Disseminated Toxoplasmosis, Resulting from Infection of Allograft, after Orthotopic Liver Transplantation: Usefulness of Quantitative PCR

Disseminated toxoplasmosis is a life-threatening disease inliver transplant recipients that can result from an organ-transmittedinfection. We report here a case of fatal disseminated toxoplasmosisafter orthotopic liver transplantation from a seropositive donor(immunoglobulin G [IgG]⁺ and IgM^-) in a patient who was nonimmunefor toxoplasmosis prior to transplantation. Quantitative PCRanalyses of various clinical specimens, including serum samples,appeared retrospectively to be a valuable diagnostic tool thatmight guide therapeutic attitudes.

INTRODUCTION

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Introduction

Toxoplasmosis is a worldwide infectious disease caused by theprotozoan Toxoplasma gondii. Disseminated life-threatening infectionis more common in immunocompromised individuals, such as patientswith human immunodeficiency virus infection or transplant recipients.In these situations, early treatment significantly reduces theextent of the damage (5). However, the classical diagnosis oftoxoplasmosis based on serological tests is inefficient andinadequate in these patients (5, 7). Therefore, the diagnosisis based on the direct demonstration of the parasite in tissuesor biological fluids or detection of Toxoplasma DNA by PCR.We report here a case of disseminated fatal toxoplasmosis afterorthotopic liver transplantation in a patient who had negativetoxoplasmosis serology, and we also consider the contributionof quantitative PCR in various clinical specimens as a valuablediagnostic tool.

CASE REPORT

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Case report

A 27-year-old French female, a native of Israel, underwent anorthotopic liver transplantation for end-stage liver cirrhosis(biliary cirrhosis due to autoimmune cholangitis) on 6 October2000. The donor was a 29-year-old French female who died ofaneurysm rupture. The immune serologic profile of the donorwas consistent with a prior infection (i.e., the presence ofimmunoglobulin G [IgG] and the absence of IgM), whereas therecipient had a negative serology prior to transplantation (Table1). The recipient maintenance immunosuppressive regimen includedtacrolimus, mycophenolate mofetil, and methyl-prednisolone.At 12 days posttransplantation, fever and fatigue were noted.Thereafter, an explorative laparotomy was performed becauseof suspicion of peritonitis. Culture of the peritoneal fluidspecimen indicated the presence of methicillin-resistant staphylococci.Antimicrobial therapy consisting of targocid, fosfomycin, andtazocillin was immediately started. Biological parameters showedpancytopenia and hypoxia. A bronchoalveolar lavage (BAL) wasperformed at day 26 after transplantation, and cytomegaloviruswas diagnosed and treated with ganciclovir.

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TABLE 1. Quantitative T. gondii real-time PCR test analysis of sequential samples from a patient with disseminated toxoplasmosis after orthotopic liver transplantation

Despite this therapy, there was no improvement in the patient'scondition. Specific screening tests, including blood culturesand urine cultures, were negative. At day 40 after transplantation,the patient developed acute respiratory failure with shock.Upon admission at the intensive care unit, the patient presentedwith severe dyspnea, unstable hemodynamics, and hyperthermia.Tazocillin was stopped and tienam, fluconazole, and filgrastimwere started. A chest radiograph showed alveolar pneumopathywith pleural effusion. A second BAL was promptly performed.Direct microscopic examination of the BAL fluid specimen (byGiemsa staining) revealed several structures that were pathognomonicof tachyzoites of T. gondii, and therapy with pyrimethamineand sulfadiazine was initiated. Nevertheless, the patient died5 days after receiving this specific anti-Toxoplasma treatment(day 50 after transplantation).

More than 9 months after transplantation, other recipients whoreceived organs from the same donor (heart, lungs, kidneys,and cornea) remain in good health, with no evidence of infection.These recipients had positive serology for toxoplasmosis beforetransplantation.

MATERIALS AND METHODS

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Materials and Methods

PCR analyses. Detection and quantitative evaluation of Toxoplasma DNA wereattempted retrospectively by a PCR test on various clinicalspecimens from the patient. The newly developed, commerciallyavailable LightCycler system was used for the PCR analyses.This system allows real-time detection and quantification ofspecific PCR products by fluorescence resonance energy transfer(2).

Nucleic acid extraction. Nucleic acid was extracted from 0.2 ml of each specimen by usingthe High Pure PCR Template preparation kit (Roche MolecularBiochemicals, Meylan, France) according to the manufacturer'sprotocol. DNA was eluted from the column with 50 µl ofPCR-grade H₂O and then amplified by LightCycler PCR (LC-PCR;Roche).

LC-PCR. LC-PCR was targeted at the T. gondii B1 gene, according to aprotocol described in detail elsewhere (2). The primers foramplification were the 23-mer 5'-GGAGCACTGGCAACCTGGTGTCG-3'(sense) and the 25-mer 5'-TTGTTTCACCCGGACCGTTTAGCAG-3' (antisense).The two hybridization probes (Tib MolBiol, Berlin, Germany)were the 25-mer 5'-ACGGGCGAGTAGCACCTGAGGAGAT-3', which was labeledat the 5' end with LC-red 640 and phosphorylated at the 3' end,and the 27-mer 5'-CGGAAATAGAAAGCCATGAGGCACTCC-3', which waslabeled at the 3' end with fluorescein.

For quantification, one 10-fold serial dilution of T. gondiiDNA (RH strain) was prepared that ranged from 0.1 to 10 parasites/µl.This series of 10-fold dilutions was included in each amplificationrun.

LC-PCRs were performed in a final volume of 20 µl by usingthe DNA Master Hybridization Probes Kit (Roche), with a 0.5µM concentration of each primer, a 0.25 µM concentrationof each probe, and 5 µl of extracted DNA sample. Carryovercontamination was prevented by using heat-labile uracil-DNA-glycosylase(UNG; Roche Molecular Biochemicals). The amplification was carriedout in a LightCycler Instrument (Roche) according to a previouslydescribed protocol (2). Quantitative results of the PCR wereexpressed by plotting the fluorescence signal of each positivesample against the standard curve obtained with known concentrationsof the parasites, and the parasitic load was calculated by interpolationfrom this standard curve.

Clinical samples. Several serum, whole-blood, and BAL samples from the patientswere consecutively collected for serology (serum), light microscopy(BAL), or culture (whole blood). All samples were frozen within4 h of receipt in the laboratory and kept stored at -20°Cuntil retrospective analyses by LC-PCR.

Serology antibody. Titers against T. gondii in transplant recipients were measuredby an enzyme-linked immunosorbent assay (ELISA) for IgG andIgM (Vidas; Biomérieux, Marcy l'Étoile, France),with the addition of an immunosorbent agglutination assay forIgM (Biomérieux) and a direct agglutination assay (Toxoscreen;Biomérieux) for IgG. A dye test was retrospectively performedto detect IgG in selected serum samples to monitor the sensitivityof these reactions.

RESULTS

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Results

The PCR and serology results obtained in various specimens beforeand after transplantation are summarized in Table 1. IgG andIgM antibodies were not detected prior to transplantation (day-28). After transplantation, IgG antibodies were weakly positivebut without detection of IgM.

The retrospective and simultaneous analysis of nine serum samplesby LC-PCR showed that the first PCR-positive serum sample wascollected on day 31 after transplantation and on day 27 in BALfluid.

DISCUSSION

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Discussion

Disseminated toxoplasmosis after liver transplantation is arare event but in most cases results in a fatal outcome (1,3, 5, 7, 10). The rarity of the disease and the nonspecificityof the symptoms have led to a general lack of awareness amongclinicians and, hence, a high mortality rate among transplantedpatients due to the delayed initiation of therapy.

In the case described here, toxoplasmosis was probably transmittedvia an infected allograft since all serologic reactions, includingthe highly sensitive dye test, did not detect specific IgG antibodiesin the recipient prior to transplantation. This assumption isalso supported by the serologic status of the donor, who wasseropositive for toxoplasmosis. In addition, the weak and delayedappearance of IgG antibodies after transplantation, detectedby both ELISA and dye test, could be considered as a seroconversion,despite the absence of IgM, in a severely immunocompromisedpatient, as previously reported in otherwise immunosuppressedpatients (5). Nevertheless, serologic diagnosis failed to demonstratea severe Toxoplasma infection clearly and early in our patient.Thus, in immunocompromised patients diagnosis is usually basedon the direct demonstration of the parasites in biological specimensby light microscopy or tissue culture (9). However, these techniquesare often time-consuming, lack sensitivity and, as illustratedhere, may delay the time of diagnosis and initiation of treatment.In contrast, the PCR technique allows a simple, rapid, and highlysensitive detection of T. gondii DNA in various specimens andrepresents a valuable diagnostic tool for assessing disseminatedtoxoplasmosis (2, 4, 6). Additionally, the LC-PCR system providesreal-time detection and quantification of amplified productsin less than 3 h, including the DNA extraction steps (11). Thus,postmortem PCR assays applied to serum samples could retrospectivelyassess a disseminated infection as early as day 31 after transplantation,i.e., 14 days before specific anti-Toxoplasma treatment hadbeen administered. As shown in Table 1, despite much lower parasiticloads in serum than in whole-blood samples, it is noteworthythat specific PCR products could be detected from unconcentratedserum samples. Subsequent samples showed increasing parasiticloads, a finding that is probably consistent with the progressionof the disease. The high level of parasitic load in the firstBAL sample also illustrates the sensitivity of PCR, althoughlight microscopy failed to detect tachyzoites, even after acareful reexamination of BAL smears.

As suggested by others, this case raises the question of specificsurveillance and prophylaxis of toxoplasmosis after liver transplantation(5, 8). Although it is recommended that human immunodeficiencyvirus-positive people who are also Toxoplasma antibody positivereceive toxoplasmosis prophylaxis, there are currently no recommendationsfor the management of transplant recipients. Serologic screeningof donor and recipient may not be relevant since disseminatedtoxoplasmosis can result either from reactivation of a latentinfection or from a contaminated graft (5). Additionally, inotherwise seronegative but severely immunocompromised recipients,such as the patient described here, serologic methods lack sensitivityand may fail to clearly show a seroconversion. Since early initiationof specific anti-Toxoplasma therapy is a critical prognosisfactor, highly sensitive PCR methods that can be applied directlyto serum samples should be of great help in cases of unexplainedfever in immunocompromised recipients. In addition, the real-timequantitative LC-PCR would be useful for monitoring treatment.

FOOTNOTES

* Corresponding author. Mailing address: Parasitology and Mycology Department, Le Kremlin-Bicetre Hospital, 78, rue du Général Leclerc, Paris XI University, F 94275 Le Kremlin-Bicetre, France. Phone: (33) 1-45-21-33-21. Fax: (33) 1-45-21-33-19. E-mail: francoise.botterel{at}bct.ap-hop-paris.fr.

REFERENCES

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