Did you look for Trichomonas vaginalis (protozoan -genital flagellate)?

Motile trophozoits can be seen in microscopic examination of discharge

If there is delay pap smear also can be done

giemsa stain also is usefulin demonstration

Culture can be done by using CPLM medium

Dear Dr Kondapaneni 

Case had about 5 litr discharge. In direct wet smear there was no sign of protozoan, also smear for gram, modified zeil nelson and zeil nelson was negative and was full of WBC. Molecular 16srRNA was negative for bacteria but discharge seems like bacterial discharge, by my  knowledge in protozoan cases it should be chocolate shape!! I am woundring what I have to do for further diagnosis???? 

Are you sure, it's infectious? Can you rule out a malignancy?

van it be a malignancy? it appearance is looking like infectious, no smell, white, full of WBC, nothing more in direct smear, no responses to antibiotics (Vanco), no growth aerobic anaerobic fungi. I am wondering what I can do furthermore for this patients in my lab?!

I used general 16srRNA for possible Viable but unculturable strains, I am worry that low number of bacteria may cause negative result but control isolate(K.pneum) was positive?! our Infectious disease specialist told me on his believe it is infectious and not malignancy!?!? 

site of infection was not mentioned. Was it vaginal discharge?

It is peritoneal abscess, direct smear for protozea is negative and fubi culture in SCC is negative. 

yes, he was treated with vancomycin, but it dosent effect on the procedure of treatment and also if there was any bacteria it should be raised in 16srRNA test.  

Dear Eva

In fact, I am looking for a way to find its etiological agent, if 16srRNA is not able to identify bacterial etiological agent so what I can do then, currently there is a publication on low power of PCR and culture on detection of bacterial etiologic agents of cellulitis (Clin Infect Dis. 2015 Aug 3. pii: civ655), I think my case has a bacterial agent but how I can find it, Big problem!!!!!???

Dear Eva

We sent it for LJ culture, because of safety issues we don't do it in our lab, but If antibiotics killed infection agents, shouldn't be bacterial debris and dead cells there and shouldn't we detect their genome by 16srRNA??

again thanks for your kind replies.

Hello,

I'm really interested in this kind of difficult cases. Eva Hamilton answer is a really good one, plus I have some questions for you:

The WBC are all Neutrophyl?

Have you considered other agents, like toxins, maybe produced in the intestinal tract that spreads into the blood but with no bacteria spread?

I also like to know more about the clinical hystory of your patient.

Thank you in advance,

Ramona.

Dear Romana

WBCs were almost Neutrophyl, we didnt consider any other possible agents because it appearance was completely similar to infectious discharge, she was a 53 years old patient with pain in stomach and hospitalized in infectious disease department and discharges cleared by surgery and sample were sent to my department for diagnosis. Cultures for anaerobic, aerobic and fungi negative direct smear was clearly neutrophyl and nothings more (no bacteria or fungi) and PCR negative!!!!!! What we are worry is recurrence of abccess in somewhere else. Z_N negative and LJ culture reseults are not available yet. vancomycin had no effect for treatment and vanco plus sxt was the choosen treatment. What I got from the discourses here I have no any other way to reach diagnosis. 

Hi Hossein

Did you exclude the possibility of a Chlamydia trachomatis  infection? It's an obligate intracellular organism so wouldn't grow in the conventional  culture media and wouldn't be detected by 16srRNA  PCR detection since it is genetically very different from the  other Eubacteria.  

It really sounds like an abscess or recurrence somewhere else... As João Rodrigues said, could be an intracellular pathogen... Chlamydia, Rickettsia spp? Mycoplasma spp? Therapy with quinolones, macrolides and tetracycline should be recommanded in this case.

Let me know, this case is intriguing me.

Chlamydia trachomatis  and Mycoplasma spp possibility 

I didnt consider their possibility, but it sounds interesting and I will try to evaluate their possiblity since i didnt know that 16srRNA is not able to identify these pathogens.

I have an ampol remained in refrigerator, do you think staining will help me for diagnosis or culturing or PCR should be done??

 Can you try modified ZN staining? Try viral panel even though you said the WBC are neutrophils.

What do youmean by viral panel?? which viruses are you meaning??

I mean considering viral infections such as herpes simplex, CMV etc

staining is less sensitive. Better to try PCR. 

by your experiences,whichprimersare suitable for identifying Chlamydia and Mycoplasma infections?

Sorry,I do not have experience in the technique. 16s r RNA  is used for all bacteria.

There may be more specific gene for Chlamydia trachomatis which can be tried.

ex: Non variable portion of MOMP gene (plasmid coded).

I'm surprised that the 16s PCR is negative, as discharges are seldom sterile. However, such samples may be highly inhibitory. I would advise reanalysing the sample with a small amount (ca. 2 ng) of bacterial DNA added. If it still comes up negative, the sample is inhibitory (try further purification, or just dilute it). If it comes up positive, your sample is probably free of bacteria. 

Note however, that some bacteria may amplify poorly with the 16s primers. 

Are mycobacteria hardly identifying by 16srRna?i am looking to see how Lj medium will grow?becausr this patient have highly discharge around spinal column and distored vertebra it should be positive for bacteria in 16s however our positive contol was positive, brucella, mycobacteria, staph, gram negatives were possible agents, this patient us not able to walk ro move and no responses to vanco and methro, strated to treat with anti Tb drugs, after removing all discharges currently she is not bad but no any significant improvment