MICROBIOLOGÍA BÁSICA
Performance of the Ogawa-Kudoh method for isolation of mycobacteria in a laboratory with large-scale workload
C. Rivas*, C. Coitinho, V. Dafond, M. Corbo, M. Baldjian
Comisión Honoraria para la Lucha Antituberculosa y Enfermedades Prevalentes. Centro de Referencia Nacional para
Micobacterias. Departamento Laboratorio. Avda.18 de Julio 2175 6p. Montevideo, Uruguay.
*Correspondence. E-mail: cmrivas1@yahoo.com
ABSTRACT
In Uruguay (population 3,323,906; notified tuberculosis incidence 18.4/100,000), virtually all 30,000 samples yearly collected for mycobacterial culture countrywide are processed in a central laboratory. An average of 110 samples are routinely shipped daily and maintained 48-96 hours at room temperature until cultured on Löwenstein-Jensen slants using the standard NALC-NaOH decontamination procedure. The much simpler Kudoh decontamination/culture method -swab and Ogawa (acidified) medium- was compared with NALC-NaOH/Löwenstein-Jensen for isolation of mycobacteria from sputa under routine conditions. To this aim, 784 sputum samples were cultured by both methods in the summertime. Gross agreement was 0.99, kappa: 1. Kudoh performance was as follows: sensitivity 100% and accuracy 98.9%. Assays using a modified culture medium, different decontamination times and NaOH concentrations showed the versatility of this procedure. Thus, the Kudoh method is suitable for culturing mycobacteria from naturally contaminated samples even when processing is deferred two to four days after collection.
Key words: Kudoh; Mycobacterium tuberculosis; Culture; Sputum samples
RESUMEN
Rendimiento del método de Ogawa - Kudoh para el aislamiento de micobacterias en un laboratorio con trabajo a gran escala. En Uruguay (3 323 906 habitantes; incidencia notificada de tuberculosis 18,4/100 000), más del 95% de los cultivos para micobacterias de todo el país (30 000 por año) son procesados en un laboratorio central. Un promedio de 110 muestras de expectoración diarias son enviadas y mantenidas a temperatura ambiente durante 48-96 horas hasta ser procesadas por el método de descontaminación con NALC-NaOH y cultivadas en medio Löwenstein-Jensen. Con el objeto de evaluar el método de Kudoh (hisopo y medio de Ogawa acidificado) como una alternativa más sencilla para el cultivo de micobacterias en este tipo de muestras, se procesaron por ambos métodos 784 esputos durante los meses de verano, a fin de comparar los resultados obtenidos. La concordancia bruta fue de 0.99; el coeficiente kappa fue 1. El método de Kudoh mostró una sensibilidad del 100% y una exactitud del 98,9%. Algunos ensayos en los que se empleó un medio modificado y diferentes concentraciones de NaOH y tiempos de descontaminación demostraron la gran versatilidad del método. Se concluye que el método de Kudoh puede ser aplicado para el cultivo de muestras naturalmente contaminadas, aun después de haber permanecido 2 a 4 días a temperatura ambiente antes de ser cultivadas.
Palabras claves: Kudoh; Mycobacterium tuberculosis; Cultivo; Muestras de expectoración
Uruguay -a country with 3,323,906 inhabitants- has
a notified tuberculosis (TB) incidence of 18.4/ 100,000
(13). Pulmonary forms constitute up to 85% of the cases;
of which 80 to 85% are bacteriologically confirmed by
microscopy and/or culture (13). More than 95% of the
sputum samples collected countrywide for TB diagnosis
and treatment control are processed in one laboratory:
the National Reference Center for Mycobacteria
(CERNAMY). The average number of clinical specimens
cultured for M. tuberculosis is 30,000 per year (range:
28,000 - 32,000); the total cost per year is US$ 92,000.
Sputum samples are shipped at room temperature without
the addition of any antiseptic and processed between
2 and 4 days after collection. After decontamination, digestion
and concentration by the standard N-acetyl-LCysteine-
NaOH (NALC-NaOH) method (8, 9), the specimens
are cultured on an egg-based Löwenstein-Jensen
medium according to IUATLD recommendation. Culture
contamination varies within a range of 5-9%, depending
on the season of the year, the highest rates being registered
in the summer months; the rate of culture-positive
samples is 5.5% (5). Different methods for culturing mycobacteria
are used worldwide. Some of them were developed
aiming at simplicity, low cost and a better chance
of accomplishment at network level. A simple and inexpensive
method for the culture of just-emitted sputum
samples was reported by Kudoh and Kudoh in 1974 (10).
Briefly, each of two sterile swabs impregnated with a useful
particle of sputum sample is submerged into a tube
containing 2 ml of 4% NaOH during two minutes for decontamination and immediately inoculated onto a slant
of Ogawa culture medium (acidified egg-based medium).
Recently, a multicentric study for evaluation of this technique
was performed in Latin America with very promising
results (6). Still, there is insufficient information about
the accuracy of the Kudoh method under routine conditions,
i.e., in non sterile samples that remain unrefrigerated
during 2 or more days before being processed. The aim
of this study was to evaluate the performance of the Kudoh
method for the culture of mycobacteria in sputum samples,
after being maintained and shipped at room temperature
without the addition of any substances to prevent
the overgrowth of contaminating flora.
The study was prospective and blind, with clinical sputum
samples shipped from all over the country and assays
performed by different technicians under routine
conditions. All macroscopically purulent or muco-purulent
sputum samples received in the TB Central Laboratory
during the study period were included. All culture
media were prepared in-house, and quality controlled by
WHO/PAHO standard procedures (14). Performance of
Kudoh was evaluated using the NALC-NaOH method as
gold standard.
A total of 784 clinical samples were cultured by both
methods, performed according to Kudoh et al. (10) and
the NALC-NaOH method, respectively (8, 9). After decontamination,
each specimen was inoculated onto 2 slants
of each Ogawa and Löwenstein-Jensen (IUATLD modification)
media. The study was performed during the summer
at an average temperature of 25 °C (minimum 22 °C -
maximum 32 ºC); cultures were performed by technicians
under routine conditions, incubated at 37 °C (normal atmosphere)
and read after 7, 21 and 56 days of incubation.
The acidified IUATLD medium was compared with the
Ogawa medium for culture after the Kudoh decontamination
procedure. One hundred smear - positive samples
were inoculated onto 2 slants of Ogawa medium and 2
slants of IUATLD acidified Löwenstein-Jensen medium;
this latter medium was acidified to pH 6.4 with the addition
of citric acid (1.35 g/l of medium). The composition of
both egg-based media differs slightly: the Ogawa formula
omits magnesium salts and asparagine, and includes
higher concentrations of glycerol (2% vs 0.75%) and
malachite green (2% vs 1.25%). Mycobacterial growth
was evaluated as described above.
The effect of variations in decontamination time and
NaOH concentration on mycobacterial recovery rates was
assessed. In other set of experiments, a total of 25 isolated
smear-positive sputum samples were comparatively
cultured with the Kudoh method and increasing NaOH
concentration of up to 6% and decontamination time up
to 4 minutes. The original description of the Kudoh method
(2-minute decontamination and 4% of NaOH) was considered
the gold standard. Mycobacterial growth was
quantified according to the WHO/PAHO semi-quantitative scale (14). Colony size, growth rate and aspect of
colonies (smooth or rough) were also recorded.
Cultures were considered contaminated when microorganisms
other than mycobacteria grew on the slants
with or without media liquefaction. The contamination of
only one tube was recorded as partial contamination.
Mycobacterial growth was quantified according to the
semi-quantitative scale proposed by the WHO/PAHO (14).
Kudoh and NALC-NaOH methods performed similarly
regarding contamination and mycobacterial growth (Table
1). All positive cultures were identified as Mycobacterium
tuberculosis and there was a very good correlation
between size, growth rate, number of colonies and direct
microscopy results. Similar percentages of contamination
were observed after combining partial contamination
with total contamination. The diagnostic performance of
Kudoh (vs. NALC-NaOH as gold standard) for positive
and non-positive (negative + contaminated) categories
was: sensitivity 100% and diagnostic accuracy 98.9%.
Agreement was 0.99; kappa: 1. Both methods showed
identical results improving the diagnostic sensitivity by
22% over microscopic examination alone.
Table 1. Comparative results of cultures from 784 sputum
samples, processed by the Kudoh and NALC-NaOH methods.
(*) N: negative, C: contaminated, P: positive, 1t: only one culture tube, 2t:
two culture tubes. Data for the combinations N(1t)-P(1t) and C(1t)-P(1t)
were null with both methods.
(**)Fourteen samples were positive to the direct microscopic examination
Results of assays with different decontamination times and NaOH concentrations are shown in Table 2. Mycobacterial growth was slightly affected when increasing decontamination times and NaOH concentrations. However, independently of the number of colonies, positive cultures were obtained in all time/NaOH concentration combinations. Different growth rates could be partially explained by the difficulty to standardize the inocula with this method.
Table 2. Degree of positivity of 25 positive cultures according to different decontamination procedures applied
to the Ogawa-Kudoh method.
(*) Results obtained with the original Ogawa-Kudoh method (2 minute-decontamination - 4% of NaOH). All samples had positive
microscopy for mycobacteria. Colony counts were done according with the WHO/PAHO semiquantitative scale (12). Col: colonies.
A better morphology (rough and dry) and size of the
colonies and more luxurious growth was observed when
the acidified IUATLD culture medium was used instead
of the Ogawa one.
Mycobacterial culture is still essential for TB diagnosis,
being more specific and sensitive than microscopy.
In Uruguay 14 - 20% of TB cases are confirmed by this
method (5). In addition, culture provides strains for drug
susceptibility testing and epidemiological surveillance.
The main disadvantages of conventional cultures are: delay in obtaining mycobacterial growth, high costs, complexity,
and biosafety requirements. The Kudoh method
is extensively used in several Asian countries, including
Japan (8). Several reports in Latin America about the use
of the Ogawa medium (12) and the swab Ogawa-Kudoh
method documented good results (2, 3, 11). Jaspe et al.,
working in field conditions, reported similar performances
when this method was compared with the standard Petroff
digestion-decontamination procedure (7). In certain states
of Brazil, the Ogawa-Kudoh swab method is routinely
applied for primary culture of sputum samples (1, 4).
The centralization of bacteriological TB diagnosis in
Uruguay generates some problems inherent to large-scale
workload, namely the need of a high budget, an
increased risk of intra-laboratory cross-contamination
and culture contamination with overgrowing flora. This
last issue is especially crucial in Uruguay, since our laboratory
receives samples that have been obtained at least
48 hours earlier and transported at room temperature.
Delays and lack of refrigeration can be the cause of
culture contamination reaching 9% in the warmest
months. Although the NALC-NaOH method is the reference
procedure for decontamination and digestion, the
adoption of a simpler technique with acceptable levels
of contamination and recovery of mycobacteria may be
an alternative for the routine culture of large numbers of
respiratory samples.
The original Kudoh method has shown to be very useful
for peripheral laboratories which process small numbers
of samples daily, but decontamination time (2 minutes)
may be difficult to maintain when 80 samples are
serially processed by one technician; thus we considered
of relevance to know how long it is possible to extend
decontamination times without affecting mycobacteria
growth. Our results showed that the decontamination time
of the procedure duration is not critical and acceptable
outcomes can still be obtained by increasing it up to 4
minutes. Furthermore, if a stronger decontaminant action
is needed (i.e. deferred processing of naturally contaminated
samples), it is also possible to increase the
NaOH concentration with acceptable recovery of mycobacteria
in culture.
Other issues to be considered are colony morphology
and growth time, since these primary cultures are further
used for phenotypic identification of mycobacterial species,
drug-susceptibility testing and molecular identification
techniques. In our hands, the substitution of Ogawa
for the acidified IUATLD medium in primary Kudoh culture
resulted in a more luxurious growth that granted a
better handling of further bacteriological procedures.
The advantages of the Kudoh method using the Ogawa
medium or another acidified egg-base medium are evident:
it is inexpensive (its use would save 30,000 US
dollars per year to the National TB Program in Uruguay),
it is very simple to perform and does not require agitation
and centrifugation steps. Therefore, we can also expect
a reduction of equipment, intra-laboratory cross contamination
rates and fewer biosafety requirements for laboratory
personnel.
In conclusion, the Kudoh method for primary isolation
of mycobacteria may be implemented in laboratories that
work with large scale workload and turned out to be applicable
for naturally contaminated respiratory samples
that are maintained at room temperature during 2 to 4
days before being cultured, even in the summer months.
The Kudoh method is simpler and cheaper than the NALC-NaOH
one and performs similarly.
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Recibido: 15/12/09
Aceptado: 30/03/10