albino rats are fed with infected patient meat & are observed for around 14 days

Answer 94

Beck’s Xenodiagnosis

Question 95

• rats are then killed in a month or so later and checked (particularly in the diaphragm) for the presence of
  Trichinella spiralis larvae

Answer 95

Beck’s Xenodiagnosis

Question 96

• Observe for the presence of female worms in the duodenum & the larvae in the muscles

Answer 96

Beck’s Xenodiagnosis

Question 97

• rarely requested and is not available in most clinical laboratory

Answer 97

Beck’s Xenodiagnosis

Question 98

• lab animal is examined

Answer 98

Beck’s Xenodiagnosis

Question 99

• massive hypergammaglobulinemia with elevated serum IgE

Answer 99

Serological tests

Question 100

• Confirmatory test

Answer 100

Serological tests

Question 101

 Bentonite flocculation test (BFT)
 Latex flocculation test (LFT)
 IFAT
 ELISA

Answer 101

Trichinella spiralis Confirmatory test

Question 102

– (+): recent infection

Answer 102

Bentonite flocculation test (BFT)

Question 103

– - (+): recent infection

Answer 103

Latex flocculation test (LFT)

Question 104

– T. spiralis antibody can be detected using TSL-1 secreting antigens obtained from the
infective stage larvae

Answer 104

ELISA

Question 105

o Westernblot
o Latex agglutination

Answer 105

ELISA

Question 106

• hypersensitivity test

Answer 106

Bachman intradermal test

Question 107

• 1:5,000 or 1:10,000 dilution of the larval antigen

Answer 107

Bachman intradermal test

Question 108

 erythematous wheal (undulation) appears in positive cases within ?

Answer 108

15- 20 minutes

Question 109

 rest remains positive for (?) after infection

Answer 109

years

Question 110

1. antigen is administered

Answer 110

 rest remains positive for years after infection

Question 111

 PCR

Answer 111

Molecular methods

Question 112

– species identification

Answer 112

PCR

Question 113

 X-ray

Answer 113

Radiological examination

Question 114

– Calcified cysts may be demonstrated

Answer 114

X-ray

Question 115

 Eosinophilia and leukocytosis may also serve as indicators for disease.

Answer 115

Other laboratory findings

Question 116

 Elevated serum muscle enzyme levels, such as, may also aid in T. spiralis diagnosis.

Answer 116

Other laboratory findings

Question 117

o lactate dehydrogenase
o aldolase
o creatine phosphokinase
o Myokinase w/ eosinophilia

Answer 117

Biochemical tests

Question 118

– elevated enzymes as indicators for diagnosis

Answer 118

Biochemical tests

Question 119

1. Microscopic identification of eggs
2. Macroscopic identification of adults
3. Serology

Answer 119

Ascaris lumbricoides

Question 120

 Definitive diagnosis of ascariasis

Answer 120

Microscopic identification of eggs

Question 121

 demonstration of the characteristic
fertilized and/or unfertilized eggs in stools

Answer 121

Microscopic identification of eggs

Question 122

 Reproductive rate: 200,000 eggs per day

Answer 122

Microscopic identification of eggs

Question 123

 Life span of adult worm: 1 year

Answer 123

Microscopic identification of eggs

Question 124

a. Direct fecal smears (saline and
iodine-stained DFS)
b. Kato thick smear (KTS)
c. stool concentation techniques
(sedimentation and flotation)
d. Kato-Katz (KK) technique

Answer 124

Ascaris lumbricoides
Microscopic identification of eggs

Question 125

 Entangled mass in GIT cause obstruction

Answer 125

Macroscopic identification of adults

Question 126

 extraintestinal ascariasis

Answer 126

Ascaris lumbricoides Serology

Question 127

a. Indirect hemagglutination (IHA)
b. Indirect fluorescent antibody
(IFA)
c. ELISA

Answer 127

Ascaris lumbricoides Serology

Question 128

 Sedimentation concentration test is
recommended instead of flotation.
Unfertilized eggs of Ascaris lumbricoides do not easily float.

Answer 128

Ascaris lumbricoides

Question 129

 Adult worms of Ascaris lumbricoides are occasionally passed in the stool or through the mouth or nose and are recognizable by
their macroscopic characteristics.

Answer 129

Ascaris lumbricoides

Question 130

 Intestinal disease can often be diagnosed
from radiographic studies of the gastrointestinal tract, in which the worm in
the intestinal tract may be visualized .

Answer 130

Ascaris lumbricoides

Question 131

 Larvae of Ascaris lumbricoides can be
identified in sputum or gastric aspirate
during the pulmonary migration phase
(examine fixed organisms for morphology).

Answer 131

Ascaris lumbricoides

Question 132

1. Microscopic examination

Answer 132

Enterobius vermicularis

Question 133

A. Graham’s scotch adhesive tape swab
B. NIH swab

Answer 133

Enterobius vermicularis Microscopic examination

Question 134

• demonstration of eggs or
  adult female worms

Answer 134

Graham’s scotch adhesive tape swab

Question 135

• cellophane tape, or cellulose tape, or adhesive tape

Answer 135

Graham’s scotch adhesive tape swab

Question 136

• Specimens
• perianal region
• early morning, before going to the toilet or bathing

Answer 136

Graham’s scotch adhesive tape swab

Question 137

• Preferred

Answer 137

Graham’s scotch adhesive tape swab

Question 138

• Yields the highest % of + results and the greatest no. of eggs seen

Answer 138

Graham’s scotch adhesive tape swab

Question 139

• Placed on the perianal region

Answer 139

Graham’s scotch adhesive tape swab

Question 140

MATERIALS
 Clear cellulose tape
 Microscope slide
 Tongue depressor or a similar material

Answer 140

Graham’s scotch adhesive tape swab

Question 141

1. Place a [?] (adhesive side
   down) on a microscope slide as follows:

Answer 141

strip of clear cellulose tape

Question 142

 starting at (?) from one end,
run the tape toward the same end

Answer 142

1.5 cm

Question 143

 wrap the tape (?) to
the opposite end

Answer 143

around the slide

Question 144

 ? even with the end of
the slide.

Answer 144

Tear the tape

Question 145

 ? to the tape at the
end torn flush with the slide.

Answer 145

Attach a label

Question 146

2. To obtain a sample from the perianal area, (?) by gripping the labeled end, and, with the
   tape looped (adhesive side outward) over a wooden
   tongue depressor that is held firmly against the slide
   and extended about 2.5 cm beyond it, press the tape
   firmly several times against the right and left perianal
   folds.

Answer 146

peel back
the tape

Question 147

3. Smooth the tape back on the slide, (?) down.

Answer 147

adhesive side

Question 148

4. Label with ?.

Answer 148

patient name and date

Question 149

5. Submit the (?) to the laboratory in a plastic bag.

Answer 149

tapes and slides

Question 150

6. Examine the slide directly under the (?) of the microscope. To make the eggs more
   visible, lift the tape from the slide and add a drop of
   xylene or toluene to the slide. Press the tape back
   down on the slide. Examine with low light intensity
   on low power.

Answer 150

low-power objective

Question 151

• named after National Institutes of Health, United States of America
  (USA

Answer 151

NIH swab

Question 152

 consists of a glass rod at one end of which a piece of
transparent cellophane is attached with a rubber band

Answer 152

NIH swab

Question 153

 glass rod is fixed on a rubber stopper and kept in a wide test
tube

Answer 153

NIH swab

Question 154

 cellophane part is used for swabbing by rolling over the
perianal area

Answer 154

NIH swab

Question 155

 It is returned to the test tube and sent to the laboratory,
where the cellophane piece is detached, spread over a glass
slide and examined microscopically

Answer 155

NIH swab

Question 156

 Eggs are rarely found in the stool (in only about 5% of
infected persons) because release is external to the
intestine. Therefore, stool is NOT the specimen of
choice.

Answer 156

Enterobius vermicularis

Question 157

 The perianal swabs may also be collected late in the
evening, when the patient has been sleeping for several
hours.

Answer 157

Enterobius vermicularis

Question 158

 The eggs may sometimes be demonstrated in the dirt
collected from beneath the finger nails in infected
children.

Answer 158

Enterobius vermicularis

Question 159

 Care must be taken during specimen collection and
examination because eggs, if present, are already
infectious.

Answer 159

Enterobius vermicularis

Question 160

 A minimum of four to six consecutive negative slides is
required to rule out the infection.

Answer 160

Enterobius vermicularis

Question 161

 Alternatively, commercial “Swube tubes” (a paddle
coated with adhesive material) can also be used for
collection.

Answer 161

Enterobius vermicularis

Question 162

1. Microscopic examination
2. Coproculture

Answer 162

Hookworms

Question 163

– demonstration of the characteristic hookworm
eggs in stools

Answer 163

Hookworms Microscopic examination

Question 164

a. direct fecal smears (saline and iodine-stained DFS)
b. Kato thick smear (KTS)
c. stool concentation techniques (sedimentation and flotation)
d. Kato-Katz (KK)

Answer 164

Hookworms Microscopic examination

Question 165

 Eggs of N. americanus and A. duodenale cannot be differentiated by morphology, thus, is reported as” hookworm species eggs”

Answer 165

Hookworms

Question 166

 If the stool remains unpreserved for over 24 h, the eggs may continue
to develop and the larvae may hatch. These larvae must be
differentiated from those of Strongyloides stercoralis (will be
discussed later), since therapies for the two infections are different.

Answer 166

Hookworms

Question 167

 Kato-Katz or kato thick smear preparation must be examined within 1 hour after preparation. Allowing the slides to stand for a long period
of time will cause drying and thin shells of hookworm egg will become
too transparent and will be difficult to see.

Answer 167

Hookworms

Question 168

 Adult hookworms are rarely seen in feces but may be recovered
following treatment. Examination of the buccal capsule is necessary to
determine the specific hookworm species.

Answer 168

Hookworms

Question 169

 Harada-Mori technique

Answer 169

Coproculture

Question 170

• use of certain fecal culture methods (sometimes referred to as coproculture) is
  especially helpful for detection of light infections with hookworm (also Strongyloides
  stercoralis and Trichostrongylus spp)

Answer 170

Coproculture

Question 171

• is also carried out to demonstrate larvae from feces which helps in distinguishing N.
  americanus and A. duodenale

Answer 171

Coproculture

Question 172

• filter paper test tube culture technique

Answer 172

Harada-Mori technique

Question 173

• initially introduced by Harada and Mori in 1955
  and was later modified by others

Answer 173

Harada-Mori technique

Question 174

• The technique employs a filter paper to which fecal material is
  added and a test tube into which the filter paper is inserted

Answer 174

Harada-Mori technique

Question 175

• Moisture is provided by adding water or saline to the tube.

Answer 175

Harada-Mori technique

Question 176

• The water continuously soaks the filter paper by capillary action.

Answer 176

Harada-Mori technique

Question 177

• Incubation under suitable conditions favors hatching of ova
  and/or development of larvae.

Answer 177

Harada-Mori technique

Question 178

 Appropriate PPE (gloves, gown) should always be worn to minimize
the risk of transdermal penetration when working with stool
specimens or larval cultures.

Answer 178

Hookworms

Question 179

 Fecal specimens to be cultured should not be refrigerated, since some
parasites (especially Necator americanus) are susceptible to cold and
may fail to develop after refrigeration.

Answer 179

Hookworms

Question 180

 Rhabditiform (L1) larvae that hatch from eggs are 250-300 µm long
and approximately 15-20 µm wide. They have a long buccal canal and
an inconspicuous genital primordium. Rhabditiform larvae are usually
not found in stool, but may be found if there is a delay in processing
the stool specimen. If larvae are seen in stool, they must be
differentiated from the L1 larvae of Strongyloides stercoralis.

Answer 180

Hookworms

Question 181

 Filariform (L3) larvae are 500—700 µm long. They have a pointed tail
and are ensheathed, with esophagus that is one-third the length of
the body. Some subtle morphological differences exist between N.
americanus and A. duodenale at this stage. These L3 are found in the
environment and infect the human host by penetration of the skin..

Answer 181

Hookworms

Question 182

 Diagnosis is based mainly on history and clinical manifestations.

Answer 182

Zoonotic hookworms

Question 183

1. Microscopic examination
2. Coproculture
3. Serology
4. Molecular diagnosis

Answer 183

Strongyloides stercoralis

Question 184

a. Direct stool microscopy
b. Baermann technique
c. Microscopic examination of duodenal and respiratory specimens

Answer 184

Strongyloides stercoralis Microscopic examination

Question 185

 Demonstration of the rhabditiform larvae in freshly passed stools

Answer 185

Direct stool microscopy

Question 186

 most important method of specific diagnosis

Answer 186

Direct stool microscopy

Question 187

 may be recovered in direct fecal smears or stool concentration techniques

Answer 187

Direct stool microscopy

Question 188

 It may be difficult to observe morphologic details in rapidly moving larvae; a drop of iodine or formalin or slight heating can be used to
kill the larvae.

Answer 188

Strongyloides stercoralis

Question 189

 Larvae found in old, unpreserved stools have to be differentiated from larvae hatched from hookworm eggs.

Answer 189

Strongyloides stercoralis

Question 190

 In addition to rhabditiform larvae, eggs of S. stercoralis, often indistinguishable from those of the hookworm, may be present in stool
samples from patients suffering from severe diarrhea.

Answer 190

Strongyloides stercoralis

Question 191

 Note that formalin-ethyl acetate concentration may remove larvae and reduce sensitivity. Sedimentation methods have a typically
higher recovery rate than those in which a flotation technique has been used.

Answer 191

Strongyloides stercoralis

Question 192

 Baermann technique is another sedimentation method of examining a stool specimen suspected of containing small numbers of
Strongyloides larvae.

Answer 192

Baermann technique

Question 193

 This uses a funnel apparatus and relies on the principle that active larvae will migrate from a fresh fecal specimen that has been
placed on a wire mesh with several layers of gauze which are in contact with tap water.

Answer 193

Baermann technique

Question 194

 Larvae migrate through the gauze into the water and settle to the bottom of the funnel, where they can be collected and examined.

Answer 194

Baermann technique

Question 195

 Specimens that have been refrigerated or preserved are not suitable for culture. Larvae of certain species are susceptible to cold
environments.

Answer 195

Strongyloides stercoralis

Question 196

 Besides being useful for diagnosis from stool specimens directly, it can be enhanced by culture if no larvae are seen in 2-3 hours.

Answer 196

Strongyloides stercoralis

Question 197

 Infective larvae may be found any time after the fourth day and occasionally after the first day in heavy infections. Caution must be
exercised in handling the fluid, gauze pad, and beaker to prevent infection. Wear gloves when using this technique.

Answer 197

Strongyloides stercoralis

Question 198

 Make sure that the end of the tubing is well inside the beaker before releasing the pinch clamp. Release the pinch clamp slowly to
prevent splashing.

Answer 198

Strongyloides stercoralis

Question 199

 Besides being used for patient fecal specimens, this technique can be used to examine soil specimens for the presence of larvae.

Answer 199

Strongyloides stercoralis

Question 200

 Beale’s string test

Answer 200

Microscopic examination of duodenal and respiratory specimens

Question 201

 Duodenal aspiration

Answer 201

Microscopic examination of duodenal and respiratory specimens

Question 202

 Aspiration of duodenal fluid or use of the less invasive Entero-test (commonly called a string test) may be useful to detect larvae in
patients with negative stool samples.

Answer 202

Microscopic examination of duodenal and respiratory specimens

Question 203

 In disseminated strongyloidiasis, filariform larvae may be detected in sputum, bronchial washings or pleural fluid.

Answer 203

Microscopic examination of duodenal and respiratory specimens

Question 204

 Principle:
o Active larvae will migrate out of a fecal specimen that has been placed on a wire mesh covered with several layers
of gauze.

Answer 204

Microscopic examination of duodenal and respiratory specimens

Question 205

a. Harada-Mori technique
b. Agar plate cultures
c. Charcoal culture method

Answer 205

Strongyloides stercoralis
Coproculture

Question 206

• discussed previously in hookworm species.

Answer 206

Harada-Mori technique

Question 207

 These are also recommended for the recovery of S. stercoralis larvae and tend to be a more sensitive method than the usual direct
smear or fecal concentration methods.

Answer 207

Agar plate cultures

Question 208

 Stool is placed onto agar plates, and the plates are sealed to prevent accidental infections and held for 2 days at room temperature.

Answer 208

Agar plate cultures

Question 209

 As the larvae crawl over the agar, they carry bacteria with them, thus creating visible tracks over the agar .

Answer 209

Agar plate cultures

Question 210

 The plates are examined under the microscope for confirmation of the presence of larvae.

Answer 210

Agar plate cultures

Question 211

 Another way to culture hookworm, Strongyloides, and trichostrongyle larvae is by using a granulated charcoal culture.

Answer 211

Charcoal culture method

Question 212

 The conditions of this culture provide an environment for larval development that mimics conditions in nature.

Answer 212

Charcoal culture method

Question 213

 It provides an efficient way to harvest large numbers of infective-stage larvae for use in experimental infections.

Answer 213

Charcoal culture method

Question 214

 Most antibody detection tests employ antigens derived from Strongyloides stercoralis (or from closely-related S. ratti or S.
venezuelensis) filariform larvae.

Answer 214

Strongyloides stercoralis Serology

Question 215

 Although indirect fluorescent antibody (IFA), indirect hemagglutination (IHA) and antigen-linked fluorescent and magnetic bead tests
are are available, enzyme immunoassay (EIA) is recommended because of its greater sensitivity.

Answer 215

Strongyloides stercoralis Serology

Question 216

 The commercial EIA kits that are currently available have sensitivity of 95%.

Answer 216

Strongyloides stercoralis Serology

Question 217

 PCR and LAMP methods may be used to detect strongyloidiasis in fresh, frozen, or non-formalin fixed stool specimens.

Answer 217

Strongyloides stercoralis
Molecular diagnosis

Question 218

 Sensitivity and specificity vary depending on the reference test used to calculate such characteristics; false negatives and false
positives do occur.

Answer 218

Strongyloides stercoralis
Molecular diagnosis

Question 219

 Diagnosis of toxocariasis relies mostly on indirect means,
particularly serology, since larvae are trapped in tissues and
not readily detected morphologically.

Answer 219

Toxocara spp.

Question 220

 While visualization of larvae in histologic sections provides
unequivocal diagnosis, the probability of capturing a larva in a
small biopsy specimen is low.

Answer 220

Toxocara spp.

Question 221

 For both VLM and OLM, a presumptive diagnosis rests on
clinical signs, compatible exposure history, laboratory
findings (including eosinophilia), and the detection of
antibodies to Toxocara.

Answer 221

Toxocara spp.

Question 222

 Detection of microfilariae in blood is the traditional method of diagnosis of most filarial nematode infections, which include those caused by W. bancrofti, Brugia species, L. loa, M. perstans, and M. ozzardi.

Answer 222

FILARIA
Microscopy

Question 223

 The notable exception to the use of blood specimens is for the diagnosis of onchocerciasis and M. streptocerca infection, which involves examination of skin snips.

Answer 223

FILARIA
Microscopy

Question 224

 For more sensitive detection of microfilariae in blood, it is important to understand the periodicity of a given species or geographical variant.

Answer 224

Blood examination
o Periodicity

Question 225

 This determines the optimal time their microfilariae will be circulating in peripheral blood and, therefore, the best time of the day to obtain blood specimens for examination.

Answer 225

Blood examination
o Periodicity

Question 226

 To determine microfilarial periodicity in an individual, it is necessary to examine measured quantities of blood collected at consecutive intervals of 2 or 4 hours over a period of 24-30 hours.

Answer 226

Blood examination
o Periodicity

Question 227

Methods used for the detection of microfilariae in the blood

Answer 227

i. Stained blood films
ii. Wet mount of capillary blood
iii. Microhematocrit method
iv. Quantitative buffy coat (QBC) method
v. Concentration procedures

Question 228

i. Stained blood films

Answer 228

Preparation of thick blood films
Staining of thick blood films
Microscopic examination

Question 229

 The examination of thick blood films is the most widely used method in field surveys of filarial infection.

Answer 229

Preparation of thick blood films

Question 230

 Carefully measured blood samples of at least 20 µl and preferably 60 µl in volume are recommended.

Answer 230

Preparation of thick blood films

Question 231

 At least two thick films should be made. Identification of microfilariae in thin films is not recommended because the microfilariae are shrunken, distorted and difficult to recognize.

Answer 231

Preparation of thick blood films

Question 232

 Giemsa and Delafield’s hematoxylin are the preferred and most widely used for preparing permanently-stained thick blood films in the detection of microfilariae.

Answer 232

Staining of thick blood films

Question 233

 With Giemsa stain, nuclei of microfilariae will stain blue to purple, and the sheath will stain pink (B. malayi).

Answer 233

Staining of thick blood films

Question 234

 Hematoxylin enhances nuclear detail in microfilaria, and stains the sheath greyish-blue (W. bancrofti, B. timori, L. loa).

Answer 234

Staining of thick blood films

Question 235

 Because microfilariae are large, they can be detected by screening at a lower magnification.

Answer 235

Microscopic examination

Question 236

 The entire thick blood film should be examined with the 10x low-power objective.

Answer 236

Microscopic examination

Question 237

 When microfilariae are seen, morphologic details can be seen using the 40x high dry objective, and then examined at a higher magnification (×100 with oil) for species-level identification.

Answer 237

Microscopic examination

Question 238

 A drop of blood directly on a slide is added with an equal-sized drop of NSS, and examined under the microscope (10x objective).

Answer 238

ii. Wet mount of capillary blood

Question 239

 Microscopic examination of fresh blood has limited usefulness.

Answer 239

ii. Wet mount of capillary blood

Question 240

 The whiplike motion of the microfilariae are easily spotted because they cause movement of surrounding red blood cells, but species identification is not possible.

Answer 240

ii. Wet mount of capillary blood

Question 241

 However, in regions where only one species is found, its presence and density in the blood can be determined with reasonable accuracy by this means.

Answer 241

ii. Wet mount of capillary blood

Question 242

PROCEDURE:
(1) Three-quarters fill a microhaematocrit capillary tube with the blood. Seal one end of the tube.
(2) Centrifuge in a microhaematocrit centrifuge at 10 000g for 2 minutes.
(3) Lay the capillary tube on a slide and secure the two ends with adhesive tape.
(4) Examine the dividing line between the blood cells and the plasma under the microscope using the 10x objective.
 Motile microfilariae will be seen at the bottom of the column of plasma, just above the layer of leukocytes and erythrocytes.
 The tube can be snapped at the bottom of the column of plasma.
 Use the first drop from each piece of the broken tube to prepare a thick film.
 Stain the film as to identify the species.

Answer 242

iii. Microhematocrit method

Question 243

(1) ? fill a microhaematocrit capillary tube with the blood. Seal one end of the tube.

Answer 243

Three-quarters

Question 244

(2) Centrifuge in a microhaematocrit centrifuge at ?.

Answer 244

10 000g for 2 minutes

Question 245

(3) Lay the capillary tube on a slide and secure the two ends with ?.

Answer 245

adhesive tape

Question 246

(4) Examine the (?) between the blood cells and the plasma under the microscope using the 10x objective.

Answer 246

dividing line

Question 247

 ? will be seen at the bottom of the column of plasma, just above the layer of leukocytes and erythrocytes.

Answer 247

Motile microfilariae

Question 248

 The tube can be snapped at the bottom of the ?.

Answer 248

column of plasma

Question 249

 Use the (?) from each piece of the broken tube to prepare a thick film.

Answer 249

first drop

Question 250

 ? as to identify the species.

Answer 250

Stain the film

Question 251

 The utilization of quantitative buffy coat tube coated with acridine orange (discussed in malaria diagnosis) has been reported to be an acceptable rapid diagnostic test for detection of microfilariae, with a sensitivity equivalent to that of thick blood film.

Answer 251

iv. Quantitative buffy coat (QBC) method

Question 252

v. Concentration procedures

Answer 252

Knott’s concentration method
Membrane filtration technique

Question 253

 These procedures are preferred when the microfilarial load is low.

Answer 253

v. Concentration procedures

Question 254

 They can be used for the enhanced detection of microfilariae in blood or other body fluids.

Answer 254

v. Concentration procedures

Question 255

PROCEDURE:
(1) Collect 1 ml of blood (whole or citrated) by venipuncture and place in a 15-ml centrifuge tube containing at least 10 ml of 2% formalin; stopper the tabe and shake vigorously. The formalin lyses RBCs, fixes blood protozoa, and kills and straightens the bodies of microfilariae.
(2) Centrifuge at at approximately 300 g for 2 minutes. If a centrifuge is not available, place the tube in an upright position for 12 hours for gravitational sedimentation.
(3) Decant the supernatant fluid (the small amount remaining in the tube is allowed to flow back on to the sediment).
(4) Remove the sediment with a Pasteur pipette and examine a drop of sediment on a slide under a coverslip with the low power objective of the microscope.
(5) A portion of the sediment may be spread on a glass slide as a thick film and allowed to dry thoroughly. Stain the film with Giemsa or hematoxylin stain.

Answer 255

Knott’s concentration method

Question 256

(1) Collect (?) of blood (whole or citrated) by venipuncture and place in a 15-ml centrifuge tube containing at least 10 ml of 2% formalin; stopper the tabe and shake vigorously. The formalin lyses RBCs, fixes blood protozoa, and kills and straightens the bodies of microfilariae.

Answer 256

1 ml

Question 257

(2) Centrifuge at at approximately (?). If a centrifuge is not available, place the tube in an upright position for 12 hours for gravitational sedimentation.

Answer 257

300 g for 2 minutes

Question 258

(3) Decant the [?] (the small amount remaining in the tube is allowed to flow back on to the sediment).

Answer 258

supernatant fluid

Question 259

(4) Remove the sediment with a Pasteur pipette and examine a drop of sediment on a slide under a coverslip with the (?) of the microscope.

Answer 259

low power objective

Question 260

(5) A (?) may be spread on a glass slide as a thick film and allowed to dry thoroughly. Stain the film with Giemsa or hematoxylin stain.

Answer 260

portion of the sediment

Question 261

 This allows for removal of elements by filtration through a membrane of desired pore size.

Answer 261

Membrane filtration technique

Question 262

 Cellulose-mixed-ester filters (e.g., Millipore filters) and polycarbonate filters (e.g., Nucleopore filters), 25 mm in diameter, are most common filters used.

Answer 262

Membrane filtration technique

Question 263

 Although a membrane filter of 5-µm porosity is ideal for isolation of most microfilariae in the blood, it is unsatisfactory for the isolation of Mansonella microfilariae because of their small size. A 3-µm-pore-size filter could be used for recovery of this organism.

Answer 263

Membrane filtration technique

Question 264

PROCEDURE:
(1) Collect fresh blood in sodium citrate or EDTA
(2) Add 1 ml of the blood to 10 ml of 10% Teepol-saline solution (Teepol lyses the red blood cells;
prepared by adding 50 g Teepol concentrate to 450 ml saline).
(3) Place a membrane filter over supporting filter paper moistened with water securely into a filter
holder.
(4) Remove plunger from the barrel of a 20-ml syringe and connect the barrel of the syringe to the
filter holder.
(5) Pour the blood-Teepol mixture (see step 2) into the barrel of syringe, replace the plunger in the
syringe, and gently force the solution through the filter.
(6) Remove the syringe from the filter holder, draw up 10 ml of water into syringe, reattach the
filter holder, and gently wash the filter by flushing the water through it.
(7) Force two syringe-volumes of air through filter to expel excess water and make microfilariae
more adherent to filter.

Answer 264

Membrane filtration technique

Question 265

(1) Collect fresh blood in sodium citrate or ?

Answer 265

EDTA

Question 266

(2) Add 1 ml of the blood to (?) Teepol-saline solution (Teepol lyses the red blood cells;
prepared by adding 50 g Teepol concentrate to 450 ml saline).

Answer 266

10 ml of 10%

Question 267

(3) Place a (?) over supporting filter paper moistened with water securely into a filter
holder.

Answer 267

membrane filter

Question 268

(4) Remove plunger from the barrel of a (?) and connect the barrel of the syringe to the
filter holder.

Answer 268

20-ml syringe

Question 269

(5) Pour the (?) (see step 2) into the barrel of syringe, replace the plunger in the
syringe, and gently force the solution through the filter.

Answer 269

blood-Teepol mixture

Question 270

(6) Remove the syringe from the filter holder, draw up (?) into syringe, reattach the
filter holder, and gently wash the filter by flushing the water through it.

Answer 270

10 ml of water

Question 271

(7) Force (?) of air through filter to expel excess water and make microfilariae
more adherent to filter.

Answer 271

two syringe-volumes

Question 272

(8) Pass (?) through the filter to fix the microfilariae.

Answer 272

3 ml of methanol

Question 273

(9) Remove the (?) from the holder and place on a glass slide; allow to dry thoroughly.

Answer 273

filter

Question 274

(10) Stain with (?) per the normal procedure.

Answer 274

Giemsa or hematoxylin stain

Question 275

(11) Dip the slide in (?) to avoid air bubble formation.

Answer 275

toluene

Question 276

(12) Add a drop of (?) and a coverslip.

Answer 276

mounting medium

Question 277

(13) Examine the slide when ?.

Answer 277

dry

Question 278

 ? is the gold standard test used for the detection of microfilariae of Onchocerca volvulus and Mansonella streptocerca.

Answer 278

Skin snip examination

Question 279

 A very small piece of the patient’s skin is collected. The skin snips are generally collected from several sites on the body to maximize detection sensitivity. The preferred site is the skin over bony prominences of the iliac crest (top of the buttocks) and the scapular area (shoulder blades), and in lower calves (legs). If the patient has nodules, the specimen is taken from the skin i n t h e c e n t e r o f t h e n o d u l e . I t i s recommended that six
specimens (two snips from all three of these sites on each side of the body of the individual) should be examined before reporting a negative result.

Answer 279

Skin snip examination

Question 280

 The specimen should not be bloodstained. When snips are cut too deeply, small capillaries may be lacerated and the snip may be contaminated by microfilariae that might be present in the patient’s blood.

Answer 280

Skin snip examination

Question 281

 Skin snips are obtained in one of two ways:

Answer 281

1. Using a sterile needle
2. Using a sclerocorneal biopsy punch

Question 282

Pierce the skin to a depth of 2-3 mm, and elevate a piece of skin with the point of the needle and shave it off with a scalpel, as close to the needle as possible.

Answer 282

Using a sterile needle

Question 283

This takes snips of uniform diameter (approximately 2.3-2.5 mm)

Answer 283

Using a sclerocorneal biopsy punch

Question 284

 The skin snips are examined under the microscope as:

Answer 284

1. Wet mount preparation
2. Permanent stained preparation

Question 285

o Place the fragment of skin in the drop of NSS on the slide. Do not flatten the piece of skin; if only one microfilaria is present, it might be damaged.
o Cover with a coverslip. If any part of the specimen is not in the coverslip with a Pasteur pipette, until the whole area underneath the coverslip is wet.
o After 30 minutes to 3 hours, examine t h e p r e p a r a t i o n u n d e r t h e microscope using the 10x low power objective.
 Microfilariae are highly motile. If any are present, they will be seen moving towards the NSS.
 If negative, continue incubating overnight at 37°C.

Answer 285

Wet mount preparation