Sequelae of osteomyelitis variolosa: a case report.

Osteomyelitis variolosa is an osteoarticular complication of small pox infection. We are presenting a case of osteomyelitis variolosa, who presented to us with fracture of ankle on the involved side with other orthopedic sequelae of small pox infection, which he contracted during his childhood period. We have discussed the distinguishing clinical and radiological features of osteomyilitis variolosa.

Stem cell transplantation: brief review and current status.

In recent years stem cell has come up as a great advance in therapy for a number of illnesses and has potential for revolutionising the medical field. Right from myelodysplastic syndrome to amyloidosis it has been tried. The present review is a modest endeavour to acquaintain in brief about current status of stem cells.

Clinical evaluation of selected Yogic procedures in individuals with low back pain.

The present study has been conducted to evaluate selected yogic procedures on individuals with low back pain. The understanding of back pain as one of the commonest clinical presentations during clinical practice made the path to the present study. It has also been calculated that more than three-quarters of the world's population experience back pain at some time in their lives. Twelve patients were selected and randomly divided into two groups, viz., group A yogic group and group B control group. Advice for life style and diet was given for all the patients. The effect of the therapy was assessed subjectively and objectively. Particular scores drawn for yogic group and control group were individually analyzed before and after treatment and the values were compared using standard statistical protocols. Yogic intervention revealed 79% relief in both subjective and objective parameters (i.e., 7 out of 14 parameters showed statistically highly significant P < 0.01 results, while 4 showed significant results P < 0.05). Comparative effect of yogic group and control group showed 79% relief in both subjective and objective parameters. (i.e., total 6 out of 14 parameters showed statistically highly significant (P < 0.01) results, while 5 showed significant results (P < 0.05).

Puberphonia: A novel approach to treatment.

Puberphonia affects the adolescent males. Authors have used a novel approach by using a Macintosh laryngoscope for stretching of vocal cords, which gives immediate and permanent relief. This procedure, which was incidentally found to be useful, while doing a laryngoscopic examination of a puberphonic patient, was effective and superior to any method used in the past 26 cases treated during 1991 to 2005 had been followed with the excellent results.

DNA melting analysis for detection of single nucleotide polymorphisms.

BACKGROUND: Several methods for detection of single nucleotide polymorphisms (SNPs; e.g., denaturing gradient gel electrophoresis and denaturing HPLC) are indirectly based on the principle of differential melting of heteroduplex DNA. We present a method for detecting SNPs that is directly based on this principle. METHODS: We used a double-stranded DNA-specific fluorescent dye, SYBR Green I (SYBR) in an efficient system (PE 7700 Sequence Detector) in which DNA melting was controlled and monitored in a 96-well plate format. We measured the decrease in fluorescence intensity that accompanied DNA duplex denaturation, evaluating the effects of fragment length, dye concentration, DNA concentration, and sequence context using four naturally occurring polymorphisms (three SNPs and a single-base deletion/insertion). RESULTS: DNA melting analysis (DM) was used successfully for variant detection, and we also discovered two previously unknown SNPs by this approach. Concentrations of DNA amplicons were readily monitored by SYBR fluorescence, and DNA amplicon concentrations were highly reproducible, with a CV of 2.6%. We readily detected differences in the melting temperature between homoduplex and heteroduplex fragments 15-167 bp in length and differing by only a single nucleotide substitution. CONCLUSIONS: The efficiency and sensitivity of DMA make it highly suitable for the large-scale detection of sequence variants.

Human peripheral blood mononuclear cell substrate for propagating wild type HIV-1.

Molecularly derived HIV proteins fail to induce broadly neutralizing antibodies (NAB) and cytotoxic T cell (CTL) responses, which are considered necessary for any effective vaccination. Natural virion proteins of the primary isolates of HIV-1 (pi-HIV), grown in human peripheral blood mononuclear cells (PBMC) followed by viral inactivation and pooling to reflect the antigenic diversity of the prevalent strains in a given population, would provide a novel polyvalent HIV vaccine (HIVAX) capable of inducing both HIV-NAB and CTL. Proven technologies are harnessed to provide a framework for advancing human blood-derived HIVAX. Fresh leukocytes, recovered by gravity flow elution from leukocyte depletion blood filters, or "buffy coats", routinely removed in preparing blood components for transfusion, provide an abundant and safe cell substrate following ficoll-separation of viable PBMC. The low content of wild-type HIV (wt-HIV) in infected plasma can be chaperoned by dendritic cells through their DC-SIGN receptor for gp120 and efficiently expanded in co-culture with CD4-enriched cell substrate in a medium containing human AB serum as a substitute for foetal calf serum. Dimethyl methylene blue (DMMB), ethylene diiimine (EDI), or psoralens can be used to inactivate the virion RNA selectively and the unbound chemicals and their products are removable by molecular sieving. Additional inactivation by physical methods would include proven hydrostatic pressure cycling technology (PCT) and flash pasteurization at 60 degrees C for less than 10 minutes to totally inactivate infectivity of the virions. Our empirical strategy is to pool five different HIV isolates of the prevalent subtype B (U.S.A.) or C (Southern Africa), augmented by other subtypes A, C/B, D, E, and 'X' (new emerging subtypes), to provide a polyvalent HIV vaccine (HIVAX). Immunochemical quantification of the gp120, gp41, p24 and p31 antigen content of HIVAX ensures consistency of the product, and safety is ensured by failure to amplify HIV nucleic acid sequences by RT-PCR and to demonstrate infectivity in animal models. Ultimate efficacy HIVAX must be shown by human clinical trials in the high-risk populations.

Hepatitis B virions isolated with antibodies to the pre-S1 domain reveal occult viremia by PCR in Alaska Native HBV carriers who have seroconverted.

BACKGROUND: Occult viremia occurring before the appearance of HBsAg or after the disappearance of HBsAg is detectable by gene amplification technologies whose efficiency depends on nucleic acid preparation. STUDY DESIGN AND METHODS: To isolate HBV DNA from viremic plasma, immunoaffinity capture (IAC) of intact HBV with biotinylated pre-S1 antibodies coupled to streptavidin-coated magnetic beads was evaluated. IAC was compared with a silica-gel method (Qiagen [QSG]) and its two modifications wherein the samples were heated with lysis buffer at 60(o)C for 10 minutes (QSG-60) or at 58 degrees C for 60 minutes with proteinase-K (QSG-PK). Each HBV DNA sample was tested by heminested PCR amplification of the HBV gene sequences. A total of 36 coded serum samples were tested, including three HBsAg-positive controls and 33 former chronic HBV carriers who had seroconverted (developed antibody to HBsAg [anti-HBs]). Commercially available seroconversion panels (PHM 907, 911, and 922) were similarly tested for window-period viremia. RESULTS: In the 33 former chronic HBV carriers who had seroconverted, IAC revealed HBV DNA in 17 samples, whereas it was revealed in only 11 samples by QSG-PK (p = 0.031), 10 by QSG-60 (p = 0.016), and 9 by QSG (p = 0.0078). However, HBV DNA was not amplified from the 17 samples at 1-in-10 dilutions; thus, they were considered to have low-level viremia. IAC revealed HBV DNA as early as or earlier than the other methods in PHM 907, 911, and 922 panels. CONCLUSION: IAC is apparently an optimal method of sample preparation for amplification of HBV DNA in patients in the pre-HBsAg window period, and for detecting low-level viremia persistent in several individuals who were former chronic HBV carriers who had seroconverted (developed anti-HBs).

Immunobiology of persistent blood-borne viral infections.

Host-virus interactions have co-evolved to play an interactive role in the pathogenesis of viral infections and their disease outcome. Host responses to viral infections, including the cell-mediated and humoral immune responses, have been a subject of intensive research in virology and immunology. Definition of specific cellular receptors for cellular entry of the agents, the rates of their intracellular viral replication, the rates of turnover of circulating virions, persistence of viral infection possibly due to inadequate immune responses, and continued formation of circulating immune complexes provide the framework for our current understanding of the immunopathology of virally induced disease. Among the multiple blood-borne viruses (BBV) transmissible through transfusion, hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency viruses (HIV-1/-2) are relatively more important than several other viruses. Not only do they establish asymptomatic persistent infections with occasional oncogenic sequelae, but they also cause significant morbidity and mortality when transmitted through transfusion of blood and blood products. Molecular characterization of these agents and their in vitro inactivation and removal from blood have become key issues in contemporary transfusion safety since the advent of AIDS. Because many of the BBV are associated with white blood cells that have no therapeutic benefit in haemotherapy, simple filtration-removal of leukocytes from donated blood confers a dual benefit of immunological and virological safety in transfusion medicine.

Properties of cyanovirin-N (CV-N): inactivation of HIV-1 by sessile cyanovirin-N (sCV-N).

Cyanovirin-N (CV-N) is a novel anti-HIV protein isolated and characterized from a cyanobacterium Nostoc ellipsosporum. CV-N protein is a single 101 amino acid chain containing two intrachain disulphide bonds and considerable internal sequence duplication, but no significant homology to previously described proteins or to the transcription products of known nucleotide sequences. In solution, CV-N exists largely as a beta-sheet protein with internal two-fold pseudosymmetry. CV-N irreversibly inactivates diverse laboratory strains, primary isolates and clades of HIV-1, as well as strains of HIV-2 and simian immunodeficiency virus (SIV). CV-N binds with extremely high affinity to highly conserved binding site(s) on the viral envelope glycoprotein gp120, preventing virus-to-cell fusion, viral entry and infection of cells. The CV-N binding site appears to overlap, but is not identical with, the unique carbohydrate-dependent epitope 2G12, and may lie predominantly within an immunologically "silent" region of gp120. CV-N is undergoing preclinical development for topical anti-HIV prophylactic (e.g., microbicidal) applications to prevent sexual transmission of HIV. Since CV-N may be immunogenic in humans, methods for using CV-N for ex vivo inactivation of HIV in blood, plasma, or putative vaccines preferably would allow for its exclusion from biologicals for parenteral use. To explore this concept we biotinylated CV-N (bCV-N) and coupled it to streptavidin coated magnetic beads to provide a product which we termed sessile CV-N (sCV-N). When reacted with a laboratory strain and a primary isolate of HIV- 1, the sCV-N completely inactivated 100 TCID50 of the virus. However RT-PCR of the viral extracts indicated that only a fraction of the virus was removed by the sCV-N, leaving behind a relatively larger fraction of non-infectious virus in the supernatant which we designated as replication incompetent virions (RIV). It would be worthwhile investigating the role of RIV as a putative HIV vaccine.

A prospective study of the risk of transfusion-acquired viral infections.

The risk of transfusion-transmitted viral infections may be estimated by several methods, but only prospective studies of transfusion recipients can directly measure the incidence, with associated 95% upper confidence bound, of these infections. From 1989 through 1995, 764 recipients of allogeneic or autologous red blood cell transfusions were enrolled; 486 (64%) provided both pretransfusion and 6-month follow-up specimens. Both specimens were tested for anti-HBc, anti-HCV, anti-HTLV-I and anti-HIV, with appropriate confirmatory testing. Thirty-nine (8.0%) subjects had seroprevalent anti-HBc, 19 (3.9%) subjects had seroprevalent anti-HCV, three (0.6%) subjects had seroprevalent anti-HTLV-I/II, and one (0.2%) subject had seroprevalent anti-HIV. There were no seroconversions for any agent among the 34 patients who received only autologous blood, and no confirmed seroconversions for anti-HTLV-I or anti-HIV among all subjects. There were three seroconversions for anti-HBc (incidence 1.04 x 10(-3); 95% confidence interval (CI) 2.15 x 10(-4), 3.05 x 10(-3) per allogeneic unit transfused), and two confirmed seroconversions for HCV (incidence 6.94 x 10(-4); 95% CI 8.34 x 10(-5), 2.51 x 10(-3) per allogeneic unit transfused). One of the two anti-HCV seroconversions occurred in March 1994, after the institution of HCV EIA 2.0 screening of donated blood. Transfusion-associated seroconversions to hepatitis B and C markers were observed at low rates in the early 1990s despite testing donors for markers of both viruses, whereas seroconversions to HTLV-I or HIV were less than 1.04 x 10(3) per allogeneic unit transfused, based upon the upper 95% confidence interval of the zero incidence in this study.

Neutralizing antibodies against HIV determined by amplification of viral long terminal repeat sequences from cells infected in vitro by nonneutralized virions.

Based on the earliest intracellular synthesis of nascent HIV-1 long terminal repeat (LTR) fragments, we have established a heminested polymerase chain reaction (HNPCR) amplification of the 5' LTR sequences (LTR-HNPCR) for molecular assay of virus-neutralizing antibodies (VNAb). We incubated HIV antibodies with virus isolates for an hour, followed by addition of lymphoid cells (H9 or peripheral blood mononuclear cells [PBMC]) and further incubation for an hour. After washing the cells three times for thorough removal of free virions and antibodies, LTR-HNPCR consistently revealed HIV DNA in H9 cells after 15 minutes, in PBMC after 4 hours, and corresponding virion expression after 7 days in culture. Replication-competent HIV detected by LTR-HNPCR following overnight culture of infected PBMC for 16 to 18 hours was comparable with tissue culture infectivity measured by p24 antigen expression at 7 days. After establishing a molecular assay for in vitro HIV neutralization by HIV Ig, a panel of five HIV isolates tested with 6 monoclonal antibodies and HIV Ig revealed that LTR-HNPCR was comparable with other VNAb assays. These preliminary data indicate that the molecular assay for HIV neutralization has a clear-cut end point, is specific, reliable, and more rapid than other VNAb assays. Therefore, it offers potential utility in evaluating immune response to candidate vaccines.

Synthesis and anti-HIV activity of some non-nucleoside 2,3-disubstituted quinazoline derivatives (Part-V).

Several [2-phenyl-4(3H)-oxo-3-quinazolinylamino]-N-substituted- arylacetamides (1a-j) have been synthesized and tested at the National Cancer Institute, Bethesda, Maryland, USA, for their anti-HIV activity against susceptible human host cells (CEM cell line) over a wide range of concentrations (6.35 x 10(-8) to 2.00 x 10(-4) M). The highest protection observed is 45.67%. The structures of these compounds have been established on the basis of elemental analysis and spectral data.

Transmission of HIV-1 in infants born to seropositive mothers: PCR-amplified proviral DNA detected by flow cytometric analysis of immunoreactive beads.

The diagnosis of HIV infection in newborns is established by amplification of proviral DNA using the polymerase chain reaction (PCR). We developed a nonisotopic method for heminested PCR using a biotinylated primer among sets of three oligonucleotides, each selected from the HIV long terminal repeat (LTR) and gag sequences. An internal probe incorporating digoxigenin-dUTP was also synthesized by PCR. The PCR products, hybridized with LTR region or gag region probes, were captured with streptavidin-coated magnetic beads and detected by fluorescein isothiocyanate-labeled antidigoxigenin in flow cytometric analysis. This immunoreactive bead assay (PCR-IRB) detected about three copies of HIV proviral DNA. A panel of 50 coded DNA specimens of infants previously assayed by conventional PCR and with known clinical results revealed that the PCR-IRB findings using LTR, but not gag, were in agreement. A double-blind prospective study of blood samples from 14 mother-infant pairs using the PCR-IRB amplification of LTR gave results similar to the commercial Amplicor HIV-1 PCR test and were consistent with the clinical outcomes. PCR-IRB results were positive for 11 mothers and three infants, one at birth, one at 2 weeks after birth, and one at 8 weeks after birth. PCR-IRB is a simple, reliable, specific, and automatable assay useful in the early diagnosis of perinatal HIV infection in clinical practice and regional screening programs.

Editorial summary of the Pre-symposium workshop on the Contemporary Assessment of Technologies.

The following is the editor's summary of the Pre-symposium workshop on Contemporary Assessment of Technologies presented at the Symposium on Molecular Approaches to Laboratory Diagnosis at San Francisco in February 1995. This workshop was moderated by Dr Joel M. Palefsky, and Dr Michael P. Busch. We have briefly summarized the presentations by: (1) Dr Indira Hewlett of the Center for Biologics Evaluation and Research, Food and Drug Administration entitled 'Technology overview'; (2) Dr John J. Sninsky of Roche Molecular Systems Inc. entitled "Polymerase Chain Reaction'; (3) Dr Terrance Walker of Becton Dickinson Research Center entitled 'Strand Displacement Amplification'; (4) Dr Mickey Urdea of Chiron Corporation entitled 'bDNA assay' and (5) Dr Robert H. Singer of University of Massachussetts Medical Center entitled 'Image analysis of in situ hybridization'. Although it was not possible to list all the references to the primary literature, we have attempted to provide the key references as far as possible.

Magnesium-mediated reversal of the apparent virucidal effect of ascorbic acid or congo red reacted in vitro with the human immunodeficiency virus.

The mechanism of in vitro inactivation of cell-free human immunodeficiency virus (CFHIV) with ascorbic acid (M) or Congo red (CR) was investigated with specific regard to the impact of an excess of magnesium ions on the viral inactivation. Quadruplicate reaction mixtures containing CFHIV were mixed with a virus-inactivating dose of 500 micrograms/ml ascorbic acid in RPMI medium devoid of fetal bovine serum and incubated for 3 h at 4 degrees C in two parallel sets of experiments. AA-free CFHIV and virion-free AA were included in each experiment as the positive and negative controls, respectively. After adding 10(6) MT2 cells to capture the surviving virons, the mixtures were incubated for 1 h at 37 degrees C. The cells from the first set were washed three times with Hanks balanced salt solution (HBSS) only, and those from the second set were washed with HBSS fortified with MgCl2 (1.0 mg/ml). Similarly, inactivation of CFHIV by increasing amounts of CR ranging between 12.5-100 micrograms/ml was also tested for the effect of MgCl2, except that (i) the assay was performed in subdued light, (ii) CFHIV-CR mixtures were incubated at 37 degrees C for 1 h in the dark and (iii) H9 cells were used instead of the MT-2 cells to capture the surviving virions in the test mixtures. The cells were cultured in RPMI with 20% FBS for 5 days at 37 degrees C. The absence of p24 antigen in the culture supernatant of MT2 or H9 cells indicated HIV inactivation by AA or CR, respectively. Remarkably, the cultured cells that were washed with HBSS + MgCl2 consistently expressed p24 antigen at levels comparable with those from the untreated virus control. Therefore, the apparent in vitro inactivation of CFHIV by either AA or CR was reversible as validated by washing of the cells with HBSS + MgCl2 following capture of the virions from CFHIV-AA or CFHIV-CR inactivation mixtures. These observations underscore the need for including extra magnesium ions as a control in validating various protocols used for assessing the in vitro virucidal activity of reverse transcriptase inhibitors, membrane binding dyes, or other candidate chemical agents.

Multicenter evaluation of the 3% paraformaldehyde method for white cell counting in leukocyte-reduced red blood cells. BEST (Biomedical Excellence for Safer Transfusion) Working Party of the International Society of Blood Transfusion.

BACKGROUND/AIM: The 3% paraformaldehyde (PFA) method is a simple technique for counting residual white blood cells (WBC) in leukocyte-depleted red blood cells (RBC). Preliminary data suggested that its sensitivity is at least equal to PCR and flow cytometry. We report the results of a multicenter study conducted by the BEST Working Party to determine precision and accuracy of the 3% PFA method. STUDY DESIGN: In the 7 participating laboratories, 5 sets of samples containing nominal concentrations of 200, 100, 50, and 10 WBC/ml were prepared by diluting whole blood into 'WBC-free' RBC. Ten milliliters of each sample were processed using the 3% PFA method, which is based on erythrocyte lysis and WBC concentration into 5% of the original sample volume; a Nageotte chamber is used to count concentrated WBC. RESULTS: The precision of the technique varied according to the nominal concentration, ranging from a CV of 12% at 200 WBC/ml to 57% at 10 WBC/ml. The technique measured fewer than the nominal WBC concentrations (mean of all laboratories, -12.4%); underestimation was probably due to cell loss during sample manipulation. Overall accuracy was however acceptable, because statistical considerations establish that the actual WBC concentration would unlikely exceed 2 times the estimated count. CONCLUSIONS: The 3% PFA method is suitable for the enumeration of residual WBC at concentrations > or = 50/ml. It represents a useful tool for evaluation of high performance filters by reference laboratories.

Flow cytometric detection of human immunodeficiency virus type 1 proviral DNA by the polymerase chain reaction incorporating digoxigenin- or fluorescein-labeled dUTP.

Serological assays are routinely used in the laboratory diagnosis of human immunodeficiency virus type-1 (HIV-1) infection, but the polymerase chain reaction (PCR) is ultimately the most sensitive and direct method for establishing definitive diagnosis. As an alternative to the conventional radioactive PCR procedure we have developed and evaluated a pair of rapid nonradioisotopic flow cytometric detection methods. Using heminested PCR we directly incorporated fluorescein-12-dUTP (fluo-dUTP) or digoxigenin-11-dUTP (dig-dUTP) into the PCR-amplicons. The labeled amplicons were hybridized with biotinylated antisense and sense probes, followed by capture of the hybrid DNA using streptavidin-coated beads which were finally analyzed in a flow cytometer by 1) direct detection of the fluorescence intensity of the amplicons incorporating fluo-dUTP and 2) immunodetection of the amplicons incorporating dig-dUTP by anti-digoxigenin IgG labeled with fluorescein isothiocyanate (FITC). Although both assays were functionally comparable with radiolabeled probe in reliably detecting as low as five copies of HIV-1 proviral DNA sequences, the immunodetection of dig-dUTP consistently yielded higher mean channel fluorescence and gave a stable signal over an extended period of 12-14 weeks. In testing a panel of 20 pedigreed PBMC specimens from blood donors with or without HIV-1 infection, the results of both flow cytometric assays were identical with those of the conventional radioactive procedure. Therefore, we conclude that the dig-dUTP incorporation in amplicons, hybridization with a pair of sense-antisense biotinylated probes and immunodetection of hybrids by flow cytometric analyses is the nonisotopic method of choice for PCR-diagnosis of HIV-1 infection.