Comparison of the extraction efficiencies of different leaching agents for reliable assessment of bio-accessible trace metal fractions in airborne particulate matter

A. Mukhtar^{1, 2} and A.Limbeck¹

¹ Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria,

alimbeck@mail.zserv.tuwien.ac.at

²Division of Science and Technology, University of Education, Lahore, Pakistan

ch_mazam@hotmail.com

Abstract

In present study, an in-vitro physiologically based extraction test has been applied for extraction of bio-accessible trace metal fractions in airborne particulate matter (APM) samples collected from different urban sites in Austria and Pakistan using the leaching agents H₂O, sodium chloride, ammonium acetate, ammonium citrate, synthetic gastric juice and artificial lung fluids. Obtained extracts were then measured using an ETV-ICP-OES procedure which allowed highly sensitive measurement of dissolved analytes even in the presence of leaching agents. Derived results indicated that the investigated leaching agents extract different amounts of trace metals. In general, leaching agents with organic nature yielded comparatively greater extractable and thus bio-accessible trace metal fractions to that of simple solvents like H₂O or aqueous NaCl solution. With water, only 26.3±4.0% of Cd was found to be bio-accessible whereas 88.4±24.8 of Cd was obtained as bio-accessible fraction with the use of synthetic gastric juice. The concentrations of bio-accessible metal fractions varied from 0.4 ng m^-3 (Cd) to 714 ng m^-3 (Zn) and 0.3 ng m^-3 (Cd) to 190 ng m^-3 (Zn) for PM10 samples collected from Karachi (Pakistan) and Graz (Austria) respectively.

Key words: Bio-accessibility; trace metals; airborne particulate matter; synthetic body fluids

Introduction

In recent years, many toxicological studies have implicated metal contents as a possible harmful component of APM (Prieditis and Adamson, 2002) since they can be absorbed into human tissues during breathing especially particles with an aerodynamic diameter less than 10 microns (PM10). The toxicity of metal depends upon its species that are present. Thus, for risk assessment of metal toxicity, it is important to determine bio-accessible concentrations instead of total metal contents (Michelozzi et al., 1998). 

Particles in the 2.5- to 10-μm size fraction are in most cases deposited in the tracheal and the bronchial region after inhalation, from where they are transported within hours by the so-called mucociliary clearance adoral and are mainly swallowed. This fraction reaches the gastrointestinal tract (GIT), where it comes into contact with gastric juice (Hamel et al., 1998). On the other hand particles less than 1 µm can enter into the alveolar region of lungs where they interact with the lung fluid (Jianjum et al., 2010 and Song et al., 2011). For risk assessment of metal toxicity the determination of bio-accessible fractions is recommended. For this purpose extraction procedures with different leaching agents such as water, buffer solutions or synthetic body fluids were reported in literature. Since the chemical composition of these leaching agents is different it is expected that inconsistent amount of bio-accessible metal fractions is obtained via the use of applied leaching agents. Therefore, it is highly needed to evaluate the extraction efficiencies of various commonly used leaching agents for true estimation of bio-accessible trace metal fractions in APM.

In present study, an in-vitro physiologically based extraction test (PBET) was employed for extraction of APM samples from Graz (Austria) and Karachi (Pakistan) with the leaching agents water, sodium chloride, ammonium acetate, ammonium citrate, synthetic gastric juice, artificial lysosomal fluid and Gamble solution, followed by measurement of resulting extracts using electrothermal vaporization inductively-coupled plasma atomic emission spectrometry (ETV-ICP-OES). Derived results were discussed in order to gain more detailed information about the extraction behavior of the investigated leaching agents.

Materials and Methods

Reagents and materials

High purity water was obtained by purifying de-ionized water (reverse osmosis/ion exchange combination Euro 20 plus, SG Water Systems, Germany) with an Easypure 2 system (Thermo Barnstead, USA). All used chemicals and reagents were of analytical reagent grade and were procured from Merck (Darmstadt, Germany). Pepsin from porcine stomach mucosa (456 units mg−1 solid) was purchased from Sigma Aldrich (Chemie GmbH, Steinheim, Germany). Certified multielement standard solutions of In, As, Ba, Cd, Co, Cu, Mn, Ni, Pb and Zn (1000 mg/L) were purchased from Merck (Darmstadt, Germany) and diluted with 1% HNO₃ (v/v) in order to prepare various working standard solutions. A 1 mol l^-1 solution of ammonium acetate was prepared by dissolving 77.0825 g weight in one litre of bi-distilled water and pH of the solution was maintained at 7. Similarly, 0.1 mol l^-1 ammonium citrate solution was prepared by dissolving 22.619 g in one litre of high purity water and pH of the solution was maintained at 4.4 with the use of HCl. The synthetic gastric juice was prepared according to a US Pharmacopeia methodology by dissolving 2 g NaCl in 50 ml of high purity water followed by addition of 7 ml of conc. HCl in order to maintain pH of resulting solution at 2-2.5. To this solution, 3.2 g of pepsin were added, dissolved well and finally the volume was make up to 1 litre using bi-distilled water. Artificial lysosomal fluid and Gamble solution were prepared according to Colombo et al., 2008. Indium at level of 1 ppm was also added to the prepared leaching agents as an internal standard in order to overcome non-spectral interferences as well as errors related to manual sample handling steps. The prepared leaching agents were stored in refrigerator at 4°C until further use.

Instrumentation

An iCAP 6500 series ICP-OES spectrometer (Thermo Scientific, USA) has been used for simultaneous multielement analysis of As, Ba, Cd, Co, Cu, Mn, Ni, Pb, Zn. For sample introduction by electrothermal vaporization, an ETV system model 4000A ETV (Spectral Systems, Fürstenfeldbruck, Germany) was used corresponding in essence to a longitudinally heated graphite tube furnace. A detailed description of instrumentation and optimized method parameters can be found in Mukhtar and Limbeck, 2011.

Collection of PM10 samples

Sampling of size segregated APM samples (PM10) was performed at an urban site in Karachi, during March-April 2009 (20 * 25 cm) using high volume sampler, with an intake volume of approximately 1223 m³. Whereas sampling at Graz was done during July-August, 2006 (147 * 147 mm) with the help of an automated sampling device (Leckel, Germany) containing a PM10 pre-separation head, with an intake volume of Graz samples was 650 m³.   Quartz fiber filters (PALL Life-sciences, Michigan, USA) were used as sampling substrates.

In-Vitro physiological based extraction test (PBET) and determination of residual/total metal contents

For determining the bio-accessible trace metal fraction present in APM, an in-vitro physiological based extraction test was performed. For this purpose, aliquots with a diameter of 10 mm were punched out from each collected PM10 sample. Six punches from each PM10 sample were taken into pre-cleaned polypropylene tubes followed by addition of 700 mg of leaching agent. From each sample three replicates have been prepared with each type of leaching agent. Closed tubes were treated in an ultrasonic bath (Sonorex TK30, Bandelin, Germany) at 37°C for 1 h in order to extract soluble trace metal fractions. After cooling down the sample solutions to room temperature, the derived extracts were centrifuged (Hettich, Zentrifugen-EBA 20) at 5000 rpm for 10 min for separation of undissolved material and remaining filter substrate . The supernatant clear sample solutions were transferred to new 3 ml polypropylene tubes and stored until further analysis.

The remaining eight aerosol filter punches (diameter 12 mm) were used for determination of total trace metal contents. For sample digestion the filter punches were transferred into pre-cleaned Bernas type Teflon lined bombs followed by addition of 1 ml conc. HNO₃, 1 ml HCl and 50 µl of HClO₄.  The Teflon lined bombs were then placed in indigenously developed refractory oven and treated at 130 °C for 1 h in order to dissolve total metal contents. Finally, the temperature of the refractory oven was increased to 150 °C and maintained for other 30 min for evaporation of excessive amount of HNO₃ and HCl. After cooling the insoluble filter material including the small droplet of HClO₄ remaining in the Teflon lined bombs were transferred in new PP tubes and . diluted to a final mass of approximately 2 g with 1% (v/v) HCl. Simultaneously a defined amount of In as internal standard was added. After centrifugation the supernatant solutions were removed and stored in new PP tube at 4°C until ananlysis.

ETV-ICP-OES Analysis of standard solutions and PM10 samples

Measurement of standard solution and prepared PM10 extracts was carried out according to Mukhtar and Limbeck, 2011. Briefly, 40 µl of the prepared extracts/digests were pipetted into precleaned graphite boats and dried using an IR-lamp. For analysis, the graphite boats were inserted into the graphite furnace tube of the ETV system and the furnace program was started by the ICP-OES software and the emission spectra of the vapor introduced into plasma was measured.

Results and Discussion

Total metal concentrations in PM10 samples from Karachi ranged from few ng m^-3 to some hundred ng m^-3. The highest concentrations among the measured elements were observed for Zn ranging from 361 ng m^-3 to 918 ng m^-3 whereas lowest concentrations were observed for trace element Cd varying from 1.9 ng m^-3 to 4.2 ng m^-3. As and Co revealed results below their detection limits i.e., less than 0.5 ng m^-3. These results were found in accordance to literature findings as reported by Venkataraman et al., 2002, and Salam et al., 2003 for mega south Asian cities Mumbai and Dhaka respectively. Similar observations were found for PM 10 samples collected from Graz, with concentrations of Zn varying from 71 ng m^-3 to 300 ng m^-3 and Cd concentrations ranging from 0.9 to 1.5 ng m^-3. These findings were in agreement with the results reported in literature from various sites in central Europe. For example, Limbeck et al., 2009 have reported concentrations of Cd and Zn in the order of 0.1 ng m^-3 and 200 ng m^-3 respectively in PM10 samples collected from various urban sites in Vienna. The results indicated clearly that atmosphere of Karachi is significantly more contaminated with toxic trace elements as compared to Graz.   

Bio-accessible fractions were found to be lower than the corresponding total metal concentrations, indicating that only a fraction of metal is soluble in various leaching agents. Since bio-accessibility test has been performed with different leaching agents, variable amounts of bio-accessible trace metal fractions have been released which reflect differences in their ionic strength and composition. Therefore a question arises about the trueness of bio-accessible fractions. It was found that lowest bio-accessible trace metal fractions were obtained with the use of water and NaCl as compared to leaching agents with organic composition like synthetic gastric juice and artificial lysosomal fluid (Figure 1a and 1b). However, the Gamble solution released comparable quantities of bio-accessible trace metal fractions to that of water and NaCl. Furthermore, the low leaching ability of Gamble solution as compared to

 

 

Fig. 1 Trace metal fractions extractable with different leaching agents (%), results are average of twenty investigated PM10 samples.

synthetic gastric juice and artificial lysosomal fluid could be explained on the basis that it is neutral (pH_˜7), thus the interaction of Gamble solution with PM10 metal particles is not so aggressive. In contrast, synthetic gastric juice and artificial lysosomal fluid being having complex organic nature and acidic pH causes the metals to release easily, thereby posing serious health risks when trace fractions become part of body fluid. It can also be deduced from above figure that the metal extracting behavior of applied leaching agents is quite similar for PM10 samples collected from two entirely different sites i.e., Graz (Central Europe) and Karachi (South Asia), indicating that bio-accessibility is element as well as leaching agent dependent.

Using synthetic gastric juice for assessment of bio-accessible trace metal fractions concentrations in PM10 ranging from 0.4 ng m^-3 (Cd) to 714 ng m^-3 (Zn) were found in aerosol samples collected in Karachi, whereas  bio-accessible trace metal concentrations in PM10 samples from Graz varied from 0.3 ng m^-3 (Cd) to 190 ng m^-3 (Zn). Comparison of bio-accessible fraction in PM10 samples reported in present study with literature data is not possible, since current study is carried out for the first time where an attempt has been made to provide a guideline for estimation of actual bio-accessible trace metal fractions in APM.

Conclusion

In this study an attempt has been made for the first time in order to propose a model for the estimation of true bio-accessible trace metal fractions in APM which could be used as a guideline for future studies. In present study, an in-vitro PBET was applied for extraction of bio-accessible metal fractions present in APM using various leaching agents followed by subsequent measurement of gastric extracts using a recently developed ETV-ICP-OES procedure. The obtained results indicated severe differences in the extraction efficiencies of the investigated leaching agents. Highest bio-accessible trace metal fractions are obtained with the use of synthetic body fluids, lowest results were observed for water and sodium chloride solution. Generally it was found that the presence of organic complexing agent as well as acidic conditions improve the solubility of trace metals significantly. Therefore, for future studies it is highly recommended to use synthetic body fluids for estimation of bio-accessible trace metal fractions in APM, since they enable a more reliable assessment of bio-accessible trace metal fractions in PM10 than pure inorganic solutions.

               

Acknowledgements

Azam Mukhtar acknowledges the Higher Education Commission (HEC), Pakistan and the Austrian Exchange Service (ÖAD) for providing a Ph.D. scholarship for the period 2007-2011.

References

Limbeck A, Handler M, Puls C, Zibral J, Bauer H and Puxbaum H. Impact of mineral components and selected trace metals on ambient PM10 concentrations. Atmos Environ 2009; 43: 530-538.

Mukhtar A and Limbeck A. Development of an ETV-ICP-OES procedure for assessment of bio-accessible trace metal fractions in airborne particulate matter, J Anal At Spectrom 2011; 26: 2081-2088.

Colombo C, Monhemius A J and Plant JA. Platinum, palladium and rhodium release from vehicle exhaust catalysts and road dust exposed to simulated lung fluids,  Ecotoxicology and Environmental Safety 2008; 71: 722–730.

Prieditis H and Adamson IYR. Comparative pulmonary toxicity of various soluble metals found in urban particulate dusts, Exp Lung Res 2002; 28: 563-572.

Jianjun N, Rasmussen P E, Hassan NM and Vincent R. Concentration Distribution and Bioaccessibility of Trace Elements in Nano and Fine Urban Airborne Particulate Matter: Influence of Particle Size, Water Air Soil Pollut 2010; 213: 211–225.

Michelozzi P, Forastiere F, Fusco D, Perucci CA, Ostro B, Ancona C and Palloti G. Air Pollution and Daily Mortality in Rome, Italy, Occup Environ Med 1998; 55: 605-610.

Hamel SC, Buckley B and Lioy PJ. Bioaccessibility of metals in soils for different liquid to solid ratios in synthetic gastric fluid, Environ Sci Technol 1998; 32: 358–362.

Song S, Lee K, Lee Y M, Lee JH, Lee S, Yu SD and Paek D. Acute health effects of urban fine and ultrafine particles on children with atopic dermatitis, Environ. Res., 3 (2011), pp. 394-399.

Salam A, Bauer H, Kassin K., Mohammad US, and Puxbaum H. Aerosol chemical characteristics of a mega-city in southeast Asia (Dhaka-Bangladesh), Atmos Environ 2003; 37: 2517-2528.

Venkataraman C, Reddy CK, Josson S, and Reddy M S. Aerosol size and chemical characteristics at mumbai, india, during the indoex-ifp (1999), Atmos Environ, 2002; 36: 1979

Management of male urethral discharge

Patient complains of urethral discharge or dysuria

If history of receptive oral or anal

sex take 2 swabs of throat or anus

1. NAAT Chlamydia

2. MC&S

Take history, examine patient

YES Visible urethral discharge present? NO

Immediate treatment for Gonorrhoea and Chlamydia

Azithromycin 1g and amoxycillin* 3g, and probenecid* 1g orally

(*use ceftriaxone 250mg IMI instead if partner from outside Top End or Central Australia)

Education and counselling. Promote / provide condoms

Arrange full check-up and same treatment for sex partner/s

Follow up test results

Review in one week to give results of tests and ensure symptoms have resolved and contact tracing done

If symptoms not better re-examine, re-test. Consult with Medical Officer at local Sexual Health Unit

SHBBVU Guidelines May 2008

33

Management of epididymo-orchitis

(swollen painful testes)

Causes

The causes of epididymo-orchitis vary with age.

Among younger sexually active men gonorrhoea and chlamydia are common (usually <35

years but can be <45 years in Indigenous men in remote communities).

Among older men, organisms associated with urinary tract infections (

E. coli, Pseudomonas,

Klebsiella

the cause.

Many references use age 35 years as a cut off point to guide treatment. However, for men

living in remote NT communities or whose sexual networks relate to remote communities

with high rates of gonorrhoea and chlamydia, 45 years may be more appropriate.

species) occur. It is not possible to tell by clinical examination which organism is

History, examination and tests

Ask about:

•

suddenly, or whether there was any trauma preceding the pain;

how long the pain and swelling have been present, whether it started slowly or

•

any nausea or vomiting;

•

recent symptoms of urethral discharge or dysuria;

•

other possible STI symptoms (eg. sores, warts or lumps, rash, sore throat);

•

sexual partners and if any of them are from outside the NT.

Examination

Look for urethral discharge and fever especially (see Male examination, p 10).

In all men with epididymo-orchitis

•

trichomonas; and

Take a first void urine for gonorrhoea culture and NAAT for chlamydia and

•

a midstream urine for MC&S.

For men in whom a urethral discharge is present regardless of age

Take 2 swabs of the discharge:

•

media); and

First swab for MC&S (roll onto glass slide and let dry in air and put swab in transport

•

pathology company instructions (dry tube for PCR, transport media tube for TMA, see

p 13).

Torsion of the testis can be very similar to epididymo-orchitis and is an emergency.

If there is any doubt about the diagnosis, the patient should be referred immediately to

hospital. Nurses and Aboriginal health workers should always consult with a doctor.

Second swab for NAAT for gonorrhoea, chlamydia and trichomonas according to

34

For all men under 35 (under 45 if remote community connection

)

•

HbcAb)

Take a blood test for syphilis serology, HIV +/- Hepatitis B (HBsAg, HBsAb, and

See hepatitis B, p 14.

Treatment

If there is any doubt about the diagnosis, consult with a specialist and consider

referring immediately to hospital to rule out torsion of the testis.

For men of any age in whom a urethral discharge is present or for men under 35 years of

age (under 45 years in remote communities) gonorrhoea or chlamydia are likely causes.

•

ceftriaxone 250mg IMI stat; and

azithromycin 1g

orally stat; and

doxycycline 100mg

bd for 14 days.

•

For men over 35 years of age (over 45 years in remote communities) in whom a urethral

discharge is not present a urinary tract infection is the likely cause.

Repeat the azithromycin on day 8 if any doubt about ability to take the doxycycline.

•

cephalexin 1g bd for 14 days, or

•

amoxycillin+clavulinate 875/125mg bd for 14 days, or

•

norfloxacin 400mg bd for 14 days, or

•

ciprofloxacin 500mg bd for 14 days.

If allergic to the medication, consult the local SHU.

For all men

•

Paracetamol for the pain and advise rest in bed.

•

Wearing firm underpants can support the scrotum and help reduce the pain.

•

infections found.

Check the results of the tests for other infections and the antibiotic sensitivity of any

•

consider sending to hospital.

See him on days 3 and 8 - if he is not improving, consult with the local SHU and

For men treated for an STI

•

given single dose treatment for gonorrhoea and chlamydia.

Ensure that all sexual partner/s from the last 3 months are checked for STIs and

•

to reduce their risk of pelvic infection and infertility.

Explain that all partner/s need to be treated as well so he doesn’t get re-infected and

•

treatment.

Advise him not to have sex until 1 week after both his treatment and his partner/s

•

Discuss condoms and safe sex.

•

give

On day 8 if there is any doubt about whether he is able to take all the doxycycline,azithromycin 1g by mouth once again.

For men treated for a urinary tract infection

•

Check the organism found on culture and its antibiotic sensitivity.

•

When the infection is resolved, arrange renal investigations (eg. ultrasound).

SHBBVU Guidelines May 2008

35

Differential diagnosis: Scrotal swelling: epididymo-orchitis

or torsion of the testis?

Scrotal swelling may be due to either epididymo-orchitis or torsion of the testis. It can be very

difficult to tell the difference between the two on clinical grounds. Torsion of the testis

requires surgery and, if there is a delay of more than several hours, can lead to loss of the

testis. Therefore a quick decision is needed.

Nurses and Aboriginal Health Workers should always consult with a doctor. Doctors should

consider consulting a specialist.

If there is any doubt about the diagnosis refer immediately to hospital.

The table below provides an outline to assist in making a diagnosis.

Torsion

Epididymo-orchitis

Age

Can be any age but more

usually under 1 year or

between 10-25 years.

Rare in boys before puberty.

Usually young sexually active

men or older men.

Onset

Usually sudden but can

be gradual. Sometimes

related to recent trauma.

Gradual.

Pain

Always present usually

moderately strong or severe.

Usually mild to moderate.

Other symptoms

May have abdominal pain,

and vomiting.

May have abdominal pain,

dysuria or urethral discharge.

Fever

Either no fever or less than

37.5

Usually more than 37.5

but may be absent.

On examination

Scrotum often swollen,

red and warm. Testicle within

the scrotum also swollen and

tender. Affected testicle may

be sitting higher than the

other and/or lying sideways.

Scrotum often swollen, red

and warm. Testicle within

the scrotum also swollen

and tender. Urethral discharge

may be present. May be nitrites

on urinalysis.

Effect of lifting the scrotum

Either no change in pain

or worsens the pain.

May relieve the pain.

oC.oC

36

Take 2 swabs of discharge if present:

MC&S and NAAT Gono/Chlamydia/Trichomonas

Take separate urines:

First-Void Urine MC&S and NAAT

Gono/Chlamydia/Trichomonas and

Mid-Stream Urine for MC&S

Take blood for HIV, syphilis serology, +/-Hep B

(if not immune and can do follow up see p 14)

Take separate urines:

First-Void Urine for MC&S and

NAAT Gono/Chlamydia/ Trichomonas and

Mid-Stream Urine for MC&S

Cephalexin 1g bd for 14 days, OR

amoxycillin+clavulinate 875/125mg

bd for 14 days, OR norfloxacin

400mg bd for 14 days, OR

ciprofloxacin 500mg bd for 14 days

Review on Days 3 and 7. Check test results.

If not improving consider sending to hospital

Management of epididymo-orchitis

Nurses and Aboriginal health workers

should always consult with a doctor

Paracetamol

Rest in bed

Scrotal support

(eg firm underpants)

Consider torsion of testis: if any doubt,

Take history, examine patient

send immediately to hospital

Visible urethral discharge present

or patient under 35 years ?

(under 45 in remote community)

YES

Probable STI

NO

Probable urinary

tract infection

Azithromycin 1g orally and ceftriaxone 250mg IMI

and doxycycline 100mg bd for 14 days

Review on Day 3: if no improvement consult with

local Sexual Health Unit and consider sending to

hospital. Check test results

Review on Day 7. If no improvement send to

hospital. If improving, give azithromycin 1g if any

doubt about compliance with doxycycline

Education and counselling

Promote / provide condoms

Arrange full check-up and treatment

for gonorrhoea and chlamydia for sex partner/s

When infection resolved, arrange renal

investigations

Patient with swollen, red, painful scrotum

SHBBVU Guidelines May 2008

37

Management of genital ulcers

Causes

The common causes will vary with the population group.

Among non-Aboriginal people the commonest cause is herpes although syphilis does occur.

Among Aboriginal people syphilis is relatively common especially in remote communities

although herpes also occurs. Donovanosis is mainly found in remote communities and while

increasingly rare, still occurs.

The possibility of malignancy as a cause of genital ulceration should not be overlooked

especially if there is poor response to treatment. Increased rates of vulval carcinoma have

been observed in the East Arnhem region in recent years.

History and examination

•

How long the ulcers have been present and have they had them before?

•

discharge, dysuria, rash, sore throat)

Ask about other possible STI symptoms (eg. warts or lumps, urethral or vaginal

•

Central Australia.

Ask about sexual partner/s and if any of them are from outside the Top End or

•

See Sexual history p 5.

Examination

It is important to look inside the vagina with a speculum in women, under the foreskin and

scrotum in men and in the peri-anal region in both sexes.

(see Male examination, p 10; Female examination, p 11).

Genital Herpes

May present as multiple, painful or itchy small blisters, which become ulcers, then scabs and

then heal. There may be tender lymph nodes in the groin. The first or primary episode is

always the most severe episode and can last 2-3 weeks. It is often associated with flu like

symptoms and headache and there can be severe localised genital swelling, pain and

retention of urine, requiring hospitalisation. Herpes can recur. If so, the ulcers are not usually

as severe and heal within a week.

Syphilis

Usually presents as a single (occasionally 2)

rolled edge and the base of the ulcer is firm (‘indurated’) although they can often be atypical

in appearance. Without treatment the sore will go away in 4-6 weeks but the person will still

have syphilis.

painless ulcer/s which are red, round with a

Donovanosis

Usually presents as a red, beefy, raised, raw, painless lesion. Can be painful if secondary

infection is present. Without treatment the ulcer will not heal, and can spread slowly outwards

and become very large, eroding normal tissue. Ulcer/s may be present for months or years. It

can be difficult to tell syphilis and donovanosis apart.

These infections can vary greatly in the way they look. It is frequently not possible to

tell by looking which organism is the cause.

38

Investigations

Do a full STI check

•

herpes, syphilis and donovanosis.

(For herpes like sores: if blisters are present gently burst with a sterile needle and

swab the fluid, for other sores just swab the sore or scab).

Using a dry swab, swab the base of the ulcer. REQUEST: genital ulcer NAAT for

•

REQUEST: MC&S and NAAT chlamydia, gonorrhoea and (in women only)

trichomonas. (If doing a speculum examination in a woman taking endocervical and

high vaginal swabs is better).

Take 2 self collected vaginal swabs or a urine test in women and a urine test in men.

•

HbcAb).

Take a blood test for syphilis serology, HIV +/- Hepatitis B (HBsAg, HBsAb, andSee hepatitis B, p14.

Treatment

Treatment should be given straight away - do not wait for test results to come back.

•

below).

If the sores are completely typical of herpes, then manage as for herpes only (see

•

p 39).

If not completely typical of herpes, then manage as for syphilis and donovanosis (see

•

Talk to your local SHU about any pregnant woman with genital ulcers.

Herpes management

Primary herpes

Primary herpes is the person’s first episode of genital herpes.

•

daily for 5 days.

Give valaciclovir 500 mg twice daily for 5 - 10 days, or famciclovir 125 mg 3 times

•

them (or any drying agent).

Keep the sores clean with salt water washes and/or put betadine on the sores to dry

•

Give paracetamol 2 tablets by mouth every 4 hours as needed for pain.

•

prior to passing urine.

Lignocaine gel may be helpful during the first few days to reduce pain particularly

•

Note: Both valaciclovir and famciclovir require authority prescriptions and are approved for

recurrent herpes. Both are effective in primary herpes but the PBS will only authorise

valaciclovir for primary episodes.

Talk to your SHU if the woman is pregnant or can’t pass urine.

Recurrent herpes

Recurrent episodes are usually less severe and heal within a week. Usually only keeping

them clean and mild pain relief is needed. If the episodes are more severe or frequent,

specific treatment may be needed. For maximum benefit, treatment should be commenced

within 24 hours of the onset of symptoms.

•

5 days

If the person is getting 6 or more recurrent episodes per year, they may benefit from taking

long term daily medication to prevent or reduce the recurrences. Seek advice from your local

SHU.

Give valaciclovir 500 mg twice daily for 3 days or famciclovir 125 mg twice daily for

SHBBVU Guidelines May 2008

39

Follow up at 1 week

•

A positive test confirms genital herpes.

•

if the sores come back).

A negative test does not exclude genital herpes (ask them to return for another swab

•

Provide herpes information and advice about safe sexual behaviour.

•

Offer sexual partners information on herpes and a full STI screen.

Any woman who has herpes or whose partner has herpes should be advised to tell

their doctor of this if they get pregnant in the future (risk of neonatal herpes).

Blood tests for antibodies to herpes are available. However, their interpretation and

application to the clinical situation is complex. Practitioners are advised to seek advice from

the local Sexual Health Unit medical officer before considering their use.

Syphilis and donovanosis management

•

Give benzathine penicillin IMI 1.8gm or 2.4 million units.

•

Azithromycin 1gm orally.

•

the same treatment.

Contact trace all sexual partners in the last 3 months and offer a full STI screen and

Syphilis and donovanosis are notifiable diseases. Contact your local SHU if treating

for syphilis or donovanosis.

Follow up at 1 week

•

Check test results.

•

If the ulcer is still present or the donovanosis test is positive keep giving

azithromycin 1g

orally once a week until the sore has healed. An alternative is

azithromycin 500mg

per day for 7 days (there is less evidence for this treatment).

•

If possible examine the ulcer each week until it is fully healed.

•

biopsy to investigate other causes.

The ulcer must be examined at 4 weeks. If no response to treatment consider a

•

for a recurrence.

Examine the person at 3 months and 6 months after treatment is completed to look

•

for RPRs on both specimens to be ‘run in parallel’.

If syphilis was diagnosed, repeat syphilis serology 6 months after treatment and ask

Safe sex advice

•

condoms do not always cover the ulcer. Advise the person not to have sex until the

sores have healed.Advise the person about safer sexual practices and condom use. Remember

Management of lower abdominal

Management of lower abdominal

pain / pelvic inflammatory disease

Woman complains of lower abdominal pain

Education and counselling

Promote / provide condoms

Arrange full check-up and

treatment for gonorrhoea,

chlamydia and trichomonas

for sex partner/s

If no VE and no history of discharge,

consider treatment if age < 35 and

other risk indicators present : deep

dyspareunia, intermenstrual

bleeding, STI or PID in past

12 months

Take history, examine patient

Take 2 endocervical swabs: MC&S, NAAT Gono/Chlamydia/Trichomonas

Take 1 high vaginal swab: MC&S

Take blood for HIV, syphilis serology, +/-Hep B (if not immune and can do follow up see p 14)

If dysuria or urinary frequency present take a mid-stream urine for MC&S

YES

YES

NO

NO

Manage for other conditions

Are any of the following present?

Missed, overdue or delayed period

Recent delivery, miscarriage or abortion

Abdominal guarding, rigidity or rebound tenderness

Abdominal mass or swelling

Active vaginal bleeding

Patient is pregnant

Patient is very unwell or Temp >38°C

Day 1: ceftriaxone 250mg IV/IM and azithromycin 1g oral

Day 2: start doxycycline 100mg bd and metronidazole 400mg bd for 14 days

(daily roxithromycin 300mg if breastfeeding instead of doxycycline; see p 27 re doxycycline)

Immediate transfer to hospital

Review on day 3: if no improvement, send to hospital

Review on day 8: if no improvement send to hospital

Repeat azithromycin 1g

Continue medications to day 14

Review on day 14 with full examination. Consult

local SHU or gynaecologist if not fully improved

*

*

30

Management of male urethral discharge or

dysuria

Causes

Usually caused by gonorrhoea or chlamydia but is sometimes caused by trichomonas or

other organisms.

It is not possible to tell by clinical examination which organism is the cause

.

History, examination and tests

•

sore throat, see p 5).

Ask the man about other possible STI symptoms (eg. sores, warts or lumps, rash,

•

outside the Top End or Central Australia (see p 14).

Ask about his sexual partners in the past 3 months and whether any of them are from

Examination

(see Male examination, p10)

Investigations

(see Investigations-males, p 15)

Do a full STI check

If discharge is present

Take 2 swabs (no need to swab inside the penis):

•

swab in transport media);

First swab for MC&S for gonorrhoea (roll onto glass slide and let dry in air and put

•

pathology company instructions (dry tube for PCR, transport media tube for TMA, see

p 13);

Second swab for NAAT for gonorrhoea, chlamydia and trichomonas according to

•

for gonorrhoea, chlamydia and trichomonas.

If discharge is not present, send a first void urine for gonorrhoea culture and NAAT

If history of receptive oral or anal sex

Take 2 swabs of throat or anus:

•

First swab for MC&S for gonorrhoea,

•

Second swab for NAAT for chlamydia.

For men over 40 years of age who have dysuria and no discharge

•

Collect midstream urine MC&S to check for a urine infection (UTI);

•

Blood test for syphilis serology, HIV +/- hepatitis B (HBsAg, HBsAb, and HbcAb)

See hepatitis B, p 14.

SHBBVU Guidelines May 2008

31

Treatment

Treat immediately for gonorrhoea and chlamydia even if no discharge is present.

•

Give oral amoxycillin 3g and probenecid 1g and azithromycin 1g once only.

•

If allergic to penicillin contact the local Sexual Health Unit.

•

Australia (see p 14) - then give

instead.

If he has had a recent sexual partner who is from outside the Top End or Centralceftriaxone 250mg IMI and azithromycin 1g orally

•

given the same treatment.

Make sure that sexual partner/s from the last 3 months are checked for STIs and

•

Explain that all partner/s need to be treated as well so he doesn’t get re-infected.

•

treatment.

Advise him not to have sex until 1 week after both his treatment and his partner/s

•

Discuss safe sex and condom use.

Follow up

•

test results for other infections and discuss safe sex and condom use again.

If resources permit, ask him to come back in 1 week to be sure he is better, check the

•

If no improvement, discuss with the local SHU.

If he has symptoms 1 week after treatment

It may be re-infection, resistant infection, trichomonas or another organism.

•

- if trichomonas is present then treat him and his partner/s with 1 dose of

Check the results of the tests taken initially:

metronidazole 2g

- if culture for gonorrhoea was positive, check the antibiotic sensitivity.

or tinidazole 2g orally (don’t give tinidazole to pregnant women),

•

back or never got better in the first place.

If he did not have trichomonas, ask whether his symptoms got better and then came

•

Check the original treatment was taken properly. Repeat if it was not.

•

Make sure all sexual partners were tested and treated.

•

If re-infection is likely, repeat the STI check-up and treatment.

•

(see p 14).

Ask if he had sex with someone from outside the Top End or Central Australia

•

necessary to do an intra-urethral swab to confirm a diagnosis of urethritis and

maximise the chance of culturing gonorrhoea (see technique below).

Talk with the local SHU about what further tests or treatment are needed. It may be

Doing a urethral swab (ie. from inside the penis)

If an antibiotic resistant gonorrhoea is suspected then another specimen for MC&S is

needed.

•

If discharge is present take a swab of it for MC&S for gonorrhoea.

•

- moisten the tip of a thin urethral swab with sterile saline (ie. the wire stem swab not

the wooden stem swab);

- gently insert the tip of the swab 1-2cm into the urethra, leave it in place for a few

seconds and then withdraw it.

If there is no discharge then:

•

Gently roll the swab on a glass slide and let dry in the air.

•

Put the swab in transport medium (charcoal is best but Stuarts medium can be used).

•

Keep the swab at room temperature: do not refrigerate or let it get too hot.

•

Write ‘MC&S for gonorrhoea’ on the form and get it to the lab as soon as possible.

32

Take blood for HIV, syphilis serology, +/-Hep B

(if not immune and can do follow up see p 14)

Take 2 swabs of discharge for:

MC&S, NAAT Gono/Chlamydia/Trichomonas

Take first void urine for:

MC&S, NAAT Gono/Chlamydia/Trichomonas

Management of male urethral discharge

Patient complains of urethral discharge or dysuria