2011-2018年山东省淄博市"三热"病人疟原虫血片质量分析

目的　分析山东省淄博市“三热”病人疟原虫血片质量,为制订和调整该市消除疟疾后监测策略和措施提供科学依据。方法　对2011—2018年淄博市市级镜检站复核的全部“三热”病人疟原虫阴性血片和辖区内全部阳性血片进行复核,对血片制作、染色、清洁度以及复核结果进行分析。结果　2011—2018年淄博市市级镜检站共复核疟原虫阴性血片2 141张,阳性血片39张。阴性血片制片合格率为99.44%,染色合格率为97.62%,清洁度合格率为93.65%,复核一致率为100%,未发现漏检;复核阳性血片39张,血片制片合格率为46.15%,染色合格率为61.54%,清洁度合格率为76.92%,复核一致率为97.44%,其中虫种分型错误1张。淄博市各区（县）血片制作合格率和染色合格率差异均有统计学意义（P值均 ＜ 0.05）,血片清洁度合格率差异无统计学意义（[χ2] = 13.72,P > 0.05）;2011—2018年淄博市各年度血片制作、染色和清洁度合格率差异均有统计学意义（P值均＜ 0.05）。结论　淄博市各区（县）血片质量较高,但市级血片质量有待提高。今后应进一步加强基层疟原虫镜检培训力度和血涂片质量控制,促进血片质量不断提高,以保证疟疾消除后监测阶段疟原虫镜检能力     

Quality control of smear microscopy for acid-fast bacilli: the case for blinded re-reading.

SETTING: Quality control of sputum smear microscopy, which is essential for ensuring correct tuberculosis (TB) diagnosis, is often performed through the unblinded rereading of all positive slides and a sample of negative slides. OBJECTIVE: To assess misclassification error introduced by knowledge of prior results. METHODS: The Southern Vietnam Regional TB Laboratory prepared three gold-standard sets of 750 slides: an unblinded set, an unblinded set in which 13% of negative slides were replaced by weakly positive slides purposefully mislabelled as negative, and a blinded set. Six provincial technicians who normally perform district quality control each reread 125 slides from each set. RESULTS: In the three sets only one negative slide was misread as positive. In the unblinded set (referent), 2.9% (9/311) positive slides were misread as negative, compared with 18.7% (57/305) in the blinded set (prevalence ratio [PR] = 6.5; 95% confidence interval [CI] 3.3-12.8; P < 0.001), and 11.3% (33/293) in the unblinded set with mislabelled slides (PR = 3.9; 95%CI 1.9-8.0; P < 0.001). CONCLUSIONS: False-negative error was more common than false-positive error. Knowledge of prior reading influences re-reading. Blinded re-reading of systematically selected slides would appear preferable, although this method requires high levels of proficiency among quality control technicians.     

Prognostic significance of skin and subcutaneous fat sequestration of parasites in severe falciparum malaria

Intradermal blood smear, histopathologic and immunohistologic studies were performed in severe malaria (n=10) and uncomplicated malaria (n=10) patients during positive parasitemia and within 6 hours after negative parasitemia by finger prick smears. Intradermal blood smears showed asexual forms and intraleukocytic pigments when finger prick blood smears showed negative results; however intradermal blood smear did not indicate disease severity within 6 hours after negative parasitemia by finger prick. Histopathologic findings showed 15 fold higher parasitized red blood cells sequestered in vessels of subcutaneous fatty tissue in severe malaria than in uncomplicated malaria (p<0.001) and may indicate disease severity. A panel of polyclonal antibodies against cytokines applied to skin biopsies clearly detected a higher titer against tumor necrosis factor-alpha (TNFalpha) and interleukin-10 (IL-10) in dermal vessels and stratum granulosum respectively, in severe malaria compared with uncomplicated malaria. Results of the study suggest that histopathology and immunohistology of skin and subcutaneous fatty tissue may indicate prognostic severity of malaria and may be associated with focal accumulation of cytokines.     

2014年常州市“三热”病人疟原虫检测血涂片质量分析

目的 目的 了解常州市 “三热” 病人疟原虫检测血涂片制作质量, 为消除疟疾后的监测提供技术保障。方法 方法 抽取 2014年常州市各市 （区） 不少于3%的已检 “三热” 病人疟原虫检测阴性血涂片和全部阳性血涂片, 市级疟疾镜检站对血涂 片的制作、 染色、 清洁度和镜检结果进行复核, 并对复核结果进行统计分析。结果 结果 共复核阴性血涂片996张, 复核率为 4.52%, 血涂片制作、 染色、 清洁度合格率分别为92.87%、 93.27%和94.48%; 复核阳性血涂片34张, 未发现误检和漏检。7 个市 （区） 血涂片制作、 染色合格率均在90%以上; 除戚墅堰区清洁度合格率为81.36%外, 其他市 （区） 均＞90%。一级、 二 级、 三级医院血涂片制作、 染色、 清洁度合格率均＞90%。血涂片质量缺陷以沉渣、 血膜制作不规范及厚膜脱落为主, 分 别占25.91%、 21.76%和19.17%。结论 结论 常州市各市 （区） 疟原虫血涂片质量较好, 今后要进一步加强培训和指导, 以保证 消除后监测阶段的疟原虫镜检能力。     

An international study of the performance of sample collection from patients

BACKGROUND AND OBJECTIVES: Collection of a blood sample from the correct patient is the first step in the process of safe transfusion. The aim of this international collaborative study was to assess the frequency of mislabelled and miscollected samples drawn for blood grouping. MATERIALS AND METHODS: Hospitals in 10 countries provided data on sample error rates during a period of at least 3 months, including the last quarter of 2001. Mislabelled samples were defined as those not meeting local criteria for acceptance by the laboratory. Miscollected samples [wrong-blood-in-tube (WBIT)] were defined as samples in which the blood group result differed from the result on file from prior testing. WBIT rates were corrected for the proportion of repeat samples and for undetectable errors occurring as a result of chance collection of blood from the wrong patient with the same ABO group. Participants also completed a questionnaire on current policies regarding sample collection. RESULTS: A total of 71 hospitals completed surveys describing policies related to sample collection. Sixty-two hospitals provided usable data on the frequency of mislabelled and miscollected samples. Mislabelled and miscollected samples were common. Based on results from over 690,000 samples, the median hospital performance resulted in a rate for mislabelling of 1 in every 165 samples (6.1 per 1000; interquartile range 1.2-17 per 1000). The presence of national patient identification systems in Sweden and Finland was associated with rates of miscollected samples that were too low to estimate. Outside these nations, miscollected samples demonstrating WBIT occurred at a median rate of 1 in every 1986 samples (0.5 per 1000; interquartile range <0.3-0.9 per 1000). There was great variation worldwide in the reported frequency of mislabelled samples, probably resulting from variation in policies for sample acceptance. Miscollected samples occurred at a more constant rate. CONCLUSIONS: The rate of mislabelled samples and miscollected samples is 1000-10,000-fold more frequent than the risk of viral infection. Rates of mislabelled samples and WBIT can be tracked as key indicators of performance of an important step in the clinical transfusion process. WBIT episodes represent important 'near-miss' errors. By providing baseline performance data for the collection of patient blood samples, this study may be useful in formulating future national standards of performance for sample collection from patients.     

Terminal deoxynucleotidyl transferase in acute leukemias by direct immunofluorescence

TdT (terminal deoxynucleotidyl transferase) can be detected by radio enzymatic assay, biochemical assay in cell extracts, serum or plasma, and intracellularly in the smear by indirect immunofluorescent methods. The IgG fraction of anti-TdT serum is conjugated with fluoresceinisothiocyanate and used directly on the cytospin smears of methanol fixed bone marrow/blood smears. The mice thymocytes and peripheral mononuclear cells of healthy donors were used as positive and negative controls, respectively, for TdT. 64% of our cases of ALL were found to be TdT+. The lymphoblasts of L1 morphology (FAB classification) were more frequently positive for TdT as compared to blasts with L2 morphology. 71% of our cALLa positive blasts in acute lymphoblastic leukemias were TdT+ve as compared to 58% of T-ALL blasts. 75% of PAS positive ALL cases were positive for TdT as well. Only 57% of the cases when acid phosphatase showed unipolar positivity (T type) were positive for TdT. 12% of cases with acute myeloid leukemia (6/47) were TdT+ve and 33% of CML in blastic crisis had TdT+ve blasts. Biochemical assay and IF assay for TdT were in good correlation in our study.     

Comparison of two versus three smears in identifying culture-positive tuberculosis patients in a rural African setting with high HIV prevalence.

SETTING: Karonga district, northern Malawi. OBJECTIVE: To compare the sensitivity and specificity of two versus three smears for the diagnosis of pulmonary tuberculosis in a setting with high HIV prevalence. DESIGN: A total of 1992 pulmonary tuberculosis suspects with three sputum smears taken over a 2-7 day period and at least one culture result were studied. Smears were auramine stained and examined using fluorescence microscopy, and positives were confirmed with Ziehl-Neelsen staining and light microscopy. Cultures were set up on L?wenstein-Jensen media. True negative and positive status was defined on the basis of culture. The sensitivity, specificity, and positive and negative predictive values of two and three smears were compared. RESULTS: Compared to culture, the sensitivity, specificity, and positive and negative predictive values of three smears were 70%, 98%, 92%, and 92%, respectively. Restriction to the first two smears gave similar results. Of those detected as smear-positive using three smears, at least 97% would have been detected by two. Among those with HIV serology results available, the sensitivity of two smears for detecting culture-positive tuberculosis was identical to that using three. CONCLUSION: In this setting, using fluorescence and light microscopy, collecting two smears rather than three would only marginally reduce sensitivity and would slightly improve the specificity of diagnosis of tuberculosis; this is unaffected by HIV status. The potential for improving specificity is important because of the costs of misdiagnosis. In practice, both sensitivity and specificity may be increased due to the time saved by examining two rather than three smears.     

Cellular interferon-gamma based assay for diagnosis of pulmonary tuberculosis

A major breakthrough in recent years has been the development of an in vitro assays that measure T-cell release of interferon gamma (IFN-gamma) in response to stimulation with antigens specific to Mycobacterium tuberculosis (MTB) such as early secreted antigenic target 6 (ESAT6) and culture filtrate protein 10 (CFP10). This study aimed at evaluating the diagnostic potential of IFN-gamma in vitro production assay for diagnosis of pulmonary tuberculosis. The study included 40 patients from Abbasia Chest Hospital, Cairo, Egypt. Thirty patients had the provisional clinical and radiological diagnosis of pulmonary tuberculosis (TB), twenty of them had positive acid fast (AF) sputum smears (group I), and ten had negative smears (group II). Bacteriological confirmation of TB was based on cultivation on L.J solid media (group I & II), and by BACTEC radiometric assay (group II). Ten patients with non tuberculous chest diseases were also included as a control group (group III). Effector T cells, within the peripheral blood mononuclear cells, which secrete IFN-gamma in response to stimulation by antigens specific for MTB (ESAT-6 and CFP-10) were analyzed in all subjects using a commercially available assay based on the enzyme linked immunospot technique. We demonstrated that 24 out of 30 (80%) suspected tuberculous patients were bacteriologically confirmed based on positive AF smears and/or culture. In vitro IFN-gamma release assay was positive in 22 of them giving a sensitivity of 91.7%. In the remaining 6 who were not confirmed bacteriologically, the assay showed positive results in 4 (66.7%) of them. Based on bacteriological diagnosis as the gold standard for diagnosis of pulmonary TB, the specificity of the assay was found to be 75%. However the assay results were negative in all non tuberculous patients (group III). It is concluded that the used in vitro IFN-gamma release assay has the potential to become a useful diagnostic tool for active tuberculosis.     

Blinded rechecking of sputum smears for acid-fast bacilli to ensure the quality and usefulness of restaining smears to assess false-positive errors.

SETTING: Microscopy Centres, Velliyur Tuberculosis Unit, Tiruvallur District, Tamil Nadu, India. PROCEDURES: Twelve microscopy centre laboratory technicians examined 41978 direct sputum smears for acid-fast bacilli. Senior Tuberculosis Laboratory Supervisors (STLS) checked all positive smears (4696) and 10% of negative smears (4776) in an unblinded fashion as per Revised National Tuberculosis Control Programme (RNTCP) guidelines. Ten per cent of the positive and negative slides and another 10% of unchecked negative slides were selected systematically for blinded rereading at the Tuberculosis Research Centre (TRC); 422 slides were reread without and with restaining. RESULTS: Unblinded checking by STLS of the smears read by the laboratory technicians yielded 95 to 100% agreement. Blinded rereading at the TRC revealed that false-negative errors were greater among the laboratory technicians (2-7%) than the STLS (0-3%). Restaining and blinded rereading of slides reduced false-positive errors from 27% to 7%. CONCLUSIONS: Blinded rereading at the reference centre facilitates assessment of laboratory technicians and STLS. Restaining before rereading the smears was found to be useful for precise estimation of false-positive errors.     

Rapid diagnosis of bacteremia in adults using acridine orange stained buffy coat smears

The use of acridine orange stained buffy coat smears was assessed as a rapid screening test for bacteremia in adults. A total of 356 consecutive blood cultures were submitted with simultaneous anticoagulated blood samples, from which a buffy coat smear was prepared and stained with acridine orange (100 mg/L; pH 3.0). Forty-one of 356 blood samples (12%) yielded organisms in the blood culture system. Compared to blood culture, the overall sensitivity of acridine orange stained buffy coat smears was 16%, specificity 88%, and positive predictive value 13%. There was no statistically significant difference in performance of the test among patients who had fever greater than 39°C and/or shock. The low sensitivity and specificity of the test makes it unsuitable as a means of rapid screening for adults with suspected bacteremia.     

Comparison of visual inspection and Papanicolau (PAP) smears for cervical cancer screening in Honduras: should PAP smears be abandoned?

OBJECTIVE: To compare visual inspection with acetic acid (VIA) to Papanicolau (PAP) smears in a community setting in a developing nation. METHODS: Women undergoing cervical cancer screening in Honduras received either VIA and PAP smears (VIA/PAP group) or PAP smears alone (PAP-only group). Local healthcare providers performed PAP screening. A VIA-trained nurse performed VIA exams. All PAP smears were processed in Honduras. PAP smears from the VIA/PAP group were reviewed in the United States. Women with positive VIA or PAP tests were offered colposcopy. We compared the relative accuracy of PAP smears and VIA and the proportions of women completing follow-up colposcopy after positive screening tests. RESULTS: In total, 1709 PAP smears were performed including women from both the VIA/PAP and PAP-only groups. Nine PAP smears were positive (0.5%). Three women completed colposcopy (33%). All three had biopsy-confirmed dysplasia. In the VIA/PAP group (n = 339), 49 VIA exams were abnormal (14%) and two PAP smears were abnormal when read in Honduras (0.6%). When reviewed in the United States, 14 of the 339 PAP smears were abnormal (4%). Forty women (83%) completed follow-up colposcopy after a positive VIA exam. Twenty-three had biopsy-proven dysplasia. All 23 dysplasia cases had negative PAP smear readings in Honduras; four PAP smears were reclassified as positive in the United States. CONCLUSIONS: Although few developing countries can maintain high-quality PAP smear programmes, many governments and charitable organizations support cervical cancer screening programmes that rely on PAP smears. This study underscores the need to promote alternative technologies for cervical cancer screening in low-resource settings.     

How many sputum smears are necessary for case finding in pulmonary tuberculosis?

We reviewed the laboratory registers of 42 tuberculosis (TB) diagnostic centres in the southern region of Ethiopia to determine the value of submitting serial sputum samples for the diagnosis of pulmonary TB (PTB) and estimate the proportion of suspects that are smear positive. A total of 15,821 TB suspects submitted three smears each (47,463 smears) in 2000 with a median of 228 per centre. The smear positivity rate (two or more positive smears) was 25%, with a range of 16.8-36.4% per zone. This exceeds the international recommendations of examining 10 suspects to identify one case. A total of 4099 (26%) of the suspects had at least one positive smear with 3753 (91.6%) of the first specimens being positive. A further 303 (7.4%) were negative in the first specimen but had a positive second specimen and 42 (1%) suspects had two negative specimens followed by a positive third smear. The value of the third sputum is negligible as 99% of the cases were identified from the first and second specimens. Reducing the number of specimens to two or even one would have multiple advantages in countries where laboratories are usually over-burdened and are not easily accessible to the population. Submission of two specimens on the same day could improve compliance in submitting samples and collecting results as the number of diagnostic visits would be reduced without significant loss of sensitivity.     

Characteristics and outcome of tuberculosis patients whose sputum smears are positive at or after 5 months of treatment.

A country-wide survey was carried out to assess the management of new smear-positive pulmonary TB (PTB) patients whose sputum smears were recorded as positive 5 months or later during treatment. During 2000 and 2001, there were 250 patients, of whom 161 (64%) had positive smears at 5 months and 89 at 7 months. Several inconsistencies and inadequacies in management were identified which need to be remedied: 7% of patients were assessed on one sputum specimen instead of two, and 17% on the basis of one positive smear result; 47% of patients with 5-month positive smears and 52% with 7-month positive smears had sputum smears examined too early or too late; 14% of patients with 5-month positive smears continued treatment, and over 60% of these were recorded as 'cured'.     

Clinical diagnosis of cutaneous leishmaniasis: a comparison study between standardized graded direct microscopy and ITS1-PCR of Giemsa-stained smears

Parasitological diagnosis of cutaneous leishmaniasis is absolutely necessary before treatment. Direct microscopy of scrapings taken from the margins of skin lesions is the most commonly used method for clinical diagnosis of leishmaniasis. In this study to evaluate the usage of stained smears as samples for PCR and the possible advantage of PCR, we compared the sensitivity of the diagnosis of Giemsa-stained skin scrapings by standardized graded direct microscopy with that of ITS1-PCR with the material of the same area of the slide. Three 5mm x 5mm squares were marked on each of the 20 Giemsa-stained touch smears from 20 clinically diagnosed Palestinian patients. Out of the 60 squares scanned for amastigotes under 100x oil-immersion light microscopy, 45 (75%) gave usable results and 23 of these were positive for Leishmania. Fifteen (25%) squares could not be scanned microscopically, 12 because of staining that was too thick and 3 because of inadequate staining. DNA from each scanned square was extracted separately after microscopy and run through ITS1-PCR. Of the 23 microscopy-positive squares, 20 (87%) of these were positive by PCR. Of the three that were negative, one failed to extract for DNA, the second showed only one amastigote in the entire square, and the third was normally graded as +1 but was not amplified for unknown reasons. Of the 22 squares negative for microscopy, 18 (82%) were ITS1-PCR positive. Additionally, all three improperly stained squares were ITS1-PCR positive. Of the 12 darkly stained squares, 11 were positive. A negative control group of 15 German individuals from which Giemsa-stained slides containing three squares each was prepared and these slides were also microscopically scanned and tested by ITS1-PCR. Both tests were negative with both methods. Compared to microscopy (data in parenthesis), PCR showed a sensitivity of 87% (37%) and a specificity of 100% (100%). We have concluded that Giemsa-stained smears are a readily usable sampling method for PCR and that ITS1-PCR is far more sensitive than microscopy.     

Bleach-digested sputum smears for the diagnosis of TB in HIV-infected individuals

We describe the performance of bleach-digested Zeihl-Neelsen (ZN) smears in TB suspects with/without HIV. In total, 51 (26%) and 62 (31%) out of the first 198 spot and digested smears were positive. Seven of the 30 HIV-positive patients had TB and their ZN smears were negative, scanty or 1 +. Six of seven digested smears were scanty. Forty-two of 115 HIV-negative patients had TB. Eleven (26%) of their digested smears were negative, 12 (29%) scanty and 19 (45%) positive. Despite the lower bacilli numbers of HIV-positive patients, the technique had sensitivity and specificity similar to that in HIV-negative patients.     

Evaluation of a synthetic oligonucleotide probe for diagnosis of Plasmodium falciparum infections

A radiolabeled synthetic oligonucleotide was evaluated as a diagnostic probe specific for Plasmodium falciparum using blood samples lysed directly on nitrocellulose filters. The probe technique successfully diagnosed malaria in experimentally infected chimpanzees that had 0.001% parasitemias (50 parasites/microliter) as determined by blood smears, and in 1 chimpanzee whose blood smear was negative, but whose blood was culture-positive for P. falciparum. In a double blind study of 50 patient samples from the Philippines, the probe results correlated well with blood smear results when the autoradiographs were read after 4-8 hr exposure. The results indicate that the oligonucleotide probe may be useful in the rapid and specific diagnosis of P. falciparum infection.     

我国广西食蟹猴养殖场2种血液寄生原虫感染调查

目的 目的 调查我国广西壮族自治区某食蟹猴养殖场血液寄生原虫感染状况, 为人体血液寄生原虫的防控提供科 学依据。方法 方法 采集广西壮族自治区南宁市某食蟹猴养殖场猴血液样本993份, 全部制作FTA卡样本, 涂制薄血膜550 份。将样本混合, 以巢式PCR及普通PCR方法分别检测FTA卡保存的猴血样本中巴贝虫以及疟原虫。检测阳性组再分 别进行单个样本检测, 阳性样本对应的薄血膜进行姬姆萨染色后再镜检。 结果 结果 经巢式PCR检测, 田鼠巴贝虫检出阳 性率为6.95% （69/993）; 仅1例经PCR检出猪尾猴疟原虫阳性。22份PCR检测巴贝虫阳性的血液样本经染色镜检, 共16 份检出巴贝虫, 其显微镜下观察到红细胞内有环状体, 无疟色素。结论 结论 我国广西地区食蟹猴的田鼠巴贝虫感染率较 高, 其可能在传播中起保虫宿主的作用; 在筛查田鼠巴贝虫低密度感染时, 巢式PCR方法敏感性较高。     

Comparison of scanty AFB smears against culture in an area with high HIV prevalence.

To verify among tuberculosis (TB) suspects attending hospitals in Abuja, Nigeria, if sputum smears graded as scanty are false-positive, sputum smears from 1068 patients were graded with the International Union Against Tuberculosis and Lung Disease classification. One specimen was cultured. Eight hundred and twenty-four (26%) smears were positive, 137 (4%) scanty and 2243 negative. Of 1068 cultures, 680 (64%) were positive. One hundred and thirty (95%) scanty and 809 (98%) positive smears were culture-positive. Twelve of 18 patients with a single scanty smear and 51 of 52 with > or = 2 scanty smears were culture-positive. Fewer than < 5% scanty results, < 1% of the patients treated for TB, are false-positive.     

Quality assurance studies in eight State tuberculosis laboratories in India.

SETTING: Tuberculosis Research Centre, Chennai, India. OBJECTIVE: To undertake quality assurance studies in sputum smear microscopy at eight State-level laboratories in India using a panel of stained smears. DESIGN: Coded panels of stained smears (100 slides in rounds I-IV and 50 slides in Round V), comprising different grades of positivity, were sent to each centre at 6-monthly intervals. The results obtained were analysed for consistency of positive and negative results and overall agreement, as well as discordance. RESULTS: Consistency of positives ranged from 38% to 100%, indicating under-reading at some sites. The negative consistency was better, however, with only five of the total of 95 readers in all rounds yielding a consistency of less than 100%. Considering overall agreement, seven of the eight centres showed an agreement of over 90%. Four of the eight centres gave no false-positive result. For the remaining centres the false positivity rate varied from 2% to 7%. A wide variation was also observed in the proportion of false-negatives (3%-52%). CONCLUSIONS: This study helped in the evaluation of the performance of individual laboratories and in identifying the drawbacks of this system of proficiency testing.     

Blood and lymph node T lymphocytes in cutaneous T cell lymphoma: evaluation by light microscopy.

Cytology of peripheral blood and lymph node lymphocytes from a group of unselected patients with cutaneous T cell lymphoma (CTCL) was studied by light microscopy. Twenty of 45 patients had circulating lymphocytes with convoluted nuclei recognized in routine Wright-Giemsa-stained peripheral blood smears. Cytocentrifuge preparations of E-rosetted lymphocytes showed that greater than 10% of the T cells had convoluted nuclei in each of 16 patients with positive blood smears and in six of 17 whose blood smears were negative or inconclusive. Peripheral blood involvement with greater than 10% convoluted T cells was most frequent in patients with erythroderma (100%) including those with normal of decreased lymphocyte counts, and was not uncommon in patients with mycosis fungoides in the plaque or tumor phase (42%). The light-microscopic morphology of the abnormal cells found in the patients with the plaque or tumor phase of mycosis fungoides was not distinguishable from that of the erythrodermic patients. Increased percentages (less than 15%) of T cells having convoluted nuclei were also found in the lymph node cell suspensions from CTCL patients with adenopathy (18 of 25 patients). These results suggest that a high frequency of extracutaneous involvement occurs in patient with CTCL, the clinical significance of which remains to be determined.