alvina 95 ABSTRACT Microbiological procedures for V. cholerae isolation from clinical specimens are important factors in clinical and epidemiological management of cholera. The standard preservation medium for enteric pathogenic bacteria, including V. cholerae, is Cary-Blair medium (CB), a semisolid medium for preservation and transport of specimens containing intestinal bacteria. A special medium for Vibrio organisms is alkaline peptone water (APW), which is both a transport and an enrichment medium. The purpose of this study was to ascertain the suitability of APW supplemented with 0.5% agar (APW-0.5) as a sensititive preservation- transport medium for rectal swab specimens for isolation of V. cholerae. A total of 144 paired rectal swab specimens were collected from children and adults with acute diarrhea. Of each specimen pair, one was placed in CB and the other in APW-0.5, from which they were plated out to thiosulfate citrate bile sucrose (TCBS) agar. Altogether, from both CB and APW-0.5 transported specimens, V. cholerae non-O1 was present in 29 (20.1%) specimens, while only 2 (1.4%) specimens were positive in CB and 9 (6.3%) positive in APW-0.5 transported specimens. The number of V. cholerae non-O1 isolates from APW-0.5 transported specimens was significantly higher (p=0.000) as compared to that from Cary- Blair transported specimens. It may be concluded that for isolation of V. cholerae, specimen transport in APW-0.5 medium was more effective than transport in Cary-Blair medium. Keywords: Alkaline peptone water, agar, Vibrio cholerae, rectal, swab *Department of Pharmacology, **Department of Public Health, ***Department of Microbiology, Medical Faculty, Trisakti University Jakarta Correspondence dr. Meiyanti, SpFK Department of Pharmacology, Medical Faculty, Trisakti University Jl. Kyai Tapa No.260 Grogol - Jakarta 11440 Phone. 021-5672731 ext.2805 Univ Med 2011;30:95-101. Alkaline peptone water plus 0.5% agar suitable for transport of Vibrio cholerae Meiyanti*, Oktavianus Ch. Salim**, Julius E. Surjawidjaja***, and Murad Lesmana*** May-August, 2011May-August, 2011May-August, 2011May-August, 2011May-August, 2011 Vol.30 - No.2 Vol.30 - No.2 Vol.30 - No.2 Vol.30 - No.2 Vol.30 - No.2 UNIVERSA MEDICINA INTRODUCTION Cholera is an acute diarrheal disease caused by serogroups O1 and O139 of V. cholerae. In its severest form the disease is characterized by profuse diarrhea with rice water stools, rapidly leading to dehydration. Cholera has two special epidemiological f e a t u r e s , n a m e l y i t s t e n d e n c y t o c a u s e explosive epidemics, frequently at several foci simultaneously, and its capability for causing p a n d e m i c s t h a t p r o g r e s s i v e l y i n v o l v e numerous parts of the world, as has been the case up to the present.(1-3) 96 Meiyanti, Salim, Surjawidjaja, et al Alkaline peptone water for Vibrio cholerae In several countries this disease has become endemic, from time to time causing epidemics.(1,4-6) It is estimated that around 5.5 million cholera cases occur annually in Asia and Africa, 8% of these being of such severity as to need hospitalization, with 20% of these hospitalized cases terminating fatally, giving rise to a total annual mortality of 120,000.(7) Consequently cholera is still considered to be a serious health problem in many developing c o u n t r i e s w i t h p o o r s o c i o - e c o n o m i c c o n d i t i o n s . (8,9) C h o l e r a s u r v e y s a r e s t i l l i m p o r t a n t i n s t r u m e n t s f o r d e t e r m i n i n g behavioral trends of the disease in many areas around the globe and within a given country, in view of the fact that the majority of the reported numbers of cases are underestimates.(8,9) Among the enteric pathogenic bacteria V. cholerae is probably the easiest organism to identify, for which however a laboratory infrastructure is required. The accuracy of laboratory investigations, including the efficient use of material such as culture media,(10,11) has a considerable impact on the exactness of bacterial identification and the ensuing accuracy of the report. Although treatment of cholera cases is not based on prior identification of the causal organism, the microbiological procedures for the isolation of V. cholerae organisms from clinical specimens are important factors determining the clinical and epidemiological management of cholera.(7) The specimens, either from stools or rectal swabs, are collected at the earliest opportunity or at onset of the disease, prior to administration of antibiotics. If the specimens cannot be processed within two hours, they are placed in preservation media.(7,12) The most common preservation medium used for enteric pathogenic bacteria, including V. cholerae, is Cary-Blair medium, which is a semisolid medium for preservation and transport of specimens containing intestinal bacteria. A special medium for Vibrio organisms is alkaline peptone water (APW), which is both a transport and an enrichment medium.(7,12) However, APW is almost never used for transport of Vibrio organisms, due to concern that longtime storage of specimens in APW at room temperature encourages overgrowth by organisms such as Pseudomonas, Proteus and Vibrio non-O1, which are capable of inhibiting growth of V. cholerae.(7,11) In contrast, there are reports stating that specimens enriched in APW and incubated for more than 20 hours did not show suppression of Vibrio isolation rates by growth of commensals.(7) As a consequence, APW may presumably be used for transport of V. cholerae within a period of 20-24 hours, yielding a substantially high sensitivity of isolation, due to the enrichment features of APW. Concern that APW in its liquid form is subject to spillage, resulting in contamination and infection by specimens containing V. cholerae, may be countered by the addition of 0.5% agar, thus transforming APW into a semi-solid medium. The purpose of this study was to ascertain the suitability of APW supplemented with 0.5% agar (APW-0.5) as a safe and sensititive preservation-transport medium for rectal swab specimens for isolation of V. cholerae. METHODS Study population Subjects recruited for this study were patients with diarrhea attending the Mampang Prapatan primary health center in South Jakarta. The subjects were categorized as having diarrhea on the basis of a self-reported frequency of defecation of 3 or more times within 24 hours, with watery/liquid/soft stools.(8,10) Before collection of the fecal specimens, the subjects were informed on the purpose of the study and were requested to sign a consent form for participation in the study. In the case of children, the consent form would be signed by their caregivers. Subsequently a clinical questionnaire was to be filled out by research personnel with data obtained by interviewing the subjects, comprising data on age, gender, duration of diarrhea, type of diarrhea, and clinical symptoms. 97 Preservation and transport medium Standard Cary-Blair medium was prepared according to the manufacturer’s instructions, while APW supplemented with 0.5% agar (APW-0.5) was prepared by adding 10 g peptone, 10 g sodium chloride, and 5 g agar to 1 L of distilled water (pH 8.4). Specimen collection and transport The stool or rectal swab specimens were collected prior to administration of antibiotics. Rectal swabs were obtained by means of cotton swabs mounted on sticks. For subject comfort, the cotton swabs were wetted in transport medium before rectal insertion. To obtain a rectal specimen, the cotton swab was inserted past the anal sphincter (around 2-3 cm), slowly rotated and withdrawn, and immediately placed in transport medium. From each patient one pair of rectal swabs was taken, one of the pair being placed in Cary-Blair medium and the other in APW-0.5 medium. The specimens were then stored at room temperature prior to being sent to the Microbiology Laboratory of the Medical Faculty, Trisakti University. In the case of patients with prior antibiotic therapy, this fact w a s r e c o r d e d i n t h e q u e s t i o n n a i r e f o r consideration in the subsequent data analysis. Bacteriological procedure On arrival at the laboratory the rectal swab specimens in Cary-Blair and APW-0.5 were immediately plated out on thiosulfate citrate bile salts sucrose (TCBS) agar, a selective medium for Vibrio organisms. The TCBS plates were then incubated aerobically at 370C for 18-20 hours. Bacterial growth resembling Vibrio colonies were picked and processed according to the following standard procedures for identification of V. Cholerae,(8,12) viz. oxidase t e s t , b i o c h e m i c a l r e a c t i o n s ( c o m p r i s i n g Kligler’s iron agar, sucrose, semisolid, motility, i n d o l e , o r n i t h i n e , l y s i n e , a rg i n i n e ) , a n d serological tests with specific antiserum for determination of V. cholerae serotypes. Statistical analysis The significance of the difference in transport media was assessed by comparing the proportions of positive specimens. A p value of <0.05 was considered to indicate statistical significance. Computations were performed by means of Epi Info version 3.5.1. RESULTS Within a period of 10 months a total of 144 paired rectal swab specimens were collected from children and adults with acute diarrhea. From these paired specimens, Vibrio species were isolated from 44 (30.6%) rectal swab specimen pairs, comprising 40 isolates (90.9%) of V. cholerae non-O1 and 4 isolates (9.1%) of V. parahaemolyticus, without any isolates of V. cholerae O1 organisms being found in this study. There were therefore 100 (69.4%) paired rectal swab specimens with negative results, yielding no isolates of Vibrio species (Table 1). Among the rectal swab specimens in Cary- Blair medium, V. cholerae non-O1 organisms were present in 21.5% (31/144) specimens and V. parahaemolyticus in 2.8% (4/144) specimens. In comparison, the rectal swab specimens transported in APW-0.5 yielded the following results: V. cholerae non-O1 was present in 26.4% (38/144) specimens, while the frequency Table 1. Number of isolates of V. cholera non-O1 and V. parahaemolyticus recovered from 144 patients with diarrhea Number of isolates (%) N Bacterial isolate V. cholerae non-O1 V. parahaemolyticus Positive 44 40 (90.9%) 4 (9.1%) Negative 100 - - Univ Med Vol.30 No.2 98 Meiyanti, Salim, Surjawidjaja, et al Alkaline peptone water for Vibrio cholerae of V. parahaemolyticus isolates was identical to the Cary-Blair isolates (Table 2). Table 2 also s h o w s t h a t 2 9 ( 2 0 . 1 % ) s p e c i m e n p a i r s transported in Cary-Blair and APW-0.5 yielded positive results for V. cholerae non-O1, while only 2 (1.4%) rectal swab specimens were positive in Cary-Blair and 9 (6.3%) positive in APW-0.5 transported specimens. Results of M c N e m a r ’s t e s t i n d i c a t e d a s i g n i f i c a n t difference between number of isolates of V. cholerae in Cary-Blair and that in alkaline p e p t o n e w a t e r ( p < 0 . 0 0 5 ) . I s o l a t e s o f V. c h o l e r a e i n A P W ( 9 / 4 0 = 2 2 . 5 % ) w e r e substantially higher in number than isolates in CB (2/40 = 5%) (Table 3). DISCUSSION The specimens used for isolation of V. cholerae are stools or rectal swabs, collected at the earliest opportunity or at onset of the disease, prior to administration of antibiotics, as the numbers of V. cholerae organisms in stools decline immediately upon antibiotic a d m i n i s t r a t i o n . ( 8 ) T h e s p e c i m e n s s h o u l d preferably not be collected from bedpans, because they may have been contaminated by p r e v i o u s u s e o r m a y c o n t a i n t r a c e s o f disinfectants after cleaning, leading to false positive or false negative results. For collecting stools, sterile stool cups may be used. Rectal swab specimens give as good results as stools in the isolation of V. cholerae, particularly in the acute phase of the disease.(5) In addition, rectal swabs specimens are superior to stools for infants and field surveys, but are inadequate for convalescents or contacts.(7) Incubation for 6-8 hours is the method recommended for APW,(5) and is used in the majority of laboratories.(8,13,14) In the initial 6- 8 hours of incubation, Vibrio organisms show a rapid growth, but more than 8 hours of incubation is considered to result in growth of competing bacteria and inhibition of Vibrio organisms, impeding isolation of the latter. H o w e v e r, r e c e n t r e s e a r c h p o i n t s t o t h e contrary, in that enrichment in APW for 24 hours, although promoting the growth of nonvibrio organisms such as Proteus to greater numbers than Vibrio organisms, yet did not decrease the growth of V. Cholerae.(4) In the present study it was found that after 24 hours there was a large number of both non-vibrio and Vibrio colonies on the culture plates, the latter thus being easily picked and identified. Enrichment in APW for 6-8 hours does not yield significantly better results in comparison with enrichment in APW for 24 hours, with r e g a r d t o c a p a b i l i t y f o r i s o l a t i o n o f V. cholerae.(15) It may be concluded that APW is superior as to frequency of isolation of Vibrio species, when compared to Cary-Blair medium. CB APW + - + 29 2 - 9 104 Table 3. Number of isolates of V. cholerae non-O1 in Cary-Blair (CB) and alkaline peptone water (APW) + 0.5% agar Culture results V. cholerae non-O1 V. parahaemoluticus CB APW +0.5% Number % Number % Positive Positive 29 20.1 4 2.8 Positive Negative 2 1.4 0 0 Negative Positive 9 6.3 0 0 Negative Negative 104 72.2 1 40 97.2 Table 2. Distribution of culture results of V. cholerae non-O1 and V. parahaemolyticus from 144 rectal swab specimens in Cary-Blair (CB) vs. alkaline peptone water (APW) + 0.5% agar 99 This is presumably because APW is both an enrichment and a transport medium. In its original form, APW is a liquid medium. Supplementation with 0.5% agar minimizes s p i l l a g e o r l e a k a g e , t h u s p r e v e n t i n g e n v i r o n m e n t a l c o n t a m i n a t i o n b y e n t e r i c pathogens. However, the results of transport of V. cholerae in agar-supplemented APW for a period of more than 24 hours cannot as yet be predicted. There is a possibilty that after 48 hours the commensal organisms start to reproduce rapidly, thus inhibiting V. cholerae, ultimately leading to negative isolation results for V. cholerae.(12) In that case, specimen transit times in APW for days or even for more than o n e w e e k , a s i s c o m m o n w i t h c l i n i c a l specimens in Cary-Blair medium, will cause APW to be ineffective as transport medium for V. cholerae. In Cary-Blair medium Vibrio organisms remain viable for 4 weeks, without any possibility of inhibition by excessive growth of commensal organisms.(12) According to this line of reasoning, Cary-Blair medium is to be preferred for transport of stool or rectal s w a b s p e c i m e n s f o r i s o l a t i o n o f e n t e r i c pathogenic bacteria, including V. cholerae, whereas enrichment-transport media are to be used only when the field-collected specimens can be cultured within 12-24 hours after collection.(7,12) If it can be demonstrated that specimen transit times in enrichment-transport media of more than 48 hours or even more than one week does not impair the viability of enteric pathogenic bacteria such as Vibrio organisms, use of such media for preservation and transport of Vibrio organisms may be taken into consideration. The present study succeeded only in isolating V. cholerae non-O1 at a frequency of 27.8%, whereas no V. cholerae O1 were found. This may be due to the drastic decline in the V. cholerae O1 population in Indonesia since 2000, from 10.5% - 18.3% in the period of 1993-1998(16) to < 2% after the year 2000,(17) for unknown reasons. V. cholerae O1 could probably be isolated with a larger sample. However, because V. cholerae O1 and V. c h o l e r a e n o n - O 1 h a v e i d e n t i c a l c u l t u r e characterisitics, the results on the viability of V. cholerae non-O1 in Cary-Blair and APW may be assumed to refer also to V. cholerae O1. Both strains may be said to differ only in serology, clinical picture, and epidemiology. Serologically, V. cholerae non-O1 does not show agglutination with anti-O1 antiserum.(12) With respect to clinical manifestations, Vibrio O1 causes profuse diarrhea, while V. cholerae non-O1 causes a milder diarrhea, but tends to r e s u l t i n e x t r a - i n t e s t i n a l i n f e c t i o n s a n d bacteriemia.(7,19) Epidemiologically, V. cholerae O1 frequently causes epidemics, whereas V. cholerae non-O1 is sporadic and non-epidemic in nature.(18) Reports on evaluations of APW as an enrichment and transport medium for V. cholerae have never been encountered after the year 1997,(15) making it difficult to obtain descriptions of more recent studies on APW. In spite of this, there have presumably been few changes since the last published reports on this topic. However, the discovery of a new variant of V. cholerae O1 that is a hybrid of the classic and El Tor biotypes(20-23) poses a new threat for the occurrence of epidemics of pandemic proportions. Therefore, there should be increased efforts at maximal isolation of V. cholerae, particularly V. cholerae O1, using various culture and transport systems. The efficacy of enrichment in APW is reportedly higher for V.cholerae non-O1 as compared with O1 strains. Enrichment in APW r e s u l t s i n s i g n i f i c a n t l y h i g h e r y i e l d s o f V.cholerae non-O1 isolates, in comparison with direct plating.(15) Our study results are similar to those of previous studies on the evaluation of enrichment in APW for isolation of V. cholerae, demonstrating the superiority of enrichment to direct plating. Consequently, enrichment media with features of transport media, such as APW, have a higher effectivity a n d s e n s i t i v i t y f o r i s o l a t i o n o f Vi b r i o organisms. Univ Med Vol.30 No.2 100 Meiyanti, Salim, Surjawidjaja, et al Alkaline peptone water for Vibrio cholerae One limitation of the present study is the relatively short transport time of the specimens in APW (less than 48 hours), which cannot describe the ultimate fate of commensal organisms in the APW. Further long-term studies are necessary to resolve this question. CONCLUSION For isolation of V. cholerae, specimen transport in APW-0.5 medium is significantly more effective than transport in Cary-Blair medium. ACKNOWLEDGEMENTS The investigators thank the Dean and Vice- D e a n s o f t h e M e d i c a l F a c u l t y, Tr i s a k t i University, for the funding of this study. Thanks are also due to the doctors and staff of the Mampang Prapatan primary health center, and to all assisting parties for the successful completion of this study. REFERENCES 1. Simanjuntak CH, Larasati W, Arjoso S. Cholera in Indonesia in 1993-1999. Am J Trop Med Hyg 2001;65:788-97. 2. Lesmana M. 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Cholera due to altered El Tor strains of Vibrio cholerae O1 in Bangladesh. J Clin Microbiol 2006;44:4211-3. Univ Med Vol.30 No.2