Screening tuberculosis suspects using two sputum smears.

SETTING: Ntcheu district hospital, Malawi. OBJECTIVE: To assess a screening strategy for tuberculosis (TB) suspects using two sputum smears. DESIGN: A strategy of screening all TB suspects with two sputum smears for 6 months (1 July-31 December 1998) was compared with the period 1 January to 30 June 1998 during which the strategy of screening TB suspects with three sputum smears was in use. All chest radiographs of patients with negative sputum smears were assessed, and in those with pulmonary cavities and extensive disease a third sputum smear was examined. Data were collected from the laboratory sputum register and the TB register. The two 6-month periods were compared. RESULTS: In the laboratory register, using a two-sputum strategy, 186 (16%) of 1152 TB suspects were smear-positive, a result that was no different than when the three-sputum strategy was used, where 173 (16%) of 1106 TB suspects were smear-positive. The clinical pattern of TB using the different screening strategies was similar, with 58% of registered patients smear-positive with the two-sputum strategy and 54% smear-positive with the three-sputum strategy. In the first 6 months 3177 sputum smears were examined compared to 2266 smears in the second 6 months, a 29% reduction in the number of smears examined. The cost of consumables using the strategy of three sputum smears was USD $731 compared with USD $521 using the strategy of two sputum smears. C O N C L U S I O N S: Screening TB suspects using two sputum smears is as effective as screening using three sputum smears, and is associated with less laboratory work and savings in costs.     

Stability for long-term storage and reproducibility of positivity in the panel test slide prepared with the polyacrylamide-based artificial sputum

OBJECTIVE: A novel artificial sputum has been developed using polyacrylamide, cultured THP-1 cell and BCG-Pasteur. Smears prepared with this artificial sputum are similar to actual sputum and has feasibility to set any positivity grades. Long-term storage and reproducibility of the positivity was examined to support further availability. METHOD: The artificial sputa were stored for up to 9 months at room temperature, 4 degrees C and -20 degrees C. Then, smears were prepared and their macroscopic and microscopic appearance were examined compared with smears from freshly prepared artificial sputum. Furthermore, smears with different positivities (+/-, 1 +, 2 + and 3 +) were prepared and examined by several trained technicians, and the reproducibility of the original sputum positivity was determined. RESULTS: Macroscopic and microscopic appearance of smears prepared from long-term stored artificial sputum showed little changes compared with smears of freshly prepared artificial sputum. The positivity of these smears fell in their original grade. A total of 91 smears were prepared from artificial sputum with different positivity and examined by trained technicians. Although 3 out of 36 +/- smears were determined as negative, all of the remaining smears were evaluated correctly. DISCUSSION: This study confirmed that the artificial sputum and the smears have long-term storage stability and reproducibility in the positivity. These results suggest that the artificial sputum can be widely used to perform external quality assessment in many countries, including high prevalence countries.     

Effects of grinding surgical tissue specimens and smear staining methods on Buruli ulcer microscopic diagnosis

To optimize Buruli ulcer (BU) microscopic diagnosis, we compared two smear preparation methods from tissue specimens: smears made with tissue suspension after grinding and smears made directly with unground tissue. We also compared two smear staining methods: auramine and Ziehl-Neelsen (ZN). IS 2404-PCR was used as reference method. One hundred and thirty-one surgical tissue specimens from patients suspected of having BU were analyzed. Both smear preparation methods and both staining methods were equivalent in any combination. Thus we recommend ZN stained smears of unground tissue for peripheral treatment centres.     

Fibreoptic bronchoscopy in smear-negative pulmonary tuberculosis

Fifty smear-negative pulmonary tuberculosis patients underwent fibreoptic bronchoscopy. Bronchial aspirate smears of twelve patients and post-bronchoscopic sputum smears of fourteen patients were positive for acid-fast bacilli (AFB). Bronchial biopsy provided the diagnosis in 9 out of 30 patients. Brush smears were positive in 28 patients, being the only positive sample in ten cases. A high yield from brush smears was obtained due to their preparation from caseous material wherever visible in the bronchi. With these results a rapid diagnosis was established in 36 of the 50 patients. When culture results were available, a definite diagnosis of tuberculosis was made in 45 of the patients. The yield from brush smears was found to be significantly better when compared to bronchial aspirate smears (p less than 0.01) and post-bronchoscopic sputum smears (p less than 0.01).     

Variations in helminth faecal egg counts in Kato-Katz thick smears and their implications in assessing infection status with Schistosoma mansoni

Examination of stool specimens by Kato-Katz (K-K) thick smears is the standard method recommended by the WHO for field diagnosis of intestinal schistosomiasis. However, there is increasing concern that this technique has low diagnostic sensitivity. In 326 study subjects, we compared the diagnostic yield of examining one, three or five Kato-Katz thick smears prepared from one stool specimen using 41.7 mg templates. In a subset of 169 subjects who had no demonstrable Schistosoma mansoni eggs in their first three Kato-Katz thick smears, we assessed the comparative advantage of examining an additional three Kato-Katz thick smears from another stool specimen, taken four weeks later, to that of cumulative yield obtained by examining all five Kato-Katz thick smears derived from the first stool specimen. For all helminth infections, single Kato-Katz thick smear-based prevalence estimates were significantly lower than those obtained from triplet or quintet Kato-Katz thick smears. Prevalence of S. mansoni infection based on single, triplet and quintet Kato-Katz thick smears from one stool specimen were 31.3%, 45.7% and 52.1%, respectively. Prevalence estimate of S. mansoni based on quintet Kato-Katz thick smears from the first day stool specimens was not different from cumulative estimate obtained with two triplet Kato-Katz thick smears from two stool specimens, 52.1% and 52.8%, respectively. In conclusion, either examination of quintet Kato-Katz thick smears from one stool specimen using 41.7 mg template or initial triplet Kato-Katz thick smears from one stool specimen, and if these are negative, followed by examination of additional triplet Kato-Katz thick smears from subsequent day stool specimen can adequately assess individuals for infection status with S. mansoni.     

A Comparative Study of Bone Marrow Squash and Wedge Aspiration Smears

Bone marrow examination entails study of aspirate smears, touch imprints and trephine biopsy. Bone marrow aspirate smears can be prepared by the squash (crush) or wedge method. Both techniques have their own advantages and disadvantages. There is paucity of studies that have compared these smear types. This study was conducted over a period of one year. Two hundred and five bone marrow aspirates were included. Both squash and wedge smears were made. Blinded slide review was done. Bone marrow cellularity, megakaryocyte number, myeloid to erythroid (M:E) ratio, morphology and final diagnosis on each smear type was compared. Chi square test, t-test and Kappa were applied to study the agreement between the wedge and squash smears. Among the 205 patients studied, squash smears showed significant over estimation of cellularity and megakaryocyte number (p < 0.05). There was no significant difference (p > 0.05) in the M:E ratio and morphological scores. Most patients (188/205 [91.7%]) showed complete diagnostic concordance while 17 (8.3%) patients had discrepancy in diagnosis between the squash and wedge smears. In 8 (3.9%) of these, major discrepancies were seen while 9 (4.4%) patients had minor discrepancies. Bone marrow cellularity and megakaryocyte numbers were underestimated in wedge smears with no differences in M:E ratio or morphology. Acceptable agreement for diagnosis was seen for patients with most disorders. Major diagnostic discrepancies were seen in patients with lesions known to have focal distribution—lymphoma, myeloma and tuberculosis (granulomas). Both squash and wedge smears should be studied for bone marrow examination.     

Comparison of the classical manual pushed wedge films, with an improved automated method for making blood smears

Comparison of apparatus for automatically spreading peripheral blood films (GENES) to manual wedge-pull technique has been performed, showing that the average size of optimal area for counting is twice larger in automated smears compared to manual ones. The variability of WBC repartition has been studied in doing differential count on manual and automated smears and both are compared to results obtain with an independent technique (Coulter STKS) for differential. The correlation coefficients for each WBC population show a closer relationship between automated smear preparations and reference instrument (STKS) than with manual spreading smears. Monocyte differential is particularly influenced by the variability of manual spreading procedures.     

Reducing the number of sputum samples examined and thresholds for positivity: an opportunity to optimise smear microscopy.

SETTING: Urban health clinic, Nairobi. OBJECTIVE: To evaluate the impact on tuberculosis (TB) case detection and laboratory workload of reducing the number of sputum smears examined and thresholds for diagnosing positive smears and positive cases. DESIGN: In this prospective study, three Ziehl-Neelsen stained sputum smears from consecutive pulmonary TB suspects were examined blind. The standard approach (A), > or = 2 positive smears out of 3, using a cut-off of 10 acid-fast bacilli (AFB)/100 high-power fields (HPF), was compared with approaches B, > or = 2 positive smears (> or = 4 AFB/100 HPF) out of 3, one of which is > or = 10 AFB/100 HPF; C, > or = 2 positive smears (> or = 4 AFB/100 HPF) out of 3; D, > or = 1 positive smear (> or = 10 AFB/100 HPF) out of 2; and E, > or = 1 positive smear (> or = 4 AFB/100 HPF) out of 2. The microscopy gold standard was detection of at least one positive smear (> or = 4 AFB/100 HPF) out of 3. RESULTS: Among 644 TB suspects, the alternative approaches detected from 114 (17.7%) (approach B) to 123 cases (19.1%) (approach E) compared to 105 cases (16.3%) for approach A (P < 0.005). Sensitivity ranged between 82.0% (105/128) for A and 96.1% (123/128) for E. The single positive smear approaches reduced the number of smears by 36% compared to approach A. CONCLUSION: Reducing the number of specimens and the positivity threshold to define a positive case increased the sensitivity of microscopy and reduced laboratory workload.     

Comparison of the classical manual pushed wedge films, with an improved automated method for making blood smears

Comparison of apparatus for automatically spreading peripheral blood films (GENES) to manual wedge-pull technique has been performed, showing that the average size of optimal area for counting is twice larger in automated smears compared to manual ones. The variability of WBC repartition has been studied in doing differential count on manual and automated smears and both are compared to results obtain with an independent technique (Coulter STKS) for differential. The correlation coefficients for each WBC population show a closer relationship between automated smear preparations and reference instrument (STKS) than with manual spreading smears. Monocyte differential is particularly influenced by the variability of manual spreading procedures.     

Microscopic counts carried out on Mycobacterium leprae and M. tuberculosis suspensions. A comparison of three staining procedures

Standard smears of heat-killed Mycobacterium leprae and M. tuberculosis H37Rv were counted microscopically following staining by the Ziehl-Neelsen, auramine, and silver-methenamine methods. The numbers of stained bacillary bodies were consistently higher in the silver-methenamine stained smears compared to the Ziehl-Neelsen and auramine stained smears. The auramine smears were examined under ultraviolet illumination and permitted the enumeration of the brightly fluorescent bacilli against a black background. The auramine counts were not as high as those obtained using the silver-methenamine stained preparations but were consistently higher than those obtained with the Ziehl-Neelsen preparations. Both the auramine and silver-methenamine stained preparations clearly outlined the cell walls of the bacilli in the smears and this greatly facilitated the counting process, especially if the cell suspension was badly clumped.     

Efficacy and safety of short-term bleach digestion of sputum in case-finding for pulmonary tuberculosis in Ethiopia.

BACKGROUND: In many settings, the diagnosis of pulmonary tuberculosis depends on sputum microscopy. However, this technique has low sensitivity. We studied the efficacy and safety of sputum digestion with bleach prior to Ziehl-Neelsen (ZN) staining. METHODS: Positive sputum smears were assessed for staining quality and viability of mycobacteria after varying bleaching times. Two hundred smears were then prepared from the first, second and third sputum sample of tuberculosis suspects. Equal amounts of 5% bleach were added to the remaining first sputum and ZN smears were prepared. FINDINGS: Optimal quality and staining was achieved with 30-45 min of bleaching. No growth was observed from positive samples after 15 min. Bleached specimens had 26% (52/200) positivity compared to 17.5% (35/ 200) of unbleached smears (P < 0.001). The bleached smears had 92.3% sensitivity, 93.4% specificity, 78.3% and 97.7% positive and negative predictive values, respectively, against a case definition. Ten patients failed to submit a second or third sputum. Six patients were positive on either the standard or bleach-digested smears, or both. INTERPRETATION: Bleach digestion is simple, cheap and kills mycobacteria. Its positivity rate is as good as three standard smears. This method has the potential to improve over-burdened services in developing countries.     

Stainable iron in aspirated and needle-biopsy specimens of marrow: a source of error.

Marrow iron was assessed in needle biopsy sections and in simultaneously obtained aspirated smears and aspirated clot sections from 251 patients. Significantly different amounts of stainable iron were observed in needle biopsy sections and aspirated smears in 15% of the specimens. The usual difference consisted of significantly less stainable iron in needle biopsy sections as compared to the aspirated smears (13%). Of clinical importance was the finding of absent stainable iron in 8% of the needle biopsy sections, in contrast to the definite deposits observed in the corresponding aspirated smear. Of similar interest was the observation that 6% of the needle biopsy sections had significantly less stainable iron than corresponding hemosiderotic smears. The amounts of stainable iron in needle biopsy sections were similar to the amounts observed in the corresponding aspirated clot sections. Evaluation of marrow iron using needle biopsy sections as well as aspirated clot sections differs from results observed in aspirated smears and could be a source of significant error in the assessment of iron stores.     

Increased prevalence of squamous intraepithelial lesions in systemic lupus erythematosus: association with human papillomavirus infection

OBJECTIVE: To compare the prevalence of abnormal Pap smears in patients with systemic lupus erythematosus (SLE) with that in a large group of healthy controls, and to determine whether SLE itself is an independent risk factor. The association of human papillomavirus (HPV) infection and the use of immunosuppressive agents with abnormal Pap smears in SLE was also assessed. METHODS: Eighty-five SLE patients participated in this cross-sectional study. A sample of cervical cells was collected from each patient for routine cytologic examination. HPV was typed by restriction and sequencing analysis. A structured questionnaire was administered to the subjects to ascertain the possible behavioral and biologic risk factors associated with cervical atypia. Data on 2,080 healthy female subjects were retrieved for comparison. RESULTS: The mean (+/-SD) age of SLE patients and controls was 42 +/- 9 years and 44 +/- 10 years, respectively. The prevalence of abnormal Pap smears was significantly increased in SLE patients compared with controls (16.5% versus 5.7%). The prevalence of squamous intraepithelial lesions was increased approximately 6-fold in SLE patients (11.8%) compared with controls (2.0%). SLE itself remained an independent risk factor for abnormal Pap smears (odds ratio 3.5, 95% confidence interval 1.8-6.9). The overall prevalence of HPV infection in SLE patients and controls was 11.8% and 7.3%, respectively. However, 10.6% of SLE patients were infected with at least 1 high-risk type of HPV, compared with 4.2% of controls. Multiple infections were also more common in SLE patients than in controls (4.7% versus 1.1%). There were no significant differences in the use of immunosuppressive agents between SLE patients with normal Pap smears and those with abnormal Pap smears. CONCLUSION: Abnormal Pap smears were more common among SLE patients than controls, even after adjusting for HPV status. SLE-associated immunosuppression increases susceptibility to high-risk HPV infection and multiple HPV infections. The use of immunosuppressant agents was not associated with abnormal Pap smears.     

A comparison of the use of Papanicolaou-stained cervical cytological smears with Gram-stained vaginal smears for the diagnosis of bacterial vaginosis in early pregnancy

Our objective is to compare the efficacy of using Papanicolaou (PAP)-stained cervical cytology smears with a standardized method of interpreting Gram-stained vaginal smears for the diagnosis of bacterial vaginosis (BV) in pregnancy. High vaginal smears were Gram-stained and examined by a single observer to characterize 3 grades of vaginal flora and diagnose BV. Cervical smears were PAP-stained and examined for characteristic patterns of vaginal flora including evidence of BV by either a number of cytotechnicians or a single cytopathologist. The results of the 2 methods were compared. Seven hundred and forty-seven women attending an antenatal clinic in a district general hospital who consented to have a smear of vaginal secretions and cervical cytology in early pregnancy. The main outcome measure is the diagnosis of BV by different methods in a pregnant population. Compared with the Gram-stain method for the diagnosis of BV, there was good agreement between PAP-stain interpretation by a single observer but the agreement was not as good with PAP-stain interpretation by multiple cytotechnicians. When the grades were consolidated to normal (grade I) and abnormal flora (grades II and III), compared to Gram-stained smears, PAP cytology undertaken by several cytotechnicians had a sensitivity of 80.7% and a specificity of 90.7%. The sensitivity and specificity increased to 87% and 97%, respectively, when the PAP-stained smears were read by a single cytopathologist. Using kappa scores, only those readings made by a single cytopathologist were reliable. The setting in a cytopathology laboratory comprises multiple cytotechnicians, so that PAP-stain analysis of vaginal smears for the diagnosis of BV is likely to provide results which are less reliable than those obtained by Gram staining. The latter should be the first choice and every effort should be made to set up this service.     

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.     

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.     

A computerized reminder system improves compliance with Papanicolaou smear recommendations in an HIV care clinic

Current guidelines call for Papanicolaou (Pap) smear screening of HIV-infected women at least annually. After the initiation of a weekly computer based Pap smear reminder list in an HIV care clinic, the prevalence of scheduled women with up-to-date Pap smears was calculated for the one-year project period and was compared to the prevalence preceding the project. The prevalence of scheduled women with up-to-date Pap smears increased from 61.4% to 73.2% (P <0.001) during the project period. Including Pap smears that were performed elsewhere, the final up-to-date Pap smear rate was 82.7%. The improved rate of up-to-date Pap smears showed no sign of attenuation over time. A computerized report generated from data in the hospital information system increased rates of compliance with Pap smear screening recommendations. Creative utilization of hospital data environments may be an inexpensive route to improved compliance with practice guidelines.     

Cutaneous leishmaniasis in Guatemala: comparison of diagnostic methods

A comparison was made of methods used to diagnose suspected cutaneous leishmaniasis in Guatemala. The most sensitive method was a combination of thin smears made from superficial scrapings of the ulcers and inoculation of culture medium with either aspirates or scrapings. The diagnosis was confirmed in 252 (70%) of 362 patients. Ability to cultivate Leishmania was correlated with the concentration of amastigotes seen on thin smears. Leishmania were cultured in 42 (27%) of 153 patients with no amastigotes found in 400 oil-immersion fields and in 174 (83%) of 209 patients with at least 1 amastigote. No difference in diagnostic outcome was found when we compared smears or cultures taken from the center or the border of the ulcer or from an incision made tangential from the ulcer. We found no difference when we compared smears obtained with scalpels, capillary tubes, or dental broaches. The use of scrub brushes soaked in iodine neither decreased the rate of culturing parasites nor decreased contamination rates.     

The Loeffler's methylene blue stain: An inexpensive and rapid method for detection of Helicobacter pylori

Abstract Loeffler's methylene blue, commonly used as a counter-stain for acid fast bacilli, was used to detect Helicobacter pylori in paraffin sections and touch smears of gastric mucosal biopsies from 15 patients with duodenal ulcer and 35 with non-ulcer dyspepsia, and the results were compared with the modified Giemsa stain. The time taken to stain smears by Loeffler's methylene blue was approximately 10 min and the results correlated well with those stained by the modified Giemsa stain. However, the Loeffler's methylene blue method was found to be simpler, quicker and cheaper than the modified Giemsa stain.     

Usefulness of core roll preparations in immediate assessment of neoplastic lung lesions: comparison to conventional CT scan-guided lung fine-needle aspiration cytology

INTRODUCTION: CT scan-guided fine-needle aspiration (FNA) of lung nodules is a well-established diagnostic technique. In our institution, both FNA and tissue core biopsy (using a 20-gauge needle) under CT scan guidance are routinely performed during the same procedure, and a preliminary diagnosis (an immediate assessment) is rendered. We compared core roll preparations (CRPs) with aspirate smears in the immediate assessment of pulmonary lesions and also assessed whether CRP resulted in the alteration of the histopathology of the core biopsy. STUDY DESIGN: Twenty-five cases of neoplastic pulmonary lesions diagnosed in CT scan-guided lung FNA specimens, with core biopsies performed sequentially at the same visit for each patient, were evaluated. CRPs were made by lightly rolling the tissue core on a glass slide, followed by air-drying. Only stained slides (Diff-Quik; Mercedes Medical; Sarasota, FL) were reviewed, and were scored for cellularity and morphology. RESULTS: CRPs in seven cases (28%) scored more diagnostic points than FNA smears, and were found to be better for cellularity and morphology compared to the corresponding FNA smears. The FNA smears scored more than CRPs in 10 cases (40%), while in 8 cases (32%) both CRPs and FNA smears scored equal diagnostic points. Using both CRP and FNA smears in the immediate assessment of lung biopsy specimens, we could assign a specific malignant histologic cell type in 23 of 25 cases (92%). In comparison, if the FNA smears were evaluated alone, we could assign a specific malignant histologic cell type in only 16 of 25 cases (64%). The CRP did not alter the histopathology of the core biopsy specimens in any of the above cases. CONCLUSION: The CRP complements the CT scan-guided lung FNA procedure in the immediate assessment of neoplastic lung lesions without altering the histopathology of core biopsy specimens.