Microbiology Case Study: Specimen Referral from a 20 Month Old Male

Case History

A 20 month old male presented to an outside hospital with symptoms unknown to our laboratory. That laboratory sent us the specimen recovered from a diaper (Image 1).

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Image 1.

Discussion

The nematode Ascaris lumbricoides is one of the most common helminth infections in the United States. It can grow to be 20-35 cm long. Infection occurs when an egg is ingested, usually in a small child eating dirt contaminated with human feces. When the larvae hatch they penetrate the duodenal wall. From there, the larvae go into the blood stream and eventually end up in the pulmonary circulation where the larvae grow in the alveoli.  In about three weeks, the larvae are coughed up from the lungs and swallowed.  The worms then mature in the jejunum (primarily).  Infection most often shows no symptomatology. If symptoms are present, they can range from mild abdominal discomfort to intestinal blockage and even cough as the worms migrate to the lungs [1].

Diagnosis can be made by examining concentrated stool for knobby-coated, bile-stained eggs that are oval [2].  However, some of the adult worms can pass with the feces.

References

  1. https://www.cdc.gov/parasites/ascariasis/index.html
  2. Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology, Seventh Edition. Elsevier Health Sciences; 2012.

 

-Angela Theiss, MD is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

Microbiology Case Study: An 18 Year Old with Vaginal Discharge

Case Presentation

An 18 year old girl presents to her pediatrician with her mother for her pre-college check-up. She has no past medical history. After her mother leaves the room for the social history component, the girl admits to having sex with her boyfriend for the first time two weeks ago and complains of a yellow green malodorous vaginal discharge that started a week ago. She endorses mild pelvic pain. A pelvic exam is performed and mild cervical tenderness is noted. The cervix is pink, nulliparous, inflamed and is covered by small red punctate spots. A thin yellow green frothy discharge of fishy odor is also detected. A wet prep is made and reveals squamous cells and numerous motile organisms.

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Figure 1.  Trichomonas vaginalis in a Pap test. The protozoa are often found next to squamous cells. (ThinPrep)

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Figure 2.  Collection of Trichomonas vaginalis parasites eating at a squamous cell in a Pap (ThinPrep)

Discussion

Our patient was diagnosed with Trichomonas vaginalis (TV). TV is a flagellated parasitic protozoan for which humans are the only known host. It is 10-20 um long and 2-14 um wide with multiple flagella projecting from the anterior and posterior sides. It has a single trophozoite stage and does not survive well outside of its host. TV is a predatory obligate parasite that eats bacteria, vaginal epithelial cells, and red blood cells. It uses fermentative metabolism to produce the carbohydrates needed for fuel. TV is a sexually transmitted disease; however, because it is not reportable to local health departments, the true epidemiologic incidence rate is unknown. Its prevalence is highly variable by population and location. For example, some studies cite a prevalence of 3.1% of American pre-menopausal women (2.3% of adolescents) [1], while in certain high-risk populations the rate might be as high as 47% [2]. Most affected patients are asymptomatic; about a third of females become symptomatic within six months of infection. Symptoms for females include vulvar and vaginal irritation and itching, pain with urination and a diffuse, malodorous, yellow-green vaginal discharge. The cervix becomes reddened in a punctuated fashion causing the well-known strawberry cervix seen on colposcopy. In males, urethritis can develop. TV is often diagnosed via wet mount microscopy, where the protozoa can be seen moving around (Video 1). However, the sensitivity is relatively low, especially among males. Detection by nucleic acid probe from urine, endocervical, and vaginal swabs are considered more sensitive. TV can also be incidentally discovered on Pap tests (Figures 1 and 2). Treatment typically consists of a single dose of metronidazole [1,2]. It is critical that partners be treated as well, because otherwise reinfection may occur.

 

References

  1. Kissinger P. Trichomonas vaginalis: a review of epidemiologic, clinical and treatment issues. BMC Infectious Diseases. 2015; 15(307): 1-8.
  2. Meites E et al. A review of evidence-based care of symptomatic trichomoniasis and asymptomatic Trichomonas vaginalis infections. Clinical Infectious Diseases. 2015; 61(S8): S837-48.

 

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-Amanda Strickland, MD, is a 2nd year Anatomic and Clinical Pathology Resident at UT Southwestern Medical Center.

Erin McElvania TeKippe, PhD, D(ABMM), is the Director of Clinical Microbiology at Children’s Medical Center in Dallas Texas and an Assistant Professor of Pathology and Pediatrics at University of Texas Southwestern Medical Center.

 

Microbiology Case Study: A 22 Year Old Female with Recent Travel

Case History

A 22 year old female with recent travel to Nicaragua noted passage of a 10-12 cm long worm in her stool. She also noted some intermittent hematochezia over the past several days and had developed an itchy eczematous rash on her extremities.

Laboratory Diagnosis

Stool sample was submitted for ova and parasite exam. Stool sediment exam showed the presence of multiple fertilized eggs measuring 50 microns (Image 1). Based on the size of the egg, and the presence of the thick and yellow mammelated coat, she was diagnosed with an Ascaris lumbricoides infection.

 

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Image 1.

Discussion

Ascaris is the largest of the common nematode parasites of humans with females measuring 20-35 cm long and males measuring 15-31 cm. Notably, males have a curved posterior end. Infection is acquired through ingestion of the embryonated eggs from contaminated soil. In the larval migration phase of infection, diagnosis can be made by finding the larvae in sputum or in gastric washings. One female worm can lay up to 20,000 eggs, therefore enumeration of eggs does not correlate with worm burden. Both fertilized and unfertilized eggs can be easily be recovered using the sedimentation concentration from a fecal sample. It is estimated that 25% of the world population is infected with Ascaris and since transmission depends on fecal contamination of the soil, in areas where infection rates are high, mass population treatment plans with Abendazole have been successful.

 

-Agnes Balla, MD is a 3rd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.

Microbiology Case Study: A 50 year Old Man with Dysuria and Hematuria

Case History

A 50 year old healthy man presented with dysuria and hematuria for 4 months. He had briefly lived in the Middle East 3 years ago. The patient underwent cystoscopy which demonstrated a solid mass in the lateral wall of the bladder. Bladder biopsies were performed and showed invasive squamous cell carcinoma associated with ova consistent with Schistosoma haematobium (Figures 1&2).

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Figure 1. H&E of bladder biopsy showing invasive squamous cell carcinoma and ova of Schistosoma haematobium.

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Figure 2. High power view of Schistosoma haematobium ova.

Discussion

Schistosomiasis is caused by blood flukes of the genus Schistosoma. There are three major species related to human disease: S. haematobium, S. japonicum, and S. mansoni. Clinical presentation of schistosomiasis depends on the species. S. haematobium infection causes urinary schistosomiasis. Urinary schistosomiasis can range from asymptomatic to gross hematuria and possible obstruction resulting in renal failure.  S. haematobium is geographically distributed primarily in Africa and the Middle East. Transmission to humans requires direct contact with water harboring snails infected with S. haematobium. The cercaria that are released from infected snails penetrate human skin and then migrate to venules of the bladder and ureters. The cercaria develop into adult male and female flukes.  The adult schistosomes reside in the bloodstream and lay eggs that pass through the urine. The eggs are highly immunogenic and produce an intense inflammatory response resulting in hematuria and dysuria. Progression to fibrosis, renal failure and carcinoma may occur as in our patient with squamous cell carcinoma of the bladder. In addition to detection in surgical specimens, S. haematobium may be detected by identification of ova in urine. The ova of S. haematobium are oval and 112-170 µm x 40-70 µm in size with a characteristic terminal spine.  In patients with a high clinical suspicion of S. haematobium, serology may be useful when ova are not identified in urine or surgical specimens. The recommended treatment for schistosomiasis is praziquantel.  The timing of treatment is important because praziquantel is most effective against the adult worm and requires a mature antibody response to the parasite. The Centers for Disease Control and Prevention recommend starting treatment for infected travelers at least 6-8 weeks after the last exposure to contaminated water.

 

-Jill Miller, MD is a 4th year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.

Microbiology Case Studies: Babesia vs. Malaria

Patient History: Case 1

A 55 year old Asian woman presented to an emergency department in southern New England in September with complaints of a high fever with chills for the past 5 days. She noted feelings of excessive tiredness, muscle aches, and headache. She also described a decrease in appetite and nausea with vomiting and diarrhea. On physical exam, she was febrile (103.8°F) and scleral icterus was identified. Laboratory workup revealed findings suggestive of hemolysis including increased LDH (401 U/L) and increased unconjugated bilirubin (1.7 mg/dL), despite hemoglobin & hematocrit values in the normal range (13.7 g/dL & 39.3%, respectively). Elevated liver enzymes were also noted; AST 81 U/L and ALT 72 U/L. When questioned regarding traveling history, she reported a trip to Spain and Portugal 5 months earlier. Though she acknowledged living in a rural area of the Northeastern U.S. and indicated that her husband was diagnosed with Lyme disease one year earlier, she denied both recent time outdoors and arthropod or mosquito bites.

Patient History: Case 2

A 31 year old African American woman with a history of sickle cell trait presented to an emergency department in southern New England in September complaining of fevers of 5 days duration. She described being asymptomatic in the mornings followed by high spiking fevers with muscle aches and dull frontal headaches in the evenings. A physical exam revealed a fever (103°F), but no evidence of meningismus. Laboratory workup revealed a mild, microcytic anemia (hemoglobin & hematocrit: 10.7 g/dL & 32.5%, MCV: 76.3 fL), a decreased absolute lymphocyte count and increased band neutrophils. When questioned regarding recent travel, she reported having returned from Africa 10 days earlier. While abroad, she had primarily been in Nigeria’s capital, but she had also visited rural areas. She did not recall having been bitten by mosquitos, but she did not take any anti-malarial prophylaxis. Further, she denied both recent travel to the woods in the Northeastern U.S. and recent arthropod bites.

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Figure 1. Peripheral blood smear from patient 1 showing ring-like forms which contain a small amount of cytoplasm and a chromatin dot as illustrated by the arrows. Both intra-erythrocytic and extra-cellular forms are present. Platelets are denoted by arrowheads.

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Figure 2. BinaxNOW lateral flow assay from patient 1 is negative for the various Plasmodium spp.

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Figure 3. Peripheral blood smear from patient 2 showing ring forms and trophozoites within red blood cells as denoted by arrows. Inset illustrates a scattered gametocyte. Platelets are denoted by arrowheads.

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Figure 4. BinaxNOW lateral flow assay from patient 2 is positive for non-falciparum malaria species as indicated by the faint positive reaction denoted by the red arrow in the T2 region.

 

For patient 1, given the results from the peripheral blood (Figure 1), the negative BinaxNow results (Figure 2) and her lack of recent travel to malaria endemic regions, her illness was attributed to infection by Babesia spp. Further serologic testing was positive for Babesia microti. She was seronegative for Anaplasma phagocytophilum, Borrelia burgdorferi, Ehrlichia chaffeenesis. This finding was confirmed by PCR of her blood, which detected B. microti, but failed to detect B. duncani or B. divergens/MO-1. Approximately 3% of her red blood cells contained intracellular parasites.

For patient 2, her disease was most consistent with an infection by a non-falciparum species of malaria, including P. ovale, P. vivax or P. malariae, given her recent travel to Nigeria and advanced forms seen in the peripheral blood (Figure 3). Further speciation was uncertain due to low parasitemia levels (<1%) and the findings were unable to exclude a mixed infection with a low P. falciparum burden.

Discussion

The clinical and laboratory presentations of babesiosis and malaria are quite similar despite the fact that each infection is caused by a distinct and highly unique microorganism. As seen in the two cases above, both illnesses often begin insidiously with fevers, headache and muscle & joint aches. The non-specific nature of the patient’s symptoms results in an unclear etiology unless key elements of the patient’s history, including exposure to insect and arthropod vectors and travel or habitation in endemic areas, are provided.

Examination of thick and thin blood smears is useful in the diagnosis of these two diseases. While both organisms have a very similar sized lifecycle forms which selectively infect red blood cells and prompt hemolysis, there are a few useful distinguishing characteristics. In the case of babesiosis, which is transmitted by the Ixodes scapularis tick in the United States, there are small ring like structures, both within red blood cells and extra-cellularly. The diagnostic tetrad form, known as a Maltese Cross, is helpful if identified but is not frequently observed in human infections. No advanced forms or pigment is present. In the case of malaria, which is transmitted via the female anopheline mosquito, protozoa are only found within red blood cells and advanced forms, including schizonts or gametocytes, are helpful in further speciation, if present. Other features, such as size of the infected red cell, number of merozoites, level of parasitemia and gametocyte shape, are helpful in the morphologic assessment of the Plasmodium spp.

Due to the pathogenic severity of P. falciparum, it is important that the microbiology laboratory has the ability to make the diagnosis in real time across all shifts. The BinaxNOW is an FDA approved lateral flow assay that is simple to perform and provides rapid diagnostics, though it isn’t as sensitive as microscopy. The test is comprised of two antigens: one specific to P. falciparum (T1) and one antigen common to all Plasmodium spp. (T2). The test will be positive for levels of parasitemia greater than 5,000 parasites per microliter.

As utilized in the above cases, other various laboratory modalities can aid in the diagnosis of babesiosis and malaria, including serologic tests and PCR, however, these tests may not be available in STAT situations. Using a variety of tests and obtaining a thorough travel history, will help the provider arrive at the correct diagnosis of blood protozoa.

 

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-JP Lavik, MD, PhD, is a 3rd year Anatomic and Clinical Pathology Resident at Yale New Haven Hospital.

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-Lisa Stempak, MD, is an Assistant Professor of Pathology at the University of Mississippi Medical Center in Jackson, MS. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. Currently, she oversees testing performed in both the Chemistry and Microbiology Laboratories. Her interests include infectious disease histology, process and quality improvement and resident education.

Microbiology Case Study: A 34 Year Old Pregnant Woman with Evidence of Fetal Anomalies

Case History:

A 34 year old G3P3 woman presents with PPROM at 30+4 weeks gestational age. Her pregnancy had been complicated by fetal hydrops and intrauterine growth restriction with evidence of multiple fetal anomalies and placenta previa.

Prenatal infectious disease testing was significant for:

  • CMV IgG+/IgM+
  • Toxoplasmosis IgG+/IgM+
  • Parvovirus B19 IgM-/IgG+
  • HIV screen negative (ELISA)
  • HSV IgM-
  • syphilis screen negative (RPR)
  • rubella immune (IgG+)
  • negative serologies for Hepatitis A, B and C
  • Testing for VZV was not performed

Clinically, the fetal hydrops and IUGR were thought to be due to congenital CMV. She underwent caesarian section, and a fetus was delivered with APGAR 0/1/1/2/1, with eventual fetal demise at two hours of life. The placenta was sent to the laboratory for surgical pathology examination. The mother declined fetal autopsy.

Laboratory Work-Up:

Surgical pathology received a singleton placenta (13 x 13 x 4 cm) with attached umbilical cord and fetal membranes. The placental disc weighed 346 grams (<10th percentile for gestational age). Otherwise, the placental disc, umbilical cord and fetal membranes were negative for any gross abnormalities.

Routine microscopic sections demonstrated round to elongate cysts within the amnion of the fetal membranes (Figures 1a & 1b) and within the Wharton’s jelly of the umbilical cord (Figure 2). These cysts measured approximately 50 microns in diameter and had a thin, translucent cyst wall. Within the cysts were innumerable small round “dot-like” forms which could best be appreciated by focusing up and down through the plane of the section.

Tissue gram stain (Brown & Brenn) was negative for significant bacterial infiltrate, and immunohistochemistry for CMV was negative for CMV inclusions.

Figure 1a: Amnion of the fetal membrane: Round to elongate cysts measuring approximately 50 microns in diameter. There are innumerable small round “dot-like” structures within the cysts (H&E, 600X).
Figure 1a: Amnion of the fetal membrane: Round to elongate cysts measuring approximately 50 microns in diameter. There are innumerable small round “dot-like” structures within the cysts (H&E, 600X).
Figure 1b: Amnion of the fetal membrane: Round to elongate cysts measuring approximately 50 microns in diameter. There are innumerable small round “dot-like” structures within the cysts (H&E, 600X).
Figure 1b: Amnion of the fetal membrane: Round to elongate cysts measuring approximately 50 microns in diameter. There are innumerable small round “dot-like” structures within the cysts (H&E, 600X).
Figure 2: Wharton’s jelly of the umbilical cord: Round cyst measuring approximately 50 microns in diameter. There are innumerable small round “dot-like” structures within the cyst (H&E, 600X).
Figure 2: Wharton’s jelly of the umbilical cord: Round cyst measuring approximately 50 microns in diameter. There are innumerable small round “dot-like” structures within the cyst (H&E, 600X).

Discussion:

The histologic features are diagnostic of congenital Toxoplasmosis. The case was sent to the reference laboratory, where immunohistochemical staining for Toxoplasmosis demonstrated positive staining within the tissue cysts.

Toxoplasmosis is caused by the protozoa Toxoplasma gondii, a member of the protozoan subgroup coccidia, which also includes the GI pathogens Cryptosporidium, Isospora and Cyclospora. Cats of the family Felidae (including but not limited to domestic cats) are the only definitive host, while virtually any mammal can serve as an intermediate host. Humans can become incidentally infected in which case they act as “incidental” intermediate hosts.

The life cycle of Toxoplasma involves sexual reproduction in the definitive host (cats), as well as asexual reproduction in the intermediate host. Toxoplasma is “facultatively heteroxenous,” in that reproduction in the intermediate host is not necessary for completion of the life cycle. Unsporulated oocysts are shed in the cat feces and become infective after 1-5 days. Cats may ingest the infective oocysts, leading to sexual reproduction and completion of the life cycle within the intestinal epithelium. Alternatively, intermediate hosts such as rodents or birds ingest infective oocysts and subsequently develop infective tissue cysts. If the intermediate host is eaten by a cat, the infective tissue cysts are ingested, leading to sexual reproduction in the cat and completion of the life cycle.

The life cycle within the intermediate host involves two morphologically distinct stages, the tachyzoite and the bradyzoite. When infective oocysts are ingested by an intermediate host, they transform into tachyzoites, which are able to invade the intestinal epithelium and then widely distribute throughout the body. Tachyzoites are crescent-shaped, non-encysted and measure from 3-7 microns in length by 2-4 microns in diameter. They migrate preferentially to the muscle and neural tissues, where they eventually develop into tissue cysts, which are known as bradyzoites. Bradyzoites are much larger than tachyzoites (approximately 50 microns), are round to elongate and contain numerous “dot-like” parasitic forms encased within a thin cyst wall.  Tachyzoites are eventually cleared following acute infection, but the intermediate host remains chronically infected with bradyzoites. If the host becomes immunocompromised, the bradyzoites differentiate into tachyzoites, which then recirculate through the body leading to reactivation of latent disease.

It is estimated that 10-20% of the U.S. population is chronically infected with Toxoplasma. Humans can become infected through one of five mechanisms: (1) ingestion of infective oocysts, either from cat feces or from infected water or other environmental sources, (2) ingestion of infective tissue cysts in undercooked meat, (3) vertical transmission to the fetus from a mother acutely infected with Toxoplasma, (4) through organ transplantation and (5) through blood transfusion. Epidemiologically, it is not clear whether the majority of infections occur through ingestion of infective oocysts or whether tissue cysts in undercooked meat are the major source of infection.

Vertical transmission from mother to fetus requires a first-time exposure during pregnancy. In primary/acute infection, tachyzoites widely disseminate and are able to invade the developing fetal tissues. By contrast pregnant women who are chronically infected with Toxoplasma harbor only tissue cysts (bradyzoites) and will not transmit infection to the fetus.

Acute infection is self-limited and usually asymptomatic, however some patients may have mild flu-like symptoms. A smaller subset of patients present with moderate to severe acute infection which can mimic mononucleosis: fever, sore throat, myalgias and cervical lymphadenopathy. Biopsy of inflamed lymph nodes reveals the classic histologic triad of follicular hyperplasia, monocytoid B-cell hyperplasia and epithelioid histiocytic aggregates. Once acute infection has passed, chronic infection is usually asymptomatic, unless the host becomes immunocompromised, in which case reactivation of latent disease can occur.

Treatment for immunocompetent patients in not indicated as acute infections are self-limited and chronic infection is asymptomatic. Immunosuppressed patients with CD4 counts <100 cells/mm3 should receive Toxoplasma prophylaxis with trimethoprim-sulfamethoxazole (TMP-SMX). Reactivation of latent disease can occur in immunosuppressed patients who are not taking prophylaxis, in which case first line treatment includes combination therapy with sulfadiazine and pyrimethamine.

 

-Javier De Luca-Johnson, MD is a 3rd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.

Microbiology Case Study: A 47 Year Old Male with Abdominal Pain and Diarrhea

A 47 year old male of Jamaican origin with no known past medical history presented to a clinic with abdominal pain and diarrhea. He has been working as a seasonal farmer and plans to return back to Jamaica by the end of the month. Stool samples were obtained and sent for culture and ova and parasite exam.

Rhabditiform larvae of Strongyloides stercoralis from the wet mount of O&P exam
Rhabditiform larvae of Strongyloides stercoralis from the wet mount of O&P exam
Blood agar plate demonstrating tracks made by crawling larvae
Blood agar plate demonstrating tracks made by crawling larvae

Strongyloides stercoralis is the primary species of the Strongyloides genus that causes human disease. The larvae are small and can reach around 1.5mm in length. The primary mode of infection is through contact with soil that is contaminated with larvae. The larvae are able to penetrate the skin and migrate through the body to the small intestine where they burrow and lay their eggs. The eggs hatch into larvae in the intestine, unlike other helminths. Of these larvae, most will be eliminated in feces, but some may shed and immediately re-infect the host. This is achieved either by burrowing into the intestinal wall, or by penetrating the perianal skin. The process is called auto-infection, and if the patient is not treated, they may continue to be infected throughout their life.

Strongyloides is generally found in warm and moist areas, as well as areas associated with agricultural activity. The majority of people infected with Strongyloides are asymptomatic, and those who do develop symptoms have generalized symptoms such as abdominal pain, bloating and diarrhea.

The time of exposure is usually unknown, although a local rash can occur at exposure. People exposed to Strongyloides can also develop a cough several days post exposure. Abdominal symptoms usually occur about 2 weeks later, and larvae can be found in the stool after 3-4 weeks. Strongyloides is treated with ivermectin as a first-line drug. Thiabendazole can also be effective.

 

-Mustafa Mohammed, MD is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.