tel: (215) 898-2118/2120, fax: (215) 898-8780, e-mail: droos@sas.upenn.edu (updated by Dr. Florence Dzierszinski on 8-20-2005 )

***Safety Precautions for Working with Toxoplasma gondii in the Roos Lab*** 

Toxoplasma gondii is an obligate intracellular protozoan parasite, which infects a diverse range of tissues and organs within many animal species. Although the sexual cycle of this organism occurs only in cats, the asexual forms are capable of infecting man. T. gondii infection can be contracted by three routes:

- Ingestion of sporulated oocysts from material contaminated with feline feces.

- Oral ingestion of bradyzoites (tissue cysts) from infected animal tissue.

- Direct introduction of tachyzoites (or infected cells) into the bloodstream, or direct contact with the eye.

In most cases human infection is readily controlled by the cellular immune response and remains asymptomatic (approximately one-third of all Americans become infected at some point in their life without ever realizing it, usually from cat litter boxes, gardening, or eating rare meat). Individuals unable to mount an effective immune response are potentially at risk, however. This includes:

- Pregnant women -- while not themselves unusually prone to T. gondii infection or clinical disease, parasite invasion across the placenta can result in severe neurological abnormalities in the fetus; congenital toxoplasmosis is a leading cause of birth defects in the US.

- Individuals infected with the Human Immunodeficiency Virus (HIV; the AIDS virus). Toxoplasmic encephalitis (in most cases a reemergence of latent bradyzoite cysts rather than a new infection) is a leading cause of death in AIDS patients.

- Other individuals who are immunosuppressed, either through infection of the immune system, or by treatment with immunsuppressive drugs for cancer chemotherapy, treatment of allergic disorders, organ transplant, etc.

Although unusual, even otherwise healthy, fully immunocompetent individuals can develop acute toxoplasmosis, particularly if the infection is heavy or highly virulent, as may well be the case in a laboratory accident. We therefore take the following precautions working with T. gondii parasites in the lab:

- No one is permitted to work with parasites without being cleared by Dr. Roos. Extreme caution is required for work with pyrimethamine-resistant parasites!!!

- Individuals who are immunosuppressed, HIV-positive, pregnant, planning a pregnancy, or uncomfortable with the prospect of an abortion in the event of seroconversion following an accidental pregnancy, should not work with live parasites.

- All individuals must be tested for seropositivity prior to working with live parasites, and a frozen serum sample will be retained in the laboratory.

- Live parasites should always be treated with respect; exercise extreme care whenever

using sharps (needles, forceps, pasteur pipettes, etc). Also note that hemocytometer coverslips may have sharp edges! Special blunt needles are available for routine needle passage of infected cells ... sharp needles should only rarely be required (e.g. for mouse infection).

- All infected material must be decontaminated (in alcohol, bleach or by autoclaving) after use. Please pay particular attention to sharps and hemocytometer cover glasses.

- Mice are sacrificed prior to parasite harvest.

- Work with oocysts must be carried out using containment facilities, and requires additional precautions and special permission from D.S. Roos.

- Any possible infections, no matter how small or unlikely, must be reported immediately, and treated as outlined below.

In Case of Possible Laboratory Infection with Toxoplasma gondii

- Don't panic. The time course for progression to clinical toxoplasmosis is days, not minutes.

- Contact Dr. Roos immediately, at any time of day or night, and wherever he may be (home phone: 215-476-3618).

At Penn Occupational Medicine (Hospital of the University of Pennsylvania, Ground Ravdin

3400 Spruce Street , Philadelphia, PA 19104 ), contact Dr. Judith G. McKenzie, MD, MPH (Director, Clinical Practice; Associate Residency Director) at 1-800-789-PENN (7366).

 Blood samples should be drawn for a critical blood count (CBC) and to check for Toxoplasma antibody titer. CBC should be repeated twice weekly to check for blood toxicity of the chemotherapy. Check anti-Toxo titer again 1-2 months after the incident.

- Prophylactic chemotherapy should be initiated as soon as possible after contacting a physician; the recommended treatment for a possible laboratory infection with wild-type T. gondii is as follows ( but see below for special considerations concerning pyrimethamine-resistant parasites):

25 mg pyrimethamine (diaprim, Burroughs-Wellcome) once daily

1 g sulfadiazine (Lilly) 4x daily DO NOT TAKE IF ALLERGIC TO SULFONAMIDES!!!

5 mg folinic acid (calcium-leukovorin, Lederle) once daily

All medication can be taken by mouth.

Continue treatment for one week if patient is seropositive, two weeks if negative.

Note: This treatment is highly effective against T. gondii and normally produces no side-effects, but be sure to contact your physician should problems arise (fever, swollen lymph nodes, whatever). Try to minimize exposure to avoid intense sunlight when taking sulfonamides, and be alert for any possible allergic reaction, such as a rash or breathing difficulty. If such symptoms appear discontinue drug use and contact physician immediately.

- All of the required drugs are routinely available from the HUP outpatient pharmacy. If any problems arise obtaining medication, we have dealt with pharmacist Dr. Freddy Grimm at HUP (215-662-2900).

- Reimbursement for any expenses incurred can be arranged through the business office (you will have to fill out a workman's compensation form). Folinic acid (Ca-Leukovorin) is very expensive, but the manufacturer (Lederle) has a policy of providing the medication free of charge for laboratory accidents. Start with medication from the outpatient pharmacy at HUP or the emergency lab supply, and contact the Lederle oncology representative, Kathy Siegel, at 215-848-8579 for replacement. If at the HUP emergency room, mention that this is a work injury.

- If questions or complications arise, you should contact Dr. Benjamin Luft at 516-444-1660 (home: 516-689-7214). Dr. Luft is chairman of the Department of Medicine at SUNY, Stonybrook, and a leading clinical expert on toxoplasmosis. Other emergency contacts include Dr. Keith Joiner (The University of Arizona; 520-626-4555) or Dr. Rima McLeod (The University of Chicago; 773-834-4130).

- Special considerations for use of pyrimethamine-resistant parasites . Experimental infections in mice are completely refractory to treatment with pyrimethamine, but remain sensitive to sulfonamides, clindamycin, azithromycin and atovaquone. Tetracycline, and arprinocid provide alternative therapeutic options, although these have not been tested in vivo. One case of accidental human infection with pyrimethamine-resistant RH-strain Toxoplasma (a pTgDHFR-TSc3/M2M3 insertional mutant) was effectively controlled in a previously seronegative individual using sulfadiazine + clindamycin (note: this unfortunate experience did not occur in the Roos lab!). In the event of possible infection with such mutant strains, contact Dr. Luft (see above), and initiate treatment with:

1.5 - 2.0 g sulfadiazine (Lilly) 4x daily (unless allergic to sulfonamides )

600 mg clindamycin (Upjohn) 4x daily (unless allergic )

750 mg atovaquone 4x daily

Note that these are very high drug doses, and require careful monitoring by a physician, paying particular attention to the possible development of hypersensitivity!