Suspected New Species of Chronic Roundworm Parasite, Cryptostrongylus pulmoni, Associated with CFS in Blinded Trials
Lawrence A. Klapow PhD, Journal of Chronic Fatigue Syndrome, 1999 5(3/4): 247-248
OBJECTIVES: 1) Describe the roundworm and how to find it. 2) Determine its occurrence in CFS and non-CFS controls in blinded trials.
METHODS: Coded three-day sputum collections, preserved in 50 % ethanol, from 24 patients and 16 controls were examined microscopically. Patients were drawn from the CFS clinic at Brigham and Women's Hospital (Harvard Medical School), and from three primary care practices. Identification required screening of numerous candidate specimens, clearing in glycerin, 1200X magnification (a 12X video mounted to the 10X ocular of a microscope set at the 10X objective), and the use of two polarizers (one in the light source the other in the image path over the ocular lens). Careful examination of photo-micrographs of both sides of the specimens, at multiple focal-depths, was required for reliable identification. Male reproductive organs (item 5-9 below), and female mouth parts (3,4,5,and 9-11), proved most useful for identification.
MALE (200 to 350 microns long): 1. Red in formalin, colorless in ethanol. 2. Nerve ring is visible in most glycerin cleared specimens surrounding a bulbless esophagus, using polarize/analyzed light. 3. A cervical flange, an exterior membrane, winds around the anterior (lost or fragmented in most specimens). 4. A pair of lateral ridges with triangulate crests extends the length of the body. 5. Spicules are very large (visible anatomy, other than terminal bifurcation, is indistinct). 6. Dorsal genital structures consist of a pair of projections spanned by a membrane, resembling a sail. 7. Ventral genital structures consist of a pair of long pointed projections, twisted like a corkscrew at their distal termini. 8. The dorsal lobe of the copulatory bursa is very large, supported by a bifurcated Ray 8, giving rise to four long raylettes. 9. Lateral lobes of the bursa are symmetrical: Ray 7 is very short and indistinct; Ray 6 is long and twists under the edge of the membrane, emerging beyond the border as a distinct point; Ray 5 is short and sigmoid; Ray 4 is long, straight, and robust, with its tip much closer to ray 3 than to ray 5; Rays 3 and 2 are long, straight, slender, and closely spaced.
FEMALE (550 to 950 microns long): 1. Only the front half is usually recovered (275 to 475 microns). 2. Vulva is mid-body. 3. Bulbless esophagus. 4. Nerve ring is visible in most glycerin cleared, specimens under polarized/analyzed lighting. 5. A cervical flange, an exterior membrane, winds around the anterior and is usually fragmented or absent in recovered specimens. 6. Cephalic vesicle is absent. 7. Oblique cuticle ridges forming chevrons are minimal, faint, and rarely seen in a few specimens. 8. Caudal area has paired papillae, and terminal cones. 9. Buccal capsule is asymmetrical. 10. Single amphid gland tubule. 11. The mouth has six prehensile lips. Numbered in a clockwise direction: lips 1 and 2 are short, followed by 3 which is long, followed by the amphid tubule, the asymmetrical buccal capsule, and three long lips, 4,5 and 6. 12. Cuticle striations are visible with a polarized light source and eyepiece optical analyzer.
RESULTS: Decayed specimens of C. pulmoni were found in 50 percent of three-day sputum samples from CFS patients (12 in 24), but not in 16 controls (chi-square association, P<0.003). The infection rate, corrected for test sensitivity, is conservatively estimated at 66 percent (95% CL, > 44% to > 84%). Two positive patients re-tested two years later were both still positive. Late larval stages were also found in sputum. Approximately 50 to 100 hours were needed to isolate and identify each specimen.
CONCLUSION: Cryptostrongylus pulmoni infects a large percentage of CFS patients, estimated at 66 percent in the current study, and is significantly associated with the syndrome (chi-square P<0.003) in blinded analyses. It appears to have chronic properties. Microscopic identification is difficult and time consuming due to the decayed state of most specimens, their small size, and extreme rarity.
Specialized, though inexpensive and simple, imaging techniques are needed for positive identification. Research to develop a DNA marker is in progress.
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