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(Radiology. 1999;211:507-512.)
© RSNA, 1999

Nuclear Medicine

Intracranial Mass Lesions: Sequential Thallium and Gallium Scintigraphy in Patients with AIDS1

Victor W. Lee, MD, Vittorio Antonacci, MD, Shripad Tilak, MD, FRCS, Jon D. Fuller, MD and Timothy P. Cooley, MD

1 From the Departments of Radiology (V.W.L., V.A., S.T.) and Medicine (J.D.F., T.P.C.), Boston University School of Medicine, Boston Medical Center, 818 Harrison Ave, Boston, MA 02118. Received January 30, 1998; revision requested April 7; final revision received August 19; accepted November 6. Address reprint requests to V.W.L.


   Abstract
TOP
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
PURPOSE: To determine the efficacy of sequential thallium and gallium scintigraphy to differentiate intracranial neoplasms (lymphoma and glioma) from other nonmalignant intracranial mass lesions among patients with acquired immunodeficiency syndrome (AIDS).

MATERIALS AND METHODS: The authors reviewed the cases of 40 patients with human immunodeficiency virus (HIV) who underwent thallium and gallium scanning to evaluate intracranial mass lesions from October l991 through November 1997. There was a definitive final diagnosis of the nature of the mass lesions in 21 of these cases. In these 21 cases, the scintigraphic patterns were reviewed and were compared with the final diagnosis.

RESULTS: On the basis of results at thallium and gallium scanning, the patients were divided into three groups. Group A included 13 patients (11 with brain tumors [lymphomas and gliomas] and two with progressive multifocal leukoencephalopathy [PML]) with thallium-positive, gallium-positive scans. Group B included five patients with intracranial infections (tuberculosis, Cryptococcus, bacteria) with thallium-negative, gallium-positive scans. Group C included three patients (one with PML and two with infarcts) with thallium-negative, gallium-negative scans. All patients with lymphomas were in group A. The sensitivity and specificity of the thallium-positive, gallium-positive pattern for intracranial malignancy were 100% and 80%, respectively.

CONCLUSION: Sequential thallium and gallium scanning helped differentiate tumors from nonmalignant intracranial mass lesions and may help differentiate infections from PML or infarcts.

Index terms: Acquired immunodeficiency syndrome (AIDS), 10.2068 • Brain, infarction, 10.4352 • Brain, infection, 10.2054, 10.23, 10.298 • Brain, radionuclide studies, 10.12163, 10.12178 • Brain neoplasms, radionuclide studies, 10.12163, 10.12178 • Fluorine, radioactive, 10.12163 • Gallium, radioactive • Lymphoma, AIDS-related, 10.34 • Thallium, radioactive • Progressive multifocal leukoencephalopathy, 10.8721


   Introduction
TOP
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Since its recognition as a distinct clinical entity in 1981, acquired immunodeficiency syndrome (AIDS) continues to be a major health problem universally. As of June 1996, more than 600,000 people had been diagnosed with AIDS in the United States. About 30% of AIDS patients present with neurologic symptoms, and almost 70% develop central nervous system (CNS) involvement during the course of their disease (1–4). The brain may be affected in generalized fashion by the human immunodeficiency virus (HIV), producing generalized neurologic changes that progress to dementia. Discrete mass lesions in the brain may result from opportunistic infections or neoplasms in these immunodeficient patients (1–6).

The most common cause of CNS mass lesions among HIV-infected people is infection with Toxoplasma gondii, which occurs in as many as 40% (3,4). Primary CNS lymphoma is the second most common cause of CNS mass lesions in this population, occurring in 10%. Progressive multifocal leukoencephalopathy (PML), tuberculosis, and Cryptococcus or other opportunistic infections are less common (1,3–5). Given this variety, acquisition of a precise diagnosis is very important to ensure optimal medical care of these patients.

The clinical distinction between CNS pathologic conditions in patients with AIDS is often difficult, as neurologic signs and symptoms are not sufficiently specific to differentiate diagnoses. In addition, the computed tomographic (CT) and magnetic resonance (MR) imaging findings are often nonspecific and show frequent overlap. For example, toxoplasmosis infections typically appear as multiple ring-enhancing lesions, usually less than 2 cm in diameter, and are located in frontal, parietal, and basal ganglia areas. A typical primary CNS lymphoma lesion is usually solitary, often cavitated, usually larger than 2 cm in diameter, and located in the periventricular areas. Similar appearances are so common, however, that definitive distinction between these two common pathologic conditions is very difficult at CT and MR imaging (7–12).

Lymphomas are known to be metabolically more active than infections such as toxoplasmosis, and this difference in metabolism has been exploited for their differential diagnosis. Early reports indicated that 2-[fluorine-18]fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) is a promising technique to differentiate tumors from infections, even though PET technology is still expensive and not easily available in most hospitals (12,13). More recently, O'Doherty et al (14) found in their larger series with FDG PET in patients with AIDS that many infectious foci were also FDG avid. Therefore, FDG PET may be useful in detecting both infections and malignancy, but it is of limited value in distinguishing them.

Recent reports of using thallium single photon emission CT (SPECT) to make this distinction are more encouraging, although the number of cases reported is still small (15,16). Thallium is a biochemical analogue of potassium and has been shown to concentrate in many tumors, including lymphomas (17–19). More important, thallium 201 concentration in infectious and inflammatory foci is relatively low, especially when scanning is delayed until 3 hours after injection (19–21). Both lymphomas and infections are known to be gallium avid. This difference in metabolism is the biochemical basis for using multitracer scintigraphy to differentiate tumors from infections. Sequential thallium and gallium SPECT has been used successfully to differentiate AIDS-related neoplasms from infections in general, although most past reports have referred to extracranial lesions. For example, in the thorax or abdomen, infections are typically gallium avid and thallium negative, whereas lymphomas are avid for both tracers (18,22–24). Although reports of success with sequential thallium and gallium scanning in AIDS patients have largely focused on extracranial pathologic conditions, a small number of brain lesions have also been documented (15,16,22). In our study, we retrospectively reviewed our experience since 1991 with sequential thallium and gallium scanning to evaluate disease in HIV-infected patients with known mass lesions in the brain. The purpose of this study was to evaluate the usefulness of sequential thallium and gallium scanning to differentiate primary CNS malignancy from CNS infection or other pathologic conditions.


   MATERIALS AND METHODS
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Patients
We retrospectively reviewed the departmental records of all patients with positive HIV serology or AIDS-defining illness who underwent sequential thallium and gallium SPECT of the brain at our hospital from October 1991 through November 1997. In all patients, brain mass lesions were depicted at CT or MR imaging before thallium and gallium SPECT. The cases of patients without HIV infection or AIDS were excluded from our review.

Scintigraphy
Thallium SPECT was performed after intravenous injection of 3–5 mCi (111–185 MBq) of 201Tl chloride. Two patients underwent only planar scanning, whereas all others underwent SPECT. Early scanning was performed within 15 minutes of injection, whereas delayed scanning was performed 3–4 hours after injection. The SPECT examinations were performed with a three-detector scanner (Trionix, Cleveland, Ohio) with high-resolution collimators. A circular orbit was used for SPECT with the "stop and shoot" mode. Each detector went through 30 4° stops, each lasting 40 seconds. The standard matrix of 64 x 128 was used. Tomographic images were reconstructed by means of backprojection in the standard planes (coronal, sagittal, transaxial), with use of a Butterworth filter with a high cutoff frequency of 0.350 cycles per centimeter.

Gallium scanning was performed 48–72 hours after intravenous injection of 7–9 mCi (259–333 MBq) of gallium 67 citrate. Three-detector scanners (Trionix) were used with medium-energy collimators. Because of the higher energy peaks of gallium, all gallium SPECT was performed 2–5 days after thallium SPECT to avoid Compton scatter (22).

In each case, the scintigrams, MR images, and CT scans were reviewed retrospectively and independently by three of the authors (V.W.L., V.A., S.T.). Disagreements were resolved by consensus after discussion. The MR images and CT scans were evaluated for the location of the abnormality, and the scintigrams were then evaluated for the presence or absence of uptake of thallium or gallium.

Patient Diagnosis
Among the 40 cases included in our review, the diagnoses in 21 were established by means of biopsy, autopsy, or bacterial and microscopic cultures, or were confirmed on the basis of additional conventional contrast material–enhanced angiographic studies for infarct and hemorrhage. In the remaining 19 cases, the diagnosis was established only indirectly, on the basis of the hospital course clinically in response to therapy (radiation, antitoxoplasmosis regimens), and they were excluded from further analysis.

The details in the 21 study cases (17 men and four women; age range, 28–72 years; mean age, 37 years) with proved diagnoses are shown in the Table.


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  		Diagnosis in 21 Mass Lesions
 
Statistical Analysis
The scintigraphic pattern of thallium-positive, gallium-positive scans as a sign of malignancy (lymphoma or brain tumors) was evaluated by correlating the scintigraphic findings and the final diagnosis. Sensitivity and specificity were calculated.


   RESULTS
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Scintigraphic Findings
For the 21 cases, results at thallium and gallium SPECT were correlated with the final diagnosis, and the cases were divided into three groups. On the basis of our past experience that delayed thallium SPECT was more specific for differentiating lymphoma from infections, the thallium SPECT results were based on the 3–4-hour delayed studies.

In group A, the brain lesions were both thallium and gallium avid (thallium-positive, gallium-positive scans). There were 13 patients (11 men and two women; age range, 30–72 years; mean age, 38 years) in this group. The diagnosis in 11 of the 13 cases was a neoplastic lesion (non-Hodgkin lymphoma in nine, anaplastic astrocytoma in one, glioblastoma multiforme in one). The diagnosis in the remaining two cases was PML (Fig 1).



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  		Figure 1a. Group A (thallium-positive, gallium-positive scans). Brain images obtained in a 33-year-old man with known history of AIDS who was admitted because of grand mal seizures after a recent history of sinusitis. (a) Initial coronal gadolinium-enhanced, T1-weighted (repetition time msec/echo time msec = 655/16) MR image demonstrates a 4-cm-diameter mass (arrow) in the left occipital pole with a ringlike contrast enhancing pattern and extensive surrounding edema. (b) Thallium scans obtained at 3 hours (left column) and gallium scans obtained at 48 hours (right column) (top row, transaxial; middle row, sagittal; bottom row, coronal). Lesion (arrows) was both thallium and gallium avid. Findings at biopsy confirmed the diagnosis of B cell non-Hodgkin lymphoma.

 


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  		Figure 1b. Group A (thallium-positive, gallium-positive scans). Brain images obtained in a 33-year-old man with known history of AIDS who was admitted because of grand mal seizures after a recent history of sinusitis. (a) Initial coronal gadolinium-enhanced, T1-weighted (repetition time msec/echo time msec = 655/16) MR image demonstrates a 4-cm-diameter mass (arrow) in the left occipital pole with a ringlike contrast enhancing pattern and extensive surrounding edema. (b) Thallium scans obtained at 3 hours (left column) and gallium scans obtained at 48 hours (right column) (top row, transaxial; middle row, sagittal; bottom row, coronal). Lesion (arrows) was both thallium and gallium avid. Findings at biopsy confirmed the diagnosis of B cell non-Hodgkin lymphoma.

 
In group B, the brain lesions were thallium negative and gallium avid (thallium-negative, gallium-positive scans). There were five patients in this group (four men and one woman; age range, 28–52 years; mean age, 38 years), and each had an infection (bacterial abscess in three, Cryptococcus infection in one, tuberculosis in one) (Fig 2).



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  		Figure 2a. Group B (thallium-negative, gallium-positive scans). Brain images obtained in a 40-year-old man with known history of AIDS for 3 years. He was admitted because of three episodes of grand mal seizures and 3 days of fever. Meningism was present at admission. (a) Initial coronal T2-weighted (908/90) MR images reveal a mass lesion (arrows) in the medial portion of the right temporal lobe, extending into the temporal horn of the ventricle. (b) Top row: Early thallium scans demonstrate high uptake in the parenchymal lesion (arrowheads) and diffuse increased uptake in the subarachnoid space (arrows), especially in the interhemispheric cistern. Middle row: Thallium scans obtained at 4 hours show only background activity, and the areas of increased uptake are cleared almost completely. Bottom row: Gallium scans show high uptake in the parenchymal lesion (arrowheads), ventricle, and subarachnoid space (arrows). The final diagnosis of Cryptococcus neoformans meningitis, ventriculitis, and encephalitis was established when cerebrospinal fluid showed numerous Cryptococcus organisms. These scans demonstrate the importance of delayed versus early thallium scanning to differentiate tumors from infections. All thallium and gallium scans were obtained in the transaxial plane.

 


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  		Figure 2b. Group B (thallium-negative, gallium-positive scans). Brain images obtained in a 40-year-old man with known history of AIDS for 3 years. He was admitted because of three episodes of grand mal seizures and 3 days of fever. Meningism was present at admission. (a) Initial coronal T2-weighted (908/90) MR images reveal a mass lesion (arrows) in the medial portion of the right temporal lobe, extending into the temporal horn of the ventricle. (b) Top row: Early thallium scans demonstrate high uptake in the parenchymal lesion (arrowheads) and diffuse increased uptake in the subarachnoid space (arrows), especially in the interhemispheric cistern. Middle row: Thallium scans obtained at 4 hours show only background activity, and the areas of increased uptake are cleared almost completely. Bottom row: Gallium scans show high uptake in the parenchymal lesion (arrowheads), ventricle, and subarachnoid space (arrows). The final diagnosis of Cryptococcus neoformans meningitis, ventriculitis, and encephalitis was established when cerebrospinal fluid showed numerous Cryptococcus organisms. These scans demonstrate the importance of delayed versus early thallium scanning to differentiate tumors from infections. All thallium and gallium scans were obtained in the transaxial plane.

 
In group C, the brain lesions were thallium and gallium negative (thallium-negative, gallium-negative scans). There were three patients (two men and one woman; age range, 29–46 years; mean age, 35 years) in this group. In one, PML was proved at biopsy. In the remaining two, cerebral infarct or hemorrhage was proved at conventional angiography and CT (Fig 3).



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  		Figure 3a. Group C (thallium-negative, gallium-negative scans). Brain images obtained in a 30-year-old woman with a 2-year history of AIDS. She had lower extremity weakness for 6 months and was admitted because of depression and suicide attempts. (a) Axial T2-weighted (3,000/120) MR images demonstrate multiple mass lesions (arrows) of various sizes. (b) Transaxial thallium scans obtained at 4 hours (left two scans) and gallium scans obtained at 48 hours (right two scans). In each scan, lesion uptake was negative. The diagnosis of PML was made at brain biopsy.

 


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  		Figure 3b. Group C (thallium-negative, gallium-negative scans). Brain images obtained in a 30-year-old woman with a 2-year history of AIDS. She had lower extremity weakness for 6 months and was admitted because of depression and suicide attempts. (a) Axial T2-weighted (3,000/120) MR images demonstrate multiple mass lesions (arrows) of various sizes. (b) Transaxial thallium scans obtained at 4 hours (left two scans) and gallium scans obtained at 48 hours (right two scans). In each scan, lesion uptake was negative. The diagnosis of PML was made at brain biopsy.

 
No case included the pattern of thallium-positive, gallium-negative scans.

Data Analysis
With the scintigraphic pattern of thallium-positive, gallium-positive scans (group A ) as a sign of malignancy (lymphoma and brain tumors), there were 11 true-positive (TP) and two false-positive (FP) cases. All patients with lymphoma were in group A. There were no false-negative (FN) cases.

The eight patients in groups B and C did not have a malignancy. In all eight cases, findings were true negative (TN).

The overall diagnostic sensitivity (TP/[TP + FN] = 11/[11 + 0]) and specificity (TN/[TN + FP] = 8/[8 + 2]) of thallium and gallium scintigraphy to differentiate tumors from nontumors were 100% and 80%, respectively.


   DISCUSSION
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The definitive diagnosis of CNS mass lesions seen at MR imaging or CT in HIV-infected patients remains elusive because clinical presentations and anatomic imaging findings are not sufficiently specific to differentiate the most common pathologic conditions, lymphoma and intracranial infections (1, 7–9). For this reason, stereotactic brain biopsy may be considered before therapy is started. However, brain biopsy is an invasive procedure that can be technically difficult or nondiagnostic due either to sampling errors or acquisition of necrotic tissue (25,26). Consequently, since toxoplasmosis is the most common intracranial mass lesion among AIDS patients, a therapeutic trial of therapy for intracranial infection or toxoplasmosis is often used. If this empiric trial fails after 2–3 weeks, patients may then be reevaluated for brain biopsy or alternative empiric treatment. Similarly, irradiation of the brain may be performed empirically if the clinical and imaging suspicion for lymphoma is high, especially if biopsy is technically difficult (27–31). However, trials of toxoplasmosis or radiation therapies are not without problems. Side effects of irradiation of the nonlymphomatous brain are well known (29–31), and trials of antitoxoplasmosis therapy can result in the delay of appropriate therapy for 2–3 weeks in patients with lymphoma. In addition, adverse effects of antitoxoplasmosis therapy are not uncommon and can be serious, adding further morbidity in patients who may be very ill. Delay in acquisition of a definitive diagnosis while waiting for response to therapeutic trials also means prolonged hospital stay and increased expense (2,27,28).

Results at sequential thallium and gallium scanning could help in these cases by expediting accurate diagnosis while reducing patient morbidity and cost. Among the 40 cases we reviewed, the diagnosis was proved directly in 21 but was established only indirectly on the basis of the clinical course and response to treatment in 19. The number of cases is small in our study, but our preliminary results indicate that the diagnostic sensitivity of thallium and gallium scanning for neoplasm (lymphoma and gliomas) is 100%, and specificity is 80%. All lymphomas are thallium and gallium avid.

Our results are similar to or better than those previously reported with thallium SPECT alone or FDG PET as modalities to differentiate lymphoma from toxoplasmosis (12–16,32). In addition to including more patients, our study also has important technical differences in comparison with previous reports. In our study, we emphasized the findings of delayed thallium SPECT and also used gallium scanning in addition to thallium SPECT (18,22,24). In previous studies, the addition of gallium scanning was found to improve diagnostic specificity in the characterization of several extracranial lesions (eg, pulmonary Pneumocystis carinii pneumonia versus Kaposi sarcoma, tuberculosis, Mycobacterium avium intracellulare) (18,22–24). Our current results indicate a similar benefit from the addition of gallium scanning to thallium scanning to investigate intracranial mass lesions. In group C (thallium-negative, gallium-negative scans), one patient had PML and two had cerebral infarcts. Recently, cerebral infarcts were found to occur more frequently among AIDS patients, even if they are children, and more cerebral infarcts will probably be seen in this group of younger patients in the future (3,33). If thallium SPECT alone had been used, which yielded negative results, disease in these three patients might have been mistaken for toxoplasmosis or other intracranial infection, leading to unnecessary expense and potentially risky therapeutic trials. Our results, therefore, suggest that the addition of gallium scanning may be useful to differentiate infections from other thallium-negative lesions (PML, infarcts, hemorrhage).

From the scintigraphic point of view, PML is a very unique lesion. Of the three PML lesions, one was thallium negative and gallium negative (group C), and two were thallium positive and gallium positive (group A) and were mistaken for lymphoma. In general, the diagnosis of PML is not difficult if characteristic MR imaging findings are present. However, despite the name "multifocal," a large proportion (60%) of PML cases are unifocal in the early stage, which leads to diagnostic confusion (5,7,33–36). Some PML lesions have the appearance of a mass rather than a demyelinating region in the white-gray junction. In the Table, we separate PML from other infections because of both pathologic and practical reasons, even though PML is generally believed to be caused by reactivation of JC virus infection. Although direct proof of the pathogenesis of PML as a viral infection is still elusive, the predominant feature of PML is that of demyelination without major inflammatory changes (33–38). The process of demyelination is believed to be a result of an exceedingly complicated process due to interaction of a dozen proteins. This reaction is fundamentally more complex than that with other infections caused by bacteria or fungi (36,37). More important to clinicians and patients, the clinical course and treatment for PML are totally different from those for other infections, which are treatable and curable. At this time there is no effective treatment for PML, even though a number of experimental regimens are now being tested. The prognosis for patients with PML is grim. For these reasons, it is important that clinicians differentiate PML from other common AIDS-related CNS infections as early as possible. Among the patients with thallium-negative scans, additional findings at gallium scanning might separate PML from other infections (group C from group B), although the number of proved PML lesions among our patients is too small to verify this hypothesis. Demyelination is neither a neoplastic nor an inflammatory process, which may explain the thallium-negative, gallium-negative scans. However, it is more difficult to explain the thallium-positive, gallium-positive scans in two group A patients with PML. A possible explanation may include the presence of another coexisting pathologic condition missed at biopsy because of sampling errors. A more complete understanding of this scintigraphic variation requires further experience.

The importance of performing delayed (3–4 hours) thallium SPECT instead of the conventional early (5–15 minutes) scanning is worth mentioning. In some earlier studies of thallium SPECT for work-up of neoplasms, only early scanning was performed (32). Findings in later studies indicate that delayed thallium SPECT in extracranial lesions is more specific. This is because an infectious process can sometimes show early uptake of thallium but then clear relatively quickly in comparison with uptake by neoplastic lesions in the lungs, mediastinum, and abdomen (20,21,32). Findings in our study confirm the same pattern for intracranial lesions (Fig 2). Therefore, in a busy department, early thallium SPECT could be dispensed with, to save time, and only delayed scanning need be performed. Even with delayed thallium SPECT, the background thallium in the normal brain was still visible (Fig 3b). We rely on visual inspection alone as sufficient to differentiate the normal background from abnormal uptake, and computer-assisted quantitation is usually not necessary. With visual inspection, we compare the thallium uptake in the brain to that in the scalp and skull. Normal background thallium uptake was lower than that in the scalp, whereas abnormal uptake was higher or equal to that in the scalp and skull. Background activity was diffuse and ill defined, whereas abnormal uptake was more focal, localizing in the same sites as the mass lesions seen at MR imaging or CT.

Technically, we always performed thallium scanning before gallium scanning to avoid Compton scatter from the higher energy photons of gallium (22). If the intracranial mass lesions were large and relatively superficially located, planar imaging without SPECT might be adequate for the diagnosis. Two of our patients underwent only planar imaging, and the resultant thallium and gallium scans were diagnostic. For small, deep lesions and better correlation with MR imaging and CT findings, thallium and gallium scanning allow more confidence in the diagnosis.

The thallium-positive, gallium-negative pattern was not seen in our 21 cases. This is in contrast to patterns reported in studies of extracranial lesions in AIDS patients (18,22–24,39). A pulmonary lesion with a thallium-positive, gallium-negative pattern indicates the diagnosis of Kaposi sarcoma (18,22–24,37). Since Kaposi sarcoma is extremely rare in the CNS, the absence of the thallium-positive, gallium-negative pattern is not unexpected. In practice, therefore, gallium scanning is probably unnecessary if brain lesions are thallium avid.

In summary, sequential thallium and gallium scanning is highly sensitive and specific in the differentiation of neoplastic from nonneoplastic intracranial mass lesions in patients with AIDS. When the intracranial mass lesions are thallium avid, gallium SPECT is not necessary, as these lesions have a high probability of being lymphoma or other neoplasm. If findings at thallium SPECT are negative, gallium scanning should be performed to further characterize the lesions and to assist in differentiating infections from infarcts or PML.


   Footnotes
 
Abbreviations: AIDS = acquired immunodeficiency syndrome CNS = central nervous system FDG = 2-[fluorine-18]fluoro-2-deoxy-D-glucose HIV = human immunodeficiency virus PML = progressive multifocal leukoencephalopathy

Author contributions: Guarantors of integrity of entire study, V.W.L., V.A.; study concepts, V.W.L., S.T.; study design, V.W.L., V.A., S.T.; definition of intellectual content, V.W.L., S.T., J.D.F., T.P.C.; literature research, V.W.L., V.A., S.T., J.D.F.; clinical studies, V.W.L., S.T., J.D.F., T.P.C.; data acquisition, V.W.L., V.A., S.T., J.D.F., T.P.C.; data analysis, V.W.L., J.D.F., T.P.C.; statistical analysis, V.W.L., V.A., J.D.F.; manuscript preparation and editing, V.W.L., J.D.F.; manuscript review, V.W.L., J.D.F., S.T.


   References
TOP
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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