Evaluation of Intestinal Protozoan Morphology in Human Fecal Specimens Preserved in EcoFix: Comparison of Wheatley’s Trichrome Stain and EcoStain

As a result of disposal problems related to the use of mercury compounds, many laboratories have switched from mercuric chloride-based Schaudinn’s and polyvinyl alcohol (PVA) stool preservatives to other, non-mercury-based preservatives. A comparison of organism recoveries and morphologies of the intestinal protozoa was undertaken with PVA containing the EcoFix zinc-based Schaudinn’s preservative (Meridian Diagnostics, Inc.); both Wheatley’s modification of Gomori’s trichrome stain (WT) and EcoStain (ES) were used to stain 51 human fecal specimens. Morphology, clarity of nuclear and cytoplasmic detail, overall color differences, and the ease or difficulty in detecting intestinal protozoa in fecal debris were assessed for the two permanent stained smears. Overall, organism morphology of the intestinal protozoa stained with WT and that of protozoa stained with ES were not equal in nuclear and cytoplasmic detail or range of color. However, the same organisms were identified in stained fecal smears with either WT or ES, with the exception of situations in which organism numbers were characterized as rare. Included were 67 protozoan challenges (number of organisms): Entamoeba histolytica-Entamoeba dispar (5), Entamoeba coli (9), Entamoeba hartmanni (6), Endolimax nana (12), Iodamoeba bütschlii (8), Blastocystis hominis (19), Giardia lamblia (6), Dientamoeba fragilis (2), yeast (2), and leukocytes (2). Five specimens were negative for parasites but contained fecal debris that was compared for morphologic detail and color range. The ES produces a more gray-green monotone with very little pink or red tone; contrast among the various colors is less than that seen with WT. Stain intensity for all organisms was acceptable, and there were no problems with stain deposition. The quality of the protozoan morphology with ES was often comparable to that with WT (36 of 67 [53.7%]) and, in some cases, better (24 of 67 [35.8%]). Organisms on the WT-stained smear exhibited better morphology in a few instances (4 of 67 [6%]), and in three instances, there were discrepant organism numbers.     

Use of restriction fragment length polymorphism as a genetic marker for typing Mycobacterium avium strains.

Restriction fragment length polymorphism (RFLP) was used to study 75 clinical isolates identified as Mycobacterium avium. Two repetitive insertion sequences, IS1311 and IS900, were used as DNA probes. Although less than 25% of isolates showed RFLP patterns with IS900, all strains gave banding patterns with IS1311. M. avium strains isolated from patients with AIDS exhibited marked polymorphism with both probes.     

Evaluation of intestinal parasite morphology in polyvinyl alcohol preservative: comparison of copper sulfate and mercuric chloride bases for use in Schaudinn fixative.

As a result of disposal problems inherent in the use of mercury compounds, many laboratories have considered using copper sulfate as a substitute for mercuric chloride in polyvinyl alcohol (PVA) preservative. The primary use for PVA-preserved specimens is the permanent stained smear, the most important technique for the identification of intestinal protozoa. A comparison of organism recovery and morphology was undertaken with PVA containing either copper sulfate or mercuric chloride base. Paired fecal specimens (417 pairs) were collected and examined with the Formalin-ether concentration and Trichrome stain techniques. Numbers of organisms recovered and helminth egg and protozoan morphology were assessed from the concentration sediment. Morphology, clarity of nuclear and cytoplasmic detail, overall color differences, and the ease or difficulty in detecting organisms in fecal debris were assessed from the permanent stained smear. No significant differences were found in the numbers and morphology of organisms seen in the concentration sediment. However, when the trichrome stain was used, the overall morphology of the intestinal protozoa preserved in PVA with copper sulfate was not equal to that seen with PVA with mercuric chloride. We do not recommend switching from mercuric chloride base to copper sulfate base unless that is the only option available for the preparation of permanent stained smears.     

Identification of a mutation in synapsin I, a synaptic vesicle protein, in a family with epilepsy

A four generation family is described in which some men of normal intelligence have epilepsy and others have various combinations of epilepsy, learning difficulties, macrocephaly, and aggressive behaviour. As the phenotype in this family is distinct from other X linked recessive disorders linkage studies were carried out. Linkage analysis was done using X chromosome microsatellite polymorphisms to define the interval containing the causative gene. Genes from within the region were considered possible candidates and one of these, SYN1, was screened for mutations by direct DNA sequencing of amplified products. Microsatellite analysis showed that the region between MAOB (Xp11.3) and DXS1275 (Xq12) segregated with the disease. Two point linkage analysis demonstrated linkage with DXS1039, lod score 4.06 at theta = 0, and DXS991, 3.63 at theta = 0. Candidate gene analysis led to identification of a nonsense mutation in the gene encoding synapsin I that was present in all affected family members and female carriers and was not present in 287 control chromosomes. Synapsin I is a synaptic vesicle associated protein involved in the regulation of synaptogenesis and neurotransmitter release. The SYN1 nonsense mutation that was identified is the likely cause of the phenotype in this family.     

Chemokine receptors that mediate B cell homing to secondary lymphoid tissues are highly expressed in B cell chronic lymphocytic leukemia and non-Hodgkin lymphomas with widespread nodular dissemination

B cell neoplasms present heterogeneous patterns of lymphoid organ involvement, which may be a result of the differential expression of chemokine receptors. We found that chemokine receptor (CCR)7, CXC chemokine receptor (CXCR)4, or CXCR5, the main chemokine receptors that mediate B cell entry into secondary lymphoid tissues and their homing to T cell and B cell zones therein, were highly expressed in B malignancies with widespread involvement of lymph nodes. Conversely, those pathologies with little or no nodular dissemination showed no expression to very low levels of CCR7 and CXCR5 and low to moderate levels of CXCR4. These findings provide evidence for the role of CCR7, CXCR4, and CXCR5 in determining the pattern of lymphoid organ involvement of B tumors. Functional studies were performed on B malignancies expressing different levels of CCR7, CXCR5, and CXCR4. Multiple myeloma (MM) cells did not express CCR7 nor CXCR5 and did not migrate in response to their ligands; a moderate expression of CXCR4 on MM cells was accompanied by a migratory response to its ligand, CXCL12. By contrast, cells from B cell chronic lymphocytic leukemia (B-CLL) expressed the highest levels of these chemokine receptors and efficiently migrated in response to all ligands of CCR7, CXCR4, and CXCR5. In addition, the migration index of B-CLL cells in response to both of the CCR7 ligands correlated with the presence of clinical lymphadenopathy, thus indicating that the high expression of functional chemokine receptors justifies the widespread character of B-CLL, representing a clinical target for the control of tumor cell dissemination.     

* Immediate allergic reactions to amoxicillin

A large group of patients with suspected allergic reactions to (β-lactam antibiotics was evaluated. A detailed clinical history, together with skin tests, RAST (radioallergosorbent test), and controlled challenge tests, was used to establish whether patients allergic to β-lactam antibiotics had selective immediate allergic responses to amoxicillin (AX) or were cross-reacting with other penicillin derivatives. Skin tests were performed with benzylpenicilloyl-poly-L-lysine (BPO-PLL), benzylpenicilloate, benzylpenicillin (PG), ampicillin (AMP), and AX. RAST for BPO-PLL and AX-PLL was done. When both skin test and RAST for BPO were negative, single-blind, placebo-controlled challenge tests were done to ensure tolerance of PG or sensitivity to AX. A total of 177 patients were diagnosed as allergic to β-lactam antibiotics. We selected the 54 (30.5%) cases of immediate AX allergy with good tolerance of PG. Anaphylaxis was seen in 37 patients (69%), the other 17 (31%) having urticaria and/or angioedema. All the patients were skin test negative to BPO; 49 of 51 (96%) were also negative to MDM, and 44 of 46 (96%) to PG. Skin tests with AX were positive in 34 (63%) patients. RAST was positive for AX in 22 patients (41%) and to BPO in just 5 (9%). None of the sera with negative RAST for AX were positive to BPO. Challenge tests with AX were performed in 23 subjects (43%) to establish the diagnosis of immediate allergic reaction to AX, and in 15 cases (28%) both skin test and RAST for AX were negative. PG was well tolerated by all 54 patients. We describe the largest group of AX-allergic patients who have tolerated PG reported so far. Diagnosis of these patients can be achieved only if specific AX-related reagents are employed. Further studies are necessary to determine the exact extent of this problem and to improve the efficacy of diagnostic methods.     

Metal content of neutrophil granules is altered in chronic inflammation

The mass fraction of certain elements was measured in isolated granulocytes and isolated granulocyte granule fractions from patients with active inflammatory arthritides (N=6) and healthy controls (N=6). The patients had significantly increased amounts of Ca in the granulocytes, in the specific and light azurophil granules, but normal Ca amounts in the dense azurophil granules. Sr was below the detection limit in the granulocytes and granule fraction from controls, but it appeared in high concentrations in the granulocytes and all granule fractions from the patients. The patients had considerably increased granulocyte amounts of Mn but only slightly increased Mn concentrations in the specific granules. Mn was not detectable in azurophil granules from patients and controls. A prominent accumulation of Fe was seen in the granulocytes from the patients, together with an Fe accumulation in the specific granules. Fe was below the detection limit in azurophil granules from patients and controls. The patients had reduced granulocyte Zn and reduced amounts of Zn in the dense and light azurophil granules but normal Zn amounts in the specific granules. The results obtained indicate that (1) the granulocyte accumulation of Ca, Sr, and Fe observed during chronic inflammation is associated with corresponding granule accumulation of these metals; (2) the considerable Mn accumulation in granulocytes during inflammation is not localized in their granules; and (3) the granule subpopulations differ in their capacity to store certain metals.     

Cervical cancer screening program of Paraná: cytohistological correlation results after five years

Since its inception in November 1997, the Cervical Cancer Screening Program of Paraná (CCSPP), Brazil, has resulted in the cytological screening of 2,244,158 women, the coverage of the female population increasing from 43% to 86%. One thousand six hundred one cases screened by cytology, submitted to colposcopy, and subjected to treatment were selected. Cytopathological results were compared with those obtained on the basis of histological analyses of the loop electrical excision procedure specimens, and were subjected to statistical analyses. The data obtained were then compared with cytohistological correlation results from the first year of the program. Considering the exact correlation between cytological and histological diagnoses, the correlation index increased from 53.34% in the first year to 67.3% at the end of 5 yr of the program. Variations that occurred in each diagnostic category are discussed. This study demonstrates a significant improvement in the concordance between cytological and histological results for the 5-yr period compared with the first year of the CCSPP.     

Foxl2 disruption causes mouse ovarian failure by pervasive blockage of follicle development

FOXL2 mutations cause gonadal dysgenesis or premature ovarian failure (POF) in women, as well as eyelid/forehead dysmorphology in both sexes (the 'blepharophimosis-ptosis-epicanthus inversus syndrome', BPES). Here we report that mice lacking Foxl2 recapitulate relevant features of human BPES: males and females are small and show distinctive craniofacial morphology with upper eyelids absent. Furthermore, in mice as in humans, sterility is confined to females. Features of Foxl2 null animals point toward a new mechanism of POF, with all major somatic cell lineages failing to develop around growing oocytes from the time of primordial follicle formation. Foxl2 disruption thus provides a model for histogenesis and reproductive competence of the ovary.     

Principle and practice of in vitro fertilization. Role in the treatment of female sterility

In vitro fertilization (IVF) and embryo transfer (ET) appear to constitute a revolution in the reproductive sciences rather than merely a new technique in the treatment of sterility. Principle of IVF: IVF accomplishes in vitro the process than normally occurs in the oviduct between the ovulation of oocyte II and embryo implantation in the endometrium. This 4 day period (under normal conditions in the woman) involves 4 steps: recovery, fertilization, segmentation and transport. Performance of IVF: Recovery of the oocytes: The oocytes are recovered under celioscopic or echographic observation when they have completed cytoplasmic maturation and their first meiosis. A precise monitoring of ovulation (spontaneous or induced) should be performed using estrogen and LH assays. IVF provides an opportunity for evaluating the methods of ovulation induction and monitoring, as a function of the maturation of the oocytes recovered. Fertilization: When the oocyte has achieved maturing after several hours of incubation, fertilization is obtained 15 h contact with washed and capacitated spermatozoa (100 000/ml). This step is highly dependent on gametocyte quality: oocyte maturity and fecundity of spermatozoa, which can be estimated from the percentage of survival in the insemination medium. Segmentation occurs in culture at pH 7.28 in the presence of 5 per cent CO2 at 37 degrees C (pronucleus 15th, 2 blastomeres 26 h, 4-8 blastomeres 52 h). Embryo transfer is carried out when an embryo is present at 52 h. Only 1/10 of the embryo transfers result in successful implantation, which depends on the quality of the embryo; the quality can only be indirect criteria.(ABSTRACT TRUNCATED AT 250 WORDS)