Evaluation of a rapid test for HIV antibodies in saliva and blood.

OBJECTIVE: To test whole blood and saliva for HIV antibodies (anti-HIV) using a rapid test strip capillary flow immunoassay, and to correlate the test strip results with blood specimen results obtained from routine diagnostic anti-HIV assays. DESIGN: A prospective pilot study of selected HIV-positive and HIV-negative individuals, children and medico-legal cases from Gauteng, South Africa. METHODS: Whole blood specimens taken from every individual and medico-legal case (total study population 153) and saliva specimens taken from 76 selected cases were tested for anti-HIV using the respective Hema-Strip HIV-1/2, Sero-Strip HIV-1/2 and Saliva-Strip HIV-1/2 (Saliva Diagnostic Systems Inc.) rapid test strip methodology. All results were correlated with the currently recommended anti-HIV assays. RESULTS: The whole blood test strip results correlated 100% with the traditional diagnostic results. Only two saliva test strip results tested false-negative, both from marasmic and severely dehydrated babies, while the other results were in concordance. All test strip results on postmortem blood and saliva were fully concordant with the diagnostic assay results. CONCLUSION: The anti-HIV test strip methodology for whole blood and saliva specimens is rapid, reliable and easy to perform and interpret. Saliva specimens can be readily collected from any individual, and there is a reduction in hazard risk. Anti-HIV saliva testing using the test strip methodology is recommended for South Africa, particularly in high-risk situations such as the paediatric and forensic medicine settings. A large field study obtaining specimens from different regions in South Africa is advised.

Bloodborne viruses and occupational exposure in the dental setting.

Occupational hazards in dentistry are most commonly associated with physical, chemical and biological agents. Bloodborne viruses, notably hepatitis B virus and human immunodeficiency virus (HIV), pose a risk for occupational exposure among oral health care workers in South Africa. Although post-exposure prophylaxis can be prescribed after exposure to either or both these viruses, universal precautions and strategies must be implemented in order to protect the oral health care professional.

Directly inherited partial trisomy of chromosome 6p identified in a father and daughter by chromosome microdissection.

Cytogenetic analysis of a 4 year old girl with developmental delay and dysmorphic features showed extra chromosomal material of unknown origin on 20p (46,XX,add(20)(p13)). Familial chromosome studies showed direct inheritance of add(20)(p13) from the father, who had a similar, albeit milder, phenotype. Fibroblast chromosome studies of the father showed no karyotype mosaicism. The additional material could not be identified on the basis of the G banding pattern owing to its small size and ambiguous banding pattern. Chromosome microdissection of the unknown material was performed, the DNA was amplified and labelled using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and reverse painted to the proband's cells to show the karyotype 46,XX,der(20)t(6;20) (p23;p13), conferring partial trisomy 6p and presumed partial monosomy for 20p. Chromosome microdissection has made possible the first reported case of directly inherited partial trisomy 6p.

Improved control of high-dose-cisplatin-induced acute emesis with the addition of prochlorperazine to granisetron/dexamethasone.

PURPOSE: To evaluate the antiemetic efficacy and safety of adding the dopamine antagonist prochlorperazine to the combination of granisetron and dexamethasone in the prevention of acute nausea and vomiting following high-dose cisplatin. PATIENTS AND METHODS: Sixty patients receiving cisplatin (> or = 75 mg/m2) (median dose = 100 mg/m2) were enrolled at three sites. Patients received prochlorperazine spansule 15 mg orally, 60 minutes prior to and 12 hours after cisplatin; dexamethasone 20 mg intravenously, 45 minutes prior to cisplatin, and 10 mg intravenously or orally, 12 hours after cisplatin; and granisetron 10 micrograms/kg intravenously, 30 minutes prior to cisplatin. Efficacy was assessed during the 24-hour period after cisplatin using complete antiemetic response (no emetic episodes and no rescue antiemetics) and patient assessment of nausea and satisfaction using 100-mm visual analog scales (nausea: 0 = none, 100 = nausea as bad as it can be; satisfaction: 0 = not at all satisfied, 100 = satisfied as can be). RESULTS: Complete response (0 emetic episodes) was noted in 84% (49/58) of patients. Forty-two patients (72%) experienced no nausea. The mean change in posttreatment nausea visual analog scales from baseline was 8.9 mm. Forty-eight patients (83%) were completely satisfied with their antiemetic treatment. The mean posttreatment patient satisfaction score was 92 mm. Treatment was well tolerated, with infrequent and minor adverse events. CONCLUSIONS: This three-drug antiemetic regimen is well tolerated and highly effective in the prevention of acute nausea and vomiting arising from high-dose cisplatin. Further studies evaluating this regimen are warranted.

Assessment of commercial enzyme immunoassay for hepatitis C virus serotyping.

AIMS: To assess a commercial enzyme immunoassay (EIA) for the serotyping of hepatitis C virus (HCV) for routine use in a diagnostic laboratory setting, as well as for noting the serotype prevalence of selected specimens. METHODS: Seventy six serum specimens, submitted to the laboratory for routine hepatitis studies between May 1992 and February 1996 and stored at -20 degrees C, were evaluated. These specimens were categorised into specific hepatic, renal, and paediatric clinical conditions. The specimens all tested positive for HCV antibodies on a screening EIA, with confirmation on a recombinant immunoblot assay (RIBA). Certain specimens were also HCV RNA positive by the reverse transcription polymerase chain reaction (RT-PCR). All the specimens were serotyped using the newly developed serotyping EIA. RESULTS: Twenty seven (35.5%) specimens were typable. Type 5 predominated (56%), followed by type 1 (33%), types 1 and 6 (7%) and type 3 (4%). The serotype 5 specimens showed 85% and 90% reactivity with recombinant antigens c100-3 and c22-3c, respectively; serotype 1 specimens showed 75% and 100% reactivity with these antigens. All serotype 5 specimens reacted with the c33-c antigen, but only 60% of serotype 1 specimens reacted with this antigen. The differences in the reactivity of the serotype 5 and serotype 1 specimens for c33-c antigen in the RIBA were significant, but no significant differences in reactivity for antigens c-1-1, c100-3, and c22-3 were noted. Serotype 3 specimens showed equal reactivity with all four antigens used in the RIBA. CONCLUSION: The serotyping EIA was easy to use, rapid, and cost effective compared with molecular assays. This assay seems to be ideal for the routine diagnostic laboratory setting, but could not be used for certain clinical specimens. The demonstration of serotypes 5, 1, and 3 was not unexpected in this cohort. The occurrence of serotype 6, although concurrent and more likely to be a false cross reaction with serotype 1 peptides, requires confirmation by molecular genotyping before it can be claimed that this type is present in South Africa.

Dose-ranging evaluation of the antiemetic efficacy of intravenous dolasetron in patients receiving chemotherapy with doxorubicin or cyclophosphamide.

Selective 5-HT3 antagonists have proven to be safe and effective for the prevention of chemotherapy-induced nausea and vomiting. Dolasetron is a new highly selective addition to this class of antiemetics that has been shown to have significant antiemetic activity in patients receiving cisplatin-containing regimens. This pilot study was designed to evaluate the antiemetic efficacy of dolasetron in cancer patients receiving doxorubicin and/or cyclophosphamide. This study used an open-label, non-randomized design to evaluate the efficacy and safety of intravenous dolasetron in the prevention of emesis in patients receiving doxorubicin (25-75 mg/m2) and/or cyclophosphamide (400-1200 mg/m2). Sixty-nine patients received a single, intravenous dose of dolasetron over 15-20 min beginning 30 min prior to the start of chemotherapy. Dose levels of dolasetron studied were: 0.3, 0.6, 1.2, 1.8 and 2.4 mg/kg. Patients were monitored for emesis, nausea and adverse events for 24h after the start of chemotherapy. Overall, 61% of patients experienced complete control of emesis. No significant trend towards increased antiemetic efficacy (P = 0.076) or nausea control with increasing dolasetron dose was noted, although the power to detect significant differences was limited by the small number of patients on the 0.3-mg/kg and 2.4-mg/kg dose levels. Age, gender, and type of chemotherapy were significant predictors of complete antiemetic control. Adverse events were generally mild and included headache, chills, lightheadedness, fever, diarrhea, dizziness, and asymptomatic prolongation of ECG intervals. Intravenous dolasetron is safe and effective in the prevention of emesis induced by doxorubicin and/or cyclophosphamide.

Schedule-dependent synergism of edatrexate and cisplatin in combination in the A549 lung-cancer cell line as assessed by median-effect analysis.

The methotrexate analog edatrexate has been shown to have greater antitumor activity and an improved therapeutic index as compared with its parent compound in preclinical systems. These studies suggest that edatrexate may have a broad role in the treatment of solid tumors. Information regarding edatrexate in combination with other chemotherapeutic agents is limited. We evaluated the interaction of edatrexate with cisplatin in vitro as assessed by median-effect analysis in the A549 human lung-cancer cell line. The effects of dose, exposure time, and schedule dependence were assessed. Cytotoxicity was evaluated using the tetrazolium-based colorimetric (MTT) assay. The inhibitory concentration producing 50% absorbance (IC50 for edatrexate with 1 h exposure was 1.4 microM. For all combination experiments, the edatrexate dose was fixed at 0.2 microM (IC10) whereas cisplatin (CDDP) concentrations were varied for 1-, 3-, and 24-h exposures either before or after edatrexate treatment. Drug interactions were assessed using the combination-index method as defined by median-effect analysis. A synergistic interaction was documented in experiments when edatrexate was applied prior to CDDP (combination index, < 1). The combination studies in which edatrexate was used prior to CDDP resulted in significant reduction of all three CDDP IC50 values: 1-h IC50, from 30.0 to 3.9 microM; 3-h IC50, from 21.3 to 1.4 microM; and 24-h IC50, from 1.7 to 0.03 microM. In contrast, synergism was not observed in experiments in which edatrexate treatment occurred after cisplatin exposure. Median-effect analysis is a useful method of determining drug interactions. In the present study, the combination of edatrexate and CDDP demonstrated schedule-dependent synergism, with the synergism being observed only in the setting of edatrexate treatment before CDDP exposure. Due to the potential broad spectrum of activity of edatrexate plus CDDP, further studies are warranted to determine the mechanism responsible for the synergism and to investigate this combination in a variety of tumor models.

Fc receptor gene translocation in a t(1;19) pre-B ALL cell line.

We have recently mapped the human FCGR2 gene to chromosome 1 bands q23-q24. In situ hybridization of FCGR2 cDNA with a cell line containing a t(1:19)(q23;p13) derived from a patient with pre-B ALL has allowed a more accurate localization of this gene to chromosome 1 band q23. Furthermore, this study indicated a splitting of the FCGR2 gene or gene cluster by the t(1;19). However, Southern analysis showed no genetic rearrangement when compared with a karyotypically normal Epstein-Barr virus (EBV)-transformed cell line from the same patient. This suggests that the translocation breakpoint does not occur within the coding region of this gene.

Isodicentric X chromosomes involving the Xq13 breakpoint in myelodysplasia and acute nonlymphocytic leukemia.

Many nonrandom chromosome abnormalities, usually autosomal in nature, have been found to be associated with specific types of acute nonlymphocytic leukemia (ANLL) and myelodysplastic syndromes (MDS). Specific abnormalities involving the sex chromosomes are rare. We have recently identified a structural abnormality of the X chromosome, for example, idic(X)(q13) in three patients: two with MDS progressing to ANLL and one with ANLL de novo. All three patients were elderly females with a very aggressive form of ANLL. Six other patients with a similar abnormality have been discovered in the literature; all having either MDS or ANLL and a short survival. It is suggested that the abnormality identifies a subset of MDS and ANLL occurring in elderly females.

Localization of the G-CSF gene on chromosome 17 proximal to the breakpoint in the t(15;17) in acute promyelocytic leukemia.

The human granulocyte-colony stimulating factor gene (G-CSF) is localized at 17q11.2-q21, the region of one of the breakpoints in the 15;17 chromosome translocation specific for acute promyelocytic leukemia (APL). As G-CSF induces differentiation and loss of tumorigenicity in myeloid leukemic cells or cell lines, it was possible that the translocation in APL involved the DNA of the G-CSF coding region or its regulatory region. In situ hybridization to chromosomes with the t(15;17) from patients with the APL translocation using a G-CSF cDNA clone revealed that the coding region of this gene is proximal to the t(15;17) breakpoint on chromosome 17. Southern analysis of DNA from patients with the APL translocation showed no differences in hybridization between normal and leukemic cells. These results indicate that the G-CSF coding sequence is not disrupted by the chromosomal rearrangement characteristic of APL.

Human tumour cell lines established using clonal agar culture.

Four families of human tumour cell lines--one of uterine, and three of ovarian origin--were established at early passage level from primary biopsy specimens of terminal patients by the propagation of anchorage-independent agar clones in liquid culture. All cell lines exhibited unique and stable characteristics and retained their ability to clone in agar. However, considerable heterogeneity was evident in clonogenic capacity, karyotype, and responsiveness to growth-promoting substances even among progeny of single agar clones isolated from the one biopsy specimen. These cell lines will be made available for further study upon request.

A new human breast carcinoma cell line (PMC42) with stem cell characteristics. II. Characterization of cells growing as organoids.

A new human breast carcinoma cell line (PMC42) has been further characterized. The cells can grow either as monolayers or as floating cords of cells. The cords grow in suspension for long periods but may spontaneously attach and grow out to form a typical PMC42 monolayer. Ultrastructurally, the cells resemble breast ductal cells in many respects. Both epidermal growth factor (EGF) and prolactin induce ultrastructural changes, and lipid production is stimulated markedly by both factors. EGF also promoted the attachment of the floating cords and the growth of cells from these cords as monolayer cultures. The karyotype of the cord cells is different from that previously described for the monolayer cultures. Cord cells are hypodiploid (mode 39), whereas the monolayer cultures are subtriploid (mode 66). Although the ploidy is different, the karyotypes are related with 9 marker chromosomes being common to both populations. In addition, cultures in which cords have attached and in which cells are growing out as monolayers are bimodal with 10-20% of the cells becoming pseudotetraploid with a mode of 77.

The influence of colcemid concentration on mitotic index and length of bone marrow chromosomes.

Bone marrow cells from 25 patients with different hematological disorders were cultured overnight and then exposed to colcemid, varying both concentration and time of exposure, in order to obtain optimal results. PHA stimulated lymphocytes were treated in a similar manner for comparison. Results showed that with concentrations at or below 0.4 microgram/ml, the mitotic index of PHA stimulated lymphocytes was reduced and the metaphase spreads were of poor quality, but spreads of bone marrow chromosomes were not adversely affected at even much lower concentrations. Furthermore, both chromosome length and mitotic index were found to increase with lower concentrations. These observations, together with results obtained by comparing mitotic indices after varying exposure times, suggest that bone marrow cells may be inhibited from passing through G2 by high concentrations of colcemid. Thus by lowering the concentration of the colcemid for bone marrow cultures, we have been able to increase greatly the yield of bandable metaphases.

A new human breast carcinoma cell line (PMC42) with stem cell characteristics. I. Morphologic characterization.

A new human breast carcinoma cell line (PMC42) was established from a pleural effusion from a woman with metastatic breast cancer. The cells were significantly pleomorphic even after 3.5 years in continuous culture. Eight different cell types could be characterized morphologically in monolayer culture. Cells cloned in agar and replated in monolayer culture were equally heterogenous. Cells also grew in suspension in papillary clusters that structurally resembled glandular organoids. Electron microscopy confirmed the differentiated structure of these cells in which many of the features of lactating breast tissue were evident. It is proposed that this is a culture derived from a malignant breast stem that has retained its ability to differentiate in vitro.

Fluorodeoxyuridine synchronization of bone marrow cultures.

A method for synchronization of bone marrow cells with fluorodeoxyuridine (FdU) is presented and compared with methotrexate (MTX) synchronization. FdU has the advantage of not requiring cell washing for release of the DNA synthesis block and was found to be more beneficial for bone marrow cultures because it generally produced a higher mitotic yield and was less damaging to chromosomes. Late-replication banding, produced after releasing the FdU block with bromodeoxyuridine (BrdU), indicated that cells in midsynthesis at the time of the block release were those that showed the most increase in chromosome length and the most improvement in the quality of the metaphase spread. Therefore, because bone marrow cells have a longer cell cycle time than stimulated lymphocytes, a minimum of 7-8 hr culture after release of the block is recommended to give optimal results. It was also found that, to increase the yield of mitoses, at least 6-8 hr of growth was necessary before the addition of either of these synchronizing agents.

DNA extraction during Giemsa differentiation of chromatids singly and doubly substituted with BrdU.

Human lymphocytes were cultured in 3H-labelled BrdU. Cells were pretreated to induce differentiation, autoradiographed and Giemsa-stained. DNA extraction was deduced if grain counts were lower in differentiated mitoses compared with untreated controls.--The differentiation method involved sequential pretreatments with short wave UV and 2 x SSc at 60 degrees C. This removed 34% of label from first division cells (with TB.TB chromosomes) but relatively more (53%) from second division (TB.BB chromosomes). In second division cells, about two thirds of label was lost from pale (BB) chromatids but only one third from dark (TB) chromatids. The UV and SSC pretreatments acted in collaboration, since neither alone reduced grain counts significantly.--On testing other methods, similar preferential DNA extraction was obtained with Perry and Wolff's FPG method, and with the hot salt pretreatment of Korenberg and Freedlender. However, good Giemsa differentiation could also be obtained using Hoechst 33258 and light pretreatments without any DNA loss. Reverse differentiation patterns (TB pale, BB dark) induced by warm acids resulted in extraction of nearly two thirds of 3H-BrdU label, but relative loss was the same from pale and dark chromatin. Direct reverse staining using alkaline Giemsa did not result in any loss of label.--Thus preferential DNA loss from pale stained chromatin underlies differentiation methods using light plus hot salt pretreatments, but it is not obligatory for good differentiation using other techniques.