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Neopterin, b 2 -Microglobulin, and Acute Phase Proteins in HIV-1-Seropositive and -Seronegative Zambian Patients with Tuberculosis M. Hosp, 1 A. M. Elliott, 1 J. G. Raynes, 1 A. G. Mwinga, 1,2 N. Luo, 2 R. Zangerle, 3 J. O. M. Pobee, 2 H. Wachter, 4 M. P. Dierich, 5 K. P. W. J. McAdam, 1 and D. Fuchs 4 1 London School of Tropical Medicine and Hygiene, London, UK; 2 School of Medicine, University of Zambia, Lusaka, Zambia; 3 Department of Venerology and Dermatology, 4 Institute of Medical Chemistry and Biochemistry, and 5 Institute of Hygiene, University of Innsbruck, and 5 Ludwig Boltzmann Institute of AIDS Research, Innsbruck, Austria Abstract. Neopterin is a biochemical marker for the activation of the cell-mediated immune system. We measured neopterin, b 2 -microglobulin, and acute phase pro- teins in 31 HIV-seropositive and -seronegative Zambian patients with tuberculosis, using stored sera that had been obtained at the beginning and at end of antituber- culosis treatment. In both HIV-seropositive and -seronegative patients neopterin and acute phase proteins were elevated when tuberculosis was initially diagnosed and fell during treatment. In contrast, the mean b 2 -microglobulin level increased during antituberculous therapy in the HIV-seropositive group. Serum neopterin levels at diagnosis were correlated with other parameters of disease activity (fever, anemia, and weight loss). In both groups, patients with persistently elevated neopterin levels at the end of treatment were more likely to suffer relapse of tuberculosis or other adverse health events in the subsequent follow-up period. Neopterin can be used to monitor the response to antituberculous therapy in both HIV-seropositive and -se- ronegative patients and may have a prognostic value for the patients’ wellbeing in the follow-up period. Key words: Neopterin—b 2 -Microglobulin—Tuberculosis—Human immunodefi- ciency virus. Introduction Infection with the human immunodeficiency virus (HIV) is an important risk factor for reactivation of latent tuberculous infection. The epidemic of HIV has altered the pattern Offprint requests to: D. Fuchs, Institute of Medical Chemistry and Biochemistry, University of Inns- bruck, Fritz Pregl Strasse 3, A-6020 Innsbruck, Austria. Lung (1997) 175:265–275 © Springer-Verlag New York Inc. 1997

Neopterin, β2-Microglobulin, and Acute Phase Proteins in HIV-1-Seropositive and -Seronegative Zambian Patients with Tuberculosis

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Neopterin, b2-Microglobulin, and Acute PhaseProteins in HIV-1-Seropositive and -SeronegativeZambian Patients with TuberculosisM. Hosp,1 A. M. Elliott,1 J. G. Raynes,1 A. G. Mwinga,1,2 N. Luo,2 R. Zangerle,3

J. O. M. Pobee,2 H. Wachter,4 M. P. Dierich,5 K. P. W. J. McAdam,1 and D. Fuchs4

1London School of Tropical Medicine and Hygiene, London, UK;2School of Medicine, University ofZambia, Lusaka, Zambia;3Department of Venerology and Dermatology,4Institute of Medical Chemistryand Biochemistry, and5Institute of Hygiene, University of Innsbruck, and5Ludwig Boltzmann Instituteof AIDS Research, Innsbruck, Austria

Abstract. Neopterin is a biochemical marker for the activation of the cell-mediatedimmune system. We measured neopterin,b2-microglobulin, and acute phase pro-teins in 31 HIV-seropositive and -seronegative Zambian patients with tuberculosis,using stored sera that had been obtained at the beginning and at end of antituber-culosis treatment. In both HIV-seropositive and -seronegative patients neopterin andacute phase proteins were elevated when tuberculosis was initially diagnosed andfell during treatment. In contrast, the meanb2-microglobulin level increased duringantituberculous therapy in the HIV-seropositive group. Serum neopterin levels atdiagnosis were correlated with other parameters of disease activity (fever, anemia,and weight loss). In both groups, patients with persistently elevated neopterin levelsat the end of treatment were more likely to suffer relapse of tuberculosis or otheradverse health events in the subsequent follow-up period. Neopterin can be used tomonitor the response to antituberculous therapy in both HIV-seropositive and -se-ronegative patients and may have a prognostic value for the patients’ wellbeing inthe follow-up period.

Key words: Neopterin—b2-Microglobulin—Tuberculosis—Human immunodefi-ciency virus.

Introduction

Infection with the human immunodeficiency virus (HIV) is an important risk factor forreactivation of latent tuberculous infection. The epidemic of HIV has altered the pattern

Offprint requests to:D. Fuchs, Institute of Medical Chemistry and Biochemistry, University of Inns-bruck, Fritz Pregl Strasse 3, A-6020 Innsbruck, Austria.

Lung (1997) 175:265–275

© Springer-VerlagNew York Inc. 1997

of tuberculosis (TB), particularly in subsaharan Africa where up to 70% of the adultpopulation have been infected withMycobacterium tuberculosis[19, 25]. The diag-nosis of TB in HIV-infected patients has become more difficult since the infections cancause similar symptoms, e.g. fever and wasting. HIV-seropositive patients with TB areless likely to have a positive sputum smear, and their chest X-ray films show less oftenthe classical upper zone involvement and cavitations [10, 11].

Neopterin andb2-microglobulin (B2M) are indicators of immune activation, andboth are used widely as progression markers for HIV disease [15]. B2M in Ugandanpatients with TB has been shown to be significantly higher in HIV-seropositive than in-seronegative individuals or in patients with asymptomatic HIV infection, suggesting anegative effect of TB on HIV disease [31]. From recent investigations it seems thatimmune stimulation caused byM. tuberculosismay represent a trigger for HIV rep-lication in patients [17]. Neopterin levels reflect the activation of cellular immunity,and high serum and urinary concentrations are found in diseases where cellular immunemechanisms are part of the pathology [14]. Since T cells and macrophages play acentral role in the host response to mycobacteria [7] neopterin production should be anindicative measure of disease activity in patients with TB [34]. In HIV-seronegativepatients with pulmonary TB, neopterin has been shown to correlate with the erythro-cyte sedimentation rate, leukocyte count, percentage of band cells, and thea2 fractionof serum electrophoresis [13]. During follow-up neopterin reflected clinical changesmore rapidly than chest X-ray examinations [13]. Neopterin concentrations have alsobeen measured in pleural fluid and have been demonstrated to be increased signifi-cantly in patients with TB compared with those with neoplasma and to be of potentialvalue in the differential diagnosis of pleurisy [2, 6].

In this study we measured the serum concentrations of neopterin in Zambianpatients with tuberculous chest infection and their changes in response to chemo-therapy. We compared neopterin concentrations with concentrations of B2M and theacute phase proteins C-reactive protein (CRP) and serum amyloid A (SAA) and ex-amined their usefulness for monitoring the clinical response to antituberculosis treat-ment (ATT) in both HIV-seropositive and -seronegative patients in Africa.

Materials and Methods

Patients were selected from participants in a cohort study for which recruitment and follow-up procedureshave been described in detail elsewhere [14]. Briefly, a representative sample of 239 TB patients presentingat the University Teaching Hospital, Lusaka, Zambia, were followed prospectively. Patients were seen atapproximately 3-month intervals during ATT and for a further year after the completion of treatment.Patients were treated for TB according to the Zambian government guidelines, as follows. Patients withsputum smear-positive pulmonary TB, miliary or meningeal TB were prescribed 2 months of streptomycin,thiacetazone, isoniazid, rifampicin, and pyrazinamide followed by 6 months of thiacetazone and isoniazid(2STHRZ/6TH). Patients with other forms of TB were treated with 2 months of streptomycin, thiacetazone,and isoniazid followed by 10 months of thiacetazone and isoniazid (2STH/10TH). Compliance with therapywas difficult to assess, but patients were asked to report difficulties in obtaining medication, and urine testsfor isoniazid were carried out at clinic visits. Prednisolone was prescribed as an adjunct to treatment forpericardial effusion, massive pleural effusion, miliary or meningeal disease and in treatment of cutaneousdrug reactions [8, 9]. Patients were considered cured if they completed treatment successfully, with nosymptoms attributable to TB, and the sputum smear, if available, being negative for acid-alcohol-fast bacilli.

266 M. Hosp et al.

Following completion of treatment, outcomes that were examined in this study were death, recurrenceof TB (defined as the return of clinical or bacteriologic evidence of active TB in a patient who had previouslycompleted treatment), and adverse clinical events such as recurrence of fever or continued weight loss.

Blood samples were collected, where possible, at the beginning and end of treatment. Serum wasseparated and stored at −70°C.

Neopterin, B2M, CRP, and SAA were retrospectively measured in the sera of 31 patients at thebeginning and at the end of treatment for tuberculous chest infection. Twenty-one patients had pulmonaryTB, 11 patients pleural, and 4 patients pericardial disease. One had lymph node disease, and one hadmeningeal involvement in addition to intrathoracic disease; in all, 6 had disease at more than one site.Twenty-four patients were male with a mean age of 34 years; 7 patients were female with a mean age of 27years. Eighteen patients (58%) were seropositive for HIV-1 antibody. The diagnosis of pulmonary TB wasconfirmed by positive sputum microscopy for acid-fast bacilli and/or culture ofM. tuberculosisin 17 out of21 patients with pulmonary disease. Pleural fluid was examined for protein and white cell count; the presenceof pericardial effusion was confirmed by echocardiography. All 31 patients completed a full course of ATTand responded well to chemotherapy.

Serum neopterin levels were determined by radioimmunoassay (Immutest Neopterin, Henning-Berlin,Germany). B2M was assayed by Cobas Fara centrifugal autoanalyzer using a method described elsewhere[30]. The assay was based on immunoturbidimetry of rabbit anti-human B2M (Dakopatts, Denmark) andsample (diluted 1:1) or standard (Protein Ref Unit, Royal Hallamshire Hospital, Sheffield, UK) over 15 minat 25°C. SAA and CRP were measured with a competitive inhibition enzyme-linked immunosorbent assayas described elsewhere [16]. Normal values, based on studies of sera from European patients, were definedas follows: neopterin < 8.8 nmol/liter [33], B2M < 2.5 mg/liter [15], CRP < 10 mg/liter, SAA < 30 mg/liter.

Statistical evaluation of the data was made by Wilcoxon’s rank sum test as well as paired rank test; ax2 test was used when incidences of abnormal values were compared between groups.

Results

Demographic and Clinical Characteristics

One hundred seven patients completed treatment and were considered cured. In thispilot study paired sera from the beginning and end of treatment were examined for 31of the 107 patients. Demographic characteristics and laboratory findings at presentationand the treatment regimens prescribed are summarized in Table 1, comparing the 31patients who were included in this study with those who were not. A slightly higherproportion of patients in the study group was HIV-negative, and this was reflected ina slightly higher proportion with positive tuberculin response and slightly higher meanhemoglobin and lymphocyte count; none of these differences was statistically signifi-cant. There was also no difference between the lymphocyte counts in seropositive andseronegative patients. Recurrence rate and mortality rate after completion of treatmentwere also slightly lower in the patients studied, but again the differences were notstatistically significant (data not shown). There were no differences between the twogroups in the proportions of patients with a history of cough, fever, or diarrhea atpresentation; with fever at presentation; with generalized lymphadenopathy; or withcavitary disease demonstrated radiologically (data not shown).

Serum neopterin, B2M, CRP, SAA, and HIV Status

Serum neopterin was elevated above the normal value in patients with active TBregardless of their HIV status. In the HIV-seropositive group 17 out of 18 patients

Neopterin Monitoring in HIV-Associated TB 267

(94%) had elevated neopterin concentrations when TB was diagnosed. Similar valueswere found in HIV-seronegative patients. 85% had elevated neopterin above 8.7 nmol/liter. At the end of ATT both groups showed significantly lower serum neopterin levels(p < 0.01; Fig. 1), and the mean concentration in the seropositive group was higher(14.3 ± 8.7 nmol/liter) than in the seronegative group (10.7 ± 9.6 nmol/liter) for whichthe median value lay within the normal range (7.0 nmol/liter). It was still elevated inHIV-seropositive patients (11.5 nmol/liter). In the latter group 13 out of 18 patients(72%) showed elevated neopterin levels at the end of ATT, which was also the case in5 out of 13 HIV-seronegative patients (38%). None of the differences between HIV-seropositive and -seronegative patients was statistically significant.

Table 1. Demographic characteristics, laboratory findings at presentation, and treatment prescribed for 107TB patients who completed treatment successfully [9]. Columns represent subgroups of patients included(right) and not included (left) in this study

Patientsnot studied(n 4 76)

Patients instudy group(n 4 31)

Age (years)15–24 20 (26%) 8 (26%)25–34 42 (55%) 13 (42%)>34 14 (18%) 10 (32%)

SexFemale 29 (38%) 7 (23%)Male 47 (62%) 24 (77%)

Site of tuberculosisa

Pulmonary 55 (72%) 21 (68%)Pleural 25 (33%) 11 (36%)Pericardial 9 (12%) 4 (13%)Lymph node 7 (9%) 1 (3%)Multiple sites 15 (20%) 6 (19%)

HIV statusNegative 28 (37%) 13 (42%)Positive 48 (63%) 18 (58%)

Treatmentb

2STH/10TH 38 (50%) 15 (48%)2STHRZ/6TH 36 (47%) 16 (52%)2ETHR/10TH 2 (3%)

Prednisolone (5 missing values)No prednisolone 48 (68%) 21 (68%)Prednisolone 23 (32%) 10 (32%)

Tuberculin response (43 missing values)Negative 25 (54%) 8 (44%)Positive 21 (46%) 10 (56%)

Hemoglobin (g/dL; 10 missing values)Mean (S.D.) 10.8 (2.1) 11.2 (2.5)

Lymphocyte count (×109/liter, ln; 13 missing values)Mean (S.D.) 0.68 (0.55) 0.81 (0.51)

a Sites of TB overlapped; several patients had disease at more than one site.b S, streptomycin; T, thiacetazone; H, isoniazid; R, rifampicin; Z, pyrazinamide; E, ethambutol.

268 M. Hosp et al.

A similar pattern of changes in response to ATT was found for the acute phaseproteins CRP and SAA. Both seropositive and seronegative patients showed elevatedlevels of CRP (26/28 with elevated CRP) and SAA (24/28 with elevated SAA), whichdecreased significantly by the end of ATT (Table 2). At that point 6 out of 17 HIV-seropositive patients (35%) and none of the seronegative patients had CRP levels above10 mg/liter. At the same time SAA levels above 30 mg/liter were found in 3 out of 17(18%) seropositive and in none of the seronegative patients.

B2M concentrations did not change significantly during ATT in our patients. Atthe end of chemotherapy only we found a significant difference between seropositiveand seronegative patients (4.2 ± 1.6 mg/liter vs 2.7 ± 0.9 mg/liter:z 4 2.62,p < 0.01).Hemoglobin was the only laboratory parameter showing a significant difference be-tween HIV-seropositive and -seronegative patients when TB was diagnosed (10.2 ± 2.1g/dL vs 12.5 ± 2.5 g/dL;z 4 2.29,p < 0.05).

Correlations between Serologic Parameters

Comparing neopterin with B2M, CRP, and SAA we found significant correlationsbetween the following parameters. In the HIV-seropositive group only neopterin andB2M correlated both before treatment (rs 4 0.582,p < 0.05) and after treatment (rs 40.821,p < 0.01), and neopterin before chemotherapy correlated with neopterin afterchemotherapy (rs 4 0.571,p < 0.05). In seronegative patients neopterin levels at thebeginning of ATT correlated with B2M (rs 4 0.820,p < 0.01) but not with CRP andSAA. CRP correlated with SAA both before therapy (rs 4 0.673,p < 0.05) and aftertherapy (rs 4 0.784,p < 0.05).

Neopterin concentrations in our patients did not correlate with radiologic findings,i.e. the number of zones involved, presence of cavities, but they were associated withother parameters of disease activity. Patients presenting with high fever, weight loss,

Fig. 1. Changes of neopterin levels in patients before and after ATT.Filled circles, HIV-1-seropositivepatients;open circles,HIV-1-seronegative patients;left panel,before ATT; right panel,after ATT.

Neopterin Monitoring in HIV-Associated TB 269

and low numbers of erythrocytes and lymphocytes showed higher levels of neopterin.In all patients both neopterin and B2M correlated with anemia and body temperaturewhen TB was diagnosed. In HIV-seropositive patients neopterin also showed a sig-nificant negative correlation with the number of lymphocytes and the total white bloodcount. CRP and SAA showed a positive correlation with the total white blood countonly in HIV-seronegative patients (data not shown).

Relationship between Serologic Parameters at Completion of Treatment andSubsequent Outcome

When summarizing the clinical course of HIV-seropositive and -seronegative TB pa-tients after completion of treatment, an association between increased neopterin con-centrations at the end of treatment and adverse health events in the subsequent year offollow-up was evident (Table 3). All patients who presented with TB relapse, died, orcomplained about other adverse health events (such as recurrence of fever, continuedweight loss, respiratory symptoms) had high neopterin concentrations when ATT wascompleted. Among 15 patients who remained well in the follow-up period only 2showed elevated neopterin concentrations at the end of treatment. The associationsbetween outcome (well vs adverse outcome) were significant (HIV-seropositive:x2 413.85, p < 0.001; HIV-seronegative:x2 4 9.24, p < 0.01). All 4 patients in the

Table 2. Mean and median values of laboratory parameters before and after ATT in HIV-1-seropositiveand -seronegative patients (p values are from Wilcoxon’s paired rank test when comparing concentrationsbefore and after treatment)

HIV-1-seronegative HIV-1-seropositive

Before ATT After ATT Before ATT After ATT

Neopterin (nmol/liter)No. (S.D.) 13 13 18 18Mean (S.D.) 21.0 (11.2) 10.7 (9.6) 24.0 (13.0) 14.3 (8.7)Median (25–75%) 19.6 (13.5–27.0) 7.0 (5.3–12.3) 21.2 (16.9–25.9) 11.5 (7.8–18.5)

U 4 2.94,p 4 0.0003 U 4 2.87,p 4 0.0004CRP (mg/liter)

No. 11 11 17 17Mean (S.D.) 47.7 (37) 3.8 (3.4) 48.8 (38) 7.8 (6.9)Median (25–75%) 38 (20–62) 2 (1–8) 37 (29–52) 6 (2–14)

U 4 2.75,p 4 0.0006 U 4 3.60,p 4 0.0003SAA (mg/liter)

No. 11 11 17 17Mean (S.D.) 140 (89) 11 (11) 124 (72) 18 (25)Median (25–75%) 132 (48–246) 6 (5–13) 127 (62–173) 8 (6–13)

U 4 2.89,p 4 0.0004 U 4 3.60,p 4 0.0003B2M (mg/liter)

No. 11 11 16 16Mean (S.D.) 2.9 (1.5) 2.7 (0.9) 3.8 (1.5) 4.2 (1.6)Median (25–75%) 2.7 (1.7–3.5) 2.8 (2.4–3.1) 3.2 (2.7–5.0) 3.8 (3.2–5.5)

U 4 0.04,p 4 0.97 U 4 1.14,p 4 0.26

270 M. Hosp et al.

HIV-seronegative group with an unfavorable outcome had normal CRP: persistentlyhigh CRP in HIV-seropositives was observed in only 4 out of 12 patients presentinglater with TB relapse, death, or other adverse health events.

Discussion

In this pilot study we examined the progression of serum neopterin, acute phaseproteins, and B2M levels during treatment for TB in HIV-seropositive and -seroneg-ative patients and the relationship between neopterin levels at completion of treatmentand subsequent outcome. Patients were included only if they successfully completedtreatment for TB, so the relationship between the levels of serologic markers at diag-nosis and early mortality and initial outcome of treatment could not be assessed.Samples were analyzed for only a fraction of patients who completed treatment, and wetherefore regard our findings as preliminary.

In HIV infection neopterin has been used as a progression marker for disease.Neopterin is a pyrazinopyrimidine compound derived from guanosine triphosphate(GTP). In vitro and in vivo it has been demonstrated that activation of the cellularimmune system is invariably accompanied by increased amounts of neopterin [12].Human monocytes/macrophages release increased amounts of neopterin upon stimu-lation with g-interferon derived from activated T lymphocytes [20]. In patients in-creased serum and urinary neopterin levels can be found in diseases in which cellularimmune mechanisms play a role, thus not only in viral infections such as HIV [14], butalso in diseases associated with intracellular bacterial [13] and protozoal [27] patho-gens, in autoimmune disorders [14], allograft rejections [23], and in certain malignantdiseases [1, 26]. The role of neopterin within the immunologic defense mechanism isnot yet clearly understood.

As a marker of cell-mediated immune activation neopterin has been demonstratedto be a useful biochemical indicator of disease activity in TB [13]. In many parts of theworld TB is the predominant opportunistic infection in patients with the acquiredimmunodeficiency syndrome (AIDS). This report is the first study investigating thepotential usefulness of neopterin in monitoring the clinical response to ATT in HIV-

Table 3. Association between neopterin concentrations at the end of ATT and survival or subsequentclinical outcome. Patients were categorized as above or below 8.7 nmol/liter neopterin, which represents the95th percentile of healthy controls in Europe

Neopterin levelat end of ATT(nmol/liter)

Well TB relapse Death Other adversehealth event

Alive Dead

HIV-seropositive>8.7 1 1 1 2 8<8.8 5 0 0 0 0

HIV-seronegative>8.7 1 1 1 0 2<8.8 8 0 0 0 0

Neopterin Monitoring in HIV-Associated TB 271

seropositive African TB patients. A previous study in outpatients at a Tanzanian districthospital suggested that because of a high background of parasitic, viral, and bacterialinfections, neopterin levels were elevated in many apparently healthy African indi-viduals. That study concluded that the value of neopterin for clinical use in the tropicswas limited [28]. This was not the case in our patients who were living in the urban orperiurban environment of greater Lusaka. The median concentration of neopterin inHIV-seronegative patients at the end of ATT was within the normal range as definedin European and North American populations. Further studies are needed to define therange of neopterin levels in African populations. In HIV-seropositive individuals neop-terin concentrations are expected to be elevated by the stage of HIV disease, thoughtto be linked with the production of HIV. Development of active TB in such patientscauses stimulation of the immune system and results in a marked additional elevationof serum neopterin. The highly significant decrease during treatment underlines itsusefulness for monitoring the clinical response to ATT also in HIV-seropositive indi-viduals. 28% of the seropositive patients in our study had normal neopterin concen-trations at the end of ATT, which suggests that TB can occur in early stages of HIVinfection. Similarly, a study in Zaire found that 30.1% of HIV-seropositive patientswith TB had a CD4 lymphocyte count of 500/mL or higher [24].

Neopterin appears to have a prognostic value for the assessment of a patient’sgeneral medical condition. In those patients who relapsed in the follow-up period, wholost weight, or who did not correct their anemia, elevated neopterin levels at the end ofchemotherapy suggested that some pathologic process was still going on, and thepatient might have benefitted from further medical attention. In such cases neopterincould perhaps be used as an indicator of the need to prolong ATT. Patients with normalneopterin concentrations after completion of ATT did well in the follow-up period of12 months. CRP, SAA, and B2M at the end of treatment did not show such a predictivevalue.

There was no difference in neopterin levels at the end of treatment betweenpatients who received steroids and those who did not; there was no consistent asso-ciation between the measures of compliance used and the neopterin levels at the end oftreatment. Clearly, since we chose to examine only patients who completed treatmentand were considered cured, the information on the relation between serologic param-eters and findings at presentation was limited, and we cannot say anything about therelation between serum neopterin levels at presentation and outcome of treatment.

B2M is another progression marker for HIV disease which reflects cell turnoverand increases with stage of HIV infection. Our findings do not concur with a reportfrom Uganda showing significantly lower B2M concentrations in patients who hadbeen treated for TB for more than 60 days [31]. But in the Ugandan cross-sectionalstudy the number of patients was small, and the investigators did not compare the samegroup of patients before and during treatment. The observed decline in B2M mightresult artificially from a survivor effect if enough early deaths occurred in patients withhigh B2M [31]. In our study we did not see a significant decline with ATT; rather B2Mlevels in HIV-seropositive patients increased during the 8 or 12 months of therapy. Ourdata suggest that B2M may be relatively independent of TB activity. Since the numberof patients in these studies was small and conflicting results have been reported, furtherinvestigations are needed.

272 M. Hosp et al.

CRP is thought to be involved in the homeostatic response to tissue injury, andserum concentrations of CRP reflect the severity of inflammation and tissue destruc-tion. Patients with tuberculous cavitation have been shown to have higher CRP levelsthan TB patients without cavities [5, 19, 21], and we observed such an association tosome extent in our study. In previous reports a wide scatter of CRP concentrations wasdescribed, and only about two thirds of TB patients were found with elevated CRPlevels [3, 21, 29]. Twenty-six of our 28 patients had raised CRP levels when TB wasdiagnosed and much lower concentrations when treatment was completed. But patientswith high CRP did not consistently show high neopterin levels at the same time,reflecting the fact that different cytokines and mediators are responsible for stimulationof acute phase proteins and neopterin. Neopterin is released by macrophages prefer-entially upon stimulation byg-interferon, whereas interleukin-6 and interleukin-1 arethe main inducers of CRP and SAA synthesis in hepatocytes [4].

In previous reports from Europe [22, 29] and from developing countries [3, 5, 13,18, 21, 22] a wide scatter of CRP concentrations was described, and about two thirdsof TB patients were found with elevated CRP levels. An assessment of CRP levels atthe end of chemotherapy in 20 Indian TB patients found a proportion of 14% withabnormal values of CRP, but it was not a suitable marker for the outcome of abacteriologic relapse in patients who had quiescent disease at the end of treatment [21].In our study a very high proportion (92%) of TB patients had higher CRP values whenTB was diagnosed, and they remained elevated in a fraction of HIV-seropositivepatients after successful treatment of TB but were not associated with the outcome inthe follow-up period. Patients with high CRP did not consistently show high neopterinlevels at the same time, reflecting the fact that different cytokines and mediators areresponsible for stimulation of acute phase proteins and neopterin. Neopterin is releasedby macrophages preferentially upon stimulation byg-interferon, whereas interleukin-6and -1 are the main inducers of CRP and SAA syntheses in hepatocytes [4].

The observation that there is no strong corelation between serologic markers andthe radiologic evidence of pulomary destruction in TB is not new, and it has beenattributed to the inability of radiologic differentiation between inactive scars and areasof active inflammatory disease [5]. During successful treatment of TB, a fall of CRPand neopterin concentrations has been described to occur quicker than changes in theradiologic presentation [13, 29].

Neopterin levels directly reflect macrophage activation, and in our study theycorrelate well with clinical parameters of disease activity such as fever, weight loss,and anemia. We could not demonstrate such a correlation with the extent of radiologicchanges. The size and nature of chest x-ray lesions do not necessarily reflect thestimulation of the immune system. Also, the small number of cases investigated maynot allow the detection of a statistically significant correlation.

The acute phase protein SAA is of interest because it is the precursor of the majorprotein constituent of secondary amyloid fibrils. TB has long been associated withsecondary amyloidosis [32]. CRP and SAA are always strongly correlated if oneinvestigates a large number of patients [32]. In our HIV-seronegative TB patients CRPand SAA correlated both before and after treatment.

Our study is limited by the small number of patients and selection bias such asexclusion of defaulters and sicker patients who died before completing treatment.

Neopterin Monitoring in HIV-Associated TB 273

Samples were analyzed only for a fraction of patients who completed treatment, and wetherefore regard our data as preliminary. They show that neopterin, CRP, and SAA aremore sensitive markers of TB activity than B2M. Neopterin, CRP, and SAA showedlittle difference between HIV-seronegative and -seropositive patients and fell withtreatment in both groups, whereas B2M remained elevated in HIV-infected patients.Persistent elevation of neopterin, suggesting persistent activation of cell-mediated im-munity, occurs in a proportion of treated TB patients and is associated with a pooroutcome in both HIV-seropositive and -seronegative patients. This information is use-ful in understanding the disease process, suggesting that poor outcome after treatmentis the consequence of inadequately controlled disease rather than reemergence aftercomplete suppression.

Acknowledgments.This study was supported (in part) by the Science and Technology for Developmentprogram of the Commission of the European Communities, Contract TS2-M-0004-UK, and by the AustrianFunds Zur Fo¨rderung der wissenschaftlichen Forschung P 10776.

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Accepted for publication: 13 December 1996

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