Early impairment of gut function and gut flora supporting a role for alteration of gastrointestinal mucosa in human immunodeficiency virus pathogenesis.
ABSTRACT Our results show that impairment of the gastrointestinal tracts in human immunodeficiency virus (HIV)-positive patients is present in the early phases of HIV disease. This impairment is associated with alterations in gut microbiota and intestinal inflammatory parameters. These findings support the hypothesis that alterations at the gastrointestinal-tract level are a key factor in HIV pathogenesis.
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ABSTRACT: Fecal calprotectin is a marker of inflammatory and neoplastic disease in the lower gastrointestinal tract. A new fecal sample preparation procedure for the measurement of calprotectin has been developed, with higher calprotectin yield and lower contamination risk. Changes in the new method compared to the original [Roseth AG, Fagerhol MK, Aadland E, Schonsby H. Assessment of the neutrophil dominating protein calprotectin in feces. A methodologic study. Scand J Gastroenterol 1992;27(9):793-798] are smaller sample size, higher dilution of the sample, presence of dissociating agents in the extraction solution and procedure performed in closed disposable tubes. The extraction yield was 78% (41-100%) of total calprotectin, giving an overall five-fold increase compared to the original method. Samples with high calprotectin values were increased to a slightly higher degree, than low calprotectin samples, thus improving the separation between high and low calprotectin levels. Median calprotectin level in healthy subjects was 26 microg/g. Pathological samples with pancolitis showed levels up to 30000 microg/g. The mean C.V. (coefficient of variation) in blended feces was lower than that of unblended, suggesting uneven distribution of calprotectin. However, no significant difference between spot measurements was found when five samples from each of 47 stools were measured. Thus measurements of calprotectin in fecal samples were accurate and reproducible. No interference with foods or relevant oral pharmaceuticals or nutraceuticals was found.Clinica Chimica Acta 03/2000; 292(1-2):41-54. · 2.85 Impact Factor
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ABSTRACT: STATEMENT OF FINDINGS: We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples. This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction. The time from sample collection to result was less than 1h. RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment.Critical Care 02/2000; 4(4):255-61. · 4.93 Impact Factor
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ABSTRACT: A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5' nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5' nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% +/- 9.8% to 73.4% +/- 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% +/- 1.92% and 8.11% +/- 4.12%, respectively, versus 0.15% +/- 0.11% and 1.38% +/- 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.Applied and Environmental Microbiology 06/2005; 71(5):2318-24. · 3.68 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2008, p. 757–758
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 2
Early Impairment of Gut Function and Gut Flora Supporting a Role
for Alteration of Gastrointestinal Mucosa in Human
Immunodeficiency Virus Pathogenesis?
Andrea Gori,1,2* Camilla Tincati,2Giuliano Rizzardini,3Carlo Torti,4Tiziana Quirino,5
Monique Haarman,6Kaouther Ben Amor,6Jacqueline van Schaik,6Aldwin Vriesema,6
Jan Knol,6Giulia Marchetti,7Gjalt Welling,8and Mario Clerici9
Division of Infectious Diseases, Department of Internal Medicine, San Gerardo Hospital, University of Milano-Bicocca, Monza,
Italy1; Department of Internal Medicine, Clinic of Infectious Diseases, San Paolo Hospital, University of Milan, Milan, Italy2;
1st Division of Infectious Diseases, Luigi Sacco Hospital, Milan, Italy3; Clinic of Infectious Diseases, University of Brescia,
Brescia, Italy4; Infectious Diseases Unit, Busto Arsizio Hospital, Busto Arsizio, Italy5; Numico Research BV, Wageningen,
The Netherlands6; Department of Clinical Science, Luigi Sacco Hospital, University of Milan, Milan, Italy7; Department of
Medical Microbiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands8;
and Department of Science Technology Biomedicine, University of Milan, Milan, Italy9
Received 30 August 2007/Returned for modification 8 October 2007/Accepted 29 November 2007
Our results show that impairment of the gastrointestinal tracts in human immunodeficiency virus (HIV)-
positive patients is present in the early phases of HIV disease. This impairment is associated with alterations
in gut microbiota and intestinal inflammatory parameters. These findings support the hypothesis that alter-
ations at the gastrointestinal-tract level are a key factor in HIV pathogenesis.
The hallmark of human immunodeficiency virus (HIV) in-
fection is the depletion of CD4?T cells, which occurs through-
out the entire disease. Recent data have shown that primary
HIV infection is associated with a preferential depletion of
CD4?T cells in the gastrointestinal (GI) tract, where more
than 60% of T lymphocytes reside, suggesting a pivotal role for
the early impairment of the gut-associated lymphoid tissue in
HIV disease (4). However, the precise pathogenic mechanisms
underlying the profound CD4?T-cell loss observed in the
various stages of disease are only partially understood.
A characteristic feature of HIV disease is chronic immune
activation, which might play a crucial role in the CD4?T-cell
depletion. It has been recently hypothesized that a breakdown
of the GI mucosal barrier may contribute to chronic immune
activation (4). Thus, the exposure of peripheral immune cells
to microbial products after gut injury may result in the abnor-
mal activation of such cells.
The healthy GI tract is colonized by a large variety of com-
mensal microbes that impact on the development of the
humoral and cellular mucosal immune system (11, 12). This
microbiota is shielded from the immune system via a strong
mucosal barrier. Infections and antibiotics are known to alter
both the normal GI-tract barrier and the microbiota, which
may result in possible immune abnormalities. If HIV infection
itself impaired the GI barrier and the composition of the gut
microbiota, the breakdown of the GI mucosa would result in
the sudden and chronic exposure of peripheral lymphocytes to
an abnormal intestinal microbiota (4).
To verify this hypothesis, we studied the effect of HIV in-
fection on the commensal intestinal microbiota and on intes-
tinal inflammation by analyzing the composition of GI tract
microbiota and by measuring the levels of fecal calprotectin in
57 healthy, asymptomatic HIV-positive, antiretrovirus-naive
individuals (average CD4?T-lymphocyte count and HIV RNA
levels of 520 cells/?l and 28,393 copies/ml, respectively) en-
rolled in a trial in Italy (COPA study). The results were com-
pared to historical data from the general population not suf-
fering from HIV infection. Informed consent was obtained
from all of the patients.
The fecal microbiota of the HIV patients was analyzed by
either fluorescence in situ hybridization or quantitative real-
time PCR (Q-PCR). Preparation of fixed fecal cells and DNA
extraction were performed as described previously (6). For
fluorescence in situ hybridization, a set of specific rRNA-tar-
geted oligonucleotides probes were used to quantify Can-
dida albicans (Caal [5?-GCCAAGGCTTATACTCGCT-3?]),
bifidobacteria (Bif164 [5?-CATCCGGCATTACCA CCC]),
lactobacilli (Lab158 [5?-GGTATTAGCAYCTGTTTCCA]),
and total bacteria (Eub338 (5?-GCTGCCTCC CGTAGGA
GT)) (1, 7, 8, 10). The relative abundance of bacterial groups
was determined as the proportion of cells hybridizing with the
specific probe to the total cells positive for the Eub338 probe.
For the quantification of Pseudomonas aeruginosa, a Q-PCR-
based method was used as described by Pirnay et al. (14).
Calprotectin was measured by using a commercial enzyme-
linked immunosorbent assay kit (PhiCal, Eurospital, Italy).
Initially, we focused our attention on two opportunistic
pathogens: P. aeruginosa and C. albicans. The presence of
these species was found to be very high in this HIV-positive
patient population compared to levels reported for healthy
individuals. P. aeruginosa was identified in 92% of all fecal HIV
samples analyzed. In healthy individuals, P. aeruginosa was
* Corresponding author. Mailing address: Division of Infectious
Diseases, Department of Internal Medicine, San Gerardo Hospital,
University of Milano-Bicocca, Via Solferino 16-20052, Milan, Italy.
Phone: 39 039 2333794. Fax: 39 039 2333898. E-mail: andrea.gori
?Published ahead of print on 19 December 2007.
found in only 20% of samples (unpublished data) using the
same Q-PCR method. Moreover, P. aeruginosa accounted for
0.7% ? 0.13% of the total microbiota in our HIV positive
group and represented a 10-fold increase compared to the
levels in the healthy individuals. Similarly, C. albicans was
detected in 100% of the feces samples of our HIV-positive
group, whereas Bernhardt and Knoke (3) reported that in the
general population only 40% are positive carriers of Candida.
More importantly, the average count of C. albicans was almost
10,000-fold higher in the HIV-positive group (ranging from
[9.1 ? 1.0] ? 107to [8.9 ? 1.0] ? 107cells/g [wet weight] of
feces) than reported for the HIV-negative general population
(?104cells/g [wet weight] of feces) (3).
Further support of the hypothesis of an impairment of the
GI microbiota in HIV-infected patients was revealed by data
showing lower levels in other microbial species, e.g., bifidobac-
teria and lactobacilli, compared to the levels reported for the
general population. Both species have been shown to have a
positive influence on mucosal immune function and gut health
(2, 5, 15, 17). The relative amount of bifidobacteria in the
HIV-positive population investigated during our trial was 2.5%
(95% confidence interval ? 1.4 to 4.2%) of the total fecal
bacteria, while the lactobacilli were nearly undetectable at
0.02% (95% confidence interval ? 0.009 to 0.05%). It is well
documented that in the general population bifidobacteria can
vary between 5 and 10% of the total bacterial community,
while lactobacilli represent 1 to 2% (9, 13). Our data thus
suggest that in HIV-infected individuals the composition of the
fecal microbiota is atypical at an early stage of infection.
Levels of fecal calprotectin, a protein secreted by recruited
neutrophils in the intestinal lining indicative of intestinal in-
flammation and used as a marker of mucosal inflammatory
activity in patients with inflammatory bowel disease (4, 11, 16,
17), were also analyzed. The cutoff for this marker is at 50 ?g/g
(wet weight) of feces, with a median value in healthy individ-
uals of 26 ?g/g (16). Our results revealed that in our HIV
patient group half of the subjects (27 of 53) had increased fecal
calprotectin levels (?50 ?g/g). Even 34% (18 of 53) of the HIV
patients had levels over 100 ?g/g (wet weight) of feces, a
finding that is clearly indicative of a significant GI inflamma-
tion. Since intestinal inflammation is known to reduce the
intestinal barrier function, these data confirm the breakdown
of the intestinal barrier.
Abnormal immune activation represents one of the main
factors associated with HIV disease progression. A better un-
derstanding of the mechanisms driving chronic activation of
the immune system could shed light onto the mechanism(s)
associated with immune cell depletion. The hypothesis that
chronic activation stems from a damaged mucosal barrier and
stimulation of immune cells by microbial products is therefore
The results presented here clearly show that impairment of
the GI tract in HIV-positive patients is present already in the
early phases of HIV disease. This impairment is associated
with elevated levels of intestinal inflammatory parameters and
clear alterations in the gut commensal microbiota, confirming
a possible correlation between intestinal microbial alteration,
GI mucosal damage, and immune activation status. These find-
ings strongly support the recent hypothesis that alterations at
the GI-tract level are a key factor in the pathogenesis of
chronic HIV infection.
We thank Mauro Moroni for helpful discussion and suggestions. We
are grateful to Alan Michael Rosen for critical reading of the manu-
script and valuable grammatical advice. We are also grateful to Daria
Trabattoni for her continuous help and to Tiziana Formenti for excel-
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