Jcm.asm.org

Albendazole Stimulates the Excretion of
Larvae in Stool
Strongyloides stercoralis
Specimens and Enhances Sensitivity for
Diagnosis of Strongyloidiasis

Witthaya Anamnart, Attarat Pattanawongsa, Pewpan
Maleewong Intapan and Wanchai Maleewong
2010, 48(11):4216. DOI:
J. Clin. Microbiol.
10.1128/JCM.00852-10.
Published Ahead of Print 15 September 2010.
Updated information and services can be found at: REFERENCES
This article cites 12 articles, 5 of which can be accessed free at: CONTENT ALERTS
Receive: RSS Feeds, eTOCs, free email alerts (when newarticles cite this article), JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2010, p. 4216–4220 Copyright 2010, American Society for Microbiology. All Rights Reserved.
Albendazole Stimulates the Excretion of Strongyloides stercoralis Larvae in Stool Specimens and Enhances Sensitivity for Witthaya Anamnart,1* Attarat Pattanawongsa,2 Pewpan Maleewong Intapan,3 Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Thasala, Nakhon Si Thammarat,1 School of Pharmacy, Walailak University, Thasala, Nakhon Si Thammarat,2 and Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen,3 Thailand Received 28 April 2010/Returned for modification 9 June 2010/Accepted 7 September 2010 We succeeded in stimulation of excretion of Strongyloides stercoralis larvae in stool by oral administration of a
single dose of 400 mg albendazole to strongyloidiasis patients. This result overcame the false-negative results of
stool examination due to low larval numbers. Stool samples were collected from 152 asymptomatic strongyloidiasis
patients in the morning, prior to eating. After breakfast, they were given a dose of 400 mg albendazole, and stool
samples were collected the following morning. Agar plate culture (APC), modified formalin-ether concentration
technique (MFECT), and direct-smear (DS) methods were used to examine stool specimens within 3 h after
defecation. The results before and after albendazole was taken were compared. All APCs that were positive became
negative after albendazole administration, while MFECT showed a 1.4- to 18.0-fold increase in larval numbers in
97.4% (148/152) of the samples. The DSs were positive in 3 out of 3 smears at a larval number of >45 larvae per
g (lpg) of stool, and in 1or 2 out of 3 smears at a larval number between 35 and 44 lpg. At a larval number of <35
lpg, the DS became negative. Interestingly 90.5% (19/21) of the samples that were negative by all methods before
albendazole administration became positive by MFECT after the treatment. Thus, MFECT can be effectively used
for diagnosis of strongyloidiasis with prior administration of albendazole to the subject.
Strongyloidiasis is a helminthic infection caused by Strongy- aimed to enhance the sensitivity for the diagnosis of strongy- loides stercoralis, a worm that is particularly dangerous for loidiasis by using a single 400-mg oral dose of albendazole to immunosuppressed patients. Although many methods are stimulate the excretion of larvae into the stool for easier de- presently being used to diagnose this disease, a gold standard tection by MFECT and/or DS, particularly in cases where for the detection of larvae in the stool is still needed (14).
strongyloidiasis was strongly suspected, such as in patients with Parasitological methods include agar plate culture (APC) (10), unexplained chronic diarrhea and patients returning from ar- the Baermann method (4), the formalin-ether concentration eas where strongyloidiasis is endemic.
technique (FECT) (13), the quantitative formalin ethyl acetateconcentration technique (QFECT) (9), and the newly modifiedFECT (MFECT) (1). However, each method has its own dis- MATERIALS AND METHODS
advantages, resulting in false negativity. In addition, female S. Subjects and stool samples. One hundred fifty-two strongyloidiasis patients
stercoralis parasites, unlike other intestinal roundworms, em- referred from other research projects were invited to join our study. All patients bed in the mucosa of the small intestine, where they lay em- had previously been diagnosed with asymptomatic strongyloidiasis for a period of bryonated eggs that immediately hatch (8). Thus, the larvae 1 month to 2 years. These patients had not received any anthelmintic drugs and are often scanty and irregularly excreted (11, 15). Repeated were diagnosed by APC, together with either FECT or MFECT. The ages of thepatients ranged from 7 to 70 years. All came from the Moklan subdistrict (7 km stool examination is therefore recommended for the diagnosis from the laboratory unit of Walailak University) in the Thasala district of Nak- of strongyloidiasis (6). During an intestinal-helminth survey by hon Si Thammarat, a province in southern Thailand. The patients were asked to direct fecal smear (DS) examination, some participants tested collect the entire amount of their morning stool in a plastic container at about negative for Strongyloides larvae. After albendazole treatment 6:00 a.m., before breakfast. The samples were labeled as “stool before albenda- for Ascaris lumbricoides, Trichuris trichiura, and hookworms, zole administration.” A single dose of 400 mg albendazole was then given orallyafter breakfast, at about 9:00 a.m. The whole morning stool was then collected the stools were reexamined. Surprisingly, stools that were pre- again from each patient within 21 h after albendazole administration; these viously negative for S. stercoralis larvae became positive (data samples were labeled “stool after albendazole administration.” Subsequently not shown). It was then considered possible that albendazole another single dose of 400 mg albendazole was given after breakfast for two treatment could increase the sensitivity of stool examination consecutive days to complete the treatment. All stool samples were sent to thelaboratory without preservatives and tested within 3 h after defecation.
methods for diagnosis of strongyloidiasis. Thus, this research All 152 subjects were willing to collect their stools two times, before and after taking albendazole, to compare APC, MFECT, and DS for the detection oflarvae before and after albendazole administration (see below). However, only * Corresponding author. Mailing address: Department of Medical 17 of 152 agreed to collect their stools another six times on six different days prior Technology, School of Allied Health Sciences and Public Health, to and after taking the drug to compare the fluctuations of larval excretion versus Walailak University, Thasala, Nakhon Si Thammarat 80160, Thailand.
larval excretion after albendazole administration (see below). Stool samples were Phone: 66-75-672187. Fax: 66-75-672106. E-mail: [email protected].
collected again 14 days later for determination of the correlation between incom- ᰔ Published ahead of print on 15 September 2010.
plete cure with albendazole treatment and the amount of larval excretion (3).
ALBENDAZOLE ENHANCES DIAGNOSIS OF STRONGYLOIDIASIS This research was approved by the Ethics Clearance Committee on Human 1.4 to 18 times over those before albendazole intake, in 97.4% Rights Related to Research Involving Human Subjects, Walailak University.
(148/152) of the stool samples. On the other hand, a decline in Agar plate culture. APC was performed as described previously (10). Briefly,
the larval numbers in two samples from subcategory 2b was 3 g of each stool sample was placed at the centers of nutrient agar plates andincubated at room temperature (28°C to 33°C) for up to 5 days. Worm motility noted, and no larvae were detected in two samples from cat- tracks, larvae, and free-living adult worms were monitored by stereomicroscope egory 4 (Table 1). Likewise, the DS became positive in 56.8% on day 3 (after 48 h). In case of a negative finding, observations were continued of samples (79/139; 13 samples in category 1 were not tested) for another 2 days. Ten milliliters of 10% formalin was added to the agar surface using three smears: 48.3% (67/139) of previously negative DS of each microscopically positive dish to collect worms for species identification samples were positive in all three smears, 5.8% (8/139) of using a compound microscope (40ϫ). The APC results were qualitatively read aspositive or negative.
previously negative DS samples were positive in two smears, Modified formalin-ether concentration technique. MFECT was performed as
and 2.9% (4/139) of previously negative DS samples were pos- described previously (1). Briefly, 2 g of fresh stool sample was suspended and itive in only one smear (Table 1). The DS was positive in all stirred well in a tube containing 10 ml of 0.85% saline. The fecal suspension was three smears when the larval number by MFECT was Ն45 lpg strained through two pieces of 4- by 4-cm wire mesh into a plastic centrifugetube. The first mesh (1.2- by 1.2-mm pore size) was placed on top of the funnel, and positive in only one or two smears when the larval number while the second mesh (2- by 2-mm pore size) was used by hand. Trapped fecal materials on both meshes were washed with 3 ml of 0.85% saline. The resulting Cases that were positive only by APC, as in category 3, suspension was centrifuged at 700 ϫ g for 5 min. The supernatant was then became positive by MFECT after albendazole stimulation. In- decanted, and the volume was adjusted to 7 ml with 10% formalin without terestingly, albendazole could dramatically and effectively mixing; 3 ml of diethyl ether was immediately added. The centrifuge tube wasclosed and shaken vigorously by hand for 1 min and immediately centrifuged at stimulate the excretion of larvae in 90.5% (19/21) of the sam- 700 ϫ g for 5 min. The debris plug was loosened, and the top three layers were ples from category 4, which represented cases that were clini- poured off. Approximately 1 ml of 10% formalin was added to the sediment.
cally suspicious only and could not be diagnosed by APC, Then, the larvae in the sediment were counted and presented as larvae per g MFECT, or DS until they became MFECT positive after al- (lpg) of stool (10% formalin was used for the wet preparations for MFECT).
Direct smear. DS was performed as described previously (2, 7), with some
modifications. A drop of 0.85% saline was placed on a slide. Stool was randomly Thirty-six of 152 subjects (23.0%) showed an incomplete stuck to a wooden stick by dipping the tip approximately six times into the cure by albendazole treatment, defined as the recovery of lar- sample. Stool was dispersed in the drop of 0.85% saline using the tip of the vae 14 days after drug administration of three doses of 400 mg wooden stick coated with the stool. A 22- by 22-mm coverslip was applied, and for three consecutive days; however, the incomplete cure had the entire smear was scanned with a microscope (magnification, ϫ100). A pos-itive DS meant that at least 1 larva per smear was found. Three smears were no relationship to the increase in the number of larvae. The examined, both before and after albendazole was taken. For confirmation of S. larval number after the first dose of 400 mg albendazole was stercoralis larvae, a drop of 10% formalin was added to an edge of the coverslip still 2 to 7 times higher than that before albendazole adminis- and allowed to diffuse to the saline smear. Direct examination was performed if tration in all 36 subjects (data not shown).
the larvae were not actively motile.
Comparison of APC, MFECT, and DS for the detection of larvae before and
S. stercoralis infections show an irregular and fluctuating after albendazole administration. Upon receiving the samples, the weights of
pattern of larval excretion (Table 3). However, when albenda- stool specimens were determined, using preweighed containers, and recorded.
zole was administrated orally, larval numbers increased to the APC, MFECT, and DS were performed both before and after albendazole maximum larval excretion point, where most larvae were administration, as described above. For DS, three smears were performed.
driven out of the mucosa into the stool (Table 3). Albendazole According to the results of the three different methods before albendazole administration, we divided the subjects into four categories: (i) category 1 was had no effect on the excretion of A. lumbricoides, T. trichiura, composed of 13 samples that were APC positive, MFECT positive, and DS positive; (ii) subcategories 2a and 2b were composed of 79 and 33 samples,respectively, which were APC positive, MFECT positive, and DS negative (afteralbendazole was taken, all samples in subcategory 2a were positive by DS, DISCUSSION
whereas those in subcategory 2b were negative by DS); (iii) category 3 wascomposed of 6 samples that were APC positive, MFECT negative, and DS Albendazole, a broad-spectrum anthelmintic drug, is an ef- negative; (iv) category 4 was composed of 21 samples that were APC negative, fective treatment for uncomplicated strongyloidiasis (12).
MFECT negative, and DS negative (Table 1). The category 4 patients had However, so far, the drug has not been used for the diagnosis positive APC results with a low number of larvae and/or adult worms 1 to 2 years of strongyloidiasis. The mechanism of action of albendazole, previously during another research project but were negative on the day beforealbendazole was administered in our study.
after being metabolized in the liver into albendazole sulfoxide, Fluctuation of larval excretion versus larval excretion after albendazole ad-
is to inhibit tubulin polymerization and subsequently decrease ministration. Stool samples were collected from each subject on days 1, 2, 4, 7,
glucose uptake, thus exhibiting larvicidal, ovicidal, and adulti- 14, 28, 42, and 43. Days 42 and 43 were the days before and after albendazole cidal activities (5). This suggests that the larvae are dead and administration, respectively. All stool samples were tested by MFECT only,except that on days 42 and 43 APC, MFECT, and DS were used.
thus unable to grow on APC. There are no data on how long Statistical analysis. SPSS 13.0 for Windows was used to perform all statistical
after albendazole treatment the cultivation of larvae in stool analysis. Descriptive statistics, including mean, standard deviation (SD), and samples by APC can become positive again. Also, even without range, were generated. Nonparametric Wilcoxon signed-rank tests were used to treatment, some larvae will gradually die within 4 h after def- determine the differences in larval numbers by MFECT before and after al- ecation, which negatively influences the sensitivity of the FECT bendazole administration in 13, 79, 33, 6, and 21 pairs of categories 1, 2a, 2b, 3,and 4, respectively. A P value of Ͻ0.05 was considered statistically significant.
or MFECT (unpublished data). Thus, after albendazole intake,MFECT should be performed on fresh stool within 4 h afterdefecation. In addition, to avoid dilution in a large amount of stool, albendazole should be administered after as much stool After 400 mg albendazole intake, APC results became neg- as possible has been excreted. Furthermore, patients should ative in all cultured stool samples (Tables 1 and 2). However, limit food intake after oral administration of albendazole, and MFECT results showed an increase in the number of larvae, by meals should have a high fiber content in order to decrease TABLE 1. Comparison of APC, MFECT, and DS results before and after one dose of 400 mg albendazole in the four categories of samples After albendazole administration (APC negative, After albendazole administration (APC negative, After albendazole administration (APC negative, After albendazole administration (APC negative, After albendazole administration (APC negative, After albendazole administration (APC negative, a P values comparing the difference in the number of larvae per gram of stool by MFECT before and after albendazole administration in the four categories.
b At least 1 larva per smear was found.
c Three smears were examined; 3 smears were DS positive when the larval number by MFECT was Ն45 lpg.
d Three smears were examined; 2 smears were DS positive when the larval number by MFECT was between 35 and 44 lpg.
e Three smears were examined; only 1 smear was DS positive when the larval number by MFECT was between 35 and 44 lpg.
f NA, not applicable.
loose bowel movements and increase large particles in the bowel might have the effect of increasing the number of ex- stool. Our observations of a 9-year-old child (not included in creted larvae. We did not show MFECT data for larvae per this study) showed that 2 g of “hard to formed” stool contained defecation in this study because the amounts of stool before 210 lpg (420 larvae per defecation), contrasting with another and after albendazole intake were the same. It was also noted stool collection of 50 g mushy stool containing 10 lpg (500 that stool passed later than 24 h after albendazole intake larvae per defecation) 2 days later. In other words, the decline should be avoided, because in 5 cases in which the stool was in the amount of stool caused by water absorption in the large collected 48 h after albendazole intake, we found no larvae or TABLE 2. Qualitative comparison of APC, MFECT, and DS results before and after one dose of 400 mg albendazole ALBENDAZOLE ENHANCES DIAGNOSIS OF STRONGYLOIDIASIS TABLE 3. Comparison of the number of irregularly excreted S. stercoralis larvae per gram of stool by MFECT on seven different days before albendazole administration and on the day after albendazole administration in 17 subjects No. of larvae on day of stool collection: a The day before 400 mg albendazole administration.
b Mean number of larvae per gram of stool on seven different days before albendazole administration.
c Median number of larvae per gram of stool on seven different days before albendazole administration.
d The day after 400 mg albendazole administration.
a low number of larvae with deteriorated bodies (data not excreted and where more than one collection of stool is re- shown). The cause might have been that the excreted larvae quired (6). The sensitivity of MFECT for the detection of were dead and had already been decomposed by intestinal larvae is 18 lpg if 1 larva is found per smear (approximately 30 enzymes and the bacterial flora and thus became undetectable.
␮l per smear) and 9, 6, 4 or 5, 3, 2, and 1 lpg if 1 larva is found Our results showed clearly that a single dose of 400 mg per 2, 3, 4, 6, 9, and 18 smears, respectively. Thus, if the larval albendazole was able to stimulate the excretion of larvae into number is less than 4 lpg, more than 4 smears must be exam- stool samples, which could then be detected by MFECT; larvae could also be detected by DS when the larval number was Although albendazole could increase the number of larvae greater than 35 lpg. There were two samples in category 2b by 1.4 to 18.0 times, the use of DS was limited by the number with a lower number of larvae excreted after albendazole in- of larvae produced and excreted. In cases of light infection, the take. This might be explained by the larval number having number of parasitic females was too low to shed enough larvae already reached the maximum larval excretion point prior to for detection by DS. This study revealed that approximately albendazole intake, thus rendering the production of larvae by 50% of samples that were negative by DS before albendazole parasitic females insufficient and impeding the stimulating ef- intake could be detected by DS after albendazole intake. The fect of albendazole. Similarly, two patients with uncomplicated administration of one dose of 400 mg albendazole to stimu- strongyloidiasis, with episodes of diarrhea showing loose stools late the excretion of larvae can be employed as a supportive with larval numbers of 28 and 16 lpg by MFECT on the day method for the diagnosis of strongyloidiasis in areas where before albendazole administration, produced only 23 and 13 lpg, respectively, after the first dose of 400 mg albendazole.
In conclusion, the application of albendazole plus MFECT After taking two more doses, the patients recovered from di- for enhancing the diagnosis of human strongyloidiasis should arrhea. The larval number was possibly decreased, because be used in patients suspected of asymptomatic strongyloidiasis.
during the episodes of diarrhea, larval excretion had already This includes patients with unexplained chronic diarrhea, pa- reached the maximum point, the point at which most larvae are tients returning from areas where strongyloidiasis is endemic, driven out of the mucosa into the stool, because the diarrhea and patients with negative results by other parasitological tests.
stimulated the excretion of larvae into the stool. Two samples The technique could also be applied in areas where APC and from category 4 showed no larvae by any of the three testing MFECT are not available but DS is routinely used.
methods before and after albendazole intake, possibly for oneof the following reasons: the patients were cured naturally and ACKNOWLEDGMENTS
the parasitic females died, or the number of parasitic femaleswas too low, resulting a production of larvae too low to be This study was funded by Walailak University. P.M.I. and W.M.
detected, although excretion occurred after albendazole in- were supported by a grant from the Research and Diagnostic Centerfor Emerging Infectious Diseases, Khon Kaen University.
We thank Pawilai Dermlim, Malisa Thongrod, and Siraporn Innimit Therefore, albendazole administration plus MFECT should for their technical assistance. We also thank Nimit Morakote for com- be employed in cases where larvae are scant or irregularly REFERENCES
asis: a major roundworm infection of man. Taylor & Francis, Philadelphia,PA.
1. Anamnart, W., A. Pattanawongsa, P. M. Intapan, and W. Maleewong. 2010.
8. Grove, D. I. 1996. Human strongyloidiasis. Adv. Parasitol. 38:251–309.
Factors affecting recovery of Strongyloides stercoralis larvae: an approach to 9. Intapan, P. M., W. Maleewong, T. Wongsaroj, S. Singthong, and N. Mora-
a newly modified formalin-ether concentration technique for diagnosis of kote. 2005. Comparison of the quantitative formalin ethyl acetate concen-
strongyloidiasis. J. Clin. Microbiol. 48:97–100.
tration technique and agar plate culture for diagnosis of human strongyloidi- 2. Beaver, P. C., R. C. Jung, and E. W. Cupp. 1984. Examination of specimens
asis. J. Clin. Microbiol. 43:1932–1933.
for parasites, p. 733–758. In P. C. Beaver, R. C. Jung, and E. W. Cupp (ed.).
10. Koga, K., S. Kasuya, C. Khamboonruang, K. Sukhavat, M. Ieda, N. Takat-
Clinical parasitology, 9th ed. Lea & Febiger, Philadelphia, PA.
suka, K. Kita, and H. Ohtomo. 1991. A modified agar plate method for
3. Daenseekaew, W., V. Pipitgool, P. Sithithaworn, and S. Kitvatanachai. 1996.
detection of Strongyloides stercoralis. Am. J. Trop. Med. Hyg. 45:518–521.
Albendazole in the treatment of uncomplicated strongyloidiasis by using the 11. Liu, L. X., and P. F. Weller. 1993. Strongyloidiasis and other intestinal
agar plate method. Srinagarind Med. J. 11:169–172.
nematode infections. Infect. Dis. Clin. North Am. 7:655–682.
Ferriolli, F., Jr. 1961. Condicoes que influema na extracao de larvas do
Pitisuttithum, P., W. Supanaranond, and D. Chindanond. 1995. A random-
ized comparative study of albendazole and thiabendazole in chronic strongy-
Strongyloides stercoralis das fezes pelo metodo de Looss-Baermann modifi- loidiasis. Southeast Asian J. Trop. Med. Public Health 26:735–738.
cado. Rev. Inst. Med. Trop. Sao Paulo 3:51–60.
13. Ritchie, L. S. 1948. An ether sedimentation technique for routine stool
5. Goldsmith, R. S. 1992. Clinical pharmacology of the anthelmintic drugs, p.
examinations. Bull. U.S. Army Med. Dept. 8:326.
742, 748–765. In B. G. Katzung (ed.). Basic and clinical pharmacology.
14. Siddiqui, A. A., and S. L. Berk. 2001. Diagnosis of Strongyloides stercoralis
infection. Clin. Infect. Dis. 33:1040–1047.
6. Grove, D. I. 1980. Strongyloidiasis in allied ex-prisoners of war in Southeast
15. Uparanukraw, P., S. Phongsri, and N. Morakote. 1999. Fluctuations of larval
Asia. Br. Med. J. 1:598–601.
excretion in Strongyloides stercoralis infection. Am. J. Trop. Med. Hyg. 60:
7. Grove, D. I. 1989. Diagnosis, p. 175–197. In D. I. Grove (ed.). Strongyloidi-

Source: http://jcm.asm.org/content/48/11/4216.full.pdf