Effect of maternal nutritional status on immunological substances in human colostrum and milk.

Substances in colostrum and breast milk confer significant disease resistance to the breast-fed infant. The influence of maternal nutritional status on both immunological and nonimmunological milk factors was studied in a group of 23 Colombian women during the first 2 months of lactation. Maternal malnutrition was characterized by significantly lower weight/height ratio, creatinine/height index, total serum proteins, serum albumin, and serum IgG and IgA. The colostrum of malnourished mothers contained only one-third the normal concentration of immunoglobulin G and less than half the normal level of albumin. Significant reductions in colostrum levels of IgA and the fourth component of complement (C4) were also observed in the malnourished group. No differences were observed in colostral concentrations of lysozyme, C3 complement, or IgM. Titers of antibody in milk directed against respiratory syncytial virus were not influenced by maternal nutritional status. The differences noted above tended to disappear in mature milk, concomitant with improvement in the nutritional status of malnourished mothers during the first several weeks postpartum. We conclude that the protective qualities of colostrum and milk may be significantly influenced by maternal nutritional status.

Genotypic polymorphisms in experimental metastatic dermal leishmaniasis.

Molecular karyotype and kDNA restriction analyses were utilized to examine the genetic heterogeneity and plasticity of the Leishmania (Viannia) guyanensis strain WHI/BR/78/M5313, composed of metastatic and non-metastatic populations. Cloning revealed that the strain was constituted by multiple closely related populations that were distinguishable by restriction fragment polymorphisms in kDNA. Size polymorphisms in molecular karyotype were not detected. Passage of clones in hamsters and recovery of parasites from cutaneous metastatic lesions yielded evidence of further genetic heterogeneity among some of the progeny populations. Overall, six kDNA minicircle restriction patterns or schizodemes were observed among clones, subclones and progeny. Although the possibility that population heterogeneity was not resolved by cloning cannot be ruled out, subcloning and kDNA restriction analysis to determine whether the putative clones consisted of homogeneous populations showed the schizodeme of subclones of 3 out of 4 clones to be identical to the clone of origin, while a subclone of the fourth had a co-efficient of similarity of 0.95. Metastasis did not segregate with a particular schizodeme: all six restriction profiles were represented among populations isolated from metastatic lesions and some clones with the same restriction profile did not produce metastatic lesions. The strain from which the clones, subclones and progeny were derived had a kDNA restriction pattern identical to the most prevalent schizodeme (38%) among these subpopulations. This finding together with the reappearance of the repertoire of schizodemes found among clones in the populations recovered from metastatic lesions in hamsters inoculated with a single clone, suggest that sequence polymorphisms in kDNA can emerge during infection.

Evaluation of polymerase chain reaction, adenosine deaminase, and interferon-gamma in pleural fluid for the differential diagnosis of pleural tuberculosis.

STUDY OBJECTIVES: Pleural tuberculosis (TB) is a diagnostic challenge because of its nonspecific clinical presentation and paucibacillary nature. The inefficiency of conventional laboratory methods and the reliance on pleural biopsy have motivated the evaluation of alternative diagnostic strategies. We have evaluated polymerase chain reaction (PCR) directed to the IS6110 sequence of Mycobacterium tuberculosis, the determination of adenosine deaminase (ADA) activity, and measurement of interferon (IFN)-gamma levels in pleural fluid in the diagnosis of pleural TB. PATIENTS: ADA activity, IFN-gamma levels, and PCR were evaluated in 140 cases of pleural effusion, 42 with confirmed pleural TB, 19 with probable pleural TB, 70 with a nontuberculous etiology, and 9 having an undetermined etiology. RESULTS: ADA activity, IFN-gamma levels, and PCR were 88%, 85.7%, and 73.8% sensitive, respectively, and 85.7%, 97.1%, and 90% specific, respectively, for pleural TB that had been confirmed by either culture or pleural biopsy specimens. The combination of PCR, IFN-gamma measurement, and ADA activity determination allowed the selective increase of sensitivity and specificity for probable and confirmed cases compared to individual methods. Positive and negative predictive values for these individual or combined methods were maintained over a wide range of prevalence of pleural TB in the patient population presenting with pleural effusions. Fever and younger age were associated with tuberculous pleural effusion (p < 0. 0001), while blood in sputum and older age were associated with malignant etiology (p < 0.008). CONCLUSIONS: These clinical variables together with the use of ADA activity determination, PCR, and measurement of IFN-gamma levels provide the basis for the rapid and efficient diagnosis of pleural TB in different clinical settings.

Lack of enzyme polymorphism in Trypanosoma rangeli stocks from sylvatic and domiciliary transmission cycles in Colombia.

Although Trypanosoma rangeli is biologically and morphologically distinct from Trypanosoma cruzi, these two hemoflagellates are epidemiologically linked. We report the results of enzyme electrophoretic studies of T. rangeli stocks isolated from sylvatic and domiciliary Rhodnius prolixus, and infected humans inhabiting foci in which T. cruzi was sympatrically transmitted. T. rangeli stocks displayed electrophoretically detectable polymorphism for only a single enzyme, isocitrate dehydrogenase (ICD), in contrast with the marked phenotypic heterogeneity previously reported among the T. cruzi stocks. The relatively restricted diversity manifested by stocks from different geographic sites and ecologic habitats may reflect the existence of distinctive biologic or genetic constraints influencing T. cruzi and T. rangeli transmission.

Leishmania (Viannia) panamensis-specific IgE and IgA antibodies in relation to expression of human tegumentary leishmaniasis.

Leishmania (Viannia) panamensis-specific IgE and IgA antibodies were quantified in patients with parasitologically confirmed American tegumentary leishmaniasis using a radioallergosorbent test (RAST) and an enzyme-linked immunosorbent assay (ELISA), respectively. The RAST values, presented as the mean +/- SEM percentage of total isotope added, were significantly elevated in patients having disease evolution greater than 12 months (3.14 +/- 0.91), as compared with those with an evolution time of 12 or fewer months (1.66 +/- 0.15) (P = 0.017). A separate group of patients, those with eosinophils in the biopsy specimen of their lesion, also had elevated mean RAST values (2.55 +/- 0.58) when compared with patients who did not demonstrate these cells in their biopsy specimens (1.32 +/- 0.24) (P = 0.038). Leishmania-specific IgA levels, presented as the mean +/- SEM optical density, were significantly higher for patients with mucocutaneous disease (0.40 +/- 0.03) than for patients with cutaneous disease (0.28 +/- 0.023) (P = 0.0063). Inhibition testing with homologous and heterologous antigens confirmed the specificity of these assays, and were used to assess cross-reactivity among L. (Viannia) subspecies and other kineto-plastic hemoparasites. Results demonstrate that patients with more severe forms of American tegumentary leishmaniasis, defined either as increased duration of disease or invasion of the mucosa, have elevated levels of Leishmania-specific IgE and IgA antibodies, respectively.

Mucosal disease caused by Leishmania braziliensis guyanensis.

Three Leishmania strains were isolated from cutaneous and mucosal lesions of a Colombian male. These strains, shown to be phenotypically identical based on isoenzyme polymorphisms and monoclonal antibody reactivity, were identified as Leishmania braziliensis guyanensis. Six clones of the mucosal strain were phenotypically identical to the 3 strains. The clinical presentation of the cutaneous lesions and the geographic origin of the infection were consistent with infection by L. b. guyanensis.

Correlation between histopathology, immune response, clinical presentation, and evolution in Leishmania braziliensis infection.

Skin biopsies from 221 parasitologically confirmed cases of tegumentary leishmaniasis caused by Leishmania braziliensis spp. were evaluated with respect to histopathology, the qualitative and quantitative nature of the cellular infiltrate, and the presence of Leishmania amastigotes. These variables were cross correlated with the Leishmania-specific immune response, clinical presentation, and response to treatment. Physical evidence of prior leishmanial lesions was associated with the absence of amastigotes (P less than or equal to 0.001) and the presence of giant (P = 0.03) and epitheloid cells (P = 0.03) in the biopsy of the active lesion. The presence of amastigotes was inversely related to the duration of the lesion (P less than or equal to 0.001) and the presence of eosinophils (P less than or equal to 0.01), whereas the presence of adenopathy (P = 0.01), necrosis (P = 0.001), histiocytes (P = 0.001), and increased serum antibody titer (P = 0.02) were directly associated with the presence of amastigotes. The lymphocyte transformation response was correlated with the presence of granulomas (P = 0.001), but showed no correlation with cutaneous delayed type hypersensitivity. The presence of epithelioid (P = 0.04) and giant cells (P = 0.03) was associated with less drug being required to achieve healing. In contrast, necrosis was associated with a greater amount of drug to achieve healing (P = 0.05). The observed correlations between tissue responses and immune and clinical parameters provide further evidence for the role of antibody and other soluble mediators of the cellular immune response in the evolution of disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Leishmanin skin test standardization and evaluation of safety, dose, storage, longevity of reaction and sensitization.

Leishmanin skin test (LST) antigens prepared from Leishmania braziliensis panamensis were compared with respect to sensitivity, specificity, and side effects. Within the dose range 0.5-3.0 x 10(5) promastigotes of L. b. panamensis and 10 x 10(5) promastigotes of combined L. amazonensis and L. b. panamensis, specificity in healthy controls was nearly 100% for all antigens. Sensitivity increased minimally with increasing dose. Lot-to-lot differences were small. Side effects, such as vesiculation and ulceration at the site of LST application increased with antigen dose. Storage under harsh conditions decreased LST potency but not sensitivity while storage at 2-8 degrees C affected neither potency nor sensitivity. Eighty-five percent of parasitologically diagnosed, LST-positive cases of leishmaniasis remained LST-positive when retested six months to three years later. The LST did not sensitive 19 healthy controls who were skin tested twice or thrice.

Divergent isoenzyme profiles of sylvatic and domiciliary Trypanosoma cruzi in the eastern plains, piedmont, and highlands of Colombia.

Fifty-four stocks of Trypanosoma cruzi from vectors, mammalian reservoirs, and infected humans were characterized by enzyme electrophoresis in starch gels using Brazilian zymodeme reference strains (Z1, X-10; Z2, ESM; Z3, CAN-3) as standards. Colombian stocks were collected in three ecologically and epidemiologically distinct settings. Thirteen enzymes were included in the evaluation. Sixteen different phenotypic profiles or "zymodemes" were evident and generated three groups of closely related stocks: a sylvatic Z1-like group, a domiciliary Z1-like group, and a sylvatic Z3-like group. The number of zymodemes observed in foci of sylvatic transmission was greater than in foci of domiciliary transmission. Modified ecologic conditions associated with agriculture and the consequent reduction of biologic diversity may account for the observed pattern of zymodeme distribution and heterogeneity. The phenotypic similarity between the principal sylvatic group of stocks and domiciliary stocks contrasts with the extensive differences observed between the domestic Z2 zymodeme and sylvatic Z1 and Z3 zymodemes in Brazil and Chile.

Leishmania braziliensis from the Pacific coast region of Colombia: foci of transmission, clinical spectrum and isoenzyme phenotypes.

Tegumentary leishmaniasis is highly prevalent in the Pacific coast region of Colombia. We have identified 90 foci of transmission in this region based on 179 parasitologically diagnosed patients. Human transmission occurred in mangrove forests, secondary growth and intervened tropical rain forest. A parasitological diagnosis, that is, either isolation or visualization of Leishmania was made in 68.6% of suspected cases. Three phenotypically distinguishable groups of L. braziliensis were encountered based on isoenzymes: L. b. panamensis variants (82%), variants of L. b. braziliensis (14.5%), and stocks intermediate between L. b. panamensis and L. b. guyanensis reference strains (3.5%). The L. b. braziliensis variants produced cutaneous disease alone relatively infrequently (12% of classified cutaneous stocks) but were more frequently (38% of all mucosal stocks) isolated from mucosal lesions. Leishmania infection of the mucous membranes caused a wide spectrum of disease, severity being closely related to time of evolution. Both contiguous and metastatic spread to the mucous membranes was supported by the clinical course of 19 mucosal cases.

Diagnosis of cutaneous and mucocutaneous leishmaniasis in Colombia: a comparison of seven methods.

Seven methods of diagnosing leishmaniasis were compared in 177 patients presenting with lesions of the skin (165) or mucosa (12) in Tumaco and Cali, Colombia. The three methods of visualizing amastigotes in tissue samples (histological staining of tissue sections, impression smears of punch biopsies, and smears of dermal scraping from slits in the lesion margins) were less sensitive than the four Leishmania isolation methods (aspiration of lesion border cultured in biphasic media, aspirate inoculated into hamster nasal tissue, culture of punch biopsy macerate, and hamster inoculation of macerate). The aspirate-culture and biopsy-hamster methods employed in this study proved most sensitive of the four methods for the recovery of parasites. The combined overall sensitivity of the 7 methods was 67% for all enrolled patients and 75% for Montenegro skin test-positive patients. The individual sensitivities for the methods for all patients and Montenegro-positive positive, patients, respectively, were: histopathology 14% and 16%, impression smear 19% and 21%, dermal scraping 22% and 26%, aspirate-culture 58% and 64%, aspirate-hamster 38% and 41%, biopsy-culture 50% and 55%, and biopsy-hamster 52% and 57%. All methods were less sensitive in lesions of greater than 6 months duration than in lesions of more recent onset. Mucosal lesions were best diagnosed by the culture or hamster inoculation of a macerated mucosal biopsy. The diagnosis by inoculation of hamsters was achieved within 2 to 12 weeks, a mean of 34.5 days. Promastigotes were seen on Senekjie's medium within 3-8 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemiologic, genetic, and clinical associations among phenotypically distinct populations of Leishmania (Viannia) in Colombia.

Phenotypic characterization of 511 strains of Leishmania, subgenus Viannia, isolated from Colombian patients was conducted based on electrophoretic polymorphisms of 13 isoenzymes. Ninety-one Colombian strains of L. braziliensis were the most heterogeneous, constituting seven zymodemes while 397 L. panamensis and 22 L. guyanensis strains yielded five and three zymodemes, respectively. Phosphogluconate dehydrogenase, nucleoside hydrolase, and superoxide dismutase were the most polymorphic enzymes in this collection of strains, and together with glucose-6-phosphate dehydrogenase, allowed the discrimination of the three aforementioned species. Hierarchical cluster analysis of the zymodemes using Jaccard's coefficient of similarities revealed two clusters, one constituted by L. braziliensis zymodemes, and another by three subgroups consisting of zymodemes of L. panamensis closely related to the species reference strain, another consisting of L. guyanensis zymodemes, and a third group distinguished by new electromorphs of proline iminopeptidase and aspartate aminotransferase that reacted with the L. panamensis-specific monoclonal antibody B-11. Multiple zymodemes of L. panamensis and L. guyanensis were found to be sympatrically transmitted in foci along the Pacific coast. Leishmania braziliensis variants were ubiquitous throughout the territory of Colombia; L. panamensis was prevalent in the western region and L. guyanensis was prevalent in the Orinoco and Amazon river basins in the eastern half of the country. Distinct zymodemes of L. panamensis predominated in the northern and southern regions of the Pacific coast. Nine zymodemes of all three species were isolated from mucosal lesions. Zymodeme 1.1 of L. braziliensis had the highest frequency of mucosal involvement (10% of the cases), and disease caused by this zymodeme had the longest mean time of evolution (31 months; P = 0.002). In addition to being useful in describing epidemiologic relationships, the intraspecific heterogeneity of strains of the Viannia subgenus within and among foci can be used to understand such fundamental questions as the pathogenicity of different populations of parasites, and the induction of cross-protection against related parasites.

Infectivity of the subspecies of the Leishmania braziliensis complex in vivo and in vitro.

The infectivity of Leishmania braziliensis ssp. in relation to their growth kinetics in Senekjie's medium was determined using the human macrophage cell line U937 and inbred hamsters. In both systems, infectivity was shown to be distinctive for each subspecies. While L. b. panamensis promastigotes from 6-day-old cultures (early stationary phase) were more infective than parasites from any other culture day, L. b. guyanensis and L. b. braziliensis reached maximum infectivity on days 8-10 and day 10 (late stationary phase of growth), respectively. Although maximum infectivity occurred during stationary growth, strict growth phase dependency was not observed. The populations of parasites on these culture days were composed mostly of small, highly motile promastigotes with flagella 2-3 times the length of their cell bodies. These promastigotes resembled the infective forms transmitted by the sand fly vector. A distinct pathological picture characterized the disease caused by the different WHO reference strains for these subspecies in hamsters: L. b. guyanensis developed the most severe lesions, while moderate and inconspicuous lesions were observed when L. b. panamensis and L. b. braziliensis, respectively, constituted the inocula.

Mucocutaneous leishmaniasis in Colombia: Leishmania braziliensis subspecies diversity.

It is generally held that with rare exception Leishmania braziliensis braziliensis is the parasite responsible for the metastatic development of mucocutaneous leishmaniasis in the New World. Yet the infrequency of mucocutaneous disease compared with cutaneous manifestations, and the difficulty of isolating parasites from mucocutaneous lesions have restricted the study of the organisms involved. We here report the biologic, isoenzymatic, and monoclonal antibody specificity characteristic of eight Leishmania isolates obtained from the mucosal lesions of the same number of patients. Individually and collectively, the identifying criteria implicate at least two L. braziliensis subspecies as etiologic agents of mucocutaneous leishmaniasis in Colombia and suggest that a spectrum of intrinsically distinguishable organisms may be involved in this disease form.

Frequency of the Asn-108 and Thr-108 point mutations in the dihydrofolate reductase gene in Plasmodium falciparum from southwest Colombia.

Several point mutations in the dihydrofolate reductase (DHFR) gene of Plasmodium falciparum have been correlated with in vitro anti-folate drug resistance of laboratory and field isolates. Furthermore, two different point mutations that generate amino acid substitutions at the same position of the enzyme have been observed in all the isolates studied to date. These point mutations change a serine (Ser-108) in the wild type to an asparagine (Asn-108 mutation) or to a threonine (Thr-108 mutation). Using the polymerase chain reaction (PCR), it is possible to identify isolates that present these mutations. We used a mutation-specific PCR to screen 71 samples from several geographic locations of Colombia for the Asn-108 mutation (pyrimethamine resistance). In this initial screening 53 of 71 yielded amplification product with the DHFR mutation-specific primers. We further analyzed the 18 samples that did not amplify using a mutation-specific nested PCR. Of those 18 samples, seven amplified with primers specific for the Thr-108 mutation (proguanil resistance), one with the wild type (Ser-108), and 10 did not amplify. Of these 10 samples, three were identified as P. falciparum using a species-specific diagnostic nested PCR base on sequences from the small ribosomal RNA subunit gene. Overall, 51.6% of the samples amplified for the Asn-108 mutation, 10.9% for the Thr-108 mutation, 35.9% with the wild type specific primer, and 4.8% did not amplify with any of the DHFR primers. We observed variability in the frequency of the mutation between the different geographic location. The frequency of the Asn-108 and Thr-108 mutations in the state of Narifio was 25% each, while in Valle del Cauca the frequencies were 59% and 11%, respectively. These results contrast with observations in Brazil in which the Asn-108 mutation was found in 90% of the blood samples screened.

Haemoculture of Leishmania (Viannia) braziliensis from two cases of mucosal leishmaniasis: re-examination of haematogenous dissemination.

Leishmania parasites were isolated from peripheral blood leucocytes of 2 patients with mucosal disease among a total of 23 parasitologically confirmed cases of leishmaniasis. One had had mucosal leishmaniasis for 4 years and active pulmonary tuberculosis was also diagnosed. The other patient presented a cutaneous lesion on his right leg of 3 months duration and asymptomatic mucosal involvement. He had received intravenous antimonials before isolation of parasites. Both patients had positive indirect fluorescent antibody and Montenegro skin tests. L. (Viannia) braziliensis was isolated from both patients. This culture of parasites from leucocytes provided direct evidence for metastatic spread of Leishmania via the blood.

Behavior of Leishmania braziliensis s.l. in golden hamsters: evolution of the infection under different experimental conditions.

Reproducibility of Leishmania braziliensis s.l. metastatic behavior in hamsters was studied for 9 isolates of L.b. panamensis and 2 of L.b. guyanensis with a previous record of metastasis. Also, the influence of corticosteroids and trauma was evaluated. In the corticosteroid-treated group, metastases appeared earlier than in the nontreated group, and localization at the site of trauma was more frequent (4/9) than in the nontreated hamsters (1/5). Nine of the 11 strains (82%) were capable of reproducing metastatic behavior. Studies on dissemination of L. b. panamensis showed that the regional lymph node is invaded as soon as 5 days postinfection, with further nonhematic dissemination to other tissues and organs in less than 4 wk.

The inflammatory response promotes cutaneous metastasis in hamsters infected with Leishmania (Viannia) panamensis.

The influence of nonspecific and immunologically elicited inflammatory responses on the development of metastatic lesions was examined in the hamster model of Leishmania (Viannia) panamensis infection. Delayed type hypersensitivity (DTH) responses were induced using the contact sensitizing agent DNFB (2, 4-dinitro-1-fluorobenzene) and infection with L. panamensis followed by intradermal application of leishmanin. Nonspecific inflammatory response was achieved by the surgical excision of toes. The inductive and eliciting procedures were performed on the ears and fore and hind paws of the right side of experimental groups of hamsters that were inoculated in the snout with a highly metastatic strain of L. panamensis (MHOM/COL/84/1099). Skin metastases were detected by physical evaluation at 15-day intervals over a period of 7-8 mo. Suspected metastases were parasitologically confirmed by culture of tissue fluid aspirated from the lesion. The frequency of metastatic lesions was greater in hamsters subjected to inflammatory stimuli (14/38) than control animals (6/33; P = 0.035). Likewise, the frequency of metastases at the site of induction and elicitation of inflammation (18/22 lesions) in the experimental groups was greater than that observed at the same site in control animals (5/11 lesions; P = 0.017). These findings support a causal relationship between inflammatory response and the development of lesions in this model of secondary disease caused by L. panamensis.

Permissiveness of human monocytes and monocyte-derived macrophages to infection by promastigotes of Leishmania (Viannia) panamensis.

During natural infections, Leishmania is in contact with a variety of mononuclear phagocytic cells in different tissues, including resident macrophages and monocytes mobilized to the site of infection from the bone marrow and blood circulation. Because the functional capabilities of fully differentiated macrophages and blood monocytes differ, the outcome of infection by Leishmania may depend upon the stage of differentiation of the host cells. To address this question, we evaluated Leishmania panamensis infection of (1) the human promonocytic/histiocytic cell line U-937 before and after induction of differentiation by phorbol myristate acetate; (2) fresh human peripheral blood monocytes; and (3) macrophages derived from monocytes by differentiation in vitro. Based on the percentage of cells infected and the number of parasites per cell, macrophages derived from monocytes or by induction of differentiation of U-937 cells were significantly more permissive to infection by stationary-phase L. (Viannia) panamensis promastigotes than monocytes. Increasing time and maturation in culture prior to exposure to infective promastigotes was associated with the increased permissiveness of differentiated macrophages to infection (P<0.05). The percentage of cells infected and number of amastigotes per cell increased with time postinfection for both monocytes and macrophages but remained significantly greater for macrophages. The increased expression of CD68, CD16, and lysozyme, and decreased expression of peroxidase by macrophages cultured for 5 days in vitro compared with fresh monocytes, whether adherent or in suspension, supported the distinct maturation status of these cells.

Sensitivity to Glucantime of Leishmania viannia isolated from patients prior to treatment.

In vitro sensitivity to pentavalent antimony (SbV) as meglumine antimoniate (Glucantime) of Leishmania of the Viannia subgenus isolated prior to treatment from patients with uncomplicated cutaneous leishmaniasis was evaluated for intracellular amastigotes in the U-937 human monocytic cell line and log phase promastigotes. The 50% effective dose (ED50) of pharmaceutical and additive-free formulations of Glucantime were determined based on the kinetics of the response of Leishmania Viannia to SbV in vitro. ED50 to SbV was inversely related to time of exposure to drug. The potency of the additive-free formulation of Glucantime was significantly greater than that of the pharmaceutical formulation, irrespective of the parasite form. In vitro sensitivity to SbV ranged from < 5.3 micrograms/ml to > 170.0 micrograms/ml. Under the conditions used, 11 (39%) of 28 strains were sensitive to clinically achievable serum concentrations of SbV. No correlation was observed between the total amount of SbV required for healing of lesions and the in vitro response to the pharmaceutical formulation of Glucantime. In contrast, a significant correlation (P = 0.001) was observed between clinical response and the in vitro sensitivity of promastigotes to the additive-free formulation of Glucantime. The greater potency of the additive-free formulation of Glucantime, the correlation of in vitro sensitivity of promastigotes to this formulation and the clinical response to treatment, and the effect of time of exposure to SbV demonstrate the importance of assay conditions on the outcome and interpretation of in vitro evaluation of drug sensitivity.