Indian J Med Res 121, April 2005, pp 220-225
220
Serum neopterin levels in HIV infected patients with & without
tuberculosis
Chandra Immanuel, Lalitha Victor, K. Silambu Chelvi, C. Padmapriyadarsini, Fathima Rehman,
Sheik Iliayas & Soumya Swaminathan
Tuberculosis Research Centre (ICMR), Chennai, India
Accepted September 22, 2004
Background & objective :  Three categories of prognostic markers are best documented as
having significance in relation to prognosis of HIV infection. These include HIV viral load,
CD4 T-cell levels and plasma levels of soluble markers of immune activation. The plasma
activation markers, like neopterin, tumor necrosis factor alpha (TNF- ?? ??? ), interleukins etc., are
products of cytokine activity and represent immunologic changes throughout the body. There
is not much information available on serum neopterin estimation in patients infected with
both HIV and tuberculosis (TB), though neopterin levels are known to be elevated in pulmonary
TB patients. In this study we attempted to correlate neopterin levels with the presence of
tuberculosis in HIV infected and uninfected individuals and studied the changes after anti-
tuberculosis treatment.
Methods : Serum neopterin concentrations were measured by high performance liquid
chromatography (HPLC) in 25 HIV-seropositive (HIV-TB) and 10-seronegative (TB) patients
with tuberculosis before, during and at the end of antituberculosis therapy (ATT). S-neo was
also measured in 10 HIV-seropositive asymptomatic individuals and 10 healthy controls. The
results were correlated with clinical, bacteriological and immunological status.
Results : All TB patients regardless of HIV status had elevated s-neo concentrations at diagnosis,
which declined gradually during treatment. Patients with HIV/TB with CD4 counts < 200/mm 3
had the highest levels at baseline with a steep fall during treatment. The median level at the
end of treatment was significantly higher in HIV/TB than in TB patients, despite clinical
improvement and bacteriological clearance of  Mycobacterium tuberculosis. HIV infected
asymptomatic individuals had neopterin levels that were higher than healthy controls but
lower than HIV-TB patients.
Interpretation & conclusion : Serum neopterin levels are elevated in HIV-positive patients,
with the highest levels in those with tuberculosis and CD4 counts < 200/mm 3. Though the
levels decrease with anti tuberculosis therapy, persistently elevated levels indicate progressive
HIV disease and a poor prognosis.
Key words :  HIV - neopterin - opportunistic infections - tuberculosis
The use of surrogate end points has been more
intensely examined in HIV/AIDS than in any other
disease. Many studies have shown the value of
prognostic markers such as CD4+ T-lymphocyte
count, the viral load and the soluble immune activation
markers in predicting the course of the illness1-5. The
CD4+ T-lymphocyte count has been the principal
biological marker utilized for clinical evaluation of
221CHANDRA et al :  SERUM NEOPTERIN & HIV/TB
disease. However, measuring CD4 counts and viral
load requires expensive equipments, separate
space, trained personnel, etc. Hence there is a
need to study other markers for use in monitoring
HIV disease progression and response to
treatment.
Activation of all components of the immune
system is a major feature of HIV infection and one
manifestation of immune activation is increased
cytokine production. However, limitation in the ability
to quantitate circulating cytokines has led to the
assessment of downstream products that reflect
cytokine activity in lymphoid tissues 2.  Such
assessment includes level of soluble markers and one
such marker is neopterin. Neopterin is a pteridine
derivative of guanosine triphosphate released into
circulation from activated macrophages. Serum
neopterin (s-neo) level is an indicator of both
macrophage activation and interferon gamma
(IFN-? ) activity, a major macrophage activating
factor1,3,4. Neopterin can be measured more easily
and accurately than IFN-?  levels in serum.
Tuberculosis is one of the commonest
opportunistic infections in HIV infected patients, in
India. Previous studies have shown that s-neo levels
are elevated in patients with pulmonary
tuberculosis5,7. Serial evaluation of s-neo in patients
on antituberculosis treatment (ATT) is useful in
assessing the response to therapy8,9. As HIV and TB
both stimulate the cellular arm of immune system,
effective treatment of tuberculosis would reduce some
of the excessive immune activation. However, there
are not much data on serial estimation of neopterin in
patients infected with both HIV and tuberculosis. In
the present study, we have tried to correlate changes
in s-neo levels with clinical and bacteriological status
of patients infected with HIV and TB, and compared
these results with HIV-negative TB patients. The
changes in s-neo levels after antituberculosis
treatment were also studied in all these groups of
patients.
Material & Methods
Study subjects : Patients admitted to various
controlled clinical trials at Tuberculosis Research
Centre (TRC) clinics and subcentre at Chennai and
Madurai, between January and June 2001, were
randomly selected for serum neopterin assays. A
thorough history and clinical examination, chest X-
ray and three sputum smear examinations for acid-
fast bacilli (AFB) and culture for  Mycobacterium
tuberculosis were done. After pretest counselling
and obtaining informed consent from the patients, HIV
status was determined by using two different rapid
tests (Comb Aids-RS, Span diagnostics, India and
TRI-DOT, J. Mitra & Co, India) followed by an
ELISA (Labsystems, UK).
Twenty five HIV-positive tuberculosis patients
aged between 22-50 yr (mean + SD 31.0 + 4.6 yr)
and 10 newly diagnosed HIV-negative smear positive
pulmonary tuberculosis patients aged between 20 to
50 yr (mean + SD 26.8 + 9.8 yr) were included in the
study. All the HIV seronegative patients had culture
confirmed pulmonary TB.
HIV-TB group comprised of patients with newly
diagnosed pulmonary and extra pulmonary forms of
tuberculosis. Of the 25 patients, 19 had pulmonary
tuberculosis while 6 had extrapulmonary tuberculosis,
including 2 with TB lymphadenitis (proven by
histopathology) and 4 with pleural effusion; 11 had
sputum smears positive for AFB while 17 had sputum
culture positive for M. tuberculosis. Seven patients
had associated opportunistic infections like diarrhoea,
oral candidiasis, scabies, etc. during pre-treatment
period. The 25 HIV-TB patients were categorized
on the basis of CD4 T cell counts at presentation as
less than or greater than 200 cells/mm3. 15 patients
had CD4 T cells <200/mm3 ranging from 22-195 cells/
mm3 (mean + SD: 92.8 + 58.8) and 10 had >200 cells/
mm3 ranging between 204 to 513 cells/mm3 (mean +
SD 334.6 + 127.5).
Tuberculosis patients were started on a 6-month
short-course intermittent chemotherapy regimen
containing rifampicin (450 mg), isoniazid (600 mg),
pyrazinamide (1500 mg) and ethambutol (1200 mg)
thrice weekly for two months followed by rifampicin
(450 mg) and isoniazid (600 mg) thrice weekly for 4
months. In addition, all HIV-positive patients with
CD4 cell count < 200 cells/mm 3  were given
corimoxazole double strength one tablet daily. Ten
asymptomatic HIV-seropositive individuals aged
between 20 to 50 yr (mean + SD 30.3 + 8.0 yr) and
222
10 healthy seronegative individuals aged 20-40 yr
(mean + SD 28.8 +3.2 yr), mainly TRC laboratory
staffs, were also included in the study. All the patients
with TB became smear and culture negative by the
end of treatment and showed clinical and radiological
improvement.
The study proposal was approved by the
Institutional Ethics committee.
Study design : Blood samples (10ml) were collected
before, during and at the end of treatment for HIV-
TB and TB patients and at one time point for HIV
seropositive asymptomatic individuals and healthy
controls. Serum was separated and stored frozen at
–70oC until use.
Neopterin assay : Neopterin in serum was measured
by high performance liquid chromatography (HPLC)
as described elsewhere10. The HPLC system used
was Shimadzu vp series (Shimadzu corporation,
Japan) equipped with two pumps (LC-10ATvp),
spectrofluorimetric detector (RF-10AXL) and system
controller (SCL-10Avp). Ultra filtrate of serum
samples were prepared using the ultrafree-
microcentrifuge filters (Sigma Chemical Co., USA).
The mobile phase was a 15mM potassium phosphate
buffer (pH 6.5) with a flow rate of 1ml/min. Standards
ranging from 0 to 160nmol/l and the protein filtrate of
samples were analysed by injecting 20µl directly to a
reverse phase C8 column (LiChroCART 5µm
250x4.0mm, Merck KgaA, Germany); neopterin was
measured by its native fluorescence (350nm
excitation, 440nm emission).
Measurement of CD4+ T-lymphocyte count : CD4
lymphocyte enumeration in whole blood samples from
HIV-positive patients was done by flow cytometry
(FACSort, Becton-Dickinson, USA) before and at the
end of treatment.
Statistical analysis: The SPSS (version 8.0) was used
for statistical analysis. Using the non-parametric
Mann-Whitney U test and Wilcoxon rank test,
comparison between the groups was performed;
P < 0.05 were considered significant. The correlation
between s-neo and CD4+ T-lymphocyte count was
assessed with use of Spearman’s rank correlation
coefficient.
Results & Discussion
The median s-neo level was significantly elevated
above the normal level in patients with active
tuberculosis regardless of their HIV status. The HIV-
positive asymptomatic group had significantly lower
level (median: 13.5 nmol/l, P<0.001) than that of HIV-
TB patients (median: 37.8 nmol, P<0.001), but higher
than healthy controls (P<0.001). Among patients with
tuberculosis, the median levels of s-neopterin in HIV-
TB group with CD4 count of >200/mm3, was similar
to that of HIV uninfected TB patients. Patients with
CD4 cell count of <200/mm3, had the highest median
level and the range of 25th and 75th percentile did not
overlap with the other two groups (Table).
There was a gradual decrease in s-neo level in
all the three groups (HIV/TB with CD4 < 200 cells/
µ l, HIV/TB with CD4> no cells/µ l, and TB patients)
in response to chemotherapy. However, the median
level (25th-75th percentile) at the end of treatment was
significantly higher in both the HIV-TB groups than
in TB patients [41.1 (33.2-72.2), 31.5 (19.4-41.8) vs
13.2 (10.7-22.1) nmol/l] with a P <0.001, P<0.01,
respectively (Fig.). The median values for both HIV
Table. Baseline serum neopterin levels in different study groups
Serum Neopterin HIV-TB HIV-TB Pulmonary TB Asymptomatic Healthy control
(nmol/l) (CD4<200/mm3) (CD4>200/mm3) (n=10) HIV positive  (n=10)
(n=15) (n=10) (n=10)
Median 90.7 37.8 29.2 13.5 7.4
25th-75th  percentile 54.7-124.8 25.0-48.5 18.9-42.0 11.8-14.5 6.0-8.1
All groups significantly (P<0.001) different from each other except pulmonary TB and HIV-TB with CD4 T cell count
>200 cells/mm3
INDIAN J MED RES, APRIL 2005
223
groups were higher than the 75th percentile for TB
patients at the end of treatment. Four patients, three
with CD4+ T-lymphocyte counts < 200 and one with
>200/mm3 had increased neopterin level at the end
of treatment after a decline during treatment.
In HIV-TB patients with CD4+ T-lymphocyte
counts <200/mm3, the median decrease in neopterin
was 34 and 45 per cent during and at the end of
treatment respectively; the corresponding decrease
in patients with CD4+ T-lymphocyte counts >200/
mm3 was 6 and 19 per cent; and in TB patients it
was 45 and 55 per cent. The decrease was statistically
significant only in HIV-TB patients with CD4+ T cell
counts <200/mm3 (P<0.01) and in TB patients
(P<0.05).
Neopterin concentrations were inversely related
to CD4+ T-cell counts, both initially and at the end of
treatment, (r = -0.484 and –0.449 respectively,
P < 0.05) in patients with HIV-TB.
 Immune activation is an important feature in HIV
infection because the extent of immune activation is
correlated to the clinical outcome of the disease11. In
HIV associated tuberculosis, cellular activation is
prominent, despite the deleterious effect of HIV on
immune response12. This activation can be quantified
by measurement of plasma levels of soluble markers.
It would be useful to have a marker that could predict
prognosis when HIV and TB occur together. In the
present study, an attempt was made to examine
whether neopterin, a surrogate marker for interferon
gamma (IFN-? ) would correlate with clinical and
bacteriological response during treatment for
tuberculosis in HIV-seropositive patients. Other
indices such as CD4 T-lymphocyte counts were
correlated to s-neo concentrations both at presentation
and at the end of ATT.
 S-neo level was significantly increased initially
in all the patients with tuberculosis irrespective of HIV
status. These findings confirm our previous report7
and of others that TB patients have elevated neopterin
levels6-8. The median neopterin level was markedly
elevated in HIV-TB patients and this was higher than
that reported in Zambian patients8; this could be
because 60 per cent of our patients had CD4+ counts
< 200/mm3. However, our results for the median
neopterin level in patients with CD4 count >200/mm3
was comparable to that of Zambian patients. The
median s-neo level of HIV-TB patients with  <200
CD4 T-lymphocyte counts µ l was the highest of all
the groups, indicating that immune activation was
maximum in patients with severe immunosuppresion.
In vitro experiments to investigate the possibility
that HIV-1 infection of mononuclear phagocytes
directly induces enhanced neopterin production,
showed that the observed 6 to 12 fold increase in
neopterin was not caused by HIV infection but might
be a result of the normal response by mononuclear
phagocytes to increased level of IFN-? 13.  Further,
acute primary HIV-1 infection was characterized by
CD4 lymphocytopenia with elevated serum level of
IFN-?  and neopterin14. Aziz et al15 had reported
progressive increase in the levels of IFN- ?  and
Fig. Median  s-neo levels in HIV/TB (n=10) and 10 TB patients before, during and  end of treatment.
224
neopterin when HIV-seropositive individuals were
stratified on the basis of CD4 cell counts, and the
highest levels were seen in CD4 category of <200/
mm3.  Similarly, circulating plasma level of IFN-?  is
shown to be increased in newly diagnosed tuberculosis
patients16. In HIV-TB patients, plasma IFN-?  level is
maximum17 in patients with CD4 T cell counts of less
than 200/mm3. These findings clearly indicate that s-
neo level is an indicator of IFN-?  activity and thus is
a good surrogate marker for this cytokine.
In this study, persons with asymptomatic HIV
infection had s-neo level higher than healthy controls
but much lower than patients with tuberculosis. These
results are in agreement with the findings of Fahey
et al18,  that HIV infection produces immune activation
even in early stages of the disease. During
antituberculosis treatment, s-neo concentrations
started to decline but this decline was statistically
significant in HIV-TB patients with CD4 +
T-lymphocyte counts of <200/mm 3 and in TB
patients. It is interesting to note that three patients in
HIV-TB group had rising s-neo levels after the decline
during treatment with a corresponding decrease in
CD4+ counts. Two of these patients who had s-neo
levels >100nmol/l at the end of treatment died within
nine months after the completion of ATT due to
causes other than TB (one patient died due to AIDS
wasting and the other developed atypical
mycobacterial infection) and one patient relapsed after
four months. Similarly, one patient with CD4 counts
of >200/mm3 at presentation, had an increased serum
neopterin level at the end of treatment (120nmol/l)
with a corresponding decrease in CD4 cell counts to
<200/mm3 and this was associated with the
development of multiple opportunistic infections, over
the period of her treatment. She developed recurrent
episodes of diarrhoea, oral thrush and skin
infestations.  Although we observed a declining trend
in other patients, the level of s-neo at the end of
treatment did not reach the normal range and it was
also higher than that found in HIV-negative TB
patients despite good clinical and bacteriological
response to treatment. This shows that there is
persistent activation of cell-mediated immunity and
the higher level at the end of treatment in HIV-TB
patients indicates either co-existing opportunistic
infections or progressive HIV disease.
The advantage of combining CD4+ T-lymphocyte
counts with neopterin levels as a prognostic marker
has been documented previously12. Our data and
earlier reports show that s-neo level does not
correlate very closely with CD4+ T-lymphocyte
counts (r=0.45 vs - 0.39, -0.43, 0.41)11,19. The increase
in the s-neo level has been shown to be largely
independent of changes in CD4 T-lymphocyte counts
in HIV infection. The CD4 levels showed an inverse
relationship to the s-neopterin levels in most patients,
both at baseline and at end of treatment. The number
of patients in this study was not adequate to test the
sensitivity of s-neopterin levels for predicting the
decline in CD4 T cell count. Though s-neo estimation
is easier to perform and less expensive as compared
to CD4 count measurement, it does not replace CD4
count estimation. Combination of a single plasma
activation marker measurement like neopterin with
CD4 T-cell levels has been shown to improve the
prognostic capability17.  However, these markers,
including neopterin are non specific and could be
elevated due to a variety of causes in individuals with
HIV infection. The limitations of our study are small
sample size and a relatively short period of follow
up. More studies are required to evaluate s-neopterin
and other serum   immune markers in patients with
HIV and TB and to correlate the changes with CD4
T cell counts.
In conclusion, though neopterin is a non specific
marker of cellular immune activation, we have shown
that s-neo levels are elevated in HIV-seropositive
compared to seronegative patients with tuberculosis.
Further, the levels decline with effective anti-TB
treatment, but remain well above the normal range in
patients with HIV co-infection. The decrease in
s-neopterin is most marked in patients with severe
immunosuppression with CD4 T cell counts < 200
cells/mm3, indicating the benefits of early diagnosis
and treatment of tuberculosis in this group. Further,
rising levels of neopterin in serum are indicative of
presence of another opportunistic infection or disease
progression itself. Neopterin is stable in stored serum
and is technically easy and cheaper to measure
compared to viral load. More studies are required to
assess its role in the monitoring of patients with HIV
in developing countries, including those on anti-
retroviral therapy.
INDIAN J MED RES, APRIL 2005
225
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Reprint requests : Dr Soumya Swaminathan, Deputy Director, Division of HIV/AIDS, Tuberculosis Research Centre,
Mayor V.R.Ramanthan road, Chetput, Chennai, India
e-mail: doctorsoumya@yahoo.com
CHANDRA et al :  SERUM NEOPTERIN & HIV/TB