Rapid diagnosis of malaria by fluorescence microscopy

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Rapid diagnosis of malaria by fluorescence microscopy SIR,—There are several points raised by Dr Kawamoto (Jan 26, 200) that concern us. We disagree with the statement that fluorochrome staining to detect malaria parasites is more sensitive, easier, and less time consuming than Giemsa staining. We believe that the ’QBC II System’ does not replace Giemsa staining for malaria diagnosis.In addition, we are not surprised that Kawamoto found that red cells stained strongly blue with Giemsa and that it was difficult to see parasites, since he was examining blood films at least a year old; this is not comparable with the clinical situation, p

where

one must

know whether

or not an

individual has malaria as

possible. No details are given of the ability of this method to speciate parasites. We found that QBC II was not able to speciate parasites as effectively as conventional microscopy. Finally, we are soon as

told the number of slides evaluated, except that five films were examined to assess time taken to find the first parasite. Kawamoto has described a new method for examining fluorochrome stained parasites, which must be evaluated with fresh blood samples and compared with conventional Giemsa and Field stains. We must know how well it distinguishes between different species of parasite and how well it assesses parasitaemia. In your talking points section, you comment that for nearly 90 years clinical diagnosis of malaria has depended on identification of Plasmodium parasites by microscopy in stained blood films. We do not feel that microscopy of Giemsa stained blood films has been supplanted; it is likely to reach its centenary. not

P. L. CHIODINI A. H. MOODY A. HUNT-COOKE

Department of Parasitology, Hospital for Tropical Diseases, London NW1 OPE, UK

Right and left pulmonary angiograms and IVUS images after urokinase therapy for acute pulmonary embolism. Black arrows=IVUS catheter position, white arrows=thrombi, W=vessel wall, C= IVUS catheter, L=free lumen. In the right lung there is a large Intramural thrombus seen by both angiography and IVUS In the left lung there are small thrombi at bifurcation site.

thrombi in both main trunks of the pulmonary arteries. After urokinase (2 x 106 U/kg over 10 min followed by 106 U/kg in 1 h) and intravenous heparin 15 IU/kg/h she improved rapidly. Angiography the next day also showed improvement with residual thrombi visible in the right and left pulmonary arteries (figure). To rule out persistent mural thrombus formation a 125 cm 20 Mhz 4-5 F IVUS catheter (Boston Scientific) was introduced via a guidewire (the 8F sheath used for angiography) into the main trunk of the pulmonary artery and advanced into the left and right pulmonary trunk. The catheter was connected to a diasonics ultrasound device and sonograms were stored on videotape. The catheter could be advanced to vessels smaller than 1 cm in diameter. IVUS indicated almost complete obstruction of the right lower pulmonary trunk, partly obstructed portions of the left lower pulmonary trunk, and partly lysed, free-floating thrombi (figure). IVUS may be of value for the diagnosis of pulmonary embolism and for evaluating the success of thrombolysis. Over transoesophageal echocardiography it has the advantage of visualising not only the large but also the smaller pulmonary arteries. It should soon be possible to use an indwelling IVUS catheter to monitor thrombolytic therapy in pulmonary embolism.

II Medical Clinic,

University Hospital, D-6500 Mainz,

Germany

GÜNTER GORGE RAIMUND ERBEL STEPHAN SCHUSTER

JUNBO GE JÜRGEN MEYER

1. Nixdorff U, Erbel R, Drexler M, Meyer J. Detection of thromboembolus of the right

pulmonary artery by transesophageal two-dimensional echocardiography. Am J Cardiol 1988; 61: 488-89 2. Pandian NG Intravascular and intracardiac ultrasound imaging on the road to reality. Circulation 1989; 80: 1091-94.

an

old concept,

now

1.

Moody AH, Hunt-Cooke A, Chiodini PL. Expenence with the QBCII Rcentrifugal haemotology analyser for haemoparasites Med Hyg 1990; 84: 782.

***This letter has been shown follows.-ED. L.

to

Dr

Becton Dickinson Trans R Soc Trop

Kawamoto, whose reply

SIR,—Dr Chiodini and colleagues state that the ’QBC’ tube technique for the diagnosis of malaria will not replace the established procedures of conventional Giemsa or Field stains. However, the acridine orange (AO) interference filter system’ differs from the QBC technique about which they complain. I recommend examining the AO staining (10 Ilgjml, pH 7 ’0-7 5) with an epi-illuminated fluorescence microscope instead of the AO filter system,l whereby thin smears should be observed with combined light microscopy using weak light from the bottom of the microscope. Like the AO filter system, this method permits observation of the morphology of infected red blood cells, so that most of the criteria for species identification normally applied to conventional staining may also be applied to the AO staining, except for pigment. Parasitaemia can be evaluated easily at x 200400 since all the parasites are fluorescing in a dark background. evaluation of the AO filter system, based on 100 smears (with thick smears on the same slides) were obtained on Nov 5-8, 1990, from undiagnosed outpatients at two villages near Ifakara, Tanzania. All these smears were inspected first using the AO filter with a daylight-illuminated microscope at magnifications of x 200-400, and the time required to detect the first parasite was recorded (maximum observation 15 min). Parasite counts and species identification in positive smears were then done using the AO filter with a halogen-illuminated microscope. Finally, smears were stained with Giemsa, examined randomly after blind coding, and the results were compared with those obtained with the AO filter system. Of 100 thin smears, 75 were positive; 32 were identified as single or mixed infections with Plasmodiumfalciparum and/or P malariae by the presence of each typical stage such as signet rings, band forms, schizonts, and/or gametocytes. 2 of them were also found to possess typical amoeboid forms of P ovale with fimbrinated red blood cells. However, it was impossible to distinguish large rings of In

a recent

smears, two sets of thin

625

P falciparum from rings of P malariae since infected erythrocytes with these parasites have similar sizes, and pigments are not seen. 33 cases (parasitaemia 0-003% or more) with large rings in addition to signet rings and/or gametocytes of P falciparum were considered as having possible mixed infection with P malariae. By Giemsa staining, these cases were thought to be P falciparum infections, although differential speciation from a thin smear only was also difficult for the examiner (F. K.), who was inexperienced in diagnosing clinical cases of human malaria. 10 cases having only 1-5 could not be rings per thin smear (parasitaemia, <0-0002%) speciated accurately by both methods. Times to the finding of the first parasite

were

shorter with the AO system than with Giemsa

staining, especially at parasite counts below 0-5% (Giemsa/AO ratio above 27), and 11cases out of 15 with very low parasite counts (< 00002%) were not detected within 15 min by Giemsa staining. These results suggest that the AO filter system combined with

simple microscopes may be used as a rapid diagnostic technique in tropical countries endemic for malaria. Rapidity in the AO system may indicate the ease of confident detection of the parasites.. This may facilitate the diagnosis of malaria for inexperienced microscopists, such as community health workers in primary health care settings. A new "thick smear" method (unpublished) which allows simultaneous haemolysis and staining with AO in a few minutes, may also help in the rapid diagnosis of malaria. Further evaluations of the AO filter system should be made by field and laboratory investigators; the AO-interference filter is now available from a Japanese company at nearly cost price. This study has been done in cooperation with Dr M. Tanner and Dr T. Teuscher, Swiss Tropical Institute field laboratory in Ifakara, and Dr P. F. Billingsley, Imperial College, London, and received financial support from

UNDP/World Banks/WHO Special Programme for Research and Training in Tropical Diseases to F. K. I thank Dr Billingsley for his valuable the

comments.

Department of Medical Zoology, Nagoya University School of Medicine, Showa, Nagoya 466, Japan

FUMIHIKO KAWAMOTO

F, Kumada N. Fluorescent probes for detection of protozoan parasites. Parasitol Today 1987, 3: 284-86.

1 Kawamoto

SIR,—Iam pleased to see someone else validating the use of fluorochromes for the diagnosis of malaria.1.2 Colleagues and I have reported on the use of fluorochrome in a field study in southern Africa.’ This fluorochrome was a benzothiocarboxypurine. It is selective; it only stains intracellular malaria parasite in fresh blood and does not stain either white cells or platelets. This discrimination

improves diagnostic ability. During this field study I noted the need for an inexpensive fluorescent microscope and on my return to Portland, Oregon, I constructed a fluorescent objective (UK patent filed December, 1990). The objective (figure) contains within it an excitation dichroic and emission filter and it also houses a lateral port. This fluorescent objective screws into the turret of any light microscope, and with an

adaptor it becomes parfocal with the other objectives on the microscope. The fluorescent objective is attached, via a light pipe, to a 30-150 W tungsten light source, which can be battery operated. The fluorescent objective is rugged, simple to attach, and easy to use. Because there are very few surfaces for the tungsten light to pass through, the fluorescent objective can provide an intense signal on fluorescent stained specimens. The light source, the adaptor, and the fluorescent objective costs about one-tenth the price of an average standard fluorescent microscope attachment. Besides routine daily use in our own laboratory, four of these objectives are being tested in laboratories in the UK, South-East Asia, and Africa. So far they all appear to be working very well. Laboratory 113C, VAMC, Portland, Oregon 97201, USA

MICHAEL T. MAKLER

GT, Sodeman TM. Identification of malarial parasites by fluorescence microscopy and acridine orange staining. Bull WHO 1973; 48: 591-96. 2. Voller A, O’Neill PO. Immunofluorescence method suitable for large scale application to malaria. Bull WHO 1971; 45: 524-29. 3. Makler MT, et al Am J Trop Med Hyg 1991; 43: 11-16. 1 Shute

Expatriates treated with ivermectin SIR,-Dr Davidson (Oct 20, p 1005) and Dr Bryan (Feb 2, p 304) and their colleagues’ reports of adverse reactions in expatriates treated with ivermectin have prompted us to re-examine our data from Sierra Leone.1,2 Davidson et al describe a high rate (61 %) of such reactions in expatriates receiving their first dose of ivermectin and attribute this to their intensive monitoring of inpatients or to a more vigorous immune response to dying microfilariae than is usual in indigenous adults with long-standing infections. They suggest that, because of this immune tolerance, higher rates of adverse reactions might be seen in children than in adults treated with ivermectin in endemic areas. If this were so, it could provoke concern about the planned wide distribution of ivermectin in sub-Saharan Africa. We have tested the in-vitro cellular immune responses in 203 individuals from our study community and found some evidence of higher responses to Onchocerca volvulus in infected 10-14-year-old children than in those of other age groups.3 A similar pattern has been reported from Liberia.4 If Davidson and colleagues’ immune tolerance hypothesis is correct, we would expect 10-14-year-old children to have higher rates of adverse reactions than those in other age groups when first treated with ivermectin. The table shows the age-specific frequencies of all reactions and skin reactions to ivermectin reported in microfilaria-positive individuals in our study. Values in children were not raised and no peak rate was seen in the 10-14 age group. The frequency of adverse reactions generally rises with age and microfilarial load. However, De Sole et al5 and we? have found that the frequency of skin reactions (increased itching and/or rash) does not seem to be correlated with skin microfilarial loads, and this was borne out by our present

analysis. Thus in our indigenous population there is of adverse reactions to ivermectin in

frequency

no

increased

children, and

AGE-SPECIFIC FREQUENCY OF ADVERSE REACTIONS TO IVERMECTIN IN MICROFILARIA-POSITIVE INDIVIDUALS