Cryptostrongylus pulmoni (provisional)

[Guest guest]

Dear Patti,

If you go to this website, you can get pictures of the parasite. I've

pasted the text below. The most interesting things, I think, are in the

Mazlen interview.

I've also pasted in the procedures that Dr. Klapow uses to find the

parasite, because I was so impressed by how very, very complicated ithe lab

work is.

Maybe your new doctor will talk to Dr. Klapow about getting him to look at

your sputum. Since you lived in Malaysia for four years, I should think Dr.

Klapow would be interested. Of course, even if you've got the parasite, I

assume getting rid of it will not be a piece of cake.

I've got my fingers crossed for his new study--I hope he can get funds, and

that he will turn up something that will help a lot of people.

http://members.tripod.com/lak123

Sue B.

upstate New York

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Cryptostrongylus pulmoni (provisional)

" The Hidden Lung Worm "

(Female ~750 microns long; Male ~280 microns long)

=====================

C. pulmoni has been found in the sputum of 40 percent of CFS patients in

both open and blinded trials. Actual infection rates are likely to be

higher due to the rarity of specimens and test insensitivity. A larger

blinded trial is currently underway to attempt to statistically distinguish

CFS patients from controls. Microscopic identification is

based mainly on decayed and fragmented specimens which are naturally

coughed up in sputum. See " Find Cryptostrongylus pulmoni " link above.

CFS is a debilitating disease characterized by profound fatigue (lasting

months and typically for years), muscle pain and weakness, joint pain,

cognitive impairment, and other symptoms. It often starats suddenly with

flu-like symptoms. The course of the disease is variable. Symptoms are

usually intermittent. Recovery rates after ten years are generally low. Its

cause remains unknown. From 400,000 to 800,000 Americans meat the U.S.

Centers of Disease Control (CDC) definition for CFS. The disease appears

to have been reported in many " Western " countries in the mid 1980s,

although there may possibly have been earlier occurences, mainly in the

latter part of the 19th century. Most cases are sporadic but clusters have

occurred in Northern Nevada, Upstate New York, and New Zealand.

=======================

Journal of Chronic Fatigue Syndrome, 1999; 5 (3/4): 247-8

Roundworm like specimens in the sputum of chronic fatigue syndrome patients

and controls in open and blinded analyses

Lawrence A. Klapow, Ph.D.

OBJECTIVES: To

1) determine the occurrence of a suspected nematode parasite,

Cryptostrongylus pulmoni (provisional), in CFS patients and controls, in

both open and blind trials

2) demonstrate skill in identification through the blind analysis, and

3) preserve specimens for biochemical evaluation.

METHODS: The distinguishing features of C. pulmoni, primarily male

reporductive structures (bursa, genitals, and spicules) and female mouth

parats, were first determined from specimens found in the sputum of a

patient taking an anti-roundworm medication. Three-day sputum samples were

obtained without the use of drugs from 28 CFS patients and 6 controls

(preserved in 50% ethanol). Patients were drawn from a CFS researach

center, general medical practices and an Internet group. Most patients

filled out a medical questionnaire. Initial microscopic examination

provided candidate specimens, which were preserved in glycerin. An imaging

system was used which allowed the mounted specimens to be turned over and

photographed from both sides at high magnification (1200X), and various

focal depths (a 12X optical video mounted to the 10X ocular of a microscope

set at the 10X objective lens). A second polarizer inserted in the ocular

lens, as an optical analyzer, sometimes revealed additional structures not

visible with the single polarized light source technique.

RESULTS: Specimens of C. pulmoni in various states of decay, were found in

11 out of 28 CFS patients, but not in 6 controls, in the combined analyses.

The sample size was too small for a chi-squared contingency test. In the

blind analysis, 5 of the 11 CFS patients, but none of the 6 controls, were

positive. The probability of getting as good a result in a coin toss

(random guess) model was 0.01. Of the 11 positives, 10 contained 1

specimen: the other had 2. Re-testing of 3 initially negative patients

resulted in 1 positive. Specimens were small: 200 to 500 microns long.

Identification required careful evaluation of the photographic images.

Three positive patients reported borderline to low-grade eosinophilia

(6-9%) Stool tests were negative.

CONCLUSION: Decayed specimens of a suspected roundworm, Cryptostrongylus

pulmoni (provisional) " the hidden lung worm, " were found in the naturally

expelled sputum of 39% of CFS patients, but not in 6 controls. Low test

sensitivity suggests that actual occurrence is likely to be higher. A

larger test design will be needed to evaluate the possible association of

C. pulmoni with CFS. A random guess mode suggested that skill in

identifying C. pulmoni rather than chance governed the results of the blind

analysis. Current diagnostic methods would not be expected to detect C.

pulmoni. The usual indicators of nematode infections, high-grade

eosinophilia and positive stool tests were not apparent. In addition to

being decayed, specimens were also very small and rare, and required

specialized imaging techniques for identification. Roundworm infections can

produce immune abnormalities like those reported in CFS, including low

serum cortisol, altered anti-viral responses, eosinophil anomalies, and

altered cholinergic processes.

Presented at the Fourth International Conference of The American

Association of Chronic Fatigue Syndrome (Cambridge MA, October 10-12,

1998.)

==============================

Dr. Klapow's e-mail address: :Lak123@...

================================

IMAGING SYSTEM

The imaging system consists of a 12X optical video positioned directly over

the eye piece of a single, straight tube microscope. With the high

clearance allowed by the 10X objective, specimens can be photogrphed from

both sides at high magnification (1200X). A second polarizer placed

directly over the ocular lens, acting as an optical analyzer, sometimes

revealed additional structures not visible with the usual single light

source polarizer. Direct microscopic examination of bulk sputum usually

produces only candidate specimens, which have to be isolated, cleared in

glycerin mounted, and photographed for positive identification.

SPECIMEN COLLECTION AND PREPARATION

1. Three-day sputum collections were obtained from patients (CDC defined)

who were instructed to force themselves to cough, afater taking a warm

shower, and deposit any sputum into a vial with 15 mls of 50% ethanol

(formalin would preserve the red color of male worms, but fumes are a

problem for many patients.)

2. Samples are centrifuged and excess supernatant is pipetted off, leaving

a liquid volume equal to about three times the residual solids.

3. The solids are re-mixed with the remaining liquid, and about five drops

are deposited on each of a series of slides (3 to 40) and covered with a

glass cover slip.

[4. ?]

5. Slides are scanned at 40X. Objects within the range of 200 to 500

microns long are further evaluted at high power, 1200X Females approach

1000 microns in total length but ae usually split in half, with only the

anterior half being identifiable.

6. Likely candidate specimens are processed further. A marker pen is used

to spot their position on both the cover slip and on the backside of the

glass slide.

7. The marked slide is then frozen for from 2 to 4 days. While still

frozen, the cover slip is quickly pried up with a razor blade and turned

over onto a waiting slide.

8. Drops of 10/90 glycerol/water are placed on the marker spot of the

cover slip and the original slide, and the candidate object is relocated.

It is then pipetted to the well of a concave glass slide along with a few

additional drops of 10/90 glycerol.

9. The concave well is covered by a dust shield (a plastic cap with a

small central hole), and allowed to stand for 3-4 days while the water

slowly evaporates.

10. The isolated specimen, now in nearly pure glycerol, is pipetted onto

the final glass slide and covered with a glass cover slip. It's position is

marked.

PHOTOMICROSCOPY AND IDENTIFICATION

1. The object is viewed at 1200X, using 10X ocular, 10X objective and 12X

video, positioned directly over the halogen filament, video set at

back-lighted position.

2. Two polarizers are used. One in the light path (in the filter holder

without a diffuser element), the other in the image path (directly over the

ocular lens).

3. Rotating the ocular polarizer separates the images coming from the back

and front side of the cuticle [cubicle?] which are polarized in different

directions by optical sugars. This can reveal deep internal structures or

fine surface details separately.

4. The object is photogrphed at multiple focus depths at 1200X and optimum

polarizer positions, and turned ovr and photographed from the other side.

5. A tripod mounted 35mm SLR camera, at aperture priority of f22 with

automatic exposure (100-200 ASA film), for long exposures, produces good

results. It was positioned at minimal focal distance from the video screen

for the 50mm lens.

6. Photographs are carefully evaluated for the features of Cryptostongylus.

7. With developing skill, expect ~40% positives in samples from CDC

defined CFS.

8. The typical yield is only one identifiable specimen per sample. GOOD LUCK!