HIV-1 tolerates changes in A-count in a small segment of the pol gene.

BACKGROUND: The HIV-1 RNA genome has a biased nucleotide composition with a surplus of As. Several hypotheses have been put forward to explain this striking phenomenon, but the A-count of the HIV-1 genome has thus far not been systematically manipulated. The reason for this reservation is the likelihood that known and unknown sequence motifs will be affected by such a massive mutational approach, thus resulting in replication-impaired virus mutants. We present the first attempt to increase and decrease the A-count in a relatively small polymerase (pol) gene segment of HIV-1 RNA. RESULTS: To minimize the mutational impact, a new mutational approach was developed that is inspired by natural sequence variation as present in HIV-1 isolates. This phylogeny-instructed mutagenesis allowed us to create replication-competent HIV-1 mutants with a significantly increased or decreased local A-count. The local A-count of the wild-type (wt) virus (40.2%) was further increased to 46.9% or reduced to 31.7 and 26.3%. These HIV-1 variants replicate efficiently in vitro, despite the fact that the pol changes cause a quite profound move in HIV-SIV sequence space. CONCLUSIONS: Extrapolating these results to the complete 9 kb RNA genome, we may cautiously suggest that the A-rich signature does not have to be maintained. This survey also provided clues that silent codon changes, in particular from G-to-A, determine the subtype-specific sequence signatures.

Impact of the biased nucleotide composition of viral RNA genomes on RNA structure and codon usage.

We are interested in the influence of nucleotide composition on the fundamental characteristics of the virus RNA genome. Most RNA viruses have genomes with a distinct nucleotide composition, e.g. ranging from minimally 12.9 % to maximally 40.3 % (C- and U-count, respectively, in coronavirus HKU). We present a global analysis of diverse virus types, including plus-strand, minus-strand and double-strand RNA viruses, for the impact of this nucleotide preference on the predicted structure of the RNA genome that is packaged in virion particles and on the codon usage in the viral open reading frames. Several virus-specific features will be described, but also some general conclusions were drawn. Without exception, the virus-specific nucleotide bias was enriched in the unpaired, single-stranded regions of the RNA genome, thus creating an even more striking virus-specific signature. We present a simple mechanism that is based on elementary aspects of RNA structure folding to explain this general trend. In general, the nucleotide bias was the major determinant of the virus-specific codon usages, thus limiting a role for codon selection and translational control. We will discuss molecular and evolutionary scenarios that may be responsible for the diverse nucleotide biases of RNA viruses.

Nucleotide composition of the Zika virus RNA genome and its codon usage.

BACKGROUND: RNA viruses have genomes with a distinct nucleotide composition and codon usage. We present the global characteristics of the RNA genome of Zika virus (ZIKV), an emerging pathogen within the Flavivirus genus. ZIKV was first isolated in 1947 in Uganda, caused a widespread epidemic in South and Central America and the Caribbean in 2015 and has recently been associated with microcephaly in newborns. METHODS: The nearly 11 kb positive-stranded RNA genome of ZIKV was analyzed for its nucleotide composition, also in the context of the folded RNA molecule. Nucleotide trends were investigated along the genome length by skew analyses and we analyzed the codons used for translation of the ZIKV proteins. RESULTS: ZIKV RNA has a biased nucleotide composition in being purine-rich and pyrimidine-poor. This preference for purines is a general characteristic of the mosquito-borne and tick-borne flaviviruses. The virus-specific nucleotide bias is further enriched in the unpaired, single-stranded regions of the structured ZIKV RNA genome, thus further imposing this ZIKV-specific signature. The codons used for translation of the ZIKV proteins is also unusual, but we show that it is the underlying bias in nucleotide composition of the viral RNA that largely dictates these codon preferences. CONCLUSIONS: The ZIKV RNA genome has a biased nucleotide composition that dictates the codon usage of this flavivirus. We discuss the evolutionary scenarios and molecular mechanisms that may be responsible for these distinctive ZIKV RNA genome features.

Unexplained diarrhoea in HIV-1 infected individuals.

BACKGROUND: Gastrointestinal symptoms, in particular diarrhoea, are common in non-treated HIV-1 infected individuals. Although various enteric pathogens have been implicated, the aetiology of diarrhoea remains unexplained in a large proportion of HIV-1 infected patients. Our aim is to identify the cause of diarrhoea for patients that remain negative in routine diagnostics. METHODS: In this study stool samples of 196 HIV-1 infected persons, including 29 persons with diarrhoea, were examined for enteropathogens and HIV-1. A search for unknown and unexpected viruses was performed using virus discovery cDNA-AFLP combined with Roche-454 sequencing (VIDISCA-454). RESULTS: HIV-1 RNA was detected in stool of 19 patients with diarrhoea (66%) compared to 75 patients (45%) without diarrhoea. In 19 of the 29 diarrhoea cases a known enteropathogen could be identified (66%). Next to these known causative agents, a range of recently identified viruses was identified via VIDISCA-454: cosavirus, Aichi virus, human gyrovirus, and non-A non-B hepatitis virus. Moreover, a novel virus was detected which was named immunodeficiency-associated stool virus (IASvirus). However, PCR based screening for these viruses showed that none of these novel viruses was associated with diarrhoea. Notably, among the 34% enteropathogen-negative cases, HIV-1 RNA shedding in stool was more frequently observed (80%) compared to enteropathogen-positive cases (47%), indicating that HIV-1 itself is the most likely candidate to be involved in diarrhoea. CONCLUSION: Unexplained diarrhoea in HIV-1 infected patients is probably not caused by recently described or previously unknown pathogens, but it is more likely that HIV-1 itself plays a role in intestinal mucosal abnormalities which leads to diarrhoea.

Identification of a new genotype of Torque Teno Mini virus.

BACKGROUND: Although human torque teno viruses (TTVs) were first discovered in 1997, still many associated aspects are not clarified yet. The viruses reveal a remarkable heterogeneity and it is possible that some genotypes are more pathogenic than others. The identification of all genotypes is essential to confirm previous pathogenicity data, and an unbiased search for novel viruses is needed to identify TTVs that might be related to disease. METHOD: The virus discovery technique VIDISCA-454 was used to screen serum of 55 HIV-1 positive injecting drug users, from the Amsterdam Cohort Studies, in search for novel blood-blood transmittable viruses which are undetectable via normal diagnostics or panvirus-primer PCRs. RESULTS: A novel torque teno mini virus (TTMV) was identified in two patients and the sequence of the full genomes were determined. The virus is significantly different from the known TTMVs (< 40% amino acid identity in ORF1), yet it contains conserved characteristics that are also present in other TTMVs. The virus is chronically present in both patients, and these patients both suffered from a pneumococcal pneumonia during follow up and had extremely low B-cells counts. CONCLUSION: We describe a novel TTMV which we tentatively named TTMV-13. Further research is needed to address the epidemiology and pathogenicity of this novel virus.

The A-nucleotide preference of HIV-1 in the context of its structured RNA genome.

A bipartition of HIV-1 RNA genome sequences into single- and double-stranded nucleotides is possible based on the secondary structure model of a complete 9 kb genome. Subsequent analysis revealed that the well-known lentiviral property of A-accumulation is profoundly present in single-stranded domains, yet absent in double-stranded domains. Mutational rate analysis by means of an unrestricted model of nucleotide substitution suggests the presence of an evolutionary equilibrium to preserve this biased nucleotide distribution.

Silent codon positions in the A-rich HIV RNA genome that do not easily become A: Restrictions imposed by the RNA sequence and structure.

There is a strong evolutionary tendency of the human immunodeficiency virus (HIV) to accumulate A nucleotides in its RNA genome, resulting in a mere 40 per cent A count. This A bias is especially dominant for the so-called silent codon positions where any nucleotide can be present without changing the encoded protein. However, particular silent codon positions in HIV RNA refrain from becoming A, which became apparent upon genome analysis of many virus isolates. We analyzed these 'noA' genome positions to reveal the underlying reason for their inability to facilitate the A nucleotide. We propose that local RNA structure requirements can explain the absence of A at these sites. Thus, noA sites may be prominently involved in the correct folding of the viral RNA. Turning things around, the presence of multiple clustered noA sites may reveal the presence of important sequence and/or structural elements in the HIV RNA genome.

Rooting human parechovirus evolution in time.

BACKGROUND: The Picornaviridae family contains a number of important pathogenic viruses, among which the recently reclassified human parechoviruses (HPeVs). These viruses are widespread and can be grouped in several types. Understanding the evolutionary history of HPeV could answer questions such as how long the circulating lineages last shared a common ancestor and how the evolution of this viral species is shaped by its population dynamics. Using both strict and relaxed clock Bayesian phylogenetics we investigated 1) the substitutions rates of the structural P1 and capsid VP1 regions and 2) evolutionary timescale of currently circulating HPeV lineages. RESULTS: Our estimates reveal that human parechoviruses exhibit high substitution rates for both structural P1 and capsid VP1 regions, respectively 2.21 x 10(-3) (0.48 - 4.21 x 10(-3)) and 2.79 x 10(-3) (2.05 - 3.66 x 10(-3)) substitutions per site per year. These are within the range estimated for other picornaviruses. By employing a constant population size coalescent prior, the date of the most recent common ancestor was estimated to be at around 1600 (1427-1733). In addition, by looking at the frequency of synonymous and non-synonymous substitutions within the VP1 gene we show that purifying selection constitutes the dominating evolutionary force leading to strong amino acid conservation. CONCLUSION: In conclusion, our estimates provide a timescale for the evolution of HPeVs and suggest that genetic diversity of current circulating HPeV types has arisen about 400 years ago.

Biodistribution and radiation dosimetry of 99mTc-HMPAO-labeled monocytes in patients with rheumatoid arthritis.

UNLABELLED: Rheumatoid arthritis (RA) involves the accumulation of monocyte-derived macrophages in the affected synovial tissue. This process of cell migration could be portrayed scintigraphically to monitor noninvasively the effects of therapy on the progress of the disease. For this purpose, labeling of purified autologous monocytes with 99mTc-hexamethylpropyleneamine oxime (99mTc-HMPAO) at very high specific radioactivity has recently been developed. The aim of this study was to assess the biodistribution and radiation dosimetry of 99mTc-HMPAO-labeled monocytes in adult patients with RA. METHODS: In 8 patients with RA, monocytes were isolated from 100 mL of blood and labeled with 99mTc-HMPAO to a yield of 10 Bq/cell. Multiple whole-body scans were performed up to 20 h after reinjection of an average of 200 MBq of 99mTc-HMPAO-labeled monocytes. Urine and blood samples were collected. The fraction of administered activity in 7 source organs was quantified from the attenuation-corrected geometric mean counts in conjugate views. Radiation-absorbed doses were estimated with OLINDA/EXM software. RESULTS: Autologous monocytes labeled with 99mTc-HMPAO at high intracellular yields showed in vivo kinetics comparable with labeled leukocytes, with initial trapping in the lungs followed by distribution into the liver, spleen, and bone marrow. The radiation-absorbed estimates for 99mTc-HMPAO-labeled monocytes were comparable with those for 99mTc-HMPAO-labeled mixed white blood cells, with an effective dose of 0.011 mSv/MBq. CONCLUSION: 99mTc-HMPAO-labeled monocytes have biodistribution and radiation dosimetry similar to those of 99mTc-HMPAO-labeled mixed white blood cells and might therefore be used for in vivo monitoring of immunomodulating therapy in patients with RA.

Occult hepatitis B infection: an evolutionary scenario.

BACKGROUND: Occult or latent hepatitis B virus (HBV) infection is defined as infection with detectable HBV DNA and undetectable surface antigen (HBsAg) in patients' blood. The cause of an overt HBV infection becoming an occult one is unknown. To gain insight into the mechanism of the development of occult infection, we compared the full-length HBV genome from a blood donor carrying an occult infection (d4) with global genotype D genomes. RESULTS: The phylogenetic analysis of polymerase, core and X protein sequences did not distinguish d4 from other genotype D strains. Yet, d4 surface protein formed the evolutionary outgroup relative to all other genotype D strains. Its evolutionary branch was the only one where accumulation of substitutions suggests positive selection (dN/dS = 1.3787). Many of these substitutions accumulated specifically in regions encoding the core/surface protein interface, as revealed in a 3D-modeled protein complex. We identified a novel RNA splicing event (deleting nucleotides 2986-202) that abolishes surface protein gene expression without affecting polymerase, core and X-protein related functions. Genotype D strains differ in their ability to perform this 2986-202 splicing. Strains prone to 2986-202 splicing constitute a separate clade in a phylogenetic tree of genotype D HBVs. A single substitution (G173T) that is associated with clade membership alters the local RNA secondary structure and is proposed to affect splicing efficiency at the 202 acceptor site. CONCLUSION: We propose an evolutionary scenario for occult HBV infection, in which 2986-202 splicing generates intracellular virus particles devoid of surface protein, which subsequently accumulates mutations due to relaxation of coding constraints. Such viruses are deficient of autonomous propagation and cannot leave the host cell until it is lysed.

Euclidean Distance Analysis Enables Nucleotide Skew Analysis in Viral Genomes.

Nucleotide skew analysis is a versatile method to study the nucleotide composition of RNA/DNA molecules, in particular to reveal characteristic sequence signatures. For instance, skew analysis of the nucleotide bias of several viral RNA genomes indicated that it is enriched in the unpaired, single-stranded genome regions, thus creating an even more striking virus-specific signature. The comparison of skew graphs for many virus isolates or families is difficult, time-consuming, and nonquantitative. Here, we present a procedure for a more simple identification of similarities and dissimilarities between nucleotide skew data of coronavirus, flavivirus, picornavirus, and HIV-1 RNA genomes. Window and step sizes were normalized to correct for differences in length of the viral genome. Cumulative skew data are converted into pairwise Euclidean distance matrices, which can be presented as neighbor-joining trees. We present skew value trees for the four virus families and show that closely related viruses are placed in small clusters. Importantly, the skew value trees are similar to the trees constructed by a "classical" model of evolutionary nucleotide substitution. Thus, we conclude that the simple calculation of Euclidean distances between nucleotide skew data allows an easy and quantitative comparison of characteristic sequence signatures of virus genomes. These results indicate that the Euclidean distance analysis of nucleotide skew data forms a nice addition to the virology toolbox.

Different rates of (non-)synonymous mutations in astrovirus genes; correlation with gene function.

BACKGROUND: Complete genome sequences of the Astroviridae include human, non-human mammalian and avian species. A consensus topology of astroviruses has been derived from nucleotide substitutions in the full-length genomes and from non-synonymous nucleotide substitutions in each of the three ORFs. Analyses of synonymous substitutions displayed a loss of tree structure, suggesting either saturation of the substitution model or a deviant pattern of synonymous substitutions in certain virus species. RESULTS: We analyzed the complete Astroviridae family for the inference of adaptive molecular evolution at sites and in branches. High rates of synonymous mutations are observed among the non-human virus species. Deviant patterns of synonymous substitutions are found in the capsid structural genes. Purifying selection is a dominant force among all astrovirus genes and only few codon sites showed values for the dN/dS ratio that may indicate site-specific molecular adaptation during virus evolution. One of these sites is the glycine residue of a RGD motif in ORF2 of human astrovirus serotype 1. RGD or similar integrin recognition motifs are present in nearly all astrovirus species. CONCLUSION: Phylogenetic analysis directed by maximum likelihood approximation allows the inclusion of significantly more evolutionary history and thereby, improves the estimation of dN and dS. Sites with enhanced values for dN/dS are prominent at domains in charge of environmental communication (f.i. VP27 and domain 4 in ORF1a) more than at domains dedicated to intrinsic virus functions (f.i. VP34 and ORF1b (the virus polymerase)). Integrin recognition may play a key role in astrovirus to target cell attachment.

Labeling of autologous monocytes with 99mTc-HMPAO at very high specific radioactivity.

Rheumatoid arthritis of joints involves the accumulation of monocyte-derived macrophages in the affected synovial tissue. This process of cell migration can be portrayed scintigraphically in order to monitor noninvasive effects of therapy on the progress of the disease. Scintigraphic detection of inflammation by means of technetium 99m-hexamethylpropylene amine oxime (99mTc-HMPAO)-labeled leukocytes provides a classic example. Present state-of-the-art methods in cell biology allow the isolation of cells like lymphocytes or monocytes, which are less abundant than main blood constituents but, instead, harbor particular functions like specific homing properties. To facilitate scintigraphic imaging of the cell functions involved, the relatively small population of cells must be labeled to radioactive yields as high as possible. We demonstrate that autologous monocytes isolated from 100 ml of peripheral blood can be radiolabeled to a yield of 10 (instead of 1) Bq per cell, allowing scintigraphic analysis of rheumatoid arthritis up to 20 h post injection of patients. The method is based on the instantaneous distribution of lipophilic 99mTc-HMPAO between the hydrophobic inside of cells and the hydrophilic (aqueous) surrounding of cells, followed by decomposition of the radiopharmaceutical into compounds that are unable to cross the cellular membrane. The procedure provides a method of choice for cell-mediated scintigraphy at low availability of cells with the correct homing properties.

Radiopharmaceutical management of 90Y/111In labeled antibodies: shielding and quantification during preparation and administration.

BACKGROUND: The combined application of potent beta-emitting isotopes for therapy with remitting isotopes for scintigraphy requires a profound regimen concerning team member safety and radionuclide quantification. METHODS: We have developed materials and methods for a proper and easy manipulation of 90Y during preparation and administration of 90Y/111In pharmaceuticals used for radioimmunotherapy. RESULTS: The efficacy of the shielding measures is documented. Protocols for the calibration of gamma-dose calibrators with respect to 90Y are extended to the assessment of quench-corrected liquid scintillation counting of 90Y. The contribution of 90Y backscatter to 111 In counting is quantified. Newly developed shielding equipment allows an adequate administration of relatively large volumes (100 ml) of 90Y/111In labeled pharmaceuticals to patients. CONCLUSIONS: The procedures described combine pharmaceutical (Good Manufacturing Practice) and radiation safety requirements with an accurate logging of relevant data.

A Novel Astrovirus-Like RNA Virus Detected in Human Stool.

Several novel clades of astroviruses have recently been identified in human faecal samples. Here, we describe a novel astrovirus-like RNA virus detected in human stools, which we have tentatively named bastrovirus. The genome of this novel virus consists of 6,300 nucleotides organized in three open reading frames. Several sequence divergent strains were detected sharing 67-93 per cent nucleotide identity. Bastrovirus encodes a putative structural protein that is homologous to the capsid protein found in members of the Astroviridae family (45% amino acid identity). The virus also encodes a putative non-structural protein that is genetically distant from astroviruses but shares some homology to the non-structural protein encoded by members of the Hepeviridae family (28% amino acid identity). This novel bastrovirus is present in 8.7 per cent (35/400) of faecal samples collected from 300 HIV-1-positive and 100 HIV-1-negative individuals suggesting common occurrence of the virus. However, whether the source of the virus is infected human cells or other, for example, dietary, remains to be determined.

Preparation, radiochemical purity control and stability of 99mTc-mertiatide (Mag-3).

BACKGROUND: Scintigraphic image analysis of 99mTc-mertiatide (Mag-3, mercaptoacetyltriglycine) clearance provides the determination of the blood flow, the tubular transit time and the excretion as well from both kidneys. Radiopharmaceutical routine recommends a radiochemical purity control before administration of the product to a patient. The main objective of this study is to develop a Mag-3 labeling procedure that fits better than the previous one in our daily routine production of radiopharmaceuticals. METHODS: Increasing proportions of 99mTc-Mag-3 were measured during the heating and cooling steps of the Mag-3 labeling procedure. HPLC analysis was used to confirm the results of a rapid radiochemical quality control assay on standard ITLC-SG paper. RESULTS: The reconstitution time takes 20-25 minutes from the harvest of pertechnetate to a ready-for-use calibrated patient syringe. The HPLC profile of 99mTc-Mag-3 including its minor impurities remains unchanged for 24-48 hours after reconstitution. CONCLUSIONS: The application of a programmable Peltier-directed device for heating/cooling provides a better control of the temperature course. The procedure proposed fully meets the labeling criteria recommended by the supplier and can be performed with a minimum of attention within a time-span that we formerly needed for solely the radiochemical purity control assay. Moreover, 99mTc-Mag-3 prepared in this way seems to be considerably more stable than mentioned in the manufacturer's instructions.

On the biased nucleotide composition of the human coronavirus RNA genome.

We investigated the nucleotide composition of the RNA genome of the six human coronaviruses. Some general coronavirus characteristics were apparent (e.g. high U, low C count), but we also detected species-specific signatures. Most strikingly, the high U and low C proportions are quite variable and act like communicating vessels, C goes down when U goes up and vice versa. U ranges among virus isolates from 30.7% to 40.3%, and C makes the opposite movement from 20.0% to 12.9%, respectively. The nucleotide biases are more pronounced in the unpaired regions of the structured RNA genome, which may suggest a certain biological function for these distinctive sequence signatures. Coronaviruses have an atypical codon usage that has been linked to mutational events operating on the viral RNA genome on an evolutionary time scale. We suggest that the atypical nucleotide bias may serve a distinct biological function and that it is the direct cause of the characteristic codon usage in these viruses. The relevance for evolution of the novel human pathogens MERS and SARS is discussed.

In silico prediction of ebolavirus RNA polymerase inhibition by specific combinations of approved nucleotide analogues.

BACKGROUND AND OBJECTIVE: The urgency of ebolavirus drug development is obvious in light of the current local epidemic in Western Africa with high morbidity and a risk of wider spread. We present an in silico study as a first step to identify inhibitors of ebolavirus polymerase activity based on approved antiviral nucleotide analogues. STUDY DESIGN: Since a structure model of the ebolavirus polymerase is lacking, we performed combined homology and ab initio modeling and report a similarity to known polymerases of human enterovirus, bovine diarrhea virus and foot-and-mouth disease virus. This facilitated the localization of a nucleotide binding domain in the ebolavirus polymerase. We next performed molecular docking studies with nucleotides (ATP, CTP, GTP and UTP) and nucleotide analogues, including a variety of approved antiviral drugs. RESULTS AND CONCLUSIONS: Specific combinations of nucleotide analogues significantly reduce the ligand-protein interaction energies of the ebolavirus polymerase for natural nucleotides. Any nucleotide analogue on its own did not reduce ligand-protein interaction energies. This prediction encourages specific drug testing efforts and guides future strategies to inhibit ebolavirus replication.

Specific mutations in the C-terminus domain of HBV surface antigen significantly correlate with low level of serum HBV-DNA in patients with chronic HBV infection.

BACKGROUND: To define HBsAg-mutations correlated with different serum HBV-DNA levels in HBV chronically-infected drug-naive patients. METHODS: This study included 187 patients stratified into the following ranges of serum HBV-DNA:12-2000 IU/ml, 2000-100,000 IU/ml, and >100,000 IU/ml. HBsAg-mutations were associated with HBV-DNA levels by applying a Bayesian-Partitional-Model and Fisher-exact test. Mutant and wild-type HBV genotype-D genomes were expressed in Huh7 cells and HBsAg-production was determined in cell-supernatants at 3 days-post-transfection. RESULTS: Specific HBsAg-mutations (M197T,-S204N-Y206C/H-F220L) were significantly correlated with serum HBV-DNA <2000 IU/ml (posterior-probability>90%, P < 0.05). The presence of Y206C/H and/or F220L was also associated with lower median (IQR) HBsAg-levels and lower median (IQR) transaminases (for HBsAg:250[115-840] IU/ml for Y206C/H and/or F220L versus 4300[640-11,838] IU/ml for wild-type, P = 0.023; for ALT:28[21-40] IU/ml versus 53[34-90] IU/ml, P < 0.001). These mutations were localized in the HBsAg C-terminus, known to be involved in virion and/or HBsAg secretion. The co-occurrence of Y206C + F220L was found significant by cluster-analysis, (P = 0.02). In addition, in an in-vitro model Y206C + F220L determined a 2.8-3.3 fold-reduction of HBsAg-amount released in supernatants compared to single mutants and wt (Y206C + F220L = 5,679 IU/ml; Y206H = 16,305 IU/ml; F220L = 18,368 IU/ml; Y206C = 18,680 IU/ml; wt = 14,280 IU/ml, P < 0.05). CONCLUSIONS: Specific HBsAg-mutations (compartmentalized in the HBsAg C-terminus) correlated with low-serum HBV-DNA and HBsAg-levels. These findings can be important to understand mechanisms underlying low HBV replicative potential including the inactive-carrier state.

Lymphatic drainage routes of the gastric cardia visualized by lymphoscintigraphy.

UNLABELLED: This study was undertaken to assess the feasibility of lymphoscintigraphy of the gastric cardia and to identify the incidence of paraesophageal lymphatic drainage, precluding total gastrectomy with esophagojejunostomy as a potentially curative therapy for gastric cardia cancer. METHODS: Ten patients scheduled for esophagectomy with high-grade dysplasia or with esophageal cancer at least 3 cm above the esophagogastric junction were enrolled in this study. Preoperatively, 111 MBq of(99m)Tc-labeled nanocolloid (n = 5) or sulfur colloid (n = 5) were injected into the submucosa of the tumor-free cardia. Subsequently, lymphoscintigraphy in combination with CT was obtained. Locoregional lymph node stations were measured for radioactivity by a gamma-probe intraoperatively and ex vivo in the resection specimen. RESULTS: In each patient, at least 1 radioactive lymph node station was detected. In total, 42 radioactive lymph node stations were detected by gamma-probe. Of those 42 areas, 38 (90%) were visible at preoperative lymphoscintigraphy. In the group of 5 patients in whom nanocolloid was used, a median of 2 (range, 1-4) node stations per patient was identified, whereas when sulfur colloid was administered a median of 6 (range, 4-8) active lymph node stations per patient could be detected (P < 0.002). Paraesophageal drainage was identified in 1 patient. CONCLUSION: Lymphoscintigraphy of the gastric cardia is feasible and can accurately determine the location of radioactive lymph nodes. Early paraesophageal lymphatic drainage is rare.