Rev Inst Adolfo Lutz

Comunicação Breve/ Brief Communication

Microbiological quality of water and dialysate from

haemodialysis units in Southern Brazil

Qualidade microbiológica da água e dialisato em clínicas

de hemodiálise do Sul do Brasil
RIALA6/1638

Izabel Cristina FIGEL2, Patricia do Rocio DALZOTO1, Ida Chapaval PIMENTEL1*

*Endereço para correspondência: 1Departamento de Patologia Básica, Setor de Ciências Biológicas, Universidade
Federal do Paraná, Centro Politécnico. Av. Francisco H. dos Santos, Jardim das Américas, Curitiba, PR, Brasil,
CEP: 80530-900. E-mail: idinha.ufpr@gmail.com
Laboratório de Microbiologia e Toxicologia, Instituto de Tecnologia do Paraná (Tecpar), Curitiba, PR, Brasil.
2

Recebido: 17.11.2014 - Aceito para publicação: 27.02.2015

ABSTRACT
Infectious diseases in renal patients may be associated with the dialysis water quality, which may
be contaminated with microorganisms. In Brazil, the water quality is evaluated by analyzing total
coliforms, heterotrophic bacteria, and bacterial endotoxin, but not Pseudomonas sp. and fungi.
Water samples from haemodialysis units in Curitiba/PR were investigated on their conformity with
the standard established by the Brazilian Health Ministry. Total coliforms, heterotrophic bacteria,
P. aeruginosa and fungi counts were performed according to APHA, and LAL methodology for
detecting bacterial endotoxin. All of the samples showed the total coliforms counts ≤1.1 MPN/100 mL,
and ≥ 95 % of analyzed samples complied with the standards for heterotrophic bacteria counting.
P. aeruginosa was recovered from 4 % of samples. In 15 % of samples, bacterial endotoxin was
detected in values above the limit established by legislation. Yeasts were isolated from 26 % samples
and filamentous fungi from 58 %, being 46 % characterized as melanized fungi. The fungi genera were
Cladosporium spp., Penicillium spp., Beauveria spp., Exophiala spp., Fusarium spp., Aspergillus spp.,
Trichoderma spp, Acremonium spp. and Rinocladiella spp.. The study highlights the significance of
P. aeruginosa and fungi detection in those systems, as these microorganisms are potentially pathogenic
to immunocompromised patients.
Keywords. Pseudomonas aeruginosa, coliforms, heterotrophic bacteria, endotoxins, fungi.

RESUMO
Doenças infecciosas em pacientes renais podem ser associadas à qualidade da água de diálise, que
pode apresentar contaminação com microrganismos. Pelos padrões brasileiros, a qualidade da
água é avaliada analisando-se coliformes totais, bactérias heterotróficas e endotoxinas bacterianas.
Pseudomonas sp. e fungos não são investigados. Amostras de água de clínicas de hemodiálise em
Curitiba/PR foram avaliadas quanto à conformidade com os padrões do Ministério da Saúde.
Contagens de coliformes totais, bactérias heterotróficas, Pseudomonas aeruginosa e fungos foram
realizadas seguindo-se APHA e detecção de endotoxina pela metodologia LAL. Todas as amostras
tiveram contagens de coliformes totais abaixo de 1,1 MPN/100 mL e ≥ 95 % das amostras apresentaram
padrões aceitáveis para bactérias heterotróficas. P. aeruginosa foi encontrada em quatro amostras.
Em 15 % das amostras, endotoxinas bacterianas foram detectadas em valores acima dos permitidos
pela legislação. Leveduras foram isoladas em 26 % das amostras e fungos filamentosos em 58 %,
sendo 46 % melanizados e 27 % hialinos. Os gêneros fúngicos detectados foram Cladosporium spp.,
Penicillium spp., Beauveria spp., Exophiala spp., Fusarium spp., Aspergillus spp., Trichoderma spp,
Acremonium spp. e Rinocladiella spp.. Foi evidenciada a importância da detecção de P. aeruginosa
e fungos nestes sistemas, uma vez que estes podem ser potencialmente patogênicos para pacientes
imunocomprometidos.
Palavras-chave. Pseudomonas aeruginosa, coliformes, bactérias heterotróficas, endotoxinas, fungos.

66 Rev Inst Adolfo Lutz. 2015; 74(1):66-70

Figel IC, Dalzoto PR, Pimentel IC. Microbiological quality of water and dialysate from haemodialysis units in Southern Brazil. Rev Inst Adolfo Lutz. São
Paulo, 2015;74(1):66-70.

INTRODUCTION be transferred to the patient blood compartment

during dialysis. The exposure to high levels of
The Brazilian Society of Nephrology endotoxins is obviously associated with pyrogenic
estimates that about 10 million people are affected by reactions and septicemia5. In Brazil, standards
different levels of Chronic Renal Disease in Brazil1. had been established for the certification of water
About 89.4 % of these patients had been submitted quality in those systems6. The microbial standards
to hemodialysis and 10.6 % to peritoneal dialysis. for water for dialysis includes absence of coliforms,
The mortality observed was 15.2 % per year mostly heterotrophic bacteria count limits of 200 CFU/mL
due to the cardiovascular (37 %) and brainvascular and endotoxin limits of is 2 EU/mL; and for
(10 %) complications, infectious (26 %) and others dyalisate, heterotrophic bacteria count limit, the
not identified (27 %)2. only parameter predicted for, of 2000 CFU/mL7.
Infectious diseases in renal patients can be Although those parameters are important and
associated to the dialysis water quality, in which necessary, others microorganisms and metabolites
several microorganisms, such as bacteria, viruses, should be investigated in dialysis water systems,
fungi, protozoa, algae and also bacterial toxins such as Pseudomonas sp., mycobacteria, fungi and
can be assessed, mostly due to the inefficacy in mycotoxins8.
the water treatment process 3. Figel et al4 detected We aimed the evaluation of the microbial
the presence of fungi in 66 % of water samples quality of water for dialysis provided by Hemodialysis
assayed from dialysis units in Southern Brazil, Units in Curitiba, Paraná, Brazil, in attempt to verify
and observed the high prevalence (46 %) of black the accomplishment of the standards demanded
fungi, that can be associated to several diseases by Brazilian Health Ministry. In addition, counts
related to immunocompromised patients. The of Pseudomonas aeruginosa and fungi were also
bacterial cytokine-inducing substances, as performed in attempt to highlight its importance
lipopolysaccharides (endotoxins), exotoxins and and propose its inclusion in future revisions of the
peptidoglycans, are also a concern, as they can standards for water for dialysis.

Figure 1. Points collected in clinical hemodialysis of samples of tap water, treated water for dialysis and dialysate (Figel et al, 2013)

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Figel IC, Dalzoto PR, Pimentel IC. Microbiological quality of water and dialysate from haemodialysis units in Southern Brazil. Rev Inst Adolfo Lutz. São
Paulo, 2015;74(1):66-70.

MATERIAL AND METHODS plate, which was incubated at 35 oC ± 1 °C for 48 h.

At the end of the incubation period, the recovered
Water sampling colonies from each plate were enumerated and the
Samples of tap water (point 1), treated results were expressed in colonies forming units
water for dialysis (point 2) and dialysate into the (CFU) per mL9. The experiments were conducted
dialysis system (point 3) were obtained from six in duplicate.
Hemodialysis Units in Curitiba, PR, Brazil (Figure 1).
The collections were performed monthly Total coliforms
from October 2009 to November 2010. The The total coliforms counts were assessed by
sampling points were previously disinfected by the most probable number (MPN) method9. 10-mL
applying 70 % alcohol to faucet and 400 mL of aliquots of the samples were added to a set of 10 tubes,
water flowed freely after the treatment, to prevent each containing double-strength Lauryl Sulphate
sample contamination with the disinfectant utilized. broth added of blue bromocresol (Hymedia) and an
The samples were collected to sterile 250 mL glass inverted Durham tube. All tubes were incubated at
flasks after 30 seconds of free flow. To the flask for 35 oC ± 1 °C for 48 h. Following incubation, the tubes
the point 1 had been added 10 % sodium thiosulfate that were media acidification was observed and/
and the flasks for bacterial endotoxin analysis were or gas was noticed inside the Durham tubes were
previously depyrogenated. submitted to confirmation test for total coliform.
The microbiological analysis of the samples Each presumptively positive tube was subcultured
(217) were performed within 12-h after collection, into a Brilliant Green Bile 2 % broth (Hymedia)
at the Laboratory of Microbiology and Toxicology and incubated at 35 oC ± 1 °C for 48 h. Positive
of the Paraná Institute of Technology – TECPAR results indicate the presence of total coliforms
and LabMicro/UFPR. and the count was expressed as MPN/100 mL9.
The research was approved by the
Committee of Ethics in Research of Federal Endotoxins
University of Parana (CEP/SD: 799.134.09.09 The bacterial endotoxin test was
CAAE: 0059.0.091.091-09). performed using the Limulus amebocyte lysate
(LAL) by gel-clot limit test. Each sample was
Methods diluted usin water for Bacterial Endotoxins Test
Regarding the standard parameters until the maximum valid dilution (MVD), that was
for dialysis water in Brazil6, we performed the calculated from equation MVD = endotoxin limit
heterotrophic bacteria count test by pour plate for water for dialysis/λ [where λ is the labeled
method, total coliforms count test by multiple- sensitivity of LAL reagent in the gel-clot technique].
tube method in 100 mL9, and detection of bacterial Equal volumes of sample and LAL reagent were
endotoxin by the Limulus amebocyte lysate (LAL) transferred to test tubes, mixed and incubated at
method10. Although not necessary according to 37 oC ± 1 °C for 1 h in water bath, in order to verify
Brazilian standards, other parameters were also a firm gel formation at the bottom of the tubes.
evaluated: P. aeruginosa count test by multiple- Positive control for endotoxin production was CSE
tube method and fungi count by spread plate and Standard Endotoxin E. coli 055:B5. The results
membrane filter method9. were expressed by endotoxins units (EU)/mL10.

Heterotrophic bacteria count Pseudomonas aeruginosa

10 mL of the sample was added to a P. aeruginosa count was performed by
diluent to make a 1:10 dilution. 1-mL aliquots of MPN/100 mL9. A 10-mL aliquot of the sample was
the sample and the 1:10 dilution preparation were inoculated into 10 tubes containing double-strength
separately added to Petri dishes. The molten PCA asparagin broth (Hymedia). The inoculated tubes
medium (0.5 % peptone, 0.25 % yeast extract, 0.1 % were incubated at 35 oC ± 1 °C for 48 h. Tubes showing
glucose, 1.5 % agar; pH 7.0) was added to each turbidity and/or pigmentation were submitted to

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Figel IC, Dalzoto PR, Pimentel IC. Microbiological quality of water and dialysate from haemodialysis units in Southern Brazil. Rev Inst Adolfo Lutz. São
Paulo, 2015;74(1):66-70.

confirmation. 0.1 mL of cultures in positive tubes of filamentous fungi were inoculated on Sabourad
were inoculatedinto acetamide broth (Hymedia) and dextrose agar (Hymedia), incubated at 25 ºC ± 1 ºC,
onto the surface of cetrimide agar, all incubated at and the single-spore colonies were selected for
35 oC ± 1 °C for 48 h. The development of a purple color identification, carried out using macro and microscopic
at acetamide broth and a presence of a green fluorescent features. The assays were performed in duplicates.
pigment at cetrimide agar indicated the presence of
P. aeruginosa. The results were expressed in MPN/mL. RESULTS AND DISCUSSION

Fungi More than 95 % of the samples were

Fungi were counted by spread plate method considered in accordance with the standards required
and by membrane filter technique9. by valid Brazilian legislation in 2009 in regard of
heterotrophic bacteria counts. All samples resulted
Spread plate method in absence of total coliforms (< 1.1 MPN/100 mL),
10 mL of the sample was added to a as predicted by legislation. Abbas et al12 analyzed
diluent to make a 1:10 dilution. 0.1-mL aliquots total coliforms in 225 samples from hemodialysis
of the sample and the 1:10 dilution preparation units in Greece and observed that 4,9 % of the
were separately transferred onto Petri dishes samples assayed showed counts not acceptable for
containing Potate Dextrose Agar (Hymedia) the stablished standards.
added of tartaric acid 10 %, pH 3.5, and the P. aeruginosa was recovered from 4 % of
samples were spread over the surface of the the samples (8/217) and this result confirm De
culture medium using a sterile Drigalski spatel. Filippis et al3 suggestion that P. aeruginosa as an
The plates were incubated at 25 oC ± 1 °C additional parameter for assuring the quality of water
for 5 days (faster growth fungi) and 20 days for dialysis.
(slower growth fungi). The experiment was conducted 15% of the samples showed bacterial
in duplicates. endotoxin > 2 EU/mL. However, 32 % of the samples
showed values < 0.125 EU/mL, in accordance to the
Membrane filter method parameters established by Brazilian legislation and
100 mL of sample were filtered through a standards from Italy, Sweden, Germany and Norway.
sterile 47-mm, 0.45-µm-pore-diam membrane filter. Hyaline yeasts were recovered from 26 % of
The membrane was placed on PDA medium, the samples (57/217) and filamentous fungi, from
pH 3.5 (Hymedia) supplemented with tartaric acid 58 % of the samples (125/217) - 46 % characterized
10 % in Petri dish, that wasincubated at 25 ºC ± 1 ºC as melanized fungi and 27 % as hyalines.
for 15 days. Colonies were enumerate and the results The higher isolation was observed when the
were expressed CFU/100 mL9. Positive cultures membrane filter method was employed (Table 1).

Table 1.Percentage of yeasts and filamentous fungi verified in water for dialysis using spread plate and membrane
filter methods
Spread plate Membrane filter
Collecting points
Yeasts
All points 9 % (19/217) 26 % (56/217)
Point 1 (tap water) 1 % (1/72) 12 % (9/72)
Point 2 (dialysis water) 14 % (10/72) 39 % (28/72)
Point 3 (dialyzate) 14 % (8/58) 31 % (18/58)
Point 4 (dialysis water) 0 % (0/15) 7 % (1/15)
Filamentous fungi
All points 21 % (46/217) 52 % (112/217)
Point 1 (tap water) 31 % (22/72) 61 % (44/72)
Point 2 (dialysis water) 18 % (13/72) 60 % (43/72)
Point 3 (dialyzate) 16 % (9/58) 36 % (21/58)
Point 4 (dialysis water) 13 % (2/15) 27 % (4/15)

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Figel IC, Dalzoto PR, Pimentel IC. Microbiological quality of water and dialysate from haemodialysis units in Southern Brazil. Rev Inst Adolfo Lutz. São
Paulo, 2015;74(1):66-70.

3. De Filippis P, Spitaleri G, Damiani F, Panà A. Microbiological

Despite of the fungi contamination
quality of haemodialysis water in various hospitals and
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the most of the international legislation do not
Pubbl.2007;63(1):21-9.
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Trichoderma spp, Acremonium spp. and Differences in the permeability of high-flux dialyzer membranes
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fungi from the water inlet of the municipal 6. Brasil. Resolução RDC nº 154, de 15 jun. 2004. Estabelece o
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