AIM--To assess the value of detecting human papillomavirus (HPV) DNA in false negative archival cervical smears in population based screening programmes for cervical cancer. METHODS--Cytomorphologically classified false negative archival Pap smears (n = 27) taken from 18 women up to six years before cervical cancer was diagnosed were blindly mixed with 89 smears from hospital patients with a variety of gynaecological complaints and tested for HPV by the polymerase chain reaction (PCR). Corresponding cervical cancer biopsy specimens were also available for HPV analysis. Neither the examining cytopathologist nor the molecular biologist was aware of the study design. RESULTS--HPV DNA was detected in the smears of 16 patients with cervical cancer missed previously by cytology. HPV 16 and 18 were found predominantly in those smears taken up to six years before the diagnosis of cervical cancer. The smears of the two remaining patients were reclassified as inadequate for cytology or contained no suitable DNA for PCR. In 15 patients the same HPV type could be found in the smears and the cervical cancer biopsy specimens. CONCLUSIONS--The results indicate that high risk HPV types can be detected in archival smears classified as false negative on cytology and that cytological screening errors may be reduced if combined with PCR testing for HPV.

The efficiency of a freeze-thaw method, a proteinase K/Tween 20 lysis method and a guanidinium isothiocyanate/silica beads method for DNA extraction from fixed and Papanicolaou-stained cells from the cervical cancer cell line Siha was measured by beta-globin polymerase chain reaction (PCR). The GTC/silica beads method, which appeared superior, revealed a human papillomavirus (HPV) general primer-mediated PCR sensitivity of 50-500 copies of HPV 16 per sample using dilutions of fixed and stained Siha cells. Application to archival cervical smears (n = 116) revealed that the yield and size of amplifiable DNA decreases with storage time. The longer the storage time, the more repetitions of the whole procedure, including the lysis step, were required to extract sufficient amplifiable DNA. In this way, an overall beta-globin PCR positivity for 98% of the smears was reached. Further analysis revealed that a maximum size of 200 bp could be amplified from smears stored for up to 9 years. The method was validated by demonstrating by PCR the same HPV types in archival smears and corresponding cervical biopsies of cervical cancer patients. In conclusion, the GTC/silica beads method appears suitable to process archival cervical smears for HPV detection by PCR. provided that stepwise adjustments are made until beta-globin PCR positivity is obtained and primers are chosen which amplify a maximum of about 200 bp.

Airway care interventions may prevent accumulation of airway secretions and promote their evacuation, but evidence is scarce. Interventions include heated humidification, nebulization of mucolytics and/or bronchodilators, manual hyperinflation and use of mechanical insufflation-exsufflation (MI-E). Our aim is to identify current airway care practices for invasively ventilated patients in intensive care units (ICU) in the Netherlands. A self–administered web-based survey was sent to a single pre–appointed representative of all ICUs in the Netherlands. Response rate was 85% (72 ICUs). We found substantial heterogeneity in the intensity and combinations of airway care interventions used. Most (81%) ICUs reported using heated humidification as a routine prophylactic intervention. All (100%) responding ICUs used nebulized mucolytics and/or bronchodilators; however, only 43% ICUs reported nebulization as a routine prophylactic intervention. Most (81%) ICUs used manual hyperinflation, although only initiated with a clinical indication like difficult oxygenation. Few (22%) ICUs used MI-E for invasively ventilated patients. Use was always based on the indication of insufficient cough strength or as a continuation of home use. In the Netherlands, use of routine prophylactic airway care interventions is common despite evidence of no benefit. There is an urgent need for evidence of the benefit of these interventions to inform evidence-based guidelines.

In recent years, general primer-mediated PCR assays have been developed to detect a broad spectrum of human papillomavirus (HPV) genotypes. In this study, a procedure enabling a simple group-specific differentiation of high-risk (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -54, -56, and -58) and low risk (HPV-6, -11, -34, -40, -42, -43, and 44) HPVs following an HPV general primer-mediated (GP5+/GP6+) PCR is presented. By computer-assisted sequence analysis, oligonucleotides (30-mers) specific for 19 different HPV genotypes were selected from the internal part of the 150-bp GP5+/GP6(+)-amplified region. These oligo probes were tested for specificity in a Southern blot analysis of PCR products derived from the same panel of HPV types. No cross-hybridizations were found. The sensitivities of the oligo probes varied from the femtogram level for the well-amplified HPV types like HPV-16 and -18 to the picogram level for the less-well amplified HPV types like HPV-39 and -51. These sensitivities were reached when the oligo probes were applied both individually and in a cocktail. On the basis of these results, two cocktail oligo probes that enabled a specific and sensitive differentiation between low- and high-risk HPV types were composed.

The aim of this study was to investigate the distribution of 27 mucosotropic human papillomavirus (HPV) genotypes (HPV 6, 11, 13, 16, 18, 30, 31, 32, 33, 35, 39,4 0, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61 and 66) in cytomorphologically abnormal cervical scrapes (Pap IIIa‐Pap IV; n = 1, 373) using the polymerase chain reaction (PCR) method on crude cell suspensions. The scrapes were analyzed for the presence of HPV DNA by HPV general‐primer‐mediated PCR (GP‐PCR), which allows the detection of a broad spectrum of HPV types at the subpicogram level. Subsequently, 2 HPV typing procedures based on either type‐specific PCR (for HPV 6, 11, 16, 18, 31 and 33) or characterization of GP‐PCR products by hybridization (for HPV 13, 30, 32, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61 and 66) were applied. Increasing total HPV prevalence was found with increasing severity of dysplasia from 71% in Pap IIIa to 100% in Pap IV scrapes (carcinoma in situ). The scrapes which were positive by type‐specific PCR included 47% cases of Pap IIIa, 71% cases of Pap IIIb and 90% cases of Pap IV. Moreover, 12% of Pap IIIa scrapes, 6% of Pap IIIb scrapes and 8% of Pap IV scrapes revealed positivity for one or more of the remaining HPV types, as determined by successive hybridizations of the GP‐PCR products. Taking the typing data together, we noted that the level of HPV heterogeneity decreased from 22 different HPV types (HPV 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 54, 55, 56, 58, 59, 61 and 66) detected in the group of Pap IIIa scrapes to 13 (HPV 6, 11, 16, 18, 31, 33, 35, 45, 51, 52, 58, 59 and 61) and 10 HPV genotypes (HPV 6, 16, 18, 31, 33,45, 51, 52, 54 and 58) in the Pap IIIb and Pap IV classes, respectively. An increasing prevalence rate from Pap IIIa to Pap IV was found for HPV 16, 18, 31, 33, 45 and 54. The prevalence rate of identified HPV genotypes increased from 59% in Pap IIIa to 98% in Pap IV, indicating that almost all high‐risk HPV genotypes related to cervical cancer in The Netherlands have been characterized.