Exploring the mechanisms of host-specificity of a hyperparasitic bacterium (Pasteuria spp.) with potential to control tropical root-knot nematodes (Meloidogyne spp.): insights from Caenorhabditis elegans.

Plant-parasitic nematodes are important economic pests of a range of tropical crops. Strategies for managing these pests have relied on a range of approaches, including crop rotation, the utilization of genetic resistance, cultural techniques, and since the 1950's the use of nematicides. Although nematicides have been hugely successful in controlling nematodes, their toxicity to humans, domestic animals, beneficial organisms, and the environment has raised concerns regarding their use. Alternatives are therefore being sought. The Pasteuria group of bacteria that form endospores has generated much interest among companies wanting to develop microbial biocontrol products. A major challenge in developing these bacteria as biocontrol agents is their host-specificity; one population of the bacterium can attach to and infect one population of plant-parasitic nematode but not another of the same species. Here we will review the mechanism by which infection is initiated with the adhesion of endospores to the nematode cuticle. To understand the genetics of the molecular processes between Pasteuria endospores and the nematode cuticle, the review focuses on the nature of the bacterial adhesins and how they interact with the nematode cuticle receptors by exploiting new insights gained from studies of bacterial infections of Carnorhabditis elegans. A new Velcro-like multiple adhesin model is proposed in which the cuticle surface coat, which has an important role in endospore adhesion, is a complex extracellular matrix containing glycans originating in seam cells. The genes associated with these seam cells appear to have a dual role by retaining some characteristics of stem cells.

Hexapeptides from mammalian inhibitory hormone hunt activate and inactivate nematode reproduction.

BACKGROUND: Biopurification has been used to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. A (rat) bioassay-guided physicochemical fractionation using ovine materials yielded via Edman degradation a 14-residue amino acid (aa) sequence. As a 14mer synthetic peptide (EPL001) this displayed antiproliferative and reproduction-modulating activity, while representing only a part of the native polypeptide. Even more unexpectedly, a scrambled-sequence control peptide (EPL030) did likewise. METHODS: Reproduction has been investigated in the nematode Steinernema siamkayai, using a fermentation system supplemented with different concentrations of exogenous hexapeptides. Peptide structure-activity relationships have also been studied using prostate cancer and other mammalian cells in vitro, with peptides in solution or immobilized, and via the use of mammalian assays in vivo and through molecular modelling. RESULTS: Reproduction increased (x3) in the entomopathogenic nematode Steinernema siamkayai after exposure to one synthetic peptide (IEPVFT), while fecundity was reduced (x0.5) after exposure to another (KLKMNG), both effects being dose-dependent. These hexamers are opposite ends of the synthetic peptide KLKMNGKNIEPVFT (EPL030). Bioactivity is unexpected as EPL030 is a control compound, based on a scrambled sequence of the test peptide MKPLTGKVKEFNNI (EPL001). EPL030 and EPL001 are both bioinformatically obscure, having no convincing matches to aa sequences in the protein databases. EPL001 has antiproliferative effects on human prostate cancer cells and rat bone marrow cells in vitro. Intracerebroventricular infusion of EPL001 in sheep was associated with elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless have the foregoing biological effects in common in mammalian systems, while being divergently pro- and anti-fecundity respectively in the nematode Caenorhabditis elegans. Peptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro, with reproductive upregulation demonstrated previously in fish and frogs, as well as nematodes. EPL001 encodes the sheep neuroendocrine prohormone secretogranin II (sSgII), as deduced on the basis of immunoprecipitation using an anti-EPL001 antibody, with bespoke bioinformatics. Six sSgII residues are key to EPL001's bioactivity: MKPLTGKVKEFNNI. A stereospecific bimodular tri-residue signature is described involving simultaneous accessibility for binding of the side chains of two specific trios of amino acids, MKP & VFN. An evolutionarily conserved receptor is conceptualised having dimeric binding sites, each with ligand-matching bimodular stereocentres. The bioactivity of the 14mer control peptide EPL030 and its hexapeptide progeny is due to the fortuitous assembly of subsets of the novel hormonal motif, MKPVFN, a default reproductive and tissue-building OFF signal.

Deciphering the mechanism of anhydrobiosis in the entomopathogenic nematode Heterorhabditis indica through comparative transcriptomics.

The entomopathogenic nematode, Heterorhabditis indica, is a popular biocontrol agent of high commercial significance. It possesses tremendous genetic architecture to survive desiccation stress by undergoing anhydrobiosis to increase its lifespan-an attribute exploited in the formulation technology. The comparative transcriptome of unstressed and anhydrobiotic H. indica revealed several previously concealed metabolic events crucial for adapting towards the moisture stress. During the induction of anhydrobiosis in the infective juveniles (IJ), 1584 transcripts were upregulated and 340 downregulated. As a strategy towards anhydrobiotic survival, the IJ showed activation of several genes critical to antioxidant defense, detoxification pathways, signal transduction, unfolded protein response and molecular chaperones and ubiquitin-proteasome system. Differential expression of several genes involved in gluconeogenesis - ß-oxidation of fatty acids, glyoxylate pathway; glyceroneogenesis; fatty acid biosynthesis; amino-acid metabolism - shikimate pathway, sachharopine pathway, kyneurine pathway, lysine biosynthesis; one-carbon metabolism-polyamine pathway, transsulfuration pathway, folate cycle, methionine cycle, nucleotide biosynthesis; mevalonate pathway; and glyceraldehyde-3-phosphate dehydrogenase were also observed. We report the role of shikimate pathway, sachharopine pathway and glyceroneogenesis in anhydrobiotes, and seven classes of repeat proteins, specifically in H. indica for the first time. These results provide insights into anhydrobiotic survival strategies which can be utilized to strengthen the development of novel formulations with enhanced and sustained shelf-life.

FOLFIRINOX as First-line Chemotherapy in Japanese Patients Suffering from Metastatic Pancreatic Cancer (KOBE FOLFIRINOX Study).

Background/Aim: FOLFIRINOX (oxaliplatin, irinotecan, 5-fluorouracil, and leucovorin) combination chemotherapy is the gold-standard therapy for advanced pancreatic cancer. In this study, FOLFIRINOX dosages for Japanese patients were established enabling FOLFIRINOX therapy optimization for efficient use. Patients and Methods: Patients with advanced pancreatic cancer were treated with varying doses of FOLFIRINOX to determine the optimum dosage for highest remission outcomes with the least post-chemotherapy toxicities. Results: Patients given 180 mg of irinotecan and a 400 mg bolus of 5-fluorouracil (5-FU) showed a marked difference in outcome when compared to irinotecan 180 mg given without the 5-FU bolus, with the overall response rate being 28%, a survival time of 6.4 months and progression-free survival time of 4.5 months. Conclusion: The optimum dose of FOLFIRINOX was a dosage combination of oxaliplatin 85 mg/m 2 , irinotecan 180 mg/m 2 , l-leucovorin 400 mg/m 2  and 5-FU 2,400 mg/m 2 , administered as a continuous 46-h infusion.

Exploring Bacillus thuringiensis as a model for endospore adhesion and its potential to investigate adhesins in Pasteuria penetrans.

AIMS: Phytonematodes are a constraint on crop production and have been controlled using nematicides; these are highly toxic and legislation in Europe and elsewhere is prohibiting their use and alternatives are being sought. Pasteuria penetrans is a hyperparasitic bacterium that form endospores and have potential to control root-knot nematodes (Meloidogyne spp.), but their attachment to the nematode cuticle is host-specific. Understanding host specificity has relied upon endospore inhibition bioassays using immunological and biochemical approaches. Phylogenetic analysis of survey sequences has shown P. penetrans to be closely related to Bacillus and to have a diverse range of collagen-like fibres which we hypothesise to be involved in the endospore adhesion. However, due to the obligately hyperparasitic nature of Pasteuria species, identifying and characterizing these collagenous-like proteins through gain of function has proved difficult and new approaches are required. METHODS AND RESULTS: Using antibodies raised to synthetic peptides based on Pasteuria collagen-like genes we show similarities between P. penetrans and the more easily cultured bacterium Bacillus thuringiensis and suggest it be used as a gain of function platform/model. Using immunological approaches similar proteins between P. penetrans and B. thuringiensis are identified and characterized, one >250 kDa and another ~72 kDa are glycosylated with N-acetylglucosamine and both of which are digested if treated with collagenase. These treatments also affected endospore attachment and suggest these proteins are involved in adhesion of endospores to nematode cuticle. CONCLUSION: There are conserved similarities in the collagen-like proteins present on the surface of endospores of both P. penetrans and B. thuringiensis. SIGNIFICANCE AND IMPACT OF STUDY: As B. thuringiensis is relatively easy to culture and can be transformed, it could be developed as a platform for studying the role of the collagen-like adhesins from Pasteuria in endospore adhesion.

Bacillus subtilis and B. licheniformis Isolated from Heterorhabditis indica Infected Apple Root Borer (Dorysthenes huegelii) Suppresses Nematode Production in Galleria mellonella.

PURPOSE: Heterorhabdits indica successfully controlled apple root borer Dorysthenes huegelii in the orchards, but nematode-infected cadavers revealed the presence of non-symbiotic bacterial B. subtilis and B. licheniformis, and no subsequent generations of H. indica were produced (hampered recycling phenomenon). Intrigued, we tested the effect of the two Bacillus species on symbiotic association of H. indica-Photorhabdus luminescens. METHODS: One-to-one competitive parallel line in vitro assays were carried out between P. luminescens and the two Bacillus spp., while in vivo H. indica development was studied on the test insect Galleria mellonella which were fed with Bacillus mixed diet, followed by nematode exposure. RESULTS: Where P. luminescens was flanked by either of the two Bacillus species, only B. subtilis significantly suppressed its growth, while in reversed assays both the Bacillus growth was unaffected. Heterorhabditis indica was able to kill Galleria larvae pre-fed with the two Bacillus spp.; these cadavers did not develop the characteristic evenly distributed brick red coloration. Besides P. luminesecns, both Bacillus spp. were found to coexist in these cadavers. Development of hermaphrodites was not affected, but second-generation females, and final nematode progeny was reduced significantly. Monozenic lawns of B. subtilis and B. licheniformis did not support H. indica development. CONCLUSION: These results show the reduced development of H. indica by the presence of the non-symbiotic bacteria in G. mellonella is likely to affect their ability to recycle in other insect larvae. Reduced recycling caused by non-symbiotic bacteria will reduce the overall long-term pest control benefits and have implications in the development of application strategies using entomopathogenic nematodes (EPNs) as insect control agents.

Plant Root-Exudates Recruit Hyperparasitic Bacteria of Phytonematodes by Altered Cuticle Aging: Implications for Biological Control Strategies.

Phytonematodes are globally important functional components of the belowground ecology in both natural and agricultural soils; they are a diverse group of which some species are economically important pests, and environmentally benign control strategies are being sought to control them. Using eco-evolutionary theory, we test the hypothesis that root-exudates of host plants will increase the ability of a hyperparasitic bacteria, Pasteuria penetrans and other closely related bacteria, to infect their homologous pest nematodes, whereas non-host root exudates will not. Plant root-exudates from good hosts, poor hosts and non-hosts were characterized by gas chromatography-mass spectrometry (GC/MS) and we explore their interaction on the attachment of the hyperparasitic bacterial endospores to homologous and heterologous pest nematode cuticles. Although GC/MS did not identify any individual compounds as responsible for changes in cuticle susceptibility to endospore adhesion, standardized spore binding assays showed that Pasteuria endospore adhesion decreased with nematode age, and that infective juveniles pre-treated with homologous host root-exudates reduced the aging process and increased attachment of endospores to the nematode cuticle, whereas non-host root-exudates did not. We develop a working model in which plant root exudates manipulate the nematode cuticle aging process, and thereby, through increased bacterial endospore attachment, increase bacterial infection of pest nematodes. This we suggest would lead to a reduction of plant-parasitic nematode burden on the roots and increases plant fitness. Therefore, by the judicious manipulation of environmental factors produced by the plant root and by careful crop rotation this knowledge can help in the development of environmentally benign control strategies.

Evidence for diversifying selection of genetic regions of encoding putative collagen-like host-adhesive fibers in Pasteuria penetrans.

Pasteuria spp. belong to a group of genetically diverse endospore-forming bacteria (phylum: Firmicutes) that are known to parasitize plant-parasitic nematodes and water fleas (Daphnia spp.). Collagen-like fibres form the nap on the surface of endospores and the genes encoding these sequences have been hypothesised to be involved in the adhesion of the endospores of Pasteuria spp. to their hosts. We report a group of 17 unique collagen-like genes putatively encoded by Pasteuria penetrans (strain: Res148) that formed five different phylogenetic clusters and suggest that collagen-like proteins are an important source of genetic diversity in animal pathogenic Firmicutes including Pasteuria. Additionally, and unexpectedly, we identified a putative collagen-like sequence which had a very different sequence structure to the other collagen-like proteins but was similar to the protein sequences in Megaviruses that are involved in host-parasite interactions. We, therefore, suggest that these diverse endospore surface proteins in Pasteuria are involved in biological functions, such as cellular adhesion; however, they are not of monophyletic origin and were possibly obtained de novo by mutation or possibly through selection acting upon several historic horizontal gene transfer events.

Impact of (18)f-fluorodeoxyglucose positron emission tomography/computed tomography scan on initial evaluation of head and neck squamous cell carcinoma: our experience at a tertiary care center in India.

The efficacy of the whole body (WB) (18)F-fluorodeoxyglucose ((18)F-FDG) positron emission tomography-computed tomography (PET-CT) as a part of conventional initial staging in all cases of head and neck squamous cell carcinoma (HNSCC) is still controversial with various studies in literature giving contradictory reports. We conducted this study at a government tertiary care oncology center in India to identify the impact of WB (18)F-FDG PET-CT scan on HNSCC staging and treatment. A prospective clinical study of patients of HNSCC who were evaluated and treated at our center was performed. The patients included in the study were HNSCC of the oral cavity, oropharynx, hypopharynx, larynx, nasopharynx, and carcinoma of unknown primary site (CUPS) with cervical metastasis. The study design was to evaluate the cases of HNSCC initially by staging with conventional investigations followed by staging with the information derived from WB (18)F-FDG PET-CT scan. At the end of the conventional investigations, a tumor, node, metastasis (TNM) staging as per AJCC 7(th) edition, and a detailed treatment plan as per NCCN 2012 guidelines was decided in consultation with the multidisciplinary oncology team of the hospital. WB (18)F-FDG PET-CT scan was carried out in all these patients. The findings of WB (18)F-FDG PET-CT were then interpreted with the staging with conventional investigations to identify the cases with change in staging and also those in whom the treatment protocol would be affected. Descriptive analysis of demographic data and analytical analysis of the sensitivity and specificity of WB (18)F-FDG PET-CT scan and also the change in staging and treatment plan after WB (18)F-FDG PET-CT scan was analyzed using SPSS version 18. A total of 131 patients met the inclusion criteria, which included 123 males and 8 females. The various sites involved among the study group are oral cavity 11 (8.3%), oropharyn × 39 (29.7%), hypopharyn × 31 (23.6%), laryn × 34 (25.9%), nasopharyn × 4 (3%), and CUPS 12 (9.1%). The majority of cases studied were of T2 and T3 stage, and changes in T staging after WB (18)F-FDG PET-CT scan were minimal and not statistically significant (P > 0.5). In the nodal staging after WB (18)F-FDG PET-CT scan, there was a statistically significant change in identification of nodal metastasis in N0 group and also identification of additional multiple/bilateral nodes (N2b and N2c). 3 (2.2%) patients had a change in M status with identification of distant metastasis in lungs (2 patients) and in the liver and lung (1 patient). Of the 131 patients, 75 (57.25%) underwent surgical management with or without adjuvant treatment (Group I) and 56 (42.74%) patients underwent nonsurgical management (Group II). There was no significant statistical difference in sensitivity and specificity of (18)F-FDG PET-CT scan in detecting cancer among the two groups. Considering all the patients in this study, WB (18)F-FDG PET-CT scan showed an overall sensitivity of 95.2% and specificity of 80%. In this study, change in TNM staging after WB (18)F-FDG PET-CT was seen in 22 (16.8%) patients and an alteration in the treatment in 21 (16.1%) patients, which were both found to be statistically significant (P < 0.5). In our study, WB (18)F-FDG PET-CT scan has shown to have an impact on initial staging of disease affecting the change in treatment protocol in a significant number of patients. The effect of this change in staging and treatment on the eventual morbidity and mortality rates is not known. In practice, the use of (18)F-FDG PET-CT scan is limited, owing to the high cost and low availability. A realistic evaluation of cost versus benefit needs to be undertaken to identify the impact of using (18)F-FDG PET-CT scan as a mode for initial evaluation of HNSCC.

Tracheal stenosis: our experience at a tertiary care centre in India with special regard to cause and management.

Tracheal stenosis (TS), a challenging problem, is a known complication of prolonged intubation and tracheostomy. The management involves a multidisciplinary approach with multiple complex procedures. In this study we discuss our experience with severe TS with regards to patient characteristics, cause and management. A retrospective analysis of 20 patients of severe TS treated at a tertiary care centre was evaluated. Inclusion criteria were all patients with severe TS who required surgical intervention. Exclusion criteria were patients with associated laryngeal stenosis and TS due to cancer. Demographic data was recorded and findings relating to aetiology, characteristics of stenosis and the various aspects of therapeutic procedures performed are discussed with review of literature. Descriptive analysis of data were performed SPSS 18. Results of the 20 patients, 17 patients (85 %) developed TS post tracheostomy, or post intubation and subsequent tracheostomy. 13 Patients (65 %) had true stenosis of which 7 patients (35 %) had simple web or circumferential fibrosis and 6 patients (30 %) had complex stenosis. Seven patients (35 %) had granulations causing severe TS which were mostly suprastomal (5 patients), stomal (5 patients) and combined stomal and suprastomal (3 patients). The average length of stenosis was 3.57 cm (0.5-8 cm). Montgomery t tube insertion was a common procedure in 18 patients (90 %) pre or post intervention. Each patient underwent an average of 3.4 procedures during their course of treatment which included rigid bronchoscopy and mechanical debulking, Nd YAG laser, KTP laser, balloon dilatation and use of stents. Among the 7 patients with granulations 100 % successful decanulation was noted with endoscopic management whereas in 13 patients with true stenosis, 10 patients (76.9 %) required open surgical management (8 tracheal resection and anastomosis and 2 tracheoplasty) with 80 % successful decanulation, 2 patients (15.4 %) were treated with endoscopy with 100 % successful decanulation and 1 patient (7.7 %) was a non surgical candidate on stent. Of the total 20 patients with severe TS in this series, 17 (85 %) of patients who were decanulated, asymptomatic on routine daily activities with normal FFB were considered cured. TS is a challenging condition requiring a highly skilled multidisciplinary team for adequate management. Prolonged intubation and tracheostomy are the common causes leading to tracheal stenosis. Simple tracheal stenosis is easier to manage than a complex stenosis which usually requires an open surgical procedure for successful management. Presence of conditions like tracheoesophageal fistula and long segment tracheomalacia are poor factors for successful management. In our cases successful decanulation was possible in 85 % of the patients following a systematic multidisciplinary approach.

Pasteuria endospores from Heterodera cajani (Nematoda: Heteroderidae) exhibit inverted attachment and altered germination in cross-infection studies with Globodera pallida (Nematoda: Heteroderidae).

The Pasteuria group of Gram-positive, endospore-forming bacteria are parasites of invertebrates and exhibit differences in host specificity. We describe a cross-infection study between an isolate of Pasteuria from pigeon pea cyst nematode, Heterodera cajani, which also infects the potato cyst nematode, Globodera pallida, from the United Kingdom. A proportion of the attached endospores, 13% on H. cajani and 22% on G. pallida adhere to the cuticle in an inverted orientation. Inverted and conventionally attached endospores germinated and produced bacillus-like rods that completed their life cycle in < 15 weeks within females of G. pallida. This is the first example in which the life cycle of a Pasteuria population was systematically followed in two different nematode genera. A 1430-base pair fragment of the 16S rRNA gene sequence of the Pasteuria isolate from H. cajani revealed 98.6% similarity to the orthologous gene in Pasteuria nishizawae. Additionally, their respective endospore sizes were not significantly different, in contrast their host ranges are. Potential reasons for this remain unclear and are discussed.

Identification of new single nucleotide polymorphism-based markers for inter- and intraspecies discrimination of obligate bacterial parasites (Pasteuria spp.) of invertebrates.

Protein-encoding and 16S rRNA genes of Pasteuria penetrans populations from a wide range of geographic locations were examined. Most interpopulation single nucleotide polymorphisms (SNPs) were detected in the 16S rRNA gene. However, in order to fully resolve all populations, these were supplemented with SNPs from protein-encoding genes in a multilocus SNP typing approach. Examination of individual 16S rRNA gene sequences revealed the occurrence of "cryptic" SNPs which were not present in the consensus sequences of any P. penetrans population. Additionally, hierarchical cluster analysis separated P. penetrans 16S rRNA gene clones into four groups, and one of which contained sequences from the most highly passaged population, demonstrating that it is possible to manipulate the population structure of this fastidious bacterium. The other groups were made from representatives of the other populations in various proportions. Comparison of sequences among three Pasteuria species, namely, P. penetrans, P. hartismeri, and P. ramosa, showed that the protein-encoding genes provided greater discrimination than the 16S rRNA gene. From these findings, we have developed a toolbox for the discrimination of Pasteuria at both the inter- and intraspecies levels. We also provide a model to monitor genetic variation in other obligate hyperparasites and difficult-to-culture microorganisms.

Direct infection of Spodoptera litura by Photorhabdus luminescens encapsulated in alginate beads.

Actively growing cultures of Photorhabdus luminescens were encapsulated in sodium alginate beads and examined for their ability to infect insect hosts. These beads, containing approximately 2.5 x 10(7)Photorhabdus cells per bead, when mixed with sterilized soil and exposed to Spodoptera litura larvae resulted in 100% mortality in 48 h, while the use of alginate encapsulated Heterorhabditis nematode resulted in 40% mortality after 72 h. The bacteria were reisolated from the dead insect thus proving Koch's postulates and demonstrating the ability of P. luminescens to kill the insect host on their own, independent of the symbiont nematode. The LC(50) dose of Photorhabdus cells was estimated at 1010 cells per larva for killing S. litura 6th instar larvae in 48 h.

Medium pH-dependent redistribution of the urease of Helicobacter pylori.

Helicobacter pylori is an aetiological agent of gastric disease. Although the role of urease in gastric colonization of H. pylori has been shown, it remains unclear as to where urease is located in this bacterial cell. The purpose of this study was to define the urease-associated apparatus in the H. pylori cytoplasm. H. pylori was incubated at both a neutral and an acidic pH in the presence or absence of urea and examined by double indirect immunoelectron microscopy. The density of gold particles for UreA was greatest in the inner portion of the wild-type H. pylori cytoplasm at neutral pH but was greatest in the outer portion at acidic pH. This difference was independent of the presence of urea and was not observed in the ureI-deletion mutant. Also, the eccentric shift of urease in acidic pH was not observed in UreI. After a 2 day incubation period at acidic pH, it was observed that the urease gold particles in H. pylori assembled and were associated with UreI gold particles. Urease immunoreactivity shifted from the inner to the outer portion of H. pylori as a result of an extracellular decrease in pH. This shift was urea-independent and UreI-dependent, suggesting an additional role of UreI in urease-dependent acid resistance. This is the first report of the intracellular transport of molecules in bacteria in response to changes in the extracellular environment.

A highly sensitive chemiluminescent reverse transcriptase assay for human immunodeficiency virus.

A simple and highly sensitive reverse transcriptase (RT) assay was developed by combining a previously reported non-radioisotopic RT assay with the use of a template-primer-immobilized microplate, an enzyme capture protocol, product digestion and a chemiluminescent substrate. The assay was able to detect directly the RT activity in serum samples, plasma and cell culture medium without the need for concentration and extraction of the enzyme. The assay was able to detect RT activity equivalent to 100 virions/ml of HIV-1. These results suggest that this highly sensitive chemiluminescent RT assay can be used not only for virological investigation but also for routine screening of biopharmaceuticals.

A new improved method for the concentration of HIV-1 infective particles.

Improvement of the sensitivity of detection systems for human immunodeficiency virus-1 (HIV-1) has been carried out. One approach to improve the sensitivity is purification and/or concentration of the virus from a specimen. In this study, a method for concentrating HIV-1 using polyethylene glycol (PEG) has been re-evaluated and the optimal protocol for concentrating the virus from low-titer specimens was determined. That is, to obtain a virus pellet, a mixture of equal volumes of a specimen and 20% PEG 20,000 solution in saline is incubated at 4 degrees C for 16 h and then centrifuged at 17860 x g in a microcentrifuge for 20 min. HIV-1 in the pellet could be detectable by HIV-1 p24 antigen capture assay for viral protein, reverse transcriptase (RT) assay for viral enzyme, reverse transcriptase polymerase chain reaction (RT-PCR) assay for viral RNA and a virus infectivity assay.