Giemsa-stained Smears Research Articles

Gastric Tuberculosis

To highlight the utility of endoscopic brush smears in the diagnosis of gastric tuberculosis in clinically unsuspected cases. A retrospective analysis of endoscopic brush smears from 210 patients with gastric symptoms. In seven of these patients (3.3%) the possibility of gastric tuberculosis was suggested in Giemsa-stained smears. Biopsy was available in all cases. Ziehl-Neelsen stain to demonstrate tubercle bacilli was used in brush smears and biopsies in seven and three cases, respectively. Endoscopically the sites involved were antrum (two), pylorus (two), pylorus and duodenum (three). One patient had an ulcerative lesion, and six had growths. Granulomas and/or epithelioid cells were seen in brush smears in all cases. Tubercle bacilli could be demonstrated in cytologic smears in four cases. Endoscopic biopsy showed granulomas in five cases and non-specific gastritis in two. Tubercle bacilli could not be demonstrated in any of the biopsy sections. On further clinicoradiologic investigation, two patients were found to be follow-up cases of pulmonary and nodal tuberculosis. Enzyme-linked immunosorbent assay for HIV, done in three cases, was negative. A final diagnosis of primary gastric tuberculosis in five patients and secondary in two was considered. Six patients responded to antituberculosis treatment and showed healing of the lesions on repeat endoscopy after six months of therapy, while one was a recent case with four weeks' follow-up. Endoscopic brush cytology is a reliable modality for the diagnosis of gastric tuberculosis.

Langerhans cell histiocytosis in lymph nodes - cytomorphological diagnosis and pitfalls

Background Langerhans cell histiocytosis (LCH) is a rare disorder of unknown aetiology that may present as a multisystem or unisystem disease. The Lymph nodes can be involved as part of disseminated disease, as a metastatic site draining a focus of LCH or may be a unisystem involvement. Paucity of literature on the cytomorphology of LCH in lymph nodes led us to undertake this study. Materials and methods Nine cases with a confirmed histological diagnosis of LCH and a prior lymph node aspirate were retrieved over a 12 year period (1988–1999). Five more cases were reviewed where the cytological diagnosis of LCH was rendered on a background of clinical and radiological findings. Papanicolaou and May Grunwald–Giemsa-stained smears were examined. S-100 protein staining was available in four cases. Results and conclusions Nine cases had multisystem involvement, while in five cases only lymph nodes were involved. There were eleven males and three females; age ranged from five months to 27 years. The cytological diagnosis of LCH had been rendered in six, suspected in four and missed in four. Of the latter, two were reclassified as LCH on review, one as reactive lymphadenitis and in one a necrotising lesion was suspected. The pathognomonic ‘LCH cell’ was identified in 12 of 14 cases along with varying numbers of eosinophils, polymorphs and lymphocytes. Giant cells were seen in only six cases. In conclusion lymph node involvement by LCH can be identified on aspirates. However, LCH must be differentiated from dermatopathic lymphadenitis, sinus histiocytosis with massive lymphadenopathy and Hodgkin's disease.

Cutaneous and paranasal aspergillosis in an immunocompetent patient

A 26-year-old Libyan woman presented with asymptomatic nodulo-ulcerative skin lesions present for 1 year. Three years prior to presentation, she had experienced a nasal discharge followed by the development of a nodule in the nasal cavity and a plaque on the hard palate. These lesions had gradually increased in size and ulcerated, resulting in perforation of the nasal septum and palate. Two years later, the patient noticed the appearance of skin lesions: a nodule on the right thumb and numerous nodulo-ulcerative lesions on the extremities. General physical examination was normal with no significant lymphadenopathy. Examination of the oral cavity revealed perforation of the distal nasal septum, with a perforated nodular plaque involving the entire palate, associated with subluxation of the upper incisors (Fig. 1a). On skin examination, multiple firm nodules and nodulo-ulcerative lesions with a central eschar and raised margins were observed. The lesions ranged in size from 0.5 to 5 cm and were distributed on the right hand and fingers, left upper arm (Fig. 1b), left calf, and right thigh. Routine laboratory investigations (liver function tests, serum calcium, electrolytes, lipid profile, urine and stool culture studies) were normal. Immunoelectrophoresis disclosed normal levels of immunoglobulins IgG, IgA, and IgM. Serologic studies for human immunodeficiency virus (HIV) and syphilis, and a tuberculin test, were all negative. A Giemsa-stained tissue smear was negative for Leishmania tropica organisms. Radiological studies disclosed a slight haziness of the maxillary sinuses with perforation of the nasal septum. A chest X-ray was normal. Histopathologic examination of biopsies taken from both the palate and from ulcerated and nonulcerated skin lesions was performed, and all showed similar findings. The biopsy of a nonulcerated skin lesion showed pseudoepitheliomatous epidermal hyperplasia with neutrophilic microabscesses (Fig. 2a). A dermal diffuse and nodular granulomatous mixed infiltrate of lymphocytes, histiocytes, giant cells, numerous eosinophils, and neutrophilic microabscesses was seen in all tissues examined. Septate hyphae were present both within giant cells and free in the dermis (Fig. 2b). The hyphae were branching at a 45 degrees angle and were positive on periodic acid-Schiff and Grocott methenamine silver stains (Fig. 2c). Fungal culture studies of material taken from an ulcerated skin lesion grew Aspergillus flavus. Blood cultures were negative for Aspergillus sp. or other microorganisms. The patient was treated with intravenous amphotericin B, but the medication was discontinued due to her intolerance to the drug. She was subsequently lost to follow-up.

How prevalent is Plasmodium malariae in Rondônia, western Brazilian Amazon?

We have compared results of Plasmodium species identification obtained with conventional on-site microscopy of Giemsa-stained thick smears (GTS) and a semi-nested polymerase chain reaction (PCR) in 96 malaria patients from Rondônia, Western Brazilian Amazon. Mixed-species infections were detected by PCR in 30% patients, but no such case had been found on GTS. Moreover, P. malariae infections were detected in 9 of 96 patients (10%) by PCR, but were not identified by local microscopists. The potential impact of species misidentification on malaria treatment and control is discussed.

Rapid extraction of DNA from archival clinical specimens: our experiences.

The analysis of DNA extracted from archival clinical specimens using polymerase chain reaction represents the basis of a variety of research and diagnostic protocols in medicine. However, the selection of optimal DNA extraction method is critical if such an analysis is to be successful. Recently, we have evaluated a number of rapid DNA extraction protocols in order to find the most suitable method for routine processing of the most common archival materials in pathological and cytological laboratories: paraffin-embedded tissues and Papanicolaou- or Giemsa-stained smears. Our results demonstrate that rapid DNA extraction methods have comparable DNA extraction efficiencies with standard DNA isolation protocols on archival clinical specimens with the exception of Giemsa-stained smears.

HIV viral load and response to antileishmanial chemotherapy in co-infected patients.

To investigate whether clearance of Leishmania parasites from tissue aspirate smears in patients with HIV and visceral leishmaniasis (VL) co-infection treated with pentavalent antimonials is influenced by initial HIV viral load and to assess the effect of active VL on HIV viral load and replication in vivo. Leishmania parasites were identified in Giemsa-stained smears prepared from tissue aspirates. Parasite index was determined by quantifying Leishmania donovani bodies in smears. HIV-1 RNA was quantitated by using the nucleic acid sequence-based amplification technique with a limit of detection of 500 copies/ml. All patients were treated with pentavalent antimonials at 20 mg pentavalent antimony (Sb(V))/kg daily for a total of 28 days. None of the patients received specific anti-retroviral therapy. Seventeen patients (73.9%) showed good initial response to anti-leishmanial treatment and the remaining six (26.1%) had very poor response. Among the good responders, 11 (64.7%) had no demonstrable Leishmania donovani bodies in post-therapy tissue aspirate smear preparations, and in the remaining six (35.3%) their parasite loads were reduced to very low levels. Patients with poor response had persistently high parasite index despite completion of anti-leishmanial chemotherapy. Poor responders had pre-treatment median HIV viral load that was >160-fold higher than responders to anti-leishmanial chemotherapy; [410000 copies/ml (quartile range, 33000-530000) and 2500 copies/ml (quartile range 500-297500), respectively]. Furthermore, compared with pre-treatment viral concentrations, patients with good response showed marked reduction in post-treatment viral load. In contrast, post-treatment HIV viral concentrations were markedly increased among patients with poor response to anti-leishmanial therapy. The results suggest that pre-treatment HIV viral load influences response to anti-leishmanial chemotherapy and active VL is associated with increased viral replication in vivo, supporting the notion that dual infection plays an important role in the pathogenesis and disease progression of either infection.

Diagnostic and prognostic potential of a competitive enzyme-linked immunosorbent assay for leishmaniasis in India.

A Leishmania donovani species-specific monoclonal antibody (monoclonal antibody D2) was evaluated for its diagnostic and prognostic potential by a competitive enzyme-linked immunosorbent assay (C-ELISA) in sera from Indian patients with visceral leishmaniasis (VL) and seven patients with post-kala-azar dermal leishmaniasis (PKDL). These results were compared with those obtained by microscopy with Giemsa-stained tissue smears and a direct enzyme-linked immunosorbent assay (direct ELISA) with crude parasite antigen. Of 121 patients with clinically diagnosed VL examined, 103 (85.1%) were positive and 11 (9.1%) were negative by all three methods. An additional 7 (5.8%) who were negative by microscopy were positive by both C-ELISA and direct ELISA. Seven PKDL patients were also examined and were found to be positive by all three methods. Analysis of the chemotherapeutic response to sodium antimony gluconate of these 110 serologically positive VL patients showed that 57 (51.8%) were drug responsive and 53 (48.2%) were drug resistant. The C-ELISA with sera from 20 longitudinally monitored VL patients before and after chemotherapy showed a significant decrease in percent inhibition of monoclonal antibody D2 in drug-responsive patients. However, in drug-unresponsive patients, the percent inhibition of D2 was unchanged or was slightly increased. Our results therefore indicate (i) the applicability of L. donovani species-specific monoclonal antibody D2 for sensitive and specific serodiagnosis by C-ELISA, (ii) that the C-ELISA is more sensitive than microscopy, especially for early diagnosis, (iii) that L. donovani is still the main causative agent of VL, irrespective of the chemotherapeutic response, and (iv) that the C-ELISA can be used to evaluate the success of drug treatment.

Infectivity and cross-immunity studies of Theileria lestoquardi and Theileria annulata in sheep and cattle : II. In vitro studies

In the studies previously reported, the tick-borne protozoan parasites Theileria lestoquardi and Theileria annulata were shown to differ in their capacity to infect sheep and cattle. In the studies presented here, these findings were further supported. In vitro infectivity of T. lestoquardi and T. annulata sporozoites for peripheral blood mononuclear cells of sheep and cattle were determined by analysis of cell cultures for cell proliferation, the detection of parasites in Giemsa-stained cytospin smears and the establishment of continuously growing schizont-infected cell lines. In the same way, the development of schizont-infected cells into continuously growing cell lines was studied with material isolated ex vivo from the sheep and cattle undergoing primary infections described elsewhere. Comparisons were also made between development of ex vivo cell lines from animals undergoing primary infections with those of the animals undergoing challenge infection with the other parasite species. Theileria species specific primers were used in a PCR to determine the identity of the parasites in the cell lines. These in vitro studies confirmed earlier observations that T. lestoquardi was unable to infect cattle, whereas infection of all sheep with T. annulata was proven. Moreover, earlier indications of the development of partial cross-immunity in sheep of T. annulata to T. lestoquardi and vice versa were strengthened. These findings may thus have consequences for the understanding of the epidemiology of T. lestoquardi infections of sheep. On the other hand, since piroplasms were not demonstrated in sheep infected with T. annulata, such sheep will not be infective to ticks and will consequently be unlikely to play a role in the maintenance and transmission of T. annulata to cattle.

Clinical and laboratory study of Kala-azar in children in Nepal

Reports are scanty regarding kala-azar in children in Nepal. In this communication we document 20 children diagnosed to have kala-azar who were admitted and treated at B. P. Koirala Institute of Health Sciences, Dharan, Nepal. The children were between 2 and 14 years old. The duration of illness varied between 12 days and 24 months with a majority (65 per cent) of children being ill for less than 6 months. Hepatomegaly and splenomegaly were seen in 95 and 90 per cent of cases respectively. Splenomegaly was not found in two (10 per cent) children. Anaemia, leucopenia, and thrombocytopenia were seen in 95, 60, and 75 per cent of children respectively. Amastigotes of Leishmania donovani (LD bodies) were demonstrated in Giemsa-stained smears of bone marrow aspirates in 16 (80 per cent) children. All the children responded to treatment with sodium stibogluconate. No mortality was observed. This study emphasizes the importance of kala-azar in children in endemic areas of eastern Nepal.

Leishmania spp: completely defined medium without serum and macromolecules (CDM/LP) for the continuous in vitro cultivation of infective promastigote forms.

The elimination of serum or of serum-derived macromolecules that supplant the fetal calf serum requirement from Leishmania culture media could decrease costs and improve the feasibility of large-scale production of well-defined parasite material. We report a completely defined medium, without serum-derived protein and/or macromolecules as a serum substitute, of common, available, and inexpensive constituents that can be used in place of serum-supplemented media for the continuous in vitro cultivation of promastigote forms of various Leishmania species. Typical promastigote morphology was observed in Giemsa-stained smears, regardless of the strain analyzed. Electrophoretic analysis showed that the proteinase patterns of aserically grown promastigote forms were similar to those obtained in serum-supplemented RPMI 1640 medium for all Leishmania studied. Similar antigenic profiles were recognized in immunoblots by sera from hosts with visceral or cutaneous leishmaniasis after growing promastigotes in the two different culture media. For parasites causing both cutaneous and visceral leishmaniasis, the absence of serum and macromolecules in the culture medium did not markedly change their in vitro infectivity for resident mouse macrophages and their virulence in animals compared with parasites cultivated in nondefined medium. Serum-free technology will be increasingly important in providing stability and reproducibility as research using promastigote moves closer to therapeutic applications.

Cutaneous leishmaniasis in Multan, Pakistan.

Cutaneous leishmaniasis (CL) is endemic in certain areas of Pakistan. This study was carried out to determine the incidence and clinical pattern of the disease in Pakistan. CL patients presenting in the out-patient clinic of the Department of Dermatology, Nishtar Hospital, Multan from January 1995 to June 1997 were included in the study. The patients were diagnosed clinically and confirmed by laboratory demonstration of the parasite in a Giemsa-stained smear from the lesion. All important clinical details were recorded on specially designed forms. Three hundred and five patients with 681 lesions were seen during 30 months. All the lesions were of the dry type. Most of the lesions (97%) were present on exposed areas of the body. One hundred and thirty-four (44%) patients had one and 75 (24%) had two lesions. Eight five per cent of the sufferers were less than 30 years of age. The disease was slightly more preponderant in males (60%). Most cases presented during winter. Lack of wet type lesions may be due to complete absence of Leishmania major in this area. This is in contrast to other areas of Pakistan where both dry and wet types of CL are present.

An investigation on the ecology of Triatoma vitticeps (Stal, 1859) and its possible role in the transmission of Trypanosoma cruzi, in the locality of Triunfo, Santa Maria Madalena municipal district, state of Rio de Janeiro, Brazil.

From January 1989 to April 1995, 465 specimens of Triatoma vitticeps were collected in the locality of Triunfo, 2nd District of Santa Maria Madalena municipal district, State of Rio de Janeiro. The bugs were found indoors by local residents with predominance of adults. The flight activity was high in hot months when the incidence in the domicile also increased. Two hundred and two bugs (111 alive and 91 dead) were examined for Trypanosoma cruzi infection. This was detected in 31 of the dead bugs (34%) and 88 (79%) of the live bugs examined. With a view to investigate the possible vertebrate hosts of the T. cruzi isolates, the blood of 122 mammals was examined through Giemsa-stained smears, hemocultures and xenodiagnosis. T. cruzi was detected in three specimens of Didelphis marsupialis and T. (M.) theileri was detected in one specimen of Bos taurus. The parasites were isolated from triatomine feces, xenoculture and hemoculture. No evidence of human infection was detected in 58 inhabitants examined, as evaluated by indirect imunofluorescence technique using T. cruzi epimastigotes as antigens. These results show that T. vitticeps is still a sylvatic species although nymphs have been found inside the domicile. Thus, an epidemiological vigilance is necessary to know the behaviour of this species following the continuous modifications promoted by the presence of man.

Aspiration Cytology of Tubular Adenomas of the Breast

To analyze fine needle aspirates from tubular adenomas of the breast and elucidate cytologic features unique to these tumors. Fine needle aspiration (FNA) cytology smears from eight tubular adenomas were reviewed, with special attention given to architectural features and individual cell characteristics. Smears were then compared with those from 25 fibroadenomas and 2 tubular carcinomas for analyzing the distinguishing features. Aspirates from tubular adenomas revealed cells arranged as small, three-dimensional balls or clusters, tubules of different shapes and less frequently as closely approximated acini. Cells were uniform, with pale cytoplasm, which showed magenta granules in the Giemsa smears from two tumors. Straight tubules, closely approximated acini and intracytoplasmic granules were not seen in aspirates from fibroadenomas. The tubules in tubular adenomas always showed myoepithelial cells and were more open than the angulated tubules of tubular carcinoma. Tubular adenomas on FNA cytology specifically show cells arranged as small, three-dimensional balls, tubules or closely approximated acini. Cells are uniform and sometimes show intracytoplasmic granules in Giemsa-stained smears.

Diagnosis of mastocytosis by fine‐needle aspiration cytology

We report a case of a 28-yr-old man in whom mastocytosis was diagnosed by fine-needle aspiration (FNA) of enlarged periaortic lymph nodes. FNA cytology revealed numerous mast cells characterized by cells having round to oval eccentrically placed nuclei and associated coarsely granular cytoplasm, most conspicuous on Diff-Quik- and Giemsa-stained smears. Lymphocytes, histiocytes, and occasional plasma cells were present in the background. We believe this paper to be the first reported case of mastocytosis diagnosed by FNA. The case demonstrates the utility of FNA in evaluating lymphadenopathy occurring in mastocytosis. Diagn. Cytopathol. 1998;18:368–370. © 1998 Wiley-Liss, Inc.

Malaria in Non-Endemic Kuwait: Detection of Very Low Level Plasmodium falciparum Infections Using Polymerase Chain Reaction

Objective: Imported malaria continues to be a problem in Kuwait since most patients present with very low levels of parasitaemia. The diagnosis is often delayed or missed because of the low sensitivity of the standard stained blood smear test used for the identification of malarial parasites. In this study, we standardized a highly sensitive polymerase chain reaction (PCR) to detect malarial parasites. Methods: A total of 100 individuals presenting with various forms of infection were screened with the standard Giemsa-stained smears and were compared with the PCR for diagnosis and the sensitivity of the two methods. Results: Our results show that PCR is a highly sensitive and specific technique to detect low levels of Plasmodium falciparum parasites in clinical samples, and at least 6 cases (12%) who were microscopically negative had PCR-detectable parasitaemia. Conclusion: The PCR is a highly sensitive assay which can detect very low levels of malarial parasites and thus is of great value to obtain accurate detection of chronic, residual and sub-patent infections in Kuwait.

Morphological features suggesting a sexual process in a trypanosomatid of the genus Herpetomonas

Summary Morphological features resembling those described in Herpetomonas megaseliae, and likewise suggesting a sexual process, were also found in a axenic cultures of a Herpetomonas sp. isolated from the salivary glands of a phytophagous hemipteran. These features have been frequent over a 3-year period and were quantitatively evaluated during the parasite growth in LIT medium at 27 °C. In Giemsa-stained smears, the more noticeable findings were: (1) pairs of apposed promastigotes joined by the posterior ends, where a ring-like line and/or an enlargement occurs; (2) bell-shaped cells, either free or in pairs with promastigotes; (3) in several pairs, features suggesting nucleus migration and fusion. Indirect immunofluorescence of β-tubulin and scanning electron microscopy suggested cellular fusion in these pairs.

Leishmania major: Parasite Interactions Suggesting Sexuality

In five experiments, Leishmania (Leishmania) major (MRHO/SU/59/P-strain) grew poorly when seeded in FYTS medium supplemented with 15% fetal calf serum, but presented several peculiar pairs of promastigotes diametrically opposed and attached at their posterior ends (5.8-13.5%). As seen in Giemsa-stained smears, a ring-like line and/or an enlargement, generally occurred at the parasite junction. A close proximity of nuclei, which sometimes were difficult to distinguish from each other, was also observed at this junction. Several of these pairs appeared to be composed of fused cells in which the nuclei could be apparently fused, as shown by fluorescence microscopy to detect s-tubulin and DNA, and by scanning electron microscopy. Under other culture conditions these pairs were absent or occurred at very low rates (0.2-2.2%). Such pairs differ markedly from longitudinally dividing cells and resemble those described in two other Leishmania species, as well as in Herpetomonas megaseliae and Phytomonas davidi, suggesting steps of a putative sexual process

Culture of the causative organism of donovanosis (Calymmatobacterium granulomatis) in HEp-2 cells.

We report successful culture of Calymmatobacterium granulomatis by standard cell culture methods. Swabs were obtained from lesions in three patients with a clinical diagnosis of donovanosis. For two patients, there was histological confirmation of the disease (i.e., the presence of Donovan bodies in Giemsa-stained smears). Specimens were inoculated onto cycloheximide-treated HEp-2 cell monolayers in RPMI 1640 medium (supplemented with fetal calf serum, NaHCO3, vancomycin hydrochloride, and benzylpenicillin). At 48 h, organisms resembling Donovan bodies were identified in monolayer cultures from all three specimens. The organisms appeared as pleomorphic bacilli with characteristic bipolar staining and "safety pin" appearance. Using a PCR designed to differentiate C. granulomatis from the Klebsiella species (which have a high degree of molecular homology), we were able to demonstrate that the cultured organisms produced a PCR product identical to that obtained from the original swab specimens. It is now possible to test in vitro susceptibility of C. granulomatis to antibiotics and to provide a ready source of DNA and antigenic material to enable the development of serological tests and, possibly in the future, a vaccine.

Typhoid Fever

<h3>CASE PRESENTATION</h3> A 35-year-old Maryland woman came to her local hospital on December 4, 1994, complaining of fever and chills for 1 week. She was 25 weeks pregnant. Four weeks earlier, she and her husband and young daughter had visited relatives in a city about 320 km northwest of Bombay, India. She had not received pretravel vaccinations or malaria prophylaxis, and she did not take any specific food or water precautions while in India. She had felt well during the trip. She had returned to Maryland on November 24. On November 29, she developed fever and chills. Her private physician examined her that day and prescribed oral ampicillin to treat a sore throat. When her fever persisted, she was admitted to the local hospital on December 4. Evaluation included blood cultures and multiple Giemsa-stained thick smears for malaria. On December 5, a physician diagnosed malaria after identifying a<i>Plasmodium falciparum</i>parasite

Morphometry of normal and teratozoospermic canine sperm heads using an image analyzer: work in progress

Combining the traditional morphologic evaluation of spermatozoa with computer assisted image analysis adds randomness, objectivity, repeatability and accuracy to morphometric measurements. We collected semen from 10 fertile, normospermic dogs aged 1 to 7 yr and from 3 teratozoospermic breed-matched dogs. Sperm head morphology was examined in Giemsa-stained smears by light microscopy, using a computer-assisted image analyzer and by transmission electron microscopy. We found significant variation in sperm head area, length, width and degree of roundness among normospermic individual dogs, indicating that it would be necessary to examine many more dogs before the size and shape of normal dog spermatozoa could be determined. The normospermic dogs were used as controls for the teratozoospermic cases. Case 1: A 2-yr-old subfertile Cavalier King Charles Spaniel had semen with small and narrow-based sperm heads and a proximal cytoplasmic droplet in most of the spermatozoa. With the image analysis system, sperm heads were shown to be smaller and more oval than in normospermic dogs. The variatons in size and shape were similar in magnitude to those of control dogs. An examined infertile half-brother had similar semen quality. Case 2: A 3-yr-old Petit Basset Griffon Vendeen with 2 unsuccesfull matings exhibited spermatozoa with severe abnormalities. Measured by image analyzer, sperm heads were irregular in shape and very small in area. One of the two littermates examined had semen of the same quality as the case dog. Case 3: A 3-yr-old fertile Golden Retriever had semen with giant sperm heads in about 50% of spermatozoa. Image analyzing results revealed 2 populations of different sized sperm heads. Giant heads consisted of 52.2% of all spermatozoa. The results of the study reported here suggest that the image analysis technique may be useful in evaluating structural changes in sperm morphology, supplementing visual assessment that is used in conventional methods.