Factors contributing to false‐negative and potential false‐negative cytology reports in SurePath™ liquid‐based cervical cytology

N. Gupta, D. John, N. Dudding, J. Crossley and J. H. F. Smith
Factors contributing to false‐negative and potential false‐negative cytology reports in SurePath ? liquid‐based cervical cytology Objectives: The characteristics of false‐negative conventional cervical cytology smears have been well documented, but there is limited literature available for liquid‐based cytology (LBC), especially SurePath? samples. We aimed to assess the characteristics of false‐negative SurePath LBC samples. Methods: Over a period of 5 years, an audit of false‐negative reports in SurePath cervical cytology was undertaken. In a workload of 183, 112 samples, 481 (0.3%) false negatives were identified using two routes: those detected by routine laboratory internal quality control (rapid pre‐screening) (n = 463) and those reported as normal (true false negatives) with concurrent high‐grade cervical histology (n = 18). Ninety‐five false‐negative cases with a subsequent biopsy reported as at least cervical intraepithelial neoplasia grade 2 (CIN2+) were reviewed for a number of different cytomorphological features. Results: Of 95 samples with subsequent CIN2+, 30.5% predominately contained microbiopsies/hyperchromatic crowded cell groups (HCGs), 27.3% sparse dyskarytotic cells, 4.2% pale cell dyskaryosis, 6.3% small dyskaryotic cells; 3.2% were misinterpreted cells, 8.4% contained other distracting cells, 7.4% were low contrast, 5.3% were unexplained and 7.4% were true negatives. The mean number of microbiopsies/HCGs in that category was 4.6. The mean number of abnormal cells in the sparse dyskaryotic cell category was 13.8. Conclusions: Microbiopsies/HCGs were the commonest reason for false negatives. They were usually present in sufficient numbers to be detected but interpretation could be problematic. Dispersed single abnormal cells were usually not identified because of their scarcity or the presence of distracters.     

p16INK4a/Ki‐67 dual labelling as a marker for the presence of high‐grade cancer cells or disease progression in urinary cytopathology

E. Piaton, A. S. Advenier, C. Carré, M. Decaussin‐Petrucci, F. Mege‐Lechevallier and A. Ruffion
p16 ^INK4a /Ki‐67 dual labelling as a marker for the presence of high‐grade cancer cells or disease progression in urinary cytopathology Objective: Overexpression of p16^INK4a independent of the presence of E6–E7 oncoproteins of high‐risk papillomaviruses has been identified in bladder carcinoma in situ lesions with or without concurrent papillary or invasive high‐grade (HG) urothelial carcinoma. As p16^INK4a and Ki‐67 co‐expression clearly indicates deregulation of the cell cycle, the aim of this study was to investigate the frequency of p16^INK4a/Ki‐67 dual labelling in urinary cytology samples. Methods: Immunolabelling was performed in demounted, destained Papanicolaou slides after ThinPrep^® processing. A total of 84 urinary cytology samples (18 negative, 10 low grade, 19 atypical urothelial cells and 37 high grade) were analysed for p16^INK4a/Ki‐67 co‐expression. We assessed underlying urothelial malignancy with cystoscopy, histopathology and follow‐up data in every case. Results: Compared with raw histopathological results, p16 ^INK4a/Ki‐67 dual labelling was observed in 48 out of 55 (87.3%) HG lesions and in 11 out of 29 (37.9%) negative, papillary urothelial neoplasia of low malignant potential or low‐grade carcinomas (P = 0.05). All cases with high‐grade/malignant cytology were dual labelled. Sixteen out of 17 (94.1%) carcinoma in situ cases and eight out of 14 (57.1%) cases with atypical urothelial cells matching with HG lesions were dual labelled. Extended follow‐up allowed three cases of progression to be diagnosed in dual‐labelled cases with negative/low‐grade cytology results after a 9‐ to 11‐months delay. Conclusions: The data show that p16^INK4a/Ki‐67 co‐expression allows most HG cancer cells to be detected initially and in the follow‐up period. Additional studies are needed in order to determine whether dual labelling can be used as a triage tool for atypical urothelial cells in the urine.     

Chloroplast transfer in Nicotiana based on metabolic complementation between irradiated and iodoacetate treated protoplasts

Protoplasts of a light sensitive plastome mutant of Nicotiana tabacum (2 n=48) were irradiated and fused with iodoacetate-treated Nicotiana plumbaginifolia (2 n=20) protoplasts. Treated parental protoplasts were unable to divide. Metabolic complementation, however, helped the recovery of interspecific fusion products which survived and formed calli. Altogether 40 clones were investigated. N. plumbaginifolia plants were obtained in 15 clones (38%), somatic hybrids in 23 clones, and both types of regenerates were found in 2 clones. Irradiation therefore significantly increased the frequency of segregant formation with the non-irradiated N. plumbaginifolia nuclei (the frequency was 1.4% in the absence of irradiation). Regenerated plants in most cases (31 out of 34) contained chloroplasts from the irradiated parent. In 6 clones plants were obtained with both types of chloroplast. Thus, irradiated N. tabacum chloroplasts had an improved chance of dominating the heterokaryonderived cells, many of which contained N. plumbaginifolia nucleus. The system described should be generally applicable for the transfer of chloroplasts without the use of selectable genetic markers.     

Fine needle aspiration cytodiagnosis of clinically suspected tuberculosis in tissue enlargements

A retrospective study of the use of fine needle aspiration (FNA) cytology to confirm a clinical suspicion of tuberculosis in tissue enlargements was performed, using 70 cases. The criteria required to make an FNA cytodiagnosis of tuberculosis were reassessed, and the sensitivity and predictive value of cytology for diagnosing such aspirates was determined. All but 2 of the 70 aspirates contained adequate cellularity. The adequate samples were diagnosed as 40 cases of caseating tuberculosis, 11 cases of noncaseating tuberculosis and 17 cases of acute necrotizing granulomatous inflammation suspicious for tuberculosis. Subsequent histologic study verified the cytologic diagnosis in 27 of 27 biopsied caseating lesions, 4 of 7 biopsied noncaseating cases and 5 of 8 necrotizing cases. The six cases with a false-positive cytodiagnosis of tuberculosis were histologically diagnosed as one Lennert's lymphoma, two reactive lymph nodes and three necrotizing metastatic carcinomas. The sensitivity of FNA cytology for the diagnosis of tuberculosis was 100%, with the predictive value of a positive result being 88%. The findings in this study emphasize that all criteria for the diagnosis of tuberculosis in FNA samples must be utilized and that particular caution should be exercised in making a diagnosis of acute necrotizing tuberculosis.     

Insectivory by Gorilla gorilla gorilla in Southeast Cameroon

Our study extends quantitative analyses of insect-eating by gorillas (Gorilla gorilla gorilla) to Cameroon. During a 2-mo period (May–June 2001), we recorded and analyzed feeding traces on plants and insects and in gorilla feces. We found 180 feeding traces, 17% of which involved insectivory. Seventy-eight percent of the fecal samples (n = 36) contained insects. Ants were found in 61% of the samples, termites in 39%, while 56% of the samples contained remains of other insects. We added 14 new species to the known insect diet of western lowland gorillas. Overall, social insects are predominant. The choice of prey by the Ntonga gorillas gives new clues for the existence of cross-cultural differences among gorilla populations. A comparison of the overall frequency of insectivory with those at other sites in Central Africa indicate a possible effect of forest disturbance on the insectivorous behavior of gorillas. The study suggests the existence of temporal variation in ant- and termite-eating by gorillas.     

Relationships between uterine culture, cytology and pregnancy rates in a Thoroughbred practice

Endometrial cytology and culture specimens (n=2123) were collected concurrently with a guarded uterine culture instrument from 970 mares (738 barren, 1230 foaling and 155 maiden mares) during three breeding seasons (2001-2004). Results were compared to the 28-d pregnancy rate for the cycle from which the samples were taken. Cytological smears were evaluated for inflammation at x100 and graded as: not inflammatory (0-2 neutrophils/field), moderate inflammation (2-5 neutrophils/field), severe inflammation (>5 neutrophils/field), or hypocellular (scant epithelial cells and no neutrophils). Uterine culture swabs were plated within 6h, incubated for 72 h and results determined at 24, 48, and 72 h. Approximately, 20% (n=423) cytology samples were positive for inflammation (>2 neutrophils), whereas approximately 11% (n=231) of cultures had microorganisms recovered. A majority (64%) of the positive cultures (147/231) had inflammation on cytology smears. Streptococcus equi subsp. zooepidemicus was associated with more positive cytology results than coliforms (P<0.01). Mares with positive cytology or culture had lower pregnancy rates than mares with normal findings (P<0.01). Lowest pregnancy rates were recorded for mares with severe endometrial inflammation (21%, versus moderate inflammation 48%). Isolation of a microorganism from mares with endometrial inflammation was not associated with a further reduction in pregnancy rates. In barren, foaling and maiden mares, cytology was positive in 28, 17, and 5%, respectively, and culture was positive in 12.2, 11.1, and 3.2%. Foaling and maiden mares had higher pregnancy rates than barren mares (62, 69, and 44%, respectively, P<0.001). In conclusion, a positive cytology was twice as common as a positive culture, and isolation of microorganisms was associated with reduced pregnancy rates, even in the apparent absence of inflammation.     

An Electrophoretic Analysis of the Seed Protein Body Proteins from Pinus Nigra

Protein bodies (PBs) of European black pine (Pinus nigra Arn.) were isolated from mature seeds. Extracted soluble matrix proteins and crystalloid proteins PBs proteins were investigated by SDS-PAGE electrophoresis in presence and absence of 2-mercaptoethanol. The proteins of molecular masses 16, 17, 18, 61 and 65 kDa were presented only in crystalloid protein samples. Only 15 kDa protein was present in soluble matrix proteins and not in crystalloid proteins. Another protein bands were present in both soluble matrix and crystalloid proteins. 20, 37, 38, 39 and 48 kDa proteins were strongly visible among crystalloid proteins. Bands of 23 and 32 kDa were more visible in soluble matrix protein samples. Different composition in crystalloid proteins was found in absence of 2-mercaptoethanol: no proteins with molecular mass 71 kDa and more proteins in soluble matrix. In case of crystalloid proteins we detected 7 protein bands in interval from 71 to 212 kDa.     

Diabetes is not associated with a change in the elemental composition of the pancreatic B cell in diabetic C57BL KsJ-db/db mice

Freeze-dried pancreas sections from 7-, 17-and 27-week-old genetically diabetic (db/db) and normal (±/±) mice were subjected to proton bombardment and the concentrations of 15 elements in B cells and exocrine pancreas were calculated from the characteristic X-rays emitted. In the 7-week-old diabetic animals, B cells contained significantly above-normal levels of Na and S, while exocrine pancreas contained subnormal levels of Ca, and excess Mn. The B cells from the 17-week-old diabetic animals contained subnormal levels of Cu and the exocrine pancreas of the 27-week-old diabetic animals was deficient in Cd. The 7-, 17- and 27-week-old, genetically diabetic (db/db) mice were hyperglycemic, hyperinsulinemic and heavier than age-matched normal (±/±) mice. Although significant changes were found in elemental composition when comparing both B cells and exocrine pancreas at different ages, the changes were not consistent. Therefore, it appears as if the measured elemental changes were random and not related to the onset of diabetes.     

<Emphasis Type="Italic">MICA</Emphasis> response to gliadin in intestinal mucosa from celiac patients

MHC class I chain-related gene A (MICA), a putative independent susceptibility gene in autoimmune diseases, encodes a surface protein present in epithelial cells that binds to NKG2D, an activating receptor of NK, and T cells, and could function as a stress-inducible activator of the innate immune response. There is no evidence of a long-term implication of MICA in the celiac autoimmune process. However, it could be that gliadin activation of MICA occurs only during the initial stages of the disease. In order to determine whether MICA is activated in response to gliadin in patients with celiac disease (CD), small intestinal mucosa biopsy samples from ten long-standing celiac patients on a gluten-free diet and from five non-celiac individuals were incubated with and without gliadin for 4 h. Total RNA was purified and MICA, IFNG and NKG2D mRNA were quantified by fluorescent real-time RT-PCR. Expression levels were calculated relative to GAPDH. MICA expression was detected in both patients and controls, but incubation with gliadin induced a strong increase in samples from the treated CD group compared with the non-CD controls (P=0.028), while no differences were observed for IFNG or NKG2D mRNA levels. The gliadin-provoked over-expression of MICA in normalized tissues from CD patients suggests a role for this stress-induced activator of the immune response in the early stages of organ-specific autoimmune destruction, probably preceding the onset of inflammation.     

Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours

U.S. Choi and D.Y. Kim Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours Objective: To investigate whether Diff‐Quik stained fine needle aspirate smears can be used to evaluate Ki‐67 expression by immunocytochemistry. Methods: Both cytological and histological samples were obtained from 24 dogs with spontaneously developed mammary gland tumours. The cytological and histological specimens were examined by Diff‐Quik and H&E stains, respectively. After examination, both samples were immunostained using the same Ki‐67 antibody. The % Ki‐67 values were calculated based on the percentage of positively stained tumour cells per 500 and 1000 tumour cells in cytology and histology specimens, respectively. Results: Ki‐67 staining was successful in 17/24 smears (71%) and 19/23 sections (83%). The correlation coefficient between the percentage of Ki‐67‐positive cells in cytological smears and in the histological sections was 0.677 (P < 0.01). These values were significantly different between histologically benign and malignant tumour groups both in cytology and histology samples (P < 0.001). The threshold value of the percentage of Ki‐67‐positive cells for distinguishing benign from malignant tumours was set at 4.85% with 90.9% sensitivity and 92.3% specificity by Receiver Operating Characteristic (ROC) curve using histopathology as the gold standard. Conclusion: Diff‐Quik‐stained cytology smears can be used to detect the presence of Ki‐67 antigen when histology sections are not available.     

Diagnostic impression cytology: a simple technique for the diagnosis of external eye disease

Diagnostic impression cytology: a simple technique for the diagnosis of external eye disease
The technique of impression cytology was used for cytologic examination of conjunctival samples from 128 patients with a variety of external eye diseases. The method of sample collection and the staining procedure are described. Microscopic examination showed 62 cases with allergic conjunctivitis, 23 with bacterial or viral infection, 26 cases with changes typical of dry eye and 17 cases with non-specific chronic inflammation. The cytologic features and numerous applications of impression cytology are discussed.     

Significance of bizarre cells in cervical screening liquid‐based cytology: A prospective study of 15 cases

Objective

The aim of this study was to assess the significance of bizarre cells (cells of squamous origin with a superficial squamous cell‐type cytoplasm and characterised by multinucleation that produces bizarre nuclear shapes) in liquid‐based cytology (LBC) Papanicoaou (pap) smears with clinical and histological follow‐up correlation.

Methods

Fifteen patients, all with LBC samples containing bizarre cells, were identified in routine ThinPrep^® LBC workload. HPV testing was performed in each case using residual LBC material. Cytological‐histological correlations were reviewed.

Results

All 15 LBC samples contained bizarre cells and tested positive for high‐risk HPV types. Ten of the 15 cases were identified as atypical squamous cells ‐ cannot exclude an HSIL (ASC‐H) with secondary diagnosis of low‐grade squamous intraepithelial lesion (LSIL), while five cases were identified as high‐grade squamous intraepithelial lesion (HSIL), and a subsequent biopsy was recommended. Additionally, 13/15 cases underwent cone biopsy or hysterectomy within 1‐11 months, of which 10 showed histologically confirmed HSIL end‐points. LSIL was present in three cases. Bizarre cells were identified in the HSIL epithelium of five cone biopsies.

Conclusions

Identification of bizarre cells in LBC is straightforward and may facilitate diagnosis. The cytology of bizarre cells is associated with HSIL in cone biopsies. We recommend assigning LBC samples containing bizarre cells as ASC‐H with secondary diagnosis of LSIL.     

Blood and urine values of free-living European wildcats in Slovenia

Hematological, serum biochemistry, and urinalysis values were determined for nine (two females and seven males) adult, free-living European wildcats (Felis silvestris) in the Kocevje Forests of the southern Slovenia. Samples were collected from August 1999 to March 2001. Cats were anesthetized with ketamine and medetomidine. Blood samples were taken by jugular venipuncture and urine samples by bladder puncture. A control group of domestic cats (F. silvestris catus) was assembled to determine if differences exist among blood and urine values between free-living European wildcats and domestic cats. Hematological, biochemical, and urine parameters were similar to those of the control group. Values of glucose, blood urea nitrogen, albumin, mean corpuscular value, basophile count, and alanine aminotransferase were significantly higher than values of the control group. All urine samples contained white blood cells and proteins, and seven of them contained red blood cells.     

Dendritic cells produce inflammatory cytokines in response to bacterial products from Staphylococcus aureus-infected atopic dermatitis lesions

Atopic Dermatitis (AD) patients often acquire secondary skin infections resulting in increased inflammation. The increased inflammation occurs through the activation of multiple cell types including dendritic cells (DC). In this study, we investigated the activity of soluble products present in infected AD lesions by measuring the ability of patients’ wash fluids from a quantitative culture of lesions to activate DC. We found that wash fluid derived from AD lesions induced cytokine production by murine bone marrow-derived DC, including IL-1β, IL-6, ΙL-10, and tumor necrosis factor-α. The lipoprotein lipoteichoic acid (LTA) from Staphylococcusaureus was implicated as a potent stimulus in the wash fluids as only wash fluid samples that contained LTA exerted this activity, and exogenous LTA triggered similar DC cytokine activation. Wash fluid- and LTA-stimulated DC cytokine production required MyD88, but not the platelet-activating factor receptor (PAF-R), despite the ability of LTA to function through this receptor in keratinocytes. Thus, our results support a role for DC in the worsening of AD inflammation due to secondary bacteria infections.     

Exosome derived from CD137‐modified endothelial cells regulates the Th17 responses in atherosclerosis

The role of exosomes derived from endothelial cells (ECs) in the progression of atherosclerosis (AS) and inflammation remains largely unexplored. We aimed to investigate whether exosome derived from CD137‐modified ECs (CD137‐Exo) played a major role in AS and to elucidate the potential mechanism underlying the inflammatory effect. Exosomes derived from mouse brain microvascular ECs treated with agonist anti‐CD137 antibody were used to explore the effect of CD137 signalling in AS and inflammation in vitro and vivo. CD137‐Exo efficiently induced the progression of AS in ApoE^?/? mice. CD137‐Exo increased the proportion of Th17 cells both in vitro and vivo. The IL‐6 contained in CD137‐Exo which is regulated by Akt and NF‐КB pathway was verified to activate Th17 cell differentiation. IL‐17 increased apoptosis, inhibited cell viability and improved lactate dehydrogenase (LDH) release in ECs subjected to inflammation induced by lipopolysaccharide (LPS). The expression of soluble intercellular adhesion molecule1 (sICAM‐1), monocyte chemoattractant protein‐1 (MCP‐1) and E‐selectin in the supernatants of ECs after IL‐17 treatment was dramatically increased. CD137‐Exo promoted the progression of AS and Th17 cell differentiation via NF‐КB pathway mediated IL‐6 expression. This finding provided a potential method to prevent local and peripheral inflammation in AS.     

Fine needle aspiration cytology in the diagnosis of sinuses and ulcers of the body surface (skin and tongue)

Both fine needle aspiration (FNA) and conventional scraping were used to obtain cytodiagnostic samples from ulcers and sinuses of the skin (18 cases) and tongue (3 cases). These included 14 cases of tuberculosis, 5 squamous-cell carcinomas and 2 cases of nonspecific infection. The FNA smears contained characteristic tuberculous granulomas or epithelioid cells in 10 of the 14 tuberculosis cases and ample diagnostic material in 4 of the 5 carcinoma cases; in contrast the scraping smears predominantly contained necrotic material on which a diagnosis was not possible. Of the 19 cases of tuberculosis and malignancy, 12 were diagnosed only by FNA, 6 were diagnosed by both methods and the FNA sample was negative while the scrape was positive in 1 case. Hence, the diagnosis was made in 18 of 19 by FNA cytology, but in only 7 of 19 cases by scraping cytology, strongly indicating the diagnostic superiority of the former in diagnosing many lesions of the body surface.     

Analysis ofcis-regulatory elements involved in the activation of a member of chalcone synthase gene family (PsChs1) in pea

Cis-regulatory elements involved in the activation of the plant defense-related gene encoding chalcone synthase 1 (PsChs1) in pea (Pisum sativum L.) were examined by transient transfection, gel mobility shift assay andin vitro DNase I-footprinting analysis. Transient transfection assay revealed that a 61 bp DNA fragment spanning from –242 to –182 ofPsChs1 was required for the maximal promoter activity and possibly involved in the enhancement of elicitor-mediated activation. Nuclear isolate from elicitor-treated pea epicotyl tissues contained some factor(s) that specifically bound to this DNA fragment to form a complex with low mobility (LMC, low mobility complex) in gel mobility shift assay. DNase I-footprinting analysis of LMC revealed that among three protected regions detected in a 61 bp DNA fragment, two regions contained identical AT-rich sequence, TAAAATACT. Site directed mutation in either or both identical sequences, TAAAATACT to TGGAATACT, resulted in the reduction or loss in the ability to form LMC. Detailed analysis of 61 bp DNA fragment demonstrated that the region from –242 to –226 containing promoter-distal TAAAATACT motif was imperative for the maximal elicitor-mediated activation ofPsChs1.     

Cytologic and biomolecular diagnosis of polyomavirus infection in urine specimens of HIV-positive patients

OBJECTIVE: To evaluate the frequency of human polyomavirus reactivation in urine specimens from HIV-positive patients; compare the sensitivity of cytology, immunohistochemistry and molecular biology; differentiate viral genotypes; and correlate the results with urinary cytologic abnormalities. STUDY DESIGN: Urine specimens from 78 unselected HIV-positive patients were evaluated by means of cytology, immunohistochemistry and nested polymerase chain reaction (n-PCR) to evaluate the presence of polyomaviruses. Restriction fragment length polymorphism (RFLP) was carried out in positive cases in order to differentiate BK virus (BKV) from JC virus (JCV). CD4 cells and serum creatinine levels were evaluated as indices of immune status and renal function, respectively, whereas the presence of red blood cells was used as an index of urogenital damage. RESULTS: Cytologic evidence of polyomavirus infection was found in 17 samples and immunohistochemically confirmed in 9; another 6 cytologically negative cases were detected by means of immunohistochemistry. In all cases, only one or two cells showed typical viral inclusions or positive staining. n-PCR identified 44 positive samples, thus confirming all of the cytologically and immunohistochemically positive cases and detecting polyomavirus genome in a further 21. RFLP detected 39 JCV, 1 BKV and 4 JCV-BKV infections. No correlation was found between the presence or type of polyomavirus and immune status, but red blood cells were found more frequently in the positive than in the negative samples. Serum creatinine levels fell within the normal range in all cases. CONCLUSION: Molecular biology is the most sensitive tool for detecting polyomavirus urinary infection in HIV-positive patients and the only reliable method of differentiating JCV and BKV viral genotypes.     

Metal uptake byCaryophyllaceae species from metalliferous soils in northern Greece

Plant samples collected from 17 populations of 9 taxa of the generaDianthus, Minuartia, Scleranthus, andSilene (Caryophyllaceae), growing in 14 metalliferous sites of N Greece, and surface soil samples from their growth area were analysed for Cu, Pb, Zn, Cd, Ni, Cr, Fe, Mn, Ca, Mg. Metal uptake varies considerably between the populations. A resistance mechanism excluding Cu and Pb from the aerial parts of the plants over a wide range of the soil concentrations up to a critical soil level appeared to exist, whereas Zn concentrations in plants were analogously related to those of the soil.Scleranthus perennis subsp.perennis showed the highest Cu concentration (205 µg/g), whereasMinuartia cf.bulgarica hyperaccumulated Pb (1175 µg/g). Ca concentrations in plants were in most cases much higher than those in soil, whereas the contrary was true for Mg. As a result the Ca/Mg ratio, which was in almost all cases lower than 1 in the soil, was much increased in the plants.     

Seasonality and toxigenicity ofVibrio cholerae non-01 isolated from different components of pond ecosystems of Dhaka City,Bangladesh

Diarrhoea due toVibrio cholerae non-01 is common in Bangladesh. Four hundred and eighty samples, including plants, water, phytoplankton and sediment, were collected from five ponds in Dhaka every 15 days for one year.V. cholerae non-01 was isolated from 181 (38%) of the samples. Two peaks were evident: one in April and the other in August/September. Forty-three (23%) of the 181 isolates were examined for toxigenicity and 19% were cytotoxic to Y1 adrenal cells. This study provides evidence of the likely infectious nature of some ponds and may have relevance to the epidemiology of diarrhoea caused byV. cholerae non-01 in Bangladesh.