A CAJM  article on the prevalence of smallpox in Zimbabwe (ex-Rhodesia)  and the use of Electron Microscope in its diagnosis.This paper reports the laboratory findings in these cases of smallpox  to indicate (1) the availability of such a medical service in Rhodesia to counter smallpox; (2) that rapid reporting can be achieved after receipt in the laboratory of the specimen; (3) that rapid positive alternative some days to reach the laboratory.
One technique used in the diagnosis of smallpox is electron-microscopy for virus particles, and some examples of the general usefulness of this technique in medical and veterinary diagnosis in Rhodesia are included in this report

Cruickshank, J.G.

Swanepoel, R.

Central African Journal of Medicine

English

IDS OpenDocs

The Central African Journal of Medicine
Editor:
M IC H A E L  G E L F A N D , C.B.E., M .D ., F .R .C .P .
A ssistan t Editor: 
JO S E P H  R IT C H K E N , M .D .
Volume Eighteen 
JANUARY - DECEMBER 
1972
Salisbury
Rhodesia
T h e C e n t r a l  A f r i c a n
Journal of Medicine
Volume 18 APRIL, 197(2 No. 4
Smallpox in Rhodesia and the 
Use of the Electron Microscope 
in the Diagnosis of This and 
Other Diseases
BY
R. SWANBPOBL and J. G. CRUICKSHANK
Department of Microbiology, University of Rhodesia.
The number of cases of smallpox reported in 
Rhodesia has fallen from 203 in 1964 to only six 
in (1970 ((Reports of the Secretary for Health, 
1964-1970). Owing largely to extensive vaccina­
tion programmes and strict surveillance at border 
posts no urban epidemics have occurred for six 
years and indeed no cases have been reported 
from Midlands province for over seven years. 
This remarkable and gratifying picture has, how­
ever, certain self-inflicted consequences.
The main one is that although smallpox is easy 
enough to recognise during an outbreak or even 
in an endemic area, sporadic cases are often
Fig. 1—Smallpox (from case 22-12-70).
Fig. 2—Orf.
difficult to diagnose. Hence undiagnosed cases 
may slip through to become the foci of out­
breaks while misdiagnosed cases cause alarm and 
initiate unnecessary and expensive activity on the 
part of the public health authorities. Paradoxical 
as it may seem, therefore, the fewer the cases the 
greater is the importance of establishing the diag­
nosis accurately in the laboratory.
Until 1969 the diagnosis of smallpox in this 
country was largely clinical, but since then the 
communicable diseases section of W.H.O. has 
expressed a wish to Obtain isolates for comparison 
with others from elsewhere. Consequently a few 
specimens have been submitted to the virus 
laboratory at the University for examination and 
virus isolation. Some of these specimens have been 
from clinically diagnosed smallpox while others 
are' from cases thought unlikely to (be smallpox 
buit in whioh laboratory confirmation would be 
desirable.
This paper reports the laboratory findings in 
these cases to indicate (1) the availability of such 
a service in Rhodesia; (2) that rapid reporting 
can be achieved after receipt in the laboratory of 
the specimen; (3) that rapid positive alternative
68
April, 1972 SMALLPOX IN RHODESIA T h e  Central African
J ournal of Medicine
diagnoses can lie established in some cases; and 
(4) that the diagnosis of smallpox can and should 
be confirmed in the laboratory in every case, 
even When the specimen may necessarily take
some days to reach the laboratory.
One technique used in the diagnosis of smallpox 
is electron-microscopy for virus particles, and 
some examples of the general usefulness of this 
technique in medical and veterinary diagnosis in 
Rhodesia are included in this report.
Materials and Methods 
Smallpox specimens are received in a form 
according to the stage at which the case is seen.
In the macular or papular stage of the rash, 
lesions are scraped with a cutting needle or scalpel 
blade and the fluid which oozes out is smeared 
on a small area on a microscopic slide. Smears 
are air-dried and taped face to face with the 
slides held slightly apart by match sticks.
Smears of vesicle fluid are prepared in similar 
fashion after puncturing vesicles with a needle or 
a scalpel blade. In  addition, fluid escaping from 
punctured vesicles is collected by applying the 
end of a capillary (e.g.,- micro-haematocrit) tube 
to the fluid and allowing it to be drawn into the 
tube by capillary action. Three or four tubes per 
patient are half or threip-quarter filled and shaken 
so that the fluid lies in the middength of the tube. 
In this way, ithe fluid is\ not drawn out When the 
tubes are placed in ai screw-capped universal 
container. |
Table 1
Smallpox Specimens Tests/R esults
Date
Received Specimen Clinical Diagnosis Origin
Immuno­
diffusion 
for variola
Electron
Microscopy
Growth 
in eggs
Comple­
ment
fixation
13/9/69 Vesicle fluid Molluscum contagi- Sby. ex Umtali Positive
*i
ND ND
*2
NA-
13/10/69 Vesicle fluid
osum?
Chickenpox /Small- Salisbury Negative ND ND NA
10/11/69 Vesicle fluid '
pox
Chickenpox / Small- Nuanetsi Positive ND ND NA
10/11/69 Pustule smear
pox?
Smallpox? Chibi NA ND Variola NA
3/4/70 Crust smear Chickenpox Bulawayo NA Negative Negative NA
24/4/70 Crusts Smallpox? Filabusi NA Negative Negative NA
11/8/70 Pustule smear Chickenpox/Small- Chiredzi NA Herpes Negative ' NA
21/9/70 Vesicle fluid &
pox?
Smallpox? Bikita Negative Negative Negative Negative
1/10/70
smear +  serum 
‘Nodule’ smear Smallpox? Bulawayo NA Negative Negative NA
14/10/70 Smear Chickenpox / Small- Bulawayo NA Herpes Negative NA
26/10/70 Vesicle smear
pox
Smallpox? Belingwe NA Herpes Negative NA
22/12/70 Pustule fluid & Smallpox? Sby. ex Bindura Negative Variola ND Negative
16/2/71
serum
Serum Smallpox Chfpinga NA NA NA Negative
26/8/71 Crusts Smallpox Chipinga NA Herpes Negative NA
*1 ND =  N at done 
*2 NA --  Not applicable
69
April, 1972 SMALLPOX IN RHODESIA T h e  Central AfricanJ ournal of M edicine
Fig. 4—Herpes simplex.
Pustules are treated as vesicles except that it 
may foe difficult to obtain capillary tube specimens 
once the lesions begin to Shrink. In the crusting 
stage three or four crusts are removed with forceps 
and placed in a screw-capped container.
All specimens are sealed into plastic bags and, 
if possible, placed in a thermos flask with ice. 
Alternatively .they are placed in a cardboard box 
with absorbent packing. Parcels are labelled 
“Pathological specimens — urgent” and dispatched 
by quickest available transport to: The Depart­
ment of Medical Microbiology, University of 
Rhodesia, P.O. Box MP 167, Mount Pleasant, 
Salisbury. Road transport firms have been found 
to provide very satisfactory service.
The three principal methods used to diagnose 
smallpox are (i) electron microscopy, (ii) agar gel 
immunodiffusion for the detection of smallpox 
antigen and '(iii) growth in eggs
Electron microscopy is performed on vesicle 
fluid, or scrapings and smears Of skin lesions or on 
ground crust material. An aqueous emulsion of 
the specimen is transferred to a copper grid, wash­
ed three times in distilled water and negatively 
stained in two per cent, sodium phosphotungstate 
at pH 7,4. The grid is dried and placed in the 
microscope. The process takes approximately 
three ito five minutes. The grid is scanned at 
about 20 000 times magnification for the presence 
of virus particles.
Agar gel immunodiffusion can be performed on 
the same types of specimen but is most likely to
be successful with vesicular or pustular fluid. 
Smallpox virus shares antigens with vaccinia virus 
and hence antivacoinial serum is used to detect 
antigen in clinical specimens.
Slides are poured with 3 ml of hot one per cent. 
Ionagar or Noble agar. After the agar has set, 
a three-hole pattern is cut, the cups being 4 mm. 
in diameter with 6,5 mm. centre to centre spacing. 
Hyperimmune antivacoinial serum is placed in one 
cup, control positive vaccinia antigen in another 
and the specimen in the third. The preparation is 
incubated at room temperature in a damp chamber 
and examined at two hours and subsequently. Gel 
diffusion tests usually give a result in two to four 
hours and certainly overnight.
Virus isolation is performed on the chorio­
allantoic membrane of the hen’s egg. Eggs are 
examined on the second day and again on the 
third day When the final report is made.
Electron microscopy for conditions other than 
smallpox is performed in similar fashion to that 
detailed above.
Results
Smallpox li’
Specimens received with^a provisional diagnosis 
of smallpox are presented m Table I. It is to be 
noted that the majority of cases reported as small­
pox since 1969 have been diagnosed purely 
dlinically and presumably laboratory confirmation 
was sought only for those cases in which the 
diagnosis was doubtful. Specimens obtained 
before 1970 were examined only by gel diffusion
Fig. 5—Human papilloma (verruca vulgaris).
70
April, 1972 SMALLPOX IN RHODESIA T h e  Central AfricanJ ournal of Medicine
or fey growth in eggs, as the electron microscope 
was not available until February, 1970.
Three of the four specimens examined in 1969 
proved to toe positive either by gel diffusion or fey 
isolation in eggs. Of the subsequent 11 cases one 
was shown by electron microscopy to be smallpox 
(Fig. 1), four specimens contained herpes or 
herpes-like particles and five showed neither 
particles in the electron microscope nor growth in 
eggs.
Complement-fixation tests undertaken in three 
cases in the hope of making a retrospective diag­
nosis, were negative. One serum was from a case 
shown to b e . smallpox by electron microscopy. 
This serum, however, had been taken in the acute 
stage of the disease when a positive result was 
unlikely.
Other viruses
Other virus diseases diagnosed by electron 
microscopy are summarised in Table II.
Table II
V iruses other than Smallpox detected 
DIRECTLY IN CLINICAL SPECIMENS BY 
Electron M icroscopy
Source Type of particle Diagnosis Figure
Human ' Pox Orf 2
Human Pox Molluscum con- 
tagiosum
3
Human Pox Cowpox
Human Herpes Herpes simplex 4
Human Herpes Chiickenpox
Human Herpes Zoster
Human Papova Verruca vul­
garis
5
Pig Pox Piigpox 6
Cattle Papova Papilloma 7
Horse Papova Papilloma 8
Sheep Pox Orf
D iscussion
The use of electron microscopy in the diagnosis 
of smallpox fulfills the criteria of good laboratory 
procedure — rapidity, simplicity, accuracy and 
sensitivity. In the 1966 Birmingham outbreak 
only one case which proved later to be positive 
was negative by electron microscopy while 40-odd 
other cases were rapidly confirmed (Cruiokshank, 
Bedson and Watson, 1966). Although it must be 
borne in mind that vaccinia, cowpox and small­
pox virus particles are indistinguishable from one 
another toy electron microscopy, the case history 
and clinical manifestations usually clarify the 
diagnosis. Generalised vaccinia is not a natural 
disease and its occurrence would in all probability
be associated with recent vaccination. Cowpox is 
an occupational hazard of people working with 
'livestock and it is unlikely to occur in generalised 
form.
The detection of herpes-like virus particles by 
electron microscopy allows an alternative diagnosis 
of ohicken pox or generalised herpes-simplex to be 
established and again the clinical manifestations 
Should make it possible to arrive at a definite 
diagnosis.
Immunodiffusion has a more restricted applica­
tion to the vesicular or pustular stages of small­
pox and as ordinarily used it does not offer any 
advance over electron microscopy in distinguish­
ing smallpox from vacoinia. It nevertheless con­
stitutes an inexpensive and rapid aid to diagnosis, 
whioh could be undertaken by most laboratories.
It is generally accepted that growth in eggs is 
the most sensitive method for isolating smallpox 
virus and specimens are always examined by this 
method irrespective of the results of other tests. 
The pocks produced on the chorioallantoic mem­
brane of eggs fey herpes-simplex, vaccinia, cow- 
pox and smallpox viruses are easily distinguished 
from each other while the herpes-zoster virus of 
chickenpox/shingles fails to grow in eggs. Variola 
minor (Alastrim), variola major and intermediate . 
strains of smallpox virus can toe distinguished 
from each other by the ceiling temperature' at 
whieh they grow in eggs (Bedson & Dumbell, 
1961).
71
fApril, 1972
Fig. 7—Bovine papilloma.
The diagnosis in every case of suspected small­
pox should be confirmed in the laboratory. Now 
that so few. cases are seen experience in the 
clinical diagnosis is decreasing and a generation 
of practitioners is growing up who have never 
and may never see the disease. The extensive 
and expensive measures required ito be undertaken , 
to trace ithe source of an outbreak and to protect 
the population at risk are such a gross inconveni­
ence to all concerned that an incorrect diagnosis 
has very far-reaching and adverse consequences.
A further reason for laboratory confirmation is 
the necessity for Rhodesia to play its part in the 
general world surveillance of this disease. Eradica­
tion is in sight and it is important to locate out­
breaks rapidly and to identify accurately the type 
of virus involved; variola major or minor (Official 
Records, W.H.O. 1970).
Finally, the virus is sufficiently hardy and stable 
for specimens in the form of smears on slides, 
vesicular or pustular fluids or crusts to remain 
usable even after a number of days “on the road”. 
There,.is thus no reason why specimens should not 
be submitted from every case no matter how 
remote geographically it may be. Even from the 
few cases presented here it is apparent that most 
cases that are doubtful turn out not to be small­
pox.
The general usefulness of the electron micro­
scope in the diagnosis of virus diseases is re­
stricted by the fact that a 'high concentration of 
virus particles is required in the clinical specimen
before they can be detected with certainty in the 
microscope. Specimens from the skin conditions 
listed in Table II are ideally suited for electron 
microscopy. Particles are plentiful in lesions from 
these conditions whether they are papillomatous, 
papular, vesicular, pustular, ulcerative .or crusting 
and a positive diagnosis can rapidly be made. In 
some instances,, as in the veterinary diseases, there
may be little doubt about the clinical diagnosis, 
but electron microscopy is nevertheless useful as 
a visual check oh the potency of the autogenous 
vaccines commonly used in these diseases.
In other diseases electron microscopy is finding 
increasing use as a means of speeding the process 
of identifying a virus once it has been grown in 
cell culture from a clinical specimen (Fig. 9). The 
problem of the morphological similarity of viruses 
with widely differing antigenic and pathogenic 
properties is being overcome by using electron- 
dense labelled antibodies; (immunoferritins) .to 
make possible the specific identification of viruses 
by electron microscopy.'However, further develop­
ment is required before the technique can be used 
routinely. |-
Su iUmary
The methods used to examine smallpox speci­
mens in the laboratory' are described. Only four 
out of 14 specimens examined in Rhodesia since 
1969 have proved to be positive for smallpox. A 
further four specimens contained herpes-like virus 
particles, and in six instances no diagnosis could 
be confirmed.
T h e  Central African
J ournal of Medicine
Fig. 8.—'Equine papilloma.
SMALLPOX IN RHODESIA
Vol. 18 No. 4 April, 1972
Fig. 9—Rhodesian isolate of Hong Kong influenza.
T h e  Central African
J ournal of Medicine
Now that the disease is so rare it is suggested 
that the diagnosis in every suspected case should 
be confirmed in the laboratory and instructions 
~ are included for the taking and submission of 
specimens.
A few examples are given of the usefulness of 
the electron microscope in the diagnosis of virus 
diseases other than smallpox, particularly diseases 
producing skin lesions in man and animals.
REFERENCES
Annual Reports of the Secretary for Health, Rhodesia. 
(1964-1970).
Bedson, H. S. & D umbell, K. R. (1961). J. Hyg. Camb. 
59, 457.
Cruickshank, J. G., Bedson, H. S. & W atson, D. S. 
(1966) Lancet, ii, 527.
Official Records of the World • Health Organisation No. 
188 p. 15. 1970. :
All electron micrographs are at a  magnification of 
about 222 000 X.
1
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Smallpox In Rhodesia And The Use Of The Electron Microscope In The Diagnosis Of This And Other Diseases

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Smallpox In Rhodesia And The Use Of The Electron Microscope In The Diagnosis Of This And Other Diseases

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